A Synopsis of Dicranum Hedw. (Dicranaceae, Bryophyta) in China, with Special References to Four Species Newly Reported and Re-Evaluation of Dicranum psathyrum Klazenga

Dicranum Hedw. is a highly diverse and widely distributed genus within Dicranaceae. The species diversity and distribution of this genus in China, however, remain not well known. A new revision of Dicranum in China using morphological and molecular phylogenetic methods confirms that China has 39 species, including four newly reported species, D. bardunovii Tubanova & Ignatova, D. dispersum Engelmark, D. schljakovii Ignatova & Tubanova, and D. spadiceum J.E.Zetterst. Dicranum psathyrum Klazenga is transferred to Dicranoloma (Renauld) Renauld as a new synonym of Dicranoloma fragile Broth. Two species, Dicranum brevifolium (Lindb.) Lindb. and D. viride (Sull. & Lesq.) Lindb. are excluded from the bryoflora of China. A key to the Chinese Dicranum species is also provided. These results indicate an underestimation of the distribution range of numerous Dicranum species, underscoring the need for further in-depth investigations into the worldwide Dicranum diversity.


Introduction
Dicranaceae are a family of mosses (Bryophyta).Dicranum Hedw.(including Orthodicranum (Bruch & Schimp.)Loeske) stands out as one of the most diverse genera within Dicranaceae, comprising approximately 98 accepted species [1].The taxonomy of this genus presents significant challenges owing to the considerable variation of gametophytic characteristics influenced by environmental conditions [2][3][4][5].Recent advancements in molecular phylogeny have led to a more precise delineation of Dicranum, including the identification of distinct groups such as Dicranum species with fragile leaves [6,7], D. fuscescens Turner complexes [8,9], D. scoparium Hedw.complexes [4,10], arctic Dicranum species [11], D. acutifolium (Lindb.& Arnell) C.E.O.Jensen complexes [12,13], and European Dicranum species [14].Despite these valuable insights, it is essential to recognize the geographical limitations of these studies, which have predominantly focused on Europe and Asian Russia, thus restricting our comprehensive understanding of Dicranum species diversity in other regions worldwide.Particularly in China, the delineation and relationships among morphological species of Dicranum remain challenging to ascertain.
In 1890-1895, Italian missionary Giuseppe Giraldi collected numerous bryophyte specimens in Shaanxi Province, China.These specimens were studied by Carl Müller, and the findings were published in the work titled Bryologia provinciae Schen-si Sinensis I-III [15][16][17].The discovery of Dicranum rectifolium Müll.Hal.and D. drummondii Müll.Hal.To advance our understanding of Dicranum diversity in China, we conducted a taxonomic revision of Dicranum in China over the last two years [7,38].Throughout this period, we encountered some intriguing specimens that did not correspond to any previously documented Dicranum species in China.To clarify the taxonomic classification of these Chinese mosses, we conducted a phylogenetic analysis by sequencing five chloroplast markers (rpoB, rps4-trnT, rps19-rpl2, trnH-psbA, and trnL-trnF) and one nuclear marker (ITS region).Through a meticulous examination of morphological characteristics and molecular phylogeny analysis, we confirmed the existence of four new records of Dicranum species in China, including D. bardunovii, D. dispersum, D. schljakovii, and D. spadiceum.
The primary objectives of this study are (1) to confirm the species diversity of Dicranum in China based on literature sources and our findings, (2) to provide photographs and descriptions of four new national records of Dicranum species, (3) to delineate the distribution of each Dicranum species within China and elucidate the differences between each species and those commonly confused species, and (4) to provide an updated key to Dicranum species in China.
(2) Ten accessions of D. dispersum form a strongly supported clade (BS ML = 99, PP BI = 0.91).Within this clade, one accession from China and one accession from Russia form a single subclade with high support values (BS ML = 99, PP BI = 0.99).(3) Two accessions of D. schljakovii collected from China form a well-supported clade with twelve other accessions (BS ML = 100, PP BI = 1).( 4) Five accessions of D. spadiceum collected from China (Qinghai and Xinjiang) are nested deeply within the other accessions from Europe and Russia (BS ML = 100, PP BI = 0.99).( 5) The accession of D. psathyrum is deeply nested within the genus Dicranoloma (Renauld) Renauld (BS ML = 99, PP BI = 0.93).
Based on the phylogenetic analysis, we conclusively confirm the newly recorded distribution of Dicranum dispersum, D. schljakovii, and D. spadiceum in China, and propose transferring D. psathyrum to the genus Dicranoloma.In addition, the tree topology allows for considering the accession of Dicranum bardunovii from China within D. acutifolium (Figure 1).Despite this, significant morphological distinctions exist between these two species (see note under D. bardunovii for details), supporting that our collection in China represents D. bardunovii rather than D. acutifolium.

20220830-30B (HSNU).
Description: Plants robust, in loose tufts, green or yellow-green in the upper part, brownish below.Stems 5-8 cm, matted with white tomentum in the upper part, rusty tomentum and rhizoids at the base.Cross-section of stem is rounded, consisting of 1-2 layers of smaller, obviously thick-walled outer cortical cells, numerous rows of larger and thinnerwalled inner cortical cells, and 6-9 rows of central strand cells.Leaves straight and erectspreading when wet, slightly curved when dry, 5.8-7.5 × 0.75-0.86mm, lanceolate, from ovate base gradually acuminate.Leaf margins are serrated in the upper 3/5, strongly serrated in the distal part, and entirely smooth below.Costa ca.200 µm wide at the leaf base, about 1/5 of leaf width at the base, the dorsal side of the costa is sharply and densely scabrose in the upper part, with one row of guide cells in the transverse section, two stereid bands, ventral epidermis not differentiated, and well differentiated dorsal epidermis.Leaf lamina is unistratose, with bistratose regions on or near the upper margins.Upper and median laminal cells are 7-18 × 7-9 µm, quadrate, irregularly angled, or short rectangular, a lot of which are strongly prorulose on the upper ends on the dorsal side of the leaf.Basal laminal cells are elongated rectangular to linear, 43-100 × 7-10 µm, and pitted.Alar cells are differentiated, brownish, decurrent, and not extending to the costa, with a group of thin-walled hyaline cells between the alar cells and costa, with 2-3 layers of cells in the transverse section.Sporophyte not seen.Dicranum bardunovii is highly similar to D. acutifolium, but can be differentiated by its sharply and densely scabrose laminal cells and costa in the distal part of the leaf, whereas the latter species has smooth cells and costa [9].Although Tubanova and Ignatova [9], Lang et al. [12], and Kiebacher and Szövényi [13] provided molecular evidence supporting distinctions between the two taxa, the phylogeny trees in their results exhibited weak clade support.Lang et al. [12] also found that one of the two accessions from the holotype specimen of D. bardunovii nested deeply within the clade of D. acutifolium and attributed it to mixed collections of these two species in the holotype specimen.However, the morphological characteristics of the type of specimen do not support their hypothesis (refer to Supplementary Table S2 in Lang et al. [12]).The phylogenetic tree in our analysis showed low branch support in this complex, with D. bardunovii collected from China deeply nested within D. acutifolium (Figure 1).However, morphologically, we are highly confident that the Chinese specimen belongs to D. bardunovii, as evidenced by the sharply and densely scabrose laminal cells and costa in the distal part of the leaf (Figure 3G).
Dicranum bardunovii is well characterized by (1) its ventral stereid band being exposed and surface cells not differentiated (Figure 3H), (2) a transverse section of leaf in the upper part like a pair of tongs (Figure 3H), (3) a sharply and densely scabrose dorsal side of laminal cells and costa in the distal part of the leaf (Figure 3G), ( 4) strongly serrated margins in the distal part (Figure 3C), and ( 5) bistratose alar cells (Figure 3H).There are no obvious differences between the morphological characteristics mentioned above from China and the illustrations based on the type of specimen in Tubanova and Ignatova [9].Description: Plants rather robust, in loose tufts, green in the upper part, and brownish below.Stems 4-8 cm, matted with white tomentum in the upper part, and rusty tomentum and rhizoids at the base.Cross-section of stem is rounded, consisting of 2-4 layers of smaller, obviously thick-walled outer cortical cells, numerous rows of larger and thinnerwalled inner cortical cells, and a number of central strand cells.Leaves somewhat flexuous, erect-spreading when wet, loosely curved when dry, usually keeled in the upper part, and appearing V-shaped or looking like a pair of tongs in the cross-section.Leaves gradually narrowed from an ovate-lanceolate base to a long acuminate apex, 9.5-12 × 1-1.2 mm.Leaf margins are serrated in the upper 3/5 to 1/2, and entirely smooth below.Costa is 1/5-1/4 of leaf width at the base and 200-300 µm wide at the leaf base, with teeth on the dorsal surface in the upper part, with one row of guide cells in the transverse section, two stereid bands, and differentiated dorsal and ventral layers of cells.Leaf laminal is unistratose, with bistratose regions on or near the upper margins.Upper and median laminal cells are 13-25(-34) × 9-11 µm, quadrate, irregularly angled, short rectangular, smooth, or slightly mammillose.Basal laminal cells are elongated-rectangular to linear, 65-120 × 7-12 µm, and slightly pitted or not.Alar cells are differentiated, brownish, extending to the costa, with 2-3(-4) layers of cells in the transverse section.Sporophyte not seen.
Dicranum dispersum has been classified as an endangered species in the Red Lists of Germany and France [40] and assessed as endangered under criteria D in The IUCN Red List of Threatened Species [39].In recent years, new distribution sites of this rare species have been discovered in Europe, Asiatic Russia, and Alaska, U.S.A. [5,41,42], and our study confirms its presence in Qinghai Province, China.To date, the known populations are highly fragmented due to habitat fragmentation, resulting in small, isolated patches that are unable to sustain minimum viable populations and are at risk of entering an extinction vortex [43,44].Furthermore, moss species, which prefer cold environments, may be more vulnerable to the global warming climate [45,46].Therefore, we recommend including this rare species on the China Species Red List and advocating for further research into the distribution and biology of high-altitude distributed Dicranum species.Description: Plants in loose tufts, light brownish green.Stems are 3-5 cm and moderately tomentose.Cross-section of stem is rounded, consisting of 2-4 layers of smaller, obviously thick-walled outer cortical cells, numerous rows of larger and thinner-walled inner cortical cells, and 5-7 rows of central strand cells.Leaves are straight and erect when wet, loosely appressed when dry, 7-8 × 0.13-0.15mm, gradually to abruptly narrowed into long and narrow tubular acumen from the ovate base, semicircular in the transverse section.Leaf margins are subentire, slightly serrulate distally, and unistratose.Costa is ca.120 µm wide at the leaf base, about 1/13 to 1/10 of leaf width at the base, smooth or weakly mammillose in the upper part on the abaxial surface, with one row of guide cells in transverse section, two stereid bands, and differentiated ventral and dorsal epidermis.Leaf lamina is unistratose and smooth.Upper and middle laminal cells are 20-41 × 9-13 µm, irregular in shape, moderately thick-walled, and slightly porose.Basal laminal cells are elongated-rectangular, 45-85 × 8-12 µm, and pitted.Alar cells are well differentiated, 2-4-stratose, brownish, and hyaline, with few or no thin-walled cells between the costa and alar groups.Sporophyte not seen.
According to the description and illustration of the lectotype specimen of Dicranum spadiceum by Ignatova et al. [5], only two layers of alar cells are present in this species.This characteristic was also noted and confirmed by Hedenäs and Bisang [2], Ireland Jr. [3], Ignatov and Ignatova [49], and Lüth [50].In our samples, three specimens from Xinjiang also exhibit bistratose alar cells (Figure 6K), while two specimens from Qinghai show partially 3-4-layer alar cells (Figure 6I).Our phylogenetic analyses indicate that these two intriguing samples from Qinghai are highly nested within the other samples (BS ML = 99, PP BI = 1; Figure 1), suggesting that they belong to D. spadiceum.We propose that this characteristic may be influenced by environmental factors, as the two specimens from Qinghai were found at high altitudes of 3737 m, whereas specimens from Xinjiang (this study), Europe, and Asian Russia were found at lower altitudes ranging from 704 m to 2430 m [5].The differences between D. spadiceum and D. schljakovii are discussed under the latter species.
Notes: This species was initially described as Dicranum fragile Hook., nom.illeg., by Hooker [52].Later, Brotherus [53] formally published the species as Dicranoloma fragile Broth.However, Klazenga [54] moved this species to the genus Dicranum due to the absence of limbidium and differences in the transverse section of the costa from all Dicranoloma species.Nevertheless, the epithet "fragile" was already in use for Dicranum fragile Brid.
According to our phylogenetic analysis, Dicranum psathyrum was found deeply nested within the genus Dicranoloma (BS ML = 99, PP BI = 0.93) (Figure 1).Consequently, Klazenga's [54] taxonomic classification should be rejected, the taxonomic status of Dicranoloma fragile needs to be reinstated, and Dicranum psathyrum should be treated as a synonym of Dicranoloma fragile.
Note: Dicranum assamicum may be confused with D. japonicum Mitt.because both species possess 2-4 layers of alar cells.However, D. assamicum can be well distinguished by the serrated costa on the upper portion of the leaf, straight capsules, smooth peristome teeth above, and the absence of minute and longitudinal point striations below [30,56,57], whereas D. japonicum is characterized by 2-3(-4) serrated ridges on the upper portion of the costa, slightly curved capsules, densely papillose peristome teeth above, and minute and longitudinal point striations below [58].
Note: Dicranum bonjeanii can be recognized by its elongated-rectangular upper cells, weakly to strongly transversely undulate upper leaf portions, and comparable orientation of apical and lower leaves.This species could only be confused with D. polysetum Sw. in China, and the differences between them are discussed under D. polysetum.
Note: Dicranum cheoi may be confused with D. fuscescens, D. hamulosum Mitt., D. bardunovii, and D. muehlenbeckii Bruch & Schimp.However, the epidermal cells on the ventral side of the costa are not differentiated in D. fuscescens, D. hamulosum, and D. bardunovii, but well differentiated in D. cheoi.In addition, the upper leaf lamina is bistratose along the margins in D. cheoi, but unistratose in D. muehlenbeckii.
Note: Dicranum groenlandicum, D. elongatum, D. himalayanum Mitt., and D. setifolium are closely related and can be easily confused; the distinctions among them are annotated under the latter three species, respectively.
Dicranum hakkodense Cardot, Bull.Herb.Boissier, sér.2, 7: 714.1907.Distribution in China: Sichuan [6].Note: Dicranum hakkodense has long been classified as D. viride var.hakkodense (Cardot) Takaki, but was subsequently reclassified as a distinct species based on molecular evidence by Ignatova and Fedosov [6].The presence of sharply denticulate leaf apices serves as a key feature for distinguishing between D. hakkodense and D. viride; while the former exhibits sharp denticulation apices, the latter has only a few blunt teeth or entirely smooth apices.Variations in comparison to D. fragilifolium and D. hengduanense are individually discussed within the context of the latter species.
Note: Dicranum hamulosum might be confused with D. cheoi and D. fuscescens, but can be distinguished by the unistratose alar cells, contrasting with the bistratose alar cells found in D. cheoi and D. fuscescens.In addition, the epidermal cells on the ventral side of the costa are undifferentiated in D. hamulosum but show differentiation in D. cheoi.
Note: Dicranum hengduanensis is one of the Dicranum species with fragile leaves.In contrast to D. hakkodense and D. psathyrum (≡Dicranoloma fragile), this species exhibits entirely smooth leaf apices instead of sharply denticulate ones.Differences from D. fragilifolium are discussed under the latter species.In addition, D. hengduanense can be distinguished from other Dicranum species with delicate leaves by the absence of stereid bands and the presence of only one layer of cells above and below the guide cells [7].
Note: Dicranum himalayanum can be confused with D. groenlandicum due to the smooth leaf margin and prosenchymatous and porose laminal cells in the upper portion of the leaf.However, these two species differ in their growth patterns: D. himalayanum forms loose tufts with branching and possesses 2-3-layered alar cells, whereas D. groenlandicum typically creates very dense cushions with minimal or no branching and has unistratose or occasionally bistratose alar cells [30,34].
Note: Dicranum kashmirense may be confused with D. scoparium but can be distinguished by the unistratose alar cells in D. kashmirense, while they are bistratose in D. scoparium.
Note: Dicranum lorifolium may be confused with D. assamicum, D. baicalense, D. bonjeanii, D. japonicum, D. nipponense, and D. scoparium in China, and it is difficult to distinguish them based solely on gametophyte.However, D. lorifolium can be well distinguished by the following unique combination of characteristics: (1) bistratose alar cells, (2) costa above with two lamellae or ridges on its back, (3) the presence of short cells above the alar cells, extending upwards along the leaf margins, and (4) always erect capsules.
Dicranum majus Turner, Muscol.Hibern.Spic.: 58.1804.Distribution in China: Widely distributed in China [35,36,66].Note: Dicranum majus is one of the largest members of the genus when well developed.This is a very variable species [2,69], in terms of leaf curvature and length, the upper leaf lamina being partly bistratose or not, and the upper laminal cells spinosely projecting or not [2].D. majus may be confused with D. scoparium, but D. scoparium has four distinct ridges on the upper back of the costa rather than furrows and serrations in D. majus.In addition, setae generally aggregate in D. majus but are solitary in D. scoparium [69].
Note: Dicranum montanum is one of the smallest species within the genus in China and is easily recognized because it looks more like Dicranoweisia cirrata (Hedw.)Lindb.than a Dicranum species.In addition, another aid in its identification is the occasional presence of small, delicate, clustered branchlets with linear leaves that are readily detachable (probably a means of asexual reproduction like Brothera leana (Sull.)Müll.Hal.), which occur near the stem apices.
Note: Dicraunm muehlenbeckii may be confused with D. fuscescens; however, the epidermal cells on the ventral side of the costa are not differentiated in D. fuscescens, whereas they are well differentiated in D. muehlenbeckii.
Distribution in China: Endemic to China, known only from Sichuan [33,34,55].Note: Dicranum papillidens was first reported by Brotherus [20] in 1924 and has remained elusive for almost a century.This species is characterized by (1) unistratose alar cells, (2) quadrate or short rectangular cells in the upper leaf, (3) a smooth abaxial surface of the upper laminal cells and costa, (4) being strongly porose in the basal and middle leaf cells, and (4) having densely papillose peristome teeth throughout.Dicranum papillidens can be easily separated from D. leiodontum by its strongly porose basal and middle laminal cells as well as its densely papillose peristome teeth.
Note: Dicranum polysetum is readily distinguishable in the field due to its distinctive leaf orientation, with erect uppermost leaves and spreading lower leaves, as well as strongly undulate leaves.This species can only be confused with D. bonjeanii; however, the orientation of the apical leaves and leaves further down are not clearly different in the latter species.
Dicranum rectifolium Müll.Hal., Nuovo Giorn.Bot.Ital., n.s.3: 98. 1896.Distribution in China: Endemic to China, known only from Shaanxi [15].Note: This species was initially reported by Müller in Shaanxi [15] but has not been rediscovered for over 125 years.Regrettably, there is no available information regarding illustrations or descriptions of this species.This species was classified as an insufficiently known species in the earlier Chinese Checklist of the Mosses [70].
Dicranum  [35,36,55].Note: Dicranum scoparium is one of the most variable Dicranum species, with both straight and falcate leaves commonly observed, and the presence of both dwarf and large male plants.In China, this species can be distinguished from other members of the D. scoparium complex (including D. baicalense, D. bonjeanii, D. japonicum, D. lorifolium, D. nipponense, and D. polysetum) by the four ridges on the dorsal surface of its costa, as opposed to the typical two ridges found in other species.
Note: Dicranum scottianum is characterized by (1) its asymmetrical leaves, with one side of the leaf being wider than the other, (2) the difference in the angle of insertion of the lamina with respect to the costa, (3) the laminal cells that are thick-walled, and (4) the wide and deep costa that has two bands of stereids.In the only specimen we examined from Taiwan, the margins are dentate in the upper part of the leaf, which can be referrable to D. canariense Hampe ex Müll.Hal.[2]; however, Price et al. [71] [35,36,55], including Heilongjiang [28], inner Mongolia [33,34,59], and Jilin [33,34].
Note: Dicranum spurium may be confused with D. linzianum in China as both species exhibit ovate-lanceolate leaves (relatively broad basal portion, becoming suddenly narrow to a short acumen).However, these species can be differentiated by the upper laminal cells: those of D. spurium are irregular, triangular, or quadrate, with prorulae on the dorsal surface, whereas D. linzianum has oval and smooth upper laminal cells.
Note: Dicranum undulatum may be confused with D. polysetum and D. bonjeanii in China due to their similarly transversely undulating leaves but can be distinguished by the irregular oval shape of the upper laminal cells, which differ from the elongated-rectangular cells found in the latter two species.

Excluded Species
Dicranum brevifolium (Lindb.)Lindb., Musci Scand.: 24.1879.Note: Sulayman et al. [72] reported the presence of this species in the Altai Mountains, Xinjiang.Although we have not seen the voucher specimen directly, based on their illustrations, it is clearly an incorrect identification: (1) the transverse section of the upper leaf looks like a pair of tongs in D. brevifolium [2,12,47], but it looks more or less like a circular outline in their illustration; (2) the ventral epidermal layer of cells, which are usually poorly differentiated in D. brevifolium [2,8,47], are distinctly differentiated in their illustration.In addition, (3) while the leaf lamina is bistratose along the margins in D. brevifolium, it is unistratose in their illustrations.In conclusion, D. brevifolium should be excluded from the moss flora of China.

Key to Dicranum Species in China
Note: Dicranum rectifolium is not included because we have no information, illustrations, or descriptions of this species, which was merely relisted without additional specimens in a checklist from 1896 and was listed as an insufficiently known species in China [70].adjusted manually.The assembled nuclear DNA was aligned with published ITS data using D. scoparium as a reference (accession number: KF423564) in Geneious v.11.1.5[78], and then annotated and extracted.
Phylogenetic analyses were conducted using maximum likelihood (ML) and Bayesian inference (BI) methods in IQtree v2.0.6 [81] and MrBayes 3.2.6 [82], respectively.IQtree was performed with the best-fitting substitution model for each DNA region (HKY+F+G4 for ITS-partition, TPM2u+F+I+G4 for rps19-rpl2-partition, rpoB-partition, rps4-trnT-partition, and trnL-trnF intergenic spacers, and TPM3+F+G4 for trnH-psbA-partition) selected by ModelFinder according to the Bayesian information criterion (BIC) [83,84], and the fast bootstrap option with 1000 replicates.For BI analyses, each DNA region was also assigned its own substitution model (HKY+G is the for best-fit model for ITS-partition; GTR+I+G for rpoB-partition and for trnL-trnF intergenic spacers; HKY+G for rps4-trnT-partition and for rps19-rpl2-partition; HKY+G for trnH-psbA-partition), as determined by the Akaike information criterion (AIC) [83,84].Two independent analyses consisting of four Markov chain Monte Carlo (MCMC) chains were run for 5,000,000 generations, with one tree sampled for every 1000 generations.The posterior distribution of trees was summarized by a >50% majority-rule consensus tree after discarding the first 25% of samples as burn-in.Convergence was assessed by examining the likelihood plots in Tracer v.1.7 [85].

Conclusions
Four species of Dicranum are newly reported for China, including D. bardunovii, D. dispersum, D. schljakovii, and D. spadiceum.Two species of Dicranum, D. brevifolium and D. viride, are proposed for exclusion from the bryoflora of China.Additionally, Dicranum psathyrum is proposed as a synonym of Dicranoloma fragile.Currently, a total of 39 Dicranum species are known in China.

Figure 1 .
Figure 1.Phylogeny of Dicranum species inferred from the combined dataset (trnH-psbA, rps4-trnT, trnL-trnF, rps19-rpl2, rpoB, and ITS).The topology derived from the best-scoring ML tree in IQtree is shown.ML bootstrap values BS ≥ 70 are shown on the left and Bayesian posterior probabilities values PP ≥ 0.90 on the right.The subject species are in red and indicated by arrows.Section division refers to Hodgetts et al. [37].

Figure 1 .
Figure 1.Phylogeny of Dicranum species inferred from the combined dataset (trnH-psbA, rps4-trnT, trnL-trnF, rps19-rpl2, rpoB, and ITS).The topology derived from the best-scoring ML tree in IQtree is shown.ML bootstrap values BS ≥ 70 are shown on the left and Bayesian posterior probabilities values PP ≥ 0.90 on the right.The subject species are in red and indicated by arrows.Section division refers to Hodgetts et al. [37].

Table 1 .
A list in chronological order of the Dicranum species described and recorded from China.Names in bold indicate being newly reported to science based on the specimens collected from China.Dicranum psathyrum (≡Dicranoloma fragile Klazenga) is not included.

Distribution in China: Yunnan
(present report), new to China.