Molecular and morphological analyses clarify species delimitation and reveal a new Betula species in section Costatae

Background and Aims: Delineating closely related and morphologically similar species with overlapping ranges can be difficult. Here, we use section Costatae (genus Betula) as a model to resolve species and subspecies boundaries in four morphologically similar trees: Betula ashburneri, Betula costata, Betula ermanii and Betula utilis (including ssp. utilis, and diploid and tetraploid races of ssp. albosinensis). Methods: We genotyped 298 individuals (20-80 per species) from 38 populations at 15 microsatellite markers and a subset of 34 individuals from 21 populations using restriction-site associated DNA sequencing (RAD-seq). Morphometric analysis was conducted to characterise leaf variation for a subset of 89 individuals. Key Results: Molecular analyses and leaf morphology found little differentiation between B. ashburneri, diploid B. utilis ssp. albosinensis and some samples of B. utilis ssp. utilis suggesting that these should be treated as a single species. By contrast, tetraploid Betula utilis ssp. albosinensis was divided into two groups with group I genetically similar to B. utilis ssp. utilis based on SNPs and group II, a very distinct cluster, which we propose as a new species, namely, Betula buggsii. Phylogenomic analysis based on 2,285,620 SNPs show a well-supported monophyletic clade of B. buggsii, forming a sister with a well-supported clade of B. ashburneri, diploid B. albosinensis and some samples of B. utilis ssp. utilis. Morphologically, Betula buggsii is characterised by elongated lenticels and a distinct pattern of bark peeling. Betula buggsii is geographically restricted to the Qinling-Daba Mountains. Conclusions: Our study reveals six genetically distinguishable species: B. ashburneri, B. buggsii, B. costata, B. utilis ssp. utilis, B. utilis ssp. albosinensis and B. ermanii. Our research demonstrates an integrative approach in delimitating species using morphological and genetic samples from their nearly entire distributions. Analyses based on subsets of species9 distributions may lead to erroneous species or subspecies delineation.