Introduction ORIGINAL ARTICLE. Michael Loizides 1 & Jean-Michel Bellanger 2 & Philippe Clowez 3 & Franck Richard 2 & Pierre-Arthur Moreau 4,5

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1 Mycol Progress (2016) 15:39 DOI /s ORIGINAL ARTICLE Combined phylogenetic and morphological studies of true morels (Pezizales, Ascomycota) in Cyprus reveal significant diversity, including Morchella arbutiphila and M. disparilis spp. nov. Michael Loizides 1 & Jean-Michel Bellanger 2 & Philippe Clowez 3 & Franck Richard 2 & Pierre-Arthur Moreau 4,5 Received: 3 January 2016 /Revised: 18 February 2016 /Accepted: 24 February 2016 # German Mycological Society and Springer-Verlag Berlin Heidelberg 2016 Abstract A detailed account of the genus Morchella in the island of Cyprus is presented, based on integrative phylogenetic, morphoanatomical, ecological, and chorological analyses. Eleven species are molecularly confirmed, nine of them previously unreported from the island. Notably, four species are recognized as new to science, including one species in Sect. Morchella and three in Sect. Distantes. Two of these are here newly described, as Morchella arbutiphila and Morchella disparilis, respectively, whilst the other two are provisionally assigned the phylogenetic codes Mes-28 and Mel-38. Following careful re-examination of the Morchella vulgaris clade, two closely related sister species are revealed, and the taxon Morchella dunensis is revived to accommodate Mes-17. A description for the widespread, yet poorly known Mediterranean species Morchella dunalii is further provided, and a detailed polythetic approach is introduced in systematics, to overcome the inherent difficulties associated with the morphological recognition of phylogenetically confirmed species. The presence on the island of five species of Section Editor: Gerhard Rambold * Michael Loizides michael.loizides@yahoo.com P.O. Box 58499, 3734 Limassol, Cyprus UMR 5175 CEFE Université de Montpellier - INSERM, Campus CNRS, 1919 Route de Mende, Montpellier, France 56 place des Tilleuls, F Pont-l Evêque, France Département des Sciences Végétales et Fongiques, Faculté des Sciences Pharmaceutiques et Biologiques, Univ Lille 2, F Lille, France Univ de Lille, EA 4483, INSERM, Institut Pasteur de Lille, F Lille, France transcontinental distribution, accounting for nearly half of the total number of species recorded, sheds new light on the genus biogeography, questioning the hypothesis of recent anthropogenic dispersals of morel species. Overall, our results place Cyprus as a worldwide hotspot of Morchella diversity, establishing the island as a place of special interest in future studies aiming to decipher the evolutionary history and ecological trends within this iconic genus. Keywords Integrative taxonomy. Island ecosystem. Mediterranean. Morchella dunalii. Morchella dunensis. Morchella vulgaris Introduction The genus Morchella Dill. ex Pers.: Fr. (Persoon 1794) has been intensively studied in recent years. The molecular confirmation of over 60 phylogenetically distinct lineages from Europe (Taşkın et al. 2010, 2012), North America (O Donnell et al. 2011), and China (Du et al. 2012a, b), has revealed considerable endemism and provincialism within the genus, which is thought to have arisen from the early diverging Rufobrunnea clade in the late Jurassic (O Donnell et al. 2011). As subsequently demonstrated by Richard et al. (2015) and Loizides et al. (2015), however, an increasing number of species (such as M. americana Clowez & C. Matherly, M. eohespera Beug et al., M. rufobrunnea Guzmán & F. Tapia, M. tridentina Bres., and the ruderal or post-fire M. eximia Boud., M. exuberans Clowez et al., and M. importuna Kuo et al.), appear to have a large-scale and in some cases worldwide distribution, difficult to explain by the single hypothesis of multiple anthropogenic introductions (as postulated by Taşkın et al. 2010, 2012, and O Donnell et al. 2011).

2 39 Page 2 of 28 Mycol Progress (2016) 15:39 Mediterranean islands are considered hotspots of biodiversity, harboring rich reservoirs of plant and animal endemism (Myers 1990; Médail and Quézel 1997; Myers et al. 2000; Médail and Myers 2004). Since most fungi form broad, or even highly specific symbiotic and endophytic associations with vascular plants, Mediterranean islands are favourable places to track fungal diversity and endemism. Still, our knowledge of fungal diversity within these rich yet vulnerable environments is fragmentary, due to climatic conditions particularly unfavourable for mushroom fruiting and poor mycological legacies, including a lack of dedicated monographic works. In the current context of climate changes in the Mediterranean basin (Giorgi 2006; IPCC 2007), documenting these ecosystems is of immediate concern for the purpose of predicting shifts in biodiversity and drafting effective policies based on carefully identified conservation priorities (Myers et al. 2000; Behrens et al. 2010). This is all the more urgent for sought-after species of high commercial value, likely to find themselves under increased pressure in the years to come. Morels have only been sporadically and fragmentarily studied in Cyprus. The first record of a Morchella species on the island dates back to 1933, when, following a number of fatal poisonings among the local population, an investigation was commissioned by the Department of Agriculture. This resulted in one of the earliest known publications on Cyprus fungi (Willimott 1933). Among other species, Willimott reported M. conica Pers., interestingly mentioning a notable oral tradition at the time, which claimed the fungus was considered inedible by the locals unless collected in dry weather. In the early twenty-first century, Viney (2005) reported M. elata Fr.: Fr. and M. esculenta (L.: Fr.) Pers., while Loizides et al. (2011) reported M. costata (Vent.) Pers., M. purpurascens (Boud.) Jacquet., M. rigida (Krombh.) Boud., M. rufobrunnea Guzmán & F. Tapia, and M. vulgaris (Pers.: Fr.) Gray. All of these early records, however, were based on traditional species concepts and had not been verified by molecular tools. Since then, the nomenclatural and taxonomic status for a large number of taxa has been phylogenetically revised. Richard et al. (2015) proposed a unified taxonomy for the genus, applying names to 30 of the 66 genealogical lineages recognized so far, including a number of neotypes, epitypes, and lectotypes for several old, validly published taxa. Thus, the names M. conica and M. costata have been shown to be illegitimate, M. purpurascens and M. vulgaris have been epitypified and redefined, and M. elata Fr. is pending redefinition itself. Following these important taxonomical revisions, Loizides et al. (2015), published molecularly confirmed collections of M. rufobrunnea and M. tridentina from Cyprus, widening the distribution range and clarifying the morphotaxonomy for these species. The taxonomical status of the previously reported Morchella species from the island, however, still remained unclear and in need of re-evaluation, as was the status of certain collections with ecological and morphological features not conforming to any of the existing species described in literature. The present study offers a conspectus of Morchella diversity in Cyprus, based on integrative phylogenetic, chorological, ecological, and morphoanatomical analyses of over 180 collections. Detailed descriptions of four species are presented below, two of which are new to science, whilst imagery, distribution maps, and phenological records are provided for all phylogenetically confirmed species. A detailed polythetic approach is introduced in systematics, aimed at overcoming the longstanding problems hindering Morchella species delimitation, and enabling their identification by nonmolecular means. The findings of this study are finally discussed, particularly with respect to the hypothesis of recent anthropogenic dispersals of morel species. Materials and methods Herbarium material and morphological observations The studied material was collected from several localities in Cyprus between 2008 and Specimens were photographed in situ and the habitat, altitude, nearby vegetation, and soil characteristics were noted. The maturity process and macromorphological characters were observed in detail from repeated collections of fresh ascocarps at various stages of development. Microscopic studies were performed on both fresh and dried material, under a Leica BM E binocular microscope and a Zeiss Axioskop microscope at 4, 10, 40,& 100 magnifications. Studies focused on the paraphyses, their shape, size, apices, and the number and orientation of their septa, the terminal elements of the sterile ridges (here termed Bacroparaphyses^), the terminal elements of the stipe cortex, the spore size, shape, and surface, and the asci size and ascus base. Measurements of microscopic structures were obtained from fresh material. Measurements from dried specimens were included only when their maximum values were found to exceed those of the fresh specimens. Studies were performed using 5 % potassium hydroxide (KOH) as a mounting medium, with Congo Red and Melzer s reagent occasionally used as necessary to stain various structures. Spore measurements were taken from naturally ejected spores directly obtained from a spore print, with normal tap water used as a mounting medium. At least 30 fully mature spores from each ascocarp were measured. The spore surface was described from mounts of dry sections of hymenium in pure glycerin stained with Waterman green ink (images) or Chlorazol black. The length of the paraphyses was measured from the apices to the basal septum, excluding the sterigmata. Terminal elements of the stipe cortex (ectal excipulum) were observed from the apex, and were measured in cases that formed well-differentiated, cylindrical or catenulate protrusions (Bhairs^). The material is deposited at LIP (Herbarium of the Faculty of

3 Mycol Progress (2016) 15:39 Page 3 of pharmacy of Lille), with duplicates in the fungal collection of the CEFE laboratory (Centre d Ecologie Fonctionnelle et Evolutive, Montpellier) and the private collections of the authors. Terminology The term Bacroparaphyses^ (= edge paraphyses, from the greek word άκρον, meaning Bedge^) is introduced for the sterile elements covering the pileus ridges. Selection strategy for sequencing A first set of specimens were selected, amongst well-characterised collections suspected to represent distinct species. A number of morphological and ecological criteria were used in the selection process, such as the putative plant host, substrate, fruiting season, altitudinal range, distribution range, and a number of macroand microanatomical characters, as outlined in BDiscussion^. Once the first set of collections was molecularly characterised, additional collections exhibiting atypical features and/or ecology were further tested, in order to refine species concepts and define their ecological/morphological boundaries. DNA extraction, amplification, and sequencing DNA extraction and polymerase chain reaction (PCR) amplification were conducted with the REDExtract-N-Amp Plant PCR Kit (Sigma-Aldrich, St. Louis, MO, USA) following the manufacturer s instructions. The internal transcribed spacers and 5.8S rdna (ITS) were amplified from each collection with the ITS-1 F/ITS-4 primer pair (Gardes and Bruns 1993). When necessary, three additional genetic loci were amplified with the following primer pairs: the RNA polymerase II largest subunit gene (RPB1) with grpb1a/arpb1c (Matheny et al. 2002), the RNA polymerase II second largest subunit gene (RPB2) with 9 F/3R (Liu et al. 1999), and the translation elongation factor 1-α gene (TEF1) with EF526F/EF3AR (Rehner and Buckley 2005). All PCR amplifications were performed as described in Richard et al (2015). Amplicons were purified and sequenced by Eurofins Genomics, Ebersberg, Germany. Raw sequence data were edited and assembled with Codon Code Aligner (CodonCode Corp., Centerville, MA, USA) and deposited in Genbank under the accession numbers indicated in Table 1. Phylogenetic analyses Phylogenetic analyses were all performed online at (Dereeper et al. 2008) and on the CIPRES Science Gateway ( org/index.php/). Because ITS is often insufficient in fully resolving all Morchella species, multilocus phylogenetic analyses (combining ITS, RPB1, RPB2, and TEF1 sequence data) were performed when necessary to assign newly sequenced collections to a known or putatively novel phylogenetic species. Multiple sequence alignments were carried out with MUSCLE 3.7 (Edgar 2004) usingfull processing mode and 16 iterations. When required, alignments were edited with Gblocks 0.91b, set to lowest stringency in the selection of conserved blocks (Castresana 2000; Talavera and Castresana 2007). Maximum likelihood (ML) phylogenetic analyses were performed with PhyML 3. 0 (Guindon et al. 2008), using the GTR + I + Γ model of evolution. Branch support was assessed using the non-parametric version of the approximate likelihood-ratio test, implemented in PhyML (SH-aLRT; Anisimova and Gascuel 2006). This recently developed metric runs much faster than the standard bootstrapping procedure (SBS) and ensures high accuracy when SH-aLRT > 0.8 (Anisimova et al. 2011; Bellanger et al. 2015). Bayesian inference of phylogeny was performed using MrBayes (Ronquist and Huelsenbeck 2003). Two runs of four Monte Carlo Markov Chains were performed for 1,000,000 generations, with tree and parameter sampling every 1,000 generations (1,000 trees). The initial burn-in was set to 25 % (250 trees). A 50 % majority-rule consensus cladogram was computed from the remaining trees; the proportions of this tree correspond to Bayesian posterior probabilities (BPP) that are reported (as percentages) on each branch of the phylograms depicted in Figs. 1, 2, and 3. Trees were annotated using TreeDyn (Chevenet et al. 2006). Results The phylogenetic analysis of 25 newly generated sequences from representative morel collections from Cyprus confirms the presence of nine Morchella species, increasing the known diversity of the genus on the island to eleven species (Table 1). Of those, nine are previously unreported from Cyprus: M. importuna Kuo et al., M. eximia Boud., M. exuberans Clowez et al., M. dunensis (Castañera, et al.) Clowez, M. dunalii Boud. and, most notably, four species found to be new to science. Two of these are represented by a single ascocarp each, and are thus provisionally assigned the new phylogenetic codes Mes-28 and Mel-38, respectively, awaiting further collections and sampling to be formally described: Mes-28 belongs to sect. Esculenta (Mes clade, O Donnell et al. 2011) andisnestedwithinthechineseandindian Mes-13/15/19 subclade, at a minimal phylogenetic distance to its closest neighbor Mes-15 representing eight nucleotide changes along the ITS (0.7 % of difference plus 5 indels, Fig. 1 and data not shown). Mel-38 belongs to sect. Elata (Mel clade, O Donnell et al. 2011) and cannot be resolved by its sole ITS sequence (Fig. 2). However, multilocus phylogenetic analysis positions the lineage within a Mel-17 to 37 subclade, having partially supported paraphyletic relationships with M. purpurascens (Mel-20), M. eohespera (Mel- 19), Mel-17 and Mel-34 (BPP = 65 %, SH-aLRT = 0.87, Fig. 3). The two other novelties belong to sect. Elata, and are here introduced as M. arbutiphila Loizides et al. and M.

4 39 Page 4 of 28 Mycol Progress (2016) 15:39 Table 1 Morchella collections subjected to phylogenetic analysis (Figs. 1, 2, and 3) Species Locality Voucher id Habitat/Vegetation Collection date Ref Genbank accession ITS RPB1 RPB2 TEF1 Sect. Elata Morchella arbutiphila sp nov Morchella disparilis sp nov Mesa Potamos Mesa Potamos Platania Platania Panayia Platania Platania Platania Ayia Paraskevi Ayia Paraskevi Ayia Paraskevi Ayia Paraskevi Amiantos ML5146MPG Arbutus andrachne, Pinus brutia 4/6/2015 This study KU KU KU KU ML5146MP2 Arbutus andrachne, Pinus brutia 4/6/2015 This study KU ML5146P2 Arbutus andrachne, Pinus brutia 4/6/2015 This study KU ML5146P3 Arbutus andrachne, Pinus brutia 4/6/2015 This study KU KU KU KU ML5148MA Arbutus andrachne, Pinus brutia 4/8/2015 This study KU KU KU KU ML51401MA (Holotype) Arbutus andrachne, Pinus brutia 4/10/2015 This study KU KU KU KU ML51431MA Arbutus andrachne, Pinus brutia, Quercus alnifolia 4/13/2015 This study KU ML51431MP Arbutus andrachne, Pinus brutia 4/13/2015 This study KU KU KU KU ALV3240 Arbutus andrachne, Pinus brutia 4/13/2011 This study KU ML21392MD (Holotype) Arbutus andrachne, Pinus brutia 3/29/2012 This study KU ML5143MDI Arbutus andrachne, Pinus brutia 4/3/2015 This study KU ML5143MD2 Arbutus andrachne, Pinus brutia 4/3/2015 This study KU KU KU KU ML51481MD Arbutus andrachne, Pinus brutia, Quercus alnifolia Morchella dunalii Souni ALV3207 Pinus brutia, Cistus parviflorus 3/15/2014 This study KU Archimandrita ML51272M Quercus coccifera spp. calliprinos 2/27/2015 This study KU Agros ML51303MDP Cistus salvifolius, Pinus brutia, Arbutus andrachne, Mirtus communis Trimiklini 4/18/2015 This study KU KU KU KU /30/2015 This study KU ML51303MC Cistus salvifolius, C. creticus, Olea europaea 3/30/2015 This study KU Mandria ML5143MDU Pinus brutia 4/3/2015 This study KU Morchella eximia Pelendri ALV year-old burned Pinus brutia forest 4/16/2009 This study KU Saittas ALV year-old burned Pinus brutia forest 3/28/2008 This study KU Morchella exuberans Morchella importuna Mandria ALV3209 Malus domestica orchard 4/2/2009 This study KU865012

5 Mycol Progress (2016) 15:39 Page 5 of Table 1 (continued) Species Locality Voucher id Habitat/Vegetation Collection date Ref Genbank accession ITS RPB1 RPB2 TEF1 Morchella sp nov (Mel-38) Morchella tridentina Sect. Esculenta Kouka ALV3205 Park, among Quercus infectoria ssp. veneris litter 4/4/2011 This study KU Lania ML51313MI Flooded river bank, among Quercus infectoria 3/31/2015 This study KU ssp. veneris litter ALV3206 Pinus brutia 3/24/2012 This study KU KU KU KU Pera Vasa Ayia Paraskevi ALV0150 Olea europaea 3/26/2009 Loizides et al. (2015) Trimiklini ALV0152 Olea europaea 3/26/2009 Loizides et al. (2015) Pera Pedi ALV0154 Olea europaea 3/28/2009 Loizides et al. (2015) Platres ALV0151 Platanus orientalis 3/1/2010 Loizides et al. (2015) Morchella dunensis Liencres (Spain) AH18336(Isoparatype) Ammophila arenaria & Linaria supina 4/1/1995 Castañera et al. (1996) Morchella sp nov (Mes-28) Sect. Rufobrunnea Morchella rufobrunnea Moutoullas JX JX JX JX KJ KU ALV3208 Malus domestica orchard 4/7/2009 This study KU Lania ML51313MV Ulmus canescens, Quercus infectoria ssp. veneris Lemesos Pissouri Lemesos Lemesos 3/31/2015 This study KU ALV0159 Park grass 2/23/2009 Loizides et al. (2015) ALV0156 Olea europaea 3/6/2011 Loizides et al. (2015) ALV0157 Olea europaea 3/11/2011 Loizides et al. (2015) ALV0158 Olea europaea 3/12/2011 Loizides et al. (2015) JX JX JX KJ JX *An autumnal collection (YY9002) provisionally identified as Morchella galilaea,wasunfortunatelylostandcouldnotbe confirmed by molecular testing. Morchella dunensis has been recorded on the island prior to this work, under the name M. rigida.asinglecollectionpreviouslyreportedasm. vulgaris (Loizides et al. 2011) was also lost and could not be verified by molecular testing (see Results and Taxonomy for details). Bold species names highlight Morchella species unreported in Cyprus prior to this work. Bold Genbank accessions highlight sequences generated for the present study.

6 39 Page 6 of 28 Mycol Progress (2016) 15:39 Fig. 1 Bayesian 50 % majority-rule mid-point rooted consensus tree inferred from ITS regions of 45 representative collections of Morchella sect. Esculenta. BranchsupportfromtheBayesianInferenceand Maximum Likelihood analyses are indicated when BPP < 95 % or SHaLRT 0.8. Cypriot collections and species taxonomically revised in the present tudy, are highlighted in bold and boxed in grey, respectively. T, TYPUS material. Credits: J-M. Bellanger

7 Mycol Progress (2016) 15:39 Page 7 of disparilis Loizides & P.-A. Moreau, spp. nov (Figs. 2 and 3 and Taxonomy). Multilocus phylogenetic analysis strongly supports M. disparilis as a monophyletic clade (BPP = 100 %, SHaLRT = 1), basal to a Mel-12 to 38 subclade after Bayesian inference (Fig. 3) or, aftermaximum Likelihoodanalysis andwith low statistics, in sister clade relationships with the Mes-12/13/26/ 27 lineage (SH-aLRT = 0.51, data not shown). Phylogenetically, M. disparilis is well separated from formerly described species and ITS fully resolves it, with no less than twenty-three nucleotide differences, representing 3.6 % of sequence divergence, plus five indels, when compared to its closest neighbor M. deliciosa (Fig. 2 and data not shown). The case of M. arbutiphila markedly differs, as this species isdeeplynestedwithinthebmel-17 to 34 complex^ (Richard et al. 2015), and cannot be resolved by its sole ITS sequence (Fig. 2). However, multilocus phylogenetic analysis strongly supports the species as a monophyletic lineage (BPP = 100 %, SH-aLRT = 0.97), sister to a Mel-23/24/31/32 subclade (BPP = 100 %, SH-aLRT = 0.9, Fig. 3). In its current state, M. arbutiphila includes Mel-30, represented by a single Turkish collection that differs from the Cypriot sequences by two to three nucleotide changes in the ITS1 sequence. Additional sequences from continental populations are necessary to evaluate ITS variability within the clade and test whether Mel-30 is still derived from within the M. arbutiphila clade or if the two lineages display reciprocal monophyly and deserve their own specific rank. Lastly, the analysis of sect. Esculenta performed in this work reveals that Morchella vulgaris, as morphogenetically defined in Richard et al. (2015), actually lumps together two closely related species, each with a distinct evolutionary history. The uniquely sequenced Cypriot collection that belongs to this inclusive clade nests within a subclade encompassing mostly mediterranean or coastal collections, such as the type materials of M. dunensis and M. andalusiae, as well as several Turkish collections phylogenetically identified as BMes-17^ by Taşkın et al (2012)(Fig.1). This M. dunensis subclade is separated from a sister subclade dominated by continental collections of M. vulgaris, by seven nucleotide changes mainly located at the 5 end of the ITS1 sequence. The autonomy of M. dunensis within the current, inclusive concept of M. vulgaris is further supported by (i) reciprocal monophyly of the two subclades in Bayesian Inference analysis (Fig. 1), (ii) distinct ecological and biogeographical patterns of collections in the two subclades, and (iii) subtle but consistent morphoanatomical features that usually allow the identification of the two species by non-molecular means (see BTaxonomy^). Taxonomy Morchella arbutiphila Loizides, Bellanger & P.-A. Moreau, sp. nov. (Figs. 4a-f, 5a-i, 12a, 13a, 16a). MycoBank MB Etymology: From the Greek word φίλος [= friend], in this case Bfriend of Arbutus^, reflecting this species ecological preferences. Diagnosis: Late-appearing, medium- to large-sized morel, characterised by a fleshy, umber- to olivaceous-brown, ovoid or conical pileus forming large, rounded, oblong pits, and a distinctly elongated stipe, often longer than the pileus s length. Stipe apex & sinus occasionally with purplish-pink tinges. Spores large < 31 μm long (Me = 26.5), faintly striate in cotton blue. Asci large. Paraphyses & acroparaphyses slender, polymorphic, septate in the lower third; capitate elements absent. Stipe terminal elements distinctly catenulate < 140 μm long. Under Arbutus andrachne. Holotype: Cyprus: Platania, ca 1100 m asl, in mixed forest under Arbutus andrachne, leg. M. Loizides, 10-IV-2015 (LIP ); isotype: herb. pers. M. Loizides (ML51401MA). Macromorphological description: Pileus3 6 ( 7) cm high 1 2 ( 3) cm wide, broadly conical, ovoid or subspherical at first, olivaceous-gray, pinkish-brown, umberbrown or olivaceous-brown, becoming more elongated at maturity, conical to cylindrical, attached to the stipe with a rather deep, well-defined sinus. Longitudinal primary ridges moderately spaced to rather dense and more or less concolorous with the pits at first, minutely tomentose and white-spotted, strongly anastomosed, forming roundish to elongated primary pits, sometimes with purplish or vinaceous tinges, soon blackening and displaying a sharp contrast with the pits; slightly to moderately eroding with age. Transversal secondary ridges usually sparse and inconspicuous, concolorous to the pits. Pits numerous, elongated, edges usually rounded, incorporating sparse, indistinct secondary pits. Stipe 3 7( 9) 0.9 2( 4) cm, more or less cylindrical and inflated at the base, weakly to moderately lacunose, hollow, strongly furfuraceous to punctate, white, sometimes with a purplish-pink or grayish-purple zone at the apex or at the margin, upon maturity typically equal or longer than the pileus s length. Flesh particularly tough and cartilagenous; odour strong, sweet to somewhat spermatic. Micromorphological description: Spores (22 ) ( 31) (14 ) ( 18) μm, Me = 26.5, Qm = 1.6, elliptical to subcylindrical, hyaline, thick- to medium-walled, often with external polar guttules; faintly longitudinally striate in cotton blue. Asci μm, cylindrical to clavate, hyaline, uniseriate, 8-spored, simple-septate at the base, inamyloid. Paraphyses hyaline, bifurcate, irregularly cylindrical, ( 230) ( 24) μm, often with 2 3 weakly inflated, aseptate compartments; apices polymorphic, clavate, subcapitate, bluntly fusiform or utriform, septate only at the base, or sometimes with an additional 1 2 septa in the lower third; not or only slightly constricted at the septa. Heteroparaphyses not seen. Acroparaphyses μm, fasciculate, polymorphic, cylindrical to irregularly clavate and inflated at various parts, 1 3 septate,brownpigmented at maturity, arising from a textura angularis;

8 39 Page 8 of 28 Mycol Progress (2016) 15:39 capitate elements absent. Stipe cortex resembling a textura angularis or globosa, composed of variously sized, spherical, subspherical, pyriform, subangular or subcylindrical cells, giving rise to long, scattered, or more often fasciculate, distinctly catenulate terminal elements up to ( 35) μm. Ecology & distribution: Oro-Mediterranean, xerophilic, appearing solitary or in small scattered groups late in the season on well-drained, sun-drenched slopes between April and May, on ophiolitic, igneous, or calcareous soils. So far exclusively recorded under Arbutus andrachne, at elevations ranging between m. Remarks: Unlike some Morchella species suffering from a shortage of clear-cut dichotomous characters, M. arbutiphila is morphologically well-defined, and appears to be confined to the Troodos ophiolite massif, where it is found in association with Arbutus andrachne. Its diagnostic features are its apparent exclusivity to Arbutus, its strikingly long stipe proportionately to the pileus (Fig. 4b f), and the frequent purplish band at the sinus margin or the stipe apex (Fig. 4d). Microscopically, it has unusually large spores, often reaching 30 μm andwithanaveragelengthgreaterthan26μm (Figs. 5a, 12a), displaying faint parallel striations when appropriately stained (Fig. 13a). It is further characterised by the large asci, the absence of capitate acroparaphyses (Fig. 5d), and the catenulate hairs on the stipe arising from a textura angularis/globosa (Figs. 5f, h i). Morchella arbutiphila is phylogenetically very close to Mel-30, so far known from a single collection in Turkey under Pinus and Quercus. As stated in BResults^, more thorough sampling of Mel-30 is necessary to get better insights in both the phylogenetic variability and host preferences of this species, and assess whether the latter should be considered conspecific with, or sister to M. arbutiphila. The species most likely to be confused with M. arbutiphila are M. disparilis, often occurring under the same host, and M. dunalii, another southern species of similar stature. The first one is distinguished by its smaller ascocarps, the exceptionally deep, abruptly angular sinus, its smaller and differently shaped spores < 27 μm, the often papillate-mucronate paraphyses, and its partially or markedly capitate acroparaphyses. The widespread M. dunalii occurs under Pinus, Quercus, and rarely Cistus, but has so far not been recorded under Arbutus. It typically has a short stipe, with distinctly smaller, thick-walled spores rarely exceeding μm andwithanaverage length < 22 μm, much wider paraphyses with frequently strongly inflated capitate or snakehead apices < 30 ( 40) μm, and wider, capitate or subcapitate acroparaphyses. Morchella tridentina, another Mediterranean species occurring under Arbutus, is morphologically very different and procuces pale ascocarps with non-darkening longitudinally split ridges, frequently moniliform paraphyses, and regularly cylindrical stipe hairs. Fig. 2 Bayesian 50 % majority-rule consensus tree inferred from ITS regions of 85 representative collections of Morchella sect. Elata. Branch support from the Bayesian Inference and Maximum Likelihood analyses are indicated when BPP < 95 % or SH-aLRT 0.8. Cypriot collections and species taxonomically revised in the present study are highlighted in bold and boxed in grey, respectively. T, TYPUS material. Credits: J-M. Bellanger Specimens examined (*sequenced collections): Platania, ca 1060 m asl, in mixed forest under Arbutus andrachne, leg. M. Loizides, 16-IV-2012 (ML PL); Ibidem, ca 1040 m asl, in mixed forest under Arbutus andrachne, leg.m. Loizides, 18-IV-2012 (ML PL); Mesa Potamos, ca 800 m asl, in mixed forest under Arbutus andrachne, leg. M. Loizides, 6-IV-2015 (ML5146MPG)*; Ibidem (ML5146MP2); Platania, ca 1050 m asl, in mixed forest under Arbutus andrachne, leg. M. Loizides, 6- IV (ML5146P2)*; Ibidem, ca 1050 m asl, in mixed forest under Arbutus andrachne, leg. M. Loizides, 6- IV (ML5146P3)*; Panayia, ca 1000 m asl, in mixed forest under Arbutus andrachne, leg. M. Loizides, 8- IV (ML5148MA)*; Platania, ca 1100 m asl, in mixed forest under Arbutus andrachne, leg. M. Loizides, 10-IV-2015 (LIP , holotype ; ML51401, isotype)*; Ibidem, ca 1100 m asl, in mixed forest under Arbutus andrachne, leg. M. Loizides, 13-IV-2015 (ML51431MP)*; Ibidem (ML51431MA); Karvounas, ca 1200 m asl, in mixed forest under Arbutus andrachne, leg.m.loizides,18-iv-2015 (ML KA); Spilia, ca 950 m asl, in mixed forest under Arbutus andrachne, leg. M. Loizides, 18-IV-2015 (ML SP); Platres, ca 1150 m asl, in mixed forest under Arbutus andrachne, leg. M. Loizides, 21-IV-2015 (ML PL); Troodos, ca 1400 m asl, in mixed forest under Arbutus andrachne, leg.m.loizides,28-iv-2015 (ML TR). Morchella disparilis Loizides & P.-A. Moreau, sp. nov. (Figs. 6a-f, 7a-k, 12b, 13b, 16b) MycoBank MB Etymology: Disparilis [Latin] = different or dissimilar, referring to the strikingly deep sinus, intermediate in depth between typical Distantes and half-free morels of the M. semilibera clade. Diagnosis: Late-appearing, small- to medium-sized morel, characterised by a conical pileus with olivaceous tinges, distant, flexuous-anastomosed primary ridges with rudimentary interconnecting ridges, and a distinctly deep, angular sinus. Stipe often elongated at maturity. Spores < 27 μm long (Me = 24.1), faintly creased in cotton blue. Paraphyses slender, often mucronate-papillate, mostly 1 2 septate at the base. Acroparaphyses with capitate/subcapitate elements. Stipe terminal elements poorly developed, mostly undifferentiated. In Mediterranean maquis, usually associated with Arbutus andrachne or Cupressus sempervirens.

9 Mycol Progress (2016) 15:39 Page 9 of 28 39

10 39 Page 10 of 28 Mycol Progress (2016) 15:39 Fig. 3 Bayesian 50 % majority-rule mid-point rooted consensus tree inferred from multilocus sequence data of 46 collections of Morchella sect. Elata, focused on the M. purpurascens complex (Mel-12 to 38). Branch support from the Bayesian Inference and Maximum Likelihood analyses are indicated when BPP < 95 % or SH-aLRT 0.8. Cypriot collections and species taxonomically revised in the present study are highlighted in bold and boxed in grey, respectively. T, TYPUS material. Credits: J-M. Bellanger

11 Mycol Progress (2016) 15:39 Page 11 of Fig. 4 Macromorphological variability of Morchella arbutiphila sp. nov., in situ. Credits: M. Loizides Holotype: Cyprus: Ayia Paraskevi, ca 550 m asl, in mixed forest under Arbutus andrachne, leg. M. Loizides, 29-III-2012 (LIP ). Macromorphological description: Pileus1 3 ( 4) cm high 1 2 ( 3) cm wide, conical to broadly conical or ovoid, ochraceous-brown, olivaceous-brown, olivaceous-green or olivaceous-gray, attached to the stipe with a markedly abrupt, 4 8-mm-deep angular sinus; margin inrolled when young, soon loosened and sometimes expanding outwards at maturity. Longitudinal primary ridges moderately spaced to distant, flexuous, anastomosed, initially concolorous with the pits, never purplish, soon blackish and often eroding with age. Transversal secondary ridges usually sparse, indistinct or occasionally absent, irregular, more or less concolorous with the pits. Pits usually large and irregular, at first concolorous and later paler than the ridges. Stipe 1 4 ( 6) ( 2.5) cm, more or less cylindrical or enlarged at the base, weakly to moderately lacunose, hollow, at maturity often equal or longer than the pileus length, whitish, often with an ochraceous-pink reflection and usually strongly furfuraceous-puncate at the apex. Flesh elastic, rather thick, tough, with a rather strong, sweet, somewhat spermatic odour. Micromorphological description: Spores (20 ) ( 27) (12 ) ( 18) μm, Me = 24.1, Qm = 1.5, elliptical, hyaline, thick- becoming thin-walled, usually with external polar guttules; spore surface faintly creased in cotton blue, with anastomoses between longitudinal wrinkles. Asci μm, cylindrical to clavate, hyaline, uniseriate or occasionally biseriate, 8-spored, simple-septate at the base, inamyloid. Paraphyses hyaline, bifurcate, irregularly cylindrical, μm, often with 2 3 inflated aseptate compartments in the upper half, 1 3-septate at the lower third or at the base, not or only weakly constricted at the septa; apices not very enlarged, variable, subcapitate, clavate, bluntly fusiform (snakehead), mucronate or papillate, ( 18) μm wide. Heteroparaphyses not seen. Acroparaphyses μm, fasciculate, polymorphic, fusiform to mucronate-papillate, but also clavate to capitate, 1 2 septate at

12 39 Page 12 of 28 Mycol Progress (2016) 15:39 Fig. 5 Micromorphological characters of Morchella arbutiphila sp. nov. Scale bar = 20 μm. (a) Spores; (b c) Paraphyses apices; (d) Acroparaphyses; (e) Paraphyses; (g) Ascus base; (f, h i) Stipe hairs. Credits: M. Loizides the base, arising from a textura globosa. Stipe cortex polymorphic, composed of variously shaped globose to irregularly clavate, cylindrical, or pyriform cells, occasionally forming irregular, poorly differenciated, short-cylindrical or catenulate protrusions measuring μm. Ecology & distribution: Appearing solitary or gregariously between late March and mid-april, often among mosses, at elevations ranging between m. So far only recorded from Cyprus and Greece (Loizides & Manatakis, unpubl. data), in warm, calcareous forests and mattoral, associated with Arbutus andrachne, Cupressus sempervirens, and possibly other Mediterranean vegetation. Remarks: The particularly deep and abruptly angular sinus, sometimes lifting outwards at maturity giving the impression of a half-free morel (Figs. 6e f, 16b), is undoubtedly the most remarkable feature of this species. The stipe, although short when young, soon becomes elongated and sometimes longer than the pileus, which has predominantly greenish-olivaceous tinges (Figs. 6a f). The flexuous-anastomosed primary ridges and rudimentary secondary ridges are additional macroscopic features defining this species (Figs. 5a, c d, 16b). Microscopically, it has larger spores than most European Distantes species (Figs. 7a, 13b), slender, often papillatemucronate paraphyses (7B F), subcapitate to capitate

13 Mycol Progress (2016) 15:39 Page 13 of Fig. 6 Macromorphological variability of Morchella disparilis sp. nov., in situ. Credits: M. Loizides acroparaphyses (7G H), and poorly developed stipe hairs (7 J K). Morchella distans Fr., an old, obscure taxon described from Sweden (Fries 1849, without iconography), is reported to have a pileus that is Badnate but distant from the stipe^, implying a wide sinus. The particularly vague, 3-lined original diagnosis states: BOmnino jungit haec nova species primam et secundam sectioneum; mitra conica basi quidem adnata, sed a stipite distante; habitus ceterum M. conica elatioris. Omnes Morchellae inter se nimis affines.^ Such a broad, non-specific description, devoid of ecological or micromorphological information, could well apply to most Distantes and is practically uninterpretable. More importantly, M. disparilis appears to have a strictly Mediterranean ecology and distribution, with its phylogenetic lineage so far not reported from northern Europe (see Taşkın et al. 2012; Richard et al. 2015). Consequently, subsequent uses of the name BM. distans^ for southern European collections with a long stipe and a shallow sinus (Boudier 1909; Jacquetant 1984; Medardi 2006) are probably misapplied, and in any case not applicable to M. disparilis. Morchella arbutiphila, also occurring late in the season under Arbutus, is the species most likely to be confused with M. disparilis. It produces larger, more robust ascocarps, with a stipe that is elongated from the start (Fig. 4f), has large oblong pits and a shallower sinus, which sometimes has pinkishviolaceous shades. Microscopically, M. arbutiphila has larger, subcylindrical spores, often reaching or exceeding 30 μm and with an average length > 26 μm (Figs. 5a and 12a), which are striate in the appropriate medium (Fig. 13a). It further has better developed and more distinctly catenulate terminal elements on the stipe reaching μm, and lacks capitate elements in the acroparaphyses. Although usually appearing earlier, the widespread Morchella dunalii sometimes co-exists in the same habitats as M. disparilis. It produces larger ascocarps that are initially pale buff or beige, with ridges that often turn vinaceous or violaceous before darkening (Figs. 8b c, 16c), has well-defined secondary ridges forming rounded, sunken, partially vertically arranged pits, as well as a shallower sinus and a short stipe. Microscopically it has smaller thick-walled spores, rarely exceeding μm and with

14 39 Page 14 of 28 Mycol Progress (2016) 15:39 Fig. 7 Micromorphological characters of Morchella disparilis sp. nov. Scale bar = 20 μm. (a) Spores; (b e) Paraphyses; (f) Asci & Paraphyses; (g h) Acroparaphyses; (i) Ascus base; (j k) Stipe hairs. Credits: M. Loizides an average length < 22 μm, but more crucially, much wider paraphyses apices often reaching 30 ( 40) μm, with septa frequently extending to the upper third, as well as polymorphic, occasionally cylindrical, or encrusted stipe hairs. Specimens examined (*sequenced collections): Ayia Paraskevi, ca 550 m asl, in mixed forest under Arbutus andrachne, leg. M. Loizides, 13-IV-2011 (ALV3240)*; Ibidem, ca550masl,inmixedforestunderarbutus andrachne, leg. M. Loizides, 29-III-2012 (LIP , holotype)*; Ibidem, ca 570 m asl, in mixed forest under Arbutus andrachne, M.Loizides,31-III-2012(ML AP); Prastio, ca 400 m asl, near Olea europaea, Pinus brutia and Cistus sp., leg. D. Markides, 12-III-2014 (DM ). Ayia Paraskevi, ca 560 m asl, in mixed forest under Arbutus andrachne, leg. M. Loizides, 3-IV-2015 (ML510243AP1)*; Ibidem, ca540masl,inmixedforestunderarbutus andrachne, leg. M. Loizides, 3-IV-2015 (ML510243AP2); Amiantos, ca 1100 m asl, in mixed forest under Arbutus andrachne, leg. M. Loizides, 18-IV-2015 (ML51481MD)*. Morchella dunalii Boud., Bull. Soc. mycol. France 3: (Figs. 8a-f, 9a-h, 12c, 13c, 16c) = Morchella fallax Clowez & Luc Martin in Clowez, Bull. Soc. mycol. France 126 (3-4): Misapplied names: Morchella purpurascens sensu Loizides et al. (2011); Morchella rielana sensu Clowez (2012) Macromorphological description: Pileus 4 8 ( 10) cm high 2 5( 8) cm wide, broadly conical, cylindrical or ovoid, at first uniformly pale beige, buff, or olivaceous-buff,

15 Mycol Progress (2016) 15:39 Page 15 of Fig. 8 Macromorphological variability of Morchella dunalii, in situ. Credits: M. Loizides gradually darkening to pale grayish-brown or olivaceous-gray, attached to the stipe with a wide, well-defined, usually rounded sinus. Longitudinal primary ridges moderately spaced to crowded, thick and fleshy, glabrous, barely eroding with age, sinuate and usually anastomosed; at first concolorous with the pits, gradually ochraceous-pink to pinkish-purple or grayish, finally black at full maturity and usually distinctly darker than the pits, creating a sharp contrast. Transversal secondary ridges few and rather irregular, often forming an irregular or partially regular ladder-like pattern at maturity, usually concolorous with the pits. Primary pits medium-sized to large, distinctly rounded to elongated, at first concolorous with the ridges, later more or less paler and usually incorporating sunken, partially vertically arranged and distinctly rounded secondary pits. Stipe 2 4( 8) 1.5 3( 4) cm, short and typically partially or entirely submerged into the substrate, more or less cylindrical and typically enlarged at the base, weakly to moderately lacunose, hollow, white, indistinctly pruinose at first, more distinctly so and sometimes ochraceous-stained with age. Flesh tough, elastic, thick, with a strong, sweet, somewhat spermatic odour. Micromorphological description: Spores (17 ) ( 26) (10.5 ) ( 16) μm, Me = 21.5, Qm = 1.5, broadly elliptical to elliptical, usually rounded at the poles, very faintly creased and without distinct striations in cotton blue, hyaline, mostly thick-walled, inamyloid, some with external polar guttules. Asci μm, cylindrical to clavate, hyaline, 8 spored, uniseriate or frequently irregularly biseriate, inamyloid, with an enlarged, distinctly rounded to clavate base. Paraphyses μm, bifurcate, cylindrical to clavate, sometimes moniliform, 2 3 ( 4)-septate, septa often extending up to the upper third; apices usually markedly to dramatically inflated ( 40) μm, clavate, capitate or bluntly fusiform (snakehead), often wider than the asci. Heteroparaphyses frequent in some collections, highly irregular. Acroparaphyses ( 40) μm, clavate, capitate, subcapitate, or pyriform, fasciculate, 2 3 septate, thick-walled, brown pigmented and often partially collapsing

16 39 Page 16 of 28 Mycol Progress (2016) 15:39 Fig. 9 Micromorphological characters of Morchella dunalii. Scale bar = 20 μm. (a) Spores; (b) Paraphyses tips; (c) Ascus base; (d e) Paraphyses; (f) Ascus; (g) Acroparaphyses; (h) Stipe hairs. Credits: M. Loizides at maturity; arising from an intricate structure resembling a textura angularis or ephemeroidea. Stipe cortex a textura globosa, composed of variously sized, irregularly arranged catenulate elements, giving rise to scattered or locally fasciculate clavate to subcapitate, sometimes encrusted terminal elements, with 1 3 septa, measuring μm. Ecology & distribution: Typically among the first morels to appear between late February and early April, usually at elevations ranging between m; fruiting solitary or in small scattered groups, rarely cespitose, in damp, mossy, open Pinus brutia forests, occasionally also in evergreen Quercus stands or Cistus mattoral, with a strong preference for calcareous soil. Also recorded from Turkey (Taşkın et al. 2010, 2012, as BMel-25^), France, and Spain (Clowez 2012), but appears to be present throughout the Mediterranean basin (Richard et al. 2015, and unpubl. data). Remarks: As shown by Moreau et al. (2011), Morchella dunalii is the correct name for this widespread and frequently occurring Mediterranean species (Figs. 14 and 15), often misidentified as BM. conica^, BM. deliciosa^, orbm. purpurascens^ due to its high chromatic, morphological and ecological plasticity (Figs. 8a f). In Cyprus and Turkey it is commonly encountered under Pinus brutia (Taşkın etal. 2010, 2012, asbmel-25^, Loizidesetal. 2011, asbm. purpurascens^), while in Spain and France it is often found under Quercus ilex (Clowez 2012, as M. fallax; Richard et al.

17 Mycol Progress (2016) 15:39 Page 17 of ; A. Palazón, pers. comm.), on calcareous soil with poor humus and scattered vegetation. Collections from Quercus coccifera stands and Cistus salviifolius/c. creticus mattoral have also been molecularly verified from Cyprus. Morphologically, M. dunalii is characterised by the following features: robust, thick-fleshed ascocarps forming a wide sinus (Fig. 8e); contrasting, purplish or blackish tinges of the anastomosed primary ridges (Figs. 8b f, 16c); sunken, partially vertically arranged rounded pits (Figs. 8e f); a short white stipe (Figs. 8a f, 16c); markedly inflated capitate or Bsnakehead^ apices of the paraphyses < 30 ( 40) μm in diameter (Figs. 9b, d e); capitate or subcapitate thick-walled acroparaphyses (Fig. 9 g); and rather small, thick-walled spores (Figs. 9a, 12c), rarely exceeding μm in length (although collections with spores up to 26 μm have been rarely seen). Even though in its typical form it can be easily recognised, its distinction from the similar M. purpurascens, M. deliciosa and, especially its rare and still poorly known sister species, M. kakiicolor (Clowez & L. Romero) Clowez et al., is not always straightforward. As noted by Loizides et al. (2015), based on the type material, M. kakiicolor appears to lack purplish tinges, has equally thick-walled, but slimmer, cylindrical paraphyses (<14 μm wide), shorter and slimmer terminal elements on the stipe ( μm) and considerably smaller spores ( μm), although the consistency of these characters needs to be verified from further collections. Morchella deliciosa Fr., as illustrated by Taşkın et al. (as BMel-26^, 2010),aswellasClowez(asBM. conica var. flexuosa^, BM. conica var. nigra^ and BM. conica var. violeipes^, 2012),andRichardetal.(2015, fromthe epitype), can also be morphologically confused with M. dunalii. Based on re-examination of molecularly confirmed collections (listed by Richard et al as M. deliciosa/ Mel-26), the latter produces more acutely conical ascocarps, often tilted at the top, with thick, longitudinally anastomosed or flexuous primary ridges, forming oblong, somewhat angular and irregular primary pits, and sparse secondary pits. Its pileus ridges remain for a long time paler than the pits, sometimes developing olivaceous or pinkish tones, and darken very late in maturity to blueish, purplish, dark grayish-black, or anthracite black. Microscopically, M. deliciosa has markedly slimmer, cylindrical, papillate to subcapitate acroparaphyses ( μm wide),which are thin-walled ( μm) or only locally thickened at the apex, slimmer paraphyses < 11 μm wide,aswell as a stipe cortex of trichoepithelial structure, consisting of 5 12 short-catenulate, subglobose to barrel-shaped elements < 18 μm wide.thesporesofm. deliciosa are very faintly or incompletely striate in optical microscopy, and larger than those of M. dunalii ( μm, Me = 25.7, Qm = 1.75, in one mature molecularly confirmed collection studied LIP PhC11). The widespread M. purpurascens (Boud.) Jacquet., as recently epitypified by Richard et al. (2015) and illustrated by Taşkın et al. (as BMel-20^, 2010), Clowez (as BM. conica var. purpurascens^ and BM. conica var. crassa^, 2012)and Richard et al. (2015, from the epitype), produces rather slender, more acutely conical ascocarps, with a shallow sinus and more pronounced pinkish or purplish tinges, lacking olivaceous colours. It has oblong, persistently pale and rather irregular pits in maturity, while the stipe becomes elongated and occasionally pink-spotted (as distinctly illustrated by Bresadola 1932, as BM. elata var. purpurascens^). Microscopically, it differs by long, clavate and thin-walled acroparaphyses (some vessel-shaped up to 45 μm wide), much slimmer paraphyses (<12 μm wide at the apices), and a trichoepithelial stipe cortex, made of irregular chains of 5 9 globose, barrel-shaped, and shortly cylindrical elements < 28 μm wide. The spores of M. purpurascens, like those of its sister species M. eohespera (Voitk et al. 2015), are distinctly longitudinally striate in SEM or under optical microscopy in cotton blue or Melzer s reagent. Morchella disparilis, sometimes co-existing in the same habitat as M. dunalii, is separated by its smaller ascocarps lacking purplish tinges, a pileus forming sparse to rudimentary and irregular secondary pits, the strikingly deep, angular sinus, and microscopically, by its slimmer, often papillatemucronate paraphyses (<18 μm wide at the apices) with low septa restricted in the lower third or at the base. Morchella arbutiphila is distinguished by its exclusive association with Arbutus, its long stipe proportionately to the pileus, its much larger < 30 μm long spores, its slimmer paraphyses with low septa restricted in the lower third or at the base, as well as noncapitate acroparaphyses. Both M. arbutiphila and M. disparilis display creased to striate mature spores in cotton blue or Melzer s, whereas the spores of M. dunalii appear to be very faintly to almost imperceptibly creased. Specimens examined (*sequenced collections): Pera Pedi, ca 550 m asl, in Pinus brutia forest, leg. M. Loizides, 20-III (ML PP); Ibidem, ca 550 m asl, in Pinus brutia forest, leg. M. Loizides, 11-III-2009 (ML PP); Trimiklini, ca 620 m asl, in Pinus brutia forest, leg. M. Loizides, 14-III-2009 (ML T); Kouka, ca 500 m asl, in Pinus brutia forest opening, leg. M. Loizides, 19-III-2009 (ML K); Ayia Paraskevi, ca 550 m asl, in Pinus brutia forest opening, leg. M. Loizides, 19-III-2009 (ML AP1); Ibidem (ML AP2); Ibidem (ML AP3); Pera Pedi, ca 550 m asl, in Pinus brutia forest, leg. M. Loizides, 22-III-2009 (ML PP1); Ibidem (ML PP2); Ibidem (ML PP3); Ibidem (ML PP4); Ibidem (ML PP5); Ayia Paraskevi, ca 530 m asl, in Pinus brutia forest, leg. M. Loizides, 28-III-2009 (ML AP1); Ibidem (ML AP2); Pera Pedi, ca 530 m asl, in Pinus brutia forest, leg. M. Loizides, 31-III-2009 (ML PP);

18 39 Page 18 of 28 Mycol Progress (2016) 15:39 Trimiklini, ca 600 m asl, in Pinus brutia forest opening, leg. M. Loizides, 24-II-2010 (ML01242T1); Ibidem, (ML01242T2); Ibidem, (ML01242T3); Ibidem, (ML01242T4); Ayia Paraskevi, ca 530 m asl, in Pinus brutia forest, leg. M. Loizides, 4-III-2011 (ML1134AP); Ibidem, ca 550 m asl, in Pinus brutia forest, leg. M. Loizides, 18-III-2011 (ML AP); Prastio, ca 575 m asl, in Pinus brutia forest, leg. M. Loizides, 5-III-2012 (ML210235PR1); Ibidem (ML210235PR2); Trimiklini, ca 600 m asl, in Pinus brutia forest, leg. M. Loizides, 1-III-2013 (ML310231T1); Ibidem (ML310231T2); Souni, ca 450 m asl, in Pinus brutia forest opening, leg. M. Loizides, 5-III-2013 (ML310235S1); Ibidem (ML310235S2); Kelefos, ca 500 m asl, in Pinus brutia forest, leg. M. Loizides, 5-III-2014 (ML410235KE1); Ibidem (ML410235KE2); Souni, ca 425 m asl, in Pinus brutia forest opening, leg. M. Loizides, 9-III-2014 (ML410239S); 7-III- 2014; Ibidem, ca 450 m asl, in Pinus brutia forest opening, leg. M. Loizides, 9-III-2014 (ML410239S); Ibidem, ca 475 m asl, in Pinus brutia forest opening, leg. M. Loizides, 15-III (ALV3207)*; Mandria, ca 800 m asl, in Pinus brutia forest, leg. M. Loizides, 2-IV-2015 (ML510242MA); Pareklisia, ca 150 m asl, in Pinus brutia forest, leg. M. Loizides, 25-II-2015 (ML PA); Prastio, ca 575 m asl, in Pinus brutia forest, leg. M. Loizides, 25-II-2015 (ML PR1); Ibidem (ML PR2); Dora, ca 600 m asl, in Pinus brutia forest opening, leg. M. Loizides, 27-II-2015 (ML D); Archimandrita, ca 400 m asl, under Quercus coccifera, leg.m.loizides,27-ii-2015 (ML51272M)*; Lythrodontas, ca 450 m asl, in Pinus brutia forest, leg. M. Cheilides, 4-III-2015 (MC5134L); Agros, ca 1100 m asl, in Pinus brutia forest, leg. M. Loizides, 30-III (ML51303MDP)*; Trimiklini, ca 650 m asl, in Cistus salvifolius and C. creticus matorral, leg. M. Loizides, 30-III (ML51303MC)*; Mandria, ca 700 m asl, in Pinus brutia forest, leg. M. Loizides, 03-IV-2015 (ML5143MDU)*. Morchella dunensis (Castañera, J.L. Alonso & G. Moreno) Clowez in Clowez, Bull Soc Mycol France 126: 261 (2012) (Figs. 10a-f, 11a-i, 12f, 13d, 16k) Basionym: Morchella esculenta f. dunensis Castañera et al. in Castañera & Moreno, Yesca 8: = Morchella andalusiae Clowez & L. Romero in Clowez, Bull Soc Mycol France 126: Fig. 10 Macromorphological variability of Morchella dunensis, in situ. Credits: M. Loizides

19 Mycol Progress (2016) 15:39 Page 19 of Fig. 11 Micromorphological characters of Morchella dunensis. Scale bar = 20 μm. (a) Spores; (b d) Paraphyses; (e) Ascus base; (f) Biseriate ascus; (g h) Acroparaphyses; (i) Stipe hairs. Credits: M. Loizides Misapplied name: Morchella rigida sensu Loizides et al. (2011) Macromorphological description: Pileus 3 8 ( 12) cm high ( 8) cm wide, irregularly spherical, ovoid or cylindrical, at first ochraceous-buff, ochraceous-yellow, ochraceous-brown, or grayish-brown, gradually fading to beige, buff, ochraceous, or ochraceous-brown, often with rust-brown or orange-brown stains, attached to the stipe with or without a sinus. Primary ridges strongly sinuate-flexuous or entirely irregular, angular, often blistered or incomplete and with rusty-brown or reddish-orange spots, severely eroding with age. Secondary ridges indistinct or absent. Pits typically deep and labyrinthoid, angular, concolorous or somewhat darker than the ridges, fading with age. Stipe ( 9) ( 4.5) cm, strongly inflated at the base, when growing on sands often irregularly fusiform and ± deeply rooting, moderately to strongly lacunose, indistinctly pruinose, whitish to ochraceous. Flesh, firm, elastic, with a sweet, somewhat spermatic odour. Micromorphological description: Spores (17 ) ( 26) (11 ) ( 16) μm, Me = 20.3, Qm = 1.56, narrowly to broadly elliptical or ovoid, hyaline, thick-walled (<1.1 μm), usually narrowing and internally microguttulate at the poles; wall surface very faintly roughened to nearly smooth in cotton blue. Asci μm, cylindrical to clavate, hyaline, 8 spored, uniseriate, frequently also irregularly biseriate,

20 39 Page 20 of 28 Mycol Progress (2016) 15:39 Fig. 12 Comparative spore deposits in water, demonstrating spore variability in size, shape, wall-thickness, and internal guttulation. Scale bar = 20 μm. (a) Morchella arbutiphila; (b) M. disparilis; (c) M. dunalii; (d) M. importuna; (e) M. tridentina; (f) M. dunensis. Credits: M. Loizides inamyloid, base often sinuous and attenuated. Paraphyses μm, numerous, bifurcate, cylindrical to clavate; apices sometimes slightly enlarged and bluntly fusiform < 18 μm, 1 3 septate and occasionally constricted at the septa; septa frequently extending to the upper half. Heteroparaphyses present in some collections, irregular. Fig. 13 Spore ornamentation of (a) Morchella arbutiphila; (b) M. disparilis; (c) M. dunalii; and (d) M. dunensis under light microscopy, mounted in pure glycerin, stained with Waterman green ink. Scale bar = 10 μm. Credits: P. Clowez Acroparaphyses μm, fusiform, clavate, or cylindrical, fasciculate, 2 4 septate and sometimes constricted at the septa; septa frequently equally distributed and extending to the upper third, pale ochraceous-brown pigmented at maturity; capitate elements absent. Stipe cortex resembling a textura angularis or prismatica, giving rise to scattered or locally fasciculate, occasionally encrusted, long-cylindrical to clavate, or rarely subcapitate terminal elements with 2 3 septa, measuring ( 140) μm. Ecology: Rare in Cyprus, recorded only three times in apple (Malus domestica) orchards, fruiting solitary or in small isolated groups, among grasses, herbs, or on bare soil. Further reported from Turkey (as BMes-17^, Taşkın et al. 2012), Spain (as BM. andalusiae^, Clowez 2012) under Fraxinus angustifolia, Castanea sativa, and Ranunculus ficaria, but also all along the Atlantic coast to the North Sea, on fixed coastal and inland dunes (Castañera and Moreno 1996; Clowez 1997). Remarks: Morchella vulgaris sensu lato displays spectacular morphological variability, which incited Clowez (2012) to distinguish various satellite taxa (namely M. acerina Clowez & C. Boulanger, M. andalusiae Clowez & L. Romero, M. anthracina Clowez & Vanhille, M. lepida Clowez & F. Petit, M. robiniae Clowez, M. spongiola Boud., and several varieties). All of the above were presented as conspecific in Richard et al. (2015), based on ITS data, matching Taşkın et al. s (2010) delimitation of BMes-17^. However, careful re-examination of the clade following additional collections, suggests that two evolutionary close yet distinct species, have been lumped together within the latter morphogenetic concept: Morchella vulgaris sensu stricto

21 Mycol Progress (2016) 15:39 Page 21 of (including the epitype collection PhC3), which is so far represented by predominantly continental collections (Fig. 1 and BResults^.); and BMes-17^, a group of sequences originating from Mediterranean xerophilic collections (Cyprus, Spain, Turkey) and the Atlantic coast [including the holotype of M. andalusiae, which matches the original concept of Mes-17 by Taşkın et al. (2010)]. The holotype of Morchella esculenta f. dunensis Castañera et al., not included in the revision by Richard et al. (2015), unfortunately failed to yield useful molecular data for the purpose of the present study; nevertheless, an isoparatype collection provided by G. Moreno (AH18336) was successfully sequenced and clustered with the Mes-17 clade, as predicted by its origin and ecology. By anteriority, the specific name Morchella dunensis (Castañera et al.) Clowez is revived here as the legitimate Linnaean name for Mes-17 in this new restricted sense, with M. andalusiae designated as a later synonym of the former. In addition to its Mediterranean-xerophilic or sandinhabiting ecology, the ascocarps of M. dunensis appear to be morphologically distinct, featuring coarser ridges and predominantly ochraceous or ochraceous-brown colours (Fig. 10a f), generally lacking the gray tinges and contrasting dark pits typically seen in M. vulgaris. Ascocarps are usually rufescent, and when found on sand they develop a long, deeply rooting and often misshapen stipe. Other common European species in sect. Morchella are usually welldifferentiated by their ecology: Morchella esculenta (L.) Pers., exceptionally found in sands, is the species most likely to be confused with M. dunensis, but displays regularly polygonal pits with more or less smooth ridges, and has a rather smooth, cylindrical, weakly inflated at the base stipe (dilated only in age or exceptional growth conditions). The recently described Morchella americana Clowez & Matherly (=M. esculentoides M. Kuo et al.) and M. fluvialis Clowez et al., can also resemble M. dunensis: The first is not or only weakly rufescent with age, contrary to the second, which is strongly rufescent; both species, however, display mostly elongate pilei with partially longitudinally arranged pits and smooth ridges, and have a typically hygrophilic ecology, in riparian forests or poplar plantations (Clowez 2012, Kuoetal.2012). Microscopically, both M. americana and M. fluvialis appear to have shorter hairs on the stipe [30 70 ( 9) ( 30) μm and μm, respectively, Clowez 2012], whereas M. fluvialis has asci with croziers (Clowez et al. 2014), a feature not seen in any of the M. dunensis collections studied by us (although irregular outgrowths arising from the basal hyphae have been occasionally observed, see Fig. 11e). A thorough assesment of M. dunensis spore surface (revealed to be smooth in some collections and faintly roughened in others, see Fig. 13d), as well as a detailed comparison with M. vulgaris, will be the object of a separate study. Specimens examined (*sequenced collections): Moutoullas, ca 900 m asl, under Malus domestica in neglected orchard, leg. S. Michailides, 7-IV-2009 (ALV3208)*; Millomeri, ca 970 m asl, under Malus domestica in orchard, leg. M. Loizides, 11-IV-2009 (ML PL); Amiantos, ca 1100 m asl, under Malus domestica on sandy bank, leg. D. Markides, 28-IV-2015 (DM ). Discussion Interpreting the data Results of the present study constitute a major contribution to our knowledge of Morchella diversity in Cyprus, but also to that of the Mediterranean region, significantly broadening our understanding of the morphology, taxonomy, and distribution of the recorded species. Out of the eleven molecularly confirmed species, eight belong to section Distantes (Elata clade): M. arbutiphila, M. disparilis, M. dunalii, M. eximia, M. exuberans, M. importuna, M. tridentina and Mel-38; two belong to section Morchella (Esculenta clade: M. dunensis and Mes-28); and one to the basal lineage Rufobrunnea, section Rufobrunnea (M. rufobrunnea). The presence of at least three previously unreported lineages (Mes-28, Mel-38 and M. disparilis) confirms the high levels of endemism and provincialism within Morchella (O Donnell et al. 2011; Richard et al. 2015). Equally striking, on the other hand, is the confirmation of five species of transcontinental distribution (M. eximia, M. exuberans, M. importuna, M. rufobrunnea and M. tridentina), most of which appear to be fairly widespread and naturally occurring on the island. In addition to the above confirmed species, a striking autumnal collection of three ascocarps harvested from an urban garden (YY9002) appeared to fit well the concept of Morchella galilaea Masaphy & Clowez (Clowez 2012, Taşkın et al. 2015). Unfortunately, the ascocarps of this collection have been lost and could not be subjected to molecular testing; this collection is therefore provisionally listed as BMorchella cf galilaea^ in the footnotes of Table 1 and illustrated (Fig. 16l), but excluded from the discussion. Out of the previously unreported lineages, Mel-38 and Mes-28 (Figs. 1, 2 and 3) are apparently rare and so far known from single collections of one ascocarp each; these species formal descriptions are therefore postponed until further collections are available and their morphological variability is thoroughly evaluated. A polythetic system of classification as an answer to polymorphism Traditional morel literature recurrently points out the difficulties in interpreting the various taxa as a result of their high morphological plasticity, and reversely provides only chief details on their ecology and chorology. Identification becomes

22 39 Page 22 of 28 Mycol Progress (2016) 15:39 further complicated, due to the fact that many macro- and micromorphological characters evolve during the life cycle of ascocarps (Clowez 2012; Loizides et al. 2015). Whilst it is certain that most morel species can rarely be identified from exsiccatae alone, combined morphological and molecular analyses have significantly elucidated which macro- and microcharacters can be useful in discriminating each species from its close genealogical relatives and lookalikes, and to what extent these features can vary or overlap. As demonstrated by Loizides et al. (2015), a polythetic approach is necessary in contemporary Morchella taxonomy, taking into account a wide range of elements, none of which are nonetheless strict prerequisites for identification. These include the biogeographical distribution of the molecularly confirmed species, the ecological preferences and putative plant associations, macromorphological characteristics (including the maturity process and developmental stages of fresh ascocarps), and a number of important, yet often neglected microscopic characters. Such ecological, macro- and micromorphological features were so far scattered in literature, but never before compiled into a workable system of identification. As suggested by previously published SEM images (Malloch 1973; Сухомилин et al. 2007), and more recent studies by Kuo et al. (2012), and Loizides et al. (2015), micromorphological features such as the spore shape and surface, the apices and septation of the paraphyses, the shape of the sterile ridges elements (for which the term Bacroparaphyses^ is here introduced), as well as the terminal elements (or Bhairs^) of the stipe cortex, have been grossly underestimated in traditional literature, and will undoubtedly prove significant in the morphological characterization of most taxa. Our observations have revealed that a number of species have predominantly capitate acroparaphyses (M. disparilis, M. dunalii, M. exuberans, M. purpurascens), while others appear to lack capitate elements (M. arbutiphila, M. deliciosa, M. dunensis, M. importuna). Morchella dunalii, as well as its sister species M. kakiicolor, are additionally characterized by uniformly thick-walled acroparaphyses. Our attention was further drawn to the shape and width of the paraphyses and their apices, but also the number and orientation of their septa, which in some cases have proven to be important (e.g. M. tridentina vs. M. rufobrunnea, in Loizides et al. 2015). Septa seem to be often (but not always) randomly distributed across the length of the paraphyses in M. dunalii, M. importuna, and M. tridentina, contrary to species like M. arbutiphila, M. disparilis, or M. rufobrunnea, which have paraphyses with fewer septa, that are nearly always restricted to the base or the lower third. Jacquetant (1984) made special mention of occasionally thickened and contorted paraphyses, which he termed Bheteroparaphyses^, attributingtothemtaxonomicvalue. Although seemingly more frequent in species of sect. Esculenta, we have randomly observed heteroparaphyses in a number of species, often in intermediate or highly irregular forms and variable distribution; we therefore putatively regard these structures as teratological forms of normal paraphyses, and consider their diagnostic significance questionable. It should be noted that such contorted, teratological forms can occasionally be seen in additional microscopic features in morels, such as the asci or the acroparaphyses. In contrast, the structure and length of the terminal elements of the stipe cortex, as previously noted by Jacquetant (1984) and occasionally by Boudier (1897, 1909), can provide valuable clues: some species form well-developed, cylindrical or clavate terminal elements (or Bhairs^) on the stipe surface (M. dunensis, M. exuberans, M. importuna, M. tridentina), while others have only poorly differentiated, thick, polymorphic, irregular, or sometimes short-catenulate protrusions (M. disparilis, M. rufobrunnea). At least one species (M. arbutiphila) forms distinct, long-catenulate hairs, whilst sharply fusiform stipe terminal elements have so far only been seen in M. rufobrunnea. Spores are very late maturing in Morchella and rarely diagnostic, mostly ranging between μm long, and sometimes fluctuating in size from one collection to the next (Clowez 2012). However, when observed en masse from a print of naturally ejected fully mature spores that subtle differences in size, shape, wall thickness, and internal guttulation can be seen between species (Fig. 12). A number of species have considerably larger spores, approximating or exceeding 30 μm, such as M. anatolica, M. angusticeps, M. arbutiphila, M. brunnea, M. sceptriformis and M. snyderi. On the other hand, M. dunensis, M. dunalii, M. purpurascens and M. vulgaris have comparatively short spores, mostly ranging between μm and rarely exceeding μm. Overall, the average length (Me) is more important than the length/ width ratio (Qm), which does not appear to significantly differ between most species. Although the thickness of the spore wall is generally inconsistent and likely depends on the maturity stage of the ascospore, some species, like M. dunensis and M. dunalii, appear to have consistently thick-walled spores (Figs. 12c and f, respectively). Moreover, as confirmed by recently published scanning electron microscope (SEM) images (Voitk et al. 2015), most Morchella species appear to have striate, creased, or roughened spore surfaces (Fig. 13a d), a trait that has been rarely documented in literature, but could prove useful in discriminating between similar taxa. Such spore-surface ornamentations are observable even via light microscopy in the appropriate medium (Chen and Liu 2005; Isiloglu et al. 2010, and this study), and need to be carefully evaluated for most other taxa. It is as-yet unclear whether the external polar guttules, observed in naturally ejected spores of several collections (Fig. 12), constitute a general feature in Morchella, or are more commonly present in some species than others. A number of species have occasionally biseriate asci, a feature that may result from accumulating hydrostatic pressure

23 Mycol Progress (2016) 15:39 Page 23 of Fig. 14 Distribution maps of (a) Morchella arbutiphila;(b) M. disparilis;(c) M. dunalii; (d) Mel-38; (e) M. exuberans;(f) M. importuna;(g) M. eximia; (h) M. tridentina; (i) M. rufobrunnea; (j) Mes-28; (k) M. dunensis; (l) M. cf galilaea. Credits: M. Loizides within relatively wide asci proportionately to the spores. This character has so far been observed in molecularly confirmed collections of M. disparilis and M. exuberans, and more frequently in M. dunalii and M. dunensis (Fig. 11f), but not seen in M. arbutiphila, M. eximia or M. importuna. A larger number of fresh specimens need to be evaluated, to verify whether this can be considered as a useful discriminating character between certain taxa (see vital vs herbarium taxonomy, Baral 1992). Out of the macromorphological characters, the evolution of the apothecium colours, from pale to dark or vice versa, is critical, as is the process of darkening of the sterile ridges. Virtually all Distantes except M. tridentina develop progressively darkening ridges at maturity, as pigmentation in the acroparaphyses darkens and becomes more diffused with age. Some species often develop pinkish, vinaceous, or purplish hues on the sterile ridges before darkening (M. dunalii, M. purpurascens and, occasionally, M. arbutiphila), while others do not (M. disparilis, M. eximia, M. exuberans, M. importuna). Other species are rufescent, often developing reddish or orange stains during their life cycles (M. fluvialis, M. dunensis, M. palazonii, M. rufobrunnea, M. tridentina). The absence or presence of a sinus at the insertion of the pileus with the stipe, including its shape and its depth, is important and sometimes diagnostic (e.g. M. disparilis, M. semilibera), as is the density and orientation of the primary and secondary ridges, together with the size and shape of the primary and secondary pits. For instance, M. dunalii displays very characteristic, partially longitudinally arranged rounded pits (Figs. 8b f, 16c), as opposed to the rectungular ladderlike pits of the pyrophilic lineages (Fig. 16e g) andthe

24 39 Page 24 of 28 Mycol Progress (2016) 15:39 Morchella arbutiphila Morchella disparilis Morchella dunalii Mel-38 Morchella exuberans Morchella importuna Morchella eximia Morchella tridentina Morchella rufobrunnea Mes-28 Morchella dunensis Morchella cf galilaea Fig. 15 Phenological graphs demonstrating species fruitings compared to altitude (Y axis) and month (X axis). Holotype collections in red. Clockwise: Morchella arbutiphila; M. disparilis; M. dunalii; Mel-38; M. exuberans; M. importuna; M. eximia; M. tridentina; M. rufobrunnea; Mes-28; M. dunensis; M. cf galilaea. Credits: F. Richard more irregular, oblong, or angular pits of most Distantes species. The surface of the stipe and stipe length proportionately to the pileus provide further clues: Morchella tridentina for example, has a smooth or only weakly lacunose pure white stipe (Fig. 16h), while the similar M. rufobrunnea has a strongly lacunose, graypruinose stipe (Fig. 16i). Other species, like M. arbutiphila, M. disparilis, and M. pulchella have an elongated stipe, usually equal or longer than the pileus length, while M. dunalii has a particularly short stipe, typically less than 1 3 or 1 of the pileus length 4 (Figs. 8a f and 16c). Cyprus confirmed as a worldwide hotspot of Morchella diversity Compared to its modest cover area of 9,200 km 2, morel species richness in Cyprus (n = 11) is remarkable, and congruent to the increased diversity (n = 20) reported from neighbouring Turkey (Taşkın et al. 2010, 2012). If we take into account the total land coverage of each country, Cyprus in fact harbours the highest recorded concentration of Morchella species in the world, with species per square kilometer, while species richness in Turkey and China is lower, at and , respectively. However, as also pointed out by

25 Mycol Progress (2016) 15:39 Page 25 of Fig. 16 Representative ascocarps from all phylogenetically confirmed species in situ (*Morchella cf galilaea not molecularly tested). (a) Morchella arbutiphila; (b) M. disparilis; (c) M. dunalii; (d) Mel-38; (e) M. exuberans; (f) M. importuna; (g) M. eximia; (h) M. tridentina; (i) M. rufobrunnea; (j) Mes-28; (k) M. dunensis; (l) M. cf galilaea; Credits: M. Loizides, except (l) M. cf galilaea, credit Y. Yiangou Taşkın et al. (2012), the increased diversity reported from Turkey compared to other countries likely represents an artifact of intensive sampling, and we would expect comparable diversity to be present throughout the Mediterranean basin. This increased diversity and endemism in the Mediterranean region, also documented for plants and other organisms (Médail and Quézel 1997; Myers et al. 2000; Médail and Myers 2004), may largely be the result of refugia from the Pleistocene glaciation, where dramatic drops in global temperatures and fluctuations of the ice sheets may have resulted in disjunct distribution of species and localized concentrations of biodiversity (Hewitt 2000; Norstedt et al. 2001; Médail and Diadema 2009; Gavin et al. 2014; Du et al. 2015). The predominance of black morels (sect. Distantes) in southern and Mediterranean regions, as opposed to yellow morels (sect. Morchella) being more widespread in temperate northern and continental regions, has been documented in previous studies (Taşkın et al. 2010, 2012), and our findings are consistent with this pattern. The biogeographical division of black and yellow morels is further reflected in their ecological preferences and putative plant associations, with approximately 70 % of species from each lineage associated with