Cantharellus violaceovinosus, a new species from tropical Quercus forests in eastern Mexico

Abstract During explorations of tropical oak forests in central Veracruz (eastern Mexico), the authors discovered a Cantharellus species that produces basidiomes with strikingly violet pileus and a hymenium with yellow, raised gill-like folds. It is harvested locally and valued as a prized edible wild mushroom. Systematic multiyear sampling of basidiomes allowed the recording of the morphological variation exhibited by fresh fruit bodies in different growth stages, which supports the recognition of this Cantharellus species from others in the genus. Two molecular phylogenetic analyses based on a set of sequences of species of all major clades in Cantharellus, one including sequences of the transcription elongation factor 1-alpha (tef-1α) and a combined tef-1α and nLSU region (the large subunit of the ribosome), confirm the isolated position of the new species in a clade close to C. lewisii from USA, in the subgenus Cantharellus. Detailed macroscopic and microscopic descriptions, accompanied by illustrations and a taxonomic discussion are presented.


Introduction
The diversity of species in Cantharellus Adans.: Fr., in combination with their ectomycorrhizal nature (mycobionts of several plant lineages), as well as their highly prized value as edible wild mushrooms, have attracted the attention of specialists from different fields worldwide (Smith and Morse 1947, Trappe 1962, Chandra 1989, Molina et al. 1992, Homola 1993, Thoen 1993, Watling 1997, Danell 1999, Pilz et al. 2002, Lee et al. 2003,Yun and Hall 2004, Boa 2005, Agerer 2006, Arora and Dunham 2008, Kumari et al. 2011, Wilson et al. 2012. Cantharellus encompasses fungi with long-lived, gymnocarpic, fleshy, variedly coloured, trumpet-shaped basidiomes with nearly smooth, veined, gill-like folded to distinctly lamellate hymenophore, pileipellis poorly differentiated, cystidia lacking, smooth and thin-walled spores, with or without clamps (Wilson et al. 2012. In many cases, basidiomes of members of closely related species or inclusive, unrelated look-alike species are difficult to identify in a strict sense, especially if there is not an accurate record of the variation of morpho-anatomical characters and colours in fresh condition. Few microscopic features in the genus had been considered discriminative, especially clamps (presence or absence), wall thickness of the terminal elements of the pileipellis hyphae and the basidiospore features (size and form).
The two former features are considered amongst the most taxonomically informative at subgeneric level and the latter used to distinguish species Buyck 2001, Buyck et al. 2014). Additionally, it has been hypothesized that there are cryptic species still undefined taxonomically, even amongst the best known Cantharellus species, especially from tropical regions but also from temperate regions (Smith and Morse 1947, Corner 1966, Heinemann 1958, Smith 1968, Petersen and Ryvarden 1971, Petersen 1976, Bigelow 1978, Petersen and Mueller 1992, Eyssartier et al. 2003, De Kesel et al. 2011, Tian et al. 2012, Wartchow et al. 2012, Buyck and Randrianjohany 2013, Foltz et al. 2013, 2016a, c, Leacock et al. 2016.
Taxonomic research on Cantharellus has increased substantially in the last decade, especially by combining DNA and morphological information to support the definition of early recognised species and others recently discovered , Tibuhwa et al. 2012, Wilson et al. 2012, 2016a, 2016b, Foltz et al. 2013, Shao et al. 2014, Shao et al. 2016, Leacock et al. 2016.
The earliest description of Cantharellus in Mexico dates from Fries (1855), who proposed C. mexicanus based on a specimen with "… pileo carnoso turbinato-infundibuliformi glabro griseofusco… lamellis augustissimis longe decurrentibus". It was collected by F.M. Liebmann at El Mirador, Veracruz and, years later, considered by Corner (1966) as "… incert. sed. (? Gomphus)…" no longer recorded in the literature. From the same region (Orizaba, relatively near to the current study site), Craterellus confluens Berk. & M.A. Curtis was described by Berkeley (1867). This species is characterised by its yellow basidiomes, it is closely related to Cantharellus lateritius (Berk.) Singer, with which it has been confused or even with other yellow chanterelles, such as C. cibarius Fr. and Craterellus odoratus (Schwein.) Fr. (Heim 1954, Corner 1966, Petersen 1979, Guzmán and Sampieri 1984. After such descriptions of new Cantharellus species from Mexico, only some additional records of about ten species described from other latitudes have been mentioned to occur in different forest ecosystems in the country, including Quercus forests (Guzmán and Sampieri 1984, Guzmán 1985, Guevara et al. 2004, Perez-Moreno et al. 2008, Garibay-Orijel et al. 2009). The identity of these records, however, has not been confirmed with molecular evidence. Recently, C. coccolobae Buyck, Moreau & Courtecuisse was described from the Caribbean (Guadeloupe), including two collections from Yucatán, Mexico (Buyck et al. 2016c).
During the authors' long term explorations in tropical oak forests in central Veracruz, a Cantharellus species was found with a striking habit, distinctive when compared to the previous records from Mexico. In fact, this fungus is unique because the fresh basidiomes in different growth stages possess a strikingly violet pileus and yellow, raised gill-like folded hymenophore, in combination with ellipsoid basidiopores and terminal elements of pileipellis slightly thick-walled. The macro-and micromorphological features depicted in this fungus, as well as its distinct position in two phylogenetic analyses, one of tef-1α and other of a combined tef-1α+nLSU sequences datasets, allowed its recognition as a new species. This Cantharellus species is locally considered a prized edible mushroom.

Sampling and morphological study
Cantharellus basidiomes were collected during June-October, through six consecutive years (2012-2017) including some collections in 2009 and 2011, in tropical oak forests from Zentla (837-850 m a.s.l.) and Alto Lucero (400-500 m a.s.l.) counties in central Veracruz (eastern Mexico). In these oak forests, Quercus oleoides is dominant and even forms pure stands. In the Zentla locality, however, Q. glaucescens and Q. sapotifolia are also present and, at times, also form monodominant small patches. Descriptions of morpho-anatomical features were achieved based on fresh samples and following Largent (1973). The colour notations indicated in the descriptions follow Kornerup and Wanscher (1978) and Munsell colour chart (1994). Basidiomes were dried in a hot air dehydrator (45 °C). Microscopic features were observed and measured after tissues were rehydrated in 3 % potassium hydroxide (KOH) and stained with 1 % Congo red or analysed in Melzer´s solution. At least thirty-five basidiospores per collection were measured in length and width. Mean ranges denoted as X m and the length/width ratio (Q -) of basidiospores, in side view, are given as an interval of mean values per collection (n=15 collections). The form of the basidiospores was interpreted after calculating the Q values, following Bas (1969

DNA extraction, PCR and sequencing
DNA was isolated from fresh material using DNAeasy Plant Mini Kit (QIAGEN, Hilden, Germany) following the manufacturer´s recommendations. The transcription elongation factor 1-alpha (tef-1α) was amplified using the primers tef1F and tef1R (Morehouse et al. 2003) and the large subunit of ribosome (nLSU) using the primers LR0R and LR7 (Vilgalys and Hesler 1990). PCR conditions were performed with an initial denaturation at 94 °C for 3 min; 35 cycles of 1 min 94 °C, 1 min at 55 °C and 2 min at 72 °C; and final elongation at 72 °C for 7 min. Amplified PCR products were purified with the DNA Clean & Concentrator Kit (Zymo Research, USA) following the manufacturer's instructions. Cycle sequencing reactions were made using BigDye Terminator 3.1 Cycle Sequencing kit (Applied Biosystems, USA); reactions were purified with ZR DNA Sequencing Clean-up Kit (Zymo Research, USA) and run in a sequencer, ABIPrism 310 Genetic Analyzer (Applied Biosystems). Sequences obtained were assembled and edited in BioEdit (Hall 1999) and deposited at GenBank database (Benson et al. 2017) ( Table 1).

Phylogenetic analysis
Six tef-1α and nLSU sequences obtained in this study, together with 113 sequences of Cantharellus species from all major clades across the genus (after Buyck et al. 2014) and with the highest similarity scores from the results of BLAST (Altschul et al. 1997) were downloaded from GenBank (http://www.ncbi.nlm.nih.gov/) and used to construct two datasets. One dataset consisted of tef-1α and other combined tef-1α+nLSU sequences. Craterellus tubaeformis and Hydnum repandum were included as outgroup taxa (Table 1 and alignment in TreeBASE S21920). Both datasets were assembled in the data editor PhyDE v.0.995 programme (Müller et al. 2010). They were aligned using Muscle (Edgar 2004) with inconsistencies corrected manually. A phylogeny of each dataset was constructed under maximum likelihood (ML) and Bayesian Inference (BI) methods. The best evolutionary model for both datasets was calculated with Mega 6.06 (Tamura et al. 2013). ML analyses were also performed using Mega 6.06 with 500 replicates of bootstrap. BI analyses were implemented with MrBayes on XSEDE (3.2.6) on CIPRES portal (Miller et al. 2010) with settings as described in Montoya et al. (2014). The phylogenies from ML and BI analyses were displayed using Mega 6.06 and FigTree v 1.3.1 (Rambaut 2009), respectively.

Results
Sixty fresh collections were obtained of the violet Cantharellus species, including basidiomes in different growth stages, most of them detected between August-October, in both localities explored. Six new tef-1α and nLSU sequences from three collections were generated in this study (Table 1). In the inferred molecular phylogenies (from tef-1α and tef-1α+nLSU sequences datasets) (Figs 1-2), the generated sequences from the Mexican specimens, clustered in a terminal clade, strongly supported only bootstrap values ≥70 and posterior probabilities ≥0.90 were considered and indicated (BS/BPP) on the branches of each tree. Both trees were congruent and the sequences of the Mexican Cantharellus cluster in a sister clade to C. lewisii from USA, in the subgenus Cantharellus ). Based on the distinctive morphological features and colour variation of the studied Cantharellus specimens, as well as the isolated position of the samples in the phylogenies obtained, it was concluded that this should be proposed as a new Cantharellus species, which inhabits the tropical Quercus forests in eastern Mexico.
Although Cantharellus amethysteus (Quél.) Sacc. (subg. Cantharellus) from Europe, may appear superficially similar to some forms of C. violaceovinosus, the former however, especially has a pileus surface covered with vinous or lilac, small scales. The authors studied two specimens of C. amethysteus from France (BB 07.284 and BB 07.309 at PC) displaying elongate basidiospores, 9.5-12 (-12.5) × 5-7 µm (X m = 11-11.2 × 5.9-6.4 µm; Q -= 1.76-1.86), as Eyssartier and Buyck (2000) reported [(9-) 9.5-10.37-11.5 (-12.5) × 6-6.5-7 µm], resulting in being larger and more elongate than in the Mexican species. Also, it is interesting that one sequence of tef-1α of a specimen from Thailand (GB coded KX857062, Table 1) identified as "C. cf. subamethysteus", appeared close to C. violaceovinosus (Fig. 1). Cantharellus subamethysteus indeed is phylogenetically related to C. lewisii  and differs from C. violaceovinosus in the shorter basidiomes (pileus 20-65 mm; stipe 42-57 × 5-11 mm), deep and bright yellow pileus surface, covered with squamules even with rather brown to dark brown tinges, shorter basidiospores [7-8 (8.75) × (4.75) 5-6 µm] and wider pileipellis elements (8-15 µm width) (Eyssartier et al. 2009). Additionally, the hymenophore of this species is rugose to faintly veined (as depicted in the picture accompanying the description). Cantharellus goossensiae (Beeli) Heinem., C. cyanoxanthus R. Heim ex Heinem., C. subcyanoxanthus Buyck, Randrianjohany & Eyssart. and C. longisporus Heinem. represent African species with basidiomes displaying violaceous tinges  therefore, at some stages their basidiomes could resemble those of C. violaceovinosus. However, the three former species have the pileipellis with thin-walled hyphal extremities, thus differing from members of subgenus Cantharellus, including the new species here described. Moreover, the four African taxa have basidiospores distinctly narrowly ellipsoid to elongate (Q>1.70) and often slightly reniform, curved or even somewhat peanut-shaped (Beeli 1928, Heinemann 1958, 1959. Cantarellus violaceovinosus was recorded as a common fleshy mushroom, during the multiyear sampling developed in the tropical Quercus forests studied. It was found in ectomycorrhizal association with native trees of Quercus species. This mushroom was very often recorded in pure stands of Q. oleoides and less frequently in Q. glaucescens and Q. sapotifolia patches. This violet pigmented chanterelle shares the same habit preferences as C. lateritius, also found in the study sites. A similar co-ocurrence has been reported between C. lewissi (the sister relative of C. violaceovinosus) and C. lateritius in the State of Texas in the USA . Basidiomes of C. violaceovinosus and C. lateritius are abundant in the local oak forests studied and both are considered choice wild mushrooms although the latter is more highly prized. They are even more appreciated than species of Amanita or Lactarius, representing an income source for wild mushroom collectors. Benefits from mushrooms harvesting, as well as other ecosystemic services, are motivating some owners to conserve relicts of the tropical Quercus forest of the region.