Three new species of Rhytidhysteron (Dothideomycetes, Ascomycota) from Mexico

Abstract The genus Rhytidhysteron is characterised by forming navicular to apothecial hysterothecia, exposing the green, yellow, orange, red, vinaceous or black colours of the hymenium which generally releases pigments in the presence of KOH. The exciple is smooth or striated, the asci bitunicate and ascospores have 1–5 transverse septa. To date, twenty-six Rhytidhysteron species have been described from the Tropics. The present study aims to describe three new species in the Neotropics of Mexico based on molecular methods and morphological features. Illustrations and a taxonomic key are provided for all known species of this genus. Rhytidhysteroncozumelense from the Isla Cozumel Biosphere Reserve, R.esperanzae from the Sierra Juárez, Oaxaca and R.mesophilum from the Sierra Madre Oriental, Hidalgo are described as new species. With the present study, the number of species of Rhytidhysteron known from Mexico is now increased to eight.

The distribution of the genus is Pantropical. It has been reported as an endophytic fungus (Rashmi et al. 2019) and causes mycosis in humans (Chowdhary et al. 2008;Mishra et al. 2014;Mahajan et al. 2014;Chander et al. 2016).
The species with the largest distribution is Rhytidhysteron rufulum. It has been described from various places, with slight morphological differences depending on where it was found. R. rufulum have hysterothecia 1500-2000 µm long, ascospores of (19-)26-36(-43) µm and the colour of the red epithecium in Melzer's Reagent changes to bright orange (Samuels and Müller 1979). According to Kutorga and Hawksworth (1997), the length of the hysterothecia ranges from 2500-4000 µm, ascospores from (22-)25-35(-39) µm and has dark brown to reddish epithecium in potassium hydroxide (KOH) which changes to pale greenish-brown or from red wine to intense pink. On the other hand, in the description made by Almeida et al. (2014), the size of the ascomata ranges from 800-2500 µm, ascospores from 21-32 µm and has black or red epithecium without extractable KOH pigment. The specimens from Thailand have ascomata from 900-2350 µm, ascospores from 28-36 µm and black or red epithecium are not reported to have a reaction with any reagent (Thambugala et al. 2016). Finally, Cobos-Villagrán et al. (2020), for the Mexican specimens, report ascomata of 1000-3000 µm, ascospores of 22.4-30.4 µm and orange-reddish, yellow or black epithecia changing to magenta in reaction with KOH. These morphological variations within R. rufulum have caused confusion in various fungal collections around the world and, as a result, they have been grouped into a complex of species (Boehm et al. 2009b;Murillo et al. 2009;Yacharoen et al. 2015;Doilom et al. 2016;Thambugala et al. 2016;Soto-Medina and Lücking 2017).
Twenty-six species are known worldwide according to the Fungorum Index (2021) and, in the last two years, it has had greater relevance, since at least seven species have been described. In the present work, morphological and molecular analyses of distinct specimens of Rhytidhysteron obtained from different locations in Mexico were performed. Phylogenetic relationships were inferred based on internal transcribed spacer (ITS), nuclear large subunit ribosomal DNA (LSU) and elongation factor 1-alpha (tef1). Additionally, a dichotomous key is provided with all the species described so far.

Study zone
The specimens have been found from three different sites: one from Cozumel Island Biosphere Reserve, Quintana Roo, which is located between coordinates 20°35'20" and 20°17'16" north latitude (N) and -86°43'55" and -87°00'07" west longitude (W). The climate, according to the Köppen system, modified by García (1981), is of the AmW (I) type, warm humid with abundant rain in summer. The average annual temperature is 25.5 °C. Average annual rainfall is 1570 mm (INEGI 2013;García-Martínez et al. 2021). The type of vegetation present in the town of San Gervasio is tropical dry forest, at 0 m above sea level.
The second specimen from La Esperanza, Santiago Comaltepec, Chinantla was collected from the Sierra de Juárez in the State of Oaxaca, between coordinates 17°32' and 17°44' north latitude (N) and -96°16' and -96°36' west longitude (W); altitude between 100 and 3200 m a.s.l. La Esperanza presents different types of climates, the main ones, according to the Köppen system, modified by García (1981), are temperate humid with abundant rain in summer, C (m) and semi-warm humid with rain all year round. The temperature range is 10-26 °C. The range of precipitation is 800-4000 mm (INEGI 2008). The type of vegetation present in the town of La Esperanza is tropical cloud forest, at 1600 m a.s.l.
The last of the specimens is from Laguna de Atezca, Molango de Escamilla, which is located in the Sierra Madre Oriental in the State of Hidalgo, between the coordinates 20°42' and 20°59' of north latitude (N) and -98°41' and -98°53' of West longitude (W), altitude between 300 and 2200 m a.s.l. The Laguna de Atezca presents different types of climates, the main ones, according to the Köppen system, modified by García (1981), are semi-warm humid with rain throughout the year, ACf and temperate humid with abundant rain in summer, C (m). The average annual temperature is 17 °C. Average annual rainfall is 1438 mm (INEGI 2009). The type of vegetation present in the town of Laguna de Atezca is tropical cloud forest, at 1281 m a.s.l.

Morphological study
The specimens were obtained by searching for dry or fallen branches in each of the localities. The material was examined following traditional techniques in mycology (Cifuentes et al. 1986). Photographs were taken using a digital camera (Nikon, D7000, Tokyo, Japan) with an 85 mm macro lens (Nikon, Tokyo, Japan). The fresh collected specimens were used to obtain morphological data such as the colour of the epithecium, growth habit and habitat. Ascomata were measured by a stereomicroscope (Zeiss 475002, Jena, Germany). Cross sections were made in the middle part of the ascomata and mounted on temporary slides in 70% alcohol and 10% KOH. Sections were observed under an optical microscope (Zeiss K-7, Jena, Germany) for the measurement of the characters of taxonomic importance.

DNA extraction, amplification and sequencing
The DNA of each specimen of Rhytidhysteron spp. was obtained using the cetyltrimethylammonium bromide (CTAB) method, according to Doyle and Doyle (1987). Three molecular markers were used, the ribosomal large subunit (LSU), the internal transcribed spacer rDNA-ITS1 5.8S rDNA-ITS2 (ITS) and translation elongation translation factor 1-α (tef1). The primers used for LSU were LOR0f and LR5r (Vilgalys and Hester 1990), for ITS, these were ITS1f and ITS4r (White et al. 1990;Schoch et al. 2012) and tef1 EF1-B-F1 and EF1-B-R . DNA amplifications were performed in a GeneAmp PCR System 9700 thermal cycler (Thermo Fisher Scientific), following recommendations by White et al. (1990) for ITS, Vilgalys and Hester (1990) for LSU and Wu et al. (2014) for tef1. The PCR products were verified by agarose gel electrophoresis. The gels were run for 1 h at 95 V cm -3 in 1.5% agarose and 1× TAE buffer (Tris Acetate-EDTA). The products were then dyed with GelRed (Biotium, USA) and viewed in a transilluminator (Infinity 300 Vilber, Loumat, Germany). Finally, the products were purified using the ExoSap Kit (Affymetrix, USA) according to the manufacturer´s instructions and were prepared for the sequencing reaction using the BigDye Terminator Cycle Sequencing Kit v. 3. 1 (Applied BioSystems). Sequencing was carried out in a genetic analyser (Sanger sequencing) by Macrogen Inc. (Seoul, Korea). The sequences of both strains of each sample were analysed, edited and assembled using BioEdit v. 1.0.5 (Hall 1999) to create consensus sequences. The consensus sequences were compared with those in the GenBank database of the National Center for Biotechnology Information (NCBI) using the BLASTN 2.2.19 tool (Zhang et al. 2000).

Phylogenetic analyses
In order to study phylogenetic relationships, our newly produced sequences of six individuals of Rhytidhysteron were added to reference sequences of ITS, LSU and tef1 (Table 1) deposited in the NCBI database (http://www.ncbi.nlm.nih.gov/ genbank/). Each gene region was independently aligned using the online version of MAFFT v7 (Katoh et al. 2002(Katoh et al. , 2017Katoh and Standley 2013). Alignments were reviewed in PhyDE (Müller et al. 2005), followed by minor manual adjustments to ensure character homology between taxa. The matrices were formed for ITS by 28 taxa (667 characters), for LSU by 31 taxa (875 characters); while the tef1 consisted of 24 taxa (896 characters). Gloniopsis calami was used as the outgroup. The aligned matrices were concatenated into a single matrix (31 taxa, 2438 characters). Five partitioning schemes were established: one for the ITS, one for the LSU, and three to represent the three codon positions of the tef1 gene region, which were established using the option to minimize the stop codons with Mesquite v3.2 (Maddison and Maddison 2017). The best evolutionary model for alignment was sought using PartitionFinder (Lanfear et al. 2014(Lanfear et al. , 2017Frandsen et al. 2015). Phylogeny was performed with Bayesian inference using MrBayes v3.2.6 x64 (Huelsenbeck and Ronquist 2001). The information block for the matrix includes two independent runs of the MC3 chains using 10 million generations (standard deviation ≤0.1). The convergence of the chains was displayed in Tracer v1 ). The highest credibility phylogram of the clades recovered with TreeAnnotator v. 1.8 (Bouckaert et al. 2014) was chosen with a 25% burn-in.
Distribution. Known from a single locality in a forest in La Esperanza, Mexico.  Notes. Rhytidhysteron esperanzae, is characterised by a brown, dull-green to black exciple and dark green to black epithecium that, in reaction with 10% KOH, changes to yellow colouration. This colouration with KOH is very different than those of of R. rufulum and R. neorufulum which are magenta and violet, respectively. R. esperanzae have larger ascospores than R. rufulum (22.4-30.4 × 8-9.6 µm) and R. mexicanum Etymology. The epithet refers to the type of vegetation (mountain mesophilic forest) it was collected from.

Discussion
The genus Rhytidhysteron is a highly diverse group with a mainly Pantropical distribution (Samuels and Müller 1979). The morphological characteristics that have, so far, helped in the segregation of the species are: shape and border of the hysterothecium, ornamentation of the exciple, colour and reaction of the epithecium, and size of the ascospores which only, in some cases, have helped delimiting species, as in the case of Rhytidhysteron columbiense Soto-Medina & Lücking and R. neohysterinum Cobos-Villagrán, Hern-Rodr., R. Valenz. & Raymundo. Therefore, species in which the size of spores overlap, have been clarified by molecular methods and the use of molecular markers, such as ITS, LSU, elongation factor 1 alpha (TEF1), amongst others. For example, in the case of R. rufulum, catalogued as a species complex based on morphology, the fungal barcodes have been helpful in describing different species that are morphologically similar (Boehm et al. 2009b;Murillo et al. 2009;Yacharoen et al. 2015;Doilom et al. 2016;Thambugala et al. 2016;Soto-Medina and Lücking 2017). In recent years, part of the taxonomy has been resolved using collections from different countries around the globe. In the present study, we observed that R. cozumelense is phylogenetically close to R. hongheense, R. camporesii and R. chromolaenae. The four species are similar in terms of ascospore size in the range of 23-30 × 8-13 µm and have a margin smooth to slightly striate. R. hongheense has slightly longer ascospores (20-33 × 9-13 µm). However, they have ascomata of contrasting sizes. R. chromolaenae forms smaller navicular hysterothecia, 750-885 µm diam., with orange epithecium, turning purple in KOH and is described from Chiang Rai Province, Thailand (Mapook et al. 2020). R. camporesii has hysterothecial ascomata of 800-1100 µm long with black epithecium that changes to magenta in KOH and it is described from Yunnan Province, China ). Finally, R. hongheense has ascomata hystherothecial 1200-2000 µm long with reddishorange epithecium and it is described from Honghe County, Yunnan Province, China (Wanasinghe et al. 2021). R. cozumelense produces longer ascomata, hysterothecial to apothecial, 2500 to 3500 µm long with reddish brown to black epithecium that changes to greyish magenta in KOH and it grows on Tabebuia rosea DC. (Bignoniaceae).
R. esperanzae is phylogenetically close to R. mexicanum, both species described from Mexico presenting similar hysterothecial to apothecial ascomata, sizes of 2000-4500 × 1200-2500 µm and a perpendicularly striate margin. However, they differ by the colour of the ascomata and the epithecium: in R. esperanzae, the ascomata is brown, the exciple dull-green to black, and the epithecium dark green to black, with a yellow reaction in KOH. In contrast, in R. mexicanum, the exciple is completely black and the epithecium yellowish green to pistachio green when fresh, light green, pale green to lemon yellow when dry, becoming ocher to yellow gold in KOH. Another difference is the size of the ascospores which are longer and wider in R. esperanzae: they are (42-)45-47(-49) × (15-)17-19(-23) µm, while in R. mexicanum, they are 34-40(-44) × 10-12(-15) µm (Cobos-Villagrán et al. 2021).
In Mexico, the tropical dry forest is the best represented vegetation with four Rhytidhysteron species: R. cozumelense, R. neorufulum, R. rufulum and R. neohysterinum. This is followed by the xerophilous scrub with R. thailandicum, R. rufulum and R. neohysterinum, and only R. mexicanum in Quercus forest. Finally, in this study, we describe R. esperanzae and R. mesophilum in a tropical cloud forest, which is a vulnerable ecosystem and therefore these species are in danger of extinction. With the present study, the number of Rhytidhysteron species known from Mexico reaches a total of eight and together with Thailand, they form the countries with the most species diversity of the genus.