Aquatic and Terrestrial Oviposition in a Treefrog

Reproductive mode plasticity: Aquatic and terrestrial oviposition in a treefrog

Justin Charles Touchon* and Karen Michelle Warkentin

Department of Biology, Boston University, 5 Cummington Street, Boston, MA 02215

Edited by David B. Wake, University of California, Berkeley, CA, and approved March 31, 2008 (received for review December 8, 2007) Diversification of reproductive mode is a major theme in animal tation above water and develop for 3–4 days, then aquatic evolution. Vertebrate reproduction began in water, and terrestrial tadpoles fall into the water upon hatching (Fig. 1A) (1, 2, 10, 22, eggs evolved multiple times in fishes and amphibians and in the 23). Using mesocosm experiments and observations of natural amniote ancestor. Because oxygen uptake from water conflicts clutches, we report the discovery of reproductive mode plasticity with water retention in air, egg adaptations to one environment in D. ebraccatus, and we argue that this plasticity is most likely typically preclude development in the other. Few animals have adaptive. Adult D. ebraccatus are capable of laying eggs either variable reproductive modes, and no vertebrates are known to lay aquatically, both at the surface of the water and fully submerged, eggs both in water and on land. We report phenotypic plasticity of or terrestrially, and they choose their reproductive mode based reproduction with aquatic and terrestrial egg deposition by a frog. on factors that affect risk of terrestrial egg desiccation. The treefrog Dendropsophus ebraccatus, known to lay eggs terrestrially, also lays eggs in water, both at the surface and fully Results submerged, and chooses its reproductive mode based on the shade Congruent with previous reports (2, 10, 22, 23), over 5 years at two above a pond. Under unshaded conditions, in a disturbed habitat forest ponds (Bridge and Ocelot Ponds) near Gamboa, Panama, we and in experimental mesocosms, these frogs lay most of their egg observed only terrestrial oviposition (350 closely monitored masses aquatically. The same pairs also can lay eggs terrestrially, clutches from 2003–2005, plus many additional observations during on vegetation over water, even during a single night. Eggs can Ͼ90 nights of field work between 2003 and 2007). In 2006, at a pond survive in both aquatic and terrestrial environments, and variable in an old gravel quarry (Quarry Pond) that lacks forest canopy over mortality risks in each may make oviposition plasticity adaptive. oviposition sites, we observed D. ebraccatus laying eggs in the water, Phylogenetically, D. ebraccatus branches from the basal node in a supported by aquatic vegetation. Some clutches were laid at the clade of terrestrially breeding species, nested within a larger water surface or across the air–water interface so that some eggs lineage of aquatic-breeding frogs. Reproductive plasticity in D. were submerged, others were in contact with both air and water, ebraccatus may represent a retained ancestral state intermediate and some were in air (Fig. 1B). Other clutches were submerged (Fig. in the evolution of terrestrial reproduction. 1C) at depths ranging from just under the water to Ϸ4 cm below the surface. Of the 148 clutches that we found at Quarry Pond during aquatic egg-laying ͉ evolution of reproductive mode ͉ Hyla ebraccata ͉ 2006–2007, 28% were submerged (all eggs completely under wa- phenotypic plasticity ͉ climate change ter), 48% were laid at the water surface, and 24% were laid terrestrially (no eggs in contact with water). The terrestrially laid he evolution of terrestrially developing eggs from ancestral eggs at Quarry Pond had significantly higher mortality from Taquatic eggs is a repeated trend in both invertebrates and desiccation (20 Ϯ 6%, mean Ϯ SEM) than terrestrial eggs at Bridge vertebrates (1–10). In both groups, aquatic predators and con- (8 Ϯ 2%) or Ocelot Ponds (0.4 Ϯ 0.2%) presumably because of straints on oxygen uptake are hypothesized to select for terres- differences in shading (Kruskal–Wallis test: nQuarry ϭ 27, nBridge ϭ 2 Ϫ9 trial eggs (2, 7–11). Terrestrial eggs can improve the embryonic 151, nOcelot ϭ 73, ␹ ϭ 37.97, P ϭ 5.69 ϫ 10 ; pairwise Wilcoxon respiratory environment, allow oviposition over fast-moving rank-sum tests, Quarry–Bridge, P ϭ 0.00023, Quarry–Ocelot, P ϭ streams where aquatic eggs might be swept away, and allow 5.0 ϫ 10Ϫ6, Bridge–Ocelot, P ϭ 0.0013) (Fig. 2). Mortality of eggs animals to colonize habitats without permanent water bodies (2, laid in the water at Quarry Pond (including submerged and surface 4, 7–10). However, terrestrial eggs experience new risks from clutches) was, however, significantly lower (45 Ϯ 3%, n ϭ 112) than desiccation and terrestrial predators (2, 4, 7–10). Because that of flooded terrestrial eggs previously observed at Bridge and aquatic and terrestrial environments select for different traits, Ocelot Ponds (61 Ϯ 6%, n ϭ 52; Wilcoxon rank-sum test, P ϭ eggs are usually well adapted to only one environment (2, 4). 0.028).

Adaptations for terrestrial oviposition have evolved indepen- Our observation of 76% aquatic oviposition by D. ebraccatus EVOLUTION dently in several groups [e.g., gastropods (8, 12), insects (9, 13), at Quarry Pond and only terrestrial oviposition by frogs at nearby and fishes and amphibians (1, 2, 4, 10)]. In all of these organisms, forest ponds (Ocelot and Bridge) could reflect either local the divergence in reproductive mode [oviposition site and type genetic differentiation, with some polymorphism among indi- of egg development (1, 2, 10)] occurred long ago, and it is thus viduals at Quarry Pond, or individual plastic responses to difficult to directly assess selective pressures that may have differences in their environments. To test for plasticity, we influenced such evolution. Closely related species or populations manipulated exposure to forest canopy shade, a variable that that vary in their reproductive modes [e.g., between viviparity affects egg desiccation rate and clearly differs between our study and oviparity (14–18)] offer the best opportunity to study the ponds (see Materials and Methods for details). We quantified selective pressures leading to reproductive mode diversification. oviposition choices of frogs from all three ponds in shaded and Although some foam-nesting frogs are reported to place nests in diverse locations (10, 19, 20), there are no vertebrate species known to deposit eggs both into water and on land. Author contributions: J.C.T. and K.M.W. designed research; J.C.T. performed research; We studied the reproductive mode of the Neotropical treefrog J.C.T. analyzed data; and J.C.T. and K.M.W. wrote the paper. Dendropsophus ebraccatus [formerly Hyla ebraccata (21)] in The authors declare no conﬂict of interest. mesocosms and at three ponds near Gamboa, Panama. D. This article is a PNAS Direct Submission. ebraccatus is a common, well studied Neotropical treefrog known *To whom correspondence should be addressed. E-mail: [email protected]. to have semiterrestrial reproduction; its eggs are laid on vege- © 2008 by The National Academy of Sciences of the USA

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0711579105 PNAS ͉ May 27, 2008 ͉ vol. 105 ͉ no. 21 ͉ 7495–7499 Downloaded by guest on September 27, 2021 A B C

Fig. 1. Aquatic and terrestrial oviposition by a treefrog. (A–C) D. ebraccatus laying eggs terrestrially as is typical in shaded habitats (A), at the water surface (B), and under water (C). Aquatic oviposition occurs in unshaded sites where terrestrial eggs desiccate rapidly. (B) Vegetation ranges from a few centimeters below the water surface (top right, rotting leaves with mass of eggs suspended in water column above them) to emergent (rosette above frogs). Arrows indicate some eggs that are partially air-exposed on leaves at the water surface. The ovipositing female is sitting in water (note reﬂection from ﬂash on the water surface). (C) Entire clutch is under water on a partially submerged leaf. Note the D. ebraccatus tadpole, an aquatic egg predator. Photographs were taken by the authors.

unshaded experimental mesocosms. Terrestrial clutches were model, F1,65 ϭ 2.3, P ϭ 0.13), and water-surface, submerged, and laid completely out of the water on emergent vegetation (e.g., terrestrial egg mass sizes did not differ between environments Fig. 1A). Aquatic clutches were laid both at the water surface (F2,287 ϭ 0.74, P ϭ 0.48). However, pairs in unshaded mesocosms and under water (e.g., Fig. 1 B and C). partitioned their eggs into significantly smaller masses (49 Ϯ 3 Frogs in unshaded mesocosms laid more water-surface and eggs) than pairs in shaded mesocosms (62 Ϯ 2 eggs; linear model, submerged egg masses than did frogs in shaded mesocosms, F1,287 ϭ 12.94, P ϭ 0.0004). Pairs in unshaded mesocosms also where the majority of egg masses were laid terrestrially (n ϭ 67 laid their terrestrial clutches closer to the water (5.8 Ϯ 1.3 cm pairs tested, 293 egg masses laid; multinomial logistic regression, high) than did pairs in shaded mesocosms (13.8 Ϯ 1.2 cm high; ␹2 ϭ Ͻ ϫ Ϫ16 2,290 111.81, P 2.0 10 ) (Fig. 3). On average, linear model, F1,151 ϭ 12.43, P ϭ 0.0006). water-surface egg masses were 35 Ϯ 4% in contact with or under water and 65 Ϯ 5% above the water. Frogs from all three ponds Discussion laid eggs aquatically in unshaded mesocosms, and 48% of pairs To the best of our knowledge, no vertebrates have been described laid both aquatic and terrestrial egg masses in a single night. to plastically lay eggs both in water and on land. In this study, we There was neither a main effect of pond nor a significant report such plasticity for D. ebraccatus. Natural variation in ovipo- interaction of pond with shade treatment, indicating that ovi- sition site is not due to local genetic differentiation between ponds position site is plastic at all ponds tested (see Materials and or polymorphism within Quarry Pond, but instead reflects the Methods for details). There was no difference in the total number plastic behavioral responses of frogs to their different environ- of eggs laid per pair in shaded or unshaded mesocosms (linear ments. Most previously documented examples of natural variation in reproductive mode within vertebrate species occur only among genetically differentiated populations, such as lizards (14–16) and 0.3

1 Terrestrial Aquatic (water-surface) Aquatic (submerged) 0.2 0.8

0.6

0.1 0.4

Clutch mortality mortality Clutch (proportion) 0.2 0 Quarry Bridge Ocelot

Proportionof egglaid clutches 0 Fig. 2. Desiccation of terrestrial egg clutches of treefrogs, D. ebraccatus,at Shaded Unshaded three ponds near Gamboa, Panama. Quarry Pond (n ϭ 27 clutches) where desiccation is highest is least shaded, Bridge Pond (n ϭ 151) is intermediate, Fig. 3. Terrestrial and aquatic oviposition by treefrogs, D. ebraccatus,in and Ocelot Pond (n ϭ 73) has the most shade over terrestrial oviposition sites. shaded and unshaded experimental mesocosms. Frogs laid most of their egg The ponds are located within 2 km of each other and were monitored masses in the water (in surface positions and submerged to Ϸ4-cm depth) (Fig. concurrently, so clutches at all ponds experienced the same weather. Most 1 B and C) in unshaded mesocosms and most terrestrially (Fig. 1A) in shaded eggs at Quarry Pond are laid aquatically, thus few terrestrial clutches were mesocosms. Data are proportion of egg masses in each category (n ϭ 293 egg available to monitor. Data are mean Ϯ SEM. masses laid by 67 pairs from three ponds).

7496 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0711579105 Touchon and Warkentin Downloaded by guest on September 27, 2021 a salamander (17) that vary in oviparity/viviparity and a frog that eggs and their ability to survive under water are likely due to produces foamy and nonfoamy egg masses (24). A normally sexual their small size [1.2–1.4 mm in diameter (23)]. D. ebraccatus eggs lizard and a normally sexual shark also have both shown a presum- are smaller than most terrestrial anuran eggs (10), which both ably plastic capacity for parthenogenesis by females isolated from reduces their oxygen demand and increases their surface area- males in captivity (25, 26). to-volume ratio, thereby enhancing oxygen diffusion under water Many Old World treefrogs of the family Rhacophoridae lay (50). Highlighting this point, Wells (10) referred to D. ebraccatus their eggs arboreally within foam nests (2, 10), and nests clutches as ‘‘little more than aquatic egg masses transferred to attributed to single species have been found in diverse locations, terrestrial oviposition sites.’’ including on tree trunks, on the ground near stream banks, and Given spatial and temporal variability in factors affecting even floating on top of water (10, 19, 20, 27–32). Although terrestrial desiccation risk and aquatic oxygen availability, as well embryos in foam nests are protected from desiccation and as in environment-specific predators, aquatic/terrestrial repro- supplied with oxygen from the foam, rhacophorid foam nest ductive plasticity may be adaptive. We observed higher desic- placement might vary with environmental conditions such as cation mortality of terrestrial eggs at Quarry Pond than at either humidity, temperature, or forest cover; to the best of our Bridge or Ocelot Ponds likely because of less shade above knowledge, this has not yet been tested. Nest placement could, clutches (Fig. 2). In contrast, mortality of aquatically laid eggs at however, simply reflect the availability of oviposition sites (20, Quarry Pond was lower than that of flooded eggs previously 29). Several Old and New World frogs lay eggs adjacent to water, observed at Bridge and Ocelot Ponds. Aquatic egg predators, on stream banks or moss, in excavated nests, or on low-lying including several species of fish and tadpoles, are abundant in all vegetation. These eggs can become inundated with water after three ponds. When clutches laid on terrestrial vegetation become heavy rains (10, 11), as can terrestrially laid D. ebraccatus flooded, they are exposed to these predators in the water clutches, so that the embryos then experience an aquatic envi- column. However, most clutches laid in the water at Quarry Pond ronment. Eggs laid near the air–water interface may therefore are on or in a dense mat of floating vegetation (mostly Salvinia) experience more environmental variation over development that may shield eggs from detection and attacks by predators in than is reflected in their initial oviposition sites. the more open water below. In addition, hypoxia is more likely Reproductive mode plasticity is poorly documented in any for flooded clutches, which may be deep in the water column, taxon, and, to our knowledge, only two other species, a mosquito than for eggs associated with floating vegetation, which remain (33) and a dragonfly (34), are known to lay both terrestrial and near the better-oxygenated surface of the water even as water aquatic eggs. Like D. ebraccatus, they choose oviposition sites in level fluctuates. response to factors affecting egg desiccation risk. These insects Variation among and even within ponds in factors such as tree are likely not the only invertebrates capable of such plasticity. canopy, aquatic vegetation, depth, and predator communities Similarly, D. ebraccatus may not be the only amphibian capable affects aquatic and terrestrial egg risks, altering selection on of both aquatic and terrestrial reproduction. Researchers study- aquatic and terrestrial oviposition. Reproductive mode variation ing amphibian reproduction, ourselves included, had not previ- in D. ebraccatus occurs at the level of the individual, not across ously documented plasticity in aquatic/terrestrial oviposition (1, populations (14–17), so that morphologically and physiologically 2, 10, 23, 35–37) perhaps because reproductive mode was similar eggs naturally experience a wide range of conditions. This considered fixed. However, the range of oviposition sites used by species therefore offers an excellent opportunity to study the D. ebraccatus and the environmentally induced variation in how ecological factors influencing the evolution of terrestrial and eggs are distributed across these sites suggests that terrestrial and aquatic reproduction. aquatic oviposition are not fully dichotomous. Rather, they may Reproductive plasticity also may occur in other species, par- represent ends of a continuum, and oviposition site plasticity ticularly in clades within which terrestrial-breeding animals may be an intermediate stage in the evolution of obligate evolved from aquatic-breeding ancestors and extant species vary terrestrial eggs. in reproductive mode. D. ebraccatus belongs to the D. leucophyl- D. ebraccatus eggs, although apparently not very well adapted latus species group, a lineage of eight Central and South Amer- to development either in air or under water, can develop in both ican species (21). D. ebraccatus is the sister species of all other environments and appear physiologically more flexible than terrestrially breeding frogs in the lineage, and all species branch- obligate aquatic or terrestrial eggs. Aquatic eggs exposed to air ing from more basal nodes breed aquatically (Fig. 4) (1, 21, 35, typically dry out rapidly and die (e.g., 38), although some 51, 52). Reproductive plasticity in D. ebraccatus could therefore salamander egg masses can survive prolonged terrestrial strand- represent an intermediate stage in the evolution of terrestrial ing (39). Desiccation mortality of terrestrial amphibian eggs is, reproduction, combining a retained ancestral capacity for however, generally low because clutches are laid in humid aquatic development with a derived ability for terrestrial ovipo- locations and are surrounded by a protective, water-rich jelly (2, sition and development. If reproductive plasticity was present in EVOLUTION 4, 10). During this study, desiccation killed an average of 20% of the common ancestor of D. ebraccatus and its relatives, it may D. ebraccatus eggs (Fig. 2). During drier periods within the rainy have helped facilitate the transition to obligate terrestrial egg season, we have observed Ͼ50% desiccation mortality of ter- development. Alternatively, a plastic capacity for aquatic repro- restrial clutches at the same ponds (J.C.T. and K.M.W., unpub- duction may have evolved secondarily from terrestrially breeding lished data). This rate is far greater than the desiccation mor- ancestors of D. ebraccatus. tality reported for many other terrestrially breeding frogs (40– The genus Dendropsophus contains Ϸ90 species of treefrogs 43). In contrast, D. ebraccatus embryos are more capable of in nine species groups [plus some unassigned to any species aquatic development than other terrestrial amphibian eggs, group (21)], among which terrestrial reproduction has evolved which die if submerged before hatching competence (40, 42, independently at least four times (Fig. 4) (1, 35, 53, 54). A 44–48). D. ebraccatus eggs can develop normally under water, complete phylogeny of the genus is lacking, as is information although developmental retardation and death occur if eggs are about the reproductive mode of nearly half the species. Thus, deep in the water column, where oxygen is lower. Other more additional origins of terrestrial reproduction and/or other strictly aquatic eggs also can suffocate if they fall to the pond reproductively plastic species may exist among these poorly bottom [e.g., Hypsiboas rosenbergi (49)], and many aquatic- studied taxa. It also is possible that other well studied species, breeding amphibians attach their eggs to vegetation or other like D. ebraccatus, harbor undocumented flexibility in egg structures in the water to hold them near the water surface (2, deposition site and the capacity of embryos to develop in 10, 23). Both the rapid desiccation of terrestrial D. ebraccatus different environments. Further research within this genus and

Touchon and Warkentin PNAS ͉ May 27, 2008 ͉ vol. 105 ͉ no. 21 ͉ 7497 Downloaded by guest on September 27, 2021 Reproductive increase the mortality of terrestrially laid eggs (45), but Mode reproductive mode variation may help D. ebraccatus to persist D. marmoratus group (8) despite these changes. Frogs from all three ponds laid a small D. garagoensis group (4)‡ subset of their eggs aquatically even in shaded mesocosms, suggesting that aquatic oviposition also may occur at a low D. parviceps group (15) frequency in forest ponds, perhaps as a bet-hedging strategy D. columbianus group (3) (Fig. 3). This would provide a buffer against unpredictable environmental variation, protecting a subset of eggs from the D. labialis group (3) possibility of desiccation. Additionally, frogs from ponds D. minutus group (4) where only terrestrial oviposition had been observed laid most D. minimus group (4) of their egg masses aquatically in unshaded mesocosms, suggesting that such plasticity may be widespread in this species D. microcephalus group (33) (Fig. 3). To the extent that the cues for aquatic oviposition D. anceps accurately predict egg desiccation risk, this plasticity should improve D. ebraccatus’ ability to survive in disturbed habitats D. elegans† or under altered rainfall patterns. Frogs in unshaded meso- D. ebraccatus cosms laid smaller egg masses, which would allow them to

group (8) + D. triangulum spread eggs among more different microsites, but probably

† also increases desiccation by increasing the ratio of edge to D. leucophyllatus central eggs in masses. Terrestrial egg masses were, however, D. bifurcus † laid closer to the water in unshaded mesocosms, which would increase their chances of transitory flooding in nature. This D. sarayacuensis

D. leucophyllatus behavior may be similar to that of amphibians that lay eggs § D. rossalleni adjacent to water in the anticipation of flooding (11). Aquatic egg laying The proximate cues that stimulate D. ebraccatus adults to repro- duce terrestrially or aquatically are unknown, but candidates in- Terrestrial egg laying clude light level, temperature, and humidity. Clearly, cues are Both aquatic and terrestrial egg laying in lineage ascertained shortly before oviposition because frogs moved from their native pond to experimental mesocosms responded to their Fig. 4. Phylogeny of the D. leucophyllatus group, showing the distribution new environment within a single night. If light level is the primary of known reproductive modes and relationships with other Dendropsophus cue, then the frogs will likely respond appropriately to forest species groups. Aquatic reproduction is ancestral in the genus and the pre- dominant reproductive mode, but terrestrial eggs have evolved at least four clearing, but not to changing rainfall patterns. However, if air times. Symbols indicate species groups where only aquatic or terrestrial egg temperature or humidity is the indicator of open habitats, then laying is known and where both reproductive modes are known for different plasticity, as well as bet-hedging, might improve D. ebraccatus’ species within a group. D. ebraccatus is the only species known to exhibit both chances of persistence in a changing climate. reproductive modes. The reproductive mode is unknown for ca. half the species of Dendropsophus. Some of these species also may have reproductive Materials and Methods plasticity, and species with described reproductive modes may have unde- Field Monitoring. We located aquatically and terrestrially laid egg clutches scribed plasticity. Numbers in parentheses indicate number of species in each at Quarry Pond and terrestrial egg clutches at Ocelot and Bridge Ponds the group. The phylogeny is based on ref. 21, except as follows: †, based on ref. 63, morning after oviposition during 2006–2007 and checked them twice daily which agrees with ref. 21 on placement of the members of the D. leucophyl- for the first 48 h. Afterward, aquatically laid eggs were hatching- latus group they have in common; ‡, tentative placement (indicated by dashed competent, making the fates of missing eggs ambiguous. Clutches were lines) based on morphological data from ref. 64; and §, tentative placement categorized based on their contact with water and air (air only, terrestrial; (indicated by dashed lines) based on morphological data from ref. 65. Repro- water and air, surface; water only, submerged). Terrestrial clutches were ductive modes are from refs. 1, 35, 53, 54, 64, and 66–69. The reproductive found on vegetation overhanging water and emergent vegetation. At mode of D. rossalleni is speculated to be terrestrial (54), but is currently Quarry Pond, aquatic egg clutches were primarily attached to Salvinia unknown. vegetation; this plant did not occur at Ocelot Pond and was in very low density at Bridge Pond. Surface clutches included eggs in contact with water and exposed to air on surface or near-surface leaves. Submerged phylogenetic reconstructions of ancestral traits functionally clutches were suspended to Ϸ4 cm deep in gaps between plants or attached related to terrestrial and aquatic egg development (e.g., egg to roots or other underwater vegetation. At each observation, we recorded size and clutch structure) should improve our understanding of the number of eggs that had died from desiccation (for terrestrial eggs), both the evolution of terrestrial reproduction and the role of from hypoxia (for aquatic eggs, characterized by developmental retarda- plasticity in evolution. An interesting candidate for compari- tion before death), or missing because of predation (for all eggs). We son to Dendropsophus is the diverse African treefrog genus similarly monitored fates of terrestrially laid clutches at Ocelot and Bridge Hyperolius, which contains Ͼ100 species, some of which breed Ponds that had been flooded during a different time period when pond levels fluctuated more (2003–2005). aquatically, others terrestrially, at least one that lays water- surface eggs in between the leaves of floating plants, as well as Testing for Reproductive Mode Plasticity. We constructed twelve 1.3-m- many species for whom the reproductive mode is not yet known diameter pond mesocosms containing both floating aquatic vegetation (55–58). and emergent vegetation and placed half under thick forest canopy The risk of terrestrial egg desiccation will likely increase in (shaded) and the rest nearby in an open field (unshaded). On eight nights the future (59), both because of habitat disturbance and in 2006 and 2007, we collected pairs of frogs that had not yet begun laying because rainfall patterns are changing in Panama (60, 61). By eggs from all three ponds (no more than four pairs per pond each night; n ϭ 2080–2099, the air temperature during June, July, and August, 21, 23, and 23 total pairs from Quarry, Ocelot and Bridge Ponds, respec- tively) and allowed them to breed in the mesocosms overnight. Only one peak breeding months for D. ebraccatus, is predicted to have pair was used per mesocosm per night, thereby preventing competition for increased at least 2.0–2.5°C, whereas precipitation is predicted oviposition sites and allowing us to identify all egg masses laid by each pair. to have decreased 10–15% and become more sporadic (61). Because variation in weather conditions might affect oviposition choices, Less predictable rainfall and more disturbed habitats will we always tested frogs from all three ponds on each night. We recorded the

7498 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0711579105 Touchon and Warkentin Downloaded by guest on September 27, 2021 2 2 2 number of egg masses laid and the location of each mass (terrestrial, ϭ 6.51; pair, ␹ 2,284 ϭ 0.73; cage, ␹ 2,282 ϭ 2.20; date, ␹ 2,280 ϭ 0.45; shade ϫ 2 surface, or submerged). For a subset of surface masses (n ϭ 66 masses by 28 pond interaction, ␹ 4,276 ϭ 3.89). pairs), we also counted the number of eggs in terrestrial, surface, and submerged positions within the mass. ACKNOWLEDGMENTS. We thank C. Schneider and M. Hughey for comments Oviposition site was modeled by using a multinomial logistic regression on the manuscript, J. Urbina for field assistance, and M.J. West-Eberhard and (MLR) in R version 2.6.0 (62). Predictors were: (i) shade, (ii) pond of origin, W. Wcislo for advice. This work was conducted at the Smithsonian Tropical Research Institute under permits from the Panamanian National Environmen- ϫ (iii) mating pair, (iv) cage, (v) date, and (vi) a shade pond of origin tal Authority, and was funded by National Science Foundation Grants IBN- interaction. Shade was the only significant factor, thus other variables were 0234439 and DDIG-0508811, Boston University, the Smithsonian Institution, 2 2 excluded from the final model (MLR: shade, ␹ 2,290 ϭ 111.81; pond, ␹ 4,286 and the Animal Behavior Society.

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Touchon and Warkentin PNAS ͉ May 27, 2008 ͉ vol. 105 ͉ no. 21 ͉ 7499 Downloaded by guest on September 27, 2021