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MINISTÉRIO DA EDUCAÇÃO
UNIVERSIDADE FEDERAL DO RIO GRANDE DO NORTE PRÓ-REITORIA DE PÓS-GRADUAÇÃO
UNIDADE ACADÊMICA ESPECIALIZADA EM CIÊNCIAS AGRÁRIAS - UAECIA
PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS FLORESTAIS
ECOLOGIA REPRODUTIVA, DIVERSIDADE GENÉTICA E SISTEMA
REPRODUTIVO DE Copernicia prunifera (ARECACEAE)
RICHELIEL ALBERT RODRIGUES SILVA
Macaíba – RN
2017
2
RICHELIEL ALBERT RODRIGUES SILVA
ECOLOGIA REPRODUTIVA, DIVERSIDADE GENÉTICA E SISTEMA
REPRODUTIVO DE Copernicia prunifera (ARECACEAE)
Dissertação de mestrado apresentada ao Programa de
Pós-Graduação em Ciências Florestais da
Universidade Federal do Rio Grande do Norte, como
pré-requisito para obtenção do título de Mestre.
Orientador: Prof. Dr. Fábio de Almeida Vieira
Macaíba - RN
2017
Universidade Federal do Rio Grande do Norte - UFRN
Sistema de Bibliotecas - SISBI
Catalogação de Publicação na Fonte. UFRN - Biblioteca Setorial da Escola Agrícola Jundiaí - EAJ
Silva, Richeliel Albert Rodrigues.
Ecologia reprodutiva, diversidade genética e sistema reprodutivo de Copernicia prunifera (ARECACEAE) / Richeliel
Albert Rodrigues Silva. - Macaíba, 2017. 51f.: il.
Dissertação (Mestre) Universidade Federal do Rio Grande do
Norte, Unidade Acadêmica Especializada em Ciências Agrárias,
Programa de Pós-Graduação em Ciências Florestais.
Orientador: Fábio de Almeida Vieira.
1. Carnaúba - Dissertação. 2. Fenologia reprodutiva -
Dissertação. 3. Estruturas reprodutivas - Dissertação. 4. ISSR -
Dissertação. 5. Taxa de cruzamento - Dissertação. I. Vieira,
Fábio de Almeida. II. Título.
RN/UF/BSPRH CDU 633.9
ECOLOGIA REPRODUTIVA, DIVERSIDADE GENÉTICA E SISTEMA
REPRODUTIVO DE Copernicia prunifera (ARECACEAE)
Richeliel Albert Rodrigues Silva
Dissertação avaliada e aprovada pela banca examinadora:
Banca Examinadora:
Data de aprovação:
16/02/2017
Macaíba - RN
2017
DEDICO
A minha mãe Maria da Piedade Rodrigues Silva.
AGRADECIMENTOS
A Deus, por ter proporcionado tantos momentos bons na minha vida.
Aos meus pais e irmãos, pelo amor e apoio em todos os momentos.
Ao Programa de Pós-Graduação em Ciências Florestais da UFRN.
Ao Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), pelos materiais e
equipamentos adquiridos por meio de projetos.
À Fundação de Apoio à Pesquisa do Rio Grande do Norte (FAPERN), pela concessão das bolsas
de estudo.
À Unidade Acadêmica Especializada em Ciências Agrárias, pela estrutura disponibilizada para
realização das minhas atividades de mestrado.
Ao professor Dr. Fábio de Almeida Vieira, pela orientação, atenção e incentivo durante a minha
vida acadêmica.
À Professora Drª Cristiane Gouvêa Fajardo, pela amizade, carinho e orientação acadêmica.
Ao Professor Dr. Murilo Malveira Brandão, por fazer parte da minha banca.
A todos que fazem parte do Laboratório de Genética e Melhoramento Florestal da UFRN
(LabGeM), pela amizade, carinho e colaboração durante o meu mestrado.
Aos professores do Programa de Pós-Graduação em Ciências Florestais da UFRN.
Aos meus amigos Jardel, André, Anny, Jéssica e Nicinha, pelo companheirismo e apoio durante
todos os momentos da minha vida.
SUMÁRIO
RESUMO ........................................................................................................................................ 7
ABSTRACT .................................................................................................................................... 8
INTRODUÇÃO ............................................................................................................................... 9
REFERÊNCIAS ............................................................................................................................ 11
CAPÍTULO 1: REPRODUCTIVE ECOLOGY OF THE Copernicia prunifera, A NATIVE
PALM FROM BRAZILIAN SEMIARID ..................................................................................... 14
ABSTRACT. ................................................................................................................................. 14
INTRODUCTION ......................................................................................................................... 14
MATERIAL AND METHODS..................................................................................................... 16
RESULTS ...................................................................................................................................... 19
DISCUSSION ................................................................................................................................ 23
CONCLUSION ............................................................................................................................. 25
ACKNOWLEDGMENTS ............................................................................................................. 25
REFERENCES .............................................................................................................................. 25
CAPÍTULO 2: MATING SYSTEM OF Copernicia prunifera (ARECACEAE) ......................... 29
ABSTRACT .................................................................................................................................. 29
INTRODUCTION ......................................................................................................................... 29
MATERIAL AND METHODS..................................................................................................... 31
RESULTS ...................................................................................................................................... 34
DISCUSSION ................................................................................................................................ 39
IMPLICATIONS FOR CONSERVATION AND MANAGEMENT .......................................... 41
ACKNOWLEDGMENTS ............................................................................................................. 42
REFERENCES...............................................................................................................................42
CONCLUSÕES GERAIS...............................................................................................................51
7
RESUMO
O presente estudo teve como objetivos descrever as características reprodutivas da palmeira
Copernicia prunifera, investigar a diversidade genética e o sistema de reprodução de uma
população natural por meio de marcadores ISSR no estado do Rio Grande do Norte, Brasil.
Foram observadas inflorescências múltiplas, constituídas de flores hermafroditas, com
coloração clara. Além disso, as flores são compostas por 3 sépalas, 3 pétalas, 6 estames e 3
carpelos. O percentual médio de pólens viáveis foi de 62%. Existem divergências nas
fenofases reprodutivas entre as populações avaliadas, sendo observada atividade contínua na
produção de flores e frutos maduros na população de Parnamirim, e descontínua na população
de Macaíba. Os marcadores utilizados para analisar a diversidade genética e o sistema
reprodutivo da Copernicia prunifera foram mediamente informativos e apresentaram elevado
polimorfismo. Os valores dos índices de diversidade entre os indivíduos adultos e as
progênies não diferiram estatisticamente (He = 0,319 e I = 0,470; He = 0,337 e I = 0,505),
respectivamente. No teste de hipóteses para detecção de gargalo genético, nos modelos IAM
(alelos infinitos) e SMM (passos de mutações), observaram-se ocorrência de redução
populacional. As taxas de cruzamento em nível de população (n = 247) apontaram
cruzamento multiloco (tm) de 0,878 e entre indivíduos não aparentados (ts) de 0,738,
indicando que a Copernicia prunifera é uma espécie de cruzamento misto, sendo
preferencialmente alógama. A diferença entre a taxa de cruzamento uniloco e multiloco (tm -
ts) foi reduzida, sinalizando baixo cruzamento entre indivíduos aparentados. O índice de
fixação entre as árvores matrizes (F) foi negativo (- 0,200), apontado ausência de endogamia.
A correlação de autofecundação (rs) evidenciou valor elevado (0,914). Os resultados
encontrados nesse estudo geraram informações sobre a ecologia reprodutiva da espécie, como
também para adoção de estratégias de manejo, conservação e melhoramento genético da
palmeira Copernicia prunifera.
PALAVRAS-CHAVES: Carnaúba, Fenologia reprodutiva, Estruturas reprodutivas, ISSR,
Taxa de cruzamento
8
ABSTRACT
The present study aimed to describe the reproductive characteristics of the palm Copernicia
prunifera, investigating the genetic diversity and the system of reproduction of a natural
population by using ISSR markers in the state of Rio Grande do Norte, Brazil. Were observed
multiple inflorescences, constituted of hermaphroditic flowers, with clear coloration. In
addition, the flowers are composed of 3 sepals, 3 petals, 6 stamens and 3 carpels. The average
percentage of viable pollen was 62%. There are differences in the reproductive phenophases
between populations evaluated, being observed continuous activity in the production of
flowers and ripe fruit in the population of Parnamirim, and discontinuous observation in the
Macaíba population. The markers used to analyze the genetic diversity and reproductive
system of Copernicia prunifera were usually informative and presented high polymorphism.
The values of the indices of diversity among the adults and the progenies did not differ
statistically (He = 0.319 and I = 0.470; He = 0.337 and I = 0.505), respectively. In the
hypothesis test for detection of genetic bottleneck, IAM models (infinite alleles) and SMM
(steps of mutations), observed occurrence of population reduction. Outcrossing rates in
population level (n = 247) pointed multilocus outcrossing rate (tm) of 0.878 and single locus
outcrossing rate (ts) of 0.738, indicating that the Copernicia prunifera is a species of mixed
mating system, and preferentially alogamous. The mating among relatives rate (tm - ts) has
been reduced, indicating low outcrossing between closely related individuals. The fixation
index between seed tree (F) was negative (- 0.200), pointed to the absence of inbreeding. The
correlation of selfing (rs) showed high value (0.914). The results found in this study generated
information on the reproductive ecology of the specie, but also to adopt management
strategies, conservation and genetic improvement of palm Copernicia prunifera.
KEYWORDS: Carnaúba, Reproductive phenology, Reproductive structures, ISSR,
Outcrossing rate
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INTRODUÇÃO
A família Arecaceae possui cerca de 200 gêneros e 2.000 espécies (SOUZA e
LORENZI, 2008), onde no Brasil ocorrem cerca de 40 gêneros e 283 espécies (LEITMAN et
al., 2015), incluindo representantes dioicos e monoicos, de morfologia floral variada, com
inflorescências interfoliares ou infrafoliares na antese em forma de espiga, juntamente com a
presença de poucas ou muitas ráquilas (HENDERSON et al., 2000). As suas raízes podem ser
subterrâneas ou aéreas (LORENZI et al., 1996). Os estipes podem ser solitários ou cespitosos
e raramente escandentes, aéreos ou subterrâneos. Quando aéreo, o estipe pode apresentar-se
liso ou densamente coberto por espinhos (MIRANDA et al., 2001). As plântulas possuem
folhas inteiras, bífidas e pinadas (MIRANDA et al., 2001).
Dentre as espécies que constituem a família Arecaceae, destaca-se o gênero
Copernicia que compreende aproximadamente 13 espécies. No Brasil, este gênero é
representado por duas espécies nativas, Copernicia prunifera e Copernicia alba, que ocorrem
em regiões bem distintas (SOUZA et al., 2005). A palmeira Copernicia prunifera (Miller) H.
E. Moore, conhecida popularmente como carnaúba, é uma espécie nativa da Caatinga, com
ocorrência predominante nos estados do Piauí, Ceará e Rio Grande do Norte (LEITMAN et
al., 2015). Observa-se que a Copernicia prunifera ocorre predominantemente em áreas
alagáveis com solos halomórficos, incluindo-se áreas de vegetação ciliar (ARRUDA e
CALBO, 2003). Além disso, é uma espécie conhecida como “árvore da vida”, com várias
utilidades na indústria e na construção civil (PEREIRA et al., 2014), como também a extração
do pó cerífero e exploração das folhas para o artesanato (COSTA e GOMES, 2016).
O estudo fenológico é um importante instrumento na caracterização da dinâmica
florestal, facilitando o entendimento de processos como a polinização, reprodução,
regeneração e estabelecimento de espécies no seu ambiente natural (TANNUS et al., 2006).
Além disso, a caracterização fenológica é relevante, devido à obtenção de informações sobre a
biologia reprodutiva da espécie de interesse, de maneira a compreender e elaborar estratégias
sustentáveis de uso da mesma (CAMPOS et al., 2013; CESÁRIO e GAGLIANONE, 2008).
Diante disso, espera-se que as informações sobre os eventos reprodutivos da Copernicia
prunifera sejam úteis para o entendimento da dinâmica da população e melhoramento
genético da espécie.
Outra abordagem importante refere-se ao conhecimento sobre as estruturas
reprodutivas de uma espécie, sendo fundamental para descrição do seu sucesso reprodutivo
(LENZI e ORTH, 2004). O entendimento da biologia reprodutiva é relevante no sentido de
10
subsidiar novos trabalhos de manejo, melhoramento genético e domesticação de espécies
nativas, além de informações relevantes sobre os padrões de cruzamentos (OLIVEIRA et al.,
2003; OLIVEIRA et al., 2002; VIEIRA et al., 2010; ARRUDA et al., 2015). Adicionalmente,
o sistema de reprodução pode modificar a dinâmica genética das populações, interferindo na
composição genética das gerações subsequentes (OOSTERMEIJER et al., 2003).
As avaliações dos sistemas reprodutivos das espécies florestais podem ser realizadas
pelo método direto, que compreende a observação da dispersão de pólen e sementes, ou
através do método indireto, que consiste na análise dos genótipos dos indivíduos nas
populações, com o auxílio de marcadores moleculares (BROQUET e PETIT, 2009). Tais
métodos fornecem informações relevantes sobre o sistema reprodutivo de uma espécie.
O método indireto pode ser aplicado em estudos que visam detectar o sistema
reprodutivo das espécies vegetais, através do uso de marcadores dominantes (MULUVI et al.,
2004; MUCHUGI et al., 2008) e co-dominantes (RAMOS et., 2011; ABREU et al., 2012;
PICANÇO-RODRIGUES et al., 2015), sendo os marcadores dominantes relevantes para
estimar as taxas de cruzamento (GAIOTTO et al., 1997). Dentre os marcadores dominantes,
há os ISSR (Inter Simple Sequence Repeats), onde um único marcador na amplificação do
DNA, resultando em múltiplos fragmentos de diversos comprimentos (SLOTTA e PORTER,
2006), além de não ser necessário o conhecimento prévio do genoma da espécie de interesse
(OLIVEIRA et al., 2014).
Estruturalmente, a presente dissertação está dividida em dois capítulos, os quais
organizados em artigos gerados de estudos desenvolvidos com a palmeira Copernicia
prunifera.
O primeiro capítulo “Reproductive ecology of the Copernicia prunifera, a native palm
from brazilian semiarid” foi enviado para revista Floresta e Ambiente (Qualis CAPES B1), no
qual foram descritas as características reprodutivas da Copernicia prunifera.
O Segundo capítulo “Mating system of Copernicia prunifera (Arecaceae)” será
submetido à revista Biochemical Systematics and Ecology (Qualis CAPES A2) e teve como
objetivo investigar o sistema de reprodução e a diversidade genética da espécie, gerando
informações para o entendimento dos mecanismos de reprodução.
11
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I.; COELHO, R. M.; COLOMBO, C. A. The genetic structure and mating system of
Acrocomia aculeata (Arecaceae). Genetics and Molecular Biology, n. 35, v. 1, p. 119-121,
2012.
ARRUDA, C. C.; SILVA, M. B.; SEBBENN, A. M.; KANASHIRO, M.; LEMES, M. R.;
GRIBEL, R. Mating system and genetic diversity of progenies before and after logging: a
case study of Bagassa guianensis (Moraceae), a low-density dioecious tree of the Amazonian
forest. Tree Genetics & Genomes, v. 11, p. 3, 2015.
ARRUDA, G. M. T.; CALBO, M. E. R. Efeitos da inundação no crescimento, trocas gasosas
e porosidade radicular da carnaúba (Copernicia prunifera (Mill.) H.E. Moore). Acta Botânica
Brasileira, v. 18, p. 219-224, 2003.
BROQUET, T.; PETIT, E. J. Molecular estimation of dispersal for ecology and population
genetics. Annual Review of Ecology, Evolution, and Systematics, Palo Alto, v. 40, n. 1, p.
193-216, 2009.
CAMPOS, A. M.; FREITAS, J. L.; SANTOS, E. S.; SILVA, R. B. L. Fenologia reprodutiva
de Bertholletia excelsa Bonpl. em floresta de terra firme em Mazagão, Amapá. Biota
Amazônia, v. 3, n. 1, p. 1-8, 2013.
CESÁRIO, L. F.; GAGLIANONE, M. C. Biologia floral e fenologia reprodutiva de Schinus
terebinthifolius Raddi (Anacardiaceae) em Restinga do Norte Fluminense. Acta Botanica
Brasilica, v. 22, n. 3, p. 828-833, 2008.
COSTA, V. L. S.; GOMES, J. M. A. Crédito e conservação ambiental no extrativismo da
carnaúba (Copernicia prunifera (Mill.) H. E. Moore) no nordeste brasileiro no período de
2007 a 2012. Interações, v. 17, n. 1, p. 4-14, 2016.
GAIOTTO, F. A.; BRAMUCCI, M.; GRATTAPAGLIA, D. Estimation of outcrossing rate in
a breeding population of Eucalyptus urophylla with dominant RAPD and AFLP markers.
Theor. Appl. Genet, v. 95, p. 842-849, 1997.
HENDERSON, A.; FISCHER, B.; SCARIOT, A.; PACHECO, M. A. W. & PARDINI, R.
Flowering phenology of a palm community in a central Amazon forest. Brittonia, v. 52, p.
149-159, 2000.
LEITMAN, P.; SOARES, K.; HENDERSON, A.; NOBLICK, L.; MARTINS, R. C.
Arecaceae in Lista de Espécies da Flora do Brasil. Jardim Botânico do Rio de Janeiro,
2015. Disponível em: <http://floradobrasil.jbrj.gov.br/jabot/floradobrasil/FB15706>.
LEITMAN, P.; SOARES, K.; HENDERSON, A.; NOBLICK, L.; MARTINS, R. C.
Arecaceae in Lista de Espécies da Flora do Brasil. Jardim Botânico do Rio de Janeiro,
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LENZI, M.; ORTH, A. I. Fenologia reprodutiva, morfologia e biologia floral de Schinus
terebinthifolius Raddi (Anacardiaceae), em restinga da Ilha de Santa Catarina, Brasil.
Biotemas, v. 17, n. 2, p. 67-89, 2004.
LORENZI, H.; SOUZA, H. M.; MEDEIROS-COSTA, J. T.; CERQUEIRA, L. S. C.; BEHR,
N. Palmeiras do Brasil: exóticas e nativas. Nova Odessa: Editora Plantarum, p. 1-20, 1996.
MIRANDA, I. P. A.; RABELO, A.; BUENO, C. R.; BARBOSA, E. M.; RIBEIRO, M. N. S.
Frutos de Palmeiras da Amazônia. Manaus: MCT INPA, p. 7-10, 2001.
MUCHUGI, A.; MULUVI, G. M.; SIMONS, A. J.; WACHIRA, F. N.; JAMNADASS, R. H.
Estimation of out-crossing rate in a natural breeding population of Warburgia ugandensis
using AFLP marker. African Journal of Biotechnology, v.7, p. 139-146, 2008.
MULUVI, G. M.; SPRENT, J. I.; POWELL, W. Estimates of outcrossing rates in Moringa
oleifera using Amplified fragment length polymorphism (AFLP). African Journal of
Biotechnology, v. 3, n. 2, p. 146-151, 2004.
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OLIVEIRA, A. F.; CARVALHO, D.; ROSADO, S. C. S. Taxa de cruzamento e sistema
reprodutivo de uma população natural de Copaifera langsdorffii Desf. na região de Lavras
(MG) por meio de isoenzimas. Revista Brasileira de Botânica, v. 25, n. 3, p. 331-338, 2002.
OLIVEIRA, M. S. P., COUTURIER, G., BESERRA, P. Biologia da polinização da palmeira
tucumã (Astrocaryum vulgare Mart.) em Belém, Pará, Brasil. Acta Botanica Brasilica. 17,
343-353, 2003.
OLIVEIRA, N. N. S.; VIANA, A. P.; QUINTAL, S. S. R.; PAIVA, C. L.; MARINHO, C. S.
Análise de distância genética entre acessos do gênero Psidium via marcadores ISSR. Revista
Brasileira de Fruticultura, v. 36, n. 4, p. 917-923, 2014.
OOSTERMEIJER, J. G. B.; LUIJTEN, S. H.; DEN NIJS, J. C. M. Integrating demographic
and genetic approaches in plant conservation. Biology Conservation, v. 113, p. 389-398,
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PEREIRA, D. S.; SOUSA, J. E. S.; PEREIRA, M. S.; GONÇALVES, N. R.; BEZERRA, A.
M. E. Emergence and initial growth of Copernicia prunifera (Arecaceae) as a function of fruit
maturation. Journal of Seed Science, v. 36, n. 1, p. 009-014, 2014.
PICANÇO-RODRIGUES, D.; ASTOLFI-FILHO, S.; LEMES, M. R.; GRIBEL, R.;
SEBBENN, A. M.; CLEMENT, C. R. Conservation implications of the mating system of the
Pampa Hermosa landrace of peach palm analyzed with microsatellite markers. Genetics and
Molecular Biology, v. 38, n. 1, p. 59-66, 2015.
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CONTIM, L. A. S.; CLEMENT, C. R.; RODRIGUES, D. P.; BERNARDES, L. G.
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Determination of the mating system of Tucumã palm using microsatellite markers. Crop
Breeding and Applied Biotechnology, v. 11, n. 2, p. 181-185, 2011.
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I. remota (Malvaceae): implications for species delimitation. Botanical Journal of the
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famílias de fanerógamas nativas e exóticas no Brasil, baseado no APG II. Nova Odessa:
Instituto Plantarun, 2. ed., 2008.
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famílias de Angiospermas da flora brasileira, baseado em APG II. Nova Odessa, SP:
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Capítulo 1: REPRODUCTIVE ECOLOGY OF THE Copernicia prunifera, A NATIVE
PALM FROM BRAZILIAN SEMIARID
Artigo submetido à Revista Floresta e Ambiente (Qualis CAPES B1)
ABSTRACT The objective of this study was to describe the reproductive characteristics of
the palm Copernicia prunifera in different locations, in the state of Rio Grande do Norte,
Brazil. We evaluated the reproductive events (flower buds, anthesis, immature and ripe fruit).
The structure of the inflorescence was also described, and we estimated the percentage of
viable pollen. Differences were observed in the reproductive patterns among the populations
evaluated, throughout continuous observation of production of flowers and ripe fruit in the
Parnamirim population, and discontinuous observation in the Macaíba population.
Hermaphroditic flowers have multiple inflorescences with light coloration. In addition, the
flowers are composed of 3 sepals, 3 petals, 6 stamens and 3 carpels. The average percentage
of viable pollen was 62%. The characterization of the reproductive ecology of Copernicia
prunifera rendered important information for future studies of germplasm conservation of the
species.
Keywords: Caatinga, Arecaceae, Carnaúba, Reproductive events, Reproductive structures.
Introduction
The Caatinga biome is found predominantly in the Northeastern region of Brazil,
occupying an area of approximately 844,453 km2
or 54.53% of the area of the region (IBGE,
2005). Due to a dearth of studies about the biome, the devastation of the Caatinga continues,
due to both extensive livestock and agricultural production systems, and by the indiscriminate
15
extraction of wood and plywood (Santana and Souto, 2006). This process of environmental
degradation has caused a great loss of the forest resources of the biome (Santos et al., 2011;
Silva et al., 2009).
Among the hundreds of species that can be found in the Caatinga, the Arecaceae
family stands out, of which globally there are, approximately, 200 genera and 2,000 species
(Souza and Lorenzi, 2008). Due to their botanical characteristics, consist in very interesting
plant group, in addition to having great ornamental, nutritional, and economic value
(Bauermann et al., 2010).
The palm Copernicia prunifera (Miller) H. E. Moore stands out, a species native to the
semiarid region of northeastern Brazil. Its distribution occurs in a geographic area of the
states of Tocantins, Maranhão, Piauí, Ceará, Rio Grande do Norte, Paraíba, Pernambuco,
Alagoas, Sergipe, Bahia, and Mato Grosso (LEITMAN et al., 2015). Copernicia prunifera
individuals primarily can be found in the northeastern river valleys (D'alva, 2007). According
to Carvalho (2008), the economics of Copernicia prunifera consist of the set of activities that
make use of the leaves, stem, fiber, fruit and roots of this palm tree for the manufacture of
numerous industrial and handicraft products. Beyond this, there are no studies related to the
reproductive ecology of the species.
Phenological study is an important tool in the characterization of forest dynamics,
facilitating the understanding of processes such as pollination, reproduction, regeneration and
establishment of species in their natural environment (Tannus et al., 2006). The time, duration
and degree of synchrony of phenological stages have important implications on the quantity
and quality of resources available to the consumer (pollinators, seed dispersers and predators)
(Williams et al., 1999).
Moreover, knowledge of the reproductive structures of a species is essential for
description of its reproductive success (Lenzi and Orth, 2004; Vieira et al., 2010). Thus, the
16
understanding of the reproductive biology is important in the sense of financing new works by
management, breeding and domestication of native species (Oliveira et al., 2003; Vieira et al.,
2012).
The knowledge of the floral biology, reproductive organs and pollinators of a species
is essential to support taxonomy of work, management, breeding and domestication of native
species while providing the interpretation of mechanisms for the pollination and elucidation
of the relationship between plants and the environment (Vieira et al., 2010). Silberbauer-
Gottsberger (1990) and Henderson (1986) argue that it is unlikely the hypothesis of
anemophily be the only type of pollination occurring in the family Arecaceae, given the
importance of insects pollination reproduction of family representatives. In fact, the most
common pollen dispersal agents in palm trees are beetles, followed by bees and flies (Barfod
et al., 2003). The protandry where the anthers mature before the stigma being receptive, it is
quite common in Arecaceae, which favors the cross-fertilization (Mantovani and Morellato,
2000). According Silberbauer-Gottsberger (1990) to protandry it should be related to
anemophily and entomophily.
Studies of reproductive biology with species of the genus Copernicia are nonexistent,
and are of great importance for the ecological characterization of a species native to the
semiarid Northeast. Overall, the objective of this study was to describe the reproductive
characteristics of the palm Copernicia prunifera.
Material and methods
Areas of study
Copernicia prunifera populations were sampled in three locations. The first was a
natural population in the municipality of Lagoa de Pedras, RN. The second was another
natural population in the municipality of Macaíba, RN, and the third was an urban plantation
17
on the edge of the Cotovelo Beach, in the municipality of Parnamirim, RN. According to the
Köppen climate classification (Peel et al., 2007), the locations studied feature tropical climate
with a rainy season (As).
The population in Lagoa de Pedras is situated in a rural area of the municipality, in the
state of Rio Grande do Norte. The site is located in Northeast Brazil, with coordinates 6° 12'
33.51"S, 35° 27' 38.24"W and an altitude of 105 meters. The second population is located in
the area belonging to the Unidade Acadêmica Especializada em Ciências Agrárias, UAECIA -
UFRN, in the municipality of Macaíba, RN. The area is located in the Northeast region of
Brazil, with coordinates 5° 53' 57"S, 35° 59' 22"W, altitude of 26 meters. Individuals of
Cotovelo Beach are situated in the municipality of Parnamirim, RN, on the coastal region of
the state, with coordinates 5° 57' 59.14"S and 35° 08' 34"W and altitude of 12 meters.
For characterization of reproductive events of Copernicia prunifera, individuals were
sampled populations of Macaíba and Cotovelo Beach, RN. The study of reproductive biology
was developed in the population in the municipality of Lagoa de Pedras.
Reproductive events
We evaluated 20 and 29 individuals in each population, respectively. As a rule of
inclusion, we evaluated only reproductive individuals, systematically and on trails throughout
the area population using the methodology of D'eça-Neves and Morellato (2004).
The evaluations were carried out in the period between the months of October 2011
and december 2012, at intervals of 15 days in the populations of Macaíba and Cotovelo
Beach. The ranges of assessments were defined as the dynamics of changes of the
reproductive events, where sites with larger variations in phenophases were evaluated more
frequently. The following reproductive events were observed: floral buds, flower, immature
fruit and ripe.
18
The phenophases were quantified through the activity index, by evaluating the
presence or absence of reproductive event; and intensity of Fournier (1974), by means of a
scale of five semiquantitative categories (0 to 4), separated at intervals of 25%. Reproductive
events were reported for each population. For data analysis, Excel spreadsheets were used.
Reproductive biology
Three individuals were selected to provide the data on the structure of the
inflorescence and two for pollen viability. Then collected the reproductive parts (flower buds
and flower), were subsequently packaged in Falcon tubes containing the solution of the FAA
50 (10% formaldehyde, 85% ethyl alcohol and 5% acetic acid). The reproductive structures
were collected in february of 2014.
The structure of the inflorescence was characterized by the length of the rachis (cm);
the number of rachillae up in inflorescence; the position of the rachilla, which is subdivided
into three distinct areas, basal, intermediate and apical region; the number of multiple
inflorescences and blooms at rachilla (Figure 1). The sexual type and flower morphological
characterization was determined with the aid of a stereoscopic microscope Medilux®.
For pollen viability analysis, there were eight repetitions in blades. The pollen grains
were stained with acetic orcein solution 1% (Dafni, 1992; Kearns and Inouye, 1993). Then the
pollen grains once stained were covered with coverslipping mountant for observation in an
optical microscope, using the magnifying lens of 40X. In order to obtain a random sampling
of stained pollen grains, we counted 100 pollen grains per blade.
The pollen grains were analyzed and classified normal/viable, with cytoplasm stained
recorded as normal and abnormal/inviable recorded for those with little or no cytoplasm
evidenced. The percentage of viable pollen was calculated by the equation: pollen Viability
(%) = number of colored grains/grain number counted * 100.
19
Results
It was found that the species presents multiple inflorescences, being made up of
hermaphroditic flowers, with a yellowish coloration (Figure 1B). In addition, the flowers are
composed of 3 sepals, 3 petals, 3 stamens and 6 carpels. The average percentage of viable
pollen was 62% (Figure 1C).
Fig. 1 Reproductive structures of Copernicia prunifera. A: Rachis (a), rachilla (b) and portion of rachilla (c). B:
multiple inflorescences of C. prunifera. C: Pollen grains of palm C. prunifera, viewed in the objective lens 40X.
The reproductive phase of Copernicia prunifera proved to be subannual, with more
than one episode of flowering per year. The occurrence of buds and flowers in individuals of
Parnamirim occurred throughout the year, with higher intensities in the months of october and
november (34.48% and 48.28%, respectively) (Figure 2B and D). The population of Macaíba,
20
in december, with buds and flower emission rates of 26.25% and 42.50%, respectively
(Figure 2A and C).
Fig. 2 Indexes of activity and intensity of reproductive phenology of Copernicia prunifera.
Floral budding in populations Macaíba (A) and Parnamirim (B) and flower in populations of
Macaíba (C) and Parnamirim (D).
Immature fruit production occurred throughout the period evaluated in the population
of Parnamirim, featuring greater intensity from december to january, with average of 60.77%
(Figure 3B). However, the same did not occur in the population of Macaíba, where the same
immature fruit production presented higher than in the months of January and February, with
average of 43.75% (Figure 3A). However, the population of Cotovelo beach had a production
of mature fruits continuously during the evaluated period, with high intensity in the months of
February to May 2012, with rates of 14.65% and 16.38%, respectively (Figure 3D).
21
Fig. 3 Indices of activity and intensity of reproductive phenology of Copernicia prunifera.
Immature fruit in populations of Macaíba (A) and Parnamirim (B) and ripe fruit in
populations of Macaíba (C) and Parnamirim (D).
Despite the occurrence of immature fruit production in the population of Macaíba,
most of these fruits had not reached the final stage of maturation, causing low rate of ripe
fruits, observed during the months of november through april, though with greater maximum
intensity in the months of February/2011 (13.75%) and March 2012 (30.00%) (Figure 3C).
The length of the rachis averaged 1.29 m; the ratio of the number of rachilla by
inflorescence ranged from 4.00 to 15.00. The rachilla exhibited greater length in the basal
portion, averaging 62.50 cm. The number of subrachilla and flowers by rachilla were
concentrated with higher proportions in the basal portion, with average values of 6.17 and
1,735.50, respectively (Table 1).
22
Table 1 Rachis length, quantitative rachilla per inflorescence, length of rachilla, number of
subrachilla, number of flowers per rachille in basal, intermediate and apical Copernicia prunifera. (n),
sample size; mean; maximum and minimum values.
Characters Portion n Minimum Mean Maximum
Rachis length (m) Total
3
1.05
1.29
1.60
Quantitative
rachilla per
inflorescence
Total
3
4.00 10.67
15.00
Length of rachilla
(cm)
Basal 3 33.00 62.50 80.00
Intermediate 3 18.00 38.17 65.00
Apical 3 6.00 19.17 54.00
Number of
subrachilla
Basal 3 4.00 6.17 9.00
Intermediate 3 3.00 5.50 8.00
Apical 3 1.00 2.00 5.00
Number of flowers
per rachille
Basal 2 803.00 1,735.50 2,668.00
Intermediate 2 871.00 1,649.00 2,427.00
Apical 2 225.00 1,014.00 1,803.00
In the studied population showed the sanhaçú do coqueiro (Tangara palmarum)
visiting the tops of some individuals (Figure 4A). Floral visits were also recorded from the
maribondo-caboclo (Polistes canadensis Linnaeus) and of irapuá (Trigona spinipes Fabricius)
(Figures 4B and 4C).
23
Fig. 4 Tangara palmarum (A), Polistes canadensis (B) and Trigona spinipes (C) in individual
Copernicia prunifera.
Discussion
Studies that compare the reproductive events of Copernicia prunifera palm in distinct
populations are nonexistent, and the results of this work are relevant, especially in regards to
defining the ideal period for gathering fruits and seeds, in addition to the dispersal and
pollination mechanisms of the species. The frequency of reproductive events observed in a
population of Copernicia alba were divergent to those of Copernicia prunifera, with higher
peak flowering between july and december and fruiting from december to may (Salis and
Mattos, 1994).
Rocha et al. (2015), after correlating the phenological data of Copernicia prunifera
with climatic variables, in the same population, found that the mature fruits showed a
significant negative correlation with the relative humidity in the studied population,
demonstrating greater number of trees with ripe fruits in periods with low humidity. In
addition, there was no significant correlation with precipitation in the evaluated period.
24
The fact that the fruits do not reach the final stage of maturation in the population of
Macaíba can be linked to environmental factors, mainly to low rainfall, high
evapotranspiration and pollinators (Vilela et al., 2008; Nazareno and Reis, 2012).
Additionally, the occurrence and intensity of some reproductive events usually are associated
with factors abiotic factors, such as, temperature, precipitation, humidity, soil; and/or biotic
factors, such as pollinators (Spina et al., 2001). Thus, the availability of water becomes an
essential factor to produce fleshy fruits (Tabarelli et al., 2003).
Souza et al. (2002) reported that pollen viability in forest species is only considered
high for values above 70%. In relation to the rate of floral visitors, it is estimated that
probably, the low frequency of visits in the population adversely interferes with pollination
rate. Information on pollination in palm trees are incipient and under the existing entomophily
(pollination by insects) and wind (pollination by wind action), have been reported as the main
systems of pollination, with highlight to entomophily (Oliveira et al., 2003).
Regarding the observation of insects in individuals of Copernicia prunifera, there have
been more frequent in the population of Parnamirim. Nevertheless, Silveira et al. (2010), in a
study conducted with individuals from Vaccinium myrtillus, family Ericaceae, identified that
species Trigona spinipes is harmful to the species, especially at the time of flowering, fruiting
and fruit with reduced size. The presence of insects observed in the population is an indication
that they are the likely pollinators of the species (Pina-Rodrigues and Piratelli, 1993).
In tropical vegetation, the zoochory is the dominant dispersal syndrome (Bollen et al.,
2004). Purificação et al. (2015) verified that in individuals from Schefflera morototoni
(Araliaceae), the Tangara palmarum presents itself as one of the main dispersers of the fruits
of this species. Additionally, in remarks carried in individuals of Cecropia pachystachya,
noted that Tangara palmarum is omnivorous, with a habit of visiting and reap the rewards in
plants (Gonçalves and Vitorino, 2014).
25
Conclusion
There are differences in reproductive patterns among populations evaluated, observed
continuously in the production of flowers and ripe fruit in the population of Parnamirim, and
discontinuously in the population of Macaíba. The Copernicia prunifera flowers are
hermaphroditic. Relatively low pollen viability was observed, and may lead to low fruit
production. Suggested the pollen viability studies in other natural populations, as also the
record of pollinators and seed dispersers.
Acknowledgments
The Fundação de Apoio à Pesquisa do Rio Grande do Norte (FAPERN), for providing
the fellowship, and Conselho Nacional de Desenvolvimento Científico e Tecnológico
(CNPq). We thank Carley Nichole Fuller for English editing of the manuscript.
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29
Capítulo 2: MATING SYSTEM OF Copernicia prunifera (ARECACEAE)
ABSTRACT
Understanding the genetic diversity and reproductive mating system of forest species is
important in assessing the genetic factors associated with, and effects of, forest fragmentation.
The objective of this study is to investigate the genetic diversity and mating system of a
population of Copernicia prunifera using ISSR (Inter-Simple Sequence Repeat) markers. We
found that the markers used presented high polymorphism and were considered informative.
The values of the diversity indices among adults and progenies did not differ statistically (He
= 0.319 and I = 0.470; He = 0.337 and I = 0.505, respectively). In testing for the presence of
genetic bottlenecks using the infinite allele model (IAM) and stepwise mutation model
(SMM), we observed a reduction in the effective population, as well as a deficit in
heterozygosity (P < 0.0001). Outcrossing rates at the population level (n = 247) produced a
multilocus outcrossing rate (tm) of 0.878 and single locus outcrossing rate (ts) of 0.738,
indicating that Copernicia prunifera has a mixed mating system that is preferentially
allogamous. The rate of mating among relatives (tm - ts) was low, indicating limited
outcrossing between closely related individuals. The fixation index between seed trees (F)
was negative (- 0.200), suggesting an absence of inbreeding, while the correlation of selfing
(rs) was high (0.914). The results of this study inform management strategies for the
conservation and genetic improvement of the Copernicia prunifera palm.
Key words: Caatinga; Genetic diversity; ISSR; Carnauba palm; Outcrossing rate
INTRODUCTION
The palm Copernicia prunifera (Miller) H. E. Moore, commonly known as carnaúba,
belongs to the Arecaceae family and the species is native to the Caatinga biome that occurs
across the states of Tocantins, Maranhão, Piauí, Ceará, Rio Grande do Norte, Paraíba,
Pernambuco, Alagoas, Sergipe, Bahia, and Mato Grosso (LEITMAN et al., 2015). The
species can be used for a variety of purposes, from urban forestry (MACHADO et al., 2006)
to wax extraction from its leaves, the main product of the species, which is used in cosmetics,
varnishes, and even for polishing fruit (JACOMINO et al., 2003; MOTA et al., 2006; SILVA
et al., 2009).
Due to the economic and social importance of Copernicia prunifera, determining its
mating system is vital, because it is an aspect that must be considered in the management,
30
conservation, and genetic improvement of the species (ARRUDA et al., 2015; VIEIRA et al.,
2010). Mating systems can alter the genetic dynamics of populations by influencing the
genetic composition of subsequent generations (OOSTERMEIJER et al., 2003). In addition,
the mating system determines the magnitude of inbreeding in the descendent population
(MORI et al., 2013). It is important to consider how the recombination of genes in each
reproductive event is expressed in descendant populations (MORI et al., 2013). Thus,
knowledge of the mating system is necessary to determine the genetic composition of
populations as it is the source of the distribution of genetic diversity and subdivisions within
and across populations (HAMRICK, 1982).
In this context, approaches to assessing the mating systems of forest species have
important implications for understanding the genetic factors (SAMANT et al., 2013) and
effects of forest fragmentation (SEOANE et al., 2005). Additionally, population genetics
studies indicate that progenies from fragmented populations are more likely to be generated
by selfing or from mating between few individuals (SEOANE et al., 2000; CASCANTE et al.,
2002; FUCHS et al., 2003).
The mating system of hermaphroditic species can combine selfing with outcrossing,
through which both random or correlated mating occurs (MORI et al., 2013). In addition,
most palm species present mixed mating systems, being preferentially allogamous (CONTE et
al., 2008; RAMOS et al., 2011; ABREU et al., 2012; NAZARENE and REIS., 2012;
OTTEWELL et al., 2012; PICANÇO-RODRIGUES et al., 2015). Due to the effects of
anthropization in the region, and because the species occurs in monodominant groups at high
densities in the studied area, it is expected that Copernicia prunifera presents a mixed mating
system, with a high rate of outcrossing between related individuals.
Analyses of the mating system of forest species can be performed using dominant
(GAIOTTO et al., 1997; SANTOS and NETO, 2011; FERREIRA et al., 2010) or co-dominant
markers (GAIOTTO et al., 2003; ALVES et al., 2015; WADT et al., 2015). To overcome
limitations in the analysis of individual genotypes, Ritland (2002) developed the multilocus
model, which includes dominant markers in the evaluation of the mating system of plant
species. Among the dominant markers, inter-simple sequence repeat (ISSR) markers are
usefull (HAN et al., 2009; SOUZA et al., 2012): they are effective in detecting
polymorphism, easily reproduced, and have lower costs than SSR markers (SANTANA et al.,
2011). In addition, along with microsatellites, ISSR markers amplify genomic fragments that
are abundant and widely distributed throughout the genome of eukaryote individuals, and do
not require sequencing (GE, 2005).
31
The aim of this study is to investigate the genetic diversity and mating system of a
natural population of Copernicia prunifera using ISSR markers, generating information that
can help us understand the reproduction mechanism of the species.
MATERIAL AND METHODS
Plant Sampling
The sampled population of Copernicia prunifera is located in the municipality of São
Miguel do Gostoso, Rio Grande do Norte, Brazil (5° 07' 18'' S and 35° 41' 02" W) (Figure 1).
The municipality is located in a microregion of the northeastern coast, with a tropical climate
with dry season (As), according to the Köppen climate classification (ALVARES et al.,
2013). The vegetation of the study area is hipoxerophytic caatinga, made up of shrubs and
thorny trees. In addition, the site presents high levels of anthropization, mainly due to the
expansion of wind power plants. The linear distance from the population to the coast is
approximately 1.5 km.
To study the mating system, leaf samples and fruits were collected from 16 adult
reproductive individuals, in a 0.55 ha area. Due to the limited number of fruits available for
some individuals, the number of progenies ranged from 4 to 20 (Figure 1). Progenies of
Copernicia prunifera were obtained based on the seed germination methodology described by
Araújo et al. (2013). The population was georeferenced using GPS and individuals were
mapped with a tape measure for greater accuracy.
32
Figure 1: Geographical location of the Copernicia prunifera individuals, in the municipality
of São Miguel do Gostoso, Rio Grande do Norte, Brazil. n = number of progenies.
Leaf tissue samples of adult individuals were placed in 2 mL plastic tubes containing
CTAB 2X (cationic hexadecyl trimethylammonium bromide), labelled and transported to the
lab. For progenies, we collected the first leaves to develop which were then stored in a freezer
at - 20°C until DNA extraction.
The PCR reactions were carried out in a Veriti thermocycler, in a volume of 12 μL,
containing diluted genomic DNA, 10X PCR buffer, 1.0 mg.ml-1
BSA, 2.5 mM dntp, 50 mM
MgCl2, 5 U.µL-1
Taq DNA polymerase, 2 µM primer, and ultra pure water. The PCR protocol
consisted of an initial denaturation for 2 min at 94 °C, followed by 37 amplification cycles of
15 seconds at 94 °C, 30 seconds at 47 °C, 1 min at 72 °C, a final extension for 7 min at 72 °C,
and cooling to 4 °C.
The PCR products were stained with GelRedTM
and analyzed using horizontal
electrophoresis, separated on 1.5% agarose gel, in a solution of 1X TAE (Tris-Acetate-
EDTA), at 100 V for 2.5 hours. We used a molecular weight marker (Ladder) of 10,000 bp
and the gels were photographed in ultraviolet light in an E-Box VX2.
m
m
33
Statistical analysis
Diversity, identity, and genetic distance
To determine the genetic diversity parameters, we built a binary array based on the
presence (1) and absence (0) of loci in gels. The data were used to calculate the percentage of
polymorphic loci, number of effective alleles, number of observed alleles, Nei's genetic
diversity (He), and Shannon diversity index (I). The adult individuals and progenies were
evaluated and analyses were carried out using the program POPGENE v. 1.32 (YEH et al.,
1997). The results from the diversity indices were submitted to analysis of variance
(ANOVA) using the program ASSISTAT 7.7 (SILVA, 2014).
Genetic identity was obtained using the program NTSYS (ROHLF, 1993), with the
goal of constructing a dendrogram of Unweighted Pair Group Method with Arithmetic mean
(UPGMA) for the 16 seed trees, based on Nei's genetic identity (1978). Analysis of Nei's
genetic distance was conducted with the POPGENE program v. 1.32 (YEH et al., 1997).
Value of PIC
The polymorphic information content (PIC) was used to test the efficiency of the ISSR
markers to detect polymorphism between two individuals, through the presence or absence of
loci. According to Botstein et al. (1980), molecular markers are classified as satisfactory in
informational content when the PIC value is greater than 0.5. Values from 0.25 to 0.5 are
moderately informative, and values below 0.25 have little information value. For this, we
used the formula proposed by Anderson et al. (1993): ∑
, where Pij is the
frequency of allele "j" at marker "i".
Genetic bottleneck detection
To verify the presence of a genetic bottleneck that resulted in a reduction in the
effective size of the population over generations, we used the infinite allele model (IAM),
according to Kimura and Crow (1964), and the stepwise mutation model (SMM), according to
Kimura and Otha (1978). The analyses were performed using the program Bottleneck 1.2.02
(CORNUET and LUIKART, 1996).
Analysis of the mating system
The mating system was assessed using the mixed mating model (RITLAND and JAIN,
1981) and correlated mating model (RITLAND, 1989). The standard deviations of the
estimates were obtained by 1,000 bootstraps. The estimated parameters were: a) multilocus
34
outcrossing rate (tm); b) single locus outcrossing rate (ts); c) rate of mating among relatives (tm
- ts); d) selfing rate (s = 1 - tm); e) fixation index between seed trees (F); f) expected fixation
index F = [(1 - tm) / (1 + tm)]; g) correlation of selfing (rs); h) multilocus paternity correlation
(rp(m)); i) single locus paternity correlation (rp(s)); j) correlation of the estimate of tm (rt); and k)
the relatedness between pollen donor trees (rp(s) - rp(m)). The parameters were obtained using
the program MLTR (RITLAND, 2004). The standard deviations were obtained by 1,000
bootstraps.
RESULTS
Polymorphism and PIC
The markers show a large numbers of loci, as well as good resolution for the analyzed
fragments (Figure 2). Eight ISSR markers were used, producing 104 loci with 100%
polymorphism (Table 1). The number of loci ranged between 10 and 17, with an average of
13 per marker. The PIC of markers ranged from 0.416 to 0.500, with an average of 0.477.
Figure 2: Pattern of ISSR amplification fragments resulting from UBC 825 primer for 19
progenies of Copernicia prunifera. L = Ladder 1 kb.
35
Table 1: Nucleotide sequence of the ISSR markers, number of loci, and the PIC value for each
primer.
ISSR Primer Sequence (5’ - 3’) Loci PIC
UBC 825 (AC)8-T ACA CAC ACA CAC ACA CT 17 0.498
UBC 827 (AC)8-G ACA CAC ACA CAC ACA CG 11 0.484
UBC 840 (GA)8-YT GAC AGA GAG AGA GAG AYT 14 0.500
UBC 851 (GT)8-YG GTG TGT GTG TGT GTG TYG 12 0.416
UBC 857 (AC)8-YG ACA CAC ACA CAC ACA CYG 13 0.492
UBC 859 (TG)8-RC TGT GTG TGT GTG TGT GRC 13 0.439
UBC 860 (TG)8-RA TGT GTG TGT GTG TGT GRA 10 0.494
UBC 873 (GACA)4 GAC AGA CAG ACA GAC A 14 0.495
Average 13 0.477
R = purine (A or G) and Y = pyrimidine (C or T).
Diversity and genetic identity
For the parameters of genetic diversity (Table 2), the adults showed 84.62%
polymorphic loci, while the progenies presented 100% polymorphism. The number of alleles
observed (Na) and the number of effective alleles (Ne) were higher among progenies than
adults, 2.000 (± 0.000) and 1.575 (± 0.020), respectively. We found no statistical difference in
the results for Nei's genetic diversity (He), assuming Hardy-Weinberg equilibrium, and the
Shannon index (I) between adults and progenies.
Table 2: Genetic diversity parameters for the population of Copernicia prunifera.
Population n Lp (%) Na Ne He I
Adults 16 84.62 1.846±0.090 1.558±0.087 0.319±0.044 0.470±0.061
Progenies 251 100 2.000±0.000 1.575±0.020 0.337±0.009 0.505±0.011
Total 267 100 2.000±0.000 1.607±0.018 0.353±0.008 0.526±0.010
Sample size (n), percentage of polymorphic loci (Lp%), number of alleles observed (Na),
number of effective alleles (Ne), Nei's genetic diversity index (He), Shannon index (I). Values
represent the average ± standard error.
Based on Nei's genetic identity (1978), the UPGMA dendrogram grouped the
population into two groups: one formed by individuals 1, 3, 5, and 9; and the other made up
of the remaining individuals (Figure 3). Individuals 9, 10, and 12 showed less genetic
similarity in relation to the others.
36
Figure 3: Dendrogram of Nei's genetic identity between Copernicia prunifera individuals.
Estimates of Nei's genetic distance between individuals are shown in Table 3. We
observed that individuals who have less genetic similarity based on the identity analysis
showed greater genetic distance: primarily between individuals 9 and 2 (0.838), 10 and 3
(0.693), 10 and 5 (0.693), 10 and 11 (0.501), 12 and 9 (0.732), 12 and 15 (0.637).
37
Table 3: Estimates of Nei's genetic distance (1978) between Copernicia prunifera individuals. Values in bold represent greater divergence
between individuals that are less genetically similar.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
1 0
2 0.425 0
3 0.288 0.288 0
4 0.753 0.354 0.601 0
5 0.288 0.486 0.214 0.713 0
6 0.486 0.288 0.486 0.202 0.550 0
7 0.517 0.214 0.486 0.226 0.655 0.167 0
8 0.455 0.262 0.340 0.275 0.486 0.190 0.190 0
9 0.382 0.838 0.637 0.732 0.410 0.753 1.034 0.794 0
10 0.517 0.550 0.693 0.250 0.693 0.314 0.396 0.368 0.501 0
11 0.470 0.301 0.327 0.425 0.440 0.226 0.354 0.354 0.584 0.501 0
12 0.637 0.470 0.713 0.340 0.713 0.567 0.410 0.470 0.732 0.440 0.693 0
13 0.486 0.368 0.486 0.470 0.486 0.262 0.396 0.425 0.567 0.486 0.134 0.567 0
14 0.470 0.382 0.410 0.455 0.410 0.327 0.470 0.410 0.455 0.470 0.190 0.486 0.070 0
15 0.517 0.396 0.396 0.410 0.455 0.288 0.455 0.396 0.501 0.396 0.156 0.637 0.167 0.134 0
16 0.455 0.425 0.396 0.470 0.425 0.340 0.517 0.425 0.410 0.455 0.112 0.567 0.123 0.091 0.101 0
In the analysis to determine a reduction in the effective population, both the IAM and the
SMM detected genetic bottleneck in the population (Table 4). Additionally, the signal test
showed a significant deficit of heterozygosity based on the evaluated models (P < 0.0001).
Table 4: Tests to verify the reduction of effective population size of Copernicia prunifera using
the models IAM and SMM.
Population IAM SMM
N Hd/He P n Hd/He P
Adults 50.59 31/73 0.00001⃰ 49.35 34/70 0.00003⃰
Progenies 38.59 15/89 0.00000* 44.86 23/81 0.00000⃰
Total 38.56 11/93 0.00000⃰ 44.65 16/88 0.00000⃰
n = expected number of loci with excess heterozygosity under the respective model; Hd/He =
number of loci with deficit of heterozygosity / number of loci with excess heterozygosity; P =
probability; * = significant at 1% probability.
Mating system
Estimates of population-level outcrossing (Table 5) showed rates of tm = 0.878, ts = 0.738,
and s = 0.122. Mating among relatives (tm – ts) was positive (0.140). The main coefficient of
selfing between seed trees was negative (-0.200), and lower than expected (0.065).
For selfing and multilocus and single-locus paternity correlation, we found high rates of
direct selfing correlation (0.914) and low rates of single-locus paternity correlation (0.017). The
level of relatedness between pollen donors in the population was -0.296.
Table 5: Estimates of mating system parameters for the Copernicia prunifera population.
Parameters Average
Multilocus outcrossing rate: tm 0.878 (0.037)
Single locus outcrossing rate: ts 0.738 (0.037)
Mating among relatives rate: tm - ts 0.140 (0.037)
Selfing rate: s = 1 - tm 0.122
Fixation index between seed tree: F - 0.200 (0.023)
Fixation index expected: F = (1 - tm) / (1 + tm) 0.065
Correlation of selfing: rs 0.914 (0.110)
Multilocus paternity correlation: rp(m) 0.313 (0.043)
Single locus paternity correlation: rp(s) 0.017 (0.030)
Correlation of the estimate of tm: rt 0.597 (0.095)
Relatedness between pollen donors: rp(s) - rp(m) - 0.296 (0.041)
( ) Standard deviation
39
DISCUSSION
The number of loci evaluated in this study was high (n = 104) in comparison with other
studies on the genetic diversity of palms using dominant markers, with results ranging between
47 and 93 (OLIVEIRA et al., 2012; VIEIRA et al., 2015; CHAGAS et al., 2015). Using ISSR
markers to study the palm species Phoenix dactylifera and Mauritia flexuosa, the percentage of
polymorphic found by Mirbahar et al. (2014) and Rossi et al. (2014) was similar to that found in
the present study. However, Chagas et al. (2015) found low levels of genetic polymorphism in a
population of Elaeis guineenses. Thus, estimates of the level of genetic variability in a population
can be influenced by the percentage of polymorphic loci.
The PIC value determines the effectiveness of molecular markers in identifying molecular
polymorphism between individuals (RESENDE et al., 2009). Thus, the markers used in this study
were moderate, according to the classification by Botstein et al. (1980). Vieira et al. (2015),
testing seven ISSR markers for Copernicia prunifera, found PIC values ranging from 0.079 and
0.444, with an average of 0.277. The PIC may vary depending on the type of molecular marker
used. According to Goudet et al. (1996) and Buonaccorsi et al. (1999), among all genetic
markers, microsatellite markers offer greater information content.
In relation to the Shannon index (I) and Nei's genetic diversity index (He), the results can
range from 0 to 1, with 0 showing an absence of diversity and 1 suggesting maximum genetic
diversity (GIUSTINA et al., 2014). Therefore, the results obtained in the present study (I = 0.526
and He = 0.353) can be considered moderate. The rate of He in this study was higher than
expected for long-lived perennial species and outcrossing (He = 0.25 and 0.27, respectively)
(NYBOM, 2004).
Our results are very similar to those seen in other natural population of Copernicia
prunifera (I = 0.44 and He = 0.228; VIEIRA et al., 2015), and relatively higher than the Shannon
index of a natural population of Phoenix dactylifera, with values between 0.290 and 0.097
(MARSAFARI and MEHRABI, 2013). As such, the differences between the values of genetic
diversity indices reflect the interaction of several processes, such as: forest fragmentation
(YOUNG et al., 1996), spatial genetic structure (ERBANO et al., 2015), and outcrossing rate
(ARRUDA et al., 2015). All these factors may result in the loss of rare alleles, a reduction in
heterozygosity, and increased inbreeding (ROSSI et al., 2014).
40
The population analyzed presented genetic bottleneck, based on IAM and SMM (P <
0.01). Therefore, there is no balance between mutation and drift in the sampled population. This
result is similar to that found for a population of Elaeis guineenses where the authors also
reported a reduction in the effective population size (CHAGAS et al., 2015). A reduction in the
effective population size can be the result of human intervention in the region, through the
installation of wind turbine towers and the introduction of cattle in the study area. These
disturbed environments can lead to an increased risk of extinction of local populations, as well as
decrease the evolutionary potential of species due to changes in the natural environment
(HAMRICK, 2004; JUMP and PEÑUELAS, 2006). Clearly, the detection of recent bottlenecks
in a population suggests the species is at risk of extinction (GONÇALVES et al., 2016).
The mating system parameters based on the mixed mating and correlated mating model
(RITLAND and JAIN, 1981; RITLAND, 1989) indicate that Copernicia prunifera is a mixed
mating species (t < 0.95), that is preferentially allogamous (tm = 0.878). In addition, the single
locus outcrossing rate was high (ts = 0.738). These values are consistent with those found for
other tropical palms, which are predominantly outcrossing, such as Acrocomia aculeata (ABREU
et al., 2012) and Hermosa landrace (PICANÇO-RODRIGUES et al., 2015). Ward et al. (2005) in
36 studies surveyed found > 90% outcrossed mating for 45 hermaphroditic or monoecious
species. Another parameter that defines allogamy is the rate of selfing (s); the result from present
study (s = 0.122) falls within the range expected for a predominantly allogamous species (s <
20%) (OLIVEIRA et al., 2002; WINN et al., 2011).
The outcrossing rate in hermaphroditic species, such as Copernicia prunifera, depends on
factors including: pollinator behaviour, which is influenced by the density of flowering
individuals in the population; selective abortion of fruits and seeds from outcrossing; presence
and intensity of self-incompatibility mechanisms; and the degree of protogyny and protandry
(MURAWSKI and HAMRICK, 1991; MENEZES and OLIVEIRA, 2011).
The rate of mating among relatives (tm - ts) showed that, although outcrossing in the
population is high, some individuals are the product of mating between relatives (0.140).
Picanço-Rodrigues et al. (2015) found a similar result for the palm Hermosa landrace.
Additionally, the correlation of selfing was high (rs = 0.914), suggesting that some plants produce
more descendants from selfing than outcrossing.
41
The fixation index between seed trees (F = - 0.200) indicates an absence of inbreeding
among reproductive individuals. Ramos et al. (2011) and Abreu et al. (2012) also identified an
absence of inbreeding in natural populations of the palms Astrocaryum aculeatum and Acrocomia
aculeata. The lack of inbreeding in the study population is consistent with the low level of mating
among relatives. Therefore, despite the occurrence of Copernicia prunifera individuals in an
anthropogenized area, in monodominant clusters, and at high densities, we can reject the
assumption of a high rate of outcrossing between related individuals expected for the study
population. In addition, we found low levels of relatedness between pollen donor trees (rp(s) - rp(m)
= - 0.296).
IMPLICATIONS FOR CONSERVATION AND MANAGEMENT
The study area suffers constant anthropogenic pressure, primarily through the
advancement of wind energy facilities in the region. Without proper planning, these facilities can
have a negative impact on the studied Copernicia prunifera population, which can lead to habitat
fragmentation and a loss of genetic variability. The genetic bottleneck detected through our
analysis may be associated with anthropogenic interventions in the study population (CHAGAS
et al., 2015).
With the aim of supporting species conservation, our study shows that the local
population and wind energy companies must be better informed about the importance of
maintaining the existing population. Furthermore, the exploitation of the species must be carried
out in a sustainable manner. For this, government programs should be developed to reduce the
anthropogenic impacts on Copernicia prunifera. Considering future genetic improvement studies
and programs for the species, we propose the formation of a base population with seeds of
different populations, collecting preferably in the study population, seeds from individuals 9, 10,
and 12, as they are the most diverse individuals in the population. For that, indicates the
methodology used by Sebbenn et al. (2003). In addition, in order to conserve the current genetic
diversity, we suggest the creation of a genebank, based on these divergent genotypes. In relation
to the results obtained for the reproductive system of Copernicia prunifera, our study shows a
clear need for conservation of populations with large numbers of individuals.
42
ACKNOWLEDGMENTS
The authors thank the Fundação de Apoio à Pesquisa do Rio Grande do Norte (FAPERN)
for providing a scholarship and the Conselho Nacional de Desenvolvimento Científico e
Tecnológico (CNPq) for the financial assistance, process n° 471099/2012-0. We also thank Dr.
Evelyn R. Nimmo for assistance in editing the manuscript.
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CONCLUSÕES GERAIS
Existem divergências nos padrões reprodutivos entre as populações avaliadas, sendo
observadas atividade contínua na produção de flores e frutos maduros na população de
Parnamirim, e descontínua na população de Macaíba.
As flores da Copernicia prunifera são hermafroditas, fornecendo como recurso pólen para
possíveis insetos polinizadores (Trigona spinipes e Polistes canadensis).
Foi observada viabilidade polínica relativamente baixa, podendo acarretar baixa produção de
frutos.
Observaram-se elevados percentuais de locos polimórficos, com valores acima de 84%, além
de índices de diversidade genética de Shannon (I) e Nei (He) intermediários.
Observaram-se decréscimos populacionais, conforme modelos IAM e SMM, entretanto não
houve endogamia, com base na baixa taxa entre indivíduos aparentados.
De acordo com os resultados obtidos do sistema reprodutivo da Copernicia prunifera, a
espécie apresenta sistema misto de reprodução, sendo preferencialmente alógama.
