Discovery of Backusella paraconstricta sp. nov. (Mucorales, Mucoromycota) in an upland forest in northeastern Brazil with an identification key for Backusella from the Americas

Abstract During a survey of mucoralean fungi in soil from an upland forest area located in Pernambuco, Brazil, a strain of Backusella (URM 8637) was isolated. Based on morphological, physiological, and molecular data [internal transcribed spacer (ITS) and large subunit (LSU) ribosomal DNA regions], it was recognized that this Backusella differed from all other species in the genus. Morphologically, the new species is characterized as forming varied-shaped columellae, including elongated, basally constricted, unisporate (rare) and multisporate sporangiola, and ellipsoidal sporangiospores. The maximum temperature growth of URM 8637 on malt extract agar and potato dextrose agar was 36 °C. In the phylogram, it was closely related to B. constricta. Based on the evidence from the analyzed datasets, a new species of Backusella is proposed. An updated identification key for Backusella from the Americas is provided.


Introduction
The genus Backusella was established by Ellis and Hesseltine in 1969 and typified with B. circina J.J. Ellis and Hesselt.Members of this genus can be found on various substrates, including excrement, invertebrates, leaf litter, soil, toads, and wood (Benny & Benjamin 1975;Walther et al. 2013;Lima et al. 2016;Nguyen & Lee 2018;Nguyen et al. 2021;Urquhart et al. 2021).Backusella belongs to Backusellaceae K. Voigt & P. M. Kirk, however it was previously associated with Mucoraceae Dumort.
Species of this genus were later transferred to Thamnidiaceae Fitzp.due to morphological similarities with Thamnidium Link (Pidoplichko & Milko 1971;Wanasinghe et al. 2018).Species of Backusella are known to occur in Australia, Brazil, China, Japan, South Korea, the United States of America, and Thailand (Zheng et al. 2013;Lima et al. 2016;Nguyen et al. 2021;Urquhart et al. 2021;Hurdeal et al. 2022;de Lima et al. 2022).
Species of Backusella form sporophores that are transiently curved when young, and erect when mature.They may or not form laterally unispored and multispored pedicellate sporangiola, which have persistent walls.Short, simple, or sympodially branched sporangiophores proliferating only multispored and/or unispored sporangiola may form near the substrate (Benny 2005;Walther et al. 2013;de Souza et al. 2014).Based on the recognition of transiently curved sporophores as a distinctive feature of Backusella, Walther et al. (2013) transferred some species from Mucor to Backusella.As of January 2023, 34 species have been accepted in Backusella (Wijayawardene et al. 2022;Hurdeal et al. 2022;de Lima et al. 2022;Cordeiro et al. 2023).
During a study on the diversity of mucoralean fungi in an upland forest area in Pernambuco, northeastern Brazil, a strain of Backusella was found.Its identity was confirmed using morphological and molecular data, which included internal transcribed spacer (ITS) and large subunit (LSU) of ribosomal DNA (rDNA).In this work we describe and illustrate this new species.This is not the first new species of Backusella discovered in Brazil; Backusella azygospora, B. brasiliensis, B. constricta, B. obliqua, and B. pernambucensis were previously isolated and described for the first time in this country.This indicates that Brazil is a tropical hotspot for discovering new mucoralean fungi (de Lima et al. 2022).In this study, we updated the identification key of Backusella from the Americas provided by de Lima et al. (2022) with two additional species.

Collection site
Soil samples were collected in April 2022 in the district of Jenipapo, municipality of Sanharó (8°17'08.6"S 36°30'53.9"W), located in the state of Pernambuco, Brazil.The local vegetation comprises of subdeciduous and deciduous forests.The climate is tropical and rainy with dry summers.The rainy season starts in January/February and ends in September, but it can continue until October.The average annual temperature is 31 °C, with an average annual rainfall of 496 mm (MME 2005).Using sterilized spatulas, soil samples were collected at a depth of 5 cm, packed in sterile plastic bags and stored in styrofoam boxes with ice for transport to the Laboratory of Fungos Zigospóricos at the Universidade Federal de Pernambuco (UFPE).

Isolation, purification and morphological description
Five milligrams of soil was added to wheat germ agar culture medium (Benny 2008), supplemented with chloramphenicol (80 mg L -1 ), contained in Petri plates.Colony growth was monitored for 72 h at room temperature (26 ± 2 °C).Mycelial fragments were removed directly from the Petri dishes under a Leica EZ4 stereomicroscope (Leica Microsystems, Wetzlar, Germany) and transferred to malt extract agar (MEA) plates (Benny 2008).At least 50 measurements were made for each fungal structure from plates incubated at 25 °C for seven days on MEA in the dark.Mycelial fragments from the specimens were transferred to slides with 2% KOH or lactophenol blue and observed using a light microscope (Leica DM500).A slide corresponding to the holotype of the new species (URM 8367) was deposited in the Herbarium URM, and the ex-type living culture of the new species (URM 8367) was deposited in the URM Culture Collection of the Universidade Federal de Pernambuco.

Growth experiments and macro and microscopy
URM 8637 was grown in triplicate on both MEA and potato dextrose agar (PDA; HiMedia, Vadhani, India) and incubated at 15, 20, 25, 30, 35, and 40 °C in the dark for morphological analysis.Colony growth was measured every 24 h and monitored for 10 d.The maximum growth temperature was determined by growing the strains on MEA at one degree increments.For morphological observation, culture slides (with fragments of the fungal mycelia) were prepared, stained with 2% KOH or lactophenol blue, and observed using a light microscope (Leica DM500).Colony color was determined according to Kornerup and Wanscher (1978).

DNA extraction, amplification, purification, and sequencing
Fungal biomass was obtained from MEA slant cultures incubated at 28 °C for up to five days and was transferred to 2-mL microtubes with screw caps.To each tube, 0.5 g of acid-washed glass beads (Sigma-Aldrich, Darmstadt, Germany) of two different diameters (150-212 μm and 425-600 μm, 1:1) were added and the fungal biomass was crushed by stirring at high speed in a FastPrep homogenizer (FastPrep-24, MP Biomedicals, California, USA).Genomic DNA was extracted as described by de Oliveira et al. (2016), whereby the mycelium was homogenized in CTAB lysis buffer [2% cetyltrimethylammonium bromide, 20 mM EDTA, 0.1 M Tris-HCl (pH 8.0), 1.4 M NaCl (Doyle & Doyle 1987;1990)], and washed with chloroform: isoamyl alcohol (24:1).The DNA-containing supernatant was then separated from the hyphal residues.The supernatant was mixed with an equal volume of isopropanol followed by DNA precipitation after incubation at -20 °C for 30 min.After centrifugation at 13,000 rpm for 15 min, the resulting DNA pellet was washed with 70% ethanol and resuspended in 50 μL ultrapure water.
For the amplification of ITS and LSU rDNA, the primer pairs ITS1/ITS4 and LR1/LSU2 (White et al. 1990;van Tuinen et al. 1998;Santiago et al. 2014) were used, respectively.The final amplicons were purified with the NucleoSAP enzymatic mix (Molecular Biotecnologia, Belo Horizonte, Brazil) and sequenced at Plataforma de Tecnologia Genômica e Expressão Gênica of the Centro de Ciências Biológicas -UFPE (Pernambuco, Brazil).

Sequence alignment and phylogenetic analysis
The sequences of the URM 8637 strain were used as queries to perform BLASTn in the GenBank database to identify the closest matching sequences.Raw reads were edited to remove ambiguous bases at both ends.Two separate datasets, one for ITS and one for LSU, were assembled using sequences of all available described species in the database.The datasets were aligned using MAFFT v.7 (https://mafft.cbrc.jp/alignment/server)(Katoh & Standley 2013) for each molecular marker.The sequences were manually edited using MEGA version 7 (Kumar et al. 2016).The ITS and LSU rDNA region alignments were concatenated before the phylogenetic analyses (Supplementary Material 01).Bayesian inference (BI) and maximum likelihood (ML) analyses were performed with MrBayes v.3.2.2 (Ronquist et al. 2012) on XSEDE and RAxML-HPC BlackBox v.8.2.8 (Stamatakis et al. 2008;Stamatakis 2014), respectively, using the CIPRES Science Gateway (http://www.phylo.org/)(Miller et al. 2010).The ML analysis was performed using the GTR+I+G standard nucleotide substitution model, and BI was performed using the best nucleotide model selected by AIC in MrModeltest 2.3 (Nylander 2004).Bayesian inference analysis was conducted using 1 × 10 6 generations with a tree burn-in value of 25%.Phylogenetic trees were viewed and arranged using the Interactive Tree of Life (iTOL) v4 (https://itol.embl.de/)(Letunic & Bork 2019).Values less than 0.95 BI posterior probability and 70% ML bootstrap were not considered.The newly obtained sequences were deposited in the GenBank database.GenBank accession numbers are listed in Tab. 1.
Etymology: referring to the phylogenetic proximity to Backusella constricta.
Habitat: Soil.Distribution: Pernambuco state (Brazil).Media and temperature test: On MEA, at 10 °C -no growth; at 15 °C -slow growth (6 cm in diameter after 168 h); at 20 °C -good growth (8 cm in diameter after 120 h); at 25 °C -excellent growth (9 cm in diameter after 96 h); at 30 °C -good growth (9 cm in diameter after 144 h); at 35 °C -slow growth (9 cm in diameter after 192 h); at 40 °C -no growth.Backusella paraconstricta exhibited similar growth and development of reproductive structures on MEA and PDA culture media.Maximum temperature growth on both MEA and PDA was 36 °C.

Discussion
In this study, we describe the novel species Backusella paraconstricta URM 8637.Our ML and BI phylogenetic analyses demonstrated that this species is genetically distinct from all other species of Backusella and sister to B. constricta in the tree inferred using a concatenated ITS/LSU dataset.The new species belongs to a major clade containing also B. variabilis, B. thermophila and B. indica. Cordeiro et al. (2023) proposed that the maximum growth temperature represents a valuable taxonomic feature in Backusella, and that temperature plays an important role in the evolution of this genus.They identified seven Backusella spp.capable of growing at temperatures ≥36 °C, four of which (B. constricta, B. variabilis, B. thermophila and B. indica.)were placed in the same clade in the inferred phylogenies.Herein, we present a new species that grows at 36 °C and also belongs to this clade (Fig. 1).This further confirms that maximum growth temperature is a taxonomically relevant character in Backusella.
Morphologically, B. paraconstricta differs from B. constricta by forming sporangia up to 70 μm in diameter, whereas those of the latter reach 100 μm in diameter.Backusella constricta forms columellae that are conical (majority) or cylindrical, and slightly or strongly constricted in the center (Lima et al. 2016).Backusella paraconstricta, instead, forms columellae that are mostly conical, subglobose, infrequently applanate, and very rarely elongate to ellipsoid, cylindrical and with a slight constriction at the base.The new species predominantly forms ellipsoidal sporangiospores, although some are subglobose, globose, and irregular, whereas B. constricta only forms sporangiospores that are subglobose to ellipsoidal and slightly irregular.Cordeiro et al. (2023) observed that most species of Backusella form subglobose to broadly ellipsoidal sporangiospores, and that ellipsoidal sporangiospores occur in Backusella species that are in the deeper branches of the ITS and RPB1 phylogenetic trees, namely B. indica, B. oblongielliptica, B. oblongispora, B. parvicylindrica, B. recurva, B. thermophila, and B. variabilis.This was also observed in our ITS/LSU phylogeny, which includes B. paraconstricta.Finally, B. paraconstricta can grow at temperatures up to 36 °C, whereas B. constricta can grow up to 39 °C.
In conclusion, our results demonstrate that B. paraconstricta is morphologically and genetically different from the other Backusella species described to date.Therefore, it was described as new.This study contributes to our knowledge of the distribution of mucoralen fungi.

Figure 1 .
Figure 1.Phylogenetic tree of Backusella inferred from the combined internal transcribed spacer (ITS) and large subunit (LSU) ribosomal DNA (rDNA) sequences.Support values on the branches represent maximum likelihood bootstrap support and Bayesian inference posterior probabilities in this order.Bootstrap values lower than 70% or 0.95 are marked with "*".New taxa are in bold font.Mucor indicus CBS 226.29 was used as outgroup.Ex-type, ex-epitype, and ex-lectotype strains are marked with T, ET, and LT, respectively.

Table 1 .
Specimens used in the phylogenetic analyses with their GenBank accession numbers.