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Article

Inocybe istriaca sp. nov. from Brijuni National Park (Croatia) and Its Position within Inocybaceae Revealed by Multigene Phylogenetic Analysis

by
Ana Pošta
1,†,
Ditte Bandini
2,
Armin Mešić
1,*,
Lucia Pole
1,
Ivana Kušan
1,
Neven Matočec
1,
Olga Malev
1 and
Zdenko Tkalčec
1,†
1
Laboratory for Biological Diversity, Ruđer Bošković Institute, Bijenička Cesta 54, HR-10000 Zagreb, Croatia
2
Panoramastrasse 47, D-69257 Wiesenbach, Germany
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Diversity 2023, 15(6), 755; https://doi.org/10.3390/d15060755
Submission received: 28 April 2023 / Revised: 31 May 2023 / Accepted: 6 June 2023 / Published: 8 June 2023
(This article belongs to the Special Issue Diversity, Ecology and Economic Use of Macrofungi)
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Abstract

:
Integrative taxonomic studies of macrofungal diversity in the Brijuni National Park (Istria County, Croatia) led to the discovery of a second species of Inocybe (Agaricales, Inocybaceae) new to science. Inocybe istriaca sp. nov. is described on the basis of morphological, ecological, and multigene phylogenetic analyses, and its placement within the family Inocybaceae is discussed. The combination of most important morphological characters that distinguish I. istriaca from the other similar Inocybe species are smooth, (sub)amygdaliform, (sub)phaseoliform, or ellipsoid basidiospores (ca. 8.5–12 × 5–7 μm), large basidia (36–45 × 9–15 μm), mostly (sub)fusiform and weakly thick-walled (up to 1.5 μm) metuloid pleurocystidia, and lamellar edge and stipe apex partially covered by a dark resinous substance. The species was collected on the edge of grassland and Mediterranean evergreen holm oak (Quercus ilex) forest. In this study, a total of 14 DNA sequences from four Inocybe species were generated. Two-gene (ITS, LSU) and four-gene (ITS, LSU, rpb2, tef1) phylogenetic analyses confirmed the status of I. istriaca as an independent species.

1. Introduction

The Mediterranean Basin is one of 35 global biodiversity hotspots characterized by outstanding concentrations of endemic species and a high level of habitat loss [1]. The region is home to 245 tree taxa (210 species and 35 subspecies), which is almost 200 taxa more than recorded in central Europe [2]. The Croatian part of the Adriatic Sea (northern Mediterranean) is distinguished by more than 600 islands and islets, a highly indented coastline, and high and steep orography in the hinterland [3].
The Brijuni archipelago consists of 14 islands and islets (total surface area: 7.4 km2) situated near the southwest coast of the Istrian peninsula in the northern Adriatic [4]. It was officially protected as a national park in 1983 and is home to nearly 700 native and exotic plant species. It is characterized by a northern Mediterranean climate with an annual average temperature of 13.9 °C, an annual average precipitation of 817 mm, and a relatively high average air humidity of 76% [4,5]. The largest island of the archipelago is Veli Brijun (5.7 km2), which is covered mainly by Mediterranean evergreen holm oak (Quercus ilex) forests and maquis. Lawns and landscape parks with holm oaks, Aleppo pines (Pinus halepensis), stone pines (P. pinea), Mediterranean cypresses (Cupressus sempervirens), and cedars (Cedrus spp.) are also well represented on the island.
During the fall seasons in 2014, 2015, 2016, and 2020, Croatian mycologists made initial field trips aiming to explore the fungal diversity of Brijuni National Park. In total, 184 macrofungal specimens were collected and deposited in the Croatian National Fungarium (CNF) in Zagreb, Croatia. Members of the ectomycorrhizal basidiomycete genus Inocybe (Fr.) Fr. sensu lato (s.l.) (Agaricomycetes, Agaricales, Inocybaceae) were frequently found, and 28 specimens were sampled. The currently accepted taxonomic framework of the family Inocybaceae [6,7], based on the results of phylogenetic analyses from a six-gene dataset [8], includes seven genera: Auritella Matheny and Bougher, Inocybe sensu stricto (s.s.), Inosperma (Kühner) Matheny and Esteve-Rav., Mallocybe (Kuyper) Matheny, Vizzini and Esteve-Rav., Nothocybe Matheny and K.P.D. Latha, Pseudosperma Matheny and Esteve-Rav., and Tubariomyces Esteve-Rav. and Matheny. The largest genus of the family is Inocybe s.s., with ca. 1000 accepted species [7]. Its members are characterized by the presence of pleurocystidia (often thick-walled and crystalliferous), hyaline basidia (without necropigment), and amygdaliform to ellipsoid, subcylindrical, angular, nodulose, or spiny basidiospores with a distinct apiculus [8].
The initial taxonomic study of Inocybe s.l. specimens from the island of Veli Brijun led to the description of a new species, I. brijunica Mešić, Tkalčec, and Haelew. [5]. Its basidiomata are macroscopically characterized by the presence of an orange to orange-red-brown membranaceous layer in the basal part of the stipe, which is an unusual feature in the genus. Further integrative taxonomic studies on Inocybe specimens from the island of Veli Brijun, combining morphological, molecular phylogenetic, and ecological characters, resulted in the discovery of another species new to science, described here as I. istriaca sp. nov.

2. Materials and Methods

2.1. Morphological Study

The species description is based on a single collection consisting of seven basidiomata. For documentation of macroscopic features, a Canon EOS 5D digital camera equipped with a Canon MR-14EX macro ring flash (Canon Europe, Uxbridge, UK) was used. Microscopic characters were observed with a BX51 optical microscope (Olympus, Hamburg, Germany) using the brightfield technique under magnifications of up to 1500× and photographed with a Canon EOS M50 digital camera. Description and images of microscopic characters were made from rehydrated specimens mounted in 2.5% potassium hydroxide (KOH), except for cystidia that were observed in 3% or 10% ammonium hydroxide (NH4OH). Micromorphological terminology mostly follows Clémençon [9]. Line drawings were made from printed photographs using a light table.
Amyloid and dextrinoid reactions of basidiospores were tested in Melzer’s reagent [10]. Basidiospores from photographs of lamellae mounts were randomly selected and measured using Motic Images Plus 2.0 software (Motic Europe, Barcelona, Spain). The length/width ratio of basidiospores is given as the “Q” value. Average basidiospore, basidia, and pleurocystidia lengths, widths, and Q values are shown in italics. Numbers in square brackets [X/Y/Z] denote X elements measured in Y basidiomata of Z collections. Measurements of cystidia do not include apical crystals when present. The type material was preserved by drying on a flow of hot air at a temperature of about 45 °C. The holotype is deposited at CNF, and an isotype is deposited at STU (State Museum of Natural History, Stuttgart, Germany).

2.2. DNA Extraction, PCR Amplification, and Sequencing

Dried specimens of Inocybe species were ground in microcentrifuge tubes under liquid nitrogen freezing using pestles, and genomic DNA was extracted using the EZNA® HP Fungal DNA Kit (Omega Bio-tek, Norcross, GA, USA) following the manufacturer’s protocol. Three nuclear gene regions, SSU (18S small subunit of ribosomal DNA), ITS (internal transcribed spacer region), and LSU (28S large subunit of ribosomal DNA), and two protein-coding regions, rpb2 (second largest subunit of the DNA-directed RNA polymerase II) and tef1 (translation elongation factor 1-alpha), were sequenced and analyzed. The 25 μL PCR mixtures contained 9.5 μL of ddH2O, 12.5 μL of GoTaq® G2 Green Master Mix (Promega, Madison, WI, USA), 1 μL of DNA template, and 1 μL of each forward and reverse primer. The following primer pairs were used for PCR amplification and sequencing: NS1/NS6 [11], ITS1F/ITS4 [11,12], LR0R/LR5 [13], bRPB2-6F/bRPB2-7.1R [14], EF1-983F/EF1-2218R [15,16]. PCR amplification for the SSU gene region was performed as described by Haelewaters et al. [17]. PCR amplification for ITS and LSU gene regions was performed using a touchdown program: initial denaturation at 95 °C for 2 min; followed by 5 cycles of denaturation at 95 °C for 30 s, annealing at 55 °C for 45 s (add −1 °C per cycle), extension at 72 °C for 1.5 min; 30 cycles of denaturation at 95 °C for 30 s, annealing at 52 °C for 45 s, extension at 72 °C for 1.5 min; and a final extension at 72 °C for 5 min. PCR amplification of rpb2 was performed as described by Mešić et al. [5] and of tef1 as described by Rehner and Buckley [16], with modification of the maximum annealing temperature to 64 °C. Successful PCR products were purified using ExoSAP-IT™ (Thermo Fisher Scientific, Waltham, MA, USA) purification reagent according to the manufacturer’s protocol and sent to Macrogen Europe (Amsterdam, The Netherlands) for bidirectional Sanger sequencing.

2.3. Sequence Alignment and Phylogenetic Analysis

Sequence reads were assembled and edited using Geneious Prime 2023.0.4. (https://www.geneious.com, accessed on 19 January 2023, Biomatters, Auckland, New Zealand), and the obtained sequences were deposited at the National Center for Biotechnology Information (NCBI) GenBank database. Two separate datasets were selected for phylogenetic analyses (Table 1). The SSU gene region was excluded from the phylogenetic analyses due to the limited number of available sequences for Inocybaceae species in the NCBI GenBank nucleotide database and a lack of species-level resolution in the genus Inocybe.
Phylogenetic dataset 1 comprised a total of 241 sequences of four gene regions (ITS, LSU, rpb2, and tef1) from 67 species, covering the genetic diversity of the family Inocybaceae and four outgroup taxa. Sequences were aligned by each locus using MAFFT v7.450 [18,19], available as a Geneious Prime plugin. After being aligned and trimmed, concatenation of ITS, LSU, rpb2, and tef1 was done using Geneious Prime 2023.0.4. The combined phylogenetic dataset 1 contained 3758 characters, including gaps, with 887 characters for ITS, 979 characters for LSU, 738 for rpb2, and 1154 for tef1. The outgroup taxa Crepidotus prostratus, Pleuroflammula tuberculosa, Simocybe phlebophora, and S. serrulata were selected following Matheny et al. [8].
Phylogenetic dataset 2 comprised 138 sequences of two nuclear gene regions (ITS and LSU) from 66 taxa covering the genetic diversity of the genus Inocybe and three outgroup taxa (Pseudosperma fascinosum, P. huginii, and P. notodryinum).
Table
Table 1. Species included in phylogenetic analyses, associated strain/voucher numbers, countries of origin, and GenBank accession numbers. Newly generated sequences are in bold. Abbreviations: HT = holotype, ET = epitype, IT = isotype, PT = paratype.
Table 1. Species included in phylogenetic analyses, associated strain/voucher numbers, countries of origin, and GenBank accession numbers. Newly generated sequences are in bold. Abbreviations: HT = holotype, ET = epitype, IT = isotype, PT = paratype.
TaxaStrain/VoucherCountryITS LSU RPB2 TEF1 Phylog. Dataset Refs.
Auritella hispidaTH10009 PTCameroonKT378203KT378207KT378215MK4261791[8,20]
Auritella spiculosaTH9866 PTCameroonKT378204KT378206KT378214MK4261821[8,20]
Crepidotus prostratusPBM3463/PERTH:08242135AustraliaHQ728537HQ728538 HQ728540 MK426172 1 [8,20]
Inocybe adorabilisSMNS-STU-F-0901582 HTAustriaOK057159OK057159OK078903—1, 2[21]
Inocybe adorabilisSMNS-STU-F-0901641 PTAustriaOK057161OK057161——2[21]
Inocybe aeruginascensJG310508/TENN063936GermanyGU949591MH220256MH249787—1[22]
Inocybe agglutinataWTU:1094
PBM1352		USAKY990521AY038312AY509113—1[14,23,24]
Inocybe agroteraeSMNS-STU-F-0901680 HTGermanyON003436ON003436——2[6]
Inocybe alcisSMNS-STU-F-0901712 ITFinlandOP164083OP164083——2[25]
Inocybe aphroditeanaSMNS-STU-F-0901678 HTGermanyON003432ON003432——2[6]
Inocybe asterospora cf.ZRL20152002ChinaLT716046KY418862KY419008KY4190641[26]
Inocybe astraianaSMNS-STU-F-0901240 HTGermanyMN512321MN512321——2[27]
Inocybe athenanaSMNS-STU-F-0901238 HTGermanyMN512320MN512320——2[27]
Inocybe audensSMNS-STU-F-0901251 HTGermanyMW647616MW647616——2[28]
Inocybe aurantiobrunneaSMNS-STU-F-0001816 ITSpainOP164016OP164016——2[25]
Inocybe beatificaSMNS-STU-F-0901261 HTGermanyMW845857———2[29]
Inocybe bellidianaSMNS-STU-F-0901473 HTGermanyMW845860MW845860——2[29]
Inocybe cacaocolorPBM3790/TENN:067022 ITAustraliaKJ778845KJ756464KJ756422—1[30]
Inocybe caesaraugustaeAH 56200 HTSpainOL352083———2[31]
Inocybe carissimaSMNS-STU-F-0901701 HTGermanyOP164058OP164058——2[25]
Inocybe carolinensisPBM3906/TENN067756 PTUSAKP636853KP171055KM555147—1[32]
Inocybe chalcodoxanthaWTU F-043333 ITCanadaNR_119900———2[33]
Inocybe coriaceaSMNS-STU-F-0901683 HTGermanyON003439ON003439——2[6]
Inocybe corydalinaAM10687
TURA6488		Russia
Belgium		MH216083AY038314AY337370—1[32]
Inocybe cuniculinaKR-M-0043257 HTNetherlandsMN625273MN625273——2[27]
Inocybe curcuminaKR-M-0042332 HTGermanyMH366621———2[34]
Inocybe cygneaSMNS-STU-F-0901671 HTGermanyON003447ON003447——2[6]
Inocybe derbschiiKR-M-0005011 HTGermanyMG012466———2[34]
Inocybe devinaSMNS-STU-F-0901659 HTGermanyON003423ON003423——2[6]
Inocybe drenthensisSMNS-STU-F-0901477 HTNetherlandsMW845869MW845869——2[29]
Inocybe dryadianaSMNS-STU-F-0901259 HTGermanyMW845873MW845873——2[29]
Inocybe dulciolensPBM2646/TENN 062477 HTUSAMH216088MH220265MH249796—1[32]
Inocybe dvalinianaSMNS-STU-F-0901559 HT
CNF 1/8916 IT		AustriaMW647624MW647624OQ587951—1, 2[28], This study
Inocybe elysiiSMNS-STU-F-0901682 HTGermanyON003438ON003438——2[6]
Inocybe erinaceomorphaJV14756F/TURA7645SwedenMH216089MH220266MH249797—1[32]
Inocybe flavoalbidaPBM3768/TENN:067000 ITAustraliaKJ729873KJ729901KJ729932MK4261831, 2[30]
Inocybe flocculosaEL10605FinlandAM882992AM882992——2[34]
Inocybe flocculosa cf.ZRL20151789ChinaLT716045KY418861KY419007KY4190631[26]
Inocybe freyaeSMNS-STU-F-0901673 HTGermanyON003431ON003431——2[6]
Inocybe fuscicothurnataPBM3980/TENN:068940USAMF487844KY990485MF416408MK4261841[23]
Inocybe fuscidulaEL9505FinlandAM882886AM882886——2[35]
Inocybe ghiblianaSMNS-STU-F-0901256 HTGermanyMW845878MW845878——2[29]
Inocybe glaucescensLVK12144/TENN073754 HTUSAMH216097MH220273MH249804—1[32]
Inocybe grammopodiaKR-M-0044138GermanyMH366590———2[34]
Inocybe griseotardaJ. Poirier n 19901119-01 HTFranceMF361839———2[36]
Inocybe griseovelataEL20906FranceFN550931FN550931——2[35]
Inocybe griseovelataSMNS-STU-F-0901568 ETGermanyMW845942MW845942——2[29]
Inocybe grusianaSMNS-STU-F-0901262 HTGermanyMW845884MW845884——2[29]
Inocybe heterosemenXC98091209 ITFranceOK057119———1, 2[21]
Inocybe humidicolaPBM3719/TENN:066955AustraliaKP171126KJ801181KJ811575MK4261851[8,30]
Inocybe inodoraEL2405NorwayAM882834AM882834——2[35]
Inocybe inodoraSMNS-STU-F-0901438AustriaMT101874MT101874——1, 2[37]
Inocybe iseranensisTR gmb 00981 HTFranceOK057141OK057141——1, 2[21]
Inocybe istriacasp. nov.CNF 1/7323 HTCroatiaOQ550176OQ550175OQ587954OQ5963311, 2This study
Inocybe knautianaSMNS-STU-F-0901491 HTGermanyMW845887MW845887——2[29]
Inocybe kuberaeSMNS-STU-F-0901668 HTGermanyON003427ON003427——2[6]
Inocybe lampetianaSMNS-STU-F-0901494 HTGermanyMW845891MW845891——2[29]
Inocybe langeiKR-M-0038101GermanyOK057121OK057121——1, 2[21]
Inocybe langeiSMNS-STU-F-0900983GermanyOK057205OK057205——2[21]
Inocybe lanuginosaPBM3023/TENN:062780USAHQ232480KP170923KM245992MK4261861[8,30]
Inocybe lasseroidesPBM3749/TENN:066979AustraliaKP171145KP170924KM245993MK4261871[8,38]
Inocybe laurinaSMNS-STU-F-0901247 HTGermanyMN512325MN512325——2[27]
Inocybe lechianaSMNS-STU-F-0901268 HTAustriaMN512330MN512330——2[28]
Inocybe lucisSMNS-STU-F-0901616 HTGermanyON003441ON003441——2[6]
Inocybe luteifoliaAHS6557 IT
PBM2642		USAFJ436331EU307814EU307816MK4261881, 2[8,39]
Inocybe magnifoliaMCA2441 HTGuyanaJN642228JN642244EU600899MK4261891[40]
Inocybe melanopusPBM3975/TENN:068973USA—MH220276MH249807MK4261901[8,32]
Inocybe morganaeSMNS-STU-F-0901459 HTAustriaOK057143———1, 2[21]
Inocybe morganaeSMNS-STU-F-0901608GermanyOK057201OK057201——2[21]
Inocybe morteniiDB19-9-20-5 PTAustriaOP164049OP164049——2[25]
Inocybe mycenoidesSMNS-STU-F-0901647GermanyOK057156OK057156OK078899—1[21]
Inocybe napipesEL6105
PBM 2376		NorwayAM882926AY239024AY337390—1[14,35]
Inocybe ochroalbaSMNS-STU-F-0901590FinlandOK057137OK057137OK078918—1[21]
Inocybe ochroalbaEL5704SwedenAM882882AM882882——2[34]
Inocybe orioliSMNS-STU-F-0901703 HTGermanyOP164074OP164074——2[25]
Inocybe orionisSMNS-STU-F-0901455 HTGermanyMW845898MW845898——2[29]
Inocybe pallidicremeaPBM2039
PBM2744/TENN:06252		USAKY990553AY380385AY337388MK4261911[8,14]
Inocybe perchtanaSMNS-STU-F-0901245 HTAustriaMN512326MN512326——2[27]
Inocybe persicinipesPBM2197/PERTH:07676727 HTAustraliaKF977215EU600837EU600836MK4261921[8,41]
Inocybe pholiotinoidesSMNS-STU-F-0901702GermanyOP164095———2[25]
Inocybe pileosulcataTBGT:10742IndiaKP308810KP170979KM406218MK4261931[8,30,38]
Inocybe pipilikaeSMNS-STU-F-0901539 HTAustriaMW647629MW647629——2[28]
Inocybe pluvialisPBM3228/TENN:067042 PTAustraliaKF871777KF853401KF891954MK4261941[8,30]
Inocybe pseudodestrictaKR-M-0043223NetherlandsMH366594———2[34]
Inocybe pseudodestrictaPRM716231 HTCzechiaMG012468———2[34]
Inocybe pseudoscabelliformisSMNS-STU-F-0901634GermanyOK057172OK057172OK078908—1[21]
Inocybe pusio cf.DB16-8-14-24GermanyMH366588———2[34]
Inocybe queletiiKR-M-0038286GermanyMT101893———1, 2[37]
Inocybe relicinaIB19920112
JV10258		New Zealand
Finland		AF325664AY038324AY333778—1[24,42]
Inocybe roseifoliaCO5576USAMH578026MK421968MH577441MK4261951[8,32]
Inocybe roseipes cf.MCVE 9856ItalyJF908143———2[43]
Inocybe rufobadiaNLB885/PERTH:08320454 HTAustraliaKF977213KF915290KF991385MK4261961[8,30]
Inocybe scolopacisSMNS-STU-F-0901527 HTGermanyMW845913MW845913——2[29]
Inocybe serrataPBM3235/TENN:069659AustraliaKP636810KP171012KM555111MK4261971[8,30]
Inocybe solianaSMNS-STU-F-0901664 HTGermanyON003425ON003425——2[6]
Inocybe somaeSMNS-STU-F-0901652 HTGermanyOK057148OK057148OK078901—1[21]
Inocybe spadiceaPBM2203/E7051 PTAustraliaKP636866EU600865—MK4261981[8,30,41]
Inocybe sphaerospora cf.ZRL20151281ChinaLT716044KY418860KY419006KY4190621[26]
Inocybe subexilisACAD11680
PBM2620		Canada
USA		MH578001EU307845EU307847MK4261991[8,39]
Inocybe subhirtellaSMNS-STU-F-0901586GermanyOK057133OK057133OK078915—1[21]
Inocybe substramineaMCVE 21445ItalyJF908170———1, 2[43]
Inocybe tardaSMNS-STU-F-0901730 ETGermanyOP164094OP164094——2[25]
Inocybe thailandicaDED8049 HTThailandGQ893013GQ892968KM656129MK4262001[8,38]
Inocybe tiburtinaSMNS-STU-F-0901565 HTGermanyMW845939MW845939——2[29]
Inocybe torresiaeTENN:067011 PT
PBM2157/E6978 HT		AustraliaKP641635EU600874EU600873—1[30,38,41]
Inocybe trolliiCNF 1/8917 ITGermanyOQ550174OQ550177OQ587952OQ5963331This study
Inocybe trolliiSMNS-STU-F-0901674 HTGermanyON003430ON003430——2[6]
Inocybe tubarioidesTENN61324
PBM2550		USAEU439453AY732211EU307855MK4262011[8,39]
Inocybe tyriiSMNS-STU-F-0901679 HTGermanyON003434ON003434——2[6]
Inocybe urceolicystisSMNS-STU-F-0901615FinlandOK057175OK057175OK078914—1[21]
Inocybe venustissimaKR-M-0042322 HTAustriaMH366625———1, 2[34]
Inocybe venustissimaKR-M-0042323 PTAustriaMH366626———2[34]
Inocybe venustissimaKR-M-0042323 PT
CNF 1/8918		AustriaOQ550173OQ550172OQ587953OQ5963321This study
Inocybe venustissimaSFC20200716-08South KoreaON059521———1, 2[44]
Inocybe venustissima
(as I. auricoma)		UBC F19796CanadaHQ604526HQ604526——1, 2unpubl.
Inocybe woglindeanaSMNS-STU-F-0901435 HTGermanyMT101882MT101882——1, 2[37]
Inocybe zethiSMNS-STU-F-0901456 HTGermanyON003440ON003440——2[6]
Inosperma calamistratumSAT9826004USAJQ801387JQ815410JQ846467MK4262041[8,45]
Inosperma rimosoidesPBM2459USADQ404391AY702014DQ385884DQ4357901[8,46]
Inosperma virosumTBGT753 PTIndiaKT329452KT329458KT329446MK4262081[8,47]
Mallocybe myriadophyllaJV19652FFinlandDQ221106AY700196AY803751DQ4357911[46]
Mallocybe terrigenaEL11704
JV16431		Sweden
Finland		AM882864AY380401AY333309—1[14,35]
Mallocybe tomentosulaPBM4138/TENN:071837USAMG773814MK421969MH577506MK4262101[8,32]
Nothocybe distinctaCAL1310 HT
ZT9250 PT		IndiaKX171343EU604546EU600904MK4262121[8,41,48]
Pseudosperma bulbosissimumDBG19916USAMH024849MH024885MH249788MK4262131[8,32]
Pseudosperma fascinosumSMNS-STU-F-0901666 HTGermanyON003426———2[6]
Pseudosperma huginiiSTU:SMNS-STU-F-0901564 HTAustriaMW647628———2[28]
Pseudosperma notodryinumCO4463/CSU 01252USAMH578028MK421970MH577509MK4262161, 2[8,32]
Pseudosperma sororiumMCA859
PBM3901		USAJQ408772MH220278MH249810MK4262181[8,32]
Simocybe phlebophoraPBM3089/PDD:97898New ZealandMK421963MK421967MK415449—1[8]
Simocybe serrulataPBM2536USADQ494696AY745706DQ484053GU1877551[49]
Tubariomyces inexpectatusAH25500 PT
AH20390 HT		SpainGU907095EU569855GU907088—1[38,41,50]
Tubariomyces sp. BB6018ZambiaMK421965EU600887EU600886MK4262201[8,38,41]
Sequences were aligned by each locus, and concatenation was done as indicated above. The concatenated alignment of ITS and LSU (Phylogenetic dataset 2) sequences contained 1766 characters, including gaps, with 832 characters for the ITS and 934 for the LSU gene region.
Phylogenetic analyses of concatenated ITS–LSU–rpb2–tef1 and ITS–LSU sequence alignments were conducted using Maximum likelihood (ML) analysis in IQTREE v1.6.12 [51,52] and Bayesian inference (BI) analysis in MrBayes 3.2.6 (Geneious plugin, [53]). The best model was selected by ModelFinder implemented in IQ-TREE, considering separately the corrected Akaike and Bayesian Information Criterion (cAIC, BIC). GTR + F + I + G4 was selected as the best model for both phylogenetic datasets. ML analyses were executed by applying the ultrafast bootstrap approximation with 1000 replicates. BI analyses were executed for 10,000,000 generations, sampling trees and other parameters every 10,000 generations. The default number of chains (four) and heating parameters were used. Posterior probabilities (BPP) were calculated after burning the first 25% of the posterior sample. Phylogenetic trees were visualized and annotated using iTOL v6.5.4 [54] and FigTree 1.4.3 (http://tree.bio.ed.ac.uk/software/figtree/, accessed on 17 Februrary 2023).

3. Results

3.1. Molecular Phylogenetic Analyses

A total of 14 DNA sequences (three ITS, three LSU, four rpb2, three tef1, and one SSU) from four Inocybe species were newly generated in this study. In addition to sequencing five gene regions (SSU, ITS, LSU, rpb2, and tef1) of I. istriaca (CNF 1/7323), the isotype of I. trollii (voucher CNF 1/8917, holotype SMNS-STU-F-0901674) and the paratype of I. venustissima (CNF 1/8918, part of KR-M-0042323) were resequenced for ITS and LSU and newly sequenced for rpb2 and tef1 gene regions. The isotype of I. dvaliniana (CNF 1/8916, holotype SMNS-STU-F-0901559) was sequenced for the rpb2 gene region. The accession numbers of all newly generated sequences used in phylogenetic analyses are marked in bold (Table 1). The ITS sequence from the holotype of Inocybe istriaca (accession number: OQ550176) was BLAST searched against NCBI GenBank’s nucleotide database. The closest two hits were sequences of I. venustissima SFC 20200716-08 (accession number: ON059521, identity 80.96%) and I. trollii SMNS-STU-F-0901674 (accession number: ON003430, identity 80.03%), considering data from published sources only.
Phylogenetic trees generated from BI and ML analyses of the concatenated ITS–LSU–rpb2–tef1 sequence alignment were identical in topology and were presented as a single phylogenetic tree in Figure 1. Phylogenetic trees generated from BI and ML analyses of the concatenated ITS–LSU sequence alignment were also identical in topology and were presented as a single phylogenetic tree in Figure 2. Only significant branch support values were presented at the nodes (Bayesian posterior probability (BI-PP ≥ 0.95) and ultrafast bootstrap support (ML-BP ≥ 70%)).
A four-gene region (ITS, LSU, rpb2, tef1) phylogenetic analysis has shown a total of seven strongly supported branches (BI-PP ≥ 0.95, ML-BP ≥ 70) representing seven genera within the family Inocybaceae, which recovered as two monophyletic groups (Inocybe-Nothocybe-Pseudosperma and Inosperma-Mallocybe-Tubariomyces-Auritella) (Figure 1). The genus Inocybe was recovered as a strongly supported (BI-PP = 1, ML-BP = 100) monophyletic group, including many poorly supported (BI-PP < 0.95, ML-BP < 70) short internodes in both phylogenetic analyses (Figure 1 and Figure 2).
Among the 53 Inocybe species in the four-gene analyses and the 66 Inocybe species in the two-gene analyses, I. istriaca was recovered as a single stem lineage, confirming its status as an independent new species. In both analyses, I. istriaca was nested in a strongly supported (BI-PP = 1, ML-BP = 100) monophyletic clade that included I. venustissima and its sister species, I. chalcodoxantha (analyzed only in the ITS-LSU phylogeny). In the four-gene phylogeny, I. dvaliniana and I. trollii clustered together with I. adorabilis, I. pseudoscabelliformis, and I. urceolicystis (Figure 1) in a strongly supported monophyletic group (BI-PP = 1, ML-BP = 100) sister to a clade composed of I. venustissima and I. istriaca.

3.2. Taxonomy

Inocybe istriaca Mešić, Tkalčec, Pošta, Pole and Bandini, sp. nov. (Figure 3, Figure 4 and Figure 5).
Mycobank MB847017
Typification: CROATIA. ISTRIA COUNTY: Brijuni National Park, Veli Brijun Island, 44.90711° N, 13.75436° E, on the edge of lawn and forest of Quercus ilex, 15 November 2016, A. Mešić and Z. Tkalčec (holotype, CNF 1/7323; isotype, SMNS-STU-F-0901784). GenBank (ex-holotype DNA isolate): SSU = OQ598554, ITS = OQ550176, LSU = OQ550175, rpb2 = OQ587954, tef1 = OQ596331.
Etymology: referring to the Istria peninsula, where the holotype was collected.
Pileus 19–35 mm wide, convex, campanulato-convex, or plano-convex with a broadly subumbonate center, margin mostly deflexed (slightly inflexed when young), entire, occasionally shortly radially splitted, surface dry, rather finely to coarsely radially fibrillose, sparsely woolly-squamulose in places, partially fibrillose-rimulose near margin, smooth to subtomentose-subsquamulose around the center, pale to light yellowish- to orangish-brown, fibrils and tufts often darker, medium orange- to red-brown, when young with whitish, narrow patches of universal veil at the margin, later evanescing, velipellis faint in mature basidiomata. Lamellae narrowly adnate to deeply emarginate, ventricose, moderately crowded, white at first, then pale greyish-brown (beige), later light brown, edge entire to eroded, whitish, concolorous, or brownish. Stipe 25–30 × 4–7 mm, subcylindrical with a slightly to distinctly broadened base (up to 10 mm, often submarginate), solid to narrowly fistulose, surface dry, longitudinally fibrillose-striate, pale to light brown, flocculose, and white at the apex. Context: white to whitish, not changing color on bruising. Smell weak, acidic fruity when cut. Taste is not recorded.
Basidiospores [200/4/1] (7.8–)8.4–10.2–11.9 × 5.2–6.2–7.2 μm, averages of different basidiomata 10.1–10.3 × 6.1–6.3 μm, Q = (1.30–)1.37–1.64–1.95(–2.05), av. Q = 1.64–1.65; in frontal view mostly ellipsoid, also ovoid or oblong, with rounded to subacute base and rounded to acute apex; in side view (sub)amygdaliform, (sub)phaseoliform, ellipsoid or oblong, with rounded to acute base and apex; smooth, germ-pore apical and indistinct (visible as a lighter spore wall) or absent, thin-walled to slightly thick-walled (up to 0.6 μm), pale yellow-brown in KOH and H2O, non-amyloid and non-dextrinoid.
Basidia [50/4/1] 36–40.2–45 × 9–11.4–15 μm, Q = (2.7–)3.0–3.6–4.2(–4.6), clavate, predominantly 4-spored, occasionally 2-spored, thin-walled, mostly hyaline, rarely brownish. Pleurocystidia metuloid, [60/4/1] 50–65.2–80 × 14–20.3–34 μm, Q = 1.59–3.36–4.50, scattered, very variable in shape, but mostly (sub)fusiform, also clavate to broadly clavate, (sub)utriform, (elongate) ellipsoid, subcylindrical, or somewhat deformed (e.g., curved to one side), usually without or with only a short neck, with a short to very long tapering pedicel, in alkaline solutions mostly (sub)hyaline, sometimes with pale yellow-brown cytoplasmic pigment, with strongly to weakly developed crystals at the apex (soluble in KOH, rarely lacking), moderately thick-walled to thick-walled (up to 1 μm in the middle, up to 1.5 μm at the apex). The lamellar edge mostly sterile, at places covered with abundant dark brown to black resinous substance. Cheilocystidia of two types: (a) metuloid, similar to pleurocystidia in size and shape, sometimes with crystals at the apex, abundant; and (b) paracystidia, mostly clavate or subcylindrical, hyaline, thin-walled to moderately thick-walled (up to 0.8 μm), scattered to abundant. Pileipellis a cutis, composed of a superficial layer of repent, thin-walled, (sub)hyaline, cylindrical, 2–5 μm wide velipellis hyphae and a lower layer of gradually shorter and wider, thin-walled hyphae with parietal to encrusted brown pigment. Stipitipellis a cutis, composed of repent, thin-walled, ca. 2–10 μm wide hyphae, sometimes with brown, parietal to minutely encrusted pigment. Caulocystidia very abundant in the upper 2–3 mm of stipe length, in clusters or in dense groups, gradually becoming rare, more simple-shaped, or as caulocystidioid hairs toward the middle of the stipe, absent from the bottom half of the stipe; at places heavily agglutinated by a dark brown to black resinous substance; similar to cheilocystidia, very variable, fusiform, narrowly to broadly utriform, (sub)cylindrical, clavate, sometimes septate (up to 3-celled), apex occasionally (sub)capitate, sometimes with apical crystals, hyaline, sometimes with brown parietal pigment, thin- to moderately thick-walled (up to ca. 1 μm); 15–60 × 5–20 μm. Clamp connections present, conspicuous, and rather abundant in all tissues.
Distribution and ecology: Known only from the holotype collection. Found on the island of Veli Brijun in Brijuni National Park, the northern Adriatic Sea, the Mediterranean region of Croatia, Europe. Ectomycorrhizal, on the edge of the mature thermophilous Quercus ilex forest and a lawn grazed by large herbivores (fallow deer [Dama dama], axis deer [Axis axis], and European mouflon [Ovis gmelini musimon]). Basidiomata growing epigeous on calcareous soil (terra rossa on limestone bedrock) covered with short grasses, mosses, and sparse holm oak litter, about 130 m from the sea coast.

4. Discussion

Inocybe istriaca, described here as new to science, has some remarkable micromorphological characters that distinguish it well from the other members of the genus. Most Inocybe s.s. species possess basidia that are 20–35(–40) × 7–12 μm in size (with a few exceptions, e.g., 28–43 × 8–12 μm in I. fraudans (Britzelm.) Sacc. [55,56], while basidia of I. istriaca are mostly larger (36–45 × 9–15 μm). Additional striking characters are the presence of a dark brown to black resinous substance on the lamellar edge and stipe apex, which covers and agglutinates cheilocystidia and caulocystidia. However, these features need to be evaluated more thoroughly when additional collections of this species become available. Other important morphological characters are: pale to light brown, radially fibrillose pileus with faint velipellis; apically flocculose, subcylindrical stipe with slightly to distinctly broadened, often submarginate base; color of the context unchanged upon bruising; weak, fruity acidic smell; medium-sized, smooth, in frontal view mostly ellipsoid and in side view amygdaliform or phaseoliform basidiospores (ca. 8.5–12 × 5–7 μm); pleurocystidia metuloid, crystalliferous, very variable, mostly (sub)fusiform, with short to very long pedicel, without or with short neck, walls apically up to 1.5 μm wide and not yellowing in alkaline solutions; cheilocystidia of two types (metuloid and leptocystidia); and presence of abundant clustered caulocystidia only in the upper 2–3 mm of stipe length.
In addition to the large basidia and the dark resinous substance on the lamellar edge and stipe apex, the most important taxonomic characters used to distinguish I. istriaca and related species are presented in Table 2. The molecular analyses performed in this study show that I. venustissima Bandini and B. Oertel and I. chalcodoxantha Grund and D.E. Stuntz are phylogenetically most closely related to I. istriaca and form a well-supported sister clade. Inocybe venustissima has somewhat larger basidiomata (pileus 20–50 mm broad, stipe 30–100 mm long), waxy shiny glabrous to rim(ul)ose pileus surface, stipe mostly with large roundish bowl-shaped bulbous base and often pruinose on the entire length (though sparsely in the lower half), somewhat smaller spores, and on average shorter pleurocystidia (50 μm vs. 65 μm in I. istriaca). It is known from montane to subalpine forests in Austria, growing near small brooks or rivulets under Picea abies on acidic soil, and from Canada near Tsuga heterophylla [34]. Inocybe chalcodoxantha Grund and D.E. Stuntz, known from coniferous forests in Canada and the USA (Washington), has a much longer stipe (up to 100 mm), a strong spermatic smell, somewhat smaller basidiospores, and thicker-walled pleurocystidia (up to 3.3 μm) [57].
In addition, from other morphologically similar species, I. adorabilis Bandini, B. Oertel, and U. Eberh. differs by having an entirely pruinose stipe (but sparsely so in the lower half), a spermatic smell, somewhat smaller basidiospores, and shorter pleurocystidia with an often rounded base and thicker walls (up to 3.5 (–4.5) μm). It is known only from subalpine areas in Austria, where it grows near Picea abies [21]. Inocybe audens Bandini, Christan, and Dondl differs by larger basidiomata (pileus 20–60 mm broad, stipe 30–80 mm long), a more glabrous pileus surface, somewhat shorter spores, and pleurocystidia with much thicker walls (up to 5.0(–6.0) μm). It occurs under coniferous trees (Picea abies, Abies alba, Larix, etc.) and develops basidiomata very early in the year (April–May) [28].
Inocybe inodora Velen. has an entirely pruinose stipe (but often sparsely so in the lower half), larger spores, and thicker-walled pleurocystidia (up to 3.0(–4.0) μm). It is widely distributed in Europe, growing mostly in ectomycorrhiza with deciduous trees [37,55,56,59,60]. Inocybe morganae Bandini, B. Oertel, and U. Eberh has an entirely pruinose stipe (but often sparsely so in the lower half), a smell reminding of bitter almonds, and somewhat shorter pleurocystidia, which are yellowish-greenish in KOH. It is known from the montane regions of Austria and Germany where it occurs near Picea abies [21]. Inocybe queletii Konrad differs by having larger basidiomata (pileus 30–60 mm broad), a spermatic smell, and thicker-walled pleurocystidia (up to 3.0 μm). It forms ectomycorrhizae with Abies alba in montane areas [55,56,62]. Inocybe substraminea Alessio differs by much larger basidiomata (pileus 50–80(–120) mm broad, stipe 60–100 × 8–15 mm), an equal or hardly widened stipe base (never submarginate), and thicker-walled pleurocystidia. It is known from submontane habitat with Fagus sylvatica in Italy [63]. Inocybe trollii Bandini and B. Oertel has a minutely lanose (woolly) pileus surface and smaller pleurocystidia with thicker walls turning yellow-green in KOH. It is known only from the type locality in Austria, where it grows with Pinus sylvestris, Corylus avellana, and Populus sp. [6]. Inocybe woglindeana Bandini, Vauras, and Weholt has an abundant velipellis, somewhat longer basidiospores (up to 14.3 μm), and often somewhat “sac-shaped” pleurocystidia with a rounded or truncate base. It grows mostly under deciduous trees on sandy or gravelly soil, always with Salix (mostly S. caprea) nearby [37]. Further three taxa, I. langei R. Heim, I. iseranensis E. Ferrari, and I. heterosemen Carteret and Reumaux, can be easily distinguished from I. istriaca by their smaller basidiospores (up to ca. 9.5 × 6 μm) and shorter pleurocystidia (up to ca. 60 μm), which are usually thicker-walled [21,58,61].

Author Contributions

Conceptualization, A.P., A.M. and Z.T.; methodology, A.P., D.B., A.M. and Z.T.; formal analysis, A.P., D.B., A.M., L.P. and Z.T.; investigation A.P., D.B., A.M., L.P. and Z.T.; resources A.M., I.K., N.M. and Z.T.; data curation A.P., A.M. and Z.T.; writing—original draft preparation, A.P. and A.M.; writing—review and editing, A.P., D.B., A.M., L.P., I.K., N.M., O.M. and Z.T.; visualization, A.P., Z.T. and D.B.; supervision, A.M. and Z.T.; project administration, A.M.; funding acquisition, A.M. All authors have read and agreed to the published version of the manuscript.

Funding

This work was fully supported by the Croatian Science Foundation under the ForFungiDNA project grants HRZZ-IP-2018-01-1736 (to A.P., A.M., L.P., I.K., N.M. and Z.T.), HRZZ-DOK-2018-09-7081 (to A.P.), and HRZZ-DOK-2021-02-4010 (to L.P.).

Institutional Review Board Statement

Not applicable.

Data Availability Statement

Sequences generated in this study are submitted to the GenBank database of NCBI (https://www.ncbi.nlm.nih.gov/genbank/, accessed on 20 January 2023).

Acknowledgments

A.M. and Z.T. are grateful to Sandro Dujmović, former director of Brijuni National Park, for research support, to Martina Hervat for help with the literature, and to Alena Sprčić for allowing us to use photographs from the Public Institution Brijuni National Park website for the preparation of Figure 2 (background photo produced by 4Film company).

Conflicts of Interest

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analysis, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

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Figure 1. Phylogenetic tree of the family Inocybaceae based on Bayesian Inference (BI) and Maximum Likelihood (ML) analyses of the concatenated four-gene (ITS, LSU, rpb2, tef1) sequence alignment. Significant branch support values, Bayesian posterior probability (BI-PP ≥ 0.95), and ultrafast bootstrap support (ML-BP ≥ 70%), are presented at the nodes. The newly proposed species, Inocybe istriaca, is marked in red and in bold font. Abbreviations: HT = holotype; IT = isotype; PT = paratype.
Figure 1. Phylogenetic tree of the family Inocybaceae based on Bayesian Inference (BI) and Maximum Likelihood (ML) analyses of the concatenated four-gene (ITS, LSU, rpb2, tef1) sequence alignment. Significant branch support values, Bayesian posterior probability (BI-PP ≥ 0.95), and ultrafast bootstrap support (ML-BP ≥ 70%), are presented at the nodes. The newly proposed species, Inocybe istriaca, is marked in red and in bold font. Abbreviations: HT = holotype; IT = isotype; PT = paratype.
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Figure 2. Phylogenetic tree of the genus Inocybe based on Bayesian Inference (BI) and Maximum Likelihood (ML) analyses of the concatenated two-gene (ITS, LSU) sequence alignment. Significant branch support values, Bayesian posterior probability (BI-PP ≥ 0.95) and ultrafast bootstrap support (ML-BP ≥ 70%), are presented at the nodes. The newly proposed species, Inocybe istriaca, is marked in white and bold font. Abbreviations: HT = holotype; ET = epitype; IT = isotype; PT = paratype.
Figure 2. Phylogenetic tree of the genus Inocybe based on Bayesian Inference (BI) and Maximum Likelihood (ML) analyses of the concatenated two-gene (ITS, LSU) sequence alignment. Significant branch support values, Bayesian posterior probability (BI-PP ≥ 0.95) and ultrafast bootstrap support (ML-BP ≥ 70%), are presented at the nodes. The newly proposed species, Inocybe istriaca, is marked in white and bold font. Abbreviations: HT = holotype; ET = epitype; IT = isotype; PT = paratype.
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Figure 3. Inocybe istriaca sp. nov. (CNF 1/7323, holotype). Basidiomata. Bar = 10 mm. Authors: A. Mešić and Z. Tkalčec.
Figure 3. Inocybe istriaca sp. nov. (CNF 1/7323, holotype). Basidiomata. Bar = 10 mm. Authors: A. Mešić and Z. Tkalčec.
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Figure 4. Inocybe istriaca sp. nov. (CNF 1/7323, holotype). (A,B) Basidiospores. (C) Pleurocystidia. (D,E) Cheilocystidia. (F) Caulocystidia. (G) Black resinous substance among caulocystidia. Bars: (A,B) = 5 μm; (C–F) = 20 μm; (G) = 50 μm. Author: A. Mešić.
Figure 4. Inocybe istriaca sp. nov. (CNF 1/7323, holotype). (A,B) Basidiospores. (C) Pleurocystidia. (D,E) Cheilocystidia. (F) Caulocystidia. (G) Black resinous substance among caulocystidia. Bars: (A,B) = 5 μm; (C–F) = 20 μm; (G) = 50 μm. Author: A. Mešić.
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Figure 5. Inocybe istriaca sp. nov. (CNF 1/7323, holotype). (A) Basidiospores. (B) Pleurocystidia. (C) Cheilocystidia of metuloid-type. (D) Cheilocystidia of paracystidia-type. (E) Caulocystidia of metuloid-type. (F) Caulocystidia of paracystidia-type. Bars: (A) = 10 μm; (B–F) = 50 μm. Del. D. Bandini.
Figure 5. Inocybe istriaca sp. nov. (CNF 1/7323, holotype). (A) Basidiospores. (B) Pleurocystidia. (C) Cheilocystidia of metuloid-type. (D) Cheilocystidia of paracystidia-type. (E) Caulocystidia of metuloid-type. (F) Caulocystidia of paracystidia-type. Bars: (A) = 10 μm; (B–F) = 50 μm. Del. D. Bandini.
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Table
Table 2. Overview of the main taxonomic characters used for delimitation between Inocybe istriaca and related species.
Table 2. Overview of the main taxonomic characters used for delimitation between Inocybe istriaca and related species.
SpeciesSpore Size (μm)Pleurocystidia Size (μm)Pleuroc. Thick at Apex (μm), Walls Colour (KOH) HabitatReferences
Inocybe istriaca8.4–10.2–11.9 × 5.2–6.2–7.2 50–65–80 × 14–20–34up to 1.5, (sub)hyalineMediterranean forest of Quercus ilex, edge with grassland This study
I. adorabilis8.0–8.9–9.9 × 4.6–5.1–5.6 37–54–69 × 11–15–22 up to 3.5(–4.5), yellowish-greenishsubalpine forest, Picea abies[21]
I. audens7.8–9.2–10.5 × 5.0–5.8–6.741–60–72 × 11–16–25up to 5.0(–6.0), (sub)hyaline to light yellowish-greenishunder coniferous trees, Picea abies, Abies alba, Larix, etc.[28]
I. chalcodoxantha7.5–10 × 5–6.5, mostly 9 × 5.550–72 × 13–211.0–3.3, hyalineunder conifers, in moss or needles [57]
I. heterosemen6.5–7.6–8.1 × 3.5–4.2–4.829–38–49 × 12–16–20 up to 2.5(–3.5), yellowish-greenishmostly deciduous forests, Salix, Betula pubescens, Populus tremula, Alnus, etc.[21,58]
I. inodora9.0–11.0–12.8 × 5.2–6.2–7.4 *
10.0–14.0 × 5.5–7.0 **		44–59–68 × 12–18–25up to 3.0(–4.0), yellowish-greenishmostly under deciduous trees[37], [55] **, [56] *, [59,60]
I. iseranensis7.5–8.3–9.4 × 4.7–5.0–5.737–46–58 × 14–16–18up to 1.5(–2.5), yellowish-greenishalpine regions, Salix herbacea, Betula nana, B. pubescens[21,61]
I. langei6.4–7.0–8.0 × 3.8–4.4–5.0 *
7.0–9.0 × 4.5–5.0(–5.5) **		35–47–57 × 9–12–15 *
40–60 × 13–20 **		up to 3.0(–3.5), (pale) yellowish-greenishmostly with deciduous, but also coniferous trees, Quercus, Salix, Alnus, Picea, Pinus, etc.[21] *, [56] **
I. morganae8.6–9.7–11.2 × 4.9–5.6–6.135–52–66 × 10–16–27 up to 1.5(–2.0), yellowish-greenishmontane regions, Picea abies[21]
I. queletii(8–)8.5–12(–13) × 5.8–756–75 × 13–22(–25)up to 3, hyalinemontane regions, Abies alba[55,56,62]
I. substraminea(9–)10–12(–13.5) × 5–655–75 × 15–22n/asubmontane forest, Fagus sylvatica[63]
I. trollii8.0–9.6–11.1 × 5.0–5.6–6.644–53–60 × 11–14–19up to 2.0(–2.5), yellow-greenunder Pinus sylvestris, Corylus avellana, Populus sp.[6]
I. venustissima7.3–8.9–10.9 × 4.6–5.3–6.7 35–50–76 × 11–16–23up to 1.5(–2.0), weak, pale yellowish-greenishmontane to subalpine forests, Picea abies (Austria); Tsuga heterophylla (Canada); Larix kaempferi (Korea)[34,44]
I. woglindeana8.0–10.2–13.0 × 4.9–5.9–7.1 *
9.0–11.3–14.3 × 5.3–6.3–7.4 **		35–57–77 × 12–19–31 *
51–67–82 × 15–20–30 **		up to 1.0, pale yellowmostly under deciduous trees, always Salix (S. caprea), mixed with Betula, Populus, Pinus sylvestris, etc.[37] Germany *, Finland **
* and ** are connected with the references.
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Pošta, A.; Bandini, D.; Mešić, A.; Pole, L.; Kušan, I.; Matočec, N.; Malev, O.; Tkalčec, Z. Inocybe istriaca sp. nov. from Brijuni National Park (Croatia) and Its Position within Inocybaceae Revealed by Multigene Phylogenetic Analysis. Diversity 2023, 15, 755. https://doi.org/10.3390/d15060755

AMA Style

Pošta A, Bandini D, Mešić A, Pole L, Kušan I, Matočec N, Malev O, Tkalčec Z. Inocybe istriaca sp. nov. from Brijuni National Park (Croatia) and Its Position within Inocybaceae Revealed by Multigene Phylogenetic Analysis. Diversity. 2023; 15(6):755. https://doi.org/10.3390/d15060755

Chicago/Turabian Style

Pošta, Ana, Ditte Bandini, Armin Mešić, Lucia Pole, Ivana Kušan, Neven Matočec, Olga Malev, and Zdenko Tkalčec. 2023. "Inocybe istriaca sp. nov. from Brijuni National Park (Croatia) and Its Position within Inocybaceae Revealed by Multigene Phylogenetic Analysis" Diversity 15, no. 6: 755. https://doi.org/10.3390/d15060755

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