Plagiotheciumschofieldii, a new species from the Aleutian Islands (Alaska, USA)

Abstract Plagiotheciumschofieldiisp. nov. is described from the Aleutian Islands, Alaska, U.S.A. Some morphological features of this species correspond to P.lamprostachys (Southern Hemisphere species); however, Plagiotheciumschofieldii is genetically and morphologically different from this and other common Northern Hemisphere species e.g., P.denticulatum, P.platyphyllum, or P.ruthei. The most important distinguishing morphological features differentiating this species are: the arrangement of the leaves on the stem; dimensions, concavity and symmetry of the leaves; dimensions of cells and their areolation; orientation of capsules. Additionally, due to the strong concavity of the leaves, they are very often badly damaged under the microscope. We present the results of DNA research of the analyzed samples, and a detailed description of the morphological features. The new species is illustrated, and its ecological preferences and currently known geographical distribution are presented. Additionally, the authors propose to add this species to Plagiothecium section, which is confirmed by morphological features and genetic analysis.


Introduction
Over the last several years, our perception has changed not only of Plagiothecium Schimp., but also of the whole family of Plagiotheciaceae M.Fleisch. (e.g., Hedenäs 2001, 2002;Wynns et al. 2018). The use of molecular methods has not only helped to understand many taxa previously considered problematic, but has also allowed for the description of a number of new taxa (e.g., Zuo et al. 2011;Wynns et al. 2018;Ignatova et al. 2019;Wolski and Nowicka-Krawczyk 2020). Nevertheless, for decades the taxonomic status of many species of this genus has been unclear and ambiguous, and those taxa currently require detailed morphological, genetic and taxonomic studies.
Although the Northern Hemisphere seems to be relatively well researched, there are still many areas (e.g., central Asia, Middle East) which remain as gaps on the world distribution map of Plagiothecium . The results of taxonomic revisions conducted in recent years indicate the underestimation of the species richness of individual parts of the world. As a consequence of this research, many countries and regions have increased their number of known taxa of the described genus (e.g., Ellis et al. 2019a;Ellis et al. 2019b;Ellis et al. 2020Ellis et al. , 2021Müller and Wynns 2020;Wolski and Nowicka-Krawczyk 2020;Wolski 2020).
The Aleutian Islands, Alaska, U.S.A., are one of the many under-explored regions of the Northern Hemisphere. As a result of the taxonomic revision of Plagiothecium specimens from this area it was possible to describe a new species from this genus; the results are presented below.

Taxonomic analyses
Material from the Missouri Botanical Garden (MO), The New York Botanical Garden (NY) and the University of British Columbia (UBC) was analyzed during the revision of Plagiothecium from the Aleutian Islands. For selected specimens intended for DNA analysis, appropriate consent was obtained from NY (NY02589541) and MO (MO5135779, MO5140205, MO5148015).

DNA isolation, amplification and sequencing
The molecular research was based on nuclear and chloroplast DNA markers: ITS (from the 3' end of the hypervariable nuclear spacer ITS1, through the 5.8S gDNA, to the 5` end of the ITS2 spacer); and rpl16 cpDNA gene encoding ribosomal protein L16. Markers were selected based on Wynns et al. (2018), Ignatova et al. (2019) and Wolski and Nowicka-Krawczyk (2020) from Plagiothecium-focused studies.
Leafy stems of mosses were cut from dried material. Approximately 20 mg of dry tissue from each specimen in duplicates was placed in a 1.5 ml Eppendorf Safe-Lock tube and frozen (-20 °C) for homogenization. Tissue homogenization was performed using a hand-held stainless steel homogenizer (Schlüter Biologie, Eutin, Germany). Total DNA was extracted using the GeneMATRIX Plant & Fungi DNA Purification Kit (Eurx, Gdansk, Poland) following the manufacturer's protocol. DNA extracts were quantified with a BioDrop DUO Spectrophotometer (BioDrop Ltd, Cambridge, U.K.). From the duplicates, the sample with the higher quality DNA (1.7-1.9 OD 260 / OD 280 ) was selected for further analysis.
For each sample, all markers were amplified by PCR in a few replicates to obtain high quality amplicons for sequencing. PCR was performed using primers and reaction conditions as described in Wolski and Nowicka-Krawczyk (2020), with a 50 µl reaction volume with 25 µl of Color Taq PCR Master Mix (2×) (Eurx, Gdansk, Poland).
PCR products were visualized on an agarose gel (1.5%, 90V, 40 minutes) stained with GelRED fluorescent dye (Biotum, Fremont, CA, U.S.A.) and two replicates of each marker per sample were chosen for sequencing. Amplicons from the PCR reaction were cleaned using Syngen Gel/PCR Mini Kit (Syngen Biotech, Wrocław, Poland) according to the manufacturer's protocol. Samples were sequenced with Sanger sequencing using primers from amplification by SEQme s.r.o. company (Dobris, Czech Republic). The obtained sequences were assembled in Geneious 11.1.5 (Biomatters Aps, Aarhus, Denmark) (http://www.geneious.com). The sequences were submitted to the NCBI GenBank database (www.ncbi.nlm.nih.gov) under the accession numbers MW936654-MW936657 for ITS and MW935831-MW935834 for rpl16.

Phylogenetic analyses
Phylogenetic analyses of studied specimens and other species in the Plagiothecium group were performed based on a concatenated ITS-rpl16 sequence matrix. Voucher information for the specimens included in this study, with corresponding GenBank accession numbers, is presented in Table 1. Sequences were aligned using the MAFFT v. 7 web server (Katoh et al. 2017) (http://mafft.cbrc.jp/alignment/server/) where the auto strategy was applied, the scoring matrix of 200PAM with Gap opening penalty of 1.53, UniREf50 for Maft-homologs and Plot and alignment with threshold of 39 score were set. The obtained alignments were checked for poorly and ambiguously aligned regions and small corrections were made by eye. The evolutionary models were calculated using PartitionFinder 2 software (Lanfear et al. 2016) chosen according to the Akaike Information Criterion (Table 2).
Phylogenetic calculations were performed using maximum likelihood analysis (ML) in the IQ-TREE web server (Trifinopoulos et al. 2016) (http://iqtree.cibiv.univie.ac.at/) with the ultrafast bootstrap (UFBoot) pseudo likelihood algorithm (Hoang et al. 2018)  four million generations each, with trees sampled every 1000 generations. The average standard deviation of split frequencies in both cases remained below 0.01 for the last 1000 generations and posterior probabilities were estimated from the 50% majorityrule consensus tree after elimination of the first 25% of samples as burn-in. Raw data sequences, the alignment and tree files were submitted to the figshare online database (https://doi.org/10.6084/m9.figshare.14443697.v1). Haplotype network analysis was performed using Median Joining Network in PopART v. 1.7 with gap coding as a single event irrespective of length and haplotypes` geographic distribution (Leigh and Bryant 2015). The analysis included species of Plagiothecium sect. Plagiothecium with representatives of P. longisetum and P. angusticellum (sect. Orthophyllum).

Results and discussion
Phylogenetic analyses based on the concatenated ITS-rpl16 matrix placed studied specimens within the branch of a Plagiothecium sect. Plagiothecium clade, or sister to it; however, the branch support was very low (BS = 49). The next branch down is to representatives of sect. Orthophyllum Jedl. and even more distant to sect. Leptophyllum Jedl. clade (Fig. 1). After branching off from the Orthophyllum clade, the internal division of sect. Plagiothecium was well supported by Bayesian inference (PP ≥ 0.98). Maximal support from both maximum likelihood and Bayesian Inference was recorded in the clade of Plagiothecium schofieldii, where the representatives create a monospecific clade (PP = 1).
The haplotype network (Fig. 2) also showed internal diversity in sect. Plagiothecium. At the center, the analysis grouped haplotypes from the Northern Hemisphere (P. denticulatum and P. ruthei). Three branches extending from the center apply to haplotypes from Central America and the Southern Hemisphere. The position of P. schofieldii haplogroup is fairly isolated and consists of two haplotypes: the first refers to three specimens from Simeonof Island, while the second to a representative from Adak Island (Table 1); however, as it grows in the Aleutian Islands, Alaska, the branch vector points out the same direction as haplotypes from the Northern Hemisphere.
The individual taxonomic features of Plagiothecium are related to a specific level of detail in our analyses, and for example: superficial layer of the stem (epidermis layer) of large, thin-walled cells; shortly pointed leaves; serration (if present) only at apex; absence of pseudoparaphyllia; leaves clearly decurrent at the base -distinguish this genus from other genera belonging to the Plagiotheciaceae. Within Plagiothecium, the shape of decurrent alar regions, and the shape of their cells distinguishes the species of individual sections, while the shape and dimensions of leaf cells are the most important features distinguishing species from each other (Iwatsuki 1970;Lewinsky 1974;Noguchi 1994;Smith 2001). Therefore, based on the fact that the analyzed specimens have decurrent alar cells that are rounded, inflated, and form distinct auricles, as well as the shape and size of the leaf cells of Plagiothecium schofieldii, we believe that this species belongs to Plagiothecium sect. Plagiothecium. This is also confirmed by molecular and haplotype network analyses.
Leaves of P. schofieldii are julaceous and imbricate -very closely arranged on the stem, while in other species the leaves are strongly complanate, flaccid, and spreading on the stem. In the Northern Hemisphere only in P. denticulatum shoots are rarely julaceous (Lewinsky 1974;Smith 2001;Li and Ireland 2008;Wynns 2015). The appearance of the mats and the arrangement of the leaves on the stem are more similar to those features in P. cavifolium (Brid.) Z. Iwats. (which belongs to Plagiothecium sect. Orthophyllum).
Stem leaves of Plagiothecium schofieldii are very strongly concave, to such an extent that under the microscope they are clearly damaged and cracked from being flattened by the coverslip. The leaves of the closely related species are rather flat. Only in the case of P. denticulatum are the leaves more or less concave, but never to such an extreme  (Lewinsky 1974;Smith 2001;Li and Ireland 2008). Plagiothecium schofieldii is characterized by symmetrical leaves, and from other members of sect. Plagiothecium only P. platyphyllum has more or less symmetrical leaves, but this is the only feature common to both species. Symmetrical leaves are typical, e.g., for species from sect. Orthophyllum (e.g., P. nemorale, P. cavifolium) (Lewinsky 1974;Smith 2001;Li and Ireland 2008;Wynns 2015;Wolski 2020). Also, leaves of P. schofieldii are clearly longer and wider than those of all the species mentioned above (Smith 2001;Li and Ireland 2008).
Plagiothecium schofieldii is clearly distinguished from P. denticulatum, P. platyphyllum and P. ruthei by the length and width of it laminal cells. The cells located in the central part of the leaf are long and very wide (88-190 × 13-29 µm), which makes the cell areolation very loose. None of the above-mentioned species has such long and broad cells, and thus their cell areolation is tighter (Lewinsky 1974;Smith 2001;Li and Ireland 2008).
Another feature that clearly distinguishes this newly described species from the previous species in sect. Plagiothecium is the orientation of the capsules. In the studied specimens of P. schofieldii, the capsules are orientated most often more or less vertically, i.e., erect, rarely inclined. Plagiothecium denticulatum, P. platyphyllum, and P. ruthei have inclined capsules (Lewinsky 1974;Smith 2001;Li and Ireland 2008;Wynns 2015).
On the other hand, in terms of morphology, P. schofieldii looks more like P. lamprostachys (Hampe) A. Jaeger -a Southern Hemisphere species (Ireland 1992;Wynns 2015) -than the common Northern Hemisphere species mentioned above. Both the morphological features and molecular analyses indicate the distinctiveness of the species (Figs. 1-4).
Additionally, Wynns (2015) pointed out that P. lamprostachys forms a clade within P. denticulatum sensu lato, which is also confirmed by our research (Fig. 1). Phylogenetic analyses of concatenated nuclear and chloroplast markers placed P. schofieldii within sect. Plagiothecium next to P. denticulatum; however, the branch support was very low. After branching off from the Leptophyllum clade (BI = 0.70), Bayesian inference highly supported the phylogenetic relations within sect. Plagiothecium indicating the separateness of a P. schofieldii clade (as well as a sect. Orthophyllum clade). This separation was also visible in the haplotype network, where the analysis extended the Alaskan clade far from the center of the network grouping species of Plagiothecium from the Northern Hemisphere.
Etymology. The present species is named in honor of Wilfred "Wilf " Borden Schofield , who spent decades studying northern regions of North America, including the Aleutian Islands, and who on July 19, 1996, collected the specimen (No. 106119), chosen here as the holotype of Plagiothecium schofieldii. According to Stephen Talbot (pers. comm.), Schofield recognized this plant as distinct in the field.
Distribution and ecology. Plagiothecium schofieldii so far has only been recorded from Adak Island, Attu Island and Simeonof Island in Alaska. In this area it has been recorded on wetlands and hills, wet cliff chimney, open, moist crevice of a cliff, shaded face of hole on slope, shaded humid outcrop, along creek and adjacent slope, near saddle between hills and near base of mountain.