Persoonia 46, 2021: 1–25 ISSN (Online) 1878-9080 www.ingentaconnect.com/content/nhn/pimj RESEARCH ARTICLE https://doi.org/10.3767/persoonia.2021.46.01 Diversity of Backusella (Mucoromycotina) in south-eastern Australia revealed through polyphasic taxonomy A.S. Urquhart1, J.K. Douch1, T.A. Heafield1, A.G. Buddie 2, A. Idnurm1 Key words Abstract Here we explore the diversity of one morphologically distinguishable genus in the Mucoromycotina, Backusella, in south-eastern Australia. We isolated more than 200 strains from locations across the states of Victoria Backusella and Tasmania. Characterization of these strains using a combination of approaches including morphology, sucrose genome sequencing utilization and whole genome sequencing for 13 strains, revealed 10 new species. The genetic basis for interspecies invertase variation in sucrose utilization was found to be the presence of a gene encoding an invertase enzyme. The genus Mucorales Backusella is revised and a new key for species identification produced. Given that we have more than doubled new taxa the number of species in this genus, this work demonstrates that there may be considerable undiscovered species polyphasic taxonomy diversity in the early diverging fungal lineages. zygospore Citation: Urquhart AS, Douch JK, Heafield TA, et al. 2021. Diversity of Backusella (Mucoromycotina) in south-eastern Australia revealed through polyphasic taxonomy. Persoonia 46: 1–25. https://doi.org/10.3767/persoonia.2021.46.01. Effectively published online: 21 September 2020 [Received: 5 June 2020; Accepted: 17 August 2020]. INTRODUCTION from single specimens, in the last 30 yr (Urquhart et al. 2017, Urquhart & Idnurm 2020). Advances in DNA sequencing have increased the rate of dis- Two hypotheses might explain the lack of reported diversity in covery of new fungal species to over 2 000 species per year. Australia. One is that the continent is depauperate in Muco­ However, despite their evolutionary and ecological significance, romycotina species and the second is the consequence of the basal fungal lineages not in the Dikarya represent only limited sampling. In this study we set out to address this dearth 1 % of this figure or just 24 species described in 2017 (Willis of knowledge by examining diversity in the genus Backusella 2018). Of these lineages, species in the order Mucorales are in south-eastern Australia. Backusella is a convenient genus significant decomposers in natural ecosystems, and some within the Mucorales to study because it can be readily dis- species are pathogens both to humans (Ribes et al. 2000) tinguished in culture by its recurved juvenile sporangiophores and other animals, such as Mucor amphibiorum of amphibians (Walther et al. 2013). Currently, the genus consists of 14 spe- and platypuses (Obendorf et al. 1993). Many Mucorales spe- cies: B. circina (Ellis & Hesseltine 1969), B. constricta (Lima et cies produce spores that are not optimized for dispersal in air, al. 2016), B. gigacellularis (De Souza et al. 2014), B. granuli­ which might account for the development of greater endemic spora and B. johoriensis (Loh et al. 2001), B. lamprospora diversity compared to many ascomycetes and basidiomycetes. (Benny & Benjamin 1975), B. locustae (Wanasinghe et al. However, our understanding of the biogeography of these spe- 2018), B. grandis, B. indica, B. oblongielliptica, B. oblongispora, cies is currently limited, and this hypothesis is largely untested. B. recurva, B. tuberculispora, and B. variabilis (Walther et al. Despite being considered a ‘megadiverse’ country (Williams 2013). It has previously been noted that B. grandis is likely to et al. 2001) only a handful of Mucorales species have been be a synonym of B. variabilis (Walther et al. 2013). The only described as being unique to Australia. After some initial work at report of Backusella in Australia is of Backusella recurva (strain the end of the 19th century by Cooke and Massee (discovery of CBS 673.75), isolated from north Queensland. Spinellus gigasporus (Cooke 1889) and Pilobolus pullus (Mas- Here, through sampling from over 25 locations in south-eastern see 1901)) the next productive period, in terms of describing Australia more than 200 strains of Backusella were isolated diversity, was in the 1970–1980s (for example Halteromyces and then analysed. Using a polyphasic approach integrating radiatus (Shipton & Schipper 1975), Mucor amphibiorum (Schip- whole-genome-sequencing-based molecular phylogenies, mor- per 1978), Umbelopsis ovata and U. fusiformis (Yip 1986b), phology and physiology we identify 10 new species, one new U. swartii and U. westeae (Yip 1986a), and Mucor laxorrhizus combination (Backusella dispersa) and suggest synonymisation var. ovalisporus (Schipper 1989)). However, since this time new for two previously described species (B. johoriensis = B. circina; species reports have slowed, despite rapid progress elsewhere B. variabilis = B. grandis), and discuss how the taxonomy of in fungal taxonomy. Only two new species, Pilaira australis and B. granulispora does not conform to a modern morphological Syncephalastrum contaminatum, have been described, both understanding of the genus. Collectively, the new species and refinements to existing taxa adjusts the total number of species in the genus from 14 to 23. As such, these findings provide 1 School of BioSciences, the University of Melbourne, VIC 3010, Australia; an example in which diversity in a single genus is markedly corresponding author e-mail: [email protected]. 2 Centre for Agriculture and Bioscience International (CABI), Wallingford, increased by sampling in Australia, a potential indicator of a Oxfordshire, OX10 8DE, UK. high level of diversity among Australian fungi. © 2020-2021 Naturalis Biodiversity Center & Westerdijk Fungal Biodiversity Institute You are free to share - to copy, distribute and transmit the work, under the following conditions: Attribution: You must attribute the work in the manner specified by the author or licensor (but not in any way that suggests that they endorse you or your use of the work). Non-commercial: You may not use this work for commercial purposes. No derivative works: You may not alter, transform, or build upon this work. For any reuse or distribution, you must make clear to others the license terms of this work, which can be found at http://creativecommons.org/licenses/by-nc-nd/3.0/legalcode. Any of the above conditions can be waived if you get permission from the copyright holder. Nothing in this license impairs or restricts the author’s moral rights. 2 Persoonia – Volume 46, 2021 MATERIALS AND METHODS Table 1 Gene regions used in the multigene phylogeny; ID refers to B. circina FSU 941. Isolation of strains Leaf litter and soil samples were collected from locations in Gene ID Function based on homology the Australian states of Victoria and Tasmania under permits 185987 WD40-repeat-containing subunit of the 18S rRNA processing complex 10008557 (Victorian Department of Environment, Land, Water 205947 GatB/YqeY domain-containing protein and Planning) or FL 18158 (Tasmanian Department of Primary 216514 rRNA-processing protein FCF1 Industries, Parks, Water and Environment). Samples of soil 220627 Nucleolar ATPase Kre33 (c. 7 g each) were mixed with sterilized water and then plated 225083 Mitochondrial ribosomal protein onto potato dextrose agar (PDA) supplemented with cefotaxime 228697 DNA replication licensing factor (100 µg/mL) and chloramphenicol (100 µg/mL) to inhibit bacte- 234491 Carbohydrate kinase rial growth. After 2–4 d growth at ambient temperature, colonies 234892 Transport protein particle (TRAPP) complex subunit displaying the characteristic recurved juvenile sporangia were 235092 Mitochondrial DNA-directed RNA polymerase RPO41 selected and plated onto fresh medium. All isolates were purified 237414 Argininosuccinate lyase by single spore isolation to ensure a homogeneous culture. 238565 WD40-repeat-containing subunit of the 18S rRNA processing complex 241757 Golgi SNAP receptor complex member DNA extraction and amplicon sequencing 242843 WD40-repeat-containing DNA was extracted from fungal material scraped from agar cul- 246934 Translocation protein sec63 ture as described previously (Pitkin et al. 1996). Taq polymerase 249721 DUF323 domain-containing protein purified from the pTaq plasmid (Desai & Pfaffle 1995) was used 251931 WD40-repeat-containing protein for polymerase chain reaction (PCR) following standard proce- 252236 Ribonuclease III dures. The internal transcribed spacers (ITS) were amplified 252238 MIR motif-containing protein with primers ITS1 and ITS4 (White et al. 1990); the large sub- 252550 Molecular chaperone (ABC1) unit rRNA (LSU) was amplified with primers NL1 (Kurtzman & 257118 Dynein heavy chain Robnett 1997) and LR3 (Vilgalys & Hester 1990); and a partial 260240 WD40-repeat-containing subunit of the 18S rRNA processing complex arginosuccinate lyase gene fragment (argA) was amplified using 264113 PCI domain containing protein primers AP52 (5´ TGGGGAGGTCGYTTCTCC 3´) and AP53 268790 Cysteinyl-tRNA synthetase (5´ TATCAGGRTTCTTCTTTTGAGG 3´), designed based on 272587 SAM-dependent methyltransferases examination of the whole genome sequencing data described 282013 Mevalonate pyrophosphate decarboxylase in the following sections. PCR products were purified with a gel 282268 Dihydroorotate dehydrogenase purification kit (Qiagen) and Sanger-sequenced at the Austra- 282537 Uridine 5´- monophosphate synthase/orotate phosphoribosyltransferase lian Genome Research Facility