﻿Didymodonmanhanensis (Pottiaceae, Bryophyta), a new species from Inner Mongolia steppe, China and its phylogenetic position, based on molecular data

﻿Abstract Inner Mongolia steppe is one of the suitable habitats for Didymodon species and a new species, Didymodonmanhanensis C. Feng & J. Kou from Manhan Mountain in semi-arid region in Inner Mongolia, China is described and illustrated. It is characterised by leaves incurved and slightly twisted when dry, spreading when moist, narrowly lanceolate from an ovate base; subulate and fragile leaf apices; distally bistratose leaf margins that are recurved in proximal 2/3–3/4; excurrent costa with guide cells in 2–3 layers and without ventral stereids; smooth laminal cells and red KOH laminal colour reaction. Our morphological analyses and molecular results, based on DNA sequences of ITS, rps4 and trnM-trnV, conﬁrm that D.manhanensis belongs to a group that includes D.obtusus J. Kou, X.-M. Shao & C. Feng and D.daqingii J. Kou, R.H. Zander & C. Feng. This new species is compared with similar species and its phylogenetic position and ecology are discussed.


Introduction
Inner Mongolia, situated in Inner Eurasia, is located in the northern part of China and presents a strip distribution from the northeast to the west. The district habitats are temperate continental monsoon climate. The annual mean temperature is 8 °C, which increases from east to west and the annual precipitation is 35-530 mm, which decreases from southeast to northwest (Miao 2017). The area of grassland accounts for 60% of the whole Inner Mongolia and more than one-quarter of the total area of grassland in China. The grassland in Inner Mongolia is divided into three types: meadow steppe formed by, for example, Stipa baicalensis Roshev, Leymus chinensis (Trin.) Tzvelev; typical steppe formed by, for example, Stipa grandis P. Smirn., Stipa krylovii Roshev, Leymus chinensis (Trin.) Tzvelev and desert steppe formed by, for example, Stipa klemenzii Roshev, Stipa glareosa P.A. Smirn., Stipa breviflora Griseb (Hua et al. 2021). The main vegetation types of the steppes present distinct zonal features (Wu et al. 2005). The Inner Mongolia steppe is a suitable habitat for the Didymoodn Hedw. species and several new species were recently discovered (e.g. Kou et al. 2016a;Kou et al. 2019;Feng et al. 2022).
The taxonomy of genus Didymodon is complicated, involving the differentiation from related genera, such as Barbula Hedw. and the circumscriptions of its infrageneric sections (Zander 1993;Zander 2007;Zhang et al. in press). A recent important event associated with Didymodon s. lat. was the split of the genus into seven smaller genera: Aithobryum R.H. Zander, Didymodon s. str., Exobryum R.H. Zander, Fuscobryum R.H. Zander, Geheebia Schimp., Trichostomopsis Card. and Vinealobryum R.H. Zander, based on macro-evolutionary analysis and the dissilient genus concept applied (Zander 2013;Zander 2019). Although initially this revolutionary concept was considered unnecessary or unsupported (Blockeel and Kučera 2019), it has later been supported by molecular phylogenetic data, but with some alterations (Jiménez et al. 2022;Zhang et al. in press) and has gained acceptance by some other authors (e.g. Osman et al. 2021;Feng et al. 2022). During our continuous investigations of xerophilic mosses, especially Pottiaceae Hampe, in China (e.g. Feng et al. 2016a, b;Kou et al. 2016bKou et al. , 2019, many specimens have been collected from different provinces. Amongst them, two samples collected from Manhan Mountain in Inner Mongolia of Didymodon s. lat. from stony habitats are different from species previously reported in the area (Li et al. 2001). They mostly resemble Didymodon obtusus J. Kou, X.-M. Shao & C. Feng. To clarify their taxonomic identity, we conducted phylogenetic analysis and confirm that these samples belong to the genus Didymodon s. str. (Zander 2013), but do not match with any species known in the genus. Here, we describe this unknown moss as a new species.

Morphological observations
Over 3000 specimens of the genus Didymodon s. lat. were examined during our revision of Pottiaceae in China. More than 50 field investigations were conducted in past years and the specimens included in this study were housed in the Herbaria at IFP, KUN and NMAC. Microscopic examinations and measurements were taken with a ZEISS Primo Star light microscope and photomicrographs were obtained with a Canon EOS 70D camera, mounted on this microscope. Specimens were examined in 2% potassium hydroxide (KOH). Three plants were dissected from each collection and, for each shoot, every possible structure from the gametophyte had to be examined and a record kept of what was found for each individual species. Specific morphological and anatomical features of taxonomic importance were assessed mainly following Zander (1993). Leaves were always taken from the upper and middle parts of the stem and cross-sections were made in the middle part of the stem. Measurements of leaf width were taken at the base, mid-leaf and upper part. Cross-sections were made at mid-leaf.

Phylogenetic analyses
To test the phylogenetic position of the new species, two specimens collected from Manhan Mountain were sampled. Due to its great similarity with D. obtusus and Didymodon daqingii J. Kou, R.H. Zander & C. Feng, the isotypes of the two species were added to the dataset. We employed one nuclear (ITS) and two chloroplast markers (rps4 and trnM-trnV), which had been used successfully in previous phylogenetic studies in Didymodon s. lat. and enabled the re-use of earlier results and easier interpretation of new data (Werner et al. 2004(Werner et al. , 2005(Werner et al. , 2009Kučera and Ignatov 2015;Kučera et al. 2018;Ronikier et al. 2018;Jiménez et al. 2022;Zhang et al. in press). Phylogenetic trees are created and shown separately. The complete list with sample names and GenBank accession numbers is presented in Tables 1 and 2. DNA extraction, PCR amplification and sequencing procedure followed the protocols described by Wang et al. (2010).
The sequences were aligned by using MAFFT 7.222 (Kazutaka and Daron 2013) and then edited in BioEdit 7.0.1 (Hall 1999). The concatenation of individual rps4 and trnM-trnV fragments was performed by our custom Perl script. Phylogenetic analyses were performed by using the Bayesian Inference (BI) and Maximum Likelihood (ML) methods. MrBayes 3.2.6 (Ronquist et al. 2012) was used for BI analyses under the GTR substitute model. The following was used: two Markov Chain Monte Carlo (MCMC) searches were run for 10 million generations each, with a sampling frequency of 1000. The first 25% of the trees were discarded as burn-in. The convergence between runs in all cases dropped below

Results
The chloroplast (cp) and ITS alignments comprised 1313 and 1364 nucleotide sites, respectively. The BI and ML phylogenetic trees have a consistent topology, although there are different levels of support depending on the method. Hence, only the topologies with branch lengths from the BI trees are presented, with added support from the ML method on the respective trees (Figs 3-4). Although the inference from analysed chloroplast regions (Fig. 3) and the ITS (Fig. 4) agrees in most aspects, the position of the new species is different between the two above phylogenetic trees and, thus, both of them are reserved.

Discussion
As indicated by Zander (1993), Didymodon s. lat. is heterogeneous and could be profitably split. In our phylogenetic analyses, this genus is polyphyletic and its species can be classified within several well-supported monophyletic clades, which correspond to other phylogenetic studies of the genus (Feng et al. 2022;Jiménez et al. 2022;Zhang et al. in press). Our results reveal a close relationship between D. manhanensis and two recently-described species in China: D. daqingii and D. obtusus. Although the latter two species were considered identical by Sollman et al. (2020), they are not closely related in our phylogenetic analyses, based on both ITS and chloroplast data. Didymodon manhanensis is distinguished from all congeners by the following combination of diagnostic features: leaves incurved and slightly twisted when dry, spreading when moist, narrowly lanceolate from an ovate base; subulate and fragile leaf apices; distally bistratose leaf margins that are recurved in proximal 2/3-3/4; costal guide cells in 2-3 layers and without ventral stereids, smooth laminal cells and red KOH laminal colour reaction. This combination of characters suggests the placement of D. manhanensis in the sect. Didymodon (Zander 1978(Zander , 1993(Zander , 1998. Following the recent revolutionary work on the genus Didymodon s. lat. by Zander (2013Zander ( , 2019, morphologically, it belongs in the amended genus Didymodon s. str. Its systematic position in Didymodon s. str. was also confirmed by our phylogenetic analyses, based on both ITS and chloroplast data. Chloroplast data support that D. manhanensis is closely related to D. cordatus and sister to both D. daqingii and D. anserinocapitatus. However, D. manhanensis differs morphologically from D. cordatus by the costa with guide cells in 2-3 layers and without ventral stereids and smooth laminal cells. It differs from D. daqingii by the leaves that are narrowly lanceolate from an ovate base, smooth laminal cells and red KOH laminal colour reaction; it differs from D. anserinocapitatus by the distally bistratose leaf margins and lack of swollen and deciduous leaf apex (Zander 2007). In the ITS analyses, there is successive branching of clades, including D. obtusus J. Kou Kop. Amongst these species, D. manhanensis is most similar to D. obtusus, a species that was recently described from Tibet in China ), but the former can be distinguished from the latter by its narrowly lanceolate leaves from an ovate base and spreading when moist, subulate and somewhat fragile leaf apex and unistratose distal lamina.
There are three species distributed in China that have excurrent costa and smooth laminal cells may be confused with the new species. Didymodon ditrichoides (Broth.) X.-J. Li & S. He, a species known from North American, Asia (China) and the Atlantic Islands (Iceland) (Li et al. 2001;Zander 2007), differs from the new species by the unistratose leaf margins, costa with 1-2 layers of guide cells and with 0-1 layer of ventral stereids and yellowish KOH laminal colour reaction (Zander 2007).
Didymodon validus Limpr. can be separated from D. manhanensis by the twisted and incurved leaves when dry, unistratose leaf margins, costa with 1 layer of guide cells and with 1-3 layers of ventral stereids and yellowish-green KOH laminal colour reaction (Shuayib et al. 2017).
The lanceolate to long-lanceolate leaves with a widely ovate base, distally bistratose leaf margins, excurrent costa and epapillose laminal cells are likewise found in Didymodon ochyrarum J.A.Jiménez & M.J.Cano, a species described from tropical South America (Jiménez and Cano 2019), which may be confused with the new species. However, D. ochyrarum can be separated from D. manhanensis by its plane leaf margins, marginal basal cells running up the margin forming a distinctly differentiated area of transversely thick-walled cells and yellowish KOH laminal colour reaction. Diagnosis. It is distinguished from all congeners by the following combination of diagnostic features: leaves incurved and slightly twisted when dry, spreading when moist, narrowly lanceolate from an ovate base; subulate and fragile leaf apices; distally bistratose leaf margins that are recurved in proximal 2/3-3/4; costal guide cells in 2-3 layers and without ventral stereids, smooth laminal cells and red KOH laminal colour reaction.