Abstract
The rarely encountered giant burrowing frog, Heleioporus australiacus, is distributed widely in a variety of sclerophyll forest habitats east of the Great Dividing Range in south-eastern Australia. Analyses of variation in nucleotide sequences of the mitochondrial ND4 gene and thousands of nuclear gene SNPs revealed the presence of two deeply divergent lineages. Multivariate morphological comparisons show the two lineages differ in body proportions with > 91% of individuals being correctly classified in DFA. The two lineages differ in the number and size of spots on the lateral surfaces and the degree by which the cloaca is surrounded by colour patches. The mating calls are significantly different in number of pulses in the note. The presence of a F2 hybrid in the area where the distribution of the two taxa come into closest proximity leads us to assign subspecies status to the lineages, as we have not been able to assess the extent of potential genetic introgression. In our sampling, the F2 hybrid sample sits within an otherwise unsampled gap of ~90km between the distributions of the two lineages. The nominate northern sub-species is restricted to the Sydney Basin bioregion, while the newly recognised southern subspecies occurs from south of the Kangaroo Valley in the mid-southern coast of New South Wales to near Walhalla in central Gippsland in Victoria. The habitat of the two subspecies is remarkably similar. Adults spend large portions of their lives on the forest floor where they forage and burrow in a variety of vegetation communities. The southern subspecies occurs most commonly in dry sclerophyll forests with an open understory in the south and in open forest and heath communities with a dense understory in the north of its distribution. The northern subspecies is also found in dry open forests and heaths in association with eroded sandstone landscapes in the Sydney Basin bioregion. Males of both taxa call from both constructed burrows and open positions on small streams, differing from the five Western Australian species of Heleioporus where males call only from constructed burrows. Using the IUCN Red List process, we found that the extent of occupancy and area of occupancy along with evidence of decline for both subspecies are consistent with the criteria for Endangered (A2(c)B2(a)(b)).
References
Anderson, E.C. & Thompson, E.A. (2002) A model-based method for identifying species hybrids using multilocus genetic data. Genetics, 160, 1217−1229. https://doi.org/10.1093/genetics/160.3.1217
Anstis, M., Price, L.C., Roberts, J.D., Catalano, S.R., Hines, H.B., Doughty, P. & Donnellan, S.C. (2016) Revision of the water-holding frogs, Cyclorana platycephala (Anura: Hylidae), from arid Australia, including a description of a new species. Zootaxa, 4126 (4), 451–479. https://doi.org/10.11646/zootaxa.4126.4.1
Anstis, M. (2017) Tadpoles and frogs of Australia. New Holland Publishers Pty Limited, pp. 1–828.
Bailey, W.J. & Roberts, J.D. (1981) The bioacoustics of the burrowing frog Heleioporus (Leptodactylidae). Journal of Natural History, 15, 693–702. https://doi.org/10.1080/00222938100770491
Beeman, K. (1998) Digital signal analysis, editing, and synthesis. In: Animal Acoustic Communication. Springer, pp. 59–103. https://doi.org/10.1007/978-3-642-76220-8_3
Bilney, R.J. (2015) Observations of giant burrowing frogs Heleioporus australiacus (Limnodynastidae) in the Mitchell River catchment, East Gippsland, Victoria. Victorian Naturalist, 132, 128–133.
Bryant, D., Bouckaert, R., Felsenstein, J., Rosenberg, N.A. & RoyChoudhury, A. (2012) Inferring species trees directly from biallelic genetic markers: bypassing gene trees in a full coalescent analysis. Molecular Biology and Evolution, 29, 1917–1932. https://doi.org/10.1093/molbev/mss086
Chifman, J. & Kubatko, L. (2014) Quartet inference from SNP data under the coalescent model. Bioinformatics, 30, 3317–3324. https://doi.org/10.1093/bioinformatics/btu530
Cogger, H.G., Cameron, E.E. & Cogger, H.M. (1983) Amphibia and Reptilia. Zoological Catalogue of Australia. Vol. 1. Australian Government Publishing Service, Canberra 313 pp.
Daly, G. (1996) Observations of the Eastern Owl Frog Heleioporus australiacus (Anura: Myobatrachidae) in southern New South Wales. Herpetofauna, 26, 33–42.
Daly, G. (2019) The distribution and status of the Giant Burrowing Frog Heleioporus australiacus in the Shoalhaven region of south-eastern New South Wales. Australian Zoologist, 40, 256–271. https://doi.org/10.7882/AZ.2018.011
de Queiroz, K. (2011) Branches in the lines of descent: Charles Darwin and the evolution of the species concept. Biological Journal of the Linnean Society, 103, 19–35. https://doi.org/10.1111/j.1095-8312.2011.01634.x
de Queiroz, K. (2020) An updated concept of subspecies resolves a dispute about the taxonomy of incompletely separated lineages. Herpetological Review, 51, 459–461.
Donnellan, S.C., Hutchinson, M.N. & Saint, K.M. (1999) Molecular evidence for the phylogeny of Australian gekkonoid lizards. Biological Journal of the Linnean Society, 67, 97–118. https://doi.org/10.1111/j.1095-8312.1999.tb01932.x
Earl, D.A. (2012) Structure harvester: A website and program for visualizing structure output and implementing the Evanno method. Conservation Genetics Resources, 4, 359–361. https://doi.org/10.1007/s12686-011-9548-7
Edgar, R.C. (2004) MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Research, 32, 1792–1797. https://doi.org/10.1093/nar/gkh340.
Evanno, G., Regnaut, S. & Goudet, J. (2005) Detecting the number of clusters of individuals using the software STRUCTURE: a simulation study. Molecular Ecology, 14, 2611–2620. https://doi.org/10.1111/j.1365-294X.2005.02553.x
Fry, D. B. (1914) On a collection of reptiles and batrachians from Western Australia. Records of the Western Australian Museum, 1, 174–210.
Gerhardt, H. C. & Huber, F. (2002) Acoustic Communication in Insects and Anurans. Common problems and Diverse Solutions. The University of Chicago Press, Chicago, Illinois, 513 pp.
Georges, A., Gruber, B., Pauly, G.B., White, D., Adams, M., Young, M.J., Kilian, A., Zhang, X., Shaffer, H.B. & Unmack, P.J. (2018) Genome wide SNP markers breathe new life into phylogeography and species delimitation for the problematic short-necked turtles (Chelidae: Emydura) of eastern Australia. Molecular Ecology, 27, 5195–5213. https://doi.org/10.1111/mec.14925
Gillespie, G. (1990) Distribution, habitat and conservation status of the giant burrowing frog (Heleioporus australiacus) in Victoria. Victorian Naturalist, 5, 144–153.
Gruber, B., Unmack, P.J., Berry, O.F. and Georges, A., (2018) dartr: An r package to facilitate analysis of SNP data generated from reduced representation genome sequencing. Molecular Ecology Resources, 18, 691–699. https://doi.org/10.1111/1755-0998.12745
Hahn, C., Bachmann, L. & Chevreux, B. (2013) Reconstructing mitochondrial genomes directly from genomic next-generation sequencing reads—a baiting and iterative mapping approach. Nucleic Acids Research, 41, e129. https://doi.org/10.1093/nar/gkt371
Harrison, L. (1922) On the breeding habits of some Australian frogs. Australian Zoologist, 3, 17–34.
Hayek, L.-A.C., Heyer, W.R. & Gascon, C. (2001) Frog morphometrics: a cautionary tale. Alytes: International Journal of Batrachology, 18, 153–177.
Hillis, D.M. (2019) Species delimitation in herpetology. Journal of Herpetology, 53, 3–12. https://doi.org/10.1670/18-123
Hillis, D.M. (2020) The detection and naming of geographic variation within species. Herpetological Review, 51, 52–56.
Hoang, D.T., Chernomor, O., von Haeseler, A., Minh, B.Q. & Vinh, L.S. (2018) UFBoot2: Improving the ultrafast bootstrap approximation. Molecular Biology Evolution, 35, 518–522. https://doi.org/10.1093/molbev/msx281
Hoskin, C., Grigg, G., Stewart, D. & MacDonald, S. (2015) Frogs of Australia. 1.1 (4614) [Mobile application software]. Retrieved from http://www.ugmedia.com.au (accessed 13 July 2021)
IUCN Standards and Petitions Subcommittee (2016) Guidelines for Using the IUCN Red List Categories and Criteria. Version 12. Prepared by the Standards and Petitions Subcommittee, IUCN, Gland. Available from: http://s3.amazonaws.com/iucnredlistnewcms/staging/public/attachments/3151/redlistguidelines.pdf (accessed 13 July 2021)
Jombart, T., Devillard, S. & Balloux, F. (2010) Discriminant analysis of principal components: a new method for the analysis of genetically structured populations. BMC Genetics, 11, 94. https://doi.org/10.1186/1471-2156-11-94
Kalyaanamoorthy, S., Minh, B.Q., Wong, T.K.F., von Haeseler, A. & Jermiin, L.S. (2017) ModelFinder: fast model selection for accurate phylogenetic estimates. Nature Methods, 14, 587–589. https://doi.org/10.1038/nmeth.4285
Katsikaros, K. & Shine, R. (1997) Sexual dimorphism in the tusked frog, Adelotus brevis (Anura: Myobatrachidae): the roles of natural and sexual selection. Biological Journal of the Linnean Society, 60, 39–51. https://doi.org/10.1111/j.1095-8312.1997.tb01482.x
Kavanagh, R.P. & Webb, G.A. (1998) Effects of variable-intensity logging on mammals, reptiles and amphibians at Waratah Creek, south-eastern New South Wales. Pacific Conservation Biology, 4, 326–347. https://doi.org/10.1071/PC980326
Kearse, M., Moir, R., Wilson, A., Stones-Havas, S., Cheung, M., Sturrock, S., Buxton, S., Cooper, A., Markowitz, S. & Duran, C. (2012) Geneious Basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics, 28, 1647–1649. https://doi.org/10.1093/bioinformatics/bts199
Kilian, A., Wenzl, P., Huttner, E., Carling, J., Xia, L., Blois, H., Caig, V., Heller-Uszynska, K., Jaccoud, D., Hopper, C., Aschenbrenner-Kilian, M., Evers, M., Peng, K., Cayla, C., Hok, P. & Uszynski, G. (2012) Diversity arrays technology: A generic genome profiling technology on open platforms. Methods in Molecular Biology, 888, 67–89. https://doi.org/10.1007/978-1-61779-870-2
Köhler, J., Jansen, M., Rodríguez, A., Kok, P., Toledo, L., Emmrich, M., Glaw, F., Haddad, C. & Rödel, M. & Vences, M. (2017) The use of bioacoustics in anuran taxonomy: theory, terminology, methods and recommendations for best practice. Zootaxa, 4251 (1), 1–124. https://doi.org/10.11646/zootaxa.4251.1.1
Kopelman, N.M., Mayzel, J., Jakobsson, M., Rosenberg, N.A. & Mayrose, I. (2015) CLUMPAK: a program for identifying clustering modes and packaging population structure inferences across K. Molecular Ecology Resources, 15, 1179–1191. https://doi.org/10.1111/1755-0998.12387
Kumar, S., Stecher, G. & Tamura, K. (2016) MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets. Molecular Biology Evolution, 33, 1870–4. https://doi.org/10.1093/molbev/msw054.
Lardner, B. & bin Lakim, M. (2002) Tree-hole frogs exploit resonance effects. Nature, 420, 475. https://doi.org/10.1038/420475a
Leaché, A.D., Banbury, B.L., Felsenstein, J., de Oca, A.N. & Stamatakis A. (2015) Short tree, long tree, right tree, wrong tree: new acquisition bias corrections for inferring SNP phylogenies. Systematic Biology, 64, 1032–1047. https://doi.org/10.1093/sysbio/syv053
Lee, A. (1967) Studies in Australian amphibia II. Taxonomy, ecology and evolution of the genus Heleioporus Gray (Anura: Leptodactylidae). Australian Journal of Zoology, 15, 367–439. https://doi.org/10.1071/ZO9670367
Lee, J.C. (1982) Accuracy and precision in anuran morphometrics: artefacts of preservation. Systematic Biology, 31, 266–281. https://doi.org/10.1093/sysbio/31.3.266
Lemckert, F. & Brassil, T. (2003) Movements and habitat use by the giant burrowing frog, Heleioporus australiacus. Amphibia-Reptilia, 24, 207–211. https://doi.org/10.1163/156853803322390453
Lemckert, F., Brassil, T. & McCray, K. (1998) Recent records of the giant burrowing frog (Heleioporus australiacus) from the far south coast of NSW. Herpetofauna, 28, 32–39.
Lemmon, A.R., Emme, S.A. & Lemmon, E.M. (2012) Anchored Hybrid Enrichment for massively high-throughput phylogenomics. Systematic Biology, 61, 727–744. https://doi.org/10.1093/sysbio/sys049
Littlejohn, M.J. & Martin, A. (1967) The rediscovery of Heleioporus australiacus in eastern Victoria. Proceedings of the Royal Society of Victoria, 80, 1–35.
Littlejohn, M.J. (1987) Calls of Victorian Frogs. University of Melbourne,
Lleonart, J., Salat, J. & Torres, G.J. (2000) Removing allometric effects of body size in morphological analysis. Journal of Theoretical Biology, 205, 85–93. https://doi.org/10.1006/jtbi.2000.2043
Luedeling, E. (2019) chillR: statistical methods for phenology analysis in temperate fruit trees. R Package. Version 0.70.21. [program]
Lunney, D. & Barker, J. (1986) Survey of reptiles and amphibians of the coastal forests near Bega, NSW. Australian Zoologist, 22, 1–9. https://doi.org/10.7882/AZ.1986.001
Mace, G.M. & Lande, R. (1991) Assessing extinction threats: toward a re-evaluation of IUCN threatened species categories. Conservation Biology, 5, 148–157. https://doi.org/10.1111/j.1523-1739.1991.tb00119.x
Mahony, M.J., Moses, B., Mahony, S., Lemckert, F. & Donnellan, S.C. (2020). A new species of frog in the Litoria ewingii species group (Anura: Pelodryadidae) from south-eastern Australia. Zootaxa, 4858 (2), 201–230. https://doi.org/10.11646/zootaxa.4858.2.3
Main, A., Lee, A. & Littlejohn, M. (1958) Evolution in three genera of Australian frogs. Evolution, 1958, 224–233. https://doi.org/10.2307/2406033
Main, A.R. (1965) Frogs of southern Western Australia. Western Australian Naturalists Club, Perth, 60 pp.
Miller, M.A., Pfeiffer, W. & Schwartz, T. (2010) Creating the CIPRES Science Gateway for inference of large phylogenetic trees. In: Proceedings of the Gateway Computing Environments Workshop (GCE), New Orleans, Louisiana, 14 November 2010, pp. 1–8. https://doi.org/10.1109/GCE.2010.5676129
Mitchell, B.A., Callaghan, C.T. & Rowley, J.J.L. (2020) Continental-scale citizen science data reveal no changes in acoustic responses of a widespread treefrog to an urbanisation gradient. Journal of Urban Ecology, 2020, 1–12. https://doi.org/10.1093/jue/juaa002
Moore, J.A. (1961) The frogs of eastern New South Wales. Bulletin of the American Museum of Natural History, 121, 1–385.
Morgan, M.J., Roberts, J.D. & Keogh, J.S. (2007) Molecular phylogenetic dating supports an ancient endemic speciation model in Australia’s biodiversity hotspot. Molecular Phylogenetics and Evolution, 44, 371–385. https://doi.org/10.1016/j.ympev.2006.12.009
Mulcahy, D.G. (2008) Phylogeography and species boundaries of the western North American Nightsnake (Hypsiglena torquata): Revisiting the subspecies concept. Molecular Phylogenetics and Evolution, 46, 1095–1115. https://doi.org/10.1016/j.ympev.2007.12.012
Nguyen, L.-T., Schmidt, H.A., von Haeseler, A. & Minh, B.Q. (2015) IQ-TREE: A fast and effective stochastic algorithm for estimating maximum likelihood phylogenies. Molecular Biology Evolution, 32, 268–274. https://doi.org/10.1093/molbev/msu300
Penman, T., Lemckert, F. & Mahony, M. (2004) Two hundred and ten years of looking for giant burrowing frog. Australian Zoologist, 32, 597–604. https://doi.org/10.7882/AZ.2004.005
Penman, T., Lemckert, F., Slade, C. & Mahony, M. (2005) Non-breeding habitat requirements of the giant burrowing frog, Heleioporus australiacus (Anura: Myobatrachidae) in south-eastern Australia. Australian Zoologist, 33, 251–257. https://doi.org/10.7882/AZ.2005.022
Penman, T., Lemckert, F., Slade, C. & Mahony, M. (2006a) Description of breeding sites of the giant burrowing frog Heleioporus australiacus in south-eastern NSW. Herpetofauna, 36, 102–105. https://doi.org/10.1071/WR04081
Penman, T., Lemckert, F.L. & Mahony, M. (2006b) A preliminary investigation into the potential impacts of fire on a forest dependent burrowing frog species. Pacific Conservation Biology, 12, 78. https://doi.org/10.1071/PC060078
Penman, T., Mahony, M., Towerton, A. & Lemckert, F. (2007a) Spatial models of giant burrowing frog distributions. Endangered Species Research, 3, 115–124. https://doi.org/10.3354/esr00311
Penman, T.D., Lemckert, F.L. & Mahony, M. (2007b) Heleioporus australiacus predation. Herpetological Review, 38, 185–186. https://doi.org/10.1071/ZO08077
Penman, T.D., Keith, D.A., Elith, J.U., Mahony, M.J., Tingley, R., Baumgartner, J.B. & Regan, T. (2015) Interactive effects of climate change and fire on metapopulation viability of a forest-dependent frog in south-eastern Australia. Biological Conservation, 190, 142–153. https://doi.org/10.1016/j.biocon.2015.05.020
Penna, M. & Solis, R. (1996) Influence of burrow acoustics on sound reception by frogs Eupsophus (Leptodactylidae). Animal Behaviour, 51, 255–263. https://doi.org/10.1371/journal.pone.0134498
Pritchard, J.K., Stephens, M. & Donnelly, P. (2000) Inference of population structure using multilocus genotype data. Genetics, 155 (2), 945–959. https://doi.org/10.1093/genetics/155.2.945
Rambaut, A. & Drummond, A. (2007) Tracer. Version 1. 4: MCMC trace analyses tool. [software]
Rawlinson, P. (1974) Biogeography and ecology of the reptiles of Tasmania and the Bass Strait area. In: Biogeography and Ecology in Tasmania. Springer, The Hague, pp. 291–338. https://doi.org/10.1007/978-94-010-2337-5_12
Rescei, J. (1996) The eastern owl frog, Heleioporus australiacus. In, Ehmann, H. Threatened frogs of New South Wales: Habitat, status and conservation. Frog and Tadpole study Group of NSW, South Sydney, pp. 56–65.
Roberts, J.D. (1997) Call evolution in Neobatrachus (Anura: Myobatrachidae): speculations on tetraploid origins. Copeia, 1997, 791–801. https://doi.org/10.2307/1447296
Ronquist, F. & Huelsenbeck, J.P. (2003) MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics, 19, 1572–1574. https://doi.org/10.1093/bioinformatics/btg180
Rowley, J.J.L., Callaghan, C.T., Cutajar, T., Portway, C., Potter, K., Mahony, S, Trembath, D.F., Flemons, P. & Woods, A. (2019) FrogID: Citizen scientists provide validated biodiversity data on frogs of Australia. Herpetological Conservation and Biology, 14, 155–170. https://doi.org/10.3897/zookeys.912.38253
Sanders, J. & Davies, M. (1984) Burrowing behavior and associated hind limb myology in some Australian hylid and leptodactylid frogs. Australian Zoologist, 21, 123–142. https://doi.org/10.1002/ece3.6784
Shaw, G. & Nodder, F. (1795) Rana australiaca. Naturalist’s Miscellany London, 6, 200.
Skerratt, L.F., Berger, L., Speare, R., Cashins, S., McDonald, K.R., Phillott, A.D., Hines, H.B. & Kenyon, N. (2007) Spread of chytridiomycosis has caused the rapid global decline and extinction of frogs. Ecohealth, 4, 125. https://doi.org/10.1007/s10393-007-0093-5
Sparks, A.H., Padgham, M., Parsonage, H. & Pembleton, K. (2017) bomrang: Fetch Australian Government Bureau of Meteorology Weather Data. The Journal of Open Source Software, 2 (17), 411-1–411-2. https://doi.org/10.21105/joss.00411
Sparks, A.H., Carroll, J., Goldie, J., Marchiori, D., Melloy, P., Padgham, M., Parsonage, H. & Pembleton, K. (2020) bomrang: Australian Government Bureau of Meteorology (BOM) Data Client. R Package Version 0.7.1. Available from: https://CRAN.R-project.org/package=bomrang (accessed 13 July 2021)
Speare, R. & Berger, L. (2005) Chytridiomycosis status of wild amphibians in Australia. Available from: http://www.jcu.edu.au/school/phtm/PHTM/frogs/chy-au-status.htm (accessed 13 July 2021)
Stauber, A. (2006) Habitat requirement and habitat use of the Red-crowned Toadlet Pseudophryne australis and Giant Burrowing Frog Heleioporus australiacus in the Sydney Basin. PhD Thesis. University of Technology, Sydney, 263 pp.
Swofford, D.L. (2003) PAUP*: phylogenetic analysis using parsimony, version 4.0 b10.
Thackway, R. & Cresswell, I. (1995) An Interim Biogeographic Regionalisation for Australia: a framework for establishing the national system of reserves, Version 4.0. Australian Nature Conservation Agency, Canberra, 88 pp.
Thorpe, R.S. (1976) Biometric analysis of geographic variation and racial affinities. Biological Reviews, 51, 407–452. https://doi.org/10.1111/j.1469-185X.1976.tb01063.x
Trifinopoulos, J., Nguyen, L.T., von Haeseler A. & Minh, B.Q. (2016) W-IQ-TREE: a fast online phylogenetic tool for maximum likelihood analysis. Nucleic Acids Research, 44 (W1), W232–W235. https://doi.org/10.1093/nar/gkw256
Umbers, K.D.L., Lehtonen, J. & Mappes, J. (2015) Diematic displays. Current Biology, 25, 58–59. https://doi.org/10.1016/j.cub.2014.11.011
Watson, G. & Martin, A. (1973) Life history, larval morphology and relationships of Australian leptodactylid frogs. Transactions of the Royal Society of South Australia, 97, 33–35.
Watters, J.L., Cummings, S.T., Flanagan, R.L. & Siler, C.D. (2016) Review of morphometric measurements used in anuran species descriptions and recommendations for a standardized approach. Zootaxa, 4072 (4), 477–495. https://doi.org/10.11646/zootaxa.4072.4.6
Webb, G. (1991) A survey of the reptiles and amphibians of Bondi State Forest and surrounding areas, near Bombala, New South Wales. Australian Zoologist, 27, 14–19. https://doi.org/ 10.7882/AZ.1991.002
Wells, K.D. (2007) The Ecology and Behavior of Amphibians. University of Chicago Press, 1148 pp. https://doi.org/10.7208/chicago/9780226893334.001.0001
Williams, C.R., Brodie Jr., E.D., Tyler, M.J. & Walker, S.J. (2000) Antipredator mechanisms of Australian frogs. Journal of Herpetology, 34, 431–443. https://doi.org/10.1023/A:1025686418909