Mucor minutus (Baijal & B.S. Mehrotra) Schipper: a rare mucoralean fungi isolated for the first time in northeastern Brazil

Mucor minutus (Baijal & B.S. Mehrotra) Schipper is described for the first time from northeastern Brazil. The specimen was isolated from the soil of Florestas do Jussará, an upland forest fragment in Pernambuco state. In Brazil, M. minutus has previously been isolated only from the state of São Paulo from the soil of the Atlantic Forest. The occurrence of M. minutus in Florestas do Jussará is presented and discussed, and a detailed description and illustration of the specimen are presented. This article contributes to the knowledge of mucoralean distribution.

Mucor minutus (Baijal & B.S. Mehrotra) Schipper may be morphologically differentiated from other species by concomitantly forming long, sympodially and monopodially branched sporangiophores, in addition to small, repeatedly sympodially branched sporangiophores, obovoid to ellipsoidal or infrequently pyriform columellae, as well as subspherical or slightly elipsoidal sporangiospores (Schipper 1975 Girlanda et al. 2001), and Vietnam (from soil, ITS sequence available in GenBank, EU076933: unpublished data) (Fig. 1A). In Brazil, M. minutus was previously isolated from an area of the Atlantic Forest, located in the municipality of Cubatão, state of São Paulo (Schoenlein-Crusius et al. 2006) (Fig. 1B) During a study on the diversity of Mucorales in an upland forest fragment in Pernambuco, northeastern Brazil, a specimen morphologically similar to M. minutus was isolated. The identity of the species was confirmed by sequencing the ITS region of rDNA. Here, we present and discuss the occurrence of M. minutus. We also provide the description and illustration this rare species isolated from soil samples in the semiarid region of Brazil.

Methods
Soil collection. Soil samples were collected in January 2018 from Florestas do Jussará, an upland forest area in the semiarid region of Brazil, located in the municipality of Gravatá (08°12′S, 035°32′W), 85.5 km from Recife (Fig. 1B). The climate is tropical with a dry summer, and an average annual temperature of 22 °C. The average annual rainfall is 725 mm, with rains concentrated between March and July (Siqueira-Filho and Machado 2001). Using sterilized spatulas, soil samples up to 5 cm deep were collected from 10 different points, respecting a minimal distance of 50 m between each on. Samples were placed in clean plastic bags and stored in polystyrene boxes with ice for transport to the laboratory of the Universidade Federal de Pernambuco (UFPE).

Isolation, purification, and identification.
A 5 mg sample of soil collected at each one of 10 sampling points was added to Petri dishes containing wheat germ agar culture medium (Benny 2008) plus chloramphenicol (NeoFenicol-Neo Química; 100 mg/L), in triplicate. The plates were stored on a laboratory bench at room temperature (26 ± 2 °C) for 7 days. To purify the specimens, fragments of mycelia were removed from the Petri dishes under a stereomicroscope (Leica EZ4) and transferred to Petri dishes with malt extract agar (MEA) (Benny 2008) plus chloramphenicol (80 mg/L). Fragments of mycelia were transferred to slides with 2% KOH or lactophenol blue and observed using light microscopy (Leica DM500). Each specimen was identified by comparing the macroscopic aspect (colony color and appearance) and microscopic structures (shape and size of the sporangiophores, columellae, sporangia, and sporangiospores), according to the description of Schipper (1975). Seventyfive measurements were made for each fungal structure from plates incubated at 25 °C for 7 days in the dark on MEA. A living culture (URM 7894) is deposited in the culture collection Micoteca URM of the Universidade Federal de Pernambuco.
DNA extraction, PCR, sequencing and phylogenetic analysis. Cultures grown in test tubes containing MEA were incubated at 28 °C for 6 days to obtain fungal biomass. After the material was transferred to 2 mL microtubes with screw caps, 0.5 g of acid-washed glass beads of two different diameters (150-212 μm and 425-600 μm, 1:1; Sigma, USA) were added to each tube. The material was crushed by stirring at high speed in a Fast-Prep homogenizer. Genomic DNA was extracted as described by de Oliveira et al. (2016). The mycelium was washed with chloroform: isoamyl alcohol (24:1) and then homogenized in 2% cetyltrimethylammonium bromide buffer. The DNA was precipitated in isopropanol, washed with 70% ethanol, and resuspended in 50 μL of ultrapure water. To amplify the ITS region both primers ITS1 and ITS4 where used (White et al. 1990). Polymerase chain reactions were performed as described by de Oliveira et al. (2014). The newly obtained sequence was deposited in the GenBank database (MW436702). The fungal sequences were aligned with ClustalX (Larkin et al. 2007) and edited with BioEdit (Hall 1999). Prior to phylogenetic analysis, the optimal nucleotide replacement model was estimated using TOPALi 2.5 (Milne et al. 2004). Phylogenetic analysis based on the ITS region was performed to reconstruct a phylogenetic tree, under a maximum likelihood (with support estimated by a bootstrap analysis with 1000 replicates) and Bayesian inference (two runs over 2 × 10 6 generations with a burnin of 2500) analyses were performed with PhyML 3.0 (Guindon et al. 2010) and MrBayes 3.2.2 (Ronquist et al. 2012), respectively. The phylogenetic tree was visualized using Treeview (Page 1996) and edited using Inkscape v. 1.1 (https://inkscape.org/en/).

Discussion
We report the first occurrence of the rare Mucor minutus (URM 7894) in northeastern Brazil, isolated from soil in an upland forest fragment. This species was first described as a variety of Mucor griseo-ochraceous Naumov (as M. griseo-ochraceous var. minutus Baijal & B.S. Mehrotra) in India (Baijal & B.S. Mehrotra 1966). Nine years later, Schipper (1975) proposed the new combination M. minutus (Baijal & B.S. Mehrotra) Schipper. As far as we know, this species has only been reported in Brazil, Canada, China, India, Nigeria, Mauritius, South Korea, Switzerland, and Vietnam. Occurrence data from many of these countries is restricted to ITS sequences deposited in GenBank, with no information as to where specimens were collected. Although published manuscripts reported M. minutus from soil in Brazil, India, Malaysia, South Korea, and Switzerland, detailed information on collection sites, including soil type, local vegetation, temperature, and precipitation are missing, except for Brazil. Like other specimens of Mucor, M. minutus is a probably a soil saprobe, as a parasitism interaction has not yet been reported for this species.
Both morphology and phylogeny show that URM 7894 is M. minutus, and the morphological characteristics of this specimen are in good agreement with the description by Schipper (1975). However, some morphological differences were observed between URM 7894 and the ex-type. Long sporangiophores up to 20 μm in diameter were observed in our specimen; these are smaller than those described by Schipper (1975), who described the long sporangiophores as up to 30 μm in diameter. The sporangia of the long sporangiophores of URM 7894 were smaller than those described by Schipper (1975), which are up to 175 μm in diameter. Additionally, URM 7894 exhibits columellae of the long sporangiophores that are obovoid, globose, infrequently conical and flattened, and up to 85 × 95 μm, while Schipper (1975) reported columellae of the long sporangiophores as obovoid to ellipsoid, infrequently pyriform, and up to 135 × 97 µm. The columellae of short sporangiophores described here are conical and subglobose, unlike those reported for the extype, which are cylindrical to ellipsoid, some conical to flattened. Nguyen et al. (2017) observed the presence of one to three septa below the columellae, unlike our isolate and the ex-type (Schipper 1975), which have no septum below the columellae.
This manuscript contributes to a better understanding of the distribution of M. minutus, a rare species reported from only 10 countries so far. Although most mucoralean species are saprobic and commonly found in soil (Benny et al. 2016), information on the distribution and ecologic niches of these fungi is still scarce . Unfortunately, there are just a few published manuscripts reporting the occurrence of M. minutus (all from soil samples), with little or no information about the biomes where these fungi were found. Thus, new surveys of this species in other substrata, such as dung, decomposing vegetables, and food spoilages in different biomes, may help the understanding on the biology and ecology of this species.