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et al. . 2007, Mueller & et al. 2007, Mueller & ectomycorrhizas ectomycorrhizas Key words: microscopy confocal laser scanning Lactarius maquis molecular phylogeny Russulaceae IMA FUNGUS · 7(2): 275–284 (2016) 275–284 · 7(2): FUNGUS IMA 1998, Basso (Heilmann-Clausen et al. 1998, Basso and L. Russulaceae are associated as ECM mycobionts , containing about 20 species of woody, evergreen , containing about 20 species of woody, Several form form Lactarius and ; Considered together, Cistus " # \ " # . The ECM identity was . of Cistaceae or semideciduous shrubs (Comandini et al. 2006). Cistus species are found in semi-arid areas from the Canary Islands 1999, Van de Putte et al. 2016). 1999, Van one of the most prominent groups of ectomycorrhizal of ectomycorrhizal one of the most prominent groups 1999, Rinaldi (ECM) basidiomycetes (Hutchinson 2008, Comandini et al. 2012b). With more than 450–500 E " in a vast range of role as mycobionts of trees and shrubs forests to temperate ecosystems, from boreal coniferous Mediterranean-type maquis, from Mesoamerican Neo- Asia, passing tropics to the rainforests of South-East Africa (Comandini et al. 1998, Eberhardt through tropical et al. 2000, Comandini et al. 2004, Nuytinck et al. 2004, Montoya & Bandala 2005, 2008, Le Comandini et al. 2010, & Walleyn Halling 2010, Verbeken Arzú et al. 2012). In Europe, some 100–110 2012a, Flores Lactarius species are recognized, depending how long- standing controversies on synonymies are resolved, and nine of ; L. rugatus mycorrhiza is the
3 Gerardii, Russula, ; , Pseudogymnocarpi subgen. constitute the newly constitute the newly # " " Russulopsis, , and Andrea C. Rinaldi Andrea C. , and 2 ; mycorrhizal species. Our study extends knowledge of Cistus (De and Pseudogymnocarpi (De , a genus of about 20 species of woody shrubs typical of the species of woody shrubs typical of the , a genus of about 20 resulted in a new infrageneric resulted in a new infrageneric Cistus on 2011, 2012). A more recent multi- A 2012). et al. 2011,
; , Ornella Comandini 1 Gymnocarpi, ; < ; , constituting the Russularia, and Plinthogalus constituting the was introduced; the genus also includes the rare was introduced; the genus also includes the rare We describe the morpho-anatomical features of the ectomycorrhizas (ECMs) formed features of the ectomycorrhizas (ECMs) describe the morpho-anatomical We 7 " E \ #
! "" # $ have shown that neither of these two two Lactarius have shown that neither of these ; 5 # O J # \ " _L=*=?/ !\G$ _ Z " L P ¨ Department of Life, Health and Environmental Sciences, University of L’Aquila, L’Aquila, Italy L’Aquila, of L’Aquila, and Environmental Sciences, University Department of Life, Health Monserrato (CA), Italy Environmental Sciences, University of Cagliari, Department of Life and by © 2016 International Mycological Association © 2016 International Mycological conditions: distribute and transmit the work, under the following are free to share - to copy, You Attribution: on 3 1 2 Marco Leonardi Peering into the Mediterranean black box: into the Mediterranean Peering Non-commercial: No derivative works: if you get Any of the above conditions can be waived 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. # # Nothing in this license impairs or restricts the author’s moral rights. permission from the copyright holder. of L. rugatus mycorrhizas on Cistus The description Mediterranean maquis. X\ belonging to description of a species Abstract: Article info: J P U J /='UZ G P > D /='UZ P '= D /='U tesquorum host-depending ECM features known for Cistus, principal features of their ECMs, which share roots. Comparison of ; but are otherwise distinguishable on the host ECM with on Fagus reveals elevated intrageneric diversity those formed by L. vellereus and L. piperatus supported by analysis of ITS sequences of relevant of mycorrhizal structures. Such a diversity is species within European ; " # relationships in ECM fungi. presence of abundant, long “bottle-shaped” cystidia on mantle surface. Indeed, the overwhelming long “bottle-shaped” cystidia on mantle surface. presence of abundant, is the third ; =; mycorrhiza This majority of milkcap mycorrhizas are acystidiate. , the others being Lactarius cistophilus to have been described associated with Cistus " G # classic genera is monophyletic. In the case of classic genera is monophyletic. In the has undergone has undergone The basidiomycete family Russulaceae the last decade. a dramatic taxonomic revision during of both Russula Studies based on multigene phylogenies and INTRODUCTION " # " Compactae subsect. Ochricompactae were found to be for which the new generic name a monophyletic entity, Multifurca 2008). American species Multifurca furcata (Buyck et al. 2008). Lactarius was found to comprise two clades, with subgenera Piperites, larger newly circumscribed genus Lactarius, and subgenera Lactariopsis, and the former Lactarius sect. Edules recognized genus ; (Buyck et al. 2010, Stubbe et al. 2012, Verbeken gene analysis of # " P Lactariopsis, Crop et al. 2017). P'= ++*9Q # " /='U => =/ => VOLUME 7 · NO. 2 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 ?99 7 " " " # 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 # /= "Q q OJG plectenchymatous throughout, hyphal cells hyaline, clamps !7 M J $ lacking, abundant cystidia present (Fig. 2A). Outer mantle \j 7 layers plectenchymatous, net-like, bearing abundant 6 min of the initial denaturation at 95 °C, followed by 30 cystidia (type D, Agerer 1995). Hyphae frequently branched, cycles of 94 °C for 30 s, 55 °C for 30 s, 72 °C for 1 min, and 3.5–5 μm diam, hyphal segments 8–15 μm long, the basal E # >/ \ # '= \j roundish part of the cystidia 4–6 μm diam (Figs 2B, 3A). were visualized through 1 % agarose gel electrophoresis Middle mantle layers plectenchymatous, very close to the 7 outer mantle layers. Lactifers not observed. Inner mantle " ¡_G \j { !¡ " layers plectenchymatous, with a gelatinous matrix between Milan, Italy) and then directly sequenced using the same the hyphae, hyphae arranged net-like to ring-like, frequently primers pair. branched (Figs 2C, 3B). Hyphae generally 2.5–4.5 μm thick, not uniform in diam. In some mantle preparations, a few Phylogenetic inference hyphae with content (lactifers?) can be observed. Very tip hyphal arrangement and characteristics as in the other parts The sequences of the ITS1, 5.8S, and ITS2 regions of the of the mantle. nuclear rDNA obtained were compared with those present in GenBank ( PQQ " QOqGJ7Q) and Anatomical characters of emanating elements. Rhizomorphs UNITE ( PQQ Q ) databases using the not observed. Emanating hyphae 3–4 μm thick, hyaline, BLASTN search (Altschul et al. 1990). Besides sequences clamps lacking, Cystidia hyaline, sometimes cylindrical, from ; ECM morphotype and basidiome, ITS mainly bottle-shaped, 25–30(–40) μm long and 4–5 μm diam sequences from basidiomes of ; # (Fig. 2D). Cystidia tips blunt and straight, but the remaining and L. cistophilus, both collected in Mediterranean-type part sinuous. Hyphal walls less than 1 μm thick in the maquis in Sardinia, were also obtained. Sequences are upper part, becoming thicker toward the base; no contents deposited in GenBank under accession numbers KU885433– observable by light microscope, but content inside the KU885436. After excluding the ambiguous regions at the 5’ \ " # and 3’ ends of the chromatograms, sequences were edited (Fig. 3C); septa present towards the base, sometimes also using BioEdit v. 7.2.5 (Hall 1999) and aligned by MUSCLE in the other parts of the cystidia; cystidia bases generally (Edgar 2004). Sequence statistics, nucleotide diversity, and roundish, 5–7 μm diam (Fig. 3D) but sometimes more distance based analyses were performed using MEGA v. 6 elongated (Fig. 2D). (Tamura et al. 2013). The best substitution model with the lowest BIC scores (Bayesian Information Criterion) was Anatomical characters, longitudinal section. Mantle (30–) chosen with the default settings. A phylogenetic tree was ;+M++!MU+$ 3 ## P obtained by the Maximum Likelihood (ML) method based layer, 25–30 μm thick, formed by a loose net of very long on the two-parameter distance model of Kimura (1980), cystidia (see above); underlying layer about 15–25 μm selecting “Nearest-Neighbor-Interchange (NNI)” as the ML thick, formed by the roundish bases of the cystidia, 2–3 heuristic method in the tree inference options. Bootstrap μm diam, and by hyphal cells to 30 μm long and 3 μm diam; tests were performed using 1000 replicates. innermost layer 8–10 μm thick, very compact and formed by hyphal cells, longitudinally orientated, 1–3 μm thick, scarce hyphae with granular content (lactifers?) may be observed; mantle of tip about 20 μm thick, hyphal organization as the remaining part, but structures more compact and single # Tannin cells not observed.
VOLUME 7 · NO. 2 277 Leonardi . ARTICLE
Fig. 2. Anatomical characters of ; ectomycorrhizas (G\jL/='+Q'LX). A. Outer mantle surface, characterized by abundant cystidia. B. Plectenchymatous outer mantle mantle layer formed by frequently branched hyphae, hyphal segments and the basal roundish part of cystidia. C. Inner mantle layer with a densely plectenchymatous structure; a few hyphae with content (lactifers?) can be observed in some mantle preparations. D. Cystidia, mainly bottle-shaped, sinuous, with blunt tips; cell walls become thicker toward the base which is generally roundish, but sometimes more elongated. Bars = 5 μm.
Cortical (epidermal) cells of 1–2 rows, radially orientated phylogenetic analysis, ITS sequences of 21 different 20–28(–35) × 8–11 μm. Hartig net paraepidermal, of one European ; and Lactarius species were imported row of roundish, 1–4 μm diam hyphal cells, palmetti-type, from the GenBank and UNITE databanks, and the ITS lobes 1–1.5 μm wide. sequences from basidiomes of ; # and Lactarius cistophilus # " Molecular and phylogenetic analyses study from samples collected in Mediterranean-type maquis in Sardinia, were also considered. Russula werneri and R. The ITS sequences of the basidiomes of ; rugatus insignis were chosen as outgroup. According to availability, and of the ectomycorrhizas found below them were identical, one to four sequences were chosen for each species. The " # sequence alignment contained 1027 characters of which 7 # " # 402 were variable basepairs and 297 were parsimony 565 bp containing the complete ITS1-5.8S-ITS2 sequence. informative. K2P distances ranged between 0.002 and 0.28. In order to assemble a multiple sequence alignment for A tree was constructed using the Maximum Likelihood (ML)
278 IMA FUNGUS mycorrhizas on ARTICLE
Fig. 3. Anatomical characters of ; ectomycorrhizas (G\jL/='+Q'LX), A. Outer mantle surface viewed with confocal laser scanning microscopy (CLSM), the basal roundish parts of the cystidia are visible. B. Plectenchymatous inner mantle layer viewed with a light microscope. C. # \qJ Z \ D. Outer mantle layer viewed with the light microscope; a single bottle-shaped cystidium with a roundish base is indicated by the arrow. Bars = 5 μm.
(Fig. 4). Nodes with bootstrap values lower than 50 % were species has long been confused with L. volemus, and some eliminated. The phylogenetic analysis shows that all the authors have considered L. rugatus a Mediterranean vicariant European species belonging to ; are well delineated species of L. volemus (Galli 2006), but molecular studies and the bootstrap values support the segregation of the reveal it as more closely related to the North American L. different taxa in subgenera and sections as delineated by De hygrophoroides and African species such as L. rubiginosus Crop et al. (2017) (Table 1). and L. volemoides (Verbeken et al. 2012). The distinctness of L. rugatus and L. volemus is also supported by the results of our analysis of ITS sequence data of European taxa DISCUSSION !M " ?$P ; " multigene analysis recently presented by Van de Putte et al. ; is a typically Mediterranean and well- (2016), and the analysis of De Crop et al. (2017) showed characterized milkcap, with a wrinkled, vividly orange cap that the two species actually belong to different subgenera. (Fig. 1), and known from southern Europe (Portugal, Spain, Hesler & Smith (1979) treated L. rugatus as a variant of L. France, Italy, and Greece) and northern Africa (e.g. Morocco, hygrophoroides " D G Algeria, Tunisia) (Kühner & Romagnesi 1954, Malençon with Mediterranean L. rugatus is most certainly to be excluded. 1974, Bertault 1978, Alessio 1979, Lalli & Pacioni 1992, ; var. rugatus (Hesler & Smith 1979) Pierotti 2002, Nounsi et al. 2014, Dimou et al. 2016). The probably simply represents an aspect of the natural variability
VOLUME 7 · NO. 2 279 Leonardi .
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Fig. 4. Maximum Likelihood tree obtained from the alignment of ITS rDNA region sequences. Bootstrap values are indicated next to relevant nodes. The scale indicates the number of substitution per site. Sequences of species in boldface were obtained during this study. The different colours in genus ; " # " 5 \ et al. 2017 (see also Table 1).
of L. hygrophoroides (Lalli & Pacioni 1992). Verbeken et al. Quercus in the Mediterranean eco-region. Even the original (2012) treated L. hygrophoroides and L. rugatus as distinct P OJ Quercus suber, á la Réghaïa, aux taxa, and the molecular analysis by De Crop et al. (2017) environs d’Alger” (Kühner & Romagnesi 1954). 7 ' In recent years, we have been carrying out a long- schemes for L. rugatus and related taxa. L. rugatus is by no term study on the distribution, ecology, phylogeny and means a mycobiont linked exclusively to Cistus, although it ectomycorrhizal biology of milkcap species occurring # _ in selected ecosystems in Europe and the Neotropics also associated with other members of the Cistaceae, such (Comandini et al. 1998, Eberhardt et al. 2000, Comandini et as Halimium halimifolium (Comandini & Rinaldi, unpubl.). al. 2012a, Flores Arzú et al. 2012, Nuytinck et al. 2004). We O " explore the milkcap ECM diversity of host trees or shrubs in a of several other authors (Basso 1999, Mua & Casula 2012, given ecologically important ecosystem, and relevant milkcap Nounsi et al. 2014), this species also grows in association with ECM types are fully characterized from a morpho-anatomical
280 IMA FUNGUS mycorrhizas on
Table 1. j # ; and related species. ARTICLE Heilmann-Clausen et al. (1998) Basso (1999) Verbeken et al. (2011, 2012) De Crop et al. (2017) Genus Lactarius Genus Lactarius Genus ; Genus ; Subgenus ; Subgenus ; Subgenus ; Subgenus ; Section ; Section ; Section ; Section ; L. volemus L. volemus L. volemus L. volemus ( 1) Section Tomentosi L. subvolemus 3 (L. luteolus 1) L. luteolus L. oedematopus 3 Subgenus Lactariopsis Section Albati Section Phlebonemi Section Piperati Section Albati L. vellereus L. brunneoviolascens 2 L. piperatus L. vellereus L. bertillonii Subgenus Lactariopsis L. glaucescens L. bertillonii Subgenus Lactarius Section Albati Subgenus Pseudogymnocarpi Subgenus Lactarius Section Lactarius L. vellereus Section Pseudogymnocarpi L. piperatus L. piperatus L. bertillonii L. glaucescens L. glaucescens Subgenus Piperati Subgenus Gymnocarpi Section Piperati Section Luteoli L. piperatus L. brunneoviolascens L. glaucescens Subgenus Lactariopsis Section Albati L. vellereus L. bertillonii 1 Neither L. rugatus nor L. luteolus (i.e. L. brunneoviolascens) were included in this treatment which concerned milkcaps from northern Europe. 2 Previously named ; # # , which is now known to be the correct name for a North American species (Verbeken et al. 2012, De Crop et al. 2017). 3 A recent study recognized three cryptic species within the morphospecies ; volemus; L. volemus, L. oedematopus, and L. subvolemus (Van de Putte et al. 2016).
and molecular points of view, also comparing ECM anatomical hyphal net present on the pseudoparenchymatous mantle characters with those known from related taxa. Morphological surface. and molecular data obtained for related milkcap taxa, living Three milkcap mycorrhizas associated with Cistus have either in the same habitat or linked to other hosts, are used to " P L. cistophilus, a member of Lactarius clarify the systematic position of the species of interest. Thus, subgen. Piperites sect. Uvidi subsect. Uvidini (Comandini & the combination of morphological characters of basidiomes Rinaldi 2008), L. tesquorum, a member of Lactarius subgen. and mycorrhizas and their molecular features contribute Piperites sect. Piperites subsect. Piperites (Nuytinck et al. to a reliable taxonomy in the genus (Nuytinck et al. 2004, 2004), and ; . The structure of these three Eberhardt et al. 2000). Such a combined approach could ECM morphotypes is quite diverse (Table 2), which makes it be of general help when dealing with the characterization of possible to distinguish them on Cistus roots. Only ; given ECM associations, showing the relevance mycorrhiza rugatus possesses numerous and characteristic cystidia descriptions still have for the recognition of natural groups, if that cover mantle surface; Lactarius cistophilus presents # a pseudoparenchymatous outer mantle layer formed by The main distinguishing character of L. rugatus ECM is epidermoid cells; and L. tesquorum has a plectenchymatous the presence of abundant cystidia resembling the terminal mantle formed by a loose net of hyphae. The three LactariusQ cells in the pileipellis of the correspondent fungal symbiont ; ectomycorrhizas only share the common host- (Lalli & Pacioni 1992, Basso 1999). Not many of the milkcap dependent ectomycorrhizal features described so far for ECM described so far present cystidia on the mantle surface, CistusP # and, when present, the characteristics and abundance ectomycorrhizal tips, and a thin mantle. The diversity of the of these elements are variable. Besides L. rugatus, only principal features of the ECMs formed by LactariusQ; mycorrhizas of Lactarius acris (Brand 1991) and L. lignyotus mycobionts with Cistus \ " (Kraigher et al. 1995), both belonging to Lactarius subgen. between the three species involved (Fig. 4). Plinthogalus, present a mantle surface completely covered Two other ECMs belonging to ; have been by “latex-containing hyphal ends resembling cystidia” or # P L. vellereus, and L. piperatus. “short cystidia-like hyphal ends”, respectively. Moreover, all This offers the possibility of comparing ECMs formed by the three species present a plectenchymatous outer mantle a species each from subgenera ; (L. piperatus), layer. Other Lactarius ECMs are reported to possess cystidia, Lactariopsis (L. vellereus), and Pseudogymnocarpi (L. such as Lactarius rubrocinctus and L. camphoratus (Brand rugatus$ " 1991), but cystidia here are infrequent and grow from a by De Crop et al. !/='>$ !7 '$ G
VOLUME 7 · NO. 2 281 Leonardi .
Table 2. Comparison of structural features of the described mycorrhizas of ; and either host- or phylogenetically-related ; QLactarius species. Ectomycorrhizhal type System/appearance SV ML IV RHIZ Host Reference ; m-pyr, short spiny cys + pl pl pl Q Cistus present work Lactarius cistophilus simple (pinnate), smooth ps dpl + lact dpl Q Cistus Comandini & Rinaldi 2008
ARTICLE Lactarius tesquorum Q L pl dpl + lact dpl hd Cistus Nuytinck et al. 2004 ; m-p, woolly hh + ps Q Q dif Fagus Beenken 2004
; # var. simple (monopodialpinnate), hh + pl ps ps + lact Q Shorea Kumar & Atri 2016 volemus Q ; ## L Q " hn + ps Q pl + lact undif Fagus Grebenc 2005, Grebenc et al. 2009
SV, surface view; ML, middle layer; IV, inner view; RHIZ, rhizomorphs; cys, cystidia; dpl, densely plectenchymatous; hd, highly differentiated; hh, extramatrical hyphae; hn, hyphal net; lact, lactifers; m-p, monopodial pinnate; m-pyr, monopodial pyramidal; pl, plectenchymatous; ps, pseudoparenchymatous; undif, undifferentiated.
of L. vellereus mycorrhizas was provided by Ceruti et According to De Crop et al. (2017) these taxa belong to al. (1988). An ECM from L. vellereus was described by different sections of subgenus ; . Brand & Agerer (1986), but later proved to be formed by _ QX\ Q Lactarius blenniusZ molecular data, comparison of the compounds produced (Brand 1987). The only reliable description of L. vellereus by LactariusQ; species could have, at least ECM became available more recently (Grebenc 2005, potentially, some value in assessing phylogenetic distances Grebenc et al. 2009). The main character of this ECM type of scrutinized species (Frisvad et al. 1998). For example, is a pseudoparenchymatous outer mantle layer formed by LactariusQ; species are well known to produce a epidermoid cells and with a hyphal net lying on it; abundant vast array of sesquiterpenes of several kinds (Vidari & Vita- lactifers are present in the inner plectenchymatous Finzi 1995, Clericuzio et al. 2008). These compounds are mantle, and infrequent type B rhizomorphs have been usually stored in laticiferous hyphae as inactive precursors, observed (Table 2). A partial description, with comments and are converted to biologically active compounds and pictures, of L. piperatus ECM has been provided by when the fungus is injured. Relevant to our discussion on Beenken (2004). An earlier description by Luppi & Gautero ; < is the presence of stearoyl-velutinal and break- (1967) did not include enough information for a critical down products velleral and isovelleral in L. vellereus and comparison. The outer mantle layer of this ; ECM L. piperatus (Camazine & Lupo 1984, Vidari & Vita-Finzi is pseudoparenchymatous, with angular cells and abundant 1995), responsible for the peppery taste of these milkcaps. extramatrical thick-walled hyphae, Russula-type rhizomorphs No velutinal esters or other sesquiterpenes could be are present, and lactifers have not been observed (Table detected either in intact or injured basidiomes of L. rugatus 2). Finally, while this study was under review, a description (Clericuzio et al. 2008), and the same substances are also of the ECM formed by L. volemus var. volemus on Shorea absent in L. volemus, which exudates permanently mild robusta (Dipterocarpaceae) from India became available milk and is particularly rich in sugars (Reis et al. 2011). No (Kumar & Atri 2016). Their description includes pictures and " # drawings prepared from cross and longitudinal sections, in L. brunneoviolascens, but that species has mild milk and which are per se less informative than direct observations \ !O '***$ of the mantle surfaces; in addition, no molecular data of In conclusion, the present study enhances our knowledge either the basidiomes or ECMs were provided to verify of Cistus mycorrhizal biology, indicating that LactariusQ the connection. Nevertheless, the outer mantle layer of ; harbour multiple mycobionts linked to this host, L. volemus var. volemus ECM is plectenchymatous with thriving in an ecologically specialized ecosystem. In the extramatical hyphae. The middle and inner mantle layers future, it would be interesting to test the proposed number of are pseudoparenchymatous, the inner being heterogeneous L or more or less pseudoparenchymatous; lactifers are also LactariusQ; – Cistus case, as compared to other host present in the inner mantle layer. genera pre-eminent in the Mediterranean ecotone, such as < " # Quercus and Pinus (Newton & Haigh 1998). Moreover, close four ; ECM, which is in line with their phylogenetic examination of ECM features revealed differences amongst distance as indicated by molecular analysis. ; ; species, a diversity supported by molecular and L. piperatus ECMs both lack lactifers in the mantle, which 7 " # # are also absent in all Russula species (Beenken 2004). mycorrhizal structure, when joined to evidence coming from Interestingly, the absence of lactifers in ECM of L. rugatus other approaches, in resolving phylogenetic relationships in contrasts with the presence of abundant, white and mild latex ECM fungi (Nuytinck et al. 2004, Mleczko & Ronikier 2007, which exudates from basidiomes. ; and L. Benkeen 2011). volemus var. volemus ECMs, however, share an important character; the presence of extramatrical hyphae (Table 2).
282 IMA FUNGUS mycorrhizas on
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