Taxonomic Reevaluation of Didymodon Nigrescens (Pottiaceae) in Japan

Hattoria 11: 61–75. 2020

Taxonomic reevaluation of Didymodon nigrescens (Pottiaceae) in Japan

Yuya INOUE1, 2, Juan A. JIMÉNEZ3, Takumi SATO2, Hiromi TSUBOTA1 & Tomio YAMAGUCHI1

1 Program of Basic Biology, Graduate School of Integrated Sciences for Life, Hiroshima University, Kagamiyama 1–3–1, Higashihiroshima, Hiroshima 739–8526, Japan 2 Hattori Botanical Laboratory, Obi 6–1–26, Nichinan, Miyazaki 889–2535, Japan 3 Departamento de Biología Vegetal, Facultad de Biología, Universidad de Murcia, Campus de Espinardo 30100, Murcia, Spain Author for correspondence: Yuya INOUE, [email protected]

Abstract Based on morphological investigation and molecular phylogenetic analysis, we ﬁnd that the previous concept of Didymodon nigrescens (Mitt.) K.Saito sensu Saito in Japan includes D. nigrescens and D. subandreaeoides (Kindb.) R.H.Zander, the latter newly reported from Japan. Morphological and phylogenetic data from Japanese material clearly segregate these species from each other. In addition, Japanese D. nigrescens shows two groups, with morphological traits supporting the phylogeny, requiring further morpho-molecular evaluation based on broad sampling. Descriptions with analytical illustrations are provided based on Japanese material. Andreaea takakii Sakurai is proposed as a synonym of D. subandreaeoides.

Introduction Didymodon nigrescens (Mitt.) K.Saito is a member of a group of Holarctic mosses whose range penetrates deeply into the tropics, including Central America and East Africa (Bednarek- Ochyra 2018). According to the monograph of Japanese Pottiaceae (Saito 1975), the species grows on calcareous rocks or cliffs in montane to subalpine zones, and was known from several localities from Central Honshu and Shikoku. Saito circumscribed the species by the following characters: (1) plants dark brown to blackish-green, (2) stems more or less branched, (3) basal cells of the leaves rhomboidal, sclerenchymatous near the costa, (4) seta moderately thick, strongly flattened when dry, and (5) perichaetial leaves about two times as long as the stem-leaves. The species was very variable and the extreme forms of variation superficially appear to be different species, based on Japanese and Chinese materials. In the course of floristic research in the Japanese Southern Alps in Central Honshu and Mt. Tsurugi in Shikoku, we collected several specimens of D. nigrescens (sensu Saito 1975) with variable morphological characters as discussed by Saito. After detailed morphological

61 and molecular investigation, using dried herbarium specimens, we found that his concept of D. nigrescens included two distinct species. One is D. nigrescens and the other D. subandreaeoides (Kindb.) R.H.Zander, the latter distributed in the Beringian part of Arctic Russia, northwestern North America, Central Europe, and also East Asia (e.g. Cao & Gao 1995; Kučera & Köckinger 2000), but has not been previously recognized for Japan. In the present paper, we newly report D. subandreaeoides from Japan and revised descriptions of these two species are provided based on Japanese material. The phylogenetic positions of the Japanese plants are inferred based on the nuclear ITS sequences with maximum likelihood and Bayesian inferences.

Materials and Methods Morphological investigation The morphological investigation was made based on fresh samples included in the molecular phylogenetic analyses and additional dried herbarium specimens deposited in CBM, H, HIRO, KOCH, MAK, NUM, MUB, NICH, and S.

Molecular phylogenetic analyses Nuclear internal transcribed spacers 1 and 2 including 5.8S (ITS) were selected for the present analyses. Total DNA was extracted from leaves or shoot tips following the method by Suzuki et al. (2013) or Doyle & Doyle (1987) with some minor modifications. At the first author’s laboratory, the conditions of PCR amplification followed Inoue & Tsubota (2014). Primers for PCR amplification and DNA sequencing followed Oguri et al. (2003). PCR products were purified by using NucleoSpin Gel and PCR Clean-up kit (Macherey-Nagel, Duren) following the manufacturer’s protocols. Purified PCR products were sequenced by Macrogen Japan (Kyoto, Japan). At the second author’s laboratory, the PCR amplification was performed in a 25 µl volume containing 1 µl Taq DNA Polymerase (1 U/µl; Biotools, Madrid, Spain), 2.5 µl of Mg2＋ buffer provided by the manufacturer, 2 µl of 2.5 mM dNTP mix, 1.5 µl of each primer (10 µM) and 1 µl of the DNA extract. Primers for PCR and sequencing followed Stech & Frahm (1999). PCR conditions were: 95°C for 4 min linked to 38 cycles at 94°C for 30 sec., 52°C for 30 sec., and 72°C for 1 min. with a final extension of 72°C for 10 min. After visualization of PCR products with 1.5% agarose gel, successful amplicons were purified using the GenElute PCR Clean-Up kit (Sigma-Aldrich, St. Louis, Missouri), and sequenced at Macrogen Spain (Madrid, Spain). Sequences obtained in the present study have been submitted to the DNA Data Bank of Japan (DDBJ), a member of the International Nucleotide Sequence Database Collaboration (INSDC). Based on the results by Werner et al. (2005) and Kučera & Ignatov (2015), we selected published sequences of subsect. Fuscobryum (R.H.Zander) Jan Kučera as ingroup, and a species of sect. Didymodon as outgroup, using D. rigidulus Hedw. A total of 25 ITS sequences were examined in the present analyses, as shown in Appendix. Sequences were aligned using the program MAFFT ver. 7.463 (Katoh & Standley 2013) with some manual adjustment on the sequence editor of MEGA ver. 7.0.21 (Kumar et al. 2016). Gaps were treated as missing data. Prior to the phylogenetic reconstruction, the

62 software ModelTest-NG (Darriba et al. 2019) was used to determine the appropriate substitution model for our data based on corrected Akaike information criterion (AICc: Sugiura 1978). Phylogenetic analyses were performed based on maximum likelihood (ML) and Bayesian inference (BI) methods. RAxML-NG ver. 0.9.0 (Kozlov et al. 2019) was used for ML analysis using the TIM3ef＋I＋G4 model with a rapid bootstrap analysis with 10,000 replicates. MrBayes ver. 3.2.7a (Ronquist et al. 2012) was used for BI using the SYM＋I＋G4 model with 10,000,000 generations, sampling trees every 1,000 generations. Convergence was assessed using Tracer ver. 1.7.1 (Rambaut et al. 2018). A 50% majority-rule consensus tree was calculated after the convergence of the chains and discarding 25% of the sampled trees as burn-in.

Results & Discussion Molecular phylogeny The data matrix had a total length of 746 bp, of which 94 (13%) were variable, and 70 (74% of the variable sites) were parsimony-informative. No topological conflict was detected between ML and BI trees which differed only at poorly supported nodes. Figure 1 shows the ML tree with supporting values from bootstrap and Bayesian posterior probabilities (BP/PP). The monophyly of subsect. Fuscobryum was well supported (100/1.00), and each analyzed species formed moderately to well supported clades as shown by Kučera & Ignatov (2015). The generic circumscription of Didymodon was recently reevaluated based on the macroevolutionary analysis using morphological data, and the genus were segregated into seven genera: Aithobryum R.H.Zander, Didymodon s.str., Exobryum R.H.Zander, Fuscobryum R.H.Zander, Geheebia Schimp., Trichostomopsis Cardot and Vinealobryum R.H.Zander (Zander 2013, 2019). If this were followed, all ingroup species of the present study would be treated as the genus Fuscobryum: F. nigrescens (Mitt.) R.H.Zander, F. perobtusum (Broth.) R.H.Zander and F. subandreaeoides (Kindb.) R.H.Zander. However, there are still not available comprehensive molecular data for evaluating this proposed systematic rearrangement, and here we use Didymodon in the broad sense. The Japanese samples were resolved as separate D. nigrescens and D. subandreaeoides clades, supporting the notion that both species occur in Japan. In D. nigrescens, two subclades were confirmed: Bhutan–Japan (93/0.98) and Nepal–China–Japan–U.S.A. (80/1.00), and Japanese samples showed different morphotypes corresponding to these subclades as discussed below. In D. subandreaeoides, Japanese samples were resolved in one clade and sister to the Russian sample (-/0.92).

Taxonomic treatment Based on our investigation, the following taxonomic treatment is presented. Descriptions are based on Japanese materials, and synonyms of each species include only basionyms and heterotypic synonyms described from Japan.

63 Figure 1. Maximum likelihood tree based on ITS sequences, depicted by RAxML-NG. Supporting values more than 50% obtained by RAxML-NG for bootstrap probabilities (BP) and MrBayes for Bayesian posterior probabilities (PP) are shown on each branch (BP/PP). The root is arbitrarily placed on the branch leading to D. rigidulus. (A) indicates plants with acute to narrowly acuminate leaf apex and not spurred costa (Figs. 2A–B, 2E; 3), and (B) with rounded-obtuse to obtusely acute leaf apex and weakly spurred costa (Figs. 2F; 4).

1. Didymodon nigrescens (Mitt.) K.Saito, J. Hattori Bot. Lab. 39: 510. 1975. Figs. 2–4 ≡Barbula nigrescens Mitt., J. Proc. Linn. Soc., Bot. 1: 36. 1859. ＝Andreaea kai-alpina Sakurai & Takaki, J. Jap. Bot. 29: 111. 2. 1954. Type: Japan. Honshu, Mt. Kitadake, 3,100 m elev., 8 Aug. 1953, Takaki 14439 in herb. Sakurai 35073 (holotype: MAK!), Takaki 14439 ex herb. Noguchi 71417 (isotype: NICH- 182424!) For further synonyms, see Saito (1975), Cao & Gao (1995), Allen (2002), Zander (2007), Aziz & Vohra (2008), and Zhao et al. (2018).

64 Figure 2. Didymodon nigrescens and D. subandreaeoides in Japan. A, B: Habitat of D. nigrescens at Mt. Toyoguchi. C, D: Habitat of D. subandreaeoides at Mt. Kitadake. E: Dry habit of D. nigrescens (group A, Inoue 5293). F: Dry habit of D. nigrescens (group B, Inoue 3664). G: Dry habit of D. subandreaeoides (Inoue 3652). Arrowheads indicate ﬂagelliform innovations.

Plants reddish to blackish brown throughout or blackish green above and blackish brown below when fresh, 0.5–2.0 cm high, forming dense turfs. Stems erect or procumbent, branched, smooth, rounded-pentagonal in cross section; central strand weakly differentiated or undifferentiated; sclerodermis weakly differentiated; hyalodermis undifferentiated; axillary hairs filiform 3–4 cells long, with 1 brown basal cell and hyaline upper cells. Rhizoids sparse

65 Figure 3. Didymodon nigrescens (group A). A: Cross section of stem. B–D: Vegetative leaves. E: Perichaetial leaf. F: Leaf apex (abaxial view). G: Basal laminal cells of vegetative leaf. H: Adaxial epidermal cells of costa (upper part). I–M: Cross sections of vegetative leaf. N: Cross section of seta. O: Deoperculate capsule. P: Mouth of deoperculate capsule. Q: Operculum. A, E, O, P drawn from Inoue 5293; B–D from Inoue 3665; F, G–M from Inoue 5298; N, Q from Inoue 3645a.

66 Figure 4. Didymodon nigrescens (group B). A: Cross section of stem. B–D: Vegetative leaves. E: Perichaetial leaf. F: Perigonial leaf. G: Leaf apex (abaxial view). H: Basal laminal cells of vegetative leaf. I: Adaxial epidermal cells of costa (upper part). J–N: Cross sections of vegetative leaf. A–D, F–N drawn from Inoue 3664; E from Inoue 3645b.

at base, reddish brown, smooth. Leaves appressed when dry, erect-patent when moist, monomorphic, long-ovate to lanceolate from an oblong, diﬀerentiated base tapering to an acute to narrowly acuminate or rounded-obtuse to obtusely acute apex, 0.8–1.7 mm long and 0.30–0.65 mm wide, not keeled, not channeled adaxially; lamina unistratose throughout, dark red in KOH; margins entire or crenulate at the upper part, recurved from base to 2/3 of the

67 leaf, not sinuous, unistratose, unbordered, decurrent on stem; costa stout, (20–)30–50(–60) µm wide at base, ending below the apex or percurrent, not spurred or weakly spurred above midleaf; epidermal cells of costa quadrate to shortly rectangular, smooth or papillose with 1–3 large, blunt papillae above midleaf and rectangular, smooth in basal part on adaxial side, subquadrate to oblate, papillose with 1–2 large, blunt papillae above midleaf and rectangular, smooth in basal part on abaxial side; costal cross section below midleaf semicircular; guide cells 3–4 in a single row; hydroids absent or not well differentiated; stereids 0–1 stratose on adaxial side, 1 stratose on abaxial side; epidermis well differentiated on adaxial and well or less differentiated on abaxial sides; upper and middle laminal cells thick-walled, subquadrate or oblate, (2.0–)5.0–9.0(–12.0)×(4.0–)5.0–7.5(–9.0) µm, papillose on both adaxial and abaxial sides with 1–2 simple papillae per lumen; basal laminal cells differentiated, thick- walled, smooth, rectangular or occasionally quadrate, 5.0–22.0(–32.0)×3.5–7.5(–10.0) µm at juxtacostal part, quadrate to oblate, (3.5–)5.0–10.0×5.0–10.0 µm at marginal part, not forming differentiated boundary. Asexual reproduction absent. Sexual condition dioicous. Perichaetia terminal; perichaetial leaves enlarged, more sheathing than vegetative leaves, oblong-lanceolate with narrowed apex, 1.70–3.0×0.43–0.80 mm. Perigonia terminal; perigonial leaves smaller than vegetative leaves, ovate, concave, 0.30–0.95(–1.20) mm long. Seta erect, 4.5–8.5 mm long, blackish brown, sinistrorsely twisted throughout when dry, with elongate thick-walled epidermal cells. Capsule stegocarpous, long-exserted, erect or slightly inclined, blackish brown; urn cylindrical, 1.1–1.7×0.4–0.5 mm; exothecial cells smooth, thick walled, rectangular at middle part, 45–90×12–22 µm; stomata phaneroporous, 11–15 at base of capsules; annulus persistent, consisting of 2–3 rows of vesiculose cells; peristome of 16 teeth divided completely to 32 filiform prongs, ca. 1.3 mm long, dextrorsely twisted, densely papillose, with basal membrane 35–40 µm in height; operculum conic-rostrate, 0.70–0.85 mm long, with dextrorsely arranged cells. Calyptra cucullate, ca. 2.3 mm long, smooth. Spores 10.0–12.5 µm in diam., yellowish brown, minutely papillose. Habitat in Japan: Growing on dry sunny or semi shaded limestone in subalpine regions around 1,800–3,000 m elev. Distribution: North America, Central America, East and South Asia, and Africa (e.g. Bednarek-Ochyra 2018). Taxonomic notes: Among Japanese populations, we confirmed two lineages with corresponding morphological characters: (A) leaves with acute to narrowly acuminate apex and costa not spurred (Figs. 2A–B, 2E; 3), and (B) leaves with rounded-obtuse to obtusely acute apex and weakly spurred costa (Figs. 2F; 4). The type of Andreaea kai-alpina Sakurai & Takaki well fitted with the plants of group B. Although we could not obtain morphogical data from the type of Barbula nigrescens Mitt. collected in India (Mitten 1859), these morphotypes were also confirmed in exotic specimens as noted in specimens examined. We could not compare sporophytic characters between these two groups due to a lack of sporophytes in group B. However, the perichaetial leaves of group B were shorter than those of plants from group A with a more narrowly acute apex. In the present study, we recognize these differences as morphological variation within the one species until more detailed morpho-molecular evidence is available. Representative specimens examined: JAPAN. Honshu, Saitama Pref., Chichibu City,

68 Azusashira-iwa, 1,830 m elev., on limestone, 15 Sep. 2019, Furuki s.n. in hb. Inoue 7823 (dupl. in HIRO, group A, DNA voucher); Yamanashi Pref., Minami Alps City, Mt. Kitadake, 35°40′16″N, 138°14′16″E, 3,010 m elev., on limestone boulder at open site, 11 Sep. 2015, Inoue 3645a (HIRO, group A, DNA voucher), Inoue 3645b (HIRO, group B, DNA voucher); ibid, 35°40′16″N, 138°14′21″E, 3,000 m elev., on limestone boulder at open site, 11 Sep. 2015, Inoue 3664 (HIRO, group B, DNA voucher), Inoue 3665 (HIRO, group A, DNA voucher); Nagano Pref., Ina City, Mt. Shiroiwa, 15 Aug. 1909, Ishiba s.n. (H-BR 3203052, group B); Shimoina Country, Oshika Village, 35°33′20″N, 138°07′38″E, 2,220 m elev., on limestone boulder at open site, 17 May 2017, Inoue 5293 (HIRO, group A), Inoue 5298 (HIRO, group A, DNA voucher), Inoue 5299 (HIRO, group A, DNA voucher); INDIA. Sikkim, Himalaya, Darjeeling, Onglak Thang, 4,180 m elev., 12 Oct. 1908, Long 16 (H-BR 0247001, group B); BHUTAN. s.d., collector unspecified in herb. Griffith 54 (H-BR 0247006, group A); USA. Alaska, Izembek NWR, Cold Bay, 54°40′N, 162°40′W, old abandoned brick house, 20 Aug. 1997, Schofield 109554 (H-3229378, group B); Aleutians West Country, Southern outskirt of Unalaska City, 0 m elev., 53°50′48.2″N, 166°30′28.3″W, old concrete foundation, 12 Aug. 2007. Schofield 127031 (H-3226386, group B); CANADA. British Columbia, Peterson Creek, 58°40′N, 125°48′W, limestone cliff face, forming spreading clones, 17 Jul. 1976, Schofield & Otto 62718 (H-3048180, group A).

2. Didymodon subandreaeoides (Kindb.) R.H.Zander, Phytologia 41: 23. 1978. Figs. 2 & 5 ≡Barbula subandreaeoides Kindb., Rev. Bryol. 32: 36. 1905. Type: Canada. British Columbia, Joho Valley, 6 Aug. 1904, Macoun 175 (lectotype: S-B3378!, designated by Kučera & Köckinger 2000). ＝Andreaea takakii Sakurai, J. Jap. Bot. 29: 111. 1. 1954; syn. nov. Type: Japan. Honshu, Mt. Kitadake, 3,100 m elev., 8 Aug. 1953, Takaki 14418 in herb. Sakurai 35072 (holotype: MAK!). ＝Grimmia andreaeoides auct. non Limpr.; Takaki, Bot. Mag. Tokyo 64: 180. 1951. For further synonyms, see Cao & Gao (1995) and Jiménez (2006).

Plants blackish or greenish brown above, blackish brown below when fresh, 0.6–1.3 cm high, forming dense turf. Stems erect, simple or branched, with deciduous flagelliform innovations, erect, smooth, rounded-pentagonal in cross section; central strand weakly differentiated or undifferentiated; sclerodermis weakly differentiated; hyalodermis undifferentiated; axillary hairs filiform, 3–5 cells long, with 1 brown basal and hyaline upper cells. Rhizoids sparse at base, reddish brown, smooth. Leaves dimorphic; leaves of main stem appressed when dry, erect-patent, not undulate when moist, ovate to ovate-lanceolate or lanceolate from an oblong base tapering to a rounded to obtuse apex, 0.45–0.95 mm long, 0.20–0.35 mm wide, not keeled, not channeled adaxially; lamina unistratose throughout or bistratose near the apex, dark red in KOH; margins plane or lightly recurved from base to ca. 3/5 of the leaf, entire or papillose-crenulate at upper part, uni- or bistratose near the apex, decurrent on stem; costa, stout, 20–35 µm wide at base, ending below the apex or percurrent, not spurred; epidermal cells of costa rounded to quadrate, papillose with 1–2 large blunt papillae above midleaf and rectangular, smooth in basal part on both adaxial and abaxial sides;

69 Figure 5. Didymodon subandreaeoides. A: Cross section of stem. B–D: Vegetative leaves of main stem. E–H: Leaves of ﬂagelliform innovation. I: Perichaetial leaf. J: Perigonial leaf. K: Leaf apex (abaxial view). L: Basal laminal cells of vegetative leaf. M: Adaxial epidermal cells of costa (upper part). N–R: Cross sections of vegetative leaf. S: Cross section of seta. T, U: Operculate and deopeculate capsules. V: Mouth of deoperculate capsule. A, E drawn from Inoue 4070; B–D, F–V from Inoue 3652.

70 costal cross section below midleaf semicircular; guide cells 2 in a single row; hydroids undifferentiated; stereids undifferentiated on adaxial side, 0–1-stratose on abaxial side; epidermis well differentiated on both adaxial and abaxial sides; upper and middle laminal cells thick-walled, rounded, quadrate or shortly rectangular, 5.0–7.5×5.0–7.5 µm, papillose on both adaxial and abaxial sides, with 1(–2) simple or bifurcate papillae per lumen, occasionally smooth; basal laminal cells differentiated, thick walled, smooth, rectangular or occasionally quadrate, 5.0–12.5×2.5–7.5 µm at juxtacostal part, quadrate to oblate, 2.5–12.5×4.0–7.5 µm at marginal part; leaves of flagelliform innovations appressed when dry, erect-patent when moist, cochleariform, ovate or orbicular, with rounded to obtusely apiculate apex, 0.16–0.32× 0.14–0.23 mm; lamina unistratose throughout, dark red in KOH; margins entire, plane, unistratose; costa, weak, 10–20 µm wide at base or occasionally absent, ending below the apex, not spurred. Sexual condition dioicous. Perichaetia terminal; perichaetial leaves enlarged, more sheathing than vegetative leaves, oblong-lanceolate with narrowed apex, 1.20– 1.75×0.30–0.70 mm. Perigonia terminal; perigonial leaves smaller than vegetative leaves, ovate, concave, 0.24–0.38 mm long. Seta erect, straight, 2.3–2.8 mm long, yellowish brown, sinistrorsely twisted throughout when dry, with elongate, thick walled epidermal cells. Capsule stegocarpous, long-exserted, erect or slightly inclined; urn cylindrical, 0.73–0.93× 0.38–0.41 mm, yellowish brown; exothecial cells smooth, thin walled, rectangular, (20–)30– 50(–70)×12–20(–42) µm, stomata phaneroporous, 8–10 at base of capsules; annulus consisting of 2–3 rows of vesiculose cells; peristome lacking; operculum conic-rostrate, 0.25– 0.30 mm long, with erectly arranged cells. Calyptra cucullate, ca. 1.25–1.45 mm long, smooth. Spores not seen. Habitat in Japan: Growing on dry sunny or semi shaded limestone in montane to subalpine regions around 1,800–3,000 m elev. Distribution: North America, North, East and South Asia (new to Japan), and Europe (e.g. Kučera & Köckinger 2000, Sollman 2005, Jiménez 2006, Zander 2007). Taxonomic notes: Didymodon subandreaeoides is distinguished from D. nigrescens by its stems with deciduous flagelliform innovations, dimorphic leaves, costal cross section showing two guide cells in a single row and no adaxial stereids, and eperistomate capsule. In the monograph of Japanese Pottiaceae (Saito 1975), the concept of D. nigrescens apparently included D. subandreaeoides since he described stems with small buds in leaf axils. However, these species can be separated molecularly as well as morphologically. In the present study we found sporophyte bearing plants among Japanese populations. Sollman (2005) reported fruiting material of D. subandreaeoides from Nepal, but the identification is doubtful since he noted peristome teeth which are lacking in Japanese material (even in operculate capsules). In previous phylogenetic studies D. subandreaeoides was resolved in the clade corresponding to subsect. Fuscobryum in the present analyses. A high degree of morphological polymorphism was reported by Kučera & Köckinger (2000) in D. subandreaeoides. Plants from Mt. Tsurugi (Shikoku) showed smaller plant size, although all Japanese populations shared a single haplotype (Fig. 1). Kučera & Ignatov (2015) discussed that an interesting autapomorphy of the subsection Fuscobryum, might include the flattened, spirally twisted seta as seen in D. nigrescens, the type species of the subsection. Our observation revealed that the seta of D. subandreaeoides is also flattened and spirally twisted when dry.

71 Based on a collection from Mt. Akaishidake in Japan, Takaki (1951) first reported Grimmia andreaeoides Limpr. (now a synonym of D. subandreaoides, see Kučera & Köckinger 2000), and his report has been adopted in the checklists of Japanese mosses [Sakurai 1954 as G. andreaeoides; Iwatsuki & Noguchi 1973; Iwatsuki 1991, 2004; Suzuki 2016 as D. rigidulus subsp. andreaeoides (Limpr.) Wijk & Margad.]. In the present study we were able to examine the voucher specimen (Takaki 6585 in NUM, labeled as Grimmia andreaeoides Limpr.) and concluded that the specimen was identical to D. icmadophilus (Schimp. ex Müll.Hal.) K.Saito since the plants had monomorphic leaves of ovate-lanceolate or triangular shape, acute leaf apices, excurrent costa, and papillose and quadrate to subquadrate upper laminal cells. The voucher specimen also includes a handwritten annotation “Didymodon constrictus (Mitt.) Saito” by H. Deguchi in 1977, suggesting he also apparently excluded this material from D. subandreaeoides and his revisional study of Japanese Grimmia Hedw. (Deguchi 1979). Zander & Caners (2017) argued that Saito’s (1975) concept of D. constrictus included plants having quadrate adaxial costal cells and spherical gemmae, clearly belonging to D. icmadophilus. Thus, D. subandreaeoides has not previously been recognized correctly in Japan, and here we report the species from the country for the first time. Saito (1975) synonymized Andreaea takakii Sakurai with D. nigrescens. Detailed examination of the type specimen proved that it should instead be synonymized with D. subandreaeoides characterized by its deciduous flagelliform innovations and dimorphic leaves. Representative specimens examined: JAPAN. Yamanashi Pref., Mt. Kitadake, 35°40′14″N, 138°14′16″E, 3,010 m elev., on limestone crevices at open site, 11 Sep. 2015, Inoue 3652 (HIRO, DNA voucher); ibid, on limestone boulder at open site, 11 Sep. 2015, Inoue 3649 (HIRO, DNA voucher); Nagano Pref., Shimoina Country, Oshika Village, Mt. Toyoguchi, 35°33′20″N, 138°07′38″E, 2,220 m elev., on limestone boulder at open site, 17 May 2017, Inoue 5295 (HIRO, DNA voucher); Tokushima Pref., Mt. Tsurugi, 33°51′20″N, 134°05′30″E, 1,790 m elev., on limestone cliff at open site, 19 July 2016, Inoue 4070 (HIRO, DNA voucher); ibid, Ohtsurugi Shrine, 1,850 m elev., on limestone cliff, 10 Aug. 1957, Seki 13057 (KOCH and HIRO). FRANCE. Savoie. Bonneval, Col de L’Iseran, 2,750 m elev., 25 Jul. 2001, Skrzypezak 01376 (MUB-41244). CANADA. British Columbia, Summit Lake, dryish cliff shelf, growing associated with D. nigrescens, 24 Jul. 1977, Schofield 66211 (H-3048183).

Acknowledgements We are indebted to the curators of the herbaria at CBM, H, KOCH, MAK, NUM, and S for arranging the examination of the specimens. We thank Dr. R. D. Seppelt for checking the English text and valuable suggestions on the manuscript. This study was partly supported by a Nakatsuji Foresight Foundation Research Grant to YI, and a Grant-in-Aid for Scientiﬁc Research (Specially Designated Research Promotion) of Japan Society for the Promotion of Science (JSPS) to HBL.

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Appendix Alphabetical list of taxa used for the phylogenetic analysis with specimen data and accession numbers. Newly obtained accession numbers are shown in bold. Didymodon nigrescens (Mitt.) K.Saito: U.S.A. Alaska, KP307512; NEPAL. Langtang Valley, KP307498; BHUTAN. Bumthang Road, KP307505; CHINA. Yunnan, Shevock & Ma 49174 (MUB-55039), LC545516; ditto, Shevock & Ma 48951 (MUB-55035), LC545515; ditto, Shevock & Ma 49104 (MUB 55036), LC545514; JAPAN. Saitama, Inoue 7823, LC545510; Yamanashi, Inoue 3645a, LC545506; ditto, Inoue 3645b, LC545507; ditto, Inoue 3664, LC545508; ditto, Inoue 3665, LC545509; Nagano, Inoue 5298, LC545504; ditto, Inoue 5299, LC545505. Didymodon perobtusus Broth.: RUSSIA. Buryatia, KP307490; Irkutskaya, KP307523.

74 Didymodon rigidulus Hedw.: ENGLAND. Derbyshire, AY437106; CZECH. C. Budejovice, KP307473. Didymodon subandreaeoides (Kindb.) R.H.Zander: FRANCE. Savoie, AY437108; SWITZERLAND. Schwyz, KP307483; CANADA. NWT, KP307484; RUSSIA. Altai Mts., Zolotukhin & Koroleva s.n. (MUB), LC545517; CHINA. Yunnan, KP307519; JAPAN. Yamanashi, Inoue 3652, LC545511; Nagano, Inoue 5295, LC545513; Tokushima, Inoue 4070, LC545512.