Khoyollomyces ramosus Hanafy, Vikram B. Lanjekar, Prashant K. Dhakephalkar, T.M. Callaghan, Dagar, G.W. Griff., Elshahed & N.H. Youssef, sp. nov.

MycoBank number: MB 830742; Index Fungorum number: IF 830742; Facesoffungi number: FoF;

Typification: The holotype (FIG. 8j) was derived from the following: USA. OKLAHOMA: Oklahoma City, 35.524°N, 97.472°W, ~300 m asl, 3-d-old culture of isolate ZS-33, originally isolated from freshly deposited feces of a Grevy’s zebra (Equus grevyi), May 2018, Radwa Hanafy. Ex-type strain: ZS-33. GenBank: MK881981 (D1–D2 28S rDNA).

Etymology: The species epithet “ramosus” (Latin for branched) refers to the observed branched sporangiophores bearing two to four sporangia in K. ramosus type strain ZS-33 (FIG. 8i–k).

Obligate anaerobic fungus that produces spherical monoflagellate zoospores. Zoospores encyst and germinate producing germ tube that develops into a highly branched anucleate rhizoidal system. Both narrow, 0.5 to 2.5 µm W, and broad hyphae, 3 to 12.5 µm W, are produced; intercalary swellings are frequently encountered in the broad hyphae. Both endogenous and exogenous sporangia were observed. Endogenous sporangia vary in shape and size, with small endogenous sporangia mainly subglobose (ranging from 20 to 60 μm diam) and large endogenous (from 80 to 160 μm L × from 35 to 65 μm W) sporangia mainly ellipsoidal. Exogenous sporangia ranged in size between 80 and 270 μm L × 35 and 85 μm W and display a wide range of morphologies, e.g.,   heart-shaped,   ovoid,   and   pyriform.   Displays a multisporangiate thallus, with the majority of sporangiophores being branched and bearing two to four sporangia. Unbranched sporangiophores with single sporangia are less frequently encountered (approximately 30% of observed sporangiophores). Zoospores are liber- ated through a wide apical pore at the top of the sporangia. The sporangia stay intact after the discharge. Mature sporangia frequently detach from hyphae or sporangiophores. Produces small, yellow to yellowish brown, irregularly shaped colonies on agar. In liquid media, the fungal growth is loose and exhibited a sand-like appearance. The clade is defined by the sequences MK882019 (ITS1) and MK881981 (D1–D2 28S rDNA).

Additional specimens examined: USA. OKLAHOMA: Oklahoma City, 35.524°N, 97.472°W, ~300 m asl, 3-d-old culture of isolates ZC-31, ZC-32, ZC-33, ZC-41, ZC-42, ZC-43, ZC-51, ZC-53, ZS-21, ZS-22, ZS-31, ZS-32, ZS-41, ZS-42, and ZS-43, originally isolated from freshly deposited feces of a Grevy’s zebra (Equus grevyi), May 2018, Radwa Hanafy,   GenBank   (D1–D2   28S   rDNA):   ZC-31 (MK881979), ZC-32 (MK881980), ZC-33 (MK881981), ZC-41 (MK881982), ZC-42 (MK881983), ZC-43 (MK881984), ZC-51 (MK881985), ZC-53 (MK881986), ZS-21 (MK881987), ZS-22 (MK881988), ZS-31 (MK881989), ZS-32 (MK881990), ZS-41 (MK881992), ZS-42 (MK881993), and ZS-43 (MK881994). WALES: Aberystwyth; 52.4156°N, −3.8878°W, 3-d-old culture of isolates HoCal4.A2, HoCal4.A2.2, and HoCal4.A4, origin- ally isolated from freshly deposited feces of one horse (Equus caballus), Aug 2013, and strains Tmc003.6a and TMC3.6b from freshly deposited feces of another host in the same location, Nov 2013, Tony Callaghan.

Figure 8. Microscopic features of Khyollomyces ramosus (clade 6, zebra-horse) strain ZS-33 (Z-OK) (a–q) and distinct resting stage structure from strain HoCal4.A2.2 (r). Light (a–e, g, i–j, l, q) and scanning electron (f, h, k, m, n–p) micrographs. d. DAPI staining for visualizing nuclei using a fluorescence microscope equipped with a Brightline DAPI high-contrast filter set. e. Overlay image. a. A monoflagellate zoospore. b. Zoospore cyst after shedding of the flagellum. c. Germinating zoospore cyst producing a germ tube (arrow). d–e. Monocentric thalli, with nuclei occurring in sporangia, not in rhizoids or sporangiophores. f. Hyphal structures with intercalary swellings in wide hyphae (arrows). g–h. Endogenous sporangial development: g. Young subglobose sporangium with single rhizoidal system. i. Mature ellipsoidal sporangium with two main rhizoidal systems. i–m. Exogenous sporangial development: i. Multisporangiate thallus with two sporangia. j. Multisporangiate thallus with four sporangia. k. Heart-shaped sporangium. l. Ovoid sporangium (S) on a wide flattened sporangiophore (Sp). m. Pyriform sporangium. n. Zoospores are released through apical pore. o– p.  Empty sporangia with intact sporangial walls after zoospores discharge. q. Mature sporangia detached from hyphae or sporangiophores. r. Resting stages from strain HoCal4.A2.2. Bars: a–c, f–g = 20 μm; d–e, k, m–q = 50 μm; h–j, l = 100 μm.