The gilled fungi and boletes of Iran: diversity, systematics, and nrDNA data

INTRODUCTION

Gilled fungi and boletes (often referred to as ‘mushrooms’) are among the most prevalent groups of macrofungi. Traditionally they are the best-studied fungi in many parts of the world probably on account of their potential value as a food source but also their conspicuous and often eye-catching fruitbodies. The number of described species worldwide now exceeds the ca. 14,500 species of Chang & Miles (2004) and Kirk et al. (2008). Gilled fungi, also commonly known as agarics, share a structural characteristic, i.e., vertical plates, denoted as lamellae or more colloq-uially as gills (with varying shapes, sizes, thicknesses, distances from each other) which serve as spore-producing tissue on the undersurface of the cap and are a highly efficient way of increasing the surface area of the fertile part of the fungus. Boletes have tubes which are, in effect, simply rolled up gills, with some members of the Boletales having true gills e.g., the genus Phylloporus. According to molecular evolutionary studies, the formation of gills has evolved several times and therefore, is considered to have been a characteristic worth preserving to assist in spore

dispersal and species survival (Hibbett et al. 1997).

Gilled fungi and boletes are very diverse, morphologically, chemically and ecologically. They range in size from a few millimeters in diameter (e.g., species of Mycena, Marasmius and Coprinellus), to more than a metre across the cap (Macrocybe titans, Phlebopus marginatus). Important variable characteristics include the presence or absence of a stipe, of veils, or veil remnants. The species exhibit numerous colors, odors and flavours. Chemically, some gilled taxa e.g., in the genera Amanita, Galerina, and Cortinarius contain a number of the most toxic natural compounds namely amatoxins, phallotoxins and orellanine; boletes do not have such lethal compounds although the genus Rubroboletus has a toxin (bolesatine) that causes severe gastrointestinal problems. In contrast, various species, e.g. Agaricus campestris, Amanita caesarea, and Boletus edulis are considered edible. There are other taxa of the genera Psilocybe, Copelandia, Gymnopilus, Inocybe, Panaeolus, Pholiotina and Pluteus, that produce hallucinogenic substances such as psilocybin and/or psilocin. Ecologically, as symbiotic partners involved in plant development (mycorrhizae), and as decomposers, with enzymes breaking down lignin and cellulose in woody and herbaceous debris, they are critical for carbon recycling and ecosystem functioning. Some species e.g. Armillarialuteobubalina and Moniliophthora perniciosa are pathogenic and/or parasitic and cause destruction of eucalypt forests and cacao crops respectively (Shearer & Tippett 1988, Meinhardt et al. 2008). The substrates also vary, as they grow on soil, wood, dung and litter. Many agarics and boletes also provide food and medicines for humans, and nutrition and habitat for other organisms. Biogeographically, they are widespread in temperate, equatorial, arctic and sub-antarctic ecosystems, possibly wherever a vegetation layer exists.

From a systematic viewpoint, the first comprehensive phylogenetic studies on agarics were published by Moncalvo et al. (2002) and Matheny et al. (2006). They were, and still are, the basis for the next studies of individual clades and groups of agarics worldwide. Concerning boletoid genera, the study by Wu et al. (2014) opened a broad field for defining many new genera. A ten-year phylogenetic overview of the Basidiomycota and related phyla in relation to DNA-based phylogeny was published by Zhao et al. (2017). An extensive study dealing with global patterns of mushroom evolution was recently published by Varga et al. (2019). According to the most recent account of a systematic outline of the Basidiomycota (He et al. 2019), the class Agaricomycetes currently contains 22 orders, of which agarics and boletes are distributed across the six orders Agaricales, Boletales, Cantharellales, Hymenochaetales, Polyporales, and Russulales.

Iran, with a wide elevational range, from 26 m b.s.l. along the southern coastline of the Caspian Sea, to 5,671 m a.s.l. at Damavand Mt. in Central Alborz, harbors diverse climates and habitats. Vast hot and dry deserts with a precipitation of less than 25 mm/yr in central Iran, coincide with narrow sub-tropical humid regions at the northern parts of the country with precipitation exceeding 1,800 mm/yr. In Iran three macrobioclimates have been recognized, namely, mediterranean, tropical and temperate, largely correlating with the three biogeographical regions Irano-Turanian, Saharo-Sindian and Euro-Siberian, respectively (Djamali et al. 2011). Large parts of the country are characterized by continentality, with hot and dry summers, cold and harsh winters, and low precipitation, while forests are mainly confined to the northern and western borders (Zohary 1973, Djamali et al. 2012).

From a biodiversity viewpoint, Iran is at the crossroad of two global biodiversity hotspots, i.e. the Irano-Anatolian and the Caucasus (Mittermeier et al. 2005, Manafzadeh et al. 2017, Noroozi et al. 2019), covering the major mountain ranges of the country. Diverse topography and climate have resulted in the development of diverse flora and vegetation types. More than 8,000 vascular plant species are known from Iran with ca. 32% endemism (Noroozi et al. 2019). The number of woody plants in Iran is about 956 species (Mozaffarian 2005). Desert and semi-desert steppes, halophytes, montane grasslands, shrublands and woodlands, deciduous temperate forests, wetlands and alpine habitats are among the major vegetation types of the country (Noroozi 2020).

Hyrcanian forests, on the northern slopes of the Alborz mountain range, are at the southeastern reaches of the Caucasus biodiversity hotspot. Despite its small area (ca. 5% of the total area of Iran), the majority of the Iranian macromycetes come from the Hyrcanian region (e.g. Ghobad-Nejhad 2011, Ghobad-Nejhad & Hallenberg 2012). Broad-leaved dense forests have developed due to the high precipitation (ca. 700 to 2000 mm/yr) and mild climate of the region. These forests are known to belong to the northern hemisphere Pleistocene glacial refugia, and contain several Arcto-Tertiary elements, such as Celtis australis, Parrotia persica, Pterocarya fraxinifolia and Zelkovacarpinifolia as well as evergreen plants (Zohary 1973, Frey et al. 1999, Akhani et al. 2010, Gholizadeh et al. 2020). The Zagros forests in western Iran are comprised of xerophytic open woodlands of deciduous trees and shrubs mainly dominated by Quercus species and constitute less than 40% of the forest cover in Iran (Sagheb Talebi et al. 2014). Among the few coniferous forests native to Iran, Juniperus excelsa woodlands are noteworthy, sparsely covering the subalpine zone of the Irano-Turanian parts of the Iranian mountains, mostly distributed at 2000 to 3000 m a.s.l.

Macrofungi in Iran are far less studied compared to microfungi. Historically, mycology in Iran has been divided up into five distinct periods, with publications starting from 1860 (Ershad & Zare 2014). While the majority of studies are characterized by records on microascomycetes, the second period in Iranian mycology (1860–1941) also harbors publications containing some agarics and boletes. Interestingly, the oldest publication related to the fungi of Iran (Buhse 1860) contains eight species of agarics and boletes from the country, most of them later recorded in other publications (Table 1). The second oldest literature on Iranian fungi (Rabenhorst 1871) enumerates two species of this group. Later, publications by Petrak (1941, 1940, 1949) alone and jointly with the first Iranian mycologist E. Esfandiari (Petrak & Esfandiari 1941), appear, recording a few species of agarics and boletes from Iran (Table 1).

Most of the fungi of Iran recorded until 2008 (excluding lichenized fungi, clinical fungi, and some other taxa) have been listed in a book by Ershad (2009). In this book, a list of 3229 fungal species has been sorted in alphabetical order, but with no attempt at taxonomic classification. Therefore, to figure out the number of any fungal groups from this book, a full systematic categorization of the records is needed.

With regard to the number of macrobasidiomycetes in Iran, no clear figure is available, but up to now ca. 430 aphyllophoroid species are known from the country, covering polypores (poroids), corticioids, hydnoids, clavarioids and heterobasidiomycetes (Ghobad-Nejhad & Hallenberg 2012, Ghobad-Nejhad & Langer 2017); of these, 132 species are polypores (Ghobad-Nejhad & Bernicchia 2019). Statistics on agarics and boletes recorded from the country are largely lacking. Therefore, the aim of this study is to provide a first account on the diversity of gilled fungi and boletes in Iran, with annotations on their distribution, classification, and nrDNA.

MATERIALS AND METHODS

Species data

The study group includes species commonly known as gilled fungi (agarics) and boletes. Basically, the taxa such as Schizophyllum commune and Daedalea/Daedaleopsis/Lenzites which have some sort of 'gills' (but studied among aphyllophoroid fungi), are not included. Data on agarics and boletes in this study were obtained from a thorough survey of scientific journal articles and books published up to the year 2020 and specimens collected by the first author. Moreover, sequences of Iranian material in GenBank and UNITE databases were incorporated after full identity check (see below). The basidiomata specimens were collected during 2000–2019 in different provinces of Iran. Samples were dehydrated using a mushroom drier or were air-dried. Macro- and micro-morphological characters of the specimens were studied under a binocular and a light microscope, using bright field or phase contrast optics. Microscopy routines generally followed Largent et al. (1977). Several identification keys (e.g., Knudsen & Vesterholt 2008, Justo et al. 2011, Antonín et al. 2013, Zervakis et al. 2014, Mahdizadeh et al. 2016, Voto et al. 2019, etc.), and some of the literature in He et al. (2019) for specific genera were used. A full list of examined specimens is available in Suppl. Table. Vouchers were deposited at Iranian Cryptgamic Herbarium (Index Herbariorum acronym ICH). Species current names and species authorities mostly follow MycoBank (www.mycobank.org), and Index Fungorum (www.indexfungorum.org). Recent or otherwise relevant taxonomic revisions and phylogenetic publications were also consulted. Records published in journals not approved by the Iranian Ministry of Science, Research and Technology (‘black-listed journals’) are not included here.

Molecular study

A clean portion of the spore-producing part of the dried basidiomata was taken for molecular study. For DNA extraction, PCR and sequencing, we generally followed the protocol by Bußkamp et al. (2020): from each basidioma, 1–2 mg of tissue was suspended in 100 μl TE buffer in a 1.5 ml tube. Immediately, for cell lysis, a microwave (600 W) was used twice for 1 min each, with a pause of 30 s followed by cooling the tubes to −20 °C for 20 minutes. The tubes were centrifuged at 10,000 rpm for 5 minutes. For PCR, 100 times diluted portion of the supernatant was used. The PCR primers for amplification of ITS1, 5.8S and ITS2 regions were ITS1F/ITS4 or ITS1/ITS4 (White et al. 1990, Gardes & Bruns 1993). For PCR, a 45 μl master mix (QIAGEN, Hilden, Germany) was used, adding 5 μl of extracted DNA. PCR was performed with initial denaturation at 94°C for 3 min, followed by 29 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 45 s, and extension at 72°C for 60 s. Final elongation was performed at 72°C for 7 min. The D1–D4 domain of nuclear ribosomal large subunit (LSU; 28S) was amplified using primers LR0R and LR5 (Vilgalys & Hester 1990). The PCR thermal cycling for LSU differed from the ITS only by an increased cycle extension time (90 s per cycle). PCR products were visualized on a 1% agarose gel stained with GelRed fluorescence dye (Biotium, Hayward, CA, USA). Bands were cut out and cleaned with QIAquick PCR Purification Kit (QIAGEN, Hilden, Germany). Purified products were sequenced using Sanger sequencing method (Sanger et al. 1977). The obtained DNA sequences were edited using MEGA6 (Tamura et al. 2013) and DNA Sequence Assembler v4 (Heracle BioSoft 2013, www.DnaBaser.com), checked with BLASTn (Altschul et al. 1990) and UNITE (Nilsson et al. 2019), and submitted to GenBank.

We also searched through GenBank and UNITE databases extracting DNA sequences of agarics and boletes obtained from Iranian material, with a special focus on the ITS sequences. The sequences’ associated metadata were carefully checked, and BLASTn searches were performed against sufficiently identified and reliable sequences in those databases. In UNITE, the Species Hypothesis (SH) with 0–1.5% threshold was accepted. Accession numbers with incorrect, ambiguous, or otherwise unverifiable identity were excluded. Selected ITS accession numbers were inserted in Table 1.