The effects of inference method, population sampling, and gene sampling on species tree inferences: an empirical study in slender salamanders (Plethodontidae: Batrachoseps).

Species tree methods are now widely used to infer the relationships among species from multilocus data sets. Many methods have been developed, which differ in whether gene and species trees are estimated simultaneously or sequentially, and in how gene trees are used to infer the species tree. While these methods perform well on simulated data, less is known about what impacts their performance on empirical data. We used a data set including five nuclear genes and one mitochondrial gene for 22 species of Batrachoseps to compare the effects of method of analysis, within-species sampling and gene sampling on species tree inferences. For this data set, the choice of inference method had the largest effect on the species tree topology. Exclusion of individual loci had large effects in *BEAST and STEM, but not in MP-EST. Different loci carried the greatest leverage in these different methods, showing that the causes of their disproportionate effects differ. Even though substantial information was present in the nuclear loci, the mitochondrial gene dominated the *BEAST species tree. This leverage is inherent to the mtDNA locus and results from its high variation and lower assumed ploidy. This mtDNA leverage may be problematic when mtDNA has undergone introgression, as is likely in this data set. By contrast, the leverage of RAG1 in STEM analyses does not reflect properties inherent to the locus, but rather results from a gene tree that is strongly discordant with all others, and is best explained by introgression between distantly related species. Within-species sampling was also important, especially in *BEAST analyses, as shown by differences in tree topology across 100 subsampled data sets. Despite the sensitivity of the species tree methods to multiple factors, five species groups, the relationships among these, and some relationships within them, are generally consistently resolved for Batrachoseps.

Biogeography and body size shuffling of aquatic salamander communities on a shifting refuge.

Freshwater habitats of coastal plains are refugia for many divergent vertebrate lineages, yet these environments are highly vulnerable to sea-level fluctuations, which suggest that resident communities have endured dynamic histories. Using the fossil record and a multi-locus nuclear phylogeny, we examine divergence times, biogeography, body size evolution and patterns of community assembly of aquatic salamanders from North American coastal plains since the Late Cretaceous. At least five salamander families occurred on the extensive Western Interior Coastal Plain (WICP), which existed from the Late Cretaceous through the Eocene. Four of these families subsequently colonized the emergent Southeastern Coastal Plain (SECP) by the Early Oligocene to Late Miocene. Three families ultimately survived and underwent extensive body size evolution in situ on the SECP. This included at least two major size reversals in recent taxa that are convergent with confamilial WICP ancestors. Dynamics of the coastal plain, major lineage extinctions and frequent extreme changes in body size have resulted in significant shuffling of the size structure of aquatic salamander communities on this shifting refuge since the Cretaceous.

Phylogeography of the Brownback Salamander reveals patterns of local endemism in Southern Appalachian springs.

The Appalachian Mountains of eastern North America are characterized by high faunal diversity and many endemic species, especially in the unglaciated southern latitudes where lineages have been accumulating for tens of millions of years. The Brownback Salamander, Eurycea aquatica, is an enigmatic species that dwells in isolated springs in southeastern North America. Eurycea aquatica have often been dismissed as simply robust spring-adapted ecomorphs of the widespread and more gracile species Eurycea cirrigera. We sequenced the mitochondrial gene encoding NADH dehydrogenase subunit-2 (ND2; 753 bp) and the nuclear recombination activating gene-1 (Rag1; 1201 bp) for E. aquatica (ND2 n = 72; Rag1 n = 17) from across their presumed distribution and compared them to E. cirrigera (ND2 n = 23; Rag1 n = 10) from nearby populations. Using phylogenetic and morphological analyses we explicitly test if E. aquatica in the Southern Appalachians is simply a local spring-adapted ecomorph of E. cirrigera or a single lineage that resulted from fragmentation of (or dispersal to) spring habitats. We found that E. aquatica from isolated springs form a well-supported monophyletic group that is nested among E. cirrigera, E. wilderae, and E. junaluska. Furthermore, we uncovered three very divergent lineages of E. aquatica that we estimate have been isolated from each another since the early Pliocene to late Miocene (2.5-6.1 Myr) and may each represent distinct species. The distribution of these lineages is coincident with the distribution of other endemic spring-dwelling vertebrates, and suggests that this region may be a relictual habitat for an unexpected diversity of unrecognized endemics.