Phylogenomic species delimitation of studfishes (Fundulidae: Fundulus): evidence for cryptic species in agreement with the central highlands vicariance hypothesis

The Central Highlands ecoregion of the eastern United States represents a hotspot of freshwater biodiversity, with replicated patterns of vicariant speciation east and west of the Mississippi River. Previous phylogeographic investigation of the studfishes (Fundulus subgenus Fundulus) revealed evidence for vicariant speciation in the Central Highlands, but data were limited to a small number of gene sequences generated with Sanger sequencing. We used double digest restriction-site associated DNA sequencing (ddRADseq) to improve resolution of phylogeographic patterns and better characterize population genetic variation. Our sample design included individuals from the Fundulus catenatus species group (F. catenatus, F. bifax, and F. stellifer) and two outgroup taxa (F. julisia and F. rathbuni). Phylogenetic analyses support a monophyletic F. catenatus complex and a sister relationship with Mobile Basin studfishes (F. bifax and F. stellifer). Population genomics and species delimitation tests provide evidence for three species-level subdivisions of F. catenatus. We describe F. catenatus as limited to the Tennessee River and its drainages, F. caddo sp. nov., in the Ouachita Highlands, and F. cryptocatenatus sp. nov., occupying the remainder of the range. Modally, F. catenatus was characterized by fewer left pectoral rays (16 vs. 17). Fundulus caddo sp. nov. had modally fewer anal rays (15 vs. 16) and lateral scale rows (13 vs. 14). Fundulus cryptocatenatus sp. nov. was characterized by modally higher dorsal rays (15 vs. 14) and fewer caudal rays (16 vs. 17). The geographic distribution is likely the result of multiple pre-Pleistocene vicariance events congruent with the Central Highlands Vicariance Hypothesis as well as separate, possibly subsequent, dispersal events. Overall, results of this study corroborate previous evidence for a complex biogeographic history of taxa endemic to rivers of the Central Highlands ecoregion. The improved resolution of genomic variation among studfish populations will guide future studies of morphological variation and will improve conservation plans for rare and endemic taxa in a freshwater biodiversity hotspot.