- Email: [email protected]
Syncephalastrum contaminatum, a new species in the Mucorales from Australia
Journal Pre-proof Syncephalastrum contaminatum, a new species in the Mucorales from Australia Andrew S. Urquhart, Alexander Idnurm PII:
S1340-3540(20)30018-8
DOI:
https://doi.org/10.1016/j.myc.2020.02.003
Reference:
MYC 480
To appear in:
Mycoscience
Received Date: 2 September 2019 Revised Date:
17 February 2020
Accepted Date: 19 February 2020
Please cite this article as: Urquhart AS, Idnurm A, Syncephalastrum contaminatum, a new species in the Mucorales from Australia, Mycoscience (2020), doi: https://doi.org/10.1016/j.myc.2020.02.003. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © [Copyright year] Published by Elsevier B.V. on behalf of the Mycological Society of Japan.
Short Communication
Syncephalastrum contaminatum, a new species in the Mucorales from Australia
Andrew S. Urquhart a, Alexander Idnurm a*
a
School of BioSciences, University of Melbourne, VIC 3010, Australia
* Corresponding author. School of BioSciences, University of Melbourne, VIC 3010, Australia. E-mail address: [email protected] (A. Idnurm).
Text: 14 pages; tables: 0; figures 4
1
Abstract
A new species is described in the Mucorales family Syncephalastraceae: Syncephalastrum contaminatum, isolated as an in vitro culture from a laboratory contaminant. The species has variable copies of the internal transcribed spacer (ITS) regions, requiring cloning of these regions prior to Sanger sequencing before subsequent use in phylogenetic comparisons with other fungi. The genome of the strain was sequenced using short paired-reads to yield a draft genome of 28.6 Mb. Syncephalastrum contaminatum is distinguished by diverse DNA sequences at several loci from the other species of Syncephalastrum, including only 81% sequence identity with its ITS regions to that of S. racemosum. Its merosporangium produces four or more asexual spores and the genome sequencing information suggests that the species is heterothallic. The identification of this species highlights the limited knowledge about the early lineages of fungi both in Australia and globally.
Keywords: mating type, Merosporangium, Mucoromycota, phylogenomics, Syncephalastrum verruculosum, Zygomycete
2
Although there may be many million fungal species and many are being described on a regular basis (Hawksworth & Lücking, 2017), few species outside of the Ascomycota and Basidiomycota phyla are described each year. For example, in an estimate from the Royal Botanic Gardens Kew just 24 non-Dikarya species were described in 2017 (Willis, 2018). There are a number of reasons for this discrepancy that are not necessarily a reflection of species paucity. One is the limit in resources available to identify and then describe this biodiversity. A second challenge is that the internal transcribed spacer (ITS) regions associated with the ribosomal RNA genes, which are used extensively as a phylogenetic marker (Schoch et al., 2012), can be variable in the non-Dikarya lineages thereby hampering rapid identification. In this study we identify a new Mucorales species in the genus Syncephalastrum (Syncephalastraceae). Syncephalastrum is easy to recognize because of its distinctive merosporangium that produces the asexual spores. A number of Syncephalastrum species have been described; however, there is considerable intraspecific variation in morphology between strains and hence those preparing monographs on the genus have consistently placed these strains into S. racemosum Cohn (Benjamin, 1959; Schipper, & Stalpers, 1983). A second species that produces a merosporangium containing a single spore, S. monosporum R.Y. Zheng, G.Q. Chen & F.M. Hu, is also recognised (Zheng, Chen, & Hu, 1988). While aiming to culture a species in the Cordycipitaceae family, a contaminating Mucorales strain, as initially determined by its rapid growth of coenocytic hyphae, was isolated in culture on potato dextrose agar (Fig. 1). The strain (named UoMD18-1) produced the striking asexual sporulation structure, termed the merosporangium, that is characteristic of species of the genus Syncephalastrum.
3
Genomic DNA was extracted from freeze-dried mycelium of strain UoMD18-1 (Pitkin, Panaccione, & Walton, 1996). The internal transcribed spacer (ITS) regions were amplified. Direct PCR and Sanger sequencing of the ITS regions did not produce a clean chromatogram, indicating a mix of amplicons. The ITS amplicons were therefore cloned into the pCR2.1-TOPO plasmid (Invitrogen), multiple plasmids were purified, and the inserts sequenced. Comparison of the sequences revealed three variations of ITS (submitted to GenBank as accessions MK799840– MK799842). The genome of strain UoMD18-1 was sequenced to obtain additional sequences to construct a multigene phylogeny and as a resource for others studying the evolution and genomics of species in the Mucorales, by using Illumina short (125 nucleotides) paired-reads on a HiSeq2500 at the Australian Genome Research Facility. Just over 8 million pairs were sequenced. The reads were assembled in Geneious (Kearse et al., 2012) using Velvet (Zerbino & Birney, 2008) with a k-mer of 75, selected after a Velvet optimization analysis, and a cut off minimum contig length of 1 kb. Three hundred seventy three contigs were produced, for a total size of 26.8 Mbp. The original reads were remapped onto this assembly, with 92% of the total aligning. The raw sequences and assembly are available from GenBank under BioProject PRJNA541789. The morphological identification of strain UoMD18-1 within Syncephalastrum based on its production of merosporangia was supported by a multi-gene phylogeny including Mucorales species for which full genome sequences are available from MycoCosm (Ma et al., 2009; Wang, Wu, Xu, & Li, 2013; Grigoriev et al., 2014; Schwartze et al., 2014; Chibucos et al., 2016; Corrochano et al., 2016; Lastovetsky et al. 2016; Mondo et al., 2017a, 2017b; Uehling et al., 2017). MycoCosm provides a comparison of genes using a Markov cluster algorithm (MCL);
4
using the “Mucoromycotina MCL. 2984” (created 28 Feb 2019) dataset, parameters were set to identify genes present as single copies in 54 Mucorales species, and these then used to search the genome sequence of strain UoMD18-1 (referred hereafter as the novel species S. contaminatum – see below) by BLAST for homologs. Eight (corresponding to protein IDs in the S. racemosum genome 530022, 521890, 563448, 493224, 496917, 483206, 483391 and 440562) were chosen based on having a phylogenetically informative level of sequence variation, aligned using MUSCLE (Edgar, 2004), and then trimmed manually to remove areas of uncertain nucleotide alignment. This resulted in a final alignment of 10.5 kb, which is deposited in TreeBASE (accession URL: http://purl.org/phylo/treebase/phylows/study/TB2:S25812). Phylogenies based on Bayesian inference were generated using MrBayes using the general time reversible (GTR) substitution model, gamma variation, and sequences from Mortierella elongata Linnem. as the outgroup (Huelsenbeck & Ronquist, 2001). This eight-gene phylogeny (Fig. 2) placed the new strain as sister to S. racemosum strain NRRL 2496, which was previously sequenced (Mondo et al., 2017a). Given the opinion that the variability in growth and spore numbers in the merosporangium for S. racemosum render traditional morphological traits uninformative (Benjamin, 1959), we investigated DNA sequence differences between S. contaminatum and previously described species to determine if S. contaminatum was indeed a new species. The ITS and large subunit (LSU) matches to the nearest described Syncephalastrum species assessed by BLAST were 76% and 97%, respectively, which is a greater level of divergence than expected due to intraspecific variability (Vu et al., 2019). Furthermore, the actin and transcription elongation factor 1 alpha (TEF) regions showed only 92% and 98% similarity, respectively, to the closest Syncephalastrum deposited in GenBank. An ITS sequence deposited in GenBank
5
(MG751268) from an undescribed Syncephalastrum from Brazil shows highest similarity to S. contaminatum (93%), and might represent a second isolate of this species. Examination of the ITS phylogeny (Fig. 3), whose alignment is deposited in TreeBASE (accession URL: http://purl.org/phylo/treebase/phylows/study/TB2:S25812), supports the separation of S. verruculosum P.C. Misra (Misra, 1975) from the other described species of Syncephalastrum, despite Schipper and Staplers (1983) considering it to be a synonym of S. racemosum. As such, we suggest that the genus Syncephalastrum should now be considered to include four species: S. racemosum, S. monosporum, S. verruculosum and Syncephalastrum contaminatum sp. nov. However, there is likely greater undescribed species diversity in the genus Syncephalastrum already represented in culture collections owing to the historical difficulty associated with morphological species delimitation in this species. For example, FSU 6155 is clearly separated on our tree and has been noted as a possible cryptic species previously (Hoffmann et al. 2008). The strains currently assigned to S. racemosum do not form a monophyletic lineage and therefore likely represent multiple species: as such additional DNA markers should be explored to resolve potential cryptic species within the strains currently assigned to S. racemosum. The sequenced S. racemosum strain NRRL 2496 is mating type (+). The Mucorales characterized to date contain either a single genetic sex locus encoding high mobility group (HMG) transcriptional regulators that differ by mating type if the species are heterothallic, or two loci with both copies of the genes encoding the HMG-domain proteins if the species are homothallic, as reviewed by Schulz et al. (2016). Examining the genome sequence of strain NRRL 2496 (Mondo et al., 2017a) revealed a candidate sex locus. Comparison of this region to S. contaminatum strain UoMD18-1 defines a similar region, albeit differing in the HMG-domain
6
protein, and therefore the prediction is that UoMD18-1 is mating type (–). Furthermore, no homolog of the opposite mating type gene, sexP, could be identified in the genome sequence by BLAST. Using this information, it is possible to predict the mating type (sex) locus region of the two strains (Fig. 4). The species is presumably heterothallic given the structure of the sex locus; furthermore, no zygospores developed during culturing. Given that S. contaminatum was isolated as a laboratory contaminant, its geographical origin and ecological preferences are unclear. As we have noted previously (Urquhart, Coulon, & Idnurm, 2017), there is considerable potential to explore fungal diversity in Australia due to its history of geographical separation.
Taxonomy
Syncephalastrum contaminatum Urquhart A.S. & A. Idnurm, sp. nov.
Fig 1.
MycoBank no.: MB 830627. Diagnosis: Colonies initially white becoming olive grey with age on potato dextrose agar medium. Sporangiophores (average width 7.3 µm, range 4–11 µm, n = 20) with frequent short lateral branches bearing terminal swellings (average diameter 22.0 µm, range 10–28 µm, n = 10) to which the merosporangia are attached. Merosporangia frequently four-spored but greater numbers also observed. Sporangiospores globose, 4.9 µm average diameter (range 3.5–7.0 µm, n = 20). Differentiated from other Syncephalastrum species by DNA sequences, given the difficulty associated with morphological similarities of species in this genus. Type: AUSTRALIA, Melbourne, Victoria. A dried specimen preserved on a filter paper (Whatman®) comprising of mycelia, sporangia and sporangiospores from an axenic in vitro
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culture on potato dextrose agar (holotype, MEL 2447220; ex-type cultures, UoMD18-1 = CBS 146107 = JMRC:SF 013913 = NRRL 66911). Gene sequences ex-holotype: Whole genome as GenBank BioProject accession PRJNA541789. Etymology: The epithet “contaminatum” (Latin) reflects that the species was isolated as a Habitat and distribution: Unknown.
Disclosure
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).
Acknowledgments
We thank Travis Heafield and Allison Van de Meene for assistance with the electron microscopy. This research was supported financially by the Australasian Mycological Society and the University of Melbourne.
References
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Benjamin, R. K. (1959). The merosporangiferous Mucorales. Aliso, 4, 321–433. Chibucos, M. C., Soliman, S., Gebremariam, T., Lee, H., Daugherty, S., Orvis, J., et al. (2016). An integrated genomic and transcriptomic survey of mucormycosis-causing fungi. Nature Communications, 7, 12218. Corrochano, L. M., Kuo, A., Marcet-Houben, M., Polaino, S., Salamov, A., VillalobosEscobedo, J. M., et al. (2016). Expansion of signal transduction pathways in fungi by extensive genome duplication. Current Biology, 26, 1577–1584. Edgar, R. C. (2004). MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Research, 32, 1792–1797. Grigoriev, I. V., Nikitin, R., Haridas, S., Kuo, A., Ohm, R., Otillar, R., et al. (2014). MycoCosm portal: gearing up for 1000 fungal genomes. Nucleic Acids Research, 42, D699–D704. Hawksworth, D. L., & Lücking, R. (2017). Fungal diversity revisited: 2.2 to 3.8 million species. Microbiology Spectrum, 5, https://doi.org/10.1128/microbiolspec.FUNK-0052-2016. Hoffmann, K., Telle S., Walther G., Eckart M., Kirchmair M., Prillinger H., et al. (2008). Diversity, genotypic identification, ultrastructural and phylogenetic characterization of zygomycetes from different ecological habitats and climatic regions: limitations and utility of nuclear ribosomal DNA barcode markers. In: Y. Gherbawy, R. L. Mach, & M. Rai (Eds.), Current Advances in Molecular Mycology (pp. 263–312). Hauppauge: Nova Science Publishers. Hoffmann, K., Pawłowska J., Walther G., Wrzosek M., de Hoog G. S., Benny G. L., et al. (2013). The family structure of the Mucorales: a synoptic revision based on comprehensive multigene-genealogies. Persoonia, 30, 57–76.
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Huelsenbeck, J. P., & Ronquist, F. (2001). MRBAYES: Bayesian inference of phylogenetic trees. Bioinformatics, 17, 754–755. Kearse, M., Moir, R., Wilson, A., Stones-Havas, S., Cheung, M., Sturrock, S., et al. (2012). Geneious Basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics, 28, 1647–1649. Lastovetsky, O. A., Gaspar, M. L., Mondo, S. J., LaButti, K. M., Sandor, L., Grigoriev, I. V., et al. (2016). Lipid metabolic changes in an early divergent fungus govern the establishment of a mutualistic symbiosis with endobacteria. Proceedings of the National Academy of Sciences USA, 113, 15102–15107. Ma, L. J., Ibrahim, A. S., Skory, C., Grabherr, M. G., Burger, G., Butler, M., et al. (2009). Genomic analysis of the basal lineage fungus Rhizopus oryzae reveals a whole-genome duplication. PLoS Genetics, 5, e1000549. Misra, P. C. (1975). A new species of Syncephalastrum. Mycotaxon, 3, 51–54. Mondo, S. J., Dannebaum, R. O., Kuo, R. C., Louie, K. B., Bewick, A. J., LaButti, K., et al. (2017a). Widespread adenine N6-methylation of active genes in fungi. Nature Genetics, 49, 964–968. Mondo, S. J., Lastovetsky, O. A., Gaspar, M. L., Schwardt, N. H., Barber, C. C., Riley, R., et al. (2017b). Bacterial endosymbionts influence host sexuality and reveal reproductive genes of early divergent fungi. Nature Communications, 8, 1843. Pitkin, J. W., Panaccione, D. G., & Walton, J. D. (1996). A putative cyclic peptide efflux pump encoded by the TOXA gene of the plant-pathogenic fungus Cochliobolus carbonum. Microbiology, 142, 1557–1565.
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Schipper, M. A. A., & Stalpers, J. A. (1983). Spore ornamentation and species concept in Syncephalastrum. Persoonia, 12, 81–85. Schoch, C. L., Seifert, K. A., Huhndorf, S., Robert, V., Spouge, J. L., Levesque, C. A., et al. (2012). Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for Fungi. Proceedings of the National Academy of Sciences USA, 109, 6241–6216. Schulz, E., Wetzel, J., Burmester, A., Ellenberger, S., Siegmund, L., & Wöstemeyer, J. (2016). Sex loci of homothallic and heterothallic Mucorales. Endocytobiosis and Cell Research, 27, 39–57. Schwartze, V. U., Winter, S., Shelest, E., Marcet-Houben, M., Horn, F., Wehner, S., et al. (2014). Gene expansion shapes genome architecture in the human pathogen Lichtheimia corymbifera: an evolutionary genomics analysis in the ancient terrestrial mucorales (Mucoromycotina). PLoS Genetics, 10, e1004496. Uehling, J., Gryganskyi, A., Hameed, K., Tschaplinski, T., Misztal, P. K., Wu, S., et al. (2017). Comparative genomics of Mortierella elongata and its bacterial endosymbiont Mycoavidus cysteinexigens. Environmental Microbiology, 19, 2964–2983. Urquhart, A. S., Coulon, P. M. L., & Idnurm, A. (2017). Pilaira australis sp. nov. (Mucorales, Mucoromycota) isolated from emu faeces in Australia. Phytotaxa, 329, 277–283. Vitale, R. G., de Hoog, G. S., Schwarz, P., Dannaoui, E., Deng, S., Machouart, M., et al. (2012). Antifungal susceptibility and phylogeny of opportunistic members of the order Mucorales. Journal of Clinical Microbiology, 50, 66–75. Vu, D., Groenewald, M., de Vries, M., Gehrmann, T., Stielow, B., Eberhardt, U., et al. (2019). Large-scale generation and analysis of filamentous fungal DNA barcodes boosts
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coverage for kingdom fungi and reveals thresholds for fungal species and higher taxon delimitation. Studies in Mycology, 92, 135–154. Walther, G., Pawłowska, J., Alastruey-Izquierdo, A., Wrzosek, M., Rodriguez-Tudela, J. L., Dolatabadi, S., et al. (2013). DNA barcoding in Mucorales: an inventory of biodiversity. Persoonia, 30, 11–47. Wang, D., Wu, R., Xu, Y., & Li, M. (2013). Draft genome sequence of Rhizopus chinensis CCTCCM201021, used for brewing traditional Chinese alcoholic beverages. Genome Announcements, 1, e0019512. Willis, K. J. (2018). State of the World's Fungi 2018. Kew: Royal Botanic Gardens. Zerbino, D. R., & Birney, E. (2008). Velvet: algorithms for de novo short read assembly using de Bruijn graphs. Genome Research, 18, 821–829. Zheng, R. Y., Chen G. Q., & Hu, F. M. (1988). Monosporous varieties of Syncephalastrum. Mycosystema, 1, 35–52.
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Figure legends
Fig. 1 – Morphological properties of Syncephalastrum contaminatum (strain UoMD18-1) cultured on potato dextrose agar. Light microscopy images of sporangiospores (A) and terminal enlargement born on short lateral branch from the main sporangiophore (B). Scanning electron micrograph showing attachment of the merosporangia to the terminal enlargement of the sporangiosphore, which is deflated as a result of sample preparation (C). Obverse and reverse of colony growing on a 9 cm diam PDA plate after 7 d (D and E). Bars: A, B 50 µm; C 10 µm.
Fig. 2 – Phylogeny of species in the Mucorales places strain UoMD18-1 within the genus Syncephalastrum. Phylogeny based on a 10.5 kb nucleotide alignment from a concatenation of eight gene fragments was inferred using a Baysian approach implemented through MrBayes. Families indicated are based on previous work (Hoffmann et al., 2013), with Mortierella elongata used as the outgroup.
Fig. 3 – Phylogeny of the ITS regions supports the separation of Syncephalastrum contaminatum from the other described Syncephalastrum species. Phylogeny was inferred using a Baysian approach implemented through MrBayes. Sequences other than from S. contaminatum were obtained from GenBank (Hoffmann et al., 2008; Vitale et al., 2012; Walther et al., 2013; Vu et al., 2019). Absidia idahoensis and Zychaea mexicana are used as outgroups.
Fig. 4 – Comparison of the predicted sex loci of Syncephalastrum contaminatum strain UoMD18-1 (–) and S. racemosum strain NRRL 2496 (+). Each strain contains either the gene
13
encoding the SexM or SexP HMG-domain protein, flanked by conserved genes (smcA and rnhA). There is sufficient similarity in sequence to provide a prediction about the maximum size of the idiomorphic region (grey bars) in each strain. Dashes separate intervals of 500 bp.
14
• • •
New species Syncephalastrum contaminatum was isolated as a laboratory contaminant. A draft whole genome sequence was generated for this zygomycete. The species differs from others in Syncephalastrum based on DNA sequence.
S1340-3540(20)30018-8
DOI:
https://doi.org/10.1016/j.myc.2020.02.003
Reference:
MYC 480
To appear in:
Mycoscience
Received Date: 2 September 2019 Revised Date:
17 February 2020
Accepted Date: 19 February 2020
Please cite this article as: Urquhart AS, Idnurm A, Syncephalastrum contaminatum, a new species in the Mucorales from Australia, Mycoscience (2020), doi: https://doi.org/10.1016/j.myc.2020.02.003. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © [Copyright year] Published by Elsevier B.V. on behalf of the Mycological Society of Japan.
Short Communication
Syncephalastrum contaminatum, a new species in the Mucorales from Australia
Andrew S. Urquhart a, Alexander Idnurm a*
a
School of BioSciences, University of Melbourne, VIC 3010, Australia
* Corresponding author. School of BioSciences, University of Melbourne, VIC 3010, Australia. E-mail address: [email protected] (A. Idnurm).
Text: 14 pages; tables: 0; figures 4
1
Abstract
A new species is described in the Mucorales family Syncephalastraceae: Syncephalastrum contaminatum, isolated as an in vitro culture from a laboratory contaminant. The species has variable copies of the internal transcribed spacer (ITS) regions, requiring cloning of these regions prior to Sanger sequencing before subsequent use in phylogenetic comparisons with other fungi. The genome of the strain was sequenced using short paired-reads to yield a draft genome of 28.6 Mb. Syncephalastrum contaminatum is distinguished by diverse DNA sequences at several loci from the other species of Syncephalastrum, including only 81% sequence identity with its ITS regions to that of S. racemosum. Its merosporangium produces four or more asexual spores and the genome sequencing information suggests that the species is heterothallic. The identification of this species highlights the limited knowledge about the early lineages of fungi both in Australia and globally.
Keywords: mating type, Merosporangium, Mucoromycota, phylogenomics, Syncephalastrum verruculosum, Zygomycete
2
Although there may be many million fungal species and many are being described on a regular basis (Hawksworth & Lücking, 2017), few species outside of the Ascomycota and Basidiomycota phyla are described each year. For example, in an estimate from the Royal Botanic Gardens Kew just 24 non-Dikarya species were described in 2017 (Willis, 2018). There are a number of reasons for this discrepancy that are not necessarily a reflection of species paucity. One is the limit in resources available to identify and then describe this biodiversity. A second challenge is that the internal transcribed spacer (ITS) regions associated with the ribosomal RNA genes, which are used extensively as a phylogenetic marker (Schoch et al., 2012), can be variable in the non-Dikarya lineages thereby hampering rapid identification. In this study we identify a new Mucorales species in the genus Syncephalastrum (Syncephalastraceae). Syncephalastrum is easy to recognize because of its distinctive merosporangium that produces the asexual spores. A number of Syncephalastrum species have been described; however, there is considerable intraspecific variation in morphology between strains and hence those preparing monographs on the genus have consistently placed these strains into S. racemosum Cohn (Benjamin, 1959; Schipper, & Stalpers, 1983). A second species that produces a merosporangium containing a single spore, S. monosporum R.Y. Zheng, G.Q. Chen & F.M. Hu, is also recognised (Zheng, Chen, & Hu, 1988). While aiming to culture a species in the Cordycipitaceae family, a contaminating Mucorales strain, as initially determined by its rapid growth of coenocytic hyphae, was isolated in culture on potato dextrose agar (Fig. 1). The strain (named UoMD18-1) produced the striking asexual sporulation structure, termed the merosporangium, that is characteristic of species of the genus Syncephalastrum.
3
Genomic DNA was extracted from freeze-dried mycelium of strain UoMD18-1 (Pitkin, Panaccione, & Walton, 1996). The internal transcribed spacer (ITS) regions were amplified. Direct PCR and Sanger sequencing of the ITS regions did not produce a clean chromatogram, indicating a mix of amplicons. The ITS amplicons were therefore cloned into the pCR2.1-TOPO plasmid (Invitrogen), multiple plasmids were purified, and the inserts sequenced. Comparison of the sequences revealed three variations of ITS (submitted to GenBank as accessions MK799840– MK799842). The genome of strain UoMD18-1 was sequenced to obtain additional sequences to construct a multigene phylogeny and as a resource for others studying the evolution and genomics of species in the Mucorales, by using Illumina short (125 nucleotides) paired-reads on a HiSeq2500 at the Australian Genome Research Facility. Just over 8 million pairs were sequenced. The reads were assembled in Geneious (Kearse et al., 2012) using Velvet (Zerbino & Birney, 2008) with a k-mer of 75, selected after a Velvet optimization analysis, and a cut off minimum contig length of 1 kb. Three hundred seventy three contigs were produced, for a total size of 26.8 Mbp. The original reads were remapped onto this assembly, with 92% of the total aligning. The raw sequences and assembly are available from GenBank under BioProject PRJNA541789. The morphological identification of strain UoMD18-1 within Syncephalastrum based on its production of merosporangia was supported by a multi-gene phylogeny including Mucorales species for which full genome sequences are available from MycoCosm (Ma et al., 2009; Wang, Wu, Xu, & Li, 2013; Grigoriev et al., 2014; Schwartze et al., 2014; Chibucos et al., 2016; Corrochano et al., 2016; Lastovetsky et al. 2016; Mondo et al., 2017a, 2017b; Uehling et al., 2017). MycoCosm provides a comparison of genes using a Markov cluster algorithm (MCL);
4
using the “Mucoromycotina MCL. 2984” (created 28 Feb 2019) dataset, parameters were set to identify genes present as single copies in 54 Mucorales species, and these then used to search the genome sequence of strain UoMD18-1 (referred hereafter as the novel species S. contaminatum – see below) by BLAST for homologs. Eight (corresponding to protein IDs in the S. racemosum genome 530022, 521890, 563448, 493224, 496917, 483206, 483391 and 440562) were chosen based on having a phylogenetically informative level of sequence variation, aligned using MUSCLE (Edgar, 2004), and then trimmed manually to remove areas of uncertain nucleotide alignment. This resulted in a final alignment of 10.5 kb, which is deposited in TreeBASE (accession URL: http://purl.org/phylo/treebase/phylows/study/TB2:S25812). Phylogenies based on Bayesian inference were generated using MrBayes using the general time reversible (GTR) substitution model, gamma variation, and sequences from Mortierella elongata Linnem. as the outgroup (Huelsenbeck & Ronquist, 2001). This eight-gene phylogeny (Fig. 2) placed the new strain as sister to S. racemosum strain NRRL 2496, which was previously sequenced (Mondo et al., 2017a). Given the opinion that the variability in growth and spore numbers in the merosporangium for S. racemosum render traditional morphological traits uninformative (Benjamin, 1959), we investigated DNA sequence differences between S. contaminatum and previously described species to determine if S. contaminatum was indeed a new species. The ITS and large subunit (LSU) matches to the nearest described Syncephalastrum species assessed by BLAST were 76% and 97%, respectively, which is a greater level of divergence than expected due to intraspecific variability (Vu et al., 2019). Furthermore, the actin and transcription elongation factor 1 alpha (TEF) regions showed only 92% and 98% similarity, respectively, to the closest Syncephalastrum deposited in GenBank. An ITS sequence deposited in GenBank
5
(MG751268) from an undescribed Syncephalastrum from Brazil shows highest similarity to S. contaminatum (93%), and might represent a second isolate of this species. Examination of the ITS phylogeny (Fig. 3), whose alignment is deposited in TreeBASE (accession URL: http://purl.org/phylo/treebase/phylows/study/TB2:S25812), supports the separation of S. verruculosum P.C. Misra (Misra, 1975) from the other described species of Syncephalastrum, despite Schipper and Staplers (1983) considering it to be a synonym of S. racemosum. As such, we suggest that the genus Syncephalastrum should now be considered to include four species: S. racemosum, S. monosporum, S. verruculosum and Syncephalastrum contaminatum sp. nov. However, there is likely greater undescribed species diversity in the genus Syncephalastrum already represented in culture collections owing to the historical difficulty associated with morphological species delimitation in this species. For example, FSU 6155 is clearly separated on our tree and has been noted as a possible cryptic species previously (Hoffmann et al. 2008). The strains currently assigned to S. racemosum do not form a monophyletic lineage and therefore likely represent multiple species: as such additional DNA markers should be explored to resolve potential cryptic species within the strains currently assigned to S. racemosum. The sequenced S. racemosum strain NRRL 2496 is mating type (+). The Mucorales characterized to date contain either a single genetic sex locus encoding high mobility group (HMG) transcriptional regulators that differ by mating type if the species are heterothallic, or two loci with both copies of the genes encoding the HMG-domain proteins if the species are homothallic, as reviewed by Schulz et al. (2016). Examining the genome sequence of strain NRRL 2496 (Mondo et al., 2017a) revealed a candidate sex locus. Comparison of this region to S. contaminatum strain UoMD18-1 defines a similar region, albeit differing in the HMG-domain
6
protein, and therefore the prediction is that UoMD18-1 is mating type (–). Furthermore, no homolog of the opposite mating type gene, sexP, could be identified in the genome sequence by BLAST. Using this information, it is possible to predict the mating type (sex) locus region of the two strains (Fig. 4). The species is presumably heterothallic given the structure of the sex locus; furthermore, no zygospores developed during culturing. Given that S. contaminatum was isolated as a laboratory contaminant, its geographical origin and ecological preferences are unclear. As we have noted previously (Urquhart, Coulon, & Idnurm, 2017), there is considerable potential to explore fungal diversity in Australia due to its history of geographical separation.
Taxonomy
Syncephalastrum contaminatum Urquhart A.S. & A. Idnurm, sp. nov.
Fig 1.
MycoBank no.: MB 830627. Diagnosis: Colonies initially white becoming olive grey with age on potato dextrose agar medium. Sporangiophores (average width 7.3 µm, range 4–11 µm, n = 20) with frequent short lateral branches bearing terminal swellings (average diameter 22.0 µm, range 10–28 µm, n = 10) to which the merosporangia are attached. Merosporangia frequently four-spored but greater numbers also observed. Sporangiospores globose, 4.9 µm average diameter (range 3.5–7.0 µm, n = 20). Differentiated from other Syncephalastrum species by DNA sequences, given the difficulty associated with morphological similarities of species in this genus. Type: AUSTRALIA, Melbourne, Victoria. A dried specimen preserved on a filter paper (Whatman®) comprising of mycelia, sporangia and sporangiospores from an axenic in vitro
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culture on potato dextrose agar (holotype, MEL 2447220; ex-type cultures, UoMD18-1 = CBS 146107 = JMRC:SF 013913 = NRRL 66911). Gene sequences ex-holotype: Whole genome as GenBank BioProject accession PRJNA541789. Etymology: The epithet “contaminatum” (Latin) reflects that the species was isolated as a Habitat and distribution: Unknown.
Disclosure
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).
Acknowledgments
We thank Travis Heafield and Allison Van de Meene for assistance with the electron microscopy. This research was supported financially by the Australasian Mycological Society and the University of Melbourne.
References
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Figure legends
Fig. 1 – Morphological properties of Syncephalastrum contaminatum (strain UoMD18-1) cultured on potato dextrose agar. Light microscopy images of sporangiospores (A) and terminal enlargement born on short lateral branch from the main sporangiophore (B). Scanning electron micrograph showing attachment of the merosporangia to the terminal enlargement of the sporangiosphore, which is deflated as a result of sample preparation (C). Obverse and reverse of colony growing on a 9 cm diam PDA plate after 7 d (D and E). Bars: A, B 50 µm; C 10 µm.
Fig. 2 – Phylogeny of species in the Mucorales places strain UoMD18-1 within the genus Syncephalastrum. Phylogeny based on a 10.5 kb nucleotide alignment from a concatenation of eight gene fragments was inferred using a Baysian approach implemented through MrBayes. Families indicated are based on previous work (Hoffmann et al., 2013), with Mortierella elongata used as the outgroup.
Fig. 3 – Phylogeny of the ITS regions supports the separation of Syncephalastrum contaminatum from the other described Syncephalastrum species. Phylogeny was inferred using a Baysian approach implemented through MrBayes. Sequences other than from S. contaminatum were obtained from GenBank (Hoffmann et al., 2008; Vitale et al., 2012; Walther et al., 2013; Vu et al., 2019). Absidia idahoensis and Zychaea mexicana are used as outgroups.
Fig. 4 – Comparison of the predicted sex loci of Syncephalastrum contaminatum strain UoMD18-1 (–) and S. racemosum strain NRRL 2496 (+). Each strain contains either the gene
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encoding the SexM or SexP HMG-domain protein, flanked by conserved genes (smcA and rnhA). There is sufficient similarity in sequence to provide a prediction about the maximum size of the idiomorphic region (grey bars) in each strain. Dashes separate intervals of 500 bp.
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• • •
New species Syncephalastrum contaminatum was isolated as a laboratory contaminant. A draft whole genome sequence was generated for this zygomycete. The species differs from others in Syncephalastrum based on DNA sequence.