Mahabalella pernambucana, a new hyphomycete from Brazil with notes on its phylogenetic positioning

ABSTRACT A new species of Mahabalella B. Sutton & S. D. Patil was isolated from decaying leaves of an unidentified plant, during a survey in the Charles Darwin Ecological Refuge in Igarassu, Pernambuco, Brazil, in June 2018. The new species is morphologically distinguishable from the other Mahabalella species by the size of their conidia, conidiogenous cells and setae. In this study, the morphological descriptions, illustrations and cultural characteristics for Mahabalella pernambucana are provided, as well as a key to species of this genus. Total DNA from the isolate was extracted, and four genes were partially sequenced (nLSU, ITS cluster, TEF1 alpha, and beta tubulin). Based on the nLSU analysis, the phylogenetic position of the new species was determined within the Sordariomycetes, Ascomycota, as a sister genus to Neotracylla, in the Tracyllaceae family.


Study area, sampling and morphological analyses
Sampling of leaf litter was carried out at the Ecological Refuge Charles Darwin, a rain forest fragment located in the municipality of Igarassu, north coast of the state of Pernambuco, Brazil.The site is within an area of about 60 ha, crossed by the Jacoca River (also called Tabatinga River), which is perennial and belongs to the main drainage basin of the municipality.Individual collections were placed in plastic bags, taken to the laboratory, and treated according to Castañeda-Ruiz et al. (2016).Mounts were prepared in polyvinyl alcohol-glycerol (8 g PVA in 100 ml water, plus 5 ml glycerol) and lactofuchsin (0.1 g acid fuchsin, 100 ml 85% lactic acid) following Carmichael (1955) or in lactic acid (90%).Measurements were taken at a magnification of ×1000 under an Olympus CX41 microscope with phase contrast, and photomicrographs were taken using a Nikon Eclipse Ni-U microscope with DIC optics and a Nikon DS-Fi2 camera.Fungal reproductive structures were isolated in water-agar, pH 6.3 with chloramphenicol, and after 2-5 days the colonies were transferred to V8 agar, pH 6.3 for DNA extraction.The holotype was deposited in the Herbarium URM and a culture voucher in the Micoteca URM of the Universidade Federal de Pernambuco, Recife, Brazil.

DNA extraction, PCR amplification and sequencing
The mycelia collected from culture on V8 media (pH 6.3) were used for total DNA extraction performed using the method of Góes-Neto et al. (2005).DNA was precipitated with isopropanol, washed with 70% ethanol, dried at 37 °C and re-suspended in ultrapure water.DNA concentration was estimated by electrophoresis in 1% agarose gel, at 3 V cm-1 in 1X Tris-Acetic acid-EDTA (TAE), by comparison with the 1 Kb plus DNA size marker (Thermo Fisher Scientific).
Amplification reactions of the fungal ITS, nLSU rDNA, beta-Tubulin and Elongation Factor 1-alpha were carried out in a final volume of 25 µl using the Top Taq Master Mix kit (Qiagen) and the primers ITS4-ITS5 (White et al. 1990), LR0R-LR5 (Vilgalys & Hester 1990), BT2A-BT2B (Glass & Donaldson 1995) and TEF-1 alpha EF-1 -TEF-1 alpha EF-2 (O'Donnell et al. 1998), respectively, at 0.40 µM each, and 25 ng of DNA template.All amplification had an initial denaturation at 94 °C for 5 min and final extension at 72 °C for 7 min, and the cycling program consisted of 94 °C for 30 s, 53 °C for 30 s, 72 °C for 1 min (37 cycles) for ITS, 94 °C for 30 s, 48 °C for 30 s, 72 °C for 1 min (35 cycles) for nLSU, 94 °C for 30 s, 58 °C for 30 s, 72 °C for 1 min 30 s (37 cycles) for beta-tubulin and 95 °C for 30 s, 62 °C for 30 s, 72 °C for 1 min (35 cycles) for TEF1alpha.The product of the amplification was visualized in agarose gel (1%).Purification was carried out with the GeneJet PCR purification kit (Thermo Scientific) according to the manufacturer instructions and the products were sent for sequencing in the DNA Sequencing Platform of the Universidade Federal de Pernambuco, in an ABI-310 Capillary Sequencer (PerkinElmer, Wellesley Massachusetts, USA).Primers ITS4/ITS5, LR0R/LR5, BT2A/BT2B and TEF-1αEF-1/TEF-1αEF-2 were used for sequencing the respective genes.The sequences were deposited in the GenBank (Benson et al. 2013).

Phylogenetic analysis for taxonomical positioning
The sequences obtained in this study were used to BLAST for sequences of the closest related taxa in the GenBank.The best matches for the new sequences were retrieved from GenBank, along with relevant sequences, including Saccharata proteae (GenBank EU552145) that was used as the outgroup based on Crous et al. (2018) and added to the final matrix.The final nLSU dataset was aligned with Clustal X (Thompson et al. 1997) and manually adjusted as necessary with BioEdit (Hall 1999).The best nucleotide substitutions model for each gene was estimated using the AIC (Akaike Information Criterion).Two phylogenetic routines were used to reconstruct the relationships' hypothesis, using the dataset: Maximum Likelihood (ML) and Bayesian Inference (BI).ML analysis was carried out using MEGA 7.0 (Kumar et al. 2016), applying the GTR+GAMMA+I model, with all parameters estimated by the software.The best-scored likelihood tree from all searches was kept.To access the reliability of the nodes, multiparametric bootstrapping replicates (1000) under the same model were computed.The bootstrap values were then annotated to the best likelihood tree found.
BI was performed using Mr. Bayes as implemented in the Topali v2.5 (Milne et al. 2004).The analysis was implemented by two MCMC independent runs, each one starting from random trees and with four simultaneous independent chains, performing 10 million generations, keeping one tree every 1000th generation.Five rate categories were used to approximate the gamma distribution, and the nucleotide substitutions rates were fixed to the estimated values.The first 25% of the sampled trees was discarded as burn-in, while the remaining ones were used to reconstruct a 50% majority-rule consensus tree and to estimate Bayesian Posterior Probabilities (BPP) of the branches.A node was considered to be strongly supported if it showed a BPP ≥ 0.95 and/or BS ≥ 90%, while moderate support was considered when BPP ≥ 0.9 and/or BS ≥ 70%.
Cultural characters on V8 agar -After 15 days at room temperature, in the dark, the colonies developed dark aerial mycelium, yellowish brown, velvety, flat and sparse, entire margin; reverse dark brown.In the culture collection, the specimens are preserved in PDA under mineral oil and using Castellani's method.
Distribution and habitat: Mahabalella spp.are known from decomposing leaf litter in Austria (temperate forest), Cuba (tropical forest), and Japan (subtropical forest), and it was now found in the Northeast of Brazil (tropical forest).The Ecological Refuge Charles Darwin is a private property of about 60 ha, crossed by the river Jacoca.This perennial watercourse is part of the main basin in the municipality, the river Botafogo catchment.This wildlife refuge is used as shelter for animals rescued by IBAMA and presents a main unpaved road for visiting and service cars, and accessory trails destined to visitors hiking to denser forest (Santiago & Barros 2003).The vegetation is characteristic of Pernambuco's Atlantic Forest zone, with coastal hygrophilous broadleaf evergreen forest formation composed of an arboreal stratum varying from 8 to 15 meters in height and a shrubby herbaceous stratum.The climate is Am-type with transition to As-type (Andrade-Lima 1960).The average temperature in the region between 2018 and 2019 ranged from 20.5 ºC (Jul/2018) to 24.2 ºC (May/2019).And the average monthly rainfall ranged from 86.8 mm (Jul/2018) to 62.5 mm (May/2019).
Conservation status: The distribution and size population of M. pernambucana are still unknown to adequately assess its conservation status under the IUCN (2012) categories and criteria.Thus, M. pernambucana is here categorized as Data Deficient (DD), until more information from the species becomes available.However, the wildlife refuge where the species was recorded is immersed in the metropolitan area matrix and suffers for plausible threats (e.g.air pollution, high population density, sugarcane plantation) that may compromise the maintenance of species.
Etymology: Latin, pernambucana, based on the name of the Pernambuco State of Brazil, where the fungus was found.
The closest hit for the beta-tubulin gene sequence is a species in the Eurotiomycetes (Tab.1).Talaromyces siamensis is considered nomen invalidum, according to the Art.40.1 of the Nomenclature Code, Melbourne (Index Fungorum 2022), however, other sequences from species of the same genus, like T. cnidii and T. aculeatus are also good matches, being these in the Aspergillaceae, Eurotiales.

Discussion
The main morphological characteristics of Mahabalella pernambucana are aligned with those of the genus, like the absence of conidiophores, straight non-septate setae, phialidic conidiogenous cells and conidia straight with appendages at each end.
Of the four species that have been previously reported in this genus, Mahabalella acutisetosa B. Sutton & S.D. Patil., M. cubensis R.F.Castañeda, and M. dimorpha Matsush.are currently accepted while M. stiriaca Melnik, Scheuer & Heftb., although the description matches the genus, has not been validly published (Nom. inval., Art. 40.6, Shenzhen, Turland et al. 2018;Index Fungorum 2022).M. dimorpha, however, presents some morphological features that support its exclusion from the genus, differing from the other species by having narrow cylindrical, sometimes branched, conidiogenous cells and septate setae (Mel'nik et al. 2003).Mel'nik et al. (2003) also comment on the conidiogenous cells that can also be produced in short conidiophores, which suggest this species to be non-congeneric.Our species differs from Mahabalella acutisetosa by its smaller conidia, conidiogenous cells and setae.
Mahabalella pernambucana also differs from closed related genus Minimidochium B. Sutton by the shape of the sporodochia, the shape of the conidiogenous cells and shape of conidia.
The best inferred phylogenetic position for M. pernambucana to date is with the Tracyllaceae, however, more DNA sequencing of related species are necessary to better support these findings.
The recently erected Order Tracyllales presents to date one family and two genera (Crous et al. 2018;2019) that remained resolved in the phylogenetic tree when the new Mahabalella sequence was included in the analysis (Fig. 2) within Sordariomycetes.The closest sequence to the TEF 1-α gene is also a species of the Sordariomycetes (Myxospora crassiseta, Stachybotryaceae, Hypocreales).With our choice of primers it was possible to sequence completely the second and third exons of the TEF 1-α gene, considering the sequence of nucleotides and peptide presented by Nakazato (2005) for Metarhizium anisopliae (GenBank AY445082).The regions identified as introns in the sequenced fragment show some variability.However, the conserved domains of the putative peptide (Fig. 3) that was possible to derive from the sequence with confidence using ExPASy (Artimo et al. 2012), aligned perfectly with Metarhizium anisopliae, Trichoderma virens and Dactylonectria novozelandica (GenBank AY445082, KU301729 and MK602815, respectively), indicating high conservation over these two different orders.

Figure 2 .
Figure 2. Phylogenetic analysis of Mahabalella pernambucana sp.nov.and related fungi.The tree is a maximum likelihood cladogram constructed with the GTR model plus gamma distribution and invariant sites factors and is based on an alignment of 811 bases of the LSU ribosomal region.Numbers closest to the branches indicate % bootstrap recovery/Bayesian posterior probabilities.

Table 1 .
Some of the most similar sequences recovered from BLAST search for each of the four genes sequenced for Mahabalella pernambucana sp.nov.