Mycologia, 107(4), 2015, pp. 874–888. DOI: 10.3852/15-050 # 2015 by The Mycological Society of America, Lawrence, KS 66044-8897

New trichomycete species from China and additional information on Gauthieromyces D.B. Strongman*

(Lichtwardt 1986). DNA sequence data exist for a relatively small number of species and these are used to elucidate phylogenetic relationships and define related groups among taxa (Wang et al. 2013, 2014), but the lack of axenic cultures for many species makes obtaining sufficient quantities and quality of DNA difficult. Trichomycetes are distributed globally (Lichtwardt et al. 2001, Nelder et al. 2006). There are several reports from Asia, specifically Japan (Lichtwardt et al. 1987; Sato 2013a, b), central and northwestern China (Adler et al. 1996, Strongman and Xu 2006, Strongman et al. 2010, Wang et al. 2010), Taiwan (Chien and Hsieh 2001), India (Rajagopalan 1967, Misra and Tiwara 2008) and Thailand (Hapsari et al. 2009). The inventory of trichomycetes from Asia includes many cosmopolitan harpellid species such as Harpella melusinae, Zancudomyces (Smittium) culisetae and Stachylina grandispora, but 15 species are known only from this region (TABLE I). Several species reported from Asia are otherwise known only from distant localities (e.g. Smittium elongatum from Japan, Australia and USA and Stachylina longa from Japan, France). The other trichomycete orders, Asellariales, Eccrinales and Amoebidiales, are also well represented in Asia with common and apparently endemic taxa reported (TABLE I). Most reports from Asian countries are from the past 10 y and compiled from individual inventories limited in time and geographic scope. However, the diversity is broad and the records provide valuable information about the biogeographical distribution of trichomycete species. Past collections from China resulted in the description of a new genus (Sinotrichium) and eight new species, mostly from larval dipterans (Strongman and Xu 2006, Strongman et al. 2010, Wang et al. 2010). The Chinese collections were focused on the Qinling Mountains in central China near Xi’an, but collections were made in a broad time frame (2006–2009) and from different areas of the country, from central (Shaanxi Prov.) and southern China (Yunnan Prov.) to the Tianshan mountains in the extreme northwestern region (Xinjiang Prov). In this paper we add information on trichomycete diversity from additional collections in central China, describing three new species, Caudomyces typhella, Genistelloides torrentis and Orphella sinica. In addition, six species are new records for Asia, Graminella bulbosa Le´ger & Gauthier ex Manier, Simuliomyces microsporus Lichtw., Smittium

Department of Biology, Saint Mary’s University, Halifax, Nova Scotia, Canada B3H 3C3

Juan Wang No. 20 Middle School, Urumqi, Xinjiang, PR China 830002

Abstract: We describe three new species of gut fungi (Kickxellomycotina, Harpellales) from aquatic insects, including Caudomyces typhella from a crane fly larva and Genistelloides torrentis and Orphella sinica from stonefly nymphs. Details on trichomycete species previously reported from Asia, including China, are provided. Recent descriptions of two new species of Gauthieromyces provide a more complete understanding of the genus; it is emended to reflect this new information and an epitype is designated. The description of G. indicus is emended based on an examination of the type specimen and additional collections of this species from China. Key words: aquatic insects, Diptera, endobionts, Plecoptera, taxonomy

INTRODUCTION Trichomycetes are fungal and protist endobionts living obligately within the guts of aquatic insects and other arthropod hosts such as crustaceans, diplopods and springtails (Lichtwardt 1986). Specifically two orders in the original class Trichomycetes were fungal (Harpellales, Asellariales) and two were protists (Eccrinales, Amoebidiales). Members of the fungal orders were assigned to the Kickellomycotina by Hibbett et al. (2007). Approximately 326 described species of trichomycetes are divided among 66 genera (Lichtwardt 2012). Trichomycete classification is based largely on morphology using measurements of trichospores (asexual) and zygospores (sexual). Spore shape, appendage number and arrangement on the thallus also are considered. To a lesser extent the holdfast structure and thallus characters are used to distinguish species, but most cannot be accurately identified to species, without, minimally, detailed trichospore morphology Submitted 21 Feb 2015; accepted for publication 30 Apr 2015. * Corresponding author. E-mail: [email protected]

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STRONGMAN AND WANG: CHINESE TRICHOMYCETES minutisporum Lichtw., Siri & M.M. White, Stachylina pedifer M.C. Williams & Lichtw., Stachylina subgrandis M.M. White & Strongman and Zygopolaris borealis Lichtw. & M.C. Williams. Several other species (e.g. Gauthieromyces indicus J.K. Misra & V.K. Tiwari, Smittium shaanxiense Juan Wang, Strongman & S.Q. Xu, and Caudomyces japonicus Lichtw., Kobayasi & Indoh, collected during previous visits to the same sites in the Qinling Mountains) were recorded again and are reported in this paper (Strongman and Xu 2006, Strongman et al. 2010). Gauthier (1960) described a species of trichomycete from France in mayfly nymphs as Genistella microspora. Lichtwardt (1983) proposed a new genus, Gauthieromyces, with the type G. microsporus, providing a Latin diagnosis and reproducing the original drawings from Gauthier (1960) as the holotype after unfruitful attempts to find new specimens in the same region of France (Lichtwardt 1983). The original collections in France made by Gauthier provided few details on spore morphology because no mature released trichospores were observed (Lichtwardt 1983). Valle et al. (2013) found a specimen of G. microsporus in Italian collections of a baetid host, but the material was immature and no additional morphological details were observed. Misra and Tiwari (2008) described a new species, Gautheriomyces indicus, from streams in Uttaranchal, India. Strongman and Xu (2006) also collected this species from baetid mayflies in the Hao River, China. Valle et al. (2008) added a third species to the genus, G. viviparus L.G. Valle, M.M. White & Cafaro, from collections made in Mexico. The description of the holotype of G. indicus differed somewhat from our observations on the Chinese collections, and the Mexican species also had some characters (trichospores with no collar and basal vs eccentric attachment to the sporogenous cell) not seen in the type species, G. microsporus. Both G. indicus and G. viviparus also have vegetative cells, named propagule-like cells in Valle et al. (2008), although the original descriptions of G. indicus and G. microsporus did not mention this character. Because the characters of the three species assigned to the genus broaden the scope of the original definition considerably, it is emended below to accommodate the full range of traits. An epitype is designated here because the original description of the type species, G. microspores, does not include information about mature, released trichospores (Lichtwardt 1983) or the presence of what appear to be vegetative propagules (bulbils). In addition, the description of G. indicus is emended to more adequately describe its morphology, namely the trichospore appendage and collar characteristics and the presence of vegetative cells.

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MATERIALS AND METHODS Immature, aquatic insects were collected from the following sites in China: Feng Yu River in Feng Yu Forest Park (Site 1) and the Nui Bei Liang conservation area (Site 2) in the Qinling Mountains, near Xi’an, Shaanxi Prov.; Lan River near An Kang (Site 3); Chang’an River adjacent to Huoditang research station (Site 4) northwest of Ningshan, another stream near the city of Ningshan (Site 5) off highway G5; Ping He Liang (Site 6), the highest at 2074 m, north of Ningshan adjacent to route G210, all in southern Shaanxi Prov. Collections also were made outside Kun Ming City (Site 7) in Yunnan Prov., 1250 km south of Xi’an. GPS coordinates and collection dates for all sites are provided (TABLE II). Insect specimens were collected by kick-sampling, which involves disrupting the stream bed with the feet and dragging a dip net (50 cm gape, 0.5 mm mesh) downstream to catch dislodged insects (Lichtwardt 1986). In the lab insect guts were removed and dissected with forceps and insect pins as described in Lichtwardt (1986) and Lichtwardt et al. (2001). Wet mounts of the trichomycetes in the guts were prepared and digital photomicrographs were taken from the semipermanent voucher slides made by infiltrating specimens with a drop of lactophenol cotton blue before sealing the edges of the cover slip with clear fingernail polish (Lichtwardt et al. 2001). Details of handling and photographing specimens are in Strongman et al. (2010). All measurements in the species descriptions below were made from material stained with lactophenol cotton blue. Taxa were identified with the LUCID keys for trichomycetes hosted on www. lucidcentral.org (Lichtwardt 2004). Type slides for new species described are deposited in the Farlow Herbarium (FH), Harvard Univ., Cambridge, Massachusetts. The type (FH–81102) and isotype (FH–81103) slides for Gauthieromyces indicus were examined and photographed with permission of FH and compared to collections of this species in China.

TAXONOMY Caudomyces typhella Strongman and J. Wang, sp. nov. FIGS. 1–3 MycoBank MB812031 Typification: CHINA. SHAANXI PROV.: Collected by J. Wang from Lan River near An Kang on N side of Daba Mountains (Site 3) in southern Shaanxi Prov., lactophenolcotton blue stained slide prepared from the gut of a crane fly larva (Tipulidae), 5 Jul 2006 (holotype FH 00313530). Slide from same host and collection (isotype FH 00313531). Etymology: A diminutive form of the Latin name for bullrush (Typha), because the trichospore with its long extension is reminiscent of a little bullrush.

Thallus attached to the hindgut of crane fly (Tipulidae) larvae, sparse, multiple branches arising from a bulbous holdfast 18–20 mm diam attached to the hindgut lining, basal branches 1 or 2 times branched, then produce trichospores at the apical branchlets. Trichospores cylindrical, 40–48 3 4–6.5 mm, hyaline,

876 TABLE I.

MYCOLOGIA Trichomycete species reported from Asia Species

Harpellales Bojamyces repens Longcore Caudomyces japonicus Lichtw. Kobayasi & Indoh Gauthieromyces indicus Misra & Tiwari Glotzia ephemeridarum Lichtw. Harpella asiana Hapsari, M.M. White & K.D. Hyde Harpella melusinae Le´ger & Duboscq Harpellomyces eccentricus Lichtw. & S.T. Moss Legeriomyces elegans Strongman, J. Wang & S.Q. Xu Legeriomyces grandis J. Wang, Strongman & S.Q. Xu Legeriomyces ramosus Pouzer

Distribution Japan, Spain, USA Japan, China China, India China, USA Thailand Cosmopolitan/China, India, Japan, Thailand

Reference Lichtwardt et al. 2001, Sato 2013b Lichtwardt et al. 1987, Strongman and Xu 2006, Strongman et al. 2010 Misra and Tiwari 2008, Strongman and Xu 2006, Strongman et al. 2010 Lichtwardt et al. 2001, Strongman et al. 2010 Hapsari et al. 2009 Adler et al. 1996, Hapsari et al. 2009, Lichtwardt et al. 2001, Misra and Tiwari 2008, Sato 2013a, Takaoka and Adler 1997 Lichtwardt et al. 1987, Lichtwardt et al. 2001

England, Japan, Norway, Sweden China

Strongman et al. 2010

China

Strongman et al. 2010

Cosmopolitan/China, India

Pennella angustispora Lichtw. Pennella montana Lichtw. Sinotrichium chironomidarum J. Wang, S.Q. Xu & Strongman Smittium chinliense Strongman & S.Q. Xu Smittium culicis Manier Smittium elongatum Lichtw. Smittium hecatei L.G. Valle & Santam. Smittium naiadis Strongman & S.Q. Xu Smittium nodifixum Strongman & S.Q. Xu Smittium rupestre Lichtw. Smittium shaanxiense J. Wang, Strongman & S.Q. Xu Smittium simulii Lichtw.

Cosmopolitan/Japan Costa Rica, Mexico, Thailand China

Lichtwardt et al. 2001, Misra and Tiwari 2008, Strongman et al. 2010 Lichtwardt et al. 1987, Sato 2013a Hapsari et al. 2009, Lichtwardt et al. 2001 Wang et al. 2010

China

Strongman and Xu 2006

Cosmopolitan/China Australia, Japan, USA Canada, China, Spain

Lichtwardt et al. 2001, Strongman et al. 2010 Lichtwardt et al. 1987, Lichtwardt et al. 2001 Lichtwardt et al. 2001, Strongman and Xu 2006

China

Strongman and Xu 2006

China

Strongman and Xu 2006

Australia, China China

Lichtwardt et al. 2001, Wang et al. 2010 Strongman et al. 2010

Cosmopolitan/China, Japan

Stachylina chironomidarum Lichtw. Stachylina grandispora Lichtw. Stachylina gravicaudata Siri, White & Lichtw. Stachylina longa Le´ger & Gauthier Stachylina macrospora Le´ger & Gauthier Stachylina magna Indoh, Lichtw. & Kobaysi Stachylina nana Lichtw.

India, USA Cosmopolitan, India, Japan China, USA

Lichtwardt et al. 1987, Lichtwardt et al. 2001, Sato 2013a, Strongman and Xu 2006, Strongman et al. 2010 In Misra and Tiwari 2008, Lichtwardt et al. 2001 In Misra and Tiwari 2008, Lichtwardt et al. 1987 Lichtwardt et al. 2001, Strongman et al. 2010

France, Japan France, India, USA(Alaska)

Lichtwardt et al. 1987, Lichtwardt et al. 2001 Lichtwardt et al. 2001, Misra and Tiwari 2008

Japan, USA

Lichtwardt et al. 1987, Lichtwardt et al. 2001

Cosmopolitan/Japan, Thailand Cosmopolitan/China, Japan

Hapsari et al. 2009, Lichtwardt et al. 1987, Lichtwardt et al. 2001 Lichtwardt et al. 1987, Lichtwardt et al. 2001, Strongman and Xu 2006, Strongman et al. 2010 Lichtwardt et al. 1987, Lichtwardt et al. 2001

Stachylina penetralis Lichtw.

Stachylina prolifica Lichtw. Kobayasi & Indoh

Japan, Spain

STRONGMAN AND WANG: CHINESE TRICHOMYCETES TABLE I.

877

Continued Species

Stachylina tianensis J. Wang, S.Q. Xu & Strongman Stipella vigilans Le´ger & Gauthier Zancudomyces culisetae Y. Wang, Tretter, Lichtw. & M.M. White Zygopolaris ephemeridarum Lichtw. & M.C. Williams Asellariales Asellaria armadillidii Tuzet & Manier ex Manier Asellaria ligiae Tuzet & Manier ex Manier Eccrinales Enterobryus cingaloboli Rajagopalan Enteromyces callianassae Lichtw. Enteropogon formosensis Chien & Hsieh Palavascia sphaeromae Tuzet & Manier ex Manier Parataeniella flavospora Chien & Hsieh Taeniella carcini Le´ger & Duboscq Amoebidiales Amoebidium parasiticum Cienkowski Paramoebidium bacillare Strongman, J. Wang & S.Q. Xu Paramoebidium curvum Lichtw.

Distribution

Reference

China

Wang et al. 2010

Armenia, England, France, Spain, Thailand Cosmopolitan/Japan (as Smittium culisetae) Canada, India, Mexco, USA

Hapsari et al. 2009, Lichtwardt et al. 2001

Cosmopolitan/Japan, Taiwan Cosmopolitan/Japan, Taiwan

Lichtwardt et al. 1987, Lichtwardt et al. 2001 Lichtwardt et al. 2001, Misra and Tiwari 2008

Chien and Hsieh 2001, Lichtwardt et al. 1987, Lichtwardt et al. 2001 Chien and Hsieh 2001, Lichtwardt et al. 1987, Lichtwardt et al. 2001

India Cosmopolitan/Japan Taiwan Cosmopolitan/Japan

Rajagopalan 1967 Lichtwardt et al. 1987, Lichtwardt et al. 2001 Chien and Hsieh 2001 Lichtwardt et al. 1987, Lichtwardt et al. 2001

Taiwan Cosmopolitan/Japan, Taiwan

Chien and Hsieh 2001 Chien and Hsieh 2001, Lichtwardt et al. 1987, Lichtwardt et al. 2001

Cosmopolitan/Japan China

Lichtwardt et al. 1987, Lichtwardt et al. 2001 Strongman et al. 2010

Cosmopolitan/Japan

Lichtwardt et al. 1987, Lichtwardt et al. 2001

attached to the generative cell by a long, straight extension, 50–100 3 2–2.5 mm, which remains attached to the released trichospore. Trichospore and extension linear, with no appendage. No zygospores found. Other specimens examined: CHINA. SHAANXI PROVINCE: Feng Yu Forest Park, Feng Yu River on the north side of the Qinling Mountains (Site 1) from the gut of a crane fly larva (Tipulidae), 9 May 2007.

Notes.—Two other described species are in this genus, Caudomyces japonicus and C. longicollis Strongman. Caudomyces japonicus has trichospores 25–30 3 3–3.5 mm, produced on narrow extensions 45–66 mm long that are often bent (Lichtwardt et al. 1987), while C. longicollis has trichospore dimensions 34–38.5 3 3.5–4 mm, and they are attached to the thallus by straight extensions 91–110 mm long (Strongman 2007). Trichospores of C. typhella are longer and wider (40–48 3 4–6.5 mm) than the other two species but attached to the thallus by long, straight extensions like C. longicollis. Genistelloides torrentis Strongman & J. Wang, sp. nov. FIGS. 4–6 MycoBank MB812032

Typification: CHINA. SHAANXI PROV.: Feng Yu River in Feng Yu Forest Park (Site 1), Qinling Mountains south of Xi’an, from hindgut of stonefly nymphs collected by J. Wang on 21 Dec 2007 and 23 Oct 2007. Lactophenol-cotton blue-stained thalli on a microscope slide containing the dissected hindgut from a stonefly (Plecoptera) collected 21 Dec 2007 from Feng Yu (holotype FH 00313532). Another specimen from stonefly nymphs collected by J. Wang on 23 Oct 2007 at the same site as the holotype was prepared and is deposited at FH 00313533 (paratype). Etymology: torrens (L.), swift stream.

Thallus attached to the hindgut of stonefly (Plecoptera) nymphs, with 2–3 main axes emerging from a knobby holdfast about 15 mm diam embedded in a dark amorphous substance secreted from the basal cell, main axis branched repeatedly with apical branchlets bearing 2–4 trichospores per fertile tip. Trichospores 33–42.5(–51) 3 7–10 mm, long obpyriform, with two fine appendages, not coiled in generative cell. No zygospores found. Notes.—Trichospores of G. torrentis are similar in length but wider than those of G. communis M.M. White and Lichtw. (Lichtwardt 2004). They are broader and longer than the trichospores of

878 TABLE II.

MYCOLOGIA Trichomycete species recorded from each location and dates of collection Species

Caudomyces japonicus Caudomyces typhellab Gauthieromyces indicus Genistelloides torrentis Glotzia ephemeridarum Graminella bulbosac Harpella melusinae Orphella sp. Orphella sinica Paramoebidium spp. Pennella montana Simuliomyces microsporusc Smittium shaanxiense Smittium minutisporumc Smittium spp. Stachylina pediferc Stachylina prolifica Stachylina penetralis Stachylina nana Stachylina subgrandisc Stachylina spp. Zygopolaris borealisc Unknown sp.

Sitea

Collection dates

2, 7 1, 3 1, 2, 5 1 1, 2, 4, 5 1 5 2 2 1–7 5 5 1 6 1 7 6 1 4 4 2, 6 1 1

22 Jul 2007; 10 Jun 2008; 5, 7 May 2009 5 Jul 2006; 9 May 2007 18, 27 Jun; 23 Oct 2007; 5, 6, 7, 13 May 2009 21 Oct, 23 Dec 2007 26 Mar 2008; 5, 6, 13 May 2009 23 Oct, 23 Nov 2007 13 May 2009 10 Jun 2008 10 Jun 2008 Multiple dates 2007, 2008, 2009 13 May 2009 13 May 2009 23 Dec 2007 13 May 2009 23 Nov, 21, 23 Dec, 2007; 29 Feb 2008 19 Apr 2007 29 Jul 2007 13 May 2009 25 Apr 2007 13 May 2009 10 Jun 2009, 13 May 2009 23 Oct 2007 21 Dec 2007

a

Site 1 Feng Yu (33.55.40N 108.50.37E), Site 2 Nui Bei Liang (33.48.47N 108.59.49E), Site 3 An Kang (32.40.57N 109.00.14E), Site 4 Huoditang research station (33.26.07N 108.26.49E), Site 5 Ningshan (33.24.35N 108.15.27E), Site 6 Ping He Liang (33.28.15N 108.30.01E) Site 7 Kun Ming (25.09.05N 102.47.09E). b New species in boldface. c New records for Asia.

G. hibernus S.W. Peterson, Lichtw. & B.W. Horn and the appendages of G. torrentis are not coiled in the generative cell as is typical for G. hibernus. The trichospores are shorter than those of G. amplispora M.M. White & Lichtw. and G. helicoides M.C. Williams & Lichtw., which also have coiled appendages (Lichtwardt 2004). Orphella sinica Strongman nov. MycoBank MB812033

&

J.

Wang, sp. FIGS. 728

Typification: CHINA. SHAANXI PROV.: Species collected by J. Wang from Hao River, Nui Bei Liang Conservation Area on the south side of the Qinling Mountains (Site 2). Microscope slide containing lactophenol-cotton bluestained thalli and hindgut of a stonefly (Plecoptera) collected on 10 Jun 2008 (holotype FH 00313534). This slide also has the thallus of a different unidentified Orphella sp. with what appear to be straight trichospores that are immature. Etymology: sinica (L.), referring to the country of origin, China.

Thalli attached to the hindgut of stonefly (Plecoptera) nymphs, approximately 2002300 mm long with

lateral branches in the basal area surrounded by secreted amorphous material. Trichospore and associated support cells (trichospore dissemination unit) produced in spore heads composed of 1–2 branches at the apex of the thallus, composed of clusters of clavate, basal cells 12217 3 628 mm bearing 223 trichospore dissemination units, each comprising a supporting generative cell 223 mm long, generative cell and terminal cell 22226 mm long, trichospores allantoid, 20.5223 3 3.524 m m, formed from a generative cell, 8211 3 324.5 mm. Homothallic zygospores mixed with trichospores in the same spore head, helicoid, 42245 around the coil, with the terminal end projecting prominently from the coil. Coil at the widest part 12.5213.5 mm diam, zygospore width 5.527 mm. Conjugation occurring from cells originating from the basal cells, intermediate cell (ic) strongly triangular, 12215 mm long, 15217 mm across the top, tapered to 425 mm at the base, sometimes asymmetrical, zygosporophore pyriform, 9212 3 327 mm, with a terminal cell 11223 mm. Notes.—Four described Orphella species have allantoid trichospores, but those of O. sinica are smaller,

STRONGMAN AND WANG: CHINESE TRICHOMYCETES

879

FIGS. 1–3. Caudomyces typhella. 1. Thallus with trichospore (arrow) attached. 2. Spent thallus with globular holdfast (arrows). 3. Released trichospores (arrowheads) with long extensions (arrows). Bars 5 20 mm.

880

MYCOLOGIA

FIGS. 4–6. Genistelloides torrentis. 4. Fertile tips of thallus branches with attached (arrows) and released trichospores. 5. Thallus basal cells showing secreted holdfast material (arrow). 6. Released trichospores with two appendages (arrows). Bars 5 20 mm.

STRONGMAN AND WANG: CHINESE TRICHOMYCETES

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FIGS. 7, 8. Orphella sinica. 7. Sporulating heads of thallus with allantoid trichospores (arrows), helical zygospores (white arrows) arising from T-shaped intermediate cells (white arrowheads). 8. Base of thallus with many lateral branches (black arrowhead) and secreted material (white arrowhead). Bars 5 20 mm.

20.5223 3 3.524 mm, than any other species. The helical zygospores in O. sinica (12.5213.5 3 5.527 mm) also are narrower across the coil than in any of the other species (Lichtwardt 2004). Orphella hiemalis S.W. Peterson, Lichtw. & Huss is most like O. sinica because it has broad T-shaped intermediate cells, which are 18223 3 8213 mm at the base, and produce zygosporophores; in O. sinica the ic is 15217 mm across the top, tapered to 425 mm at the base, and

more triangular. The trichospore and zygospore dimensions and the intermediate cell size and shape allow O. sinica to be distinguished from all other Orphella spp. (Lichtwardt 2004). Other species.—At least 23 species were recorded in our collections, 200622009 (TABLE II). Many trichomycetes were collected from sites previously sampled or are species known from other countries in the region (TABLE I), but specimens of six species,

882

MYCOLOGIA

FIGS. 9–11. Other trichomycetes collected. 9, 10. Paramoebidium sp. with conspicuous papilla (FIG. 9 arrow) at terminal end of the thallus. 11. Spore head of a possible Orphella sp. with clavate basal cells (arrowhead) and long, clavate trichospores (arrow).

Graminella bulbosa, Simuliomyces microsporus, Smittium minutisporum, Stachylina pedifer, St. subgrandis and Zygopolaris borealis, expand their known geographical distribution (Lichtwardt et al. 2001). Three additional species had distinctive morphological features but either too little material was collected or the characters visible were too immature to allow definitive identification or description. Paramoebidium is a protist trichomycete that is common in the hindguts of aquatic host insects, but the simple unbranched thallus and paucity of other distinctive characters do not provide sufficient morphological features to differentiate among a large number of potential species. We collected a specimen of Paramoebidium from a mayfly nymph at Feng Yu (Site 1) with a wide thallus and a distinctive prominent papilla at the terminal end (FIGS. 9, 10). Three

Paramoebidium spp. have a terminal papilla (Lichtwardt 2004), but the papilla is smaller than on the Chinese specimen. One hindgut from a stonefly contained the new species, Orphella sinica, intermingled with what could be a different Orphella sp. This specimen had a terminal spore head with typical basal cells but arising from these were elongated, clavate cells (FIG. 11). These may have been trichospores without the generative and terminal cells normally found on other species with straight trichospores (Lichtwardt 2004). This specimen was immature so it is impossible to determine what the final spore head morphology would look like. A third unique thallus was retrieved from the hindgut of another stonefly collected at Feng Yu. This specimen had a bulbous, possibly bifurcate basal holdfast cell (FIG. 12), and a spore head composed of 324

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883

FIGS. 12–15. Unidentified trichomycete. 12. Thallus mass with a bifurcate holdfast (arrows). 13. A cluster of septate (arrows) fertile branches. 14, 15. Spore heads with possible trichospores forming at the tips of septate branchlets (arrows). Bars 5 20 mm.

branches, each producing a cluster of 324 terminal branchlets (FIGS. 13215). Each of these was divided by septa into 425 short cells and there appeared to be a trichospore forming at the tip of the apical cells (FIG. 15). The overall structure resembled Zygopolaris spp. or Pteromaktron spp., but these are known only from mayfly hosts and the arrangement of the apical cells and fertile branches (Lichtwardt 2004) differ from our specimen. No identification to genus or species was possible based only on thallus features. Gauthieromyces was proposed for the unusual, invalidly published trichomycete species, Genistella microspora (Lichtwardt 1983). The genus was monotypic until a second species was described by Misra and Tiwari (2008) and another species, G. viviparous, was added by Valle et al. (2008). The addition of new

species and newly observed morphological characters (trichospore collar, appendage and vegetative propagules) not seen in the type species allows a more complete description of the genus; it is emended to reflect this new information. Examination of the type slide for G. indicus revealed additional features not in the protologue and that species is emended as well. Gauthieromyces Lichtw., emend. Strongman Thallus arbusculate with a bulbous basal cell narrowing apically into an axial trunk with lateral branches, also branching from the bottom of the basal cell. Vegetative propagules produced from the basal cell and from the base of the axial trunk. Fertile branches bearing curved trichospores attached eccentrically (basolaterally) or basally to their generative cells. In hindgut of Baetidae (Ephemeroptera) nymphs.

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Type species: Gauthieromyces microsporus Lichtw. Epitype, designated here: Gauthieromyces indicus on slide FH 00081103, Farlow Herbarium Harvard Univ. Gauthieromyces indicus Misra & Tiwari, emend. Strongman FIGS. 16–23 Thallus in hindgut of Baetis sp. (Ephemeroptera) nymphs, arbusculate, up to 300 mm long, with a broad basal holdfast, 34–54 mm 3 13–22 mm, and a stout, dichotomously branched main axis, tapering from the base to an apex, 2.5–5 mm wide. Secondary branches not tapering, 2.5 mm diam. Fertile branches 25–60 3 2.5–5 mm, bearing 3–15 irregular, horseshoe-shaped trichospores 5.5–7 mm across, from the point of attachment to the generative cell, 2–4.5 mm wide, 16– 20 mm around the U-shape, attached eccentrically to generative cells that are 2.5–7.5 3 2.5 mm. When released, trichospores with a collar 2–4.5 3 1–2 mm and single long appendage, often tightly wound or tangled near the collar. Propagules 38–48 3 12.5–15 mm at the distal end formed from a basal cell or from branches emerging from the swollen base, with a thickened cell wall at the apex and tapered to 5.5–8 mm where attached to the thallus. Zygospores not seen. DISCUSSION Many species of trichomycetes are reported from Asia (TABLE I) despite the few collectors in the region. The most extensive collections are from Japan (20 species), recorded over a 6 y period (Lichtwardt et al. 1987) and China, documenting 28 species including those in this paper, 2005–2009 (Strongman and Xu 2006, Strongman et al. 2010, Wang et al. 2010). The species from Asia shows trichomycetes that are cosmopolitan in distribution and some that are known only from one or more Asian countries (TABLE I). Of note some species are reported from markedly disparate locations, either from hosts with low vagility e.g. G. ephemeridarum (China, USA) and B. repens (Japan, Spain, USA) from mayflies or more vagile hosts such as blackflies, e.g. P. montana (Costa Rica, Mexico, Thailand, China) or chironomids such as S. hecatei (Canada, Spain, China) and S. macrospora (France, India, Alaska). Misra (2001) and Lichtwardt (2012) described groups of tropical trichomycete species, including some from Thailand and India, and noted that there were also also cosmopolitan species found along with some known only from the tropics. Other examples show that some trichomycete species, although rare, can be found in widely separated geographic locations. For example, Lancisporomyces nemouridarum Strongman & M.M. White described from stoneflies in eastern Canada (Strongman and

White 2006) was found in Mexico (Valle et al. 2008) and Allantomyces zopilotei L.G. Valle, M.M. White & Cafaro, described in the latter paper from Mexico, was recorded from eastern Canada (White and Strongman 2012). As inventories from different parts of the world are compiled, a clearer picture of the actual distribution of these obligate gut inhabitants will emerge. Structures that are vegetative in nature, producing new thalli in the same host gut, have been described variously as vegetative spores in Ejectosporus spp., allantoid auxiliary spores in Allantomyces caenidarum M.C. Williams & Lichtw. and bulbils or propagules in Gauthieromyces spp. and Graminella spp. (Lichtwardt et al. 2001). Morphologically these vegetative structures are different (Lichtwardt et al. 2001). Vegetative spores in Ejectosporus spp. are produced on terminal branches and look like trichospores but eject the sporoplasm in situ (Lichtwardt et al. 1991, Strongman 2005). Allantoid auxiliary spores in A. caenidarum also are produced on fertile branches, often intermingled with trichospores, but these propagules are released into the gut lumen where they germinate to form more thalli attached to the gut lining (Williams and Lichtwardt 1993). The bulbils or propagules of Graminella spp. are produced from the swollen basal cell or from branches emerging from this base and either fall off, as they do in G. bulbosa, or eject sporoplasm, leaving the wall of the bulbil attached to the thallus, as in Graminella microspora S.T. Moss & Lichtw. (Lichtwardt and Moss 1981, Lichtwardt 2004). The description of the type species G. microsporus makes no mention of vegetative propagation (Lichtwardt 1983), but the other two Gauthieromyces spp. produce vegetative propagules. In G. indicus clavate cells with a conspicuous thickening of the apical end are attached to the swollen basal cell, and primary branches arising from the base were common in the type material although not described in the protolog (FIG. 19). Collections of this species from China (FIGS. 20, 22, 23) frequently had the same type of cell. Valle et al. (2008) described elongate propagulelike cells associated with the basal cell and branches in G. viviparus and also mention the thickened cell wall observed in these cells. Some features of the propagules of G. indicus make them distinctively different from other known vegetative cells. The thickening of the bulbous apex of these cells in G. indicus is more pronounced (FIGS. 19, 22, 23) than that described for G. viviparus. Lichtwardt and Moss (1981) illustrated propagules in G. microspora extruding in situ through the apical part of the cell but no thickening was seen as in G. indicus propagules. Perhaps the thickened apical wall in the propagules of G. indicus aids in the release of cell contents, but we have collected it many times in China

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FIGS. 16–19. Gauthieromyces indicus from the holotype and isotype slides (FH). 16. Whole thallus showing swollen base (white arrow) with basal branches and fertile apical branches with attached c-shaped trichospores (black arrow). 17. Attached and released trichospores. A collar (white arrow) and single appendage (black arrow) are visible on the released trichospore. 18. Released trichospore with a single appendage (black arrow) that is tangled at the base of the collar (white arrow). 19. Thick-walled propagules (white arrow) formed from branches arising from the swollen base (black arrow). Bars 5 20 mm.

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FIGS. 20–23. Gauthieromyces indicus from China collections. 20. Whole thallus, fertile branches with trichospores attached and two released (arrowhead). Propagules attached to the swollen base and branches (arrow). 21. Released C-shaped trichospores with a collar (arrowheads ) and single appendage (arrows). 22. Propagule with thickened apex (arrow), produced from a cell where three branches come together. 23. Propagules produced from a cell in the swollen base (arrowhead) and from branches arising from the base. Note the three branches (arrows) either fused or emerging from where the propagule is produced. Bars 5 20 mm.

STRONGMAN AND WANG: CHINESE TRICHOMYCETES and never observed extrusion in the gut like that seen in G. microspora. In the Asian species these propagules are produced from the swollen basal cells and from the branches emerging from the basal cells, a condition also noted for G. viviparus. A cell on a branch producing a propagule can have multiple associated branches (FIGS. 22, 23). It is difficult to determine whether these adjacent branches contribute to the formation of the propagule and thus constitute homothallic conjugation. Does the morphology of the propagules in G. indicus and the arrangement of the branches contributing to the formation of these cells suggest they are sexual spores? Homothallic zygospore production is widespread in harpellid species (Lichtwardt et al. 2001), and the recognition of unusual forms of sexual spores such as the helical zygospores in Orphella spp. (Valle and Santamaria 2005) or turbinate zygospores in Zygopolaris spp. (Lichtwardt et al. 2001) support this theory. Presently, however, there are no irrefutable data that show whether these structures are vegetative propagules or sexual spores. Evidence that these cells can form by heterothallic conjugation would support a sexual-spore interpretation. Cytoplasmic extrusion of these unusual cells and subsequent adhesion and growth of a thallus in the gut would demonstrate a vegetative function. ACKNOWLEDGMENTS We appreciate assistance from Dr Shengquan Xu, College of Life Science, Shaanxi Normal Univ., Xi’an, PR China, for supervising and financially supporting JW during her graduate studies and providing support and participating in the collecting trips to the Qinling Mountains. DBS also received support and work space from the College of Life Science, Shaanxi Normal Univ., while collecting in China and travel funds from Saint Mary’s Univ. Thanks to Carolyn Bird, Chester Basin, NS for advice on the Latin names. The Farlow Herbarium, Harvard Univ., Cambridge, Massachusetts, provided a loan and permission to photograph the type and isotype slides of G. indicus.

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