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Speciation with gene flow in a narrow endemic West Virginia cave salamander (Gyrinophilus subterraneus)


Due to their limited geographic distributions and specialized ecologies, cave species are often highly endemic and can be especially vulnerable to habitat degradation within and surrounding the cave systems they inhabit. We investigated the evolutionary history of the West Virginia Spring Salamander (Gyrinophilus subterraneus), estimated the population trend from historic and current survey data, and assessed the current potential for water quality threats to the cave habitat. Our genomic data (mtDNA sequence and ddRADseq-derived SNPs) reveal two, distinct evolutionary lineages within General Davis Cave corresponding to G. subterraneus and its widely distributed sister species, Gyrinophilus porphyriticus, that are also differentiable based on morphological traits. Genomic models of evolutionary history strongly support asymmetric and continuous gene flow between the two lineages, and hybrid classification analyses identify only parental and first generation cross (F1) progeny. Collectively, these results point to a rare case of sympatric speciation occurring within the cave, leading to strong support for continuing to recognize G. subterraneus as a distinct and unique species. Due to its specialized habitat requirements, the complete distribution of G. subterraneus is unresolved, but using survey data in its type locality (and currently the only known occupied site), we find that the population within General Davis Cave has possibly declined over the last 45 years. Finally, our measures of cave and surface stream water quality did not reveal evidence of water quality impairment and provide important baselines for future monitoring. In addition, our unexpected finding of a hybrid zone and partial reproductive isolation between G. subterraneus and G. porphyriticus warrants further attention to better understand the evolutionary and conservation implications of occasional hybridization between the species.

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Data availability

All data generated or analyzed during this study are included in this published article and its supplementary information files. The cytochrome-b sequence data for 59 samples (SRA), under BioProject PRJNA824885 (samples: SRR19240940-SRR19241032) will be available from GenBank [ON524106-ON524164] and Illumina sequence data are available on the Sequence Read Archive (SRA).


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This research was funded by the Amphibian Research and Monitoring Initiative (ARMI) program of the U.S. Geological Survey. Portions of the laboratory and computational work were conducted in and with the support of the L.A.B. facilities at the National Museum of Natural History. The computations in this paper were conducted on the Smithsonian High Performance Cluster (SI/HPC; G Capshaw and KP Mulder were supported by Smithsonian Institution Fellowships. We thank B Mosher, L Pekurny, R Bernard, J Fleming, K Oxenrider, and M Powell for help conducting the 2018 survey of the General Davis Cave and The Nature Conservancy for granting access. John R. Holsinger was instrumental for the General Davis Cave expedition of 1988. We thank D Portik for advice on running δaδi. We are grateful to three anonymous reviewers and the associate editor who provided constructive feedback to improve the manuscript. This is contribution number 803 of the ARMI program.


Any use of trade, firm, or product names is for descriptive purposes only and does not imply endorsement by the U.S. Government.


Funding information is not applicable/No funding was received.

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EHCG, KPM, ABB and RCB designed the study; EHCG, KPM, ABB, DBC, AW, GC, MLN, JGP, JFJ, SRK, and RCB contributed data; EHCG, KPM, DBC and RCB analyzed data; EHCG, KPM and RCB wrote the first draft of the manuscript; all authors contributed to revisions. All authors whose names appear on the submission: (1) made substantial contributions to the conception or design of the work; or the acquisition, analysis, or interpretation of data; or the creation of new software used in the work; (2) drafted the work or revised it critically for important intellectual content; (3) approved the version to be published; and (4) agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

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Correspondence to Evan H. Campbell Grant.

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Animal research

Research was conducted with approval from the U.S. Geological Survey Patuxent Wildlife Research Center ACUC.

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Grant, E.H.C., Mulder, K.P., Brand, A.B. et al. Speciation with gene flow in a narrow endemic West Virginia cave salamander (Gyrinophilus subterraneus). Conserv Genet 23, 727–744 (2022).

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  • Endangered species
  • Hybrid zone
  • Sympatric speciation
  • Amphibian decline
  • Troglobitic