Abstract
Woodland salamanders of the genus Plethodon are characterized by strong ecological and morphological conservatism. One assemblage, the Wehrle’s salamander (Plethodon wehrlei Fowler & Dunn) species group, is distributed from New York to Tennessee, USA, and includes several morphological variants, four of which are sufficiently distinct to have been recognized as species in the past. For many years after two of these species were placed in synonymy, only P. wehrlei and P. punctatus Highton were recognized. A recent phylogeographic study using mitochondrial DNA and nuclear DNA uncovered considerable genetic diversity within the group and conservatively resurrected one of the previously synonymized forms (P. dixi Pope & Fowler). However, their analysis could not resolve all relationships among remaining populations of P. wehrlei, leaving the taxon paraphyletic. We re-evaluated the evolutionary history of this group using genomic data, recovered strong support for at least five distinct clades, and corroborated previously reported relationships. We also collected morphological data and demonstrated morphological distinctiveness for four of the five clades that we herein recognize as species. We resurrect the synonymized name P. jacksoni Newman to represent the southern clades of P. wehrlei in southwestern Virginia and North Carolina exclusive of P. dixi. In addition, we describe a yellow-spotted form of P. wehrlei endemic to the Cumberland Plateau as a new species. Although our proposed changes rectify the paraphyly of P. wehrlei, our sampling was not sufficient to resolve potential taxonomic issues remaining within the species herein recognized as P. jacksoni.
References
Adams, D.C., Berns, C.M., Kozak, K.H. & Wiens, J. (2009) Are rates of species diversification correlated with rates of morphological evolution? Proceedings of the Royal Society B, 276, 2729–2738.
https://doi.org/10.1098/rspb.2009.0543
Amphibia Web (2018) Amphibia Web. University of California, Berkeley, California. Available from: https://amphibiaweb.org (accessed 17 December 2018)
Anderson, J.A. & Tilley, S.G. (2003) Systematics of the Desmognathus ochrophaeus complex in the Cumberland Plateau of Tennessee. Herpetological Monographs, 17, 75–110.
https://doi.org/10.1655/0733-1347(2003)017[0075:SOTDOC]2.0.CO;2
Astley, H.C., Astley, V.E., Brothers, D. & Mendelson, J.R. III (2017) Digital analysis of photographs for snake length measurement. Herpetological Review, 48, 39–43.
Bayona-Vásquez, N.J, Glenn, T.C., Kieran, T.J, Pierson, T.W., Hoffberg, S.L., Scott, P.A., Bentley, K.E., Finger, J.W., Jr., Louha, S., Troendle, N., Díaz-Jaimes, P., Mauricio, R. & Faircloth, B.C. (2019) Adapterama III: Quadruple-indexed, triple-enzyme RADseq libraries for about 1 USD per sample (3RAD). bioRxiv. [published online]
http://dx.doi.org/10.1101/205799v3
Berendzen, P.B., Simons, A.M. & Wood, R.M. (2003) Phylogeography of the northern hogsucker, Hypentelium nigricans (Teleostei: Cypriniformes): genetic evidence for the existence of the ancient Teays River. Journal of Biogeography, 30, 1139–1152.
https://doi.org/10.1046/j.1365-2699.2003.00888.x
Bishop, S.C. (1947) Handbook of Salamanders: the Salamanders of the United States, of Canada, and of Lower California. Comstock Publishing Company, Ithaca, New York, 555 pp.
Bogert, C.M. (1954) Amphibians and reptiles of the world. In: Drimmer, F. (Ed.), The Animal Kingdom. Vol. 2. Book III. Doubleday & Co, New York, pp. 1189–1390.
Bryant, D. & Moulton, V. (2004) Neighbor-net: an agglomerative method for the construction of phylogenetic networks. Molecular Biology and Evolution, 21, 255–265.
https://doi.org/10.1093/molbev/msh018
Camp, C.D., Tilley, S.G., Austin, Jr. R.M. & Marshall, J.L. (2002) A new species of black-bellied salamander (genus Desmognathus) from the Appalachian Mountains of northern Georgia. Herpetologica, 58, 471–484.
https://doi.org/10.1655/0018-0831(2002)058[0471:ANSOBS]2.0.CO;2
Camp, C.D. & Wooten, J.A. (2016) Hidden in plain sight: cryptic diversity in the Plethodontidae. Copeia, 104, 111–117.
https://doi.org/10.1643/OT-14-150
Cochran, D.M. (1961) Type specimens of reptiles and amphibians in the US National Museum. Bulletin of the United States National Museum, 220, 1–291.
https://doi.org/10.5962/bhl.part.26967
Conant, R. (1958) A Field Guide to Reptiles and Amphibians of the United States and Canada East of the 100th Meridian. Houghton Mifflin Co, Boston, 366 pp.
Conant, R., Cagle, F.R., Goin, C.J., Lowe Jr., C.H., Neill, W.T., Netting, M.G., Schmidt, K.P., Shaw, C.E., Steggins, R.C. & Bogert, C.M. (1956) Common names for North American amphibians and reptiles. Copeia, 1956, 172–185.
https://doi.org/10.2307/1440446
Crother, B.I., Bonett, R.M., Boundy, J., Burbrink, F.T., de Queiroz, K., Frost, D.R., Highton, R., Iverson, J.B., Jockusch, E.L., Kraus, F., Krysko, K., Leaché, A.D., Moriarty Lemmon, E., McDiarmid, R.W., Mendelson III, J.R., Meylan, P.A., Reeder, T.W., Ruane, S. & Seidel, M.E. (2017). Scientific and standard English names of amphibians and reptiles of North America north of Mexico, with comments regarding confidence in our understanding, eighth edition. Society for the Study of Amphibians and Reptiles Herpetological Circular, 43, 1–102.
Cupp, P.V. Jr. & Towles, D.T. (1983) A new variant of Plethodon wehrlei in Kentucky and West Virginia. Transactions of the Kentucky Academy of Science, 44, 157–158.
Eaton, D.A.R. (2014) PyRAD: assembly of de novo RADseq loci for phylogenetic analyses. Bioinformatics, 30, 1844–9.
https://doi.org/10.1093/bioinformatics/btu121
Eaton, D.A.R. & Overcast, I. (2019) ipyrad: Interactive assembly and analysis of RAD-seq data sets. [in preparation]
Felix, Z.I., Wooten, J.A. & Rigsby, J. (2011) Taxonomic affinities of the yellow-spotted isolates of the Wehrle’s salamander (Plethodon wehrlei): preliminary data. Southeastern Biology, 58, 406–407.
Fontaneto, D., Flot, J. & Tang, C.Q. (2015) Guidelines for DNA taxonomy, with a focus on the meiofauna. Marine Biodiversity, 45, 433–451.
https://doi.org/10.1007/s12526-015-0319-7
Fowler, J. (1951) Preliminary observations on an aggregation of Plethodon dixi. Herpetologica, 7, 147–148.
Fowler, H.W. & Dunn, E.R. (1917) Notes on salamanders. Proceedings of the Academy of Natural Sciences of Philadelphia, 69 (1), 7–28.
Fujita, M.K., Leaché, A.D., Burbrink, F.T., McGuire, J.A. & Moritz, C. (2012) Coalescent-based species delimitation in an integrative taxonomy. Trends in Ecology and Evolution, 27, 480–488.
https://doi.org/10.1016/j.tree.2012.04.012
Funk, D.J. & Omland, K.E. (2003) Species-level paraphyly and polyphyly: frequency, causes, and consequences, with insights from animal mitochondrial DNA. Annual Review of Ecology, Evolution, and Systematics, 34, 397–423.
https://doi.org/10.1146/annurev.ecolsys.34.011802.132421
Graham, M.R. (2007) Distribution and conservation genetics of the Cow Knob salamander, Plethodon punctatus Highton (Caudata: Plethodontidae). MS thesis, Marshall University, Huntington, West Virginia, 76 pp.
Graham, C.F., Glenn, T.C., McArthur, A.G., Boreham, D.R., Kieran, T., Lance, S., Manzon, R.G., Martino, J.A., Pierson, T., Rogers, S.M. & Wilson, J.Y. (2015) Impacts of degraded DNA on restriction enzyme associated DNA sequencing (RADSeq). Molecular Ecology Resources, 15, 1304–1315.
https://doi.org/10.1111/1755-0998.12404
Guttman, S., Carlin, A.A. & Labanick, G.M. (1978) A biochemical and morphological analysis of the relationship between Plethodon longicrus and Plethodon yonahlossee (Amphibia, Urodela, Plethodontidae). Journal of Herpetology, 12, 445–454.
https://doi.org/10.2307/1563348
Hall, R.J., and Stafford, D.P. (1972) Studies in the life history of Wehrle’s Salamander, Plethodon wehrlei. Herpetologica, 28, 300–309.
Hanken, J. (1999) Why are there so many new amphibian species when amphibians are declining? Trends in Ecology and Evolution, 14, 7–8.
https://doi.org/10.1016/S0169-5347(98)01534-1
Highton, R. (1962) Revision of North American salamanders of the genus Plethodon. Bulletin of the Florida State Museum, 6, 235–367.
Highton, R. (1972) Distributional interactions among eastern North American salamanders of the genus Plethodon. In: Holt, P.C. (Ed.), The distributional history of the biota of the southern Appalachians. Part III. Vertebrates. Virginia Polytechnic Institute and State University, Blacksburg, Virginia, pp. 139–188.
Highton, R. (1987) Plethodon wehrlei Fowler and Dunn. Catalogue of American Amphibians and Reptiles, 402, 1–3.
Highton, R. (1988) Plethodon punctatus Highton. Catalogue of American Amphibians and Reptiles, 414, 1–2.
Highton, R. (1989) Biochemical evolution in the slimy salamanders of the Plethodon glutinosus complex in the eastern United States. Part I. Geographic protein variation. Illinois Biological Monographs, 57, 1–78.
https://doi.org/10.5962/bhl.title.49905
Highton, R. (1997) Geographic protein variation and speciation in the Plethodon dorsalis complex. Herpetologica, 53, 345–356.
Highton, R. (1999) Geographic protein variation and speciation in the salamanders of the Plethodon cinereus group with the description of two new species. Herpetologica, 55, 43–90.
Highton, R., Hatings, A.P., Palmer, C., Watts, R., Hass, C.A., Culver, M. & Arnold, S.J. (2012) Concurrent speciation in the eastern woodland salamanders (genus Plethodon): DNA sequences of the complete albumin nuclear and partial mitochondrial 12s genes. Molecular Phylogenetics and Evolution, 63, 278–290.
https://doi.org/10.1016/j.ympev.2011.12.018
Highton, R. & Larson, A. (1979) The genetic relationships of the salamanders of the genus Plethodon. Systematic Biology, 28, 579–599.
https://doi.org/10.2307/sysbio/28.4.579
Highton, R. & MacGregor, J.R. (1983) Plethodon kentucki Mittleman: A valid species of Cumberland Plateau woodland salamander. Herpetologica, 39, 189–200.
Highton, R. & Peabody, R.B. (2000) Geographic protein variation and speciation in salamanders of the Plethodon jordani and Plethodon glutinosus complexes in the Southern Appalachian Mountains with the description of four new species. In: Bruce, R.C., Jaeger, R.G. & Houck, L.D. (Eds.), The Biology of Plethodontid Salamanders. Springer, Boston, pp. 31–93.
https://doi.org/10.1007/978-1-4615-4255-1_3
Huelsenbeck, J.P. & Ronquist, F. (2001) MRBAYES: Bayesian inference of phylogenetic trees. Bioinformatics, 17, 754–755.
https://doi.org/10.1093/bioinformatics/17.8.754
Huson, D.H., Klöpper, T., Lockhart, P.J. & Steel, M.A (2005) Reconstruction of reticulate networks from gene trees. In: Annual International Conference on Research in Computational Molecular Biology. Springer, pp. 233–249.
https://doi.org/10.1007/11415770_18
IBM Corporation (2016) IBM SPSS Statistics for Windows. Version 24.0. IBM Corporation, Armonk, NY. [program]
Jaekel, M. & Wake, D.B. (2007) Developmental processes underlying the evolution of a derived foot morphology in salamanders, Proceedings of the National Academy of Sciences of the United States of America, 104, 20437–20442.
https://doi.org/10.1073/pnas.0710216105
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
Juterbock, J.E. (1984) Evidence for the recognition of specific status for Desmognathus welteri. Journal of Herpetology, 18, 240–255.
https://doi.org/10.2307/1564077
Köhler, J., Vieites, D.R., Bonett, R.M., García, F.H., Glaw, F., Steinke, D. & Vences, M. (2005) New amphibians and global conservation: a boost in species discoveries in a highly endangered vertebrate group. BioScience, 55, 693–696.
https://doi.org/10.1641/0006-3568(2005)055[0693:NAAGCA]2.0.CO;2
Kozak, K.H., Weisrock, D.W. & Larson, A. (2006) Rapid lineage accumulation in a non-adaptive radiation: phylogenetic analysis of diversification rates in eastern North American woodland salamanders (Plethodontidae: Plethodon). Proceedings of the Royal Society B, 273, 539–546.
https://doi.org/10.1098/rspb.2005.3326
Kozak, K.H. & Wiens, J. (2006) Does niche conservatism promote speciation? A case study in North American salamanders. Evolution, 60, 2604–2621.
https://doi.org/10.1111/j.0014-3820.2006.tb01893.x
Kozlov, A., Darriba, D., Flouri, T., Morel, B. & Stamatakis, A. (2018) RAxML-NG: A fast, scalable, and user-friendly tool for maximum likelihood phylogenetic inference. bioRxiv. [published online]
https://doi.org/10.1101/447110.s
Kuchta, S.R., Brown, A.D., Converse, P.E. & Highton, R. (2016) Multilocus phylogeography and species delimitation in the Cumberland Plateau salamander, Plethodon kentucki: incongruence among data sets and methods. PLoS ONE, 11, e0150022.
https://doi.org/10.1371/journal.pone.0150022
Kuchta, S.R., Brown, A.D. & Highton, R. (2018) Disintegrating over space and time: paraphyly and species delimitation in the Wehrle’s salamander complex. Zoologica Scripta, 47, 285–299.
https://doi.org/10.1111/zsc.12281
Lefébure, T., Douady, C.J., Gouy, M. & Gibert, J. (2006) Relationship between morphological taxonomy and molecular divergence within Crustacea: proposal of a molecular threshold to help species delimitation. Molecular Phylogenetics and Evolution, 40, 435–447.
https://doi.org/10.1016/j.ympev.2006.03.014
Mora, C., Tittensor, D.P., Adl, S., Simpson, A.G.B. & Worm, B. (2010) How many species are there on earth and in the ocean? PLoS Biology, 9, e1001127.
https://doi.org/10.1371/journal.pbio.1001127
Moritz, C., Schneider, C.J. & Wake D.B. (1992) Evolutionary relationships within the Ensatina eschscholtzii complex confirm the ring species interpretation. Systematic Biology, 41, 273–291.
https://doi.org/10.1093/sysbio/41.3.273
Narum, S.R. (2006) Beyond Bonferroni: less conservative analyses for conservation genetics. Conservation Genetics, 7, 783–787.
https://doi.org/10.1007/s10592-005-9056-y
NatureServe (2018) NatureServe Web Service. Arlington, Virginia. Available from: https://www.natureserve.org (accessed 31 December 2018)
Newman, W.B. (1954) A new plethodontid salamander from southwestern Virginia. Herpetologica, 10, 9–14.
Petranka, J.W. (1998) Salamanders of the United States and Canada. Smithsonian Institution Press, Washington, 587 pp.
Pope, C.H. & Fowler, J.A. (1949) A new species of salamander (Plethodon) from southwestern Virginia. Natural History Miscellanea Chicago Academy of Sciences, 47, 1–4.
Posada, D. & Crandall, K.A. (1998) MODELTEST: testing the model of DNA substitution. Bioinformatics, 14, 817–818.
https://doi.org/10.1093/bioinformatics/14.9.817
Puillandre, N., Modica, M.V., Zhang, Y., Sirovich, L., Boisselier, M.C., Cruaud, C., Holford, M. & Samadi, S. (2012) Molecular Ecology, 21, 2671–2691.
https://doi.org/10.1111/j.1365-294X.2012.05559.x
de Queiroz, K. (1998) The general lineage concept of species, species criteria, and the process of speciation: a conceptual unification and terminological recommendations. In: Howard, D.J. & Berlocher, S.H. (Eds.), Endless Forms: Species and Speciation. Oxford University Press, New York, pp. 57–75.
de Queiroz, K. (2005) Ernst Mayr and the modern concept of species. Proceedings of the National Academy of Sciences of the United States of America, 102, 6600–6607.
https://doi.org/10.1073/pnas.0502030102
R Core Team (2018) R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna. Available from: https://www.R-project.org/ (accessed 30 April 2019)
Redmond, W.H. & Jones, R.L. (1985) Plethodon wehrlei. Herpetological Review, 16, 31.
Rissler, L.L. & Apodaca, J.J. (2007) Adding more ecology into species delimitation: ecological niche models and phylogeography help define cryptic species in the black salamander (Aneides flavipunctatus). Systematic Biology, 56, 924–942.
https://doi.org/10.1080/10635150701703063
Rohlf, F.J. (2010) tpsDig, digitize landmarks and outlines. Version 2.16. Department of Ecology and Evolution, State University of New York, Stony Brook. [program]
Ronquist, F., Teslenko, M., van der Mark, P., Ayres, D.L., Darling, A., Höhna, S., Larget, B., Lui, L., Suchard, M.A. & Huelsenbeck, J.P. (2012) MrBayes 3.2: Efficient Bayesian phylogenetic inference and model choice across a large model space. Systematic Biology, 61, 539–542.
https://doi.org/10.1093/sysbio/sys029
Schliep K.P. (2011) phangorn: phylogenetic analysis in R. Bioinformatics, 27, 592–593.
https://doi.org/10.1093/bioinformatics/btq706
Schliep, K., Potts, A.J., Morrison, D.A. & Grimm, G.W. (2017) Intertwining phylogenetic trees and networks. Methods in Ecology and Evolution, 8, 1212–1220.
https://doi.org/10.1111/2041-210X.12760
Tilley, S.G. (1981) A new species of Desmognathus (Amphibia: Caudata: Plethodontidae) from the southern Appalachian Mountains. Occasional Papers of the Museum of Zoology, University of Michigan, 695, 1–23.
Tilley, S.G., Erikson, R.L. & Katz, L.A. (2008) Systematics of dusky salamanders, Desmognathus (Caudata, Plethodontidae) in the mountain and Piedmont regions of Virginia and North Carolina, USA. Zoological Journal of the Linnean Society, 152, 115–130.
https://doi.org/10.1111/j.1096-3642.2007.00336.x
United States Environmental Protection Agency (2006) Level III Ecoregions of the Continental United States. National Health and Environmental Effects Resource Lab, Corvalis, Oregon, map M-1.
Wake, D.B. (2009) What salamanders have taught us about evolution. Annual Review of Ecology, Evolution, and Systematics, 40, 333–352.
https://doi.org/10.1146/annurev.ecolsys.39.110707.173552
Waldron, J.L., Pauley, T.K., Felix, Z.I., Humphries, W.J. & Longenecker, A.J. (2001) The herpetofauna of the Bluestone National Wild and Scenic River. Proceedings of the West Virginia Academy of Science, 73, 1–10.
Wiens, J.J., Engstrom, T.N. & Chippindale, P.T. (2006) Rapid diversification, incomplete isolation, and the “speciation clock” in North American salamamanders (Genus Plethodon): testing the hybrid swarm hypothesis of rapid radiation. Evolution, 60, 2585–2603.
https://doi.org/10.1111/j.0014-3820.2006.tb01892.x
Wynn, A.H., Highton, R. & Jacobs, J.F. (1988) A new species of rock-crevice dwelling Plethodon from Pigeon Mountain, Georgia. Herpetologica, 44, 135–143.