Defining evolutionary boundaries across parapatric ecomorphs of Black Salamanders (Aneides flavipunctatus) with conservation implications.

The accurate delimitation of evolutionary population units represents an important component in phylogeographic and conservation genetic studies. Here, we used a combined population assignment and historical demographic approach to study a complex of ecomorphologically distinctive populations of Black Salamanders (Aneides flavipunctatus) that are parapatrically distributed and meet at a three-way contact zone in north-western California. We used mitochondrial tree-based and multilocus clustering methods to evaluate a priori two- (Northern and Southern) and three (Northern, Coast and Inland) population hypotheses derived from previous studies. Mitochondrial results were consistent with the two- and three-population hypotheses, while the nDNA clustering results supported only the two-population hypothesis. Historical demographic analyses and mtDNA gene divergence estimates revealed that the Northern and Southern populations split during the Pliocene (2-5 Ma). Subdivision of the Southern population into Coast and Inland populations was estimated to be late Pleistocene (0.24 Ma), although our mtDNA results suggested a Pliocene divergence. Effective gene flow estimates (2N(e)m) suggest that either the two- or three-population hypotheses remain valid. However, our results unexpectedly revealed that the Northern population might instead represent two parapatric populations that separated nearly 4 Ma. These results are surprising because the Pliocene divergence between these ecomorphologically conservative forms is similar or older than for the ecomorphologically divergent Coast and Inland sister populations. We conclude that Black Salamanders in north-western California belong to at least three or four populations or species, and these all meet criteria for being Evolutionary Significant Units or 'ESUs' and therefore warrant conservation consideration.