Two New Species of Backusella (Mucorales, Mucoromycota) from Soil in an Upland Forest in Northeastern Brazil with an Identification Key of Backusella from the Americas

During a survey of Mucorales from a forest located in Pernambuco state, Brazil, two new Backusella species were discovered and described based on morphological and molecular data (internal transcribed spacer and large subunit ribosomal DNA sequences). Both species were characterized as unbranched sporangiophores and sporangia with columellae of varied shapes forming. Multispored sporangiola were frequent, whereas unispored sporangiola were rare. URM 8395 forms sporangiophores that may support hyaline, slightly curved or circinate pedicels with multispored sporangiola at their apical portion, and abundant giant cells and chlamydospores. Columellae of sporangia are hyaline, conical (majority), or ellipsoidal with a truncate base, globose to subglobose or subglobose to conical, and, rarely, with slight medial constriction. URM 8427 does not form sporangiola from pedicels, giant cells are not observed, and columellae of sporangia are globose to subglobose, cylindrical with a truncate base, some with a slight constriction, applanate, obovoid, ellipsoidal, or, rarely, conical. Some columellae may have one side more swollen than the other and some are arranged obliquely on the sporangiophores. Sterile sporangia may or may not be formed on short sporophores. The detailed description and illustration of both novel species as well as an identification key for Backusella from the Americas are provided.


Introduction
Backusella Hesseltine & J.J. Ellis belongs to the Backusellaceae family (established by K. Voigt and PM Kirk) and comprises fungi characterized by the production of sporangiophores that are transiently curved when young and erect when mature. These fungi possess non-apophysate and multisporate, deliquescent-walled sporangia [1], and most species form uni-and/or multispored sporangiola with a persistent wall [2]. Backusella species are saprobic in nature and are found in soil, litter, plant debris, herbivore dung, decaying wood, Fragaria, diseased roots, and Medicago sativa [3,4].
Backusella has been described as the member of the Mucoraceae family [5], with B. circina J.J. Ellis & Hesselt. as the type species of this genus. Subsequently, species of this genus were transferred to Thamnidiaceae Fitzp. [6], owing to their morphological similarity with Thamnidium Link [3]. However, Benny and Benjamin [7]  that produced sporangia and sporangiola into this genus [7].
During a study on the diversity of Mucorales in an upland forest located in the municipalities of Arcoverde and Águas Belas, Pernambuco, Brazil, two species of Backusella that varied morphologically and genetically (via ITS and LSU rDNA analysis) from other species were isolated. The aim of this study was to describe and illustrate these new species. We also provide a key to the Backusella species from the Americas.

Sampling Site
Soil samples were collected from an upland forest in the city of Arcoverde (at 1000 m altitude), district of Mimoso (08 • 39 688 S 036 • 90 013 W), and at Serra do Comunaty (09 • 05 441 S 037 • 05 438 W), in the municipality of Águas Belas (at 900 m altitude), both of which are located in the state of Pernambuco, Brazil. Mimoso has a tropical rainy climate with dry summers, an average temperature ranging between 12 • C and 25 • C, and an average annual rainfall of 653 mm [13,14]. The vegetation consists of deciduous and semideciduous forests. The municipality of Águas Belas also has a tropical rainy climate with dry summers and an average annual rainfall of 373 mm. The temperature ranges between 18 and 34.5 • C [15]. The vegetation is composed of semideciduous and deciduous forests [16].

Isolation, Purification, and Identification
Five milligrams of soil were added to wheat germ agar medium [17] containing chloramphenicol (200 mg·L −1 ) in Petri dishes in triplicate. Colony growth was monitored for 7 days at room temperature (28 ± 2 • C). To purify Backusella spp., mycelial fragments taken from the edge of the growing colony were transferred to malt extract agar (MEA) medium [17]. The species were identified by observing their macroscopic (appearance, color, and diameter of colonies) and microscopic (microstructures) characteristics, according to the descriptions of Benny and Benjamin (1975)

Experiments
Pure cultures were grown in triplicate in both MEA and potato dextrose agar (PDA) media [17] and incubated at 10,15,20,25,30,35, and 40 • C for 15 days. For morphological identification, fragments of colonies were obtained from the plates for examination of fungal structures. These fragments were placed together with a drop of potassium hydroxide (KOH, 3%) or lactophenol blue on microscope slides and observed under a light microscope (Carl Zeiss Axioscope 40, Göttingen, Germany). Photomicrographs were captured using the Leica DM 500 microscope combined with the Leica ICC50 camera (Leica, Heerbrugg, Switzerland) and Leica Application Suite software (v. 3.4). The color designation of the colonies was performed according to the terminology of Kornerup and Wanscher [18].

DNA Extraction, Amplification, Cloning, and Sequencing
Fungal biomass was extracted from MEA cultures in test tubes incubated at 28 • C for up to 5 days. The material was transferred to 2 mL microtubes with screw caps. To each tube, 0.5 g of acid-washed glass beads of two different diameters (150-212 µm and 425-600 µm, 1:1; Sigma, St. Louis, MO, USA) were added. The material was crushed by stirring at high speed in a FastPrep homogenizer (FastPrep-24; MP Biomedicals, Irvine, CA, USA). The genomic DNA extraction procedure was conducted as described by de Oliveira et al. [19]. Briefly, the mycelium was homogenized in cetyltrimethylammonium bromide (C TAB) lysis buffer (2% CTAB, 20 mM ethylenediaminetetraacetic acid (EDTA), 0.1 M Tris-HCl, pH 8.0, 1.4 M NaCl) [20,21], followed by washing with chloroform:isoamyl alcohol (24:1) to subsequently separate the DNA-containing supernatant 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 of ultrapure water.
For amplification of the ITS and LSU rDNA, we used the primer pairs ITS1/ITS4 and LR1/LSU2 [22][23][24], respectively. Thermal cycling parameters were as follows: 5 min at 95 • C (one cycle), 45 s at 94 • C, 1 min at 60 • C, 1 min at 72 • C (39 cycles), and a final elongation of 7 min at 72 • C. The final amplicons were purified with the enzymatic mix NucleoSAP (Cellco Biotech of Brazil, São Carlos, Brazil) and used for sequencing at the Genomic Technology and Gene Expression Platform of the Center for Biological Sciences of the Federal University of Pernambuco-UFPE (Pernambuco, Brazil). Direct sequencing of the ITS region from PCR products of strain URM 8427 failed. PCR products were cloned using the pGEM-T Easy Vector (Promega, Madison, WI, USA), following the manufacturer's instructions. These clones were sequenced using the primers M13F forward (5 -GTAAAACGACGGCCAGT-3 ) and M13R reverse (5 -GCGGATAACAATTTCACACAGG-3 ).
Sequence data were compared with similar sequences available in the National Center for Biotechnology Information GenBank database using Nucleotide BLAST (BLASTn). The newly obtained sequences were deposited in the GenBank databases (Table 1).

Phylogenetic Analyses
The phylogenetic relationship of the new Backusella species and related species was determined by analysis of concatenated sequence datasets of two loci (ITS and LSU). Sequences were aligned via multiple alignment using Fast Fourier Transform (MAFFT) v.7 (https://mafft.cbrc.jp/alignment/server, accessed on 21 July 2022) [25,26] and manually improved in Molecular Evolutionary Genetics Analysis (MEGA) v.7 [27]. The analysis of the concatenated sequence datasets of the two loci was performed in MEGA v.7. Maximum likelihood (ML) analysis was performed in Randomized Axelerated Maximum Likelihood (RAxML)-HPC v. 8.2.8 Black Box [28] using the general time-reversible (GTR)+G+I model test with 1000 bootstrap (BS) replicates on the CIPRES Science Gateway Portal (https://www.phylo.org/portal2/, accessed on 21 July 2022) [29]. Bayesian inference (BI) was performed using MrBayes v. 3.2.2 [30] on The Extreme Science and Engineering Discovery Environment (XSEDE) through CIPRES using the GTR+I+G model as selected by jModelTest v.2.1.10 under the Akaike information criterion for BI [31,32]. BI analysis was conducted with 1 × 10 6 generations with a burning value of 25%. Phylogenetic trees were viewed and arranged using Interactive Tree of Life v4 (https://itol.embl.de, accessed on 21 July 2022) [33]. Values less than 0.90 BI posterior probability (BPP) and 70% ML bootstrap (MLBS) are not shown. Sequence data were compared with those of similar sequences available in the GenBank database using BLASTn.

Phylogenetic Analyses
Phylogenetic analyses of the combined ITS and LSU sequence data identified 53 taxa, including the newly generated sequences and outgroup (   . Columellae of sporangia of varied shapes, some arranged obliquely on the sporangiophores, some with one side more swollen than the other; sporangiospores globose to subglobose B. obliqua Columellae of sporangia conical and cylindrical, sometimes constricted at the center, never arranged obliquely on the sporangiophores nor with one side more swollen than the other; sporangiospores subglobose to ellipsoidal, some slightly irregular
Habitat Media and temperature tests: On PDA, at 10 • C, slow growth (2 cm in 120 h) with poor sporulation; at 15 • C, better growth (5 cm in 120 h) than that at 10 • C with good sporulation; at 20 • C, good growth (7 cm in 120 h) with good sporulation; at 25 • C, better growth (9 cm in 96 h) with good sporulation; at 30 • C, good growth (9 cm in 144 h) and good sporulation; at 35 • C, slow growth (3 cm in 120 h) with lack of sporulation; and at 40 • C, lack of growth. A similar growth pattern was observed on MEA and PDA at all temperatures.

Discussion
Backusella brasiliensis (URM 8395) and B. obliqua (URM 8427) were described in this study based on their morphological and molecular characteristics that make them different from the other closely related species; therefore, they were noted as new species. Phylogenetically, B. brasiliensis formed a clade distant from other Backusella species but closer to B. dispersa and to a clade containing URM 8427, B. azygospora, B. mclennaniae, B. liffmaniae, B. psychrophila, B. lamprospora and Backusella as 'group X' (Figure 1). However, these species are morphologically different. Backusella brasiliensis is differentiated from B. azygospora as it does not produce azygospores, which are common in the later species. Besides, chlamydospores and sterile branches in long sporangiophores are not observed in B. azygospora [11]. Backusella dispersa forms smaller sporangia (30.2-47.5 × 28.3-46.1 µm) and larger columellae (19.5-38.7 × 18.5-33.4 µm) than those observed for the new species. In addition, sporangiospores of B. dispersa are globose to broadly ellipsoidal (8-12 × 7-10 µm), and are different from those of the new species, which are globose to subglobose (majority), 4-12 (-17) µm diameter or irregular, 20-30 × 12-17 µm. Although no chlamydospores are formed in B. dispersa [8], they are common in URM 8395. The new species is easily differentiated from B. mclennaniae, B. liffmaniae and B. psychrophila by forming giant cells and chlamydospores. Yet, B. brasiliensis forms columellae that are conical (majority), ellipsoidal with truncate base, globose to subglobose or subglobose to conical, and, rarely, with a slight constriction at center, whereas columellae of B. mclennaniae and B. liffmaniae and B. psychrophila are globose, ellipsoidal or applanate.
Backusella brasiliensis and B. lamprospora present morphological similarities, such as the following: the main axes of their sporangiophores support hyaline, slightly curved or circinate pedicels with multispored sporangiola at their apical portion; short and simple or sympodially branched sporophores bearing only multispored and/or unispored sporangiola (rare) are formed near the substrate [7]. However, the main sporangiophore axes of B. lamprospora are simple or sympodially branched, differing from those of the new species that are unbranched or once-branched. Backusella lamprospora is characterized by forming sporangiophores with hemispherical to globose or ovoid columellae, differing from those found in B. brasiliensis, which are conical, ellipsoidal with a truncate base, globose to subglobose, subglobose to conical, conical, or cylindrical, including some with a slight constriction at the center. Furthermore, the sporangiospores of B. brasiliensis are globose, subglobose, and irregular, whereas those of B. lamprospora are only subglobose [7].
The formation of giant cells and chlamydospores is not common in Backusella species, and as far as we know, in addition to the new species, giant cells have been cited only in B. gigacellularis and B. oblongielliptica, whereas chlamydospores have only been observed in B. chlamydospora. Backusella gigacellularis does not produce unispored sporangiola, and its sporangia are larger (40-131 µm diameter) than those of B. brasiliensis. Moreover, sporangiospores of the new species are globose, subglobose, and irregular, whereas those of B. gigacellularis are ellipsoidal and, rarely, irregular [9]. Backusella oblongielliptica can be easily differentiated from B. brasiliensis by the lack of chlamydospore and sporangiola formation and the production of oblongly ellipsoidal sporangiospores [2,34]. Backusella chlamydospora differs from B. brasiliensis as it produces abundant unispored sporangiola, larger sporangia (35-80 × 35-75 µm), and subglobose, conical, ellipsoidal, cylindrical, hemispherical, or nearly pyriform columellae, which are sometimes bell-shaped, long conical, or constricted at the center [4]. In contrast, B. brasiliensis rarely produces unispored sporangiola and does not form cylindrical or pyriform columellae.
Phylogenetically, B. obliqua (URM 8427) is close to the Backusella species of 'group X', which is a group of a putative new species not yet proposed [8], but also close to mboxemphB. lamprospora and B. psychrophila. Morphologically, Backusella 'group X' forms sporangiophores up to 8 µm diameter, unlike B. obliqua, which forms sporangiophores up to 12 µm diameter. The columellae produced by B. obliqua are globose to subglobose, subglobose to irregular, cylindrical with a truncate base (some with slight constriction), flattened, obovoid, ovoid, ellipsoidal, or, rarely, conical, some arranged obliquely on the sporangiophores, and some with one side more swollen than the other, while Backusella 'group X' forms only globose, ellipsoidal, or applanate columellae. The sporangiospores of B. obliqua are globose to subglobose, unlike that of Backusella 'group X', which forms globose to broadly ellipsoidal sporangiospores. Backusella obliqua can be differentiated from B. lamprospora by forming sporangiophores without lateral pedicellate sporangiola. Columellae of B. lamprospora are hemispherical to globose or ovoid, differing from those of B. obliqua [7]. This new species forms sporangiophores that are simple or sympodially branched up to three times, whereas sporangiophores of B. psychrophila are occasionally branched. In addition, sporangiospores of B. obliqua are globose to subglobose, whereas those of B. psychrophila are broadly ellipsoidal to ellipsoidal [8].
In conclusion, B. obliqua and B. brasiliensis are morphologically and genetically different from other Backusella species, justifying their recognition as novel species. Brazil is a tropical country with areas considered global biodiversity hotspots that support high fungal diversity [12, [35][36][37]. Voigt et al. [38] showed that of the 74 Mucoromycota species newly described between 2015 and 2020, 54 (74%) were isolated from Brazil, South Korea, Australia, and China, with 17 new species described in Brazil, confirming the great potential for the discovery of new taxa of Mucoromycota in this country. Regarding Backusella, of the 26 accepted species, only six (B. azygospora, B. brasiliensis, B. constricta, B. gigacellularis, B. lamprospora, and B. variabilis) were isolated from Brazil, including from areas of upland forest [10][11][12]39]. Considering that a high diversity of fungi is expected in tropical hotspots, we believe that this number of known Backusella species in Brazil is underestimated and it could increase with more investments in research and further training of Brazilian taxonomists studying Mucoromycota.