Range fragmentation poses challenges for species persistence over time and may be caused by both historical and contemporary processes. We combined genomic data, phylogeographic model testing, and paleoclimate niche modeling to infer the evolutionary history of the Pine Barrens Treefrog (Hyla andersonii), a seepage bog specialist, in eastern North America to better understand the historical context of its fragmented distribution. We sampled H. andersonii populations across the three disjunct regions of the species’ range: Alabama/Florida (AF), the Carolinas (CL), and New Jersey (NJ). Phylogenetic relationships within H. andersonii were consistent between the nuclear species tree and mitochondrial analyses, indicating divergence between AF and CL/NJ (Atlantic clade) ~0.9 million years ago and divergence of the NJ clade ~0.15 mya. Several predictions of northeastern expansion along the Atlantic coast were supported by phylogeographic analyses. Model testing using genome-wide SNP data and species distribution models both provided evidence for multiple disjunct refugia. This comprehensive phylogeographic study of H. andersonii demonstrates a long history of range fragmentation within an endemic coastal plain species and highlights the influence of historical climate change on the current distribution of species and their genetic diversity.

Tissue samples were collected by hand from adult frogs and total genomic DNA was purified from toe clips. Samples were processed using anchored hybrid enrichment (Pseudacris v.1 probe set). Mitochondrial genomes were assembled as sequencing 'bycatch'. Nuclear and mitochondrial assemblies were separately aligned in MAFFT v.7.023b. Two nuclear datasets were generated: one with sequence alignments and one with extracted single nucleotide polymorphisms (SNPs). Divergence times were estimated in BEAST v.1.10.1 using the mitochondrial alignment. Nuclear phylogenetic relationships were estimated in SVDQuartets and ASTRAL. Tests for northeastern expansion and estimates of ancestor location, dispersal rate, and migration direction were done in PhyloMapper using mitochondrial data. The R package 'delimitR' tested phylogeographic models using the nuclear SNP data. Species distribution models were also generated using Maxent for contemporary and three historical time points; however, no GPS coordinates are provided publicly (request directly from the corresponding author).

ReadMe file included that describes all files. Aligned datasets (one mitochondrial and two nuclear) as input files to BEAST, SVDQuarterts, and ASTRAL. Input files for Phylomapper and delimitR are also included. Only input files for MaxEnt are not included (request directly from the corresponding author).