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Two new bryophilous agarics from India
m y c o s c i e n c e x x x ( 2 0 1 4 ) 1 e6
Available online at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/myc
Full paper
Two new bryophilous agarics from India K. P. Deepna Latha, K.N. Anil Raj, Raihana Paramban, Patinjareveettil Manimohan* Department of Botany, University of Calicut, Kerala 673 635, India
article info
abstract
Article history:
Two new bryophilous agarics, Galerina indica and Rickenella indica, are described from Kerala
Received 11 February 2014
State, India. Comprehensive descriptions, photographs, and comparisons with pheneti-
Received in revised form
cally similar and phylogenetically related species are provided. Inferences of their phylo-
26 March 2014
genetic relationships within the respective genera are provided based on the sequences of
Accepted 26 March 2014
nuclear ribosomal internal transcribed spacer region. This forms the first record of the
Available online xxx
genus Rickenella from the entire tropics and the first report of a bryophilous Galerina from Kerala. Also, this is the first report of the association of the genera Galerina and Rickenella
Keywords: Basidiomycota
with the moss genera Leucobryum and Campylopus respectively. ª 2014 The Mycological Society of Japan. Published by Elsevier B.V. All rights reserved.
Bryophyta Systematics Taxonomy
1.
Introduction
Fungi associated with bryophyte substrates are taxonomically diverse but their diversity remains understudied (Felix 1988; Hawksworth 2001; Davey and Currah 2006). About 400 species of obligate bryophilous fungi have been described so far, ska most of which are ascomycetes (Do¨bbeler 1997; Ptaszyn et al. 2009). Only a few bryophilous basidiomycetes have been reported and many of them are agarics (Kost 1988). Our knowledge on bryophilous agarics is based almost entirely on studies made in temperate and far northern regions. Very little is known about the diversity of these fungi in the tropics. During our studies on the agarics of Kerala State, India, we came across two new and remarkable bryophilous agarics that are described here along with inferences of their phylogenetic relationships based on the sequences of nuclear ribosomal internal transcribed spacer (ITS) region.
2.
Materials and methods
2.1.
Morphological studies
The bryophilous fungi described here were collected from a moist deciduous forest (Muthanga Wildlife Sanctuary) of Kerala State, India. Conventional morphology-based taxonomic methods as well as molecular phylogenetic methods were employed for this study. Microscopic observations were made on material stained with 1% aqueous solutions of phloxine and Congo red and mounted in 3% aqueous KOH. Melzer’s reagent was used to observe whether the basidiospores and tissues were amyloid. Lactophenol-cotton blue was used for observing plage of the basidiospores. For evaluation of the range of spore-size, 20 basidiospores each from one specimen of each collection cited were measured. In the description of the basidiospores, the abbreviation Q
* Corresponding author. Tel.: þ91 9744778277. E-mail addresses: [email protected], [email protected] (P. Manimohan). http://dx.doi.org/10.1016/j.myc.2014.03.004 1340-3540/ª 2014 The Mycological Society of Japan. Published by Elsevier B.V. All rights reserved.
Please cite this article in press as: Latha KPD, et al., Two new bryophilous agarics from India, Mycoscience (2014), http:// dx.doi.org/10.1016/j.myc.2014.03.004
2
m y c o s c i e n c e x x x ( 2 0 1 4 ) 1 e6
represents the range of ratio of spore length to spore width calculated for each spore and Qm is the average of the Q values. Color codes used in the descriptions are from Kornerup and Wanscher (1978). The examined collections are deposited at Kew (Mycology) Herbarium and the Kew accession numbers (e.g., K(M) 190552) are indicated.
2.2.
Phylogenetic analysis
2.2.1.
DNA extraction, PCR and sequencing
The entire ITS region (ITS1, 5.8S rRNA gene, and ITS2) was analyzed in this study. DNA was extracted from dried specimens of the two agarics employing the procedure described by Izumitsu et al. (2012). PCR reactions were performed with primers ITS1 and ITS4. Amplification reactions were performed in a GeneAtlas thermal cycler (Astec, Fukuoka, Japan). Amplified PCR products were purified using column purification (GeneJet PCR purification kit, Thermo Fisher Scientific, Mumbai, India) as per manufacturer’s guidelines and were subjected to automated DNA sequencing on ABI3730xl DNA analyzer (Applied Biosystems, Foster City, CA, USA) using the same primers used for PCR. The generated sequences were edited manually using BioEdit sequence alignment editor version 7.0.9.0 (Tom Hall, Ibis Biosciences, Carlsbad, CA, USA). The edited sequences were then used separately for BLAST search in the GenBank database (www. ncbi.nlm.nih.gov). The newly generated sequences were deposited in GenBank.
2.2.2.
Sequence analysis
The newly generated ITS sequence (KJ187768; 677 bp) of the newly discovered Galerina species along with those retrieved from GenBank (31 ITS sequences) based on the result from a BLAST search were aligned using MUSCLE version 3.8.31 (Edgar 2004). A final set of sequences from 33 species of Galerina were aligned. All sequences were trimmed at the ends to a final length of 629 bp. The pre-existing sequences of Galerina species from GenBank were selected based on their similarity indices. Panaeolus sphinctrinus (Fr.) Que´l. (Gulden et al. 2005) was selected as outgroup taxon for rooting purpose. Maximum parsimony (MP) analysis was conducted using MEGA version 5, with 1000 heuristic bootstrap (BS) replicates, using random step-wise addition, and holding one tree at each step. Maxtrees were set to 1000. TBR branch swapping algorithm was used to assess branch support. All characters were treated as unordered and gaps were treated as missing data. The aligned sequence data matrix has been deposited in TreeBase (http://purl.org/phylo/treebase/ phylows/study/TB2:S15288). No phylogenetic analysis was carried out with the ITS sequence (KJ187769; 661 bp) of the newly discovered Rickenella species owing to the very limited number of Rickenella ITS sequences available in the GenBank.
3.
Results
3.1.
Taxonomy
Galerina indica K.P.D. Latha & Manim., sp. nov. Fig. 1.
MycoBank no.: MB 807699. Diagnosis: Characterized by small, mycenoid basidiomata; a brownish orange, pellucid-striate, glabrous pileus; emarginate lamellae; ellipsoid to amygdaliform, ornamented basidiospores with smooth plage and a loose myxosporium; 2spored basidia; versiform cheilocystidia; utriform or lageniform pleurocystidia with a somewhat subcapitate apex; cutistype pileipellis and stipitipellis with spiral encrustations; and presence of clamp connections. Differing from Galerina salicicola P.D. Orton in having smaller basidiomata, 2-spored basidia, and a bryicolous habitat. Type: India, Kerala State, Wayanad District, Muthanga Wildlife Sanctuary, on moss bed (Leucobryum sp., Dicranaceae, Bryopsida), in small groups, 7 September 2012, K. P. Deepna Latha (holotype, K(M)190552). Gene sequence ex-holotype: KJ187768 (ITS). Etymology: Specific epithet refers to India, the country from where this species was first described. Basidiomata small, mycenoid. Pileus 2e7 mm diam., initially convex, becoming conico-convex to almost campanulate with a small umbo; surface brownish orange (6C8) on umbo and striations, golden yellow (5B7) elsewhere, weakly hygrophanous and becoming paler, eventually developing a greenish hue towards margin, pellucid-striate, glabrous to the naked eye, finely sulcate towards margin; margin slightly incurved or somewhat decurved, crenate. Lamellae 10e20, emarginate, close, grayish orange (6B4), up to 2 mm wide, with lamellulae of 3 lengths; edge entire or slightly crenate, concolorous with the sides. Stipe 7e12 0.5e1.5 mm, central, terete, equal or slightly tapered towards base, solid; surface brownish orange (7C5), glabrous to the naked eye, finely appressed fibrillose all over as well as finely pruinose towards apex and base under a lens; base with pale brown mycelial cords. Odor and taste not distinctive. Basidiospores 7.5e10 4.5e6 (8.8 0.61 5.2 0.44) mm, Q ¼ 1.5e2, Qm ¼ 1.69, ellipsoid to amygdaliform, with a conspicuous plage, a golden brown wall with verruculose ornamentation and a loose wrinkled myxosporium. Basidia 14e23 5e7 mm, clavate, 2-spored, thin-walled, hyaline or pale yellow; sterigmata up to 4 mm long. Lamella-edge heterogeneous. Cheilocystidia 11e44 4.5e5 mm, versiform: globose, pyriform, utriform, lageniform or tibiiform, thin- to slightly thick-walled, pale yellow or hyaline. Pleurocystidia 30e42 12e16 mm, utriform or lageniform, with a somewhat subcapitate apex, thin- to slightly thick-walled, hyaline or pale yellow. Lamellar trama composed of both narrow and inflated hyphae; hyphae 4e24 mm wide, with cylindrical or subfusoid elements, thin-walled, hyaline or pale yellow. Pileus trama subregular; hyphae 4e12 mm wide, with cylindrical or subfusoid elements, inflated up to 30 mm, thin-walled, hyaline or pale yellow. Pileipellis a cutis; hyphae 4e7 mm wide, with cylindrical or subfusoid elements, thin- to slightly thick-walled, with a pale yellow wall showing spiral encrustations. Stipitipellis a cutis; hyphae 3e24 mm, with cylindrical or subfusoid elements, thin- to slightly thick-walled, with a pale yellow wall showing spiral encrustations. Clamp connections seen on all hyphae. Habitat and distribution: On moss bed (Leucobryum sp., Dicranaceae, Bryopsida), India.
Please cite this article in press as: Latha KPD, et al., Two new bryophilous agarics from India, Mycoscience (2014), http:// dx.doi.org/10.1016/j.myc.2014.03.004
m y c o s c i e n c e x x x ( 2 0 1 4 ) 1 e6
3
Fig. 1 e Galerina indica (K(M)190552). A: Basidiomata. B: Basidiospores with myxosporium (arrow). C: Basidiospore with plage (arrow). D: Basidium. E: Pleurocystidium. F: Cheilocystidia. G: Pileipellis. H: Stipitipellis. Bars A 10 mm; BeH 10 mm.
Note: The mycenoid basidiomata, the presence of clamp connections, the golden brown, ornamented basidiospores with a distinct smooth plage, and the thin-walled pleurocystidia with broadly rounded apices suggest that the present species is referable to the genus Galerina subgenus Galerina section Physocystis (Smith and Singer 1964). However the key to the species of section Physocystis (Smith and Singer 1964) does not lead the present one to any species. None of the species described in the section Physocystis are bryicolous. Comparison of ITS sequence data derived from the present Galerina species with the nucleotide sequences of taxa available in GenBank suggests that the present Galerina is a distinct
species. In a megablast search of the GenBank nucleotide database using the ITS sequence, the closest hit was Galerina salicicola (GenBank AJ585493; identities ¼ 597/624 (96%), Gaps ¼ 13/624 (2%)) followed by G. badipes (Pers.) Ku¨hner (GenBank JF908012; identities ¼ 628/688 (91%), Gaps ¼ 24/688 (3%)). Subsequent phylogenetic analysis (Fig. 2) placed the new species close to G. salicicola which was previously placed in the section Physocystis of subgenus Galerina (Smith and Singer 1964). Galerina salicicola shares a few characters such as the similar shape and size of the basidiospores and similar shapes of the cheilo- and pleurocystidia with the present species. However, G. salicicola has consistently 4-spored basidia and is
Please cite this article in press as: Latha KPD, et al., Two new bryophilous agarics from India, Mycoscience (2014), http:// dx.doi.org/10.1016/j.myc.2014.03.004
4
m y c o s c i e n c e x x x ( 2 0 1 4 ) 1 e6
Fig. 2 e The phylogenetic tree obtained from the MP analysis using ITS sequence data. Values in the branches indicate the BS support of the clades. BS values greater than 50% are shown. GenBank accession numbers are given after the name of each taxon.
lignicolous. Galerina pruinatipes A.H. Sm. differs from the present species in having larger-sized basidiospores, pileocystidia and lignicolous basidiomata. According to Gulden et al. (2005) Galerina is a polyphyletic genus and their molecular phylogenetic analysis revealed three or four separate monophyletic lineages among the species traditionally considered as belonging to that genus. In their analysis, Gulden et al. (2005) found G. salicicola to belong to the “naucoriopsis” lineage. Rickenella indica K. P. D. Latha & Manim., sp. nov. Fig. 3. MycoBank no.: MB 807702. Diagnosis: Characterized by very small, omphalinoid basidiomata; a pubescent pileus and stipe; deeply decurrent
lamellae; ellipsoid basidiospores; capitulate cystidia in the cortical layer and hymenium; and presence of clamp connections. Differing from Rickenella fibula (Bull.) Raithelh. in having a distinctly shorter stipe, lamellae with a pinkish tint, slightly broader and shorter basidiospores, capitate cystidia, and lacking inflated elements in the lamellar trama. Type: India, Kerala State, Wayanad District, Muthanga Wildlife Sanctuary, on moss bed (Campylopus sp., Dicranaceae, Bryopsida), scattered or in small groups, 5 July 2013, K. P. Deepna Latha (holotype, K(M)190585). Gene sequence ex-holotype: KJ187769 (ITS). Etymology: Specific epithet refers to India, the country from where this species was first described.
Please cite this article in press as: Latha KPD, et al., Two new bryophilous agarics from India, Mycoscience (2014), http:// dx.doi.org/10.1016/j.myc.2014.03.004
m y c o s c i e n c e x x x ( 2 0 1 4 ) 1 e6
5
Fig. 3 e Rickenella indica (K(M)190585). A: Basidioma. B: Basidiospore. C: Basidium. D: Pleurocystidium. E: Cheilocystidium. F: Lamellar trama showing air pockets (arrow). G: Pileipellis with pileocystidia. H: Stipitipellis with caulocystidium. Bars A 10 mm; BeH 10 mm.
Basidiomata small, omphalinoid. Pileus 1e7 mm diam., convex with a shallow central depression; surface orange (6B8) or reddish orange (7B8) at the center and golden yellow (5B7) elsewhere when young, becoming brownish orange (5C6) or brownish yellow (5C7) all over with age, hygrophanous and becoming paler, faintly pellucid-striate, glabrous to the naked eye, finely pubescent under a lens, shallowly sulcate-striate towards margin; margin slightly incurved when young, becoming almost decurved with age, somewhat wavy. Lamellae 10e15, decurrent to deeply decurrent, occasionally furcate or transvenose, close, white with a pinkish tint, up to 2 mm wide, with lamellulae of 3 lengths; edge entire to the naked eye, finely torn under a lens, concolorous with the sides. Stipe 3e22 0.5e1 mm, central, terete, equal or slightly
tapered towards apex, cartilaginous, solid; surface orange gray (5B2), somewhat translucent, glabrous to the naked eye, finely pubescent all over under a lens; base with whitish mycelial cords. Context soft, 0.5 mm wide, orange (6B8). Odor and taste not distinctive. Basidiospores 3.5e6 3e4 (4.93 0.83 3.27 0.36) mm, Q ¼ 1.14e2.16, Qm ¼ 1.51, ellipsoid, thin-walled, hyaline, smooth, inamyloid. Basidia 12e20 4e6 mm, clavate, 4spored, thin-walled; sterigmata up to 3 mm long. Lamellaedge heterogeneous. Cheilocystidia 30e53 9e12 mm, narrowly lageniform to obclavate often with subcapitate apex, hyaline, thin-walled. Pleurocystidia 31e53 6.5e13 mm, subcapitate, thin-walled, hyaline, inamyloid. Lamellar trama subregular with lots of irregular air pockets; hyphae 2e15 mm
Please cite this article in press as: Latha KPD, et al., Two new bryophilous agarics from India, Mycoscience (2014), http:// dx.doi.org/10.1016/j.myc.2014.03.004
6
m y c o s c i e n c e x x x ( 2 0 1 4 ) 1 e6
wide, with cylindrical or subfusoid elements, thin-walled with oleiferous hyphae, hyaline, inamyloid. Pileus trama subregular; hyphae 7e22 mm wide, with cylindrical or subfusoid elements, thin-walled, hyaline, inamyloid. Pileipellis a cutis with scattered pileocystidia; hyphae 4e7 mm wide, with cylindrical or subfusoid elements, thin-walled, hyaline. Pileocystidia similar to cheilocystidia in all aspects. Stipitipellis a cutis with scattered caulocystidia; hyphae 3e14.5 mm wide, with cylindrical elements, hyaline, thin-walled. Caulocystidia similar to cheilocystidia in all aspects. Clamp connections observed on all hyphae. Additional specimens examined: India, Kerala State, Wayanad District, Muthanga Wildlife Sanctuary, on moss bed (Campylopus sp., Dicranaceae, Bryopsida), scattered or in small groups, 6 Sep 2012, K. P. Deepna Latha (K(M)190586). Habitat and distribution: On moss bed (Campylopus sp., Dicranaceae, Bryopsida), India. Note: The combination of characters such as the very small, brightly colored, omphalinoid basidiomata growing on moss, a pubescent pileus and stipe, deeply decurrent lamellae, and the presence of both cystidia and clamp connections indicates that this species belongs to Rickenella (Basidiomycota, Hymenochaetales, Repetobasidiaceae). The present species has some macro- and micromorphological characteristics in common with European Rickenella fibula (Bulliard 1784; Raithelhuber 1973) in the golden yellow omphalinoid basidiomata with a pubescent pileus and stipe, the decurrent lamellae, the ellipsoid basidiospores, the capitulate cystidia in the cortical layer and hymenium and the presence of clamp connections. However, there are several characters that show minor deviations in the Kerala collection and these include: 1) slightly broader and shorter basidiospores with a lower median Q value; 2) cystidia that are mostly distinctly capitate; 3) distinctly shorter stipe; 4) pileus that never becomes plano-convex or applanate or infundibuliform; 5) lamellae with a distinct pinkish tint; and 6) absence of inflated elements in the lamellar trama. In a megablast search of the GenBank nucleotide database using the ITS sequences of the Kerala collections, the closest hit was Rickenella fibula. (GenBank JQ694100; identities ¼ 649/ 659 (98%), Gaps ¼ 4/659 (0%)). As R. fibula showed only 98% ITS identity, the Kerala collections do not seem to be conspecific with that species. Additionally, R. fibula is so far known only from the temperate regions of the world. Considering the morphological differences, the distinct ITS sequences and the geographical factor, the Kerala collections are considered as sufficiently different from R. fibula to warrant erection of a new species.
4.
Discussion
All species of Rickenella and some species of Galerina are bryophilous (Kost 1988). However, the relationships between the respective agarics and their hosts are difficult to categorize. The exact nature of the association of the present Galerina and Rickenella species with their respective hosts is not known. So far, only a single species of moss-associated Galerina (G. vinolenta (Berk.) A.H. Sm. & Singer) has been reported from India (Berkeley 1852, as Agaricus vinolentus Berk.). The present report forms the second record of a bryophilous Galerina from
India. Also, this is the first report of the association of the genera Galerina and Rickenella with the moss genera Leucobryum and Campylopus respectively.
Disclosure The authors declare no conflict of interest.
Acknowledgments We are thankful to Dr Manju C. Nair, Guruvayurappan College, Calicut for identifying the moss genera. KPDL acknowledges support from the Kerala State Council for Science, Technology and Environment (KSCSTE) in the form of a PhD fellowship (Grant No. 001/FSHP/2011/CSTE). KNAR acknowledges support from the University Grants Commission (UGC), India, in the form of a Rajiv Gandhi National Fellowship (Grant No. F. 142(SC)/2009 (SA-III)).
references
Berkeley MJ, 1852. Decades of fungi. Sikkim and Khassya fungi. Hooker’s Journal of Botany and Kew Garden Miscellany 4: 130e140. Bulliard JBF, 1784. Champignon de la France. Agaricus fibula. Herbier de la France 4: 186. Davey ML, Currah RS, 2006. Interactions between mosses (Bryophyta) and fungi. Canadian Journal of Botany 84: 1509e1519; http://dx.doi.org/10.1139/b06-120. Do¨bbeler P, 1997. Biodiversity of bryophilous ascomycetes. Biodiversity and Conservation 6: 721e738. Edgar RC, 2004. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Research 32: 1791e1797; http://dx.doi.org/10.1093/nar/gkh340. Felix H, 1988. Fungi on bryophytes, a review. Botanica Helvetica 98: 239e269. Gulden G, Stensrud Ø, Shalchian-Tabrizi K, Kauserud H, 2005. Galerina Earle: a polyphyletic genus in the consortium of darkspored agarics. Mycologia 97: 823e837; http://dx.doi.org/ 10.3852/mycologia.97.4.823. Hawksworth DL, 2001. The magnitude of fungal diversity: the 1.5 million species estimate revisited. Mycological Research 105: 1422e1432; http://dx.doi.org/10.1017/S0953756201004725. Izumitsu K, Hatoh K, Sumita T, Kitade Y, Morita A, Gafur A, Ohta A, Kawai M, Yamanaka T, Neda H, Ota Y, Tanaka C, 2012. Rapid and simple preparation of mushroom DNA directly from colonies and fruiting bodies for PCR. Mycoscience 53: 396e401; http://dx.doi.org/10.1007/s10267-012-0182-3. Kornerup A, Wanscher JH, 1978. Methuen handbook of colour, 3rd edn. Methuen, London. Kost G, 1988. Interactions between Basidiomycetes and Bryophyta. Endocytobiosis and Cell Research 5: 287e308. _ ska A, Mułenko W, Zarnowiec Ptaszyn J, 2009. Bryophytes microniches inhabited by microfungi. Annales Universitatis Mariae Curie e Skłodowska 64: 35e43; http://dx.doi.org/10.2478/ v10067-010-0012-y. Raithelhuber J, 1973. Zur abgrenzung der gattungen Gerronema, Omphalina, Clitocybe und Haasiella. Metrodiana 4: 61e73. Smith AH, Singer R, 1964. A monograph on the genus Galerina Earle. Hafner Publishing Co, New York.
Please cite this article in press as: Latha KPD, et al., Two new bryophilous agarics from India, Mycoscience (2014), http:// dx.doi.org/10.1016/j.myc.2014.03.004
Available online at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/myc
Full paper
Two new bryophilous agarics from India K. P. Deepna Latha, K.N. Anil Raj, Raihana Paramban, Patinjareveettil Manimohan* Department of Botany, University of Calicut, Kerala 673 635, India
article info
abstract
Article history:
Two new bryophilous agarics, Galerina indica and Rickenella indica, are described from Kerala
Received 11 February 2014
State, India. Comprehensive descriptions, photographs, and comparisons with pheneti-
Received in revised form
cally similar and phylogenetically related species are provided. Inferences of their phylo-
26 March 2014
genetic relationships within the respective genera are provided based on the sequences of
Accepted 26 March 2014
nuclear ribosomal internal transcribed spacer region. This forms the first record of the
Available online xxx
genus Rickenella from the entire tropics and the first report of a bryophilous Galerina from Kerala. Also, this is the first report of the association of the genera Galerina and Rickenella
Keywords: Basidiomycota
with the moss genera Leucobryum and Campylopus respectively. ª 2014 The Mycological Society of Japan. Published by Elsevier B.V. All rights reserved.
Bryophyta Systematics Taxonomy
1.
Introduction
Fungi associated with bryophyte substrates are taxonomically diverse but their diversity remains understudied (Felix 1988; Hawksworth 2001; Davey and Currah 2006). About 400 species of obligate bryophilous fungi have been described so far, ska most of which are ascomycetes (Do¨bbeler 1997; Ptaszyn et al. 2009). Only a few bryophilous basidiomycetes have been reported and many of them are agarics (Kost 1988). Our knowledge on bryophilous agarics is based almost entirely on studies made in temperate and far northern regions. Very little is known about the diversity of these fungi in the tropics. During our studies on the agarics of Kerala State, India, we came across two new and remarkable bryophilous agarics that are described here along with inferences of their phylogenetic relationships based on the sequences of nuclear ribosomal internal transcribed spacer (ITS) region.
2.
Materials and methods
2.1.
Morphological studies
The bryophilous fungi described here were collected from a moist deciduous forest (Muthanga Wildlife Sanctuary) of Kerala State, India. Conventional morphology-based taxonomic methods as well as molecular phylogenetic methods were employed for this study. Microscopic observations were made on material stained with 1% aqueous solutions of phloxine and Congo red and mounted in 3% aqueous KOH. Melzer’s reagent was used to observe whether the basidiospores and tissues were amyloid. Lactophenol-cotton blue was used for observing plage of the basidiospores. For evaluation of the range of spore-size, 20 basidiospores each from one specimen of each collection cited were measured. In the description of the basidiospores, the abbreviation Q
* Corresponding author. Tel.: þ91 9744778277. E-mail addresses: [email protected], [email protected] (P. Manimohan). http://dx.doi.org/10.1016/j.myc.2014.03.004 1340-3540/ª 2014 The Mycological Society of Japan. Published by Elsevier B.V. All rights reserved.
Please cite this article in press as: Latha KPD, et al., Two new bryophilous agarics from India, Mycoscience (2014), http:// dx.doi.org/10.1016/j.myc.2014.03.004
2
m y c o s c i e n c e x x x ( 2 0 1 4 ) 1 e6
represents the range of ratio of spore length to spore width calculated for each spore and Qm is the average of the Q values. Color codes used in the descriptions are from Kornerup and Wanscher (1978). The examined collections are deposited at Kew (Mycology) Herbarium and the Kew accession numbers (e.g., K(M) 190552) are indicated.
2.2.
Phylogenetic analysis
2.2.1.
DNA extraction, PCR and sequencing
The entire ITS region (ITS1, 5.8S rRNA gene, and ITS2) was analyzed in this study. DNA was extracted from dried specimens of the two agarics employing the procedure described by Izumitsu et al. (2012). PCR reactions were performed with primers ITS1 and ITS4. Amplification reactions were performed in a GeneAtlas thermal cycler (Astec, Fukuoka, Japan). Amplified PCR products were purified using column purification (GeneJet PCR purification kit, Thermo Fisher Scientific, Mumbai, India) as per manufacturer’s guidelines and were subjected to automated DNA sequencing on ABI3730xl DNA analyzer (Applied Biosystems, Foster City, CA, USA) using the same primers used for PCR. The generated sequences were edited manually using BioEdit sequence alignment editor version 7.0.9.0 (Tom Hall, Ibis Biosciences, Carlsbad, CA, USA). The edited sequences were then used separately for BLAST search in the GenBank database (www. ncbi.nlm.nih.gov). The newly generated sequences were deposited in GenBank.
2.2.2.
Sequence analysis
The newly generated ITS sequence (KJ187768; 677 bp) of the newly discovered Galerina species along with those retrieved from GenBank (31 ITS sequences) based on the result from a BLAST search were aligned using MUSCLE version 3.8.31 (Edgar 2004). A final set of sequences from 33 species of Galerina were aligned. All sequences were trimmed at the ends to a final length of 629 bp. The pre-existing sequences of Galerina species from GenBank were selected based on their similarity indices. Panaeolus sphinctrinus (Fr.) Que´l. (Gulden et al. 2005) was selected as outgroup taxon for rooting purpose. Maximum parsimony (MP) analysis was conducted using MEGA version 5, with 1000 heuristic bootstrap (BS) replicates, using random step-wise addition, and holding one tree at each step. Maxtrees were set to 1000. TBR branch swapping algorithm was used to assess branch support. All characters were treated as unordered and gaps were treated as missing data. The aligned sequence data matrix has been deposited in TreeBase (http://purl.org/phylo/treebase/ phylows/study/TB2:S15288). No phylogenetic analysis was carried out with the ITS sequence (KJ187769; 661 bp) of the newly discovered Rickenella species owing to the very limited number of Rickenella ITS sequences available in the GenBank.
3.
Results
3.1.
Taxonomy
Galerina indica K.P.D. Latha & Manim., sp. nov. Fig. 1.
MycoBank no.: MB 807699. Diagnosis: Characterized by small, mycenoid basidiomata; a brownish orange, pellucid-striate, glabrous pileus; emarginate lamellae; ellipsoid to amygdaliform, ornamented basidiospores with smooth plage and a loose myxosporium; 2spored basidia; versiform cheilocystidia; utriform or lageniform pleurocystidia with a somewhat subcapitate apex; cutistype pileipellis and stipitipellis with spiral encrustations; and presence of clamp connections. Differing from Galerina salicicola P.D. Orton in having smaller basidiomata, 2-spored basidia, and a bryicolous habitat. Type: India, Kerala State, Wayanad District, Muthanga Wildlife Sanctuary, on moss bed (Leucobryum sp., Dicranaceae, Bryopsida), in small groups, 7 September 2012, K. P. Deepna Latha (holotype, K(M)190552). Gene sequence ex-holotype: KJ187768 (ITS). Etymology: Specific epithet refers to India, the country from where this species was first described. Basidiomata small, mycenoid. Pileus 2e7 mm diam., initially convex, becoming conico-convex to almost campanulate with a small umbo; surface brownish orange (6C8) on umbo and striations, golden yellow (5B7) elsewhere, weakly hygrophanous and becoming paler, eventually developing a greenish hue towards margin, pellucid-striate, glabrous to the naked eye, finely sulcate towards margin; margin slightly incurved or somewhat decurved, crenate. Lamellae 10e20, emarginate, close, grayish orange (6B4), up to 2 mm wide, with lamellulae of 3 lengths; edge entire or slightly crenate, concolorous with the sides. Stipe 7e12 0.5e1.5 mm, central, terete, equal or slightly tapered towards base, solid; surface brownish orange (7C5), glabrous to the naked eye, finely appressed fibrillose all over as well as finely pruinose towards apex and base under a lens; base with pale brown mycelial cords. Odor and taste not distinctive. Basidiospores 7.5e10 4.5e6 (8.8 0.61 5.2 0.44) mm, Q ¼ 1.5e2, Qm ¼ 1.69, ellipsoid to amygdaliform, with a conspicuous plage, a golden brown wall with verruculose ornamentation and a loose wrinkled myxosporium. Basidia 14e23 5e7 mm, clavate, 2-spored, thin-walled, hyaline or pale yellow; sterigmata up to 4 mm long. Lamella-edge heterogeneous. Cheilocystidia 11e44 4.5e5 mm, versiform: globose, pyriform, utriform, lageniform or tibiiform, thin- to slightly thick-walled, pale yellow or hyaline. Pleurocystidia 30e42 12e16 mm, utriform or lageniform, with a somewhat subcapitate apex, thin- to slightly thick-walled, hyaline or pale yellow. Lamellar trama composed of both narrow and inflated hyphae; hyphae 4e24 mm wide, with cylindrical or subfusoid elements, thin-walled, hyaline or pale yellow. Pileus trama subregular; hyphae 4e12 mm wide, with cylindrical or subfusoid elements, inflated up to 30 mm, thin-walled, hyaline or pale yellow. Pileipellis a cutis; hyphae 4e7 mm wide, with cylindrical or subfusoid elements, thin- to slightly thick-walled, with a pale yellow wall showing spiral encrustations. Stipitipellis a cutis; hyphae 3e24 mm, with cylindrical or subfusoid elements, thin- to slightly thick-walled, with a pale yellow wall showing spiral encrustations. Clamp connections seen on all hyphae. Habitat and distribution: On moss bed (Leucobryum sp., Dicranaceae, Bryopsida), India.
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Fig. 1 e Galerina indica (K(M)190552). A: Basidiomata. B: Basidiospores with myxosporium (arrow). C: Basidiospore with plage (arrow). D: Basidium. E: Pleurocystidium. F: Cheilocystidia. G: Pileipellis. H: Stipitipellis. Bars A 10 mm; BeH 10 mm.
Note: The mycenoid basidiomata, the presence of clamp connections, the golden brown, ornamented basidiospores with a distinct smooth plage, and the thin-walled pleurocystidia with broadly rounded apices suggest that the present species is referable to the genus Galerina subgenus Galerina section Physocystis (Smith and Singer 1964). However the key to the species of section Physocystis (Smith and Singer 1964) does not lead the present one to any species. None of the species described in the section Physocystis are bryicolous. Comparison of ITS sequence data derived from the present Galerina species with the nucleotide sequences of taxa available in GenBank suggests that the present Galerina is a distinct
species. In a megablast search of the GenBank nucleotide database using the ITS sequence, the closest hit was Galerina salicicola (GenBank AJ585493; identities ¼ 597/624 (96%), Gaps ¼ 13/624 (2%)) followed by G. badipes (Pers.) Ku¨hner (GenBank JF908012; identities ¼ 628/688 (91%), Gaps ¼ 24/688 (3%)). Subsequent phylogenetic analysis (Fig. 2) placed the new species close to G. salicicola which was previously placed in the section Physocystis of subgenus Galerina (Smith and Singer 1964). Galerina salicicola shares a few characters such as the similar shape and size of the basidiospores and similar shapes of the cheilo- and pleurocystidia with the present species. However, G. salicicola has consistently 4-spored basidia and is
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Fig. 2 e The phylogenetic tree obtained from the MP analysis using ITS sequence data. Values in the branches indicate the BS support of the clades. BS values greater than 50% are shown. GenBank accession numbers are given after the name of each taxon.
lignicolous. Galerina pruinatipes A.H. Sm. differs from the present species in having larger-sized basidiospores, pileocystidia and lignicolous basidiomata. According to Gulden et al. (2005) Galerina is a polyphyletic genus and their molecular phylogenetic analysis revealed three or four separate monophyletic lineages among the species traditionally considered as belonging to that genus. In their analysis, Gulden et al. (2005) found G. salicicola to belong to the “naucoriopsis” lineage. Rickenella indica K. P. D. Latha & Manim., sp. nov. Fig. 3. MycoBank no.: MB 807702. Diagnosis: Characterized by very small, omphalinoid basidiomata; a pubescent pileus and stipe; deeply decurrent
lamellae; ellipsoid basidiospores; capitulate cystidia in the cortical layer and hymenium; and presence of clamp connections. Differing from Rickenella fibula (Bull.) Raithelh. in having a distinctly shorter stipe, lamellae with a pinkish tint, slightly broader and shorter basidiospores, capitate cystidia, and lacking inflated elements in the lamellar trama. Type: India, Kerala State, Wayanad District, Muthanga Wildlife Sanctuary, on moss bed (Campylopus sp., Dicranaceae, Bryopsida), scattered or in small groups, 5 July 2013, K. P. Deepna Latha (holotype, K(M)190585). Gene sequence ex-holotype: KJ187769 (ITS). Etymology: Specific epithet refers to India, the country from where this species was first described.
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Fig. 3 e Rickenella indica (K(M)190585). A: Basidioma. B: Basidiospore. C: Basidium. D: Pleurocystidium. E: Cheilocystidium. F: Lamellar trama showing air pockets (arrow). G: Pileipellis with pileocystidia. H: Stipitipellis with caulocystidium. Bars A 10 mm; BeH 10 mm.
Basidiomata small, omphalinoid. Pileus 1e7 mm diam., convex with a shallow central depression; surface orange (6B8) or reddish orange (7B8) at the center and golden yellow (5B7) elsewhere when young, becoming brownish orange (5C6) or brownish yellow (5C7) all over with age, hygrophanous and becoming paler, faintly pellucid-striate, glabrous to the naked eye, finely pubescent under a lens, shallowly sulcate-striate towards margin; margin slightly incurved when young, becoming almost decurved with age, somewhat wavy. Lamellae 10e15, decurrent to deeply decurrent, occasionally furcate or transvenose, close, white with a pinkish tint, up to 2 mm wide, with lamellulae of 3 lengths; edge entire to the naked eye, finely torn under a lens, concolorous with the sides. Stipe 3e22 0.5e1 mm, central, terete, equal or slightly
tapered towards apex, cartilaginous, solid; surface orange gray (5B2), somewhat translucent, glabrous to the naked eye, finely pubescent all over under a lens; base with whitish mycelial cords. Context soft, 0.5 mm wide, orange (6B8). Odor and taste not distinctive. Basidiospores 3.5e6 3e4 (4.93 0.83 3.27 0.36) mm, Q ¼ 1.14e2.16, Qm ¼ 1.51, ellipsoid, thin-walled, hyaline, smooth, inamyloid. Basidia 12e20 4e6 mm, clavate, 4spored, thin-walled; sterigmata up to 3 mm long. Lamellaedge heterogeneous. Cheilocystidia 30e53 9e12 mm, narrowly lageniform to obclavate often with subcapitate apex, hyaline, thin-walled. Pleurocystidia 31e53 6.5e13 mm, subcapitate, thin-walled, hyaline, inamyloid. Lamellar trama subregular with lots of irregular air pockets; hyphae 2e15 mm
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wide, with cylindrical or subfusoid elements, thin-walled with oleiferous hyphae, hyaline, inamyloid. Pileus trama subregular; hyphae 7e22 mm wide, with cylindrical or subfusoid elements, thin-walled, hyaline, inamyloid. Pileipellis a cutis with scattered pileocystidia; hyphae 4e7 mm wide, with cylindrical or subfusoid elements, thin-walled, hyaline. Pileocystidia similar to cheilocystidia in all aspects. Stipitipellis a cutis with scattered caulocystidia; hyphae 3e14.5 mm wide, with cylindrical elements, hyaline, thin-walled. Caulocystidia similar to cheilocystidia in all aspects. Clamp connections observed on all hyphae. Additional specimens examined: India, Kerala State, Wayanad District, Muthanga Wildlife Sanctuary, on moss bed (Campylopus sp., Dicranaceae, Bryopsida), scattered or in small groups, 6 Sep 2012, K. P. Deepna Latha (K(M)190586). Habitat and distribution: On moss bed (Campylopus sp., Dicranaceae, Bryopsida), India. Note: The combination of characters such as the very small, brightly colored, omphalinoid basidiomata growing on moss, a pubescent pileus and stipe, deeply decurrent lamellae, and the presence of both cystidia and clamp connections indicates that this species belongs to Rickenella (Basidiomycota, Hymenochaetales, Repetobasidiaceae). The present species has some macro- and micromorphological characteristics in common with European Rickenella fibula (Bulliard 1784; Raithelhuber 1973) in the golden yellow omphalinoid basidiomata with a pubescent pileus and stipe, the decurrent lamellae, the ellipsoid basidiospores, the capitulate cystidia in the cortical layer and hymenium and the presence of clamp connections. However, there are several characters that show minor deviations in the Kerala collection and these include: 1) slightly broader and shorter basidiospores with a lower median Q value; 2) cystidia that are mostly distinctly capitate; 3) distinctly shorter stipe; 4) pileus that never becomes plano-convex or applanate or infundibuliform; 5) lamellae with a distinct pinkish tint; and 6) absence of inflated elements in the lamellar trama. In a megablast search of the GenBank nucleotide database using the ITS sequences of the Kerala collections, the closest hit was Rickenella fibula. (GenBank JQ694100; identities ¼ 649/ 659 (98%), Gaps ¼ 4/659 (0%)). As R. fibula showed only 98% ITS identity, the Kerala collections do not seem to be conspecific with that species. Additionally, R. fibula is so far known only from the temperate regions of the world. Considering the morphological differences, the distinct ITS sequences and the geographical factor, the Kerala collections are considered as sufficiently different from R. fibula to warrant erection of a new species.
4.
Discussion
All species of Rickenella and some species of Galerina are bryophilous (Kost 1988). However, the relationships between the respective agarics and their hosts are difficult to categorize. The exact nature of the association of the present Galerina and Rickenella species with their respective hosts is not known. So far, only a single species of moss-associated Galerina (G. vinolenta (Berk.) A.H. Sm. & Singer) has been reported from India (Berkeley 1852, as Agaricus vinolentus Berk.). The present report forms the second record of a bryophilous Galerina from
India. Also, this is the first report of the association of the genera Galerina and Rickenella with the moss genera Leucobryum and Campylopus respectively.
Disclosure The authors declare no conflict of interest.
Acknowledgments We are thankful to Dr Manju C. Nair, Guruvayurappan College, Calicut for identifying the moss genera. KPDL acknowledges support from the Kerala State Council for Science, Technology and Environment (KSCSTE) in the form of a PhD fellowship (Grant No. 001/FSHP/2011/CSTE). KNAR acknowledges support from the University Grants Commission (UGC), India, in the form of a Rajiv Gandhi National Fellowship (Grant No. F. 142(SC)/2009 (SA-III)).
references
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