AR TICLE Peering Into the Mediterranean Black

IMA FUNGUS · 7(2): 275–284 (2016) P'=++*9Q#"/='U=>=/=> Peering into the Mediterranean black box: \ectomycorrhizas ARTICLE on Marco Leonardi1, Ornella Comandini2, and Andrea C. Rinaldi3 1Department of Life, Health and Environmental Sciences, University of L’Aquila, L’Aquila, Italy 2Department of Life and Environmental Sciences, University of Cagliari, Monserrato (CA), Italy 35#OJ#\"_L=*=?/!\G$_Z"LP¨ Abstract: We describe the morpho-anatomical features of the ectomycorrhizas (ECMs) formed Key words: by ; on Cistus, a genus of about 20 species of woody shrubs typical of the confocal laser scanning microscopy Mediterranean maquis. The description of L. rugatus mycorrhizas on Cistus [ X\ Lactarius description of a species belonging to ; subgen. Pseudogymnocarpi. The ECM identity was maquis [ " G # L. rugatus mycorrhiza is the molecular phylogeny presence of abundant, long “bottle-shaped” cystidia on mantle surface. Indeed, the overwhelming Russulaceae majority of milkcap mycorrhizas are acystidiate. This is the third ;=; mycorrhiza to have been described associated with Cistus, the others being Lactarius cistophilus and L. tesquorum7"E\# principal features of their ECMs, which share host-depending ECM features known for Cistus, but are otherwise distinguishable on the host roots. Comparison of ; ECM with those formed by L. vellereus and L. piperatus on Fagus reveals elevated intrageneric diversity of mycorrhizal structures. Such a diversity is supported by analysis of ITS sequences of relevant species within European ;species. Our study extends knowledge of Cistus mycorrhizal "##"" relationships in ECM fungi. Article info:JPUJ/='UZGP>D/='UZP'=D/='U INTRODUCTION Considered together, Lactarius and ; form one of the most prominent groups of ectomycorrhizal The basidiomycete family Russulaceae has undergone (ECM) basidiomycetes (Hutchinson 1999, Rinaldi et al. a dramatic taxonomic revision during the last decade. 2008, Comandini et al. 2012b). With more than 450–500 Studies based on multigene phylogenies of both Russula E" and Lactarius have shown that neither of these two role as mycobionts of trees and shrubs in a vast range of classic genera is monophyletic. In the case of Russula, ecosystems, from boreal coniferous forests to temperate " # [ " Mediterranean-type maquis, from Mesoamerican Neo- Compactae subsect. Ochricompactae were found to be tropics to the rainforests of South-East Asia, passing a monophyletic entity, for which the new generic name through tropical Africa (Comandini et al. 1998, Eberhardt Multifurca was introduced; the genus also includes the rare et al. 2000, Comandini et al. 2004, Nuytinck et al. 2004, American species Multifurca furcata (Buyck et al. 2008). Montoya & Bandala 2005, 2008, Le et al. 2007, Mueller & Lactarius was found to comprise two clades, with subgenera Halling 2010, Verbeken & Walleyn 2010, Comandini et al. Piperites, Russularia, and Plinthogalus constituting the 2012a, Flores Arzú et al. 2012). In Europe, some 100–110 larger newly circumscribed genus Lactarius, and subgenera Lactarius species are recognized, depending how long- Lactariopsis, ;< ;, Russulopsis, Gerardii, standing controversies on synonymies are resolved, and and the former Lactarius sect. Edules constitute the newly nine of ; (Heilmann-Clausen et al. 1998, Basso recognized genus ; (Buyck et al. 2010, Stubbe et 1999, Van de Putte et al. 2016). al. 2012, Verbeken et al. 2011, 2012). A more recent multi- Several Russulaceae are associated as ECM mycobionts gene analysis of ; resulted in a new infrageneric of Cistus"#\"# [ # "P ;, Cistaceae, containing about 20 species of woody, evergreen Lactariopsis, Gymnocarpi, and Pseudogymnocarpi (De or semideciduous shrubs (Comandini et al. 2006). Cistus Crop et al. 2017). species are found in semi-arid areas from the Canary Islands © 2016 International Mycological Association You are free to share - to copy, distribute and transmit the work, under the following conditions: Attribution: !""#$ Non-commercial: # No derivative works: # For any reuse or distribution, you must make clear to others the license terms of this work, which can be found at http://creativecommons.org/licenses/by-nc-nd/3.0/legalcode. Any of the above conditions can be waived if you get permission from the copyright holder. Nothing in this license impairs or restricts the author’s moral rights. VOLUME 7 · NO. 2 275 Leonardi . ARTICLE Fig. 1. ;(G\jL/='+Q'), from the Mediterranean maquis of Sardinia, Italy. A. Basidiomes. B. Habit of ectomycorrhiza with Cistus sp.; the abundant short cystidia give a light and hyaline appearance to the mycorrhizal surface. throughout the Mediterranean basin to the Caucasus, where Several Cistus species (C. creticus, C. salvifolius, and C. "#"" monspeliensis) were present in the collection sites, so that ecosystems, often forming extensive swards (Ellul et al. it was not possible to identify the host(s) of L. rugatus at 2002, Guzmán et al. 2009). Obligate seeders, Cistus species species level (all attempts to trace mycorrhizas directly are early-stage colonizers that follow disturbance, particularly to roots of possible hosts failed). Soil cores (about 20 × ["G 20 × 20 cm) were excavated from beneath basidiomes include physical seed dormancy, high seed longevity, and and immersed overnight in water, and ectomycorrhizal small and light seeds, allowing the generation of persistent roots were carefully separated under a dissecting soil seed banks; the sharp rise in temperature generated microscope. Several tips were immediately transferred [ into 50 % EtOH and stored at –20 °C for subsequent DNA to germination (Bastida & Talavera 2002). Overall, these j##!G\jL/='=QU ecological characteristics make Cistus mycorrhizal biology G\jL/='?QU G\jL/='+Q'$ !G\jL particularly interesting. /='=QULX G\jL/='+Q'LX$ Here we describe the morpho-anatomical features of the of the Department of Biomedical Sciences, University of mycorrhizas, collected in Sardinia, Italy, formed by ; Cagliari, Cagliari, Italy. rugatus with Cistus. The identity of the ECMs was also [ " 7 " Microscopy X\#"" recognized genus ; subgen. Pseudogymnocarpi. # # E Furthermore, we compare the ECM anatomical features on microscope slides with polyvinyl lactophenol for light with those of Lactarius Cistus-[ microscopy. Observations were made with a Zeiss Axioplan 2 Lactarius cistophilus and L. tesquorum, and also with those "[q´U formed by other taxa in Lactarius and ;. Images were acquired with a Leica DFC290 digital camera. For longitudinal sections (2.5 mm thick), ectomycorrhizas were embedded in LR White resin (Multilab Supplies, Surrey, MATERIALS AND METHODS UK), cut with a Leica Ultracut R ultramicrotome and stained with toluidine blue in 1 % sodium borate for 15 s at 60 °C. For Study site and fungal collections # " E (4 % glutaraldehyde) were mounted in Vectashield Antifade Basidiomes of ;(Fig. 1A) were harvested Mounting Medium (Burlingame, CA) and then examined by in the vast forested area that extends between Capoterra TCS SP5 Leica confocal microscopy (Leica Microsystems, and Santadi (39°8’30” N, 8°53’24” E, 227 m asl), about Mannheim, Germany) equipped with an inverted microscope 20 km south-west of Cagliari (Sardinia, Italy), and in a DMI 6000 CS (Objective HCX PL APO CS 40×1.3 oil) and sandy area close to Gonnesa (39°15’8” N, 8°24’44” E, 94 a VIS Argon laser. The laser excitation wavelength was m asl), about 70 km west of Cagliari; basidiomes were E?997""" [[# used to characterize ectomycorrhizas follows Agerer (1986, of macroscopic and microscopic characters (Basso 1999). 1987–2006, 1991, 1995). Munsell Soil Color Charts (2000) Specimens were collected from under Cistus spp. in a low- were used as reference for the descriptions of the colours of density Quercus suber wood and in treeless area covered ectomycorrhizas. Q"" " 276 IMA FUNGUS \ mycorrhizas on ]^_|}"! RESULTS rDNA region ARTICLE Description of ectomycorrhizas Genomic DNAs of the basidiomata were isolated from 20 mg of each dried sample using DNeasy Plant Mini Kit Morphological charactersP Mycorrhizal system to 4–5 mm (Qiagen, Hilden, Germany) according to the manufacturer’s long, monopodial-pyramidal or coralloid, 2–3 orders of instructions. Extracts were eluted in 50 μL of sterile water [Main axes 0.4–0.5 mm diam. >[ and their DNA concentration estimated using a NanoDrop straight to slightly bent, to 1.5 (–1.8) mm long and 0.3–0.4 D5L'=== J !7 M J mm diam. Mycorrhizas !/+>Q;$ " _$7_7J[#" yellow short cystidia give a lighter and hyaline appearance ITS1F and ITS4 primers pair (White et al. 1990, Gardes & to the mycorrhizal surface (Fig. 1B); older mycorrhizas pale Bruns 1993) following the protocol reported by Leonardi et brown. [ short spiny, with sporadic al. (2005). A direct PCR approach was applied to identify all longer extramatrical hyphae. Soil particles and hyphal mats ECM morphotypes isolated from soil samples. One to three often stuck on the mycorrhizal surface; not secreting latex representative ECM tips per morphotype were selected as when injured; mantle not transparent. Rhizomorphs and PCR targets. A little fragment of ECM mantle was excised sclerotia lacking. from each selected tip as described by Iotti & Zambonelli !/==U$[+=)q\j" Anatomical characters of mantle in surface views. Mantle _7J'MQ_7J? 7 # /= "Qq OJG plectenchymatous throughout, hyphal cells hyaline, clamps !7MJ$ lacking, abundant cystidia present (Fig. 2A). Outer mantle \j7[ layers plectenchymatous, net-like, bearing abundant 6

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