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Nidicolous development in Limnonectes limborgi (Anura, Dicroglossidae)

In: Amphibia-Reptilia
Authors:
Jodi J.L. Rowley 1Australian Museum, 6 College Street, Sydney, NSW, 2010, Australia

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Ronald Altig 2Department of Biological Sciences, Mississippi State University, MS 39762, USA

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The Southeast Asian frog Limnonectes limborgi and the closely related L. hascheanus have been considered direct developers for almost 50 years. We report that rather than having direct development, L. limborgi has nidicolous development (= eggs oviposited terrestrially and larvae are free-living but nonfeeding) from large, nonpigmented eggs laid in a terrestrial nest constructed by the male. Thirteen nests were observed at four sites in Cambodia and Vietnam, five of which contained clutches of up to 15 eggs or tadpoles. Calling males were in attendance regardless of the presence of or developmental stage of their progeny. Large yolk reserves present as late as stage 37 and the lack of organic material in the gut indicate that these tadpoles do not feed. It is likely that L. hascheanus also has nidicolous development.

Limnonectes limborgi and L. hascheanus are closely related dicroglossine frogs from Southeast Asia (Inger and Stuart, 2010). Both species oviposit eggs in terrestrial nests and have been assumed to be direct developers (Taylor, 1962; Dubois, 1987 “1986”; Manthey and Grossman, 1997; Ohler, Grosjean and Hoyos, 1999; Inger and Stuart, 2010). This assumption is based upon observations of L. limborgi (Thailand and Vietnam, incorrectly reported as L. hascheanus: Taylor, 1962; Ohler, Grosjean and Hoyos, 1999) and a species referred to as L. hascheanus (Borneo: Manthey and Grossman, 1997). Previous observations did not involve the entire developmental sequence even though Taylor (1962: 415) noted that the “tadpole stage is undergone in the egg, and there is no free swimming stage”. More complete observations verify a different developmental mode.

Observations and photographs of L. limborgi nests, eggs and tadpoles were made at two sites in Cambodia (Virachey National Park, Ratanakiri Province, 14.200°N, 107.383°E, 700 m elevation; Seima Biodiversity Conservation Area, Mondulkiri Province, 12.317°N, 107.099°E, 560 m elevation) and two sites in Vietnam (Nui Ong Nature Reserve, Binh Thuan Province, 12.027°N, 107.725°E, 230 m elevation; Pu Hoat Proposed Nature Reserve, Nghe An Province, 19.709°N, 104.754°E, 800 m elevation). All sites were within the known range of L. limborgi and outside the range of L. hascheanus. Identifications were confirmed morphologically and molecularly for adult specimens collected at Virachey National Park at the same time as nests were observed by Inger and Stuart (2010). Specimens from other sites deposited at the Australian Museum (AMS) were identified morphologically (see Appendix for specimens examined). Some specimens currently at the AMS will be deposited at the University of Science, Ho Chi Minh City (UNS) and have been cross-catalogued at both institutions. In these instances, voucher numbers are reported as UNS/AMS. All observations were made during the rainy season (March-August).

Figure 1
Figure 1

(A) Adult male of Limnonectes limborgi in the process of building a nest, (B) adult male within a completed nest, (C) an embryo at about stage 19, (D) clutch of 12, recently oviposited, nonpigmented eggs in a nest without free water, (E) 15 nonpigmented tadpoles at stage 28, and (F) seven tadpoles at about stage 37 in a small volume of fluid. This figure is published in colour in the online version.

Citation: Amphibia-Reptilia 33, 1 (2012) ; 10.1163/156853812X626179

Limnonectes limborgi nests were all located in evergreen forest more than 10 m away from any pool or stream. All nests were found by focusing on the male’s calls from within the nests that were completely covered in leaf litter; other nests without calling males may have gone undetected. Calling males (= nests) were spaced more than 1 m apart at all sites. When leaf litter was removed carefully, nine of the males jumped away from the nests and four remained in the nests. There was no obvious correlation with those remaining in the nests and the presence or absence of eggs or tadpoles. The males fit tightly within the confines of the nests (fig. 1A and B), and based on the average body size of male L. limborgi (33 mm SVL; Inger and Stuart, 2010) and other objects in the images, we estimated that the nests were 3-4 cm diameter and 2-3 cm deep. Ambient temperatures taken less than 10 cm from the nest sites were 25.5-28.7°C and relative humidity was 73.4-100% (Kestrel 3500 hand-held weather meter; Nielsen-Kellerman, Inc., Boothwyn, Pennsylvania, USA). The substrate was typically clay-like in appearance and moist, so the relative humidity experienced by the clutch under the leaf litter would likely be constantly close to 100%.

Of five nests found at Virachey National Park on 20-22 June 2006, one nest was empty, one had 12 eggs, and three others had 9, 10, and 15 tadpoles. Five empty nests found at Nui Ong Nature Reserve on 23-25 May 2009 were occupied by calling males. Two nests at Seima Biodiversity Conservation Area found on 1 August 2009 were empty, and one nest found at Pu Hoat Proposed Nature Reserve on 14 June 2010 had 2 eggs (other eggs may have been displaced while uncovering the nest). The comparable numbers of eggs and tadpoles observed in each nest (apart from the nest at Pu Hoat Proposed Nature Reserve) indicate that survival probability within individual nests is high.

In four cases, eggs or tadpoles were observed in the nests that spanned Gosner (1960) stages of about 4-37 (cleavage planes not visible in nonpigmented ova; fig. 1D). Each ovum had one jelly layer that appeared to be embedded in a large volume of very clear jelly, although such a clutch structure is unknown; one would have to observe oviposition to verify whether each egg has a large amount of outer jelly that melds with that of other eggs (a mass as defined by Altig and McDiarmid, 2007), or if an unstructured volume of jelly that encompasses all the ova is released as one unit at some point during oviposition. The figure of eggs of Babina adenopleura in Liu (1950) appear similar to those reported here and include one egg that is not melded into the mass. Eggs from a nest in Pu Hoat Proposed Nature Reserve (fig. 1C) measured about 4 mm in ovum diameter. All nests observed with eggs or tadpoles lacked free water, and the one nest partially filled by rain did not have either eggs or tadpoles. Nest construction under sufficient leaf litter to avoid the nest being flooded by rain may reflect the quality of the nest and the choices made by the attendant male. The small amount of fluid in the nests with eggs and tadpoles is likely derived from the large volume of jelly being partially liquefied by secretions from the tadpole’s hatching glands.

Embryos at stage 19 (fig. 1C) sit on top of the yolk as is characteristic of development of large, yolky eggs. Motile tadpoles at about stage 28 (about 10 mm total length) were nonpigmented and had large yolk reserves (fig. 1E); the nonpigmented tadpole tissues appeared pinkish relative to the white yolk. Tadpoles at about stage 37 (about 15 mm total length) were slightly mottled brown dorsally with a nonpigmented tail, and large yolk stores remain (fig. 1F). Tadpoles remained relatively motionless and feeding was never observed. Our observations verify that development is endotrophic and nidicolous (= eggs oviposited terrestrially and larvae are free-living but nonfeeding).

There is a wide range of reproductive strategies within the ‘ranoids’ of southeastern Asia compared to similar taxa from the New World and Europe. The genus Limnonectes includes a number of reproductive modes, such as the deposition of small tadpoles that infers internal fertilization (Iskandar and Tjan, 1985; Iskandar and Erdelen, 2006), endotrophic development in terrestrial nests, arboreal clutches that may be attended or not, and the typical anuran life cycle in both lotic and lentic water. Within this range, a continuum of modes involving nonaquatic breeding exists: building nests or laying eggs under leaf litter adjacent to or away from streams (L. arathooni in Sulawesi, Brown and Iskandar, 2000; L. palavanensis and L. finchi [including tadpole transport by the adult] from Sulawesi, Inger and Stuebing, 2005) and shallow nests or depressions in stream beds (L. leporinus and L. malesianus in Borneo, Inger and Stuebing [2005], L. leporinus reported as Rana blythii by Emerson [1992]; L. poilani in Vietnam reported as L. blythii by Orlov [1997]). Terrestrial egg attendance has also been reported in L. arathooni in Sulawesi (Brown and Iskandar, 2000), and L. palavanensis and L. finchi from Borneo (Inger and Stuebing, 2005). Depositing eggs away from permanent water in streams is likely an attempt at avoiding aquatic predators (Cochran, 1955; Crump, 1974) that is made possible by high soil water content and a humid microhabitat on the forest floor. The isolation of a given male in a nest may also isolate him from overt competition from other males. This mode is quite different from Neotropical hylids that construct nests in sandy soil (Martins, 1993) and have tadpoles with typical development that escape to a larger body of water at about stage 25. These eggs, laid as a surface film, do not survive if they sink because of rain or other physical disturbances.

Attendance by a calling male at the nests of L. limborgi suggest that a given male may attract multiple mates to the same nest, but we never observed embryos of two ages in one nest. Skin secretions from the attendant male that are deposited during nest construction and future attendance may also inhibit fungal or bacterial infections (Harris et al., 2006; Woodhams et al., 2007; Little et al., 2008). Choice of nest location (e.g., proper soil and moisture regimen; Mitchell, 2001) and nest construction (e.g., Lehtonen and Wong, 2009) by the males warrants further study. Closer examinations over time are needed for eggs of various taxa.

Direct development, a developmental mode not known for Limnonectes, was apparently assumed by previous observers based simply on the presence of large, nonpigmented, terrestrial eggs. Our observations change the thesis of Marmayou et al. (2000) because the direct development in the genus Philautus from arboreal eggs on leaves differs greatly from the nidicolous development of Limnonectes (as Taylorana) in terrestrial nests made and guarded by males. Because of the high energy density required in an endotrophic egg, clutch sizes are never large in those species that have nidicolous development, but the proximity of nests that surely vary in quality also suggests that the presence of polyandry should be questioned (Byrne and Keogh, 2009). Much more data will be required (e.g. clutches/time, egg energy content and survivorship) before we can understand the entire reproduction of endotrophic species.

Acknowledgements

H.E. Dr Mok M., Senior Minister of Environment, H.E. Chay S. General Director of Administration for Nature Conservation and Protection, Peov S. from the Forestry Administration, Seng B. and D. Emmett from Conservation International, Neang T. from the Ministry of Environment and Flora and Fauna International Cambodia and staff at the Wildlife Conservation Society Cambodia kindly facilitated fieldwork in Cambodia. The Vietnamese Ministry of Agriculture and Rural Development, Vinh University, the University of Science Ho Chi Minh City, the Ministry of public security (Nghe An Province), and Forest Protection Department and protected areas staff kindly facilitated surveys in Vietnam. The research was supported by grants from ADM Capital Foundation, Ocean Park Conservation Foundation Hong Kong, The John D. and Catherine T. MacArthur Foundation, the Lawrence Foundation, Conservation International, and the Annie Alexander Endowment, the Museum of Vertebrate Zoology, University of California, Berkeley. Neang T., Hoang D.H., Le T.T.D., Dau Q.V., D. Emmett, Cao T.T., C. Minshew, Phung T.H., Nguyen T.L., Hong S., Thoy R., Somm S., Kin S., Hun S., Ith S., Chey K., Le N.T., Hoang D.T., and Le T.T. assisted with field work. D.T. Iskandar and an anonymous reviewer made many helpful comments on earlier drafts of the manuscript. For all this assistance we are most grateful.

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Appendix

Appendix: Limnonectes limborgi specimens examined

Seima Biodiversity Conservation Area, Mondulkiri Province, Cambodia: AMS R 173983-173984.

Nui Ong Nature Reserve, Binh Thuan Province, Vietnam: AMS R 173216, AMS R 173217, AMS R 173243, AMS R 173244, AMS R 173379, UNS 00215/AMS R 173245, UNS 00216/AMS R 173248, UNS 00217/AMS R 173255, UNS 00218/AMS R 173302, UNS 00219/AMS R 173300.

Pu Hoat Proposed Nature Reserve, Nghe An Province, Vietnam: AMS R 176088, AMS R 176190, AMS R 176221.

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