1887

Abstract

The anaerobic gut fungi (AGF, ) represent a basal zoosporic phylum within the kingdom . Twenty genera are currently described, all of which were isolated from the digestive tracts of mammalian herbivores. Here, we report on the isolation and characterization of novel AGF taxa from faecal samples of tortoises. Twenty-nine fungal isolates were obtained from seven different tortoise species. Phylogenetic analysis using the D1/D2 region of the LSU rRNA gene, ribosomal internal transcribed spacer 1, and RNA polymerase II large subunit grouped all isolates into two distinct, deep-branching clades (clades T and B), with a high level of sequence divergence to their closest cultured relative (). Average amino acid identity values calculated using predicted peptides from the isolates’ transcriptomes ranged between 60.80–66.21  % (clade T), and 61.24–64.83  % (clade B) when compared to all other AGF taxa; values that are significantly below recently recommended thresholds for genus (85%) and family (75%) delineation in the . Both clades displayed a broader temperature growth range (20–45 °C, optimal 30 °C for clade T, and 30–42 °C, optimal 39 °C for clade B) compared to all other AGF taxa. Microscopic analysis demonstrated that strains from both clades produced filamentous hyphae, polycentric rhizoidal growth patterns, and monoflagellated zoospores. Isolates in clade T were characterized by the production of unbranched, predominantly narrow hyphae, and small zoospores, while isolates in clade B were characterized by the production of multiple sporangiophores and sporangia originating from a single central swelling resulting in large multi-sporangiated structures. Based on the unique phylogenetic positions, AAI values, and phenotypic characteristics, we propose to accommodate these isolates into two novel genera ( and ), and species ( and ) within the order . The type species are strains T130A () and B1.1 ().

Funding
This study was supported by the:
  • National Science Foundation (Award 2029478)
    • Principle Award Recipient: CarrieJ. Pratt
  • This is an open-access article distributed under the terms of the Creative Commons Attribution License. This article was made open access via a Publish and Read agreement between the Microbiology Society and the corresponding author’s institution.
Loading

Article metrics loading...

/content/journal/ijsem/10.1099/ijsem.0.005921
2023-05-30
2024-06-18
Loading full text...

Full text loading...

/deliver/fulltext/ijsem/73/5/ijsem005921.html?itemId=/content/journal/ijsem/10.1099/ijsem.0.005921&mimeType=html&fmt=ahah

References

  1. Hess M, Paul SS, Puniya AK, van der Giezen M, Shaw C et al. Anaerobic fungi: past, present, and future. Front Microbiol 2020; 11:584893 [View Article] [PubMed]
    [Google Scholar]
  2. Orpin CG. Studies on the rumen flagellate Neocallimastix frontalis. J Gen Microbiol 1975; 91:249–262 [View Article] [PubMed]
    [Google Scholar]
  3. Hanafy RA, Dagar SS, Griffith GW, Pratt CJ, Youssef NH et al. Taxonomy of the anaerobic gut fungi (Neocallimastigomycota): a review of classification criteria and description of current taxa. Int J Syst Evol Microbiol 2022; 72: [View Article] [PubMed]
    [Google Scholar]
  4. Le M, Raxworthy CJ, McCord WP, Mertz L. A molecular phylogeny of tortoises (Testudines: Testudinidae) based on mitochondrial and nuclear genes. Mol Phylogenet Evol 2006; 40:517–531 [View Article] [PubMed]
    [Google Scholar]
  5. Barboza PS. Digesta passage and functional anatomy of the digestive tract in the desert tortoise (Xerobates agassizii). J Comp Physiol B 1995; 165:193–202 [View Article] [PubMed]
    [Google Scholar]
  6. Hatt J-M, Clauss M, Gisler R, Liesegang A, Wanner M. Fiber digestibility in juvenile Galapagos tortoises (Geochelone nigra) and implications for the development of captive animals. Zoo Biol 2005; 24:185–191 [View Article]
    [Google Scholar]
  7. Yuan ML, Dean SH, Longo AV, Rothermel BB, Tuberville TD et al. Kinship, inbreeding and fine-scale spatial structure influence gut microbiota in a hindgut-fermenting tortoise. Mol Ecol 2015; 24:2521–2536 [View Article] [PubMed]
    [Google Scholar]
  8. Pratt CJ, Chandler E, Youssef NH, Elshahed M. Uncovering novel hosts for anaerobic gut fungi. In Presented at the 90th meeting of the Mycological Society of America Gainesville, FL July 10th-13th 2022
    [Google Scholar]
  9. Calkins S, Elledge NC, Hanafy RA, Elshahed MS, Youssef N. A fast and reliable procedure for spore collection from anaerobic fungi: application for RNA uptake and long-term storage of isolates. J Microbiol Methods 2016; 127:206–213 [View Article] [PubMed]
    [Google Scholar]
  10. Hungate RE. A roll tube method for cultivation of strict anaerobes. Meth Microbiol 1969; 3:117–132
    [Google Scholar]
  11. Theodorou MK, Williams BA, Dhanoa MS, McAllan AB, France J. A simple gas production method using A pressure transducer to determine the fermentation kinetics of ruminant feeds. Anim Feed Sci Technol 1994; 48:185–197 [View Article]
    [Google Scholar]
  12. Hanafy RA, Elshahed MS, Liggenstoffer AS, Griffith GW, Youssef NH. Pecoramyces ruminantium, gen. nov., sp. nov., an anaerobic gut fungus from the feces of cattle and sheep. Mycologia 2017; 109:231–243 [View Article] [PubMed]
    [Google Scholar]
  13. Wang X, Liu X, Groenewald JZ. Phylogeny of anaerobic fungi (phylum Neocallimastigomycota), with contributions from yak in China. Antonie van Leeuwenhoek 2017; 110:87–103 [View Article] [PubMed]
    [Google Scholar]
  14. Elshahed MS, Hanafy RA, Cheng Y, Dagar SS, Edwards JE et al. Characterization and rank assignment criteria for the anaerobic fungi (Neocallimastigomycota). Int J Syst Evol Microbiol 2022; 72: [View Article] [PubMed]
    [Google Scholar]
  15. Schloss PD, Westcott SL, Ryabin T, Hall JR, Hartmann M et al. Introducing mothur: open-source, platform-independent, community-supported software for describing and comparing microbial communities. Appl Environ Microbiol 2009; 75:7537–7541 [View Article] [PubMed]
    [Google Scholar]
  16. Katoh K, Rozewicki J, Yamada KD. MAFFT online service: multiple sequence alignment, interactive sequence choice and visualization. Brief Bioinform 2019; 20:1160–1166 [View Article] [PubMed]
    [Google Scholar]
  17. Hoang DT, Chernomor O, von Haeseler A, Minh BQ, Vinh LS. UFBoot2: improving the ultrafast bootstrap approximation. Mol Biol Evol 2018; 35:518–522 [View Article] [PubMed]
    [Google Scholar]
  18. Nguyen L-T, Schmidt HA, von Haeseler A, Minh BQ. IQ-TREE: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Mol Biol Evol 2015; 32:268–274 [View Article] [PubMed]
    [Google Scholar]
  19. Fu L, Niu B, Zhu Z, Wu S, Li W. CD-HIT: accelerated for clustering the next-generation sequencing data. Bioinformatics 2012; 28:3150–3152 [View Article] [PubMed]
    [Google Scholar]
  20. Hanafy RA, Wang Y, Stajich JE, Pratt CJ, Youssef NH et al. Phylogenomic analysis of the Neocallimastigomycota: proposal of Caecomycetaceae fam. nov., Piromycetaceae fam. nov., and emended description of the families Neocallimastigaceae and Anaeromycetaceae. Microbiology 2022 [View Article]
    [Google Scholar]
  21. Rodriguez-R LM, Konstantinidis KT. The enveomics collection: a toolbox for specialized analyses of microbial genomes and metagenomes. PeerJ Preprints 2016 [View Article]
    [Google Scholar]
  22. Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M et al. Fiji: an open-source platform for biological-image analysis. Nat Methods 2012; 9:676–682 [View Article] [PubMed]
    [Google Scholar]
  23. Meili C, Jones A, Arreola A, Habel J, Pratt C et al. Patterns and determinants of the global herbivorous mycobiome. bioRxiv [View Article]
    [Google Scholar]
  24. Mackie RI, Rycyk M, Ruemmler RL, Aminov RI, Wikelski M. Biochemical and microbiological evidence for fermentative digestion in free-living land iguanas (Conolophus pallidus) and marine iguanas (Amblyrhynchus cristatus) on the Galápagos archipelago. Physiol Biochem Zool 2004; 77:127–138 [View Article] [PubMed]
    [Google Scholar]
  25. Liggenstoffer AS, Youssef NH, Couger MB, Elshahed MS. Phylogenetic diversity and community structure of anaerobic gut fungi (phylum Neocallimastigomycota) in ruminant and non-ruminant herbivores. ISME J 2010; 4:1225–1235 [View Article] [PubMed]
    [Google Scholar]
  26. Stanford CB, Iverson JB, Rhodin AGJ, Paul van Dijk P, Mittermeier RA et al. Turtles and tortoises are in trouble. Curr Biol 2020; 30:R721–R735 [View Article] [PubMed]
    [Google Scholar]
  27. Hanafy RA, Youssef NH, Elshahed MS. Paucimyces polynucleatus gen. nov, sp. nov., a novel polycentric genus of anaerobic gut fungi from the faeces of a wild blackbuck antelope. Int J Syst Evol Microbiol 2021; 71: [View Article] [PubMed]
    [Google Scholar]
  28. Letcher PM, Powell MJ, Chambers JG, Longcore JE, Churchill PF et al. Ultrastructural and molecular delineation of the Chytridiaceae (Chytridiales). Can J Bot 2005; 83:1561–1573 [View Article]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/ijsem/10.1099/ijsem.0.005921
Loading
/content/journal/ijsem/10.1099/ijsem.0.005921
Loading

Data & Media loading...

Supplements

Supplementary material 1

PDF
This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error