Two new species of the liverwort genus Hygrobiella Spruce (Marchantiophyta) described from the North Pacific based on integrative taxonomy

The molecular and morphological variability of Hygrobiella laxifolia (Hook.) Spruce across its distributional area was investigated to verify its genetic entity. We sequenced nuclear ITS1-5.8S rDNA and three plastid genomic loci (trnL-F, trnG-intron and matK) of 31 specimens referred to H. laxifolia and gathered from remote regions: northwestern European and Far Eastern parts of Russia, as well as western USA. Three robustly supported clades within "H. laxifolia'' were obtained by maximum parsimony, maximum-likelihood analyses and Bayesian approach. Haplotype network reconstruction also revealed three haplotype groups separated from each other by a row of missing haplotypes. The majority of Far Eastern accessions were composed of two genetic entities and were contrary to a third one containing all samples from northwestern European Russia, western USA and Bering Island as well. As a result of critical morphological reinvestigation for each of the discovered genetic entities, diagnostic characters were provided and a hypothesis of cryptic speciation in H. laxifolia was not supported. Accessions from removed localities united in a third clade were classified as typical H. laxifolia, whereas Far Eastern specimens from two other clades were applicable to new species-Hygrobiella intermedia Bakalin & Vilnet sp. nova and H. squamosa Bakalin & Vilnet sp. nova, which are described and illustrated here. The re-evaluation of species morphology indicated that the presence of secondary pigmentations, form of perianth and leaves, stem cross section anatomy and length of underleaves appear to be the main differentiation features. Available data suggest sympatric distribution of three Hygrobiella species in the northern Pacific region and the origin of two new species under the strong impact of volcanism.