2021 年 62 巻 5 号 p. 331-335
Absidia healeyae is a new species described in the Mucorales genus Absidia after screening 16 strains of Absidia isolated from seven locations in the state of Victoria in Australia. After initial analysis of the large ribosomal subunit sequence, the genomes of representative strains from two clades were sequenced using short paired-reads. Additional taxonomic markers extracted from the genome sequencing data support the novelty of A. healeyae. The identification of a new species in the genus Absidia, from a relatively small collection of isolates, hints at an unexplored diversity in the early diverging lineages of fungi in Australia.
The genus Absidia currently consists of approximately 25 species for which molecular data are available. With the advent of molecular techniques to identify variation between species, a clearer understanding of the composition of the genus has emerged through a series of phylogenetic studies, resulting in the transfer of many of the species formerly considered to be within Absidia (notably the animal pathogen Absidia corymbifera (Cohn) Sacc. & Trott.) into the genera Lichtheimia and Lentamyces (Hoffmann, 2010).
For most genera in the order Mucorales, in addition to the species considered in phylogenetic studies, there are a large number of validly published species for which no DNA sequence information or ex-type material exists. Given that many descriptions date from the 19th and early 20th centuries they often include insufficient detail to permit “rediscovery”. How these names should be treated when describing a new species remains an open question (Dayarathne et al., 2016). Nevertheless, new Absidia species are being described, including Absidia bonitoensis C.L. Lima, D.X. Lima, Hyang B. Lee & A.L. Santiago (Lima et al., 2021), Absidia pernambucoensis D.X. Lima, C.M. Souza-Motta & A.L. Santiago, Absidia cornuta D.X. Lima, C.A. de Souza, H.B. Lee & A.L. Santiago (Lima et al., 2020), Absidia multispora T.R.L. Cordeiro, D.X. Lima, Hyang, B. Lee & A.L. Santiago, Absidia saloaensis T.R.L. Cordeiro, D.X. Lima, Hyang B. Lee & A.L. Santiago (Cordeiro et al., 2020), Absidia pararepens Jurjević, M. Kolařík & Hubka (Crous et al., 2020), A. terrestris Rosas de Paz, Dania García, Guarro, Cano & Stchigel (Crous et al., 2018), A. panacisoli T. Yuan Zhang, Ying Yu, He Zhu, S.Z. Yang, T.M. Yang, Meng Y. Zhang & Yi X. Zhang (Zhang et al., 2018), A. jindoensis Hyang B. Lee & T.T.T. Nguyen (Wanasinghe et al., 2018), A. stercoraria Hyang B. Lee, H.S. Lee & T.T.T. Nguyen (Li et al., 2016), A. koreana Hyang B. Lee, H.W. Lee & T.T.T. Nguyen and A. caatinguensis D.X. Lima & A.L. Santiago (Ariyawansa et al., 2015). However, thus far, no Absidia species have been discovered in Australia, which likely reflects our poor understanding of the early diverging fungi in this continent (Urquhart, Coulon, & Idnurm, 2017; Urquhart & Idnurm, 2020; Urquhart, Douch, Heafield, Buddie, & Idnurm, 2021). In this study we set out identify Absidia strains from the Australian state of Victoria. In a total of 16 strains (Table 1), we identified three species, at least one of them new and described here as A. healeyae.
a Format is day, month, year (20##).
Sixteen strains showing whorled sporangia consistent with the genus Absidia were obtained from leaf litter samples taken from seven nature reserves in the Australian state of Victoria (Supplemental Fig. S1). Approximately 7 g of leaf litter were suspended in sterile water to a total volume of 50 mL, and a range of aliquot volumes (50 µL to 2 mL) was spread onto potato dextrose agar (PDA) plates supplemented with cefotaxime (100 µg/mL) to inhibit bacterial growth. Each strain was purified by single spore isolation on PDA to ensure homogeneity. Cultures were maintained on PDA at 22 °C on a laboratory bench. Three distinct groups of isolates were immediately evident based on colony pigmentation (purple, brown or green).
Genomic DNA was extracted from lyophilised mycelium, using a 1% cetyl trimethylammonium bromide buffer incubated at 65 °C for 30 min before one chloroform extraction and precipitation with an equal volume of isopropanol (Pitkin, Panaccione, & Walton, 1996). A fragment of the large ribosomal subunit (LSU) was amplified from each strain using primers NL1 5´-GCATATCAATAAGCGGAGGAAAAG-3´ (Kurtzman & Robnett, 1997) and LR3 5´-GGTCCGTGTTTCAAGAC-3´ (Vilgalys & Hester, 1990) using ExTaq DNA polymerase (Takara, Otsu, Japan). PCR conditions were 94 °C for 2 min followed by 32 cycles of 94 °C for 20 s, 55 °C for 20 s and 72 °C for 1 min, in an Eppendorf Mastercycler PCR machine (Eppendorf South Pacific, Macquarie Park, Australia). These PCR products were sequenced using Sanger chemistry at the Australian Genome Research Facility.
Analysis of the LSU sequences suggested two clades potentially distinct from previously described species of Absidia. A representative of each clade (strains UoMAU1 and UoMAU379) was chosen for genome sequencing, using Illumina sequencing at the Australian Genome Research Facility as 125 bp paired end reads. Sequences were assembled using Velvet (Zerbino & Birney, 2008) with the k-mer value set to 71 bp and a cut off minimum contig length of 500 bp. Assembly statistics are given in Table 2. The raw sequences and assemblies are available from the NCBI database under BioProjects PRJNA630489 (strain UoMAU1) and PRJNA630491 (strain UoMAU379).
Sequences for the internal transcribed spacer (ITS), translation elongation factor 1 alpha (TEF1) and actin were extracted from the genome assembly. Sequences were aligned using CLUSTAL Omega (Sievers et al., 2011). Phylogenetic analysis was then conducted using a maximum likelihood analysis in MEGA 10.2.4 (Kumar, Stecher, Li, Knyaz, & Tamura, 2018) using the Tamura-Nei model (Tamura & Nei, 1993) with gamma distribution.
The LSU tree grouped the 16 isolates into three clades (Fig. 1; TreeBASE accession URL: http://purl.org/phylo/treebase/phylows/study/TB2:S28061). The clades were congruent with the colony colours of the strains: UoMAU1, UoMAU2, UoMAU3, UoMAU380, UoMAU382, UoMAU385 and UoMAU386 were purple; UoMAU379 was brown; and the remaining strains were green. The clade consisting of the purple strains was distinct from LSU sequences previously deposited in GenBank while the green strains belonged to A. glauca. Green colony colour is a known characteristic of A. glauca (Ellis & Hesseltine, 1965). The third clade, containing only UoMAU379, is closely related to Absidia inflata NRRL 6591 (LSU sequence constructed from whole genome sequencing data, BioProject accession PRJNA519280) as well as unpublished sequences in GenBank (accessions LR606139 and LR606138). We hypothesize that these isolates may belong to the same species, however, further detailed taxonomic work will be required to confirm this. We have deposited the sequence of the region containing the 18S rRNA gene, 5.8S rRNA gene and 28S rRNA gene in GenBank (accession MT436029) along with those for actin and TEF1 (MW861732 and MW861734).
The three additional regions of the genome of strain UoMAU1 were examined and further support the separation from previously described species, and we thus named it as a new species A. healeyae. BLAST (Altschul, Gish, Miller, Meyers, & Lipman, 1990) comparison to the GenBank nr database revealed that ITS of A. healeyae was only 77% similar to the highest match, Absidia sp. SH1 accession EU816583.1, which is well below the expected similarity between strains of the same species (Vu et al., 2019). Similarly, the A. healeyae TEF1 and actin sequences were only 87% and 92% similar to the top BLAST results of A. padenii accession AF157238.1 (O’Donnell, Lutzoni, Ward, & Benny, 2001) and A. repens FSU4726 accession AY944763 (Hoffmann, Discher, & Voigt, 2007). While the molecular data presented in this paper clearly establish A. healeyae as a distinct species, our understanding of the phylogenetic structure of this genus is hampered by the limited number of regions available for phylogenetic studies. By providing whole genome sequencing data we hope that future researchers will be able to utilise a greater set of regions.
Absidia healeyae A.S. Urquhart & A. Idnurm, sp. nov. Fig. 2.
MycoBank no.: MB 835491.
Description: Colonies 31 mm diam after 4 d at 22 °C on PDA, initially white with purple pigmentation apparent by 7 d. Growth inhibited at temperatures above 30 °C. Sporangiophores 3.5–5.2 µm, 4.3 µm on average, in width below sporangia, branching typically into whorls of short branchlets each bearing a terminal sporangium, with a single septum below the sporangium. Sporangia 25.7 µm × 32 µm to 30.4 µm × 39.1 µm, 34.8 µm × 26.9 µm on average, pyriform, apophysate. Sporangiospores spherical, 2.3–3.5 µm diam, 2.8 µm on average. Columellae hemispherical above the apophysis, sometimes with a small protrusion at the apex. Zygospores not observed.
Type: AUSTRALIA, Tarra-Bulga National Park, Victoria. A dried specimen from an in vitro culture on potato dextrose agar, preserved on Whatman® filter paper, comprising of mycelia, sporangia and sporangiospores (holotype in the National Herbarium of Victoria, Melbourne, Australia, MEL 2417184; ex-type cultures, UoMAU1 = CBS 144487 = JMRC:SF:013685 = NRRL 66765).
Genome sequence ex-holotype: BioProject accession PRJNA630489.
Gene sequences ex-holotype: MT436027 (LSU), MT436028 (18S rRNA gene, 5.8S rRNA gene, 28S rRNA gene, including ITS), MW861733 (TEF1), MW861731 (actin).
Etymology: healeyae in recognition of Kara Moana Healey, Victoria’s first female national park ranger, of Tarra-Bulga National Park (Shingles, 2001).
Habitat: Leaf litter, wet native Australian forests.
Distribution: Isolates thus far obtained from three nature reserves in the Australian state of Victoria: Tarra-Bulga NP, Jack Cann Reserve and Macedon Regional Park. The distance between the farthest sites is approximately 220 km.
Notes – Eight other species of Absidia producing exclusively spherical-spores have been described. Five of these are available in culture collections and have corresponding molecular data: A. glauca Hagem, A. caerulea Bainier, A. californica J.J. Ellis & Hesselt. A. padenii (Hesselt. & J.J. Ellis) Milko and A. macrospora Váňová. Absidia healeyae is distinct from these species as the molecular data show a high degree of dissimilarity to these species across all four gene regions. Furthermore, A. healeyae can be distinguished from these species based on its unique combination of purple colony colouration and spherical spores, which are somewhat smaller than those produced by A. caerulea [3–5 µm, mostly about 3.5 µm (Ellis & Hesseltine, 1965)]. Three other species with spherical spores have been recognised as uncertain species since no ex-type strain is available (Ellis & Hesseltine, 1965): A. septata Tiegh., A. scabra Cocc. and A. reflexa Tiegh. Absidi healeyae can be distinguished from A. septata because A. healeyae is most likely heterothallic due to the lack of zygospore formation by individual strains and because analysis of the genome sequence indicates strain UoMAU1 contains only the sexM gene, positioned between homologs of algL and sagA that flanking the sex locus in other Mucorales species whereas A. septata is homothallic, from A. scabra because A. healeyae possesses a cross-wall in the sporagiophore below the apophysis, and from A. reflexa because the sporangiophores are not circinate in A. healeyae.
The authors declare no conflicts of interest. The fungal material was collected under permit number 10007429 issued by the Department of Environment, Land, Water and Planning (State Government of Victoria).
Travis Heafield and Dr. Allison Van de Meene assisted with the electron microscopy.