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
evolution. Vertebrate reproduction began in water, and terrestrial
eggs evolved multiple times in fishes and amphibians and in the
amniote ancestor. Because oxygen uptake from water conflicts
with water retention in air, egg adaptations to one environment
typically preclude development in the other. Few animals have
variable reproductive modes, and no vertebrates are known to lay
eggs both in water and on land. We report phenotypic plasticity of
reproduction with aquatic and terrestrial egg deposition by a frog.
The treefrog Dendropsophus ebraccatus, known to lay eggs ter-
restrially, also lays eggs in water, both at the surface and fully
above a pond. Under unshaded conditions, in a disturbed habitat
and in experimental mesocosms, these frogs lay most of their egg
masses aquatically. The same pairs also can lay eggs terrestrially,
on vegetation over water, even during a single night. Eggs can
survive in both aquatic and terrestrial environments, and variable
mortality risks in each may make oviposition plasticity adaptive.
Phylogenetically, D. ebraccatus branches from the basal node in a
clade of terrestrially breeding species, nested within a larger
lineage of aquatic-breeding frogs. Reproductive plasticity in D.
ebraccatus may represent a retained ancestral state intermediate
in the evolution of terrestrial reproduction.
aquatic egg-laying ? evolution of reproductive mode ? Hyla ebraccata ?
phenotypic plasticity ? climate change
vertebrates (1–10). In both groups, aquatic predators and con-
straints on oxygen uptake are hypothesized to select for terres-
trial eggs (2, 7–11). Terrestrial eggs can improve the embryonic
respiratory environment, allow oviposition over fast-moving
streams where aquatic eggs might be swept away, and allow
animals to colonize habitats without permanent water bodies (2,
4, 7–10). However, terrestrial eggs experience new risks from
desiccation and terrestrial predators (2, 4, 7–10). Because
aquatic and terrestrial environments select for different traits,
eggs are usually well adapted to only one environment (2, 4).
Adaptations for terrestrial oviposition have evolved indepen-
dently in several groups [e.g., gastropods (8, 12), insects (9, 13),
and fishes and amphibians (1, 2, 4, 10)]. In all of these organisms,
the divergence in reproductive mode [oviposition site and type
of egg development (1, 2, 10)] occurred long ago, and it is thus
difficult to directly assess selective pressures that may have
influenced such evolution. Closely related species or populations
that vary in their reproductive modes [e.g., between viviparity
and oviparity (14–18)] offer the best opportunity to study the
selective pressures leading to reproductive mode diversification.
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.
We studied the reproductive mode of the Neotropical treefrog
Dendropsophus ebraccatus [formerly Hyla ebraccata (21)] in
mesocosms and at three ponds near Gamboa, Panama. D.
to have semiterrestrial reproduction; its eggs are laid on vege-
he evolution of terrestrially developing eggs from ancestral
aquatic eggs is a repeated trend in both invertebrates and
tation above water and develop for 3–4 days, then aquatic
tadpoles fall into the water upon hatching (Fig. 1A) (1, 2, 10, 22,
23). Using mesocosm experiments and observations of natural
clutches, we report the discovery of reproductive mode plasticity
in D. ebraccatus, and we argue that this plasticity is most likely
adaptive. Adult D. ebraccatus are capable of laying eggs either
aquatically, both at the surface of the water and fully submerged,
or terrestrially, and they choose their reproductive mode based
on factors that affect risk of terrestrial egg desiccation.
Congruent with previous reports (2, 10, 22, 23), over 5 years at two
observed only terrestrial oviposition (350 closely monitored
in an old gravel quarry (Quarry Pond) that lacks forest canopy over
supported by aquatic vegetation. Some clutches were laid at the
water surface or across the air–water interface so that some eggs
were submerged, others were in contact with both air and water,
surface. Of the 148 clutches that we found at Quarry Pond during
2006–2007, 28% were submerged (all eggs completely under wa-
ter), 48% were laid at the water surface, and 24% were laid
terrestrially (no eggs in contact with water). The terrestrially laid
eggs at Quarry Pond had significantly higher mortality from
(8 ? 2%) or Ocelot Ponds (0.4 ? 0.2%) presumably because of
differences in shading (Kruskal–Wallis test: nQuarry? 27, nBridge?
151, nOcelot? 73, ?2? 37.97, P ? 5.69 ? 10?9; pairwise Wilcoxon
rank-sum tests, Quarry–Bridge, P ? 0.00023, Quarry–Ocelot, P ?
5.0 ? 10?6, Bridge–Ocelot, P ? 0.0013) (Fig. 2). Mortality of eggs
laid in the water at Quarry Pond (including submerged and surface
that of flooded terrestrial eggs previously observed at Bridge and
Ocelot Ponds (61 ? 6%, n ? 52; Wilcoxon rank-sum test, P ?
Our observation of 76% aquatic oviposition by D. ebraccatus
forest ponds (Ocelot and Bridge) could reflect either local
genetic differentiation, with some polymorphism among indi-
viduals at Quarry Pond, or individual plastic responses to
differences in their environments. To test for plasticity, we
manipulated exposure to forest canopy shade, a variable that
affects egg desiccation rate and clearly differs between our study
ponds (see Materials and Methods for details). We quantified
oviposition choices of frogs from all three ponds in shaded and
Author contributions: J.C.T. and K.M.W. designed research; J.C.T. performed research;
J.C.T. analyzed data; and J.C.T. and K.M.W. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
*To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
© 2008 by The National Academy of Sciences of the USA
May 27, 2008 ?
vol. 105 ?
no. 21 ?
unshaded experimental mesocosms. Terrestrial clutches were
laid completely out of the water on emergent vegetation (e.g.,
Fig. 1A). Aquatic clutches were laid both at the water surface
and under water (e.g., Fig. 1 B and C).
Frogs in unshaded mesocosms laid more water-surface and
submerged egg masses than did frogs in shaded mesocosms,
where the majority of egg masses were laid terrestrially (n ? 67
pairs tested, 293 egg masses laid; multinomial logistic regression,
?22,290 ? 111.81, P ? 2.0 ? 10?16) (Fig. 3). On average,
water-surface egg masses were 35 ? 4% in contact with or under
water and 65 ? 5% above the water. Frogs from all three ponds
laid eggs aquatically in unshaded mesocosms, and 48% of pairs
laid both aquatic and terrestrial egg masses in a single night.
There was neither a main effect of pond nor a significant
interaction of pond with shade treatment, indicating that ovi-
position site is plastic at all ponds tested (see Materials and
of eggs laid per pair in shaded or unshaded mesocosms (linear
model, F1,65? 2.3, P ? 0.13), and water-surface, submerged, and
terrestrial egg mass sizes did not differ between environments
(F2,287? 0.74, P ? 0.48). However, pairs in unshaded mesocosms
partitioned their eggs into significantly smaller masses (49 ? 3
F1,287? 12.94, P ? 0.0004). Pairs in unshaded mesocosms also
laid their terrestrial clutches closer to the water (5.8 ? 1.3 cm
high) than did pairs in shaded mesocosms (13.8 ? 1.2 cm high;
linear model, F1,151? 12.43, P ? 0.0006).
To the best of our knowledge, no vertebrates have been described
to plastically lay eggs both in water and on land. In this study, we
report such plasticity for D. ebraccatus. Natural variation in ovipo-
sition site is not due to local genetic differentiation between ponds
or polymorphism within Quarry Pond, but instead reflects the
plastic behavioral responses of frogs to their different environ-
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
(B), and under water (C). Aquatic oviposition occurs in unshaded sites where terrestrial eggs desiccate rapidly. (B) Vegetation ranges from a few centimeters
(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.
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
Quarry Bridge Ocelot
Clutch mortality (proportion)
three ponds near Gamboa, Panama. Quarry Pond (n ? 27 clutches) where
desiccation is highest is least shaded, Bridge Pond (n ? 151) is intermediate,
and Ocelot Pond (n ? 73) has the most shade over terrestrial oviposition sites.
The ponds are located within 2 km of each other and were monitored
concurrently, so clutches at all ponds experienced the same weather. Most
eggs at Quarry Pond are laid aquatically, thus few terrestrial clutches were
available to monitor. Data are mean ? SEM.
Desiccation of terrestrial egg clutches of treefrogs, D. ebraccatus, at
Proportion of egg clutches laid
shaded and unshaded experimental mesocosms. Frogs laid most of their egg
1 B and C) in unshaded mesocosms and most terrestrially (Fig. 1A) in shaded
mesocosms. Data are proportion of egg masses in each category (n ? 293 egg
masses laid by 67 pairs from three ponds).
Terrestrial and aquatic oviposition by treefrogs, D. ebraccatus, in
www.pnas.org?cgi?doi?10.1073?pnas.0711579105Touchon and Warkentin
a salamander (17) that vary in oviparity/viviparity and a frog that
produces foamy and nonfoamy egg masses (24). A normally sexual
lizard and a normally sexual shark also have both shown a presum-
ably plastic capacity for parthenogenesis by females isolated from
males in captivity (25, 26).
Many Old World treefrogs of the family Rhacophoridae lay
their eggs arboreally within foam nests (2, 10), and nests
attributed to single species have been found in diverse locations,
including on tree trunks, on the ground near stream banks, and
even floating on top of water (10, 19, 20, 27–32). Although
embryos in foam nests are protected from desiccation and
supplied with oxygen from the foam, rhacophorid foam nest
placement might vary with environmental conditions such as
humidity, temperature, or forest cover; to the best of our
knowledge, this has not yet been tested. Nest placement could,
however, simply reflect the availability of oviposition sites (20,
29). Several Old and New World frogs lay eggs adjacent to water,
on stream banks or moss, in excavated nests, or on low-lying
vegetation. These eggs can become inundated with water after
heavy rains (10, 11), as can terrestrially laid D. ebraccatus
clutches, so that the embryos then experience an aquatic envi-
ronment. Eggs laid near the air–water interface may therefore
experience more environmental variation over development
than is reflected in their initial oviposition sites.
Reproductive mode plasticity is poorly documented in any
taxon, and, to our knowledge, only two other species, a mosquito
(33) and a dragonfly (34), are known to lay both terrestrial and
aquatic eggs. Like D. ebraccatus, they choose oviposition sites in
response to factors affecting egg desiccation risk. These insects
are likely not the only invertebrates capable of such plasticity.
Similarly, D. ebraccatus may not be the only amphibian capable
of both aquatic and terrestrial reproduction. Researchers study-
ing amphibian reproduction, ourselves included, had not previ-
ously documented plasticity in aquatic/terrestrial oviposition (1,
2, 10, 23, 35–37) perhaps because reproductive mode was
considered fixed. However, the range of oviposition sites used by
D. ebraccatus and the environmentally induced variation in how
aquatic oviposition are not fully dichotomous. Rather, they may
represent ends of a continuum, and oviposition site plasticity
may be an intermediate stage in the evolution of obligate
D. ebraccatus eggs, although apparently not very well adapted
to development either in air or under water, can develop in both
environments and appear physiologically more flexible than
obligate aquatic or terrestrial eggs. Aquatic eggs exposed to air
typically dry out rapidly and die (e.g., 38), although some
salamander egg masses can survive prolonged terrestrial strand-
ing (39). Desiccation mortality of terrestrial amphibian eggs is,
however, generally low because clutches are laid in humid
locations and are surrounded by a protective, water-rich jelly (2,
4, 10). During this study, desiccation killed an average of 20% of
D. ebraccatus eggs (Fig. 2). During drier periods within the rainy
season, we have observed ?50% desiccation mortality of ter-
restrial clutches at the same ponds (J.C.T. and K.M.W., unpub-
lished data). This rate is far greater than the desiccation mor-
tality reported for many other terrestrially breeding frogs (40–
43). In contrast, D. ebraccatus embryos are more capable of
aquatic development than other terrestrial amphibian eggs,
which die if submerged before hatching competence (40, 42,
44–48). D. ebraccatus eggs can develop normally under water,
although developmental retardation and death occur if eggs are
deep in the water column, where oxygen is lower. Other more
strictly aquatic eggs also can suffocate if they fall to the pond
bottom [e.g., Hypsiboas rosenbergi (49)], and many aquatic-
breeding amphibians attach their eggs to vegetation or other
structures in the water to hold them near the water surface (2,
10, 23). Both the rapid desiccation of terrestrial D. ebraccatus
eggs and their ability to survive under water are likely due to
their small size [1.2–1.4 mm in diameter (23)]. D. ebraccatus eggs
are smaller than most terrestrial anuran eggs (10), which both
reduces their oxygen demand and increases their surface area-
(50). Highlighting this point, Wells (10) referred to D. ebraccatus
clutches as ‘‘little more than aquatic egg masses transferred to
terrestrial oviposition sites.’’
Given spatial and temporal variability in factors affecting
as in environment-specific predators, aquatic/terrestrial repro-
ductive plasticity may be adaptive. We observed higher desic-
cation mortality of terrestrial eggs at Quarry Pond than at either
Bridge or Ocelot Ponds likely because of less shade above
clutches (Fig. 2). In contrast, mortality of aquatically laid eggs at
Quarry Pond was lower than that of flooded eggs previously
observed at Bridge and Ocelot Ponds. Aquatic egg predators,
including several species of fish and tadpoles, are abundant in all
flooded, they are exposed to these predators in the water
are on or in a dense mat of floating vegetation (mostly Salvinia)
that may shield eggs from detection and attacks by predators in
the more open water below. In addition, hypoxia is more likely
for flooded clutches, which may be deep in the water column,
than for eggs associated with floating vegetation, which remain
near the better-oxygenated surface of the water even as water
Variation among and even within ponds in factors such as tree
canopy, aquatic vegetation, depth, and predator communities
affects aquatic and terrestrial egg risks, altering selection on
aquatic and terrestrial oviposition. Reproductive mode variation
in D. ebraccatus occurs at the level of the individual, not across
populations (14–17), so that morphologically and physiologically
species therefore offers an excellent opportunity to study the
ecological factors influencing the evolution of terrestrial and
Reproductive plasticity also may occur in other species, par-
ticularly in clades within which terrestrial-breeding animals
evolved from aquatic-breeding ancestors and extant species vary
in reproductive mode. D. ebraccatus belongs to the D. leucophyl-
latus species group, a lineage of eight Central and South Amer-
ican species (21). D. ebraccatus is the sister species of all other
terrestrially breeding frogs in the lineage, and all species branch-
ing from more basal nodes breed aquatically (Fig. 4) (1, 21, 35,
51, 52). Reproductive plasticity in D. ebraccatus could therefore
represent an intermediate stage in the evolution of terrestrial
reproduction, combining a retained ancestral capacity for
aquatic development with a derived ability for terrestrial ovipo-
sition and development. If reproductive plasticity was present in
the common ancestor of D. ebraccatus and its relatives, it may
have helped facilitate the transition to obligate terrestrial egg
development. Alternatively, a plastic capacity for aquatic repro-
duction may have evolved secondarily from terrestrially breeding
ancestors of D. ebraccatus.
The genus Dendropsophus contains ?90 species of treefrogs
in nine species groups [plus some unassigned to any species
group (21)], among which terrestrial reproduction has evolved
independently at least four times (Fig. 4) (1, 35, 53, 54). A
complete phylogeny of the genus is lacking, as is information
about the reproductive mode of nearly half the species. Thus,
additional origins of terrestrial reproduction and/or other
reproductively plastic species may exist among these poorly
studied taxa. It also is possible that other well studied species,
like D. ebraccatus, harbor undocumented flexibility in egg
deposition site and the capacity of embryos to develop in
different environments. Further research within this genus and
Touchon and Warkentin PNAS ?
May 27, 2008 ?
vol. 105 ?
no. 21 ?
phylogenetic reconstructions of ancestral traits functionally
related to terrestrial and aquatic egg development (e.g., egg
size and clutch structure) should improve our understanding of
both the evolution of terrestrial reproduction and the role of
plasticity in evolution. An interesting candidate for compari-
son to Dendropsophus is the diverse African treefrog genus
Hyperolius, which contains ?100 species, some of which breed
aquatically, others terrestrially, at least one that lays water-
surface eggs in between the leaves of floating plants, as well as
many species for whom the reproductive mode is not yet known
The risk of terrestrial egg desiccation will likely increase in
the future (59), both because of habitat disturbance and
because rainfall patterns are changing in Panama (60, 61). By
2080–2099, the air temperature during June, July, and August,
peak breeding months for D. ebraccatus, is predicted to have
increased at least 2.0–2.5°C, whereas precipitation is predicted
to have decreased 10–15% and become more sporadic (61).
Less predictable rainfall and more disturbed habitats will
increase the mortality of terrestrially laid eggs (45), but
reproductive mode variation may help D. ebraccatus to persist
despite these changes. Frogs from all three ponds laid a small
subset of their eggs aquatically even in shaded mesocosms,
suggesting that aquatic oviposition also may occur at a low
frequency in forest ponds, perhaps as a bet-hedging strategy
(Fig. 3). This would provide a buffer against unpredictable
environmental variation, protecting a subset of eggs from the
possibility of desiccation. Additionally, frogs from ponds
where only terrestrial oviposition had been observed laid most
of their egg masses aquatically in unshaded mesocosms, sug-
gesting that such plasticity may be widespread in this species
(Fig. 3). To the extent that the cues for aquatic oviposition
accurately predict egg desiccation risk, this plasticity should
improve D. ebraccatus’ ability to survive in disturbed habitats
or under altered rainfall patterns. Frogs in unshaded meso-
cosms laid smaller egg masses, which would allow them to
spread eggs among more different microsites, but probably
also increases desiccation by increasing the ratio of edge to
central eggs in masses. Terrestrial egg masses were, however,
laid closer to the water in unshaded mesocosms, which would
increase their chances of transitory flooding in nature. This
behavior may be similar to that of amphibians that lay eggs
adjacent to water in the anticipation of flooding (11).
The proximate cues that stimulate D. ebraccatus adults to repro-
duce terrestrially or aquatically are unknown, but candidates in-
clude light level, temperature, and humidity. Clearly, cues are
ascertained shortly before oviposition because frogs moved from
their native pond to experimental mesocosms responded to their
new environment within a single night. If light level is the primary
cue, then the frogs will likely respond appropriately to forest
clearing, but not to changing rainfall patterns. However, if air
temperature or humidity is the indicator of open habitats, then
plasticity, as well as bet-hedging, might improve D. ebraccatus’
chances of persistence in a changing climate.
Materials and Methods
Field Monitoring. We located aquatically and terrestrially laid egg clutches
at Quarry Pond and terrestrial egg clutches at Ocelot and Bridge Ponds the
morning after oviposition during 2006–2007 and checked them twice daily
for the first 48 h. Afterward, aquatically laid eggs were hatching-
competent, making the fates of missing eggs ambiguous. Clutches were
categorized based on their contact with water and air (air only, terrestrial;
water and air, surface; water only, submerged). Terrestrial clutches were
found on vegetation overhanging water and emergent vegetation. At
Quarry Pond, aquatic egg clutches were primarily attached to Salvinia
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
the number of eggs that had died from desiccation (for terrestrial eggs),
from hypoxia (for aquatic eggs, characterized by developmental retarda-
tion before death), or missing because of predation (for all eggs). We
similarly monitored fates of terrestrially laid clutches at Ocelot and Bridge
Ponds that had been flooded during a different time period when pond
levels fluctuated more (2003–2005).
Testing for Reproductive Mode Plasticity. We constructed twelve 1.3-m-
diameter pond mesocosms containing both floating aquatic vegetation
and emergent vegetation and placed half under thick forest canopy
(shaded) and the rest nearby in an open field (unshaded). On eight nights
in 2006 and 2007, we collected pairs of frogs that had not yet begun laying
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
pair was used per mesocosm per night, thereby preventing competition for
Because variation in weather conditions might affect oviposition choices,
D. marmoratus group (8)
D. garagoensis group (4)
D. parviceps group (15)
D. columbianus group (3)
D. labialis group (3)
D. minutus group (4)
D. minimus group (4)
D. microcephalus group (33)
D. leucophyllatus group (8)
Aquatic egg laying
Terrestrial egg laying
Both aquatic and terrestrial egg laying in lineage
of known reproductive modes and relationships with other Dendropsophus
species groups. Aquatic reproduction is ancestral in the genus and the pre-
dominant reproductive mode, but terrestrial eggs have evolved at least four
times. Symbols indicate species groups where only aquatic or terrestrial egg
laying is known and where both reproductive modes are known for different
reproductive modes. The reproductive mode is unknown for ca. half the
species of Dendropsophus. Some of these species also may have reproductive
plasticity, and species with described reproductive modes may have unde-
scribed plasticity. Numbers in parentheses indicate number of species in each
which agrees with ref. 21 on placement of the members of the D. leucophyl-
lines) based on morphological data from ref. 64; and §, tentative placement
(indicated by dashed lines) based on morphological data from ref. 65. Repro-
ductive modes are from refs. 1, 35, 53, 54, 64, and 66–69. The reproductive
mode of D. rossalleni is speculated to be terrestrial (54), but is currently
Phylogeny of the D. leucophyllatus group, showing the distribution
www.pnas.org?cgi?doi?10.1073?pnas.0711579105 Touchon and Warkentin
number of egg masses laid and the location of each mass (terrestrial,
surface, or submerged). For a subset of surface masses (n ? 66 masses by 28
pairs), we also counted the number of eggs in terrestrial, surface, and
submerged positions within the mass.
Oviposition site was modeled by using a multinomial logistic regression
(MLR) in R version 2.6.0 (62). Predictors were: (i) shade, (ii) pond of origin,
(iii) mating pair, (iv) cage, (v) date, and (vi) a shade ? pond of origin
excluded from the final model (MLR: shade, ?22,290? 111.81; pond, ?24,286
? 6.51; pair, ?22,284? 0.73; cage, ?22,282? 2.20; date, ?22,280? 0.45; shade ?
pond interaction, ?24,276? 3.89).
ACKNOWLEDGMENTS. We thank C. Schneider and M. Hughey for comments
on the manuscript, J. Urbina for field assistance, and M.J. West-Eberhard and
W. Wcislo for advice. This work was conducted at the Smithsonian Tropical
tal Authority, and was funded by National Science Foundation Grants IBN-
0234439 and DDIG-0508811, Boston University, the Smithsonian Institution,
and the Animal Behavior Society.
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Touchon and Warkentin PNAS ?
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vol. 105 ?
no. 21 ?