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ECOLOGÍA DE MACROALGAS MARINAS EXÓTICAS:
APROXIMACIÓN A LOS FACTORES QUE REGULA LA
COLONIZACIÓN DE CAULERPA CYLINDRACEA EN EL MEDITERRÁNEO Y SU
INTERACCIÓN CON LOS HÁBITAS BENTÓNICOS
(PRADERAS DE POSIDONEA OCEÁNICA)
Jaime Bernardeau Esteller
OCT · 2015
JAIME BERNARDEAU ESTELLER
Ecología de Macroalgas Marinas Exóticas:
aproximación a los factores que regulan la
colonización de Caulerpa cylindracea en el Mediterráneo
y su interacción con los hábitats bentónicos
(praderas de Posidonia Oceanica).
TESIS DOCTORAL
DEPARTAMENTO DE CIENCIAS DEL MAR Y BIOLOGÍA APLICADA
FACULTAD DE CIENCIASGRUPO DE ECOLOGÍA DE ANGIOSPERMAS MARINAS (GEAM)
CENTRO OCEANOGRÁFICO DE MURCIA. INSTITUTO ESPAÑOL DE OCEANOGRAFÍA
Ecología de Macroalgas Marinas Exóticas: aproximación a los factores que regulan la colonización
de Caulerpa cylindracea en el Mediterráneo y su interacción con los hábitats bentónicos
(praderas de Posidonia Oceanica).
JAIME BERNARDEAU ESTELLER
Memoria presentada para aspirar al grado de:
DOCTOR POR LA UNIVERSIDAD DE ALICANTE
MENCIÓN DE DOCTOR INTERNACIONAL
DOCTORADO EN CIENCIAS DEL MAR
Dirigida por:
Juan Manuel Ruiz Fernández
Investigador Titular del Instituto Español de Oceanografía
Codirigida por:
José Luis Sánchez Lizaso
Profesor Titular de Universidad de Alicante
Lázaro Marín Guirao
Investigador Titular del Instituto Español de Oceanografía
Exotic Marine Macroalgae Ecology:approach to factors that regulate colonization
of Caulerpa cylindracea in the Mediterranean Sea and interaction with benthic habitats
(Posidonia oceanica meadows)
A mi Luca y a mi amore
Al Mediterráneo
A la red de Posidonia oceanica de la región de Murcia
Y al Windsurf!
Quién podría vivir en la tierra
si no fuera por el mar
Luis Cernuda. El joven marino
Cuando Hans Reiter vio por primera vez un bosque de
algas se emocionó tanto que se puso a llorar debajo del
agua. Esto parece difícil, que un ser humano llore mien-
tras bucea con los ojos abiertos (…)
Roberto Bolaño. 2666
¿Por qué nos gusta el mar? Es porque tiene una podero-
sa capacidad para hacernos pensar cosas que nos gusta
pensar.
Robert Henri
Life is passing time as gracefully as possible
Miki Dora
Los ecosistemas reflejan el ambiente físico en el que se
han desarrollado y los ecólogos reflejan las propiedades
de los ecosistemas en que han crecido y madurado.
Ramón Margalef. Perspectivas de la teoría ecológica.
· Un punto indefinido entre Villaricos y San Juan de Terreros. 12:00 am (aproximadamente). Agosto de 1991.
Mi padre apaga el motor mientras el tío Pepe termina de afianzar el ancla. Un sol radiante y el mar
hecho una balsica. Otro día de otro verano eterno. Mientras mis primos y hermanos revoletean por la
zodiac, se tiran al agua y juguetean, mi padre y yo lo tenemos claro. Gafas, aletas y nuestros maltrechos
fusiles de pescasub amateurs. Por delante un par de horas buenas de buceo. De ese pausado y sin pre-
tensiones, seguros de que al día siguiente y al otro habrá más. Sigo a mi padre desde el claro de arena
donde hemos fondeado hacia unas rocas que quedan más hacia costa. Se mueve tranquilo, acostum-
brado a ese medio en el que ha pasado ya media vida, y como siempre con su fusil sin cargar, ya llegara
el momento, si es que tiene que llegar. Conforme nos acercamos a las rocas comienzo a ver como
todo el fondo está recubierto por un “alga” de hojas verduzcas alargadas. En mi corta vida de apneista,
acostumbrado a los fondos más desnudos y rocosos de la zona de Mojacar o al cascajo infinito marme-
roniense, no había visto algo igual. El “alga” forma un denso tapete que lo cubre todo y se mece al son
del mar, de forma embriagadora, en un baile sin fin que aleja tu mente de la realidad y la hace fluir.
· Facultad de biología de la Universidad de Murcia. Noviembre de 2000.
Deambulo por el hall de la facultad. Con la mente espesa después de una noche un poco pasada me
resisto a tirar para clase. Pierdo el tiempo entre charla y charla, como buscando una excusa que diluya
definitivamente la sensación de culpabilidad. Un cartel en la pared llama mi atención, es un anuncio
de la lectura de una tesis de un tal Juan Manuel Ruiz Fernández. No sé qué historias sobre Posidonia
oceanica. En el cartel una foto de una pradera y diversos artefactos que parecen sacados de una peli de
ciencia ficción de serie B (bueno, incluso C). Que disparate es este, pienso. Definitivamente es la excusa
perfecta que andaba buscando. La sala de grados está a tope y me acurruco en una butaca buscando
pasar desapercibido entre tanto fruto seco con estética al más puro estilo biólogo desarrapado. Al rato
aparece el Juan Manuel este, con unas gafas de pasta escandalosas, y comienza a hablar. Su discurso
me atrapa rápido, durante algo menos de una hora se suceden imagines de experimentos imposibles,
sombreros gigantes, graficas y mas graficas, términos y cosas que apenas soy capaz de entender. Foto-
síntesis, eutrofización, balances energéticos, carbohidratos, impactos. Cuando acaba, mi cuerpo apenas
sobresale de la butaca. Ha sido apabullante, brutal. Buf, esto debe ser ciencia, pienso.
· AP-7, a la altura de Villajoyosa. Marzo o quizás abril de 2005.
Otro día duro de agua. Es media tarde, el sol aun aprieta y la hora y pico de vuelta a Murcia desde Altae
se hace larga. De no ser por la conversación incesante y sincopada de Lázaro sería del todo insoporta-
ble. Sin embargo hoy hay algo distinto, su voz esta algo más apagada y fija la mirada al frente mientras
sujeta con fuerza el volante de su Ford Sierra. Por un momento hay un silencio que se lo come todo, y
entonces explota. Mientras alguna lagrima se escapa me cuenta que no puede seguir con este trabajo,
que por las noches no puede dormir bien, que aquello se aleja absolutamente de las razones que lo
han llevado a la investigación y que ser consecuente con los principios y valores de cada uno es muy
importante y al final es lo único que nos queda. Sus palabras golpean con fuerza en mi tierno cerebro
pseudocientífico, y ya nunca saldrán.
· Murcia. Diciembre de 2008.
Cena en familia en un restaurante de Murcia. A mitad de cena una llamada rompe la cordialidad. En la
pantalla del movil pone Rossss, y me da un cierto sobresalto por la hora (más allá de las 9 de la noche) y
el día (viernes). Por un momento pienso en no contestar, y seguir entre vinos y quesos, pero al final me
pueden los remordimientos y me levanto teléfono en mano. Rocío me cuenta que hay un contrato de un
año para un experimento que llevan en marcha en estos momentos sobre salinidad y Posidonia, y que
había pensado en mí. Por un momento pienso en mi trabajo actual, en mis horarios y en mi seguridad,
en mi edad un poco pasada para meterme en estos jaleos. Se me pasa por la cabeza decirle que nece-
sito un poco de tiempo para darle vueltas, tiempo para buscarme las excusas necesarias para pensar
que estoy mejor así. Pero ese día hay algo distinto, de mi interior nace una necesidad de aprovechar
esa oportunidad que durante tanto tiempo busqué. Este es mi momento, me digo, y entre risas (sobre
todo la de Rosss, que como siempre se lo come todo) me sorprendo a mi mismo diciéndole que cuenten
conmigo. Al volver a la mesa ataco sin miramientos, les cuento a todos las novedades, mi madre se
queda muda durante un momento, pero rápidamente fagocita sus miedos protectores y me dice, “si es
lo que te gusta y tu quieres, adelante”.
· Zulo del Geam. Un viernes cualquiera de 2010.
Hoy es viernes y toca Vangelis. El Chemi me pregunta las contraseñas del spotify. Esta mañana se ha
levantado innovador y duda entre la BSO de The Bounty o la de 1492. Le paro los pies, “Chemilín, déjate
de rollos y pon Blade Runner”. Por los altavoces suena ya imponente “Love theme” y nuestras mentes
despiertan. Diseños experimentales, análisis estadísticos, revisiones de textos, diseño y construcción de
estructuras, bandejas, filtros y cualquier otra cosa que sea necesaria. No hay desafío que pueda achan-
tar la mente de un molinero.
· Dirección general del Geam. Enero 2015.
- ¿Cómo se llama este grupo?
- Aranzazu, no me lo puedo creer. ¿Otra vez? si lo oímos ayer, y antes de ayer
- ¿Si? Uf, no me acuerdo, ¿Cómo se llama?
- Son de the Windy Hills
- Ah, es verdad, si lo tengo apuntado en mi lista. Este es el grupo del surfista, director de docus y demás,
¿no?
- Exactamente
- Oye
- Dime
- Luego pon el disco ese de Neil Young, ese tan tranquilito
-Jejeje, vale. Pero me tienes que hacer un mapa.
- ¿Otra vez?
- Si, pero esta vez c
· Una noche entre 2011 y 2015. 4 a.m.
Me despierto. Sobresaltado. Todavía es noche cerrada. Mi cabeza rápidamente se activa, llena de temo-
res, inseguridades. Vuelta hacia un lado, vuelta hacia al otro. La almohada estirada, con un doblez, con
dos. Boca arriba, boca abajo, de lado. Por un momento pienso en saltar de la cama. En ese momento
Maria me abraza, me susurra algo que no soy capaz de entender y me besa en la mejilla. Respiro pro-
fundamente. El sueño vuelve.
· Playa de las Cañas, Calblanque. 21 de junio de 2015.
Al final todo llega a su fin, y es hora de mirar atrás y agradecer a todos los que habéis estado ahí apo-
yándome, de una forma o de otra. Ya sabéis que mi memoria nos es muy buena, y algún nombre se me
pasará, pero no puedo dejar de acordarme especialmente de algunos de vosotros:
De mi familia, por supuesto, por todo.
Del Geam, al completo (eso va también por ti, Maridolis), mi otra familia, bueno, mi familia también.
De Jose Luis, por su confianza. Eternamente agradecido.
Del Charton, por ponerme en vereda y contribuir de una manera u otra que todo esto empezase.
De Juan y Tamara, me hubiera encantado compartir todo esto con vosotros hasta al final, mil gracias
por todos los momentos que pasamos juntos y por toda vuestra ayuda.
De Fiona, por su infinita paciencia.
Del equipo de la Universidad de Sassari, Giulia, Stefania, Prof. Cosu. ¡Que grandes días!.
Y de toda la plantilla del IEO, Fina, Colache, Iñaki , Vera, Julio, Fernando, Ricardo, Silverio, los pesqueros
de solera (Javi, Antonio, Angelopoulos), los pesqueros gastronómicos (Miguel, Ester, Encarni), los pes-
queros del más allá (Belli, la piccolina, Lolo, Iosu) y las pesqueras del más acá (Elena y Cris), la cúpula
dorada (Paco, Jorge, Mr. Rocamora, Lola, Geli), los contaminantes contaminados (Victor G y Victor L,
Juan Antonio, Juliana, Cristóbal, Pencho, Cristina, David, Emily, Carlos, Nané, Concha, Ines, Juanjo), los
molusquistas hermanos (Marina, Esmeralda, Paco, Diana, Carmen), Dani y sus mejunjes levanta espíri-
tus, las pobrecicas que han visto dar con sus huesos en la recepción (Alejandra, Encarna, Juani, Paqui,
Marina, Esther y la terremoto de San Javier, la mismísima Maria Antonia), los becarios (ufa, cuantos
han sido en estos años, ¡grandes!) super Rocio informatica (y su Nayara) y por supuesto mi queridísima
Mara (que ha tenido que soportar mi inmundicia día si y día también). Muchísimas gracias a todos por
vuestras sonrisas y compañía durante todos estos años. Espero que sigamos juntos muchos más.
Por último agradecer al Servicio de Pesca de la región de Murcia su confianza total en el proyecto “Red
de Seguimiento de las praderas de P. oceánica de la Región de Murcia”, marco en el que se engloban
todos los trabajos de esta tesis.
Introducción general
Chapter 1. Recent spread of the invasive alga Caulerpa
cylindracea (Bryopsidales, Chlorophyta) along the Medi-
terranean coast of the Murcia Region (SE Spain)
Chapter 2. Photosynthesis and daily metabolic carbon
balance of the invasive Caulerpa cylindracea (Chloro-
phyta:Bryopsidales) along a depth gradient
Chapter 3. Resistance of Posidonia oceanica seagrass
meadows to the spread of the introduced green alga
Caulerpa cylindracea: assessment of the role of light
Chapter 4. Photoacclimation of Caulerpa cylindracea:
light as a limiting factor in the invasion of native Medite-
rranean seagrass meadows
Discusión general
Conclusiones
Anexo. Assessment of long-term interaction between
the endemic seagrass Posidonia oceanica and Caulerpa
cylindracea in the Mediterranean Sea
Bibliografía
INDICE
01
19
31
45
69
95
121
113
135
INTRODUCCIÓNGENERAL
I N T R O D U C T I O N
p. 03
1. La introducción de especies: un fenómeno global
La dispersión de los organismos es un proceso natural implicado en los fenómenos de distribución y de-
sarrollo de la biodiversidad en el planeta. Sin embargo, la existencia de barreras naturales (p.e. geográ-
ficas), condiciona su capacidad colonizadora y determina la composición de la flora y fauna específicas
de cada región. La intervención humana en los ecosistemas ha permitido a muchas especies superar
estas barreras que impiden su dispersión, acelerando e intensificando los procesos de introducción de
especies a escala global. Los sistemas de transporte humanos, ya sea de forma voluntaria o involun-
taria, han favorecido la dispersión de cientos de especies fuera de sus áreas naturales de distribución,
fenómeno que se ha visto acelerado en los últimos siglos como consecuencia del importante desarrollo
tecnológico (Di Castri 1989). Como resultado de esta situación la biota del planeta se encuentra some-
tida a un proceso de cambio y homogenización sin precedentes (Crooks and Suarez 2006).
Una especie introducida o exótica puede ser definida como aquella que cumple las siguientes caracte-
rísticas, a saber, (i) coloniza nuevas áreas donde previamente no estaba presente, (ii) este nuevo rango
de distribución esta relacionado de manera directa o indirecta a la actividad humana, (iii) presentan
una discontinuidad geográfica con el área natural de distribución de la especie, y (iv) son capaces de
reproducirse dentro de estas nuevas áreas de distribución sin la ayuda del hombre (Carlton 1985, Bou-
douresque and Verlaque 2002). Cuando estas especies introducidas son capaces de transformar signi-
ficativamente la estructura y función de los ecosistemas receptores, amenazar su biodiversidad, y tener
incluso consecuencias a nivel socioeconómico y de la propia salud humana, se les considera Especies
Exóticas Invasoras (EEI) o simplemente especies invasoras (NISC 2006).
Las invasiones biológicas se manifiestan como el crecimiento masivo de las especies una vez han sido
introducidas. De todas las especies introducidas solo una pequeña fracción tiene potencial invasor y
puede ser considerada una amenaza real para la biodiversidad y el funcionamiento de los ecosistemas
afectados (Norse 1993, Carlton, 2000, Primack 2004, Mooney et al. 2005; ver revisión en Mack et al.
2000).
La identificación de las especies introducidas, la determinación de su potencial invasor, así como el
análisis de los patrones de propagación y de los mecanismos y factores que determinan su éxito en los
ecosistemas invadidos son un tema de interés central en ecología, no solo por sus implicaciones en la
gestión de los ecosistemas y recursos marinos (Rejmaneck 2000), sino también porque son una oportu-
nidad única para el estudio de procesos fundamentales relacionados con el funcionamiento de los eco-
sistemas. (ver revisión en Sax et al. 2005 y Cadotte et al. 2006). En efecto, el estudio de la capacidad de
las especies invasoras para establecerse en una comunidad y sus efectos sobre la biota autóctona han
proporcionado información sobre aspectos fundamentales de la Ecología, tales como el conocimiento
de los factores que limitan la distribución de especies (Richardson y De Bonos 1991) o la importancia
de la identidad de las especies y el papel que desempeñan en el funcionamiento de los ecosistemas
(Vitousek y Walker 1989).
p. 04 p. 05
INTRODUCCIÓN GENERAL
TESIS DOCTORAL
2. Los macrófitos marinos como objeto de estudio en los fenómenos de introducción de especies
Los ecosistemas marinos costeros están considerados como uno de los ambientes más afectados por
la introducción de especies (Carlton 1996). La presente tesis doctoral centra su interés en el estudio de
macroalgas introducidas que han demostrado un gran potencial invasor en las nuevas áreas marinas
costeras colonizadas (Schaffelke et al. 2006), como es el caso del clorófito Caulerpa cylindracea (Son-
der) (en adelante C. cylindracea) en el Mediterráneo.
En los ecosistemas marinos costeros los macrófitos (angiospermas marinas y macroalgas) son un com-
ponente clave de la estructura y funcionamiento de las comunidades bentónicas que integran dichos
sistemas. Este grupo taxonómico y funcional integra un elevado número de especies introducidas o
exóticas, entre las que destacan varias especies de algas verdes sifonales como Caulerpa taxifolia, Cau-
lerpa cylindracea o Codium fragile, con un potencial invasor muy elevado (Williamson y Smith, 2007).
La capacidad de estas especies de macroalgas invasoras de transformar los paisajes colonizados, y sus
posibles consecuencias ambientales y socio-económicas, ha motivado una especial preocupación por
parte de gestores, científicos y público general en las áreas costeras invadidas (p.e. Mediterráneo). A
pesar de ello, los estudios que analizan su impacto en los ecosistemas marinos costeros son hasta la
fecha escasos, están realizados a una escala espacial y temporal reducida, y abarcan un número muy
reducido de especies (Grosholz 2002, McQuaid and Arenas 2006).
También es muy limitado el conocimiento de los mecanismos y factores que determinan y controlan la
capacidad invasora de las macroalgas. Diversas características de estas especies de macroalgas invaso-
ras, como su elevada capacidad de propagación, tasas de crecimiento o plasticidad fenotípica, parecen
explicar dicho potencial y, por tanto, su habilidad para competir y desplazar las especies nativas (In-
derjit et al. 2006, Schaeffelke et al. 2006). Por otro lado, la capacidad de algunas de estas especies de
actuar como especies ingenieras (sensu Crooks 2002) puede provocar la aparición de profundos cam-
bios en las características de los ecosistemas receptores derivados de la alteración en mayor o menor
medida de los regímenes sedimentarios, las condiciones oceanográficas, la estructura del hábitat y/o
de la red trófica de dichos ecosistemas (Dukes y Mooney, 2004, Wallentinus y Nyberg 2007, Deudero
et al. 2011).
3. Dinámica de la introducción de algas invasoras
Igual que se ha descrito para la mayoría de las introducciones documentadas, en la dinámica de intro-
ducción de macrófitos marinos bentónicos se pueden definir cuatro fases diferenciadas según la escala
temporal y espacial en la que se desarrollan y las barreras y vectores implicados (Theoarides y Dukes
2007, Blackburn et al. 2011; Fig. 1):
(i) Fase de transporte: Esta fase implica el transporte interregional de la especie, asociado a deter-
minadas actividades humanas de forma accidental, involuntaria o deliberada, a través de largas
distancias, salvando barreras geográficas de diversa naturaleza. En el caso concreto de los macró-
fitos marinos las vías o vectores de entrada en esta fase están principalmente relacionadas con el
(i) transporte marítimo, ya sea formando parte del fouling en los cascos de embarcaciones y otras
estructuras marítimas o en aguas de lastre y, (ii) la acuicultura (Williams y Smith 2007). Los grandes
puertos son por tanto considerados una de las principales vías de introducción de este tipo de orga-
nismos marinos (Ruiz et al. 2000, Hewitt et al. 2004).
(ii) Fase de introducción: La fase de introducción afecta exclusivamente a especies transportadas de
forma deliberada y que son cultivadas o mantenidas en cautividad fuera de sus rangos naturales de
distribución. Estas especies se enfrentan a barreras físicas asociadas a sus formas de confinamiento
que limitan su introducción en la nueva región.
(iii) Fase de establecimiento: una vez la especie llega a una nueva región debe ser capaz de sobrevi-
vir y desarrollar tasas positivas de crecimiento que le permitan desarrollar poblaciones viables que
perduren en el tiempo. En la mayoría de los casos, estas nuevas especies no consiguen superar esta
fase y desaparecen (especies ocasionales), mientras que un número muy pequeño de especies in-
troducidas (ca. 10%, Williamson and Filter 1996) son capaces de desarrollar poblaciones viables y
naturalizarse (especie naturalizada/establecida). Las barreras a las que se enfrenta la nueva especie
y que pueden determinar su éxito de supervivencia y/o reproductivo son de naturaleza muy diversa
y están asociadas a factores relacionados con las propias características de la especie (p.e. tasas de
crecimiento o reproductivas), y propiedades abióticas y bióticas de la nueva zona (p.e. característi-
cas ambientales o fenómenos de resistencia biótica).
(iv) Fase de propagación: tras el establecimiento, la especie puede comenzar un proceso de dispersión
hacia regiones adyacentes a la zona de introducción, ocupando una mayor o menor variedad de
hábitats y condiciones ambientales y desarrollando en algunos casos un carácter invasor. Durante
esta fase, en el caso de los macrofitos y otros organismos marinos, además de los vectores comen-
tados en la primera fase, intervienen otros de carácter más local como por ejemplo las embarcacio-
nes deportivas, las redes de pesca o las corrientes marinas. En esta fase o etapa, la especie puede
extinguirse de forma natural tras un periodo de expansión muy activo, o bien puede persistir con
fluctuaciones más o menos intensas y regulares de su abundancia.
Fig. 1. Dinámica de la introducción de especies exóticas (adaptado de Blackburn et al. 2011)
p. 06 p. 07
INTRODUCCIÓN GENERAL
TESIS DOCTORAL
4. Factores que afectan a la introducción y al éxito invasor
La introducción de especies es un proceso multifactorial cuyo éxito depende de la interacción entre el
organismo introducido y el ecosistema receptor a lo largo de una escala espacial y temporal amplia
(Londsale 1999, Shea y Chesson 2002). A pesar de que han sido identificados como fenómenos al-
tamente idiosincrásicos y dependientes de las condiciones locales donde se producen (Meiners et al.
2004, McQuaid y Arenas 2009), se pueden diferenciar tres grandes grupos de factores que controlan el
éxito de los procesos de introducción e invasión (Londsale 1999, Theoarides y Dukes, 2007):
(i) el numero de eventos y tasas de introducción de organismos, denominado de forma general
como “esfuerzo de introducción”,
(ii) las características y atributos de las especies que son introducidas,
(iii) la susceptibilidad (o resistencia) de los hábitats a ser colonizados por la nueva especie.
En el caso particular de los macrófitos marinos, los principales atributos a los que se atribuye el éxito
de su introducción son una elevada capacidad de crecimiento, el desarrollo de fases microscópicas y
de resistencia, la presencia de mecanismos de reproducción sexual y/o asexual (esporas y propágulos)
que facilitan su dispersión, ciclos vitales poliploides y contenidos genómicos reducidos, así como una
elevada capacidad para mantener un estatus biológico adecuado en un amplio rango de condiciones
ambientales, o lo que es lo mismo, una amplia tolerancia ambiental (Smith y Walters 1999, Nyberg y
Wallentinus 2005, Inderjit y Drake 2006, Schaeffelke et al. 2006, Varela-Álvarez et al. 2012).
Las especies exóticas con una mayor tolerancia ambiental serán capaces de resistir condiciones cam-
biantes y/o desfavorables durante su transporte desde las zonas de origen (Hewitt y Hayes 2002, Hewitt
et al.2007). Por otro lado, esta mayor tolerancia puede dotar a la nueva especie de una mayor capaci-
dad de aclimatación a las condiciones ambientales de la nueva zona así como facilitar la colonización
de una gran variedad de hábitats y gradientes ambientales. Así, por ejemplo, la capacidad de crecer en
un amplio rango de temperatura e irradiancia ha sido identificado como uno de los factores determi-
nantes en la introducción de Caulerpa taxifolia en el Mar Mediterráneo (Meinesz et al. 1993), alga que
a pesar de su origen tropical ha sido capaz de colonizar las aguas costeras mediterráneas de marcado
carácter templado (al menos en sus fases iniciales). El grado de tolerancia ambiental y la capacidad de
aclimatación están condicionados por la variabilidad genética, o polimorfismo genético de la pobla-
ción, y por la plasticidad fenotípica individual del organismo, es decir, la capacidad para modificar sus
características fisiológicas, morfológicas o de su ciclo vital en respuesta a señales ambientales y dentro
de un periodo de tiempo inferior a una generación (Harvell 1986, Schlichting y Pigliucci 1998, DeWitt
y Scheiner 2004). Aunque la relación entre plasticidad fenotípica y éxito en el proceso de introducción
no ha sido todavía estudiada en profundidad en macrófitos marinos, existen numerosas evidencias de
su importancia en ecosistemas terrestres (Parker et al. 2003) y en ecosistemas acuáticos no marinos
(Hastwell et al. 2008, Hyldgaard y Brix 2012).
La mayor o menor susceptibilidad (o resistencia) de un hábitat o biocenosis a ser colonizado por una
especie exótica es el resultado de la acción simultánea de las condiciones abióticas del medio, del ré-
gimen de perturbaciones y las interacciones bióticas con las especies nativas (Londsale 1999, Davis et
al. 2000). Las perturbaciones del medio, tanto de origen natural como antrópico, pueden modificar la
resistencia de las comunidades nativas a la colonización de una especie introducida (Planty-Tabacchi et
al. 1996, Burke y Grime 1996, Arrontes 2002, Valentine y Johnson 2003, Bulleri et al. 2011). Esto puede
suceder, por ejemplo, como consecuencia de la alteración de la cantidad de recursos disponibles (p.e. a
través de la eliminación de posibles competidores o por el enriquecimiento del medio), o por los efectos
de las perturbaciones físicas sobre la estructura del hábitat (p.e. fragmentación de la vegetación bentó-
nica por la influencia de temporales) (Davis et al. 2000, Sánchez y Fernández 2006).
Respecto a las interacciones de la especie introducida con la biota nativa, éstas pueden actuar tan-
to inhibiendo como facilitando el proceso de introducción, a través de mecanismos diversos como la
competencia, la depredación, los daños derivados de patógenos, el mutualismo o la facilitación. Dos
grandes hipótesis han sido postuladas para explicar el efecto de las interacciones bióticas sobre la sus-
ceptibilidad de las comunidades nativas a la introducción de nuevas especies:
1. The Enemy Releases Hipotesis (ERH) según la cual la ausencia de enemigos naturales,
tales como predadores o patógenos permite el desarrollo de las especies introducídas en su
nuevo rango de distribución. Esta teoría fue formulada por primera vez por Darwin (1859) para
explicar como algunas especies que son consideraras raras o poco abundantes en sus áreas
originales son especialmente abundantes en otras áreas donde son introducidas.
2. The Biotic Resistance Hipotesis (BRH), propuesta por primera vez por Elton en 1958 es-
tablece, a grandes rasgos, que los fenómenos de competencia desarrollados por las especies
nativas pueden impedir la introducción de especies. Esta teoría esta fundamentada sobre la
idea de que los factores bióticos del medio interaccionan con las condicionas abióticas restrin-
giendo el nicho ecológico de las especies (Hutchinson 1957).
La teoría definida por Elton asume que aquellas comunidades con una mayor diversidad taxonómica
serán menos susceptibles a la introducción de especies como consecuencia de una utilización de los
recursos disponibles más eficiente y completa (hipótesis del uso complementario de los recursos según
Hooper (1998)). Sin embargo, a pesar de que estudios posteriores han corroborado esta idea (Naeem
et al. 2000, Stachowicz et al. 2007), en las últimas décadas diversas investigaciones no solo no han
encontrado una clara relación positiva entre diversidad especifica y resistencia a la invasión, sino que
en algunos casos identifican una relación negativa entre ambos factores (Dukes 2001, Stohlgren et al.
1999, Capers et al. 2007). Estos resultados contradictorios han llevado a sugerir la existencia de otros
factores clave que afectan a la vulnerabilidad de las comunidades nativas a la introducción dentro del
contexto de la interacción entre comunidades nativas y especies exóticas. Algunos investigadores han
postulado que son los atributos funcionales de una o pocas especies clave dentro de la comunidad,
y el estricto control que establecen sobre los recursos y factores ambientales, los que condicionan la
p. 08 p. 09
INTRODUCCIÓN GENERAL
TESIS DOCTORAL
disponibilidad de recursos a la especie exótica y que hace a las comunidades nativas competidores
superiores por dichos recursos. (Prieur-Richard and Lavorel 2000, Symstad 2000). Diversos estudios rea-
lizados en comunidades de algas bentónicas parecen apoyar esta hipótesis; dichos estudios sugieren
que el control que ejercen determinadas especies de macrófitos formadores de doseles vegetales (ca-
nopy-forming species) sobre factores primarios como la luz o el sustrato, constituyen un mecanismo
clave en el control de los procesos de introducción (Arenas 2006, Britton-Simmons 2006). Este tipo de
comunidades bentónicas actuarían, por tanto, como barreras naturales efectivas contra la introducción
y propagación de especies invasoras. El conocimiento del funcionamiento de estos hábitats nativos, y
la medida en la que transforman el medio es, por tanto, un aspecto clave para determinar la resistencia
de los ecosistemas receptores a la introducción de especies exóticas.
5. Macrófitos invasores del Mediterráneo
El Mediterráneo representa el 0,82% de la superficie total de los océanos del planeta, pero es considera-
do uno de los “puntos calientes” de la biodiversidad marina, siendo su biodiversidad global un 6,3% de
la estimada a nivel mundial (Coll et al. 2010, Costello et al. 2010). De todas las especies macroscópicas
marinas conocidas del Mediterráneo, un 8,9% son macrófitos, repartidos en 277 especies de algas par-
das, 657 algas rojas, 190 algas verdes y 5 angiospermas marinas, siendo un 22% de ellas endémicas de
este mar. En contraste con esta excepcional biodiversidad, estudios recientes indican que desde princi-
pios del siglo XX el número de especies exóticas se ha ido duplicando cada 20 años aproximadamente,
lo que sitúa al Mediterráneo como una de las regiones con mayores tasas de introducción a nivel global
(Boudouresque y Verlaque 2002). Teniendo en cuenta solo el número de especies de macrófitos intro-
ducidos, Williams y Smith (2007) identifican el Mediterráneo como la región más invadida del planeta.
Esta situación se explica en buena medida por la confluencia de múltiples vías de introducción como la
acuicultura, el denso tráfico marítimo y la conexión con el Mar Rojo (región Indopacífica en general) a
través del Canal de Suez (Galil 2009). De las más de 90 especies de macrófitos catalogadas como intro-
ducidas en el Mediterráneo, 10 han sido descritas como invasoras (según Otero et al. 2013 y Rodríguez
Prieto et al. 2013). Éstas son las algas verdes Caulerpa taxifolia, Caulerpa cylindracea, y Codium fragile
subsp. fragile, las algas rojas Acrothamnion preissii, Lophocladia lallemandii, Asparagopsis taxiformis,
Asparagopsis armata y Womersleyella setacea, las algas pardas Sargassum muticum y Stypopodium
schimperi y la angiosperma Halophila stipulacea.
6. Invasión de Caulerpa cylindracea en el Mar Mediterráneo
El género Caulerpa, incluido en la familia Caulerpaceae del orden Bryopsidales (Clase Ulvophyceae,
phylum Clorophycophyta) incluye 86 especies (Guiry y Guiry 2007) de las cuales 6 han sido descritas en
el Mar Mediterráneo:
(i) Caulerpa prolifera (Forsskål) Lamouroux, única especie del genero nativa del Mediterrá-
neo
(ii) Caulerpa scalpelliformis R. Brown ex (Turner), especie introducida detectada por primera
vez en las costas de Israel y Libia (Ryass 1941)
(iii) Caulerpa mexicana Sonder ex. (Kützing), especie introducida de origen lessepsiano (Ma-
yhoub 1976)
(iv) Caulerpa sertularioides (SG Gmelin), especie introducida proveniente del Mar Rojo (Ol-
sen et al. 1998)
(v) Caulerpa taxifolia (M. Vahl) C. Agardh, especie tropical introducida en las costas de Fran-
cia en los años 90 procedente de su cultivo en acuario (Jousson et al. 1998) y protago-
nista de episodios invasivos en zonas del Mediterráneo occidental (Boudoruesque 1995).
Recientemente, Jongma et al. (2012) han identificado en aguas del Mediterráneo una
nueva variedad procedente del suroeste de Australia y que ha sido denominada como
Caulerpa taxifolia var. Distichophylla
(vi) Caulerpa chemnitizia (Esper) J.V. Lamouroux . En este taxón se incluye un hibrido en-
tre las variedades conocidas anteriormente como Caulerpa racemosa var turbinata (J.
Agardh) Eubank y Caulerpa racemosa var. uvifera (C. Agardh) J. Agardh, presente al me-
nos desde 1926.
(vi) Caulerpa lamourouxii (Turner) C. Agardh (Forsskål) J. Agardh, especie ampliamente
distribuida en regiones tropicales y templadas de todo el planeta y cuya presencia se
conoce desde los años 50.
(vii) Caulerpa cylindracea (Sonder) [anteriormente conocida como Caulerpa racemosa (For-
sskål) J. Agardh var cylindracea (Sonder) Verlaque, Huisman et Boudouresque (de aquí
en adelante C. cylindracea)], taxón que ha demostrado un un fuerte carácter invasor en
el Mediterráneo.
C. cylindracea es una especie de aguas templadas y subtropicales procedente probablemente del su-
roeste de Australia (Verlaque et al. 2003, Belton et al. 2014)) cuyo importante desarrollo como espe-
cie invasora justifica su catalogación entre las “100 peores especies invasoras del Mar Mediterráneo“
(Streftaris y Zeneteos 2006). En los siguientes apartados se profundiza sobre diversos aspectos de la
invasión protagonizada por esta especie en el Mediterráneo y las características biológicas y ecológicas
que parecen explicar su elevado potencial invasor.
6.1. Introducción y dispersión Los mecanismos a través de los cuales se produjo la introducción en el Mediterráneo son todavía objeto
de especulación, si bien el tráfico marítimo (a través de las aguas de lastre) y el comercio asociado a
la acuariofilia han sido considerados como posibles vectores de entrada (Klein y Verlaque 2008) C.
cylindracea fue detectada por primera vez en las costas de Libia en 1990 (Nizamudin, 1991). A partir de
esa primera observación se registra una primera fase de dispersión sobre la cuenca oriental, en la que
alcanza de forma sucesiva las costas de Grecia, Albania y Chipre. Posteriormente se registra una segun-
p. 10 p. 11
INTRODUCCIÓN GENERAL
TESIS DOCTORAL
da fase en la que el alga se desplaza en sentido oeste hacia la cubeta occidental a través del estrecho
de Sicilia, colonizando progresivamente las costas italianas, francesas y españolas, así como algunos
países de la ribera africana como Túnez y Argelia (ver revisión sobre este tema en Piazzi et al. 2005b y en
Klein y Verlaque 2008). En la actualidad se considera presente prácticamente en todo el Mediterráneo,
si bien la escasez de citas sobre su desarrollo en las costas mediterráneas africanas está posiblemente
relacionada con un menor esfuerzo de muestreo en estas zonas. Así mismo, también ha sido detectada
en aguas de las Islas Canarias (Verlaque et al. 2004), lo que evidencia también una posible dispersión
del alga en el océano Atlántico.
La primera observación en las costas españolas se produce en las islas Baleares en 1998 (Ballesteros et
al. 1999) alcanzando en 1999 la costa este peninsular, más concretamente las aguas de la Comunidad
Valenciana (Aranda et al. 1999) desde donde se inicia un proceso de dispersión en sentido suroeste que
alcanza la Región de Murcia en 2005 (Ruiz et al. 2011, Capítulo 1de esta tesis) y continua por las costas
de Andalucía y Ceuta en 2007 y 2008 (Rivera-Ingraham et. al. 2010). En este mismo año es identificada
en aguas de Cataluña, lo que determina su presencia en todo el litoral español (no existen referencias).
Los patrones de distribución del alga observados en el Mediterráneo se caracterizan por la aparición de
poblaciones aisladas y separadas entre sí por distancias relativamente largas, lo que refleja la influencia
de la actividad humana en su dispersión a gran escala (Ould-Amhed y Meisnez 2007, Flagella et al.
2008, Papini et al. 2013).
6.2. Morfología y biología Al igual que el resto de especies de este orden, C. cylindracea se caracteriza por ser una alga de natura-
leza cenocítica y por tanto con estructura sifonal. Presenta un desarrollo característico a través de esto-
lones horizontales de 1-2 mm de diámetro de los que surgen múltiples y delgados rizoides, que permiten
el anclaje del alga al sustrato, y frondes aislados de tamaño pequeño (inferiores a 15cm normalmente
aunque se han detectado ejemplares con longitudes próximas a los 20 cm) divididos en pinnas de as-
pecto vesicular o claviformes denominadas ramuli con una disposición radial o dística y orientados ha-
cia arriba. La longitud de estos ramuli oscila entre 1,5 y los 7 mm mientras que su diámetro varía entre 1
y 3mm. En cualquier caso, los diferentes estudios realizados en el Mediterráneo indican una importante
variación y plasticidad morfológica asociada a factores como la batimetría, los cambios estacionales o
la localización geográfica (ver revisión en Klein y Verlaque 2008).
En relación a la biología de esta variedad en aguas del Mediterráneo, C. cylindracea presenta un ciclo de
vida endopoliploide con una presencia dominante de clones haplofásicos capaces de producir gametos
y un contenido genómico reducido (Varela-Álvarez et al. 2012). Los fenómenos de producción de ga-
metos tanto masculinos como femeninos indican la posibilidad de que existan eventos de reproducción
sexual, si bien la formación de zigotos solo ha sido descrita en laboratorio (Panayotidis y Žuljević 2001).
La producción de estos gametos es holocárpica (Panayotidis y Žuljević 2001), lo que determina que
todo el citoplasma esté implicado en la formación de dichos gametos y por tanto una vez expulsados se
produce la degradación del estolón. La colonización y dispersión de la especie se produce principalmen-
te mediante reproducción vegetativa o asexual, mediante tres mecanismos distintos relacionados con
su constitución sifonal: (i) estolonización, (ii) fragmentación y (iii) formación de propágulos (Ceccherelli
y Piazzi et al. 2001a, Renoncourt y Meinesz 2002).
6.3. Ecología En el Mediterráneo, C. cylindracea no ha mostrado unos requerimientos ecológicos demasiado estric-
tos, lo que le ha permitido colonizar una amplia variedad de sustratos y profundidades y tolerar las
marcadas variaciones estacionales de las condiciones ambientales (Klein y Verlaque 2008). Así, por
ejemplo, en algunas zonas ha sido observada hasta una profundidad de 70 metros y en regiones como
el golfo de León llega a soportar variaciones de entre 8 y 28ºC de temperatura. Esta elevada tolerancia
ambiental también se muestra en relación a otros factores abióticos como la salinidad, siendo capaz
de colonizar lagunas litorales caracterizadas por importantes fluctuaciones en este parámetro (Mastro-
totaro et al. 2003).
Las tasas de crecimiento, estimadas como velocidad de elongación del estolón, pueden alcanzar valo-
res que oscilan entre los 1,3 y 2 cm día -1 (Piazzi y Cinelli 1999; datos propios). Esta elevada capacidad
explica que el alga sea potencialmente capaz de formar densos tapices sobre el sustrato (y las especies
nativas que lo colonizan) en los que los estolones forman estructuras tridimensionales de hasta 15 cm
de grosor (Klein y Verlaque 2008). En estos casos la longitud de estolón por unidad de superficie puede
llegar a alcanzar valores de hasta 2600 m m-2 (Žuljević et al. 2003) y niveles de biomasa que pueden
rondar los 1.260 g PS m-2 (Iveša y Devescovi 2006).
Diversos estudios han identificado un periodo máximo de abundancia y crecimiento entre finales de
verano y principios de otoño, y un periodo de crecimiento mínimo en invierno, asociado s ha impor-
tantes fenómenos de regresión poblacional, (Ruitton et al. 2005b, Lenzi et al. 2007). Durante la época
favorable. Este aparente patrón estacional de crecimiento mostrado en algunas zonas del Mediterráneo
es coherente con la estrecha correlación positiva observada entre la temperatura y el metabolismo y
Fig. 2.C. cylindracea en aguas de la Región de Murcia.
p. 12 p. 13
INTRODUCCIÓN GENERAL
TESIS DOCTORAL
crecimiento del alga en laboratorio (Flagella et al. 2008). Esto sugiere que el alga desarrolla mecanis-
mos de anticipación como estrategia de aclimatación fisiológica a los cambios estacionales del medio
(“seasonal anticipator species” sensu Kain (1989); Flagella et al. 2008). Sin embargo, la ausencia de
patrones estacionales de abundancia observada en diversos estudios (Giaccone y Di Martino 1995,
Cebrian y Ballesteros 2009) indica a su vez que otros factores abióticos y bióticos del medio como el
hidrodinamismo (Klein y Verlaque 2008), la herbivoría (Tomas et al. 2011) o las perturbaciones de
origen antrópico (Bulleri et al. 2011, Gennaro y Piazzi 2013) pueden jugar un papel determinante en el
desarrollo del alga a escala local.
C. cylindracea ha mostrado tener una elevada capacidad de aclimatarse a las importantes variaciones
espacio-temporales de la irradiancia submarina, como las que tienen lugar entre épocas del año y a lo
largo de gradientes de profundidad. Las poblaciones del alga muestran un mayor desarrollo entre los 5
y los 30m (ver revisión en Klein y Verlaque 2008), aunque se ha llegado a observar a profundidades tan
extremas como 70 metros. Desde un punto de vista fisiológico, los escasos estudios realizados indican
una elevada plasticidad para fotoaclimatarse a ambientes con condiciones lumínicas muy diferentes.
Así, por ejemplo, se ha observado que es capaz de reorganizar su aparato fotosintético en respuesta a
variaciones de la luz asociadas a gradientes de profundidad, a las causadas por el sombreado producido
por el dosel vegetal de otros macrófitos (p.e. Cymodocea nodosa), y a las que tienen lugar a lo largo de
ciclos diarios y estacionales (Raniello et al. 2004, 2006).
El tipo de sustrato es un factor determinante para la colonización del alga, prefiriendo sustratos esta-
bles, duros o con cierto grado de consolidación, frente a los sedimentos arenosos inestables. De esta for-
ma, las comunidades bentónicas más invadidas por el alga son los fondos rocosos fotófilos dominados
por macroalgas autóctonas cespitosas, los fondos detríticos con o sin presencia de rodolitos calcáreos
(maërl), y la mata muerta de Posidonia oceanica (L.) Delile (ver revision en Klein y Verlaque 2008) . Sin
embargo, determinadas biocenosis dominadas por algas de porte erecto y las praderas de angiosper-
mas marinas, en especial las de P. oceanica, a pesar de ofrecer sustratos de colonización relativamente
estables, han mostrado una mayor resistencia a ser colonizados por el alga invasora (Ceccherelli et al.
2000, Piazzi et al. 2001a, Ceccherelli et al. 2002, Ceccherelli y Campo 2002, Bulleri et al. 2010, Katsa-
nevakis et al. 2010, Infantes el al 2011).
En cualquier caso, se desconocen los factores o mecanismos que determinan la interacción entre el alga
invasora y las comunidades nativas y la mayor o menor resistencia de estas últimas a ser invadidas. Cec-
cherellli et al. (2002b) sugieren que las comunidades con especies de porte erecto, formadoras de es-
tructuras tridimensionales o doseles foliares, son las que muestran mayor resistencia a la colonización,
a pesar de no identificar los mecanismos y factores que determinan dicha resistencia. De hecho, las
praderas de P. oceanica han mostrado, como se comentaba anteriormente, ser una de las comunidades
con una mayor resistencia a ser penetradas por los estolones del alga, que normalmente está ausente
en el interior de los densos doseles foliares de dichas praderas. Mediante experimentos manipulativos in
situ, se ha comprobado que la reducción de la densidad de haces favorece el desarrollo del alga dentro
de la pradera (Ceccherelli et al. 2000), lo que sugiere la existencia de una serie de factores asociados
a la estructura del dosel vegetal que limitan el crecimiento de C. cylindracea dentro de las praderas de
P. oceanica, o lo que es lo mismo la existencia de algún tipo de interacción competitiva a favor de la an-
giosperma. De forma similar, praderas que han experimentado algún tipo de alteración de su estructura
a consecuencia de impactos antrópicos parecen ser más vulnerables a la invasión por C. cylindracea
que aquellas en las que su estructura permanece en buen estado de conservación (Montefalcone et
al. 2010, Lenzi et al. 2013). Ceccherelli et al. (2000) plantean la hipótesis de que la disponibilidad de
sustrato dentro de la pradera es uno de dichos factores. Sin embargo, el papel de otros factores prima-
rios clave para el crecimiento y supervivencia algal como la disponibilidad de luz no han sido todavía
investigados, a pesar de la dramática reducción de la luz que los doseles foliares de P. oceanica causan
sobre el fondo (Dalla Via et al. 1998).
6.4. Impactos sobre las comunidades nativasLos fondos colonizados por C. cylindracea pueden llegar a experimentar profundas transformaciones de
sus características físico-químicas y biológicas. Los densos tapices que el alga puede desarrollar tienen
una elevada capacidad de retención de partículas capaz de modificar profundamente sus característi-
cas biogeoquímicas (Holmer et al. 2009, Hendriks et al. 2010). Los efectos asociados a esta alteración
han sido comparados con los generados por el incremento de las tasas de sedimentación sobre las co-
munidades de macroalgas bentónicas (Piazzi et al. 2005a) e incluyen reducciones en la diversidad y en
la cobertura de especies nativas (Piazzi et al. 2001b, Balata et al. 2004, Piazzi et al. 2005a). La presencia
del alga ha sido también relacionada con cambios en la diversidad funcional de estas comunidades,
favoreciendo el desarrollo de especies cespitosas en detrimento de otras de porte erecto o mayor com-
plejidad estructural (Bulleri et al. 2010).
En relación al impacto sobre las angiospermas marinas, se han detectado cambios en las tasas de
floración y producción de Zoostera noltii Hornem. y Cymodoecea nodosa (Ucria) Aschers. en praderas
mixtas colonizadas por el alga (Ceccherelli y Campo 2002). En el caso de P. oceanica, como ya ha sido
comentado, el alga parece ser incapaz de colonizar el interior de sus densos doseles foliares y parece
que su expansión se encuentra limitada a los bordes de la pradera en contacto con poblaciones del alga
desarrolladas sobre otros tipos de habitas adyacentes (observaciones personales). En cualquier caso,
Dumay et al. (2002a) identifican cambios en el ciclo vegetativo de praderas de P. oceanica invadidas
por el alga que incluyen reducciones en la longitud foliar e índice de área foliar así como un aumento
de la tasa de recambio foliar. Estos efectos han sido relacionados con fenómenos de interacción entre
ambas especies asociados a la producción de sustancias alelopáticas (caulerpenina) por parte del alga.
De hecho, experimentos realizados con extractos del alga han demostrado la actividad fitotóxica de la
caulerpenina sobre el rendimiento fotosintético de la fanerógama marina Cymodocea nodosa (Raniello
et al. 2007). Si bien, se trata de un aspecto que todavía no ha sido estudiado en profundidad, especial-
mente en relación a su repercusión a largo plazo sobre las praderas (reducción de la resiliencia de las
praderas de áreas colonizadas a largo plazo y, por tanto, aumento de su vulnerabilidad).
Los estudios realizados sobre los efectos de C. cylindracea en las comunidades de invertebrados bentó-
nicos muestran resultados contradictorios en relación a índices de diversidad y abundancia de especies,
p. 14 p. 15
INTRODUCCIÓN GENERAL
TESIS DOCTORAL
aunque todos coinciden en la elevada capacidad del alga de cambiar su estructura y dinámica (Buia
et al. 2001, Vázquez-Luis et al. 2008, Pacciardi et al. 2011). Aunque la información disponible sobre
el impacto causado por el alga en niveles tróficos superiores es muy reducida, C. cylindracea ha sido
también relacionada con la disminución en la abundancia de especies de macrofauna como esponjas
(Baldacconi y Corriero 2009) y gorgonias (Cebrian et al. 2012). Por otro lado, algunos estudios han do-
cumentado el consumo activo del alga por parte de especies de peces, sugiriendo la posibilidad de ge-
nerarse cambios estructurales en la cadena trófica derivados de los efectos negativos a nivel fisiológico
del consumo de caulerpenina contenido en el alga (Terlizzi et al. 2011, Deudero et al. 2011, Felline et al.
2012) y que explicarían los cambios detectados en la comunidad íctica en zonas con una alta presencia
de C. cylindracea (Bernardeau-Esteller y Martínez-Garrido 2010).
Por último, y aunque no hay estudios concretos sobre este aspecto, cabe indicar que la capacidad de
C. cylindracea de desarrollar praderas monospecíficas mas o menos continuas puede ser considerada
como una fuente potencial de impacto sobre las características a gran escala de los paisajes en algunos
ecosistemas marinos afectados por la introducción de alga.
7. Justificación de la tesis
A pesar del importante número de estudios realizados sobre la biología y ecología de C. cylindracea en
aguas del Mediterráneo existen todavía importantes carencias sobre el conocimiento de los factores
implicados en su éxito invasor, lo que dificulta en último término un análisis global y riguroso sobre
el alcance y consecuencias de la invasión así como el desarrollo de estrategias adecuadas de gestión
destinadas a limitar y controlar el impacto del alga en esta región (Klein y Verlaque 2008).
Como se ha descrito en los primeros apartados de esta introducción, el éxito invasor de una especie
introducida está relacionado con multitud de factores, como la capacidad de aclimatarse a las nuevas
condiciones ambientales y la interacción con las comunidades nativas que juegan un papel determi-
nante.
Aunque existen evidencias de que C. cylindracea presenta una elevada plasticidad fisiológica en res-
puesta a variaciones en las condiciones ambientales, no ha sido todavía evaluado el grado en el que los
mecanismos de aclimatación desarrollados por el alga ante factores abióticos clave (p.e. luz) inciden so-
bre su capacidad productiva y por tanto, sobre su crecimiento. A su vez, las investigaciones sobre la inte-
racción entre el alga y las comunidades nativas han sido en general desarrolladas en escalas temporales
cortas (inferiores a dos años) dificultando la evaluación de los fenómenos competitivos desarrollados
por el alga. En el caso concreto de las praderas de P. oceanica, los resultados obtenidos hasta la fecha
parecen evidenciar una elevada resistencia de esta comunidad a la invasión, sin embargo no existen
estudios que analicen la interacción entre ambas especies en un marco temporal amplio en el que otros
fenómenos ya descritos para el alga, como el deterioro de las condiciones del sustrato o efectos por fito-
toxicidad alelopática, pueden jugar un papel determinante. A su vez, el grado de conocimiento sobre los
factores que pueden estar relacionados con dicha resistencia sigue siendo muy reducido. Estos aspectos
son especialmente relevantes para poder establecer posibles impactos y escenarios futuros en base a la
evolución de las propias praderas en el Mediterráneo y a su relación con otros fenómenos potenciales de
perturbación sobre estas comunidades (p.e. cambio global, impactos de origen antrópico).
7.1. Objetivo y estructura de la tesis. El objetivo general de la presente tesis doctoral es analizar el establecimiento y ecología de C. cylin-
dracea en el Mar Mediterráneo bajo la perspectiva de su potencial colonizador, profundizando en el
estudio de la interacción del alga con las praderas de P. oceanica y en el análisis del papel de la luz como
factor determinante en los procesos de invasión del alga en esta región.
Los objetivos específicos de la tesis y que constituyen los cuatro capítulos que la conforman son los
siguientes:
Fig. 3.Colonización de C. cylindracea sobre fondos rocosos.
Fig. 4.C. cylindracea en el limite de una pradera de Posidonia oceanica.
p. 16
INTRODUCCIÓN GENERAL
TESIS DOCTORAL
1. Análisis del establecimiento y dispersión de C. cylindracea en litoral de la región de Murcia
Los mecanismos usados por C. cylindracea para su dispersión en aguas del Mediterráneo han sido am-
pliamente discutidos y relacionados con su éxito invasor. Sin embargo, la dinámica de propagación del
alga a escala local y regional y su capacidad de expansión una vez asentada en una determinada zona
ha sido escasamente documentada y los resultados existentes están, en general, restringidos a unas po-
cas áreas del Mediterráneo. Los objetivos del capítulo 1 son (i) documentar la presencia y dispersión de
C. cylindracea en las costas de la Región de Murcia y (ii) analizar de manera cuantitativa la capacidad
de expansión y grado de desarrollo del alga tras su introducción. Este capítulo define además el marco
general en el que se establecen los estudios de la presente tesis doctoral, describiendo las poblaciones
del alga y los hábitats sobre los que se desarrollan los trabajos que la integran. Para conseguir los ob-
jetivos de este capítulo se investigo la presencia y superficie de ocupación del alga en 42 localidades
localizadas a lo largo de la costa murciana entre los años 2005, año de primera detección, y 2007.
Además, en algunas de las localidades seleccionadas se analizó la abundancia en términos de biomasa
y características biométricas con el fin de caracterizar y definir el grado de desarrollo vegetativo de las
poblaciones.
2. Estudio de la capacidad fotosintética de C. cylindracea a lo largo de un gradiente de profundidad
y su repercusión sobre el metabolismo del carbono
El objetivo de este capítulo es determinar la repercusión que los mecanismos de fotoaclimatación de-
sarrollados por C. cylindracea ante variaciones naturales en los regímenes lumínicos tienen sobre la
capacidad productiva del alga. Con este fin se evaluó la capacidad fotosintética y productiva (mediante
una aproximación basada en la estima del balance de carbono diario) del alga en tres poblaciones
naturales desarrolladas a diferente profundidad bajo regimenes lumínicos significativamente distintos
que representan, por tanto, una aproximación a las condiciones determinadas por un gradiente de pro-
fundidad. La hipótesis analizada es que la plasticidad fotosintética del alga constituye un mecanismo
efectivo para optimizar su capacidad productiva ante las variaciones lumínicas definidas por gradientes
de profundidad.
3. Valoración del papel de luz en la resistencia de las praderas de P. oceanica a la invasión de
C. cylindracea
El objetivo general de este capítulo es identificar y evaluar los mecanismos y factores implicados en la
resistencia a la invasión mostrada por P. oceanica. Esta angiosperma marina genera un dosel vegetal
de elevada complejidad cuya estructura tridimensional modifica intensamente las condiciones ambien-
tales en su interior. Esta modificación, como ya ha sido comentado anteriormente en esta introducción,
es especialmente relevante en el caso del ambiente lumínico, que sufre una profunda alteración tanto
a nivel cuantitativo (se ha estimado que la reducción en la disponibilidad puede llegar a ser de hasta el
5% de la irradiancia superficial) como cualitativo (perdida de longitudes de onda de bajo rango ener-
gético). Con el fin de evaluar la posible implicación de la disponibilidad lumínica dentro de las praderas
de P. oceanica en su alta resistencia a la colonización se llevó a cabo un análisis comparativo de los
regímenes de luz, abundancia, características fotosintéticas y capacidad productiva entre poblaciones
de C. cylindracea desarrolladas dentro y fuera de praderas de P. oceanica. La hipótesis planteada es
que las condiciones de luz bajo el dosel vegetal de P. oceanica constituye un factor determinante para
el crecimiento y supervivencia del alga y por lo tanto para el desarrollo de su potencial invasor en este
valioso ecosistema mediterráneo.
4. Estudio experimental del efecto de la disponibilidad de luz sobre la fotosíntesis, metabolismo del
carbono y crecimiento de C. cylindracea: la luz como factor limitante en la colonización de praderas
de P. oceanica
Los resultados obtenidos en el estudio previo evidenciaron la implicación de la luz en los fenómenos
de resistencia a la colonización de las praderas de P. oceanica. Sin embargo, le metodología aplicada
en dicho estudio, basada en una análisis de poblaciones in situ, dificultaba la capacidad de diferenciar
el efecto de este factor del generado por otros factores ambientales relacionados las características
del dosel vegetal (pe. el hidrodinamismo o la disponibilidad de nutrientes) y que también pudiesen
estar implicados en el proceso de colonización. El objetivo de este capítulo es por tanto establecer si
las condiciones de luz dentro de la pradera son capaces de explicar de forma aislada los fenómenos
de resistencia observados y parte de una hipótesis similar a la del capitulo anterior. Con este fin se ha
estudiado la respuesta fotoaclimatativa y la capacidad de producción y crecimiento de C. cylindracea
en dos experimentos manipulativos complementarios, desarrollados en condiciones de mesocosmos y
campo, en los que las condiciones lumínicas fueron controladas.
Ademas se incorporan en una anexo los resultados sobre la Evaluación de la interacción a largo plazo
entre C. cylindracea y las praderas de P. oceanica, estudio que ha sido recientemente enviado para
su publicación y que por su relación con los aspectos tratados en la presente tesis se ha considerado
oportuno su inclusión.
En este Anexo se presentan los resultados (2007-2014) obtenidos en el estudio sobre la interacción a
largo plazo entre ambas especies que se está desarrollando en aguas del litoral de Murcia en el contexto
de la Red de Seguimiento de las Praderas de P. oceanica de esta región. La hipótesis planteada en este
estudio es que, a pesar de la alta resistencia a la invasión mostrada por las praderas de P. oceanica, C.
cylindracea es capaz de competir con la angiosperma marina, de manera que puede provocar cambios
estructurales en la pradera, reducir su resiliencia ante otros fenómenos de perturbación e incrementar
su capacidad colonizadora. El seguimiento de la interacción entre ambas especies se está realizando
en tres zonas en las que los fondos colonizados por C. cylindracea están en contacto con praderas de
P. oceanica, llegando a colonizar los primeros centímetros de dichas praderas.
CHAPTER 1Recent spread of the invasive alga
Caulerpa cylindracea (Bryopsidales,
Chlorophyta) along the Mediterranean
coast of the Murcia Region (SE Spain)
p. 21
Recent Spread of the Invasive Alga Caulerpa cylindra-cea (Bryopsidales, Chlorophyta) along the Mediterra-nean coast of the Murcia Region (SE Spain).
Abstract
The aim of this paper is to document the recent
appearance and spread of the green alga Cau-
lerpa cylindracea along the coast of Murcia in
south-east Spain. This is the westernmost sigh-
ting of the invasive alga in the Mediterranean
Sea. It was found for the first time in the area in
2005 and over the next two years the number of
new sightings increased almost exponentially. At
some of the invaded stations the alga increased
its surface area 6.5- to 44-fold in one year. In the
period 2005–2007 the total surface area coloni-
sed by the alga in the region was estimated to be
at least 265 ha. Benthic assemblages colonised
by the alga were rocky photophilic algae, dead P.
oceanica rhizomes, infralittoral and circumlittoral
soft bottoms and maërl beds. No penetration of
the alga was observed in P. oceanica meadows,
except in one locality. Biometric analysis indica-
ted high vegetative development in the establi-
shed colonies in comparison to those described
in other Mediterranean areas. The results of this
study reveal that the rapid spreading dynamics
of C. cylindracea in the region of Murcia are a po-
tential threat for the native benthic communities.
Introduction
The biological characteristics of Caulerpa cylin-
dracea Sonder. (hereinafter C. cylindracea) (high
rates of vegetative dispersal, production of alle-
lopathic substances, etc.) determine its high colo
nisation potential and its extraordinary ability to
outcompete and alter native benthic assembla-
ges, which make this species a particular poten-
tial threat for the Mediterranean coastal ecosys-
tem (Piazzi et al. 2005b).
C. cylindracea was observed in the Eastern Medi-
terranean Sea for the first time along the coast
of Libya in 1990 (Nizamuddin 1991), being the
origin of this invasive variety still unknown (Verla-
que et al. 2003, Durand et al. 2002, Panayotidis
2006). Since then, the species has spread rapidly,
gradually invading the Mediterranean Sea. This
has been well-documented in the western basin
along the coasts of Italy, France and North Afri-
ca (Piazzi et al. 2005b). Along the Mediterranean
coast of Spain, the species was first sighted in the
Balearic Islands in 1998 (Ballesteros et al. 1999).
It reached the east coast of the Iberian Peninsula
(Castellón) in 1999 (Aranda et al. 1999) and be-
gan to spread quickly southward, being sighted in
Alicante (SE Spain) in 2000 (Aranda et al. 2003).
At that point, the algal spread seemed to stabili-
se (Fig. 1), but its presence was confirmed in the
Murcia region in 2005, indicating that the coloni-
sing process was continuing southward.
Precise studies documenting the presence of the
alga in newly colonised areas (i.e. colony size,
depth range, substrate type, morphometric data
and invaded native communities) are fundamen-
Publicado en: Ruiz JM, Marín-Guirao L, Bernardeau-Esteller J, Ramos-Segura A, García-Muñoz R, Sandoval-Gil JM
(2011) Spread of the invasive alga Caulerpa racemosa var. cylindracea (Caulerpales, Chlorophyta)
along the Mediterranean coast of the Murcia region (SE Spain). Anim Biod Conserv 34(1): 73-82.
p. 23p. 22
CHAPTER 1
TESIS DOCTORAL
tal to elucidate its colonising potential, spreading
dynamics and mechanisms (vectors) at local and
large spatial scales (Klein and Verlaque 2008).
Cartographic methods make it possible to mea-
sure the extent of the spread and can assist in
helping to predict potential impacts and future
scenarios (Meinesz 2007). Detailed informa-
tion on the spreading dynamics and extent of
C. cylindracea is available for a limited number
of Mediterranean regions (Piazzi et al. 1997b;
Ruitton et al. 2005a). The goals of the present
study were: (1) to document the spreading dyna-
mics of C. cylindracea along the coast of Murcia
(SE Spain) from its appearance in 2005 to 2007,
both at regional and local scales; (2) to provide
some quantitative estimate of the colonised sur-
face area. Furthermore, the work includes several
characteristics of the invaded sites (colonised as-
semblages, colonization depth) together with the
vegetative development of several colinies in this
geographical area.
Material and Methods
Study Area and Field Sampling Programme
This study was carried out on the Mediterranean
coast of Murcia, SE Spain (Fig. 1). After C. cylin-
dracea was first sighted in the region in 2005, an
active detection programme was established to
map the distribution of the alga and its spreading
dynamics over time (Meinesz 2007, Ruitton et al.
2005a). To this end we initially selected 42 sam-
pling stations uneven distributed along 224 km
of the Murcia coastline through a depth range
from 2 to 30 m (Fig. 1). These stations were selec-
ted from different long-term sampling program-
mes that had already been initiated in the region
for different purposes (scientific monitoring of
P. oceanica meadows, environmental impact as-
sessments and scientific projects), but since they
were visited at least once a year by specialised
divers this ensured reliable information about the
date of appearance of the alga. Of course, this
sampling strategy resulted in a non-systematic
sampling design, but it allowed us an insight into
the colonisation process in a representative area
of the Murcia coast. The period covered by this
sampling programme was 2005–2007 i.e. the
first three years of the colonisation process of C.
cylindracea in the Murcia region.
Once the alga was detected at a given station, di-
vers from our research team surveyed a total sur-
face area of 0.5 ha to characterise the colonised
area (depth range, types of colonised substrate
and benthic assemblages) and to estimate its
surface area (i.e. colonisation levels sensu Ruitton
et al. 2005a). Based on the field data obtained,
invaded localities were assigned to one of the
following five categories of colonisation level:
(I) one or few small colonies covering a surface
area of less than 10 m2; (II) colonies of varying
sizes covering a total surface area between 10
and 104 m2; and (III) meadows covering surfa-
ce areas between 104 and 105 m2, (IV) 105 and
106 m2 and (V) greater than 106 m2. For cate-
gories I and II, the surface area was estimated in
a single survey within the sampling station using
quadrats and transects. For cases belonging to
categories III–V, where the colonised area ex-
tended beyond the area surveyed by divers at a
single station, additional dives were necessary to
determine the limits of the total colonised area.
These additional dives were performed at nei-
ghbouring points separated from the sampling
station by several hundreds of metres and at di-
fferent depths and directions (a specific sampling
design was established in each case). Once the
limits of the invaded area were identified these
were determined by GPS and input into a Geo-
graphic Information System (Arcview microcom-
puter programme Version 9.0, Esri ©) to estimate
the surface area of the polygon thus generated.
Biometric Analysis
Biometric analysis of C. cylindracea colonies was
performed using data from summer 2007 (June
28th to August 9th), a season in which the ve-
getative development of the alga was close to
its annual maximum (Klein and Verlaque, 2008).
Samples were collected at three of the most in-
vaded stations: station 1 (–10 m), station 14
(–25 m), and station 25 (–22 m) (Fig. 1). Fronds,
stolons and rhizoids were carefully collected by
hand within six replicated 1,600 cm 2 quadrats
that were randomly placed within fully colonised
areas (i.e. 100% cover) along a 50 m transect.
Samples were processed in the laboratory to de-
termine the following biometric variables as des-
cribed by Capiomont et al. (2005) and Ruitton et
al. (2005b): the total length of stolons (m m-2),
number of stolon apices (no. apices m-2), number
of fronds (no. fronds m-2) and frond height (cm).
Total biomass (g dw m-2) was determined by dr-
ying the samples at 70 ºC until constant weight.
Results
Distribution and Estimated Colonised Area
Field data obtained at the invaded localities are
Fig. 1.
Recent spread of C. cylindracea in the
Western Mediterranean basin (A). Dis-
tribution of sampling stations on the
Murcia coast (B), and in the Marine
Reserve Cabo de Palos-Islas Hormigas
(C). Invaded stations are indicated by
black circles, the size of which corres-
ponds to one of the five categories of
colonisation level (see legend and me-
thods section). Information related to
the 42 sampling stations is included
in Appendix 1.
p. 25p. 24
CHAPTER 1
TESIS DOCTORAL
summarised in Figure 1 and Table 1. C. cylindra-
cea was first detected in station 14 (locality of
Cablanque) in 2005 as dispersed patches cove-
ring a total surface area of more than 104 m2. By
2007, the colony had formed a more homoge-
nous meadow of at least 2.5·106 m2. After 2005,
the number of new invaded localities increased
almost exponentially: two in 2006 and six in 2007
(Table 1). In 2006, the population of station 4 (lo-
cality of Isla Grosa) was first found as a few small
patches over a total surface area of 221 m2 that
increased to 104 m2 in 2007 (Figs. 1b and 2). In
the station 25 (locality of Cabo Tiñoso) the initial
surface area in 2006 was estimated as 13,724 m2
and this increased to 89,187 m2 in 2007. In 2007,
all new sightings were concentrated along the
easternmost coast of the region (stations 1, 6, 9,
12, 13 and 19) with very different colonisation
levels, ranging between categories I and III (Fig.
1b and c, Table 1). The cumulated field data gave
a gross estimation of the total invaded area of
265 ha in 2007, which is probably an underesti-
mation of the real colonised area since informa-
tion on areas deeper than 30 m was not available
and some coastal zones were excluded from the
survey.
Characteristics of the Colonised Areas
The depth of invaded areas ranged from 2–30 m,
but the maximum colonised depth was greater
than 30 m since deeper stands continued fur-
ther into this isobath (Table 1). Shallow colonies
(<10m) were the least frequent while most of the
studied colonies fell within 10–30 m. The alga
colonised a wide range of substrates and native
assemblages: rocky photophilic algae (boulders
and vertical walls), infralittoral and circumlitto-
ral soft-bottoms, dead mats of P. oceanica, and
mäerl beds (Table 1). In most localities, C. cylin-
dracea formed compact multilayered mats up to
12 cm thick over the substrate. Cabo Tiñoso was
the only locality where the P. oceanica meadow
was partially invaded by the alga, but no penetra-
tion of the seagrass canopy was observed at the
other localities.
Biometric Characterization of the Colonies
Table 2 summarises the biometric characteris-
tics of C. cylindracea colonies at three selected
stations: 4, 14 and 25. The total biomass varied
between 9.4 and 135.9 g dw·m-2. The number of
stolon apices ranged from 150 to 3,756 m-2, while
the total number of fronds ranged from 937 to
9,018.7 m-2. In addition, the total length of sto-
lons ranged from 1,684 to 5,777 m·m-2, while the
height of fronds varied between 0.3 and 9.5 cm.
Discussion
C. cylindracea was observed for the first time on
the coast of Murcia as an isolated colony at the
locality of Calblanque (station 14) in 2005. The
origin and the introducing vector of the alga in
the region is unknown, but two hypothesis can
be advanced: (1) dispersion from the nearest
colonies, located in the province of Alicante 90
km to the north, and (2) introduction through the
nearby harbour at Cartagena, which is a crucial
point for the very dense maritime traffic suppor-
ted by this part of the Mediterranean Sea (Fig.
1b). Further regional dispersion in subsequent
years occurred in an almost exponential manner
and new colonies appeared without a clear spa-
Station no.
41425
Locality
Isla GrosaCalblanque
Cabo Tiñoso
Depth (m)
102522
Total biomass
(g dw · m-2)62.7 ± 42.716.9 ± 7.3
49.7 ± 21.0
Number of apices
(No. apices · m-2)1.133 ± 958323 ± 187
2.238 ± 832
Number of fronds
(No. fronds · m-2)5.401 ± 2.6311.260 ± 589
6.256 ± 1.316
Total stolon length
(m · m-2)3.487 ± 1.0733.913 ± 1.1334.528 ± 798
Frond height
(cm)1.6 ± 0.43.1 ± 0.31.9 ± 0.7
Station no.
1469
1213141925
Locality
La MangaIsla Grosa
Piles IIsla Hormiga
La BarraLos Punchosos
CalblanqueCabo Negrete
C. Tiñoso
colonization
year
200720062007200720072007200520072006
2005
(m2)
000000
30000
2006
(m2)
0221
0000
NA0
13,724
2007
(m2)
10104
11
104
4.5·104
2.5·106
104 - 105
9·105
Level of
colonization
IIIIIIIIIIV
IIIIV
Table 1.
Sampling stations colonised by C. cylindracea along the coast of Murcia. Stations correspond to Figure 1 and Appendix 1. Level of
colonisation as explained in the methods section.
Table 2. Biometric analysis of the C. cylindracea populations studied. Mean values ± SD.
Fig. 2.
Distribution and estimated surface area colo-
nised by C. cylindracea in station 4 (locality of
Isla Grosa) and station 25 (locality of Cabo Ti-
ñoso) in 2006 and 2007. Black arrows indicate
the presence of new small patches of the alga.
p. 27p. 26
CHAPTER 1
TESIS DOCTORAL
tial pattern in localities separated by hundreds
of metres to tens of kilometres. This rapid and
discontinuous regional spread is similar to that
described by Langar et al. (2002) on the Tuni-
sian coast and by Ruitton et al. (2005a) along
the French Mediterranean coast. This pattern of
spread has been attributed to the efficient repro-
ductive mechanisms reported for C. cylindracea,
both sexual (Panayotidis and Zulevic 2001) and
vegetative (Renoncourt and Meinesz 2002), that
determine its higher colonisation potential relati-
ve to other invasive Caulerpales (e.g. C. taxifolia,
Meinesz 2007).
In 2007 the most widespread population of C.
cylindracea was found in station 14 at the loca-
lity of Calblanque, the site where it was first sigh-
ted. However, there was no relationship between
the actual colony size and the time elapsed since
it was first observed, as indicated by the large
variation in the estimated surface area between
new colonies detected in 2006 and 2007 (1 to
105 m2). This is because at some localities the
alga appeared before the date of its first sigh-
ting, but was not detected in the preceding year.
The alga was probably already present as one or
a few small inconspicuous patches that would
be difficult to find, even by trained divers, but
this implies that C. cylindracea is able to spread
over a surface area of at least 1 ha in a 1-year
period, which represents a very fast colonisation
rate. Station 25 at the locality of Cabo Tiñoso is
a good example: the invaded area increased 44-
fold in 1 year (i.e. 7.5 ha year-1). Similary, the alga
colonised a surface area of almost 1 ha in 2007 in
station 4 from only few small patches found one
year earlier (Fig. 2).
Similar spreading dynamics have been reported
from other Mediterranean localities (Piazzi et
al. 1997b, Piazzi and Cinelli 1999, Piazzi et al.
2001a, Ruitton et al. 2005b), showing that once
the alga arrives at a locality, substrate colonisa-
tion can be a very rapid process. This could be
due to the high stolon elongation rate (up to 2
cm·day-1) and reproductive capacity of the alga
(Panayotidis and Zuljevic 2001, Ceccherelli and
Piazzi 2001, Renoncourt and Meinesz 2002, Ruit-
ton et al. 2005b), but habitat characteristics such
as substrate type and the complexity of native
communities are also determinant. As described
for other Mediterranean localities (Ruitton et al.
2005a, Zuljevic et al. 2003, Piazzi et al. 2005b,
Piazzi and Balatta 2009), the alga fully colonised
a variety of substrates and biocenoses present
throughout its depth distributional range (i.e.
rocky photophilic algae, dead rhizomes of P. oce-
anica, detritic soft bottoms and maërl beds), with
the exception of P. oceanica meadows. Similarly,
penetration of C. cylindracea in P. oceanica mea-
dows has rarely been reported and only occurs in
low density canopies (Piazzi et al. 1997a, Piazzi
and Cinelli 1999; Ceccherelli et al. 2000, Monte-
falcone et al. 2007).
Biometric analysis of C. cylindracea meadows
indicated a high degree of vegetative develop-
ment in the studied colonies. The mean values of
biometric descriptors were within the ranges ob-
served in other invaded localities of the Western
Mediterranean at a similar depth range (Buia et
al. 2001, Ruitton et al. 2005b, Capiomont et al.
2005, Klein and Verlaque 2008). However, the
mean values of total stolon length found in this
study (3–4.5 km m-2) were higher than the maxi-
mum values reported elsewhere in the season of
maximum algal development (summer–autumn:
1–2.6 km m-2; Capiomont et al. 2005, Ruitton et
al. 2005b, Zuljevic et al. 2003). This extensive
stolon development indicates overgrowth over
the colonised substrate (Capiomont et al. 2005)
and hence, a high potential impact on the native
assemblages, particularly in those with lower ver-
tical stratification (Balata et al. 2004, Piazzi and
Balata 2009) such as the maërl beds observed in
deeper waters of our study area.
The recent appearance of C. cylindracea on the
coast of Murcia and its appearance in Algeria
in 2006 (Ould-Ahmed and Meinesz 2007) re-
presents the most recent spread of the invasive
alga documented in the Western Mediterranean
Sea and indicates that its geographical propa-
gation is still continuing in a westerly direction.
In fact, at the time of writing (2008–2009), new
sightings of the alga confirm its presence along
the west coast of Murcia and in the neighbou-
ring province of Almeria. The estimated rate of
spread of the alga in the Murcia region (at least
265 ha in three years) is higher than that repor-
ted in the Marseille region (France) over six years
(350% increase; Ruitton et al. 2005a), but is si-
milar to the highest values reported for southern
latitudes (coast of Tuscany, Italy) for a similar
time period (1993–1997; Piazzi et al. 1997b).
This evidence, together with the high degree of
stolon development reported in this study, sug-
gest that the highly invasive nature of C. cylin-
dracea on the SE coast of Spain may be favou-
red by the higher temperatures and irradiance
characteristic of these southern Mediterranean
latitudes. However, interpretation of these regio-
nal variations must be made with caution due to
the low number of studied cases, and the use of
different methodologies and experimental condi-
tions (Klein and Verlaque, 2008). It is clear that
the invasion by this and other introduced species
represents a serious potential threat for nati-
ve marine communities, but the real ecological
consequences, and their economic impact (local
fisheries, tourism), are subjects that need to be
addressed further.
p. 28
CHAPTER 1
TESIS DOCTORAL
Station no.
123456789
101112131415161718192021222324252627282930313233343536373839404142
Locality
La MangaTomás Maestre
Isla GrosaIsla GrosaCala Túnez
Piles IPiles II
Bajo de en medioIs. Hormiga
C. Escalera-someraC. Escalera-profunda
La BarraLos Punchosos
CalblanqueCalblanqueCalblanqueCalblanqueCalblanque
Cabo NegreteCabo Negrete
El GorguelCabo de Agua
Punta del AguilónIsla de las Palomas
C. TiñosoC. TiñosoC. TiñosoLa AzohíaMazarronMazarrónBolnuevoBolnuevoBolnuevoCalnegreCalnegreCalnegre
CalabardinaIsla del FrailePunta PardaPunta PardaPunta PardaPunta Parda
Depth (m)
26-275-74-8
4-125-8
18-22 (20)10-2010-2010-2510-1220-24
2-45-13 (7)25-26
272529272727
5-175-175-17
19-225-300-12
21-2517-2117-2117-21
2124-2517-1816-1719-2024-257-15
14-1630303030
X703019700822701766701985703513704710704991706008707082703946703966702954702866700052693773694689695176697283697404697569687416683908682494673129664394663125663172661074656472655306646505645186644435643447642439641492632933629651622786622625621972621615
RF
C
CNCNCNCNC
CC
NCNCNC
CNC
NC
RS
NC
C
NC
IS
NC
NC
NC
NCNC
DC
NC
NC
NCNCC
NC
NCNCNCNCNCNCNCNC
DM
C
CNCNCNC
CNC
C
NCNCNCNC
P
NCNC
NCNC
NC
C
NC
NCNC
PU
NCNC
NCNC
NC
PDNC
NCNCNCNCNCNCNC
NC
NC
NCNCNCNCNCNCNCNC
NCNCNCNC
PM
C
NC
Y418244441794424178400417794641681614168591416875641684094170103416762941675434167457416676641618474160728416020841594984159339415936041595194160406415852841589274160784415652041567044156648415799941595104159294415727341561164155026415459441533624151921414298641415824137131413698141361364136042
UTM Habitat/Biocenosis
Appendix 1.
Summary of the information related to the 42 sampling stations: localities, coordinates UTM, surveyed depth (in brakets colonized
depth) and habitat/Biocenosis present (C = colonised, NC = not colonised).
CHAPTER 2Photosynthesis and daily metabolic
carbon balance of the invasive Caulerpa
cylindracea (Chlorophyta:Bryopsidales)
along a depth gradient
p. 33
Photosynthesis and daily metabolic carbon balance of the invasive Caulerpa cylindracea (Chlorophyta:Bryop-sidales) along a depth gradient.
Abstrac
The photosynthetic plasticity of the invasive
green alga Caulerpa cylindracea has been pro-
posed as a relevant mechanism determining its
successful performance on Mediterranean ben-
thic assemblages over broad depth gradients. In
the present study, the photosynthetic performan-
ce of C. cylindracea was evaluated through a car-
bon balance approach in three invaded sites with
contrasting depths (11, 18 and 26 m) and light
regimes. At each sampling depth, photosynthesis
vs irradiance (P vs E) curves were performed on C.
cylindracea fronds and daily net productivity va-
lues were obtained by the numerical integration
of P vs E models with continuous recording of irra-
diance measured on the sea floor. Photosynthe-
tic responses were consistent with those typically
exhibited by shade-adapted macroalgal species
and other Mediterranean populations of C. cylin-
dracea: a significant reduction in maximum pho-
tosynthesis (Pmax
) occurring at an intermediate
depth (18 m) and a higher photosynthetic effi-
ciency (α) and lower dark respiration rate (Rd) at
the deepest sampling depth. Mean values of dai-
ly net C balance obtained in the deepest site were
only 15% lower than that of the shallower depth,
despite the severe reduction in light availability.
Mean daily net carbon balances obtained from
the deepest site were only 15% lower
than those obtained from the shallower depth,
despite the severe reduction in light availability.
This daily net carbon gain was ca. 29% higher
than what would be expected if photosynthetic
adjustments did not occur in the deepest algal
population. The evidence provided by this data
support the hypothesis of photoacclimation in C.
cylindracea as an effective mechanism to opti-
mise algal productivity across depth gradients in
the Mediterranean Sea.
Introduction
In the colonized sites Caulerpa cylindracea
Sonder. (hereafter C. cylindracea) is able to de-
velop high biomasses over different substrate
types, constraining the diversity of native ben-
thic assemblages (Argyrou et al. 1999, Piazzi et
al. 2001b, Balata et al. 2004, Piazzi and Balatta
2008, Vázquez-Luis et al. 2008, Klein and Verla-
que 2009).
Whilst many studies have focused on spatial pat-
terns and temporal dynamics of the distribution,
phenology and biomass of C. cylindracea, only
a few have dealt with the potential competitive
mechanisms responsible for its ecological success
in sublittoral Mediterranean environments (see
Klein and Verlaque, 2008 for a review). Among
Publicado en:Bernardeau-Esteller J, Marín-Guirao L, Sandoval-Gil JM, Ruiz, JM (2011) Photosynthesis and daily me-
tabolic carbon balance of the invasive Caulerpa racemosa var. cylindracea (Chlorophyta: Caulerpales)
along a depth gradient. Sci Mar 75(4): 803-810.
p. 34 p. 35
CHAPTER 2
TESIS DOCTORAL
other plant traits (e.g. vegetative and sexual re-
productive success, production of allelopathic
substances, physiological resistance to stress),
morphological and physiological plasticity has
been suggested as a likely adaptive feature ena-
bling acclimation to a wide range of environmen-
tal conditions in this (Klein and Verlaque, 2008)
and other C. cylindracea varieties (Peterson 1972,
Riechert and Dawes 1986, Ohba et al. 1992). The
capacity of the alga to photoacclimate to varying
light regimes has special relevance in this con-
text, since C. cylindracea has been shown to be
able to develop down to 70 m depth (Klein and
Verlaque 2008), colonize the understory of ma-
crophyte canopies (Cecherelli and Campo 2002)
and maintain biomass through time - even during
conditions of severe light limitation (e.g. deep
populations in winter: Cebrian and Ballesteros
2009). However, our knowledge of the photo-ac-
climative capacity of Mediterranean populations
of C. cylindracea is sparse at best (Raniello et al.
2004, 2006). Raniello et al. (2004, 2006) repor-
ted interesting data showing how C. cylindracea
is able to re-organize its photosynthetic pigment
system in response to varying light conditions
caused by depth gradients, seagrass canopies,
as well as daily and seasonal cycles. Regarding
depth (Raniello et al. 2006), changes in pigment
composition were thought to represent algal
photoacclimation responses in order to optimize
light capture (increase in α) and photosynthetic
performance (decrease in Ek) as light becomes
limiting. These are common responses seen in
some macroalgae species able to develop over
broad depth gradients (Ramus et al. 1977, Mar-
kager and Sand-Jensen 1992, Gómez et al. 1997,
Johansson and Snoeijs 2002). Nonetheless, the
extent to which the ability of Mediterranean
populations of C. cylindracea to photoacclima-
te effectively is responsible for productivity and
potential colonization success remains unknown.
In the present study we analyzed the phosynthe-
tic responses of C. cylindracea in order to assess
the pattern of algal productivity along a depth
gradient. To this end a carbon balance approach
was taken, based on the numerical integration
of Photosynthesis vs Irradiance (P vs E) models
throughout continuous measurement of instan-
taneous irradiance recorded at the sea floor. This
mechanistic approach has been previously de-
monstrated to provide reliable estimates of pri-
mary productivity in marine macrophytes (Matta
and Chapman 1991, Zimmerman et al. 1994).
Photosynthesis and respiration rates of C. cylin-
dracea fronds, together with continuous irradian-
ce field data, were measured at three different
locations of contrasting depth and light on the
coast of the Murcia Region of SE Spain, a part of
the Spanish Mediterranean coast invaded by the
alga since 2005 (Ruiz et al. 2011).
Material and Methods
Study area
The present study was performed at three sam-
pling stations, each located at three separate
locations at different depths on the coast of the
Murcia Region of SE Spain: shallow station (S,
11m, Isla Grosa; N37º43’, E00º42’), interme-
diate station (I, 18 m, Cabo Tiñoso; N37º32’,
E00º44’) and deep station (D, 26 m, Calblanque;
N37º32’N, E1º07’) (Fig. 1). These depths are re-
presentative of the current bathymetric range of
C. cylindracea on the Murcian coast (10-30 m,
Ruiz et al. 2011). At the time of sampling, the se-
lected stations were located in the most invaded
areas (in terms of colonized surface area) of the
Murcian coast, with the alga present at stations
I and D since 2005 and at station S since 2006.
The most commonly invaded benthic communi-
ties are the unvegetated sediments and photo-
philic macroalgal assemblages on hard substra-
tes found at station S and the coastal detritic
sediments found at stations I and D, the latter
being dominated by rhodoliths.
Field measurements:
PAR irradiance and algal biomass
All field work was performed by SCUBA divers at
the end of summer (August 2008), a time of year
in which the development of the alga is close to
maximum in many areas of Murcia (personal
observation) and other Mediterranean regions
(Klein and Verlaque 2008, but see Cebrian and
Ballesteros 2009). Incident irradiance on the
sea floor was determined at each sampling sta-
tion from continuous recording of instantaneous
PAR irradiance (E, μmol quanta m-2 s-1, 400-700
nm; Kirk 1994), obtained via the deployment of
a spherical 4π quantum sensor (Alec Electronics,
MDS MK5, Japan) for a period of 21 days (12th
August-1st September). All sensors were placed at
a height of 5 cm from the sea floor. During the
measuring period, the accumulation of epiphytes
or particles on the sensor surface was observed
to be negligible and no associated decline in in-
cident light with time was observed. Quantum
sensors were programmed to record at 10 mi-
nute intervals (n = 144 measurements per day).
A mean daily light cycle was obtained for each
sampling station, characterized by maximum
instantaneous irradiance at noon and the inte-
grated daily irradiance (mol quanta m-2 s-1). In
addition, measurement of sub-surface instanta-
neous irradiance (Eo) was performed at noon on
five standard days (i.e. those with full sunlight, a
cloudless sky and calm weather) using a flat, co-
sine-corrected 2π sensor connected to a LI-COR
quantum meter (model LI-190SA). Mean Eo and
noon instantaneous irradiance values measured
at the sea floor on the same days were used to
Fig. 1.
Location of sampling stations: shallow (-11 m, S), intermediate (-18 m, I) and deep (-26 m, D).
p. 36 p. 37
CHAPTER 2
TESIS DOCTORAL
calculate both the percentage of Eo reaching
the seabed and the water-column attenuation
coefficient (Kd, m-1; Kirk 1994) for each sampling
station. Measurements of both sensor types were
intercalibrated in the laboratory and showed a
very strong linear relationship (R2=0.998) with a
constant factor of 1.176.
At each sampling station, the abundance of C.
cylindracea was determined by measuring its to-
tal biomass (fronds, stolons and rhizoids) in six re-
plicate quadrats of 400 cm2, randomly positioned
within a surface area of 25 m2 colonized by the
alga. Plant material of each sample was placed
in dark plastic bags before being sorted, dried at
70ºC (24 h) and weighed for biomass quantifica-
tion (g dw m-2) in the laboratory.
Measurement of photosynthesis and dark res-
piration rates
Samples of C. cylindracea collected at each sta-
tion were transported to the laboratory in cooled
and aerated containers for the measurement of
photosynthetic and respiration rates and the de-
termination of photosynthetic parameters. Prior
to photosynthetic measurement, plants were
held overnight in dark conditions and at a contro-
lled temperature. Photosynthesis vs irradiance (P
vs E) curves of C. cylindracea assimilatory fronds
were generated by following the incubation me-
thods described by Walker (1985) and Cayabyab
and Enríquez (2007). Photosynthetic and respi-
ration rates were obtained by measuring oxygen
flux evolution with a Clark-type O2 electrode ins-
talled in a DW3 chamber (Hansatech, UK) con-
nected to a controlled temperature circulating
bath. The incubation medium was filtered seawa-
ter at a temperature equal to that measured in
the field during plant collection: 23ºC for stations
S and I and 18ºC for station D. Dark respiration
rate was calculated after an initial incubation in-
terval of 15 min in darkness (initial Rd), with net
oxygen production (P) subsequently determined
for 13 light levels between of 14 and 1500 μmol
quanta m-2 s-1 provided by a LS2 tungsten-halo-
gen light source (Hansatech, UK). After exposure
to the final level of light intensity, the frond was
returned to darkness and the final dark respira-
tion rate determined (final Rd). No significant
differences were found between initial and final
respiration rates in all incubations performed.
Four C. cylindracea frond replicates of ca. 2 cm
length were incubated for each sampling station.
Prior to incubations, NaCO3 was added to the in-
cubation chamber up to a concentration of 5 mM
to prevent carbon limitation and N2 bubbled into
water to maintain oxygen concentrations within
a saturation range of 20-80%.
Rates of oxygen flux were normalized to biomass
(fresh weight, fw) and plotted against E values to
construct the P vs E curve, from which the pho-
tosynthetic parameters were then derived. The
maximum rate of net photosynthesis (net-Pmax
)
was estimated by averaging the maximum P
values obtained at saturating irradiances, with
gross photosynthesis (gross-Pmax
) then obtained
by adding Rd to net-P
max. Photosynthetic efficien-
cy (α) was estimated from the slope of the regres-
sion line fitted to the initial linear part of the cur-
ve. The point of compensation irradiance (Ec) was
calculated as the intercept on the irradiance axis
and the saturation irradiance (Ek) as the quotient
between and net-Pmax
. Mean values of c and Ek
obtained at each sampling station were then em-
ployed to calculate the light-compensation (Hc)
and light-saturation (Hk) periods, respectively, for
each daily light curve obtained from continuous
light measurement at the sea floor.
Daily metabolic carbon balance.
For the calculation of sampling station carbon
balance, the net photosynthetic rate (P) was de-
rived for each irradiance value of the entire light
time series obtained for that station using the fo-
llowing Michaelis-Menten function fitted to each
P vs E data set:
P = [gross-Pmax
·E/(E+Ek)] + R
d (Baly, 1935)
where gross-Pmax
, Ek and R
d are mean maximum
gross photosynthesis, saturation irradiance and
dark respiration rate obtained from the P vs E cur-
ves, respectively. This model is the best of a num-
ber of functions that have been previously found
to fit well with P-E plots obtained for Caulerpa
species (Gatusso and Jaubert 1985, Chisholm
and Jaubert 1997). The accuracy of this appli-
cation was evaluated by non-linear least-squa-
res regression at 95% probability (Sigma-Plot,
Jandel Scientific). Daily net photosynthesis was
obtained by numerical integration of calculated
P values over 24 h periods, before being trans-
formed into equivalent carbon units (daily net C
gain, mg C g-1 fw d-1) using the ratio g C:g O2 = 0.3,
assuming a photosynthetic quotient of 1.0 (Ma-
tta and Chapman 1991, Rosenberg et al. 1995).
If photoacclimation does occur, the photoac-
climation efficiency can be estimated as the
proportion of daily net photosynthesis due to
changes in photosynthetic parameters (Ruiz and
Romero 2001, Cayabyab and Enríquez, 2007).
For this purpose, we considered that if photoac-
climation does not occur at the deeper stations
(I and D), then the P vs E curve obtained from
these stations must be equal to that obtained for
plants from the shallower station, S. Therefore,
by integration of the P vs E curve from station S
with light data from stations I and D, we can si-
mulate the daily net photosynthesis that would
be expected for plants from these deeper sites if
photoacclimation did not occur. The photoaccli-
mation efficiency can be then calculated as the
relative percent difference between the simula-
ted values and real values, which were previously
obtained using the respective P vs E curves.
Data analysis
A one-way ANOVA (Quinn and Keough 2002) was
used to assess the significance of differences in
irradiance, algal biomass and photosynthetic pa-
rameters between sampling depths. Prior to this,
data were transformed when the assumption of
homoscedasticity was not fulfilled. A post-hoc
multiple pairwise comparison of means - the
Student-Newman-Keuls test (SNK; Quinn and
Keough 2002) - was used when ANOVA revealed
significant differences between depths. All signi-
ficant effects were tested at a probability level of
p = 0.05.
Results
The irradiance measurements of the studied
stations present a well-defined gradient related
to depth (Fig. 2, Table 1). Relative to their mean
value obtained at station S, both the noon ins-
tantaneous irradiance and the integral of daily
irradiance decreased significantly at the other
two stations: by 20 and 19%, respectively, at sta-
tion I and by 42 and 36% at station D (1-way
ANOVA, p<0.05; Table 1). Both the mean noon
sub-surface irradiance (Eo) and the water-column
attenuation coefficient (Kd) showed very similar
values between sampling stations, with no signi-
ficant differences observed (Table 1). Mean noon
instantaneous irradiance at the sea floor repre-
sented 33.6, 26.2 and 21.6% of Eo for stations S,
I and D, respectively.
Total algal biomass showed significant differen-
ces between sampling depths (F=8.3, P<0.05; Fig.
3). The highest mean value was obtained at the
shallowest depth (station S; 62.6 ± 17.4 g dw m-2),
decreasing by 20% at station I (49.7 ± 8.5 g dw
m-2) and by 73% at the deepest station (D, 16.9
± 2.9 g dw m-2).
The P vs E curves of C. cylindracea fronds ob-
tained at each station are presented in Figure
4, with mean values of the photosynthetic pa-
rameters summarized in Table 2. All photosyn-
p. 38 p. 39
CHAPTER 2
TESIS DOCTORAL
thetic parameters showed significant variation
between sampling depths. Mean net and gross-
Pmax
and Ek showed a substantial and significant
decrease with depth, although no significant
differences were found between mean values
of these parameters at stations I and D (SNK,
p>0.05), which were significantly lower than tho-
se obtained at the shallowest station (S) (Table
2). These differences were greater for the deepest
station (D) (38% for net-Pmax
, 37% for gross-Pmax
and 57% for Ek) than for station I (28, 25 and
38%, respectively). A similar general pattern was
found for Rd and E
c, but here the significant di-
fferences were caused only by the low mean va-
lues of these parameters obtained at the deepest
station (D), which were both ca. 33% lower than
those obtained at stations S and I. This variation
in mean photosynthetic and respiration rates
resulted in the gross-Pmax
:Rd ratios measured at
stations I (6.25) and D (7.39) being very close to
that obtained at station S (7.78). Photosynthe-
tic efficiency (α) was significantly higher at the
deepest station (D) than at the shallower two.
The average daily periods of light compensation
(Hc) and saturation (H
k) obtained by intercepting
the mean Ec and E
k derived from the P vs E curves
(Table 2) with the daily light curves (Fig. 2) are
also shown in Table 2. While mean Hc was rather
similar at each sampling depth, mean Hk showed
a slight but significant increase with depth, with
the absolute difference between stations S and
D being almost 2 hours. The daily metabolic car-
bon balance was highly positive at all sampling
depths, although the mean net C gain was 26%
and 16% lower at stations I and D, respectively,
relative to the mean values calculated for the
shallowest station S (Table 2). When considering
a scenario of no photoacclimation (Table 2), the
mean Hc values of stations I and D were very si-
milar to those obtained via their respective P-E
models. However mean Hk values recalculated at
these stations were ca. 27 and 52% lower, res-
pectively. The mean daily net C gain calculated
for station I under the assumption of no photoac-
climation was similar to that obtained using the
P-E model for this station, but was ca. 29% lower
at station D.
Discussion
Photosynthetic responses observed along the
studied depth gradient essentially consisted of:
a) an increase in photosynthetic efficiency at
sub-saturating irradiances (α); b) a decrease in
the maximum photosynthetic rates at saturating
irradiances (gross- and net-Pmax
); and c) a reduc-
tion of the respiratory demand (Rd). Clearly, the
simplicity of the sampling design employed in
this study (i.e. a comparison between the three,
single sampling sites) precludes interpretation
of the reported photosynthetic behaviour of C.
cylindracea across sampling depths exclusively
in terms of photoacclimation. In fact, despite the
clear pattern of light reduction (Fig. 2 and Table
1), other local factors not controlled by the ex-
perimental design can also vary with depth and
influence photosynthetic performance. However,
the photosynthetic responses observed in this
study are in good agreement with general meta-
bolic strategies used by sublittoral macroalgae to
optimise light use and productivity under varia-
ble light conditions (Markager and Sand-Jensen
1992, Kirk 1994, Pérez-Lloréns et al. 1996, Gómez
et al. 1997, Lobban and Harrison 1997, Gómez
2001). Furthermore, our results are highly consis-
tent with differences in photosynthetic behaviour
reported by other studied Mediterranean popula-
tions of C. cylindracea between shallow and deep
habitats (Raniello et al. 2006). Importantly, these
authors also interpreted there results as due to
photoacclimation.
The pattern of variation of photosynthetic para-
meters was not uniform between sampling dep-
ths. The decline in photosynthetic rates (net and
gross Pmax
) and Ek represents the most significant
photosynthetic response of fronds at the interme-
diate depth (I, 18 m). No further decline in pho-
tosynthetic rate was observed at the deepest sta-
tion (D, 26 m), but the lowest light requirements
(Ek and E
c mean values) were recorded here. This
is likely due to the increase in photosynthetic effi-
ciency (α) and decrease in respiration activity Rd,
both considered clear adaptations for the growth
and survival of macroalgae under limiting light
conditions (Markager and Sand Jensen 1992, Gó-
mez 2001).
Table 1.
Summary of irradiance measurements at each sampling station, determined from continuous light measu-
rement at the sea floor during a 21 day period (i.e. daily light curves; Fig. 2) and instantaneous irradiance
just below the surface measured at noon on standard sunny days (n = 5). Data are presented as means ±
standard error. MS = means squares, F = F statistic, *p<0.05, **p<0.01, n.s. = not significant.
Measurement:Daily light curves at sea floor:Noon instantaneous irradiance(μmol quanta m-2 s-1)Integrated daily irradiance(mol quanta m-2 d-1)Sub-surface irradiance:Noon instantaneous irradianceE
o (μmol quanta m-2 s-1)
Water-column attenuation coefficientK
d (m-1)
% Eo
Sampling station One way ANOVA
S (- 11 m)
442 ± 28
11.74 ± 0.56
1710 ± 78
0.098 ± 0.01
36.68
I (-18 m)
354 ± 21
9.47 ± 0.30
1677 ± 224
0.082 ± 0.01
26.24
D (-26 m)
254 ± 17
7.52 ± 0.24
1659 ± 219
0.095 ± 0.01
21.6
MS
40686
0.047
42432
0.000007
-
F
146.86**
19.56**
0.44 n.s.
0.25 n.s.
-
Fig. 2.
Daily variation in sea floor irradiance at
each sampling station. Data are presen-
ted as the average of 21 d (see methods
section). These curves were used to esti-
mate the daily period of light saturation
(Hk) and compensation (H
c) from satura-
tion (Ek) and compensation (E
c) irradian-
ces obtained in the P vs E curves (see
Table 2).
p. 40 p. 41
CHAPTER 2
TESIS DOCTORAL
Higher photosynthetic efficiencies (α) is a com-
mon feature of macroalgae to reduce light
requirements for photosynthesis in low light
environments and is usually achieved through
adjustment of light-harvesting pigments; i.e. the
size of the absorption cross section (Gómez et al.
1997, Lobban and Harrison 1997, Falkowski and
Raven 2007). Accordingly, high ratios of both
chlorophyll b and siphonoxantin to chlorophyll
a have been reported for a number of Caulerpa
species living in deep water environments (Yoko-
hama and Misonou 1980, Riechert and Dawes
1986, Williams and Dennison 1990, Raniello et
al. 2006), although no changes in α and pigment
contents was found for the other Mediterranean
invader C. taxifolia across a depth range simi-
lar to that of this study (Chisholm and Jaubert
1997). Studying a Mediterranean C. cylindracea
population, Raniello et al. (2006) found increased
concentrations of these accessory pigments with
depth parallel to an increment in α and a decline
in Ek derived from ETR curves, which is consistent
with the photosynthetic behaviour of the alga
reported in this study for the deepest station (D)
using P vs E curves. All these evidence suggest
that the plasticity of α can be an important pho-
toacclimatory mechanism of C. cylindracea to
cope with light reduction with depth.
A low respiration rate is also considered a com-
mon strategy to minimize light requirements and
carbon losses in macroalgal species growing in
deep water habitats (Littler et al. 1986, Marka-
ger and Sand-Jensen 1992, Gómez et al. 1997,
Johanson and Snoeijs 2002). In our study, the
reduced dark respiration observed at the deepest
station (D) enabled net-Pmax
and the P:Rd ratio
to be maintained at levels similar to those ob-
served at the shallower intermediate station (I).
This reduction in dark respiration has not been
previously documented in Mediterranean popu-
lations of C. cylindracea although it is consistent
with that observed in a tropical population of C.
racemosa across a similar depth gradient (Rie-
chert and Dawes 19867). The lower ambient
temperature of the deepest station (D; 18ºC) in
relation to the shallower intermediate station (I;
21ºC) could also have contributed to the decrea-
se in respiratory activity (e.g. Terrados and Ros
1992). However, previous experimental evidence
for this (Flagella et al. 2008) and other species
of this genus (Gattuso and Jaubert 1985) indi-
cates a low sensitivity of Rd to this factor within
this narrow temperature range. Similarly, plants
of the congeneric C. taxifolia collected in summer
showed very constant dark respiration rates when
incubated across the temperature range between
Table 2.
Photosynthetic parameters derived from P vs E curves and daily net carbon gain calculated for each sampling station. Data are presented
as mean ± standard error. Different letters indicate groups of homogeneous means obtained in the post-hoc test SNK (p<0.05). MS = means
squares, F = F statistic, *p<0.05, **p<0.01. Mean values of Hk, H
c and daily C gains calculated under the assumption of no photoacclimation are
indicated in the lower part of the table, as well as the photoacclimation efficiency (see the Materials and Methods section).
Variablenet-P
max
(μmol O2 g fw-1 h-1)
Rd
(μmol O2 g fw-1 h-1)
gross-Pmax
(μmol O
2 g fw-1 h-1)
α(μmol O
2 g fw-1 h-1/μmol quanta m-2 s-1)
Ek
(μmol quanta m-2 s-1)E
c
(μmol quanta m-2 s-1)H
k
(h)H
c
(h)Daily net C gain(mg C g FW-1 d-1)
Hk
(h)H
c
(h)Daily net C gain(mg C g FW-1 d-1)
Sampling station (depth) One way ANOVA
S (11 m)
12.00 ± 0.8 a
1.76 ± 0.15 a
13.77±0.92 a
0.05 ± 0.01a
229.50 ± 17.9 a
22.60 ± 3.4 a,b
6.4 ± 0.4 a
11.9 ± 0.1a
0.45 ± 0.03 a
I (18 m)
8.60 ± 0,1b
1.64 ± 0.04 a
10.25 ± 0.16 b
0.05 ± 0.01a
141.70 ± 35.0 b
37.30. ± 8.8 a
7.8 ± 0.2 b
10.8 ± 0.1 b
0.33 ± 0.01 b
I (18 m)
5.7 ± 0.3
11.5 ± 0.05
0.36 ± 0.02
I (18 m)
-26.9%
-3.6
+9%
D (26 m)
7.50 ± 0.4 b
1.17 ± 0.41 b
8.65 ± 0.50 b
0.08 ± 0.01 b
98.90 ± 4.8 b
15.20 ± 7.0 b
8.3 ±0.2 b
11.9 ± 0.1 a
0.38 ± 0.01 b
D (26 m)
4.0 ± 0.3
11.5 ± 0.01
0.27 ± 0.01
D (26 m)
-52%
-3.6
- 28.9%
MS
0.04
0.01
24.35
0.004
0.48
0.25
20.50
8.70
0.07
F
27.0**
5.80*
13.72**
7.20*
12.7**
5.00*
12.4**
69.8**
9.80**
simulation of no photoacclimation: photoacclimation efficiency:
Fig. 3.
Total biomass (g DW m-2) of C. cylindra-cea at each sampling station. Data are
presented as means and standard errors
(n = 6). Different letters indicate groups
of homogeneous means obtained in the
post-hoc test SNK (p<0.05).
Fig. 4.
Photosynthesis vs Irradiance (P vs E)
curves determined from C. cylindracea fronds at sampling stations S (full circles),
I (full squares) and D (empty triangles).
Points are means ± SE (n = 4). The solid
line represents the curve model fitted to
experimental data (see methods section).
p. 42 p. 43
CHAPTER 2
TESIS DOCTORAL
15 and 25ºC (Gacia et al. 1996b). Thus, reduction
in metabolic demands could effectively reflect a
strategy of the alga to maximize carbon gains
under more limiting light conditions.
The photosynthetic responses reported in this
study support the hypothesis that photosynthe-
tic plasticity can be an important mechanism
accounting for the success of C. cylindracea
across the depth gradients found in Mediterra-
nean coastal waters (Raniello et al. 2006), and
that this can be achieved through optimisation
of carbon fixation as light becomes more limiting
(Markager and Sand Jensen, 1992, Markager and
Sand-Jensen 1994, Gómez 2001). Carbon balan-
ce calculations support this hypothesis for the
studied C. cylindracea populations. In the inter-
mediate station I, reductions in incident light and
daily net productivity were similar in magnitude
(28% and 26%, respectively, relative to station
S). However, at the deeper station D, where there
is a more severe light reduction (41%), the daily
net C gain was only 15% lower than that esti-
mated for the shallower station S. This result can
only be explained by reduced light requirements
and respiratory losses (and higher photosynthe-
tic efficiency) in C. cylindracea fronds from the
deepest population, which yielded mean P:R ra-
tios and Hk and H
c periods very close to (or even
higher than) those obtained for the shallow sta-
tion S. In the absence of photoacclimation, Hk va-
lues should be considerably shorter (4 h) and me-
tabolic carbon balance ca. 29% lower than the
values actually measured in fronds from station
D. Therefore, the reported photosynthetic res-
ponses effectively reverted during optimisation
of C. cylindracea productivity in the deepest site.
In conclusion, our results provide evidence su-
pporting the capacity of C. cylindracea to cope
with changes in the light regime caused by depth
variations through photosynthetic adjustments.
Furthermore, this coping mechanism enables
deep-water C. cylindracea populations to achieve
positive carbon balances close to those quantified
for shallower populations: at least during a period
of the year when growth conditions are optimal
(i.e. the end summer, see for example Ruitton et
al. 2005b). However, generalization of this con-
clusion must be made with caution until confir-
mation by similar studies at broader spatial and
temporal scales. Other questions arise from our
results that should be addressed by further future
research. For example, the lower respiration rates
observed for C. cylindracea fronds from the dee-
pest station (D) suggest reduced growth rates at
this depth (Gómez 2001), which would allow for
carbon storage to support algal growth and bio-
mass under situations of severe light limitation in
these deeper waters, such as during the winter.
Although evidence for this metabolic adaptation
has been reported in Caulerpa species (Robledo
and Freile-Pelegrín, 2005), and has been sugges-
ted for macroalgae species which display nega-
tive carbon balances in winter (see Dunton and
Shell 1986, Gómez 2001), this metabolic adap-
tation has not yet been demonstrated for Medi-
terranean C. cylindracea populations. Moreover,
the existence of such adaptive mechanisms could
limit the capacity of this species to accumulate
biomass in deep assemblages (as suggested by
the lower algal abundance measured in station
D). In this sense, much more research is neces-
sary to determine which factor(s) are responsible
for uncoupling C. cylindracea net productivity
and biomass. In particular, attention must be
paid to the multiple environmental (abiotic and
biotic) factors that can influence vertical patterns
of algal abundance (Piazzi et al. 2001a, Ruitton
et al. 2005a,Bulleri and Benedetti-Cecchi 2008,
Cebrian and Ballesteros 2009, Klein and Verlaque
2008, 2009, Tomas et al. 2011), as well as the an-
nual carbon balance of the algae.
CHAPTER 3Resistance of Posidonia oceanica
seagrass meadows to the spread of the
introduced green alga Caulerpa cylin-
dracea: assessment of the role of light
p. 47
Resistance of Posidonia oceanica seagrass meadows to the spread of the introduced green alga Caulerpa cylin-dracea: assessment of the role of light.
Abstract
Posidonia oceanica seagrass meadows are one
of most resistant Mediterranean habitats to in-
vasion by the green alga Caulerpa cylindracea.
We evaluated the hypothesis that light reduction
caused by the seagrass canopy can limit algal
photosynthesis and growth and hence potentia-
lly explain this resistance. To this end, we analy-
sed light regimes and C. cylindracea biomass and
photoacclimative variables measured outside
and within P. oceanica meadows at different si-
tes and during contrasting times. The success of
photoacclimatory responses was assessed using
an ecophysiological, carbon balance approach.
C. cylindracea abundance significantly varied
depending on the sampling site and time, but its
biomass was always 10- to 50-fold higher out-
side the meadow. Outside the canopy, C. cylin-
dracea showed characteristic morphological and
photosynthetic plasticity closely related to the
spatio-temporal variation in light regimes, which
varied as expected with depth and season. Under
these conditions, the alga was able to perform
successful photoacclimation, although some
degree of light limitation was observed at the
deepest sites and in winter conditions, as indica-
ted by near-zero carbon balance and lower algal
abundances. Within the P. oceanica canopy, light
was reduced by 60–89% relative to that outside
and was at its lowest levels recorded (1–7% of
the sub-surface irradiance), close to the minimum
light requirements for growth. Light limitation
was evident inside the canopy in the winter sam-
pling, when the photosynthetic plasticity of the
alga appears to be exceeded and when carbon
balances were clearly negative. Therefore, light
appears to play a key role in the apparent inca-
pacity of C. cylindracea to penetrate within P.
oceanica meadow edges.
Introduction
The green alga Caulerpa cylindracea (Sonder)
(hereinafter, C. cylindracea), described as one
of the most successful invaders of the Medite-
rranean Sea (Streftaris and Zenetos 2006) has
successfully colonized a wide variety of soft and
hard substrata, including dead Posidonia oceani-
ca rhizomes or ‘‘matte’’ (i.e. the compact bioge-
nic structure resulting from growth of rhizomes
intertwined with roots and autochthonous and
allochthonous detritus; Boudouresque and Meis-
nez, 1982). It is found at depths between 0 to
60 m, being most abundant between 0 and 30
m (Klein and Verlaque 2008). Overgrowth by C.
cylindracea on Mediterranean benthic commu-
nities can alter biodiversity (Argyrou et al. 1999,
Piazzi et al. 2001b, Balata et al. 2004, Piazzi and
Publicado en:Marín-Guirao L, Bernardeau-Esteller J, Ruiz JM, Sandoval-Gil JM (2015) Resistance of Posidonia oceani-
ca seagrass meadows to the spread of the introduced green alga Caulerpa cylindracea: assessment of
the role of light. Biol Inv 17 (7): 1989-2009.
p. 48 p. 49
CHAPTER 3
TESIS DOCTORAL
Balatta 2008, Vázquez-Luis et al. 2008, Klein and
Verlaque 2009), but the degree and extent of this
impact depends on many factors, including the
type of assemblage (Lonsdale 1999, Arenas et al.
2006).
At present, little is known about the variation in
the resistance of natural Mediterranean commu-
nities to C. cylindracea invasion, but it has been
proposed that benthic assemblages dominated
by canopy-forming species are more resistant to
invasion since the canopy might limit resources,
especially light and space (Piazzi et al. 2001a,
Ceccherelli et al. 2002, Klein and Verlaque 2008,
Bulleri and Benedetti 2008, Bulleri et al. 2010).
However, the mechanisms underlying the resis-
tance to invasion of benthic assemblages have
rarely been investigated and are poorly unders-
tood (Lonsdale 1999, Arenas et al. 2006, Brit-
ton-Simmons 2006). Meadows of P. oceanica are
one of the Mediterranean infralittoral biocenoses
that is more resistant to invasion by C. cylindra-
cea (Klein and Verlaque 2008). The invasive alga
is not usually found within P. oceanica meadows,
whereas it has often been found at the edges of
meadows or in very sparse or patchy meadows
(Occhipinti-Ambrogi and Savini 2003, Piazzi et
al. 1997a, b, Piazzi and Cinelli 1999, Ceccherelli
et al. 2000, Montefalcone et al. 2007, Katsane-
vakis et al. 2010, Infantes et al 2011, Ruiz et al.
2011). This resistance to the invasion of the alga
has been related to P. oceanica shoot density,
suggesting that some factors correlated with the
canopy structure must be involved in the reduced
capacity of C. cylindracea to penetrate the mea-
dows, such as space limitation, water motion, nu-
trient supply or canopy shading (Ceccherelli et al.
2000). In the present study we examine the role
that light may play in determining the resilience
of P. oceanica to this highly invasive alga.
P. oceanica is an ecosystem engineer (Koch
2001) that forms conspicuous and extensive
meadows from near the surface, to depths of
30–40 m and its ecological importance is widely
recognised (e.g. Pergent et al. 2012). P. oceani-
ca is a clonal plant consisting of a basal rooted
rhizome, with shoots of vertical and horizontal
growth, bearing 5 to 10 blade-like leaves, 12 mm
broad and more than 1 m long. This large shoot
size, together with the high shoot densities of P.
oceanica meadows (400–1,000 shoots m-2; Ba-
lestri et al. 2003, Procaccini et al. 2003), creates
a highly complex canopy structure. In fact, the
mean leaf area index (LAI) of P. oceanica mea-
dows can reach values as high as 13 m2.m-2 (e.g.
Romero 1985, Balestri et al. 2003), which is com-
parable with the maximum values measured in
terrestrial forest canopies (Scurlock et al. 2001).
These LAI values are also very high when com-
pared with those obtained for other seagrass
species of similar architecture (e.g. Thalassia tes-
tudinum, LAI = 0.65–4.34 m2 m-2; Enríquez and
Pantoja-Reyes 2005). These seagrass meadows
thus strongly modify the environmental condi-
tions within their leaf canopies, particularly the
light climate (Enríquez et al. 1992, Dalla Via et
al. 1998, Zimmerman 2006). As previously shown
in other canopy-forming plant communities, such
as terrestrial (Canhan et al. 1990) and kelp (Clark
et al. 2004) forests, modification of the light envi-
ronment has been shown to be involved in the de-
termination of the structure of the understorey.
Accordingly, most algal species that inhabit the
basal part of P. oceanica meadows are sciaphi-
lic, and a large number of them are undeveloped
and do not grow beyond juvenile stages (Templa-
do et al. 2004).
Previous studies have demonstrated the high
photosynthetic plasticity of Mediterranean po-
pulations of C. cylindracea, which could allow
the alga to acclimate to reduced light conditions
(Bernardeau-Esteller et al. 2011, Raniello et al.
2004, 2006). C. cylindracea has been shown to
be able to colonise and photoacclimate the ba-
sal substratum of C. nodosa meadows, another
common Mediterranean seagrass, the canopy of
which is less complex than that of P. oceanica re-
ducing any shading effect (Raniello et al. 2004).
However, the capacity of the alga to photoaccli-
mate to the more severe light reductions created
by P. oceanica leaf canopies has not yet been in-
vestigated. Furthermore, the extent to which the
photoacclimatory responses elicited by the alga
effectively compensate for imbalances of the
metabolic carbon budget, which ultimately de-
termines the availability of resources for survival
and growth under reduced-light conditions (Mal-
ta and Chapman 1991), has also been neglected.
The general aim of the present study was to con-
tribute to the understanding of the mechanisms
underlying the resistance by native P. oceanica
meadows to the spread of C. cylindracea. We
specifically examined the hypothesis that the
light regime within P. oceanica leaf canopies
might limit C. cylindracea growth and survival
under canopies of this seagrass species. To this
end, we performed a comparative analysis of li-
ght regimes and C. cylindracea variables related
to its abundance (total biomass) and photoac-
climative capacities (frond length, pigments and
photosynthetic parameters), characterised outsi-
de and within P. oceanica meadows of different
highly-invaded sites of the southeastern coast of
Spain (Ruiz et al. 2011). We used an ecophysio-
logical, carbon balance approach by integrating
daily light curves and photosynthesis-irradiance
(P-E) models to obtain the average daily net pro-
ductivity of the alga (Bernardeau et al 2011).
Material and Methods
Experimental design
The present study was conducted in 2009 on the
Mediterranean coast of Murcia (SE Spain), where
the invasive alga C. cylindracea was observed for
the first time in 2005 (Ruiz et al. 2011). Canopy
properties, light regimes and algal abundance
were sampled within and outside P. oceanica
leaf canopies of highly-invaded areas identified
in this region (Ruiz et al. 2011; Bernardeau et al
2011). Within this area, sampling was done at
different sites and times to assess a variety of en-
vironmental situations and encompass as much
as possible the spatio-temporal variability of this
habitat. The three sites, Isla Grosa (IG; 37º43’N,
00º42’E), Cabo Tiñoso (CT; 37º32’N, 00º44’E)
and Calblanque (CB;37º32’N, 01º07’E), had con-
trasting depths (11, 18 and 26 m, respectively),
with a range that encompassed most of the verti-
cal distribution of P. oceanica and C. cylindracea
in this region. The three sites were in an area with
similar climate and oceanography (Vargas-Yáñez
et al. 2010) and substratum type (i.e. detritic soft
bottoms mixed with dead P. oceanica “matte”;
Calvín-Calvo et al. 1998, Ruiz et al. 2011). At all
sites, the substratum outside the P. oceanica
meadow was almost totally covered by dense
C. cylindracea stands, from which some stolons
penetrated inside the seagrass leaf canopy, al-
though only up to the first 25–40 cm from the
meadow edge. As for the outside, within the P.
oceanica canopy, the stolons of the alga coloni-
sed both sediments and basal seagrass rhizomes
(dead and alive). There was no apparent discon-
tinuity in the nature of the substratum or any
other environmental feature that could be rela-
ted to the position of the transition between the
meadow and the algal stand. To assess temporal
variation, sampling of all three sites was done in
both January and July, times that represented
the two extremes of the seasonal environmental
variation: winter (T1) and summer (T2). In order
to avoid temporal resampling of the sites, the
sampling of the selected variables at each time
p. 50 p. 51
CHAPTER 3
TESIS DOCTORAL
and site was done in a randomly-selected mea-
dow area 50 meters in length and 10 meters in
width (i.e. a sampling area of 500 m2), with the
long axis of the rectangle centered on the sea-
grass meadow edge.
For each sampling site and time, algal variables
and light regimes were determined at two posi-
tions relative to the edge of the seagrass mea-
dow: i) an outer position (OUT), on the adjacent
densely-invaded detritic sediments within 1-2 m
from the meadow edge and ii) at an inner posi-
tion (IN), 25–40 cm from the meadow edge. In
addition, general descriptive data of the P. ocea-
nica meadow structure were also collected.
Caulerpa cylindracea biomass, frond height and
pigments
Samples of C. cylindracea were gathered in five
randomly-selected sampling locations separated
by 10 m at each site, time and position. In each
sampling location, fronds, stolons and rhizoids of
C. cylindracea were carefully collected by hand
within three 400 cm2 square frames randomly
distributed in the area (Ruitton et al. 2005b).
Samples were transported to the laboratory in
plastic bags together with seawater, in chilled
containers. After removing the sediment, debris
and other algal species, total C. cylindracea bio-
mass (g DW m-2) was determined by drying the
samples at 70°C to constant weight. The three
biomass values determined in each sampling lo-
cation were then averaged to obtain five replica-
tes (n = 5) for each site, time and position combi-
nation. The frond height (cm) was determined by
measuring the height of 10 algal fronds rando-
mly selected from each sample. Measurements
were averaged per sample and then per sampling
location to constitute one of the five replicates (n
= 5) of each site, time and position combination.
The pigment content was analysed in 2 rando-
mly-selected healthy and non-epiphytised C.
cylindracea fronds of approximately 3 cm in hei-
ght from each C. cylindracea sample. Results from
the analyses were averaged per sample and sam-
pling location so the final number of replicates
was five (n = 5) for each site, time and position.
The analysis was conducted spectrophotometri-
cally after manual extraction of a homogenised
suspension using 90% acetone (Dennison 1990),
with MgCO3 added as a chlorophyll stabiliser. The
acetone extracts (10 ml) were stored at 4°C in the
dark for 24 h and centrifuged. The chlorophyll a
and b content was computed using the equations
of Lichtenthaler & Wellburn (1983).
Posidonia oceanica meadow and leaf canopy
structure
To characterise the structure of the three selected
meadows, the shoot density and the percentage
of meadow cover were measured at all sampling
sites and times, following standard methods adop-
ted for this seagrass species (Ruiz et al. 2010 a,b).
Shoot density (shoots m-2) was estimated in six
randomly-selected locations in each meadow by
counting the number of shoots within two 400-
cm2 quadrats randomly placed within each loca-
tion. The average of each pair of measurements
was the individual, independent replicate (n = 6).
The percentage of meadow cover was visually es-
timated as the percentage of the bottom covered
by seagrass patches within 1,600-cm2 square fra-
mes subdivided into four 20×20 cm squares. Visual
estimations were performed every meter along
three (n = 3), 10-m linear transects randomly selec-
ted within the meadow at each visit.
The leaf area index (LAI) and the canopy height
were used to characterise the leaf canopy. In
each site and time, 5 shoots were collected in
four randomly-selected locations. The total leaf
surface area (based on one side) was calculated
for each shoot by measuring the length and wid-
th of all leaves per shoot and averaged for each
sampling location. LAI (m2 m-2), as a descriptor
of the degree of leaf packing within the canopy
(Enriquez and Pantoja-Reyes 2005), was then cal-
culated by multiplying the total leaf area of each
location by the averaged shoot density of each
meadow, so the total number of replicates was
four (n = 4) in each site and time. The canopy
height (cm) was estimated in situ by divers, using
a ruler and taking two measurements at six di-
fferent locations along the meadow edges. The
average value of the two measurements in each
location constituted each one of the replicates
per site and time (n = 6).
Irradiance measurements
The light field at each site, time and position was
characterised 5 cm above the bottom using sphe-
rical quantum sensors (Alec MDS MK5). Sensors
were programmed to record irradiance values
every 10 min and recorded data for at least two
weeks in each season. Maximum instantaneous
irradiance at noon (Emax
, μmol quanta m-2 s-1) and
total daily irradiance values (Etotal
, mol quanta m-2
d-1) were obtained from the diurnal irradiance cy-
cles.
Light attenuation coefficients were determined
for both the water column (water-Kd, m-1) and
the meadow canopy (canopy-Kd, m-1) under stan-
dard conditions (i.e. between 12:00 h and 14:00
h on standard sunny days with minimal water
movement and sediment resuspension; Enrí-
quez and Pantoja-Reyes 2005). Water column
down-welling irradiance was measured using a
cosine-corrected quantum sensor (LI-190SA; LI-
COR). Irradiance data were recorded for each m
from the sea subsurface (E0) to the sea bottom
(Ez), integrating the values obtained over 10 s on
three different days in both summer and winter.
Irradiance within the seagrass canopy was me-
asured using spherical quantum sensors (Alec
MDS MK5) at every 5 cm from the base of the
meadow to the top of the canopy using a marked
vertical bar for reference. Data were averaged for
a 10-s period (one measurement s-1) at each hei-
ght. Light attenuation coefficients (water-Kd and
canopy-Kd) were estimated using the Beer-Lam-
bert equation: Ez=E
0e-Kdz; where E
z and E
0 are the
irradiance values at a given depth (z in m) and at
the sea subsurface/top canopy, respectively; and
Kd is the light attenuation coefficient (Kirk 1994).
The percentage of subsurface irradiance that
reached the sea bottom was calculated from E0
(subsurface irradiance) measured at noon on
calm sunny days using the LI-COR quantum sen-
sor and the corresponding sea floor values within
and outside the meadows measured using the
spherical quantum sensors. Both sensor types
were intercalibrated in the laboratory, showing a
strong linear relationship with a constant factor
of 1.02.
C. cylindracea photosynthetic variables
Prior to photosynthetic measurements, C. cylin-
dracea samples were kept overnight in the dark
under controlled temperature in natural seawa-
ter taken from the collection site. Photosynthesis
and dark respiration rates were measured using
a polarographic oxygen electrode and a mag-
netic stirrer (DW3, Hansatech Instruments Ltd)
under controlled temperature (Bernardeau et al.
2011). Incubation was carried out at the same
temperature as that measured in the field during
sampling: 13°C in winter for all three sites; and
24°C at IG and 22°C at CT and CB in summer.
Apical segments of non-epiphytised C. cylindra-
cea fronds of approximately 2 cm in length were
used for the measurements. Two replicated algal
segments from three of the five sampling loca-
tions (see above) were randomly selected and
p. 52 p. 53
CHAPTER 3
TESIS DOCTORAL
employed for the photosynthetic measurements
(n = 3). Dark respiration rates were measured by
maintaining the fronds in the dark for 15 min. Net
oxygen production was then determined at 13 di-
fferent light intensities (from 14 to 2,271 μmol
quanta m-2 s-1) using a high-intensity light sour-
ce (LS2, Hansatech Instruments Ltd). Net pho-
tosynthetic rates were plotted against the light
intensities (P-E curves), and the photosynthetic
parameters were calculated as follows: the maxi-
mum rate of net photosynthesis (net-Pmax
, μmol
O2 g-1 FW h-1) was determined by averaging the
maximum values above the saturating irradian-
ce (Ek). The photosynthetic efficiency (α, μmol O
2
g-1 FW h-1/μmol quanta m-2 s-1) was calculated as
the slope of the regression line fitted to the initial
linear part of the P-E curve, and the compensa-
tion irradiance (Ec) as the intercept on the X-axis.
Ek was calculated as the ratio P
max/α. Mean daily
compensation (Hc) and saturation (H
k) periods
were calculated from each daily light curve as the
number of h per day that irradiance values excee-
ded Ec and E
k mean values, respectively.
Daily metabolic carbon balances
Daily carbon balance, as a predictor of plant li-
ght limitation (Dennison and Alberte 1985), was
calculated according to the Michaelis-Menten
function (P = [gross-Pmax
E/(E+Ek)] + R [Baly 1935])
previously applied to C. cylindracea (Gatusso and
Jaubert 1985, Bernardeau et al. 2011), where P
is net photosynthesis, gross-Pmax
is the maximum
gross photosynthetic rate, E is the irradiance me-
asured in the field, Ek is the saturation irradiance,
and R is the respiration rate. Photosynthetic para-
meters obtained from P-E curves and continuous
recordings of field irradiance measurements were
entered into the function to generate estimates
of net production, which were integrated across
24 h periods to yield daily net production values
(n = 3). If the photosynthetic quotient is assumed
to equal unity, and the ratio g C:g O2 = 0.3 (Matta
and Chapman 1991), then the net productivity
in oxygen units can be multiplied by 0.012 to ob-
tain the equivalent carbon units (mg C g FW-1).
This calculation presumes constant dark respira-
tion throughout the day and does not consider
other carbon losses (exudation, grazing) or gains
(light-independent carbon fixation).
Data analyses
The spatio-temporal variation of P. oceanica
meadow structure descriptors was evaluated
using a two-way ANOVA with sampling sites
(three levels: IG, CT and CB) and times (two levels:
T1, in winter, and T2, in summer) as fixed factors.
For the analysis of C. cylindracea variables a ran-
domized-block design was applied for each sam-
pling time separately, defining sites as blocks and
position (two levels: IN vs OUT) as main factor.
For both designs, prior to carrying out the ANO-
VA, the data were tested for heterogeneity of
variance using Cochran’s C-test and transformed
when necessary. Where variance remained hete-
rogeneous, untransformed data were analysed,
as ANOVA is a robust statistical test and is rela-
tively unaffected by the heterogeneity of varian-
ces, particularly in balanced designs (Underwood
1997). The Student-Newman-Keuls (SNK) test
was used for a posteriori pairwise comparisons of
means. A probability level of 0.05 was regarded
as significant except when data transformation
was not possible. In such cases, the level of signi-
ficance was reduced to P < 0.01 to minimize type
I errors. Randomized block analysis also assumes
that there are no interactions between blocks
and the main factor. To test this assumption plots
of dependent variables versus blocks were exami-
ned (Quinn and Keough 2002). Regression analy-
sis was used in order to describe the relationship
between irradiance and plant variables with the
depth of the site. Univariate statistical analysis
was performed using the statistical package STA-
TISTICA (StatSoft Inc. 2001, version 6.0).
We also employed a multivariate approach to
explore photoacclimatative response patterns
between sites, times and positions, but based on
the integration of the multiple univariate respon-
ses obtained in each case. Principal Components
Analysis (PCA) was carried out on the correlation
matrix of photoaclimatation variables, following
fourth square transformation of the data. This
analysis provides a measure of association be-
tween each original variable and the resulting
principal components. Multivariate analysis was
conducted using the software package CANOCO
version 4.5 for Windows (Ter Braack and Šmilauer
2002).
Results
Light regimes
At both sampling times, mean noon subsurface
irradiance (E0) was similar between sites, but with
higher values in the summer sampling (T2) than
in the winter sampling (T1) (Table 1). The water
column attenuation coefficients (water-Kd) were
also similar between sites and times (two-way
ANOVA, P > 0.05), with mean values ranging from
0.082 to 0.124 m-1.
At T1, the mean total daily irradiance (Etotal
) and
the noon maximum irradiance (Emax
) obtained at
the bottom in the OUT-position varied by an or-
der of magnitude between sites, with mean Emax
values representing 4 to 27% of E0. Irradiance
showed a high and negative exponential relation
with the depth (z) of the sampling site (e.g. Etotal
= 1149.5·e-0.13z , R2 = -0.994), with highest mean
values recorded at the shallowest site IG, and the
lowest at the deepest, CB. At T2, these irradian-
ce mean values were 13–41% of Eo and 2–5 fold
higher than at T1, and also showed a similar, but
linear negative relation with site depth (Etotal
=
939- 27.9·z ; R2 = -0.992).
At both sampling times, Etotal
and Emax
mean va-
lues obtained inside the seagrass canopy (IN-po-
sition) at the three sites were reduced (82–88%
at T1 and 60–89% at T2), relative to those deter-
mined in the OUT-position. At T1, between-site
variation of irradiance mean values in the IN-po-
sition reflected that of external light availability
(i.e. OUT-position mean values), but not in T2
where mean values determined at the IN-posi-
tion showed very small variation between sites
(1.59–2.30 mol quanta m-2 s-1). The sharp light
extinction associated with the seagrass canopy
shelf-shading corresponded to canopy-Kd mean
values that ranged between 5.8 and 8.5 m-1. Sites
showed significant differences between mean ca-
nopy-Kd values (two-way ANOVA, P < 0.001), with
the deepest meadow CB always showing the
lowest values (Table 1).
Seagrass meadow structure
Meadow structure descriptors showed significant
variation between sampling sites and times, ex-
cept in the case of meadow cover, for which diffe-
rences were only significant among sites (Fig. 1,
Table 2). Spatial variation was the major source
of variation of shoot density (54.7%) and mea-
dow cover (97.6%), and temporal variation in the
case of LAI (70.0%) and canopy height (87.2%).
For shoot density, LAI and canopy height, the pa-
ttern of variation among sites differed between
sampling times, as indicated by the significant
effect of the ANOVA interaction term (Table 2).
At both sampling times, spatial variation showed
a high significant negative correlation with the
depth of the sampling sites in the case of the
shoot density (R2 = 0.58 – 0.72, β = -0.76 – -0.85,
P < 0.001, N = 18), meadow cover (R2 = 0.78 –
0.88, β = -0.88 – -0.94, P < 0.01, N = 9) and LAI
(R2 = 0. 53 – 0.93, β = -0.73 – -0.96, P < 0.01, N =
12). In all these cases, maximum values of the
variable were found at the shallowest site and
minimum values at the deeper one.
p. 54 p. 55
CHAPTER 3
TESIS DOCTORAL
Total algal biomass
The position of the alga relative to the meadow
edge showed a significant effect on the total
alga biomass at both times (Fig. 2, Table 3). The
stands of C. cylindracea growing outside the sea-
grass meadow showed up to a 50-fold higher bio-
mass than that of stands growing beneath the
leaf canopy, except at T1 at the shallower IG site
where this variable was similar in OUT and IN-po-
sitions. Algal biomass was also significantly affec-
ted by sites at both times (Table 3) and showed a
strong negative correlation with the depth of the
sampling site (R2 = 0.64, β = -0.80, P < 0.001, N =
15), but only at T2 at the OUT-position.
Caulerpa cylindracea frond variables
In relation to the height of the fronds, the posi-
tion of C. cylindracea stands was the factor that
explained the major component of its total va-
riance in both sampling times (59-66%), and on
average, fronds growing within the meadows (i.e.
IN-position) were almost twice as tall as those
growing in the OUT-position. The blocking factor
‘site’ had also a significant effect on this variable
at both sampling times (Fig. 3, Table 3), which
showed a significant positive correlation with the
depth of the sampling sites outside the leaf ca-
nopy at T2 (R2 = 0. 0.90, β = 0.95, P < 0.001, N
= 15).
Position also had significant effects on the pig-
ment content (Chl a, Chl b and the molar Chl b/a
ratio) of algal fronds in both sampling times, with
higher percentages of total explained variance
in T2 (Fig. 3, Table 3). In general, those fronds
growing within the meadows (i.e. IN-position)
had significantly higher content in chlorophyll
a and b, as well as higher Chl b/a molar ratios,
than those growing in the OUT-position. The
chlorophyll content also significantly differed
among sites at both sampling times and showed
a negative correlation with the depth of the sam-
pling site for the Chl a and b content at T1 at the
OUT-position, and for Chl b and Chl a/b ratio at
T2 at the IN-position due to the significantly hi-
gher mean values observed for these variables at
the shallower sites relative to the deeper ones.
In the T2 sampling time all photosynthetic va-
riables derived from P-E curves were significantly
affected by the position of C. cylindracea stands,
except for maximum photosynthetic rate (Pmax
),
which in addition was the only photosynthetic va-
riable that significantly differed among positions
in T1 (Fig. 4, Table 3). In the summer sampling
(T2), the significant lower respiratory demands
(65% in average) exhibited by C. cylindracea at
the IN-positions significantly increased their pho-
tosynthetic efficiency (i.e. α) and reduced their Ec
and Ek values with respect to fronds growing at
the OUT-positions. The factor ‘site’ also signifi-
cantly affected the photosynthetic parameters
Pmax
, Rd, and α at the winter T1 sampling and only
α at the summer T2 (Fig. 4, Table 3). For Pmax
and
Rd this spatial variability showed a close, negative
and significant correlation with the depth of the
sampling site at T1 in both positions (R2 = 0.54 –
0.82, β = -0.69 – -0.82, P < 0.05, N = 9). Whereas,
between-site differences in photosynthetic effi-
ciency (α) inside the leaf canopy had a negative
and significant correlation with the depth of the
sampling site at both sampling times (R2 = 0.50 –
0.61, β = -0.71 – -0.78, P < 0.05, N = 9).
The mean daily period of photosynthetic com-
pensation (Hc) and the mean daily period of
photosynthetic saturation (Hk) were both signi-
ficantly affected by the factors ‘position’ and
‘site’ in both sampling times; the former factor
explaining in general the higher percentages of
the total variance (Fig. 5, Table 3). Under full illu-
mination conditions (i.e. at the outside positions)
CB (26m)
1084 ± 171506 ± 138
0.106 ± 0.0120.095 ± 0.010
40 ± 6 (3.7%E0)204 ± 15 (13.5%E0)
0.63 ± 0.104.75 ± 0.34
5.79 ± 0.546.37 ± 0.70
7 ± 1 (0.7%E0)82 ± 7 (5.4%E0)
0.11 ± 0.021.59 ± 0.15
Variablea) water column:
noon subsurface irradiance(E
0, μmol quanta m-2 s-1)
water column Kd (m-1)
b) seabed OUT-position:
max. noon bottom irradiance–OUT(E
max, μmol quanta m-2 s-1)
total daily bottom irradiance–OUT(E
total, mol quanta m-2 d-1)
c) seabed IN-position
canopy Kd (m-1)
max. noon bottom irradiance–IN(E
max, μmol quanta m-2 s-1)
total daily bottom irradiance–IN(E
total, mol quanta m-2 d-1)
Site
Time
T1T2
T1T2
T1T2
T1T2
T1T2
T1T2
T1T2
IG (11m)
1059 ±1131496 ± 63
0.122 ± 0.0080.124 ± 0.020
286 ± 17 (27.0%E0)622 ± 51 (41.5%E0)
5.06 ± 0.3115.74 ± 0.92
8.43 ± 0.566.69 ± 0.51
51 ± 4 (4.8%E0)101 ± 9 (6.7%E0)
0.71 ± 0.061.78 ± 0.13
CT (18m)
1017 ± 341305 ± 124
0.082 ± 0.0100.082 ± 0.010
100 ± 13 (9.8%E0)459 ± 13 (35.2%E0)
1.61 ± 0.2310.50 ± 0.30
8.52 ± 0.717.96 ± 0.83
17 ± 3 (1.7%E0)90 ± 16 (6.9%E0)
0.20 ± 0.042.30 ± 0.26
Table 1.
Characteristics of light regimes determined at sampling sites, times and positions from irradiance measurements performed across
vertical profiles of the water column and the Posidonia oceanica seagrass canopy and from continuous light measurements perfor-
med on the seabed (see methods). Data are presented as means ± standard error.
T1 = winter sampling time, T2 = summer sampling time, IN = inside canopy position, OUT = outside canopy position, Kd = light atte-
nuation coefficient, %Eo = proportion of the subsurface irradiance reaching the seabed.
Fig. 1.
Mean and standard error
of Posidonia oceanica
meadow structure varia-
bles obtained for each
combination of sampling
site (IG, CT and CB) and
time (T1 and T2). Di-
fferent letters indicate
groups of homogeneous
means obtained in the
post-hoc SNK test (P <
0.05).
p. 56 p. 57
CHAPTER 3
TESIS DOCTORAL
the highest light levels (i.e. at T2, in the outside
position) are positioned on the right extreme of
the axis (the most positive values), whereas those
exposed to the lowest irradiances (i.e. T1, inside
position) are at the opposite position (the most
negative values). Moreover, the position of the
objects on the PC1 axis showed a high and positi-
ve significant correlation (r = 0.82, P < 0.001) with
the mean total daily irradiance (Etotal
, Table 1; Fig.
6B); the lineal regression model fitted to these
data revealed that this factor explained 68% of
the total variation along the PC1 axis (Fig. 6B).
The vectors depicted in the plot (Fig. 6A), with the
arrow pointing to the higher values of the varia-
ble, indicate that this first axis had strong positive
correlations (scores > 0.8) with Ek, E
c, and R
d, and
negative correlations (scores < -0.8) with the con-
centration of chlorophyll a and b (Table 4). These
strong correlations identified the importance of
this set of responses in the photoacclimative res-
ponse of C. cylindracea to cope with the shady
conditions found within the meadows.
Discussion
Outside the seagrass leaf canopy, between-site
variability of the light regime showed a high ne-
gative correlation with the depth of the site that
was consistent with the characteristic pattern
of light extinction that occurred together with
water column vertical profiles (Kirk, 1994). Sites
had similar mean water-Kd values and were also
very similar in many other climatic, geological
and oceanographic features (Marín-Guirao et al
pers. obs.; Vargas-Yáñez et al. 2010). Therefore,
the possibility that the reported spatial variation
in light regimes was caused by other local factors
apart from depth, is assumed to be very low. Si-
milarly, the pronounced variation in irradiance
between sampling times (about one order of
magnitude), matched typical differences in un-
derwater irradiance between winter and summer
at similar depths and latitudes (e.g. Enríquez et
al. 2004, Raniello et al. 2004, Vargas et al. 2010).
Therefore, it can be considered that the reported
differences in light regime are representative
of the typical spatio-temporal variation of this
factor associated with depth and seasonality,
at least in benthic macroalgal assemblages wi-
thin the depth range and region considered in
this study. The invasive C. cylindracea has been
shown to be able to photoacclimate and persist
across the environmental light gradient asso-
ciated with depth and season in sublittoral Me-
diterranean environments (Raniello et al. 2004,
2006; Bernardeau et al. 2011), which has been
invoked as one of the key mechanisms involved
in its colonisation success in native habitats. In
support of this, many of the significant effects
associated with sampling sites and times ob-
served in the analysed variables of algal stands
Hc ranged between 7 and 13 h and H
k varied be-
tween 1 to 10 h; in contrast, these daily periods
were significantly and consistently shortened by
43% and 72%, respectively, at the inside posi-
tions (Fig. 5). Minimum mean values (1–7 h for Hc
and 0–1.1 h for Hk) were usually found in C. cylin-
dracea fronds of the inside position at the winter
T1 sampling, but also at T2 at the deepest site CB
(Fig.5). Regarding the variation among sites, Hc
and Hk periods were in general shorter at deeper
sites than at shallower ones (Fig. 5).
According to these results, daily metabolic carbon
balances showed significant differences among
positions only for the winter T1 sampling, when
carbon balances within the meadow canopy (i.e.
IN-position) were consistently negative in all sites
(-0.14 – -0.20 mg C g-1 FW d-1) but positive (site
IG: 0.52 mg C g-1 FW d-1) or close to zero (sites CT
and CB: -0.01 mg C g-1 FW d-1) at the OUT-posi-
tion (Fig. 5, Table 3). C. cylindracea carbon balan-
ces were also significantly affected by the factor
‘site’, with the shallowest site IG showing signifi-
cantly higher carbon balances than the deepest
ones CT and CB.
Multivariate analysis
The PCA performed using the selected C. cylin-
dracea photoacclimative variables (frond height,
pigment content and photosynthetic parame-
ters) yielded eigenvalues of 0.759 and 0.132
for the PCI and PC2 axes, respectively (Table 4).
The first PCA axis (PC1) explained 75.9% of the
variance in the original data set. The ordination
of the objects along this axis (Fig. 6A) appears
to relate to the reported differences in light re-
gime, since those cases that were exposed to
Table 2.
Summary of the two-way ANOVA test per-
formed to assess the effect of sampling sites
and times on Posidonia oceanica meadow
structure variables.
df = degrees of freedom, MS = Mean Squares,
%Var. = percentage of explained variance,
F = F-statistics, P = P value; ns = not signifi-
cant, *P < 0.05, **P < 0.01, ***P < 0.001.
Shoot density
Site (S)Time (T)SxTResidual
LAI
Site (S)Time (T)SxTResidual
Meadow cover
Site (S)Time (T)SxTResidual
Canopy height
Site (S)Time (T)SxTResidual
df
2
1
2
30
df
2
1
2
18
df
2
1
2
12
df
2
1
2
30
MS
167066
104275
30087
4085
MS
261.9
835.1
91.4
5.0
MS
621.2
2.2
6.7
6.6
MS
372.6
3700.7
146.3
26.1
%Var.
54.7
34.1
9.8
1.3
%Var.
21.9
70.0
7.7
0.4
%Var.
97.6
0.3
1.1
1.0
%Var.
8.8
87.2
3.4
0.6
F
40.9
25.5
7.4
F
52.2
166.6
18.2
F
93.8
0.3
1.0
F
14.3
141.6
5.6
P
***
***
**
P
***
***
***
P
***
ns
ns
P
***
***
**
Fig. 2.
Mean and standard error of Caulerpa
cylindracea total biomass determined in-
side (IN-position, black bars) and outside
(OUT-position, grey bars) Posidonia ocea-
nica seagrass meadows for each combina-
tion of sampling site (IG, CT and CB) and
time (T1 and T2).
p. 58 p. 59
CHAPTER 3
TESIS DOCTORAL
growing outside the seagrass canopy reflected
photoacclimatory responses previously observed
in response to variability in the light regime.
During the summer sampling (T2), C. cylindracea
stands growing outside the canopy showed in ge-
neral highest Pmax
and Rd rates, which is consistent
with the highest irradiance levels (well above Ec
and Ek mean values) and day-length recorded in
this time. This high light availability for photosyn-
thesis and growth could explain the lack of signi-
ficant differences in most of individual photosyn-
thetic characteristics (P-I curves) between sites,
as it was also reflected in the integrative multiva-
riate analysis (PCA, Fig. 6B), with cases belonging
to T2-OUT occupying a very close position in the
PCI axis despite their differences in light climate
(Etotal
, Fig. 6B). Accordingly, the alga showed high
Hk and carbon balance mean values except in the
deepest site (CB) with the lowest light availabili-
ty and total biomass. This suggest the existence
of some degree of light limitation at this site in
summer, which is supported by the considerable
enhancement of frond height, a typical morpho-
logical adaptation of this and other macroalgal
species to light-limiting conditions (Calvert 1976,
Ohba and Enomoto 1987, Kirk 1994). In addition,
these results were also consistent with those ob-
tained in a previous study performed at the same
sampling sites in a similar sampling time (Bernar-
deau-Esteller et al. 2011), confirming a more limi-
ted capacity of colonization by the alga in these
deepest areas.
Effect Biomass (g FW m-2) Position Site error Frond height (cm) Position Site error Chlorophyll a (mg g-1 FW) Position Site error Chlorophyll b (mg g-1 FW) Position Site error Chlorophyll b/a (molar ratio) Position Site errorPmax (mmol O2 g
-1 FW h-1) Position Site error Rd (mmol O2 g
-1 FW h-1) Position Site error α (mmol O2 g
-1 FW h-1/mmol quanta m-2 s-1) Position Site error Ek (mmol quanta m-2 s-1) Position Site error Ec (mmol quanta m-2 s-1) Position Site error Hc (h) Position Site error Hk (h) Position Site error Carbon balance (mg C g-1 FW d-1) Position Site error
df
12
26 12
26 12
26 12
26 12
26 12
14 12
14 12
14 12
14
12
14 12
14 12
14
12
14
MS
2193.22553.9
98.1
19.1625.3730.108
3183.54994.1664.0
1996.1950.4184.7
0.0090.0010.000
42.2576.625.75
0.653.810.20
0.0020.0240.002
1118.6369.6137.2
21.424.28.4
77.327.20.5
44.719.71.5
0.4960.1540.024
MS
12401.61241.1128.8
56.2557.1590.559
13670.21021.9128.3
9910.501649.28238.153
0.0580.0110.000
31.222.910.6
30.540.021.53
0.0200.0090.002
12068.8
986.1741.6
2345.0
28.752.7
78.253.45.8
112.725.11.6
0.3830.7020.125
%Var
22.351.825.9
58.632.98.6
10.532.856.7
22.921.955.2
56.76.6
36.7
15.355.529.2
5.9
69.125.1
2.0
63.234.8
29.619.650.8
11.425.762.9
55.539.15.47
42.637.719.7
43.627.129.4
%Var
68.013.618.4
66.116.817.1
71.810.717.5
81.210.68.13
69.926.43.7
13.920.465.8
58.70.1
41.2
32.429.338.4
49.48.1
42.5
74.71.8
23.5
29.440.230.4
61.127.211.8
10.839.749.5
F
22.3526.02
177.749.8
4.7957.521
10.8095.147
40.2282.342
7.3413.32
3.2819.29
0.81612.73
8.152.69
2.532.87
141.950.0
30.313.4
20.76.4
F
96.309.64
100.5812.80
106.68.0
259.817.0
490.592.4
2.952.17
19.960.014
11.805.34
16.31.3
44.50.5
13.69.3
72.616.1
3.075.61
P
<0.001<0.001
<0.001<0.001
<0.05<0.01
<0.01<0.05
<0.001n.s.
<0.05<0.001
n.s.<0.001
n.s.<0.001
n.s. (0.012)n.s.
n.s.n.s.
<0.001<0.001
<0.001<0.001
<0.001<0.05
P
<0.001<0.001
<0.001<0.001
<0.001<0.01
<0.001<0.001
<0.001<0.001
n.s.n.s.
<0.001n.s.
<0.01<0.05
<0.01n.s.
<0.001n.s.
<0.01<0.01
<0.001<0.001
n.s.<0.05
SNK-test
IN<OUTIG=CB<CT
IN>OUTIG<CT<CB
IN>OUTIG>CT=CB
IN>OUTIG>CT=CB
IN>OUT - -
IN<OUTIG>CT=CB
- -IG>CT=CB
- -IG>CT=CB
- - - -
- - - -
IN<OUTIG>CT=CB
IN<OUTIG>CT=CB
IN<OUTIG>CT=CB
SNK-test
IN<OUTIG=CT>CB
IN>OUTIG=CT<CB
IN>OUTIG=CT>CB
IN>OUTIG>CT>B
IN>OUTIG>CT=CB
- - - -
IN<OUT - -
IN>OUTIG=CT>CB
IN<OUT - -
IN<OUT - -
IN<OUTIG=CT>CB
IN<OUTIG=CT>CB
- -
IG=CT>CB
T1 (Winter) T1 (Summer)Table 3.
Summary ANOVA test performed to assess the effect of the position (P) and sampling site (S) on all Caulerpa cylindracea variables at
the two studied sampling times (T1 & T2). df = degrees of freedom, MS = Mean Squares, %Var. = percentage of explained variance,
F = F-statistics, P = P value, SNK-test = SNK post-hoc analysis. ns = not significant.
Fig. 3.
Mean and standard
error the height
and pigment con-
tent (chlorophyll a,
b and b/a) of Cau-
lerpa cylindracea
fronds determined
inside (IN-position,
black bars) and out-
side (OUT-position,
grey bars) Posido-
nia oceanica sea-
grass meadows for
each combination
of sampling site
(IG, CT and CB) and
time (T1 and T2).
p. 60 p. 61
CHAPTER 3
TESIS DOCTORAL
Fig. 4.
Mean and standard error of photosynthetic characteristics deri-
ved from P-E curves obtained from Caulerpa cylindracea fronds
inside (IN-position, black bars) and outside (OUT-position, grey
bars) Posidonia oceanica seagrass meadows for each combina-
tion of sampling site (IG, CT and CB) and time (T1 and T2).
Fig. 5.
Mean and standard error of light compensation (Hc) and sa-
turation (Hk) periods and daily carbon balance estimated for
Caulerpa cylindracea fronds inside (IN-position, black bars) and
outside (OUT-position, grey bars) Posidonia oceanica seagrass
meadows for each combination of sampling site (IG, CT and CB)
and time (T1 and T2).
p. 62 p. 63
CHAPTER 3
TESIS DOCTORAL
During the winter sampling (T1), physiological
variables of C. cylindracea growing outside the
seagrass canopy reflected a major photoacclima-
tory effort (relative to T2) according to the more
reduced light availability characteristic of this
season, particularly in the deepest sites. Thus,
photosynthesis and respiration rates, as well as
Ec and E
k, were in general lower than in T2 and
showed significantly lower mean values in the
deepest sites (CT and CB), where frond height
was significantly higher than in the shallower site
IG. All these are characteristic photoacclimatory
responses of marine macrophytes to overcome
light limitation (Falkowski and Raven 2007, Lob-
ban and Harrison 1997, Littler et al. 1986, Lüning
1990) that allows the lengthening of Hc and H
k
periods and counterbalances the metabolic car-
bon budget (Dennison and Alberte 1982, 1985,
Dunton 1986, Gomez et al. 1997). Other res-
ponses were opposite to those expected under a
situation of light limitation, such as the signifi-
cant reduction in photosynthetic efficiency and
pigment content reported in the deepest sites.
However, in this case, the adjustments of the
photosynthetic metabolism (particularly in respi-
ration) probably avoided further reductions in Hc
and Hk and allowed the average carbon balance
to remain close to zero at those sites with greater
depths. Under such situation the alga can main-
tain the standing biomass but with a very limited
growth. The capacity of C. cylindracea to main-
tain biomass during winter in these deeper areas
has been reported at other sites at similar latitu-
des (Giaccone and Di Martino 1995; Cebrián and
Ballesteros 2009), but not in colder areas where
a winter decline occurs (e.g. Piazzi et al. 1997b,
Piazzi and Cinelli 1999, Buia et al. 2001, Capio-
mont et al. 2005, Ruitton et al. 2005b, Lenzi et
al. 2007). At the shallowest site (IG), a clear un-
coupling between biomass and carbon balance
was evident, and we attribute this to the effect
of abiotic factors other than light that might in-
fluence the pattern of vertical distribution of the
alga, such as winter storms (e.g. Cebrián and Ba-
llesteros 2009; Marín-Guirao et al. pers. obs.).
Within the P. oceanica leaf canopy, light availabi-
lity was drastically reduced up to levels that were
always 3.0–8.8 times lower than those recorded
outside. Such low irradiance levels are typically
measured inside P. oceanica meadows (1–7% of
Eo) and reflect the elevated K
d values associated
with the strong self-shading caused by complex
canopies formed by this seagrass species (Dalla
Via et al. 1998). The complexity of the leaf ca-
nopy showed significant spatio-temporal varia-
tion characteristic of P. oceanica meadows el-
sewhere (Romero 1989, Buia et al. 1992, Pergent
et al. 1995, Dalla Via et al. 1998, Olensen et al.
2002). On one hand, shoot density, meadow co-
ver and LAI decreased with depth, which explains
the lowest mean Kd values observed in the dee-
pest site CB; on the other hand, LAI and canopy
height were lower in the winter time, accordingly
with the seasonal variation of the seagrass pro-
duction, but Kd values were equal or even higher
than those in the summer time, contributing to
the explanation of the considerably low light le-
vels within the canopy at that time. In fact, this
variation in the canopy structure is considered
the main adaptive mechanisms of this and other
Posidonia species to offset depth-related light
reductions (Olesen et al 2002, Ralph et al. 2007,
Collier et al 2008).
Evidence provided by this study strongly suggests
that the low light levels reported within the sea-
grass canopy are more limiting for C. cylindracea
growth and survival than those recorded outside.
In general, fronds growing inside the meadow
margin, showed photoacclimatory responses al-
ready described for plants growing outside toge-
ther with other typical acclimation adjustments
Fig. 6.
A) Ordination diagram of the principal com-
ponents analysis with all selected photoac-
climative variables. B) Lineal relationship be-
tween X-axis positions and mean total daily
irradiance (Etotal
, mol quanta m-2 d-1; Table 1),
indicating the lineal regression model, slope
(i.e. regression coefficient; p< 0.05) and coe-
fficient of determination (r2). Sampling sites
= IG, CT and CB. Fh = frond height.
Table 4.
Eigenvalues of the first two axes of the PCA
(PC1 and PC2) and the scores of selected
photoacclimative variables.
Variable / axisEigenvaluesEcEkRdChl b Chl a Pmax Frond heightChl b/aα
PC1
0.759
0.957
0.940
0.887
-0.731
-0.769
0.759
-0.252
-0.214
-0.006
PC2
0.132
-0.017
-0.075
0.403
0.641
0.570
0.581
-0.183
0.455
0.939
p. 64 p. 65
CHAPTER 3
TESIS DOCTORAL
of marine macrophytes to low light conditions,
such as an increase in chlorophyll content and
in the chlorophyll b/a ratio, aimed at enhancing
the efficiency of light absorption (e.g. Kirk 1994,
Falkowski and Raven 2007, Raniello et al. 2004).
Raniello et al. (2004) reported increments in Chl
b and other complementary pigments (e.g. sipho-
noxantin) in C. cylindracea growing under dense
C. nodosa canopies, which could be linked to a
more efficient exploitation of green light, which is
the dominant light under seagrass canopies (e.g.
P. oceanica; Dalla Via et al 1998). In the summer
sampling (T2), the increment in pigment content
and Chl b/a ratio could explain the maintenance
of photosynthetic efficiencies (α) very similar to
those plants growing outside the seagrass ca-
nopy. Further to this, the inhibition of respiration
(up to 89% with respect to the fronds outside)
likely allowed C. cylindracea to attain positive car-
bon balances within the seagrass canopy at the
three sites, despite the fact that the daily satura-
tion periods were less than 4 h (i.e. 64–72% lower
than for plants growing outside the canopy). The
high correlation of this variable with the first PCA
axis (Fig. 4) suggests that the inhibition of respi-
ratory rates represent one of the most important
photoacclimatory mechanisms of C. cylindracea,
not only within the severe shading created by
the canopy, but also (as explained above) outsi-
de the canopy in winter. Although this response
might also reflect low temperature effects on
algal metabolism in winter conditions (Flagella
et al. 2008, Robledo and Freile-Pelegrín 2005, Te-
rrados and Ros 1992), it has been recognized to
be a common physiological strategy to minimize
carbon losses and allow seaweed survival under
low light regimes (Littler et al. 1986, Lüning et
al. 1990, Markager and Sand-Jensen 1994, Pé-
rez-Lloréns et al. 1996, Bernardeau et al. 2011).
In addition, low respiration rates are indicative
of limited growth (Kirk 1994, Pérez-Lloréns et al.
1996), which could further explain the large di-
fferences in algal biomass between the inner and
outer stands observed at the deepest sites (CT
and CB) during the summer, despite their almost
identical carbon balances.
Light climate within the canopy in the winter
represented the most extreme condition for the
alga, since it showed a very limited photoaccli-
matory capacity unable to maintain Hc values
and achieve Hk daily periods longer than 1.1 h at
the shallower site (IG) and of zero h at the deeper
sites. As a result, carbon balances were negative
and hence light availability in these conditions
must be below the minimum requirements for
algal growth and survival (Dennison and Alber-
te 1982, Gómez et al. 1997). In agreement with
this, and based only on water-Kd values (Table 1)
and the Beer-Lambert equation (Kirk 1994), light
levels recorded within the canopy are equivalent
to the range of maximum distributional depths
reported in the Western Mediterranean basin
(35–60 m: Piazzi et al. 2005b; Klein and Verlaque
2008; Ruiz et al. 2011). These results are also con-
sistent with those obtained in the PCA analysis
(Fig. 4), in which the ordination of the objects (i.e.
measurements obtained in each combination of
position, location and time) along the PC1 axis
was highly correlated with light availability and
mainly represented the integration of all pho-
toacclimatative responses in each case. With
respect to the objects during the winter time, it
should be noted that measurement made at the
inner position at the shallow site were very clo-
se to those at the outer position of the deepest
site. This simple observation suggests that the
extreme low light conditions observed beneath
the seagrass meadow in winter have overcome,
or are close to, the limit of the photosynthetic
plasticity of C. cylindracea. For instance, it can be
seen that, particularly in deepest sites, values of
Pmax
and Rd values (and E
c and E
k) of C. cylindracea
plants growing inside the seagrass canopy did not
differ from those showed by plants growing out-
side, suggesting a limit for the plasticity of these
variables under more limiting light conditions. In
such a situation, the maintenance of algal bio-
mass observed underneath the seagrass canopy
in winter is only possible by using carbon storage
reserves during summer, when the carbon balan-
ce was shown to be positive and growth arrested.
This is a strategy to survive light-limiting periods
previously reported for this (e.g. Terrados and
Ros 1992, Robledo and Freile-Pelegrín 2005) and
other seaweed species (e.g. Rosemberg and Ra-
mus 1982, Gagne et al. 1982, Dunton and Schell
1986, Gómez and Wiencke 1998, Lobban and
Harrison 1997). Other possible mechanisms of C.
cylindracea survival beyond its photoautotrophic
limits might be carbon acquisition by heterotro-
phy, as reported for the congenerous C. taxifolia
(Chisholm and Jaubert 1997), or sharing of re-
sources between shaded and illuminated parts of
the coenocytic stolons (Collado-Vides and Roble-
do 1999, De Senerpont Domis et al. 2003).
Our results show that the development of C.
cylindracea biomass is consistently limited inside
the P. oceanica canopy, irrespective the sampling
site and time considered in this study. A monito-
ring study performed at the same sampling sites
(unpubl. data) has demonstrated that this sharp
biomass gradient is stable over years (i.e. 2007–
2013) without any symptoms of seagrass mea-
dow deterioration. In fact, this is consistent with
the observation that P. oceanica meadows are
one of the least-invaded habitats elsewhere and
the idea that P. oceanica can be considered as an
effective “ecological barrier” against the spread
of this highly invasive alien species. Nonetheless,
these types of generalizations must be subjec-
ted to future evaluations of possible long-term
interactions between the alga and the seagrass
through, for instance, phytotoxic allelochemical
effects (Dumay et al. 2002a, Raniello et al. 2007)
or deterioration of substrate conditions (Holmer
et al. 2009). This study provides extensive and
consistent evidence supporting the hypothesis
that light plays a key role in explaining the high
resilience of the P. oceanica meadow with regard
to the C. cylindracea bioinvasion. As reported in
this and other studies using similar ecophysio-
logical approaches, C. cylindracea has a great
physiological and vegetative plasticity allowing it
to adapt to a wide range of environmental con-
ditions and light climates (Raniello et al. 2004,
2006; Bernardeau-Esteller et al 2011), which in
turn is one of the traits contributing to explain
its highly invasive character (Klein and Verlaque
2008). However, results obtained in this study has
shown that the extremely low light levels within
P. oceanica meadows can be below the minimum
light requirements for C. cylindracea growth, sur-
passing its plastic capacity to acclimate to further
light reductions. However, much more research
must be done before attaining some robust con-
clusions about this topic. First of all, other factors
could be involved, or interact with light availabili-
ty, that should be investigated. In the case of this
particular study, the substrate type was the same
at both sides of the seagrass meadow edge (i.e.
P. oceanica “matte”), and hence other kind of fac-
tors such as nutrients, sedimentation, water mo-
vement or space limitation should be considered.
Secondly, given the experimental design used in
this study, results obtained here must be corro-
borated with similar studies in other regions and
using complementary experimental work in the
field and in the laboratory. Furthermore, other
basic aspects should be addressed, such as the
potential of early recruitment phases (e.g. spo-
res) to colonize the seagrass meadows, in addi-
tion to the acclimation capacity of the adult sta-
p. 66 p. 67
CHAPTER 3
TESIS DOCTORAL
ges. Regardless the factors involved, the apparent
ecological resistance of P. oceanica meadows
seem to be linked to its complex canopy struc-
ture. Therefore, and considering that recovery
of damaged P. oceanica meadows is a very slow
process (Duarte et al. 2006), the conservation of
its integrity against anthropogenic disturbances
must be a priority of environmental policies con-
cerned with the control of bioinvasions in the Me-
diterranean Sea, such as the Marine Strategy EU
Directive or the Ecosystem Approach.
CHAPTER 4Photoacclimation of Caulerpa cylindracea: light
as a limiting factor in the invasion of native
Mediterranean seagrass meadows
p. 71
Photoacclimation of Caulerpa cylindracea: light as a li-miting factor in the invasion of native Mediterranean seagrass meadows.
Abstract
Reduction in light availability caused by the ca-
nopy of the Mediterranean seagrass Posidonia
oceanica has been suggested as a critical me-
chanism to resist the invasion of the exotic ma-
croalga Caulerpa cylindracea. We experimentally
evaluated the role of light as a limiting factor
on the capacity of colonization and spread of
this invasive seaweed in P. oceanica meadows
by assessing photoacclimation responses and
productivity and growth capacity of C. cylindra-
cea in mesocosm and in situ light manipulation
experiments. Despite the high photoacclimative
plasticity developed by the alga, the light regime
within the seagrass meadow during the study pe-
riod was close to the minimum light requirements
for growth, restricting the development capacity
of this species. In addition, while increases in li-
ght availability resulting from canopy alteration
also enhanced the productive capacity of the
invasive seaweed in the field, such increase was
not followed by gains in biomass production. Our
results thus support the hypothesis that light
availability has a major role in the underlying
resistance of seagrass meadows to the invasion
by C. cylindracea, but also indicate that there are
additional factors related to the canopy of P. oce-
anica that further hinder the growth and coloni-
zation capacity of the alga.
1. Introduction
A main goal for ecologists is to understand the
factors and mechanisms that determine inva-
sive success of introduced species. Phenotypic
plasticity has been recognized as an important
mechanism related to successful invasion proces-
ses. Plasticity enhances ecological niche breadth
and allows organisms to express advantageous
phenotypes in a broader range of environmen-
tal conditions, which contributes to maintain
positive population growth and increases the
likelihood of invasiveness (Richards et al. 2006).
In addition, native communities strongly differ in
their resistance to invasions (Londsale 1999). Di-
fferences in susceptibility to invasion have been
linked, among other processes, to biotic resis-
tance derived from interspecific competition for
resources between native and introduced species
(Theoharides and Dukes 2007; Branch and Ste-
ffani 2004).
Exotic seaweeds are a major threat to coastal
marine habitats worldwide as they often have
negative effects on the structure and diversity of
native communities (Williams and Smith 2007).
Among all the factors influencing macrophyte
communities, light is key in regulating produc-
tivity, abundance and distribution (Lobban and
Harrison 2004, Kirk 1994, Breeman 1988). The
Publicado en:Bernardeau-Esteller J, Ruiz JM, Tomas F, Marín-Guirao L (2015) Photoacclimation of Caulerpa cylindra-
cea: Light as a limiting factor in the invasion of native Mediterranean seagrass meadows. J Exp Mar Biol
Ecol 465:130-141.
p. 72 p. 73
CHAPTER 4
TESIS DOCTORAL
minimum light requirement for algal growth is
reached when captured light allows to balan-
ce loss processes within the tissue (e.g. respira-
tion and exudation, Markager and Sand-Jensen
1992). If available light in a habitat is below or
close to those minimum requirements, develop-
ment capacity and therefore invasive potential of
exotic macrophytes will be hampered. This occurs
for example in the case of the tropical red alga
Womersleyella setacea in the Mediterranean Sea,
as light requirements of this species reduce dra-
matically its invasion capacity at depths greater
than 35 meters (Cebrian and Rodriquez-Prieto
2012). A high plasticity in photoacclimation me-
chanisms allows an exotic alga to develop an effi-
cient photosynthetic response (that determine
an efficient use of light) in a wide range of light
conditions, that could enhance its competitive
capacity and colonizing potential in new habitats
(Raniello et al. 2006, Bernardeau-Esteller et al.
2011;, Marín-Guirao et al. 2015).
Interspecific competition for light is especially
relevant in structured communities dominated
by large-sized species (canopy formers) since
they generate intense changes in the quality and
quantity of light available at the understory la-
yers (Middelboe and Binzer 2004; Reed and Fos-
ter 1984). Such changes in light availability may
be an important mechanism underlying invasion
resistance of these communities if shading by
the canopy creates light conditions near the mi-
nimum light requirements of the introduced spe-
cies (Arenas et al. 2006). For instance, a decrease
in survival of the invasive japanese seaweed Sar-
gassum muticum in nearshore marine communi-
ties in the western coast of USA has been linked
to shading effects produced by the native canopy
species (Britton-Simmons 2006). The structure of
macrophyte assemblages can be modified due
to the action of natural (high water movements,
herbivory) and human-induced (pollution, fishe-
ries) stressors (Lobban and Harrison 1996). A
reduction in abundance of canopy-forming spe-
cies following these disturbances will result in an
increase in the available light in the lower layers
of the community, which can promote growth of
species constrained by limited light levels (Reed
and Foster 1984). Therefore, it can be assumed
that disturbance of canopy species could promo-
te success of introduced species whose growth
capacity is limited by light conditions.
The Mediterranean Sea, recognized as a hotspot
of biodiversity, is one of the seas most affected
by species introductions (Coll et al. 2010). To
date, 3.3% of total described species in the Me-
diterranean Sea (more than 900, Zenetos et al.
2010) are considered exotic, of which 85 are ma-
crophytes. Within this functional group, the green
alga Caulerpa cylindracea (Sonder) has a strong
invasive character, rapidly colonizing most of the
Mediterranean Sea (Piazzi et al. 2005b, Klein and
Verlaque 2008). Under certain conditions, the
alga is able to develop large biomass, which has
been linked with significant changes in physico-
chemical and community characteristics of recei-
ving habitats (see Klein and Verlaque 2008 and
literature cited therein). The ecological success of
this species in invading the Mediterranean Sea
has been linked, in addition to other traits (e.g.
vegetative and sexual reproductive success, high
growth rates) to high morphological and physio-
logical plasticity (Gacia et al. 1996a, for a review
see Klein and Verlaque 2008). The alga has been
described in a wide range of depths (between
0-60m), suggesting a large capacity of photoac-
climation. In this regard, previous studies have
reported stable populations of the alga in depths
close to 30 meters as well as under the leaf ca-
nopy of macrophytes, indicating a high toleran-
ce of the species to low light regimes. However,
C. cylindracea has shown a reduced capacity to
colonize healthy meadows of the dominant me-
diterranean seagrass Posidonia oceanica (Katsa-
nevakis et al. 2010, Bulleri et al. 2011, Ceccherelli
et al., 2014), the greater structural complexity of
which determines more intense shading condi-
tions (Enriquez et al. 1992, Dalla Via et al. 1998 ).
Recently, Marín-Guirao et al. (2015) analyzed the
photosynthetic and productive characteristics of
natural C. cylindracea populations growing insi-
de and outside of leaf canopies of P. oceanica
in a highly invaded area. Results obtained in this
study suggest that light availability inside the
meadow exceeds the photoacclimation capacity
of the alga and seem to be close to the minimum
light requirements for growth, suggesting that
this factor can play an important role as a me-
chanisms of resistance of P. oceanica habitats to
invasion. However, the methodological approach
used in that study (i.e. non-experimental) preclu-
ded isolating the effect of light availability from
the influence of other environmental factors that
can be related to the development capacity of
the algae and that thus may also be altered by
canopy structure (e.g. water movement, nutrient
availability).
The aim of this study was therefore to experi-
mentally examine the role of light availability in
the colonization of the meadows of P. oceanica
by the alga, testing whether reduced light regi-
mes within this habitat are able per se to explain
the resistance phenomena observed. In order
to evaluate this hypothesis two complementary
experimental approaches were used. We studied
photoacclimation responses (through analysis of
photosynthetic performance and pigment con-
tent) and productive and growth capacity (by
assessment of carbon balance, starch content,
apical elongation and variation in stolon bio-
mass) of C. cylindracea in a mesocosm and a field
experiment in which different light regimes were
experimentally manipulated.
2. Material and methods
2.1. Mesocosm Experiment
Use of a mesocosm system allowed maintaining
controlled environmental conditions (temperatu-
re, light, salinity, pH), enabling us to isolate the
effect of light from the influence of other fac-
tors whose variation could affect the response
of the studied variables. The mesocosm system
consisted of 24 glass independent aquaria of
100 l capacity. Each aquarium had its own light
system (400W halogen lamp, Aqua Medic aquali-
ght-400), water circulation and filtration system,
and contained a plastic tray (22 x 40 cm base
and 10 cm high), filled with previously washed
coarse sediments.
Eight light treatments (L1 to L8) were established
in a range of daily photon flux values (i.e. integra-
ted daily irradiance) comprised between 0 and
13.61 mol quanta m-2 d-1 (Table 1), which inclu-
de light regimes of all natural habitats in which
the alga is found in the study area (unpublished
data). Each light treatment was assigned to 3
randomly selected aquaria. Daily photon flux for
each treatment was determined through daily
integration of instantaneous irradiance values re-
corded in the aquarium on a daily cycle of 12:12
h. These values were obtained using a submersi-
ble light sensor (PAR spherical quantum sensor
MDS MK5, Alec Electronics, Japan) located at
the same depth as the tray, with continuous rea-
dings every ten minutes. The illumination system
is not able to simulate the natural, bell-shaped
light curve (e.g. Fig. 1). Instead, the daily ‘light
curve’ in aquariums had a rectangular shape with
a constant instantaneous irradiance throughout
the illumination period (12 h) that was reached
in a few minutes once the lamps were switched
p. 74 p. 75
CHAPTER 4
TESIS DOCTORAL
on and fell down to total darkness immediately
after the lamps were switched off. As an exam-
ple, the artificial light curve of the L6 treatment
is represented in Figure 1. Unavoidably, the diffe-
rence between the natural and the artificial daily
light curves can have consequences in determi-
nation of photosynthetic parameters (e.g. com-
pensation and saturation light periods, Hc and
Hk, respectively), which should be considered for
the interpretation of the results obtained in the
mesocosm and in the field. In this same context,
differences in light quality between artificial and
natural light sources must be also considered.
High quality natural seawater from a nearby, oli-
gotrophic and unpolluted area was employed in
the mesocosms. Environmental conditions inside
the tanks were similar to those prevailing in the
selected areas for of the stolons of C. cylindracea
(see below) during the time of year in which the
experiment took place. For that purpose, a pe-
riodic analysis of nutrients by colorimetric test
(phosphorus and nitrogen; Merck®), continuous
recording of pH with specific electrodes (Aqua
Medic AT-Control) and a daily monitoring of
the salinity of the water with a conductivimeter
WTW (Model Cond. 197i) were carried out in
each aquarium. Salinity values were maintained
constant (37.5 PSU) by osmosis water addition.
Water temperature during the experiment was
19 ± 0.1 ° C and was controlled by automatic
cooling system (see Marín-Guirao et al. 2011 for
more details).
C. cylindracea stolons were collected by hand in
a nearby population located on the southwest
coast of the Region of Murcia (Isla Grosa, UTM
X: 0701991, Y: 4177942, H 30S) at -11 m deep.
Collection of stolons was done randomly in a
large area (c.a. 1000m2) to capture the natural
variability. The colonized area is an infralittoral
well-illuminated bottom composed by a mosaic
of sand and dead P. oceanica rhizomes (Ruiz et
al. 2011); mean noon irradiance was 195.66 ±
3.99 μmol quanta m-2 s-1 during the experimental
period (i.e. OUT treatment in Fig. 1). Collection
was performed in November 2011, a period of
high vegetative development (Bernardeau-Es-
teller, unpublished results). Immediately after
collection, the cut end of the stolon was sealed
with very small plastic clothes pegs to avoid loss
of internal content and was put in a black plas-
tic bag to prevent overexposure to light. Stolons
selected were transported in refrigerated contai-
ners with seawater to the laboratory. Once here,
they were immediately transplanted into aquaria
for acclimatization for 3 days before the start
of the experiment. Four stolons were planted in
each aquarium, and were of similar characteris-
tics, with an initial length of 25-30 cm, a number
of fronds ranging from 10-15, and a single apical
meristem. During the acclimatization process, ex-
perimental units were subjected to daily photon
fluxes similar to those recorded in the field (ca
4.43 ± 0.05 mol quanta m-2 d-1). After the tran-
sitional period, light conditions were changed in
each aquarium to obtain the experimental light
treatments. Algae were exposed to these treat-
ments for 7 days.
2.2. Field experiment
Simultaneously with the mesocosm experiment,
a field experiment was conducted in the same
area where stolons of C. cylindracea were collec-
ted from for the mesocosms experiment. This
highly colonized area is adjacent to a dense P.
oceanica meadow, but C. cylindracea stolons are
not able to penetrate beyond the seagrass mea-
dow edge (Ruiz et al. 2011, Marín-Guirao et al
2015). Four experimental light treatments (two
inside the seagrass meadow and two outside)
were created: (i) within the meadow (IN), (ii) in
areas within the meadow where the height of the
leaf stratum was experimentally reduced by clip-
ping (CLIPPING), (iii) outside the meadow (OUT),
and (iv) outside the meadow but in areas whe-
re light availability was experimentally reduced
(SHADED) to be similar to those of the IN treat-
ment. The light regime of each experimental
treatment was characterized based on its noon
instantaneous irradiance and the integrated
daily irradiance obtained from daily light cycles
(Fig. 1). To this end, PAR light sensors (spherical
quantum sensors; Alec MK5 MDS) were installed
on the bottom of all experimental plots. Instan-
Table1.
Summary of irradiance measurements in
each light treatment in the mesocosm ex-
periment. Data are presented as means ±
standard error.
Noon Instantaneous Irradiance
μmol quanta m-2 s-1
0.00 ±0.00
6.00 ± 0.20
20.97 ± 0.76
29.95 ± 0.99
43.73 ± 1.09
102.50 ± 3.17
211.77 ± 4.69
314. ± 9.30
Integrated Daily Irradiance
mol quanta m-2 d-1
0.00 ±0.00
0.24 ± 0.01
0.91 ± 0.11
1.29 ± 0.01
1.89 ± 0.04
4.43 ± 0.05
9.15 ± 0.14
13.61 ± 0.52
Treatment
L1
L2
L3
L4
L5
L6
L7
L8
Measurements
Fig. 1.
Daily course of irradiance measured at
the sea floor irradiance and summary of
irradiance measurements obtained for
each field experiment. The dotted line
corresponds to the course of the daily irra-
diance in mesocosm aquaria (only the L6
treatment is represented as an example).
p. 76 p. 77
CHAPTER 4
TESIS DOCTORAL
taneous irradiance measurements were recorded
every 10 minutes during the 7 days of the expe-
riment. The integrated daily irradiance (Etotal,
mol
quanta m-2 d-1) was obtained by the integration
of these instantaneous measurements recorded
in each daily cycle. Throughout the experiment,
water temperature was recorded in situ by HOBO
Pro v2 Water Temperature Data Logger (Onset
Computer, EME Systems, Berkeley, CA, USA). The
average temperature recorded during the experi-
ment was 19.8 ± 0.1 ° C.
Based on the similarities in light regimes obtai-
ned in the IN and the SHADED treatments, we
would expect that algae under these conditions
will present an equivalent photoacclimation res-
ponse, showing a clear limitation in its production
and growth capacity. Moreover, increase in light
availability linked to the experimental manipula-
tion of the canopy (CLIPPING treatment) should
determine an approximation of the responses of
seaweeds under this treatment to those recorded
outside of the meadow (OUT treatment).
Light conditions of the SHADED treatment were
obtained by using floating structures anchored to
the substrate. These structures consisted of a PVC
frame with neutral density filters which determi-
ned that the light regime beneath them was simi-
lar to that recorded within the meadow (IN treat-
ment). A preliminary study showed that this type
of structures do not alter the physico-chemical
conditions of the bottom, minimizing the effect
of any other factor different than light (Bernar-
deau-Esteller, unpublished results).
In the plots of the CLIPPING treatment the
reduction of the leaf layer was carried out ma-
nually to obtain a final leaf length of 15 cm (the
original leaf length of P. oceanica canopy was ca
80 cm). This reduction simulated a high rate of
herbivory (e.g. Tomas et al. 2005) and determines
light conditions intermediate to those obtained
inside and outside the meadow (Fig. 1).
For each experimental treatment, four plots of
2x2 m2 were randomly selected. Five stolons with
the same characteristics as those used in the
mesocosm experiment were transplanted in each
plot. Before the start of the experiments, stolons
were collected and sealed and then transported
in darkness and refrigerated containers to the
laboratory where they were marked and initial
characterization was conducted (see section be-
low referring to the phenological variables used).
Subsequently, stolons were transported back to
the study area where they were transplanted in
the plots. Stolons were attached to the substrate
by a nylon cord anchored by two stainless steel
pegs tied at both ends. After seven days, algae
were collected and transported back to the labo-
ratory to perform all the measurements.
2.3. Alga Analyses
In both experiments (mesocosms and field) the
same response variables were considered.
2.3.1. Photosynthesis vs irradiance curves
(P vs. E curves)
Prior to photosynthetic measurements, C. cylin-
dracea samples taken from the aquarium system
or collection site were held overnight in the dark
under controlled temperature in natural seawa-
ter.
Photosynthesis and dark respiration rates (Rd)
were measured using a polarographic oxygen
electrode and a magnetic stirrer (DW3, Hansa-
tech Instruments Ltd) under controlled tempe-
rature. Incubation was carried out at the same
temperature measured in the mesocosm system
and in the field (19ºC). Three replicated apical
segments of non-epiphytized C. cylindracea
fronds of approximately 2 cm height were em-
ployed for the measurements. Dark respiration
rates were measured by maintaining the fronds
in the dark for 15 min. Net oxygen production
was then determined at 9 different light inten-
sities (from 1 to 700 μmol quanta m-2 s-1) using
a high intensity light source which consists of
an array of 36 red LED’s (LH36/2R, Hansatech
Instruments Ltd). Net photosynthetic rates were
plotted against the light intensities (P vs. E cur-
ves), and the photosynthetic parameters were
calculated as follows: the maximum rate of net
photosynthesis (Pmax
) was determined by avera-
ging the maximum values above the saturating
irradiance (Ek). The photosynthetic efficiency (α,
μmol O2 g FW-1 h-1/μmol quanta m-2 s-1) was calcu-
lated as the slope of the regression line fitted to
the initial linear part of the P vs. E curve, and the
compensation irradiance (Ec) as the intercept on
the X-axis. Ek was calculated as the ratio P
max/α.
Gross photosynthesis (gross-Pmax
) was calculated
as the sum of Pmax
and Rd
2.3.2. Pigment content
Pigment content was determined in the same
apical segments of fronds selected for obtaining
P vs. E curves (n = 3). The analysis was conducted
spectrophotometrically after manual extraction
of a homogenized suspension using 90% ace-
tone (Dennison 1990), with MgCO3 added as a
chlorophyll stabilizer. The acetone extracts (10
ml) were stored at 4 °C in the dark for 24 h and
centrifuged. Chlorophyll a (chl a), Chlorophyll b
(chl b) and carotenoid content was computed
using the equations of Lichtenthaler & Wellburn
(1983).
2.3.3. Estimated daily irradiance regimes and
daily metabolic carbon balances
Mean daily compensation (Hc) and saturation
(Hk) periods were calculated by averaging the
number of hours per day that irradiance values
exceeded the corresponding values of compensa-
tion (Ec) and saturation (E
k) irradiances, respecti-
vely. The values of Ec and E
k used in these calcula-
tions were those obtained from the P vs. E curves.
Daily carbon balance, as a proxy of plant light
limitation (Dennison and Alberte, 1985), was
calculated according to the Michaelis-Men-
ten function (P = [gross-Pmax
E/(E+Ek)] + R
d (Baly
1935)) previously applied to C. cylindracea (Ber-
nardeau-Esteller et al. 2011), where P is the net
production, gross-Pmax
is the maximum gross pho-
tosynthetic rate, E is the irradiance measured in
the field, Ek is the saturation irradiance, and R
d is
the respiration rate. Semicontinuous (i.e. every
10 min) mesocosm and field irradiance measure-
ments were entered into the function to generate
estimates of net production, which were integra-
ted across 24 h periods to yield daily net produc-
tion. If the photosynthetic quotient is assumed
to equal 1, and the ratio g C: g O2 = 0.3 (Matta
and Chapman 1991), then the net production in
oxygen units (μmol O2 g FW-1) can be multiplied
by 0.012 to obtain the equivalent carbon units
(mg C g FW-1). This calculation presumes cons-
tant dark respiration throughout the day and
does not consider other carbon losses (e.g. exu-
dation, grazing) or gains (e.g. light-independent
carbon fixation).
2.3.4. Stolon biomass balance and apical growth rate
To calculate stolon biomass balance and apical
growth rate in a particular stolon, the following
equation was used: Xx = (X
f - X
i) / t, where X
x is the
measurament for a variable (biomass or length)
expressed in units d-1, Xf and X
i are the observed
measurements of the variable at the end and be-
ginning of the experiment, and t, the time dura-
tion of the experiment. The average value of all
stolons from each tray (mesocosm experiment)
or plot (field experiment) constituted each one of
the replicates in both experiments (n=3 and n=4,
respectively).
Total biomass of a stolon was determined using
a precision scale (Metler-Toledo). Growth rate
p. 78 p. 79
CHAPTER 4
TESIS DOCTORAL
based on apical stolon length was determined
according to the methodology described by
Ruitton et al. (2005b). At the beginning of the
experiment the apical region of the stolon was
marked by placing a metal ring on the back of
the last frond before the apical meristem and
manually measured (cm). This mark was used as
a reference for measurement at the end of the
experiment. Length measurements included both
the main axis of the stolon and the stolon ramifi-
cations generated during the experiment.
2.3.5. Starch content
For starch analysis, algae from each tray (meso-
cosm experiment) and plot (field experiment)
were cleaned with distilled water, dried for 48
hours at 50 °C and ground to fine powder. Starch
content was analyzed following the method des-
cribed by Yemm and Willis (1954). Ground ma-
terial (0.150g) was washed with 80% ethanol to
remove all trace of soluble sugars and extracted
with 1 N KOH to solubilize starch. Finally, starch
was determined spectrophotometrically using an
anthrone assay. Starch content was expressed as
percentage of the dry weight of the sample. Each
of these measurements constituted a replicate of
the experiment (n = 3 for the mesocosm experi-
ment; n= 4 for the field experiment).
2.4. Statistical analysis
2.4.1. Multivariate analysis
To explore the photoacclimative response of C.
cylindracea to the different treatments in the
mesocosm experiment, Principal Component
Analysis (PCA) was performed based on the co-
rrelation matrix of photoacclimatation variables
(which includes photosynthetic parameters, dark
respiration rate and pigment content). Data were
previously transformed to achieve centralization
and standardization. PCA was performed with
the program CANOCO version 4.5 (Microcompu-
ter Power Ltd).
2.4.2. Univariate analysis
For both the mesocosm and field experiments,
differences among treatments for each varia-
ble were tested using one-way ANOVAs. Prior to
analysis, data were tested for heterogeneity of
variance using Cochran’s C-test and transformed
when necessary. Where variance remained hete-
rogeneous, untransformed data were analysed,
as ANOVA is a robust statistical test and is relati-
ve unaffected by the heterogeneity of variances,
particularly in balanced experiments (Underwood
1997). A probability level of 0.05 was regarded
as significant except when data transformation
was not possible. In such cases the level of signi-
ficance was reduced to P < 0.01 to minimize type
I error. The Student-Newman-Keuls (SNK) test
was used for a posteriori pairwise comparisons
of means. ANOVA analyzes were developed with
the program GMAV® version 5 for Windows (Un-
derwood and Chapman 1998).
In addition, for both experiments, relationships
between photoacclimation variables and light
were explored using simple regressions. These
analyzes were developed with the program Sig-
maplot 10.0 (Systat Software Inc.).
3. Results
3.1. Mesocosm experiment
The first axis of the PCA performed on Caulerpa
cylindracea’s photoacclimative response (Fig
2A) represents most of the variance explained
(67%), which had a strong correlation (r > 0.7)
with all variables except Pmax
. This correlation was
negative in the case of Ek, R and E
c, while it was
positive for pigment content and photosynthetic
efficiency (α). The second ordination axis explai-
ned 23.8% of the variation and was highly corre-
lated (r =0.91) with Pmax
(Fig. 2A).
Distribution of treatments on AXIS 1 can be in-
terpreted in terms of light availability with higher
irradiance treatments on the left side of the PCA
and lower irradiance treatments in the right side.
Indeed, the ordination of treatments along AXIS
1 showed a high and significant correlation with
the Integrated daily Irradiance (R2 = 0.5833, p <
0.0001, n =3 ; Fig. 2B).
P vs. E curves allowed the estimation of several
photosynthetic parameters, most of which (Pmax
,
Rd, E
c and E
k) having a positive and significant li-
near relationship with irradiance (Figure 3A). The
highest values of these parameters were recorded
in the treatment of highest irradiance (L8; 13.61
mol quanta m-2 d-1) and were significantly higher,
except for Ec, to the other treatments (SNK, Fig.
3A, Annex Table 1). Minimum values were obser-
ved in the low irradiance treatment L3 (0.91 mol
quanta m-2 d-1) in the case of Rd and E
c, and dark-
ness treatment (L1) in the case of Pmax
and Ek (Fig.
3A). Values recorded in these treatments repre-
sented a reduction of about 70%, in the case of
Rd and E
c, and close to 50% for P
max and E
k com-
pared to treatment L8. Photosynthetic efficiency
(α) decreased with increasing irradiance (Fig. 3A),
although significant differences were found only
between treatment L3, which yielded the highest
Fig. 2.
Multivariate Analysis of photoacclimation
responses of C. cylindracea in the meso-
cosm experiment: A. Ordination diagram
of the Principal Component Analysis (PCA)
with selected photoacclimative variables for
mesocosm experiment. B Relationship be-
tween X-axis position and Integrated Daily
Irradiance (Table 1).
p. 80 p. 81
CHAPTER 4
TESIS DOCTORAL
value (30% greater than the value recorded in
higher irradiance treatments) and the rest of the
treatments (SNK, Fig. 3A, Annex Table 1).
Chl a and chl b concentrations and the ratio chl-
b/a showed a similar pattern to that observed
for photosynthetic efficiency, characterized by a
negative linear relationship with irradiance (Fig.
3B). Higher values of chlorophyll were generally
observed in the low – intermediate irradiance
treatments, and were highest in treatment L3
(0.91 mol quanta m-2 d-1). No clear response for
carotenoid content was found regarding diffe-
rent light levels. However, the ratio of these ac-
cessory pigments in relation to chlorophyll a and
b content showed a positive linear relationship
with irradiance, registering the highest values
above 9.15 mol quanta m-2 d-1 (L7) (Fig. 3B, An-
nex Table 1).
Daily compensation period (Hc) remained at
maximum values (12h) in all treatments except
for L1 and L2, in which values were significantly
lower (0 and 10.42 h respectively, Fig. 4). Daily
saturation period (Hk) values progressively in-
creased with increasing irradiance, being close
to 0 hours for darkness treatment L1 and the low
light treatment L2 (0.24 mol quanta m-2 d-1) and
with maximum values (12h) in treatments with
more than 5 mol quanta m-2 d-1of daily irradiance
(L7 and L8) (Fig. 4, Annex Table 1). In accordan-
ce with Hc and photoacclimation variables, daily
carbon balance also showed a positive response
with increasing light. All treatments (except L3
and L4) significantly differed from each other,
with carbon balance being negative in treatment
L1 and nearly 0 in L2 (0.24 mol quanta m-2 d-1)
(Fig. 4, Annex Table 1). Stolon biomass balance
showed negative or very close to 0 values for irra
diance levels below 0.3 mol quanta m-2 d-1 (treat-
ments L1 and L2, Fig. 4). A progressive increase
in this variable was identified above these values
of irradiance, reaching maximum values from 4
mol quanta m-2 d-1 (treatment L6 and L7, Fig. 4).
Finally, a significant reduction occurred at maxi-
mum irradiance (treatment L8, 13.61 mol quanta
m-2 d-1, Fig. 4, Annex Table 1). Apical growth rate
had a similar pattern to stolon biomass balance,
although values were always positive even in the
absence of light (L1), with rates ranging from
0.4 (treatment L1) to 3.4 cm d-1 (treatment L7 )
(Fig 4). Starch content ranged from 1.99 ± 0.04
% DW in treatment L1 to 2.57 ± 0.06 % DW in
treatment L2, and while there were significant
differences among treatments, there was no sig-
nificant correlation with irradiance.
Fig. 3.
Photoacclimation response of C. cylindracea for mesocosm experiment: A. Photosynthetic
parameters derived from P vs. E curves. B. Pigment content. Symbols are mean ± standar error.
Solid lines represent the regression line fitted to data and the smoothed dashed line illustrates
the trajectory of the response variable as irradiance increases.
p. 82 p. 83
CHAPTER 4
TESIS DOCTORAL
Fig. 3.
Fig. 4.
Productive and growth capacity of C. cylindracea in the mesocosm experiment: Li-
ght-compensation period (Hc), light-saturation period (H
k), daily carbon balance, stolon
biomass balance, apical growth rates and starch content for the mesocosm experiment.
Data are mean ± standard error. The smoothed dashed line illustrates the trajectory of
the response variable as irradiance increases.
p. 84 p. 85
CHAPTER 4
TESIS DOCTORAL
Fig. 5.
Photoacclimation response of C. cylindracea for the field experiment: A. Photosynthetic
parameters derived from P vs. E curves. B. Pigment content. Data are mean ± standard
error. Solid lines represent the regression line fitted to data and the smoothed dashed line
illustrates the trajectory of the response variable as irradiance increases. Mean values ob-
tained in treatment L2 and L6 of the mesocosm experiment (asterisks) have been included
as a reference.
Fig. 5.
p. 86 p. 87
CHAPTER 4
TESIS DOCTORAL
3.2 Field experiment
Similarly to the mesocosm experiment, parame-
ters derived from P vs. E curves showed a signi-
ficant linear relationship with irradiance. This
relationship was positive in all cases, except for
photosynthetic efficiency (α), for which a signifi-
cant reduction was observed with increasing irra-
diance (Fig. 5A). SNK test detected differences
between treatments for the variables Pmax
, Ek y R
d,
in which the recorded values were significantly
higher in OUT than in the other treatments, while
contrarily α values were significantly lower in the
OUT treatment than in the IN and SHADED ones
(Fig. 5A, Annex Table 2).
With regard to pigment content, no significant
differences were observed amongst treatments.
However, the ratio chl b/a showed a positive li-
near relationship with irradiance, registering sig-
nificant differences between the conditions of
low irradiance (IN-SHADED) and the rest light
treatments. On the contrary, ratios between caro-
Fig. 6.
Productive and growth capacity of C. cylindracea in the field experiment: Light-compensation pe-
riod (Hc), light-saturation period (H
k), daily carbon balance, stolon biomass balance, apical growth rates
and starch content for field experiment. Symbols are mean ± standard error. The smoothed dashed
line illustrates the trajectory of the response variable as irradiance increases. Mean values obtained in
treatment L2 and L6 of the mesocosm experiment (asterisks) have been included as a reference.
tenoids and chlorophylls were significantly higher
at lower irradiances (IN-SHADED; Fig. 5B). While
there were no significant differences in Hc among
treatments (always exceeding 7 hours), we did
detect differences in Hk, which were significantly
higher in the OUT and CLIPPING (>5h) in compa-
rison to SHADED and IN treatments (2h). Maxi-
mum values of carbon balance were observed
outside the meadow (OUT; 0.53 mgC g-1 FW d-1),
with a significant reduction of 42% in CLIPPING
and of about 80% in lower irradiance conditions
(IN and SHADED; Fig. 6, Annex Table 2).
Algae outside the meadow (OUT) exhibited the
highest values of stolon biomass balance, apical
growth rate and starch reserves. Stolon biomass
balance was negative in the rest of treatments,
being significantly lower within the meadow (IN)
than in SHADED and CLIPPING conditions. In
contrast, no significant differences were found
between these low light availability treatments
in terms of apical growth rate and starch content
(Fig. 6, Annex Table 2).
In figures 5 and 6 mean values obtained in L2
and L6 treatments of the mesocosm experiments
are represented in order to allow some compa-
rative, graphical analysis with those obtained in
the field experiment under similar field regimes
i.e. the IN and OUT treatments. It can be seen
how most of the response variables followed si-
milar patterns in both experimental approaches
(except chl b and ratio chl b:a). However, there
were appreciable quantitative differences for
some of the variables, particularly in the high li-
ght treatments (OUT and L6). Major quantitative
differences are appreciated in Rd, α, pigment con-
tent and composition (Fig. 5), Hk, stolon biomass
and growth and starch content (Fig. 6).
4. Discussion
Under the light gradient set in the mesocosm
experiment, C. cylindracea showed a clear pho-
toacclimation response to reduction in light
availability which included (i) a reorganization
of the photosynthetic apparatus illustrated by an
increase in pigment content (and antenna size)
and changes in photosynthetic performance (i.e.
reductions in Pmax
, Ek, and E
c, and increments in α),
as well as (ii) a reduction in respiration rate (Rd).
These physiological mechanisms are considered
common strategies to overcome low light regi-
mes in marine macrophytes (Littler et al. 1986,
Lünning 1990, Kirk 1996, Lobban and Harrison
1997, Falkowski and Raven 2007) and were con-
sistent with results from previous descriptive field
studies that examined photoacclimation capaci-
ty of the alga under natural light gradients in the
Mediterranean Sea (Raniello et al. 2004, 2006,
Bernardeau-Esteller et al. 2011; Marín-Guirao et
al. 2015). While increase in pigment content and
photosynthetic efficiency (α) reflect an improve-
ment in both light harvesting capacity and ener-
gy conversion efficiency (Lobban and Harrison
1997, Hanelt and López-Figueroa 2012), reduc-
tion in Rd reveals a decrease in metabolic demand
in order to maximize carbon gains (Markager and
Sand-Jensen 1994, Peréz-Llorens et al. 1996, Ber-
nardeau et al. 2011). These responses enable
the alga to reduce light requirements for grow-
th (illustrated by the decrease in compensation
and saturation irradiance [Ec and E
k]) and extend
the daily period at which algae photosynthesizes
at saturating irradiance (Hk) in order to main-
tain the photosynthetic and productive capacity
under low light regimes (Denisson and Alberte
1982, 1985, Litter et al. 1986, Gantt 1990, Matta
and Chapman 1996, Gómez et al. 1997).
Even though there were quantitative differences
in some photosynthetic variables (mainly Ek, R
d, α
and Chlorophyll b in high light treatments), the
photoacclimative patterns described above for
the mesocosm experiment were very similar to
p. 88 p. 89
CHAPTER 4
TESIS DOCTORAL
those observed in the field experiment, except
for pigment content (Chlorophyll b) and compo-
sition (Chlorophyll b and carotenoid ratios). Such
differences could be explained by unavoidable
differences in some key factor related with the di-
fferent nature of both experimental approaches,
despite the fact that in the mesocosm system
we tried to simulate field conditions as similar
as possible. For example, carotenoids (relative to
chlorophyll a and b) differences between meso-
cosm and field data are likely explained by di-
fferences in the spectral composition of light (i.e.
change not only in quantity but also in quality of
light) related to depth and light capture within
the seagrass canopy. As it was already mentio-
ned in the methods section, we were not able to
reproduce exactly the natural light regime in the
mesocosm system. In the mesocosm system, li-
ght has a greater component of the red part of
the spectrum, while these wavelength are vir-
tually nonexistent in field conditions due to the
absorption by water column and the P. oceani-
ca leaf canopy, where there is a predominance
of green and blue wavelength. These differences
in light quality would promote the development
of accessory pigments such as some carotenoids
(e.g. siphonaxanthin) that have an enhanced
ability to absorb green light (Kirk 1994, Dalla Via
et al. 1998). In fact, increased siphonaxanthin
concentrations as a response to light reductions
have been previously described in this (Raniello
et al. 2006) and other species of the genus Cau-
lerpa (Riechert and Dawes 1986). The influence
of other key factors such as nutrients or tempera-
ture in explaining divergences between field and
laboratory results is much less probable since the-
se conditions were highly controlled in the meso-
cosm system. Seawater used in the mesocosm
was obtained from the same area where the
field experiment was performed and frequently
renewed to maintain nutrient levels. Therefo-
re, quality of seawater in both experiments can
be considered similar at least in relation to the
studied responses. Such differences in pigment
composition (or in any other photoaclimative va-
riable) were not as evident between the IN and
the SHADED treatment of the field experiment,
revealing that while shading structures success-
fully reproduced the light environment inside the
seagrass leaf canopy, they did not modify other
factors linked to meadow structure (e.g. hydro-
dinamism, nutrient availability, etc.) that also
appear to have influenced the photoacclimatory
responses observed in the field experiment.
In the mesocosm experiment maximum growth
rates and biomass production were observed in
light levels ranging between 4.43 (L6) and 9.15
(L7) mol quanta m-2 d-1. Light reductions below
these optimum light levels lead to photoacclima-
tory responses and reductions in the production
and growth capacity of the alga. When light le-
vels ranged between 1.89 (L5) and 0.91 (L3) mol
quanta m-2 d-1, photoacclimation mechanisms
allowed for an optimization of light capture and
use, maintaining positive Hk values and net car-
bon gains. In fact, while these treatments suffe-
red a reduction in light availability which ranged
between 60% and 80% compared to treatment
L6 (i.e. optimum light conditions), carbon gains
decreased only between 20% and 30% in com-
parison to L6, and allowed the alga to produce
new biomass. In this light range, reduction in
growth capacity is not only a consequence of
the decrease in carbon production but it is also
related to the costs of the process of acclimation.
Changes in photosynthetic apparatus determine
an increase in maintenance costs of the maco-
phytes (Raven 1984, Copertino et al. 2006), while
reducing respiratory rates imply a lower provision
of internal resources for growth (Kirk 1994, Pe-
réz-Llorens et al. 1996).
Under more severe light reductions (i.e. below L3
light levels), no further photoacclimation took
place, suggesting that photosynthetic plasticity
capacity of the alga was exceeded. This uncou-
pling between acclimation response and light
availability determines an inefficient use of light,
as illustrated by the extremely low values of Hk
and carbon balance (very close to 0), which in
turn determine a limitation in the capacity of
the alga to develop new biomass. According to
these results, it can be inferred that minimum
light requirements for growth under mesocosm
conditions are very close to the L2 light regime
(0.24 mol quanta m-2d-1). In fact, based on light
extinction coefficient mean values (Kd) obtained
by authors in the same experimental area (Ma-
rín-Guirao et al. 2015), we estimated that the
extremely low light levels measured inside the P.
oceanica meadow are within those prevailing at
the maximum depth range of C. cylindracea in
the Western Mediterranean Sea (but considering
that the spectral composition under seagrass ca-
nopies and maximum distributional depths can
differ). Positive growth rates based on apical sto-
lon length described in L2 and L1 (i.e. darkness)
treatments could be explained as a mechanism
of talus expansion resulting from the dilution
of internal biomass in response to light-limiting
conditions (Sand-Jensen 1988, Pérez-Llorens et
al. 1996).
In the field experiment, light availability in treat-
ments that reproduce light regime within a P.
oceanica meadow (IN and SHADED = 0.41-0.47
mol quanta m-2d-1) laid between the L2 and L3
mesocosm treatments, suggesting that in these
treatments the algae were probably close to the
limit of its photoacclimation plasticity and its
minimum light requirements for growth. In fact,
the very low (although still positive) values of Hk
and daily carbon balance registered support the
idea that this light regime overcomes the alga’s
acclimation capacity. Despite the positive values
registered for these productivity variables, stolon
biomass balance was negative in both treat-
ments, indicating that carbon fixation was not
enough to maintain new biomass production and
thus that light availability limits the development
capacity of the alga. In this field experiment, the
growth capacity of the alga was in general con-
siderably lower than that reported in the meso-
cosm experiment under comparable light levels,
suggesting an apparently higher light require-
ment under field conditions due to, for example,
an increase in maintenance costs associated with
the impacts of other environmental factors such
as grazing and mechanical damage (Markager
and Sand-Jensen 1992). However, once again di-
fferences in light regime provided in both experi-
mental approaches could also be involved in such
discrepancies in algal productivity. As explained
in the methods section, the illumination system
of the mesocosm produced rectangular-shaped
daily light ‘curves’ (Fig. 1), which necessarily re-
sulted in daily Hk periods (i.e. the daily period at
which the alga is photosynthesizing at its maxi-
mum rate) much larger that those derived from
typical, natural bell-shaped light curves. Since
Hk is crucial in determining carbon balance and
growth (Dennison and Alberte 1985, Dunton and
Shell 1986, Gómez et al. 1997) it could reasona-
bly account for the higher rates of algal growth
and biomass accumulation in the aquariums, li-
kely based on the consumption of internal resour-
ces (as indicated by the similar starch content
between light treatments). Furthermore, the hi-
gher algal productivity of the mesocosm system
is consistent with other quantitative differences
previously reported for some photosynthetic va-
riables (Ek, R
d and α) between both experimental
approaches.
p. 90 p. 91
CHAPTER 4
TESIS DOCTORAL
Increases in light availability provided by expe-
rimental manipulation of the seagrass leaf ca-
nopy (i.e. the CLIPPING treatment) allowed C.
cylindracea to achieve a carbon balance three
times higher than those recorded in the IN and
the SHADED treatments, as well as Hk daily pe-
riods longer than 5 hours. These results reinforce
the hypothesis supported by the other results ob-
tained in this study that light availability inside
the seagrass canopy limits the photosynthetic
performance of the algae, and are consistent
with recent experimental studies demonstrating
that the removal of P. oceanica leaves promotes
the establishment and spread of the invasive
seaweed (Tamburello et al. 2014). However, des-
pite the efficient use of light demonstrated by
the alga, stolons transplanted to the CLIPPING
plots presented apical growth rates and biomass
losses similar to that recorded in IN and SHADED
treatments. Furthermore, these stolons were de-
pleted in starch content relative to those in the
OUT plots. These results could be explained if car-
bon gains and internal reserves are being used to
cope with some kind of additional stress instead
of for biomass growth and maintenance. In fact,
stolons from the CLIPPING plots showed some
physical damages at the end of the experimental
period; algae stolons from IN plots also displayed
these wounds, but not those from SHADED plots,
which could explain the significant higher bio-
mass losses measured in stolons from IN treat-
ments. These unexpected results suggest that in
addition to light, other stressful factors linked to
the meadow structure can be limiting the grow-
th and development of C. cylindracea inside the
seagrass leaf canopy. Macrophyte canopies may
affect the distribution of plant understory species
through several differing effects other than sha-
ding, such as scouring (Black 1974, Velimirov and
Griffiths 1979), or exudation of chemicals subs-
tances (Fletcher 1974, Dayton et al. 1984). Since
wounds appeared in stolons just after a short,
stormy event that occurred during the experi-
mental period, a scouring effect caused by sea-
grass leaves over the bottom could be proposed
as a candidate factor. In fact, scouring (Gambi et
al. 1989, 1990) and chemical exudation (Cuny et
al. 1995) are mechanisms by which P. oceanica
can influence the understory assemblages within
the meadow. In addition, a shorter seagrass ca-
nopy can decrease protection from fish (Farina et
al. 2009), some of which being avid consumers of
this alga (Tomas et al. 2011).
In summary, our results are consistent with the
presumed high photosynthetic plasticity of C.
cylindracea and its capacity to colonize Medite-
rranean habitats within a broad range of light
regimes (Piazzi et al. 2000, Klein and Verlaque
2008). However, acclimation mechanisms de-
veloped by the alga represent an energy cost
which may affect its ability to grow in low light
environments, as illustrated by the lower abun-
dances shown by the alga at depths greater than
25-30 m (Klein and Verlaque 2008, Katsanevakis
et al. 2010, Bernardeau et al 2011). Despite the
influence of certain experimental factors (mainly
light quality and quantity) on algal productivity,
results obtained from mesocosm and field expe-
rimental approaches consistently showed that
light levels inside the P. oceanica leaf canopy
overcome the phenotypic plasticity capacity of
C. cylindracea, strongly limiting its photosynthe-
tic performance and leading to carbon balances
unable to sustain algal development. A similar
conclusion was achieved in a previous field study
under winter conditions (Marín-Guirao et al.
2015), confirming that the alga is subjected to
light regimes under or very close to its minimum
light requirements for growth over long periods
of its annual life cycle. In fact, the presence of
C. cylindracea stolons growing at the meadow
edge zone can only be explained by net carbon
gains obtained by the alga during summer, when
Variable
Pmax Rd Ec Ek α Chl a Chl b Carotenoids Chl b/a Ratio Carotneoids/chl a Ratio Carotneoids/chl b Hc Hk Carbon balance Stolon biomass balance Apical growth rate Carbohydrate content
Effect
Treat.
Res.
Treat.
Res.
Treat.
Res.
Treat.
Res.
Treat.
Res.
Treat.
Res.
Treat.
Res.
Treat.
Res.
Treat.
Res.
Treat.
Res.
Treat.
Res.
Treat.
Res.
Treat.
Res.
Treat.
Res.
Treat.
Res.
Treat.
Res.
Treat.
Res.
df
7
16
7
16
7
16
7
16
7
16
7
16
7
16
7
16
7
16
7
16
7
16
7
16
7
16
7
16
7
16
7
16
5
12
MS
7,41
1,01
0,65
0,04
8,83
0,75
149,49
11,94
0,0035
0,0002
3095,98
311,43
1357,40
79,43
198,09
24,03
0,0059
0,0001
0,0024
0,0001
0,0344
0,0005
52,90
0,0009
81,96
0,42
0,61
0,0002
0,02
0,0003
1,53
0,14
0,11
0,019
SE
0,58
0,12
0,50
2,00
0,01
10,19
51,46
2,83
0,01
0,00
0,00
0,02
0,38
0,01
0,01
0,22
F
7,35
15,48
11,73
12,52
15,25
9,94
14,09
8,24
64,28
28,66
65,01
6094,9
193,08
3300,79
60,38
10,85
5,88
P
***
***
***
***
***
***
***
***
***
***
***
***
***
***
***
***
n.s.
SNK test
L1=L2=L5=L6=L4=L7=L3<L8
L3=L2=L4=L1=L5=L6=L7<L8
L3<L2=L4=L5=L1=L6=L7=L8
L1=L2=L3=L5=L4=L6=L7<L8
L7=L1=L8=L6=L5=L4=L2<L3
L1=L6=L7=L8=L5=L4=L2<L3
L7=L8=L6=L1<L5=L4=L2<L3
L6=L1=L7=L4=L5=L8=L2=L3
L7=L8<L6<L1=L4=L5=L3=L2
L4=L6=L3=L1=L5=L2=L7<L8
L4=L3=L6=L2=L5=L1<7<8
L1<L2<L3=L4=L5=L6=L7=L8
L1=L2<L3<L4<L5<L6=L7=L8
L1<L2<L4=L3<L5<L6<L7<L8
L1=L2<L3=L8=L4=L5<L6=L7
L1=L2<L8=L3=L6=L5=L4=L7
Mesocosm Experiment
Table1.
Summary of the one way ANOVA and SNK tests performed to assess the effect of treatment on all C. cylindracea
variables in the mesocosm experiment: df = degree of freedom, MS = Mean Squares, F = F-statistics, p = P value,
SE= standard error; ns = not significant, *p < 0.05, **p < 0.01, ***p < 0.001
light availability allow highly positive carbon ba-
lances (Marín-Guirao et al.2015). Thus, results
obtained in this and previous studies support the
hypothesis that light plays a major role in deter-
mining the resistance of P. oceanica meadows to
C. cylindracea bioinvasion in the Mediterranean
Sea, but also indicates that other factors linked
to the meadow structure could also be involved in
the growth and colonization capacity of the alga.
Therefore, further experimentation would be ne-
cessary in future research to attain a better un-
derstanding of the vulnerability of this seagrass
habitat to C. cylindracea invasions.
5. Annex 1:
p. 92 p. 93
CHAPTER 4
TESIS DOCTORAL
Variable
Pmax Rd Ec Ek α Chl a Chl b Carotenoids Chl b/a Ratio Carotneoids/chl a Ratio Carotneoids/chl b Hc Hk Carbon balance Stolon biomass balance Apical growth rate Carbohydrate content
Effect
Treat.
Res.
Treat.
Res.
Treat.
Res.
Treat.
Res.
Treat.
Res.
Treat.
Res.
Treat.
Res.
Treat.
Res.
Treat.
Res.
Treat.
Res.
Treat.
Res.
Treat.
Res.
Treat.
Res.
Treat.
Res.
Treat.
Res.
Treat.
Res.
Treat.
Res.
df
3
8
3
8
3
8
3
8
3
8
3
8
3
8
3
8
3
8
3
8
3
8
3
24
3
24
3
24
3
12
3
12
3
12
MS
2,89
0,41
0,03
0,002
252,85
0,26
0,006
15,28
1,35
0,001
153,40
423,96
65,96
65,70
93,50
58,57
0,01
0,0008
0,0023
0,0002
0,11
0,001
8,23
2,47
55,50
4,41
0,30
0,019
0,0041
0,0003
0,12
0,031
28,60
1,42
SE
0,37
0,02
0,30
22,56
0,01
0,01
0,00
0,01
0,45
0,05
0,01
0,10
1,42
F
7,05
19,02
16,55
10,19
5,16
0,36
1,00
1,6
14,87
12,59
15,23
3,34
12,58
15,83
15,81
3,75
20,09
P
*
***
***
**
*
n.s.
n.s.
n.s.
**
**
**
n.s.
***
***
***
*
***
SNK test
SHADED=IN=CLIPPING<OUT
SHADED=IN=CLIPPING<OUT
SHADED=IN=CLIPPING=OUT
IN=SHADED=CLIPPING<OUT
OUT=CLIPPING=SHADED=IN
SHADED=IN<OUT=CLIPPING
CLIPPING=OUT=SHADED=IN
CLIPPING=OUT<SHADED<IN
IN=SHADED<CLIPPING=OUT
IN=SHADED<CLIPPING=OUT
IN<CLIPPING=SHADED<OUT
IN=CLIPPING=SHADED<OUT
IN=SHADED=CLIPPING<OUT
Mesocosm Experiment
Table 2.
Summary of the one way ANOVA and SNK tests performed to assess the effect of treatment on all C. cylindracea
variables in field experiment: df = degree of freedom, MS = Mean Squares, F = F-statistics, p = P value, SE= stan-
dard error; ns = not significant, *p < 0.05, **p < 0.01, ***p < 0.001.
DISCUSIÓNGENERAL
G E N E R A L D I S C U S S I O N
p. 97
1. Dispersión y dinámica poblacional de C. cylindracea a escala regional
Tras más de 20 años presente en el Mediterrá-
neo, C. cylindracrea puede ser considerada una
especie naturalizada, donde actualmente se en-
cuentra en fase de propagación, según el marco
general definido para la dinámica de especies
exóticas introducidas (Theoarides y Dukes 2007,
Blackburn et al. 2011). La observación de las pri-
meras colonias del alga en la Región de Murcia
en 2005 constata la continuación del proceso de
dispersión a través del Mediterráneo Occidental
en sentido este-oeste, que posteriormente ha
continuado hasta presentarse en la actualidad
en toda la cubeta occidental (Rivera-Ingraham et
al 2010). Los resultados de los trabajos iniciales
de seguimiento de la distribución del alga en esta
región del sureste peninsular, pusieron en eviden-
cia diversos aspectos sobre las pautas de disper-
sión y los mecanismos empleados que ya habían
sido puestos de manifiesto en otras regiones del
Mediterráneo (Klein y Verlaque 2009). Por un
lado, se observó un patrón espacial de dispersión
muy discontinuo, caracterizado por la aparición
de nuevas colonias aisladas y separadas entre si
distancias que oscilan entre centenas de metros
a decenas de kilómetros entre años sucesivos, lo
que indica (i) la importancia de los mecanismos
de reproducción vegetativa en la colonización
de nuevos hábitats y la elevada resistencia de
los fragmentos y propágulos generados y (ii) la
intervención de vectores secundarios de origen
antrópico en su dispersión a escala local y regio-
nal. Recientemente Papini et al. (2013), a través
de técnicas de Perfil Geográfico, han propuesto
un modelo para la dispersión del alga en el Me-
diterráneo en base al cual se establece que los
principales vectores secundarios implicados en
la propagación del alga a escala regional son el
fondeo de embarcaciones y el transporte asocia-
do a las artes de pesca. En efecto, la primera po-
blación detectada en Murcia se corresponde con
una zona frecuentada habitualmente por embar-
caciones de artes menores y muchos de los pun-
tos donde se ha detectado posteriormente son o
bien zonas con un elevado atractivo turístico don-
de es frecuente la presencia de embarcaciones
deportivas o zonas utilizadas habitualmente por
Las investigaciones desarrolladas en la presente tesis doctoral analizan diversos aspectos de la ecología
de C. cylindracea en el Mediterráneo aportando información sobre cuestiones relevantes relacionadas
con el éxito invasor como son (i) la dispersión y capacidad de desarrollo del alga en una nueva región,
(ii) los factores que controlan la introducción y propagación de la nueva especie y (iii) y el impacto sobre
las comunidades nativas.
En el capítulo 1 así como en el Anexo se ha examinado la capacidad de expansión del alga tras su
llegada a una nueva región, los patrones de dispersión a escala regional y la dinámica poblacional a
medio-largo plazo y en los capítulos 2, 3 y 4 se ha evaluado la influencia de factores abióticos del medio
(luz) sobre el potencial invasor del alga y la interacción con los hábitats nativos, más concretamente
sobre las praderas de P. oceanica. Los resultados obtenidos en los diferentes trabajos han sido discuti-
dos en cada capítulo pero se considera necesario presentarlos, en un contexto general, en el presente
apartado.
p. 98 p. 99
DISCUSIÓN GENERAL
TESIS DOCTORAL
la flota pesquera de la región (Ruiz et al. 2014).
Estos vectores secundarios podrían explicar tam-
bién la propagación del alga desde las colonias
más cercanas conocidas, localizadas entonces en
la provincia de Alicante. A escalas espaciales lo-
cales, factores naturales como la hidrodinámica
local pueden también contribuir a la dispersión
del macrófito debido al transporte de fragmentos
y propágulos entre zonas próximas, al igual que
ya ha sido descrito para C. taxifolia en el Medi-
terráneo (Thibaut 2001). Por último, tampoco se
puede descartar la posibilidad de otros vectores
como el tráfico marítimo pesado vinculado con
las grandes infraestructuras portuarias, que juega
un papel clave en la dispersión del alga a escala
geográfica (Papini et al 2013). De hecho, las pri-
meras colonias observadas en las localidades de
Calblanque y Cabo Tiñoso comprenden una am-
plia área marina caracterizada por una elevada
densidad de este tipo de tráfico a consecuencia
de la presencia del puerto de Cartagena y la refi-
nería de petróleo de la dársena de Escombreras,
uno de los nudos más importantes de conexión
de rutas marítimas del Mediterráneo. Al igual que
en otras zonas del Mediterráneo (ver revisión en
Klein y Verlaque 2009), C. cylindracera en las cos-
tas murcianas se ha desarrollado principalmente
en zonas dominadas por fondos detríticos y de
Mäerl, fondos rocosos con comunidades de algas
fotófilas y fondos con mata muerta de P. oceani-
ca. Se han registrado tasas de colonización muy
elevadas en los primeros tras su aparición (de
hasta 1ha año-1), similares a las reportadas pre-
viamente en otras regiones (Piazzi et al. 1997b,
De Biasi et al 1999) y que confirman la alta ca-
pacidad de expansión que puede llegar a desa-
rrollar el del alga bajo condiciones ambientales
favorables una vez se ha establecido.
Tras esta fase de dispersión inicial, se continuó
el seguimiento de la abundancia del alga en un
número limitado de localidades para conocer en
detalle la tendencia de la dinámica de las pobla-
ciones establecidas, y sus variación intra-anual
(estacional) e interanual. Los resultados recogi-
dos en el Anexo constituyen la primera serie tem-
poral a medio-largo plazo (8 años, 2007-2014)
sobre la abundancia del alga en el Mediterráneo.
La elevada frecuencia de regresiones invernales
observada en el periodo estudiado (Anexo) y los
bajos balances de carbono obtenidos durante el
invierno (Capítulo 3) sugieren un claro patrón
estacional similar al observado en otras zonas
del Mediterráneo (Ruitton et al. 2005b, Lenzi et
al 2007), definido por notables diferencias en la
abundancia del alga entre invierno y verano. Es-
tudios posteriores del ciclo de crecimiento anual
del alga realizados en la zona (Bernardeau-Es-
teller, datos no publicados y no presentados en
esta tesis) han confirmado este patrón estacio-
nal en el que se identifica una época de máximo
crecimiento y abundancia en verano y principio
de otoño, y una época en la que su crecimiento
se muestra severamente ralentizado en invierno
y principio de primavera. Aunque las poblaciones
del alga pueden persistir durante las condiciones
más adversas del invierno (Capítulo 3 y Anexo) es
probable que en esta época desfavorable su resi-
liencia se vea reducida y por tanto se incremente
la vulnerabilidad ante otras fuentes de pertur-
bación mecánica, como por ejemplo el elevado
hidrodinamismo propio de las tormentas inver-
nales. En esta época precisamente se ha podido
comprobar que las poblaciones del alga son muy
vulnerables a los efectos de los grandes tempora-
les, incluso en las zonas más profundas (hasta 26
metros), lo que explicaría las bruscas regresiones
invernales observadas en la mayoría de los años.
Sin tener en cuenta las variaciones estacionales,
durante todo el periodo de estudio C. cylindracea
ha mantenido poblaciones estables en las esta-
ciones monitorizadas, con niveles de abundancia
que oscilan dentro de los rangos ya definidos
para la especie (ver revisión en Klein y Verlaque
2009), aunque con importantes fluctuaciones
interanuales que parecen sugerir dos etapas en
su desarrollo en la región. La primera etapa está
asociada a los primeros años de la aparición del
alga, en la que se observan los valores máximos
de biomasa (hasta 70g/m2), y la segunda etapa
a los siguientes años del periodo de seguimiento,
en los que los valores de abundancia se mantie-
nen dentro de unos niveles más bajos, inferiores a
30g/m2, y más o menos constantes a lo largo del
tiempo. Esta evolución parece reflejar una diná-
mica característica de muchas especies exóticas
en fase de propagación, caracterizada por la apa-
rición de fluctuaciones importantes en la abun-
dancia a lo largo del tiempo como consecuencia
de la interacción más o menos compleja con
factores abióticos y bióticos del medio (i.e. com-
petencia, predación, etc; Boudouresque 1999,
Blackburn et al. 2011). Estas dinámicas impiden
por tanto descartar proliferaciones del alga en el
futuro tal y como se ha descrito en otras macro-
algas exóticas en el Mediterráneo (Boudoures-
que 1999). En cualquier caso parece descartarse
un declive de la especie, como ha sido sugerido
en otras zonas del Mediterráneo (Barbara et al.
2013) o una dinámica regresiva a largo plazo
como la observada para C. taxifolia (Jauber et al.
2003, Iveša et al. 2006) Los datos más recientes
del seguimiento del alga en la región de Murcia
(Ruiz et al. 2014) indican que la especie sigue
presente con abundancias apreciables en todas
las localidades monitorizadas (Capítulo 1).
p. 100 p. 101
DISCUSIÓN GENERAL
TESIS DOCTORAL
Como ya se comentaba en la introducción de la
presente tesis, el éxito en la introducción de una
especie en un nuevo hábitat o ecosistema esta
condicionado, entre otros factores, por (i) las
características y atributos de la propia especie
y (ii) la resistencia de los hábitats y ecosistemas
receptores a la colonización. Atributos como la
elevada capacidad de crecimiento o el desarrollo
de mecanismos de reproducción vegetativa han
sido ya señalados en apartados anteriores como
determinantes para comprender el éxito de esta
especie en el Mediterráneo y han sido reconoci-
dos en general como rasgos característicos de
otras especies sifonales con una alta capacidad
invasora (Willianms y Smith, 2007). Por otro lado,
existen diversas evidencias que sugieren que el
alga presenta una elevada plasticidad fenotípi-
ca a nivel fisiológico y morfológico ante factores
abióticos decisivos capaces de limitar el desarro-
llo y distribución de los macrófitos marinos como
la salinidad, la temperatura o la luz (Lobban
1997). Esta importante capacidad aclimatativa
explicaría, por tanto, la elevada tolerancia am-
biental y el amplio nicho ecológico mostrado por
C. cylindracea en muchas zonas del Mediterráneo
(ver revisión en Klein y Verlaque 2009).
En concordancia con estos estudios, los trabajos
desarrollados en el Capítulo 1 permitieron identi-
ficar poblaciones del alga en aguas de la Región
de Murcia sobre gran variedad de sustratos y con-
diciones ambientales. Sin embargo, y al igual que
ha sido descrito por otros autores (Katsanevakis
et al.2010, Bulleri et al. 2011), se observó que una
de las comunidades más resistentes a la coloniza-
ción son las constituidas por macrófitos de porte
erecto y que forman grandes doseles vegetales
(canopy formers) y más concretamente las pra-
deras de Posidonia oceanica. La susceptibilidad a
la invasión de una determinada comunidad o há-
bitat está, entre otros factores, relacionada con
la disponibilidad de los recursos abióticos y con
la forma en que las interacciones con las comu-
nidades nativas modifican dicha disponibilidad.
Aunque diversas investigaciones habían anali-
zado ya la plasticidad fotoaclimatativa del alga
ante diversos gradientes de luz en el Mediterrá-
neo (Raniello et al. 2004 y 2006), los trabajos de-
sarrollados en los capítulos 2, 3 y 4 han permitido
por primera vez (i) estudiar diversas respuestas
de fotoaclimatación bajo condiciones controla-
das de laboratorio y (ii) valorar la repercusión de
estos mecanismos sobre el crecimiento y capaci-
dad productiva del alga,. Los principales resulta-
dos obtenidos en estos capítulos son analizados
en los dos apartados siguientes.
2. Mecanismos que regulan la introducción de C. cylindracea en el Mediterráneo
a fotoaclimatación es el conjunto de respuestas
desarrollados por los organismos autótrofos ante
cambios en la condiciones lumínicas con el ob-
jetivo de mantener un balance positivo entre la
energía generada a través de la fotosíntesis y la
energía metabólicamente consumida (Kirk 1994,
Raven y Geider 2003). El análisis de los mecanis-
mos de fotoaclimatación en condiciones contro-
ladas de mesocosmos (Capítulo 4) permitió cons-
tatar que las distintas respuestas identificadas
previamente en condiciones naturales (gradiente
de profundidad y gradiente generado por el dosel
vegetal de P. oceanica; capítulos 2, 3 y 4) estaban
relacionadas inequívocamente con las variacio-
nes en los regimenes lumínicos. Estas respuestas
fotoaclimatativas (resumidas en la Tabla 1) fue-
ron fundamentalmente de tres tipos, (i) variacio-
nes a nivel morfológico, (ii) cambios en el aparato
fotosintético y (iii) ajustes en la actividad meta-
bólica. Estas respuestas reflejan la activación de
mecanismos de aclimatación habitúales en ma-
crófitos marinos (Kirk 1994), incluyendo esta y
otras especies congéneres en el Mediterráneo
(Gacia et al. 1996b, Häder et al. 1997, Raniello
et al. 2004, 2006). Curiosamente, los ajustes a
nivel metabólico expresados por la modificación
de las tasas respiratorias y que jugaron un papel
determinante en la aclimatación de la especie,
sólo habían sido descritos hasta la fecha en po-
blaciones tropicales del alga (Riechert y Dawes
1986). Algunas de estas respuestas no sólo refle-
jan cambios cuantitativos de la disponibilidad
de luz, sino también cambios cualitativos del régi-
men lumínico debido a la modificación que expe-
rimenta el espectro de luz con la profundidad o al
atravesar el dosel foliar de P. oceanica. En sínte-
sis, el objetivo último de todos estos mecanismos
es (i) mantener la capacidad absorción de luz y su
uso eficiente a nivel fotosintético y (ii) reducir la
demanda metabólica para preservar el balance
energético del alga y reducir los requerimientos
lumínicos para su crecimiento (Kirk 1994, Raven
y Geider 2003, Falkowski et al. 2007).
2.1. El papel de la luz
2.1.1. Mecanismos de fotoaclimatación de C. cylindracea
p. 102 p. 103
DISCUSIÓN GENERAL
TESIS DOCTORAL
Mecanismo de fotoaclimatación
Cambios morfológicos
Reorganización del aparato
fotosintético
Cambios metabólicos
Tipos de respuesta Indicadores Gradiente lumínico
Cambios contenido
pigmentario
Cambios funciomaniento
fotosintético
Cambios tasa
respiratoria
Longitud Fronde
Chla
Chlb
Carotenoides
Ratio pigmentos
Pmax
α
Ek
Ec
Rd
profundidad
dosel P. oceanica
mesocosmos
profundidad
dosel P. oceanica
mesocosmos
profundidad
dosel P. oceanica
mesocosmos
dosel P. oceanica
mesocosmos
profundidad
dosel P. oceanica
mesocosmos
profundidad
dosel P. oceanica
mesocosmos
profundidad
doselP. oceanica
mesocosmos
profundidad
dosel P. oceanica
mesocosmos
profundidad
dosel P. oceanica
mesocosmos
profundidad
dosel P. oceanica
Las elevadas variaciones registradas en la mayor
parte de las variables analizadas (e.g. reduccio-
nes de hasta el 70% en las tasas respiratorias
e incrementos en el contendido pigmentario de
hasta el 40% bajo condiciones severas de limita-
ción de luz) evidencian la importante plasticidad
de los mecanismos fotoaclimatativos desarrolla-
dos por la especie.
En condiciones naturales, los patrones de acli-
matación expresados mostraron divergencias
entre los distintos experimentos realizados. Se
observaron variaciones tanto cualitativas como
cuantitativas en las respuestas manifestadas
por el alga entre los distintos niveles de un gra-
diente espacial, ante gradientes similares en
épocas distintas del año e incluso ante gradien-
tes similares en la misma época del año pero en
experimentos realizados en años diferentes. Por
ejemplo, en el Capítulo 2 se puso de manifiesto
como el alga es capaz de desarrollar diferentes
estrategias fotoaclimatativas en función de los
cambios del régimen lumínico que se producen
con la profundidad. A profundidades intermedias
del rango batimétrico que ocupa C. cylindracea
en la zona de estudio, los mecanismos desarrolla-
dos por el alga para compensar la reducción en la
disponibilidad de luz y optimizar su utilización, se
basan fundamentalmente en cambios en el apa-
rato fotosintético manifestados por incrementos
en la eficiencia fotosintética (α). A profundida-
des mayores, donde la reducción de la luz es más
severa, dichos mecanismos compensatorios se
basan principalmente en estrategias de reduc-
ción de la demanda metabólica (reducción de
la respiración, Rd). Sin embargo, este patrón de
aclimatación mostraba ciertas variaciones signi-
ficativas cuando se midió al año siguiente sobre
los mismos sitios, y en la misma época (verano),
para la realización de los experimentos descritos
en el Capítulo 4. Por ejemplo, se pudo observar
como en profundidades intermedias como les
mecanismos de respuesta además de cambios en
el aparato fotosintético incorporaban también
una reducción de las tasas respiratorias. Por otro
lado, en cada nivel de profundidad, este patrón
de aclimatación varía también considerablemen-
te según la época del año. Así, por ejemplo, en
invierno se observa un mayor esfuerzo de fotoa-
climatación (reflejado por la mayor reducción de
las tasas fotosintéticas, respiratorias, así como
por los valores de Ec y Ek en relación al verano)
como consecuencia de la mayor reducción en la
disponibilidad de luz característica de esta esta-
ción, especialmente en las zonas mas profundas.
Esta variabilidad espacio-temporal de la respues-
ta se justifica si tenemos en cuenta que la acli-
matación a la luz es un proceso energéticamente
costoso condicionado por multitud de factores,
aparte de las características que definen el régi-
men lumínico, que pueden ser tanto ambientales
(temperatura, disponibilidad de nutrientes, etc.)
como inherentes al propio organismo (contenido
nutricional interno, ciclo de vida, requerimientos
para el crecimiento, reservas de carbono, etc.)
(Lobban y Harrison 1997, Hanelt et al. 2003). Por
tanto, las respuestas fotoaclimatativas expresa-
das por el alga en cada situación determinada
(profundidad, época, etc.) serán las que permitan
una mejor optimización del uso de la luz en base
a los recursos disponibles y posibilidades de la es-
pecie. En este contexto, Flagella et al. (2008) han
sugerido que C. cylindracea presenta un compor-
tamiento tipo “anticipador estacional” (seasonal
anticipator), por lo que las respuestas menciona-
das podrán también estar condicionadas por los
programas de aclimatación estacional internali-
zados por la especie.
Tabla 1.
Mecanismos de fotoaclimatación de C. cylindracea. Chla y Chlb, contenidos en clorofila a y b; Pmax, fotosíntesis neta máxima; α;
eficiencia fotosintetica; Ek, irradiancia de saturación; Ec, irradiancia de compensación; Rd, dark respiration rate.
p. 104 p. 105
DISCUSIÓN GENERAL
TESIS DOCTORAL
El objetivo final de los procesos de fotoaclimata-
ción es mantener cierta capacidad productiva y
una tasa de crecimiento positiva cuando las con-
diciones de luz son subóptimas (Kirk 1994, Raven
y Geider 2003). Por tanto, la distribución poten-
cial de un organismo en ambientes con diferen-
tes regímenes lumínicos dependerá de la mayor
o menor plasticidad de sus mecanismos de acli-
matación (Kirk 1994, Falkowski et al. 2007). Estos
aspectos son especialmente relevantes en el caso
de la introducción de macrófitos marinos, ya que
el potencial colonizador estará condicionado por
su capacidad productiva y de crecimiento. En el
Capítulo 3, mediante la simulación de la ausen-
cia de mecanismos de fotoaclimatación en las
poblaciones profundas del alga, se puso de ma-
nifiesto cómo las respuestas fotoaclimatativas
pueden haber jugado un papel determinante en
el éxito colonizador de la especie en los amplios
rangos batimétricos en que se ha desarrollado
en el Mediterráneo, ya que se traducen de forma
efectiva en una optimización de las tasas produc-
tivas bajo condiciones de luz limitante.
La plasticidad de la respuesta a las variaciones
en los regimenes de luz puede estar limitada por
diversos factores entre los que evidentemente se
incluye la propia disponibilidad del recurso (Van
Kleunen y Fischer 2005). La respuesta de aclima-
tación se encuentra condicionada por los propios
costes de producción de los elementos implica-
dos en dicha respuesta (p.e. pigmentos, enzimas,
etc.) así como por los asociados al propio mante-
nimiento de la maquinaria de aclimatación (Ra-
ven y Geider 2003). Por tanto, unos niveles de luz
muy reducidos pueden determinar una reducción
de los recursos internos disponibles, reduciendo la
capacidad del alga de invertir en mecanismo de
fotoaclimatacion y determinado por tanto una li-
mitación en su respuesta. La falta de adecuación
biológica derivada de los límites en la capacidad
de aclimatación puede a su vez determinar un
desequilibrio entre la energía generada a través
de la fotosíntesis y los propios costes metabólicos
del organismo, que son en definitiva expresados
como una inhibición de la capacidad productiva
y de crecimiento. Estas condiciones determinan
por tanto los requerimientos mínimos de luz que
permiten el desarrollo de un organismo vegetal.
En condiciones de mesocosmos, los niveles mí-
nimos de luz que permitían el crecimiento del
alga se obtuvieron en valores próximos a 0,24
mol quanta m-2d-1 (tratamiento L2, Capítulo 4).
Bajo estos niveles de irradiancia, C. cylindracea
no fue capaz de continuar desarrollando ningún
tipo de respuesta fotoaclimatativa adicional
respecto a la respuesta observada a niveles su-
periores de irradiancia. Esta incapacidad se tra-
duce en periodos de saturación (Hk) y balances
de carbono próximos a cero y, en consecuencia
una fuerte limitación de su capacidad producti-
va y de crecimiento. En condiciones naturales, se
ha comprobado que estos escenarios limitantes
para el desarrollo del alga tienen lugar dentro del
dosel de la pradera, en las estaciones de invierno
(Capítulo 3) y otoño (Capítulo 4), e independien-
temente de la profundidad, y también fuera del
dosel foliar de la pradera, pero solo en invierno y
en la estación más profunda (26m; Capítulo 3).
En muchos de estos casos, como se observa en
la tabla 2, la irradiancia total diaria presentaba
valores medios algo superiores al establecido en
condiciones de mesocosmos como limitante del
desarrollo algal, es decir, 0,24 mol quanta m-2d-1.
Sistema experimental
MESCOSMOS
CAMPO IN
OUT
Tratamiento/Localidad Irradiancia Total Diaria(mol quanta m-2d-1)
Época muestreo
L2
I. grosa (-11m)
C. Tiñoso (-18m)
Calblanque (-26m)
I. grosa (-11m)
C. Tiñoso (-18m)
Calblanque (-26m)
11.74±0.56
9.47±0.30
7.52±0.24
1.78±0.13
2.30±0.02
1.59±0.26
15.74±0.92
10.50±0.30
4.75±0.34
0.71±0.06
0.20±0.04
0.11±0.13
5.06±0.31
1.61±0.23
0.63±0.10
0.24±0.01
0.41±0.08
4.56±0.72
Verano
2008
Verano
2009
Otoño
2011
Invierno
2009
Estas divergencias entre los resultados obtenidos
en el sistema de mesocosmos y los experimentos
in situ, no son más que un reflejo de las inevita-
bles diferencias entre las condiciones de labora-
torio, más controladas, y las condiciones natura-
les, sujetas a mayor variabilidad y complejidad
(interacción con otros factores). En primer lugar,
en el mesocosmos, los niveles de luz son contro-
lados y mantenidos a niveles constantes, y en la
naturaleza experimentan cierta variabilidad cau-
sada por los numerosos factores que afectan a la
cantidad de radiación solar incidente o a la turbi-
dez de la columna de agua. Por otro lado, existen
otros factores (p.e. herbivoría, hidrodinamismo,
etc) que pueden (i) condicionar o limitar la plas-
ticidad de aclimatación a la luz (Valladares et al.
2007) y/o (ii) incrementar los costes de manteni-
miento del organismo (Markager y Sand-Jensen
1994), lo que en definitiva resulta en un incre-
mento de los requerimientos de luz necesarios
para el desarrollo.
En cualquier caso, los experimentos realizados
ponen de manifiesto que el régimen lumínico
que prevalece bajo el dosel foliar de P. oceanica
es limitante para el desarrollo del alga durante la
mayor parte del año. Además, en la época más
favorable (verano), los niveles de irradiancia, pese
a superar los requerimientos mínimos para el cre-
cimiento, son también considerablemente bajos
en comparación con los registrados fuera de la
pradera. Esta situación es consistente con la ca-
pacidad del dosel foliar de P. oceanica de mante-
ner abundancias del alga muy bajas a lo largo del
tiempo, como se observa en las praderas monito-
readas entre 2007 y 2014 (Anexo). Por tanto, los
resultados obtenidos apoyan la hipótesis inicial
de que la disponibilidad de luz dentro de las pra-
deras de P. oceanica juegan un papel relevante
para explicar la limitada capacidad colonizadora
del alga en estos hábitats. Al igual que sucede en
otras especies de Caulerpa (Terrados y Ros 1992,
Robledo y Freile-Pelegrín 2005) y otros macrófi-
tos marinos (Rosemberg y Ramus 1982, Gagne et
al. 1982, Dunton y Schell 1986, Gomez y Wiencke
1998) sometidos a ambientes lumínicos limitan-
tes de forma estacional, es probable que durante
la época favorable se favorezca la producción de
sustancias de reserva que faciliten la subsisten-
cia de la población en la época desfavorable. Es
2.1.2 Consecuencias de los procesos de fotoaclimatacion en la capacidad de colonización
de C. cylindracea.
Tabla 2.
Resumen de las medidas de irradiancia obtenidas en las poblaciones de C. cylindracea donde se observaron condiciones limi-
tantes para el crecimiento del alga.
p. 106 p. 107
DISCUSIÓN GENERAL
TESIS DOCTORAL
posible que otros mecanismos puedan estar tam-
bién interviniendo en el mantenimiento de las
poblaciones, como por ejemplo, la obtención de
carbono mediante heterotrofía, como ya ha sido
documentado en C. taxifolia (Chisholm y Jaubert
1997), o la translocación de recursos desde fuera
de la pradera gracias a la naturaleza cenocítica
del alga (Collado-Vides y Robledo 1999). La au-
sencia de este mecanismo de resistencia podría
explicar también la mayor susceptibilidad mos-
trada por otras angiospermas en el Mediterrá-
neo, que dado su menor porte determinan cam-
bios menos drásticos en los regimenes lumínicos
(ver revision en Klien y Verlaque 2009).
Por otro lado, y de acuerdo con los resultados ob-
tenidos, la disponibilidad de luz perece jugar un
papel importante no solo bajo el dosel foliar de P.
oceanica, sino también para explicar la variación
de su capacidad colonizadora en los sustratos
fuera de las praderas a lo largo de gradientes na-
turales de irradiancia asociados a la profundidad.
Si no tenemos en cuenta episodios puntuales de
regresión, entre 2007 y 2014 la abundancia de
las poblaciones más someras fuera de la pradera
fue superior al registrado en la localidad más pro-
funda (Anexo). Como se ha demostrado, durante
el invierno en esta localidad más profunda las
condiciones lumínicas son totalmente limitantes
para el desarrollo de C. cylindracea (fuera de la
pradera), lo cual limita a su vez la capacidad de
desarrollo del alga en las épocas más favorables.
Esto es consistente con el mencionado gradien-
te de biomasa del alga y sugiere que, al menos
en la zona estudiada, la capacidad colonizadora
del alga se encuentra limitada a partir de profun-
didades de 25 m. Esta idea no es incompatible
con la observación de poblaciones del alga a
profundidades superiores en ésta y otras zonas
del Mediterráneo español. De forma similar, se
ha obtenido evidencia de la importancia de la
disponibilidad de luz para explicar la distribución
vertical de otras especies invasoras como Wo-
mersleyella setacea (Cebrian y Rodríguez-Prieto,
2012). El patrón de abundancia de C. cylindracea
a lo largo de gradientes de profundidad descri-
to en la zona de estudio, ha sido observado en
otras áreas del Mediterráneo (Capiomont et al.
2005, De Biasi et al. 1999), pero no se ha des-
crito en otras zonas (Cebrian y Ballesteros 2009).
Esto es probablemente debido a la influencia
de otros factores capaces de enmascarar este
patrón de abundancia subyacente. Por ejemplo,
en las estaciones someras contempladas en este
trabajo, en algún año la abundancia del alga se
mantuvo a niveles tan bajos como las estaciones
más profundas debido a la incidencia de fuertes
temporales antes de la realización de los mues-
treos. Por otro lado, en estas zonas someras se
ha descrito que la herbivoría sobre C. cylindracea
es muy intensa y mantiene la abundancia de sus
poblaciones a niveles muy bajos. Ambos factores,
hidrodinamismo y herbivoría, están sujetos a una
elevada variabilidad interanual.
Diversas investigaciones sobre los mecanismos
asociados a la resistencia que ofrecen las comu-
nidades marinas a la introducción de macrófitos
exóticos revelan que, dicha resistencia parece
estar estrechamente ligada a la identidad y di-
versidad de los grupos funcionales de producto-
res primarios que componen la comunidad y a la
forma en que dichos grupos usan y compiten por
los recursos disponibles (Arenas et al. 2006, Brit-
ton-Simmons 2006). Estas ideas se contraponen
a las teorías basadas en los postulados de Elton
(1958) que establecen que la resistencia biótica
a la invasión de una comunidad estaría vincula-
da con su diversidad específica, de manera que
una mayor diversidad determinaría un uso más
completo de los recursos, reduciendo su dispo-
nibilidad para las nuevas especies introducidas
(teoría del uso complementario de los recursos;
Hooper 1998)). Una investigación reciente basa-
da en técnicas de metanálisis ha mostrado que
mientras en las comunidades terrestres la diver-
sidad funcional tiene un papel relevante en la
resistencia a la introducción, su papel en los eco-
sistemas marinos es menos importante ya que
parece ser determinante la presencia de ciertos
grupos funcionales con una elevada capacidad
de competencia por los recursos primarios (Kim-
bro et al. 2013).
Los estudios desarrollados en los capítulos 3 y 4
muestran que el severo control definido por el
dosel vegetal de P. oceanica sobre la disponibili-
dad de la luz es un mecanismo competitivo fun-
damental para explicar la resistencia biótica a la
colonización del alga y aportan por tanto, nue-
vas evidencias sobre el papel del grupo funcional
constituido por “canopy -former species” en los
mecanismos de resistencia de comunidades na-
tivas frente a la introducción de especies exóticas
marinas. La baja capacidad colonizadora mos-
trada por el alga durante el periodo 2007-2014
bajo el dosel foliar de P. oceanica (Anexo) indica
la prevalencia de estos mecanismos de resisten-
cia a largo plazo y refleja que las praderas de P.
oceanica en buen estado de conservación actúan
a modo de barrera ecológica frente a la disper-
sión del alga, algo que ya había sido sugerido por
diversas investigaciones en otras zonas del Medi-
terráneo (Katsanevakis et al. 2010, Bulleri et al.
2010). La eficacia de estos mecanismos de resis-
tencia implica el mantenimiento de la estructura
tridimensional definida por el dosel foliar de la
pradera. En las zonas colonizadas por C. cylindra-
cea, las praderas de P. oceanica han mostrado es-
tabilidad estructural, como evidencia el análisis
de sus tendencias poblacionales realizado en el
Anexo, que son estables o positivas, y similares a
las observadas en praderas de zonas no invadidas
de la misma zona. Estas tendencias se observan
también en praderas invadidas de otras zonas de
la Región de Murcia (Ruiz et al. 2014) así como
en praderas no invadidas de otras regiones medi-
terráneas (Sanchez-Rosas et al. 2009, Álvarez et
al. 2009, Guillén et al. 2013, González-Correa et
al. 2015) Por tanto, de acuerdo con estos resulta-
dos, en las zonas invadidas por C. cylindracea, no
parece existir algún tipo de interacción negativa
entre el alga y la angiosperma que implique un
deterioro de la vitalidad de la pradera de P. ocea-
nica, al menos a nivel estructural y poblacional en
la región estudiada. Aunque estos resultados no
se pueden generalizar y extrapolar a otras áreas
geográficas, no se dispone de evidencia científi-
ca consistente de que dicha interacción negativa
2. 2. Interacción entre C. cylindracea y P. oceanica: Resistencia biótica de las praderas
de P. oceanica
p. 108 p. 109
DISCUSIÓN GENERAL
TESIS DOCTORAL
haya tenido lugar en otras localidades mediterrá-
neas.
Estos resultados contrastan con el potencial atri-
buido a C. cylindracea de alterar el desarrollo
vegetativo de la angiosperma, bien mediante la
acción de substancias alelopáticas (Raniello et
al. 2007, Dumay et al. 2002b) o bien a través de
la anoxificación de los sedimentos que coloniza,
con la consiguiente acumulación de fitotóxicos
frente a los que P. oceanica ha mostrado cierta
vulnerabilidad (Holmer et al. 2009). Sin llegar
a cuestionar el potencial de estos mecanismos
de acción, es necesario matizar que se requiere
nueva y robusta evidencia experimental que de-
muestre de forma efectiva la relación entre di-
chos mecanismos y el deterioro de la estructura
y vitalidad de la pradera de P. oceanica. Por otro
lado, los mecanismos de acción mencionados
estén probablemente vinculados (i) al desarrollo
de grandes biomasas del alga y su persistencia
en el tiempo de manera que se puede dar una
alteración de las condiciones del sedimento y (ii)
a una elevada disponibilidad de recursos internos
que permitan la sisntesis de compuestos secun-
darios que actúen a modo de compuestos ale-
lopaticos. Sin embargo, como se ha descrito en
apartados anteriores, por un lado, dentro de las
praderas el desarrollo del alga se encuentra muy
limitado (capacidad productiva y de crecimiento
reducidas); por otro lado, fuera de las praderas,
C. cylindracea puede colonizar los sustratos adya-
centes a los límites de las mismas, pero su bioma-
sa sigue una dinámica altamente fluctuante en
el tiempo, entre años y estacionalmente dentro
de cada año.
La demostrada incapacidad de C. cylindracea
de colonizar los sustratos en el interior del es-
trato foliar de P. oceanica, contrasta también
con su mayor capacidad de invadir el interior de
las praderas de otras especies de angiospermas
mediterráneas, como C. nodosa y Z. noltii (Cec-
cherelli y Campo 2002, Raniello et al. 2004). La
estructura del dosel foliar desarrollado por estas
angiospermas es mucho menos complejo que el
formado por las hojas de P. oceanica, cuyas hojas
presentan además concentraciones de pigmen-
tos fotosintéticos mayores y absortancias de luz
incidente superiores al 85% (Sandoval-Gil et al.
2013Esto se traduce en una mayor transmitan-
cia de la irradiancia incidente (del 50%) a través
del dosel foliar de C. nodosa y Z. noltii (Raniello
et al. 2004 ) y, por tanto, en una mayor dispo-
nibilidad de la luz en el interior de las praderas
que forman que, a su vez, permitiría mayores de-
sarrollos del alga invasora dentro de las mismas.
En este caso, el desarrollo de mayores biomasas
de C. cylindracea en el interior de las praderas de
ambas especies podría hacer efectivos los meca-
nismos de acción antes mencionados y explicar
la interacción documentada experimentalmente
entre el alga y las angiospermas, negativa en el
caso de C. nodosa y positiva en el caso de Z. noltii
(Ceccherelli y Campo 2002).
Esta situación descrita para C. cylindracea es
comparable a la observada con C. taxifolia en el
Mediterráneo, donde muestra una capacidad li-
mitada de colonizar praderas de P. oceanica y de-
sarrollar un impacto negativo a largo plazo sobre
las mismas (Jaubert et al. 1999). Por el contrario,
otros macrófitos invasores como Lophocladia
lallemandi han mostrado una mayor capacidad
de afectar a la vitalidad de la angiosperma, de-
teriorando su estructura, probablemente como
consecuencia de la reducción de la disponibilidad
de luz incidente y la alteración del balance de car-
bono (Ballesteros et al. 2007). La capacidad dife-
rencial observada entre las especies de Caulerpa
y la rodofícea para interaccionar con la angios-
perma radica en su diferente naturaleza y mo-
dos de acción, de acuerdo con el modelo general
desarrollado por Thomsen et al. (2012) sobre los
impactos negativos de macrófitos marinos en
praderas de angiospermas. L. lallemandi se com-
porta en el Mediterráneo como epífita de otros
macrófitos mientras que tanto C. cylindracea
como C. taxifolia presentan un tipo de desarrollo
ligado al sustrato. El crecimiento epífito podría
permitir al alga roja eludir las limitaciones lumí-
nicas impuestas por el dosel vegetal de P. ocea-
nica, lo que favorecería el desarrollo de mayores
biomasas que incrementarían su capacidad com-
petitiva por este recurso y determinarían, a su
vez, alteraciones en las condiciones ambientales
(incrementos en las tasas de sedimentación, en el
grado de anoxificación de los sedimentos y en el
enriquecimiento orgánico de los mismos).
La perturbación de las comunidades nativas, ya
sea debida a causas naturales o antrópicas, pue-
de determinar la reducción en la abundancia de
las especies que componen dichas comunidades
o cambiar las condiciones ambientales, incre-
mentando los recursos disponibles y siendo por
tanto un factor que puede facilitar el desarrollo
de las especies exóticas (Olyarnik et al. (2009).
Los cambios en el ambiente lumínico definidos
por la pradera de P. oceanica están basados en la
estructura tridimensional del dosel foliar, de for-
ma que la perturbación o alteración física del mis-
mo podría reducir la resistencia del hábitat a la
colonización por el alga invasora. De acuerdo con
esta hipótesis, Tamburello et al. (2014) y Cecche-
relli et al. (2014) han observado como la reduc-
ción del estrato foliar simulando fenómenos de
herbivoría severos promueve la proliferación del
alga. En el Capítulo 4 se pudo comprobar como
la manipulación de la estructura de la pradera de
P. oceanica incrementaba la disponibilidad lumí-
nica por encima de los requerimientos mínimos
de crecimiento de C. cylindracea. Sin embargo, se
puso también de manifiesto que probablemente
la interacción con otros factores asociados a las
características estructurales de la pradera (p.e.
la abrasión asociada al movimiento de las hojas
durante condiciones de elevado hidrodinamismo
(fuertes temporales)) podrían también contribuir
a limitar el crecimiento del alga. En este sentido,
las perturbaciones severas que pueden causar
alteraciones significativas de la estructura de la
pradera, como por ejemplo el fondeo de embar-
caciones, han sido relacionados con un aumento
de la expansión del alga (Tamburello et al. 2014).
La propagación del alga invasora mediada por
la supresión de los mecanismos de resistencia
del hábitat apenas han sido evaluados hasta la
fecha (Ceccherelli et al. 2014), pero tal y como
se comentaba anteriormente no es descartable
que la reducción de dichos mecanismos pueda
a su vez de manera directa o indirecta promover
los mecanismos de interacción aumentando el
potencial impacto negativo sobre las praderas.
Bullleri et al. (2011) han observado a su vez que
la capacidad de resiliencia de comunidades na-
tivas de fondos rocosos que incluyen macrófitos
de porte erecto sometidos a fuentes de pertur-
bación antrópica se ve mermada como conse-
cuencia de la invasión del alga. En el caso de C.
taxifolia existen evidencias que indican que el
deterioro de la pradera de P. oceanica puede, por
un lado, reducir la resiliencia del hábitat tras el
impacto de otros factores estresantes múltiples
(Molenaar et al. 2009) y por otro, favorecer y ace-
lerar un deterioro adicional de la pradera (Vilelle
y Verlaque1995).
p. 110 p. 111
DISCUSIÓN GENERAL
TESIS DOCTORAL
Dinámica de C. cylindracea en el Mediterráneo e
impacto sobre las comunidades nativas
Aún dentro de las fases de propagación, las po-
blaciones de los macrófitos invasores pueden
reflejar importantes variaciones en el tiempo, tal
y como muestran los resultados obtenidos en el
Capítulo 2. Estas variaciones son la expresión de
las complejas relaciones que se establecen entre
las especies introducidas y los ecosistemas nati-
vos en las que intervienen factores ecológicos y
evolutivos (Diezt y Edwards 2006) y que pueden
modular los efectos de las especies invasoras a lo
largo del tiempo (Strayer et al. 2011). Los estu-
dios que han evaluado la interacción de C. cylin-
dracea con las comunidades nativas se basan en
experimentos a corto-medio plazo que por tanto
pueden ofrecer una idea errónea de los impac-
tos reales del alga en las comunidades nativas.
Por tanto, una adecuada evaluación de dichos
impactos implica el desarrollo de estudios a más
largo plazo, especialmente en aquellas comuni-
dades biológicas ecológicamente relevantes que
han mostrado una mayor susceptibilidad a la
invasión, como por ejemplo las comunidades de
algas fotófilas, las comunidades de coralígeno y
Mäerl o las comunidades de otras angiospermas
marinas como Cymodocea nodosa.
Interacción entre C. cylindracea y P. oceanica.
A pesar de los resultados y conclusiones obteni-
dos sobre la interacción entre ambas especies a
lo largo de la presente tesis, existen todavía nu-
merosos aspectos de dicha interacción que toda-
vía se desconocen y que podrían tener implica-
ciones sobre el propio estado de conservación de
la angiosperma:
• En el Capítulo 4 se mostraba como, además de
la disponibilidad de luz, es muy probable que
existan otras factores vinculados a la estructu-
ra del dosel foliar de la pradera de P. oceanica
implicados en los mecanismos de resistencia
a la colonización de C. cylindracea. La fricción
asociada al movimiento de las hojas o la pro-
ducción de sustancias químicas alelopáticas
han sido directamente relacionadas con los
factores determinantes en la composición de
las comunidades que habitan debajo del dosel
foliar de esta especie (Gambi et al. 1990, Cuny
et al. 1995)) y de los formados por otros ma-
crófitos marinos (Black 1974, Dayton et al.,
1984). El papel de estos u otros posibles me-
canismos, y las interacciones entre ellos, deben
ser evaluados para obtener un conocimiento
más completo acerca de la vulnerabilidad de
este hábitat a la invasión.
3. Futuras direcciones y perspectivas de investigación
• Como ha sido ya comentado anteriormente,
la alteración física de la pradera, ya sea por
efecto de altas tasas de herbivoría, por tem-
porales o el fondeo de embarcaciones, parece
determinar una inhibición de los mecanismos
de resistencia que favorece en última instancia
la capacidad colonizadora del alga dentro del
hábitat. Sin embargo, el papel que otras presio-
nes antrópicas asociadas tradicionalmente con
el deterioro de la pradera tienen sobre la inte-
racción entre ambas especies es todavía des-
conocido. Un caso particularmente interesante
lo representan los procesos de eutrofización
que son una de las presiones más comunes y
extendidas en las praderas mediterráneas. Los
aportes de nutrientes y materia orgánica pue-
den provocar el deterioro de la pradera como
consecuencia de la reducción en la disponibi-
lidad lumínica, la aparición de desequilibrios
metabólicos asociadas a un incremento de la
concentración de nitrógeno, la anoxificación
del sedimento o incremento de las tasas de
herbivoría (Sanchez-Lizaso et al. en revisión).
En el caso de C. cylindracea, el impacto de es-
tos fenómenos ha sido menos estudiado, pero
se ha observado como el exceso de nutrientes
facilita su dispersión en fondos de coralígeno
a través de un incremento en su capacidad de
crecimiento y mediante la reducción de la re-
sistencia a la colonización de las comunidades
nativas (Gennaro y Piazzi 2014). Por tanto, es
posible los efectos de estos fenómenos en am-
bas especies puedan actuar de forma sinérgica
sobre la colonización del alga en la pradera de
P. oceanica.
• Las pocos estudios existentes hasta el momen-
to parecen indicar una capacidad potencial de
C. cylindracea de afectar a la vitalidad de la
pradera de P. oceanica mediante procesos de
alelopatía y anoxificación del sedimento (Hol-
mer et al. 2009. Sin embargo, como se suge-
ría en el Anexo, la influencia efectiva de estos
mecanismos sobre las praderas a largo plazo
no ha sido demostrada hasta la fecha y pare-
ce estar condicionada por la baja capacidad
productiva y de crecimiento del alga Por tanto,
es necesario obtener nuevas evidencias experi-
mentales que demuestre de forma efectiva la
relación entre dichos mecanismos y el deterio-
ro de la estructura y vitalidad de la pradera de
P. oceanica.
• Otra línea de investigación muy interesante
sería analizar como los futuros escenarios aso-
ciados con el cambio global pueden afectar a
la interacción entre ambas especies. En general
el impacto que la variación en los factores re-
p. 112
DISCUSIÓN GENERAL
TESIS DOCTORAL
lacionados con el cambio climático (por ejem-
plo la temperatura o la concentración de CO2)
tienen sobre las invasiones biológicas son poco
conocidos, aunque existen estudios que sugie-
ren que se pueden ver favorecidas por estos
nuevos escenarios como consecuencia de un
incremento de su capacidad colonizadora y de
la susceptibilidad a la invasión de los ecosiste-
mas nativos (Dukes y Mooney 1999).
El impacto que estos cambios pueden tener so-
bre ambas especies no ha sido apenas inves-
tigado. Los eventos de calentamiento extremo
(olas de calor) han sido relacionados a nivel
regional con incrementos en la mortalidad de
P. oceanica (Díaz-Almela et al. 2007, Marbá y
Duarte 2010) o reducción en la producción de
hojas y el crecimiento de los rizomas (Mayot et
al. 2005), lo que reduciría la resistencia de la
pradera a la invasión del alga. En C. cylindracea
dichos eventos parecen también tener un po-
tencial efecto negativo sobre la abundancia del
alga (Bernardeau-Esteller, obs.pers.), aunque
experimentos en condiciones de mesocosmos
no muestran efectos negativos en el desarro-
llo del alga en situaciones de alta temperatura
(Flagella et al. 2008). En cualquier caso, el im-
pacto que otros factores como el incremento
en las concentraciones de CO2 o el aumento de
perturbaciones extremas como los temporales
históricos no ha sido todavía evaluado.
CONCLUSIONESC O N C L U S I O N S
p. 114 p. 115
CONCLUSIONES
TESIS DOCTORAL
Dispersión y dinámica
poblacional de C. cylindracea
1. C. cylindracea fue detectada por primera vez en aguas de la re-
gión de Murcia en el año 2005, año a partir del cual se produjo una
rápida expansión por todo el litoral de esta región, donde ha mos-
trado tasas de colonización muy elevadas.
2. El alga mostró un patrón espacial de dispersión muy disconti-
nuo, caracterizado por la aparición de nuevas colonias aisladas y
separadas entre si distancias que oscilan entre centenas de metros
a decenas de kilómetros entre años sucesivos, lo que indica (i) la
importancia de los mecanismos de reproducción vegetativa en la
colonización de nuevos hábitats y la elevada resistencia de los frag-
mentos y propágulos generados y (ii) la intervención de vectores
secundarios de origen antrópico en su dispersión a escala local y
regional.
3. Las principales comunidades bentónicas colonizadas por el alga
en las costas murcianas fueron zonas dominadas por fondos detríti-
cos y de mäerl, fondos rocosos con comunidades de algas fotófilas
y fondos con mata muerta de P. oceanica. Por el contrario, las pra-
deras de P. oceanica y los fondos sedimentarios fueron los fondos
menos colonizados
4. La dinámica poblacional de C. cylindracea parece evidenciar
un claro patrón estacional similar al observado en otras zonas del
Mediterráneo (Ruitton et al. 2005b, Lenzi et al 2007), definido por
notables diferencias en la abundancia del alga entre invierno y ve-
rano una época de máximo crecimiento y abundancia en verano y
principio de otoño, y una época en la que su crecimiento se muestra
severamente ralentizado en invierno y principio de primavera.
p. 116 p. 117
CONCLUSIONES
TESIS DOCTORAL
Mecanismos de fotoaclimatación de C. cylindracea ante
gradientes espaciales de luz y relación entre disponibilidad lumínica
y éxito invasor
5. Los mecanismos de fotoaclimatación
desarrollados por C. cylindracea ante los
gradientes espaciales definidos por la pro-
fundidad y el dosel vegetal de P. oceancia in-
cluyeron respuestas que actúan a nivel mor-
fológico (cambios en la longitud del fronde),
a nivel del aparato fotosintético (cambios en
el contenido pigmentario y el funcionamien-
to del aparato fotosintético) y a nivel meta-
bólico (cambios en las tasas respiratoria)
6. Las respuestas observadas mostraron
cambios determinados tanto por las varia-
ciones cuantitativas en el régimen lumínico
como por variaciones cualitativas (cambios
en el espectro de luz)
7. Las respuestas de aclimatación del alga
revelaron una elevada variabilidad espa-
cio-temporal (variación entre profundidades,
variaciones interanuales e intranuales), lo
que refleja la fuerte dependencia de dichas
respuestas ante las condiciones locales im-
perantes. En última instancia, las respuestas
fotoaclimatativas expresadas serán aquellas
que permitan una mejor optimización del
uso de la luz en base a los recursos disponi-
bles.
8. Los resultados obtenidos el en capitulo 2
evidenciaron que los mecanismos de fotoa-
climatación desarrollados por C. cylindracea
son un mecanismos eficaz para optimizar la
capacidad productiva del alga a lo largo de
gradientes de profundidad. Sin embargo, los
propios costes asociados a estos mecanis-
mos parecen limitar la capacidad coloniza-
dora del alga a profundidades elevadas (en
la zona de estudio esto parece observarse al
menos a partir de los 25m de profundidad).
9. Ante niveles de luz reducidos se produce
un desacople entre la capacidad de aclima-
tación del alga y la luz disponible, que deriva
en un uso ineficiente de este recurso, refleja-
do por la reducción en la capacidad produc-
tiva del alga
10. Dentro de la pradera de P. oceanica y en
distintas épocas del año se registraron regi-
menes de luz próximos a los requerimientos
mínimos de luz necesarios para el crecimien-
to de C. cylindracea. Estos resultados eviden-
cian que la disponibilidad de luz dentro de
este hábitat parece ser un factor determi-
nante en la resistencia a la colonización.
11. Además de la disponibilidad de luz, los
experimentos realizados indican que existen
otros factores relacionados con la estructura
de la pradera implicados en la resistencia a
la invasión.
p. 118 p. 119
CONCLUSIONES
TESIS DOCTORAL
Interacción entre C. cylindracea y
P. oceanica: resistencia biótica de las praderas
de P. oceanica
12. La baja capacidad colonizadora mostrada por el
alga durante el periodo 2007-2014 bajo el dosel foliar
de P. oceanica indica la prevalencia de los mecanismos
de resistencia a largo plazo y refleja que las praderas
de P. oceanica en buen estado de conservación actúan
a modo de barrera ecológica frente a la dispersión del
alga.
13. La interacción entre C. cylindracea y P. oceanica no
determino un impacto negativo en la abundancia de la
angiosperma marina, al menos en las praderas estudia-
das y durante los ocho años de estudio
ANEXOA N N E X
Assessment of long-term interaction between the endemic
seagrass Posidonia oceanica and Caulerpa cylindracea in the
Mediterranean Sea
p. 123
Assessment of long-term interaction between the ende-mic seagrass Posidonia oceanica and Caulerpa cylindra-cea in the Mediterranean Sea.
Abstract
C. cylindracea has shown a reduced capacity to
colonize healthy P. oceanica meadows throu-
ghout the Mediterranean coast, although it is
suggested that the invasive alga is able to affect
the seagrass vitality through different mechanis-
ms (e.g. allelopathic, sediment anoxia) and hen-
ce alter its population dynamics and meadow
structure in the long-term. To assess the existence
of long-term negative interactions between both
macrophytes, the abundance of both species in
invaded and non-invaded locations of was moni-
tored over an 8-years period (2007-2014). Results
indicate that in all of the invaded locations C.
cylindracea biomass present inside the seagrass
leaf canopy was about 10 to 50 –fold lower than
that measured just outside the leaf canopy. Also,
no differences were highlighted between invaded
and non-invaded meadows and all the monitored
meadows showed stable or progressive trends. In
summary, our results do not support the existence
of a long-term competitive interaction between
the invasive alga and the native seagrass, at least
in the studied meadows and at the meadow level.
C. cylindracea forms huge biomass gradients as-
sociated to the seagrass meadow edges that are
stable with time, which suggests the existence of
highly limiting conditions for algal growth and
survival under the P. oceanica leaf canopy.
1. Introduction
Exotic macrolgae can generate negative impacts
and outcompete native seagrass habitats
(Garbary et al 2004, Eklöft et al. 2006), is being
recognized as a potential threat to these ecolo-
gicaly relevant habitats in coastal areas worldwi-
de (Williams 2007). Seagrass habitats play a key
role in the functioning of Mediterranean coastal
ecosystems (Larkum et al. 2006), therefore, the
knowledge of the interactions between seagras-
ses and highly invasive species such as those of
the genus Caulerpa is an issue of major concern
among scientific and coastal managers (e.g. De
Villele and Verlaque 1995, Meines et al. 1993,
Meinesz et al. 2001, Boudouresque and Verlaque
2002, Ceccherelli et al. 2002, Dumay et al. 2002a,
Belsher et al. 2003).
The green alga Caulerpa cylindracea Sonder. (he-
reinafter C. cylindracea) has rapidly spread throu-
ghout the western Mediterranean during the last
20 years, where it has established in different
habitats including seagrass meadows (Piazzi et
al. 2005b, Klein and Verlaque 2009). The little
available evidence suggests that the capability
of the alga to invade seagrass habitats and in-
teract with its abundance and vitality largely
depends on the type of Mediterranean seagrass
species, its size, growth rate and the complexity
of the tridimensional structures that they form.
In general, the alga seems to be able to pene-
trate in seagrass canopies formed by species of
medium-small size such as Cymodocea nodosa
and Zostera noltei (Raniello et al. 2004), but not
in the more complex leaf canopies of the largest
species, Posidonia oceanica (Marín-Guirao et
al.2015). Enviado para su publicación a Marine Biology.
p. 124 p. 125
ANEXO
TESIS DOCTORAL
Fig. 1.
Location of study area and
monitored stations in the
Mediterranean sea.
(Ruitton et al 2005b,Mezgui et al 2007, Cebrian
and Ballesteros 2009. Enguix et al. 2014).
2.2. Sampling procedures
Seagrass descriptors
In each location four stainless steel pegs were
set up along the meadow edge every 10 meters.
These markers were used as a spatial reference
for all seagrass measurements in order to avoid
confounding effects by the high small-scale spa-
tial heterogeneity of the meadow structure on
interannual variations of the selected seagrass
descriptors. The percentage of meadow cover
was estimated in 10 m transects deployed in
each marker following a fixed compass bearing
(Ruiz et al. 2010a). Within each transect, a visual
estimation of the percentage of the bottom co-
vered by seagrass patches was performed inside
1,600 cm2 quadrats subdivided into four 20x20
cm squares. The values obtained were averaged
for the ten quadrats representing the percenta-
ge cover of the whole transect, which is the true
replicate (n = 4). Shoot density was estimated by
counting the number of shoots inside 400cm2
square frames randomly placed inside living sea-
grass patches. , every five meters in the same
transects used for cover measurements. The ave-
rage of the three measurements obtained along
each transect was used as an independent repli-
cate (n = 4 replicates). In addition, six permanent
plots of 1,600 cm2 were randomly set in October
2007 in the sampled meadow areaand the exact
number of shoots was counted in each one (ni).
These shoot censuses were repeated each year
in the same season for estimating the annual
net population growth (NPGy), which is the re-
lative change in shoot numbers experienced by
the meadow in a per year basis (%·year-1). This
variable was estimated following the equation:
NPGy (% year-1) = [(nf –ni) x 100)/ ni x (365 /
P)], where ni and nf are, respectively, the mean
value obtained at the beginning of the time se-
ries and at the end of each annual period, and
being P the length of that period in days. For a
given permanent quadrat, the sum of all NPGy
values obtained in the whole monitoring period
By means of a short-term manipulative experi-
ment, Cecherelli and Campo (2002) studied the
impact of C. cylindracea on mixed meadows of
the seagrasses C. nodosa and Z. noltei. These
authors showed a reduction in the shoot densi-
ty of C. nodosa in invaded experimental plots.
whereas Z. noltei showed the opposite response,
increasing its shoot density in the invaded plots.
As mentioned above, C. cylindracea has shown a
reduced capacity to colonize healthy P. oceanica
meadows throughout the Mediterranean coast
(Katsanevakis et al. 2010, Bulleri et al. 2011, Ruiz
et al. 2011; Ceccherelli et al. 2014), which repre-
sent one of the main and more extended habi-
tats of the Mediterranean infralittoral bottoms.
However, and in contrast with this apparent re-
sistance, et al. (2002a) reported significant al-
terations in the vegetative development of the
seagrass in invaded areas that were interpreted
as stress symptoms caused by allelopathic effects
of secondary metabolites produced by the alga.
From these results it could be hypothesized that
in the long term such stressful effects would in-
terfere with plant growth (Dumay et al. 2002a)
and perhaps would compromise seagrass survival
and its resilience against the invasion, but the
long-term effects of C. cylindracea on the vitality,
structure and functions of P. oceanica meadows
has yet to be evaluated.. This study represents a
first assessment of the existence of such long-
term negative interactions between the invasive
alga and the native Mediterranean seagrass. To
this end we monitored the abundance of both
macrophytes in invaded and non-invaded locali-
ties of the Southeastern coast of Spain (Murcia)
over an 8-year period. If a negative interaction
between both macrophytes exists over time, then
i) the seagrass meadow structure would decline
and ii) the abundance of the alga into the sea-
grass leaf canopy would increase
2. Material and Methods
2.1. Study area and sampling design
The present study was conducted in the Medi-
terranean coast of Murcia (SE of Spain), where
the exotic seaweed C. cylindracea was observed
for the first time in 2005 (Ruiz et al. 2011). The
study was initiated two years after (2007) in the
three most invaded localities with well-developed
P. oceanica meadows (Fig. 1; Ruiz et al. 2011):Isla
Grosa (I1, -11m), Cabo Tiñoso (I2, -18 m), and
Calblanque (I3, -25 m). For comparison, another
three non-invaded locations with well-developed
meadows were selected encompassing the depth
range of the invaded locations: Calabardina (N1,
-14m), Las Palomas (N2, -17m) and La Azohia
(N3, -20m), where the alga was completely ab-
sent. All the selected locations presented well-de-
veloped and healthy P. oceanica meadows, not
influenced by anthropogenic disturbances and of
similar oceanographic conditions (substrate type,
water quality, etc.) (Ruiz et al.2014).
In each locality, seagrass meadow descriptors
(shoot density, percentage of meadow cover and
net shoot growth in permanents plots) were me-
asured each autumn between 2007 and 2014.
All of these descriptors have demonstrated to
be effective indicators of meadow structure and
vitality and population dynamics and are widely
used in long-term monitoring programs of this
species (Krause-Jensen et al.2004, Marbá et al.
2005, Pergent-Martini et al. 2004). During the
study period, the abundance of C. cylindracea
(standing biomass,g DW m-2) was measured in
invaded localities, both within the P. oceanica
meadow and in substrates outside, adjacent to
the meadow edge; algal biomass was measured
twice a year (Autumn and Winter) over the study
period in order to assess the high seasonal and
interannual variability reported for C. cylindracea
p. 126 p. 127
ANEXO
TESIS DOCTORAL
Algal biomass
A sample collection was performed for the deter-
mination of the algal biomass in two contrasting
times of the annual growth cycle of the seaweed
in this area: October ( Autumn), when growth
rates and biomass are still at its maximum and
January (Winter), when the growth and abun-
dance of the alga is usually lower, at least in the
study area (Bernardeau-Esteller, unpublished
data). Fronds, stolons and rhizoids of C. cylin-
dracea were carefully collected by hand within
400cm2 square frames and introduced in labeled
plastic bags. In each location ten samples were
randomly collected inside the seagrass meadow
(IN) and another ten samples were obtained
in the area outside just in front of the meadow
edge (OUT) (Ruiz et al. 2011). Samples were
transported to the laboratory with seawater in
chilled containers. Sediment, debris and frag-
ments of other algal species were gently removed
from each sample before drying the alga at 60 °C
until constant weight to calculate total standing
biomass (g DW·m-2).
2.3. Statistical analysis
In order to explore the potential influence of C.
cylindracea on seagrass descriptors with time,
a three way repeated measures ANOVA was per-
formed with Condition (two levels: invaded and
non-invaded) as fixed factor, Location (three le-
vels: I1,I2 and I3 for invaded locations and N1,
N2 and N3 for non-invaded ones) as a random
factor nested within Condition. Time was inclu-
ded as the repeated measured factor (eight levels
corresponding to the eight successive annual pe-
riods). Depth was introduced as a covariate due
to the high negative relationships between dep-
th and meadow structural parameters (i.e. shoot
density and meadow cover) (J.M. Ruiz, unpubli-
shed data; Ruiz et al. 2014). Differences in NPG
between invaded and non-invaded mea dows
with Condition ( two levels, invaded, non-inva-
ded) as a fixed factor and Location (three levels)
as a random factor nested within Condition were
examined with a two-way ANOVA. Analyses was
computed using Greenhouse-Geisser adjusted
degrees of freedom when data did not meet the
assumption of sphericity (Mauchly’s test, α =
0.05). Spatio-temporal variation of C. cylindracea
biomass was analysed by a two-way ANOVA with
Position (two levels: IN and OUT) as a fixed fac-
tor and Time as a random factor. Since samples
of winter 2008, 2013 and 2014 were not carried
out, ANOVA was applied to each of these seaso-
nal periods separately.
Prior to carrying out the ANOVAs, the data was
tested for normality (Kolmogorov–Smirnov) and
equal variances (Levene test) and transformed
where necessary. Where variance remained hete-
rogeneous, untransformed data was analysed, as
ANOVA is a robust statistical test and is relatively
unaffected by the heterogeneity of variances,
particularly in balance experiments (Underwood
1997). When appropriate, a posteriori pairwise
and multiple comparisons of means was per-
formed using respectively LSD test for repeated
measures ANOVA and SNK for two way ANOVA.
A probability level of 0.05 was regarded as signi-
ficant except when data transformation was not
possible. In such cases the level of significance
was reduced to P<0.01 to minimize type I error
and special care was taken in the interpretation
of results. Furthermore, shoot density and cover
data were tested in order to assess trends of time
series by using (i) linear regression model and (ii)
non-parametric Kendall’s coefficient of rank co-
rrelation (τ) Relationships between algal biomass
values inside the meadow (autumn values) and
meadow descriptors were explored using simple
regressions. Analysis were performed using the
(2007-2014) correspond to the total net popula-
tion growth (NPGT). This variable represents the
net balance between recruitments and mortality
of the shoot population, taking positive values
when shoot recruitment is higher than mortality
(population growth) and negative values when
mortality overcome recruitment overcomes re-
cruitment (population decline).
Table 1.
Summary of three-way ANOVAs performed to assess the effect of Position, Location and Time on C. cylindra-cea biomass
SourcePositionLocationTimePositionxLocationPositionxTimeLocationxTimePositionxLocationxTimeResidual
Autumn Winter
df
1
2
7
2
7
14
14
432
df
1
2
3
2
3
6
6
216
MS
53259,27
2255,46
3385,49
1724,01
2073,25
1350,03
1464,73
107,88
MS
6375,29
2943,88
11255,51
1816,08
6532,94
23616,08
19175,27
5270,52
F
36,36
1,67
2,51
15,98
1,42
0,92
13,58
F
3,51
0,13
0,48
0,35
0,34
1,23
3,64
p
***
ns
ns
ns
ns
***
***
p
ns
ns
ns
ns
ns
ns
**
Fig. 2.
Temporal variation of biomass (g DWm-2) of C. cylin-dracea stands growing within (IN, full dots) and out-
side (OUT, empty dots) of the three studied invaded
meadows (A=I1, B=I2 and C=I3). Data are presented
as means and standard errors. Asterisks indicate signifi-
cant differences provided by the pair-wise comparison
for the “Position” Factor (two levels, IN and OUT) for
each sampling time obtained in SNK test performed
after ANOVA. *p<0.05, **p<0.01. ND= no data.
p. 128 p. 129
ANEXO
TESIS DOCTORAL
Fig. 5.
Annual net population growth (NPGy) of invaded (A) and non-invaded (B) meadows from 2007 to
2014. Data areis presented as means ± standard error.
Table 3.
Summary of the two-way Repeated Measured ANOVA performed to assess the effects of Condition,
Location and Time on Annual Net Population Growth (NPGy). ns=not significant, ***P<0.001.
SourceBetween-Subjects EffectsConditionLocation (Condition)ResidualWithin-Subjects EffectsTimeTimexConditionTimexLocation(Condition)Residual
df
1
4
30
4,53
4,53
18,12
158,55
MS
38,3088629
319,388108
520,059365
6617,36154
1343,36163
818,203926
1159,25942
F
0,11994455
0,61413779
8,08766779
1,64184207
0,70579882
p
ns
ns
***
ns
ns
Table 4.
Mean and standard error of Total Population Growth (NPGt) measurements at each location and
results of two-way ANOVA and SNK test performed to assess the effects of Condition and Location on
this variable. ns=not significant, *P<0.05.
NPGt
ANOVASourceConditionLocation(Condition)ErrorSNK: I3<N1=N3=N2=I2=I1
Variable Location
I1
27,61
± 9,28
df
1
4
30
I2
23,47
± 11,84
MS
785,94
3461,00
1176,36
I3
-24,91
± 8,90
F
0,21
2,94
N1
-18,66
± 19,65
p
ns
*
N2
15,83
± 15,39
N3
1,87
± 8,05
Fig. 3.
Shoot density of Posidonia oceanica of invaded (A) and non-invaded (B) meadows from 2007 to 2014. Data are
presented as means ± standard error.
Fig. 4.
Percentage of cover of invaded (A) and non-invaded (B) meadows from 2007 to 2014.
Data are presented as means ± standard error.
Table 2.
Summary of the two-way Repeated Measured ANOVA performed to assess the effects of Condition, Location
and Time on Shoot Density and Meadow Cover. Depth factor was include as covariate. ns=not significant,
***P<0,001.
SourceBetween-Subjects EffectsDepth [covariate]ConditionLocation (Condition)ResidualWithin-Subjects EffectsTimeTimexDepthTimexConditionTimexLocation(Condition)Residual
Shoot Density Variable Meadow Cover
df
1
1
4
18
7
7
7
28
144
df
1
1
4
18
7
7
7
28
144
MS
1.89
8,84
357,56
28,04
35,84
35,58
32,37
354,11
976,18
MS
10,07
28,24
65,62
41,90
81,86
81,53
86,28
42,59
16,79
F
0.067
0.02
12,75
0,40
0,40
0,37
1,87
0,40
F
0,240
0,43
1,57
1,92
1,91
2,03
2,54
p
ns
ns
***
ns
ns
ns
***
p
ns
ns
ns
ns
ns
ns
***
p. 130 p. 131
ANEXO
TESIS DOCTORAL
tion of this growth during the winter (Ruitton et
al. 2005b, Lenzi et al. 2007). However, this does
not seem to be a general pattern as for some
years the algal abundance did not only decline in
winter, but it had maintained close to mean va-
lues recorded in previous and following autumns
(locations I1 and I3 in 2009 and location I2 in
2011). This interannual variability could explain
the fact that other authors did not find a seaso-
nal pattern of C. cylindracea during shorter time
periods (≤ 2 years; South Italy, Giaccone and Di
Martino, 1995; Balearic Islands, Cebrian and Ba-
llesteros 2009). This algal species has demonstra-
ted the ability to balance its carbon budget in the
winter although with reduced growth rate, which
suggests that the algal biomass is able to persist
under conditions of light and temperature typical
of winter periods (Flagela et al. 2008. Marin-Gui-
rao et al. 2015, Bernardeau-Esteller et al. Unpu-
blished data). Nonetheless, in such conditions the
algae could have a reduced resilience and hence
a higher vulnerability to additional physical dis-
turbance such as, the action of hydrodynamic
forces associated to winter storms, which inten-
sity and frequency can be subjected to great in-
terannual variability. Strong hydrodinamism has
been identified as a possible factor involved in C.
cylindracea winter regressions (Klein and Verla-
que 2008), however this hypothesis must be ad-
dressed with specific experimental work.
The populations of C. cylindracea showed very
low biomass within the seagrass leaf canopy,
which highlight the low capacity of the alga to
colonize this habitat. These results are in line
with other works showing that P. oceanica mea-
dows with a good conservation state can act as
ecological barriers against the spread of the in-
troduced seaweed (Katsanevakis et al. 2010, Bu-
lleri et al. 2010). Although the factors underlying
this incapacity of of the alga to penetrate into
P. oceanica meadows are still poorly understood,
Marín-Guirao et al. (2015) and Bernardeau-Este-
ller et al. (2015) have shown that physical factors
linked to the leaf canopy structure such as light
limitation can play a relevant role, regardless the
depth and meadow structure properties asso-
ciated to that depth. Similarly, the inability of C.
cylindracea to colonize the P. oceanica meadow
was evident even in the deepest meadow of the
I3 location, probably as consequence of the clo-
se coupling between meadow structure and light
availability described for P. oceanica across dep-
th gradients (Pergent et al 1995, Ruiz et al. 2014).
Descriptors of seagrass meadow structure indica-
te that most of the monitored meadows followed
a stable or progressive trend throughout the stu-
died 8-years period (Table 5), without differences
between invaded and non-invaded meadows. In
location I3 a negative total population growth
(NPGt) was obtained in the permanent quadrats,
which contrast with the positive and stable trends
Table 5.
Summary trends.
I1I2I3N1N2N3
Shoot density Meadow Cover NPGt
++-===
Balance 2007-2014
+===++
Balance 2007-2014
=++===
Lineal Regression
==+===
Lineal Regression
=+====
Tau Kendall
==+===
Tau Kendall
===+==
Locationstatistical package SPSS version 17.0 (SPSS Inc.
Chicago, Ill) and Sigmaplot 10.0 (Systac Softwa-
re Inc.).
3. Results
C. cylindracea biomass outside of the meadow in
the autumn season were in overall significantly
higher (between 10 and 50 fold) than inside (Fig.
1, Table 1, SNK, α=0.05). Abundance outside of
the meadow ranged from 70.3 to 9.4 in I1, 54.4
to 15,4 in I2 and 23.5 to13.9 in I3. In years in
which these differences were not observed (2012
and 2014 for I1 and I2; 2010 and 2012 for I3; Fig.
4) algal biomass was low (<6 g/m2), registering in
some cases a complete regression of the popula-
tion (p.e. location I1 in autumn 2013) (Fig. 1). For
the winter season biomass ranged from 0.0 to 3.2
g/m2 in all locations and in both position (Fig. 1)
except in 2011 for I1(69.9 g/m2) and in 2009 for
I2 (53.4 g/m2) and I3 (14.5 g/m2) (Fig. 1). Only in
these years biomass outside of the meadow was
significantly higher than that recorded inside (14
to 70 fold higher; Table 1, SNK, α=0.05).
Invaded and non- invaded P. oceanica meadows
did not show differences in their structural para-
meters (shoot density and meadow cover) and
population dynamics (NPGy, NPGt) throughout
the study period (Table 2, Table 3, Table 4) and
no correlations with algal biomass within the
meadow canopy were detected in those invaded.
Both invaded and non-invaded meadows showed
significant interannual changes (LSD test,
α<0.05) in meadow descriptors. Variations up
to 50% for shoot density (location N1 between
2007 and 2008, Fig 3) and 100% for meadow
cover (location N3 between 2010 and 2011; Fig
. 4) were detected. Time also had a statistically
detectable effect on NPGy, with maximum net
gains and losses close to 40% (locations N1 and
N2 in 2007) and -20% (location N3 in 2013)(Fig.
4, Table 3). For the whole time series meadow
descriptors showed stable or progressive trend
in most locations (Table 5). In I1, N2 and N3
a significant increase in shoot density between
the first and the last year of the time series was
observed, while for meadow cover this increase
was significant in I2 and I3. In the rest of the
locations no changes for both variables were de-
tected at the end of the study period (LSD test,
α=0.05). Both meadow descriptors showed posi-
tive lineal relationships and significant positive
correlations based on Kau coefficient with time
in I3 (r = 0.42, p = 0.048; 0.88, p = 0.0004 and τ =
0.86, p = 0.001). These significant positive trends
were also observed for meadow cover in I2 (r
= 0.51, 0.028) and N1 (τ = 0.50, p = 0.042). The
rest of the locations had not shown significant
trends. NPGt showed values greater than zero in
all locations except in N1 and I3, although SNK
test only found significant differences between
I3 and other locations (Table 4).
4. Discussion
Over the study period (2007-2014), populations
of C. cylindracea outside of the meadow showed
biomass values within the ranges reported for
this species in other invaded localities at a similar
depth range (Capiomont et al. 2005, Mezgui et
al. 2007, Cebrian and Ballesteros 2009, Enguix
et al. 2014 ). Results obtained in this study sug-
gest the existence of marked fluctuations of algal
biomass in all studied locations with maximum
values in the autumn sampling time and a dras-
tic decline in the winter time, when the alga even
disappear in most years of the study period. This
seasonal pattern of algal biomass, including win-
ter regressions, has been reported in populations
of several areas of the Western Mediterranean
Sea (Buia et al. 2001, Ruitton et al. 2005b, Len-
zi et al. 2007, Enguix et al. 2014) and is consis-
tent with the greater growth rates measured in
C. cylindracea stolons in summer and the inhibi-
p. 132 p. 133
ANEXO
TESIS DOCTORAL
netheless the scientific knowledge at this level is
very low and more robust experimental evidence
is necessary to conclude something about the
operation of such competitive mechanisms in
Mediterranean seagrass habitats.
In summary, our results indicate that C. cylindra-
cea populations are well established in inffralito-
ral bottoms of the studied area, although with a
high temporal variability mainly accounted for
by seasonal forces but also by local oceanogra-
phic and climatic conditions. Our results reinforce
the idea of P. oceanica meadows as ecological
barriers against the spread of C. cylindracea, as
indicated by the observation of stable, persistent
gradients of algal biomass at both sides of the
meadow edge. The resistance mechanisms of
P. oceanica meadows to bioinvasions seems to
be linked to its highly complex canopy structure
(Ceccherelli et al. 2014, Marín-Guirao et al. 2015,
Bernardeau-Esteller et al. 2015). Interactions be-
tween C. cylindracea and Mediterranean seagras-
ses have only been demonstrated in the much less
complex leaf canopies of C. nodosa and Z. noltei
(Cecchererelli and Campo 2002) within which the
alga is able to grow and develop high biomasses
(Raniello et al. 2004).. Our results show that the
presence of C. cylindracea in invaded areas did
not affect the structure of the P. oceanica mea-
dow during the studyperiod and hence resistance
mechanisms of the seagrass to the invasion also
remained intact. Therefore, this study does not
support the existence of a long-term competiti-
ve interaction between the invasive alga and the
native seagrass, at least in the studied meadows
and at the meadow level, although more in depth
research is necessary to address the inconsisten-
cy of these results in other geographical regions
and the involved mechanisms of competitive in-
teractions.
showed by shoot density and meadow cover me-
asured in the same meadow. This apparently
contradictory result could probably reflect the di-
fferent scales at which each descriptor is measu-
red and their differential sensitivity to changes in
meadow structure. Permanent quadrats were ins-
talled very close to the meadow edges, adjacent
to the densely colonized sediments, which could
suggest a possible specific negative effect of the
algal population on seagrass shoots of the mea-
dow limit. However, this possibility is not suppor-
ted by the observation of very similar trends in
the non-invaded locality N1 (Table 4), although
it was not statistically significant in this case due
to the high variability between replicates. Moreo-
ver, when we analyse the shoot population grow-
th per annual basis (i.e. annual net population
growth, NPGy; Fig. 4) a general trend towards
negative values was observed both for most in-
vaded and non-invaded locations since 2011. In
addition, local factors could also account for the
particular declining behaviour of the shoot popu-
lation in I3 permanent quadrats. Meadow edges
are vulnerable to physical disturbances caused
by sediment dynamics and hydrodynamic forces
(Fonseca and Bell 1998, Infantes et al. 2009, Ben-
Brahin et al 2014). This could explain the particu-
larly negative NPGt values observed in I3 since
in this location was frequent the observation of
deeply buried P. oceanica shoots caused by the
migration of large sand waves. Persistent burial
of P. oceanica shoots above 3 cm have been de-
monstrated to be a cause of shoot mortality in
this seagrass species, with one of the lowest rates
of vertical growth among seagrass species (Bou-
douresque et al. 1984; Manzanera et al 2014).
Consistently, the P. oceanica meadow at the I3
location presented a fragmented seascape for-
med by patches of tens of meters spread over
the sandy bottom revealing the existence of a
particular regime of physical disturbance in this
locality (Hemminga and Duarte 2000).
Therefore, our results do not support the existen-
ce of a negative interaction between C. cylindra-
cea and P. oceanica meadows, at least at the po-
pulation/meadow level and in the studied region.
Two possible mechanisms have been suggested
as being able to cause potential effects of C.
cylindracea on P. oceanica meadows in the long
term: (i) allelopathic interaction by which the re-
lease of a phytotoxic compound would affect and
damage the competitor physiology (Dumay et al.
2002a), and (ii) modifications of sediments con-
ditions to turn these adverse to support seagrass
growth (Holmer et al. 2009). From this study the
operation of such mechanisms cannot be addres-
sed in the invaded locations, but some additio-
nal information allows us to speculate that they
could be of low relevance in this case. First of all,
concentrations of phytotoxic compounds in C.
cylindracea tissues are of 1 order of magnitude
lower than those measured in congeneric species
C. prolifera and C. taxifolia (Dumay et al. 2002b.
Box et al. 2010). This fact, together with the high
fluctuations of algal abundance reported in inva-
ded locations, considerably reduces the probabili-
ty of allelopathic, negative effects of C. cylindra-
cea populations on the seagrass meadow since it
would probably require a very high and persistent
algal biomass over time. The formation of sedi-
ment anoxia, and the consequent accumulation
of phytotoxic compounds in sediments, is a plau-
sible hypothesis, but it also probably depends on
the persistence of high algal biomasses. Consis-
tently, no sediment anoxia was observed during
sampling surveys in all monitored invaded areas
throughout the 8-years periods. Furthermore,
since the development of the alga is limited wi-
thin the P. oceanica meadow, the probability of
sediment anoxia within the leaf canopy (where
the plant is rooted) should be also very low. No-
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CODA
Seguro que fueron más, muchas más, pero estas sonaron una y otra vez du-rante los últimos compases de elaboración de esta tesis, quedando grabadas a modo de banda sonora perenne de las sensaciones y estados vividos.
The Caulerpa Remix: the role of life
1. Fjögur píanó. Sigur Ros2. Bye Bye. Destroyer3. They Harder they Come. Jimmy Cliff4. Snowflakes Falling on the Sea.The Windy Hills5. Heroes and Villains. The Beach Boys6. Via Lattea. Franco Battiato7. Is That Enough. Yo la Tengo8. Change Your Mind. Neil Young and Crazy Horse9. Le onde. Ludovico Einaudi10. Remember Our Heart. Alexander11. Stranizza d’amuri. Franco Battiato12. Ya Hey. Vampire Weekend13. Fire Scene. S Carey14. How Can You Really. Foxygen15. Sketch for a Summer. The Duruti Column16. Slash Your Tires. Luna17. Kaputt. Destroyer18. Did i Tell You. Yo la Tengo19. This Old Heart of Mine. The Isley Brothers20. Helpless. Neil Young 21. Wia. Wim Mertens22. Apple Tree. The Windy Hills
https://open.spotify.com/user/jaimito78/playlist/5Bpm7yXKhe5pGCIcE6JlTv
OCT · 2015
JAIME BERNARDEAU ESTELLER