In Press at Mycologia, preliminary version published on April 24, 2015 as doi:10.3852/14-280

Short title: New Rhizophydiales taxa A new family and four new genera in Rhizophydiales (Chytridiomycota) Peter M. Letcher1 Martha J. Powell William J. Davis Department of Biological Sciences, University of Alabama, Tuscaloosa, Alabama 35487 Abstract: Many chytrid phylogenies contain lineages representing a lone taxon or a few organisms. One such lineage in recent molecular phylogenies of Rhizophydiales contained two marine chytrids, Rhizophydium littoreum and Rhizophydium aestuarii. To better understand the relationship between these organisms, we increased sampling such that the R. littoreum/R. aestuarii lineage included 10 strains of interest. To place this lineage in Rhizophydiales, we constructed a molecular phylogeny from partial nuc 28S rDNA D1–D3 domains (28S) of these and 80 additional strains in Rhizophydiales and examined thallus morphology and zoospore ultrastructure of our strains of interest. We also analyzed sequences of the nuc rDNA region encompassing the internal transcribed spacers 1 and 2, along with the 5.8S rDNA (ITS) of our 10 strains of interest to assess sequence similarity and phylogenetic placement of strains within the lineage. The 10 strains grouped together in three well supported clades: (i) Rhizophydium littoreum + Phlyctochytrium mangrovei, (ii) three strains of Rhizophydium aestuarii and (iii) five previously unidentified strains. Light microscopic observations revealed four distinct thallus morphologies, and zoospore ultrastructural analyses revealed four distinct constellations of ultrastructural features. On the bases of morphological, ultrastructural and molecular evidence we place these strains in the new family Halomycetaceae and four new genera (Halomyces, Paludomyces, Ulkenomyces, Paranamyces) in Rhizophydiales.

Copyright 2015 by The Mycological Society of America.

Key words: chytrid, morphology, phylogeny, ultrastructure, zoospore INTRODUCTION Lineages represented by a few strains or a lone taxon or OTU can be found in many molecular phylogenies of chytrids (e.g. James et al. 2006, Letcher et al. 2006). (We use the term “strain” to designate a clonally related individual derived from the pure isolation [“isolate”] of a fungus). Because of limited and sparse sampling, potential diversity of such a lineage remains enigmatic and taxonomy of and within the lineage is uncertain. Such has been the case for two marine chytrids, Rhizophydium littoreum Amon and Rhizophydium aestuarii (Ulken) Amon. These strains have been included in molecular phylogenies of Rhizophydiales Letcher (Letcher et al. 2006, Lepelletier et al. 2014), in which they occurred as sister taxa. However the two strains are divergent in their 28S and ITS regions (Letcher et al. 2006) and occurred in sister lineages (Letcher et al. 2006) instead of as sisters in the same lineage (Lepelletier 2014) and have different zoospore ultrastructures (R. littoreum: Kazama 1972b, Amon 1984; R. aestuarii: Lange and Olson 1977). Because of this molecular divergence and ultrastructural dissimilarity, R. littoreum and R. aestuarii may not be as closely related as phylogenies with limited sampling indicate. They also are not monophyletic with Rhizophydium Schenk in molecular phylogenies (Letcher et al. 2006, Lepelletier et al. 2014) nor are their zoospores similar to those that currently characterize Rhizophydium (Longcore 2004, Letcher et al. 2006). These organisms have remained incertae sedis at the family level. As a chronological summary to illuminate this situation, Kazama (1972a) isolated a phototactic, obligate marine chytrid (designated isolate 71-1-E) from Bryopsis plumosa, a siphonaceous marine alga. When on its host, his isolate had thallus features consistent with the genus Phlyctochytrium Schroeter sensu Sparrow (1960) (an epibiotic, inoperculate sporangium, a

rhizoidal system with an apophysis and an epibiotic resting spore), but when grown on agar the apophysis was difficult to identify. The validity and applicability of rhizoidal system characters separating Phlyctochytrium (apophysate) from Rhizophydium (non-apophysate) has been questioned by numerous investigators (e.g. Couch 1932, Miller 1968, Barr 1969) because of gradation of morphological characters. Nonetheless Kazama tentatively identified his isolate as Phlyctochytrium sp. Ultrastructural analysis of the zoospore of Phlyctochytrium sp. (Kazama 1972b) revealed features that would correspond to the Rhizophydium-type zoospore that Barr (1980) characterized when he began classifying chytrid genera on the basis of zoospore ultrastructure. Also in 1972 Ulken (1972a) isolated on pine pollen a marine chytrid from North Sea estuary sediments, and because it was epibiotic, inoperculate, apophysate and had an epibiotic resting spore placed it in Phlyctochytrium as P. aestuarii Ulken. A strain of this isolate subsequently was deposited in the American Type Culture Collection (ATCC) as ATCC® 26190™. Ulken (1972b) also isolated on pollen and cattle hair another marine chytrid, this one from a mangrove swamp in Brazil and because it manifested morphological characters typical of Phlyctochytrium, yet was morphologically distinct from P. aestuarii and all other described species of Phlyctochytrium, named it Phlyctochytrium mangrovei (as mangrovii) Ulken. A strain of this isolate was deposited as ATCC® 26191™. Lange and Olson (1977) in analyzing zoospore ultrastructure of Ulken's (1972a) P. aestuarii found it to be “almost identical” [sic] to the zoospore Kazama (1972a, b) described for Phlyctochytrium sp. Lange and Olson (1977) did not synonymize the two, perhaps because P. aestuarii had “a less well-developed eyespot-like complex” [sic] (Lange and Olson 1977; “eyespot-like complex” = “microbody-lipid globule complex (MLC) fenestrated cisterna” in current terminology) for example Letcher and Powell

(2014) appressed to a lipid globule, than that of Phlyctochytrium sp., and P. aestuarii was not shown to be phototactic. A decade after Kazama (1972a) isolated his Phlyctochytrium sp. (isolate 71-1-E), Amon (1984) isolated an obligate marine chytrid (designated isolate PC) from Codium sp., a siphonaceous marine alga and in comparing its ultrastructure to that of Kazama's isolate 71-1-E, found them to be similar. Amon (1984), in considering the Rhizophydium type zoospore described by Barr (1980), named Kazama's isolate 71-1-E as Rhizophydium littoreum Amon. A strain of this isolate subsequently was deposited as ATCC® 36100™. Because of zoospore ultrastructural similarities between R. littoreum and P. aestuarii and then-recent changes in generic concepts (Barr 1980), Amon (1984) transferred P. aestuarii to Rhizophydium as Rhizophydium aestuarii (Ulken) Amon. No ultrastructural studies of P. mangrovei were performed; thus no comparison of the ultrastructure of P. mangrovei to that of R. littoreum and R. aestuarii was made. Recent molecular phylogenetic studies of Rhizophydiales (Letcher et al. 2006, Lepelletier et al. 2014) have included R. littoreum (as strain “Barr 263”) and R. aestuarii (as strain “Barr 303”), and these two strains clustered but without robust support. In these phylogenies one or both of two additional strains (PL 137, PL 157) clustered with, or were sister to, R. littoreum and R. aestuarii (Letcher et al. 2006, Lepelletier et al. 2014) but also without robust support. Here we analyze partial 28S sequences for 90 strains in Rhizophydiales. To place strains reliably in a molecular phylogeny, our sampling included 80 strains representative of all families and lineages in Rhizophydiales and 10 strains of interest (R. littoreum, R. aestuarii, P. mangrovei, PL 137, PL157 and five related strains recovered from random sampling in a variety of habitats); the current status of these strains are incertae sedis. We also analyze a combined

dataset of partial 28S plus complete ITS sequences for our strains of interest to assess molecular divergence and confirm phylogenetic placement within the lineage. We examine thallus morphology and zoospores of our strains of interest to make morphological and ultrastructural comparisons and correlate zoospore ultrastructure with our molecular phylogeny. As a result we erect the new family Halomycetaceae and four new genera (Halomyces, Paludomyces, Ulkenomyces, Paranamyces) to accommodate these strains. MATERIALS AND METHODS Strains.—Rhizophydium aestuarii (ATCC® 26190™ deposited by A. Ulken and listed in recent molecular phylogenies of Rhizophydiales as “Barr 303”, which Dr Barr received from D. A. Ulken, the isolator) (pers comm C. Babcock, Canadian Collection of Fungal Cultures [CCFC], Ottawa, Ontario, Canada) and Rhizophydium littoreum (ATCC® 36100™ deposited by way of F.Y. Kazama, the isolator, to J.P. Amon and listed in recent molecular phylogenies of Rhizophydiales as “Barr 263”, which Dr Barr received from Dr Amon) (pers comm C. Babcock) were obtained from CCFC. Phlyctochytrium mangrovei (ATCC® 26191™, isolated and deposited by A. Ulken) was obtained from ATCC. Two strains (PL 137, PL 157) that occurred in recent Rhizophydiales molecular phylogenies as relatives of R. aestuarii and R. littoreum and five additional strains (JEL 695, PL 190, WJD 150, WJD 158, WJD 193) that showed sequence similarity with R. aestuarii and R. littoreum determined via BLAST queries (http://www.ncbi.nlm.nih.gov/) were included in the sampling. These constitute our strains of interest. The strains R. aestuarii, R. littoreum, P. mangrovei, PL 157 and PL 190 were maintained on a saline medium + agar (1 g peptone, 2 g yeast extract 2 g dextrose, 25.2 g Instant Ocean sea salt [Carolina Biological Supply Co., Burlington, North Carolina] to obtain a solution of ~ 2.5% salinity, 10 g agar, 1 L distilled water modified from Ulken 1965). Strains PL 137, JEL 695, WJD 150, WJD 158 and WJD 193 were maintained on PmTG medium + agar (1 g peptonized milk, 1 g tryptone, 5 g glucose, 8 g agar, 1L distilled water). Molecular/phylogenetic analyses.—We performed two molecular/phylogenetic analyses. (i) Using a dataset of partial 28S sequences, we analyzed 90 ingroup strains in Rhizophydiales and two outgroup strains (Barr 117A Spizellomyces punctatus/Spizellomycetales [Wakefield et al.2010] and Barr 186 Rhizophlyctis rosea/Rhizophlyctidales [Letcher et al. 2008a]) from clades sister to Rhizophydiales (James et al. 2006) (SUPPLEMENTARY TABLE I). Strains from all currently described families and lineages in Rhizophydiales were

included to ascertain the phylogenetic position and affinities of R. aestuarii, R. littoreum, P. mangrovei and seven related strains (JEL 695, PL 137, PL 157, PL 190, WJD 150, WJD 158, WJD 193). (ii) Using a combined dataset of partial 28S plus complete ITS sequences, we analyzed phylogenetic relationships among our isolates of interest, with strain ARG 071 Protrudomyces laterale from a sister clade as outgroup. Partial 28S and ITS sequences were generated as described by Letcher and Powell (2005) or obtained from GenBank (http://www.ncbi.nlm.nih.gov/). Phylogenetic analyses were performed as described in Vélez et al. (2011). To summarize, sequences were aligned with Clustal X (Thompson et al. 1997) and adjusted manually in BioEdit (Hall 1998). The alignments were submitted to TreeBASE (http://purl.org/phylo/treebase/phylows/study/TB2:S17280, TB2:17290). Maximum parsimony (MP) trees were generated with PAUPRat (Sikes and Lewis 2001) and support values were generated as heuristic searches with 500 replicates, each with 10 random-addition replicates. Maximum likelihood (ML) trees were constructed as described in Vélez et al. (2011). MrModeltest 2.3 (http://www.abc.se/nylander) was used to determine the best fit model of base substitution, and GARLI 0.951 (Zwikl 2006) was used to assess ML. ML branch support was assessed with 500 bootstrapping replicates. 28S and ITS sequences of our strains of interest were assessed for sequence similarity via pairwise alignment in BioEdit. Light microscopy.—Variation in thallus morphology among our strains of interest was assessed from observations made with a Zeiss Axioskop with a Zeiss AxioCam MRc5 camera. Thallus morphologies of R. littoreum (Kazama 1972a; Amon 1976, 1984), R. aestuarii (Ulken 1972a) and P. mangrovei (Ulken 1972b) have been reported and are succinctly illustrated here from our observations to facilitate morphological comparison. A solution of 0.1% toluidine blue was used to test for differential staining of the rhizoidal system and discharge pores (Parker et al. 1982). Electron microscopy.—Fixation and observation of zoospores followed procedures described in Letcher and Powell (2005). Zoospore ultrastructures of R. littoreum (Kazama 1972b, Amon 1984) and R. aestuarii (Lange and Olson 1977) had been reported and are illustrated here from our independent fixation and observations of these strains, to facilitate comparison of ultrastructural features. Zoospore ultrastructure of P. mangrovei and seven related strains (JEL 695, PL 137, PL 157, PL 190, WJD 150, WJD 158, WJD 193) also are presented here, and ultrastructural character and character states are compared (TABLE I).

RESULTS

Phylogenetic analyses.—The partial 28S dataset for 90 strains in Rhizophydiales had 935 characters with 422 parsimony informative sites after uninformative characters were excluded with PAUP* (Swofford 2002). For MP analysis 1005 trees derived in PAUPRat were most parsimonious (L = 2096, CI = 0.378, RI = 0.789) and were used to compute a majority-rule consensus tree (> 70% branch support). MrModeltest indicated that the most appropriate model of DNA substitution was Hasegawa, Kishino and Yano with rates of substitution among sites approximated by gamma distribution (HKY + G). MP and ML (−lnL = 8746.68) phylogenies were identical with similar or equal support values. The phylogeny is presented (FIG. 1) with ML and MP bootstrap values > 70%. Rhizophydium littoreum and Phlyctochytrium mangrovei (partial 28S sequences were 97.6% similar) formed a clade with 100% support, which was sister to a clade containing three strains of Rhizophydium aestuarii (partial 28S sequences were > 99% similar), also with 100% support. However that lineage (R. littoreum + P. mangrovei + R. aestuarii) had < 70% support. Sister to the R. littoreum + P. mangrovei + R. aestuarii lineage were five strains (JEL 695, PL 157, WJD 150, WJD 158, WJD 193) with 100% support (partial 28S were > 99% similar). The lineage formed by these 10 strains had < 70% support and in the Rhizophydiales phylogeny was sister to a lineage composed of strains in Aquamycetaceae, Angulomycetaceae and Protrudomycetaceae. The partial 28S plus complete ITS dataset for our strains of interest had 1508 characters, with 407 parsimony-informative sites after uninformative characters were excluded with PAUP*. For MP analysis 1005 trees derived in PAUPRat were most parsimonious (L = 630 steps, CI = 0892, RI = 0.936) and were used to compute a majority-rule consensus tree (> 50% branch support). This multigene phylogeny (FIG. 2) of our strains of interest was consistent with the single-gene phylogeny (FIG. 1) of 90 strains in Rhizophydiales. ITS sequences for R. littoreum

and P. mangrovei were 82% similar; ITS sequences for three strains of R. aestuarii were > 99% similar; ITS sequences for five strains (PL 157, JEL 695, WJD 150, WJD 158, WJD 193) were > 99% similar. The clade composed of R. littoreum, P. mangrovei and R. aestuarii had 99% support. Light microscopy.—Observations of thalli of our strains revealed all to be epibiotic and eucarpic but with differences in the zoospore discharge apparatus and rhizoidal structure (FIGS. 3–5). Thallus features of R. littoreum (Kazama 1972; Amon 1976, 1984), R. aestuarii (Ulken 1972a), and P. mangrovei (Ulken 1972b) have been presented in detail and are summarized here from our independent observations of these strains. Rhizophydium littoreum (ATCC 36100) (FIGS. 3A, B, 5A). The sporangium of R. littoreum is spherical, 24–26 µm diam, with 2–3 “extended” [sic] (Amon 1984), slightly erumpent discharge papillae 3.4 µm average width, 2.3 µm long (FIG. 3A, B); the rhizoidal system is composed of a swollen rhizoidal axis and elongate, branched, tapering rhizoids (FIG. 5A). Phlyctochytrium mangrovei (ATCC 26191) (FIGS. 3C, D, 5B). The sporangium of P. mangrovei is spherical, 18–28 µm diam, with multiple low-discharge papillae (2–8 when grown on pollen [Ulken 1972b], 30 or more when grown on agar medium) 2.8 µm wide on average, 1.4 µm long (FIG. 3C, D) that stained with toluidine blue (FIG. 3D); the rhizoidal system is composed of a spherical apophysis and elongate, branched, tapering rhizoids (FIGS. 3C, D, 5B). Rhizophydium aestuarii (ATCC 26190, PL 137, PL 190) (FIGS. 3E, F, 5C). The sporangium of R. aestuarii is spherical, 23–41 µm diam with multiple (5–16 when grown on pollen [Amon 1984] 30 or more when grown on agar medium) “less extended” [sic] (Amon 1984), conical discharge papillae 3.6 µm average diam at the base, 2 µm at the top; the rhizoidal

system is composed of a broad apophysis and elongate, branched, tapering rhizoids (FIGS. 3E, F, 5C). Strains PL 157, JEL 695, WJD 150, WJD 158, WJD 193 (represented by strain PL 157, FIGS. 4, 5D). Thallus morphologies of these strains were similar. For PL 157 (FIG. 4A–J), germlings were initially subspherical, 4–5 µm diam, with a single tapering rhizoidal axis (FIG. 4A); as the thallus developed the sporangium became spherical and rhizoids branched from the axis (FIG. 4B). As the thallus approached maturity the rhizoidal system was composed of a dense mass of short, tapering rhizoids (FIG. 4C, D) and an irregular apophysis that stained with toluidine blue (FIG. 4D). At maturity and just before zoospore release the sporangium wall became slightly irregular in outline (FIG. 4E). Zoospores began to swarm inside the sporangium before discharge and were released individually through a single apical pore (FIG. 4F, H–J). Released zoospores frequently remained in close association with the parent sporangium and germinated in situ, causing the sporangium wall to appear ornamented with hair-like protrusions (FIG. 4G). After discharge empty sporangia remained rigid, with the single discharge pore apparent (FIG. 4I, J). Schematics of our strains of interest are illustrative of these morphological features (FIG. 5). Electron microscopy.—Observations of our strains revealed zoospores with similar morphologies (FIG. 10), although constellations of ultrastructural character states were different for each type of zoospore observed (TABLE I). Zoospore ultrastructure of R. littoreum (Kazama 1972b, Amon 1984) and R. aestuarii (Lange and Olson 1977) have been reported, and ultrastructural features from our independent observations of these strains are presented here to facilitate comparison among our strains of interest.

Rhizophydium littoreum (ATCC 36100). The zoospore of R. littoreum (FIGS. 6, 10A) is spherical, 2.7–3.6 µm diam, with the nucleus and multiple mitochondrial profiles outside the ribosomal aggregation (FIG. 6A, B, C, I). Endoplasmic reticulum ramifies through the ribosomal aggregation (FIG. 6B). The zoospore has a single lipid globule to which is appressed a MLC fenestrated cisterna and a lobed microbody (FIG. 6A–E, J). A mitochondrion is often closely associated with the kinetosome (FIG. 6A, C, I). A microtubular root composed of 4–5 parallel, stacked microtubules extends from the side of the kinetosome to the MLC fenestrated cisterna (FIG. 6E, F, I). The nonflagellated centriole lies at an angle (~ 15°) to the kinetosome (FIG. 6F– H), and the two structures are connected by a fibril bridge in which the fibrils form a narrow (0.010–0.025 µm) zone of convergence (FIG. 6G, H). Phlyctochytrium mangrovei (ATCC 26191). The zoospore of P. mangrovei (FIGS. 7, 10B) is spherical, 3–4 µm diam, with the nucleus and multiple mitochondrial profiles outside the ribosomal aggregation (FIG. 7A, B). The zoospore has a single lipid globule to which is appressed a MLC fenestrated cisterna and a lobed microbody (FIG. 7B, H). A mitochondrion is often in close association with the kinetosome (FIG. 7A, G). A microtubular root composed of 4– 5 parallel, stacked microtubules extends from the side of the kinetosome to the MLC fenestrated cisterna (FIG. 7E, F). The nonflagellated centriole is parallel to or lies at a slight angle (~ 7°) to the kinetosome (FIG. 7A, C, D), and the two structures are connected by a fibril bridge in which the fibrils form a wide (~ 0.075 µm) zone of convergence (FIG. 7C, D). The area around the kinetosome is rich in vesicles (FIG. 7I). Rhizophydium aestuarii (ATCC 26190, PL 137, PL 190). The zoospore of R. aestuarii (FIGS. 8, 10C) is spherical, 3–4 µm diam, with the nucleus and multiple mitochondrial profiles outside the ribosomal aggregation (FIG. 8A, B). The zoospore characteristically has two lipid

globules (FIG. 8B, C), the usually smaller of which has a fenestrated MLC fenestrated cisterna and a simple microbody appressed to its surface (FIG. 8B, E, F), the usually larger of which has a MLC simple cisterna and a lobed microbody appressed to its surface (FIG. 8A–C, I). A microtubular root composed of 4–5 parallel, stacked microtubules extends from the side of the kinetosome to the MLC fenestrated cisterna (FIG. 8D, K, L). The nonflagellated centriole is parallel to, or lies at an angle (15–30°) to, the kinetosome (FIG. 8G, H, J, K,), and the two structures are connected by a fibril bridge in which there is only a minimal zone of convergence in the fibrils (FIG. 8G, H, J). A region of irregularly branched vesicles is anterior to the kinetosome (FIG. 8J). Strains PL 157, JEL 695, WJD 150, WHD 158, WJD 193. The zoospore of PL 157 (FIGS. 9, 10D) (as well as JEL 695, WJD 150, WJD 158, WJD 193) is spherical, 3–4 µm diam, with the nucleus and multiple mitochondrial profiles outside the ribosomal aggregation (FIG. 9A–D). The zoospore has a single lipid globule (FIG. 9A, C) or two globules (FIG. 9B, D); a MLC fenestrated cisterna and a lobed microbody are appressed to one globule (FIG. 9A–D), and a MLC simple cisterna and lobed microbody is appressed to the second globule (FIG. 9D). A mitochondrion is often closely associated with the kinetosome (FIG. 9A, B). A microtubular root composed of 3–4 parallel, stacked microtubules extends from the side of the kinetosome to the MLC fenestrated cisterna (FIG. 9G, H). The nonflagellated centriole is parallel to the kinetosome, and the two structures are connected by a fibril bridge in which the fibrils form a wide (~ 0.075 µm) zone of convergence (FIG. 9E, F). One or more vesicles (0.25–0.35 µm diam) with reticulate contents are adjacent to the kinetosome (FIG. 9B, I) and occasionally may occur in the anterior portion of the zoospore (FIG. 9C). TAXONOMY

Here we establish the new family Halomycetaceae and four new genera (Halomyces, Paludomyces, Ulkenomyces, Paranamyces) in Rhizophydiales. Halomycetaceae Letcher and M.J. Powell, fam. nov. MycoBank MB811430 Thallus monocentric, eucarpic. Sporangium with few to many discharge papillae. Rhizoids apophysate or non-apophysate. Zoospores with a single lipid globule, a MLC fenestrated cisterna, a lobed microbody and a microtubular root. Typification: Halomyces Letcher & M.J. Powell 2015 (holotype). Halomyces Letcher and M.J. Powell, gen. nov. MycoBank MB811463 Thallus monocentric, eucarpic. Sporangium spherical, 13.8–26.5 µm diam, with few (2–3) discharge papillae. Rhizoids branched, tapering, non-apophysate but with a swollen rhizoidal axis. Zoospores spherical, 2.7–3.6 µm diam, with a single lipid globule, a MLC fenestrated cisterna, a lobed microbody, endoplasmic reticulum ramifying through the ribosomal aggregation, a narrow zone of convergence in the fibril bridge between the kinetosome and nonflagellated centriole, and a microtubular root of 4–5 stacked microtubules. Typification: Halomyces littoreus (Amon) Letcher & M.J. Powell, comb. nov. (neotype) Etymology: Greek > Latin halo-: salt or saline, reflecting the habitat of the neotype. Halomyces littoreus (Amon) Letcher & M.J. Powell, comb. nov. MycoBank MB811464 FIGS. 3A, B, 5A, 6, 10A Basionym: Rhizophydium littoreum Amon, Mycologia 76:137. 1984.

Typification: UNITED STATES. VIRGINIA: Gloucester Point, near Virginia Institute of Marine Science, York River estuary, 37°25′N, 76°5′W, ~ 1 m, 1971, F.Y. Kazama, 71-1-E, (ATCC® 36100™), from Bryopsis plumosa. Kazama, 1972a: PLATE 2: FIG. 15 (NEOTYPE). Note 1: Amon (1984:138) designated multiple images from multiple studies (FIGS. 1–32 in Kazama 1972a, FIGS. 2–10 in Amon 1976 and FIG. 12 in Amon 1984) as the type. The type may be an illustration (singular), such as a photograph depicting a feature or features of an organism (McNeill et al. 2012, Art. 8.1). In addition because the material deposited at ATCC by Amon (1984) as Rhizophydium littoreum was of necessity a strain (subculture) of Kazama's (1972) original isolate and thus an “ex-type” (McNeill et al. 2012, Art. 8B.2) it follows that Amon's deposit was not "original material", and it is assumed that no original material exists or is missing. Because "a neotype is a specimen or illustration selected to serve as nomenclatural type if no original material is extant or as long as it is missing” (McNeill et al. 2012, Art. 9.7), we are designating a single illustration (PLATE 2, FIG. 15) from Kazama (1972a) as the neotype. Note 2: This taxon also has been reported from Codium sp., Buzzard's Bay, Massachusetts, USA (Amon 1984), and likely from Codium fragile, Woods Hole, Massachusetts, USA (Porter and Smiley 1980). Gene sequences: Partial 28S sequence DQ485540, ITS sequence DQ485604. Paludomyces Letcher & M.J. Powell, gen. nov. MycoBank MB811465 Thallus monocentric, eucarpic. Sporangium spherical, 18–28 µm diam, with multiple (up to 30) discharge papillae. Rhizoids branched, tapering, with a spherical apophysis. Zoospores spherical, 3–4 µm diam, with a single lipid globule, a MLC fenestrated cisterna, a lobed microbody, a wide

zone of convergence in the fibril bridge between the kinetosome and nonflagellated centriole, and a microtubular root of 4–5 stacked microtubules. Typification: Paludomyces mangrovei (Ulken) Letcher & M.J. Powell, comb. nov. (neotype) Etymology: Latin paludem: swamp; living in wet, swampy places Paludomyces mangrovei (Ulken) Letcher & M.J. Powell, comb. nov. MycoBank MB811466 FIGS. 3C, D, 5B, 7, 10B Basionym: Phlyctochytrium mangrovei Ulken, Veroff. Inst. Meeresforsch. Bremerhaven 13:227. 1972. (As Phlyctochytrium mangrovii).

Typification: BRAZIL. SÃO PAULO, Cananéia, Santos Harbor, 25°01′S, 47°93′W, 16 m. Pine pollen and cattle hair (keratin), mangrove swamp sediment, 1969, A. Ulken s.n. (ATCC® 26191™). Ulken, 1972b: 219: FIG. 1f (NEOTYPE). Note: Although no type was explicitly designated by Ulken (1972b), the type of the name was indicated through multiple figures, with the captions stating “nov. spec.”, constituting valid and effective publication (McNeill et al. 2012; Arts. 40.1, 40.4). The type may be an illustration (singular), such as a photograph depicting a feature or features of an organism (McNeill et al. 2012, Art. 8.1). In addition, because the material deposited at ATCC by Ulken (1972b) as Phlyctochytrium mangrovei was of necessity a strain (subculture) of Ulken's (1972b) original isolate, and thus an “ex-type” (McNeill et al. 2012, Art. 8B.2), it follows that Ulken's deposit was not “original material” and it is assumed that no original material exists or is missing. Because “a neotype is a specimen or illustration selected to serve as nomenclatural type if no original material is extant or as long as it is missing” (McNeill et al. 2012, Art. 9.7), we are designating a single illustration (FIG. 1f) from Ulken (1972b) as the neotype.

Gene sequences: Partial 28S sequence KP723816, ITS sequence KP723822. Note 1: In the original description of P. mangrovei Ulken (1972b) noted that the sporangium wall was ornamented with “unbranched or branched hairs and/or short prickles”, but added that “the ornaments of the fungus alter with salinity” and that “during the cultivation time of nearly two years many of the organisms did not show hairs or prickled ornaments on the sporangium wall, but in subcultures there always were individual sporangia demonstrating that they did not loose [sic] the ability to form ornaments”. In our examination of this strain sporangial ornamentation was not observed. Note 2: Although only a single strain of P. mangrovei is available for study, it may be fairly cosmopolitan in distribution, having been reported from Brazil (Ulken 1972a), the Chukchi Sea, Arctic Ocean (Sparrow 1973) and Hawaii and Mexico (Ulken 1978, as P. mangrovis). Ulkenomyces Letcher & M.J. Powell, gen. nov. MycoBank MB811467 Thallus monocentric, eucarpic. Sporangium spherical with up to 30 discharge papillae. Rhizoids branched, extensive, with a broad subsporangial apophysis. Zoospores spherical, 3–4 µm diam, usually with two lipid globules (the larger globule having a MLC simple cisterna and a lobed microbody, the smaller having a MLC fenestrated cisterna and a simple microbody), a vesiculated region anterior to the kinetosome composed of irregularly branched vesicles, a minimal zone of convergence in the fibril bridge between the kinetosome and nonflagellated centriole, and a microtubular root of 4–5 stacked microtubules. Typification: Ulkenomyces aestuarii (Ulken) Letcher & M.J. Powell, comb. nov. (neotype)

Etymology: In honor of Dr Annemarie Ulken, the original discoverer and describer of this species. Ulkenomyces aestuarii (Ulken) Letcher & M.J. Powell, comb. nov. MycoBank MB811468 FIGS. 3E, F, 5C, 8, 10C Basionym: Phlyctochytrium aestuarii Ulken, Veröff. Inst. Meeresforsch. Bremerhaven 13:215. 1972. Synonym: Rhizophydium aestuarii (Ulken) Amon, Mycologia 76:138. 1984.

Typification: FEDERAL REPUBLIC OF GERMANY. FREE HANSEATIC CITY OF BREMEN: Bremerhaven, Weser River, Double Lock at river opening to the North Sea, 53°32′60″N, 8°34′60″E, ~ sea level. Submersed estuary mud, pine pollen, 1972, A. Ulken, s.n., (ATCC® 26190™). Ulken, 1972a: 211: FIG. 5p (NEOTYPE). Note: Although no type was designated explicitly by Ulken (1972a), the type of the name was indicated through multiple figures with the captions stating “nov. spec.”, constituting valid and effective publication (McNeill et al. 2012; Arts. 40.1, 40.4). The type may be an illustration (singular), such as a photograph depicting a feature or features of an organism (McNeill et al. 2012, Art. 8.1). In addition, because the material deposited at ATCC by Ulken (1972a) as Phlyctochytrium aestuarii was of necessity a strain (subculture) of Ulken's (1972a) original isolate, and thus an “ex-type” (McNeill et al. 2012, Art. 8B.2), it follows that Ulken's deposit was not original material and it is assumed that no original material exists or is missing. Because “a neotype is a specimen or illustration selected to serve as nomenclatural type if no original material is extant, or as long as it is missing” (McNeill et al. 2012, Art. 9.7), we are designating a single illustration (FIG. 5p) from Ulken (1972a) as the neotype. Gene sequences: Partial 28S sequence DQ485541, ITS sequence DQ485605.

Other specimens examined: (i) CANADA. BRITISH COLUMBIA, Penticton, Mahoney Lake, 49°28′33″N, 119°58′33″W, 400 m. Mud sample, merimictic saline lake rich in hydrogen sulfide, keratin, 1 Aug 2006, P.M. Letcher PL 190, University of Alabama Chytrid Culture Collection (UACCC). (ii) SOUTH AFRICA. NORTHERN CAPE PROVINCE: Namaqualand, farm impoundment, pollen, soil sample collected by J.E. Longcore, 19 Aug 2004, P.M. Letcher PL 137, University of Alabama Chytrid Culture Collection (UACCC).

Paranamyces Letcher & M.J. Powell, gen. nov. MycoBank MB811469 Thallus monocentric, eucarpic. Sporangium spherical, with a single discharge pore. Rhizoids branched dense, compact, with an irregular apophysis. Zoospores with two lipid globules, a fenestrated MLC cisterna, lobed microbodies, a microtubular root of 4–5 stacked microtubules, and one or more vesicles with reticulate contents adjacent to the kinetosome. Typification: Paranamyces uniporus Letcher & M.J. Powell 2015, sp. nov. (holotype) Etymology: Paraná River, Argentina, the location of the mud sample from which this taxon was isolated. Paranamyces uniporus Letcher & M.J. Powell, sp. nov. FIGS. 4, 5D, 9, 10D MycoBank MB811470 Thallus monocentric, eucarpic. Sporangium spherical, 20–30 µm diam, with a single discharge pore. Rhizoids branched, dense, compact, with an irregular apophysis. Zoospores spherical, 3–4 µm diam, with two lipid globules (the larger globule having a MLC simple cisterna and a lobed microbody; the smaller having a MLC fenestrated cisterna and a simple microbody), a wide zone of convergence in the fibril bridge between the kinetosome and nonflagellated centriole, a microtubular root of 4–5 stacked microtubules and one or more vesicles with reticulate contents adjacent to the kinetosome.

Typification: ARGENTINA: BUENOS AIRES PROV.: Delta del Paraná, Reserva Natural Otamendi, 34°14′13″S, 58°51′19″W, 20 m. Pollen, estuarine mud flat, 8 Jul 2005, Peter M. Letcher, PL 157, Letcher et al. 2015: FIG. 3J (HOLOTYPE); preserved at the University of Alabama Chytrid Culture Collection (UACCC) in a metabolically inactive state at −80 C or lower in cryoprotectant (McNeill et al. 2012, Art.8.4). Etymology: named for the single pore morphology of the sporangium. Gene sequences: Partial 28S sequence DQ485594, ITS sequence DQ485685. Sequences were derived from ex-type material (McNeill et al. 2012, Art. 8B2). Zoospore ultrastructure: Zoospores used for ultrastructural examination were derived from ex-type material (McNeill et al. 2012, Art. 8B2). Other specimens examined: (i) UNITED STATES, MAINE: Orono, 44°88′N, 68°67′W, 15 m. Garden soil, keratin, 12 Mar 2011, Joyce E. Longcore, JEL 695. (ii) UNITED STATES, ALABAMA: Tuscaloosa, University of Alabama, Riverside Pond, 33°12′N, 87°32′W, 68 m. Pollen, soil at waterline from a pond, Mar 2011, William J. Davis, WJD 150. (iii) UNITED STATES, ALABAMA: Oxford, 33°61′N, 85°83′W, 212 m. Pollen, wet soil around cattails (Typha sp.), 15 Jun 2011, William J. Davis, WJD 158. (iv) UNITED STATES, OHIO: Bath Township, Bath Nature Preserve, 41°15′N, 81°38′W, 280 m. Keratin, tamarack bog, 16 Aug 2011, William J. Davis, WJD 193, collected under Bath Township and University of Akron Field Station Permit No. 2011-012.

Note: When isolate PL 157 was grown on agar, at zoospore discharge the zoospores often remained with the parent sporangium and germinated in situ, giving the parent sporangial wall the appearance of being ornamented with fine hairs (FIGS. 4G). However neither PL 157 nor the other strains examined and considered here as P. uniporus had wall ornamentation when thalli were examined with light microscopy and transmission electron microscopy. TAXONOMIC KEYS Morphological key.— 1.

Discharge opening papillate ----------------------------------------------------------------------- 2

Discharge opening a pore ---------------------------------------------- Paranamyces uniporus 2.

Discharge papillae many (up to 30) -------------------------------------------------------------- 3 Discharge papillae few (2–3) ----------------------------------------------- Halomyces littoreus

3.

Discharge papillae low (1.4 µm high), stain with toluidine blue --------------------------------------------------------------------------------------------------------- Paludomyces mangrovei Discharge papillae conical (3-4 µm high), did not stain with toluidine blue ---------------------------------------------------------------------------------------------- Ulkenomyces aestuarii

Zoospore ultrastructural key.— 1.

Ribosomal aggregation ramified with endoplasmic reticulum -------- Halomyces littoreus Ribosomal aggregation not ramified with endoplasmic reticulum --------------------------- 2

2.

Zoospore with a single lipid globule --------------------------------- Paludomyces mangrovei Zoospore with two lipid globules ----------------------------------------------------------------- 3

3.

Vesicles with reticulate contents lateral to kinetosome ------------- Paranamyces uniporus A vesicular region anterior to kinetosome ----------------------------- Ulkenomyces aestuarii

DISCUSSION This research was initiated to understand better the relationship between two marine chytrids previously referred to as “Rhizophydium littoreum” (here as Halomyces littoreus comb. nov.) and “Rhizophydium aestuarii” (here as Ulkenomyces aestuarii comb. nov), which occurred as sister taxa in a monophyletic lineage in molecular phylogenies of Rhizophydiales (Letcher et al. 2006, Lepelletier et al 2014) but which have different constellations of zoospore ultrastructural features (Kazama 1972b, Lange and Olson 1977, Amon 1984). These two strains represent “orphan taxa” (Janzen and Hallwachs 1994), in that marine chytrids constitute an understudied group with no current expertise. We increased the sampling of these orphan taxa for molecular phylogenetic

analysis by including a strain of the marine chytrid Phlyctochytrium mangrovei (here as Paludomyces mangrovei) obtained from ATCC and seven unidentified strains recovered from random sampling over 10 y in a variety of habitats on three continents (North America, South America, Africa) that were related to H. littoreum and U. aestuarii, as indicated by BLAST analyses of partial 28S sequences. Three of the seven unidentified strains also had been included in phylogenies (Letcher et al. 2006, Davis et al. 2013, Lepelletier et al 2014). In addition to molecular analyses, we compared thallus morphological features and examined zoospore ultrastructure of these 10 strains, which resulted in the erection of a new family and four new genera in Rhizophydiales. Thallus morphology.—Halomyces littoreus, U. aestuarii and P. mangrovei originally were placed in Phlyctochytrium sensu Sparrow (1960) on the basis of thallus morphology: a monocentric, eucarpic thallus having an epibiotic, inoperculate sporangium, a rhizoidal system with an endobiotic apophysis and branched rhizoids and an epibiotic resting spore. However Sparrow's (1960) concept of the genus is somewhat at variance with the morphology of the type, P. hydrodictyi (A. Braun) Schroeter, one of the first chytrids observed by Braun in 1846 (Braun 1856). Phlyctochytrium hydrodictyi, a parasite of the freshwater algae Hydrodictyon and Rhizoclonium, has never been observed with rhizoids (although it has an endobiotic subsporangial apophysis), and no resting spore has been reported. The three taxa in our study originally described as Phlyctochytrium sp. (= H. littoreus), P. aestuarii and P. mangrovei do not conform to the original morphological concept of Phlyctochytrium as represented by P. hydrodictyi, in that they all manifest rhizoidal systems and thus their inclusion in the genus can be considered questionable. Phlyctochytrium sp. and Phlyctochytrium aestuarii were transferred

to Rhizophydium as R. littoreum and R. aestuarii respectively (Amon 1984) on the basis of zoospore morphology not on the basis of thallus morphological features. Molecular analyses.—The three species previously included in Phlyctochytrium sensu Sparrow (here as H. littoreus, U. aestuarii, P. mangrovei) occur in Rhizophydiales (Letcher et al. 2006) and are properly placed there on the basis of molecular and ultrastructural evidence (Letcher et al. 2006, Lepelletier et al. 2014, this study). Other species of Phlyctochytrium sensu Sparrow (P. planicorne G.F. Atk., P. bullatum Sparrow and P. aureliae Ajello) occur in molecular phylogenies of Chytridiales (e.g. Letcher and Powell 2014) and are properly placed there on the basis of molecular and ultrastructural evidence. Still other species of Phlyctochytrium sensu Sparrow (P. reinboldtae Persiel, P. californicum D.J.S. Barr, P. africanum A. Gaertn.) occur in Spizellomycetales (Wakefield et al. 2010) and are properly placed there on the basis of molecular and ultrastructural evidence. Thus the genus Phlyctochytrium sensu Sparrow is polyphyletic and in need of revision. However we cannot molecularly or ultrastructurally define Phlyctochytrium because the type P. hydrodictyi (observed perhaps only a half dozen times since its origin [Sparrow 1960]) has not been brought into culture from which molecular and ultrastructural analyses could be accomplished. In our molecular analyses our strains of interest grouped as three well-supported clades in a monophyletic lineage: (i) H. littoreus + P. mangrovei; (ii) three strains of U. aestuarii; (iii) five strains herein designated Paranamyces uniporus. Thus H. littoreus and U. aestuarii are not as closely related as indicated in molecular phylogenies with limited sampling (Letcher et al. 2006, Lepelletier et al 2014). We performed two molecular analyses with different datasets. Because different genes (and different lineages) evolve at different rates, different genes are applicable to molecular

analyses at different taxonomic levels. Our first molecular analysis was of a partial 28S dataset for 90 isolates in Rhizophydiales to reliably place our strains of interest (here as Halomycetaceae) within the order and to ascertain that none of our strains of interest grouped outside the lineage or placed within currently delineated families. In the order Rhizophydiales partial 28S sequences are quite applicable to molecular analysis, specifically because the variable regions of the sequences for most strains can be aligned with confidence (Letcher et al. 2006, 2008C). A weakness in this type of analysis is that the 28S gene may not be sufficiently variable to reliably sort taxa that are closely related. For example Letcher et al. (2006) used combined 28S + 5.8S data to examine 93 strains in Rhizophydiales and 44 of those strains formed a monophyletic clade (Terramycetaceae) but the data were too conserved to discriminate among these strains. However analysis of combined partial 28S + complete ITS1-5.8S-ITS2 sequence data resulted in clear delineation of two strongly supported clades of strains (Terramyces, Boothiomyces), each of which was coincidental with a specific zoospore morphology. As a second example Letcher et al. (2012) used combined 28S + complete ITS1-5.8S-ITS2 data to discriminate among 11 closely related strains in Alphamyces (Alphamycetaceae), resulting in the delineation of two new genera (Betamyces, Gammamyces), with each of the three genera in the family having a distinct zoospore morphology. Thus it is not in error that in our partial 28S phylogeny presented here (FIG. 1), Boothiomyces in Terramycetaceae, Alphamyces in Alphamycetaceae and Aquamyces in Aquamycetaceae each appear polyphyletic. Instead it is a result of the ordinal limits of 28S resolution among closely related strains. Our second molecular analysis was of combined partial 28S + complete ITS1-5.8S-ITS2 sequence data for our strains of interest to confirm phylogenetic relationships within the lineage (FIG. 2). Complete ITS sequences cannot be used at the ordinal level because the ITS sequences

are too variable to be aligned across the order. This analysis reflects subtle differences in ITS sequences in Ulkenomyces and Paranamyces strains, but because ITS sequences among strains in each genus are > 99% similar each genus is considered to contain a single species. Zoospore ultrastructure.—Halomyces littoreus and U. aestuarii have been studied ultrastructurally (H. littoreus: Kazama 1972b, Amon 1984; U. aestuarii: Lange and Olson 1977), and although their zoospore features were not identical they were assessed to be “related” (Lange and Olson 1977, Amon 1984) because each had a “Rhizophydium-type” zoospore (Barr 1980). The Rhizophydium-type zoospore was created from a composite of ultrastructural features found among 12 species of Rhizophydium (Barr and Hadland-Hartmann 1978b) and was useful for distinguishing zoospores characteristic of Rhizophydium from zoospores characteristic of Chytridium (Barr and Hadland-Hartmann 1978b, Barr 1980). Before our research ultrastructural data had not been revealed for P. mangrovei. Molecular-based phylogenetic hypotheses predict zoospore ultrastructural types (James et al. 2006; Letcher et al. 2008a, 2008c; Longcore and Simmons 2012; Simmons 2011). Using a constellation of character states of zoospore ultrastructural characters, we can assign an organism to an order. Fine structure analyses of zoospores of H. littoreus, U. aestuarii, P. mangrovei and seven related strains reveal four distinct constellations of ultrastructural features (four zoospore “subtypes”). Among ultrastructural features all four subtypes have lobed microbodies, two subtypes have a single lipid globule, while two subtypes have two lipid globules present; three subtypes have a mitochondrion associated with the kinetosome, three subtypes have various vesicular arrangements adjacent to the kinetosome and all four subtypes have a MLC fenestrated cisterna and an associated microtubular root.

Specific morphology of the microtubular root is a useful ultrastructural character among orders in Chytridiomycetes that have microtubular roots (e.g. Chytridiales: Letcher and Powell 2014; Cladochytriales: Mozley-Standridge et al. 2009; Rhizophydiales: Letcher et al. 2006, 2008c). In Rhizophydiales, when present, the microtubular root typically appears to be composed of 4–5 parallel, stacked microtubules. Kazama (1972b fig. 7) and Amon (1984, figs. 3, 4) reported this arrangement for H. littoreus, and we have reported it here for P. mangrovei and P. uniporus. Lange and Olson (1977 fig. 2a) reported seven parallel, stacked microtubules in the microtubular root of U. aestuarii but noted that two of the microtubules were only 60–120 nm long. Here we observed the microtubular root in U. aestuarii to be composed of four (possibly five) microtubules and think it fits with the morphological concept of the microtubular root in Rhizophydiales. In all (> 20) Rhizophydiales zoospore subtypes (Kazama 1972b; Lange and Olson 1977; Amon 1984; Longcore et al. 1999, 2011; Longcore 2004; Letcher and Powell 2005k; Letcher et al. 2006, 2008b, c, 2012; Powell et al. 2011, 2015; Lepelletier et al. 2014; Powell and Letcher 2014) the ribosomes are aggregated (rather than dispersed) in the cytoplasm. A distinguishing feature of the H. littoreus zoospore is the presence of endoplasmic reticulum ramifying through the ribosomal aggregation. Kazama's (1972b p 559) ultrastructural analysis of this taxon mentioned that “on rare occasions, membranous elements were found within the ribosomal aggregate (not illustrated)” [sic]. Amon (1984 figs. 3, 6) illustrated this feature as we do here (FIG. 5B). The terminal plate (Barr and Hadland-Hartmann 1978a), a structure posterior to the kinetosome, has been reported in numerous Rhizophydiales zoospores (e.g. Barr and HadlandHartmann 1978b; Longcore 2004; Longcore et al. 1999; Letcher et al. 2006, 2008c), including H.

littoreus, and here we report its occurrence in P. mangrovei, P. uniporus and U. aestuarii. Lange and Olson (1977) did not observe a terminal plate in U. aestuarii perhaps because it is a relatively thin structure that is not always apparent in longitudinal sections through the kinetosome region (Barr and Hadland-Hartmann 1978a). Nonetheless observing it in all four new genera here, we consider it to be an ultrastructural feature throughout Rhizophydiales. Delineation of genera in Rhizophydiales has been based on monophyletic lineages that exhibit specific zoospore morphologies (Kazama 1972b; Lange and Olson 1977; Amon 1984; Longcore et al. 1999, 2011; Longcore 2004; Letcher and Powell 2005; Letcher et al. 2006; 2008b, c, 2012; Powell et al. 2011, 2015; Lepelletier et al. 2014; Powell and Letcher 2014). Although the majority of families in Rhizophydiales are monotypic (Kappamycetaceae, Aquamycetaceae, Angulomycetaceae, Protrudomycetaceae, Operculomycetaceae, Rhizophydiaceae, Pateramycetaceae, Uebelmesseromycetaceae, Coralloidiomycetaceae, Dinomycetaceae, Gorgonomycetaceae), it is the genus instead of the family that is differentiated on the basis of zoospore morphology. Thus Terramycetaceae contains two genera (Terramyces, Boothiomyces), Alphamycetaceae contains three genera (Alphamyces, Betamyces, Gammamyces), Globomycetaceae contains two genera (Globomyces, Urceomyces), and Batrachochytriaceae contains two genera (Batrachochytrium, Homolaphlyctis); the zoospores of these genera each possess a unique suite of ultrastructural character states (Letcher et al. 2006, 2008c, 2012; Longcore et al. 1999, 2011) as do the zoospores of genera in monotypic families. Decisions concerning genus and family delineation have been based in most cases on knowledge of the material in hand at the time of delineation but are certainly subject to revision as additional strains are discovered and additional diversity within a lineage is revealed. Some families (e.g. Batrachochytriaceae, Coralloidiomycetaceae) have been delineated perhaps simply to give

formal taxonomic standing to a lineage (Doweld 2013, 2014) rather than have that lineage remain as incertae sedis although genera in those families, just as all genera in Rhizophydiales, have distinct and unique zoospore morphologies (Powell and Letcher 2014). In Powell and Letcher (2014) the characters and character states of zoospores in Rhizophydiales are listed, and how constellations of character states distinguish taxa is demonstrated in a dichotomous key using the ultrastructural features. The organisms originally known as P. aestuarii (Ulken 1972a) and Phlyctochytrium sp. (Kazama 1972a) were transferred to Rhizophydium as R. aestuarii and R. littoreum respectively (Amon 1984). Two pieces of evidence indicate that these taxa should be transferred from Rhizophydium. First, zoospore ultrastructure in Rhizophydiales (Letcher et al. 2006) indicates that “R. littoreum” and “R. aestuarii” are not members of Rhizophydium. An investigation of the epitype culture of the type of Rhizophydium, R. globosum (A. Braun) Rabenh. (Letcher et al. 2006) defined its zoospore ultrastructural configuration, and the constellations of zoospore ultrastructural character states of the zoospores of “R. littoreum” and “R. aestuarii” differ from that of R. globosum as well as all other zoospore configurations in Rhizophydiales. Such is also the case for the zoospore of P. mangrovei, as shown here in our ultrastructural analysis of that organism. Second, in molecular analyses (Letcher et al. 2006, Lepelletier et al. 2014, this study), “R. littoreum” and “R. aestuarii” did not place in Rhizophydiaceae, the family that contains the type R. globosum. Thus, because of their unique zoospore ultrastructural configurations and molecular divergence, these two strains cannot be the genus Rhizophydium or in family Rhizophydiaceae and have been placed in new families and genera in Rhizophydiales. In our molecular phylogeny the new family Halomycetaceae encompasses a monophyletic lineage, which is sister to a monophyletic lineage composed of Aquamycetaceae,

Angulomycetaceae and Protrudomycetaceae. The genera in these four families have similar zoospore configurations (Letcher et al. 2008c), although each genus has a distinctive constellation of ultrastructural character states. Ecology.—Limited sampling within the group containing H. littoreus and U. aestuarii suggested these strains are representative of a halophytic lineage (Gleason et al. 2011). In our expanded sampling Halomycetaceae (composed of H. littoreus and P. mangrovei) appears to be halophytic. This cannot be ascertained in the Ulkenomycetaceae lineage because of the three strains of U. aestuarii only two were collected in saline/marine habitats: ATCC 26190 from a mangrove swamp; PL 190 from a small saline lake. The third strain, PL 137, was collected from dry farmland soil, and the salinity of that habitat is not known. It will be interesting to see whether strains in this clade collected in the future have saline affinities and tolerances. Of the five strains in Paranamycetaceae only one (PL 157) was collected from a saline habitat (an estuarine mud flat); the other four strains were from wet but not characteristically saline environs. An interesting aspect of our story involves α-keratin (e.g. skin, feathers, hair, wool) as a fungal substrate. Αlpha-keratin is a tightly packed protein having large amounts of the sulfurcontaining amino acid cysteine, required for the disulfide bridges that confer strength, rigidity and stability to the protein. It is hypothesized that fungi that degrade keratin produce and secrete the reducing agent sulfite (Grumbt et al. 2013), which is used to break disulfide bridges in initial keratin degradation. Thus, if chytrids are degrading keratin in the environment, they preferentially would be found in areas rich in sulfur that could be used to produce sulfite. For example, mangrove swamps are generally anoxic because the soil is perpetually waterlogged; sulfides (such as hydrogen sulfide) are among compounds liberated by anaerobic bacteria in anoxic habitats.

Four of our strains of interest (Paludomyces mangrovei, Ulkenomyces aestuarii PL 190, Paranamyces uniporus WJD 193, Paranamyces uniporus JEL 695, individually discussed below) initially were isolated from keratin substrates (human hair, snakeskin) added as bait to soil or mud samples. Because we cultured these isolates on synthetic media they cannot be considered obligately keratinophilic (Karling 1946) but can be considered generally keratinophilic as natural colonizers of keratinic substrates (Filipello Marchisio 2000). Griffin (1960) and de Vries (1962) considered chytrids to be the primary colonizers, in a succession of fungi, on keratin in soil. Paludomyces mangrovei. In assessing chytrid fungi from sediment samples from a mangrove swamp in Brazil Ulken (1970) observed Rhizophydium keratinophilum Karling (1946), a putatively obligate keratinophilic organism having sporangia and resting spores ornamented with short, simple or branched spines or long, simple or branched threads. Ulken observed this organism on both pollen and mouse hair. Two years later Ulken (1972b) described Paludomyces mangrovei from the same swamp, and because its sporangial wall was ornamented with “branched or unbranched hairs and/or short prickles”, Ulken initially thought her isolate to be R. keratinophilum, but because it showed so much morphological variation during 2 y of study, named it as a new species. That isolate was observed on pollen and cattle hair. Whether P. mangrovei is conspecific with R. keratinophilum is currently unresolvable because to the best of our knowledge no cultures of R. keratinophilum are available; thus no molecular or zoospore ultrastructural comparisons can be made at this time between these two isolates having putatively similar thallus morphology.

Ulkenomyces aestuarii PL 190. This isolate was recovered from keratin bait (human hair) of an anoxic mud sample from a small saline lake containing hydrogen sulfide (Klepac-Ceraj et al. 2012). It did not exhibit sporangial ornamentation. Paranamyces uniporus isolates JEL 695 and WJD 193. Grouping in a clade sister to H. littoreus, P. mangrovei and U. aestuarii, these two isolates were recovered from keratin-baited soil samples (JEL 695 on human hair, WJD 193 on snakeskin). Neither of the isolates exhibited sporangial ornamentation. The essence of our brief ecological discussion is that sampling in targeted anoxic, saline or sulfurous habitats such as mangrove swamps, muck soils, mud flats, volcanic muds, freshwater and coastal sediments and baiting samples from these habitats with keratin and pollen may yield additional isolates and taxonomic diversity in this lineage. Our serendipitous finding of isolates in this lineage, from random sampling on a global scale, bodes well for the discovery of further diversity of these orphan taxa. ACKNOWLEDGMENTS We very much appreciate S. Pennycook for his assistance with nomenclatural issues and J.E. Longcore for providing cultures. This study was supported by the National Science Foundation through REVSYS grants DEB-0516173, DEB-0949305 and MRI grant DEB-0500766.

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LEGENDS FIG. 1. Phylogeny of 90 strains in Rhizophydiales based on partial 28S sequences. Outgroup strains Barr 117A Spizellomyces punctatus and Barr 186 Rhizophlyctis rosea represent Spizellomycetales and Rhizophlyctidales. Maximum likelihood (ML)/maximum parsimony (MP) support values > 70% are indicated on nodes. ML −lnL = 8746.68; MP tree length = 2096 steps. Arrow indicates lineage (Halomycetaceae, fam. nov.) of our strains of interest containing Halomyces, Paludomyces, Ulkenomyces and Paranamyces, a new family and four new genera in Rhizophydiales. ET = epitype, HT = holotype, NT = neotype. FIG. 2. Cladogram (MP, majority rule) of Halomycetaceae fam. nov., based on combined (partial 28S + complete ITS) data. Bootstrap values are indicated. Tree length = 630 steps, CI = 0.892, RI = 0.936. FIG. 3. Sporangial features of Halomyces littoreus (A, B), Paludomyces mangrovei (C, D) and Ulkenomyces aestuarii (E, F). A. Mature sporangium with few (2–3), slightly erumpent discharge papillae (arrows). B. Papillae (arrows) following zoospore discharge. C. Sporangium with multiple (30 or more) low discharge papillae (arrows). D. Discharge papillae (arrows) stained with toluidine blue. E. Sporangium with multiple (30 or more) conical papillae (arrows). F. Multiple papillae (arrows) following zoospore discharge. Abbreviations: Ap = apophysis, R = rhizoids, Rax = rhizoidal axis, Sp = sporangium. Bar: A–F = 10 µm. FIG. 4. Morphology of vegetative thallus development of Paranamyces uniporus. A. Germlings, each with a single rhizoidal axis. B. Germling with branching rhizoids. C. Immature thallus with compact aggregate of fine rhizoids. D. Immature thallus stained with 0.1% toluidine blue, in which an irregularly shaped subsporangial apophysis is evident. E. Mature sporangium. F. Zoospore discharge. G. Zoospore discharge after 30 min, in which multiple zoospores have adhered to the sporangium wall and germinated. H. Discharge pore visible late in zoospore discharge. I, J. Evacuated sporangia, each with a single discharge pore. Bars: A, B = 5 µm; C–J = 10 µm. FIG. 5. Schematic of thallus morphology of four genera. A. Halomyces littoreus, the sporangium having few (2–3) discharge papillae (arrows), a swollen rhizoidal axis (Rax) and branched rhizoids. B. Paludomyces mangrovei, the sporangium having numerous (up to 30) low discharge papillae (arrows), a spherical apophysis and elongate branched rhizoids. C. Ulkenomyces aestuarii, the sporangium (Sp) having numerous (up to 30) conical discharge papillae (arrows), a broad sub-sporangial apophysis (Ap) and elongate branched rhizoids (R). D. Paranamyces uniporus, the sporangium having a single discharge pore (arrow), a broad, irregular apophysis, and compact, densely branched rhizoids. Bars: A–D = 10 µm.

FIG. 6. Zoospore ultrastructure of Halomyces littoreus. A. Longitudinal section (LS) with posterior flagellum (F), the nucleus partially within ribosomal aggregation, mitochondria outside the aggregation and a lipid globule to which are appressed a microbody and fenestrated MLC cisterna. B. Transverse section (TS) with ER traversing the ribosomal aggregation. C. LS illustrating a mitochondrion adjacent to the kinetosome and the fenestrated MLC cisterna in the posterior region of the zoospore. D. Lipid globule to which are appressed a fenestrated MLC cisterna and a lobed microbody (arrows). E. LS of microtubular root extending from the side of the kinetosome to the fenestrated MLC cisterna. F. TS of microtubular root (arrow). G. LS through kinetosome and nonflagellated centriole illustrating narrow zone of convergence (arrow) in fibril bridge between the two; arrowhead indicates terminal plate. H. TS through kinetosome and nonflagellated centriole illustrating narrow zone of convergence (arrow) in fibril bridge between the two. I. LS. Mitochondrion adjacent to kinetosome, and oblique section through microtubular root. J. TS across face of fenestrated MLC cisterna. Bars: A, B, D = 0.5 µm; C, E–J = 0.25 µm. Abbreviations: ER = endoplasmic reticulum, F = flagellum; FB = fibril bridge; FC = MLC fenestrated cisterna; K = kinetosome; L = lipid globule, M = mitochondrion, Mb = microbody, Mt = microtubular root, N = nucleus; NfC = nonflagellated centriole, R = ribosomes, SC = MLC simple cisterna, VR = vesicle region, Ves = vesicle. FIG. 7. Zoospore ultrastructure of Paludomyces mangrovei. A. Longitudinal section (LS) with nucleus partially surrounded by ribosomal aggregation and mitochondria outside aggregation. B. Transverse section (TS) with a single lipid globule, to which are appressed a lobed microbody and a fenestrated MLC cisterna. C. LS through kinetosome and nonflagellated centriole, with a wide zone of convergence (arrow) in the fibril bridge connection the two; arrowhead indicates terminal plate. D. TS through kinetosome and nonflagellated centriole, with a wide zone of convergence (arrow) in the fibril bridge connection the two. E. LS of microtubular root (arrow). F. TS of microtubular root (arrow). G. LS illustrating mitochondrion adjacent to kinetosome. H. Oblique section through fenestrated MLC cisterna (arrow). I. LS through kinetosomal region illustrating vesiculate nature of peripheral cytoplasm. Bars: A, B, H, I = 0.5; µm; C–F = 0.25 µm; G = 0.15 µm. Abbreviations: ER = endoplasmic reticulum, F = flagellum, FB = fibril bridge, FC = MLC fenestrated cisterna, K = kinetosome, L = lipid globule, M = mitochondrion, Mb = microbody, Mt = microtubular root, N = nucleus, NfC = nonflagellated centriole, R = ribosomes, SC = MLC simple cisterna, VR = vesicle region, Ves = vesicle.. FIG. 8. Zoospore ultrastructure of Ulkenomyces aestuarii. A. Longitudinal section (LS) with mitochondria (M) and the nucleus (N) outside the ribosomal aggregation (R) and a microbody (Mb) appressed to a lipid globule (L). B.

Transverse section (TS) with two lipid globules, the smaller with a fenestrated microbody-lipid globule cisterna (FC). C. TS. The larger lipid globule is surrounded by a lobed microbody; the smaller lipid is surrounded by endoplasmic reticulum (ER). D. Section through a lipid globule illustrating TS of the microtubule root (arrow) as it passes through the MLC cisterna. E. Oblique section through FC (arrow). F. TS through FC (arrow). G. LS through kinetosome (K) and nonflagellated centriole (NfC); fibril bridge between the two shows minimal zone of convergence (arrow) of connecting fibers; arrowhead indicates terminal plate. H. TS through kinetosome and nonflagellated centriole; fibril bridge between the two shows minimal zone of convergence (arrow). I. Detail of ER (arrows) enclosing microbody on lipid globule surface. J. LS through kinetosome region, with a vesicular region (VR) anterior to the kinetosome, arrowhead indicates terminal plate. K. LS of microtubular root (arrow). L. TS of microtubular root (arrow). Bars: A–C, I = 0.5 µm; D–H, J–L = 0.25 µm. Abbreviations: ER = endoplasmic reticulum, F = flagellum; FB = fibril bridge, FC = MLC fenestrated cisterna, K = kinetosome, L = lipid globule, M = mitochondrion, Mb = microbody, Mt = microtubular root, N = nucleus; NfC = nonflagellated centriole, R = ribosomes, SC = MLC simple cisterna, VR = vesicle region, Ves = vesicle. FIG. 9. Zoospore ultrastructure of Paranamyces uniporus. A. Longitudinal section (LS), with ribosomal aggregation, the nucleus and mitochondria outside the ribosomal aggregation and a single lipid globule to which is appressed a lobed microbody and a fenestrated MLC cisterna. B. LS with two lipid globules, the smaller globule with a fenestrated MLC cisterna, and reticulate vesicles (Ves) with reticulate contents in the posterior region of the zoospore and adjacent to the kinetosome (K). C. Transverse section (TS) with a single lipid globule, to which are appressed a lobed microbody and a fenestrated MLC cisterna. D. TS with two lipid globules adjacent to a lobed microbody. E. LS through kinetosome region; arrowhead indicates terminal plate. F. LS through kinetosome and nonflagellated centriole, which are connected by a fibril bridge having a wide zone of convergence (arrow) where the connecting fibers meet. G. TS, illustrating wide zone of convergence (arrow) in the fibril bridge. H. LS through kinetosome illustrating oblique section through microtubular root (arrow). I. LS through kinetosome illustrating transverse section through microtubular root (arrow). J. Enlargement of vesicle characteristic of Paranamyces zoospore. Bars: A–D = 0.5 µm, E–H = 0.25 µm, I = 0.15 µm. Abbreviations: ER = endoplasmic reticulum, F = flagellum; FB = fibril bridge, FC = MLC fenestrated cisterna, K = kinetosome, L = lipid globule, M = mitochondrion, Mb = microbody, Mt = microtubular root, N = nucleus; NfC = nonflagellated centriole, R = ribosomes, SC = MLC simple cisterna, VR = vesicle region, Ves = vesicle.

FIG. 10. Schematics (medial longitudinal sections of zoospore and transverse sections of kinetosome and nonflagellated centriole) of ultrastructure of four genera. A. Halomyces littoreus, characterized by a single lipid globule with a fenestrated MLC cisterna and a lobed microbody, ER ramifying through the ribosomal aggregation, and a narrow zone of convergence in the fibrilla bridge between the kinetosome and nonflagellated centriole. B. Paludomyces mangrovei, characterized by a single lipid globule with a fenestrated MLC cisterna and a lobed microbody, a richly vesiculated area adjacent to the kinetosome, and a wide zone of convergence in the fibril bridge between the kinetosome and nonflagellated centriole. C. Ulkenomyces aestuarii, characterized by two lipid globules (the smaller with a fenestrated MLC cisterna and simple microbody, the larger with a lobed microbody), a vesicular region anterior to the kinetosome and a minimal zone of convergence in the fibril bridge between the kinetosome and nonflagellated centriole. D. Paranamyces uniporus, characterized by two lipid globules globules (the smaller with a fenestrated MLC cisterna and simple microbody, the larger with a lobed microbody), vesicles with reticulated contents (predominantly in the posterior region of the zoospore), and a wide zone of convergence in the fibril bridge between the kinetosome and nonflagellated centriole. Bars: A–D = 1.0 µm. Abbreviations: ER = endoplasmic reticulum, F = flagellum; FB = fibril bridge, FC = MLC fenestrated cisterna, K = kinetosome, L = lipid globule, M = mitochondrion, Mb = microbody, Mt = microtubular root, N = nucleus; NfC = nonflagellated centriole, R = ribosomes, SC = MLC simple cisterna, VR = vesicle region, Ves = vesicle.

FOOTNOTES Submitted 27 Oct 2014; accepted for publication 9 Apr 2015. 1

Corresponding author. E-mail: [email protected]

TABLE I. Comparison of characters and character states of zoospores of strains ATCC 36100 Rhizophydium littoreum (Halomyces littoreus comb. nov.), ATCC 26191 Phlyctochytrium mangrovei (Paludomyces mangrovei comb. nov.), ATCC 26190 Rhizophydium aestuarii (Ulkenomyces aestuarii comb. nov.) and PL 157 (Paranamyces uniporus gen. et sp. nov.) Strain

No. of lipid globules

Vesiculated region anterior to kinetosome

Zone of convergence of fibril bridge

Lobe of Endoplasmic Vesicles MLC Kinetosome mitochondrion reticulum lateral to cisterna to nonassociated ramifying kinetosome flagellated with through centriole kinetosome ribosomes position ‗‗‗‗‗‗‗‗‗‗‗‗‗‗‗‗‗‗‗‗‗‗‗‗‗‗‗‗‗‗‗‗‗‗‗‗‗‗‗‗‗‗‗‗‗‗‗‗‗‗‗‗‗‗‗‗‗‗‗‗‗‗‗‗‗‗‗‗‗‗‗‗‗‗‗‗‗‗‗‗‗‗‗‗‗‗‗‗‗‗‗‗‗‗‗‗‗‗‗‗‗‗‗‗‗‗ R. littoreum 1 Absent Narrow Present Present Absent Fenestrated ~ 15° P. mangrovei 1

Absent

Wide

Present

Absent

Present

Fenestrated

R. aestuarii

2

Present

Absent

Absent

Absent

Absent

1 fenestrated, 15–30° 1 simple

PL 157

2

Absent

Wide

Present

Absent

Present

1 fenestrated, parallel 1 simple

~ 7°

A new family and four new genera in Rhizophydiales (Chytridiomycota).

Many chytrid phylogenies contain lineages representing a lone taxon or a few organisms. One such lineage in recent molecular phylogenies of Rhizophydi...
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