Cortinarius subgenus Leprocybe in Europe: expanded Sanger and Next Generation Sequencing unveil unexpected diversity in the Mediterranean

Abstract

Molecular phylogenies in the past decade have demonstrated that the described diversity of Cortinarius is still underestimated, especially outside continental and boreal ecoregions where the genus has been historically investigated. We tackled this issue by revisiting the so far unresolved subgenus Leprocybe, and focused on the largely unexplored Mediterranean hotspot of biodiversity. The sequencing and phylogenetic analysis of 161 vouchered collections from Austria, Cyprus, France, Germany, Italy and Spain, including 16 types, allowed for the delineation of 11 species in this lineage, three of them recognised as new to science and formally introduced as C. jimenezianus, C. selinolens and C. viridans spp. nov., respectively. Interestingly, the newly described species exhibit a strict Mediterranean distribution, and one of them is putatively endemic to the island of Cyprus, highlighting the remarkable potential of this neglected ecoregion to uncover further undescribed diversity of Cortinarius in the future. The present work also unveils 23 synonymies in this subgenus, as well as previously undetected crypticism within C. venetus. Next Generation Sequencing carried out on three old and contaminated holotypes, successfully decrypts their phylogenetic identity, including that of C. leproleptopus, finally settling the long-standing controversy over the taxonomic status of this species. A brief overview of each species in the subgenus is lastly provided and a key is proposed to facilitate the identification of presently known European taxa of Leprocybe in the field. Citation: Bidaud A, Loizides M, Armada F, et al. 2021. Cortinarius subgenus Leprocybe in Europe: expanded Sanger and Next Generation Sequencing unveil unexpected diversity in the Mediterranean. Persoonia 46: 188-215. https://doi.org/10.3767/persoonia.2021.46.07.

Keywords: Cedrus brevifolia; Veneti; endemism; phylogeny; taxonomy.

Fig. 1

Mapping of the most frequent intragenomic SNPs along the ITS barcode of three Cortinarius species. Consensus sequence of the 5.8S-ITS2 rDNA fragment of the holotypes of: a. C. pseudovenetus; b. C. leproleptopus; c. C. xantholamellatus. Generated by Illumina Miseq sequencing, with SNPs found in at least 0.5 % of the total number of reads within each mOTU indicated as lower case letters above the sequence. Grey and red boxes mark, respectively, the 5.8S rDNA and positions distinguishing each species from their closest neighbour. The number of polymorphic sites is in: a. 48; b. 56; c. 50. Note that the 5.8S rDNA is almost as polymorphic as the ITS2 rDNA and that species diagnostic positions are not (with one exception) touched by these frequent SNPs.

Fig. 2

ITS phylogeny of Cortinarius subg. Leprocybe. Combined Bayesian and Maximum likelihood analyses of 217 ITS sequences falling in subg. Leprocybe, including 11 sequences belonging to sect. Veronicae, sect. Rubicunduli and other distantly related lineages in the genus. Branches with strong statistical support (SH-aLRT > 0.8 and BPP ≥ 95%) are highlighted as thick lines, others display support values as SH-aLRT/% BPP. Typus collections are highlighted in bold, those originating from the Mediterranean area (as defined in e.g., Quézel & Médail 2003) are written in blue, those taxonomically described in Bidaud et al. (2005) are marked by ‘ADC’ and those illustrated in Fig. 3, 4, 5, 6 and 7 of this study are marked by a camera symbol. Species limits, retained name and major tree hosts for the 11 European species known to date are indicated on the right of each relevant clade, with grey highlight for the three new species here introduced (cf. Taxonomy). Indicated sections are from the companion paper Ammirati et al. 2021.

Fig. 2

ITS phylogeny of Cortinarius subg. Leprocybe. Combined Bayesian and Maximum likelihood analyses of 217 ITS sequences falling in subg. Leprocybe, including 11 sequences belonging to sect. Veronicae, sect. Rubicunduli and other distantly related lineages in the genus. Branches with strong statistical support (SH-aLRT > 0.8 and BPP ≥ 95%) are highlighted as thick lines, others display support values as SH-aLRT/% BPP. Typus collections are highlighted in bold, those originating from the Mediterranean area (as defined in e.g., Quézel & Médail 2003) are written in blue, those taxonomically described in Bidaud et al. (2005) are marked by ‘ADC’ and those illustrated in Fig. 3, 4, 5, 6 and 7 of this study are marked by a camera symbol. Species limits, retained name and major tree hosts for the 11 European species known to date are indicated on the right of each relevant clade, with grey highlight for the three new species here introduced (cf. Taxonomy). Indicated sections are from the companion paper Ammirati et al. 2021.

Fig. 2

ITS phylogeny of Cortinarius subg. Leprocybe. Combined Bayesian and Maximum likelihood analyses of 217 ITS sequences falling in subg. Leprocybe, including 11 sequences belonging to sect. Veronicae, sect. Rubicunduli and other distantly related lineages in the genus. Branches with strong statistical support (SH-aLRT > 0.8 and BPP ≥ 95%) are highlighted as thick lines, others display support values as SH-aLRT/% BPP. Typus collections are highlighted in bold, those originating from the Mediterranean area (as defined in e.g., Quézel & Médail 2003) are written in blue, those taxonomically described in Bidaud et al. (2005) are marked by ‘ADC’ and those illustrated in Fig. 3, 4, 5, 6 and 7 of this study are marked by a camera symbol. Species limits, retained name and major tree hosts for the 11 European species known to date are indicated on the right of each relevant clade, with grey highlight for the three new species here introduced (cf. Taxonomy). Indicated sections are from the companion paper Ammirati et al. 2021.

Fig. 3

Cortinarius venetus, a genetically polymorphic pan-European greenish species. a–d. In situ photography of fresh basidiomata; e. transmitted light microscopy imaging; f. scanning electron microscopy imaging of spores of collections genetically belonging in the smallest subclade depicted in Fig. 2 (from: a. AB 05-09-65; b. JMB2013092405; c, e. GC18091404; d, f. JDRG20120201). — Scale bars: a–d = 5 cm, e = 50 μm, f = 5 μm. — Photos by: a. A. Bidaud; b. J.-M. Bellanger; c, e. G. Corriol; d. J.D. Reyes.

Fig. 4

Cortinarius jimenezianus, a Cistus-associated Leprocybe from southern Spain. a–b. In situ photography of fresh basidiomata; c. transmitted light microscopy imaging of the suprapellis; d. UV-light fluorescent imaging of a dried specimen; e. scanning electron microscopy imaging of spores; f. sporogram (from: a, e–f. FA4796 (isotype); b. JDRG26120401; d. JDRG17121801/FA4792). — Scale bars: a–b = 5 cm, c = 50 μm, d = 1 cm; e = 5 μm; f = 10 μm. — Photos by: a, c. F. Armada; b. J.D. Reyes.

Fig. 5

Cortinarius selinolens, a quercophilous Mediterranean C. melanotus lookalike. a–b. In situ photography of fresh basidiomata; c. transmitted light microscopy imaging of the suprapellis; d. UV-light fluorescent imaging of a dried specimen; e. scanning electron microscopy imaging of spores; f. sporogram (from: a, c, f. AB 08-10-396 (isotype); b, e. JDRG02121005; d. AB 11-11-377/JMB2011112503). — Scale bars: a–b = 5 cm, c = 30 μm, d = 1 cm; e = 5 μm; f = 10 μm. — Photos by: a, c. A. Bidaud; b. J.D. Reyes.

Fig. 6

Cortinarius viridans, a Cyprian endemic Leprocybe associated with Cedrus brevifolia. a–b. In situ photography of fresh basidiomata; c. transmitted light microscopy imaging of the suprapellis; d. UV-light fluorescent imaging of a dried specimen; e. scanning electron microscopy imaging of spores; f. sporogram (from: a, c. ML911192CV1 (isotype); d–f. ML810192CV). — Scale bars: a–b = 5 cm, c = 50 μm, d = 1 cm; e = 5 μm; f = 10 μm.— Photos by: a. P.-A. Moreau; b–c. M. Loizides.

Fig. 7

Cortinarius leproleptopus, a pan-European yellowish Leprocybe. In situ photography of fresh basidiomata from: a–d. Germany; e. Cyprus. Note the green-olivaceous tinges on some German collections, absent from the original diagnosis describing Mediterranean collections. Reaction to KOH on the flesh and pileus is shown in c and d (from: a. GH20140907; b. FR2014332; c. GH20140911b; d. SSt12-039; e. ML21211CL). — Scale bars = 5 cm.— Photos by: a. G. Hensel; b. M. Huth; c. M. Huth & G. Hensel; d. G. Saar; e. M. Loizides.

Fig. 8

Biogeographical distribution of Leprocybe species in the Mediterranean. Mapping of sequenced collections of the eight confirmed species in subg. Leprocybe around the Mediterranean basin. Colours delimit vegetation types in the Mediterranean, as defined in Quézel & Médail (2003). Note that C. pescolanensis is present in a single locality within the area, at high elevations, and that C. cotoneus, C. phrygianus and C. squamivenetus are so far not represented in the Mediterranean.