CC BY
83Contribution to the study of the genus Auricularia (Auriculariales, Basidiomycota) in Russia
V. F. Malysheva1, E. M. Bulakh2
1Komarov Botanical Institute, Prof. Popov Str., 2, St. Petersburg, 197376, Russia;
vf.malysheva@gmail.com 2Institute of Biology and Soil Science, Far East Branch of the Russian Academy of Sciences, 100-letiya Vladivostoka Prospect, 159, Vladivostok, 690022, Russia;
bulakh@ibss.dvo.ru
Abstract. The paper covers the study of the genus Auricularia in Russia. The taxonomic history of the genus and problems of its morphological systematics are described in details. The evolutionary relationships of species and species complexes were revealed based on a comparative study of the nucleotide sequences of nrDNA (ITS, rpb2). The occurrence of A. auricula-judae only in European of Russia part was confirmed by molecular data, whereas the morphologically similar species A. americana had the Far Eastern distribution in Russia. In total, 7 taxa of Auricularia have so far been recorded from Russia: known species A. mesenterica, A. minor, A. cornea, A. americana, A. auricula-judae, newly described species — A. villosula and unde-scribed Auricularia sp. An identification key to the studied Auricularia species and its morphological descriptions are also provided.
Keywords: Auricularia, molecular taxonomy, morphology, phylogeny, Russia, new species, Auricularia villosula.
К изучению рода Auricularia (Auriculariales, Basidiomycota)
в России
В. Ф. Малышева1, Е. М. Булах2
ботанический институт им. В. Л. Комарова РАН, ул. Профессора Попова, д. 2,
Санкт-Петербург, 197376, Россия; vf.malysheva@gmail.com 2Биолого-почвенный институт ДВО РАН, пр. 100-летия Владивостока, д. 159, Владивосток, 690022, Россия; bulakh@ibss.dvo.ru
Резюме. Проведено исследование представителей рода Auricularia в России. Представлен очерк таксономической истории рода, рассмотрены проблемы его морфологической систематики. На основе сравнительного изучения нуклеотид-ных последовательностей ДНК (ITS, rpb2) изучены взаимоотношения между видами и видовыми комплексами. Выяснено, что A. auricula-judae имеет лишь европейское распространение, а на Дальнем Востоке обитает морфологически сходный вид — A. americana. Исследование показало, что на территории России встречается семь таксонов Auricularia: A. mesenterica, A. minor, A. cornea, A. americana, A. auricula-judae, описываемый в статье как новый для науки A. villosula, а также неописанный вид Auricularia sp. Для всех видов приводятся подробные морфологические описания и ключ для их определения.
Ключевые слова: Auricularia, молекулярная таксономия, морфология, филогения, Россия, новый вид, Auricularia villosula.
Introduction
Auricularia Bull. is a cosmopolitan genus, consisting of nearly 10-15 described and accepted species (Lowy, 1952; Kobayasi, 1981; Kirk et al., 2008; Looney et al, 2013). Most species of this genus exhibit tropical and subtropical distribution at forest communities, and only several species inhabit temperate zone of the Northern hemisphere. All species have saprotrophic lifestyle on wood debris.
The genus was described by P. Bulliard (1785). Since 1785 more than one hundred species have been recognized in it. Currently most of them were considered as dubious species. Such species are not accepted or have been already reduced to the synonyms. Up to now Auricularia is considered to be a rather complicated taxonomic group, despite of its proven monophyletic status (Weiss, Oberwinkler, 2001). The problems with infrageneric taxonomy are based on high variability of morphological characters and scarcity of microstructural features.
The hyphal scheme of basidiome proposed by B. Lowy (1951) and comprising several characters, such as internal stratification of hyphae, abhymenial hair length and the presence/absence of differentiated medulla, has always been considered as the most reliable criterion for classification systems in Auricularia. There were several attempts to subdivide the genus into some sections at the base of these specifications (Donk, 1952; Kobayasi, 1981). However, the numerous accumulated data and special works on the investigation of the genus have shown that internal structure of basidiome, the width of hyphal zones especially, is too variable due to the environmental conditions, and the age of the basidiome as well as the time of tissue exposure in KOH (Looney et al., 2013). The mating tests, ecology, geographical patterns and molecular data could serve as additional criteria to tell the species apart in this genus. As a consequence of the taxonomic characters instability, the number of species within the genus Auricularia remains unclear.
Before our study the exact number of Auricularia species occurring in Russia was not known. This number was inconsistent depending on the data of different authors (Raitviir, 1967; Govorova, 1997). Most of them were known from the Russian Far East.
The aim of this study was to investigate the phylogenetic relationships among the species of the genus Auricularia in Russia and to verify collections from different localities, using both morphological study and molecular genetic techniques.
Materials and Methods
Morphological analysis
Macromorphological descriptions are based on fresh specimens, herbarium collections from the Mycological Herbarium of the Komarov Botanical Institute (LE, Saint Petersburg) and Institute of Biology and Soil Science (VLA, Vladivostok) as well as color photos of basidiomata. Mi-cromorphological characters were studied from cross-sections of dried basidiomata mounted in 5 % KOH. Cross-sections were photographed and measured at 10*, abhymmenial hairs at 10* and 20*, and spores and basidia at 40* using Zeiss AxiolmagerAl light microscope.
Molecular techniques
DNA was isolated from dried specimens. The data from GenBank (http://www.ncbi.nlm.nih.gov/Genbank/) were used as additional information source for comparative study.
The procedure of DNA extraction completely corresponded to the manufacture protocol of the AxyPrep Multisource Genomic DNA Mini-prep Kit (Axygen Biosciences, California, USA). The primer pairs ITS1F-ITS4B (http://www.biology.duke.edu/fungi/mycolab/primers.htm) were used both for PCR and for sequencing of ITS region. For amplification and sequencing of rpb2 locus (a single-copy nuclear protein-coding gene) the primers Aur-6F and Aur-7R (Looney et al., 2013) were used.
PCR products were purified using the Fermentas Genomic DNA Purification Kit (Thermo Scientific). Sequencing was performed with an ABI model 3130 Genetic Analyzer (Applied Biosystems) using the BigDyeTM Terminator Cycle Sequencing Ready Reaction Kit (AB). Raw data was processed using MEGA 6 (Tamura et al, 2013). The sequences were aligned with the web tool MAFFT (http://mafft.cbrc.jp/alignment/server/).
Phylogenetic reconstructions were performed with maximum likelihood (ML), maximum parsimony (MP) and Bayesian analysis (BA). The outgroups used for analysis of ITS data set included Exidia recisa (Dit-mar) Fr. and Eichleriella leveilleana (Berk. and M. A. Curtis) Burt, which have all been shown to be closely related species nonetheless locating outside the ingroup within the Auriculariales (Weiss, Oberwinkler, 2001; Looney et al., 2013). The outgroup used for analysis of rpb2 data set included Ramaria rubella (Schaeff.) R. H. Petersen.
Maximum likelihood analysis was run in RAxML servers, v.7.2.8 (http://phylobench.vital-it.ch/raxml-bb/index.php), with one hundred rapid bootstrap replicates. Only the clades with a support > 65 % were considered to be a strong support.
Maximum parsimony was performed using PAUP*4.0.b10 (Swof-ford, 2002) with a heuristic search option. Characters were unordered and
weighted equally; the number of trees retained was limited to 100 (for ITS); starting trees were obtained by stepwise addition with random sequence addition and tree bisection-reconnection (TBR) branch-swapping algorithm; one tree was held at each step during stepwise addition. Parsimony bootstrap analysis was performed with 1000 replicates. Only clades with a support > 55 % were retained for further analysis. Gaps were treated as missing characters.
Table
Sequences used in molecular phylogenetic analysis
No Species GenBank ITS GenBank RPB2 Country Herbarium Number
1 Auricularia americana JX065146 JX065180 USA, Tennessee
2 A. americana JX065163 JX065178 USA, Tennessee
3 A. americana JX065152 JX065182 USA, North Carolina
4 A. americana JX065151 JX065181 USA, Tennessee
5 A. americana JX065166 JX065179 USA, Tennessee
6 A. americana JX065154 JX065177 USA, Idaho
7 A. americana DQ200918 DQ366278 USA, Oregon
8 A. americana KJ698429 KJ698438 Russia, Far East LE 296428
9 A. americana KJ698431 KJ698439 Russia, Far East VLA M-11352
10 A. auricula-judae AF291268
11 A. auricula-judae DQ520099 DQ521419 Germany
12 A. auricula-judae JX065174
13 A. auricula-judae KJ698425 Russia, Western Caucasus LE 254030
14 A. auricula-judae KJ698426 Russia, Western Caucasus LE 254071
15 A. cornea HM448462
16 A. cornea FJ617292
17 A. cornea HM448454
18 A. cornea JX065164 JX065185 Australia
19 A. cornea FJ792587
20 A. cornea FJ617297
21 A. cornea FJ617302
22 A. cornea KJ698435 Laos LE 269791
23 A. cornea KJ698430 Russia, Far East LE 296425
24 A. cornea KJ698432 Russia, Far East VLA M-12.828
25 A. cornea KJ698424 Russia, Far East LE 262938
26 A. cornea KJ698433 Russia, Far East VLA M-11322
27 A. cornea KJ698427 Russia, Far East LE 262998
28 A. cornea KJ698428 Russia, Far East VLA M-18.531
Table (continued)
No Species GenBank ITS GenBank RPB2 Country Herbarium Number
29 A. cornea KJ698437 Russia, Far East VLA M-15691
30 A.fuscosuccinea JX065153
31 A. fuscosuccinea JX065157 JX065190 USA, Louisiana
32 A. fuscosuccinea JX065139
33 A. fuscosuccinea JX065140
34 A. fuscosuccinea JX065141 JX065193 Argentina
35 A. fuscosuccinea JX065184 Costa Rica
36 A. mesenterica AF291271
37 A. mesenterica KJ698436 Russia, Samara Region LE 254558
38 A. minor KJ698434 Russia, Far East LE 296424
39 A. nigricans JX065176
40 A. nigricans JX065172
41 A. nigricans JX065167
42 Auricularia sp. EU560401
43 Auricularia sp. EU560406
44 Auricularia sp. EU647879
45 Auricularia sp. AB615232
46 Auricularia sp. HQ388358
47 Auricularia sp. KJ698421 Russia, Republic of Buryatia LE 262806
48 Auricularia sp. HQ388355
49 Auricularia sp. KJ698423 Russia, Far East LE 296433
50 Auricularia sp. KJ698422 Russia, Far East LE 296423
51 Auricularia sp. FJ478123
52 A. villosula KJ698417 KJ698440 Russia, Far East VLA M-11291
53 A. villosula KJ698418 KJ698441 Russia, Far East LE 296422
54 A. villosula KJ698419 Russia, Far East VLA M-11348
55 A. villosula KJ698420 Russia, Far East LE 262989
56 Eichleriella leveilleana AY509553
57 Exidia recisa AF291276
58 Ramaria rubella AY786064
Note. Entries in bold are the sequences newly generated for this study.
Bayesian analysis was performed using MrBayes 3.1 (Ronquist, Huel-senbeck, 2003) software for two independent runs, each with 1 500 000 generations with sampling every 100 generations, with GTR model (for ITS) and four chains. Posterior probability (PP) value > 0.95 is considered as significant.
The evolutionary distance between sequences was assessed using MEGA 6 with substitution model Kimura 2-parameter.
All ITS and rpb2 sequences have been deposited in GenBank; their accession numbers are given in Table.
Results
The ITS data set consists of 55 Auricularia sequences and 2 sequences of an outgroup. The rpb2 data set consists of 16 Auricularia sequences and 1 sequence of an outgroup. For this study we produced twenty new ITS and five new rpb2 sequences. The final ITS alignment contained 574 characters (with gaps), of which 125 were parsimony informative. In the MP analysis 100 MPT were recovered (Tree Length = 427; CI = 0.6183; RI = 0.8787; RC = 0.5433). The final rpb2 alignment contained 580 characters (with gaps), of which 131 were parsimony informative. In the MP analysis 8 MPT were recovered (Tree Length = 363; CI = 0.7879; RI = 0.8127; RC = 0.6403).
The phylogenetic studies resulted in two tree-topologies for ITS and rpb2 data sets (Figs. 1b, 2). The topologies yielded from all analyses (MP, ML and BA) were the same. Eight/nine major monophyletic clades with high support were recovered in ITS tree. The terminal groupings did not vary in the analyses of the different genetic markers. Additional analysis of rpb2 region showed the same monophyletic clades in relation to the species involved.
All recovered phylogenetic lineages are characterized by distinctive morphological features (presence/absence medulla, spore size, length of abhymenial hairs).
Two ITS sequences of Auricularia obtained from the Russian Far East collections (LE 296428 and VLA M-11352) appeared to belong to A. americana-clade. The pairwise distance between sequences within this clade was 2.3 % that corresponded average level of intraspecific ITS variability in Basidiomycota (Nilsson et al, 2008).
Four Russian Far East collections (VLA M-11348, VLA M-11291, LE 296422, LE 262989) formed distinctly separate high supported clade based on both ITS and rpb2 data sets. These collections were characterized by unique morphological features sufficient for describing a new species — Auricularia villosula (description is given below in taxonomi-cal part).
All specimens with strongly pubescent basidiomata were nested in one clade — A. cornea. The pairwise distance between ITS sequences within this clade reaches 1.3 % that allows us to consider all members of this group as a single species.
Fig. 1. Phylogenetic relationships of Auricularia species based on dataset of
nrITS sequences.
a — Part of MP Bootstrap 50 % majority-rule consensus tree showing separate position of Auricularia minor (LE 296424); b — Tree inferred from ML analysis. Bootstrap values from Maximum Parsimony, Maximum Likelihood analyses > 55 % and Posterior probability from Bayesian analysis > 0.95 are shown over the branches (MP/ML/BA). Collections are indicated with the herbarium number (for Russian material) and GenBank accession number.
r/h
A americana JX065I79 A, americana JX065I78 A. americana jxossiso A americana JX0653K2 A. americana JX06518! I A. americana JX065I77 I A. americana DQ366278 A. americana LE 296428 A. americana VLAM-M352
-A. auricula-judae DQ52I419
A. cornea VLAM-15691 ¡A. villosulaVLAM-11291
\A. vUtosuta LE 296422
IA. fiiscosuccinea JXn65184 A. fuscosuccinea JX065193 A. fuscosttccinea JX0S5190 -Rumana rubella AY786064
Fig. 2. Result of ML-analysis, showing phylogenetic relationships of Auricularia species based on RPB2 dataset. Bootstrap values from Maximum Parsimony, Maximum Likelihood analyses > 55 % and Posterior probability from Bayesian analysis > 0.95 are shown over the branches (MP/ML/BA). Collections are indicated with the herbarium number (for Russian material) and GenBank accession number.
Some collections from the Russian Far East morphologically similar to A. auricula-judae (Bull.) Quel. were clustered in single clade 'Auricularia sp.'. The pairwise distance between ITS sequences within this clade was only 1.8 %.
The independent taxonomical position of A. minor Kobayasi was reliably confirmed with MP analysis (Fig. 1a). On ML tree this species formed a rather long branch. The phylogenetic distance between A. minor and 'Auricularia sp.' clade was more than 2 %.
The European representatives of A. auricula-judae were characterized by isolated position from Asian species group «A. americana — A. villo-sula — Auricularia sp.» inferred from the topologies.
Seven species of Auricularia (A. mesenterica (Dicks.) Pers., A. minor Kobayasi, A. cornea Ehrenb., A. americana Parmasto et I. Parmasto ex Audet, Boulet et Sirard, A. auricula-judae (Bull.) Quél., described below A. villosula Malysheva and Auricularia sp.) were identified from the specimens collected in Russia.
Key to the species of the genus Auricularia distributed in Russia
1. Hymenium merulioid and strongly lobed...................................... A. mesenterica.
— Hymenium mostly smooth or with occasional folds ........................................... 2.
2. Cortical layer hirsute with hairs 150-500 pm long................................A. cornea.
— Cortical layer velutinous to pubescent with hairs 50-200 pm long.................... З.
3. Abhymenial hairs up to S0 pm long.................................................................... 4.
— Abhymenial hairs up to 200 pm long.................................................................. 5.
4. Basidiospores up to 15 pm long. Medulla absent................................A. villosula.
— Basidiospores up to 11 pm long. Medulla present ...................................A. minor.
5. Medulla present ..............................................................................Auricularia sp.
— Medulla absent or weakly differentiated ............................................................. б.
6. Distributed in the Russian Far East .................................................. A. americana.
— Distributed in European part of Russia .....................................A. auricula-judae.
Auricularia mesenterica (Dicks. : Fr.) Pers., 1822, Mycol. Eur. 1: 97 (Plate II, 5).
= Helvella mesenterica Dicks., 1785, Fasc. pl. crypt. brit. 1: 20.
= Auricularia corrugata Sowerby, 1800, Col. fig. Engl. Fung. Mushr. 3: pl. 290.
= Auricularia lobata Sommerf., 1826, Mag. Naturvididensk. 6: 295.
Basidiomata effuse-reflexed, resupinate with free lobed and undulate margin, coalescing, mature specimens 2-5 mm thick and up to 15 cm wide; consistency firm-gelatinous, rubbery; superior surface distinctly concentrically zonate, densely pilose, folded, grey-ochre to brown; ab-hymenial hairs mattered or clustered, hyaline to pigmented, thick-walled, 700-1200 x 2-5 ^m; inferior hymenial surface smooth or veined, light-brown to purple-brown, occasionally with lilac tint.
Medulla absent. Internal hyphae 2-6 ^m wide, hyaline, with clamp connections. Basidia cylindrical, 45-60 x 3-5 ^m, with three septa. Basidiospores allantoid, hyaline, thin-walled, 12-15 x 5-6 ^m.
Habitat: on wood of deciduous trees.
General distribution: cosmopolitan (America, Eurasia, Africa, Australia).
Specimens examined: Russia, Leningrad Region, Volkhov District, broadleaf forest, on fallen trunk of Ulmus glabra, 15.09.1999, A. E. Ko-valenko, LE 208569; Samara Region, Stavropolsky District, Zhigulevsky
Nature Reserve, Shiryaevskaya valley, on a fallen trunk of deciduous tree, 17.05.2004, N. V. Psurtseva, LE 254558.
Note: This taxon is easily recognized at the base of its morphology (concentrically zonate pilose basidiome surface, veined hymenium). It cannot be confused with other species of Auricularia.
Auricularia cornea Ehrenb. : Fr., 1820, in Nees von Esenbeck (ed.), Horae Phys. Berol.: 91 (Plate I, 6, 8).
= Exidia cornea (Ehrenb. : Fr.) Fr., 1822, Syst. mycol. 2(1): 222.
Basidiomata sessile or substipitate, solitary or caespitose, excentric, infundibuliform, saucer-shaped, with undulate margin, mature specimens 0.8-2.0 mm thick and up to 10 cm wide; consistency firm-gelatinous; when dry, thin, opaque and brittle; superior surface pilose, pale ochraceous, yellow-green, olive or pinkish when fresh; abhymenial hairs dense, often aggregate in short tufts, hyaline to pigmented, thick-walled, 150-500 * 4.3-7.8 ^m; inferior hymenial surface smooth, red-brown to dark brown.
Medulla present. Internal hyphae hyaline, with clamp connections. Basidia clavate, 45-55 * 4-5 ^m, with three septa. Basidiospores allan-toid, hyaline, thin-walled, 11.5-14.0 * 5.0-6.5 ^m.
Habitat: on wood of deciduous and coniferous trees.
General distribution: Tropical America, Africa, Eastern Asia (including Russian Far East), Australia.
Specimens examined: Russia, Far East, Primorye Territory, Verkh-neussuriiskiy Stationary, on the branch of Sambucus sp., 10.08.1990, E. M. Bulakh, VLA M-11322; Marine Reserve, Gildebrand Island, on the branch of Sambucus sp., 03.06.1999, Bulakh, VLA M-12.828; Sakhalin Region, Moneron Island, on a fallen tree, 05.08.2001, V. Yu. Barkalov, VLA M-15691; Nadezhdinsky District, on fallen deciduous tree, 01.09.2002, Barkalov, VLA M-18.531; Ussuriysky Nature Reserve, Pei-shula, flood plain of Suvorovka River, mixed forest (with Pinus koraien-sis, Phellodendron amurense, Chosenia arbutifolia), on a fallen deciduous tree, 12.08.2011, V. F. Malysheva, LE 262998; Ussuriysky Nature Reserve, Peishula, flood plain of Koryavaya River, mixed forest (with Pinus koraiensis, Phellodendron amurense, Chosenia arbutifolia), on a fallen deciduous tree, 16.08.2011, Malysheva, LE 262938; Sikhote-Alin Nature Reserve, Nechet, cedar forest with fir and deciduous trees, on a fallen deciduous tree, 16.08.2012, M. N. Gromyko, LE 296425. Laos, «Tat Kuangi Sl» National Park, tropical forest, on fallen wood, 01.30.2013, O. N. Ezhov, LE 269791.
Note: A. cornea is somewhat similar in appearance to A. nigricans. It differs from A. nigricans by less pilose surface. The most distinctive feature of A. cornea is olivaceous tint of its basidiomata.
Some collections previously doubtfully recognized as A. polytricha (Mont.) Sacc. and A. papyracea Yasuda appeared to belong to A. cornea, that was concluded from our detailed examination of their morphology and molecular data. The absence of these species at the territory of Russia was confirmed by our study.
Auricularia villosula Malysheva, sp. nov. (Plate II, 2, 4).
MycoBank: MB 808546
Recognized by yellow-brown gelatinous basidiomata, very short abhymenial hairs and the absence of medulla.
Etymology: Refers to weak pubescence of the basidiome surface.
Holotype: Russia, Far East, Primorye Territory, Shkotovsky district, Bolshoy Kamen vicinities, 20.06.1991, A. G. Mikulin, LE 296422.
Basidiomata substipitate to sessile, attached to substrate by central point, solitary or caespitose, discoid or saucer-shaped, with free lobed and undulate margin, mature specimens 1-2 mm thick and up to 5 cm wide; consistency gelatinous; thin, opaque and brittle when dry; superior surface velutinous, flat or venose, yellow-brown or reddish brown when fresh, sometimes yellow-green when dry; abhymenial hairs 30-70 x 5.06.0 ^m, irregular, hyaline, thick-walled; inferior hymenial surface glabrous, pale brown to dark brown with greenish tint.
Medulla absent. Internal hyphae thin-walled, hyaline, with clamp connections. Basidia cylindrical, 40-60 x 4.0-5.0 pm, with three septa. Basidiospores allantoid, hyaline, thin-walled, 11.7-15.3 x 4.8-6.3 pm.
Habitat: on wood of deciduous trees.
General distribution: so far known only from the Russian Far East.
Specimens examined: Russia, Far East, Primorye Territory, Us-suriyskiy Nature Reserve, broadleaf forest, on a fallen deciduous tree, 03.07.1990, O. K. Govorova, VLA M-11348; Ussuriysky Nature Reserve, mixed forest, on a fallen trunk, 30.07.1992, Govorova, VLA M-11291; Ussuriyskiy Nature Reserve, vic. Peishula Reserve station, flood plain of Suvorovka River, mixed forest (with Pinus koraiensis, Phellodendron amurense, Chosenia arbutifolia), on a fallen deciduous tree, 15.08.2011, Malysheva, LE 262989.
Note: The new species is morphologically similar to Auricularia sp., A. auricula-judae and A. americana. It differs from Auricularia sp. by the absence of medulla and from other species by having very short abhymenial hairs (up to 70 pm long).
Auricularia minor Kobayasi, 1981, Bull. Natn. Sci. Mus., Tokyo, B 7(2): 50 (Plate I, 5, 7).
Basidiomata sessile or substipitate, solitary or caespitose, excentric, infundibuliform, saucer-shaped or earlike, with undulate margin, mature basidiomata 0.5-2.0 cm in diameter; consistency firm-gelatinous; when dry, thin, opaque and brittle; superior surface smooth or sometimes
venose, pilose, yellow-brown to dark olivaceous when fresh and yellow-green when dry; abhymenial hairs 50-80 x 4.5-6.5 pm, irregular, hyaline, thick-walled, without central strand; inferior hymenial surface glabrous or slightly folded, pale brown to dark brown.
Medulla present, central. Internal hyphae thin-walled, hyaline, with clamp connections. Basidia cylindrical, 40-60 x 3.5-5.0 pm, with three septa. Basidiospores allantoid, hyaline, thin-walled, 7.0-11.0 x 4.0-5.0 pm.
Habitat: on wood of deciduous trees.
General distribution: Eastern Asia (Russian Far East, Japan, Borneo, New Guinea).
Specimen examined: Russia, Far East, Primorye Territory, Kedrovaya Pad' Reserve, lowland forest, 10.10.1994, Govorova, LE 296424.
Note: This species is characterized by small basidiomata, short abhymenial hairs, small basidiospores and the presence of medulla.
Auricularia sp. (Plate II, 1, 3).
Basidiomata sessile or substipitate, solitary or caespitose, excentric, infundibuliform, saucer-shaped or earlike, with undulate margin, mature specimens 0.8-1.5 mm thick and up to 12 cm wide; consistency firm-gelatinous; when dry, thin, opaque and brittle; superior surface smooth or sometimes venose, pilose, yellow-brown to reddish brown when fresh and yellow-green when dry; abhymenial hairs 60-200 x 4.5-6.5 pm, irregular, hyaline, thick-walled, without central strand; inferior hymenial surface glabrous or slightly folded, pale brown to dark brown.
Medulla present, central. Internal hyphae thin-walled, hyaline, with clamp connections. Basidia cylindrical, 40-60 x 3.5-5.0 pm, with three septa. Basidiospores allantoid, hyaline, thin-walled, 11.5-16.3 x 4.2-6.0 pm.
Habitat: on wood of deciduous trees.
General distribution: Eastern Asia (China, Japan, Russian Far East), Siberia.
Specimens examined: Russia, Far East, Primorye Territory, Sik-hote-Alin Nature Reserve, Ust'-Serebryany, flood plain forest, on fallen trunk, 04.09.1996, O. V. Morozova, LE 296433; Khankaisky district, Novo-Kachalinsk, broadleaf forest, on a fallen trunk of Quercus sp., 26.07.1996, A. S. Leley, LE 296423; Republic of Buryatia, Kabansky district, flood plain of Vydrina River, on a fallen trunk of Populus sp., 09.08.1998, Morozova, LE 262806.
Note: It is close to A. auricula-judae and A. americana but differs from both by having the medulla and from the first by different geographical distribution.
Auricularia americana Parmasto et I. Parmasto ex Audet, Boulet et Sirard, 2003, in Boulet, Les Champignons des Arbres de l'Est de l'Amérique du Nord: 287 (Plate I, 1, 3).
= Auricularia americana Parmasto et I. Parmasto, 19S7, Bibl. Mycol. 115: 137.
Basidiomata sessile or substipitate, solitary or caespitose, saucer-shaped or earlike, with lobed and undulate margin, mature specimens 1-4 mm thick and up to S cm wide; consistency firm-gelatinous; when dry, thin, opaque and brittle; superior surface velutinous to pubescent, yellow-brown to reddish brown when fresh, sometimes yellow-green when dry; abhymenial hairs 60-120 x 4.5-5.0 pm, irregular, hyaline, thick-walled; inferior hymenial surface, glabrous or slightly folded, pale brown to dark brown.
Medulla absent. Internal hyphae thin-walled, hyaline, with clamp connections. Basidia cylindrical, 41-74 x 3.0-5.0 pm, with three septa. Basidiospores allantoid, hyaline, thin-walled, 12.5-16.4 x 5.0-6.4 pm.
Habitat: on wood of coniferous trees.
General distribution: North America (USA, Canada), Eastern Asia (Russian Far East).
Specimen examined: Russia, Far East, Primorye Territory, Sikhote-Alin Nature Reserve, Nechet, cedar forest with fir and deciduous trees, on a fallen branch of Pinus koraiensis, 16.0S.2012, Gromyko, LE 29642S; Amur Region, Khingansky Reserve, Kundur, flood plain of Karancha River, mixed forest, 10.10.1991, Mikulin, VLA M-11352.
Note: Morphologically similar A. auricula-judae is characterized by slightly larger basidiospores (15.0-17.5 x 4.6-7.0 pm), habitat on deciduous wood and European distribution. Another similar species Auricularia sp. has a distinct medulla.
Auricularia auricula-judae (Bull.) Quél., 1SS6, Enchir. fung. (Paris): 207 (Plate I, 2, 4).
= Tremella auricula-judae Bull., 17S9, Herb. Fr. 9: tab. 427, fig. 2.
= Auricularia sambuci Pers., 1S22, Mycol. eur. (Erlanga) 1: 97.
= Auricularia auricula (L.) Underw., 1902, Mem. Torrey bot. Club, 12: 15.
= Auricularia lactea (Quél.) Bigeard et H. Guill., 1913, Fl. Champ. Supér. France, 2.
= Auricularia auricularis (Gray) G. W. Martin, 1943, Am. Midl. Nat. 30: S1.
Basidiomata sessile or substipitate, solitary or caespitose, excentric, infundibuliform, saucer-shaped or earlike, with undulate margin, mature specimens 0.S-1.5 mm thick and up to 12 cm wide; consistency firm-gelatinous; thin, opaque and brittle when dry; superior surface smooth or sometimes venose, pilose, yellow-brown to reddish brown when fresh and yellow-green when dry; abhymenial hairs 60-200 x 4.5-6.5 pm, irregular, hyaline, thick-walled, without central strand; inferior hymenial surface, glabrous or slightly folded, pale brown to dark brown.
Medulla absent. Internal hyphae thin-walled, hyaline, with clamp connections. Basidia cylindrical, 50-60 x 5-6 pm, with three septa. Basidiospores allantoid, hyaline, thin-walled, 15.0-17.5 x 4.6-7.0 pm.
Habitat: on wood of deciduous trees.
General distribution: temperate regions of Europe (including European part of Russia and Caucasus).
Specimens examined: Russia, Western Caucasus, Karachaevo-Cher-kesia, Teberda State Biosphere Reserve, Arkhyz site, Bukovo vicinities, fagus forest, on a fallen branch of Fraxinus sp., 22.08.2009, Malysheva, LE 254030; Teberda State Biosphere Reserve, vicinities of Teberda town, fagus forest, on a trunk of Euonymus sp., 10.08.2009, Malysheva, LE 254071.
Note: It can be distinguished from A. americana by larger basidiospores, habitat and geographical distribution. It differs from Auricularia sp. by the absence of medulla.
Discussion
Recent extensive treatments of Auricularia in North America (Mon-toya-Alvarez et al, 2011; Looney et al, 2013) and China (Li et al, 2014) have helped to verify in some extent the distribution and phylogenetic relationships between species, but they left open the question of taxo-nomical status of Asian collections of A. auricula-judae. In these studies conducted by A. F. Montoya-Alvarez et al. (2011) and B. P. Looney et al. (2013) the investigated Chinese and Japanese collections of A. auric-ula-judae were shown to be clearly distinct from European clade (these are 'clade I' and 'clade II' in Looney et al., 2013). However, the detailed morphological studies of these East Asian collections have not been implemented by these authors. The morphological descriptions given for A. auricula-judae in the paper of Montoya-Alvarez and co-authors were based on collections from Colombia only, which were not involved into phylogenetic analyses and could belong to A. americana.
In our phylogenetic study the collections from European Russia were shown to be nested inside A. auricula-judae clade containing also collections from Germany and Switzerland. The collections from the Russian Far East and Siberia morphologically similar to A. auricula-judae appeared to be appertaining to two different clades corresponding to A. americana (on coniferous wood) and Auricularia sp. This pattern has been confirmed by two loci. The main distinguishing character of all Auricularia sp. specimens is the presence of medulla. In the previously cited work (Looney et al, 2013) the authors designated this clade, with specimens identical to our Auricularia sp. as A. auricula-judae 'clade II' comprising specimens from East Asia (China, Japan). The taxon has been
left undescribed in that work, because the authors have not examined any specimens that correspond to this clade. Given that the holotype of A. auricula-judae originates from Europe, B. P. Looney et al. (2013) refer to the Asian group as 'clade II'. In the present study we investigated thoroughly the morphology of the representatives of this clade from Siberia and the Russian Far East. There were no morphological differences between Auricularia sp., A. auricula-judae and A. americana with exception of medulla. Due to instability of such feature as the presence/absence of a medullar zone, we do not describe a new species herein, suggesting the taxon is left as a phylogenetic unit only without any supporting additional morphological characters.
The specimens with short abhymenial hairs of basidiomata and distributed at the Far East formed well supported clade in both ITS and rpb2 topologies (Figs 1, 2). We consider this clade as a new species A. villosula.
The representative material of two species, A. nigricans (Fr. : Fr.) Birkebak, Looney et Sánchez-García and A. cornea, has been analyzed for clarifying phylogenetic position of taxa, characterized by strong pubescence of basidiomata surface, in the general Auricularia phylogeny. All examined collections with strongly pubescent basidiomata appeared to be A. cornea. Noteworthy, some of these specimens were labeled initially as A. tenuis (Lév.) Farl. The problem of synonymy of A. tenuis to A. cornea has not been resolved yet. Possibly, the assumption of G. J. Wong and K. Wells (1987) about the synonymy of A. cornea to A. tenuis based on interfertility of both taxa may be true. Epitypification of A. cornea as well as thorough study of A. tenuis new collections from its type locality Borneo are needed to verify this conclusion.
The analysis of the Russian material showed the presence of some phylogeographic discrepancies within a single, morphologically uniform species complex A. auricula-judae. The Asian collections, including Far Eastern specimens, are represented by rather different taxa. Further studies with more sampling of specimens from Asian territory (Russia, China, Korea, Japan) could help to clarify taxonomical designations for these collections.
Acknowledgements
We are very grateful to Dr. Elena Voronina (Lomonosov Moscow State University) for editing English and helpful comments. This study was supported by the Russian Foundation for Basic Research (projects №12-04-33018 and №13-04-00838).
References
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Looney B. P., Birkebak J. M., Matheny P. B. 2013. Systematics of the genus Auricularia with an emphasis on species from the southeastern United States. North American Fungi. 8(6): 1-25.
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Montoya-Alvarez A. F., Hayakawa H., Minamya Y., Fukuda T. 2011. Phylogenetic relationships and review of the species of Auricularia (Fungi: Basidiomycetes) in Colombia. Caldasia. 33(1): 55-66.
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Литература
Bulliard P. 1785. Herbier de la France ou collection complette des plantes indigènes de ce royaume; avec leurs détails anatomiques, leurs propriétés, et leurs usages en médecine. Paris: Tab. 290.
[Govorova] Говорова О. К. 1997. Гетеробазидиальные грибы заповедников Дальнего Востока России. Микология и фитопатология. 31(3): 10-13.
Donk M. A. 1952. On the genera Auricularia, Hirneola and Laschia. Reinwardtia. 1: 487-500.
Kirk P. M., Cannon P. F., Minter D. W., Stalpers J. A. (eds.). 2008. Dictionary of the Fungi. 10th ed. UK: 771 p.
Kobayasi Y. 1981. The genus Auricularia. Bull. Natl. Sci. Mus. 7: 41-67.
Li L., Zhong C.-H., Bian Y.-B. 2014. The molecular diversity analysis of Auricularia auricula-judae in China by nuclear ribosomal DNA intergenic spacer. Electronic J. Biotechnol. 17(1): 27-33.
Looney B. P., Birkebak J. M., Matheny P. B. 2013. Systematics of the genus Auricularia with an emphasis on species from the southeastern United States. North American Fungi. 8(6): 1-25.
Lowy B. 1951. A morphological basis for classifying the species of Auricularia. Myco-logia. 43: 351-358.
Lowy B. 1952. The genus Auricularia. Mycologia. 44: 656-692.
Montoya-Alvarez A. F., Hayakawa H., Minamya Y., Fukuda T. 2011. Phylogenetic relationships and review of the species of Auricularia (Fungi: Basidiomycetes) in Colombia. Caldasia. 33(1): 55-66.
Nilsson R. H., Kristiansson E., Ryberg M., Hallenberg N., Larsson K.-H. 2008. In-fraspecific ITS variability in the kingdom Fungi as expressed in the International sequence databases and its implications for molecular species identification. Evolutionary Bioinformatics. 4: 193-201.
[Raitviir] Райтвийр А. Г. 1967. Определитель гетеробазидиальных грибов (Hetero-basidiomycetidae) СССР. Л.: 114 с.
Ronquist F., Huelsenbeck J. P. 2003. MRBAYES 3: Bayesian phylogenetic inference under mixed models. Bioinformatics. 19: 1572-1574.
Swofford D. L. 2002. PAUP*. Phylogenetic Analysis Using Parsimony (*and other methods). Version 4.0b10. Sunderland: 144 p.
Tamura K., Stecher G., Peterson D., Filipski A., Kumar S. 2013. MEGA6: Molecular Evolutionary Genetics Analysis Version 6.0. Molec. Biol. Evol. 30: 2725-2729.
Weiss M., Oberwinkler F. 2001. Phylogenetic relationships in Auriculariales and related groups — hypotheses derived from nuclear ribosomal DNA sequences. Mycol. Res. 105: 403-415.
Wong G. J., Wells K. 1987. Comparative morphology, compatibility, and interfertility of Auricularia cornea, A. polytricha, and A. tenuis. Mycologia. 79: 847-856.
Plate I. Basidiomata and cross-sections of Auricularia species. 1 — basidiomata of A. americana (LE 296428); 2 — basidiome of A. auricula-judae (LE 254030; 3 — cross-section of A. americana without medulla (VLA M-11352); 4 — cross-section of A. auricula-judae without medulla (LE 254071); 5 — basidiomata of A. minor (LE 296424); 6 — basidiomata of A. cornea (LE 296425); 7 — cross-section of A. minor with unclear medulla (LE 296424); S — cross-section of A. cornea with medulla (VLA M-12.828). Scale bars: 1, 2, 6 — 2 cm; 3, 4, 7, S — 200 pm; 5 — 0.5 cm.
Plate II. Basidiomata and cross-sections of Auricularia species. 1 — basidiomata of Auricularia sp. (LE 262806); 2 — basidiomata of A. villosula (LE 262989); 3 — cross-section of Auricularia sp. with medulla (LE 262806); 4 — cross-section of A. villosula with short abhymenial hairs and without medulla (VLA M-11291); 5 — basidiomata of A. mesenterica (LE 254558). Scale bars: 1, 2 — 1 cm; 3, 4 — 200 ^m; 5 — 2 cm.
