Abstract
Molecular techniques using fungal DNA barcoding (ITS) and other markers have been key to identifying the biodiversity of different geographic areas, mainly in megadiverse countries. Here, we provide an overview of the fungal diversity in Brazil based on DNA markers of phylogenetic importance generated since 1996. We retrieved fungal sequences of ITS, LSU, SSU, tef1-α, β-tubulin, rpb1, rpb2, actin, chitin synthase, and ATP6 from GenBank using different field keywords that indicated their origin in Brazil. A total of 19,440 sequences were recovered. ITS is the most representative marker (11,209 sequences), with 70.1% belonging to Ascomycota, 18.6% Basidiomycota, 10.2% unidentified, 1.1% Mucoromycota, two sequences of Olpidium bornovanus (Fungi incertae sedis), one sequence of Blastocladiomycota (Allomyces arbusculus), and one sequence of Chytridiomycota (Batrachochytrium dendrobatidis). Considering the sequences of all selected markers, only the phyla Cryptomycota and Entorrhizomycota were not represented. Based on ITS, using a cutoff of 98%, all sequences comprise 3047 OTUs, with the majority being Ascomycota (2088 OTUs) and Basidiomycota (681 OTUs). Previous numbers based mainly on morphological and bibliographical data revealed 5264 fungal species from Brazil, with a predominance of Basidiomycota (2741 spp.) and Ascomycota (1881 spp.). The unidentified ITS sequences not assigned to a higher taxonomic level represent 1.61% of all ITS sequences sampled and correspond to 38 unknown class-level lineages (75% cutoff). A maximum likelihood phylogeny based on LSU illustrates the fungal classes occurring in Brazil.
Keywords: Brazil, Brazilian List, Fungi, GenBank, ITS barcoding
Introduction
The kingdom Fungi is monophyletic and consists of eukaryotic and heterotrophic organisms with a great diversity regarding their morphological, molecular, subcellular, biochemical, and ecological aspects [1–4]. For decades, many authors have attempted to estimate the global number of fungal species. Hawksworth [5] estimated 1.5 million species based on a 5:1 ratio of fungal to plant species. This conservative number was widely accepted until the early twenty-first century. More recent estimates based on molecular analyses of environmental samples from soil communities revealed numbers of about 3.5–5.1 [6] to 6 million [7] species. Recently, Hawksworth and Lücking [8] proposed an updated estimate of 2.2–3.8 million global fungal species. However, the number of described species in the world is currently about 120,000 [8], which means that we only know 2 or 3.1–5.4% of the estimates presented by Taylor et al. [7] and Hawksworth and Lücking [8], respectively.
The taxonomy and classification of fungi have been based on different characteristics through the last two centuries, including biochemical, molecular, morphological, physical, physiological, and sexual [1, 9]. Molecular techniques using fungal DNA barcoding and other markers have been key to identifying and exploring the biodiversity of different geographic areas [10–12] and are even more useful to explore megadiverse countries [13].
The nuclear ribosomal DNA internal transcribed spacer (ITS) region has been widely used in phylogenetic studies for sequence-based classification and identification in many fungal groups [13–18], as well as for exploration of fungal diversity in diverse substrates [19–23].
The ITS region has been proposed as the universal DNA barcoding marker for fungi [24]. However, in some groups of fungi, ITS does not provide the best taxonomic resolution and other markers represent reasonable alternatives, e.g., the D1/D2 domains of the nuclear ribosomal large subunit (LSU) gene for many yeasts [25–27]; the nuclear ribosomal small subunit (SSU) gene for arbuscular mycorrhizal fungi [28]; the β-tubulin for Penicillium spp. [29]; and the translation elongation factor 1-α (tef1-α), the largest subunit of RNA polymerase II largest (rpb1), and the second largest subunit of RNA polymerase II (rpb2) for Fusarium spp. [30].
Furthermore, multi-loci analyses have been essential for a better resolution of the higher taxonomic level relationships [31–37] or species complexes [38–42], and the number of complete fungal genome sequences for genetic and phylogenomic studies has substantially increased [43–45].
The advent of generating DNA sequences for biodiversity studies brought together many new web-accessible databases and tools [46]. The International Nucleotide Sequence Database Collaboration (INSDC) is a long-standing foundational initiative that represents the most widely used sequence repository [47, 48] and comprises, among other sources, the GenBank (at the National Center for Biotechnology Information - NCBI), a comprehensive no cost and publicly available database for DNA data [49, 50]. The UNITE database, which focuses on ITS fungal sequences, has also been an important tool for the identification of environmental sequence data, mainly obtained through metabarcoding efforts [51].
The latest GenBank release (October 2019) recorded 216,763,706 sequences that have been deposited since 1982 [52] and, according to Benson et al. [49], contains almost 260,000 formally described species of diverse organisms. Hawksworth and Lücking [8] pointed out that the named fungal species with sequences in Genbank (until November 2016) represent only 29% of the 120,000 known species. The authors also reinforced the importance of obtaining sequences for the missing named accepted species, while Seifert and Rossman [53] have already highlighted the value of DNA sequences in describing new species. However, this is not a formal requirement according to the International Code of Nomenclature for algae, fungi, and plants [54].
The number of DNA sequences from Brazilian fungal samples has increased considerably over the last decades and, together with documentation of new records and species, the knowledge about the Brazilian fungal diversity has also improved [55–59]. In 2010, based on a collaborative project and as part of the Global Strategy for Plant Conservation (GSPC), the first online version of the “Catalog of Plants and Fungi of Brazil” that provided 3187 species names of fungi to Brazil, among other groups of algae, plants, and fungus-like organisms [60], was published. The online version of the “Brazilian List,” as it is popularly known, was continually updated until 2015 and it includes data about nomenclature, geographic distribution, life form, substrate, and habitat [61] although there is no molecular data associated with these records.
Based on the updated Brazilian List, Maia et al. [62] compiled a total of 5264 accepted fungal species names (excluding fungus-like organisms) which represent an increment of approximately 65% to the number of species previously presented in 2010. Despite the contribution of many researchers to update the Brazilian List, the 5264 names represent only 36–40% of the number of known fungal species in Brazil, which was suggested by Lewinsohn and Prado [63] as 13,090–14,510, and only 2–3.5% of the 150,000–263,000 species estimated as occurring in the country [63].
Considering the importance of molecular data for biodiversity studies and the potential for mycological research in Brazil, we provide an overview of the fungal diversity in Brazil represented by selected DNA markers available in GenBank since 1996, when the first DNA sequences from Brazilian samples were deposited in the database.
The main goal of this work is to characterize the Brazilian fungal diversity based on DNA sequences generated over 20 years. Therefore, we (i) determine the total number of sequences of each marker per higher taxonomic categories, and how many sequences of each marker became available per year; (ii) determine which phyla, classes, and orders are represented by each marker; (iii) determine what is the Brazilian diversity of fungi based on the ITS sequences with a cutoff of 98% similarity; (iv) access the unidentified ITS sequences to assign them to a higher taxonomic level (phylum or class); (v) determine which are the best represented genera based on the number of ITS sequences and the number of operational taxonomic units (OTUs) they represent; (vi) illustrate the Brazilian fungal diversity represented in GenBank within a phylogenetic context; and (vii) compare the diversity of the molecular data with the number of fungal species estimated and known for the country.
Material and methods
Obtaining sequences and metadata
DNA sequences of fungi from Brazil corresponding to 10 selected markers were retrieved from the NCBI GenBank database in October 2017, using query strings (Table 1) to search the sequences from Brazil, including ITS (nuclear ribosomal internal transcribed spacer), LSU (nuclear ribosomal large subunit), SSU (nuclear ribosomal small subunit), rpb1 (RNA polymerase II largest subunit), rpb2 (RNA polymerase II second largest subunit), tef1-α (translation elongation factor 1-alpha), actin, chitin synthase, ATP6 (ATPase 6), and β-tubulin. The markers were selected based on the list of DNA markers of phylogenetic importance suggested in MycoBank to register a new taxon (http://www.mycobank.org). The sequences were downloaded in a file with format GenBank (full), which was parsed using a custom Perl script (available upon request) to extract information from the following fields: “country,” “host,” “voucher,” “isolate,” “strain,” “isolation source,” and “notes.” Then, we parsed each of the records and extracted the accession numbers that contained “Brazil” or “Brasil.” Once we had the corresponding accession numbers of the sequences that represented samples exclusively from Brazil, we extracted the information on “source” and submission date, which is usually included in one of the fields labeled as “journal.”
Table 1.
Query strings used to search for DNA sequences from Brazilian fungal samples in GenBank
| Query | Query strings |
|---|---|
| Main query (country) | Fungi[ORGN] AND 300:10000[SLEN] AND Brazil OR Brasil |
| ITS* | rRNA[Title] OR ribosomal RNA[Title] |
| LSU* | rRNA[Title] OR ribosomal RNA[Title] |
| SSU* | rRNA[Title] OR ribosomal RNA[Title] |
| rpb1 | rpb1[Title] OR RNA polymerase II largest subunit[Title] NOT rpb2 NOT rpbII |
| rpb2 | rpb2[Title] OR rpbII[Title] OR RNA polymerase II second largest subunit[Title] OR RNA polymerase II second large subunit[Title] NOT rpb1 |
| tef1-α | TEF1[Title] OR EF1[Title] OR EF-1[Title] OR TEF-1[Title] OR TEF[Title] OR tef1a[Title] OR EF[Title] OR translation elongation factor 1[Title] OR EF1alpha[Title] OR EF1a[Title] OR EF1-alpha[Title] OR TEF1-alpha[Title] |
| actin | actin[Title] OR actG[Title] |
| chitin synthase | chitin synthase[Title] OR CHS[Title] OR CHS-1[Title] |
| ATP6 | ATP6[Title] OR ATPase6[Title] OR ATP synthase F0 subunit 6[Title] OR ATP synthase subunit 6[Title] OR MTATP synthase F0 subunit 6[Title] OR MT-ATP6[Title] OR MTATP6[Title] OR ATP-6[Title] |
| β-tubulin | tub[Title] OR btub[Title] OR beta-tubulin[Title] OR tubulin[Title] OR tub2[Title] |
*The module gene_parser from PifCoSM (https://github.com/RybergGroup/PifCoSm) was used to separate the ITS, LSU, and SSU regions, with additional manual inspection to remove redundant sequences
We considered the taxon name associated to the “source” field and followed the fungal classification adopted by the GenBank database, which is continually updated and currently considers nine phyla (Ascomycota, Basidiomycota, Blastocladiomycota, Chytridiomycota, Cryptomycota, Entorrhizomycota, Microsporidia, Mucoromycota, and Zoopagomycota), based on the most recent phylogenies [2, 45, 64–66].
Computational analyses
In order to recognize operational taxonomic units (OTUs), we downloaded all the ITS sequences from Brazil and used the software CD-hit [67] with a cutoff of 98%, following the sequence similarity adopted by Tedersoo et al. [11] in a global diversity study of soil fungi.
Many of the retrieved sequences were unidentified. In order to assign them to a higher taxonomic level (phylum or class), we performed a BLASTn analysis against the NCBI GenBank database, using -max_target_seqs 50 and -outfmt “7 qacc sacc evalue ppos sscinames.” The hit with the highest percentage of similarity was used as a proxy of the identity of the sequence. A few of the sequences gave unclear results, and we were not able to assign them to any known taxonomic category, so they remained as unidentified and were later clustered using CD-hit with a cutoff of 98% to estimate the number of OTUs that remained unclassified, and with a cutoff of 75%, representing class-level following the criterion adopted by Tedersoo et al. [11], to identify if these sequences belonged to different higher taxonomic levels.
Additionally, we downloaded all the LSU sequences with the aim of reconstructing a molecular phylogeny to show the Brazilian fungal diversity represented in GenBank within a phylogenetic context. When LSU sequences of particular classes occurring in Brazil were not recovered, but other gene markers were found from Brazilian samples, we downloaded LSU sequences from samples from other parts of the world, just to fully illustrate the diversity of Brazilian fungi represented in GenBank. The LSU sequences were aligned using MAFFT 6.717 [68] and the phylogenetic tree was reconstructed using RAxML 8.2.4 [69]. The maximum likelihood (ML) tree generated was displayed in FigTree v1.4.2 (http://tree.bio.ed.ac.uk/software/figtree/), and the branches were collapsed in classes of fungi or another higher taxonomic category when class was not applicable. The graphics (Figs. 1–3) were created using Microsoft Excel and the final artworks were edited on Adobe Illustrator.
Fig. 1.
Number of DNA sequences from Brazilian fungal samples for each selected marker submitted to GenBank per year
Fig. 3.
Relative proportion of the classification of unidentified Brazilian fungal ITS sequences using BLASTn search against the NCBI GenBank database. A, Ascomycota; B, Basidiomycota; C, Chytridiomycota; M, Mucoromycota; and Z, Zoopagomycota
Results
Our search led to a total of 19,440 DNA sequences of fungal samples from Brazil belonging to seven out of the nine phyla considered by the current classification adopted by GenBank. Only the phyla Cryptomycota and Entorrhizomycota were not recovered.
Ascomycota is the most well-representated phylum with a total of 14,442 sequences, which corresponds to three subphyla (Pezizomycotina, Saccharomycotina, and Taphrinomycotina) and includes 10 classes and 51 orders (Table 2). Basidiomycota has 3518 sequences corresponding to four subphyla (Agaricomycotina, Pucciniomycotina, Ustilaginomycotina, and Wallemiomycotina) and includes 12 classes and 35 orders (Table 2). Mucoromycota showed a lower number of sequences (286), being represented by three subphyla (Glomeromycotina, Mortierellomycotina, and Mucoromycotina) and seven of the eight orders currently recognized in the phylum [45]; only Endogonales was not represented (Table 2).
Table 2.
Number of DNA sequences in GenBank from Brazilian fungal samples assigned to higher taxonomic categories per selected marker
| Taxa | ITS | LSU | tef1 | β-tubulin | rpb2 | SSU | rpb1 | actin | chitin | ATP6 |
|---|---|---|---|---|---|---|---|---|---|---|
| Fungi | ||||||||||
| Unclassified | 1138 | 2 | 22 | |||||||
| Incertae sedis | ||||||||||
| Olpidium (2/6/40) | 2 | |||||||||
| Ascomycota | ||||||||||
| Unclassified | 48 | 23 | ||||||||
| Incertae sedis | ||||||||||
| Amphobotrys (1/1/1) | 2 | |||||||||
| Aporospora (1/-/-) | 1 | |||||||||
| Clathrosporium (2/-/1) | 2 | 2 | ||||||||
| Dichotomophthora (1/-/2) | 2 | |||||||||
| Phaeoramularia (1/-/-) | 1 | |||||||||
| Phaeoseptoria (1/-/19) | 1 | |||||||||
| Phialocephala (-/1/20) | 1 | |||||||||
| Pseudofusarium (1/-/-) | 1 | |||||||||
| Repetophragma (1/-/11) | 1 | |||||||||
| Radulidium (-/-/1) | 1 | |||||||||
| Sirosporium (1/-/25) | 1 | 1 | ||||||||
| Pezizomycotina | ||||||||||
| Unclassified | 3 | 1 | ||||||||
| Incertae sedis | ||||||||||
| Coremiella (-/-/1) | 2 | |||||||||
| Pyriculariopsis (-/-/5) | 1 | |||||||||
| Arthoniomycetes | ||||||||||
| Arthoniales (2/54/1538) | 2 | |||||||||
| Dothideomycetes | ||||||||||
| Unclassified | 4 | 1 | 1 | |||||||
| Asterinales (3/246/-) | 4 | 10 | 3 | |||||||
| Asterotexiales (-/-/-) | 2 | |||||||||
| Botryosphaeriales (160/19/1628) | 913 | 58 | 298 | 116 | 11 | 2 | ||||
| Capnodiales (164/98/7244) | 460 | 237 | 165 | 70 | 49 | 34 | 28 | |||
| Dothideales (7/74/350) | 33 | 37 | 1 | |||||||
| Hysteriales (-/2/69) | 3 | |||||||||
| Myriangiales (22/2/157) | 76 | 67 | 49 | 44 | 20 | 18 | ||||
| Mytilinidiales (1/1/-) | 1 | |||||||||
| Pleosporales (185/76/4764) | 603 | 35 | 11 | 121 | 16 | 13 | ||||
| Trypetheliales (-/-/192) | 35 | |||||||||
| Tubeufiales (8/10/-) | 12 | 10 | 5 | |||||||
| Venturiales (6/2/-) | 7 | 2 | 1 | |||||||
| Incertae sedis | ||||||||||
| Arxiella (1/-/2) | 1 | |||||||||
| Hyalosphaera (-/-/3) | 1 | |||||||||
| Inocyclus (1/-/7) | 3 | 3 | 3 | |||||||
| Perisporiopsis (1/-/16) | 1 | |||||||||
| Phalangispora (1/-/2) | 1 | 1 | ||||||||
| Speiropsis (3/2/7) | 6 | 5 | ||||||||
| Eurotiomycetes | ||||||||||
| Chaetothyriales (79/3/213) | 354 | 36 | 96 | 2 | 6 | 2 | 13 | 23 | ||
| Eurotiales (186/60/928) | 481 | 52 | 1 | 116 | 122 | 31 | 25 | |||
| Onygenales (13/1/271) | 153 | 43 | 2 | 1 | 30 | 1 | ||||
| Phaeomoniellales (7/-/-) | 28 | 1 | ||||||||
| Pyrenulales (-/29/538) | 1 | |||||||||
| Verrucariales (3/2/1032) | 3 | |||||||||
| Lecanoromycetes | ||||||||||
| Unclassified | 2 | |||||||||
| Baeomycetales (-/-/16) | 2 | |||||||||
| Caliciales (1/-/-) | 1 | |||||||||
| Lecanorales (29/219/5695) | 41 | 19 | 1 | 11 | 2 | 3 | ||||
| Ostropales (1/162/2753) | 1 | 33 | 2 | 2 | 5 | |||||
| Peltigerales (6/2/1208) | 19 | 6 | 4 | 4 | ||||||
| Pertusariales (-/8/901) | 1 | |||||||||
| Teloschistales (-/22/1954) | 1 | |||||||||
| Incertae sedis | ||||||||||
| Chromatochlamys (-/-/-) | 2 | |||||||||
| Phlyctis (-/-/12) | 1 | 1 | ||||||||
| Leotiomycetes | ||||||||||
| Erysiphales (15/7/769) | 32 | 8 | ||||||||
| Helotiales (24/60/3881) | 56 | 22 | 9 | 16 | ||||||
| Rhytismatales (1/2/795) | 1 | |||||||||
| Incertae sedis | ||||||||||
| Geomyces (1/-/4) | 1 | |||||||||
| Lauriomyces (1/2/5) | 1 | 1 | ||||||||
| Myxotrichum (1/-/12) | 1 | |||||||||
| Oidiodendron (1/1/28) | 1 | |||||||||
| Pseudeurotium (1/-/5) | 1 | |||||||||
| Xylogone (2/-/1) | 5 | |||||||||
| Orbiliomycetes | ||||||||||
| Orbiliales (5/1/288) | 5 | |||||||||
| Pezizomycetes | ||||||||||
| Pezizales (2/28/1683) | 2 | |||||||||
| Sordariomycetes | ||||||||||
| Unclassified | 2 | |||||||||
| Calosphaeriales (1/-/54) | 1 | |||||||||
| Chaetosphaeriales (13/26/311) | 5 | 4 | ||||||||
| Coniochaetales (8/1/76) | 9 | 4 | 1 | |||||||
| Coronophorales (1/-/87) | 1 | 1 | ||||||||
| Diaporthales (179/8/1196) | 474 | 23 | 80 | 87 | 9 | 1 | ||||
| Falcocladiales (2/-/-) | 4 | 2 | 2 | |||||||
| Glomerellales (88/-/-) | 1010 | 15 | 1 | 439 | 1 | 1 | 3 | |||
| Hypocreales (263/82/2647) | 1286 | 101 | 1006 | 335 | 227 | 84 | 111 | 31 | ||
| Magnaporthales (18/-/-) | 27 | 12 | 126 | 130 | 10 | 5 | ||||
| Meliolales (3/87/1980) | 3 | 20 | 11 | |||||||
| Microascales (22/4/397) | 103 | 3 | 10 | 34 | 5 | |||||
| Myrmecridiales (1/-/-) | 3 | 2 | ||||||||
| Ophiostomatales (8/-/341) | 133 | 1 | 89 | 39 | 5 | |||||
| Phyllachorales (26/2/1226) | 54 | 1 | 32 | |||||||
| Sordariales (38/63/854) | 94 | 8 | 5 | 1 | 4 | |||||
| Togniniales (6/-/-) | 8 | 26 | ||||||||
| Trichosphaeriales (17/2/73) | 41 | 1 | 1 | |||||||
| Xylariales (163/275/2487) | 319 | 50 | 5 | 29 | 3 | 1 | 2 | |||
| Incertae sedis | ||||||||||
| Papulosa (-/-/1) | 1 | |||||||||
| Saccharomycotina | ||||||||||
| Saccharomycetes | ||||||||||
| Saccharomycetales (237/-/906) | 867 | 936 | 12 | 6 | 8 | 8 | 12 | |||
| Taphrinomycotina | ||||||||||
| Schizosaccharomycetes | ||||||||||
| Schizosaccharomycetales (1/-/5) | 1 | |||||||||
| Basidiomycota | ||||||||||
| Unclassified | 10 | |||||||||
| Agaricomycotina | ||||||||||
| Agaricomycetes | ||||||||||
| Unclassified | 1 | 13 | ||||||||
| Agaricales (202/924/13233 | 437 | 76 | 130 | 8 | 7 | 25 | ||||
| Amylocorticiales (1/-/-) | 1 | 1 | ||||||||
| Atheliales (2/10/106) | 2 | 28 | 27 | 2 | ||||||
| Auriculariales (5/29/198) | 9 | 9 | 3 | |||||||
| Boletales (20/92/1316) | 45 | 11 | 1 | 1 | 2 | 1 | ||||
| Cantharellales (62/40/544) | 186 | 7 | 1 | 34 | ||||||
| Corticiales (4/22/136) | 4 | 5 | 1 | |||||||
| Geastrales (10/37/64) | 16 | 13 | 6 | 4 | ||||||
| Gloeophyllales (1/2/33) | 1 | |||||||||
| Gomphales (2/5/336) | 4 | 1 | 4 | |||||||
| Hysterangiales (2/6/114) | 7 | |||||||||
| Hymenochaetales (54/165/610) | 83 | 79 | 28 | 31 | ||||||
| Lepidostromatales (1/-/-) | 11 | 1 | ||||||||
| Phallales (2/35/88) | 3 | 2 | 2 | 2 | 2 | |||||
| Polyporales (116/453/1801) | 207 | 145 | 2 | 3 | 2 | 1 | 1 | |||
| Russulales (6/137/1767) | 6 | 1 | 1 | 1 | ||||||
| Sebacinales (-/-/30) | 4 | |||||||||
| Stereopsidales (1/-/-) | 1 | 3 | 3 | |||||||
| Thelephorales (7/7/269) | 9 | |||||||||
| Dacrymycetes | ||||||||||
| Dacrymycetales (1/3/101) | 1 | 3 | ||||||||
| Tremellomycetes | ||||||||||
| Cystofilobasidiales (1/-/20) | 1 | 1 | ||||||||
| Filobasidiales (2/-/4) | 3 | 6 | ||||||||
| Tremellales (66/4/341) | 200 | 232 | 26 | 22 | 65 | 29 | 2 | |||
| Trichosporonales (19/-/-) | 60 | 57 | 1 | 1 | 1 | |||||
| Pucciniomycotina | ||||||||||
| Agaricostilbomycetes | ||||||||||
| Agaricostilbales (1/-/43) | 1 | 2 | ||||||||
| Incertae sedis | ||||||||||
| Sporobolomyces (1/-/25) | 2 | 2 | ||||||||
| Cystobasidiomycetes | ||||||||||
| Unclassified | 1 | |||||||||
| Cystobasidiales (7/-/9) | 12 | 8 | ||||||||
| Erythrobasidiales (5/-/2) | 5 | 3 | ||||||||
| Incertae sedis | ||||||||||
| Symmetrospora (3/-/-) | 5 | 4 | ||||||||
| Microbotryomycetes | ||||||||||
| Sporidiobolales (14/-/83) | 22 | 42 | ||||||||
| Incertae sedis | ||||||||||
| Colacogloea (2/-/4) | 2 | 1 | ||||||||
| Curvibasidium (-/-/2) | 1 | |||||||||
| Yunzhangia (-/-/-) | 1 | |||||||||
| Pucciniomycetes | ||||||||||
| Pucciniales (9/749/7798) | 615 | 5 | 13 | 14 | 1 | |||||
| Ustilaginomycotina | ||||||||||
| Exobasidiomycetes | ||||||||||
| Exobasidiales (3/-/83) | 4 | 6 | 1 | |||||||
| Microstromatales (4/-/10) | 9 | 6 | 1 | |||||||
| Malasseziomycetes | ||||||||||
| Malasseziales (26/-/7) | 101 | 77 | ||||||||
| Moniliellomycetes | ||||||||||
| Moniliellales (-/-/-) | 7 | |||||||||
| Ustilaginomycetes | ||||||||||
| Ustilaginales (10/-/851) | 13 | 23 | 3 | 1 | 1 | 7 | 4 | |||
| Wallemiomycotina | ||||||||||
| Wallemiomycetes | ||||||||||
| Wallemiales (-/-/2) | 1 | 1 | ||||||||
| Blastocladiomycota | ||||||||||
| Blastocladiomycetes | ||||||||||
| Blastocladiales (1/17/179) | 1 | 2 | 1 | 1 | 1 | |||||
| Chytridiomycota | ||||||||||
| Chytridiomycetes | ||||||||||
| Chytridiales (-/68/494) | 1 | |||||||||
| Cladochytriales (-/-/-) | 1 | |||||||||
| Rhizophydiales (1/24/104) | 1 | |||||||||
| Spizellomycetales (-/1/27) | 1 | 1 | ||||||||
| Microsporidia | ||||||||||
| Nosema (-/-/81) | 9 | |||||||||
| Pleistophora (-/-/34) | 1 | 1 | ||||||||
| Potaspora (-/-/-) | 1 | 1 | ||||||||
| Mucoromycota | ||||||||||
| Glomeromycotina | ||||||||||
| Unclassified | 3 | 3 | 2 | |||||||
| Glomeromycetes | ||||||||||
| Archaeosporales (2/10/6) | 3 | 4 | ||||||||
| Diversisporales (24/39/74) | 38 | 46 | 44 | |||||||
| Glomerales (5/61/86) | 23 | 23 | 3 | 4 | ||||||
| Paraglomerales (-/5/3) | 4 | |||||||||
| Mortierellomycotina | ||||||||||
| Mortierellales (1/10/93) | 1 | |||||||||
| Mucoromycotina | ||||||||||
| Mucorales (35/76/205) | 60 | 20 | 2 | |||||||
| Umbelopsidales (1/-/-) | 1 | |||||||||
| Zoopagomycota | ||||||||||
| Entomophthoromycotina | ||||||||||
| Entomophthoromycetes | ||||||||||
| Entomophthorales (-/3/256*) | 2 | |||||||||
| Neozygitomycetes | ||||||||||
| Neozygitales (-/2/21**) | 2 | |||||||||
| Kickxellomycotina | ||||||||||
| Kickxellales (-/-/32) | 1 | 1 | ||||||||
| Total | 11,209 | 2863 | 2097 | 1693 | 603 | 493 | 241 | 128 | 65 | 48 |
Numbers in parentheses for each taxon represent, respectively, the number of OTUs based on ITS (98% cutoff)/the number of species in the Brazilian List [93]/the global number of known species [92]. (-/-/-), OTUs not calculated because no ITS sequences available/number of taxa not available in the Brazilian List [93]/number of known species not considered by Kirk et al. [92] because the taxon was newly described or treated in a different taxonomic rank
*Number based on Entomophthorales, excluding Neozygitaceae
**Number based on Neozygitaceae
There are few DNA sequences available for the other four phyla sampled and also fewer taxa represented (Table 2). Microsporidia has a total of 13 sequences from three species: Nosema ceranae I. Fr., F. Feng, JA da Silva, SB Slemenda & NJ Pieniazek [70]; Pleistophora hyphessobryconis Schäperclaus [71]; and the non-validly published taxon Potaspora aequidens [72]. Blastocladiomycota is represented by six sequences of Allomyces arbusculus EJ Butler (Blastocladiomycetes, Blastocladiales) [73].
Zoopagomycota has a total of six sequences of Entomophthoromycotina and Kickxellomycotina, represented by Conidiobolus lamprauges Drechsler (Entomophthorales, Entomophoromycetes) [74]; Neozygites tanajoae Delal., Humber & AE Hajek (Neozygitales, Neozygitomycetes) [55]; Coemansia brasiliensis Thaxt. ex Linder [75]; and Spiromyces sp. (Kickxellales). Finally, Chytridiomycota is represented by five sequences from four orders of Chytridiomycetes, including the following taxa: Batrachochytrium dendrobatidis Longcore, Pessier & DK Nichols (Rhizophydiales) [76]; Geranomyces variabilis (Longcore, DJS Barr & Désauln.) DR Simmons (Spizellomycetales) [77]; and Entophlyctis luteolus Longcore (Chytridiales) and Nowakowskiella multispora Karling (Cladochytriales). Two sequences of the incertae sedis taxon Olpidium bornovanus Sahtiy. ex Karling (=Leiolpidium bornovanum Doweld) [78, 79] were also recovered. A total of other 1162 unidentified sequences not assigned to any phylum completed our dataset.
The ITS region is the most representative marker, corresponding to 57.7% of the total number of sequences, and followed by LSU (14.7%), tef1-α (10.8%), β-tubulin (8.7%), rpb2 (3.1%), SSU (2.5%), rpb1 (1.2%), actin (0.7%), chitin synthase (0.3%), and ATP6 (0.2%). The first sequences from fungal Brazilian samples submitted to GenBank were SSU sequences dated October 25, 1996 (Fig. 1): U76338 Colletotrichum gloeosporioides (Penz.) Penz. & Sacc. and U76340 Sphaerodothis acrocomiae Chardón & RED Baker (=Camarotella costaricensis (F. Stevens) KD Hyde & PF Cannon). The next sequences were submitted in 1998 (Fig. 1) and represent ITS and LSU sequences (AF079596, AF079643–AF079644, AF079678, AF079725–AF079726) of some Agaricales ant symbiont [80], ITS (AF046891 and AF046900) of a phytopathogenic Diaporthales [81], and ITS (AF097913) of a new species of Trichoderma described as a parasite of the cacao witches broom pathogen [82].
The first sequences for the other markers have been submitted since 2000 for β-tubulin, 2002 for tef1-α, 2003 for actin, 2005 for rpb1 and rpb2, and 2007 for ATP6 and chitin synthase (Fig. 1). The number of ITS sequences has been continuously increasing since 2006, with a decrease in 2012, 2013, and 2015, and the highest number of sequences was submitted in 2016.
The phyla represented by each marker are not equally distributed and, although ITS is the most commonly used marker, SSU is the only marker that includes sequences of all the seven sampled phyla (Fig. 2). Also, Ascomycota is the best represented phylum for most markers, except for ATP6, which is predominantly composed of sequences of Basidiomycota, mainly phytopathogens that belong to the order Cantharellales (Agaricomycetes) [83]. The total number of ITS sequences includes 7837 sequences of Ascomycota, 2100 Basidiomycota, 1138 unidentified, 130 Mucoromycota, two sequences of Olpidium bornovanus, one sequence of Blastocladiomycota (Allomyces arbusculus), and one sequence of Chytridiomycota (Batrachochytrium dendrobatidis).
Fig. 2.
Relative proportion of DNA sequences in GenBank from Brazilian fungal samples assigned to phyla for all and each selected marker
Based only on the ITS sequences and using a cutoff of 98%, the Brazilian fungal sequences comprise a total of 3047 OTUs, of which 2088 belong to Ascomycota, 681 belong to Basidiomycota, and 69 belong to Mucoromycota. The unidentified sequences not assigned at least to the phylum rank represent 245 OTUs. When performing a BLASTn search of these unidentified sequences at > 75% sequence similarity (representing class-level), they were assigned to seven classes of Ascomycota (67.14% of the unidentified sequences), three classes of Basidiomycota (15.47%), three subphyla in Mucoromycota (1.14%), plus sequences in Chytridiomycota and Zoopagomycota; 181 sequences could not be assigned to any known class of fungi with this approach (Fig. 3). Furthermore, clustering the 181 unclassified sequences revealed 72 distinct OTUs using a cutoff of 98% and 38 higher level groups at 75% cutoff, indicating that there are many unknown deeply divergent lineages in Brazil.
For the identified ITS sequences, the classes with the greatest number of sequences are also the most diverse regarding the ITS marker (Fig. 4), corresponding to Sordariomycetes (3592 seq., 857 OTUs), Dothideomycetes (2125 seq., 563 OTUs), Eurotiomycetes (1019 seq., 288 OTUs), and Agaricomycetes (1033 seq., 498 OTUs).
Fig. 4.
ML tree including the main higher taxonomic categories occurring in Brazil from which there are molecular data available. The first number in the parentheses for each taxon represents the total of ITS sequences available or (-) when there are no ITS sequences. For the following numbers in the parentheses, see footnotes in Table 1. *Number based on Malasseziales; **number based on Entomophthorales, excluding Neozygitaceae
Hypocreales is the order of Ascomycota with the highest number of ITS sequences retrieved (1286 seq.) and also the most diverse order (263 OTUs). On the other hand, there are some orders of Ascomycota in which the relative proportion between the number of ITS sequences and the corresponding OTUs is disproportionate (Table 2), such as Glomerellales with 1010 sequences equivalent to only 88 OTUs, and Xylariales with 319 sequences that correspond to 163 OTUs. In Basidiomycota, Pucciniales has the largest number of ITS sequences (615 seq.) but they represent only 9 OTUs. Agaricales (202 OTUs) and Polyporales (116 OTUs) are the most diverse orders in Basidiomycota (Table 2).
Among the most sampled genera in Ascomycota (> 150 ITS seq.), the following are the most diverse based on the number of OTUs recognized (Table 3): Phyllosticta (109 OTUs), Penicillium (95 OTUs), Diaporthe (90 OTUs), Candida (83 OTUs), Fusarium (80 OTUs), and Colletotrichum (76 OTUs). Some genera of Ascomycota, despite the high number of ITS sequences available, represent a low diversity, such as Fonsecae (200 seq., 16 OTUs) and Lasiodiplodia (204 seq., 9 OTUs). On the other hand, OTUs in the genera Xylaria (95 seq., 52 OTUs) and Phyllachora (54 seq., 27 OTU) showed a more uniform sampling.
Table 3.
Fungal genera occurring in Brazil with the greatest number of ITS sequences and their genetic diversity based on OTUs (ITS 98% cutoff)
| Genera ordered by number of ITS sequences | Genera ordered by number of OTUs | ||
|---|---|---|---|
| Genera (Phylum)* | No. of sequences | Genera (Phylum)* | No. of OTUs |
| Colletotrichum (A) | 969 | Phyllosticta (A) | 09 |
| Candida (A) | 536 | Penicillium (A) | 95 |
| Phyllosticta (A) | 467 | Diaporthe (A) | 90 |
| Fusarium (A) | 449 | Candida (A) | 83 |
| Phakopsora (B) | 444 | Fusarium (A) | 80 |
| Trichoderma (A) | 289 | Colletotrichum (A) | 76 |
| Diaporthe (A) | 257 | Aspergillus (A) | 63 |
| Aspergillus (A) | 204 | Trichoderma (A) | 44 |
| Lasiodiplodia (A) | 204 | Rhizoctonia (B) | 38 |
| Fonsecaea (A) | 200 | Pluteus (B) | 29 |
| Penicillium (A) | 199 | Cora (B) | 24 |
| Puccinia (B) | 168 | Cryptococcus (B) | 18 |
| Cryptococcus (B) | 94 | Fonsecaea (A) | 16 |
| Rhizoctonia (B) | 90 | Ceratobasidium (B) | 16 |
| Cora (B) | 70 | Lasiodiplodia (A) | 9 |
| Ceratobasidium (B) | 56 | Puccinia (B) | 4 |
| Pluteus (B) | 56 | Phakopsora (B) | 3 |
*Genera with > 150 ITS sequences for Ascomycota (A) and > 50 ITS sequences for Basidiomycota (B)
In Basidiomycota, although Phakopsora (444 seq.) and Puccinia (168 seq.) were the most sampled genera for ITS (> 50 ITS seq.), their diversity is very low with only 3 and 4 OTUs, respectively. Rhizoctonia (38 OTUs), Pluteus (29 OTUs), and Cora (24 OTUs) are the most diverse genera among the most sampled Basidiomycota for ITS (Table 3).
The ML tree based on LSU sequences (Fig. 4) illustrates the main groups of fungi occurring in Brazil, grouped into classes, and for which there are DNA sequences available. Neozygitomycetes is not represented in the tree because there is no LSU sequence available for any representative of this class, only SSU sequences of Neozygites tanajoae Delal., Humber & AE Hajek from Brazilian samples (AY233981–AY233982).
Besides the phyla Cryptomycota and Entorrhizomycota that were not recovered in this study, the classes (or subphyla) of fungi for which there is no molecular data available from Brazilian samples include: Coniocybomycetes, Geoglossomycetes, Laboulbeniomycetes, Lichinomycetes, and Xylonomycetes (Pezizomycotina, Ascomycota); Archaeorhizomycetes, Neolectomycetes, Pneumocystidomycetes, and Taphrinomycetes (Taphrinomycotina, Ascomycota); Atractiellomycetes, Classiculomycetes, Cryptomycocolacomycetes, Mixiomycetes, Spiculogloeomycetes, and Tritirachiomycetes (Pucciniomycotina, Basidiomycota); Neocallimastigomycetes and Monoblepharidomycetes (Chytridiomycota); and Basidiobolomycetes (Entomophthoromycotina, Zoopagomycota) and Zoopagomycotina (Zoopagomycota).
Discussion
The total of 19,440 sequences recovered in this study and also the number of sequences for each selected marker supersede the total number of fungal sequences from any other country in South America and for any marker, based on a search conducted by Gazis [84], who used the words “fungi + country” as query.
Although there are no molecular data for Cryptomycota from Brazil, Maia et al. [62] recorded five species in the country. According to the Brazilian List [85] and the bibliography consulted [86, 87], there are eight names of Cryptomycota from Brazil, but only five represent validly published species: Rozella allomycis Foust, R. chytriomycetis Karling, R. cladochytrii Karling, R. endochytrii Karling, and R. rhizophydii Karling. About 20 species have been described in Cryptomycota, but molecular data and complete morphological and ultrastructure characterization for these taxa are rare [88]. According to a recent phylogenetic analysis based on SSU [88], there are only 13 sequences of Cryptomycota available, including sequences from Australia, France, Japan, and the USA, and most of them are represented by environmental, undetermined sequences. Rozella allomycis and R. rhizoclosmatii Letcher & Longcore are the only species in the phylum that have been characterized based on molecular, morphological, and ultrastructural data [73, 88–90].
For Entorrhizomycota, we did not find any record of species occurring in Brazil. Entorrhizomycota was proposed by Bauer et al. [66] based on multi-loci molecular analyses to accommodate the class Entorrhizomycetes and the single genus Entorrhiza, previously assigned to Ustilaginomycotina (Basidiomycota). Thereafter, Riess et al. [91] proposed a new order in Entorrhizomycota, viz. Talbotiomycetales, to accommodate Talbotiomyces calosporus (PHB Talbot) Vánky, R. Bauer & Begerow. Recently, Zhao et al. [37], based on a six-gene phylogenetic study with estimated divergence time, accepted the recognition of Entorrhizomycota as phylum.
Ascomycota and Basidiomycota are the most diverse phyla of fungi worldwide with ca. 64,131 and 31,515 species, respectively [92], and they also represent the phyla with the largest proportion of sequences sampled in our study (Fig. 2). Among the classes of Ascomycota recovered in this study (Fig. 4), Saccharomycetes and Schizosaccharomycetes, both comprising most of the yeast-forming fungi, have not been included in the Brazilian List [93].
For Saccharomycetes, we found a total of 1849 sequences (Table 2, Fig. 4), most of them are from several published works, including taxonomic and phylogenetic studies with new records and species from Brazil [94–101]. For Schizosaccharomycetes, the only sequence recovered (JQ726610) is part of a study with yeasts isolated from Brazilian cocoa box fermentations [102].
It is also important to point out that we found a sequence (AF457891) assigned in GenBank as Lichinomycetes, because in the “source” field it is named as Peltula crispatula (Nyl.) Egea (Lichinomycetes), a synonym of Cladonia crispatula (Nyl.) Ahti, which is currently accepted as a member of Lecanoromycetes [103]. Cladonia crispatula is listed in the Brazilian List [104] and the sequence AF457891 is also published under C. crispatula, as part of a broad phylogeny for the genus [105]. Thus, we considered this sequence in Lecanorales and recognize that there is no molecular data for Lichinomycetes from Brazil and most likely no other records for species of this class in the country. A similar misidentification was found for the sequence EU075543 that is named in GenBank as Lecidea haematites Fée, a synonym of Ramboldia haematites (Fée) Kalb. The genus Lecidea is a member of Lecideales, Lecanoromycetes [106], but according to Kalb et al. [107], the Brazilian sample (EU075543) represents a Lecanorales (Lecanoromycetes) member, whose current name is R. haematites.
In our Basidiomycota dataset, we recovered sequences of six classes that have not been included in the Brazilian List [93], most of them are taxa of yeast-forming fungi: Agaricostilbomycetes, Cystobasidiomycetes, Malasseziomycetes, Microbotryomycetes, Moniliellomycetes, and Wallemiomycetes.
Agaricostilbomycetes is represented by sequences of Bensingtonia sp. (FJ605257) [108], unpublished sequences of Sporobolomyces sp. (HQ014448–HQ014452), and Sterigmatomyces halophilus Fell (KY105555 and KY109791) [109]. Cystobasidiomycetes is represented by a total of 38 sequences of Cystobasidiales, Erythrobasidiales, and species of the incertae sedis genus Symmetrospora, most of them as part of unpublished works, except for Sette et al. [110] and Arcuri et al. [111], and representing undetermined samples at the species level.
For Malasseziomycetes, we found a total of 178 sequences (Table 2, Fig. 4), most of them as part of unpublished works and representing only nine species: Malassezia dermatis Sugita, M. Takash., A. Nishikawa & Shinoda; M. furfur (C.P. Robin) Baill.; M. globosa Midgley, E. Guého & J. Guillot; M. japonica Sugita, M. Takash., M. Kodama, Tsuboi & A. Nishikawa; M. nana A. Hirai, R. Kano, Makimura, H. Yamag. & A. Haseg.; M. pachydermatis (Weidman) C.W. Dodge, M. restricta E. Guého, J. Guillot & Midgley; M. slooffiae J. Guillot, Midgley & E. Guého; and M. sympodialis R.B. Simmons & E. Guého. Malassezia nana was proposed as new species by Hirai et al. [112] based on samples isolated from Brazil and Japan.
For Microbotryomycetes, we recovered a total of 69 sequences, most of them as part of unpublished works, except for Arcuri et al. [111] and Sperandio et al. [113], and representing species of the genera Colacogloea, Rhodosporidiobolus, Rhodotorula, Sporidiobolus, Sporobolomyces, and Yunzhangia. Sequences of Sporobolomyces sp. are also assigned to Agaricostilbomycetes (Table 2) because the genus is known to be polyphyletic [114].
Moniliellomycetes is represented by only seven unpublished and undetermined sequences of the genus Moniliella; while Wallemiomycetes is represented by only two sequences (KM196339 and KM196392) of Wallemia mellicola Jančič, HDT Nguyen, Seifert & Gunde-Cim., a species described by Jančič et al. [115].
Most of the Ascomycota and Basidiomycota classes sampled in this study, but not included in the Brazilian List, represent groups of yeast-forming fungi, which shows the need to consolidate efforts so that these groups of fungi are also incorporated in the next initiatives to inventory the Brazilian fungal diversity. Microsporidia is also absent from the Brazilian List, but sequences of Nosema ceranae, Pleistophora hyphessobryconis [71], and Potaspora aequidens [72] confirm its occurrence in Brazil.
Besides the orders in the classes discussed above, we present 26 orders of fungi (Table 2) that have not been listed before in the Brazilian List, including: two orders in Dothideomycetes (Asterotexiales and Trypetheliales); one in Eurotiomycetes (Phaeomoniellales); two in Lecanoromycetes (Caliciales and Baeomycetales); eight in Sordariomycetes (Calosphaeriales, Coronophorales, Falcocladiales, Glomerellales, Magnaporthales, Myrmecridiales, Ophiostomatales, and Togniniales); four in Agaricomycetes (Amylocorticiales, Lepidostromatales, Sebacinales, and Stereopsidales); three in Tremellomycetes (Cystofilobasidiales, Filobasidiales, and Trichosporonales); two in Exobasidiomycetes (Exobasidiales and Microstromatales); one in Ustilaginomycetes (Ustilaginales); one in Chytridiomycetes (Cladochytriales); one in Mucoromycotina (Umbelopsidales); and one in Kickxellomycotina (Kickxellales).
Some of these orders were recently proposed and therefore are not included in the latest update of the Brazilian List, such as Asterotexiales [116], Falcocladiales [117], Lepidostromatales [118], Phaeomoniellales [119], and Umbelopsidales [45].
In other cases, the taxa in the Brazilian List were considered incertae sedis, such as many genera of Glomerellales, Magnaporthales, Myrmecridiales, and Trypetheliales; or treated in a different taxonomic classification, such as Physcia considered in Teloschistales instead of Caliciales; Ceraceomyces considered in Boletales instead of Amylocorticiales; Stereopsis considered in Polyporales instead of Stereopsidales; Trichosporon considered in Tremellales instead of Trichosporonales; and Nowakowskiella considered in Chytridiales instead of Cladochytriales [93]. The records of Caronophorales [120] and Sebacinales [121] from Brazil, based on the molecular data recovered here, are also more recent than the latest update of the Brazilian List.
The other 10 orders of fungi not recorded in the Brazilian List include many groups of phytopathogens and endophytes, such as Calosphaeriales [122], Microstromatales (only unpublished sequences), Togniniales [123, 124], and Ustilaginales [125]; fungi saprobes of insects, such as Kickxellales [75], and of clinical importance for humans and other animals, such as Ophiostomatales [126–131]; other yeast-forming fungi, including Cystofilobasidiales (unpublished sequences), Exobasidiales [113, 132], Filobasidiales [111], and Ustilaginales [113, 133]; or lichen-forming fungi, such as Baeomycetales [134]. Again, we reinforce the need to consolidate efforts so that a variety of groups of fungi of agronomic and clinical importance can also be incorporated in broad inventories of Brazilian fungal diversity.
On the other hand, there are 12 orders of fungi recorded in the Brazilian List for which we did not recover molecular data: Acrospermales, Jahnulales, Microthyriales (Dothideomycetes), Candelariales, Trechisporales (Agaricomycetes), Doassansiales (Exobasidiomycetes), Urocystidiales (Ustilaginomycetes), Lobulomycetales, Rhizophlyctidales (Chytridiomycetes), Monoblepharidales (Monoblepharidomycetes), Dimargaritales (Kickxellomycotina), and Zoopagales (Zoopagomycotina). A second manually quick search in GenBank using the words “taxa + Brazil” as query, in which taxa was substituted for the orders and respective genera listed in the Brazilian List, confirmed that there are no molecular data associated with these taxa from Brazilian samples. Microthyriales is the third order of Ascomycota with the highest number of species listed from Brazil, with ca. 120 species [62], and even though it represents a diverse group occurring in the country, it has not yet been studied within a molecular context.
The number of sequences represented for each group of fungi per marker could reflect the effectiveness of using some markers for species identification and phylogenetic studies in certain groups. For instance, the relative proportion of Mucoromycota sequences is higher for SSU than the other markers (Fig. 2). SSU sequences have been widely used for the identification of species of Glomeromycotina (Mucoromycota), and the most comprehensive taxa sampling of this group was done using that molecular marker [135].
The SSU region has also been used traditionally for molecular studies in Zoopagomycota [136], although multi-loci studies have been essential for broad phylogenetic studies and protein-coding genes such as MCM7 and TSR1 have shown significant promise in the group [137]. The sequences of Zoopagamycota recovered in this study are mostly SSU sequences (Table 2). Molecular studies in Blastocladiomyota, Chytridiomycota, and Microsporidia [73, 138–140] also have been based fundamentally on SSU, and the sampling of this marker is representative among the sequences from Brazilian specimens of these groups.
The OTU clustering approach is useful to estimate the intraspecific ITS variability in some groups [141], and approximately correlates to species level, also called “species hypothesis” by Kõljalg et al. [142], on different similarity thresholds (97–99%), but it has also been shown that OTUs do not necessarily correspond to species [143]. Therefore, it is important to highlight that the weighted intraspecific ITS variability is different for each group of fungi, reaching average values of 5.63% in Chytridiomycota and 7.46% in Glomeromycotina [141]. On the other hand, the effectiveness of ITS as DNA barcode or even to determine the OTUs is relevant in most Ascomycota and Basidiomycota [11, 24, 141, 144], which represent the most sampled phyla for ITS in our study. Alternative methods such as amplicon sequence variants (ASVs) have been proposed to analyze high-throughput sequencing data in order to reduce sequencing errors, to avoid arbitrary tresholds that are usually imposed in the OTU clustering approach, and to provide an accurate measurement of diversity [145].
The total of 3047 OTUs calculated for all fungal sequences represents 57.9% of the number of species listed by Maia et al. [62] and 21–23.2% of the total estimated number of known fungal species in Brazil [63]. Compared with the number of fungal species known in the world [8], it represents 2.54%. When we consider the estimates of global fungal species [7, 8] or the species occurring in Brazil [63], the number of OTUs recovered in this study represents 0.05–0.14% or 1.16–2.03%, respectively.
Tedersoo et al. [11], in a metabarcoding study of global diversity of soil fungi, found a total of 80,486 OTUs classified as fungi, from which, excluding the OTUs represented by single sequences (35,923 OTUs), Ascomycota (48.7%) and Basidiomycota (41.8%) were the most OTU-rich plyla. In our study, the numbers of OTUs found for Ascomycota (2088) and Basidiomycota (681) represent 68.5% and 22.3% of the total, respectively.
The unidentified ITS sequences not assigned to any phylum or class of fungi represent 1.61% of all ITS sequences sampled, 2.36% of all OTUs calculated and correspond to 38 class-level lineages. In the global soil fungi study of Tedersoo et al. [11], ~ 6% of all fungal OTUs could not be assigned to any higher taxonomic level and represent 14 divergent class-fungal lineages. This comparison reinforces the status of Brazil as a megadiverse country, which is not well explored regarding its funga composition. Also, following the three major sources for unrecognized fungal diversity listed by Hawksworth and Lücking [8], we believe that all of them fit to Brazil, which includes biomes considered to be biodiversity hotspots and many understudied geographic areas, besides a considerable number of cryptic fungal species and ecologically cryptic fungi.
The most OTU-rich classes in this work are Sordariomycetes, Dothideomycetes, Eurotiomycetes, and Agaricomycetes (Fig. 4). According to the data from the Brazilian List [93], the classes occurring in Brazil with the highest number of species are Agaricomycetes (1977 spp.), Pucciniomycetes (750 spp.), Sordariomycetes (551 spp.), and Dothideomycetes (436 spp.). Among the classes sampled in this study, Agaricomycetes, Dothideomycetes, Lecanoromycetes, and Sordariomycetes are the most diverse classes regarding the global number of species [92]. It is interesting to note that Pucciniomycetes is one of the classes with more species listed in the Brazilian List, but the molecular diversity within the Brazilian samples of this class available in GenBank represents only nine OTUs; what could indicate that Pucciniomycetes in Brazil has been well studied based on morphology, but many species remain without molecular data available.
It is important to highlight that the numbers for Dothideomycetes and Eurotiomycetes should not be directly associated to species level because different species are usually grouped together within the same OTU in some taxa of these groups [24, 29, 30], mainly representatives of Hypocreales (e.g., Fusarium) and Trichocomaceae (e.g., Aspergillus and Penicillium). In our study, ITS sequences of Fusarium represent 12.5% of the total ITS sequences in Sordariomycetes, while Aspergillus and Penicillium represent 39.5% of the total ITS sequences in Eurotiomycetes.
Maia et al. [62] listed six orders of Ascomycota as the most diverse in Brazil (> 90 spp.): Xylariales, Asterinales, Lecanorales, Ostropales, Microthyriales, and Capnodiales. In our study Xylariales (163 OTUs) and Capnodiales (164 OTUs) are the most diverse based on the number of OTUs calculated here. Capnodiales is also considered the most diverse order regarding the global number of species, with ca. 7244 spp. [92].
For Basidiomycota, Maia et al. [62] listed Agaricales, Puccinales, Polyporales, Hymenochaetales, Russulales, and Boletales as the most diverse orders. In our study, Agaricales (202 OTUs), Polyporales (116 OTUs), and Hymenochaetales (54 OTUs) are among the most OTU-rich orders, together with Tremellales (66 OTUs) and Cantharellales (62 OTUs). Considering the orders of Basidiomycota recovered in this study, Kirk et al. [92] pointed out Agaricales (13,233 spp.), Pucciniales (7798 spp.), and Polyporales (1801) as the most diverse orders in the world.
For the richest genera of fungi occurring in Brazil, Maia et al. [62] listed a total of 11 genera of Ascomycota, seven of Basidiomycota, and one of Mucoromycota; all of them have at least 37 species recorded. Considering the genera with the highest number of ITS sequences recovered in this study and that also are the most OTU-rich ones (Table 3), only Pluteus (Basidiomycota) is among the list of genera of Maia et al. [62]. Based on this, Pluteus is one of the genera of fungi occurring in Brazil that has been well studied in terms of taxonomy, using both morphological and molecular data [143–161].
In Basidiomycota, we also highlight the progress on molecular studies involving the genus Cora, a group of macrolichens that has shown surprising diversity after the use of the ITS sequences in systematic studies, with an increment of almost 180 species newly described, including many occurring in Brazil [12, 92, 162].
The genus Xylaria, although has not been considered in our list for the most well-sampled genera (Table 3), was previously pointed out by Maia et al. [62] to be among the most species-rich genera occurring in Brazil and we found a total of 52 OTUs. The ITS sequences of Xylaria from Brazil, 95 in total, are mostly represented by unidentified samples and as part of general studies for exploration of fungal diversity, including few published works [20, 163–166]. In addition, we acknowledge that the intraspecific ITS variability in the genus can be up to 24.2% [141]. On the other hand, Trichoderma, a genus with known low ITS interspecific variability [167, 168], is among the richest genera occurring in Brazil (Table 3), but for which there are only four species reported in the Brazilian List [169].
For some of the other most OTU-rich genera presented here (Table 3), including Aspergillus, Candida, Colletotrichum, Diaporthe, Fusarium, Phyllosticta, Penicillium, and Rhizoctonia, we recognize that there may not be any correlation between the number of OTUs found in this study and the number of species. Many authors have shown that the ITS interspecific variability in these groups is very high and sometimes its use may yield unreliable species diagnoses or that other markers have better taxonomic resolution at the species level [25–27, 29, 30, 141, 170–176].
Conclusions
Based on the combined data of this work and the previous data from the Brazilian List, we found a total of 8 phyla, 13 subphyla, 28 classes, and 113 orders of fungi occurring in Brazil. Entorrhizomycota remains unrecorded in Brazil. Ascomycota and Basidiomycota are the most diverse phyla, respectively. The discrepancy between the number of OTUs found in this study and the number of species recorded for these phyla in previous morphological and bibliographical studies suggests that in Brazil, the Basidiomycota has been well studied within a taxonomic framework using morphologica data, while Ascomycota has been better investigated using molecular data and mainly for taxa of clinical and agronomic importance, which usually have not been listed in the Brazilian fungal inventories.
We highlight groups that are potentially better studied in the country, such as Sordariomycetes, Dothideomycetes, Eurotiomycetes, and Agaricomycetes; groups that are in need of studies to incorporate molecular data in order to have more accurate estimates of their diversity and occurrence in the country, such as some orders in Dothideomycetes, Agaricomycetes, Exobasidiomycetes, Ustilaginomycetes, Chytridiomycetes, Monoblepharidomycetes, Kickxellomycotina, Zoopagomycotina; and groups that knownly occur in Brazil but are not included in the Brazilian List, such as phytopathogens, endophytes, saprobes, fungi of clinical importance for human and other animals, and yeast-forming fungi.
Finally, considering Brazil as a megadiverse country and poorly explored regarding its funga composition, we emphasize the need of joint efforts of mycologists to update the inventories and to reach unexplored areas and understudied habitats, besides the urgent need of new biodiversity conservation strategies to avoid losing species that have not even been described.
Acknowledgments
The authors thank Dr. Genevieve Gates (Tasmanian Institute of Agriculture) for the language and pre-submission review.
Funding information
N. Menolli Jr. thanks to Coordenação de Aperfeiçoamento de Pessoal de Niv́ el Superior – Brazil (Capes), and Fundação de Amparo à Pesquisa do Estado de São Paulo (Fapesp) (grants 04/04319-2, 09/53272-2, 2018/15677-0.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
Footnotes
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Nelson Menolli Jr and Marisol Sánchez-García contributed equally to this work.
Change history
6/9/2020
Due to a processing error, there was a mistake in Table 3. The first entry in the right column should read 109. The corrected table is given below.
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