Diversity and distribution of macrofungi (Ascomycota and Basidiomycota) in Tolima, a Department of the Colombian Andes: an annotated checklist

Abstract Background Macrofungi are classified in the phylum Ascomycota and Basidiomycota and they are very important from an ecological and economic point of view. Most studies of fungi in Colombia have been carried out mainly in the Andean Region, especially in the Departments of Antioquia, Valle del Cauca and Cundinamarca. However, other Departments in the Andean Region, like Tolima, located in the Cordillera Central, are well documented for plants (4,797 species) and animals (2,983 species), but very poorly documented in terms of knowledge of fungal diversity. New information This study provides a compiled and annotated checklist of all known macrofungi in the Department of Tolima, based on published literature and on the identification of new specimens collected from five localities of the Department. All records were updated taxonomically and we include detailed information on the localities in which they are distributed in the Department. The list includes 164 taxa distributed in 15 orders (Agaricales, Polyporales, Russulales, Boletales, Hymenochaetales, Xylariales, Auriculariales, Thelephorales, Cantharellales, Hypocreales, Pezizales, Gloeophyllales, Phallales, Tremellales, Dacrymycetales) and eighteen records in a doubtful taxa section. We present 26 new reports, 19 for Tolima and nine for Colombia. We also provide genetic and phylogenetic evidence of the occurrence of Gloeoporustelephoroides and Podoscyphavenustula in Colombia. This checklist provides the basis for future studies on species diversity and taxonomy in Tolima, by identifying the least studied taxa and ecosystems and conservation priorities.


Introduction
Complex multicellular forms have evolved independently in many clades of the Eukaryota domain, including fungi, plants and animals (Torruella et al. 2015). Some groups of fungi form diverse macroscopic structures (sporomes) including mushrooms, stinkhorns, truffles, earth stars, puffballs, shelf fungi, clavarioid, coralloid fungi, discoid fungi and cup fungi. These organisms are artificially grouped as macrofungi and classified in the phylum Ascomycota and Basidiomycota. Diversity estimates derived from data using plant species/ macrofungal ratios indicate that there may be between 53,000 to 110,000 macrofungal species in the world (Mueller and Schmit 2007). Although macrofungi have perhaps the longest history of diversity studies of any group of fungi, they are still poorly studied in most of the world (Lodge et al. 2004, Mueller andSchmit 2007).
Colombia is considered the second country with the highest biodiversity on the planet with 75,947 biological records of known species in the different Kingdoms (SIB Colombia 2022a ). These data, added to high rates of endemism, place the country as a priority region for the conservation of biodiversity worldwide (Myers and De Grave 2000). Although knowledge of diversity in the country has been limited by a strong taxonomic bias towards Macrofungi were collected in three localities of the Municipality of Ibagué: 1) secondgrowth forest in San Jorge Botanical Garden (JBSJ) (4°27'06.7"N 75°13'19.8"W), which is on the border between the tropical dry forest and the premontane forest, to 1200 m a.s.l.; 2) Canyon of Combeima River (4°33'25.8"N 75°19'34.4"W -4°34'43.2"N 75°19'28.4"W), which corresponds to a low montane humid forest, between 1900 and 2350 m a.s.l.; 3) Alejandro Von Humboldt Botanical Garden (JBAVH) in Universidad del Tolima (4°25'34.89''N 75°12'46.77''W), which is a tropical dry forest at 1100 m a.s.l. They were also collected in a locality of the Municipality of Líbano, in Santa Librada Reserve (4°52'48.4"N 75°01'17.4"W), which corresponds to a tropical rainforest, at 1100 m a.s.l. Additionally, they were collected in Chicoral Village, Municipality of Espinal (4°11'56.8"N 74°59'18.1"W -4°12'35.6"N 74°58'37.1"W), in a rural area, at 390 m a.s.l.

Fieldwork
The specimens were collected by performing random sampling in five localities in the period of 2019-2022. The study has the Collection permit conceded for access to biological resources for non-commercial purposes (Permiso Marco de Recolección, Resolución 2191 de 2018, Universidad del Tolima). Sporomes were photographed in situ, completely removed, placed in paper bags and taken to the laboratory. All descriptions are based on well-developed (mature) specimens. Morphological identification was made from macroscopic and microscopic characteristics. For micromorphological analysis, free-hand sections of the sporomes were prepared on microscope slides with 3% potassium hydroxide (KOH), Red Congo or Cotton Blue. Melzer's Reagent (IKI) was used to determine presence or absence of amyloid or dextrinoid reactions. All microscopical structures were measured with the aid of an eyepiece micrometer with a subjective accuracy of 0.1 µm, using 1000x magnification. Identification was based on current literature and using dichotomous keys (e.g. Ryvarden 2004, Coelho et al. 2006, Drechsler-Santos et al. 2007, Ryvarden 2009, Montoya-Alvarez et al. 2011, Ryvarden 2015, Ryvarden 2016, Luangsa-ard et al. 2017, Westphalen et al. 2019, Wu et al. 2021. All specimens were preserved and deposited in the Fungario Universidad del Tolima (FUT). A dataset for distribution of macrofungi in Tolima was created in Excel software and then used for preparing the interactive map in ArcGIS Pro 30.3 software.

Literature review
The list of species was based on the review of scientific literature, national or international, books or book chapters and scientific notes recording macrofungi from Tolima available in public databases, such as Google Scholar, ResearchGate, Scielo and Scopus and vouchers information available in public databases, such as ColFungi and MyCoPortal. Information from unpublished data, results presented at conferences or theses were not included in the list. Lichenised fungi are also excluded. To determine the specific locations of the reports, databases of biological collections were reviewed. Species are presented in alphabetical order within the corresponding Linnean classification: phylum, order, family and genus. Accepted names agree with Index Fungorum (http://www.indexfungorum.org) as of February 2023. The following herbaria databases were consulted (Herbaria acronyms follow Index Herbariorum, Thiers (2016) onwards): Herbario de la Universidad de Antioquia (HUA), Cornell University Herbarium (CU), Field Museum of Natural History (F), Herbario Nacional Colombiano (COL), Medellín headquarters of Universidad Nacional de Colombia Herbarium (MEDEL), Museo de Historia Natural de la Universidad de los Andes (ANDES-F), The New York Botanical Garden (NY), Institute of Agricultural and Environmental Sciences of the Estonian University of Life Sciences (TAAM), University of Tartu (TUF), Museo Nacional de Historia Natural of Cuba (MNHN), University of Georgia, Julian H. Miller Mycological Herbarium (GAM), State University of New York College at Cortland (CORT) and Fungario Universidad del Tolima (FUT). Specimens recorded as new for Colombia and for the Department of Tolima are presented with a detailed morphological description. Additionally, at the end of the list, there is a section with specimens classified as doubtful taxa and presented in alphabetical order. These specimens are present as incongruent data in literature or we do not have sufficient data to confirm their identity.

Taxon sampling, DNA extraction and PCR amplification
Dried specimens of Podoscypha and Gloeoporus were selected for molecular sampling. Approximately 30 mg of tissue from each collection were ground directly in a 1.5 ml vial, using plastic pestles with liquid nitrogen (Justo et al. 2011). DNA was extracted using a 3% CTAB extraction buffer and then isolated by the sequential addition of chloroform. Finally, isopropyl alcohol was added to precipitate the DNA, which was washed with 70% ethanol and resuspended in the TE buffer (Doyle 1990). The purity and concentration of DNA was performed using μDrop™ Plate (Thermo Scientific). The DNA concentration was adjusted to 100 µg/ml. Primer pairs of ITS1F (5′-CTT GGT CAT TTA GAG GAA GTA A -3′)/ ITS4 (5′-TCC TCC GCT TAT TGA TAT GC-3′) were used to amplify a fragment of the ITS region (White et al. 1990, Gardes andBruns 1993). The PCR assay was conducted in a total volume of 25 µl consisting of 14.87 µl distilled deionised water, 5 µl of 5 of 5× colourless GoTaq® Flexi Buffer (Promega, USA), 1 μl dNTPs (1.5 mM) (Invitrogen, USA), 1 μl of each primer (forward and reverse) (10 pmol/μl), 1 μl MgCl (25 mM), 0.125 μl of 0.6 U GoTaq® Flexi DNA polymerase (Promega, USA) and 1 μl gDNA as the template. The amplification was performed in a T-100™ thermocycler (Bio-Rad, USA) with an initial denaturation step at 95°C for 3 min, followed by 35 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 2 min and a final step of extension at 72°C for 5 min. The amplicons were visualised on 2% agarose gel by electrophoresis (PowerPac™ HC, Bio-Rad, USA) using 100-bp DNA ladder Load Ready™ (Amplyus, USA). The gel was stained with HydraGreen™ (ACTGene, USA) and visualised under UV light using the ENDURO GDS gel documentation system (Labnet International, Inc., USA). Final PCR products were purified and sequenced by the Sanger method (MacroGen Ltd., South Korea).

Taxon sampling, alignment and phylogenetic inference
The electropherograms were visually inspected to ensure good sequence quality and ambiguous sequence reads were discarded. Double peaks were interpreted as true base ambiguities when they were detected in both forward and reverse sequencing electropherograms. Once assembled, consensus sequences were queried against the 2 TM entire GenBank database using BLAST (http://blast.ncbi.nlm.nih.gov/) and their pairwise identity was recorded. All newly-generated consensus sequences were deposited in GenBank. The consensus sequences generated in this study and related sequences downloaded from GenBank (www.ncbi.nlm.nih.gov/genbank, Table 1) were aligned using MAFFT v.7.299 (Katoh and Standley 2013). The ITS and 28S regions were aligned using the L-INS-I strategy (command line: mafft-localpair-maxiterate 1000). The coding regions were aligned using the E-INS-I strategy with no cost for opening gaps and equal cost for transformations (command line: mafft-genafpair-maxiterate 1000). After alignment, sequences were translated and checked for stop codons using Aliview v.1.18 (Larsson 2014). Two datasets were prepared: the first combined dataset for Gloeoporus specimen includes 14 sequences of ITS and 13 of 28S (Table 1). Bjerkandera adusta (Wild.) P. Karst. was used as the root. The second combined dataset for Podoscypha specimen includes 33 sequences of ITS and 21 of 28S (Table 2). Abortiporus biennis (Bull.) Singer was used as the root. Both phylogenetic relationship analyses were inferred in a Maximum Likelihood framework as implemented in IQTREE v.2.0 (Nguyen et al. 2015). ModelFinder (Kalyaanamoorthy et al. 2017) was used to select the optimal partition scheme and substitution models.

Notes:
The species is differentiated from other species by the size of the perithecia and the Neotropical distribution. Additionally, N. aurantiaca Luangsa-ard, Thanak. & Tasan looks morphologically similar to N. martiale, but differs in the type of ascospore. The first produce only whole (non-fragmenting) ascospores, while the latter produce ascospores either dissociated (Luangsa-ard et al. 2017). In Colombia, this is the first record of the species for Tolima.  Notes: Macrocybe titans could be confused with Clitocybe gigantea (Fr.) Quélet, but the latter presents a funnel-shaped crown and decurrent lamellae (Bigelow andKimbrough 1980, Corrales andLópez-Q 2005). Macrocybe titans is distinguished macroscopically from other species because the surface of the stipe is visibly squamulose and, microscopically, by having numerous spindle-shaped pseudocystidia, with refractive content (Pegler et al. 1998). The species has been collected normally in disturbed environments of the Neotropics and has been recorded as edible. At the moment, this species has only been collected in the Departments of Antioquia and Santander in Colombia. This is the first record of the species for Tolima.    1G and 6). Margin acute and very thin. Pore surface cream to pinkish. Pores round to angular, irregular, 5-7 per mm.
Notes: This species is separated from other species in the genus by the white to pinkish hymenophore and microscopically, by the simple septate generative hyphae. This is the first record of the species for Tolima.  (Fig. 1H). Pore surface papillate. Hyphal structure monomitic, with generative hyphae with simple septa. Basidiospores ellipsoid to subglobose, hyaline, thin-walled, 4.1-4.7 × 3.0-3.6 μm.

Notes:
The species is recognised by the long slender brown velutinate stipe and equally coloured and velutinate pileus. The species was originally described from Brazil. This is the first record of the species for Tolima. Diagnosis: Basidiomes pileate to effused reflexed (Fig. 2D). Pileus semicircular. Pileus upper surface zonate, brownish-yellow at the margin becoming darker as a thin cuticle starts to develop from the base, glabrous, strongly zonate. Margin acute. Pore surface pale isabelline to cream. Pores round, tiny, 6-9 per mm. Tubes and context woodcoloured. Hyphal structure dimitic, generative hyphae with clamps, thin-walled and hyaline; skeletal hyphae thick-walled, dominating in the tubes and context. Basidiospores globose to subglobose, hyaline, thick-walled, slightly dextrinoid, 4-6.4 × 3.8-6.4 µm.

Perenniporiella micropora (Ryvarden) Decock & Ryvarden, 2003
Notes: This species is characterised by having small pores and globose to subglobose basidiospores. It was originally described from Peru (Ryvarden 1987). This is the first record of the species in Colombia. Notes: The species is characterised by the small, thick, glabrous pilei and large truncate spores. Originally, the species was described from Australia, but currently presents a worldwide distribution. In South America, it has been recorded in Brazil. This is the first record of the species in Colombia.

Notes:
The species is microscopically separated by the dendrohyphidia and larger basidiospores from similar species. It is distributed in Puerto Rico and Honduras (type locality), but certainly has a wider distribution in the Caribbean (Ryvarden 2000). This is the first record of the species for Tolima. In this study, 38 specimens were collected and morphologically identified, which were classified as 19 new reports (Figs 1, 3) for the department of Tolima and seven new reports for Colombia (Fig. 2). In addition, the new reports include a morphological description and comments.
We keep a total of eighteen species under doubtful taxa. There is a group of species that have been recorded in the literature for the Department of Tolima (Franco-Molano et al. 2010, Peña-Venegas and Vasco-Palacios 2019, Cossu et al. 2022, Gómez-Montoya et al. 2022), but no voucher or collection was referenced. We carried out a search for vouchers of these species in the databases of the Herbarium of the Universidad de Antioquia or in the MyCoPortal. We did not have access to these specimens to review them morphologically, but we made a reference for future studies that will allow us to establish their presence in Tolima. There is another group of species recorded for the Department classified as doubtful taxa, for which we were not able to find any data regarding a voucher that could be reviewed to confirm their occurrence in the Department. In this case, after the name of the species, we leave only the bibliographical reference that cites the occurrence.
Phylogenetic inference for Gloeoporus species. For this study, we generated one consensus sequences of ITS (Table 1). In total, the ITS dataset had an aligned length of 1386 characters, of which 1072 were constant, 314 were variable and parsimonyuninformative and 194 were parsimony-informative. The best tree inferred in a Maximum Likelihood framework has a log likelihood = -4064.2346. The best fit models selected were TIM2+F+R2 for ITS and TN+F+G4 for 28S. The phylogenetic inference (Fig. 6) and the morphological analysis confirmed that the collected specimen corresponds to Gloeoporus thelephoroides (Hook.) G. Cunn. (LRD130, BS = 91, SH alRT = 95).

Phylogenetic inference for Podoscypha species.
For this study, we generated one consensus sequences of ITS (Table 2). In total, the ITS dataset had an aligned length of 1948 characters, of which 1329 were constant, 619 were variable and parsimonyuninformative and 413 were parsimony-informative. The best tree inferred in a Maximum Likelihood framework has a log likelihood = -9011.705842. The best fit models selected were TN+F+I+G4 for ITS and TN+F+R2 for 28S. The phylogenetic inference (Fig. 7) and the morphological analysis confirmed that the collected specimen corresponds to Podoscypha venustula (Speg.) D.A. Reid (BS = 96, SH alRT = 96).  Phylogenetic relationship of Podoscypha species inferred from a combined dataset of ITS+nLSU conducted by IQ-TREEE optimal tree (log likelihood = -9011.705842). The sequences generated in this study are indicated in bold. Values at nodes indicate ultrafast bootstrap (left) and the Shimodaira-Hasegawa approximate likelihood-ratio test (right); minus (-) indicates support values lower than 90%. Two codes after voucher specimens indicate the country of origin (ISO 3166 -Alpha 2). The bar indicates the number expected substitutions per position.

Discussion
Recently, Gómez-Montoya et al. (2022) reported 115 species of macrofungi of Basidiomycota for the Department of Tolima, Colombia, and Vasco-Palacios and Franco-Molano (2012) have reported only four species of Ascomycota. In this study, we managed to compile a total of 164 species of macrofungi (154 of Basidiomycota and 10 of Ascomycota), 146 being considered as good records and we placed 18 species as doubtful taxa. Additionally, new records, based on morphological and phylogenetical analyses, are presented, which makes it the most complete and critical checklist to date for Tolima.
The order Agaricales, with 76 species recorded in the Department, is considered the best represented. The 97% of the reports have been made in the Montane Rainforest and in forests dominated by Quercus. Only one species, P. cubensis ( Pulido 1983), has been recorded from the tropical dry forest, which is one of the most threatened ecosystems in Colombia (Etter et al. 2017). Although this is the best represented order in the Department, it is necessary to collect and study species of Agaricales from other municipalities and different ecosystems of Tolima.
The order Polyporales is the second-best represented order with 32 species. About 85% of the recorded species are distributed in lowland forest areas and the remaining records have been made in Montane Rainforests of Murillo, Líbano and Ibagué. Within Polyporales, we present 12 new records for the Department and five for Colombia. These results agree with those presented by Gómez-Montoya et al. (2022) in which Agaricales and Polyporales are always the best represented groups in almost all ecosystems where diversity studies of fungi have been conducted (oak forest, coniferous forest, mixed forest, Amazon, lowland and other ecosystems).
The order Hymenochaetales is represented by six species in three different families. In this study, we included two records for the tropical rainforest and premontane dry forest. We present the first record of Phylloporia chrysites for Colombia. This species was previously described in Venezuela and is found associated with the roots of living plants, possibly with a parasitic lifestyle. New samples must be collected to determine the diversity of this order in Tolima.
For the order Auriculariales, five species are reported. A very important species, from the nutritional point of view, is A. auricula-judae that was registered in Murillo Municipality ( Gómez-Montoya et al. 2022). Wu et al. (2021) documented that it is a species with European distribution and probably it is a species complex in other parts of the world. It is necessary to review the morphology of the Colombian specimens and obtain molecular and phylogenetic data that allow us to properly name this species and classify it correctly in the phylogeny of the group. Protomerulius caryae was previously recorded in Colombia for the Department of Antioquia (Vasco-Palacios and Franco-Molano 2012). It was reported for the first time in Tolima in this study.
The orders Boletales, Cantharelalles, Phallales, Russulales, Thelephorales and Tremellales were represented by 12 species or less. Species recorded in these orders were all collected in montane rainforest and oak forest, except for Dentipellicula guyanensis that was recorded in tropical dry forest. Gloeophyllum striatum and D. spathularius are the only species reported for Gloeophyllales and Dacrymycetales, respectively, both species being reported from tropical dry forest. Vasco-Palacios and Franco-Molano (2012) reported that, in Colombia, these species are distributed below 2100 m a.s.l. It is necessary to carry out studies of the diversity of these groups in other localities and types forests present in Tolima, such as tropical dry forest, tropical rainforest, paramo and wetlands.
It is important to note that there are some endemic species described from Tolima, such as Hohenbuehelia espeletiae, described from Santa Isabel paramo. This species is only known from this type locality and from the type material, which makes it an excellent candidate to evaluate its state of conservation, mainly due to the loss and destruction of the paramo ecosystems. It is a priority to carry out studies on fungal diversity and conservation in the paramos of Colombia because it is currently a threatened ecosystem. Another endemic species is Favolaschia roseogrisea. It was also described from Tolima, but has not been collected since then. The type specimen is not located in Colombia (Singer B 6035 F) and it would be very important to have new records of this species deposited in Colombian herbaria and with an exhaustive morphological and phylogenetical analyses. The non-lichenised Ascomycota fungi have been little studied in the Department of Tolima, only ten species being recorded, so new works are needed to study the diversity of this group in the Department. The humid mountain forest is the best sampled with five species, but the diversity of Ascomycota in Tolima is still unknown. It is important to note that Ascomycota is the best represented group in Colombia with 4,554 species (Sanjuan and Brothers 2022). Certainly, the very low number of records in the Department is a clear sign of a knowledge gap.
The data provided in this study constitute an important baseline for the knowledge of fungal biodiversity in the Department of Tolima; additionally, it is a contribution to increase the knowledge of fungi distributed in dry and humid forests of low altitude, which are considered very little sampled forests in the Colombian Andes regarding fungal diversity ( Vasco-Palacios and Franco-Molano 2019, Gómez-Montoya et al. 2022).