Orthotrichum moravicum (Orthotrichaceae), a new moss species from the Czech Republic VÍTĚZSLAV PLÁŠEK Department of Biology and Ecology, University of Ostrava, Chittussiho 10, Ostrava CZ-710 00, Czech Republic e-mail: vitezslav.plasek@osu.cz JAKUB SAWICKI Deparment of Botany and Nature Protection, University of Warmia and Mazury in Olsztyn, 10-728 Olsztyn, Poland e-mail: jakub.sawicki@uwm.edu.pl VĚRA TRÁVNÍČKOVÁ Kroměřı́žská 169, Kojetı́n, CZ-752 01, Czech Republic e-mail: TravnickovaV@seznam.cz MARKÉTA PASEČNÁ Department of Biology and Ecology, University of Ostrava, Chittussiho 10, Ostrava CZ-710 00, Czech Republic e-mail: vitezslav.plasek@osu.cz ABSTRACT. Orthotrichum moravicum Plášek & Sawicki, sp. nov., is a confirmed record of a new species from the Czech Republic. The new taxon is described in subgen. Pulchella and illustrated by photos. Molecular data indicating the phylogenetic placement of the species are presented, indicating a sister relationship with O. pallens. KEYWORDS. Orthotrichum moravicum, Orthotrichum scanicum, Orthotrichum vittii, Orthotrichum pallens, mosses, new species, taxonomy, Czech Republic. ¤ During a bryofloristic survey in the Moravskoslezské Beskydy Mountains in July 2006 a noteworthy epiphytic moss from the genus Orthotrichum was collected. The specimen proved to be the first record of this taxon for the Czech Republic and, upon closer examination, also to represent a species new to science. We are pleased to name the species after the province of the Czech Republic, Moravia, where the first specimen has been found. ¤ ¤ The nomenclature of the moss taxa in the text below follows Hill et al. (2006). Orthotrichum moravicum Plášek & Sawicki, sp. nov. (Fig. 1–19) Plantae usque ad 1 cm altae, cum foliis erectoadpressis, lanceolatis vel oblongo-lanceolatis, apicis late acutis. Cellulae superiores cum 1–2 papillis altis et conicis. Capsulae ad dimidium immersae, oblongae vel The Bryologist 112(2), pp. 329–336 Copyright E2009 by The American Bryological and Lichenological Society, Inc. 0007-2745/09/$0.95/0 330 THE BRYOLOGIST 112(2): 2009 Figures 1–5. Orthotrichum moravicum. 1. Habit. 2a. Leaves. 2b. Leaf apex. 3a. Cross-section of costa and leaf. 3b. Cross-section of middle leaf cells with high papillae. 4. Immersed stomata. 5a. Exostome teeth (right one with visible papillose surface) and endostome segments with remarkable marginal appendages. 5b. Detail view of endostome segments with removal of exostome teeth. Drawing from holotype by Věra Trávnı́čková. Plášek et al.: A new Czech Orthotrichum 331 Figures 6–14. SEM micrographs of Orthotrichum moravicum. 6. Habit. 7. View of exostome and endostome with double-row continuous connective membrane. 8. OPL view of exostome teeth. 9, 10. Endostome segments with appendages. 11–13. PPL view of exostome teeth. 14. Detail of pore. Photographs by V. Plášek and G. Kratošová. 332 THE BRYOLOGIST 112(2): 2009 Figures 15–19. Photomicrographs of Orthotrichum moravicum. 15. Peristome. 16. Leaf apex. 17. Immersed stomata. 18. Plant with mature capsule. 19. Detail of an endostome with some appendiculae linking contiguous segments. Photographs by V. Plášek. Plášek et al.: A new Czech Orthotrichum Figure 20. Map of central Europe marking the locality of Orthotrichum moravicum (coordinates in WGS 84: 49u269530N, 18u299140E). oblongo-cylindricae. Stomata cryptopora. Peristoma duplex; exostoma 8 segmentis appropinquatis, in sicco recurvatis, in basi papillosis, in dimidio superiore papilloso-striatis; endostoma tam-quam exostoma longum, 16 segmentis glabris, appendicis gracilibus. Calyptra oblongo-conica, nuda. TYPE. CZECH REPUBLIC. PROV. MORAVIA: Moravskoslezské Beskydy Mts., 3 km NE of Bı́lá village, valley of Chladná voda stream, 645 m, 49u269530N, 18u299140E (see Fig. 20), 31.VII.2006, M. Pasečná & V. Plášek (holotype OP, isotype NY). Description. Plants pale green, 5–10 mm high, in small sparse cushions to 1.5 cm tall. Stems rarely and shortly branched, slender; rhizoids red-brown, branched, smooth, densely gathered in lower thirdhalf. Stem leaves erect-appressed and slightly incurved when dry, erect when moist, oblonglanceolate to lanceolate, 1.6–2.5 mm long, keeled, narrowly obtuse, broadly acute to broadly apiculate; margins recurved from base to near apex, entire; costa ending near apex, in basal part (80–) 84(–92) mm wide, near apex (24–)28(–32) mm wide; laminal cells in the apex oval to elliptic, (9–) 10–12(–13) 3 (8–)9–10(–11) mm, in the middle of the leaves rounded, (9–)10–14 3 8–10 mm, 1stratose, with 1–2 fairly high conical papillae (4– 7 mm); basal laminal cells rectangular, non-nodose, 8–10 3 12–40 mm wide. Asexual propagula not 333 seen.Sexual condition cladautoicous. Setae 0.5– 1.1 mm, ochrea reaching to 1/5 of the seta, vaginula naked; capsules to K-emergent; urn oblong to oblong-cylindric when dry, oblong barrel-shaped when moist, 1.3–2 mm long, pale brown with dark brown strong 8 ribs not reaching the capsule base, contracted to 4/5 or along entire length when old; exothecial bands 4(–5) cells wide, of moderately differentiated exiguous thick-walled cells, extending from the mouth to K–2/3 of capsule length; neck concolorous with the urn, gradually narrowed to the seta; stomata immersed, mainly in central part of capsule, scarcely covered by subsidiary cells; operculum not seen; peristome double; exostome teeth 8, light brown, 210–220 mm long, reflexed when dry, often slightly cleft or perforate in upper parts, external side (OPL) evenly and densely papillose at base to papillae-striate in upper part, internal side (PPL), the lower half slightly striate, upper half sparsely and irregularly ornamented with striae and papillae; endostome segments 16 with conspicuous marginal (mainly unicellular) appendages, with some appendiculae linking contiguous segments hyaline, smooth on both sides, principal segments biseriate (at least at base), (180–)190–195(–200) mm long, intermediate ones smaller, (175–)180–188(–195) mm long and formed of 1 row of cells, basal membrane continuous, smooth, biseriate. Calyptra conicoblong, light brown, naked. Spores 10–13 mm, densely papillose. Discussion. Orthotrichum moravicum is similar to O. vittii in sporophytic characters, but they are easy to distinguish from one another by differences in their leaf apices, which are are hyaline awns in O. vittii (Lara et al. 1999). A related species, O. pallens, is distinguished mainly by lacking marginal appendages on its endostome segments. In addition O. pallens has endostome segments alternately longer and shorter, whereas in O. moravicum all segments are fairly long (almost as long as the exostome teeth). Orthotrichum scanicum, another close relative, differs from O. moravicum by having exothecial bands only 2(–3) cells wide, 16 exostome teeth (rather than 8), markedly smaller appendiculate endostome segments, and a typically denticulate leaf apex. 334 THE BRYOLOGIST 112(2): 2009 Table 1. Voucher information for the 14 collections included in a molecular assessment of the species within genus Orthotrichum. Note: OLN 5 Herbarium of the University of Warmia and Mazury in Olsztyn, Poland; TC 5 Herbarium of the Museum in Tachov, Czech Republic. Taxon Orthotrichum affine Orthotrichum anomalum Orthotrichum casasianum Orthotrichum gymnostomum Orthotrichum fastigiatum Orthotrichum lyellii Orthotrichum moravicum Orthotrichum obtusifolium Orthotrichum pallens Orthotrichum pumilum Orthotrichum rupestre Orthotrichum speciosum Orthotrichum stramineum Orthotrichum striatum Locality (herbarium) Czech Republic, Bohemia: Český les Mts., between Pivoň & Mnichov villages, 560 m (OP) Poland, Góry Bialskie Mts., between Stary Gieraltów & Goszów villages, 517 m (OP) Spain, Alava: Luquiana (VIT) Canada, Newfoundland, Notre Dame Bay (NY) Czech Republic, Moravia: Hrubý Jesenı́k Mts., NE edge of Lázně Jesenı́k town, 550 m (OP) Slovakia, Poloniny Mts., Ruský potok village, 482 m (OP) Czech Republic, Moravia: Moravskoslezské Beskydy Mts., 3 km NE of Bı́lá village, valley of Chladná voda stream, 645 m (OP) Slovakia, Nı́zké Tatry Mts., 3 km S of Liptovský Ján village, Liptovaký dvôr settlement, 900 m (OP) Poland, Góry Bialskie, between Nowy Gieraltów & Bielice villages, 632 m (OP) Poland, Mazowieckie: Chiechanów (OLN) Czech Republic, Bohemia: Branov village, Malá Pleš reserve, 600 m (TC) Czech Republic, Bohemia: Český les Mts., Ostrůvek village, Randezvous, 735 m (TC) Czech Republic, Bohemia: Český les Mts., Rybnı́k village, U Huberta, 595 m (TC) Czech Republic, Moravia: Jihlavské vrchy hills, S edge of Jihlávka village, 680 m (OP) Very similar in habitat, Orthotrichum casasianum was described from Spain (Mazimpaka et al. 1999). It differs from O. moravicum by smaller appendiculae which do not link contiguous hyaline endostomial segments and by exostome ornamentation: OPL reticulate. The leaf apex in O. casasianum has a small, 1(–2)-celled mucro or sometimes a percurrent costa, whereas O. moravicum has the leaf apex broadly acute to broadly apiculate with a subpercurrent costa. Ecology and distribution. The new species was observed growing on bark of Salix caprea, on an inclined branch, with eastern exposure, at a height of approximately 120 cm. The tree was growing within a bank of vegetation, situated between a stream and forest road. After the specimen was identified and confirmed as a new species, a detailed survey of the locality was carried out. As of July 2007, two new populations on Salix caprea trees in the proximity of the first cushion were found. The following GenBank Acc. No. ITS2 length EU072690 452 EU072691 436 EU850820 EU072687 EU072692 433 484 452 EU072689 EU072688 480 432 EU072693 484 EU072694 436 EU035537 EU072686 EU072695 435 454 452 EU072696 436 EU072697 452 associated species were recorded at the locality: Orthotrichum affine, O. speciosum, Ulota bruchii, Pterigynandrum filiforme and Hypnum cupressiforme. Molecular analysis. The list of species used in the molecular analysis, details concerning voucher data and GenBank accesion numbers are given in Table 1. Total genomic DNA was extracted from herbarium material. Single stems were ground with silica beads in a FastPrep tissue disruptor for 20 seconds and subsequently treated processed using the DNAEasyH Plant Mini Kit (Qiagen) following the manufacturer’s protocol. Extraced DNA samples were stored at 220uC. For amplification and sequencing of ITS we used the primers of Fiedorow et al. (1998). The ITS loci were amplified in a volume of 25 ml containing 20 mM (NH4)SO4, 50 mM TrisHCl (pH 9.0 at 25uC), 1.5 mM MgCl2, 1 ml BSA, 200 mM each dATP, dGTP, dCTP, dTTP, 1.0 mM of each primer, one unit of Taq polymerase and 1 ml of the genomic DNA. The reaction was processed at Plášek et al.: A new Czech Orthotrichum 335 Figure 21. Minimum Evolution tree of the ITS2 sequences. Bootstrap values are presented. 94uC for 1 min, followed by 30 cycles at 94uC for 1 min, 58uC for 1 min, and 72uC for 1.5 min, with a final extension step of 72uC for 5 min. Purified PCR products were sequenced in both directions using the ABI Prism Dye Terminator Cycle Kit (Perkin-Elmer Applied Biosystems) and then visualized using an ABI Prism 377 Automated DNA Sequencer (PerkinElmer Applied Biosystems). Electropherograms were edited and assembled using Sequencher 4.5 (Genecodes Inc.). The sequences were aligned using Muscle 3.6 (Edgar 2004) and manually adjusted with BioEdit 7 (Hall 1999). MEGA 4 (Tamura et al. 2007) was used for the Minimum Evolution (ME) analysis (Rzhetsky & Nei 1992). The evolutionary distances were computed using the Kimura 2-parameter method (Kimura 1980), and are in the units of the number of base substitutions per site. The ME tree was searched using the Close Neighbor Interchange (CNI) algorithm (Nei & Kumar 2000) at a search level of 2 and the maximum number of trees retained at each step was set to 100. Bootstrap analysis (Felsenstein 1985) was carried out with 1000 replicates. The length of the ITS2 region varied between 430–484 base pairs (Table 1). The shortest sequence was of Orthotrichum moravicum, and the longest sequence of O. obtusifolium. The alignment had a total length of 536 sites. Of the aligned sequences, 21 positions were gapped sites and excluded from further analysis. Of the remaining sites, 437 were constant, 42 variable but parsimony-uninformative, and 36 parsimony-informative. The tree based on ME analysis revealed three well-supported clades (Fig. 21). Clade A is formed by O. gymnostomum and O. obtusifolium both from subgenus Orthophyllum. Species from subg. Gymnophorus formed clade B, together with O. rupestre from subg. Phanerophorum. All taxa from this clade have superficial stomata. The new species, O. moravicum is included within clade C (ME 88% bootstrap support), which is formed by species with immersed stomata. Orthotrichum moravicum shows a direct relationship with other taxa of subgen. Pulchella, and a sister relationship specifically to O. pallens (ME 88% bootstrap support), from which it is well separated (branch length 0.00921). CONCLUSION According to Lewinsky (1993), the presence of cryptopore stomata and a connective endostomial membrane in Orthotrichum moravicum suggest its inclusion within subgenus Pulchella. This hypothesis is supported by the results of our molecular analysis. 336 THE BRYOLOGIST 112(2): 2009 Sequence data from ITS2 showed a close relationship of O. moravicum to other species of subgenus Pulchella, and especially to O. pallens and O. pumilum from section Diaphana, but the new species differs from them by four and seven substitutions, respectively. This level of inter-specific variation is even higher than observed for currently recognized species of Orthotrichum included within our study. Although molecular data are used in this case to help support the species status of this new taxon, O. casasianum, O. pallens, O. scanicum and O. vittii can be distinguished from congeners using morphological features such as obvious marginal appendages on the endostomial segments, OPL and PPL ornamentation, different exothecial band structure and other features discussed above. ACKNOWLEDGMENTS We thank Kenneth M. Cameron for helpful comments and Jan Kučera, Jarosław Proćków & Tereza Poštová for help with developing the Latin diagnosis. DNA analysis was made possible through financial support from The Lewis B. and Dorothy Cullman Program for Molecular Systematic Studies, The New York Botanical Garden. SEM micrographs were made with help of Gabriela Kratošová (VSB - Technical University of Ostrava). The field survey was supported from coffers of the Czech Science Foundation (GA CR) No. 206/07/0811. LITERATURE CITED Edgar, R. C. 2004. Muscle: multiple sequence alignment with high accuracy and high throughput. Nucleic Acid Research 32: 1792–1797. Felsenstein, J. 1985. Confidence limits on phylogenies: An approach using the bootstrap. Evolution 39: 783–791. Fiedorow, P., I. Odrzykoski & Z. Szweykowska-Kulińska. 1998. Phylogenetic studies of liverworts using molecular biology techniques. Pages 244–249. In J. Małuszyńska (ed.), Plant Cytogenetics: Spring Symposium, Cieszyn, 19–22 May 1997. 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