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, Stipabaicalensis Roshev, Leymuschinensis (Trin.) Tzvelev; typical steppe formed by, for example, Stipagrandis P. Smirn., Stipakrylovii Roshev, Leymuschinensis (Trin.) Tzvelev and desert steppe formed by, for example, Stipaklemenzii Roshev, Stipaglareosa P.A. Smirn., Stipabreviflora 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 Didymodons. lat. was the split of the genus into seven smaller genera: Aithobryum R.H. Zander, Didymodons. 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. Kou and Feng 2018; 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. 2016b, 2018, 2019; Kou and Feng 2018), 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 Didymodonobtusus 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 Didymodons. str. (Zander 2013), but do not match with any species known in the genus. Here, we describe this unknown moss as a new species.

Materials and methods

Morphological observations

Over 3000 specimens of the genus Didymodons. 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 Didymodondaqingii 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, 2005, 2009; Kučera 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).

Table 1.

New sequences used in this study, including taxa vouchers information and GenBank accession numbers.

Species	Voucher information	ITS	rps4	trnM-trnV
Didymodonmanhanensis 4	China, Inner Mongolia, Chao Feng 2016060162	OL514237	OL450506	OL450515
Didymodonmanhanensis a3	China, Inner Mongolia, Chao Feng 2016060176	OL514238	OL450507	OL450516
Didymodonobtusus	China, Tibet, Xiao-Ming Shao & Jin Kou 20140815037	OL514239	OL450508	OL450517
Didymodondaqingii	China, Inner Mongolia, Chao Feng 20170605032	OL514240	OL450509	OL450518

Table 2.

. Sequences from GenBank used in this study, including taxa and GenBank accession numbers.

Species	ITS	rps4	trnM-trnV
Acaulontriquetrum	MW398556
Aloinarigida	MW398549
Aloinellaandina	MW398550
Andinellachurchilliana	MW398720
Andinellacoquimbensis	MW398711
Andinellaelata	MW398708
Andinellagranulosa	MW398714
Andinellalimensis	MW398710
Andinellaoedocostata	MW398733
Andinellapruinosa	MW398726
Barbulaunguiculata	MW398553	HM147777	JQ890366
Bryoerythrophyllumrecurvirostrum	MW398547	JQ890468	JQ890407
Bryoerythrophyllumrubrum	MW398548
Chenialeptophylla	MW398561
Cinclidotusriparius	MW398554
Crossidiumsquamiferum	MW398558
Didymodonacutus	AY437111	KP307551	KP307667
Didymodonalpinus	MW398606
Didymodonandreaeoides	MW398768
Didymodonanserinocapitatus	MW398649	KP307545	KP307640
Didymodonasperifolius	MW398594	JQ890472	KP307600
Didymodonaustralasiae (Trichostomumaustralasiae)	MW398737	KP307571	KP307651
Didymodonbrachyphyllus (Vinealobryumbrachyphyllum)	MW398817
Didymodonbuckii	MW398578
Didymodoncaboverdeanus	MW398607
Didymodoncalifornicus (Vinealobryumcalifornicum)	MW398819
Didymodoncanoae	MW398584
Didymodoncardotii	MW398729
Didymodonchallaensis (Trichostomopsischallaensis)	MW398748
Didymodonconstrictus	MW398613
Didymodoncordatus	MW398664	KP307564	KP307668
Didymodonditrichoides	MW398642
Didymodoneckeliae (Vinealobryumeckeliae)	MW398826
Didymodonedentulus	MW398685
Didymodonepapillatus	MW398665
Didymodonerosodenticulatus	MW398792	MF536597	MF536635
Didymodonerosus	EU835148	MF536609	MF536646
Didymodonfallax (Geheebiafallax)	MW398779	KP307552	KP307663
Didymodonferrugineus (Geheebiaferruginea)	MW398796	MF536588	MF536625
Didymodonfragilicuspis	KP307482
Didymodonfuscus	MW398689	KP307537	KP307601
Didymodonaff.fuscus		KP307546	KP307615
Didymodongaochienii		KP307538	KP307658
Didymodongelidus	MW398693
Didymodongiganteus	MW398786	KP307548	KP307669
Didymodonglaucus	MW398612
Didymodonguangdongensis (Vinealobryumguangdongense)	MW398657
Didymodonhedysariformis	MW398582	KP307569	KP307629
Didymodonhengduanensis	MW398629
Didymodonhegewaldiorum	MW398739
Didymodonherzogii	MW398746
Didymodonhumboldtii	MW398667
Didymodonicmadophilus	MW398632	KP307598	KP307604
Didymodonimbricatus	MW398646
Didymodonincrassatolimbatus	MW398572
Didymodonincurvus	MW398680
Didymodoninsulanus (Vinealobryuminsulanum)	MW398811
Didymodonjaponicus	MW398757
Didymodonjimenezii	MW398622
Didymodonjohansenii	MW398589	KP307542	KP307662
Didymodonkunlunensis	MW398610
Didymodonlaevigatus	MW398618
Didymodonlainzii	MW398575
Didymodonleskeoides (Geheebialeskeoides)	MW398777	MF536604	MF536642
Didymodonluehmannii	MW398718
Didymodonluridus	AY437098	MF536587	MF536624
Didymodonmaschalogena	MW398615
Didymodonmaximus (Geheebiamaxima)	MW398784	MF536591	MF536628
Didymodonmesopapillosus	MW398758
Didymodonmolendoides	MW398687
Didymodonmongolicus	KU058175
Didymodonmurrayae	KP307513	KP307563	KP307650
Didymodonnevadensis	MW398730
Didymodonnicholsonii (Vinealobryumnicholsonii)	MW398808
Didymodonnigrescens	LC545516	KP307543	KP307611
Didymodonnorrisii	MW398830	KP307585	KP307617
Didymodonnovae-zelandiae	MW398769
Didymodonobtusus	MW398666
Didymodonoccidentalis		KP307533	KP307599
Didymodonochyrarum	MW398763
Didymodonparamicola (Trichostomopsisparamicola)	MW398740
Didymodonpatagonicus	MW398675
Didymodonperobtusus	KP307523	KP307539	KP307609
Didymodonrevolutus (Husnotiellarevoluta)	MW398569	JQ890471	KP307646
Didymodonrevolutusvar.africanus	MW398568
Didymodonrigidulus	MW398602	KP307589	KP307647
Didymodonrigidulusvar.subulatus	MW398672
Didymodonrivicola	MW398599	KP30756	KP307607
Didymodonsantessoni	MW398705
Didymodonsicculus	MW398801	MF536606	MF536643
Didymodonsinuosus	MW398567	JQ890476	JQ890410
Didymodonspadiceus (Geheebiaspadicea)	MW398795	MF536593	MF536631
Didymodonsubandreaeoides	AY437108	KP307570	KP307630
Didymodontectorum	MW398659
Didymodontibeticus	MW398638
Didymodontomaculosus	AY437114
Didymodontophaceus	MW398807	MF536607	MF536644
Didymodontophaceusvar.anatinus		MF536589	MF536626
Didymodontorquatus	MW398719
Didymodonumbrosus (Trichostomopsisumbrosa)	MW398742
Didymodonvalidus	MW398650
Didymodonvinealis (Vinealobryumvineale)	MW398815	JQ890475	KP307606
Didymodonvinealisvar.rubiginosus	MW398822
Didymodonvulcanicus	MW398636
Didymodonwaymouthii	MW398770
Didymodonwisselii	MW398655
Didymodonxanthocarpus	MW398696	KP307534	KP307638
Didymodonzanderi	MW398585	KP307535	KP307621
Dolotortulamniifolia	MW398555
Erythrophyllopsisandina	MW398546
Gertrudiellauncinicoma	MW398698
Gertrudiellauncinicomavar.serratopungens	MW398701
Guerramontesiamicrodonta	MW398543
Hennediellaheimii	GQ339750
Hennediellapolyseta	GQ339759
Leptodontiumexcelsum	MW398545
Microbryumcurvicolle		JX679986	JX679936
Microbryumdavallianum	MW398557
Pseudocrossidiumhornschuchianum	MW398551	JQ890481	JQ890420
Pseudocrossidiumrevolutum	MW398552
Pterygoneurumovatum	MW398560
Sagenotortulaquitoensis	GQ339761
Stegonialatifolia	MW398559
Syntrichiaruralis	MW398564	FJ546412	FJ546412
Tortulamuralis	MW398562	JN581679	JQ890421
Tortulasubulata	MW398563
Triquetrellaarapilensis	MW398544
Tridontiumtasmanicum	MW398750

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 0.01. ML analyses were executed in IQ-TREE 1.6.3 (Nguyen et al. 2014) under the TPM3u+F+R3 (for cpDNA) and TIM3e+I+G4 (for ITS) substitute models, respectively, selected by the ModelFinder programme (Kalyaanamoorthy et al. 2017), based on the Bayesian Information Criterion (BIC) and 1000 fast bootstrapping replicates were used. The final obtained trees were visualised and edited in FigTree v.1.4.0 (Rambaut 2014).

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. The topology of the ITS dataset shows that D.manhanensis is nested within the monophyletic group comprising Didymodonepapillatus J. Kou, X.-M. Shao & C. Feng, Didymodonmongolicus D.-P. Zhao & T.-R. Zhang, Didymodonvalidus Limpr., Didymodonwisselii (Dixon) D.H. Norris & T.J. Kop. and Vinealobryumguangdongensis C. Feng & J. Kou and is sister to D.obtusus, but with weakly-supported values. In the combined plastid dataset, D.manhanensis is nested within the group including Didymodoncordatus Jur. and is sister to D.daqingii and Didymodonanserinocapitatus (X.J. Li) R.H. Zander, with well-supported values.

Figure 3.

Phylogenetic relationships (50% majority consensus tree) from the Bayesian Inference of the concatenated rps4 and trnM-trnV datasets. Numbers above branches indicate posterior probability from the BI analysis, followed by bootstrap values for the ML analysis.

Figure 4.

Phylogenetic relationships (50% majority consensus tree) from the Bayesian Inference on the ITS dataset. Numbers above branches indicate posterior probability from the BI analysis, followed by bootstrap values for the ML analysis.

Discussion

As indicated by Zander (1993), Didymodons. 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.

Didymodonmanhanensis 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, 1993, 1998). Following the recent revolutionary work on the genus Didymodons. lat. by Zander (2013, 2019), morphologically, it belongs in the amended genus Didymodons. str. Its systematic position in Didymodons. 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, X.-M. Shao & C. Feng, D.manhanensis, D.epapillatus J. Kou, X.-M. Shao & C. Feng, D.mongolicus D.-P. Zhao & T.-R. Zhang, D.validus Limpr., Vinealobryumguangdongensis C. Feng & J. Kou and D.wisselii (Dixon) D.H. Norris & T.J. Kop. Amongst these species, D.manhanensis is most similar to D.obtusus, a species that was recently described from Tibet in China (Kou et al. 2018), 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. Didymodonditrichoides (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). Didymodonvalidus 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 Didymodonochyrarum 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.

Taxonomic treatment

. Didymodon manhanensis

C. Feng & J. Kou sp. nov.

70A98184-1F76-5333-8D1C-D128E33BD3A0

Figs 1 , 2 Chinese name: 蛮汉山对齿藓

Figure 1.

DidymodonmanhanensisA dry plants B moist plants C cross-section of stem D leaves E leaf apex F upper part of costa (dorsal) G upper part of costa (ventral) H axillary hairs. Photographed on 21 November 2021 by Chao Feng from the holotype (NMAC!).

Figure 2.

DidymodonmanhanensisA median leaf cells B basal juxtacostal cells C basal marginal cells; D–H cross-sections of leaves, sequentially from apex to base. Photographed on 21 November 2021 by Chao Feng from the holotype (NMAC!).

Type.

China. Inner Mongolia: Ulanqab City, Manhan Mountain, 40°39'19.2931"N, 112°19'36.3792"E, on soil under the grass, elevation 1417 m, 20 June 2016, Chao Feng 2016060162 (holotype: NMAC!; isotype: MO!).

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.

Description.

Plants medium, growing in turfs, green-blackish distally, brown-blackish proximally. Stems very seldom branched, 0.8–1.6 cm in length, not papillose, transverse section rounded to rounded-pentagonal, central strand developed, sclerodermis present, hyalodermis absent; axillary hairs filiform, of 4–8 hyaline cells, the basal cell brown. Leaves crowded on stem, incurved and slightly twisted when dry, spreading when moist, narrowly lanceolate from an ovate base, constricted just above the base, 1.3–2.3 × 0.43–0.55 mm, distal lamina narrowly channelled ventrally; margins plane distally, recurved in proximal 2/3–3/4 of leaf, entire, distal margins bistratose; apex subulate, somewhat fragile; leaf base ovate, not sheathing, not decurrent; costa stout, tapering distally, 57.5–75 µm wide at base, excurrent as a long, thick subula, not spurred, ventral cells of costa in upper middle part of leaf quadrate or subquadrate, smooth, 4 rows of cells across costa ventrally at mid-leaf, dorsal cells of costa in upper middle part of leaf quadrate or subquadrate, smooth, transverse section semicircular to nearly rounded, epidermis present adaxially and abaxially, not or weakly bulging, ventral stereids absent, guide cells 10–16 in 2–3 layers, 2–4 layers of dorsal stereids, reniform or crescent-shaped, without hydroids; upper laminal cells quadrate to rhombic, usually with angular lumens, 7.5–10 × 5–10 µm, smooth, slightly thick-walled, weakly convex on both surfaces, distal lamina unistratose, basal cells weakly differentiated juxtacostally, rectangular, 12.5–37.5 × 5–7.5 µm, thin-walled, smooth; basal marginal cells subquadrate or quadrate, 5–8.75 × 6.25–7.5 µm, with weakly-thickened walls, smooth. Gemmae absent. Dioicous. Sporophytes unknown. KOH laminal colour reaction red.

Additional specimens examined.

China Inner Mongolia: Ulanqab City, Manhan Mountain, on soil under the grass, 20 June 2016, Chao Feng 2016060176 (NMAC).

Etymology.

The specific epithet refers to Manhan Mountain, the type locality.

Habitat and distribution.

Manhan Mountain is situated in Liangcheng County in the southern section of the Yinshan Mountains in the middle of Inner Mongolia, with an average altitude of approximately 1500 m (Huang et al. 2014). Its soil types are mainly leaching grey, cinnamonic soil (Lyu et al. 2012). The vegetation on Manhan Mountain is typical forest shrub vegetation, including natural forest that consists of Betulaplatyphylla Sukaczev and Populusdavidiana Dode, plantation that consists of Larixprincipis-rupprechtii Mayr and Pinussylvestrisvar.mongolica Litv., natural shrubs that consists of Ostryopsisdavidiana Decne., Spiraeasalicifolia L. and Rosadavurica Pall. and the herbaceous plants including Stipabungeana Trin., Cleistogenessquarrosa (Trin.) Keng, Lespedezabicolor Turcz., Carex spp. and Leymuschinensis (Trin.) Tzvelev (Zhang et al. 2017; Li et al. 2021). Didymodonmanhanensis is currently known only from the type locality at the foot of the Manhan Mountain, north-western Liangcheng County, Inner Mongolia, China, growing on soil under the grass.

Key to species morphologically similar to D.manhanensis

1	Leaf apices apically swollen as a propagulum	D.anserinocapitatus
–	Leaf apices not swollen, usually evenly narrowing	2
2	Cells on the upper ventral surface of the costa elongate	D.wisselii
–	Cells on the upper ventral surface of the costa quadrate	3
3	Laminal cells smooth	4
–	Laminal cells papillose	10
4	Costa with 2–3 layers of guide cells and without ventral stereids	5
–	Costa with 1 layer of guide cells and with ventral stereids	6
5	Leaves patent to spreading when moist, leaf lamina bistratose	D.obtusus
–	Leaves spreading when moist, leaf lamina unistratose	D.manhanensis
6	Costa percurrent or ending before the apex	7
–	Costa long-excurrent	8
7	Leaf margins bistratose near apex	D.epapillatus
–	Leaf margins unistratose	D.mongolicus
8	Plants flagellate, leaves linear-lanceolate	D.ditrichoides
–	Plants thickly leaved, leaves short-lanceolate to long-lanceolate	9
9	Leaves appressed when dry	D.acutus
–	Leaves twisted or incurved when dry	D.validus
10	Leaf margins plane	D.tibeticus
–	Leaf margins recurved	11
11	Costa without ventral stereids	12
–	Costa with ventral stereids	13
12	Costa excurrent	D.daqingii
–	Costa ending below apex	D.imbricatus
13	Marginal basal cells forming a distinctly differentiated area of smooth and transversely thick-walled cells	D.hengduanensis
–	Marginal basal cells not forming a distinctly differentiated area	14
14	Distal laminal cell superficial walls thicker than the internal walls	D.mesopapillosus
–	Distal laminal cell superficial walls of same thickness as the internal walls	15
15	Laminal cells with low papillae over the transverse walls, which reach the two adjacent cells	16
–	Laminal cells with papillae situated over the lumina	17
16	Leaves spreading when moist	D.guangdongensis
–	Leaves erect to patent when moist	D.vulcanicus
17	Leaf margins recurved in proximal 1/4–3/4	D.icmadophilus
–	Leaf margins strongly recurved or revolute to near apex	18
18	Leaf base squared in shape, costa slender	D.tectorum
–	Leaf base usually ovate in shape, costa stout	D.cordatus

Citation

Feng C, Zhang G-L, Wu T-T, Kou J (2022) Didymodon manhanensis (Pottiaceae, Bryophyta), a new species from Inner Mongolia steppe, China and its phylogenetic position, based on molecular data. PhytoKeys 197: 41–57. https://doi.org/10.3897/phytokeys.197.80531

Funding Statement

Natural Science Foundation of China (grant no. 42001045, 32060051, 31660051), Shenzhen Key Laboratory of Southern Subtropical Plant Diversity (grant no. 99203030) and the Innovative team of China's Ministry of Education-Research on the sustainable use of grassland resources (IRT_17R59)