New Species of Bioluminescent Mycena Sect. Calodontes (Agaricales, Mycenaceae) from Mexico

Mycena section Calodontes is macromorphologically distinguished by the collybioid or mycenoid basidiome, which is pink, purple, or violet, and, rarely, reddish-brown or yellowish. It is further characterized by the presence of oxalate crystals in the basal mycelium. The section comprises approximately 40 taxa, of which only five species and one variety exhibit bioluminescence. As part of an extensive study on Mycena sect. Calodontes in Mexico, specimens belonging to this section were collected and subjected to morphological analysis. Sequences from the nuclear internal transcribed spacer (ITS) of nuclear ribosomal DNA, RNA polymerase II large subunit Rpb1 (rpb1), and translation elongation factor-1α (Tef-1α) were generated to infer the relationships within Mycena sect. Calodontes using maximum likelihood and Bayesian inference. The phylogenetic evidence, along with the macro- and micromorphological features, supported the recognition of five new bioluminescent species within Mycena sect. Calodontes. Detailed macro- and micromorphological descriptions, line-drawing illustrations, and light and dark photographs of the new species are provided.

Asia, Europe, North America Mycelium and basidiome [27] During our investigations on Mycena in Mexico, we collected specimens exhibiting morphological characteristics similar to those of M. sect.Calodontes, such as pink, violet, or purple basidiomata.This led us to believe that some of the specimens might belong to the M. pura complex.Consequently, we anticipated that they could represent new taxa, given the limited attention the genus has received in Mexico.The aims of this study were (1) to make a phylogenetic study of the Mexican species of M. sect.Calodontes, and (2) to describe potentially five new bioluminescent species, based on the phylogenetic results as well as macro-and micromorphological features.Our intention was to contribute to the understanding of bioluminescent species within Mycena, focusing specifically on the Calodontes section.

Morphological Study
The studied specimens were deposited in the fungal collection of the Herbarium of the Institute of Botany at the University of Guadalajara (IBUG), with isotypes also deposited at XAL (herbarium abbreviations according to Thiers [33]).The macroscopy terminology followed Maas Geesteranus [21], while microscopic features were based on Largent et al. [34] and Vellinga [35].Color codes in the descriptions were referenced from Kornerup and Wanscher [36].Free-hand sections were prepared from various parts of the dried basidiomata, which were first rehydrated in 70% ethyl alcohol, then mounted in a 5% solution of KOH, Melzer's reagent (to determine amyloidity, inamyloidity, dextrinoidity, or non-dextrinoidity reactions), or Congo red.A minimum of 30 measurements were taken for each taxonomic structure.Basidiospore statistics included X m , the arithmetic mean of the spore length by spore width (±SD) for n spores measured for a given specimen; Q, the ratio of spore length to spore width, expressed as a range for all spores measured; and Q m , the mean of all Q values (±SD).Basidiospores, cystidia, and other structures were drawn using a drawing tube mounted on a Leica DME light microscope (Wetzlar, Germany).All measurements represented the minimum and maximum dimensions observed, with measurements indicating values considered outside the normal range in parentheses.Microscopic structures were photographed using Axio Vision 4 software on a Zeiss Primostar 3 optical microscope.Photographs capturing bioluminescence in complete darkness were taken with the camera of a Redmi Note 8 smartphone (Beijing, China) equipped with a wide lens (32 s exposure, ISO 3200, and 1.79 f).

Edition, Alignment, and Phylogenetic Analysis
Sequence review and editing were performed using Chromas vs. 1.45 [42].The matrix was constructed and aligned using PhyDE vs. 0.9971 [43].The alignment was visually inspected and manual corrections were made when necessary.In addition, we downloaded 157 sequences available in GenBank that were generated in previous studies [1,14,[16][17][18][19][20] (Table 2).Mycena rubromarginata (Fr.)P. Kumm. was used as an outgroup for phylogenetic analysis.Three separate data matrices were created for ITS, rpb1, and Tef -1α, which were analyzed individually using the program raxmlGUI vs. 2.0 [44] to ensure that there were no conflicting topologies.Subsequently, the matrices were concatenated, and maximum likelihood (ML) and Bayesian inference (BI) analyses were conducted.ML analyses were performed in raxmlGUI vs. 2.0 [44] using the GTRGAMMA and empirically based frequency model, with 1000 bootstrap (BS) replicates with all models free and parameters estimated by raxmlGUI vs. 2.0 [44,45].For the Bayesian analysis, the most appropriate evolutionary model for each dataset was selected using jModelTest vs. 2.1.10with the Akaike criterion corrected [46].BI analyses were conducted in MrBayes vs. 3.2.7 [47].Two independent runs were performed, each consisting of ten million generations with trees sampled every 100 generations.The standard deviation of the split frequencies was examined to confirm the convergence between the independent runs.The first 25% of the samples representing the burn phase were discarded, and posterior probabilities (PP) were calculated from a 50% majority consensus tree of the remaining trees.The resulting trees were visualized using FigTree vs. 1.4.1 [48].
Although ML and BI analyses generated slightly different topologies, the placement of the five Mexican taxa on both trees was consistent.The analysis identified 24 statistically supported clades (BS ≥ 70%, PP ≥ 0.90) that separated the different members of the Mycena sect.Calodontes.The ML tree (Figure 1) revealed that the Mycena collections from Mexico did not cluster with any clades of M. pura s.l. or known species of M. sect.Calodontes.Instead, the Mexican collections formed five monophyletic lineages, four of which had high statistical support: M. luceata (BS = 100%, PP = 1), M. lucisnieblae (BS = 100%, PP = 1), M. luxmanantlanensis (BS = 100%, PP = 1), and M. sophiae (BS = 100%, PP = 1).Mycena lucisnieblae was grouped in a statistically supported clade (BS = 74%, PP = 0.99), which included M. shengshanensis Z.W. Liu, Y.P. Ge & Q.Na and M. subulata Z.W. Liu, Y.P. Ge & Q.Na, described in China, as well as M. pearsoniana, known from North America and Europe.Habitat and distribution-Gregarious, growing on leaf litter in cloud forest, Mexico (Veracruz).
Habitat and distribution-Gregarious, growing on leaf litter in cloud forest, Mexico (Veracruz).
clade formed by M. brunneoviolacea described from Africa [14], M. aff.pura clade X sensu Harder et al. from the USA [18], M. rufobrunnea from China [20], and M. seminau from Malaysia [1].Although the Mexican specimen is within this clade, it is in an independent lineage, which can be distinguished from the rest of the taxa by its morphological characteristics.Mycena brunneoviolacea is distinguished by its dark violet-brown or dark brown pileus, narrowly lageniform to lageniform cheilocystidia and pleurocystidia, and caulocystidia present [14].Mycena rufobrunnea is separated by the dark brown to reddishbrown or greyish-brown pileus and utriform cheilocystidia, sometimes clavate, and caulocystidia present [20].Mycena seminau differs in that it has a dark brown to brown pileus, yellowish-gray to reddish-gray lamellae, and narrower cheilocystidia, 32.8-56.8× 5.6-9.6 µm [1].Habitat and distribution-Gregarious, growing on leaf litter in cloud forest.Known from Mexico (Veracruz).
Notes-Mycena luciferina is characterized by a campanulate, reddish-pink to pale pink pileus, white intervenose lamellae; basidiospores averaging 7.4 × 3.8 µm, amyloid; cheilocystidia clavate to cylindrical, with a rounded apex, pleurocystidia rare, similar to cheilocystidia, an ixocutis, with simple hyphae, and by its strongly bioluminescent pileus.A morphologically similar species is M. rosea, a European species that has basidiome with a pink pileus but is distinguished by pale pink to lilaceous pink lamellae, larger cheilocystidia, 30-90 × 9-36 µm, and caulocystidia present [13,21].It is also like some of the species in the Mycena pura complex, especially those with pink pileus; however, they are easily distinguished by having larger cheilocystidia and pleurocystidia [13,21].
MycoBank MB849398.Etymology.From lux (Latin), light, -manantlanensis, referring to the place Manantlán, in reference to the bioluminescent basidiomes that were collected in the Sierra de Manantlán.
Habitat and distribution-Gregarious, growing on leaf litter in cloud forest, Mexico (Jalisco).
Additional specimens examined-Mexico, Jalisco, Municipality of Autlán de Navarro, Estación Científica Las Joyas, Sierra de Manantlán 19 Notes-Mycena sophiae is distinguished by its very small basidiome, ruby pileus, pinkish-white lamellae, relatively small amyloid basidiospores, and cylindrical to narrowly clavate cheilocystidia.Mycena sirayuktha Aravind.& Manim., described in India [12], is a morphologically similar species in its greyish-red pileus and intervenose lamellae, but is different by the pleurocystidia present and gelatinized lageniform, fusiform, clavate, or utriform cheilocystidia.Other similar species are M. clarkeana Grgur.and M. nullawarrensis Grgur., described in Australia, which have a reddish-brown pileus, but both species are different by having pleurocystidia [15].Mycena cahaya from Malaysia could be a sister group of M. sophiae; they are different because M. cahaya has a brown pileus, basidiospores averaging 7.1 × 3.8 µm, and ventricose to fusoid mucronate cheilocystidia, and pleurocystidia present [1].Mycena lucisnieblae has small basidiomata resembling those of M. sophiae in size but is distinguished by the fact that M. lucisnieblae has pink or purplish-pink basidiomata.

Discussion
This study represents the first occurrence of bioluminescent species belonging to Mycena sect.Calodontes in Mexico.With the inclusion of these five species, the global count of bioluminescent fungi increases to 108, with 15 taxa recorded in that country.The Mexican bioluminescent species of M. sect.Calodontes are exclusively found in cloud forests and can be distinguished from species in other sections by their pileus color, which can be pink, red, purple, orange, or violet, their intervenose lamellae, the presence of oxalate crystals on the basal mycelium, the absence of pleurocystidia (except in M. luciferina), the absence of terminal hyphae in pileipellis and stipitipellis, the pileipellis an ixocutis (except in M. sophiae), and bioluminescence exhibited in the mycelium or some part of the basidiomata, or both.
According to the traditional classification, M. luciferina, characterized by its hyaline cheilocystidia and pleurocystidia, could be classified under M. sect.Calodontes subsect.Purae.Mycena luxmanantlanensis, with its fusiform cheilocystidia with rostrate apex, could be placed in the subsect.Generosae.Lastly, M. luceata, M. lucisnieblae, and M. sophiae could be grouped under subsect.Violacella due to their cheilocystidia with a rounded apex and the absence of pleurocystidia.However, the presence of amyloid basidiospores differentiates them from the species within subsect.Violacella [21,23].As observed in other studies [1,18,20,25], these subsections are polyphyletic, and the micromorphological characters used in the infrageneric classification exhibit homoplasy.The presence of oxalate crystals on the basal mycelium in the Mexican species was confirmed, consistent with reports by Olariaga et al. and Clémençon for certain species of the M. sect.Calodontes from Europe [25,26].The information presented here enhances our understanding of the distribution and ecological aspects of the members of M. sect.Calodontes, specifically in the cloud forest of Mexico.
The phylogenetic analysis conducted on M. sect.Calodontes aligns with previous studies [1,18,20,25].Most of the lineages identified at the species level exhibited strong support, while shallower branches displayed weaker values, consistent with findings in other studies [20,25].Importantly, this study marks the first inclusion of specimens from M. sect.Calodontes in Mexico, affirming the presence of five distinct clades among the Mexican samples.Additionally, the 11 clades proposed by Harder et al. [16][17][18] for M. pura were indeed recovered; however, they appeared in separate linages from the newly identified species.
We must emphasize that the relationships between certain taxa within M. sect.Calodontes has shifted with the inclusion of Mexican specimens.For instance, Liu et al. [20] recovered M. polycystidiata as a sister species to M. diosma, while in our study a clustering of M. luceata and M. polycystidiata was observed, albeit without support.Similarly, in Liu et al. [20], M. pura clade IX was linked with M. cahaya; however, our phylogeny uncovered an unsupported clade of M. cahaya and M. sophiae.Furthermore, in Harder et al.'s phylogenetic analysis [18], a clade featuring Ecuadorian taxa labeled as M. aff.pura was identified as the sister group of M. pearsoniana.Likewise, Liu et al. [20] found a clade composed of Ecuadorian taxa clustered with M. pearsoniana, M. shengshanensis, and M. subulata from China.In contrast, our analysis placed the Ecuadorian taxa together with M. luxmanantlanensis from Mexico in a well-supported clade.Additionally, another supported clade included M. pearsoniana, M. shengshanensis, and M. subulata along with M. lucisnieblae.Likewise, M. luceata and M. luxmanantlanensis clustered in a large unsupported clade, along with M. lucisnieblae, M. pearsoniana, M. shengshanensis, and M. subulata.This clade represents a mixture of species with temperate affinities, such as M. pearsoniana, and tropical ones, from Ecuador and Mexico, distinct from the M. pura complex.
Mexico is one of the countries with the greatest richness of bioluminescent fungi, with a total of 15 species, most of which are exclusive to the country and with only a few known also from other countries.In that country, M. sect.Calodontes represents the section, so far, with the largest number of species of bioluminescent fungi.It is expected that the knowledge of bioluminescent species will increase as other plant communities and states in the country are explored.On other hand, our phylogenetic reconstruction allowed us to recognize the phylogenetic position of the five new species from Mexico as independent of the M. pura complex.The inclusion of the Mexican specimens in the phylogenetic analyses changed the panorama, generating new relationship hypotheses within M. sect.Calodontes.In this sense, it is expected that, as more taxa are added to the studies, the phylogenetic relationships of this section will continue changing.Finally, this work is the basis for subsequent phylogenetic studies that include Mexican taxa.

Figure 1 . 1 .
Figure 1.Phylogenetic tree of Mycena sect.Calodontes from the ML analysis of the ITS + rpb1 + Tef-1α dataset.Maximum likelihood bootstrap values (≥70%) and posterior probabilities (≥95%) are shown Figure 1.Phylogenetic tree of Mycena sect.Calodontes from the ML analysis of the ITS + rpb1 + Tef -1α dataset.Maximum likelihood bootstrap values (≥70%) and posterior probabilities (≥95%) are shown on each branch.The hyphen (-) indicates that the value is absent.Branch lengths are scaled to the expected number of nucleotide substitutions per site.The new species are marked in purple.Mycena luxmanantlanensis showed a sister relationship with a specimen from Ecuador, tentatively identified as M. aff.pura (BS = 100%, PP = 1).They formed a clade (BS = 96%, PP = 1) with other specimens from Ecuador, also identified as M. aff.pura.Mycena sophiae formed a clade with M. cahaya from Malaysia, although this relationship lacked statistical support.Furthermore, M. luceata and M. polycystidiata Z.W. Liu, Y.P. Ge, L. Zou & Q.Na from China were found to be sister species, but in an unsupported clade.For the fifth new Mexican species, M. luciferina, we only had a DNA sequence from the single collected specimen.This sequence was positioned at the base of an unsupported clade that included M. brunneoviolacea A.C. Cooper, Desjardin & B.A. Perry from the Republic of São Tomé and Príncipe, M. rufobrunnea Z.W. Liu, Y.P. Ge & Q.Na from China, and M. seminau A.L.C. Chew & Desjardin from Malaysia.

Table 1 .
Bioluminescent fungi in Mexico recorded in the literature.

Table 2 .
Specimens of Mycena employed in this study.Sequences generated for this work are in bold.