Abstract
The hip-pocket frog (Assa darlingtoni), a small terrestrial myobatrachid frog found in mid-eastern Australia, has a highly derived, unusual, reproductive mode involving a unique form of male parental care. Males have subcutaneous pouches that open near the hip, and the developing tadpoles are carried in these pouches to post metamorphosis. It is found on several isolated mountain ranges in closed forest habitats, associated with high rainfall and temperate or sub-tropical climates. We established genetic relationships among specimens sampled across the range using phylogenetic analyses of thousands of single nucleotide polymorphisms (SNPs) from the nuclear genome and mitochondrial ND2 gene nucleotide sequences. These analyses uncovered two lineages that are genetically distinct in both nDNA and mtDNA analyses and that have low levels of divergence in male advertisement calls and are morphologically cryptic. Our data support separate species status for each lineage, based on the molecular genetic data. The first, which we name as a new species, Assa wollumbin sp. nov., is restricted to a single mountain, Wollumbin (= Mount Warning), the eroded cone of an ancient shield volcano—the Tweed Volcano. The second, the nominal species A. darlingtoni, has a wider distribution in five geographically disjunct subpopulations along 430 km of the Great Dividing Range in south-eastern Queensland and north-eastern New South Wales. The distributions of the two species closely approach within 15 km of each other on the central plug and rim of the caldera of the Tweed Volcano. Assa wollumbin sp. nov. meets the conservation criteria for Critically Endangered [A3(e), B2(a,b)]. When all subpopulations of A. darlingtoni are combined the conservation assessment is Endangered [A3(e), B2(a,b)]. Because of the fragmented nature of the distribution of A. darlingtoni, combined with the genetic evidence of concordant sub-structuring, we also conducted a conservation assessment on the five subpopulations. Two were assessed as Critically Endangered (D’Aguilar Range and Conondale/Blackall Ranges), and the remainder as Endangered (Dorrigo Plateau, McPherson Ranges, and Gibraltar Ranges/Washpool).
References
Anstis, M. (2013) Tadpoles and frogs of Australia. New Holland Publishers, Sydney, 827 pp.
Beeman, K. (1998) Digital signal analysis, editing, and synthesis. In: Hopp, S.L. Owren, M.J. & Evans, C.S (Eds.), Animal Acoustic Communication. Springer, pp. 59–103. https://doi.org/10.1007/978-3-642-76220-8_3
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
Bryant, L.M. & Krosch, M.N. (2016) Lines in the land: a review of evidence for eastern Australia’s major biogeographical barriers to closed forest taxa. Biological Journal of the Linnean Society, 119, 238–264. https://doi.org/10.1111/bij.12821
Chapple, D.G., Hoskin, C.J., Chapple, S.N.J. & Thompson, M.B. (2011) Phylogeographic divergence in the widespread delicate skink (Lampropholis delicata) corresponds to dry habitat barriers in eastern Australia. BMC Evolutionary Biology, 11, 191. https://doi.org/10.1186/1471-2148-11-191
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
Clulow, S., Mahony, M.J, Elliott, L., Humfeld, S. & Gerhardt, H.C. (2017) Near-synchronous calling in the hip-pocket frog Assa darlingtoni. Bioacoustics, 26, 249–258. https://doi.org/10.1080/09524622.2016.1260054
de Queiroz, K. (1998) The general lineage concept of species, species criteria and the process of speciation. In: Howard, D.J. & Berlocher, S.H. (Eds.), Endless Forms, Species and Speciation. Oxford University Press, Oxford, pp. 57–75.
de Queiroz, K. (2007) Species concepts and species delimitation. Systematic Biology, 56, 879–886. https://doi.org/10.1080/10635150701701083
Donnellan, S.C, McGuigan, K., Knowles, R., Mahony, M.J. & Moritz, C. (1999) Genetic evidence for species boundaries in frogs of the Litoria citropa species-group (Anura: Hylidae). Australian Journal of Zoology, 47, 275–293. https://doi.org/10.1071/ZO99013
Ehmann, H. & Swan, G. (1985). Reproduction and development in the marsupial frog, Assa darlingtoni (Leptodactylidae, Anura). In: G. Grigg, R. Shine & H. Ehmann (Eds), Biology of Australasian frogs and reptiles. Surrey Beatty & Sons Pty Ltd, Chipping Norton and NSW in association with the Royal Zoological Society of New South Wales, New South Wales, pp. 279–285.
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
Familiar López, M. (2015) Distribution, ecology, disease and physiology of a mountain-top endemic frog in the face of climate change: a study on Philoria loveridgei. PhD thesis. Griffith University, Queensland, 135 pp. https://doi.org/10.25904/1912/2775
Frost, D.R. & Hillis, D.M. (1990) Species in concept and practice: herpetological applications. Herpetologica, 46, 86–104. https://www.jstor.org/stable/i376478
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
Gerhardt, H.C. & Huber, F. (2002) Acoustic communication in insects and anurans: common problems and diverse solutions. University of Chicago Press, Chicago, Illinois, 531 pp.
Greenfield, M.D. (1994) Synchronous and alternating choruses in insects and anurans: common mechanisms and diverse functions. American Zoologist, 34, 605–615. https://doi.org/10.1093/icb/34.6.605
Gruber, B., Unmack, P.J., Berry, O.F. & 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
Halsch, C.A., Shapiro, A.M., Fordyce, J.A., Nice, C.C., Thorne, J.H., Waetjen, D.P. & Forister, M.L. (2021) Insects and recent climate change. Proceedings of the National Academy of Sciences, 118, e2002543117 https://doi.org/10.1073/pnas.2002543117
Hero, J.-M., Morrison, C., Gillespie, G., Roberts, J.D, Newell, D., Meyer, E., McDonald, K., Lemckert, F.L., Mahony, M.J. & Osborne, W. (2006) Overview of the conservation status of Australian frogs. Pacific Conservation Biology, 12 (4), 313–320. https://doi.org/10.1071/PC060313
Hines, H., Mahony, M.J. & McDonald, K.R. (1999) An assessment of frog declines in wet subtropical Australia. In: Campbell, A. (Ed.), Declines and Disappearances of Australian Frogs. Biodiversity Group, Environment Australia, Canberra, pp. 44–63.
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
IUCN (2001) IUCN Red List Categories and Criteria: Version 3.1. IUCN Species Survival Commission. IUCN, Gland and Cambridge, 30 pp.
IUCN Standards and Petitions Committee. (2019) Guidelines for Using the IUCN Red List Categories and Criteria. Version 14. Prepared by the Standards and Petitions Committee. Available from: http://www.iucnredlist.org/documents/RedListGuidelines.pdf (accessed 18 August 2021)
Jombart, T. (2008) Adegenet: a R package for the multivariate analysis of genetic markers. Bioinformatics, 24, 1403–1405. https://doi.org/10.1093/bioinformatics/btn129
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
Katoh, K., Kuma, K., Toh, H. & Miyata, T. (2005) MAFFT version 5: improvement in accuracy of multiple sequence alignment. Nucleic Acids Research, 33, 511–518. https://doi.org/10.1093/nar/gki198
Kearse, M., Moir, R., Wilson, A., Stones-Havas, S., Cheung, M., Sturrock, S., Buxton, S., Cooper, A., Markowitz, S., Duran, C., Thierer, T., Ashton, B., Mentjies, P. & Drummond, A. (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
Keith, D.A., Mahony, M.J., Hines, H.B, Elith, J., Regan, T.J., Baumgartner, J.B., Hunter, D., Heard, G.W., Mitchell, N.J., Parris, K.M., Penman, T., Scheele, B., Simpson, C.C., Tingley, R., Tracy, C.R., West, M. & Akcakaya H.R. (2014) Detecting extinction risk from climate change by IUCN Red List criteria. Conservation Biology, 28, 810–819. https://doi.org/10.1111/cobi.12234
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_5.
Knowles, R., Mahony, M.J., Armstrong, J. & Donnellan, S,C. (2004) Systematics of sphagnum frogs of the genus Philoria (Anura : Myobatrachidae) in eastern Australia, with the description of two new species. Records of the Australian Museum, 56, 57–74.
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
Kriger, K.M. & Hero, J.M. (2007) The chytrid fungus Batrachochytrium dendrobatidis is non-randomly distributed across amphibian breeding habitats. Diversity and Distributions, 13, 781–788. https://doi.org/10.1111/j.1472-4642.2007:00394.x.
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
Lemckert, F.L. & Morse, R. (1999) Frogs in timber production forests of Dorigo escarpment in northern new South Wales: An inventory of species present and the conservation of threatened species. In: Campbell, A. (Ed.), Declines and Disappearances of Australian FrogsEnvironment Australia, . Canberra, pp. 72–80.
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
Loveridge, A. (1933) Four new crinine frogs from Australia. Occasional Papers of the Boston Society of Natural History, 8, 55–60.
Mahony, M.J. (2006) Amphibians of the Gibraltar Range. Proceedings of the Linnean Society of New South Wales, 127, 83–91.
Mahony, M.J., Moses, B., Mahony, S.V., Lemckert, F.L., & 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
McDonald, W. (2010) Part 1. Parks and Reserves in the Gondwana Rainforests. Chapter 4: The Tweed Caldera Group: In: Kitching, R, Braithwaite, R. & Cavanaugh, J. (Eds.), Remnants of Gondwana. A Natural and Social History of the Gondwana Rainforests of Australia. Surrey Beatty & Sons Pty Ltd. Chipping Norton, pp. 39–50.
McDonald, W. & Hunter, J. (2010) Part 2. The Natural Environment. Chapter 11. The Vegetation. In: Kitching, R, Braithwaite, R. & Cavanaugh, J. (Eds.), Remnants of Gondwana. A Natural and Social History of the Gondwana Rainforests of Australia. Surrey Beatty & Sons Pty Ltd, Chipping Norton, pp. 129–134.
McGuigan, K., McDonald, K.R., Parris, K.M. & Moritz, C. (1998) Mitochondrial DNA diversity and historical biogeography of a wet forest-restricted frog (Litoria pearsoniana) from mid-east Australia. Molecular Ecology, 7, 175–186. https://doi.org/10.1046/j.1365-294x.1998.00329.x
Moussalli, A., Hugall, A.F. & Moritz, C. (2005) A mitochondrial phylogeny of the rainforest skink genus Saproscincus, Wells and Wellington (1984). Molecular Phylogenetics and Evolution, 34, 190–202. https://doi.org/10.1016/j.ympev.2004.08.022
Murray, K., Retallick, R., McDonald, K.R., Mendez, D., Aplin, K., Kirkpatrick, P., Berger, L., Hunter, D., Hines, H.B., Campbell, R., Pauza, M., Driessen, M., Speare, R., Richards, S.J., Mahony, M., Freeman, A., Phillott, A.D. Hero, J.-M., Kriger, K., Driscoll, D., Felton, A., Puschendorf, R. & Skerratt, L.F. (2010) The distribution and host range of the pandemic disease chytridiomycosis in Australia, spanning surveys from 1956–2007. Ecology, 91, 1557–1558. https://doi.org/10.1890/09-1608.1
New South Wales National Parks and Wildlife Service. (1994) Fauna of New South Wales. Northeast Forest Biodiversity Study. National Parks and Wildlife Service, Hurstville, 124 pp.
Ollier, C. (1982). The Great Escarpment of eastern Australia: tectonic and geomorphic significance. Journal of the Geological Society of Australia, 29, 13–23.
Ostro, L.E., Young, T.P., Silver, S.C. & Koontz, F.W. (1999) A geographic information system method for estimating home range size. The Journal of Wildlife Management, 63, 748–755. https://doi.org/10.2307/3802859
Ponniah, M., & Hughes, J.M. (2004) The evolution of Queensland spiny mountain crayfish of the genus Euastacus. I. Testing vicariance and dispersal with interspecific mitochondrial DNA. Evolution, 58, 1073–1085.
Pritchard, J.K., Stephens, M. & Donnelly, P. (2000) Inference of population structure using multilocus genotype data. Genetics, 155, 945–959. https://doi.org/10.1093/genetics/155.2.945
Rambaut, A. & Drummond, A.J. (2007) Tracer. Version 1.4. Available from: http://beast.bio.ed.ac.uk/Tracer (accessed 13 October 2021)
Read, K., Keogh, J.S., Scott, I.A.W., Roberts, J.D. & Doughty, P. (2001) Molecular phylogeny of the Australian frog genera Crinia, Geocrinia, and allied taxa (Anura: Myobatrachidae). Molecular Phylogenetics and Evolution, 21, 294–308. https://doi.org/10.1006/mpev.2001.1014
Reside, A.E., VanDerWal, J., Phillips, B., Shoo, L.P., Rosauer, D.F., Anderson, B.A., Welbergen, J., Moritz, C., Ferrier, S., Harwood, T.D., Williams, K.J., Mackey, B., Hugh, S. & Williams, S.E. (2013) Climate change refugia for terrestrial biodiversity: Defining areas that promote species persistence and ecosystem resilience in the face of global climate change. National Climate Change Adaptation Research Facility, Gold Coast, 216 pp.
Reside, A.E., Welbergen, J.A., Phillips, B.L., Wardell‐Johnson, G.W., Keppel, G., Ferrier, S., Williams, S.E. & VanDerWal, J. (2014) Characteristics of climate change refugia for Australian biodiversity. Austral Ecology, 39, 887–897. https://doi.org/10.1111/aec.12146
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
Sauer, J., Domisch, S., Nowak, C. & Haase, P. (2011) Low mountain ranges: summit traps for montane freshwater species under climate change. Biodiversity and Conservation, 20, 3133–3146. https://doi.org/10.1007/s10531-011-0140-y
Shea, G. (2005) The type locality of the Sphagnum Frog Philoria loveridgei (Anura: Myobatrachidae). Memoirs-Queensland Museum, 51, 524.
Straughan, I. & Main, A. (1966) Speciation and polymorphism in the genus Crinia Tschudi (Anura, Leptodactylidae) in Queensland. Proceedings of the Royal Society of Queensland, 78, 11–28.
Swofford, D.L. (2003) PAUP*: phylogenetic analysis using parsimony, version 4.0 b10.
Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M. & Kumar, S. (2011) MEGA5: Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Molecular Biology and Evolution, 28, 2731–2739. https://doi.org/10.1093/molbev/msr121
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
Tyler, M.J. (1972) A new genus for the Australian leptodactylid frog Crinia darlingtoni. Zoologische Mededelingen. Leiden, 47, 193–201.
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
Wells, K.D. (2007) The Ecology and Behavior of Amphibians. University of Chicago Press, Chicago, Illinois, 1148 pp. https://doi.org/10.1016/j.anbehav.2008.03.001
Wiley, E.O. (1978) The evolutionary species concept reconsidered. Systematic Zoology, 27, 17–26. https://doi.org/10.2307/2412809