Mycoscience
Online ISSN : 1618-2545
Print ISSN : 1340-3540
Short communication
Leucoagaricus karjaticus (Agaricaceae), a new species from Maharashtra, India
Prashant B. Patil, Nitinkumar P. Patil, Sunita Chahar, Satish Maurya
Author information
Keywords: Lepiotoid fungi, phylogeny, taxonomy
JOURNAL OPEN ACCESS FULL-TEXT HTML

2024 Volume 65 Issue 5 Pages 244-252

Browse “Advance online publication” version
Details
Download PDF (9549K)
Download citation RIS

(compatible with EndNote, Reference Manager, ProCite, RefWorks)

BIB TEX

(compatible with BibDesk, LaTeX)

Text
How to download citation
Contact us
Abstract

A new species, Leucoagaricus karjaticus, was described from the tropical region of the Western Ghats of Maharashtra, India based on morphological characteristics and molecular phylogenetic analysis. Leucoagaricus karjaticus is characterised by its yellowish brown to reddish brown granular scales on the pileus, stipe apex exudes colourless drops, broadly ellipsoid to ellipsoid to ovoid basidiospores, clavate to cylindrical, catenulate cheilocystidia with subglobose to cylindrical ante-terminal elements. Further, the molecular markers nrITS and nrLSU sequence data analyses of Leucoagaricus karjaticus with their sister taxa revealed the distinct phylogenetic position of the new species.

Marcel V. Locquin classified Leucoagaricus as a subgenus of Leucocoprinus Pat. among the lepiotoid fungi (Locquin, 1943a, 1943b). Singer (1948) later elevated Leucoagaricus to the genus level. The small to medium-sized, slender, fragile to sturdy basidiomata with non-plicate pileus margins, free lamellae, presence of annulus, smooth or ornamented basidiospores with or without germ pore, dextrinoid and metachromatic in cresyl blue, hymenium without pseudoparaphyses, presence of cheilocystidia, and hyphae without clamp connections were the defining characteristics of the genus Leucoagaricus Locq. ex Singer. Morphologically, Leucocoprinus differs from Leucoagaricus in that the pileus has striations and pseudoparaphyses around the basidia. However, Leucoagaricus is polyphyletic, and species of Leucoagaricus are intermixed with those of Leucocoprinus, and Micropsalliota Hohn in the phylogenetic analyses (Dutta et al., 2021; Ma et al., 2022; Vellinga, 2004b; Vellinga et al., 2011).

Vellinga (2004a) stated that Leucoagaricus species are more common and diverse in the tropics, and numerous recent macrofungal studies have identified Leucoagaricus species from tropical locations (Ashraf et al., 2023; Dutta et al., 2021; Ge, 2010; Ge et al., 2015; Hussain et al., 2018; Justo et al., 2021; Khalil et al., 2023; Khan et al., 2023; Kumar & Manimohan, 2009; Latha et al., 2020; Liang et al., 2010; Ma et al., 2022; Qasim et al., 2015; Sysouphanthong et al., 2018; Sysouphanthong & Thongklang, 2022; Ullah et al., 2019; Yuan et al., 2014). In India, so far 29 species of Leucoagaricus with five varieties have been reported (Dutta, et al., 2021; Farook et al., 2013; Kumar & Manimohan, 2009; Kumari & Atri, 2013; Latha, et al., 2020; Manjula, 1983; Natarajan et al., 2005; Rajput et al., 2015; Sathe & Daniel, 1980; Senthilarasu, 2014).

During the mycofloristic survey in monsoon seasons from 2017 to 2021 in the different locations of Matheran Hills (one of the hotspots in Western Ghats, Maharashtra, India), we found an interesting fungus collected from the vicinity of termite mounds belonging to the Lepiotaceous group. In our current study, we followed the broad generic concept in the sense of Vellinga (2001) for Leucoagaricus & Leucocoprinus, and described the new species, Leucoagaricus karjaticus on the basis of morphological characteristics and molecular phylogenetic analyses.

Basidiomata were photographed in the habitat using a Canon EOS 700D Camera (Canon Inc., Japan), and their macroscopic characteristics were studied from fresh specimens in the laboratory. Colour notations of fresh mushrooms were recorded from the ‘Methuen Handbook of Colour’ (Kornerup & Wanscher, 1978). After being examined, the mushrooms were dried in a hot air oven for 24 h at 45-50 °C temperature (Hu et al., 2022). The dried holotype samples were then placed in the Ajrekar Mycological Herbarium (AMH), Pune, India, while the paratypes were kept in the Botany department of Smt. Chandibai Himathmal Mansukhani College, Thane, India. The microscopic characteristics were studied from thin hand-cut sections of dried material, then rehydrated in 5% (w/v) KOH, stained with 1% (w/v) phloxin and 0.5% (w/v) Congo red in distilled water with assistance of a compound microscope MLX-B (Olympus, Tokyo, Japan) connected with Magnus Magcam DC-5 camera (Magnus Opto Systems India Pvt. Ltd., New Delhi, India). Melzer's reagent was used to check the amyloidity of basidiospores, whereas cresyl blue and cotton blue were used to detect the metachromatic and cyanophilic reactions of basidiospores respectively. The abbreviation [50/1/3] means 50 basidiospores were measured from 1 basidiocarp of 3 collections. At least 20 each of basidia, cheilocystidia, pileal, and stipe elements were measured. The following notations were used for describing basidiospores: Xm for arithmetic mean of length by width of basidiospores (± standard deviation), Q for quotient of length divided by width of individual basidiospores and Qm stands for the mean of Q values (± standard deviation). The abbreviation La is used for Leucoagaricus, Lc for Leucocoprinus and L for Lepiota.

The CTAB (Doyle & Doyle, 1987) approach was used to extract the whole genomic DNA. Furthermore, the DNA markers nrITS and nrLSU were chosen for the molecular phylogenetic investigations based on the findings of earlier studies (Dutta et al., 2021; Ge et al., 2015; Hussain et al., 2018; Justo et al., 2021; Ma et al., 2022; Vellinga et al., 2011). For the chosen markers, the Polymerase Chain Reaction (PCR) amplification was done at Genematrix LLP (Pune, India). ITS1/ITS4 primers (White et al., 1990) were used to amplify the nrITS, whereas LROR/LR5 primers (Moncalvo et al., 2000; Vilgalys & Hester, 1990) were used to amplify the nrLSU region. Following the markers' successful amplification, the PCR products were purified and sent to Apical Scientific Sdn Bhd (Seri Kembangan, Selangor, Malaysia) for Sanger sequencing. Using Chromas (Technelisium Pvt. Ltd, Australia) software, the acquired sequences were examined for quality and then further curated using BioEdit v 7.2.5 (Hall, 1999). Both forward and reverse sequences were used to create the consensus sequences, which were then uploaded to GenBank (Table 1).

Table 1 Fungal taxa, voucher specimen numbers, localities and GenBank accession numbers for nrITS and nrLSU sequences used for the present phylogenetic analyses. “-” means information not available from GenBank database. Sequences newly generated in the present study were shown in bold.

TaxonVoucher number/strainLocalityGenBank accession numbersReferences
nrITSnrLSU
Cystolepiota pseudoseminudaKUN-HKAS 92275ChinaMN810149MN810101Hou & Ge, 2020
Cystolepiota pseudoseminudaKUN-HKAS 73969ChinaMN810144MN810100Hou & Ge, 2020
Cystolepiota seminuda4-X-1989 H.A. Huijser s.n.NetherlandsAY176350AY176351Vellinga, 2004b
Lepiota aff. furfuraceipesE.C. Vellinga 3646ThailandMN582751-GenBank
Lepiota aff. furfuraceipesE.C. Vellinga 3621Thailand-HM488778Vellinga et al., 2011
Leucoagaricus adelphicus15-XI-2001, E.C. Vellinga 2669 (UC)USAAY243622-GenBank
Lepiota cf. atrodiscaE.C. Vellinga 3164USAGU903304-Vellinga & Balsley, 2010
Lepiota cf. atrodiscaE.C. Vellinga 3415USAGU903305-Vellinga & Balsley, 2010
Lepiota cf. phaeostictaTN51705USAGU903307-Vellinga & Balsley, 2010
Lepiota flammeotinctaDuke JJ97Costa RicaU85331U85296Johnson & Vilgalys, 1998
Lepiota sp.BAB-5053IndiaKR155098-GenBank
Lepiotaceae sp. PA635PA635PanamaEF527366-Vo et al., 2009
Lepiotaceae sp. PA624PA624PanamaEF527358-Vo et al., 2009
Lepiotaceae sp. PA617PA617PanamaEF527352-Vo et al., 2009
Lepiotaceae sp. PA652PA652PanamaEF527378-Vo et al., 2009
Lepiotaceae sp. PA634PA634PanamaEF527365-Vo et al., 2009
Lepiotaceae sp. PA639PA639PanamaEF527368-Vo et al., 2009
Lepiotaceae sp. PA530PA530PanamaEF527339-Vo et al., 2009
Leucoagaricus albosquamosusCFSZ20662ChinaOM976879OM976865Ma et al., 2022
Leucoagaricus albosquamosusCFSZ22880ChinaOM976878OM976866Ma et al., 2022
Leucoagaricus amanitoidesE.C. Vellinga 3331 (UC)USAEF080869EF080873Vellinga & Davis, 2007
Leucoagaricus americanus6-VIII-2000, E.C. Vellinga 2454 (UCB)USAAY176407AF482891Vellinga, 2004b
Leucoagaricus americanusNYBG:0005USAKY350857KY350858GenBank
Leucoagaricus asiaticusLAH10012012PakistanKP164971-Ge et al., 2015
Leucoagaricus atroviridisSYAU-073ChinaOM976852Ma et al., 2022
Leucoagaricus atroviridisSYAU-074ChinaOM976853OM976869Ma et al., 2022
Leucoagaricus atroazureusHKAS 48450ChinaEU416299EU416300Liang et al., 2010
Leucoagaricus aurantioruberCFSZ18372ChinaOM976874OM976862Ma et al., 2022
Leucoagaricus aurantioruberCFSZ19756ChinaOM976875OM976863Ma et al., 2022
Leucoagaricus badhamiiMCVE:3047ItalyGQ329056-GenBank
Leucoagaricus barssiiAFTOL-ID 1899, ECV 3126USADQ911600DQ911601GenBank
Leucoagaricus bresadolaeBas7981 (L)USAAF295929-Vellinga, 2000
Leucoagaricus brunneocanusZ.L. Yang 3972ChinaKP096238-Ge et al., 2015
Leucoagaricus brunneodiscusCUH AM708IndiaMT943754MT940572Dutta et al., 2021
Leucoagaricus brunneodiscusCUH AM709IndiaMT940574MT940575Dutta et al., 2021
Leucoagaricus brunnescensR. Balsley (UC)USAGQ203804-Vellinga et al., 2010
Leucoagaricus bulbigerANGE 197Dominican RepublicMN483028-Justo et al., 2021
Leucoagaricus bulbigerANGE 197BDominican RepublicMN483029-Justo et al., 2021
Leucoagaricus callainitinctusCAL 1799IndiaMT108797MT108798Latha et al., 2020
Leucoagaricus candidusCFSZ 11287ChinaOM976876OM976861Ma et al., 2022
Leucoagaricus candidusCFSZ 20964ChinaOM976877OM976864Ma et al., 2022
Leucoagaricus centricastaneusSYAU-075ChinaOM976854OM976870Ma et al., 2022
Leucoagaricus centricastaneusSYAU-076ChinaOM976855OM976871Ma et al., 2022
Leucoagaricus cinerascens28-XII-199, P.B. Matheny 1831 (WTU)USAAY176410-Vellinga, 2004b
Leucoagaricus croceovelutinusE.C. Vellinga 3131 (UC)USAEU166351-GenBank
Leucoagaricus croceovelutinus19-IX-1998, E.C. Vellinga 2243 (L)NetherlandsAF482862-Vellinga et al., 2003
Leucoagaricus crystallifer3-IX-1998, H.A. HuijserGermanyAF482863AY176412Vellinga et al., 2003
Leucoagaricus cupresseusE.C. Vellinga 2841 (UC)USAGU136193-Vellinga et al., 2010
Leucoagaricus dacrytusTENN: 074972USAMT196954-GenBank
Leucoagaricus dyscritusE.C. Vellinga 3956 (UC)USAGU13680HM488777Vellinga et al., 2010
Leucoagaricus flammeotinctoidesE.C. Vellinga 3304 (UC)USAGQ258475-Vellinga et al., 2010
Leucoagaricus flavovirensHKAS 50024ChinaEU416295EU416296Liang et al., 2010
Leucoagaricus georginae19-IX-1998, E.C. Vellinga 2238 (L)NetherlandsAY176413AY176414Vellinga, 2004b
Leucoagaricus guatopoensisANGE 419Dominican RepublicMN483031-Justo et al., 2021
Leucoagaricus guatopoensisANGE 199Dominican RepublicMN483030-Justo et al., 2021
Leucoagaricus hesperiusE.C. Vellinga 3515 (UC)USAGU139788-Vellinga et al., 2010
Leucoagaricus irinellus21-VIII-1997, R. Chalange 97082101FranceAY243648-GenBank
Leucoagaricus jubilaei9-X-1999, J. & A. Guinberteau 99101101FranceAY243635-GenBank
Leucoagaricus karjaticusAMH 10515IndiaOR775576OR775580In this study
Leucoagaricus karjaticusMMH 1311IndiaOR775578-In this study
Leucoagaricus lacrymansP. Zhang 599ChinaKY039574-Yang & Ge, 2017
Leucoagaricus lahorensisLAH 10042012PakistanKJ701794-Qasim et al., 2015
Leucoagaricus leucothitesHMAS 88854ChinaJN944083JN940293GenBank
Leucoagaricus littoralis MCVE:702ItalyGQ329041-GenBank
Leucoagaricus majusculusMFLU 09-0164ThailandHM488764-Vellinga et al., 2011
Leucoagaricus margaritiferANGE 254Dominican RepublicMN482998-Justo et al., 2021
Leucoagaricus margaritiferANGE 509Dominican RepublicMN482997-Justo et al., 2021
Leucoagaricus melanotrichus23-IX-1998, E.C. Vellinga 2262 (L)NetherlandsAY176417AY176418Vellinga, 2004b
Leucoagaricus melanotrichus var. fuligineobrunneusH.A. HuijserNetherlandsGU903306-Vellinga & Balsley, 2010
Leucoagaricus meleagris30-VII-1996, E.C. Vellinga 1990 (L)Netherlands-AF482890Vellinga et al., 2003
Leucoagaricus meleagris18 & 19-VIII-1997, E.C. Vellinga 2095 (L)NetherlandsAF482867-Vellinga et al., 2003
Leucoagaricus naucinusCBS 387.66ArgentinaMH858835MH870472Vu et al., 2019
Leucoagaricus nivalisMYR 414ChinaOM974308OM967225Ma et al., 2022
Leucoagaricus nivalisYang 5792ChinaKY039573KY039578Yang & Ge, 2017
Leucoagaricus nympharumHMAS 99343ChinaEU416310EU681805Liang et al., 2010
Leucoagaricus orientiflavusHKAS 54260ChinaGU084262JN940290Ge, 2010
Leucoagaricus orientiflavusHKAS 54265ChinaGU084261JN940291Ge, 2010
Leucoagaricus pardalotusE.C. Vellinga 3313 (UC)USAGQ258479-Vellinga et al., 2010
Leucoagaricus pegleriCA 20Dominican RepublicMN483002-Justo et al., 2021
Leucoagaricus pegleriANGE 192Dominican RepublicMN483003-Justo et al., 2021
Leucoagaricus pilatianus16-X-1999, J. & A. Guinberteau 99101608FranceAY243626-GenBank
Leucoagaricus proximus LE 262861RussiaJX133172-Malysheva et al., 2013
Leucoagaricus proximusLE 262855RussiaJX133171-Malysheva et al., 2013
Leucoagaricus purpureolilacinus6-XI-1998, E.C. Vellinga 2291(L)NetherlandsAF482869-Vellinga et al., 2003
Leucoagaricus pyrrhulusE.C. Vellinga 3306 (UC)USAGQ258474-Vellinga et al., 2010
Leucoagaricus roseovertensANGE 196Dominican RepublicMN483005-Justo et al., 2021
Leucoagaricus roseovertensCA 19Dominican RepublicKM983716-Justo et al., 2021
Leucoagaricus rubrobrunneusLE 262863RussiaNR120166-Malysheva et al., 2013
Leucoagaricus rubrotinctusHKAS 54240ChinaJN944081JN940295GenBank
Leucoagaricus sabinaeANGE 182Dominican RepublicMN483007-Justo et al., 2021
Leucoagaricus sabinaeANGE 305Dominican RepublicKM983666-Justo et al., 2021
Leucoagaricus serenus11-IX-1995, E.C. Vellinga 1930 (L)BelgiumAF482871AY176421Vellinga et al., 2003
Leucoagaricus sericifer 1-X-1997, E.C. Vellinga 2116 (L)NetherlandsAY176426AY176427Vellinga, 2004b
Leucoagaricus silvestris ANGE 251Dominican RepublicMN482994-Justo et al., 2021
Leucoagaricus silvestrisANGE 489Dominican RepublicMN482995-Justo et al., 2021
Leucoagaricus sinicusHKAS 60647ChinaDQ182505DQ457653GenBank
Leucoagaricus sp. ECV-2010cR.B. Balsley photo 938 (UC)USAGU903308-Vellinga et al., 2010
Leucoagaricus sp. ecv2484E.C. Vellinga 2484 (UC)USAGU136182-Vellinga et al., 2010
Leucoagaricus sp. Vellinga 2561E.C. Vellinga 2561 (UCB)USAAY176430AY176431Vellinga, 2004b
Leucoagaricus sublittoralis19-IX-1998, E.C. Vellinga 2235 (L)NetherlandsAY176442AY176443Vellinga, 2004b
Leucoagaricus subpurpureolilacinusHKAS 123027ChinaOM974314OM967227Ma et al., 2022
Leucoagaricus stillatusANGE 252Dominican RepublicMN483000-Justo et al., 2021
Leucoagaricus stillatusANGE 256Dominican RepublicMN483001-Justo et al., 2021
Leucoagaricus tangerinusHKAS 50036ChinaNR155314-Yuan et al., 2014
Leucoagaricus tener23-X-1998, E.C. Vellinga 2261 (L)NetherlandsAY176444AY176445Vellinga, 2004b
Leucoagaricus tenerMCVE:751ItalyGQ329043-GenBank
Leucoagaricus tropicusCUH AM699IndiaMT669365MT669370Dutta et al., 2021
Leucoagaricus tropicusCUH AM707IndiaMT669367MT669371Dutta et al., 2021
Leucoagaricus truncatusZ.W. Ge 793ChinaKP096235-Ge et al., 2015
Leucoagaricus truncatusHKAS 49288ChinaNR155319-Ge et al., 2015
Leucoagaricus turgipesANGE 583Dominican RepublicMN483017-Justo et al., 2021
Leucoagaricus turgipesANGE 511Dominican RepublicMN483019-Justo et al., 2021
Leucoagaricus umbonatusSHL 1PakistanKU647739KU900521Hussain et al., 2018
Leucoagaricus variicolorAH 40328SpainNR120314-Muñoz et al., 2012
Leucoagaricus virensCFSZ 19869ChinaOM976881-Ma et al., 2022
Leucoagaricus virensCFSZ 19794ChinaOM976880OM976867Ma et al., 2022
Leucoagaricus viridiflavusINM-2-87722JapanKR259170KR259171GenBank
Leucoagaricus viriditinctusHKAS 50033ChinaEU419375EU419376Liang et al., 2010
Leucoagaricus wychanskyiIX/X-1987 H.A. Huijser (L)NetherlandsAF482874-Vellinga et al., 2003
Leucocoprinus aff. heinemanniiANGE 357Dominican RepublicMN483013-Justo et al., 2021
Leucocoprinus aff. heinemanniiAJ 490USAMN483012-GenBank
Leucocoprinus antillarumANGE 500Dominican RepublicMN482992-Justo et al., 2021
Leucocoprinus antillarumANGE 255Dominican RepublicMN482991-Justo et al., 2021
Leucocoprinus birnbaumiiCBS 323.80JapanMH861267MH873036Vu et al., 2019
Leucocoprinus brebissonii13-X-1991, E.C. Vellinga 1784 (L)FranceAF482859AY176446Vellinga et al., 2003
Leucocoprinus cepaestipesNY-EFM548UKU85338U85286GenBank
Leucocoprinus cf. fragilissimusPA250PanamaAF079738AF079656Mueller et al., 1998
Leucocoprinus cf. zamurensisPA415PanamaAF079753AF079671Mueller et al., 1998
Leucocoprinus cretaceus9-II-1999, T. Lassee & J. Omar TL6171MalaysiaAY176447-Vellinga, 2004b
Leucocoprinus fuligineopunctatusANGE 463Dominican RepublicMN482982-Justo et al., 2021
Leucocoprinus fuligineopunctatusANGE 421Dominican RepublicMN482983-Justo et al., 2021
Leucocoprinus griseofloccosusGE17001 (PC)FranceMH257568-
Leucocoprinus microlepisCA 31Dominican RepublicMN482987-Justo et al., 2021
Leucocoprinus microlepisANGE 190Dominican RepublicMN482988-Justo et al., 2021
Leucocoprinus scissusANGE 257Dominican RepublicMN482986-Justo et al., 2021
Leucocoprinus scissusANGE 154Dominican RepublicMN482985-Justo et al., 2021
Leucocoprinus sp.ANGE 418Dominican RepublicMN483016-Justo et al., 2021
Micropsalliota geesteraniLAPAG 520UKKM922965KM923966Parra et al., 2016
Micropsalliota geesterani23-IX-1998 E.C. Vellinga 2263 (L)NetherlandsAF482857AF482888Vellinga et al., 2003
Micropsalliota pseudoglobocystisGX20172236ChinaMT671234MT671246Li et al., 2021
Micropsalliota pseudoglobocystisGX20172228ChinaMT671245MT671233Li et al., 2021
Micropsalliota rubrobrunescensZRL3048ThailandHM436627HM436586Zhao et al., 2010
Termiticola sp.HKAS 122480ChinaON794316-GenBank
Termiticola sp.rlc-381ChinaMW374173-GenBank
Termiticola sp.WML-08ChinaOK584457-GenBank
Termiticola sp.2-II-1999, TL6106MalaysiaAY176352AY176353Vellinga, 2004b

Following the earlier studies, the ingroups and outgroups (Cystolepiota seminuda (Lasch) Bon and Cystolepiota aff. seminuda as outgroups) were retrieved from NCBI database to construct the phylogenetic Accessions (Dutta et al., 2021; Ge et al., 2015; Hussain et al., 2018; Justo et al., 2021; Ma et al., 2022; Vellinga et al., 2011). The newly generated sequences were aligned along with NCBI data using the MSA-Muscle tool available with MEGA v 7.0 (Kumar et al., 2016). Initially, the nrITS and nrLSU trees were constructed individually to check their topology. Both the trees depicted exactly same tree topology. Further, to construct the combined tree, both the markers were analysed to understand their congruence using Partition Homogeneity Test (ILD) in PAUP v 4.0b10 (Swofford & Sullivan, 2003). Based on the ILD test, both the matrix was concatenated using TaxonDNA v 1.7.8 (Vaidya et al., 2010). The combined dataset consists of 144 nrITS and 62 nrLSU sequences (including three newly generated sequences during the present study).

In order to estimate the phylogenetic position and relationship, two methods were used: Maximum Likelihood (ML) and Bayesian inference (BI). For ML analysis, IQTree v1.6.8 (Nguyen et al., 2014) was used, and the best fit evolutionary model (GTR+F+R5) was selected using ModelFinder (an inbuilt tool of IQTree; Kalyaanamoorthy et al., 2017). MrBayes v 3.2.6 (Ronquist et al., 2012) was used to generate the Bayesian tree, and the following parameters were used: the MCMCMC algorithm was run with two parallel chains for four million generations, the standard deviation of split frequency was obtained less than 0.01, and the effective sample size (ESS) for each parameter was ensured exceeding 200. However, using jModeltest, the most appropriate evolutionary model was determined to be GTR+I+G (Darriba et al., 2012). Moreover, FigTree v 1.4.2 displayed the consensus trees (Rambaut, 2014). The phylogram's statistical supports were calculated using posterior probabilities (PP) and bootstrap values (BS).

Taxonomy

Leucoagaricus karjaticus P.B. Patil, N.P. Patil, S. Chahar & S. Maurya, sp. nov.　Figs. 1, 2.

MycoBank no.: MB 850864.

Fig. 1 - Leucoagaricus karjaticus (AMH 10515, holotype). A, B: Basidiomata in the natural habitat, found close to forest termite mounds. C: Basidiomata with scale bar. D, E: Basidiomata showing lamellae with colourless drops on stipe surface. F: Pileal surface. Bars: D-F 1 cm.
Fig. 2 - Leucoagaricus karjaticus (AMH 10515, holotype). A, B: Basidiospores, some exceptionally large basidiospores as mentioned in the text. C-E: Basidia. F-H: Cheilocystidia. I, J: Pileipellis hyphae. K: Annulus hyphae from context. Bars: A, B 5 μm; C-H 10 μm; I-K 20 μm.

Diagnosis: Differs from La. tangerinus by its white, fugacious annulus, larger basidiospores and basidia, and longer, narrowly clavate to subfusiform cheilocystidia.

Type: INDIA, Maharashtra, Raigad District, Matheran Hills (18°58’48.00”N, 73°16’12.00”E, 800 m a.s.l.), collected by P. B. Patil on 22 Jul 2018. (AMH 10515, Holotype).

DNA sequence ex-Holotype (preserved holotype as per ICN rule art 8.4 and recommendation 8B.2): OR775576 (nrITS) and Paratype (OR775578).

Etymology: The species epithet “karjaticus” refers to the place “Karjat” where the holotype was collected.

Basidiomata medium to large. Pileus 45-80 mm, ovate to campanulate when young, becoming convex to applanate at maturity, dry, with or without obtusely umbonate, umbo reddish brown (8E4) to dark brown (8F4), sometimes cracks at maturity, surface covered with yellowish brown (5D5) to reddish brown (8E8) granular scales, gradually paler and sparser towards the margin, on a creamy white background, margin striate, with white floccose remnants, exceeding the lamellae, context 3-5 mm thick on disc, white, no change in colour upon bruising, lamellae free, creamy white, 2-4 mm broad, straight to slightly ventricose, moderately crowded, remote from the stipe, lamellulae of 6 different lengths, edges entire. Stipe 70-100 × 6-10 mm, central, cylindrical, fistulose, mostly curved, slightly attenuated towards apex, stipe apex often exudes colourless drops, creamy white to light brown above annulus, yellowish brown below annulus, slightly granular-scaly towards base, scales concolorous with those of pileus, basal mycelia white. Annulus superior, membranous, ascending, white on upper side, granular-scaly (similar to the pileus surface) below.

Basidiospores [50/1/3] (4)4.5-6(7) × 3-4.5(5) µm, Q = 1.22-1.68(1.75), [Xm = 5.1 ± 0.7 × 3.6 ± 0.45 µm, Qm = 1.4 ± 0.12] (some basidiospores exceptionally large up to 7.5-9 × 5-5.5 µm), broadly ellipsoid to ellipsoid to ovoid in side view, ovoid in front view, with rounded apex, smooth, no germ pore, slightly thick walled (0.5 µm thick), hyaline, dextrinoid, metachromatic in cresyl blue, with one to two guttules when observed in KOH. Basidia 14-18.3 × 6-8 µm, clavate, thin walled, hyaline, 4-spored, rarely 2-spored, sterigmata 1.7-2.5 µm long. Basidioles 12-15 × 5.8-6.5 µm, clavate, thin walled, hyaline. Pleurocystidia absent. Cheilocystidia numerous and crowded at lamella edge, 17-35 × 6.5-14 µm, broadly clavate to cylindrical to capitate-stipitate, regularly catenulate by 3-5 elements with subglobose to cylindrical ante-terminal elements, hyaline, thin walled. Hymenophoral trama regular, hyphae interwoven, hyaline, thin walled. Pileipellis a trichoderm, composed of radially arranged cylindrical hyphae, constricted at the septa, 3.5-8 µm broad, often branched, terminal elements 15-40 × 6-8 µm, oblong to cylindrical with slightly rounded apex. Annulus hyphae cylindrical, thin walled, 3-7 µm broad, interwoven, often branched. Stipitipellis consist of parallel hyphae, 8-13 µm wide. Clamp connections are absent in all tissues.

Habitat and distribution: All sample collections were free-living, found close to forest termite mounds, solitary to scattered in semi-evergreen forests dominated by tree species like Memecylon umbellatum Burm.F., Garcinia talbotii Raizada ex Santapau., Olea dioica Roxb., Xantolis tomentosa (Roxb). Raf. So far known only from Matheran Hills, Maharashtra, India.

Additional specimens (paratypes) examined: INDIA, Maharashtra, Raigad District, Matheran Hills (18°58’48.00”N, 73°16’12.00”E), 5 Aug 2019 (MMH 1311, OR775578 for nrITS and OR053821 for nrLSU), 8 Sep 2019 (MMH 1312), 14 Aug 2022 (MMH 1313), Prashant B. Patil.

Based on the overall appearance of basidiomata, phylogenetic placement and the other Leucoagaricus taxa that exude droplets at stipe, La. karjaticus appears close to La. tangerinus Y. Yuan & J.F. Liang, La. margaritifer Justo, Bizzi & Angelini, La. stillatus Justo, Bizzi & Angelini, La. brunneodiscus A.K. Dutta & K. Acharya, La. tener (P.D. Orton) Bon, La. dacrytus Vellinga and Lepiota furfuraceipes Han C. Wang & Zhu L. Yang. However, the Chinese species, La. tangerinus differs by having white, fugacious annulus, larger basidiospores (6.5-7 × 4-4.5 µm) and basidia (16-26 × 7-10 µm), and longer, narrowly clavate to subfusiform cheilocystidia (25-60 × 8-15 µm) (Yuan et al., 2014). The taxon described from Dominican Republic, La. margaritifer is distinguishable from La. karjaticus by its whitish annulus, larger and less broad, ovoid, ellipsoid to oblong basidiospores (5.5-7.5 × 3.5-4.5 µm), larger (24-74 × 8-30 µm), clavate, ventricose, mostly mucronate, lageniform or bifurcated cheilocystidia (Justo et al., 2021). Another Dominican Republic species, La. stillatus has a jagged margin, white annulus, larger basidiospores (6-8 × 4-4.5 µm) and larger clavate, ventricose cheilocystidia (22-58 × 7-20 µm; Justo et al., 2021). The Indian taxon, La. brunneodiscus differs from La. karjaticus by its larger basidiospores (7.1-7.8 × 4.3-4.8 µm) and basidia (19-24 × 7-8 µm; Dutta et al., 2021). Leucoagaricus tener has a smaller size basidiomata, turns orange when touched and larger, narrowly clavate to slightly fusiform cheilocystidia measuring 19-75 × 6.5-13 µm (Vellinga, 2001). Leucoagaricus dacrytus has smaller basidiomata (pileus 10-33 mm; stipe 20-50 × 1.5-3.5 mm), pileus surface with cobwebby patches and less broad, oblong-ellipsoid basidiospores measuring 5.9-7.4 × 2.9-4.1 µm (Vellinga & Balsley, 2010). Lepiota furfuraceipes is distinctly differs from La. karjaticus in the presence of densely covered furfuraceous to punctate squamules on the pileus and stipe surface, non-metachromatic, larger basidiospores (6-8 × 4-5 µm), longer, subfusiform, narrowly clavate cheilocystidia (17-72 × 5-18.5 µm) and much longer terminal elements of pileipellis (17-105 × 5-13 µm; Wang & Yang, 2005). Leucocoprinus lacrymans T.K.A. Kumar & Manim. and Lc. cepistipes (Sowerby) Pat. also exude droplets but differs from La. karjaticus in the basidiospores having germ pore and basidia surrounded by pseudoparaphyses (Kumar & Manimohan, 2004; Vellinga, 2001). Leucoagaricus silvestris Justo, Bizzi & Angelini (not exude drops) is morphologically related to La. karjaticus but markedly differs by having larger basidiospores (7-9 × 5-6 µm) and basidia (18-29 × 8-12 µm), and longer polymorphic cheilocystidia (18.5-50 × 9-23 µm; Justo et al., 2021). Leucoagaricus karjaticus is also morphologically close to the monotypic genus, Termiticola E. Horak comprising the single species T. rubescens, described from Papua New Guinea by E. Horak in 1979. They shared common characteristics such as growing near termite mounds, granular-scaly pileus surface, margin striate with white floccose remnants, superior, membranous annulus, clavate to subfusoid, catenulate cheilocystidia. However, T. rubescens is distinguishable from La. karjaticus in having larger basidiospores (6-7 × 3.5-4 µm) and basidia (20-28 × 5-8 µm), chained hyphae forming palisade-like pileus covering.

The combined dataset alignment contained 1491 characters, which includes the aligned sequence dataset composed of 788 bp from nrITS, and 703 bp from nrLSU for the analyses. The partition homogeneity test revealed the strong congruence between two markers with a significance value of 1.0 and suggests that the dataset can be combined for further analysis. Based on combined analysis using ML and Bayesian methods we obtained similar tree topologies.

In phylogenetic analyses, Leucoagaricus karjaticus appears as a sister to an undescribed species with three sequences from the China (Termiticola sp. HKAS 122480, rlc 381, WML-08) and one from Malaysia (Termiticola sp. TL6106) by strong statistical support values (100% MLBS, 1 PP) (Fig. 3). All three sequences were roughly indicated as Termiticola spp. In addition, the Chinese taxon, La. tangerinus, and an unknown species from Thailand (L. aff. furfuraceipes) are also sister to La. karjaticus. (Fig. 3). The Malaysian taxon (Termiticola sp. TL6106) was found growing close to termite mounds with brown spores; Vellinga (2004b) tentatively assigned it to the genus Termiticola. Furthermore, Vellinga (2004b) 's phylogenetic analysis placed this taxon in the clade of the Leucoagaricus/Leucocoprinus. In fact, the majority of ant-cultivated fungi and its closely related species belong to Leucoagaricus/Leucocoprinus clade (Heisecke et al., 2021; Mueller et al., 1998; Mueller, 2002; Vo et al., 2009). In phylogenetic analyses of Vo et al. (2009), the ITS r-DNA gene sequences of two free living Leucocoprinaceous taxa were closely related to ant-cultivated fungi, suggesting that these cultivated fungi were brought recently into the symbiosis or that the free-living counterparts recently escaped the symbiosis as hypothesized by Mueller et al. (1998). This discovery of free-living counterparts suggested that ant-cultivated fungi might have close links to free-living Leucocoprinaceous fungi (Vo et al., 2009). Our present studied species is free-living but growing near termite mounds; so, it might left symbiosis recently and completed its life cycle as free-living, but we cannot rule out the possibility of the presence of hyphal connections between fruiting bodies and termite mounds. Moreover, we cannot overlook the report of polyphyly of the genera Leucoagaricus/Leucocoprinus by the various researchers, but the lack of molecular dataset from this group continued this uncertainty (Dutta et al., 2021; Johnson, 1999; Johnson & Vilgalys, 1998; Vellinga, 2004b; Vellinga et al., 2011). Following the previous studies and a conservative approach, currently, we describe the new species under the genus Leucoagaricus.

Fig. 3 - Maximum likelihood phylogram generated from the combined dataset of two nuclear genes (nrITS and nrLSU). Bootstrap values (BS/ right side) ≥ 70 and Bayesian posterior probability (PP/ left side) ≥ 0.8 are given at the internodes. Cystolepiota seminuda and Cystolepiota pseudoseminuda are selected as outgroups. The highlighted taxa represents the basidiomata exude drops. The scale bar represents a phylogenetic distance of 0.07 nucleotide substitutions per site.

Disclosure

The authors declare no conflicts of interest.

Acknowledgements

We greatly acknowledge the Principal, Smt. C.H.M. College, Ulhasnagar, Maharashtra, India for providing the laboratory facilities. We also thank the Principal, Bajaj College of Science, Wardha, for providing laboratory facilities for us to work in, as well as the DST-Fist (grant number-SR/FST/COLLEGE-/2022/1205, dated 19th December, 2022) for funding support to the Bajaj College of Science, Wardha, in order to enhance the laboratory facilities.

References
 
© 2024, by The Mycological Society of Japan

This article is licensed under a Creative Commons
[Attribution-NonCommercial-ShareAlike 4.0 International] license.
https://creativecommons.org/licenses/by-nc-nd/4.0/deed.ja
feedback
 Top