Morphological and molecular data demonstrate the existence of <i>Betula fruticosa</i> and <i>B. middendorffii</i> and the absence of <i>B. ovalifolia</i> in northeastern China

Abstract Shrub birches are important components of massive wetlands in Northeast China, although much of these habitats have been lost over the past two decades. However, the taxonomy of shrub birches has been ambiguous owing to their morphological variation. In this study, we incorporated morphological and molecular data to address the taxonomic challenges associated with shrub birches. We characterized the morphological variation in the leaves, seeds, and bracts of 11 shrub birch populations in Northeast China and a population of Betula ovalifolia in northern Japan. We genotyped birch individuals from Northeast China at 15 microsatellite loci as well as a subset of individuals, using restriction site-associated DNA sequencing (RAD-seq). The populations in Northeast China consisted of B. fruticosa and B. middendorffii. These two species and B. ovalifolia differ in the width of their seed wings and the angle between the side lobes of their bracts, despite substantial intraspecific variation. Our genetic data revealed two distinct clusters, corresponding to B. fruticosa and B. middendorffii. Ploidy level assessment via RAD-seq revealed that B. fruticosa is diploid and B. middendorffii is tetraploid. Phylogenetic analysis revealed that B. fruticosa formed a sister clade to diploid B. humilis, and B. middendorffii formed a clade with diploid B. nana. Our results indicate the existence of B. fruticosa and B. middendorffii and the probable absence of B. ovalifolia in the northern Daxing’an Range, as all the tetraploid individuals sampled there represented B. middendorffii. In addition, our results suggest that B. humilis and B. nana may have served as diploid parents of the tetraploid species B. ovalifolia and B. middendorffii, respectively.