Aestipascuomyces dupliciliberans Marcus Stabel, Radwa Hanafy, Tabea Schweitzer, Meike Greif, Habibu Aliyu, Veronika Flad, Diana Young, Michael Lebuhn, Mostafa Elshahed, Katrin Ochsenreither, and Noha Youssef, sp. nov.

MycoBank number: MB 837526; Index Fungorum number: IF 837526; Facesoffungi number: FoF;

Typification: The holotype shown in Figure 2b in this manuscript is derived from the following: U.S.A. TEXAS: Sutton county, 30.591 N and 100.138 W ~300 m above sea level, 3-day-old culture of strain R4, which is isolated from the frozen rumen content of a female aoudad sheep (Ammotragus lervia), collected in April 2018 by Mr. Jim Austin. Ex-type strain R4 is stored on solid agar media at 39 ◦C at Oklahoma State University, Department of Microbiology and Molecular Genetics.

Etymology: duplicus = Latin for double, liberans = Latin for liberating or releasing. The species epithet highlights the dual zoospore release mechanisms.

An obligate anaerobic fungus that produces globose (5–14 µm in diameter) zoospores with 7–20 flagella (19–36 µm long). Zoospores germinate into determinate monocentric thalli with highly branched anucleated rhizoids that lack constriction and intercalary swellings. Both endogenous and exogenous thalli developments are observed. Mature endogenous sporangia are mainly rhomboid (30–70 µm L x 40–85 µm W) and elongated (25–90 µm L x 15–40 µm W). Mature exogenous sporangia range in size between 40 and 90 µm (L) and 15 and 35 µm (W), and display a variety of shapes including obpyriform, ellipsoid, globose, constricted ellipsoid, and ovoid. Sporangiophores vary in length between 10 and 300 µm. Wide flattened sporangiophores and sporangiophores ending with sub-sporangial swellings are occasionally encountered. Zoospores are released either through an apical pore or through the lysis of the sporangial wall. Sporangial walls remain intact or are completely collapsed after zoospore release. The fungus produces white filamentous colonies with a white center of sporangia (2–5 mm diameter) on agar roll tubes and heavy fungal biofilm-like growth that does nott attach to the tube’s glass surface in liquid media. The strain is defined by the sequences MW019494- MW019497 (for ITS-1, 5.8S rDNA, ITS2, D1–D2 28S rDNA).

Additional specimens examined: Radwa Hanafy strains R1 (MW019480, MW019481, MW019483, MW019484, MW019485, MW019486), R2 (MW019482, MW019487), R3 (MW019488, MW019490, MW019491, MW019492, MW019494), and R5 (MW019498, MW019489, MW019499, MW019479, MW019500) (GenBank accession number of clones in parenthesis), isolated from the same frozen rumen content of a female aoudad sheep (Ammotragus lervia) from which the type strain was isolated, in April 2018. Marcus Stabel strain A252 (MW049132-MW049145) (GenBank accession number of clones in parenthesis), isolated from the feces of an alpaca (Vicugna pacos) from the Karlsruhe Zoo, Karlsruhe, Germany in April 2019.

Author Contributions: Conceptualization, M.S.E., N.H.Y., and K.O.; methodology and formal analysis, M.S., T.S., M.G., H.A., and R.A.H.; resources, M.S.E., N.H.Y., and K.O.; writing—original draft preparation, M.S. and R.A.H.; writing—review and editing, V.F., D.Y., M.L., M.S.E., H.A., N.H.Y., and K.O.; supervision, M.S.E, N.H.Y., D.Y., V.F., M.L., H.A. and K.O.; project administration, M.S.E., N.H.Y., M.L., and K.O. All authors have read and agreed to the published version of the manuscript.

Funding: This research was funded by the National Science Foundation, grant numbers 1557102 and 2029478 to MSE and NHY, and “Landesgraduiertenförderung”, part of the bioeconomy graduate program BBW ForWerts for MS. We acknowledge support by the KIT-Publication Fund of the Karlsruhe Institute of Technology.

Acknowledgments: We thank Tammy and Jim Austin for collecting the aoudad sheep samples in Texas, USA. We also kindly thank the zoo in Karlsruhe for providing the needed feces for the isolation. We kindly thank Reinhardt Fischer and Christian Streng for their help with fluorescence microscopy. We thank Tony Callaghan and Gareth Griffith, Aberystwyth University, for sharing their method for isolation and cultivation of anaerobic fungi.

Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figure 2. Microscopic features of Aestipascuomyces dupliciliberans type strain R4.   Light (ah, kn, and pq), fluorescence (c and e) and scanning electron (i, j, and o) micrographs are shown. (b,c) and (d,e) each depict the same field with (c) and (e) showing the fluorescence field, and (b) and (d) showing the overlay of fluorescence and phase contrast micrographs. (a) A spherical polyflagellated zoospore. (be) Monocentric thalli; nuclei were observed in sporangia, not in rhizoids or sporangiophore. (fh) Endogenous sporangia: (f) ovoid sporangium with single rhizoidal system, (g) rhomboid sporangium with two adjacent rhizoidal systems, and (h) elongated sporangium. (in) Exogenous sporangia: (i) obpyriform sporangium on a flattened sporangiophore, (j) ellipsoidal sporangium on a long sporangiophore, (k) globose sporangium with sub-sporangial swelling and tightly-constricted neck, (l) ovoid sporangium with broad neck and wide port, (m) mature ovoid sporangium full of zoospores, and (n) constricted ellipsoidal sporangium. (oq) Zoospore release mechanisms: (o) an empty sporangium with intact wall after zoospore release through an apical pore (arrow), (p) zoospore release through rupturing the sporangial wall, and (q) collapse and disintegration of the sporangial wall after zoospore release. (SW), sub-sporangial swelling; (N), neck. Bar = 20 µm (a, fh, kn, and pq). Bar = 50 µm (be, i, and o). Bar = 100 µm (j).