Opuscula Philolichenum, 17: 322-329. 2018. *pdf effectively published online 17August2018 via (http://sweetgum.nybg.org/philolichenum/)

Discovery of isidiatum (, ) disjunct in northeastern Asia

LIUDMILA KONOREVA1, SERGEY CHESNOKOV2, IRINA STEPANCHIKOVA3, IVAN FROLOV4, LUDMILA GAGARINA5 AND SVETLANA TCHABANENKO6

ABSTRACT. – Coenogonium isidiatum is reported new to Russia from the Far East, constituting a considerable northern range extension for the species. Morphology, ecology and distribution of the species are discussed. Molecular data (mrSSU and nrITS DNA sequences) were obtained from the material and phylogenetic analyses recovered these as a strongly supported and monophyletic with respect to other sequenced Coenogonium species.

KEYWORDS. – Biogeography, distribution, isidia, Kurile Islands, Kamchatka, Sakhalin, sterile crustose .

INTRODUCTION

Coenogonium Ehrenb. is characterized by filamentous or crustose thalli, biatorine (sometimes zeorine) apothecia with yellow to orange or brownish discs, paraplectenchymatous exciples, partially amyloid hymenia, unitunicate asci with entirely thin walls, and two-celled (rarely simple), colorless ascospores, and trentepohlioid photobionts (Ferraro & Michlig 2013, Lücking 2008, Rivas Plata et al. 2006). Currently the comprises about 130 species of mainly tropical to subtropical lichens (Gagarina 2015). Originally the genus Coenogonium consisted of species with filamentous thalli only and those with crustose thalli were included into the separate genus Dimerella Trevis (Vězda & Poelt 1975). However, studies on morphology, anatomy and phylogeny suggested that Dimerella should be merged with Coenogonium (Kauff & Lutzoni 2002, Lücking & Kalb 2000, Lücking 2008, Rivas Plata et al. 2006).

1LIUDMILA KONOREVA – a) Botanical Garden-Institute FEB RAS, Makovskogo str., 142, Vladivostok, 690024, Russia; b) Laboratory of Flora and Vegetation, The Polar-Alpine Botanical Garden-Institute of the Kola Science Centre of Russian Academy of Sciences, Botanical Garden str., Kirovsk, 184256 Murmansk Region; c) Laboratory of Lichenology and Bryology, Komarov Botanical Institute of Russian Academy of Sciences, Professor Popov str. 2, 197376 St. Petersburg, Russia. – e-mail: [email protected] 2SERGEY CHESNOKOV – a) Laboratory of Lichenology and Bryology, Komarov Botanical Institute of Russian Academy of Sciences, Professor Popov str. 2, 197376 St. Petersburg, Russia; b) Sakhalin Branch of Botanical Garden-Institute of the Far Eastern Branch of Russian Academy of Sciences, Gorkogo str. 25, mailbox 34, 693023 Yuzhno-Sakhalinsk, Russia. – e-mail: [email protected] 3IRINA STEPANCHIKOVA – a) St. Petersburg State University, Universitetskaya emb. 7–9, 199034 St. Petersburg, Russia; b) Laboratory of Lichenology and Bryology, Komarov Botanical Institute of Russian Academy of Sciences, Professor Popov str. 2, 197376 St. Petersburg, Russia. – e-mail: [email protected] 4IVAN FROLOV – Russian Academy of Sciences, Ural Branch: Institute Botanic Garden, Vos’mogo Marta 202a str., 620144 Yekaterinburg, Russia. – e-mail: [email protected] 5LUDMILA GAGARINA – Laboratory of Lichenology and Bryology, Komarov Botanical Institute of Russian Academy of Sciences, Professor Popov str. 2, 197376 St. Petersburg, Russia. – e-mail: [email protected] 6SVETLANA TCHABANENKO – Sakhalin Branch of Botanical Garden-Institute of the Far Eastern Branch of Russian Academy of Sciences, Gorkogo str. 25, mailbox 34, 693023 Yuzhno-Sakhalinsk, Russia. – e-mail: [email protected]

322 Altogether 14 species of Coenogonium are known from extratropical Eurasia (Gagarina 2015), and two of those have been previously reported from Russia: C. luteum (Dicks.) Kalb & Lücking and C. pineti (Schrad. ex Ach.) Lücking & Lumbsch (Gagarina 2017). Both species are widely distributed in boreal and temperate forests in Russia (Gagarina 2015, 2017), with the other extratropical Eurasian species having been reported from Southeastern Asia (South Korea (Kondratyuk et al. 2016), Japan and China (Obermayer 2004). Here we report the discovery of C. isidiatum (G. Thor & Vězda) Lücking, Aptroot & Sipman in the Russian Far East, a remarkable range extension of the species into northeastern Asia.

MATERIALS AND METHODS

Field and herbarium study. – Specimens were collected by Liudmila Konoreva and Sergey Chesnokov on Sakhalin, Shikotan and Iturup Islands in the Sakhalin Region of Russia in 2017, and by Irina Stepanchikova on the Kamchatka Peninsula in the Kamchatka Territory of Russia in 2016. The specimens were deposited in the herbaria of the Komarov Botanical Institute of the Russian Academy of Sciences (LE) and the University of Helsinki (H). The material was examined by the authors in the Laboratory of Lichenology and Bryology of Komarov Botanical Institute, using standard microscopic techniques (Smith et al. 2009). High Performance Thin Layer Chromatography (HPTLC) was performed according to standard procedures (Culberson & Ammann 1979, Kranner et al. 2002), using solvent system A. Photographs of the species were taken with a Stemi-2000 CS microscope with an attached AxioCam MRc5 camera. The distribution map was prepared using MapInfo GIS software. Geographical coordinates are given in spatial reference system WGS 1984. Ludmila Gagarina revised specimens in the herbaria of the Museum of Evolution, Uppsala University (UPS) and the Swedish Museum of Natural History, Stockholm (S), including the type specimen of Coenogonium isidiatum.

Molecular data generation and analyses. – Extraction of DNA and PCR amplification were performed following Cubero et al. (1999). We used the primer pairs mrSSU1 and mrSSU3R (Zoller et al. 1999), and ITS1F (Gardes & Bruns 1993) and ITS4 (White et al. 1990) for the production of mrSSU and nrITS rDNA sequences. Amplicons were sequenced at Eurogen (Moscow)®. Chromatograms were edited in FinchTV 1.4.0 (Geospiza, Inc.; Seattle, WA, USA), then resulting sequences were assembled in BioEdit 7.2.5 (Hall 1999) and aligned online by MAFFT version 7 (Katoh & Standley 2013) with the L-INS-i method (Katoh et al. 2005). The alignment was manually checked and adjusted in BioEdit 7.2.5. Newly generated sequences were uploaded into the NCBI (GenBank); accession numbers are provided (see Table 1 in the appendix). The alignment was deposited in TreeBASE (Submission ID 23107). Our sequences of mrSSU were aligned together with all Coenogonium mrSSU sequences available in GenBank (see Table 1 in the appendix). Species of the order were selected as an outgroup (see e.g. the phylogenetic reconstruction by Resl et al. 2015). ITS sequences were not used in the phylogenetic reconstructions. Maximum likelihood reconstruction was carried out in RAxML (Stamatakis et al. 2005) through the RAxMLGUI interface (Silvestro & Michalak 2012); the GTR+G model was chosen with jModelTest 0.1.1 (Guindon & Gascuel 2003; Posada 2008). Bootstrap support values were calculated on 500 bootstrap replicates using rapid bootstrapping (“ML + rapid bootstrap” function in RAxMLGUI). The whole original alignment (including ambiguously aligned regions) was used in the analysis; gaps were treated as missing data. Pairwise genetic distances between ITS sequences were calculated in PAUP under the JC model of evolution. The mean and standard deviation of the pairwise distances were calculated in Excel.

RESULTS AND DISCUSSION

We generated four new mrSSU sequences from samples identified as Coenogonium isidiatum collected on the Kamchatka Peninsula, Sakhalin Island and Kurile Islands. The four sequences were recovered in a strongly supported clade (ML BP: 100) within a strongly supported and monophyletic Coenogonium (ML BP: 100) (Figure 1). All our mrSSU sequences were 100% identical to each other. We also generated five new nrITS sequences from the same material (see Table 1 in the appendix), and these included some variable positions. The calculated mean pairwise distance and standard deviation for our nrITS sequences (0.0054 ± 0.004) corresponds well to the reported intraspecific variability of ITS sequences in the genus , which is also placed in the order Ostropales (Muscavitch et al. 2017). Hence, molecular data support a morphologically based hypothesis that our specimens belong to one species of the genus Coenogonium. Unfortunately, no previously published sequences of C. isidiatum

323

Figure 1. Phylogeny of the genus Coenogonium and closely related groups in the Ostropomycetidae inferred from mrSSU sequences and presented as the most likely tree. Numbers on branches represent maximum likelihood bootstrap values ≥70%. Newly sequenced samples are indicated by bold text.

were available in GenBank, hence the molecular data cannot be used to confirm the identification of our samples that was based on morphological data. We did attempt to generate sequences from the Brazil specimen that we examined (see below), however these were unsuccessful, probably because of the age of the material. Given that Coenogonium is a species-rich genus, it is surprising that there are relatively little molecular data available for the group in GenBank. Assembling a comprehensive molecular dataset and phylogeny for the genus is an important avenue for future research, particularly for examining character evolution and confirming the broad geographic distributions of species such as C. isidiatum. Below we provide a taxonomic treatment of C. isidiatum, including a description of the material from the Russian Far East.

Coenogonium isidiatum (G. Thor & Vězda) Lücking, Aptroot & Sipman, Fun. Divers. 23: 297. 2006. ≡ Dimerella isidiata G. Thor & Vězda, Folia Geobot. Phytotax. 19: 72. 1984. TYPE: NEPAL: No 4 West (Nuwakot) district, 32 km NW Pokhara, 12 km NW Ghandrung, 28°22'N, 83°45'E, in mixed coniferous-deciduous forest, 3050 m, 01.xii.1979, G. Thor 1479 (S-L2268!, holotype).

FIGURE 2.

DESCRIPTION OF STUDIED SPECIMENS. – Thallus crustose, homoiomerous, thin, smooth, green-grey to grey with bluish tinge; cortex 10–30 µm thick; medulla not differentiated. Prothallus not seen (absent in type). Isidia numerous, coralloid, 0.2–0.4 mm tall, simple or slightly branched, concolorous with thallus. Apothecia scattered, sessile, rounded, constricted at the base, (0.5–)0.7–1.2(–1.9) mm in diameter (1.5–2 mm in diam. in type material), 0.4 mm tall. Margin concolorous with disc, epruinose, smooth. Disc waxy to orange red, flat or later slightly convex, epruinose. True exciple paraplectenchymatous, colorless, 85 µm

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Figure 2. Coenogonium isidiatum from Mizhulsky Ridge on Sakhalin Island in Russia (L. Konoreva 198, LE L-14441). Scale = 2 mm. Photograph by S. Chesnokov, 2018.

thick (up to 100 µm in type material), below hymenium 100–150 µm thick (up to 120 µm in type material). Epithecium colourless or yellowish, non-granular, 5–10 µm thick. Hymenium colorless, 82–113 µm high (up to 80–100 µm in type material), J+ blue. Hypothecium yellowish, 10–20 µm high. Paraphyses simple, with tips slightly enlarged up to 4 µm. Asci cylindrical, Catillaria-type, with 8 ascospores. Ascospores 1- septate, ellipsoid, colourless, 10–13 × 2.5–3.5 µm (up to 9–14 × 3–4 µm in type material), 2.5–3.5 times as long as broad. Pycnidia not seen. Photobiont Trentepohlia, cells subglobose, without characteristic chains, 9–15 µm in diameter.

CHEMISTRY. – No substances detected by HPTLC. Spot tests: (thalus cortex and medulla): K-, С-, KС- and Р-, UV-.

ECOLOGY AND DISTRIBUTION. – Until now, Coenogonium isidiatum has been reported primarily from subtropical and tropical latitudes (Figure 3), albeit primarily high elevations of mountainous regions that are unlike low elevations ecosystems at the same latitutides (e.g., the type locality in Nepal; Thor & Vězda 1984). It has been reported from diverse substrates including the bark of trees, mosses, and rocks. The previously published reports from Asia were largely based on material collected well above sea-level, ranging from approximately 1000 meters elevation (North Korea: Szerdahelyi & Lőkös 1992, Vězda 1988; South Korea: Kondratyuk et al. 2016) to between 3000 and 4300 meters elevation (China: see specimens cited herein; Thor & Vězda 1984). The report from Sri Lanka (Weerakoon & Aptroot 2014) also appears to be based on material collected from a mountainous area, although specific locality data were not provided in the publication. Collections from the New World appear to mostly be derived from low elevations (e.g.,

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Figure 3. Global distribution of Coenogonium isidiatum as presently known. Red dots = newly documented records, blue dots = records from the literature and herbaria.

U.S.A.: Seavey & Seavey 2014, from swamps near sea level; Argentina: Ferraro & Michlig 2013, from ~56 meters elevation). However, the reports from Costa Rica (Rivas Plata et al. 2006) and Brazil (Cáceres et al. 2014) did not provide specific locality data. Given the broad elevational range of the species as reported in the literature, it is noteworthy, but not necessarily surprising, that the species was found in Far Eastern Russia. Our records expand the range of the species to the boreal zone of the Northern Hemisphere (southern taiga of Sakhalin and Kurile Islands and northern taiga of the Kamchatka Peninsula). The northernmost record in Kamchatka occurs in a unique oceanic spruce forest in Central Kamchatka, which is probably a pre-glacial relict (Dirksen et al. 2013). Primeval spruce forests of the Nikolka Volcano are also refugia for the globally rare lichen Erioderma pedicellatum (Hue) P. M. Jørg. and other rare and protected stenotopic species (Vyatkina et al. 2016).

DISCUSSION. – Coenogonium isidiatum is morphologically close to C. isidiigerum (Vězda & Osorio) Lücking, Aptroot & Sipman and C. isidiosum (Breuss) Rivas Plata, Lücking, L. Umaña & Chaves. Coenogonium isidiigerum differs by the presence of a white hypothallus and in having much longer ascospores (20–24 µm vs. 10–13 µm in C. isidiatum). Coenogonium isidiosum can be distinguished by its narrower ascospores (up to 2.5–3 µm width, 4–5 times as long as broad vs. 2.5–4 µm width, 2.5–3.5 times as long as broad in C. isidiatum) and brown-yellowish apothecial disc (vs. waxy to orange red of C. isidiatum) (Rivas Plata et al. 2006, Gagarina 2015).

Additional specimens examined. – RUSSIA. SAKHALIN REGION. SAKHALIN ISLAND: vicinity of city of Yuzhno-Sakhalinsk, Mizhulsky Ridge, 47°03′01″N, 142°30′37″E, 532 m, Picea sp.- Betula sp. forest on slope, 20.v.2017, on bark of Picea sp., L. Konoreva 198 (H, LE L-14441); Tomarinsky District, protected area “Krasnogorsky”, vicinity of Lake Uglovskoe, 48°34′55″N, 141°58′38″E, 48 m, Abies sachalinensis forest with Betula sp. and Taxus cuspidata, 14.v.2017, on bark of Abies sachalinensis, L. Konoreva 102 (LE L-14440). ITURUP ISLAND: protected area “Ostrovnoy”, Stokap Volcano, Kraterny Creek, 44°51′01″N, 44°51′01″E, 122 m, rocks in valley of creek, 15.viii.2017, on mosses over rocks, L. Konoreva 606 (LE L-14436); protected area “Ostrovnoy”, Stokap Volcano, Kraterny Creek, 44°50′25″N, 147°17′44″E, 369 m, Picea sp. forest on slope, 15.viii.2017, on mosses over rocks, L. Konoreva 618 (LE L- 14437). SHIKOTAN ISLAND: Notoro Mountain, 43°46′10″N, 146°41′56″E, 232 m., rocky outcrop, 14.vi.2017, on plant debris, S. Chesnokov 167 (LE L-14439); Notoro Mountain, 43°46′13″N, 146°41′43″E, 304 m, on rocks among mosses and on soil, 14.vi.2017, S. Chesnokov 172 (LE L-14438). KAMCHATKA TERRITORY. MIL’KOVO DISTRICT: northern slope of Nikolka Mountain, 55°24′14″N, 159°49′57″E, 573 m, Picea ajanensis pristine forest with Betula ermanii, on slope, 21.viii.2016, on mossy base of old

326 spruce, I. Stepanchikova Nik-1790 (LE L-14442). BRASIL. SERGIPE: Parque dos Falcões, just E of Itabaiana, 10°44′50″S, 37°22′39″W, 400 m, transitional forest, 11.xi.2012, on tree bark, M. Cáceres & A. Aptroot 15039 (hb. Aptroot). CHINA. TIBET: Himalaya Range, 280 km SEE of Lhasa, 40 km SW of Mainling, 4300 m, Juniperus-Rhododendron forest, 12.viii.1994, on dead Juniperus bark, W. Obermayer 6072 (UPS L-153698).

ACKNOWLEDGEMENTS

We are grateful to the curators of UPS and S herbaria for permission to access their collections. We also thank A. Aptroot for providing a specimen from Brazil and the editor for significant improvement of the manuscript. The studies in the Sakhalin Region were carried out within the institutional project of the Sakhalin Branch of Botanical Garden-Institute FEB RAS “Cryptogamic biota of Pacific Asia: , biodiversity, species distribution”. The studies in Kamchatka Territory were conducted in the framework of the institutional research project (no. АААА-А18- 118031590042-0) of the Komarov Botanical Institute of the Russian Academy of Sciences “The lichen flora of the Russian Federation”. Ivan Frolov worked in the frame of the national project of the Institute Botanic Garden (Russian Academy of Sciences, Ural Branch).

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APPENDIX – MOLECULAR VOUCHER METADATA

The table below presents the specimens and GenBank accession numbers used in the molecular component of this study, together with their voucher information. New sequences generated for this study are indicated in bold.

Species Voucher; location mrSSU ITS accession accession

Absconditella lignicola Baloch et al. (2009) FJ904691 – Belonia russula Grube et al. (2004) AY648888 – Belonia russula Baloch et al. (2010) HM244735 – Coenogonium isidiatum Chesnokov LE-L-14438; Shikotan MH179140 MH179135 Coenogonium isidiatum Konoreva LE-L-14437; Iturup MH179141 MH179136 Coenogonium isidiatum Konoreva LE-L-14436; Iturup MH179142 MH179137 Coenogonium isidiatum Konoreva LE-L-14441; Sakhalin – MH179138 Coenogonium isidiatum Konoreva LE-L-14440; Sakhalin – MH179139 Coenogonium isidiatum Stepanchikova LE-L-14442; Kamchatka MH179143 – Coenogonium leprieuri Lutzoni et al. (2004) AY584698 – Coenogonium luteum Lutzoni et al. (2004) AY584699 – Coenogonium pineti Lumbsch et al. (2004) AY300884 – Coenogonium pineti Resl et al. (2015) KR017337 –

328 TABLE CONTINUED FROM PAGE 328.

Species Voucher; location mrSSU ITS accession accession

Cryptodiscus cladoniicola Pino-Bodas et al. (2017) KY661675 – Cryptodiscus cladoniicola Pino-Bodas et al. (2017) KY661674 – Cryptodiscus epicladonia Pino-Bodas et al. (2017) KY661680 – Cryptodiscus foveolaris Baloch et al. (2009) FJ904692 – Gyalecta fagicola Baloch et al. (2010) HM244753 – Gyalecta flotowii Lumbsch et al. (2004) AY300889 – Gyalecta flotowii Baloch et al. (2010) HM244740 – Gyalecta geoica Baloch et al. (2010) HM244741 – Phlyctis petraea Muscavitch et al. (2017) MF625051 – Ramonia sp. Lumbsch et al. (2004) AY300921 – radiata Lumbsch et al. (2004) AY300914 –

329