Phylogeny, species delimitation and taxonomy in Polytrichum sect. Polytrichum (Polytrichaceae Schwagr.; Bryophyta)

Abstract

The Polytrichaceae Schwägr. is a relatively a small, distinct family of mosses (Phylum Bryophyta) usually recognised in its own order and class, Polytrichales and Polytrichopsida. The presence of the characteristic “polytrichoid peristome”, lamellae on the adaxial side of the leaf, well developed conducting tissue and the occurrence of some robust species with heights of >40–60cm are some of the defining characters of the family. The type genus of Polytrichaceae, Polytrichum Hedw. comprises three sections viz. Polytrichum section Polytrichum, section Aporotheca and section Juniperifolia. The section Polytrichum is a well-circumscribed clade that includes eight species and all plants currently recognised within the species concept of Polytrichum commune Hedw., one of the most widespread and ecologically important moss species of northern temperate and boreal regions across Asia and North America and also known from southern temperate areas. Although some molecular taxonomic and monographic work has been done for certain genera within the family over the past three decades, a comprehensive integrated (molecular and morphological) taxonomic revision has not been done for Polytrichum sect. Polytrichum, which contains the type species of the genus, P. commune Hedw. This study presents the first robust molecular phylogenetic framework for Polytrichum sect. Polytrichum using both Sanger and Next Generation Sequencing (NGS) approaches. For Sanger sequencing, a robust taxon sampling (114 ingroup accessions) was done to cover a substantial geographic distribution and morphological diversity. Six molecular markers were used, including two nuclear markers (ITS1 and ITS2) and four plastid markers (rbcL, trnL-F, rpl16 and trnG). Phylogenetic relationships of the species were tested for the concatenated (3851 bp) matrix using both Maximum Likelihood (ML) and Bayesian inferences. The monophyly of five clades was strongly supported within Polytrichum sect. Polytrichum [Arctic and Subarctic P. swartzii and P. jensenii clades, a P. commune sensu stricto clade, and South American P. angustifolium and P. brachymitrium clades] while one poorly supported large clade was recovered including three morphologically and geographically distinct taxa, P. perigoniale, P. subpilosum and P. ericoides. Within this, P. ericoides and P. subpilosum were monophyletic, but ambiguously resolved in relation to elements within P. perigoniale, hence the clade is best viewed as a species complex. A haplotype network is presented from a representative subset of this clade using the ITS2 marker to illustrate and interpret the relationships of taxa and geographical populations. To investigate the unresolved species relationships further, a target enrichment with Physcomitrella RNA baits was employed to sequence 809 low copy nuclear loci for 24 representative accessions of all extant taxa within the section. The data was assembled using different approaches (de novo and reference mapping) and with different software and settings to assess their impact on downstream phylogenetic analysis. All loci were concatenated and analysed under the ML phylogenetic framework and treated as a single partition. The final NGS phylogeny showed a similar phylogenetic pattern as was inferred using Sanger Sequencing. However, a striking difference between the NGS and Sanger phylogenies was that P. ericoides was separated as a well-supported monophyletic clade sister to the species complex comprising. subpilosum, P. perigoniale and P. brachymitrium in the NGS phylogeny. Moreover, P. brachymitrium, which was sister to the species complex with the Sanger data, was nested within it in the NGS results. Possible taxonomic and phylogenetic explanations are provided to address this issue. This study presents the first worldwide monographic revision of Polytrichum sect. Polytrichum based on herbarium specimens, including all available type specimens. The unresolved nomenclatural issue of selecting a lectotype for the well-known moss taxon, Polytrichum commune Hedw., has been resolved. Interpreting the molecular phylogenetic results of the present study, it is now clearly revealed that Polytrichum perigoniale Michx. (earlier treated as P. commune var. perigoniale) is a distinct taxon at species rank which has been widely misunderstood, with many historic and recent collections from North America, Australasia, Africa and Southeast Asia erroneously named previously as P. commune. This taxonomic confusion is resolved by the molecular and morphological delimitations proposed in this study. Eight morphological species are confirmed from the study: Polytrichum angustifolium Mitt., P. brachymitrium Müll.Hal., P. commune Hedw., P. ericoides Hampe, P. jensenii I.Hagen, P. perigoniale Michx., P. subpilosum P.Beauv. and P. swartzii Hartm. New observations are reported and a taxonomic key to separate species is provided based on vegetative and reproductive characters. Typification, taxonomic descriptions, illustrations, geographic distributions, synonymy, ecological notes and new species records are provided under each species. All doubtful taxa are listed along with reasons for their exclusion. New combinations are also included under each taxon, with justifications provided. The sporophytes of P. ericoides Hampe. are reported for the first time, described and illustrated. The taxon Polytrichum commune Hedw. in its revised circumscription is excluded from Africa and China, while the geographic distribution of P. brachymitrium Müll.Hal. is expanded from Brazil to Venezuela, Argentina and Colombia. The Arctic and Subarctic taxon P. swartzii Hartm. is excluded from China, Taiwan and Japan. This study provides the first phylogenetic study of Polytrichaceae using herbarium DNA to infer phylogenetic relationships using hybrid capture methods. Although the capture was variably successful it provided much target (nuclear) and off target (plastid) data for future research. This study will open avenues for inferring phylogenetic relationships of extant genera and species of the family Polytrichaceae through the design of genus- and species-specific DNA probes, to elucidate any reticulate evolutionary trajectories such as allopolyploidy and hybridisation within the group.