Hattoria 4: 1－42, 2013

Taxonomic studies of the Trichocoleaceae in Southeast Asia. III. The genus Trichocolea Dumort.

Tomoyuki Katagiri1, Atsushi Sadamitsu1, Hiroshi Miyauchi1 Hiromi Tsubota2 and Hironori Deguchi1

1Department of Biological Science, Graduate School of Science, 1–3–1, Kagamiyama, Higashi-hiroshima-shi, Hiroshima-ken, 739–8526, Japan 2Miyajima Natural Botanical Garden, Graduate School of Science, Hiroshima University, Mitsumaruko-yama, 1156–2, Miyajima-cho, Hatsukaichi-shi, Hiroshima-ken, 739–0543, Japan

Abstract. Seven species, including two new species, of the genus Trichocolea Dumort.: T. iriomotensis T.Katag., T. japonica T.Katag., T. magna T.Katag., T. mollissima (Hook.f. & Taylor) Gottsche, T. pluma (Reinw., Blume & Nees) Mont., T. rudimentaris Steph., and T. tomentella (Ehrh.) Dumort., are recognized for Southeast Asia, including the New Guinea Islands. Distribution data, habitat, taxonomic notes, distinguishing characters of the species, and a key to the species of the genus Trichocolea are provided. Three new synonyms of Trichocolea pluma: Jungermannia tomentella var. javanica Reinw., Blume & Nees, T. samoana Steph., T. tonkinensis Steph., one new synonym of Trichocolea tomentella: T. tomentella f. nodulosa Nees, and one new synonym of Trichocolea rudimentaris: T. fragillima Herzog are proposed. Lectotypes are selected for T. australis Steph. (= T. mollissima), T. striolata Steph. (= T. pluma), T. levifolia Steph. (= T. pluma), and T. rudimentaris Steph. Phylogenetic analyses based on rbcL gene sequences revealed monophyly of Trichocoleaceae in which two clades Eotrichocolea–Leimotra clade and Trichocolea clade were resolved. It is suggested that epicoelocaules, as observed in Trichcocolea and Eotrichocolea, are independently derived from perianth-bearing shoots.

Introduction

Tournefort (1700, p. 556) originally named a plant, now called Trichocolea tomentella, as “Muscus palustris absinthii folio insipidus” in his Institutiones Rei Herbariae, which is the oldest publication of the species. Vaillant (1727, p. 140) followed Tournefort and used a shorter form “Muscus palustris absinthii folio”. Linnaeus (1753, p. 1134), in his Species Plantarum which is the starting point of the scientific binary nomenclature, listed the Vaillant’s prelinnean phrase name under Jungermannia ciliaris L. (≡ Ptilidium ciliare (L.) Hampe). Ehrhart (1783) recognized a heterogeneous element among the plants so far understood as J. cilialis, and named it Jungermannia tomentella Ehrh. (≡ T. tomentella).

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The genus Trichocolea Dumort. [type: T. tomentella (Ehrh.) Dumort.] was proposed by Dumortier (1822) to accommodate the two species: T. tomentella (Ehrh.) Dumort. and T. tomentosa (Sw.) Gottsche , although the generic name was initially given under the spelling of “Thricholea”. Reinwardt et al. (1824) studied Javanese liverworts and proposed a variety of Jungermannia tomentella: var. javanica Reinw., Blume & Nees and a new species Jungermannia pluma Reinw., Blume & Nees without recognition of the genus Trichocolea. Nees von Esenbeck (1838) accepted the genus Trichocolea and recognized two species: T. tomentella with six forms, f. tomentella, f. tomentosa (Sw.) Nees, f. javanica (Reinw., Blume & Nees) Nees, f. pluma (Reinw., Blume & Nees) Nees, f. subsimplex (Nees) Nees, and f. nodulosa Nees, and T. lanata (Hook.) Nees. Gottsche et al. (1845), in his Synopsis Hepaticarum which summarised previous works on liverworts worldwide, recognised three species: T. tomentella with six varieties corresponding to the forms mentioned above, T. lanata, and T. polyacantha (Hook.f. & Taylor) Gottsche, Lindenb. & Nees. Gottsche (1864) recognized two sections under the genus Trichocolea: sect. Hirtiflora (≡ sect. Trichocolea) and sect. Laeviflora. Three species: T. tomentella, T. mollissima (Hook.f. & Taylor) Gottsche, and T. lanata were assigned to the section Hirtiflora and T. tomentosa to the section Laeviflora which corresponds to the genus Leiomitra Lindb. [type: L. tomentosa (Sw.) Lindb.] (Lindberg 1875). The genus Eotrichocolea R.M.Schust. [type: E. polyacantha (Hook.f. & Taylor) R.M.Schust.] was segregated from Trichocolea by Schuster (1963), so three distinct genera: Trichocolea, Leiomitra, and Eotrichocolea were recognized for the former Trichocolea s. lat. The monotypic genus Castanoclobos J.J.Engel & Glenny [type: C. julaceus (Hatcher ex J.J.Engel) J.J.Engel & Glenny] from New Zealand was added by Engel & Glenny (2007) to the family Trichocoleaceae Nakai, but Katagiri & Deguchi (2012) placed it in the Pseudolepicoleaceae Fulford & J.Taylor and considered the Trichocoleaceae to comprise only three genera: Eotrichocolea, Leiomitra, and Trichocolea. The genus Trichocolea can be distinguished from the other genera by (1) regularly 1–3-pinnate habit, (2) obliquely to subtransversely inserted leaves, (3) succubous leaf insertion, (4) stem cortex of 1–5 layers of thick-walled cells, (5) epicoelocaule lacking perianth, (6) and ellipsoidal capsule dehiscing into four regular valves. In Southeast Asia, taxonomic revision of the family Trichocoleaceae has remained inadequate since Stephani (1909, 1923) who reported eleven species of Trichocolea s. lat. including eight new species. Sixteen species with two varieties have been so far reported from the area by the following studies (Reinwardt et al. 1824; Nees von Esenbeck 1830; Lindberg 1875; Stephani 1908, 1909, 1923; Herzog 1931, Horikawa 1934; Inoue 1979; Katagiri et al. 2011). The majority of these species, however, remain poorly studied. The current data on the species number of the regions investigated do not seem to reflect the actual distribution of the species. This may be due to different standards for identification and taxonomic treatment of little-known or poorly understood species. The aim of this revision is to provide the first comprehensive survey of SE Asian Trichocolea species and to determine the inter- and intra-generic relationships among the family Trichocoleaceae using molecular phylogenetic analysis.

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Materials and Methods Taxonomic revision Approximately 1400 herbarium specimens from BISH, BORH, CBM, G, H, HIRO, HO, JE, KYO, L, M, NICH, PC, S, STR, SZG, and TNS were examined in the present study. The morphological concept of the genus Trichocolea was described and SE Aisan species are fully described and illustrated. Distribution data, habitat, taxonomic notes, distinguishing characters of the species, and a key to the species of the genus Trichocolea are provided. Species are listed alphabetically and each includes their basionym and all synonyms recognized in the present study. Deviations for each character were excluded in descriptions. Structural analyses were made by manually dissecting the plant material. The distinctive characters of representative specimens were drawn with the aid of a drawing tube. Detailed surface ornamentation of spores and elaters was investigated using scanning electron microscopy (SEM). Oil-bodies were photographed based on fresh materials for each species. Taxa excluded from Trichocoleaceae are shown at the end with due discussion.

Molecular phylogenetic analysis DNA extraction, PCR amplification, and sequencing. Experimental procedures follow Tsubota et al. (2004, 2005). The design of PCR and DNA sequencing primers follows Tsubota et al. (1999, 2000, 2001) and Masuzaki et al. (2010, 2011). Taxon sampling. The phylogenetic tree was constructed based on the ribulose 1,5-bisphosphate carboxylase, large subunit (rbcL, 1428 base pairs). The data set comprised 24 OTUs based on 27 materials including nine species of Trichocoleaceae and five species for outgroups. The Japanese materials of Trichocolea japonica (AB618046 and AB751578) and European materials of T. tomentella (AB751588, AB751589, and AB751590) show identical sequences respectively, and each of them was represented as a single OTU. The outgroups were selected based on Masuzaki et al. (2010, fig. 4). The twenty OTUs among eight species were newly obtained in the present study and the other sequences were downloaded from DNA databases published in Davis (2004), He-Nygrén et al. (2004), Heslewood & Brown (2007), Glenny et al. (2009), and Cooper et al. (2011). The full species names, their GenBank accession numbers, references, and voucher specimens with their sampling localities for each species included in the present study are given in Table 1. The rbcL gene sequences were aligned manually. Phylogenetic analysis. Phylogenetic analysis using the rbcL gene sequences was performed based on maximum likelihood criteria as previously described (Tsubota et al. 2004; Ozeki et al. 2007; Masuzaki et al. 2010) with some differences as follows: Prior to the phylogenetic reconstruction, Kakusan4 (Tanabe 2011) was implemented in AICc (Sugiura 1978) to make a rational decision regarding the nucleotide-based substitution model that best fitted our data, and the approximate unbiased test (AU; Shimodaira 2002, 2004) in the final stage of the analysis scheme. Phylogenetic trees were constructed using the following six program packages to obtain the candidate topologies: (1) RAxML ver. 7.2.6 (Stamatakis 2006) with maximum likelihood (ML) method (Felsenstein 1981) using GTR + gamma model; (2) GARLI ver. 2.0 (Zwickl 2006) with ML method using GTR + gamma + proportion invariant model; (3) PAUPRat (Sikes & Lewis 2001) over PAUP ver. 4.0b10

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(Swofford 2002) with maximum parsimony (MP) method (Fitch 1971) to implement Parsimony Ratchet searches (Nixon 1999) using the Parsimony Ratchet search strategy with random weighting of each character in fifty 200-iteration runs; (4) MrBayes ver. 3.2.1 (Ronquist & Huelsenbeck 2003) with Bayesian inference (BI) method using GTR + gamma model with 8 chains for 10,000,000 generations. To construct the consensus tree, the first 25% of the trees were discarded as burn-in; (5) PhyloBayes ver. 3.3 (Lartillot & Philippe 2004) with BI method using GTR + gamma model, for 2 chains; (6) BEAST ver. 1.7.2 (Drummond et al. 2012) with BI method using GTR + gamma codon partition model for 2 chains with 10,000,000 generations. Based on the ML criteria, re-calculation was performed with GTR + gamma model by Baseml in PAML ver. 4.5 (Yang 2007), with the set of candidate topologies being evaluated by AU test and Bayesian posterior probability (PP) calculated by the BIC approximation (Schwarz 1978, Hasegawa & Kishino 1989) using CONSEL ver. 0.20 (Shimodaira & Hasegawa 2001). A 50% majority-rule condensed tree for the topologies with high ranking log-likelihood values that passed the AU test at the 0.05 significance level was also computed by MEGA5. Supporting values more than 50% were overlaid to assess the robustness of each branch of the condensed topology: AU test (AU), bootstrap probabilities (NP), and Bayesian posterior probabilities (PP) are shown on or near each branch (AU/NP/ PP in %); those with <50% support are indicated with a dash (–).

Results and Discussions Taxonomic treatments Trichocolea Dumort., Comment. Bot.: 113. 1822, “Thricholea”; corr. Nees, Naturgesch. Eur. Leberm. 3: 103. 1838, nom. & orth. cons.*1 Type: Trichocolea tomentella (Ehrh.) Dumort. (≡ Jungermannia tomentella Ehrh.) Tricholea Dumort., Anal. fam. pl.: 69. 1829, nom. illeg. (Art. 52.1). Thricolea Dumort., Syll. Jungerm. Europ.: 66. 1831, nom. illeg. (Art. 52.1). Tricholea Dumort., Recueil Observ. Jungerm.:20 1835, nom. illeg. (Art. 52.1). Trichocolea sect. Hirtiflora Gottsche, Ann. Sci. Nat. Bot. ser. 5, 1: 208. 1864, nom. inval. (Art. 22.2). Trichocolea subg. Hirtiflora (Gottsche) Schiffn., Hepat. (Engl.-Prantl): 110. 1895, nom. inval. (Art. 22.2). Plants variable in size, prostrate to loosely procumbent, usually forming loose tufts or occasionally intermixed with other bryophytes, pale to vivid green in fresh materials, light yellowish to light brown in aged herbarium materials, lacking secondary brownish pigmentation, with monopodial branching and regularly 1–3-pinnate branches; branching of Frullania-type. Stems slender to robust, with or without sparse to dense paraphyllia; cortex usually well-developed, made up of small, thick-walled cells in 1–5(–7) layers. Rhizoids usually absent, rarely poorly developed at base of the underleaves; distal ends of rhizoids digitate. Leaves succubous, distant to imbricate, obliquely to subtransversely inserted, asymmetrical, papillate-verrucose to striate-verrucose, 4–6-lobed, the principal lobes usually with smaller secondary lobes, the lobes ultimately becoming resolved into a mass of uniseriate cilia; disc (2–)3–5 cells high, with or without superficial cilia; cilia usually

4 subulate to narrowly cylindrical, usually linear or occasionally curved to arcuate, 2–6 cells long, opposed or verticillate in groups of three, the septa dilated or not dilated and occasionally constricted; underleaves as large as or slightly smaller than lateral leaves, subsymmetrical. Oil-bodies pale smoky grey, opaque, homogeneous, smooth to coarsely granular, mostly spherical to ovoidal or occasionally fusiform or ellipsoidal, 3–7 per cell in cilia, 6–8(–12) per cell in disc, with or without eye-spot. Asexual reproduction unknown except for T. rudimentaris in which frequent fragmentation of cilia is observed. Dioicous. Androecia intercalary on main stem; androecial bracts 8–20 paired, hardly differentiated from leaves except higher and with concave disc; antheridia 1–2 per each bract; cells of the antheridial stalk biseriate. Gynoecia mostly on main stem and occasionally on branches; innovation long. Epicoelocaule clavate, covered with paraphyllia and reduced bracts; unfertilized or aborted archegonia 2–4 at the apical part of epicoelocaule; bracts and bracteoles inserted at or below epicoelocaule base, similar in shape to leaves and underleaves but smaller. Perianth absent. Capsule narrow ellipsoidal to obloid, regularly dehiscing into four valves; capsule walls 6–8-layered; epidermal cells of capsule large, with or without linear to semiannular thickenings; innermost cells of capsule with annular to complete or incomplete thickenings. Seta long, becoming hollow at maturity. Spores reddish brown, granulate to finely rugose, 10–14 µm in diam.; elaters reddish brown, with two sinistrorse spiral thickenings, 100–200 µm long, 10–16 µm wide.

Key to the Southeast Asian species of Trichocolea 1. Plants less than 1.0 cm long and 0.5 cm wide including branches, 1(–2)-pinnate; leaf cilia fragile ································································································· T. rudimentaris 1. Plants more than 1.0 cm long and 0.5 cm wide including branches, 2–3-pinnnate; leaf cilia not fragile ········································································································ 2 2. Leaf cilia (1–)2–3(–4) cells long; stem with thick-walled, 5–6(–7)-layered cortex; superficial cilia usually present on leaf disc ······································································· T. magna 2. Leaf cilia (3–)4–5(–7) cells long; stem with thick-walled, 1–3(–4)-layered cortex; superficial cilia absent on leaf disc ·························································································· 3 3. Terminal cells of cilia often curved to arcuate, usually longer than the penultimate cells; distal ends of terminal cells of cilia heavily thickened ················································ T. mollissima 3. Terminal cells of cilia linear, usually as long as the penultimate cells; distal ends of terminal cells of cilia evenly thickened ······················································································ 4 4. Septa of cilia dilated; oil-bodies with eye-spot ······························································ 5 4. Septa of cilia not dilated; oil-bodies without eye-spot ······················································ 6 5. Plants usually more than 1 cm wide including branches; cells of leaf disc thick-walled; oil-bodies smooth, with a single eye-spot ··································································· T. pluma 5. Plant usually less than 1 cm wide including branches;cells of leaf disc thin-walled; oil-bodies coarsely granular, with (1–)2–4 eye-spots ·············································· T. iriomotensis 6. Cilia strongly papillate-verrucose; epidermal cells of capsule with linear to semiannular inner thickenings ························································································ T. japonica 6. Cilia striate-verrucose to weakly papillate-verrucose; epidermal cells of capsule without inner thickenings ························································································ T. tomentella

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1. Trichocolea iriomotensis T.Katag., sp. nov. Affinis Trichocoleae plumae sed differt plantae parvus 1.5–2.0(–3.5) cm longa 4.0–8.5(–1.2) mm lata, cellulae ciliarum muris tenuibus, corporibus oleosis grosse granulosus cum multis oculi-maculae. Type: Japan, Ryukyu, Iriomote Island, Ootomi Trail, between Daiichi-yamagoya and Ootomi-guchi, 150–210 m alt., on moist boulder, 23 Dec. 2009, Miyauchi 1869 (holotype: HIRO). Fig. 1 Plants prostrate to creeping, vivid to light bright green in fresh materials, becoming yellowish when dry, large, 1.5–2.0(–3.5) cm long, 4.0–8.5(–12.0) mm wide, including branches, with regularly 2(–3)-pinnate branches; branches usually determinate, the primary branches 2.0–3.0 mm long, the secondary branches 1.2–2.0 mm long. Stems slender, 0.6–0.8(–1.0) mm wide with leaves, with sparse paraphyllia, 280–320 µm wide, 14–16 cells in diam.; cortex well-developed, made up of small, thick-walled cells in 1–2 layers, epidermis striate-verrucose. Rhizoids usually absent, rarely poorly developed at base of the underleaves; distal ends of rhizoids digitate. Leaves distant to imbricate, weakly asymmetrical, 0.5–0.8 mm long, 0.8–1.2 mm wide, obliquely to subtransversely inserted, 4–6-lobed; disc 3–4 cells high, without superficial cilia; cells of disc narrowly oblong to rectangular, thin-walled, papillate-verrucose to striate-verrucose, (40–)50–60(–70) × 20–30 µm; cilia narrowly cylindrical, linear, 3–4(–5) cells long; cells of cilia rather thin-walled, weakly striate-verrucose, 80–120 × 12–16 µm, the septa dilated; underleaves as large as or somewhat bigger than lateral leaves, subsymmetrical, 0.4–0.8 mm long, 0.6–1.0 mm wide, transversely inserted, 4–6-lobed; disc of underleaves 2–3 cells high, without superficial cilia; cells of underleaves similar in size and shape to those of the lateral leaves. Oil-bodies smoky grey, coarsely granular, homogeneous, opaque, irregular in size and shape, mostly ovoidal to fusiform to ellipsoidal and 6.0–10.0 × 4.0–6.0 µm or spherical and 4.0–7.0 µm in diam., (3–)4–6(–7) per cell in cilia, (5–)6–8 per cell in disc, with (1–)2–4 eye-spots per each oil-body; eye-spot variable in size, 1.5–2.5 µm in diam. Asexual reproduction unknown. Reproductive structures unknown. Taxonomic notes. Trichocolea iriomotensis is unique in the genus by having coarsely granular oil-bodies with multiple eye-spots (Fig. 2). T. iriomotensis grows closely appressed to and creeping on the substrata, in loose mats, with thin stems and sparse branches and apparently never occurs associated with other species of Trichocolea. These life-form characters observed in T. iriomotensis could be considered as an ecological adaptation to rheophytic habitats, which are very frequently subject to flooding after heavy rains. Judging from the morphological characters mentioned above, T. iriomotenisis is closely related to Trichocolea hatcheri E.A.Hodgs., a New Zealand species. Both species share such several characters as (1) vivid green plants, (2) creeping habit, (3) leaf disc 2–4 cells high, and (4) thin-walled cilia with slightly thickened septa (Hatcher 1958, as T. mollissima; Hodgson 1965; Engel & Glenny 2008). Oil-bodies for T. hatcheri are unknown. T. hatcheri is less well distinguished from T. iriomotensis by (1) the larger plants, 3–6 cm long and 1.0–1.6 cm wide including branches, and (2) the terminal cells of the cilia are frequently longer than the penultimate cells (Hodgson 1965, Engel & Glenny 2008). Further morphological

6 investigation and molecular phylogenetic analyses of the two species are needed. Distinguishing characters. T. iriomotensis is characterized by (1) the vivid green color, (2) the small plants, 1.5–2.0(–3.5) cm long and 4.0–8.5(–12.0) mm wide including branches, (3) the thin-walled leaf disc, and (4) the coarsely granular oil-bodies usually with 2–4 eye-spots per each oil-body. Habitat. Growing on moist to wet boulders (sandstone) by streams in shaded places in subtropical forests (150–200 m alt.). Distribution. Endemic to Japan, known only from type locality (Iriomote Island). [Fig.10A] Specimens examined [Paratypes]. JAPAN. Ryukyu. Iriomote Island: Ootomi Trail, between Daiichi-yamagoya to Ootomi-guchi, Miyauchi 1865 (HIRO); ditto, Yamaguchi 477 (HIRO); transcontinental road between Nakama river and Urauchi river, Katagiri 3424 (HIRO).

2. Trichocolea japonica T.Katag., Bryologist 114: 744. 2011. Type: Japan. Honshu, Mt. Kaikoma, Southern Japanese Alps, between Kitazawa mountain pass and Sensui mountain pass, Katagiri 3198 (holotype: HIRO!; isotypes: NY!, TNS!). Description and Discussion. See Katagiri et al. (2011). Distinguishing characters. T. japonica resembles T. tomentella in having a regularly 2–3-pinnate habit, thin-walled cells of the leaf cilia without dilated septa, and lacking an eye-spot in oil-bodies. T. japonica is, however, easily distinguished from T. tomentella by (1) the strongly papillate-verrucose surface of the leaf cilia, and (2) the linear to semiannular thickenings in the epidermal cells of the capsule wall. Distribution. Endemic to Japan, known only from the central region of Honshu. T. japonica has previously been known from only the type locality (Katagiri et al. 2011). An additional distribution for T. japonica was recently reported by Inoue et al. (2012) from a locality ca. 20 km southwest of the type locality. [Fig. 10A] Habitat. Growing on wet boulders or humus-covered limestone rocks on steep mountain slopes at ca. 2000 m alt. Habitats of T. japonica are restricted to moist, partially shaded, not damp places, where water supply is ombrogenous or at least seepage through rock crevices. Specimens examined. JAPAN. Honshu. Yamanashi-ken: Mt. Kaikoma, Southern Japanese Alps, between Kitazawa mountain pass and Sensui mountain pass, Katagiri 2591(HIRO, DNA extracted). Nagano-ken: Ooshika-mura, climbing trail on Mt. Shiomi, between Torikura Mountain Entrance and Sanpuku-toge, Inoue 1274 (HIRO, DNA extracted).

3. Trichocolea magna T.Katag., sp. nov. Affinis Trichocoleae plumae sed differt caulis crassa rigidus cum cortex 5–6(–7)-stratosus, ciliium (1–)2–3(–4) cellulas longo, folium discus cum superficialis ciliium. Type: Netherlands New Guinea, Star Mts., Mt. Antares, 1500 m alt., 2 July 1959, Zanten 425 (holotype: TNS; isotypes: HIRO, L). Fig. 3 Plants brownish to reddish yellow or yellowish orange in aged herbarium materials, (6–)8–12(–16) cm long, 1.5–2.4 cm wide including branches, with regularly 2(–3)-pinnate

7 branches; branches usually determinate, the primary branches 8–14 mm long the secondary branches 2–4 mm long. Stems rigid, straight elongated, with dense paraphyllia, 1.0–1.5 mm wide with leaves, 500–700(–800) µm wide, (34–)38–40(–46) cells in diam.; cortex well-developed, made up of small, thick-walled cells in 5–6(–7) layers, epidermis striate-verrucose. Rhizoids usually absent, rarely poorly developed at base of the underleaves; distal ends of rhizoids digitate. Leaves succubous, distant, weakly asymmetrical, 0.7–1.0 mm long, 1.2–1.5 mm wide, obliquely to subtransversely inserted, 4–6-lobed; disc (3–)4–5 cells high, with or without superficial cilia on its basal and marginal parts; cells of disc narrowly oblong to rectangular, thick-walled, striate-verrucose, 60–80 × 20–26 µm; cilia subulate, linear, (1–)2–3(–4) cells long; cells of cilia thick-walled, finelly papillate-verrucose to striate-verrucose, 40–60 × 14–18 µm, the septa dilated; underleaves somewhat smaller than lateral leaves, subsymmetrical, 0.6–0.9 mm long, 1.0–1.2 mm wide, transversely inserted, 6–8-lobed; disc of underleaves 2–3(–4) cells high, with or without superficial cilia on its basal and marginal parts, 0.6–1.0 mm long, 0.8–1.2 mm wide; cells of underleaves similar in size and shape to those of the lateral leaves. Oil-bodies unknown. Asexual reproduction unknown. Reproductive structures unknown. Taxonomic notes. This species has so far been misunderstood and often misidentified with other species of Trichocolea. Specimens of Trichocolea magna have been identified and deposited in herbaria under various names, mostly as T. tomentella, T. pluma or T. breviseta, or occasionally as T. aff. tomentella, Trichocolea sp., T. tonkinensis, or T. capillata. Among these species, T. magna has the greatest morphological affinity with T. pluma. Both species share the regularly 2–3-pinnate ramification pattern, thick-walled cells of leaf, and dilated septa of cilia, but T. pluma can be easily distinguished from T. magna by the following characters: (1) shorter stems to 3–6 cm long, (2) longer cilia, usually 4–5 cells long, (3) a thick-walled, 2–3-layered stem cortex, and (4) absence of superficial cilia on the leaf disc. T. magna is unique among the SE Asian species of Trichocolea in having superficial cilia on the leaf disc. In Trichocolea s. str. this character is known in an Oceanian species, T. rigida R.M.Schust. (Engel & Glenny 2008) and a Brazilian species, T. argentea Herzog (Herzog 1925, Hatcher 1957), whereas it occurs more commonly in species currently recognized as Leiomitra (Grolle 1977, Katagiri & Deguchi 2012). In contrast to the other species of Trichocolea, the stems of T. magna are thick and rigid, hence they hardly bend due to the thick-walled, 5–6-layered cortex. In other members of the genus, cortices of stems are at most 3-layered and the stems are more flexible. Distinguishing characters. T. magna is characterized by (1) the long straight elongated stem, usually 8–12 cm in length, (2) the short cilia, usually 2–3 cells long, (3) the stem cortex consisting of 5–6 layers of thick-walled cells, and (4) the superficial cilia on the leaf disc. Habitat. Growing on decaying logs or tree trunks, or occasionally on soil, rocks or humus in evergreen forests (1000–2500m alt.). Distribution. Widely distributed in SE Asia (from Papua New Guinea to China). [Fig. 10B] Representative specimens examined [Paratypes]. CHINA. Yunnan Prov.: Mts. Ailaoshan, around Ailaoshan branch of Academia Sinica, Furuki 17558 (CBM). Taiwan: Taipei Co., Mt. Taiheizan,

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Horikawa 9381 (HIRO). PHILIPPINES. Luzon: Pampanga Prov., Mt. Pinatubo, Camp Stotsenburg, Elmer 22204 (NICH). Mindanao: Davao Prov.: Mt. Apo, Todaya, Elmer 11370 (G, L, PC). Negros: Negros Occidental Prov., Mt. Canlaon, between V.H.F. Relay station to campsite, Iwatsuki & Sharp 14657a (NICH).THAILAND. Nakhon Si Thammarat Prov.: Nakornsrithamarat, granitic massive Khao (Mt.) Luang, Touw 11904 (NICH). MALAYSIA. Borneo: Sarawak, Mt. Dulit, Herzog s.n. (NICH-233233). INDONESIA. Papua Prov. (New Guinea Island): Trikora, Jayapura, west Irian, Oizuru s.n. (TNS-75429). South Sulawesi (Celebes): Masamba, northwest spur of Kamboeno, Eyma 1343 (L). PAPUA NEW GUINEA. Morobe Prov.: Between Kasanombe and Namba, about 30 km north of Lae, Inoue 32462 (TNS-72813).

4. Trichocolea mollissima (Hook.f. & Taylor) Gottsche, Mexik. Leverm.: 118. 1863. Jungermannia mollissima Hook.f. & Taylor, London J. Bot. 3: “290” [= p. 390]. 1844. Type: New Zealand, Auckland Islands, Hooker s.n. (holotype: K, non vidi; isotypes: G-264411!, PC-103945!). Trichocolea australis Steph., Sp. Hepat. 4: 62. 1909. Type: New Zealand, Colenso 1823 (lectotype designated here: G-67963!). Fig. 4 Plants yellowish to light yellowish brown in aged herbarium materials, prostrate to ascending, usually forming mats, (1.5–)2.5–5.0(–8.0) cm long, (1.0–)1.5–2.5(–3.5) cm wide, including branches, with monopodial branching and regularly 2(–3)-pinnate branches; branches usually determinate, the primary branches (4–)6–10(–14) mm long, the secondary branches (1.5–)3.0–5.0 mm long. Stems robust, 0.8–1.4 mm wide with leaves, 360–440 µm wide, 16–18 cells in diam., with dense paraphyllia; cortex well-developed, made up of small, thick-walled cells in 3–4 layers, epidermis striate-verrucose. Rhizoids usually absent, rarely poorly developed at base of the underleaves; distal ends of rhizoids digitate. Leaves distant to imbricate, weakly asymmetrical, 0.6–1.0 mm long, 0.8–1.2 mm wide, obliquely to subtransversely inserted, 4–6-lobed; disc (2–)3–4 cells high, usually without superficial cilia, rarely with 1(–2) superficial cilia at the marginal parts; cells of disc narrowly oblong to rectangular, thin-walled, weakly striate-verrucose, 40–60 × 16–20 µm; cilia subulate to acerose, usually linear, often curved to arcuate at the apical part, 4–5 cells long; cells of cilia thick-walled, weakly striate-verrucose, 60–110 (–160) × 14–18(–20) µm, the septa dilated; terminal cell of the cilia usually longer than the penultimate cell, frequently curved to arcuate or sigmoid, tapering to a rather sharp point, conspicuously thick-walled in the tip; underleaves somewhat smaller than lateral leaves, subsymmetrical, 0.6–0.8 mm long, 0.8–1.0 mm wide, transversely inserted, 6–8-lobed; disc of underleaves 3–4 cells high, without superficial cilia; cells of underleaves similar in size and shape to those of the lateral leaves. Oil-bodies pale smoky grey, opaque, homogeneous, irregular in shape, mostly spherical to ovoid to broad-elliptic and 3–6 × 3–6 µm to 4–6 × 6–10 µm, 8–10 per cell in cilia, 8–15 per cell in disc, usually without eye-spot or occasionally with 1(–2) eye-spots. Asexual reproduction unknown. Dioicous. Androecia intercalary on main stem and branches; bracts hardly differentiated from leaves, with disk higher and concave; antheridia 1–2 per each bract; cells of the antheridial stalk biseriate. Gynoecium on main stem or branches; innovation long;

9 epicoelocaule clavate, covered with paraphyllia and reduced bracts; unfertilized or aborted archegonia 2–3 at the apical part of epicoelocaule; bracts and bracteoles inserted at or below epicoelocaule base, similar to leaves and underleaves except for smaller size. Perianth absent. Capsule ellipsoidal to obloid, black to blackish brown, regularly dehiscing into four valves; capsule walls 7–8-stratose; epidermal cells of capsule subquadrate to rectangular in surface view, without inner thickenings; innermost cells of capsule irregular in shape, irregularly oriented in surface view, with complete or incomplete semiannular inner thickenings. Seta long, becoming hollow at maturity. Spores reddish brown, with low papillae and short-vermiculate markings, 12.0–15.0 µm in diam; elaters reddish brown, to 160 µm long, ca. 12 µm thick, with two sinistrorse spiral thickenings. Taxonomic notes. Jungermannia mollissima was described from New Zealand by Hooker & Taylor (1844). This species had been buried in the synonymy of Trichocolea tomentella var. javanica (Reinw., Blume & Nees) Gottsche, Lindenb. & Nees since Gottsche et al. (1845) until Gottsche (1863). This taxonomical treatment might lead to confusion of T. mollissima (Hook.f. & Taylor) Gottsche with T. tomentella var. javanica particularly in the SE Asian region. SE Asian specimens of T. mollissima have often been kept in herbaria under the name of T. tomentella var. javanica. Our morphological investigations on the type material proved that the variety is no more than an extreme expression of T. pluma in having thin walls of the cilia of the leaf and it is quite independent of T. mollissima. Compared with Oceanian plants of T. mollissima, SE Asian plants are rather smaller (1.5–2.5 cm long and 0.8–1.4 cm wide including branches), but the morphology of the cilia agrees well with Oceanian material, including the isotypes (G, PC). As already pointed out by Schuster (1968, 2000) and Engel & Glenny (2008), further confusion regarding the name of this species arose from Hatcher (1958) in his revision for New Zealand species of Trichocolea. Hatcher (1958) used the name T. australis Steph. for this species (T. mollissima), whereas his “T. mollissima” corresponds to the species currently recognized as T. hatcheri E.A.Hodgs. Schuster (1968, 2000) and Engel & Glenny (2008) have discussed the distinction between T. mollissima and T. hatcheri. T. australis, described by Stephani (1909), has been treated as a synonym of T. mollissima without designation of a type (Schuster 1968, Engel & Glenny 2008). In Stephani’s collection (G), there are two specimens of T. australis: Colenso 1823 and Colenso 1987. The former specimen was selected here as the lectotype of T. australis, since the plants well match the original description. The latter specimen totally differs from the description in having the smaller plants, 1.2–1.5 cm long and 0.6–0.9 cm wide including branches, and proved to be T. hatcheri. T. mollissima shares the regularly 2–3-pinnate ramification and the dilated septa of cilia with T. pluma, but can be easily distinguished from the latter by the terminal cells of the cilia being frequently curved, with heavily thickened-walls at the distal ends, and the absence of eye-spot in the oil-bodies. Distinguishing characters. T. mollissima is characterized by (1) the regularly 2(–3)-pinnate habit, (2) the terminal cells of cilia usually longer than the penultimate cells, (3) the distal ends of cilia with heavily thickened-wall, and (4) the thick-walled cells of cilia with dilated septa.

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Habitat. Growing usually on decaying logs, bases of trees, tree trunks, or wet ground near streams in Nothofagus forests (100–1300m alt.), occasionally occurring on humus-covered rocks or on banks in mixed wet forests. Distribution. Oceania (Australia, New Zealand) to SE Asia (Philippines, Malaysia, Indonesia). [Fig. 10C] Representative specimens examined. INDONESIA. Java: s. loc., “Herb. bryol. C. Alb. Tärnlund”, leg. Junghuhn (NICH-223618). MALAYSIA. Borneo: North Borneo, ultrabasic area between mining camp and summit of Mt. Silam, southwest of Lahad Datu, East Coast, Iwatsuki 5380 (NICH-255380); Kinabalu, Furuki s.n. (HIRO-1016769, DNA extracted; CBM). PHILLIPINES. Negros: s. loc., Elmer 9999 (L). AUSTRALIA. Tasmania: Styx River Vally south of Maydena, Tall Tree Reserve, Styx River crossing, Katagiri 3490 (HIRO, DNA extracted). NEW ZEALAND. North Island: South Auckland Prov., Herangi Range, Whareorino Forest, near tributary of Awakino River, west of Leitch’s Road, Engel & Konrat 23949 (TNS-179423). South Island: Nelson/Westland Prov. boundary, Paparoa range, south side of Pararari River, Engel 19187 (TNS-145015).

5. Trichocolea pluma (Reinw., Blume & Nees) Mont., Voy. Bonite, Bot. 2: 238. 1846. Jungermannia pluma Reinw., Blume & Nees, Nova Acta Phys.-Med. Acad. Caes. Leop.-Carol. Nat. Cur. 12: 209. 1824. Type: Java, in montibus Parangkudjang, Prov. Bantam, leg. Blume (holotype: STR!). Jungermannia tomentella Ehrh. var. pluma (Reinw., Blume & Nees) Lindenb., Nova Acta Phys.-Med. Acad. Caes. Leop.-Carol. Nat. Cur. 14 (Suppl.): 19. 1829. Trichocolea pluma Dumort., Recueil Observ. Jungerm.: 20. 1835, nom. nud. Trichocolea tomentella (Ehrh.) Dumort. f. pluma (Reinw., Blume & Nees) Nees, Naturgesch. Eur. Leberm. 3: 106. 1838. Trichocolea tomentella (Ehrh.) Dumort. var. pluma (Reinw., Blume & Nees) Gottsche, Lindenb. & Nees, Syn. Hepat.: 237. 1845. Jungermannia tomentella Ehrh. var. javanica Reinw., Blume & Nees, Nova Acta Phys.-Med. Acad. Caes. Leop.-Carol. Nat. Cur. 12: 208. 1824, syn. nov. Type: Java, leg. Blume (holotype: STR!; isotype: G-264952!). Trichocolea tomentella (Ehrh.) Dumort. f. javanica (Reinw., Blume & Nees) Nees, Naturgesch. Leberm. Eur. 3: 106. 1838. Trichocolea tomentella (Ehrh.) Dumort. var. javanica (Reinw., Blume & Nees) Gottsche, Lindenb. & Nees, Syn. Hepat.: 237. 1845. Trichocolea striolata Steph., Leafl. Philipp. Bot. 2: 386. 1908. Type: Philippines, Luzon Island, Prov. Tayabas, Lucbon, May 1907, Elmer 9288 (lectotype designated here: G-9834/G-43832!; isolectotypes: G-283292!, G-283293!, L!, PC!). Trichocolea tonkinensis Steph., Sp. Hepat. 4: 60. 1909, syn. nov. Type: Vietnam, Tonkin, near Muong Hum, in valley Nam Pou Ho, leg. Mercier, ex herb. Paris (holotype: G-9843/G-283294!). Trichocolea levifolia Steph., Sp. Hepat. 4: 60. 1909. Type: New Caledonia, Mt. Mou, 1219 m alt., January 1906, Le Rat 47 (lectotype designated here: G-9756!). Trichocolea laevifolia Steph. ex Paris, Rev. Bryol. 37: 132. 1910, nom. nud. Trichocolea samoana Steph., Denkschr. Kaiserl. Akad. Wiss., Math.-Naturwiss. Kl. 88: 33. 1911,

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syn. nov. Type: Samoa. Upolu Island, Mt. Lanutoo, leg. Rechinger (holotype: G-9828!). Trichocolea tamina Steph., Sp. Hepat. 6: 377. 1923. Type: New Guinea, near Tami River, leg. Schultze (holotype: G-9842/G-43833!). Trichocolea obconica auct. non Steph.; Inoue, Hikobia 4: 276. 1965. Trichocolea tomentella auct. non (Ehrh.) Dumort.; Hattori, J. Hattori Bot. Lab. 5: 72. 1951. Fig. 5 Plants pale to bright green, becoming yellowish to whitish when dry, loosely procumbent, forming loose tufts, 3.0–6.0 cm long, 1.0–2.4 cm wide including branches, with regularly 2–3-pinnate branches; branches usually determinate, the primary branches 0.6–1.4 cm long, the secondary and tertiary branches 1.5–5.0 mm long. Stems robust, 0.8–1.4 mm wide with leaves, 360–420 µm wide, 20–24 cells in diam., with or without sparse paraphyllia; cortex well-developed, made up of small, thick-walled cells in (1–)2–3 layers, epidermis striate-verrucose. Rhizoids usually absent, rarely poorly developed at the base of underleaves; distal ends of rhizoids digitate. Leaves distant to imbricate, weakly asymmetrical, 0.6–1.2 mm long, 0.8–1.4 mm wide, obliquely to subtransversely inserted, 4–6-lobed; disc 3–4 cells high, without superficial cilia; cells of disc narrowly oblong to rectangular, thick-walled, papillate-verrucose to striate-verrucose, (40–)50–60(–70) × 18–20(–24) µm; cilia subulate to narrowly cylindrical, linear, (3–)4–5 cells long; cell of cilia thick-walled, weakly papillate-verrucose to striate-verrucose, the septa dilated, 80–120 × 16–20 µm; underleaves as large as or somewhat bigger than lateral leaves, subsymmetrical, 0.6–1.2 mm long, 0.8–1.4 mm wide, transversely inserted, 6–8-lobed; disc of underleaves 3–4 cells high, without superficial cilia; cells of underleaves similar in size and shape to those of the lateral leaves. Oil-bodies pale smoky grey, opaque, homogeneous, smooth, with a single eye-spot, (3–)4–5(–6) per cell in cilia, 6–8 per cell in disc, in the cells of cilia mostly spherical to ovoidal and 4–7 µm in diam., occasionally to fusiform or ellipsoidal and 6–10 × 3–4 µm. Asexual reproduction unknown. Dioicous. Androecia intercalary on main stem; bracts 8–20 paired, hardly differentiated from leaves, except with higher and concave disk; antheridia 1–2 per each bract. Gynoecium on main stem; innovation long. Epicoelocaule clavate, covered with paraphyllia and reduced bracts; unfertilized or aborted archegonia 2–4 at the apical part of epicoelocaule; bracts and bracteoles inserted at or below epicoelocaule base, without superficial cilia, similar to leaves and underleaves except for smaller size. Perianth absent. Seta long, becoming hollow at maturity. Capsule narrow ellipsoidal to obloid, black to blackish brown, regularly dehiscing into four valves; capsule walls 6–7-stratose; epidermal cells of capsule thick-walled, subquadrate to rectangular and 70–80 × 30–40 µm in surface view, without inner thickenings; innermost cells of capsule irregularly oriented, narrowly oblong to fusiform and 50–60(–80) × 10–18 µm in surface view, 6–8 µm thick in cross-section, with reddish brown, annular to complete or incomplete semiannular inner thickenings. Spores reddish brown, 10–14 µm in diam., granulate to finely rugose; elaters reddish brown, 100–160(–200) µm long, 10–12 µm thick, with two sinistrorse spiral thickenings. Taxonomic notes. Trichocolea pluma was originally described by Reinwardt et al. (1824) as Jungermannia pluma, from plants collected on Java. Various authors, however, have treated it with different taxonomic rank under T. tomentella. Lindenberg (1829), Nees

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(1838), and Gottsche et al. (1845) treated the species as a variety or form of T. tomentella. Grolle (1977) treated T. pluma as T. pluma complex including four species: T. tomentella, T. pluma, T. tamina, and T. fragillima. Piippo (1984) followed Grolle (1977) and substantially synonymized T. pluma with T. tomentella, treating both as T. tomentella s. lat. Other authors, however, considered T. pluma as a species independent from T. tomentella. Rather sharp morphological differences between T. pluma and T. tomentella have been noted (Horikawa 1934, Kitagawa 1973, Inoue 1978), namely T. pluma was distinguished from T. tomentella by (1) regularly 2-pinnate habit, (2) the terminal cells of leaf cilia usually (7.5–)8.0–10 times as long as wide, (65–)75–90(–100) × 8–12 µm, (3) weakly to distinctly dilated septa of the cilia, and (4) absence of dense paraphyllia on the stem. Further distinguishing characters between them were investigated on Japanese plants by Kikukawa & Deguchi (2007) who distinguished T. pluma from T. tomentella by the thick-walled cells of the leaf cilia and the well-developed cortex of stem. Among the distinguishing features mentioned above, only the following characters can be considered to be of taxonomic value: (1) dilated septa of the leaf, (2) thick-walled cells of the cilia, and (3) a well-developed cortex of the stem. The density of paraphyllia or length and width of cells of the cilia show a wide range of variation among populations, and therefore they are of little use in distinguishing the two species from each other. Other distinct differences can be observed in the oil-bodies. Hattori (1951b, as T. tomentella) and Furuki & Higuchi (1996) reported the presence of a single eye-spot on each oil-body in T. pluma, where these are absent in T. tomentella (Müller 1939; Breil 1970; Inoue 1979, as T. gracillima; Paton 1999; Damsholt 2002). Chemical distinction between T. pluma and T. tomentella was summarized by Asakawa (1995). Sporophytic distinction between the species was reported by Katagiri & Deguchi (2011) who showed that epidermal cells of the capsule of T. pluma are thicker-walled than those of T. tomentella (Fig. 6), although the morphological difference was based on a limited number of specimens due to the rare occurrence of sporophyte-bearing plants in Trichocolea. Further investigations of detailed morphological and anatomical characters of the sporophyte are needed for a sound understanding of their use in delimiting species of Trichocolea. The holotype of Jungermannia tomenetella var. javanica in Nees’s collection (STR) was examined in the present study and it proved to be no more than poorly developed plants of T. pluma in having rather thin-walled cells and weakly dilated septa of cilia. T. striolata Steph. and T. tamina Steph. have been synonymized with T. pluma by Kitagawa (1973). The plants of their types show a typical form of T. pluma in having a 2-pinnate branching habit and thick-walled cilia with dilated septa. For the typification of T. striolata, a specimen (G-9834/G-43832) to which Stephani’s handwriting was attached, is selected here as the lectotype, since it best fits the original description. In Stephani’s collection, there are two other specimens (G-283292, G-283293) having the same habitat and collector’s information with the lectotype and these should be considered to be isolectotypes. T. samoana Steph. and T. tonkinensis Steph. are newly synonymized with T. pluma in the present paper after due examinations of their holotypes. The type of the former represents a typical form of T. pluma. In the type specimen of T. samoana, an annotation slip is present on which a note was given by Hatcher suggesting that this species might be conspecific with T. pluma. The type for the latter species resembles T. tomentella in having thinner-walled

13 cells of the cilia than those of typical plants of T. pluma but other characters, such as dilated septa of the cilia and rather thick-walled cells of leaf disc, agree with occasional populations of T. pluma. T. levifolia was synonimized with T. pluma by Hürlimann (1983) without typification. Twelve specimens of T. levifolia were found in Stephani’s collection, all of which are identical with T. pluma. The specimen Le Rat 47 is here selected as the lectotype since Stephani cited the specimen in his Icones Hepaticarum (Stephani 1985, n. 10295) and the plants best agree with the description. T. pluma shows marked morphological plasticity, particularly in plant size and degree of thickness of cell walls in the cilia. Occasional populations or poorly developed plants of T. pluma resemble T. tomentella in having a 1–2-pinnate habit and thin-walled cells of the leaf, but they can be easily distinguished from each other by presence or absence of an eye-spot in oil-bodies and by the degree of dilation of the septa in the cilia. Distinguishing characters. T. pluma is characterized by (1) the regularly 2–3-pinnate habit, (2) the thick-walled cells of the leaf cilia and disc, (3) dilated septa of the cilia, (4) the well-developed stem cortex consisting of 2–3 layers of thick-walled cells, and (5) presence of an eye-spot in each oil-body. Habitat. Growing on boulders by streams, soil-covered boulders, humus, decaying logs, tree trunks, and bases of trees in evergreen to montane forests (0–2500 m alt.), occasionally occurring on branches in tropical rainforests. Distribution. Widely distributed in SE Asia (from Papua New Guinea to Japan). [Fig. 10D] Representative specimens examined. JAPAN. Honshu. Fukushima-ken: Nihonmatsu-shi, Dake Hot Spring, Mt. Adatara-yama, Miyauchi 1897 (HIRO, DNA extracted); Hiroshima-ken: Miyajima-cho, Mt. Misen to Tatara, Miyauchi 1389 (HIRO, DNA extracted). Sikoku. Kochi-ken: Hata-gun, Mt. Imano, leg. Inoue, Inoue ed. Bryophyta Selecta Exsiccata no. 223 (HIRO). Kyushu. Miyazaki-ken: Minaminaka-gun, Kitago-cho, Inohae valley, Katagiri 2593 (HIRO); ditto, Kitago, det. as Trichocolea tomentella by S. Hattori, Hattori 16151 (NICH); Kagoshima-ken: Ookawara, Yuukyunomori, Miyauchi 1873 (HIRO, DNA extracted); ditto, Osumi peninsular, around Tashiro-mura, Hattori 1908 (TNS); Okinawa-ken: Okinawa Island, Kunigami-gun, Kunigami-mura, Yonaha-dake, Miyauchi 1739 (HIRO, DNA extracted,). CHINA. Guangdong Prov.: Ruyuan Co., Tianjingshan, Xianxi Power Station, Zhou 280 (HIRO, DNA extracted; SZG). Guangxi Prov.: Shiwandashan, Chitng 4388 (SZG); s. loc., Wu & Lin 902-1 (NICH-374708). Hubei Prov.: Shennongjia Forest Distr., west of Hongriwan construction camp along the trail leading towards Huanghunling Mt. Elev, Wu 287 (HIRO, L). Cheking Prov.: Longquan, leg. Lin, Hepaticae sinicae Exsiccatae ser. 1, no. 47: Trichocolea tomentella (HIRO, NICH). Szechuen Prov.: Omei-shan, Hepaticae sinicae exsiccatae, ser. 1, no. 48: Trichocolea tomentella, Chen s.n. (HIRO, NICH). Hainan: Yinggeling Nature Reserve, Zhang 4096 (SZG); Guangdong, Chen et al. 661a (NICH); Bawangling, Lin & Zhang 14 (L); Kwangtung, Lin 353a, 661a (L). Taiwan: Nantou Co., Taroko National Park, Furuki 22034 (HIRO, DNA extracted; CBM). Botel Tobago: Mt. Kotou-san, Horikawa 14499 (HIRO). MALAYSIA. Malay Penin.: Pahang, Fraser Hill, Yamaguchi 25866 (HIRO). Penang Island: Pulo-Pinang, Voyage de la Bonite, leg. Gaudichaud s.n. (G-9811). Borneo: Sabah, Kinabalu Park, slope of Gunong Kinabalu, Liwagu Trail along Liwagu River, Masuzaki 989 (HIRO). PHILIPPINES. Luzon: Mountain Prov., Mt. Data, Monamon-Sur., Deguchi

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21985 (HIRO). Batan: Mt. Iraya, det. as Trichocolea obconica by H. Inoue, Hatsushima 22624 (TNS). Palawan: Mt. Salakot, about 100 m from the summit, Gruezo 6430 (KYO). Mindanao: Davao, Mt. Apo, Setagawa 14460 (HIRO). Negros: Negros Occidental, Mt. Canlaon, Edaño 56725 (TNS). Mindoro: Mt. Halcon, Edano 9291 (L). THAILAND. Nakhon Si Thammarat Prov.: Nakornsrithamat, granitic massive Khao (Mt.) Luang, Touw 11574 (KYO, L). INDONESIA. Java: Salahaetoe, Ambon, Rant 711 (NICH). Bangka: Soengai Ilan, Teysmann 2233 (NICH). Sulawesi (Celebes):Todjamboe, Kjellberg 23 (NICH). Sumatra: West Sumatra, Tindjoe Laut, leg. Micholitz, Levier 4674 (G). Ambon: Mt. Salhutu, Beccari s.n. (L-790684, L-790717). West Irian Jaya (New Guinea Island): Vogelkop, Tsuyama 13 (TNS). Papua Prov. (New Guinea Island): Netherlands New Guinea, Hollandia Distr., Cycloop Mountains, southern slope of Makanoi Range, north of Kotanica, Royen & Sleumer 6254 (TNS). West Kalimantan (Boeneo): Bentiang, Sejanjang, Pontianak, Shea 28625 (NICH).VIETNAM. Khanh Hoa Prov.: Annam, Nhatrang, Poilanec 3710 (PC). Lam Dong Prov.: Annam, Langbian, leg. Micholitz, Levier 4682 (G). Vinh-Phuc Prov.: Tam-Dao Mts., along the path to Rung Rinh summit, leg. Pócs & Ninh, Pócs 9896b (G). BHUTAN. Wangdue Phodrang: Norgompa, 46 km east of Wangdiphodrang, Würmli 52 (G). PAPUA NEW GUINEA. Morobe Prov.: Cromwell Mts., Indagen airstrip, Koponen 31486 (TNS). Central Prov.: Woitape, Takaki 45817 (TNS). West Sepik Prov.: Frieda River prospecting area of Frieda Copper Co. Kokomo Creek 2.5 km northwest of Frieda Base Camp, Koponen 35738 (TNS). Chimbu Prov.: Mt. Wilhelm, Sloover 42766 (HIRO, L). Moresbay Prov.: Mt. Mo-roka, leg. Loria, Levier 77 (G). Eastern highland Prov.: near Goroka, Gahavisuka National Park, Lizipauga Botanical Sanctuary, Thiers 3657 (L). Oro Prov.: Hygrographers Range, near Siurane village, Pullen 5611 (L). Western Highlands Prov.: Mt. Hagen, near saw-mill, Zanten 683162 (L). East New Britain Prov.: Bismarck Archipelago, Mt. Baining, leg. Parkinson, Levier 4576 (G). Milne Bay Prov.: Sudest Isl., Mt. Riu, Brass 27849 (L). MELANESIA. Viti Levu: Suva, leg. Armstrong Jr, Levier 1737 (G). Ovalau: Graeffe 584 (G). New Caledonia: Mt. Humbolt, Higuchi 27412 (TNS). MICRONESIA. Pohnpei (Ponape): Sankaku-yama, Horikawa 18690 (HIRO). Kosrae (Kusai-to): Matante-yama, Horikawa 19358 (HIRO). POLYNESIA. Tahiti: on the way to Arai, Fare Hamata, Hürlimann 1250 (PC). Moorea. North of Maatea, Toto Valley, Sloover 21137 (HIRO, TNS). Upolu: Mt. Lanutoo, Rechinger 2377 (TNS). Savai’i: s. loc., Graeffe s.n. (G-9780). Tutuila: s. loc., Graeffe 1864 (G).

6. Trichocolea rudimentaris Steph., Sp. Hepat. 6: 367. 1923. Type: New Caledonia. Tao, forest, 600 m alt., January, 1910, leg. Franc, Thériot 239 (lectotype designated here: G-9827/G-113396!; isolectotype: PC!). Trichocolea rudimentaris Steph. ex Thér., Diagn. Esp. Var. Nouv. Mouss. 8: 8. 1910, nom. nud. Trichocolea fragillima Herzog, Ann. Bryol. 4: 84. 1931, syn. nov. Type: Philippines, summit Mt. Makiling, Baker 7028 (lectotype: JE!). Trichocolea obconica auct. non Steph.; Inoue, J. Hattori Bot. Lab. 30: 54. 1967; Inoue & Miller, Bull. Nat. Sci. Mus. Tokyo, 11: 2. 1968. Fig. 7 Plants yellowish brown to pale yellow in aged herbarium materials, small, 5.0–8.0 mm long, 2.0–4.0 mm wide, including branches, with regularly 1(–2)-pinnate branches usually 1.0–2.0 mm long. Stems slender, without paraphyllia, 0.5–0.8 mm wide with leaves, 240–260 µm wide, 10–12 cells in diam.; cortex moderately developed, made up of small, thick-walled cells in 1(–2) layers, epidermis striate-verrucose. Rhizoids usually absent, rarely

15 poorly developed at the base of underleaves; distal ends of rhizoids digitate. Leaves, succubous, distant to imbricate, weakly asymmetrical, 0.4–0.6(–0.8) mm long, 0.4–0.8(–1.2) mm wide, obliquely to subtransversely inserted, 4–6-lobed; disc 2–3(–4) cells high, without superficial cilia; cells of disc narrowly oblong to rectangular, rather thin-walled, weakly striate-verrucose, 50–70 × 24–36 µm; cilia narrowly cylindrical, linear, 3–4 cells long, weakly striate-verrucose, the septa not dilated or occasionally slightly constricted; apical 1–2 cells of cilia fragile and frequently detached usually at the middle lamellae or occasionally breaking off at the middle part of the cell; cells of the cilia thin-walled, 50–80 × 14–16(–20) µm; underleaves somewhat smaller than lateral leaves, subsymmetrical, 0.3–0.6(–0.8) mm long, 0.4–0.6(–0.8) mm wide, transversely inserted, 2–4-lobed; disc of underleaves 2(–3) cells high; cells of underleaves similar in size and shape to those of the lateral leaves. Oil-bodies, homogeneous, smooth, mostly elliptical and 3.0–6.0(–10) × 2.5–3.5 µm or rarely globose and 3.0–4.0(–5.0) µm in dam., (2–)3–5(–6) per cell in cilia, with a single eye-spot of ca. 1.0 µm in diam. Asexual reproduction not confirmed except for frequent fragmentation of cilia. Reproductive structures unknown. Taxonomic notes. In the original description of Trichocolea rudimentaris Stephani (1923) gave the type locality only as “New Caledonia (Lerat legit)”, but no T. rudimentaris specimen collected by Le Rat was available in the Stephani collection (G). Only a single specimen of T. rudimentaris is kept in the collection (G-9827/G-113396), which has the following information on habitat and collector’s name: “New Caledonia, Tao, forêt, 600 m, janvier, 1910, leg. Franc, no. 239”. According to Thériot (1910, p. 8) who gave explicit information on the habitat of T. rudimentaris, the original material for the species was collected by I. Franc in January 1910 from Tao in New Caledonia. The information on habitat and collector’s name is also shown by his handwritings in the Stephani’s Icones Hepaticarum (Stephani 1985, n. 10303) with a specific number of the specimen (“ex Herb. Thériot 239, Franc legit”). The specimen kept in Stephani’s collection (G) well fits with the description and illustrations given by Stephani (1923, 1985). In these circumstances, the specimen, Thériot 239 (G-9827/G-113396), is selected here as the lectotype for T. rudimentaris. Another specimen with the same information as the lectotype in Thériot’s collection (PC) should be considered as isolectotype. Plants of the lectotype(G) and isolectotype (PC) show (1) small size (6–9 mm long and 2–3 mm wide including branches), (2) 1(–2)-pinnate habit, and (3) small leaves with fragile cilia and discs 2(–3) cells high, and proved to be no more than Trichocolea fragillima. Trichocolea fragillima was described by Herzog (1931) from the Philippines, and has been known as a Philippine endemic until Grolle (1965) extended the distribution to Papua New Guinea. Despite such unique characters among the genus as fragile leaf apices and small plants, no detailed morphological or ecological discussion has been made for the species. Grolle (1977) referred the species in the T. pluma complex which included T. pluma, T. tamina (= T. pluma), and T. tomentella. Piippo (1984) followed Grolle (1977) and treated the species complex under the species T. tomentella s. lat. T. fragillima (= T. rudimentaris) is, however, a species independent of the others recognized as the T. pluma complex or T. tomentella s. lat. and is unique in the genus in having fragile cilia serving as propagules. In

16 the Trichocoleaceae, there has so far been no record for asexual reproduction except for Cavers (1903, p. 158) who noted that T. tomentella has the same type of gemmae as in Blepharostoma trichophyllum (L.) Dumort., namely, the gemmae are spherical, 1–2-celled, formed in chains from the segments of the multilaciniate leaves, especially those from upper part of the shoot. Such gemmae have not been confirmed in any species of Trichocolea or even in the Trichocoleacae. The description on the gemmae by Cavers might be based on B. trichophyllum. Juvenile plants of T. tomentella occasionally resemble those of B. trichophyllum with deeply divided leaf discs. Fragile cilia of T. fragillima have been described by Buch (1932, p. 68) who noted that cilia of T. fragillima split at the middle lamellae. Our examinations, based on ample material, revealed that the apical 1–2 cells of cilia usually detach at the middle lamellae (Fig. 7I) or occasionally break off at the middle part of the cell (Fig. 7J). The occurrence of similar morphology of the fragile leaf apices is rare in liverworts and has been known only in Chaetocolea palmata Spruce (Pseudolepicoleaceae) and Plagiochila caduciloba H.L.Blomq. (Plagiochilaceae) (Schuster 1959, 1985). Considering the phylogenetic positions for these genera or families (Glenny et al. 2009), such leaves with caducous apices as seen in the three genera might be considered a result of convergent evolution. Another distinctive character of T. rudimentaris is the small plant size. Stems are usually less than 1.0 cm long and 12 cells in diam., whereas those for other species of the genus are longer, at least more than 2.0 cm and 14–16 cells in diam. Leaf discs are much smaller (usually 2–3 cells high and 9–14 cells wide) than those of other species (e.g., 3–4 cells high and 18–26 cells wide in T. pluma). These less well-developed, vestigial vegetative structures having the vigorous asexual productive capability with fragile cilia, as observed in T. rudimentaris, might be regarded as structures adaptive to unstable habitats in order to facilitate colonization of such finite and transient substrata as decaying logs, tree trunks, branches, or boulders by streams in tropical rainforests. Other species of Trichocolea with larger-sized plants lacking asexual reproductive structures generally grow on more stable substrata that persist for some years. According to the classification of life strategies given by During (1979), T. rudimentaris might be considered as having a perennial shuttle or colonist strategy, whereas other species of Trichocolea mostly have a perennial stayer strategy. The description of the oil-bodies is based on Malaysian plants reported as Trichocolea obconica by Inoue (1967). T. rudimentaris occasionally resembles populations of poorly developed plants of T. pluma in having a regularly 1-pinnnate ramification pattern and oil-bodies with a single eye-spot, but T. pluma can be distinguished by the dilated septa of the cilia and thick-walled cells of the cilia and disc of the leaf. Distinguishing characters. T. rudimentaris can be easily distinguished from the other species of Trichocolea by its fragile cilia. The species is also characterized by (1) small plants usually less than 1.0 cm wide including branches, (2) the 1-pinnnate ramification pattern, (3) rather thin-walled cells of the cilia without dilated septa, and (4) oil-bodies with a single eye-spot. Habitat. Growing on decaying logs, or tree trunks or branches in tropical rainforests (ca. 1000–2000 m alt.), occasionally found on wet boulders by streams in subtropical forests

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(ca. 50–100 m alt., Iriomote Island). Distribution. SE Asia, New Guinea Island (Grolle 1965, as Trichocolea fragillima), Micronesia, and New Caledonia Island. [Fig. 10E] Specimens examined. JAPAN. Ryukyu. Iriomote Island: Urauchi river, transcontinental road between Nakama river and Urauchi river, Masuzaki 1264 (HIRO). CHINA. Taiwan: Pingtung Co., Mt. Peitawushan, ex Herb. TAI, Lai 9833 (NICH). PHILIPPINES. Luzon: Between Kinabuhayan to Puroy, Mt. Banahao, Iwatsuki et al. 13550a (NICH). THAILAND. Nakornsrithamarat, granitic massive Khao (Mt.) Luang, Touw 11670 (KYO, L). MALAYSIA. Malay Penin.: Gunong Brinchang, Cameron Highlands, det. as Trichocolea obconica by H. Inoue, Inoue 16629 (TNS); Pahang, Cameron Highlands, near the summit of Mt. Berinchang, Furuki 7793 (CBM). INDONESIA. Java: Thibodas, ex Herb NY, Arnoldi & Streliu 27 (NICH). MICRONESIA. Pohnpei (Ponape): Trail from Ron Kiti to Nanalaud summit, det. as Trichocolea obconica by H. Inoue, Miller 6915(NICH).

7. Trichocolea tomentella (Ehrh.) Dumort., Syll. Jungerm. Europ.: 67. 1831, “Thricolea”. Jungermannia tomentella Ehrh., Hannover. Mag. 21: 277. 1783. Type: Europe, Hannover, 1785, leg. Ehrhart, Ehrh. Pl. Crypt. Exs. no. 8 [lectotype designated by Grolle (1976): M!; isolectotypes: HAL, LINN, MW, non vidi, fide Grolle (1976)]. Trichocolea tomentella (Ehrh.) Dumort. f. nodulosa Nees, Naturgesch. Eur. Leberm. 3: 107. 1838, syn. nov. Type: Poland, “auf dem Heerdberge bei Agnetendorf”, 1831, leg. Nees (not found in STR). Trichocolea tomentella (Ehrh.) Dumort. var. nodulosa (Nees) Gottsche, Lindenb. & Nees, Syn. Hepat.: 237. 1845. Trichocolea biddlecomiae Austin, Bot. Gaz. (Crawfordsville) 3: 6. 1878. Type: USA, near Urbana, Ohio, 1876, leg. Biddlecome (holotype: NY-236674!; isotype: G-264410!). Trichocolea gracillima auct. non Austin., Inoue, Stud. Crypt. Papua New Guinea. 28. 1979. Fig. 8 Plants green, becoming yellowish or occasionally whitish when dry, loosely procumbent, forming loose tufts, 2.0–4.0 cm long, 0.7–1.5 cm wide, including branches, with regularly 2–3-pinnate branches; branches usually determinate, the primary branches 4.0–8.0 mm long, the secondary and tertiary branches 1.2–2.6 mm long. Stems slender to robust, 0.6–1.0 mm wide with leaves, 400–440 µm wide, 15–19 cells in diam., with sparse to dense paraphyllia; cortex moderately developed, made up of small, rather thick-walled cells in 1–2 layers, epidermis striate-verrucose. Rhizoids usually absent, rarely poorly developed at the base of underleaves; distal ends of rhizoids digitate. Leaves distant to imbricate, weakly asymmetrical, 0.5–0.8 mm long, 1.0–1.6 mm wide, obliquely to transversely inserted, 6–8-lobed; disc 3–4 cells high, without superficial cilia; cells of disc narrowly oblong to rectangular, thin-walled, striate-verrucose to weakly papillate-verrucose, (40–)60–80(–90) × 24–30 µm; cilia usually narrowly cylindrical, occasionally to subulate, linear, (3–)4–6(–7) cells long; cells of cilia thin-walled, striate-verrucose to weakly papillate-verrucose, 60–100 × 18–20(–24) µm, the septa not dilated and flat or occasionally slightly constricted; underleaves as large as or somewhat bigger than lateral leaves, subsymmetrical, 0.6–1.0 mm long, 0.8–1.4 mm wide, transversely inserted, 6–8-lobed; disc of underleaves 4–5 cells high, without superficial cilia; cells of underleaves similar in size and shape to those of the lateral

18 leaves. Oil-bodies pale smoky grey, opaque, homogeneous, smooth, without an eye-spot, (3–)4–5(–6) per cell in cilia, 6–8 per cell in disc, in the cells of cilia mostly spherical and 6–7 µm in diam. to ovoidal and 8–10 × 4–5 µm. Asexual reproduction unknown. Dioicous. Androecia intercalary on main stem; bracts 4–8 paired, hardly differentiated from leaves, except higher and with a concave disk; antheridia 1–2 per bract; cells of the antheridial stalk biseriate. Gynoecium on main stem; innovation long. Epicoelocaule clavate, covered with paraphyllia and reduced bracts; unfertilized or aborted archegonia 2–4 at the apical part of epicoelocaule; bracts and bracteoles inserted at or below epicoelocaule base, without superficial cilia, similar to leaves and underleaves except for smaller size. Perianth absent. Seta long, becoming hollow at maturity. Capsule narrow ellipsoidal to obloid, black to blackish brown, regularly dehiscing into four valves; capsule walls 7–8-stratose; epidermal cells of capsule thin-walled, subquadrate to rectangular and 60–80 × 30–40 µm in surface view, without inner thickenings; innermost cells of capsule narrowly oblong to fusiform, irregularly oriented and 40–60(–80) × 10–16 µm in surface view, 6–10 µm thick in cross-section, with reddish brown, annular to complete or incomplete semiannular inner thickenings. Spores reddish brown, 10–14 µm in diam., finely rugose; elaters reddish brown, 120–200 µm long, 10–12 µm thick, with two sinistrorse spiral thickenings. Taxonomic notes. Trichocolea tomentella exhibits a wide range of habitat induced variation between populations, particularly in such characters as growth form, plant size, frequency of branching, stem thickness, density of paraphyllia on the stem, length and width of leaf cilia, and height of the leaf disc. Plants growing in dripping wet habitats in shaded places have often a more ascending habit and form loose mats, are of smaller size, have more slender stems with 1-pinnate sparse branches, sparse paraphyllia on the stem, shorter and wider cilia, and smaller leaves with lower (2–3 cells high) leaf discs than the typical form of the species. An extreme example of this expression has been described as Trichocolea biddlecomiae Austin, known only from a swamp near Urbana in North America (Biddlecome s.n., NY-236674). In humid and not always wet habitats there is a tendency to show a prostrate growth habit with the plants forming a more compact mat, larger size, having thick stems with more dense branches, dense paraphyllia on the stems, longer and narrower cilia, and larger leaves with higher (3–4 cells high) leaf discs. More stable characters of taxonomic value can be recognized in the thin-walled cells of leaves, the surface ornamentation of cilia being striate-verrucose to weakly papillate-verruose, and the non-dilated septa of the cilia. From our morphological study, no distinct differences can be recognized among populations from Europe, North America, and SE Asia due to the high phenotypic plasticity of the species. An example was shown by Kitagawa (1984) who cultured T. tomentella over four years on an agar medium (MSK-3) containing mineral salts only. The resulting plants were extremely small, with the stems 6 mm long and 6-cell rows wide at most. Newly developed leaves and underleaves were deeply 1–3-furcate or -ternate, with vestigial laminae. Trichocolea tomentella f. nodulosa Nees was described from plants collected by Nees himself in Poland (Nees 1838). The description of this form only refers to small plants bearing knob-like short branches. Such a morphological character, however, often occurs in poorly developed plants of T. tomentella and is considered as a phenovariant of the species.

19

No original specimen of this form has been found in Nees’s collection (STR). The form nodulosa is synonimized here with T. tomentella. Trichocolea biddlecomiae Austin has been treated as a synonym of T. tomentella (Horikawa 1934, Frye & Clark 1943, Hatcher 1957, Schuster 1966). The morphology of the type material includes the following: plants small, 4.5– 8.0 mm long and 2.0–3.5 mm wide including branches, 1-pinnnate with remote branches. Stems slender, 150–200 µm wide, 7–8 cells in diam., with sparse paraphyllia; cortex slightly developed, made up of small, rather thick-walled cells in a single layer. Leaves small, 300–600 µm long and 300–500 µm wide; disc 2–3 cells high, thin-walled; cilia linear, 4–6 cells long; cells of cilia thin-walled, striate-verrucose to faintly papillate-verrucose, 50–70(–85) µm long and 18–22(–26) µm wide, the septa not dilated. These characters occur frequently in poorly developed plants of T. tomenetella growing in wet swampy habitats and are not sufficient to recognize T. biddlecomiae as an independent species from T. tomentella. We examined pre-Linnean materials (in PC) named by Tournefort (1700, p. 556) and Vaillant (1727, p. 140) as “Muscus palustris absinthii folio insipidus” and “Muscus palustris absinthii folio” respectively. These proved to be identical to Trichocolea tomentella. T. tomentella resembles T. japonica in having thin-walled cells of the leaf cilia and lacking an eye-spot in the oil-bodies. However, T. japonica is easily distinguished from the other members of Trichocolea by (1) the strongly papillate-verrucose surface of the leaf cilia, and (2) the linear to semiannular thickenings in the epidermal cells of the capsule wall. Our morphological studies using SEM revealed that no distinct differences occur in spores and elaters between T. tomentella and T. japonica (Fig. 9). Less well-developed plants of T. tomentella occasionally resemble Trichocolea gracillima Austin*2, a little-known Hawaiian species, in having small plants and slender stems with sparse paraphyllia. T. gracillima has been known only from the type locality, West Maui Mountains, and appears only to be distinguished from T. tomentella by the finely papilliate-verrucose cilia. Further morphological investigation and molecular phylogenetic analyses of the two species are needed to fully elucidate the relationships of these species. Distinguishing characters. T. tomentella is characterized by (1) the regularly 2–3-pinnate habit, (2) the thin-walled leaf cells (3) the striate-verrucose to weakly papillate-verrucose surface of the cilia, (4) non-dilated septa of the cilia, (5) the absence of an eye-spot in the oil-bodies, and (6) the stem cortex consisting of 1–2 layers of rather thick-walled cells. Habitat. Growing on various substrata, usually found on soil or boulders by streams in shaded places in evergreen to montane forests [ca. 0–1500(–3000) m alt.]. It also occurs on humus, tree trunks, decaying logs, clay in swamps, cliffs of steep hillsides, or fallen twig litter. The species usually grows on substrata with a constant supply of high moisture in shaded places. It is frequently found at sites above the flooding level in streams but never on constantly submerged substrata as already suggested by Schuster (1966). It is absent from acid sites such as peat bogs but present in either calcareous or non-calcareous neutral sites. Distribution. Europe (Norway to Portugal: Söderström et al. 2002), North Africa (Tunisia: Müller 1954, fig. 174), North America (Newfoundland to Florida: Schuster 1966), and Asia (Russian Far East to New Guinea: Konstantinova & Bakalin 2009, Potemkin &

20

Sofronova 2009). [SE Asia: Fig. 10F] Representative specimens examined. UNITED KINGDOM. Wales: Monmouth, by stream above Llandogo, Taylor 498 (HIRO). FINLAND. Regio aboensis, Lojo, Lindberg 64 (HIRO). POLAND. Malopolska: Malopolska Upland, 5 km southeast of Golejów near Staszów, Ochyra 925 (HIRO). FRANCE. Centre: Cher, Foret d’Ivoy, Katagiri 3521(HIRO, DNA extracted); Surroundings of Paris (probably): s. loc., “Muscus palustris absinthii folio insipidus”, herb. Tournefort, no. 5490 (P); ditto, herb. Vaillant, no. 103 (PC-103948). GERMANY. Thuringia: Thüringer Holzland, Stadtroda, ex Herb. JE, Hentschel & Zündorf 3370 (HIRO, DNA extracted; TNS-211207). SPAIN. Asturias: Colunga, La Torre, near Fuente de Obaya, ex Herb. MA, Calvo & Carlón 4269 (HIRO, DNA extracted). CANADA. Quebec: Shawville, comté de Pontiac, Lamarre 480031(HIRO). UNITED STATES. New York: about 160 m below Potters Falls along Sixmile Creek, Ithaca, Tompkins Co., Schuster 44504 (TNS). JAPAN. Hokkaido. Ishikari Prov.: Nopporo, Sasaki s. n., Hepaticae Japonicae Exsiccatae ser. 15 (1967), n. 747 (HIRO, NICH, TNS). Honshu. Gifu-ken: Takayama-shi, Mt. Norikura, Goshikigahara, Miyauchi 715 (HIRO, DNA extracted). Sikoku. Kochi-ken: Agawa-gun, Ino-cho, near Kanpuzan tunnel, Kamegamori pass, Katagiri 3230, 3469 (HIRO). Kyushu. Kumamoto-ken: Yatsushiro-shi, Touyo-machi, Kawamata, Miyauchi 1935 (HIRO); Kagoshima-ken: Yakushima Island, along trail between Section 93 of National Forest and Hananoego, Takaki & Mizutani 152 (NICH). CHINA. Hubei Prov.: Shennongjia Forest District, west of Hongriwan construction camp along the trail leading towards Huanghunling Mt. Elev, Sino-Amer. Exped. no. 287: Trichocolea tomentella (TNS-77495, NICH-392697). Shaanxi (Schensi) Prov.: Tai-Bai-shan, Hepaticae Sinicae Exsiccatae ser.1 no. 49: Trichocolea tomentella, Lee 749 (HIRO, NICH).Yunnan Prov.: Tengchong Co., Jietou, along the trail from Dahetou to Giang Rhododendron forest, Zhang 4645 (SZG). Xizang (Tibet) Prov.: Chayu Co., National Cibagou Nature Reserve, Zhang 6785 (HIRO, DNA extracted; SZG). Hainan: Yinggeling Nature Reserve, Zhang 4140 (SZG). Taiwan: Taichung Co., en route from Shiyuan Yakou to Mt. Nanhu, between Shiyuan Yakou and New Yunleng Cabin, Yamaguchi 32617 (HIRO, DNA extracted). PHILIPPINES. Mindanao: Davao Prov., Mt. Apo, Colley 17 (TNS-50315). Luzon: Mountain Prov., Mt. Tabayok, Jacobs 627 (TNS). MALAYSIA. Borneo: Sabah, Ulu Liwagu, southeastern slope of Mt. Kinabalu, Mizutani 3621 (NICH). INDONESIA. Java: Thibodas, Arnoldi & Streliu 18 (NICH). Bangka: Soengai Slan, leg. Teysmann, herb. Hort. Bot. Bog. no. 4026 (NICH). Papua Prov. (New Guinea Island): Vogelkop Peninsula, Aifat river vally, Royen & Sleumer 6927 (TNS). PAPUA NEW GUINEA. Morobe Prov.: Mt. Kaindi, en route from Wau Ecological Institute to summit of Mt. Kaindi, as Trichocolea gracillima Austin, Inoue 24836, 24837 (TNS). Chimbu Prov.: Mt. Wilhelm, Sloover 42766(TNS), 42963 (HIRO).

Molecular phylogenetic analysis The GTR + gamma model provided the best fit model for the dataset. A total of 5094 distinct topologies were obtained in the ML, MP, and BI analyses, of which 4646 topologies passed the AU test at the 0.05 significance level. The condensed topology of the 4646 topologies is shown in Fig. 11. Trichocoleaceae including Eotrichocolea, Leiomitra, and Trichocolea appeared as a monophyletic group with high supporting values (AU/NP/PP = –/99/1.00). The Eotrichocolea– Leimotra clade and Trichocolea clade were resolved in the family. This has already been shown by Glenny et al. (2009) on the basis of the data on rbcL,

21 rps4, and trnL–F sequences. Our condensed tree also showed monophyly for each genus, although the supporting vales for that of Leiomitra are low (AU/NP/PP = –/–/–). A future multi-gene phylogenetic analysis using an ample number of species of Leiomitra and Eotrichocolea might better resolve the intergeneric relationships of Trichocoleaceae. Schuster (2000, p. 160) considered Leiomitra the most apomorphic genus in his subfam. Trichocoleoideae (= Trichocoleaceae s. str.) in having a perianth. The condensed tree, treating Trichocoleaceae and the outgroup genera only, is not conclusive to determine if the presence of a perianth is plesiomorphic or apomorphic in the Trichocoleacae. Taking the presence or absence of a perianth in other leafy liverwort genera/families into account for the phylogenetic analysis, the presence of a perianth observed in Leiomitra may be regarded as plesiomorphic and epicoelocaules observed in Trichcocolea and Eotrichocolea are apomorphic. According to Hentschel et al. (2006), based on maximum likelihood analysis of 113 rbcL sequences of leafy liverworts, the presence of a perianth is plesiomorphic and coelocaule structures evolved independently in five clades in Jungermanniales: (1) a clade corresponding to Schistochilaceae, (2) a clade corresponding to Trichocoleaceae, (3) a clade comprising Lepicoleaceae and Vetaformaceae, (4) Mastigophora within Herbertaceae, and (5) Pseudolepicoleaceae. Trichocolea japonica was resolved to be nested within a clade of T. tomentella, to which it is morphologically closely related. We consider T. japonica to be differentiated from T. tomentella. In Trichocolea, the character of non-dilated septa of the cilia observed in T. japonica, T. tomentella, and T. rigida is considered to be apomorphic. Trichocolea pluma showed a close phylogenetic relationship with T. tomentella and was recognized to be monophyletic in the present study. A Chinese sample of “T. tomentella (accession number: AY462332)” nested within the T. pluma clade has a high possibility of being identical with T. pluma and we have treated it T. pluma in the present study, although the voucher specimen He-Nygrén 1137(H) was not available for reconfirmation. Trichocolea tomentella appeared to be paraphyletic group and showed different DNA haplotype patterns between European and SE Asian populations. Among European populations analysed, there is no intraspecific variation in the rbcL region. Genetic homogeneity over the European populations of T. tomentella has been also shown by Korpelainen et al. (2004) who showed that none of five noncoding chloroplast DNA regions (atpB–rbcL spacer, accD–psaI spacer, trnT–L spacer, trnL–F spacer, and trnL intron) possessed intraspecific variation. On the other hand, among SE Asian populations, three rbcL haplotypes were recognized as different from the European haplotype. This suggests a SE Asian origin of T. tomentella assuming that the number of haplotypes and nucleotide diversity are expected to be higher in ancestral populations than derived populations (Savolainen et al. 2002).

Taxa excluded from Trichocoleaceae The following two species were described by Stephani (1923) from Ledermann’s collections from New Guinea. These species may well be excluded from the Trichocoleaceae, judging from the descriptions and unpublished illustrations in Stephani’s Icones Hepaticarum, since no materials including their types have been found in G and other herbaria. In these

22 circumstances even designation of neotypes is not possible. 1. Trichocolea ledermannii Steph., Sp. Hepat. 6: 373. 1923. Type: New Guinea, leg. Ledermann [Not found in G; Ledermann’s collection in B destroyed (fide Grolle 1977)]. = Lepidozia lacerifolia Steph.; fide Grolle, J. Hattori Bot. Lab. 30: 117. 1967. Grolle (1967) and Mizutani (1968) have treated this species as a synonym of Lepidozia lacerifolia Steph. based on the description and illustrations given by Stephani (1985, n. 10294), without detailed discussion on morphology of this species. The trilobed leaves described by Stephani have not been observed in Trichocoleaceae but they commonly occur in Lepidozia (Lepidoziaceae). 2. Trichocolea pauciseta Steph. Sp. Hepat. 6: 377. 1923. Type: New Guinea, leg. Ledermann [Not found in G; Ledermann’s collection in B destroyed (fide Grolle 1977)]. = Kurzia or Telaranea sp.; fide Golle, J. Hattori Bot. Lab. 43: 66. 1977. It is not possible to make a taxonomic decision for this species without type material due to the poor description and illustrations which refer to 4 cm long stems, 5 mm long branches, and trilobed leaves with discs 1–2 cells high Stephani (1985, n. 10301). Grolle (1977) suggested this species may belong in the genus Kurzia or Telaranea.

Annotations *1 The genus Trichocolea has been proposed by Dumortier (1822) under the spelling of “Thricholea”. Seven years later, Dumortier (1829) replaced the spelling “Thricholea” with “Tricholea”. In Sylloge Jungermannidearum (Dumortier 1831), three different spellings have been used: Thricolea (p. 24, 28, 66, 98), Tricolea (p. 99), and Tricholea (on pl.1, fig. 8). He adopted the spelling, Tricholea, for the genus, mentioning that this contracted form is better-harmonized in Latin than Trichocolea (Dumortier 1835). To clarify the confusion of the generic name, Nees von Esenbeck (1838) corrected the Dumotier’s spellings to Trichocolea. The Nees’s spelling has been widely accepted among bryologists except for Dumortier (1874) and Frye & Clark (1943). Little (1949) successfully argued the Nees’s spelling, Trichocolea, as the generic name and it has been adopted as a nomen genericum conservandum at the VIII International Botanical Congress, 1954. *2 Trichocolea gracillima Austin has been known only from the type locality. Miller (1963) had predicted that other populations of the species should be found in bog forests, but no additional locality has been reported, except for Inoue (1979) from New Guinea. Our detailed morphological investigations, however, proved that the New Guinean plants examined by Inoue (1979) are T. tomentella. The morphology of T. gracillima is described and illustrated here (Fig. 12) based on the type (holotype: NY; isotypes: BISH, G) and an authentic specimen [West Maui, valleys, alt. ca. 1000 m, Baldwin 255 (BISH)]. Trichocolea gracillima Austin, Bot. Gaz. (Crawfordsville) 3: 6. 1878. Type: Hawaii, Island of West Maui, on shaded ground, alt. ca. 1200 m, leg. D.D.Baldwin [holotype: NY-1201998!; isotypes: G-264895!, Baldwin 134! (BISH-500471)]. = Trichocolea tomentosa D.D.Baldwin, Thrum’s Hawaiian Almanac: 42. 1877, later homonym. Not Trichocolea tomentosa (Sw.) Gottsche 1863. [fide Miller (1963)]. Description. Plants yellowish to light yellowish brown in aged herbarium materials, (2.0–)2.5–4.0(–6.4) cm long, 0.8–1.2(–1.8) cm wide including branches, with regularly 2-pinnate rather sparse branches; branches usually determinate, the primary branches 3.5–6.5(–10.0) mm long, the secondary 1.5–3.0(–5.0) mm long. Stems slender, (0.5–)0.6–0.8(–1.0) mm wide with leaves, with

23 sparse paraphyllia, (260–)320–360(–420) µm wide, (11–)12–14 cells in diam.; cortex slightly developed, made up of small, rather thick-walled cells in a single layer, epidermis striate-verrucose. Rhizoids not seen. Leaves distant, asymmetrical, 0.6–0.8(–1.0) mm long, (0.5–)0.6–0.8(–1.0) mm wide, obliquely to subtransversely inserted, 4–6-lobed; disc 2–3 cells high, without superficial cilia; cells of disc rectangular to short rectangular, thin-walled, finely papillate-verrucose to striate-verrucose, 40–50(–80) × 15–20(–30) µm; cilia narrowly cylindrical, linear, 3–5 cells long; cells of cilia thin-walled, finely papillate-verrucose with well-demarcated, sharp verrucae, (60–)80–110(–130) × (10–)18–22(–24) µm, the septa not dilated or occasionally constricted; the distal end of terminal cells of the cilia not thick-walled but verrucae conspicuous; underleaves as large as or somewhat bigger than lateral leaves, subsymmetrical, 0.6–0.8(–1.0) mm long, 0.8–1.0(–1.2) mm wide, transversely inserted, (2–)3–4-lobed; disc of underleaves (1–)2–3 cells high, without superficial cilia; cells of underleaves similar in size and shape to those of the lateral leaves. Oil-bodies unknown. Asexual reproduction unknown. Reproductive structures unknown.

Acknowledgments

We are grateful to the curators and staff of BISH, BORH, CBM, G, H, HO, JE, KYO, L, M, NICH, PC, S, STR, SZG, and TNS for the loan specimens including types. We express our sincere thanks to Prof. Stephan Robert Gradstein (Museum National d'Histoire Naturelle, Paris) for valuable discussions and comments. We thank Rémi Dupré and Florient Desmoulins (Conservatoire botanique national du Bassin Parisien) for excellent field research assistance in France, and Estébanez Belén (Universidad Autonoma de Madrid), Gaik Ee Lee (Universiti Kebangsaan Malaysia), Kuei-Yu Yao (Taiwan Endemic Species Research Institute), Pierre Boudier (Muséum des Sciences Naturelles et de Préhistoire), Shinji Uchida (Miyajima Natural Botanical Garden), Tsuguoki Tango (Shimane Pref., Japan), and Yuya Inoue (Hiroshima University) for offering materials of the taxonomic studies and molecular analysis. We also thank Prof. Rod Seppelt (Australian Antarctic Division, Tasmania) for checking the English text and his helpful comments. This work was supported by KAKENHI (23370045).

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Fig. 1. Trichocolea iriomotensis T.Katag. A. Plant. B. Lateral leaf. C. Underleaf. D & E. Apices of cilia, at right surface ornamentation drawn in. F. Median cells of disc, at left surface ornamentation drawn in. G. Paraphyllia from stem. H. Cross-section of stem. I. Cilium cells with oil-bodies. A–H drawn from Miyauchi 1869 (HIRO; holotype), I from Katagiri 3424 (HIRO).

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Katagiri

tomentella . T.

D from D from . D.

pluma T. T.

, (HIRO)

. C.

iri 2593 iri

ag

Kat T. T. japonica

. B. from

C (HIRO),

T. iriomotensis 3198

i . A.

Katagir

Trichocolea

ies of ec

B from (HIRO),

Katagiri3424

(HIRO). (HIRO). Fig. 2. Oil-bodies of SE Asian sp 30 32 A photographed from photographed from A

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Fig. 3. Trichocolea magna T.Katag. A. Plant. B. Lateral leaf. C. Underleaf. D & E. Apices of cilia, at right surface ornamentation drawn in. F. Median cells of disc, at left surface ornamentation drawn in. G. Part of cross-section of stem (center indicated by a cross). H. Superficial cilium on marginal part of disc. I. Superficial cilium on basal part of disc. J. Paraphyllia from stem. Drawn from Zanten 425 (TNS; holotype).

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Fig. 4. Trichocolea mollissima (Hook.f. & Taylor) Gottsche A. Plant. B. Lateral leaf. C. Underleaf. D & E. Apices of cilia, at right surface ornamentation drawn in. F. Median cells of disc, at left surface ornamentation drawn in. G. Occasional presence of superficial cilia on marginal part of disc. H. Paraphyllia from stem. I. Part of cross-section of stem (center indicated by a cross). J. Arcuate (left) and moderately curved (right) terminal cells of cilia. Drawn from Junghuhn s.n. (NICH-223618).

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Fig. 5. Trichocolea pluma (Reinw., Blume & Nees) Mont. A. Plant with an unfertilized gynoecium. B. Lateral leaf. C. Underleaf. D & E. Apices of cilia, at right surface ornamentation drawn in. F. Median cells of disc, at left surface ornamentation drawn in. G. Paraphyllia from stem. H. Part of cross-section of stem (center indicated by a cross). I. Cilium cells with oil-bodies. Drawn from Katagiri 2593 (HIRO).

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Fig. 6. Sporophytic characters of Trichocolea pluma (A–C) and T. tomentella (D–F). A & D. Dehisced capsule. B & E. Outermost cells of capsule. C & F. Part of cross-section of capsule wall. A–C drawn from Hattori 1908 (TNS), D–F from Katagiri 3469 (HIRO).

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Fig. 7. Trichocolea rudimentaris Steph. A. Plants. B. Lateral leaf (crosses indicate detached or broken parts of cilia). C. Underleaf (crosses indicate detached or broken parts of cilia). D & E. Apices of cilia, at right surface ornamentation drawn in. F. Median cells of disc, at left surface ornamentation drawn in. G. Cross-section of stem. H. Cilium cells with oil-bodies. I. Cilia detaching (left) and detached (right) at the middle lamellae. J. Cilia broken off at the middle part. A–G & I–J drawn from Masuzaki 1264 (HIRO), H drawn based on the photograph and descriptions for Trichocolea obconica by Inoue (1967, pl.1A).

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Fig. 8. Trichocolea tomentella (Ehrh.) Dumort. A. Plant. B. Lateral leaf. C. Underleaf. D & E. Apices of cilia, at right surface ornamentation drawn in. F. Median cells of disc, at left surface ornamentation drawn in. G. Paraphyllia from stem. H. Part of cross-section of stem (center indicated by a cross). I. Cilium cells with oil-bodies. Drawn from Katagiri 3230 (HIRO).

37

ed

ph

togra

pho

A–B

(C–D).

T. japonica T.

(A–B) and

(HIRO). Trichocolea tomentella Trichocolea Katagiri 3198 res res and elaters of po C–D from , (HIRO) Katagiri Katagiri 3469 Fig. 9. SEM of images s from

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Fig. 10. Distributions of SE Asian species of Trichocolea, based on specimens (solid symbols) and literature (open symbols). A. T. iriomotensis (circle) and T. japonica (star). B. T. magna. C. T. mollissima. D. T. pluma. E. T. rudimentaris. F. T. tomentella.

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Fig. 11. Phylogenetic relationship of Trichocoleaceae based on rbcL gene sequences depicted by a 50% majority-rule condensed tree for the 4646 topologies which passed the AU test. Supporting values more than 50% obtained by the program CONSEL were overlaid: the values by the AU test (AU), bootstrap probabilities (NP), and Bayesian posterior probabilities (PP) are shown on or near each branch (AU/NP/PP; in %); those with <50% support are indicated with a dash (–).

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Fig. 12. Trichocolea gracillima Austin A. Plant. B. Lateral leaf. C. Underleaf. D–F. Apices of cilia, at left septa constricted, at right surface ornamentation drawn in. G. Median cells of disc, at left surface ornamentation drawn in. H. Paraphyllia from stem. I. Cross-section of stem. Drawn from Baldwin s.n. (NY-1201998; holotype)

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Table 1. List of species investigated for rbcL gene sequences with their respective GenBank accession numbers, references, and voucher specimens with sampling localities.

Species Acc. no. Reference Voucher information Trichocoleaceae Eotrichocolea polyacantha (Hook.f. & FJ173322 Glenny et al. Glenny 9891b (herbarium not shown) Taylor) R.M.Schust. (2009) New Zealand Leiomitra lanata (Hook.) R.M.Schust. AY462305 He-Nygrén et Glenny 8521 (herbarium not shown) al. (2004) New Zealand Leiomitra tomentosa (Sw.) Lindb. AY608040 Davis (2004) Davis 368 (DUKE) Ecuador Trichocolea iriomotensis T.Katag. AB751577 Present study Miyauchi 1869 (HIRO-269249) Japan (Iriomote) Trichocolea japonica T.Katag. AB618046 Present study Katagiri 2591 (HIRO-260484) Japan (Yamanashi) AB751578 Present study Inoue 1274 (HIRO-1016764) Japan (Nagano) Trichocolea mollissima (Hook.f. & AB751579 Present study Katagiri 3490 (HIRO-1011361) Taylor) Gottsche Australia (Tasmania) AB751586 Present study Furuki s.n. (HIRO-1016769) Malaysia (Borneo) Trichocolea pluma (Reinw., Blume & AB751580 Present study Miyauchi 1897 (HIRO-1007242) Nees) Mont. Japan (Fukushima) AB751581 Present study Miyauchi 1389 (HIRO-257104) Japan (Hiroshima) AB751582 Present study Miyauchi 1873 (HIRO-269253) Japan (Kagoshima) AB751583 Present study Miyauchi 1739 (HIRO-269119) Japan (Okinawa) AB751584 Present study Zhou 280 (HIRO-1016765) China (Guangdong) AB751585 Present study Furuki 22034 (HIRO-1009601) China (Taiwan) Trichocolea rigida R.M.Schust. AB751587 Present study Katagiri 3505 (HIRO-1011376) Australia (Tasmania) Trichocolea tomentella (Ehrh.) Dumort. AB751588 Present study Hentschel & Zündorf 3370 (HIRO-1016768) Germany (Thuringia) AB751589 Present study Katagiri 3521 (HIRO-1015691) France (Cher) AB751590 Present study Calvo & Carlón 4269 (HIRO-1016767) Spain (Asturias) AB618047 Present study Miyauchi 715 (HIRO-252809) Japan (Gifu) AB751591 Present study Zhang 6785 (HIRO-1016766) China (Tibet) AY462332 He-Nygrén et He-Nygrén 1137 (H, not available) al. (2004) China(Hunan) AB751592 Present study Yamaguchi 32617 (HIRO-1008971) China (Taiwan) Outgroup taxa Arachniopsis major Herzog EF101021 Heslewood & NSW-611656 Brown (2007) New Caledonia Bazzania tridens (Reinw., Blume & Nees) AB692997 Present study Miyauchi 1993 (HIRO-1009872) Trevis. Japan (Hiroshima) Kurzia helophila R.M.Schust. JF316300 Cooper et al. Cooper 383 (NSW) (2011) New Zealand Lepidozia vitrea Steph. AB751593 Present study Miyauchi 1833 (HIRO-269213) Japan (Okinawa) Zoopsis setulosa Leitg. EF101025 Heslewood & NSW-709890 Brown (2007) Australia (New South Wales)