Mycologia, 100(1), 2008, pp. 149–162. # 2008 by The Mycological Society of America, Lawrence, KS 66044-8897
Harpellales in the digestive tracts of Ephemeroptera and Plecoptera nymphs from Veracruz, Mexico Laia Gua`rdia Valle1
Veracruz. Mayflies and stoneflies are interesting host orders of Harpellales because they both have ancient insect origins and have not yet been observed with fungal diaspores in their (dispersive) adult stages (White et al 2006a). In this paper we use the lowercase form of trichomycetes for the gut fungi as an ecological group instead of the uppercase form for class Trichomycetes, which is now being reconsidered by the fungal community based on molecular phylogenies (Hibbett el al 2007). The traditional four orders of the Trichomycetes included two (Amoebidiales and Eccrinales) that are now recognized as Mesomycetozoan protists (Benny and O’Donnell 2000, Ustinova et al 2000, Lutzoni et al 2004, Tanabe et al 2004, Adl et al 2005, Cafaro 2005, White et al 2006b, Hibbett el al 2007). Despite higher level taxonomic flux the Harpellales are microscopic filamentous fungi that live attached to the chitinous gut lining of their arthropod hosts (Lichtwardt et al 2001), including not only nymphs of Ephemeroptera and Plecoptera but also larval stages of the aquatic lower Diptera, Coleoptera and Trichoptera (Lichtwardt 1986, Lichtwardt et al 1999, 2001, White 1999) and rarely adult freshwater Isopods (White 1999). No ephemeropteran gut fungi and only one genus (Genistelloides Peterson, Lichtw. & Horn 1981) of Harpellales inhabiting stoneflies has been cultured. Certain aspects of the symbiosis are not completely understood, and gut fungi usually are considered to be commensalistic. Mexico spans both south temperate and northern tropical forests; it is in the confluence of both nearctic and neotropical biogeographic regions (Llorente et al 1996). Veracruz, with Chiapas and Oaxaca, are the Mexican states with the highest density of species (Flores and Ge´rez 1994), making them particularly attractive locations for biological surveys and field collections. In a mycological context Mexico is known for its outstanding fungal diversity (Guzma´n 1998), although some micromycetes are yet scantily known or not surveyed (including the Harpellales). The Harpellales, the trichomycete order with the most diversity (Lichtwardt et al 2001), has been surveyed worldwide, although temperate nearctic and palearctic regions have been explored more intensively (White et al 2000). Known distributions of gut fungi are more in accordance with the distribution of past and present trichomycetologists and their institutions, as well as selected field sites and convenient
Unitat de Bota`nica, Departamento Biologia Animal, Biologia Vegetal i d’Ecologia, Facultat de Cie`ncies, Universitat Auto`noma de Barcelona, 08193-Bellaterra (Barcelona), Espan ˜a
Merlin M. White2 Boise State University, Department of Biology, 1910 University Drive, 210 S/N Building, Boise, Idaho 83725-1515
Matı´as J. Cafaro3 Departamento de Biologı´a, Universidad de Puerto Rico, Mayagu ¨ ez, Puerto Rico 00681-9012
Abstract: This is the first report of Harpellales (Zygomycota) from Mexico, including herein only the endosymbiotic species of gut fungi in the digestive tracts or shed exuviae of Plecopteran and Ephemeropteran nymphs. Four new species are described: Allantomyces zopilotei, Bojamyces olmecensis, Gauthieromyces viviparus and Graminella ophiuroidea. Among previously known Harpellales, Lancisporomyces nemouridarum and Zygopolaris ephemeridarum are southern range extremes and new records for Mexico. All species are illustrated and discussed relating to biogeographic implications of the new reports from Mexico, as well as the particular environmental circumstances of the Harpellales in the tropics. Key words: biogeography, endosymbionts, neotropics, taxonomy, Zygomycota INTRODUCTION
The data presented in this paper are part of a broader Biodiversity Surveys and Inventories (BS&I) project to examine the biodiversity of Harpellales in North America, including the first surveys of gut fungi in Mesoamerican Mexico. Adding new taxa to and expanding range extensions for known species distributions of the Harpellales fosters future biogeographic interpretations (Lichtwardt 1995, Lichtwardt et al 1993). We have combined herein our findings on the diversity of Harpellales (Zygomycota) associated with mayflies (Ephemeroptera) and stoneflies (Plecoptera) collected during two surveys in the state of Accepted for publication 24 September 2007. 1 Corresponding author. E-mail:
[email protected] 2 E-mail:
[email protected] 3 E-mail:
[email protected]
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laboratory stations. This first report, and with data from forthcoming publications, will form the baseline of trichomycete diversity in Mexico to compare with the known high diversity reported for other taxa. These collections also provide new data to be added to those from prior neoptropical studies (Alencar et al 2003, Lichtwardt 1994, 1997, White et al 2000). MATERIALS AND METHODS
The description of all taxa is based on the material collected from several localities in two distinct areas of the state of Veracruz, Mexico. Each area was sampled separately for a total duration of 35 d. Collections from the initial expedition (3 Nov–19 Nov 2005; MEX-1–19) were mostly submontane humid areas in the National Park of ‘‘Cofre de PeroteNauhcampate´ petl’’ (19u259330–19u339520N; 97u069550– 97u129520W), an inactive volcanic uplift area (elevation
TABLE I. Site
4282 m). Surrounding areas, with climates influenced by this significant formation were explored as well. Among these was the Actopan River basin (see TABLE I) and with other collections in the lowland humid cloud forest. Laboratory facilities for the first trip were provided by the Instituto de Ecologı´a A.C. (Xalapa). Collections during the second survey (24 Jul–13 Aug 2006; MEX-20–47) were in the lowland and submontane humid evergreen tropical forest of ‘‘Los Tuxtlas’’, where the substrate is predominantly acidic Pleistocenic igneous with annual average rainfall of 4725 mm (Flores-Delgadillo et al 1999). Laboratory facilities were provided by the Tropical Biological Station ‘‘Los Tuxtlas’’, which is associated with the National Autonomous University of Mexico (Universidad Nacional Auto´noma de Me´xico, UNAM). The station is within ‘‘Los Tuxtlas’’ Biological Reserve (18u349– 18u369N; 95u049–95u099W) that comprises ca. 700 ha of remnant patches of primary vegetation interspersed with local human communities (Martı´nez-Sa´nchez 2001).
Collection sites in Veracruz, Mexico, where Harpellales were recovered in Ephemeroptera and Plecoptera Date
Location, description FIRST TRIP
Coordinates
Altitude (m)
Temp C
1300
16.5
1440
14.5
400
23.5
(XALAPA)
MEX-1
5 Nov 05
Coatepec. La Pitaya (Zocoastla). Rı´o Pixquiac. Next to the convent. Former coffee plantation land. San Andre´s Tlalnelhuayocan, Rancho Viejo, Agu¨ita Frı´a. Spring fed stream, Misty forest. Emilio Zapata. El Aguaje. Rı´o El Aguaje (Actopan -affluent). Stream crossing road near small town (agricultural area, pesticide spraying noted nearby). High mayfly diversity. Xico. Road from Xico to Xico Viejo, Km. 2.5. Puente de la Virgen de Guadalupe. Cofre de Perote mountain. Xico. Xico Viejo. Xico Viejo River. Next to the little village, pasture land. Xico. Xico Viejo. Xico Viejo River.
MEX-4
6 Nov 05
MEX-5
8 Nov 05
MEX-12
11 Nov 05
MEX-13a
11 Nov 05
MEX-15
14 Nov 05
MEX-16
14 Nov 05
MEX-18
15 Nov 05
Xico. Road from Xico to Xico Viejo, Km. 2.5. Puente de la Virgen de Guadalupe. Coatepec. La Pitaya. Rı´o Pixquiac.
MEX-19
17 Nov 05
Emilio Zapata. El Aguaje. Rı´o El Aguaje.
MEX-31
30 Jul 06
(LOS TUXTLAS) Miguel Hidalgo y Costilla. El Amopal. River next to town.
MEX-33
30 Jul 06
Stream ‘‘large’’ bridge, big boulders, steep. 550 m.
MEX-34
30 Jul 06
Fast flowing creek near MEX-33, crossing the road.
MEX-51
5 Aug 06
MEX-54
8 Aug 06
´ rganos. Apparently pristine, San Andre´s Tuxtla. River at Los O although in town; upriver looks clean. ´ rganos. San Andre´s Tuxtla. River at Los O
MEX-56
10 Aug 06
´ rganos. San Andre´s Tuxtla. River at Los O
N 19 30.3819 W 96 57.6379 N 19 31.1809 W 96 59.2309 N 19 25.1279 W 96 37.0199 N 19 26.6779 W 97 02.7579 N 19 27.1119 W 96 03.4899 N 19 27.1119 W 96 03.4899 N 19 26.6779 W 97 02.7579 N 19 30.3819 W 96 57.6379 N 19 25.1279 W 96 37.0199
1667
14
1800
14
1800
14
1667
14
1300
17
400
23.5
660
21
560
22
500
21
220
23
220
23
220
23
SECOND TRIP
N 18u22.1509 W 94u57.2809 N 18u22.0219 W 94u57.9029 N 18u22.0549 W 94u58.5599 N 18u39.3499 W 95u09.1829 N 18u39.3499 W 95u09.1829 N 18u39.3499 W 95u09.1829
VALLE ET AL: MAYFLY AND STONEFLY HARPELLALES Ephemeropteran and plecopteran hosts of Harpellales were captured following the methods described by Lichtwardt et al (2001), using regular limnological nets or small aquarium nets and hosts were picked from substrates with plastic pipetters or small forceps. Hosts, with minimal native substrate and stream water from collection sites, were consolidated in small jars or plastic bags and transported to the lab on ice in portable coolers. Living hosts were dissected with the aid of stereomicroscopes. Gut-dissected endosymbionts were placed on glass slides as wet mounts, cover slips added, and living specimens were microphotographed (with Kodak 200 ISO, 35 mm daylight color film) as viewed with phase optics using an Olympus CH-2 compound scope. Specimens were fixed and stained by infiltration with Lactophenol cotton-blue (LPCB) before sealing with clear fingernail polish. Voucher slides were reexamined later with DIC optics (Zeiss). Spores and thalli were measured directly from water-mounted specimens or photographs. Photomicrographs of LPCB specimens (indicated with an ‘‘(f)’’ by figure legend numbers) were taken with a Zeiss Axiocam digital camera (see Kim et al 2007 for discussion of effects of LPCB). All measurements listed herein are from living specimens. Each collection at a location per date was given a reference number, which for simplicity we prefaced with a single geographic reference MEX. Individual dissection code labels include a second number indicating our site code (see TABLE I) and a third with a letter/number combination to correspond to the individual and their specific host dissection (sequentially numbered). Any number in the third position preceded by a letter was a dissection by Valle (L), White (W) or Cafaro (C). TAXONOMY
Allantomyces zopilotei L.G. Valle, M.M. White & Cafaro sp. nov. FIGS. 1–11 Thalli pinnatim ramosi, interdum ad termina distalia et di- et trichotome ramosi. Cellulae axis principalis ad terminum proximalem 14–20 mm diam, ad ramos distales tantum usque 3 mm. Cellula tenax in exemplis junioribus processu acuto praedita, maturitate variabiliter in secretionem tenacem tenuem reducta vel lobulata et plerumque ramis proximalibus lateralibus praedita. Trichosporae ovato-ellipticae vel subcylindricae, tumore submediano praeditae, quoad longitudinem valde variabiles, 9–24 3 3.5–5 mm, collare reflexo, 3–5 3 2.5 mm, interdum collare carentes, appendice unica, tenui, crispatiuscula praeditae. Cellulae generatoriae per ramum fertilem (1–)4–8, quoad longitudinem variabiles. Zygosporae ad typum I nominatum pertinentes, ad locum affixionis medialem curvatiusculae vel paene rectae, 53–63 3 5.5–6.5 mm, collare carentes. Post emissionem in loco affixionis area derasa in zygosporae visibilis. Zygosporophorum ex hyphis conjugantibus obscure distinctum, in dispositione V- (Y-)formi ordinatae, zygospora ad confluentiam hyphalem emergenti. Sporae allantoideae auxilliares non visae. Ad integumentum proctodaei interius nympharum Tricorythodum et Leptohyphodum (Ephemeropterorum: Leptohyphidarum) inde-
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terminatarum affixa. Holotypus, hic designatus, MEX-19W6A, in FH conservatus, in rivo El Aguaje prope Emilio Zapata lectus.
Thalli pinnately branched, sometimes with di- and trichotomous branching at distal ends (FIGS. 1–4). Main axis cells 14–20 mm diam at the proximal end, decreasing to 3 mm at distal branches. Holdfast cell tapered to a point in young specimens, variable with maturity ranging from nondifferentiated with a thin holdfast secretion to lobulate, usually with proximal lateral branches (FIG. 5). Trichospores ovate-ellipsoidal to subcylindrical with a submedial swelling, and a wide range in lengths, 9–24 3 3.5–5 mm, with an outflared collar 3–5(–7) 3 2.5 mm (sometimes absent), and a single, fine, slightly curled appendage (FIGS. 6– 8). Generative cells (1–)4–8 per fertile branch, variable in length (FIGS. 2–4). Zygospores Type I, perpendicular to and straight or slightly bent at the medial point of attachment, 53–65 3 5.5–7 mm, collarless (FIG. 9). A scar is visible on the released zygospore, where it was attached to the zygosporophore (FIG. 9). Zygosporophore not clearly delimited from the conjugating hyphae, arranged in a V- (or Y-) shaped pattern, the zygospore emerging from the vertex of conjugating hypha (FIGS. 10–11). No auxiliary allantoid spores observed. Attached to the hindgut lining of Tricorythodes sp. and Leptohyphes sp. (Ephemeroptera: Leptohyphidae) nymphs. Etymology. Local voice, zopilote 5 Mexican name for a vulture (Coragyps atratus) common in tropical and subtropical zones of America; for the mature zygospores, which resemble flying zopilotes. Specimens examined. MEX-1-: L3, L4, L5, L7, L10, L11, L12; MEX-18-: L2, L3, L4, L6, L7, L9, W6, W7; MEX-19-: L1, L2, L3, L4, L6, W3, W6A (HOLOTYPE), W7 (ISOTYPE), W9, W10, MEX-51-, MEX-54-: L12, L15, MEX-56-: L2–L9.
This second species of Allantomyces M.C. Williams & Lichtw. was found in a mayfly host, Trichorythodes (Leptohyphidae), a genus distributed throughout North and Mesoamerica. The type species A. caenidarum was found in Caenidae hosts from Australia (Williams and Lichtwardt 1993). Most of the living larvae that were examined presented immature thalli in their hindgut; few had mature thalli producing trichospores. Most of mature zygospore-producing thalli were found in the shed exuviae, which were abundant entangled in vegetation at the river margins (Site MEX-19), where flow was reduced. During the second survey a few late instar nymphs (MEX-56) had thalli with zygospores. Except for the smallest trichospores both spore types are larger in the new species compared to those from A. caenidarum (trichospores [11–]13[–16] 3 [2.5–] 3[–4] mm, zygospores [34–]42[–46] 3 [4–]5[–6] mm, according to Williams and Lichtwardt [1993]).
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FIGS. 1–11. Allantomyces zopilotei from Tricorythodes sp. 1. Thalli overview with fertile branches and trichospores (MEX-18W6). 2–4. Fertile branches with trichospores; magnified in 3 to show a loose trichospore (arrow) with a small collar (MEX-18W6). 5. Basal cell, detached from gut lining (arrow) (MEX-18-L3). 6–8. Loose trichospores, each with a collar and faint appendage (arrow) (MEX-19-L1, MEX-1-L10). 9. Detached zygospore, with a scar (arrow) after release from the zygosporophore (MEX-19-W6A). 10–11. Zygospores, attached to corresponding zygosporophore (arrows) (MEX-19-W6A). Bars 5 10 mm in 3, 5–8; 25 mm in 1–2, 4, 9–11.
Trichospores in the new species present a wideranging size (FIGS. 2, 4), but we cannot consider them to be dimorphic because no bimodality among the range could be detected; the measurements are continuous between the largest and smallest spores observed (all spores measured were released and therefore assumed to be mature). Zygospores of A. zopilotei typically are bent slightly and lack a collar
(FIGS. 9–11), whereas they are straight with a collar in A. caenidarum (Williams and Lichtwardt 1993). The conjugant hyphae of A. zopilotei unite in a V- (or Y-) shaped pattern (FIGS. 10–11), with the zygosporophore nearly indistinguishable as a short prolongation distally, with the zygospore subsequently developing directly above it (FIGS . 10–11). This is distinguished from A. caenidarum zygospores, which
VALLE ET AL: MAYFLY AND STONEFLY HARPELLALES arise from an extension of one of the conjugants (also arranged in a different manner), above a prominent zygosporophore (Williams and Lichtwardt 1993). Differences among the specimens collected in each of the trips are not significant, except for the fact that a major proportion of largest trichospores were collected in the area of Xalapa and the trichospores with largest collars in the area of ‘‘Los Tuxtlas’’. These differences are not relevant considering the wide-range of spore sizes within a single population and the variation also concerning thallial characteristics. Zygospores were slightly wider in the specimens from Tuxtlas region (2nd trip), but differences again were not significant. Bojamyces olmecensis M.M. White, L.G. Valle & Cafaro sp. nov. FIGS. 12–15 Thalli sparse ramosi, 3.5–5 mm diam. Cellula tenax indistincta, plusminusve simplex, tenuis, secreta. Trichosporae elongato-ellipticae, 30–35 3 3.5–5 mm diam, collare marginibus convergentibus 3.5–5 3 2–2.5 mm praeditae, appendice unica filiformi gerentes, in thallis orientatione fortuita effectae. Cellulae generatoriae thallum totum secus ubique sparsae, quoad longitudinem variabiles, interdum quam trichosporae longiores. Zygosporae in progressu initiali rotundae, maturitate biconicae ad typum I nominatum pertinentes, 50–60 3 5–7 mm, in zygosporophoris in medio atque ad perpendiculum affixae, in vicinitate conjugationum scalariformium effectae. Zygosporophorum conoideum, ca. 7.5 mm longum, ad basem ampliatum, apicem versus regulariter attenuans. Ad integumentum proctodaei interius nympharum Leptophlebiidarum (Ephemeropterorum) affixa. Holotypus, hic designatus, MEX-34W1A, in FH conservatus, in rivo rapide fluenti prope lectus.
Thalli sparsely branched, 3.5–5 mm diam (FIGS. 12, 15). Holdfast cell undifferentiated, simple with a thin layer of secreted holdfast material. Ellipsoidally elongate trichospores, 30–35 3 3.5–5 mm diam, with a collar of convergent margins 3.5–5 3 2–2.5 mm and a single, filiform appendage (FIGS. 12, 13, 15). Trichospores develop with random orientation on thalli. Generative cells scattered along the entire thallus, variable in length, sometimes longer than the trichospore. Zygospores biconical, Type I at maturity 50–60 3 5–7 mm, medially and perpendicularly attached to the zygosporophore, formed in the vicinity of the scalariform conjugations between adjacent branches (FIGS. 14, 15). Zygosporophore cone shaped, about 7.5 mm long, with a swollen base tapering at the attachment point of the zygospore (FIGS. 14, 15). Attached to the hindgut lining of Leptophlebiidae nymphs (Ephemeroptera). Etymology. Olmecensis 5 in reference to the Olmecs, pre-Hispanic natives of southeastern Mexico. Specimens examined. Collection site MEX-34-: W1A (HOLOTYPE), W2 (ISOTYPE), W4 (ISOTYPE).
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Both species of Bojamyces are unique among Harpellales by having a nonpolar thallus structure and scattered generative cells. The type species B. repens Longcore (1989), found originally in the hindgut of Leptophlebiidae mayfly nymphs from USA, lacks appendages and has relatively large trichospores, (30–)45(–77) 3 6–8 mm (Longcore 1989). The second species, B. transfuga Valle & Santam. (2004), found in Spanish Caenidae (mayfly) nymphs, has a single trichospore appendage as well as zygospores, (27–)42(–50) 3 8–9 mm, according to Valle and Santamaria (2004), that typically are shorter and wider than those observed in B. olmecensis. Trichospores of the new species are similar in length to those of B. transfuga, (24–)30(–36) 3 5–6 mm, Valle and Santamaria (2004), but usually are narrower and with a longer collar. Generative cells in B. olmecensis can be disarticulated in groups of few cells, as also reported for B. repens (Longcore 1989). All zygospores observed were attached to the corresponding zygosporophore, thus sexual spore appendages remain undetermined. Zygospores in A. zopilotei are spherical in early development (FIG. 14, arrows) but they progressively enlarge and become biconical. This process has been reported before (Lichtwardt et al 2001, Moss et al 1975, Valle 2007) and may be common for most, if not all, species of Harpellales. Gauthieromyces viviparus L.G. Valle, M.M. White & Cafaro sp. nov. FIGS. 16–20 Thalli arbusculati vel pinnatim in parte superna ramosi, usque 260 mm longi, basaliter aucti, ad apicem attenuantes. Cellula tenax 20–45 3 10–21 mm, interdum lateraliter atque basaliter ramosi. Trichosporae litteram V in forma extensam simulantes, longitudine arcus 7–9 mm 3 2.5–3 mm, per extremum proximale ad cellulam generatoriam affixae, collare carentes, in statu emissionis appendice unica filiformi gerentes. Cellulae generatoriae usque ad per ramum fertilem 25, 3–6 3 2.5–3.5 mm. Interdum structurae propaguloideae in cellulam basalem atque contiguam axis thalli principalis inferioris formatae, crescentes antea a thallo (parenti) principali separatae. Zygosporae ignotae. Ad integumentum proctodaei interius nympharum Baetodis sp. atque Baetis sp. (Ephemeroptera: Baetidae) affixa. Holotypus, hic designatus, MEX-31-L2, in FH conservatus, in ad flumen parvum prope oppidum El Amopal lectus.
Thalli up to 260 mm long, diversely branched, presenting dichotomous sections, with an enlarged basal cell narrowing abruptly to long branches (FIG. 16). Holdfast cell 20–45 3 10–21 mm, sometimes with lateral and basal branches (FIGS . 16, 19). Trichospores an extended V-shape, with an arc length of 7–9 mm 3 2.5–3 mm, attached to the generative cell at the proximal end, no collar (FIGS. 16–18, 20), with a single filiform appendage on release (FIGS. 17–18). Generative cells up to 25 per fertile branch, 3–6 3
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FIGS. 12–20. 12–15. Bojamyces olmecensis from Leptophlebiidae. 12. Generative cells and trichospores, note trichospore appendage sacs within generative cells (arrows) (MEX-34-W4). 13. Detached trichospores, each with a collar (arrowhead) and single appendage (arrow) (MEX-34-W2). 14(f). Mature biconical zygospores, immature spherical zygospores (arrows) and conjugation tube (arrowhead) (MEX-34-W1). 15. Three mature zygospores (arrows) and one trichospore (arrowhead) (MEX34-W1). 16–20. Gauthieromyces viviparus. 16. Thallus overview with fertile branches, attached and detached trichospores and
VALLE ET AL: MAYFLY AND STONEFLY HARPELLALES 2.5–3.5 mm. Outgrowths, which we refer to as propagule-like structures, can form on the basal and second cell of the thallus and start growing before being detached from the main (parent) thallus (FIG. 16). Zygospores not found. Attached to the hindgut lining of Baetodes sp. and Baetis sp. nymphs (Ephemeroptera: Baetidae). Etymology. Latin, viviparus: vivus 5 alive + parire 5 born; for the presence of propagules, which start their development attached to parent thallus. Specimens examined. Collection site MEX-5-L1; MEX-19-: L5, W8; MEX-31-: L1 (HOLOTYPE), L2; MEX-33-: L2 (PARATYPE), L3, L4.
Genus Gauthieromyces was monotypic until recently, but now having three species including the species described here; all being relatively rare considering the widespread nature and frequency of collection of their Baetidae hosts. The type species, Gauthieromyces microsporus Lichtw. (5Genistella microspora Gauthier), was described from Baetis hosts collected in France, and the only illustration available of the specimens is a line drawing from the author (Gauthier 1960, Lichtwardt 1983); because this species has yet to be re-encountered. The second species is in process of publication by Misra and Tiwari (pers comm). It was reported from India, also in Baetis nymphs (Misra and Tiwari pers comm, Strongman and Xu 2006), being more similar to the type species in the horseshoe-shaped trichospores and in size. In the Indian species the authors observed some released trichospores with multiple fine appendages (Misra and Tiwari pers comm). Apparently the same species also was observed subsequently in central China (Strongman and Xu 2006) but remained unnamed because the first China paper with the species description has yet to be published. We observed a single long appendage in released trichospores, often fine and difficult to discern, partially folded on initial trichospore release (FIG. 18). Gauthier (1960) could not clearly discriminate the presence of appendage structures in trichospores because only attached spores were available on that occasion. Gauthieromyces viviparus is distinguished from both G. microsporus and the Asian species by trichospore size (smaller) and shape (an open ‘‘V’’) instead of the tightly folded U-shape of the latter two species of the genus. In addition to its single appendage (FIGS. 17, 18) trichospores of G.
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viviparus are attached to the generative cell from one extreme (FIGS. 16, 20) not eccentrically as in the other two species. Generative cells of G. viviparus and Asian species are similar in size and number but comparatively shorter and more numerous than in G. microsporus (Gauthier 1960). We have observed the presence of propagule-like structures attached to the two basal-most cells. These thallial cells have a thicker wall than regular hyphal cells, especially in the septal area limiting contiguous cells, giving them the appearance of a sporangium. We have not observed how the cell content is transferred into the developing propagule, just the resulting structure, which in our specimens remained attached to the parent thallus by a short, thin tube; a septum will delimit the attached structure at the apex of this connecting tube (FIG. 16). Another septum was observed delimiting two cells in the largest propagule-like structures, which presumably correspond to the basal and upper cells of a new thallus, on detachment from the parent thallus, to complete its development attached within the same gut lining. Vegetative propagules are established more firmly and typical of Graminella, but this strategy has been noted for other Harpellales (Ejectosporus, Lichtwardt et al 1991, Strongman 2005). If properly interpreted, vegetative propagules are ideally suited for the purpose of increasing thallial growth inside the same gut before host molting. The propagule-like structures of G. viviparus, which were observed on several dissections, apparently are not developmental anomalies. Strongman (pers comm) also has shared his observations of unusual swellings on thalli of Gauthieromyces specimens collected recently in China. Thus, with future collections and confirmation of vegetative structures as developmental stages, the generic description of Gauthieromyces might need to be emended. The hosts in locality MEX-5 and -19 were Baetodes nymphs, while in locality MEX-31 and -33 only Baetis nymphs were infested with Gauthieromyces, although the former host (admittedly more scant) also was present in the latter collections. Graminella ophiuroidea M.M. White, L.G. Valle and Cafaro sp. nov. FIGS. 21–25 Thalli 200–300 3 4–10 mm, sparse ramosi, interdum dichotomi. Cellulae axis principalis 4–6 diam. Cellula
r two propagule-like structures (arrows) attached to the basal cell and lower main axis (MEX-31-L2). 17, 18. Loose trichospore with filiform appendage (arrow) (MEX-31-L1, MEX-31-L2). 19. Detached propagule with a disc-like holdfast (arrow) (MEX-5L1). 20. Dense aggregate of fertile branches and trichospores, from multiple thalli (MEX-31-L1). Bar 5 25 mm in 12–16, 19, 20; 10 mm in 17, 18.
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FIGS. 21–33. 21, 22. Graminella ophiuroidea from Baetidae. Overview of thallus attached to the hindgut lining, with trichospores and inflated basal cell (arrows) (MEX-16-W1C). 23. Detail of fertile branchlet, generative cells and attached trichospores (MEX-16-W3). 24. Detached thallus with refractive holdfast material at the basal cell (arrowhead) (MEX-16-W1A). 25. Overview of a cluster of thalli attached to the gut lining, with a visible basal cell indicated (arrow) (MEX-16-W1A). 26–30. Lancisporomyces nemouridarum from Nemouridae. 26. Overview of main axis, immature thallus (MEX-12-W3). 27. Zygospores arising from conjugation tubes (MEX-4-W15B). 28–29. Detached trichospores, each with two appendages, helically coiled upon initial release (MEX-4-W15A, B). 30. Detached zygospore with a cylindrical collar (arrow) (MEX-12-W3). 31–33.
VALLE ET AL: MAYFLY AND STONEFLY HARPELLALES basalis oblonga, 25–35 3 8–14 mm, secretione tenaci pro affixione ad velamen proctodaei interius praedita, axe principali apicaliter exorienti, axibus secundariis basaliter vel lateraliter emergentibus. Trichosporae ovato-ellipticae, 8–12 3 2.5–3.5 mm, appendice unica tenui, collare carentes. Cellulae generatoriae usque ad per ramum fertilem 20, quoad longitudinem variabiles, eae quoque in hypha axiali sub ramificationibus distalibus dichotomis fertilibus effectae. Zygosporae ignotae. Propagula vegetativa ex cellula basali orientia. Ad integumentum proctodaei interius larvarum Baetidarum (Ephemeropterorum) affixa. Holotypus, hic designatus, MEX-16-W1A, in FH conservatus, in rivulo viam transcurrenti ex Xico usque ad Xico Viejo lectus.
Thalli 200–300 3 4–10 mm, sparsely branched, sometimes dichotomous. Main axis cells 4– 6 mm diam. Basal cell oblong, 25–35 3 8–14 mm, attached to the hindgut lining by a secreted holdfast, with a main axis arising apically, and other axes emerging basally or laterally from the basal cells. Trichospores ovate-ellipsoidal, 8–12 3 2.5–3.5 mm, with a single thin appendage and no collar. Generative cells up to 20 per fertile branch, variable in length, also produced in the axial hypha below distal dichotomous fertile branch ramifications. Zygospores not found. Vegetative propagules formed from the basal cell. Attached to the hindgut lining of Baetidae (Ephemeroptera) larvae. Etymology. Latin, ophiuroidea 5 thalli similar to a sea star in the genus Ophiura (Phylum Echinodermata), for the branches arising in any direction from the inflated basal cell. Specimens examined. MEX-16-: W1A (HOLOTYPE), W2, W3, W5 (ISOTYPE), W7, W8.
The genus Graminella has included two species, G. bulbosa Le´ger & Gauthier ex Manier (1962) and G. microspora S.T. Moss & Lichtw. (1981); both are differentiated from G. ophiuroidea by the basal cell shape and also the thallial arrangement. In the two previously described species the basal cell bears apical or lateral branches (Le´ger and Gauthier 1937, Manier 1962, Lichtwardt and Moss 1981), while in G. ophiuroidea branches also arise from the bottom of the basal cell and in greater number (FIGS. 21–22, 24– 25) and in higher number. The basal cell in the new species is oblong with a constant width, at least in mature thalli (probably not fully developed in FIG. 22), and the projecting branches have a markedly smaller diameter (FIG. 21). In both G. bulbosa and G.
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microspora the branches emerging from the basal cell progressively attenuate to their tips and the basal cell itself is not so homogeneously oblong but wider at the proximal region (Le´ger and Gauthier 1937, Lichtwardt and Moss 1981). Trichospores of G. ophiuroidea (FIG. 23) are similar to those of G. bulbosa in size, shape and disposition, and also resemble those of G. microspora, although being slightly larger in G. ophiuroidea. Zygospores have not been observed, but we think that the thallial characteristics are peculiar enough to justify a new taxon. All the species of the genus have been reported from mayfly nymphs of the family Baetidae (Lichtwardt and Moss 1981, White and Lichtwardt 2004, White et al 2006a, Valle 2007). Lancisporomyces nemouridarum Strongman & M.M. White. Can J Bot 2006 FIGS. 26–30 This species recently was described from the hindguts of Amphinemura nigritta Provancher (Plecoptera, Nemouridae) nymphs in Canada (Strongman and White 2006). Of the four species of the genus L. nemouridarum most closely resembles the type species, L. vernalis Santam., described initially in Nemoura hosts from Spain (Santamaria 1997) and later noted in Amphinemura hosts (Valle 2004). This report is the first citation of L. nemouridarum outside Canada. In Mexico the specimens were found in Amphinemourid hosts from multiple collection sites (Slides MEX-4-: L8, L9, L10, L11, L13, W1, W2, W10, W11, C33; MEX-12-: W3, W6, W7, W9, W10, W13, W14; MEX-16-W12 and MEX-18-L8). The characteristics of Mexican specimens are coincident with those reported by Strongman and White (2006), with the main thallus typically pinnately branched and adnate to the gut lining with multiple peg-like projections of holdfast (or basal main axis). In our collections trichospores were 34–48 3 6–8.5(–11) mm, without a collar, and had two appendages helically folded inside the generative cell and immediately after being released. Generative cells number 1–3(–6) per fertile branch, 5.5–7.5 mm diam and are highly variable in length. Zygospores are typically lance-shaped, with a thickened conical head, 37–48 3 6–7.5(–9) mm, and a long cylindrical proximal end, 3.5–4 mm diam, in total 140–180 mm long (compared to 120 mm in L. vernalis, according to Santamaria 1997, Valle 2004). The zygospore arises from a cylindrical zygosporophore of 30–40 3 3.5–4.5 mm formed at the center of
r Zygopolaris ephemeridarum from Baetidae. 31. Zygospore attached to zygosporophore (arrow) (MEX-16-W11). 32. Loose trichospore with a short, stout appendage and a refractive ‘‘blob’’ of material at its base (arrowhead) (MEX-12-L1). 33. Trichospores on fertile branchlets (MEX-15-L1). Bar 5 10 mm in 23, 32; 25 mm in 21, 22, 24, 27–31, 33. Scale bar 5 50 mm in 25–26.
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an inflated conjugation tube. On zygospore release part of the zygosporophore can be carried by the zygospore as a cylindrical collar (about 20–30 mm long). Multiple scalariform conjugations may be produced from two parallel conjugant thalli. The differentiation of L. nemouridarum from L. vernalis is mostly by the lower number of generative cells per fertile branch and larger zygospores in the former species. Other species of the genus, L. falcatus Strongman & M.M. White in Paracapnia and L. anguilliformis Strongman & M.M. White in Allocapnia (Strongman and White 2006), have distinctive characteristics, especially the zygospore shape, that do not allow confusion with the other two species of the genus. Zygopolaris ephemeridarum S.T. Moss, Lichtw. & Manier 1975 FIGS. 31–33 This species originally was described from Baetidae in USA (Moss et al 1975). Mexican specimens also were found in Baetidae nymphs (slides MEX-12-W4; MEX-13-: L1, L3, L6, L9; MEX-15-: L1, L2; MEX-16-: L1, L2, L5). The species was not reported before from tropical regions. Specimens in our collections have these attributes: elongate-obpyriform trichospores 20–27 3 6–7.5 mm (FIGS. 32–33) ([25–]32[–38] 3 5– 8 mm in USA specimens according to Lichtwardt and Williams [1984]); often with a minute collar or ephemeral remnants of the generative cell (FIG. 32), occasionally bearing one appendage-like extension (MEX-N-16) or a single inconspicuous filiform appendage (FIG. 32) (MEX-U-21). A discoid secreted holdfast is secreted usually by a laterally projected thumb-like holdfast cell. Generative cells 5–10 3 6– 7.5 mm, 6–16 per fertile branch. Conical-pyriform zygospores, 50–60 3 15–20 mm at proximal end (FIG. 31) ([40–]55[–86] 3 [13–]15[–26] mm in USA specimens according to Lichtwardt and Williams [1984]). On release zygospores of Mexican specimens had a rounded base, with remnants of the zygosporophore still attached; zygosporophores 20–26 3 12– 17 mm, formed at the center of scalariform conjugations. In our collections many mature thalli protruded beyond the host’s anus, as explained in Lichtwardt and Williams (1984). DISCUSSION
Mexico is considered to be one of the most diverse countries on the planet (Mittermeier et al 1997, Fullera et al 2007). As a region of North America (unexplored for gut fungi) it is a country that is temperate in the north and tropical in the south. Some of Mexico’s diversity surely can be attributed to the generally recognized pattern of increasing species
richness in tropical versus temperate regions (Hawkins 2001). However not all taxonomic groups follow this pattern and either do not experience any significant variation or actually may decrease in species richness latitudinally toward the tropics (Boyero 2002). Published data concerning tropical patterns of diversity richness for aquatic insects often are contradictory (see Stout and Vandermeer 1975, Haynes 1987, Lake et al 1994). Some of the contradictions might be the result of incomplete accounting of the actual diversity of aquatic insects in neotropical regions (Boyero 2002) compared to the better studied temperate regions, especially in the northern hemisphere. The same can be said of arthropod endosymbionts such as trichomycetes. According to the current knowledge of these fungi from neotropical regions (Lichtwardt 1994, 1997, White et al 2000, Alencar et al 2003), the prevalence and diversity of Harpellales appears to be lower in the tropics compared to temperate regions. However any affirmation of this tendency would be premature with the few sustained surveys and limited territorial scope of previous surveys of gut fungi in the neotropics. In the sites surveyed in Mexico we found that the most diverse and abundant hosts were mayfly nymphs. Ephemeropterans are usually more tolerant of warm waters than Plecopterans, with the spatial distribution of the stoneflies more closely linked to (cooler waters found in) higher latitudes and altitudes (Macant 1962, Illies 1964). The Harpellales associated with Ephemeropterans and Plecopterans in Mexico were found in rivers and streams at altitudes of 200–1800 m with a temperature of 14–23 C. The species previously known from northern temperate regions (Lancisporomyces nemouridarum and Zygopolaris ephemeridarum) were collected in streams at 1440–1800 m, with water at 14–16 C. It is possible that these species might not have the ability to develop in warmer lowland tropical waters because to date they are known only from colder temperate regions. Among the four new species, all from mayflies, the new Graminella species was found at nearly 1700 m, in water at 14 C but not at lower elevations (with warmer recorded stream temperatures) sampled. The remaining three new species (Allantomyces zopilotei, Bojamyces olmecensis and Gauthieromyces viviparus) were found at elevations of 200–400 m, with temperatures of 22–24.5 C. It remains to be determined whether the latter species have adapted specifically to warmer tropical continental waters. Allantomyces has been a monotypic genus with the sole species, A. caenidarum, reported in Caenidae hosts from Australia (William and Lichtwardt 1993), incidentally living at relatively high temperatures. A remarkable feature was reported for the Australian
VALLE ET AL: MAYFLY AND STONEFLY HARPELLALES species: the production of auxiliary allantoid spores formed at generative cells, with immediate germination to spread the infestation within the same individual gut (Williams and Lichtwardt 1993). We did not observe these allantoid spores in the Mexican specimens. It is possible that the production of allantoid spores is a response to a specific condition or cue that the Mexican hosts were not experiencing at the time of collection. The species within Allantomyces provide an example of disjunct distribution, which might be the result of allopatric speciation by vicariance. However our data on Harpellales remains incomplete across vast geographic areas. The host of A. zopilotei is not a Caenidae but a Leptohyphidae (proximal to Caenidae and Ephemerellidae, all placed in the infraorder Pannota), a New World family of mayflies endemic to the western hemisphere, living in streams and rivers from the southwestern United States to Central and South America (Baumgardner 2003). These patterns continue to reinforce our understanding that some gut fungi have been coevolving with their hosts for a long time. The genus Bojamyces, with B. repens from Maine, USA, (Longcore 1989) and B. transfuga from Mediterranean Europe (Valle and Santamaria 2004), is known from localities with varied climatic conditions, both in temperate zones. Nonetheless representatives of the genus might be more widespread because Lichtwardt and Williams (1992) reported an unnamed species of Bojamyces with minimal and immature specimens, resembling B. repens in Tasmanian Leptophlebiidae nymphs. Bojamyces olmecensis might be a species that has adapted to tropical conditions. Generative cells of Bojamyces repens may disarticulate and disperse in groups of a few cells with corresponding trichospores, a characteristic that also has been observed in the Mexican species. However neither the specimens found in Tasmania (Lichtwardt and Williams 1992) nor for B. transfuga in Spain (Valle and Santamaria 2004) have presented this trait. Another unique feature that has been reported for Bojamyces is the ability of thallial growth and sporulation outside the host, living (and not simply expunged with the shed hindgut linings with each molting cycle) and growing in the extruded exuviae (Longcore 1989, Valle and Santamaria 2004). We also recorded the presence of thalli within extruded exuviae, which were not uncommon among emerging vegetation at the calmed stream margins. Most zygospore-forming thalli were obtained from fieldcollected exuviae in these microhabitats. The genus Gauthieromyces was considered to be rare, originally known only from France (Lichtwardt 1983) until recently when two successive reports
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announced the discovery of a new species in the genus from two distant Asiatic localities, in India (Misra and Tiwari pers comm) and in China (Strongman and Xu 2006). These citations indicate that the genus has a wider distribution than formerly perceived and further studies are necessary on this and other understudied taxa to elucidate their actual biogeography. Gauthieromyces viviparus is separable by spore characteristics; the pattern in which trichospores are bent in an extended V-shape and attached is distinctly different from the previously described species. Thallial characteristics of all generic species of Gauthieromyces do resemble those of Graminella, now even with the presence of a vegetative propagulelike structure that was, before present, exclusively reported only for Graminella species. Nonetheless the observation of such propagule-like structures (FIGS. 16, 19) growing while still attached to parent thalli (FIG. 16) has not been reported in Graminella and might be exclusive to Gauthieromyces. The key generic characters of Gauthieromyces have been its arbusculate thallus and sharply curved (U-shaped), eccentrically attached trichospores. The extended Vshaped trichospores, with a polar spore attachment and presence of the putatively vegetative propagule of Gauthieromyces viviparus, narrows the distance between these putative sister taxa (Gauthieromyces and Graminella). Further molecular analysis (see White 2006) will be necessary to confirm the phylogenetic relationships of these two genera. Although the vegetative propagule (and its ontogeny) of G. viviparus has not been observed previously, the structure may detach specifically from the parent cell to anchor to the gut for subsequent growth and sporulation. Gauthieromyces viviparus was found both in Xalapa and Los Tuxtlas collections with both populations presenting the same overall diagnostic characters, although appendages and propagule-like structures were observable only in the specimens collected in Los Tuxtlas, where the material was more abundant and in better condition. Graminella ophiuroidea is a novelty for a genus that before present had only two representatives from temperate regions (G. bulbosa and G. microspora). This new Graminella species is tropical in distribution, but the altitude of the locality (1700–1800 m) may provide environmental conditions or at least stream temperatures that may be propitious for Graminella species. Graminella ophiuroidea has a particular physiognomy that makes it easily recognizable, especially regarding basal cell morphology. The Mexican species apparently has the same vegetative reproductive capacity (as evidenced by the enlarged basal cells), by propagules, as already reported in the genus (Le´ger and Gauthier 1937, Lichtwardt and
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Moss 1981). Nonetheless it would be worthwhile to obtain more material of this new species to confirm the presence of vegetative propagule, and to provide a detailed description of their development. Lancisporomyces nemouridarum was described recently from Canadian streams (Strongman and White 2006). This record from ecologically and geographically distinct sites in Mexico might be indicative of a wider prevalence of this species during previous colder periods. Perhaps its occurrence is due to the moderating effect that uplifts can provide in tropical regions, as a shelter for nearctic species (Colinvaux et al 1996, Green 2004, Still et al 1999). Zygopolaris ephemeridarum has been reported from North America (Lichtwardt and Williams 1984) but is likely to be more widespread. Mexican specimens share the characteristics of northern populations, except for the rare presence in few trichospores, of a filiform appendage or an appendage-like extension (FIG. 32). In previous observations of material from the USA, both trichospores and zygospores have lacked well-developed appendages; only a blob of material at trichospore bases rarely has been observed (Moss et al 1975), which was also present in some trichospores from the Mexican material. Lichtwardt and Williams (1984) reported appendages within the generative cells of unreleased trichospores and also three short appendages in a loose trichospore of Z. ephemeridarum. The observation of a fine, relatively long appendage (rarely short and wider) in our specimens adds even more variability to the appendage condition of this species. The substance observed at the base of the zygospore in our specimens has a different aspect than in North American sexual spores, which often display a delicate skirt of clear material surrounding the base of the zygospore (Lichtwardt and Williams 1984). Because the features of appendages in both trichospores and zygospores for this species seem to be quite variable we do not consider our minor variations of these structures to be specific attributes, instead of phenotypic variations within the species. An intensive but not extensive prospecting has demonstrated that Mexico is a fertile region for new taxa of gut fungi. Considering that four of the six species reported are new, from only two host groups in just a few sites in one state in Mexico, the country holds promise for continued surveys for other gut fungi, as well as other understudied organisms. ACKNOWLEDGMENTS
We are grateful for the help of the National Science Foundation for supporting this research through a Biodiversity Surveys & Inventory (BS&I) Award DEB-0344722,
MMW, PI and LGV and MJC, senior collaborators. We appreciate being granted scientific collecting permits to work in Mexico, specifically oficio Nos. SGPA/DGVS/06258 and 13864, from Secretarı´a de Gestio´n para la Proteccio´n al Ambiente, SEMARNAT and Pesca de Fomento No. GDOPA/12030/260805/5953 issued by the Comisio´n Nacional de Acuacultura y Pesca a trave´s de su Direccio´n General de Ordenamiento Pesquero y Acuı´cola. Special thanks to Dr Ricardo Ayala Barajas and Ange´lica Narvaez for help during the process of permit issuance and Dr Jorge Sobero´n Mainero during planning stages. Senior collaborators in Mexico who helped coordinate our surveys include Drs Joaquı´n Cifuentes Blanco (UNAM), who also assisted with logistics after our survey, and Drs Francisco Lorea Hernandez and Rosario Medel Ortiz, Instituto de Ecologı´a AC (INECOL), Xalapa. The authors especially express their gratitude for the support of Dr Luis Cervantes Peredo (IEAC) who kindly received us, offered lab space, helped with logistics and also joined us in the field. Dr Jorge Valenzuela, Dr Bruce Campbell and Roberto Arce Pe´rez (IEAC) also assisted with selection of suitable sites. Dr Ricardo Garcı´a Sandoval (then a graduate student at UNAM) helped collect and process some of the material, as a trainee, during the first trip and assisted with specimens after our departure. We thank the staff of the Biological Station ‘‘Los Tuxtlas’’ for the kind reception and logistic help, especially Biol. Rosamond Coates and her postsurvey support as well. Dr Boris Kondratieff, Colorado State University, kindly provided identifications of vouchered host specimens. Patricia Eckel, Missouri Botanical Garden, provided the Latin for the new taxa.
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