Nested singletons in molecular trees: Utility of adding morphological and geographical data from digitized herbarium specimens to test taxon concepts at species level in the case of (Salicaceae).

Using the genus , we assessed the status of nested singletons: individual specimens corresponding to accepted species but in molecular trees appearing nested within clades of closely related species. Normally, such cases would be left undecided, while on the other hand, timely taxonomic decisions are required. We argue that morphological, chorological, and ecological data can be informative to illuminate patterns of speciation. Their use can provide a first step in testing taxon concepts at species level. We focused on five cases of nested singletons in trees of the genus . We employed PCA and cluster analysis to assess phenotypic differentiation. Using geocoordinates, we calculated niche space differentiation based on 19 bioclim variables, by means of PCA and niche equivalency and similarity tests and generated dot maps. We found that the singletons were morphologically distinctive in two of the five cases ( and ), relatively distinctive in two other cases ( and ), and partially overlapping in the last case (). For two cases ( and ), ecological niche space was broadly overlapping, in two cases it was found broadly nested ( and ), and in one case narrowly nested (), but in no case niche differentiation was observed. Niche overlap, similarity and equivalency showed corresponding patterns. Given these data, one would interpret and as presumably well-distinguished taxa, their narrow distribution ranges suggesting recently emerging lineages. The other three cases are not clearcut. Morphological data would suggest particularly conspecific with , but differences in the distribution are intriguing. Our approach would reject the notion of potential synonymy based on nested phylogenetic placement for at least two of the five cases. The other case also shows no complete lack of differentiation which would support synonymy.