Novel Mucor species (Mucoromycetes, Mucoraceae) from northern Thailand

Abstract Mucor species are common fast-growing fungi found in soil. Two new species of Mucor and one new geographical record of M.nederlandicus were collected from northern Thailand and are described in this study. Evidence from morphophysiological data and phylogenetic analysis supports the introduction of the new taxa. Phylogenetic analysis based on the internal transcribed spacer (ITS) and large subunit of the nuclear ribosomal RNA (LSU) data showed that the new isolates cluster distinctly from other Mucor species with high or maximum bootstrap support. Mucoraseptatophorus is characterized by aseptate sporangiophores, globose columella, resistant and deliquescent sporangia, has sympodial, and monopodial branches and shows growth at 37 °C. It also differs from M.irregularis in having smaller sporangiospores, and larger sporangia. Mucorchiangraiensis has subglobose or slightly elongated globose columella, produces hyaline sporangiospores, and resistant and deliquescent sporangia. Furthermore, this species has wider sporangiophore, smaller sporangia and lower growth than M.nederlandicus. A detailed description of the species and illustrations are provided for the novel species.


Introduction
Soil fungi play key roles in nutrient cycling and the functioning of terrestrial ecosystems (Torsvik and Øvreås 2002;Frąc et al. 2018;Peng et al. 2020;Zhou et al. 2020). To date, only a few fungal taxonomic studies are available from tropical forest soil (Sato et al. 2015;Amma et al. 2018;Cordeiro et al. 2020;Lima et al. 2020). Taxonomic studies on these groups are even scarcer in Southeast Asia despite the region harboring extraordinary genetic diversity (Amma et al. 2018). Up to 96% of fungi isolated from the northern part of Thailand are projected to be novel taxa and new species are regularly being discovered (Hyde et al. 2018;. However, the diversity of non-Dikarya soil fungi in the country remains to be fully explored (Khuna et al. 2019;Hurdeal et al. 2021;Suwannarach et al. 2021).
Mucorales are cosmopolitan fungi commonly found in soil (Pawłowska et al. 2019;Muszewska et al. 2021). It is an early-diverging fungal group in a basal position with respect to Ascomycota and Basidiomycota in the fungal tree of life (Spatafora et al. 2016;Nicolás et al. 2020). The order comprises 55 genera with over 260 species (Walther et al. 2019;Cordeiro et al. 2020;Lima et al. 2020;Nicolás et al. 2020;Wijayawardene et al. 2020;Hurdeal et al. 2021). However, for the majority of these species, their ecological roles and geographical distributions are unknown. Mucoralean fungi are usually fast-growing and produce coenocytic or irregularly septate hyphae. Septae are usually formed to delimit reproductive structures but are not dispersed in regular intervals as in Dikarya fungi (Walther et al. 2019;Wagner et al. 2020). Characters including homothallism, formation of sporangiola, and the shape of the suspensors have been previously employed to describe mucoralean taxa (Walther et al. 2019). However, the application of molecular phylogenetics has transformed the taxonomy of Mucorales as it has revealed that some of the above-mentioned morphological characters are not taxonomically informative.
Mucor is the most species-rich genus within Mucorales commonly found in soil and dung. Its species comprise mainly saprobes, but also endophytes, parasites of plants and human pathogens causing mucormycosis (e.g. M. irregularis) (Mendoza et al. 2014;Lunge et al. 2015;Mishra et al. 2019;Wagner et al. 2020). Mucor was first described by Fresenius (1850) and has over 300 species cited in previous literature (Kirk 1986;Jacobs and Botha 2008;Nguyen et al. 2016;Lima et al. 2020). The suggested number of valid taxa varies from 50 to 91 species (Walther et al. 2019;Lima et al. 2020;Wijayawardene et al. 2020). Mucor species form fast-growing colonies characterized by simple or branched sporangiophores, non-apophysate and globose sporangia, deliquescent, and incrusted sporangial wall, and zygospores on opposed or tong-like suspensors. However, morphology-based classification of Mucor is highly debated. For example, previous literature used rhizoids to demarcate Rhizomucor from Mucor ). Contrary to this, recent studies have revealed that some species of Mucor produce rhizoids (Lima et al. 2018;. Mucor comprises mesophilic species with some growing at high temperatures, but never at temperatures above 42 °C (Walther et al. 2019).
While studying the diversity of soil fungi in northern Thailand, two Mucor isolates differed morphologically and genetically from other known species. Using molecular phylogeny of ITS and LSU genetic markers along with morphological characterization, two new species of Mucor are proposed. Full description, taxonomic notes, photoplates, and phylogenetic trees are provided.

Collections and field sites
Soil samples were collected from the provinces of Chiang Mai, and Chiang Rai, Thailand. Superficial organic matter (1-3 cm deep) was manually removed and a clean shovel was used to dig the soil. Samples were placed in a zip lock bag and stored at 4 °C until further use. The collecting site in Chiang Rai comprised a maple tree plantation and the sample was collected during the winter season in December 2019. The temperature in the province during this month is usually around 13.5 °C, while the annual rainfall is 2172 mm. The samples collected, consisted of peat and grainy soil. In Chiang Mai, moist peat soil was collected from a deciduous forest during the monsoon season in October 2019. During this season, the temperature is around 31 °C with an annual rainfall of 1108 mm.

Isolation, culture and morphological studies
The dilution plating method was used for the isolation of fungal species (Senanayake et al. 2020). Soil samples were diluted to a ratio of 1:5 and 1:10 with sterilized distilled water. The mixture was shaken at 25 °C and 200 rpm for two hours using an incubator shaker. 100 μL of the suspension was then plated on agar plates. All Petri plates used herein were 90 mm. To maximize the number of fungi isolated, four different media were used: yeast malt extract (YMA) (yeast extract: 3g; malt extract: 3g; peptone: 5g; glucose: 10g; agar: 15g; distilled water: 1L), plate count agar (PCA) (enzymatic digest of casein: 5g; yeast extract: 2.5g; glucose: 1g; ; agar: 15g; distilled water: 1L), nutrient agar (NA) and modified choanephora agar (CH) (glucose: 3 g; pancreatic digest of casein: 2 g; KH 2 PO 4 : 1 g; MgSO 4 .7H 2 O: 0.5 g; thiamine HCl: 25 mg; agar: 20 g; distilled water: 1L) with or without benomyl. Benomyl (2 mg/L) was used to limit the growth of other fungi. Suspension of each soil sample was spread on four media using a flame sterilized rod spreader in triplicate. Plates were sealed and incubated at 20, 30, and 37 °C.
All plates were checked daily. Once fungal colonies were seen, the plates were screened using a microscope (400X) (Zeiss Primostar). Recovered strains were divided into morphotypes based on colony appearance and the presence of specific sporulation structures, when possible. A sterile straw was used to cut the fungal tips, which were transferred to a new agar plate. This process was done 2 or 3 days post-inoculation, before different fungal colonies overlapped on the plate. Growth experiments were performed using MEA media incubated at 8, 20, 25, 30, and 37 °C. Once grown, the cultures were examined using a compound microscope (Nikon Eclipse Ni) and pictures were taken with a Nikon DS-RI2 digital camera. The cultures were maintained in 15% glycerol at 4 °C. Herbarium and type specimens were deposited in Mae Fah Luang University (MFLU) Herbarium, Chiang Rai, Thailand as inactive dried cultures. Ex-type living cultures were deposited in the Mae Fah Luang culture collection (MFLUCC), Chiang Rai, Thailand.

DNA extraction and PCR amplification
Total genomic DNA was extracted from fungal mycelia using the Solg TM Genomic DNA Prep Kit following the manufacturer's instructions. Polymerase chain reaction (PCR) was used to amplify the partial fragments of internal transcribed spacer (ITS), and large subunit ribosomal RNA (LSU) using fungal-specific primers (Vilgalys and Hester 1990;White et al. 1990;De Hoog and Van Den Ende 1998). The PCR reaction mixture consisted of the fungal DNA, 14 μL dH 2 O, 1 μL AccuPower PCR PreMix (BioneerCorp., Daejeon, Korea) and 1.5 μL (5 pmol/μL) each of forward and reverse primers resulting in a final volume of 20 μL.
Amplification of the ITS and LSU fragments was performed using the following conditions: initial heat treatment of 5 min at 94 °C, 30 cycles with a denaturation step at 94 °C for 30 sec, annealing at 52 °C for 45 sec and an elongation step of 1 minute and 30 sec at 72 °C and a final elongation period of 7 minutes at 72 °C.
The PCR products were then purified using an Accuprep PCR Purification Kit (Bioneer). Sequencing was performed by Macrogen (South Korea) using an Applied Biosystems 3130XL DNA analyzer.

Sequence and phylogenetic analysis
Raw reads were edited by removing ambiguous bases in the ends using BioEdit. The forward and reverse trimmed reads were assembled into contigs using SeqMan Version 7.1.0. Newly generated sequences were used as queries to perform blast searches against the nucleotide database in GenBank (Altschul et al. 1990). This was done to check for possible contamination and to find the closest taxa. The taxon sampling spans the diversity of the genus with the exception of some phylogenetically closely related species in the M. circinelloides complex. Information on each species was extracted from Gen-Bank taxonomy and correlated with Species Fungorum (http://www.speciesfungorum. org/), Index Fungorum (http://www.indexfungorum.org/) and MycoBank (https:// www.mycobank.org/) to determine the validity and current name of each taxon. Datasets of the ITS and LSU genetic markers were built. The software MAFFT Version 7 available on the online server https://mafft.cbrc.jp/alignment/server/ (Katoh and Toh 2008) was used to align the sequences and each matrix was trimmed using TrimAl Table 1. Data used for phylogenetic analysis in this study and their corresponding GenBank accession numbers. Type, ex-neotype, ex-isotype, and ex-type strains are denoted by T, NT, IT, and ET, respectively. Sequences derived in this study are shown in bold letters. (Capella- Gutiérrez et al. 2009). Genetic distances were calculated using the kimura2 parameter in MEGAX with the pairwise deletion gap option. The nucleotide substitution models were evaluated for each genetic marker on the online CIPRES Portal (https://www.phylo.org/portal2) using the jModelTest2 on XSEDE and GTR + I + G was deemed as the best suited.
Maximum likelihood (ML) analysis was performed on the online CIPRES Portal using RAxML-HPC2 on XSEDE Version 8.2.12 with bootstrap support obtained from 1000 pseudoreplicates (Miller et al. 2010;Stamatakis 2014). The ML analysis was performed by partitioning the dataset according to the specific genetic marker. Bayesian inference (BI) analysis (Huelsenbeck and Ronquist 2001) was also performed on the online CIPRES Portal using MrBayes on XSEDE Version 3.2.7a. The analysis was conducted by running four simultaneous chains for 5,000,000 generations in two independent runs with a sampling frequency of 100 and temp parameter was adjusted to 0.05. At the end of the analysis the standard deviation of split frequency was less than 0.01 at which point convergence of the runs was declared.
Culture characteristics. Colonies on MEA reaching 62 mm diameter after 2 days of incubation at 25 °C. Colony white at first, becoming pale yellow with age; reverse pale yellow. Colony fully covers the Petri plate (90 mm) by the third day at 25 and 30 °C but does not reach the lid of the plate. At 20 °C, colony reaches a diameter of 70.5 mm after 3 days. Vertical growth is lower at 25 and 30 °C than at 20 °C. The colony does not reach the lid after 3 days. At 30 °C, sporulation is excellent, with branching of sporangiophore more frequent than in others. Monopodial branching more prominent but sympodial and dichotomous branches also observed. On PDA, cultures are white and pale brown in the middle with grey to pale brown sporangia. Colony reaching 64 mm diameter after 3 days of incubation at 25 °C. Optimal growth and excellent sporulation were observed on both MEA and PDA media at 30 °C. At 37 and 8 °C in MEA, growth is observed but with no sporulation. The colony reaches a diameter of 31 mm at 37 °C after 3 days. At 8 °C, the colony reaches a diameter of 14 mm after 9 days. Growth is observed at temperatures ranging from 8 to 37 °C.
Distribution. Thailand.  subglobose or slightly elongated globose columellae rather than commonly obovoidpyriform or subspherical as in M. inaequisporus. Although the shape and size of sporangiospores are often variable in both species, the majority of spores of M. chiangraiensis are ellipsoidal. Sporangiospores are usually colorless and hyaline as compared to pale or greenish-yellow in M. inaequisporus.
Culture characteristics. Day-old cultures are white, cottony, floccose, have erect sporangiophores with hyphae reaching the lid of the Petri plate in MEA. The white color of the colony persists even after 6 days. At maturity, the colony reverse is white or pale yellow. On MEA at 25 °C, the colony reaches a diameter of 51 mm after 3 days of incubation. At 20 and 30 °C, the colony reaches a diameter of 47 mm and 44.5 mm respectively after 3 days. Sporulation is excellent at 20 to 30 °C. At 30 °C, mostly unbranched sporangiophores are observed with few sympodial branches. On PDA at 25 °C, colony reaches a diameter of 48 mm after 3 days of incubation. The front and reverse of the colony are white in both MEA and PDA. Growth is observed at temperatures ranging from 8 to 30 °C. Optimal growth and excellent sporulation were observed at 25 °C on both MEA and PDA media. No growth was observed at 37 °C. At 8 °C in MEA, the colony reached a diameter of 34 mm after 9 days but with no sporulation.

Mucor nederlandicus
Culture characteristics. Colonies on MEA reaching a diameter of 60 mm after 3 days of incubation at 25 °C. At temperatures 20 and 30 °C, the colony diameter is 46 mm and 45.5 mm respectively after 3 days. Colony growth is observed at 8 to 30 °C with optimal growth at 25 °C. Unbranched or sympodial branching of sporangiophore are prominent at 30 °C. Colonies on PDA, reaching 51 mm after 3 days of incubation at 25 °C. In both culture media, colony from above and reverse: white or very pale yellow, and cottony. No growth was observed at 37 °C. At 8 °C, the colony reached a diameter of 21 mm after 9 days but with no sporulation in MEA.

Discussion
Thailand has extraordinary fungal diversity with many fungal species having been described from the northern provinces of the country (Hyde et al. 2018). The present study introduces two new species of Mucor and one new geographical record of M. nederlandicus from soil habitats in northern Thailand based on molecular and morphophysiological data. Many Mucor species have been isolated from soil and other substrates including postharvest crops, dung, and leaf litter ). The newly-described Mucor species exhibit overall characteristic morphology observed in the genus, including branched sporangiophores and globose sporangia (Hoffmann et al. 2013;Walther et al. 2019). Commonly used morphological characters for species-level delimitation are the size and shape of structures, such as sporangiospores and columella (Chai et al. 2019;Lima et al. 2020). Morphological examination revealed that the newly isolated strains differ from their known sister taxa. This includes differences in the sporangia and columellae sizes. For example, M. aseptatophorus differs from its sister taxon M. irregularis in having smaller sporangiospores, but larger sporangia. Phenotypic characters comprise a pivotal aspect of modern fungal taxonomy. Nonetheless, some Mucorales groups display morphological plasticity, hence their taxonomy cannot be solely based on morphological features as these might not always be taxonomically informative.
In our phylogenetic analyses, placement of the new species was stable with high statistical support using both ML and BI methods of inference. Genetic distance analysis of the ITS region contributed further evidence to the introduction of the two novel Mucor species. The percentage nucleotide difference in the ITS genetic marker to the sister taxa is 2.5-4.5% for M. aseptatophorus and 1-13.3% for M. chiangraiensis (Table  2), exceeding the threshold for establishing new species Jeewon and Hyde 2016).
Most studies focusing on Mucor species delimitation use ITS and LSU as these are the most widely available genetic markers (Madden et al. 2012;Lima et al. 2018;Chai et al. 2019;Lima et al. 2020). In the Mucor circinelloides complex, protein-coding genes, such as the largest subunit of RNA polymerase II (rpb1) are also available for most species (Walther et al. 2019;Wagner et al. 2020). Several genetic markers, which have been traditionally used in the taxonomy of Dikarya fungi, cannot be applied in Mucorales due to the presence of paralogous and multiple-copy genes in the latter (Walther et al. 2019;Wagner et al. 2020). In general, the number of genetic markers necessary for species delimitation varies depending on the approach (Luo et al. 2018;Bhunjun et al. 2020). For instance, automatic barcode gap discovery can be used or best suited with single locus, while at least a couple of unlinked markers from independent loci are necessary for the genealogical concordance phylogenetic species recognition (GCPSR) concept (Laurence et al. 2014). Thus, only comparatively few studies use the latter (Taylor et al. 2000;Walther et al. 2019;Wagner et al. 2020). In the case of Mucor, the availability of only ITS and LSU for most taxa precludes the use of the GCPSR concept, as both markers belong to the same locus. Recently, phylogenomics approaches using hundreds of genes have started to emerge in an effort to delineate problematic fungal taxa (Vandepol et al. 2020). Nonetheless, in the case of Mucor, using ITS and LSU to delineate species has been adequate.
Herein, M. aseptatophorus showed growth at 37 °C. Mucor irregularis, which has close phylogenetic affinity to the new species also has the ability to grow at this temperature. Mucor irregularis is an opportunistic pathogen causing cutaneous mucoromycosis mostly in immunocompromised individuals (Lu et al. 2013;Xu et al. 2018;Soare et al. 2020). Thermotolerance in Mucor usually hints at pathogenic potential, however not all thermotolerant species are pathogens. Thus, the thermotolerant ability of M. aseptatophorus warrants further investigation in order to determine whether or not it is pathogenic.
In the last decade, at least 20 new Mucor species have been introduced and described from soil, freshwater, leaf litter, and dung habitats, indicating that we are nowhere near to discovering all taxa in the genus . Information on the geographic distribution of Mucor has started to emerge. Its species have been isolated in Brazil, Great Britain, the USA, China, South Korea, Finland, Germany, and France, suggesting that Mucor has worldwide distribution occupying diverse habitats. Herewith, this study contributes to the study of Mucor in Thailand. As more species are being discovered, and the diversity of these organisms is being explored, the ecological roles of Mucor remain largely unknown and many habitats are yet to be explored. This highlights the need for more studies that explore the ecological importance, diversity, and accurate taxonomic classification of Mucorales.