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

: 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 µ m), large basidia (36–45 × 9–15 µ m), mostly (sub)fusiform and weakly thick-walled (up to 1.5 µ m) 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.


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 km 2 ) 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 km 2 ), 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,

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 (NH 4 OH). 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).

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 ddH 2 O, 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 se-quencing: 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.

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). 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

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%)).
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.
Diversity 2023, 15, x FOR PEER REVIEW 10 Etymology: referring to the Istria peninsula, where the holotype was collected. Pileus 19-35 mm wide, convex, campanulato-convex, or plano-convex with a br subumbonate center, margin mostly deflexed (slightly inflexed when young), entir casionally shortly radially splitted, surface dry, rather finely to coarsely radially fibri sparsely woolly-squamulose in places, partially fibrillose-rimulose near margin, sm to subtomentose-subsquamulose around the center, pale to light yellowish-to oran brown, fibrils and tufts often darker, medium orange-to red-brown, when young whitish, narrow patches of universal veil at the margin, later evanescing, velipellis in mature basidiomata. Lamellae narrowly adnate to deeply emarginate, ventricose, erately crowded, white at first, then pale greyish-brown (beige), later light brown, entire to eroded, whitish, concolorous, or brownish. Stipe 25-30 × 4-7 mm, subcylin with a slightly to distinctly broadened base (up to 10 mm, often submarginate), so narrowly fistulose, surface dry, longitudinally fibrillose-striate, pale to light brown culose, and white at the apex. Context: white to whitish, not changing color on bru Smell weak, acidic fruity when cut. Taste is not recorded. frontal view mostly ellipsoid, also ovoid or oblong, with rounded to subacute bas rounded to acute apex; in side view (sub)amygdaliform, (sub)phaseoliform, ellipso oblong, with rounded to acute base and apex; smooth, germ-pore apical and indi (visible as a lighter spore wall) or absent, thin-walled to slightly thick-walled (up μm), pale yellow-brown in KOH and H2O, non-amyloid and non-dextrinoid. 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 H 2 O, non-amyloid and non-dextrinoid.   -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 thickwalled (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. tered, very variable in shape, but mostly (sub)fusiform, also clavate to broadly cl (sub)utriform, (elongate) ellipsoid, subcylindrical, or somewhat deformed (e.g., curv one side), usually without or with only a short neck, with a short to very long tap pedicel, in alkaline solutions mostly (sub)hyaline, sometimes with pale yellow-brow toplasmic pigment, with strongly to weakly developed crystals at the apex (solu KOH, rarely lacking), moderately thick-walled to thick-walled (up to 1 μm in the m up to 1.5 μm at the apex). The lamellar edge mostly sterile, at places covered with dant dark brown to black resinous substance. Cheilocystidia of two types: (a) met similar to pleurocystidia in size and shape, sometimes with crystals at the apex, abun and (b) paracystidia, mostly clavate or subcylindrical, hyaline, thin-walled to mode thick-walled (up to 0.8 μm), scattered to abundant. Pileipellis a cutis, composed of perficial layer of repent, thin-walled, (sub)hyaline, cylindrical, 2-5 μm wide velipell phae and a lower layer of gradually shorter and wider, thin-walled hyphae with pa to encrusted brown pigment. Stipitipellis a cutis, composed of repent, thin-walled, 10 μm wide hyphae, sometimes with brown, parietal to minutely encrusted pigment locystidia very abundant in the upper 2-3 mm of stipe length, in clusters or in groups, gradually becoming rare, more simple-shaped, or as caulocystidioid hairs to the middle of the stipe, absent from the bottom half of the stipe; at places heavily a tinated by a dark brown to black resinous substance; similar to cheilocystidia, very ble, fusiform, narrowly to broadly utriform, (sub)cylindrical, clavate, sometimes se (up to 3-celled), apex occasionally (sub)capitate, sometimes with apical crystals, hy sometimes with brown parietal pigment, thin-to moderately thick-walled (up to ca. 1 15-60 × 5-20 μm. Clamp connections present, conspicuous, and rather abundant tissues.

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].