Reproductive and Physiological Ecology of Climbing and Terrestrial Polybotrya (Dryopteridaceae) at the La Selva Biological Station, Costa Rica

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Int. J. Plant Sci. 175(4):432–441. 2014.� 2014 by The University of Chicago. All rights reserved.1058-5893/2014/17504-0004$15.00 DOI: 10.1086/675576

REPRODUCTIVE AND PHYSIOLOGICAL ECOLOGY OF CLIMBING AND TERRESTRIALPOLYBOTRYA (DRYOPTERIDACEAE) AT THE LA SELVA BIOLOGICAL STATION, COSTA RICA

Bianca K. Canestraro,* Robbin C. Moran,† and James E. Watkins Jr.1,‡

*Universidade Federal do Parana, Curitiba-PR, Brazil; †New York Botanical Garden, 2900 Southern Boulevard, Bronx,New York 10458, USA; and ‡Department of Biology, Colgate University, Hamilton, New York 13346, USA

Editor: Adrienne Nicotra

Premise of research. Recent studies have suggested that the evolution of holoepiphytism in certain fernsmay have proceeded through a hemiepiphytic intermediary. Defining hemiepiphytism in ferns is complicatedby the presence of separate, free-living gametophyte and sporophyte stages. Currently, we lack detailed fieldobservations into fern species that have historically been referred to as hemiepiphytes, especially as to whetherthe gametophytes establish themselves on mineral soil, rotting logs, or the bases of tree trunks. In addition,nothing is known of the physiological shifts that can occur as an individual fern moves from a terrestrial habitto a climbing habit. The goal of this work is to describe the developmental ecology of species that have beentermed hemiepiphytes in Polybotrya and examine the ecophysiological changes between terrestrial and climbingindividuals.

Methodology. The research was carried out at the La Selva Biological Station in Costa Rica, where thedevelopmental ecology was studied in plots across the forest. We surveyed for presence/absence of gametophytesand sporophytes on different substrates. We then selected sporophytes in both terrestrial and climbing con-ditions and made several ecophysiological measurements in the field and lab.

Pivotal results. In one of the few studies of its kind, we describe the gametophyte morphology, whatsubstrates the gametophytes grow on (mineral soil, rotting logs, or bases of trunks), and sporophyte devel-opment and ecology of Polybotrya caudata Kunze and Polybotrya osmundacea Willd. Contrary to modernreports, neither species conformed to the classic definition of a hemiepiphyte. We propose the new term “root-climber” to refer to a species that begins as a terrestrial individual and then shifts to a climbing habit but stillmaintains its connection to the ground, using adhesive roots on the stems as the main mechanism of attachment.We also find that climbing can elicit distinctive physiological responses, and these responses can be speciesspecific. In P. osmundacea Willd., changes associated with climbing concerned water relations, not light. Therewere no discernible physiological changes in P. caudata as a function of climbing.

Conclusions. The ecology of ferns is complex given the reliance on independent gametophytes and spo-rophytes with unique habitat requirements. Studies that attempt to examine fern ecology must carefully considerboth stages of the life cycle. This is especially true in cases where taxa have been referred to as hemiepiphytes.

Keywords: ferns, climbing, hemiepiphyte, clasping roots, functional ecology.

Understanding the ecology of ferns requires observation andstudy of the entire life cycle. Studies have demonstrated theunique ecology and physiology that divide the gametophyteand sporophyte generations (Watkins et al. 2007b, 2010; Wat-kins and Cardelus 2012), yet both of these stages must workin concert to ensure a species’ long-term survival. Ferns thathave historically been referred to as hemiepiphytes pose a par-ticular challenge, as gametophytes and sporophytes may ex-perience even more radically different habitats than terrestrialand/or holoepiphytic ferns. Benzing (1990) defined hemiepi-phytes as plants that spend some time of their life disconnectedfrom the ground. He recognized two categories: (1) primary

1 Author for correspondence; e-mail: [email protected].

Manuscript received May 2013; revised manuscript received November 2013;electronically published April 8, 2014.

hemiepiphytes, whose species lack terrestrial connections earlyin life but later establish terrestrial roots, and (2) secondaryhemiepiphytes, whose species begin life as terrestrially rootedplants but later climb and lose connection with the soil. Therecontinues to be debate around the proper use of the term“hemiepiphyte,” especially as it relates to secondary hemi-epiphytism (Moffett 2000; Zotz 2013). Fitting any definitionto ferns is difficult because the independent gametophyte hasdifferent niche requirements relative to sporophytes. In fact,the situation is even more complicated in species where ga-metophytes and young sporophytes are initially epiphytic butthe sporophytes later establish contact with the soil by down-ward-growing roots (Nitta and Epps 2009; Lagomarsino et al.2012). Unfortunately, we lack detailed life cycle observationsfor most fern species. Thus, discussing this growth habit inferns is subject to problems of definition coupled with lack of

CANESTRARO ET AL.—REPRODUCTIVE AND PHYSIOLOGICAL ECOLOGY OF POLYBOTRYA 433

information about where either generation begins on theground or as an epiphyte.

Far from a simple semantic argument, properly defining thelife history of a species has broad implications. Watkins andCardelus (2012) proposed that the known physiological dif-ferences between epiphytic and terrestrial ferns are unlikely tohave arisen in one step. Rather, they argued that the radiationof terrestrial ferns into epiphytic habitats required an inter-mediate stage, one that had the necessary preadaptations re-quired for fully epiphytic life. They suggested that this stagewas a hemiepiphytic intermediary that gradually accumulatedthe necessary traits for a holoepiphytic life. This hypothesis issupported by some phylogenetic analyses in bolbitidoid fernsdemonstrating that Elaphoglossum, a largely holoepiphyticlineage, possibly arose from hemiepiphytic ancestors (Moranet al. 2010a). Primary hemiepiphytism has been reported insome filmy ferns (Hennequin et al. 2008) to include Vanden-boschia collariata (Nitta and Epps 2009). Using filmy ferns asa model to understand hemiepiphytism in ferns is complicatedby their unique morphology and physiological nature of ex-treme stress tolerance, which limit our ability to apply eco-logical patterns found in the Hymenophyllaceae to other ferngroups (Proctor 2003, 2012). Strong evidence for primaryhemiepiphytism in ferns has been reported in the genera Ela-phoglossum (Lagomarsino et al. 2012), Campyloneurum (J.E. Watkins Jr., personal observation), and Colysis (W. L. Testoand M. Sundue, personal communication). Contrary to somereports, there is little viable evidence of secondary hemiepi-phytism in ferns (Kato and Tsutsumi 2013 and referencestherein).

Unfortunately, our ability to test life form evolution for fernsis hindered by our poor understanding of their ecology. In spiteof major progress in describing gametophyte morphologyacross many disparate lineages, we know next to nothing ofthe field ecology and behavior of gametophytes and youngsporophytes, especially whether the plants grow on soil or thebases of trunks. This has resulted in widespread misuse of theterm “hemiepiphyte” as it applies to ferns. Indeed, many spe-cies that are listed as hemiepiphytes are mere climbers thatnever lose terrestrial connections (J. E. Watkins Jr., personalobservation).

Given the importance of fern radiations in angiosperm can-opies during the Cretaceous and Tertiary (Schneider et al.2004; Schuettpelz and Pryer 2009; Watkins and Cardelus2012), it is important to generate a better understanding ofthe ecophysiological changes that occur as individual plantsshift from terrestrial to “hemiepiphytic” and potentially holo-epiphytic forms (and vice versa). A number of studies haveexamined the functional changes that occur in hemiepiphyticseed plants as they make such transitions. In the case of stran-gler figs, individuals radically change their nutrient, carbon,and water relations as they shift from epiphytic to terrestrial(Putz et al. 1995; Holbrook and Putz 1996a, 1996b), yet nosuch data are currently available for ferns.

A potentially informative group of ferns in which to examinehemiepiphytism and/or terrestrial and climbing transitions isPolybotrya Willd. It includes ∼35 species, all of which areendemic to the Neotropics (Moran 1987). All the species areclimbing except for two terrestrial ones: Polybotrya fractis-erialis (Baker) J. Sm. and Polybotrya sorbifolia Kuhn. The

climbing species, which have been referred to as hemiepiphytes(Moran 1987; Young and Leon 1989; Oliveira-Dittrich et al.2005; Athayde Filho and Windisch 2006), produce long-creep-ing rhizomes. It has been suggested that some species grow onthe forest floor until they find suitable substrate on which toclimb (Moran 1987; Young and Leon 1989, 1991). Yet, noarticles have ever reported on the gametophyte generation orexamined the field ecology of potentially hemiepiphytic Poly-botrya. Thus, there is no way to know where plants actuallystart.

The goal of our study is to record the ecology of two speciesof Polybotrya in a lowland tropical rainforest in Costa Rica.We specifically examine where the gametophytes are estab-lishing (on soil, logs, or bases of trunks), sporophyte recruit-ment into the population, and physiological changes that occuras the sporophyte shifts from terrestrial to climbing. Finally,we attempt to clarify the definition of “hemiepiphyte” as itapplies to these two species.

Material and Methods

Study Site and Species

This study was conducted at La Selva Biological Station(Heredia Province) in the Atlantic lowlands (∼30 m) of north-eastern Costa Rica (lat 10�25.591′N, long 084�00.37′W) inJanuary of 2013. This station contains 1400 ha of tropicalwet forest with a mean annual rainfall total of ∼4000 mm.Precipitation peaks in June or July, but mean monthly rainfallis never below 150 mm mo�1 (http://www.ots.ac.cr/meteoro/default.php?pestacionp2).

Polybotrya is a genus of climbing ferns characterized byproducing holodimorphic fronds and stem anatomy with nu-merous meristeles surrounded by a sclerenchymatous sheath.Some species can produce fronds that reach 1 m in length, andmost all fertile fronds are produced on the apical portion ofthe stem (Moran 1987). The genus is composed of 35 Neo-tropical species (Moran 1987). Of these species, we examinedPolybotrya caudata Kunze and Polybotrya osmundaceaWilld., two species that are common and widely distributedat the La Selva Biological Station. Both occurred in primaryand secondary forest; however, P. caudata was more frequentin primary forest. These two species have the largest geograph-ical and altitudinal range of any other species in the genus.Although similar in lamina cutting and size, the species areeasily separated by indument characters: P. caudata producesdense hairs on the abaxial surface of the lamina tissue andthick, tightly appressed rhizome scales, whereas P. osmundaceais glabrous and has thinner, widely spreading rhizome scales(fig. 1; Moran 1987). In addition, P. osmundacea producesanadromous medial pinnae, whereas the medial pinnae in P.caudata are catadromous. The combination of such charactersmade field identification possible.

Field Methods

To determine where and how the species are recruiting, weestablished 25 1 # 1-m plots within a 2-ha area of primaryforest. Sample location was initially determined by findingboth species present in the 2-ha area. Plots were surveyed forpresence/absence of gametophytes and sporophytes on three

434 INTERNATIONAL JOURNAL OF PLANT SCIENCES

Fig. 1 Comparison of rhizomes and pinnules of two species of Polybotrya at the La Selva Biological Station, Costa Rica. A, C, Polybotryacaudata, with appressed rhizome scales and less divided pinnules that are pubescent on their abaxial surface. C, D, Clasping roots, which areshort, adhesive secreting, and perpendicular to the rhizome. These roots anchor the rhizome to the substrate. B, D, Polybotrya osmundacea,with spreading scales and more divided pinnules that are glabrous on their adaxial surface. In B, the whitish lines are the aerophores. In C andD, the rachis is at the right and the distal portion of the leaf is oriented upward.

different substrates: mineral soil, rotting logs, and angiospermtrunks. Plots were initially chosen at random, and we firstsampled a 1 # 1-m soil plot followed by the same area onthe nearest-neighbor rotting log and angiosperm. We thenchoose a random direction and moved 5 m away from theinitial plot and established a second sample point. This pro-cedure was repeated until we had sampled 25 plots within the2-ha sample area. When plants were discovered, we also re-

corded the laminar length of the longest frond on each indi-vidual plant.

Gametophytes were identified by finding young emergingsporophytes (sporelings) that were identifiable to speciesand still connected to gametophytes. As will be describedbelow, gametophytes were found to have a unique characterthat allowed for easy identification in the field. Field-col-lected gametophytes were brought back to the lab and pho-


tographed using both dissecting and high-powered lightmicroscopes.

Physiological Methods

To determine maximum photosynthetic rate, stomatal con-ductance, and water-use efficiency (WUE), we measured oneleaf on 30 terrestrial and 30 climbing plants of P. osmundaceaand 14 of P. caudata using a LI-6400 portable photosynthesissystem (Li-Cor, Lincoln, NE) in situ. In all cases, leaves werefully expanded and apparently of the same cohort. Ambientlight measurements were recorded at the position of the leafbeing measured using the photosynthetically active radiation(PAR) sensor on the LI-6400 portable photosynthesis system.Gas exchange measurements were recorded at a CO2 concen-tration of 400 mmol mol�1 and 800 mmol m�2 s�1 PAR. Plantswere exposed to these conditions for 420 s before measure-ment, to allow for photosynthetic parameters to stabilize. Pre-liminary light curves (not shown) indicated that these lightconditions and acclimatization times were sufficient to allowplants to achieve maximum assimilation rates and for otherphotosynthetic variables such as stomatal conductance, Ci, andso on, to stabilize.

Leaf Elemental Analysis

For determination of d13C content, tissue samples fromclimbing and terrestrial individuals were excised from frondsused in gas exchange measurements and dried at 60�C for 48h. Dried samples were then ground into a fine powder usinga Wiley mill (Thomas Scientific, Swedesboro, NJ) and passedthrough a no. 40 screen. Samples were then rolled and com-busted on a Costech carbon and nitrogen analyzer and massspectrophotometer (Valencia, CA) at Colgate University.

Statistics

One-way ANOVA, followed by post hoc Tukey tests and t-tests, was run to determine differences between habit for eachvariable measured. For all analyses, a significance level of 0.05was used. All statistics were completed using the program JMP,version 9.0.0 (SAS Institute, Cary, NC).

Results

Gametophyte Morphology

We discovered several hundred gametophytes of Polybotryain our sampling plots (figs. 2, 3A–3C). Identification to genuswas initially made by searching for a developmental series ofyoung sporophytes attached to gametophytes (fig. 3A–3F). Itwas not possible to separate gametophytes to species, whetherPolybotrya caudata or Polybotrya osmundacea; however, sev-eral characteristics made for easy identification to genus, andall characters used were verified using Nayar and Kaur (1971).In general, the gametophytes are cordiform-thalloid withbroad wings that were often unequal (fig. 2A–2C). Most strik-ingly, the thallus surface and margins were covered with co-pious unicellular papillate hairs that were not secretory (fig.2D, 2E). Unlike hairs of related species, the marginal hairswere short, dense, and evenly spaced along the margins. Iden-tical but fewer hairs were also present on the thallus surface.

In general, overall morphology was similar to that reportedfor other dryopteroid ferns. We also discovered several ga-metophytes that exhibited small cordate proliferations (fig. 2F–2H). Careful sampling of a population of several dozen ga-metophytes revealed a number of male gametophytes (26% ofthe sampled gametophytes in one population alone), whichsuggests an antheridiogen system operating in the wild (fig.2I).

Establishment

The results of our field sampling efforts demonstrated thatgametophytes and sporophytes were more frequently found onrotting logs than they were on angiosperm trunks or soil. Weencountered a single plot with one gametophyte growing onthe base of an angiosperm trunk, not on the soil (fig. 4). Ingeneral, for those plants that climbed, the climbing portionsproduced larger fronds than those portions on the soil or rot-ting logs (fig. 4B). We initially attempted to include tree fernsas a possible substrate for Polybotrya gametophyte establish-ment in our sample design; however, there were too few in-dividuals to be biologically meaningful. We did observe youngsporophytes growing on tree ferns, but it was difficult to de-termine whether these were holoepiphytic or had climbed uponto the trunk from the soil.

Physiological Ecology

Light measurements taken at leaf level were not significantlydifferent between terrestrial plants and climbing plants, norwas there a significant relationship between increased climbingheight and increased light level (r2 p 0.018, P p 0.463). Inspite of this, there were physiological differences in someplants. In P. osmundacea, climbing individuals had lower pho-tosynthetic and stomatal conductance rates and lower WUEthan terrestrial individuals (fig. 5). In P. caudata, there wereno significant differences between climbing and terrestrial mea-surements. Regression analysis revealed no significant rela-tionship with any variable and height as a continuous variablefor climbing individuals for either species (data not shown).Also, stomatal density was statistically invariant betweenclimbing individuals and terrestrial individuals within species;however, P. caudata produced significantly fewer stomata inboth terrestrial and climbing individuals compared to P. os-mundacea (fig. 6). Isotopic analysis of d13C natural abundancedemonstrated that terrestrial and climbing individuals of P.osmundacea were not significantly different. In contrast, climb-ing individuals of P. caudata were significantly higher in theheavier isotope, indicating that such individuals have greaterintegrated lifetime WUE. When compared across species andidentical habits, only climbing individuals of P. caudata weresignificantly different and again more enriched (fig. 6). Wefound no significant difference in %N or %C with compari-sons across and within species and habits (data not shown).

Discussion

Attempts to Describe Growth Habit as It Relates toHemiepiphytism and Climbing Are Complicated

Ferns that exhibit some form of stem climbing have oftenbeen described as hemiepiphytes. There is confusion in the


Fig. 2 Field-collected gametophytes of Polybotrya from the La Selva Biological Station, Costa Rica. Gametophytes are of the typicaldryopteroid type by their cordiform shape (A–C) and glandular hairs (D–F). Gametophytes sometimes branch to give rise to additional cordiformthalli (G, H). We found evidence of an antheridiogen system in the wild as evidenced by the occurrence of dozens of small male individuals indense gametophyte populations.

literature about the proper definition of “hemiepiphytism,”and most of it revolves around the use of the term “secondaryhemiepiphyte” (Moffett 2000; Zotz 2013). All definitions,however, agree in that a plant regarded as a secondary hemi-epiphyte must, at some point, be disconnected from the forestfloor (Benzing 1990; Moffett 2000; Zotz 2013). Many hemi-epiphytes often have stages that resemble climbing vines. Ittherefore requires significant understanding of a species’ field

biology—such as whether the gametophytes start growth onthe soil or bases of trees and whether the rhizome maintainsor loses its connection with the soil—to accurately distinguishbetween vines and hemiepiphytes. Unlike confusion aroundhemiepiphytism, the term “liana” strictly refers to woodyclimbing plants that rely on other plants or substrates for sup-port (Schnitzer and Bongers 2002). Because ferns lack sec-ondary growth (Ogura 1972), they are, by definition, not

Fig. 3 A, Gametophytes and young sporophytes of Polybotrya osmundacea growing on a rotting log at the La Selva Biological Station,Costa Rica. B, Dozens of gametophytes and young sporophytes colonizing a decaying woody fruit of Lecythis ampla. C–F, Developmental seriesin Polybotrya where gametophytes initially establish on rotting logs (C) form sporophytes (E) and reach large sizes but never seem to becomefertile (F). G, Terrestrial individuals of Polybotrya are frequently encountered at La Selva; however, it appears that this occurs after establishmenton a rotting log and after the log decays. H, Several individuals actively turn away from the mineral soil if the rotting log remains. I–K, In somecases sporophytes encounter suitable substrate and climb into the understory, reaching several meters tall.


Fig. 4 Location and occurrence of the gametophytes and sporo-phytes and length of the longest lamina of Polybotrya from 25 1 #

1-m plots at the La Selva Biological Station, Costa Rica. Plots weresampled for the presence and absence of gametophytes and sporo-phytes on mineral soil, woody angiosperm tree trunks, and rottinglogs. All rotting logs were in an advanced state of decay and hadalready lost their cortex.

Fig. 5 Physiological parameters of the sporophytes of climbing(Climb) and terrestrial (Ter; nonclimbing) individuals of Polybotryaosmundacea (PolOsm) and Polybotrya caudata (PolCau) at the LaSelva Biological Station, Costa Rica. WUE p water-use efficiency;Cond p stomatal conductance at maximum photosynthetic (Amax)rates.

woody, and therefore referring to them as lianas is inaccurate(Dubuisson et al. 2003). Herbaceous vines, on the other hand,exhibit marked variation in the means of climbing mecha-nisms, from leaf and stem climbers, to twiners, to root andhook climbers (Darwin 1875; Holbrook and Putz 1996b;Schnitzer and Bongers 2002). Darwin (1875) initially distin-guished climbing plants based on the mechanisms used to as-cend. The root-climber group (clinging-climbers sensu Isnardand Silk 2009) referred to taxa similar to Hedera that useadhesive secretions from the roots to adhere to their climbingsurface (Darwin 1875; Isnard and Silk 2009).

Polybotrya caudata and P. osmundacea produce two kindsof roots (figs. 1, 3). The first is for clasping the substrate. Suchroots are 1–3 cm long and extend at approximately right anglesto the rhizome (fig. 1A). The roots secrete an adhesive an-choring the rhizome to the substrate. The second kind of rootis long, mostly unbranched, and runs downward at a narrowangle to the rhizome. Upon touching the soil, it branches pro-fusely. Presumably, these long roots supply the rhizome with

most or all of its water and mineral nutrition, although thisidea has yet to be tested with physiological experiments. Theseterrestrial roots appear to secrete an adhesive. A similar kindof root dimorphy has been documented for the primary hemi-epiphyte Elaphoglossum amygdalifolium (Dryopteridaceae;Lagomarsino et al. 2012). Lagomarsino et al. (2012) calledthe short roots “clasping roots” and the long ones “feederroots.”


Fig. 6 Stomatal densities and natural abundance of d13C in thesporophytes of climbing and terrestrial (nonclimbing) individuals ofPolybotrya osmundacea (PolOsm) and Polybotrya caudata (PolCau)at the La Selva Biological Station, Costa Rica.

In the case of P. osmundacea and P. caudata, neither con-forms to Benzing’s (1990) definition of a hemiepiphyte. Hun-dreds of observations from dozens of plots found only a singlegametophyte growing on an angiosperm and none on mineralsoil. Instead, gametophytes and young sporophytes occurredmost frequently on old, decorticated, rotting logs (fig. 3). Therewere dozens of large sporophyte colonies found growing ter-restrially throughout the forest. Our best interpretation of thisobservation is that gametophytes and young sporophytes userotting logs as nurse sites that aid in establishment. Once es-tablished, sporophytes persist until the log disintegrates, leav-ing the sporophyte connected to the mineral soil. There seemedto be preference for the log substrate, as we observed severalcases where rhizomes would actively turn up and away fromthe soil to maintain contact with the log (see below). Even-tually, individuals become terrestrial and rooted in the mineralsoil, with the rhizomes at or 1–2 cm below the soil surface.Some rhizomes will encounter suitable substrate and begin toclimb. At no point does either species lose contact with theforest floor. An individual attaches to tree trunks by a strongadhesive that makes intact rhizome removal difficult. Thesespecies are not hemiepiphytes sensu Benzing (1990) because

they maintain the connection with the forest floor after theyclimb. They combine a terrestrial existence with a form ofclimbing that uses climbing rhizomes and adhesive roots. Bothspecies fit Darwin’s (1875) root-climbing category. Again thiscategory alone is insufficient because individuals may spendtheir entire lives as nonclimbing terrestrial plants. In his at-tempt to clarify concepts in canopy biology, Moffett (2000)coined the term “nomadic vine” for climbing plants whoseterrestrial portions eventually rotted away, leaving only anepiphytic stage. For Polybotrya we propose the term “root-climber” (sensu Darwin 1875) to more accurately describe thelife history described here, where the terrestrial portion of theplant persists and does not decay. There may be other speciesof Polybotrya in Brazil that are true hemiepiphytes (B. K. Ca-nestraro, personal observation), but these lack documentationfrom field studies.

The preference for rotting logs was further demonstrated byseveral individuals whose rhizomes would actively grow awayfrom the mineral soil back onto the log (fig. 3H). Benzing(1990) classifies epiphytes into several functional categoriesand defines species that inhabit rotting substrate and leaf litteras humus-adapted epiphytes. Yet, this term does not seem toapply to Polybotrya because many individuals can also persistfor decades on mineral soil (J. E. Watkins Jr., personal obser-vation). Rotting logs seem to serve as critical starting pointsfor the gametophyte, whereas the sporophyte is more plasticin its habitat requirements. The sporophyte may prefer rottinglogs but can clearly persist on mineral soils.

Currently influencing understory plant species compositionat the La Selva Biological Station is the large number of white-collared peccaries. All of the fern populations sampled wereheavily impacted by peccaries, and the soils were highly andconstantly disturbed. This might explain the absence of ga-metophytes on the forest floor mineral soil. Watkins et al.(2007a) argued that disturbance plays a key role in creatingsafe sites for terrestrial fern gametophyte establishment. How-ever, the type of disturbance caused by peccaries is likely toointense and too consistent to allow for serious establishment.We did not quantify disturbance in our study; however, wenote that rotting logs appeared largely unaffected by pigs.Thus, such sites may be the safest for gametophyte and spo-rophyte establishment.

An important way to understand the functional conse-quences of climbing and/or hemiepiphytism is to examine thephysiological shifts that occur as plants move from one stageto another. In the case of hemiepiphytes, several studies haveshown that terrestrially rooted plants have lower specific leafarea, higher stomatal conductance, and higher stomatal den-sities than epiphytic stages (Holbrook and Putz 1996a, 1996b).In addition, epiphytic stages have differences in leaf %N andd15N signatures (Watkins et al. 2007c). In the case of Poly-botrya, differences between climbing and terrestrial stageswere species specific. Climbing and terrestrial individuals of P.caudata did not differ in any physiological character exceptd13C, where climbing individuals were significantly more water-use efficient (integrated WUE; i.e., more enriched in d13C) thanterrestrial individuals. Differences were more pronounced inP. osmundacea, where climbing individuals had lower pho-tosynthetic and stomatal conductance rates and were less wa-ter-use efficient (instantaneous) than terrestrial individuals.


Leaf %N and d15N natural abundance were not different be-tween terrestrial and climbing individuals of either species.Watkins et al. (2007c) showed that epiphytic and terrestrialferns exhibit differences in d15N and %N, with epiphytic spe-cies exhibiting significantly enriched d15N signatures and lower%N relative to terrestrial species. Several other studies haveproduced similar results in angiosperms. It is reasonable toexpect some differences in d15N signatures and %N in climbingindividuals if they were disconnected from the forest floor. Thelack of such differences further supports the definition of thesetwo Polybotrya species as root climbers, not hemiepiphytes.

These results suggest that climbing can exert physiologicalpressures, but responses are species specific. Whereas climbingclearly had an impact on P. osmundacea, it had little or noneon P. caudata. The only difference in the latter species wasthat climbers were more water-use efficient than terrestrial in-dividuals. Both species are similar in gross morphology andecology. Indeed, it is difficult to visually distinguish the two,although P. osmundacea tends to have the laminae slightlymore divided than P. caudata (fig. 1C, 1D; Moran 1995). YetP. caudata produces dense pilose hairs on the abaxial surfaceof the leaves, sometimes so dense that the surface is obscured.It seems possible that this increases the boundary layer andreduces transpiration. Scales on fern laminar surfaces havebeen shown to act as a photoprotective mechanism (Watkinset al. 2006), yet little is known of the impact that hairs/scaleshave on transpiration. It may be that dense hairs in P. caudatabuffer the impacts of climbing that are more apparent in P.osmundacea. Furthermore, if plants become more waterstressed as they climb and if leaf hairs act to reduce transpi-ration, it is possible that hair density increases with plantclimbing height. Future studies should further examine theinfluence of height and hair density in this species.

Why do species such as P. osmundacea climb? Several ideasmight explain the climbing habit. These range from escapefrom herbivory and competition to increased light exposureas plants climb. Our data demonstrate that light levels did notincrease with plant height. This suggests that factors other thanincreased light select for the climbing habit. One such factor

might be spore dispersal, which is largely driven by wind, andeven small increases in height can dramatically increase dis-persal distance (Conant 1976; Peck et al. 1990). Both speciesclimb to heights over 5 m; thus, the potential for increasedspore dispersal seems significant. In species such as P. osmun-dacea that lack morphological and/or physiological mecha-nisms to reduce the impacts of climbing, the benefits that comewith increased dispersal may outweigh reduced photosyntheticcapacity. Interestingly, the climbing habit has evolved multipletimes in the ferns, and many climbing species exhibit completefertile-sterile leaf dimorphism. Examples include Blechnumfragile (Blechnaceae; Moran and Riba 1995), Lomariopsis (Lo-mariopsidaceae; Moran and Riba 1995), and bolbitidoid ferns(Arthropteris, Lomagramma, Mickelia, and Teratophyllum;Dryopteridaceae; Moran et al. 2010a, 2010b). The same ex-planation for increased efficiency in spore dispersal with in-creased climbing height might also apply to these groups.

This study demonstrates the importance that an organismalapproach—especially one that embraces both the gametophyteand the sporophyte—can have on understanding plant ecology.Many climbing fern species, including the two discussedherein, have been considered hemiepiphytes for years, yet care-ful attention to the entire life cycle is needed to accuratelyestablish this. Future work should include careful integrationof rhizome anatomy between climbing individuals and terres-trial individuals, a thorough evaluation of rhizome hydraulics.

Acknowledgments

Data from this study were collected during the Organizationfor Tropical Studies (OTS) Tropical Ferns and Lycophytescourse, January 2013, organized and taught by R. C. Moranand J. E. Watkins Jr. We thank the staff of OTS, particularlyAndres Santana, Barbara Lewis, and Pia Pabby, for makingthis course possible. This research was also partially supportedby a grant to R. C. Moran from the National Science Foun-dation (DEB 1020443).

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Documents

Reproductive and Physiological Ecology of Climbing and Terrestrial Polybotrya (Dryopteridaceae) at the La Selva Biological Station, Costa Rica