Abstract
In some environments prevailing conditions are unpredictable, thus anuran species use bet-hedging strategies and produce eggs of varied sizes. We investigated whether four species of Physalaemus (two from open, two from forest habitats) exhibit bet-hedging strategies, and if intra-clutch variation in egg size is greater for species that breed in the more unpredictable ponds of open habitats. All species studied adopted the bet-hedging strategy, with intra-clutch variation in egg size regardless of the environment; however, we found greater intra-clutch variation in the two species from open areas. The lower variation in egg-size found within forest habitats may be explained by the more stable environments that forest ponds provide for anuran eggs/embryos. Future studies in a phylogenetic context are needed to confirm the patterns detected here.
The amount of resources that a female provides affects both maternal and offspring fitness (Bernardo, 1996). When resources for reproduction are limited, there is a trade-off between fecundity and offspring survival (Trivers, 1974); several small offspring versus few large offspring (Lack, 1947; Lloyd, 1987). How females balance this trade-off has been central to life history studies for over 60 years (Lack, 1947; Crump, 1979; Yeager and Gibbons, 2013). The first model proposed to explain this trade-off predicted that a single optimal offspring size should be maintained in constant environmental conditions (Smith and Fretwell, 1974). There is evidence that in unfavorable conditions with heavy predation pressure (Petranka, Kats and Sih, 1987; Kaplan, 1992) or high levels of competition (Marshall, Cook and Emlet, 2006; Allen, Buckley and Marshall, 2008), larger individuals are favored, because they are less susceptible to predation and have greater competitive ability. On the other hand, in favorable conditions, individuals free of these risks can survive, even those that are small, so females can optimize their fitness by producing a large number of small offspring (Sibly, Calow and Smith, 1988; Tamate and Maekawa, 2000).
This model, however, assumes a constant environment, and if an offspring’s environment is variable and unpredictable, individuals should avoid producing offspring that are specialized for a particular environmental condition (Olofsson, Ripa and Jonzén, 2009). Indeed, there is theoretical and empirical evidence of variation in offspring size in such non-constant environments (Seger and Brockmann, 1987; Dziminski and Alford, 2005; Yeager and Gibbons, 2013).
Three main provisioning strategies have been described for unpredictable environments: ‘conservative bet-hedging’, a low-risk strategy in which females consistently produce larger offspring (Seger and Brockmann, 1987); ‘diversified bet-hedging’, in which females produce offspring of variable sizes, thereby at least some of the offspring (larger) will survive if unfavorable conditions prevail, whereas most will survive if conditions are favorable (Koops, Hutchings and Adams, 2003); and finally, ‘adaptive coin-flipping’ (Cooper and Kaplan, 1982), in which females randomly produce many small or fewer large offspring (Olofsson, Ripa and Jonzén, 2009). This last strategy results in variation in clutch size of the same female over years and between females in a population, such that some females may be closer to the optimal egg size than others (Kaplan and Cooper, 1984).
Organisms with complex life cycles, such as anurans with aquatic larvae and terrestrial adults, are interesting subjects for studying maternal provisioning trade-off strategies (Allen, Buckley and Marshall, 2008). Anurans also exhibit a great diversity of reproductive modes, including a great variety of environments for offspring development (Haddad and Prado, 2005).
Among anurans, the genus Physalaemus, with 47 species distributed from central Argentina to the Guyanas (Frost, 2017), is a good model to test hypotheses about maternal provisioning trade-off strategies because these species breed in a wide variety of habitats from more constant ponds in rainforests to more unpredictable habitats such as temporary water bodies in open seasonal environments of the Cerrado, Chaco, and Caatinga domains (Cei, 1980; Frost, 2017). Species of Physalaemus generally deposit eggs in foam nests and tadpoles develop in puddles (e.g. Lynch, 1971). However, some species, such as P. atlanticus, P. crombiei, P. signifier and P. spiniger, can exhibit alternative reproductive modes, such as depositing foam nests directly on the humid forest floor, on water accumulated in axils of bromeliads, or inside fallen leaves or tree holes on the forest floor (Haddad and Pombal, 1998; Haddad and Prado, 2005; Pupin et al., 2010).
Since differences in predictability of habitats and in the natural history of species may represent different challenges for females in provisioning eggs, we compared intra-clutch variation among species of Physalaemus inhabiting contrasting environments. We tested the following predictions: (1) Physalaemus species, which use primarily temporary ponds for reproduction, will exhibit intra-clutch variation in egg size; and (2) species that use more unpredictable habitats to breed, such as ponds in open seasonal areas (Cerrado and Pantanal), will produce clutches with greater variability in egg size than species reproducing in more constant habitats, such as ponds inside forests (Atlantic rainforest).
Mean ± standard deviation (SD) of egg volume (mm3) and intra-clutch coefficient of variation (CV) of egg volume in species of Physalaemus of forest and open areas. Values are given as mean ± standard deviation with range and sample size in parenthesis.
We collected clutches of four species; two that breed in ponds in open seasonal environments (P. albonotatus and P. cuvieri) and two that breed in ponds inside rainforests (P. atlanticus and P. crombiei). The clutches were fixed in 10% formalin before late cleavage when the eggs start to increase in size (Kaplan, 1979). To ensure that clutches were produced by different individuals we included only clutches collected on the same day, since there is no record of repeated deposition or clutch-splitting for any species of Physalaemus. Furthermore, the clutches collected for each species were spaced 30 cm to 1 m away from each other. Ten clutches of P. albonotatus were collected in ponds on the Santa Clara farm, municipality of Corumbá, Mato Grosso do Sul State, on October 20, 2000; five clutches of P. cuvieri were collected in a temporary pond on the Mata Negra farm, municipality of Rio Claro, São Paulo State, on November 25, 2015; five clutches of P. crombiei were collected in small puddles in a remnant of Atlantic Forest in Aracruz, Espírito Santo State, on November 23, 1994; and eight clutches of P. atlanticus were collected in ponds in the Atlantic Forest in Ubatuba, São Paulo State, on January 20, 1999. Except for the state of Mato Grosso do Sul, which is located in the Central-West Region of Brazil, the remaining sample sites are located in the Southeast Region of Brazil. We randomly chose 40 eggs per clutch and photographed each egg under a stereomicroscope (Zeiss Discovery. V20). Using photo-capturing software (Zen pro 2012), we traced the circumference of the eggs in the same software that was used to calculate the egg area. All eggs of each clutch were in the same stage of development when photographed and egg jelly capsules were not included in the measurements. We estimated egg volume following Dziminski and Alford (2005), using , where volume in cubic units, and area in square units. To test for intra-clutch variation in egg size, we performed a paired t-test comparing the smallest and the largest egg of each clutch for each species (sensu Dziminski and Roberts, 2006). We calculated the intra-clutch coefficient of variation (CV = SD/mean) for egg volume for each clutch and compared them between species from open ( clutches) and forest ( clutches) areas using the Mann-Whitney test (Zar, 1999). Results were considered statistically significant when .
The largest and the smallest egg were significantly different for all species analyzed: P. atlanticus (, ), P. crombiei (, ), P. albonotatus (, ), and P. cuvieri (, ). All Physalaemus species exhibited intra-clutch variation. The intra-clutch CV ranged from 0.08 to 0.21 when considering all species, and varied among clutches of the same species (see table 1, fig. 1B). Species from forest habitats exhibited less intra-clutch variation in egg size than species from open areas (Mann-Whitney , ) (table 1, fig. 1A).
(A) Distribution of intra-clutch coefficient of variation of egg volume (CV) for species of Physalaemus from forest and open areas. (B) Intra-clutch coefficients of variation of egg volume (CV) for each clutch for species from forest and open areas.
Citation: Amphibia-Reptilia 39, 1 (2018) ; 10.1163/15685381-00003146
Even with our limited sample, we found intra-clutch variation in egg size in all species analyzed, regardless of the environment. This suggests that none of the species employed the ‘optimal egg size strategy’ (Smith and Fretwell, 1974), but rather the ‘diversified bet-hedging strategy’ (Seger and Brockmann, 1987). There is considerable empirical and theoretical evidence that when the offspring environment is unpredictable, the bet-hedging strategy is adaptive (Seger and Brockmann, 1987; Lips, 2001; Marshall, Bonduriansky and Bussièri, 2008). Indeed, Physalaemus clutches both in open and forest habitats are deposited in temporary ponds, which can flood and dry several times during the year. Although possessing different levels of severity in open and forest habitats, these events lead to great uncertainty regarding pond duration, availability of resources, levels of competition and predation (Wellborn, Skelly and Werner, 1996; Alford, 1999), which may act as important selective forces favoring bet-hedging strategies.
Larger eggs result in earlier metamorphosis (Dziminski and Roberts, 2006), which enables hatchlings to be better competitors (Allen, Buckley and Marshall, 2008) and decreases predation risks (Richards and Bull, 1990); on the other hand, smaller eggs favor the production of a greater number of offspring (Kaplan and Cooper, 1984). Depending on prevailing conditions, one strategy can be more advantageous in relation to the other and if conditions are uncertain, as was the case for the species studied here, choosing the wrong strategy may lead to the loss of an entire clutch (Yeager and Gibbons, 2013).
Consistent with our prediction, the difference in CVs between open and forest environments was significant; the open area species, Physalaemus albonotatus and P. cuvieri, had greater intra-clutch variation (mean CVs 0.16 and 0.15, respectively), than the forest species, P. atlanticus and P. crombiei (both with CV = 0.12). Species from open habitats, such as the Cerrado and Pantanal, breed in temporary ponds exposed to direct solar radiation, low annual rainfall and a long dry season, resulting in greater uncertainty about pond duration (Prado, Uetanabaro and Haddad, 2005). In contrast, temporary ponds inside the Atlantic Forest experience high humidity and rainfall, making these more constant environments (Haddad and Prado, 2005). However, we recognize that a larger sample size is needed. Future studies should investigate a larger number of Physalaemus species to confirm the pattern we detected here.
The species studied here possess different reproductive modes, which may have had an influence on differences in intra-clutch variation. The forest species, P. atlanticus and P. crombiei, have smaller clutches with larger eggs than do other Physalaemus species (Pupin et al., 2010), a characteristic of the alternative reproductive modes exhibited by these species. Besides depositing eggs in puddles, these two species can also deposit eggs on moist leaf litter, or in water accumulated in axils of bromeliads, rock crevices and holes of fallen trees (Haddad and Pombal, 1998; Haddad and Sazima, 2004; Pupin et al., 2010). Because these alternative micro-habitats offer limited food resources for hatchlings (Caldwell and de Araújo, 1998; Ryan and Barry, 2011), a greater provisioning for eggs is necessary to improve offspring survival, which may limit the range of variation in egg size.
Evolutionary history could also explain the variation in CVs among these species from different lineages (forest: P. signifer clade; open: P. cuvieri clade; see Lourenço et al., 2015). The small number of species analyzed here precluded us from investigating egg clutch variation in a phylogenetic context, which would help elucidate if the tendencies found are due to environmental differences, behavioral differences and/or evolutionary history. Nonetheless, our work presents an approach largely unexplored for South American anurans and may inspire similar studies with other species in unpredictable environments and/or with different life histories, or with the same species in different environments.
Acknowledgements
C.P.A. Prado and C.F.B. Haddad acknowledge the São Paulo Research Foundation (FAPESP, grants #2009/12013-4 and #2013/50741-7) for financial support and CNPq for research fellowships. N.C. Pupin acknowledges CNPq for a graduate fellowship. The authors are also grateful to the anonymous reviewers and the editor for valuable comments and criticisms.
References
Alford R.A. (1999): Ecology: resource use, competition and predation. In: Tadpoles: the Biology of Anurans Larvae, p. 240-278. Mc Diarmid R.W., Altig R., Eds, University of Chicago Press, Chicago.
Allen R.M., Buckley Y.M., Marshall D.J. (2008): Offspring size plasticity in response to intraspecific competition: an adaptive maternal effect across life-history stages. Am. Nat. 171: 225-237.
Bernardo J. (1996): The particular maternal effect of propagule size, especially egg size: patterns, models, quality of evidence and interpretations. Am. Zool. 36: 216-236.
Caldwell J.P., de Araújo M.C. (1998): Cannibalistic interactions resulting from indiscriminate predatory behavior in tadpoles of poison frogs (Anura: Dendrobatidae). Biotropica 30: 92-103.
Cei J.M. (1980): Amphibians of Argentina. Monitore Zool. Ital. (NS) Monogr. 2: 1-609.
Cooper W.S., Kaplan R.H. (1982): Adaptive “coin-flippin” a decision-theoretic examination of natural selection for random individual variation. J. Theor. Biol. 94: 135-151.
Crump M.L. (1979): Intra-population variability in energy parameters of the salamandra Plethodon cinereus. Oecologia 38: 235-247.
Dziminski M.A., Alford R.A. (2005): Patterns and fitness consequences of intraclutch variation in egg provisioning in tropical Australian frogs. Oecologia 146: 98-109.
Dziminski M.A., Roberts J.D. (2006): Fitness consequences of variable maternal provisioning in quacking frogs (Crinia georgiana). J. Evol. Biol. 19: 144-155.
Frost D.R. (2017): Amphibian species of the world: an online reference. Version 6.0 (23 January, 2017). Electronic database accessible at http://research.amnh.org/herpetology/amphibia/index.html. American Museum of Natural History, New York, USA.
Haddad C.F.B., Pombal J.P. Jr. (1998): Redescription of Physalaemus spiniger (Anura: Leptodactylidae) and description of two new reproductive modes. J. Herpetol. 32: 557-565.
Haddad C.F.B., Prado C.P.A. (2005): Reproductive modes in frogs and their unexpected diversity in the Atlantic forest of Brazil. Bioscience 55: 207-217.
Haddad C.F.B., Sazima I. (2004): A new species of Physalaemus (Amphibia; Leptodactylidae) from the Atlantic forest in southeastern Brazil. Zootaxa 479: 1-12.
Kaplan R.H. (1979): Ontogenetic variation in ovum “size” in two species of Ambystoma. Copeia 1979: 348-350.
Kaplan R.H. (1992): Greater maternal investment can decrease offspring survival in the frog Bombina orientalis. Ecology 73: 280-288.
Kaplan R.H., Cooper W.S. (1984): The evolution of developmental plasticity in reproductive characteristics: an application of the “Adaptive coin-flipping” principle. Am. Nat. 123: 393-410.
Koops M.A., Hutchings J.A., Adams B.K. (2003): Environmental predictability and the cost of imperfect information: influences on offspring size variability. Evol. Ecol. Res. 5: 29-42.
Lack D. (1947): The significance of clutch size. Ibis 89: 302-352.
Lips K. (2001): Reproductive trade-offs and bet-hedging in Hyla calypsa, a Neotropical treefrog. Oecologia 128: 509-518.
Lloyd D.G. (1987): Selection of offspring size at independence and other size-versus-number strategies. Am. Nat. 129: 800-817.
Lourenço L.B., Targueta C.P., Baldo D., Nascimento J., Garcia P.C.A., Andrade G.V., Haddad C.F.B., Recco-Pimentel S.M. (2015): Phylogeny of frogs from the genus Physalaemus (Anura, Leptodactylidae) inferred from mitochondrial and nuclear gene sequences. Mol. Phylogenet. Evol. 92: 204-2016.
Lynch J.D. (1971): Evolutionary relationships, osteology and zoogeography of leptodactyloid frogs. Misc. Publ. Mus. Nat. Hist. Univ. Kansas 53: 1-238.
Marshall D.J., Cook C.N., Emlet R.B. (2006): Offspring size effects mediate competition in a colonial marine invertebrate. Ecology 87: 214-225.
Marshall D.J., Bonduriansky R., Bussièri L.F. (2008): Offspring size variation within broods as a bet-hedging strategy in unpredictable environments. Ecology 89: 2506-2517.
Olofsson H., Ripa J., Jonzén N. (2009): Bet-hedging as an evolutionary game: the trade-off between egg size and number. Proc. R. Soc. B 276: 2963-2969.
Petranka J.W., Kats L.B., Sih A. (1987): Predator-prey interactions among fish and larval amphibians: use of chemical cues to detect predatory fish. Anim. Behav. 35: 420-425.
Prado C.P.A., Uetanabaro M., Haddad C.F.B. (2005): Breeding activity patterns, reproductive modes, and habitat use by anurans (Amphibia) in a seasonal environment in the Pantanal, Brazil. Amphibia-Reptilia 26: 211-221.
Pupin N.C., Gasparini J.L., Bastos R.P., Haddad C.F.B., Prado C.P.A. (2010): Reproductive biology of an endemic Physalaemus of the Brazilian Atlantic forest and the trade-off between clutch and egg size in terrestrial breeders of the P. signifer group. Herpetol. J. 20: 147-156.
Richards S.J., Bull C.M. (1990): Size-limited predation on tadpoles of three Australian frogs. Copeia 1990: 1041-1046.
Ryan M.J., Barry D.S. (2011): Competitive interactions in phytotelmata – breeding pools of two poison-dart frogs (Anura: Dendrobatidae) in Costa Rica. J. Herpetol. 45: 438-443.
Seger J., Brockmann J.H. (1987): What is bet-hedging? In: Oxford Surveys in Evolutionary Biology, p. 182-221. Harvey P.H., Partridge L., Eds, Oxford University Press, Oxford.
Sibly R., Calow P., Smith R.H. (1988): Optimal size of seasonal breeders. J. Theor. Biol. 133: 13-21.
Smith C.C., Fretwell S.D. (1974): The optimal balance between size and number of offspring. Am. Nat. 108: 499-506.
Tamate T., Maekawa K. (2000): Interpopulation variation in reproductive traits of female masu salmon, Oncorhynchus masou. Oikos 90: 209-2018.
Trivers R.J. (1974): Parent-offspring conflict. Am. Zool. 14: 249-264.
Wellborn G.A., Skelly D.K., Werner E.E. (1996): Mechanisms creating community structure across a freshwater habitat gradient. Annu. Rev. Ecol. Syst. 27: 337-363.
Yeager C.R., Gibbons M.E. (2013): Maternal provisioning trade-off strategies of Agalychnis callidryas. J. Herpetol. 47: 459-465.
Zar J. (1999): Biostatistical Analysis. Prentice Hall, Upper Saddle River, New Jersey.
Footnotes
Associate Editor: Marc Mazerolle.
