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The genus Smittium (Trichomycetes, Harpellales) in the Iberian Peninsula

     Unitat de Botànica, Departament de Biologia Animal, de Biologia Vegetal i d’Ecologia, Facultat de Ciències, Universitat Autònoma de Barcelona, 08193-Bellaterra (Barcelona), Spain

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS NEW SPECIES PREVIOUSLY KNOWN SPECIES DISCUSSION KEY TO THE IBERIAN... LITERATURE CITED

 

A study of larval Diptera (Chironomidae, Culicidae and Simuliidae) from Spain has been carried out to fulfill a catalogue of species of the genus Smittium (Harpellales: Legeriomycetaceae) present within these hosts. Among the reported taxa, eight are new species: Smittium brevisporum, S. bulbosporo-phorus, S. gracilis, S. hecatei, S. heterosporum, S. inex-pectans, S. prostratum and S. pseudodimorphum. We also report six previously described species, which are new for the Iberian Peninsula: S. alpinum, S. dipterorum, S. megazygosporum, S. pusillum, S. typhellum and S. fecundum. Three other species (S. simulii, S. culicis and S. culisetae) previously were reported from Spain. In two of them (S. fecundum and S. culicis), we describe for the first time the presence of zygospores. Some of the included species have been artificially cultured as well as ultrastructurally studied using scanning electron microscopy (SEM), with the purpose of observing the surface of both trichospores and the trichospore collar and the morphology of the trichospore appendage.

Key words: Dipteran larvae, Legeriomycetaceae, scanning electron microscopy, Spain, taxonomy, trichospores, zygospores

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS NEW SPECIES PREVIOUSLY KNOWN SPECIES DISCUSSION KEY TO THE IBERIAN... LITERATURE CITED

 
The first species described in the genus Smittium was S. arvernense by Poisson (1936). Some other species actually related to the genus previously were reported with other generic names. Poisson (1932) described earlier the genus Dixidium, with the single species D. dixae. In agreement with what is mentioned by Lichtwardt (1986), this taxon might belong to the genus Smittium, although it was found in an undetermined larva of Dixa (Diptera, Dixidae), an unusual host for Smittium. Manier and Lichtwardt (1968) rejected the genus Dixidium for its poor description and the absence of illustrations.

Other species currently placed in Smittium formerly were described as Orphella Léger & Gauthier (Léger and Gauthier 1931, Tuzet and Manier 1947), Rubetella Tuzet, Rioux and Manier (Tuzet et al 1961, Manier et al 1961, Manier and Mathiez 1965, Manier 1963), Genistella Léger & Gauthier (Léger and Gauthier 1932, 1935) and Typhella Léger & Gauthier (Léger and Gauthier 1935, Manier and Mathiez 1965). The presence of zygospores in Smittium (Poisson 1936) initially was used as the diagnostic character to distinguish species of this genus from those of the presumably related genera. Afterward, the taxonomic revision made by Manier and Lichtwardt (1968) and Manier (1970), where many of the species conceived in other genera were transferred to Smittium, the taxonomy of the genus became more clear and reliable.

In the study of the genus Smittium, where the number of morphological attributes used for classification often is critically reduced, there is a natural tendency of weighting sporangial characters. This is justified by the mostly conservative behavior of these reproductive structures within one species, while other thallial features, such as the basal cell morphology, or branching patterns, would be more variable. The emphasis on such scarce characters was not a handicap in the past because relatively few species were described and they were more or less easily identifiable on the basis of spore morphometry. Nonetheless, to date about 60 species of Smittium are accepted, some of them being published with descriptions that are too brief and that produce low predictability because spore morphometrics, by themselves, do not allow a reliable identification. At any rate, these characters can be used for a preliminary classification, as we do in the preliminary key to the species included here, and for providing additional information of basal cell and holdfast morphology.

All of these inherent difficulties in Smittium classification have lead to an increased use of phylogenetic data, based on comparative studies of rDNA sequences, accompanying the classical phenetic taxonomy. Using molecular analyses, the poliphyletic origin of the genus Smittium recently has been postulated to include at least five distinct lineages (Lichtwardt et al 2001, Misra and Horn 2001, Gottlieb and Lichtwardt 2001).

In this study we follow the classical concept of the genus Smittium established by Poisson (1936). We have tried to include as many characters as possible in the descriptions for each of the reported species, all of them accompanied by photographs.

Twenty species of Smittium have been found in the Iberian Peninsula, eight of which correspond to new species here described: Smittium brevisporum, S. bul-bosporophorus, S. gracilis, S. hecatei, S. heterosporum, S. inexpectans, S. prostratum and S. pseudodimorphum, all them dissected from the hindgut lining of Chironomidae larvae. Among the others, some represent first reports for our territory of study.

SEM techniques for the study of Trichomycetes have not been used frequently (Moss and Lichtwardt 1976), and some of the features observed have not been reported previously (e.g., the coat of unspecified material surrounding both trichospores and trichospore collars of S. culicis Manier [FIGS. 62, 63], the verrucose surface and band-like appendage of S. heterosporum [FIGS. 38, 39], or the vesicle-like bodies adhered to the appendage surface in S. hecatei [FIGS. 26, 27]).

View larger version (162K): [in this window] [in a new window]   FIGS. 56–76. Previously known species of Smittium. 56. S. alpinum (BCB-Tr0773). Fertile branches with trichospores, some of them detached and showing its collars (arrows). 57. S. chironomi (BCB-Tr1015). Overall view of a thallus. 58–63. S. culicis (FIGS. 58–61, BCB-Tr1219; FIGS. 62–63, from axenic culture). 58. Thalli with conjugation bridges (arrowhead) and young zygospores on supporting cells (arrows). 59–60. Free trichospores showing their variability within one thallus. 61. Zygospores. 62. Trichospore and collar with their coat. 63. Some coated trichospores on generative cells. 64–68. S. fecundum (FIGS. 64–66, from axenic culture; FIGS. 67–68, BCB-Tr1268). 64. Detail of a trichospore showing the collar and a cylindrical and helically folded appendage. 65. Trichospore and its collar with smooth surface. 66. Detail of a collar with convergent margins. 67. Zygospore with collar and appendage (arrow), extruding its content. 68. Sexual conjugant hyphae with bridges (arrowhead) and supporting cells (arrows). 69–72. S. megazygosporum (FIG. 69, BCB-Tr0765; FIG. 70, BCB-Tr1194; FIGS. 71–72, from axenic culture). 69. Free trichospores with collar and appendage (arrow). 70. Overall view of a thallus. 71–72. Trichospores with broken collars and emerging cylindrical appendages. 73–74. S. pusillum (BCB-Tr1591). 73. Branches and trichospores. 74. Free trichospore with collar. 75. S. typhellum (BCB-Tr1443). Trichospore attached to the generative cell. 76. S. dipterorum (BCB-Tr1122). Overall view of a mature thallus. All figures photographed from lactophenol cotton-blue mounted slides, except FIGS. 58, 67–70, photographed from water mounted slides. FIGS. 57, 59–61, by DIC. FIGS. 58, 67–70, 76, by phase contrast. FIGS. 74–75, by bright field. FIGS. 62–66, 71–72, by SEM. Scale bar FIG. 56 = 40 µm; Scale bar FIGS. 57–58, 67, 76 = 50 µm; Scale bar FIGS. 59–60, 75 = 10 µm; Scale bar FIG. 61 = 25 µm; Scale bar FIG. 68 = 30 µm; Scale bar FIGS. 69–70, 73–74 = 20 µm; Scale bar FIGS. 62, 64, 71–72 = 2 µm; Scale bar FIGS. 63, 65 = 5 µm; Scale bar FIG. 66 = 1 µm.

View larger version (140K): [in this window] [in a new window]   FIGS. 20–39. Smittium hecatei and S. heterosporum. 20–27. S. hecatei (FIGS. 20–21, 23–25, BCB-Tr1365; FIG. 22, BCB-Tr1372; FIGS. 26–27, from axenic culture). 20. Fertile branches with trichospores (arrow) and ß (arrowhead). 21. Free trichospore showing the collar and appendage (slightly out of focus, arrow). 22. Basal cell covered with holdfast material (arrow). 23. Fertile branches. 24. Fertile branches with trichospores ß (arrowhead) and one free trichospore (arrow) 25. Overall view of a thallus. 26–27. SEM images of the collar and cylindrical appendage, with adhered globose vesicles (arrows). 28–39. S. heterosporum (FIGS. 28, 31, BCB-Tr1242, from axenic culture; FIGS. 29, 35, BCB-Tr1298, from axenic culture; FIG. 30, BCB-Tr0765; FIGS. 32–33, BCB-Tr0529; FIG. 34, BCB-Tr0598; FIGS. 36–39, from axenic culture). 28. Fertile branches. 29. Trichospores ß (arrowhead) and one free trichospore showing the verrucose surface (arrow). 30. Basal cells and holdfasts. 31. Trichospore extruding the sporangiospore. 32. Zygospore carrying its eccentric collar and appendage (arrow). 33. Detail of a zygospore with its thickened extreme, and a trichospore of type ß. 34. Fertile branch with trichospores ß (arrowhead) and one free trichospore type (arrow). 35. Detail of the verrucose surface of an empty trichospore (arrow). 36–37. Trichospores showing collars (arrows). 38. Detail of a trichospore collar from which the ribbon-like appendage comes out. 39. Detail of the verrucose ornamentation on trichospore outer wall. All FIGS. photographed from lactophenol cotton-blue mounted slides, except FIGS. 21, 28, 30–31, 33–34, photographed from water mounted slides. FIGS. 20, 22–25, 29–30, 32, 35, by DIC. FIGS. 21, 28, 31, by phase contrast. FIGS. 33–34, by bright field. FIGS. 26–27, 36–38, by SEM. Scale bar FIGS. 20, 24, 28–34 = 20 µm, FIGS. 33–34 with the same scale of FIG. 32; Scale bar FIGS. 21–22, 35–37 = 10 µm; Scale bar FIG. 23 = 40 µm; Scale bar FIG. 25 = 50 µm; Scale bar FIGS. 26–27 = 2 µm; Scale bar FIGS. 38–39 = 1 µm.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS NEW SPECIES PREVIOUSLY KNOWN SPECIES DISCUSSION KEY TO THE IBERIAN... LITERATURE CITED

Some cultured species were studied with SEM. For this purpose, the axenically cultured material of S. culicis, S. fecundum Lichtw. & M.C. Williams, S. hecatei, S. heterosporum and S. megazygosporum Manier & Coste, were fixed with glutaraldehyde 2.5% in Cacodilate buffer 2M (pH 7). The thalli were washed with the same cacodylate solution and dehydrated in ethanol series (15 min in each 10–20–40–60–80%) to absolute ethanol and immediately transferred to acetone before being critical-point dried and gold-coated. Grids were examined with an HITACHI S-570.

Axenic cultures of S. culicis, S. fecundum, S. hecatei, S. heterosporum, S. megazygosporum and S. simulii Lichtw. were grown on stationary Petri dishes with Brain-Heart Infusion agar (BHIa) 1/10, with added vitamins (biotin, thiamin), following the methods proposed by Lichtwardt (1986).

For a more consistent study, type specimens were borrowed from the Farlow Herbarium (FH) and from the Museum d’Histoire Naturelle de Paris (PC). These types have been examined and sporangial features have been measured to compare to Spanish specimens.

	NEW SPECIES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS NEW SPECIES PREVIOUSLY KNOWN SPECIES DISCUSSION KEY TO THE IBERIAN... LITERATURE CITED

 
Smittium brevisporum L.G. Valle & Santam., sp. nov. FIGS. 1–5

View larger version (158K): [in this window] [in a new window]   FIGS. 1–19. Smittium brevisporum, S. bulbosporophorus, S. gracilis and S. delicatum. 1–5. S. brevisporum (BCB-Tr1150). 1–2. Fertile branches showing terminal trichospores. 3. Free trichospore with a small collar (arrow) and an unfolded appendage. 4–5. Trichospores in different stages of release; when released, showing the thick folded appendage (arrowhead) and the minute collar (arrow). 6–11. S. bulbosporophorus (FIGS. 6, 9–11, BCB-Tr1285; FIGS. 7–8, BCB-Tr1284). 6. Peduncle, bulbous zygosporophore (arrow), appendage (arrowhead) and zygospores. 7. Fertile branches with trichospores. 8. Overall view of a thallus. 9. Free trichospore showing the collar and appendage. 10. Zygospore. 11. Sexual conjugant hyphae producing zygospores. 12–18. S. gracilis (FIG. 12, BCB-Tr0448; FIGS. 13–14, 18, BCB-Tr1331; FIGS. 15, 17, BCB-Tr0441; FIG. 16, BCB-Tr0438). 12–13. Terminal fertile branches bearing trichospores. 14. Free trichospore showing the short collar. 15–16. Thalli habitus. 17–18. Basal cells with holdfast. 19. S. delicatum (FH AUS-24-7, type). Detail of basal cell. All FIGS. photographed from lactophenol cotton-blue mounted slides. FIGS. 1, 3, 5–6, 12–14, 17–19, by DIC. FIGS. 2, 4, 7–11, by phase contrast. FIG. 16 by bright field. Scale bar FIG. 1 = 10 µm; Scale bar FIGS. 2, 6, 8, 11 = 20 µm; Scale bar FIGS. 3–5 = 5 µm; Scale bar FIGS. 7, 9–10, 12–14, 17–19 = 10 µm; Scale bar FIGS. 15–16 = 50 µm.

Thalli profusely branched from the base, with an indeterminate pattern, tightly growing to form aggregates in which curved hyphal apexes can be observed. Basal cell with a simple disk-like holdfast. Tri-chospores long-ellipsoidal to ellipsoidal, (9–)12–14.5(–19) x 2–2.5 µm, each showing, after released from generative cells, a short and thick, longitudinally folded, appendage (FIGS. 4, 5), and a nearly inconspicuous collar (FIG. 5), 0.5–1 x 1.5 µm, that becomes observable when the appendage unfolds (FIG. 3). Each fertile branch with 4–8(–12) generative cells (FIGS. 1, 2). Zygospores not found.

Etymology. – L., brevi = small, short; and sporum = spore.

Specimens examined.. SPAIN. BARCELONA: Fogars de Montclú s; Santa Fe del Montseny, Santa Fe stream, 3-IX-2000, L.G. Valle, BCB-Tr0345; same locality, 25-IX-2001, L.G. Valle, BCB-Tr1085–1087; same locality, 7-XI-2001, L.G. Valle, BCB-Tr1152 (HOLOTY PE), BCB-Tr1150 (ISOTY PE). LLEIDA: Espot; Estany de Ratera (Parc Nacional d’Aigüestortes i Estany de Sant Maurici), in a little unnamed stream, 19-VI-2001, L.G. Valle, BCB-Tr0853. All prepared from Chironomidae Orthocladiinae (Cricotopus spp. and other) larvae.

This species displays morphological affinities with S. microsporum M.C. Williams & Lichtw., an Australian species having smaller trichospores (8–10 x 2–2.3 µm) with inconspicuous appendages (Lichtwardt and Williams 1992a), different by the short and appreciable appendage of the species here described (FIGS. 3–5). The number of generative cells is larger in S. microsporum, with more than 15 cells per fertile branch. Smittium paludis M.C. Williams & Lichtw. is comparable to S. brevisporum, with its small trichospores (12–14 x 2.5–3 µm), although the typical branching pattern of S. paludis is verticillate and the trichospore appendage is inconspicuous (Lichtwardt and Williams 1990). Smittium dipterorum Lichtw. also is similar but may be separated from our species by the appendage morphology and trichospore size, longer in S. dipterorum (Lichtwardt 1997). The longer collar of S. parvum Lichtw. (2–6 µm according to our measurements from the type specimens, photomicrographs KU-204-35 Holotype [FH], and KU-204-36 Isotype [FH]) is the best character to differentiate this species from S. brevisporum.

This species has been observed growing with S. bulbosporophorus and S. inexpectans, described below.

Smittium bulbosporophorus L.G. Valle & Santam., sp. nov. FIGS. 6–11

Thallus cum sparsim ramificanti principali axe et aliquot lateralibus secundariis ramis. Basalis cellula aliquando lata facta in maturo thallo, secernens disciforme tenaculum. Trichosporae obovato-ellipsoidales ad late ellipsoidales, (9–)12–14.5(–18) x 3.5–5.5 µm, praebentes singulas conspicuas appendices atque elongatum, cylindricum vel leviter decrescens versus extremum collare, 1.5–3 µm. Unaquaeque fertilis rama cum 2–6(–8) genitalibus cellulis, 5–7.5 µm in longitudine. Zygosporae biconicae, 35–40 x 7–8 µm; zygosporophorum globosum, fere sphaericum, in extremo quadrante zygosporae longitudinis locatum. Liberae zygosporae cum globoso collari, 3.5–4 x 3.5 µm, atque facile visibili appendice. In Chironomidarum larvarum proctodaeo incoli.

Thallus with a sparsely branched main axis, occasionally pinnate, and with secondary lateral branches arising from the base proximity (FIG. 8). Basal cell often broadened in mature thalli, not differentiated in young individuals, secreting a disk-like holdfast. Trichospores obovate-ellipsoidal to broadly ellipsoidal, (9–)12–14.5(–18) x 3.5–5.5 µm, each showing, after released from generative cells, a conspicuous, easily distinguishable, appendage, measuring about three times the trichospore length and a collar that is long, cylindrical or slightly narrowing toward the end, 1.5–3 x 2 µm (FIG. 9). Each fertile branch with 2–6(–8) generative cells, 5–7.5 µm length (FIG. 7). Zygospores biconical, 35–40 x 7–8 µm; zygosporophore globose, nearly spherical, located at the last quarter of the zygospore length (FIGS. 6, 10–11). Once released, the zygospore shows a globose collar, 3.5–4 x 3.5 µm, and an easily observable appendage (FIG. 6).

Etymology. – L., bulbo = globose, bulbous; sporophorus = sporophore (referring to zygosporophore).

Specimens examined.. SPAIN. BARCELONA: Montseny, Tordera river, prepared from Chironomidae Diamesinae larvae, 13-II-2002, L.G. Valle, BCB-Tr1284 (HOLOTY PE), BCB-Tr1285 (with zygospores!), Tr1289, Tr1291 (ISO-TY PES); Fogars de Montclú s; Santa Fe del Montseny, Santa Fe stream, prepared from Chironomidae Diamesini larvae, 25-IX-2001, L.G. Valle and S. Santamaria, BCB-Tr1087, Tr1089, Tr1095, Tr1106–1107; same locality, 7-XI-2001, L.G. Valle, BCB-Tr1150, Tr1153, Tr1156. BIZKAIA: Trucíos, Agüera river, prepared from Chironomidae Orthocladiinae larvae, 29-V-2002, L.G. Valle, BCB-Tr1434–1435, Tr1438–1439, Tr1441, Tr1443. GIRONA: Agullana, La Guilla stream, prepared from Chironomidae Orthocladiinae and Diamesiinae larvae, 14-VI-2001, L.G. Valle, BCB-Tr0836–0839.

The most noticeable feature of this species probably is the presence of a globose zygosporophore (FIG. 6), that inspired its specific epithet. In other species, zygosporophores can be slightly broadened under the zygospore, as Smittium cylindrosporum Lichtw. & Arenas, but not as extremelt as in this species. On the other hand, zygospore measurements are larger in S. cylindrosporum (51–52 x 10–11 µm, according to Lichtwardt and Arenas 1996) than in S. bulbosporophorus, which represents, along with S. arvernense, the smallest zygospores described in the genus. Poisson’s species differs from ours by trichospore measurements (20–25 x 5 µm) and by zygospore characteristics, which are even slightly shorter and wider in the medial part (30–35 x 8–10 µm), with a small collar (Poisson 1936), not as eccentric as that of S. bulbosporophorus. Another difference between both species is the trichospore layout on fertile branches, being more tidily arranged in S. arvernense and untidy in S. bulbosporophorus, which shows few trichospores at the very last portions of the sparse and disarranged long branches (FIG. 8). When sexual reproduction is present, the conjugated thalli grow in such a dense manner that zygospore observation can be very difficult (FIG. 11).

Morphologically, S. morbosum Sweeney is probably the closest species. It is the only species described as pathogenic and even lethal against its hosts (Sweeney 1981a, b). Smittium bulbosporophorus, apparently, is not pathogenic to hosts and differs from S. morbosum, not only by its ecology and host relationship but also by its sporangial features, mainly by trichospore collar morphology, longer and not outwardly flared in S. morbosum but cylindrical or narrowing toward the end in the new species. In addition, the thallus is more densely branched in S. morbosum and lacks a broadened basal cell, which otherwise is typical of most mature thalli of S. bulbosporophorus.

Smittium bulbosporophorus was found sharing the host hindgut with S. brevisporum, described above.

Smittium gracilis L.G. Valle & Santam., sp. nov. FIGS. 12–18

Thallus cum (2–)3–4(–6) ramis ortis e basali cellula, hippocrepica atque laterales expansiones ferens. Secundariae atque tertiariae ramae verticillatim in supera area dispositae. Secreta tenaculi materia in concava area basalis cellulae locata. Trichosporae subcylindricae, (18–)20–26(–29) x 2–3.5 µm, cum tenuisima appendice, circa bis magisve longiores quam trichosporae longitudo, inconspicuum collare circa 1 µm longum. Unaquaeque fertilis rama cum 2–8 genitalibus cellulis. Zygosporae ignotae. In Chironomidarum larvarum proctodaeo incoli.

Thalli with (2–)3–4(–6) branches arising directly from the basal cell, which is horseshoe-shaped and has lateral expansions (FIGS. 17, 18). A small amount of secreted material of the holdfast can be seen in the concavity of the basal cell in its middle part. Secondary and tertiary branches verticillately arranged at the upper zone (FIGS. 15, 16). Basal hyphae measuring 3–4 µm diam, to 2–3 µm in distal ones. Tri-chospores subcylindrical, (18–)20–26(–29) x 2–3.5 µm, each showing, after released from generative cells, a very thin appendage about twice or more as long as the trichospore length, and an inconspicuous collar of approximately 1 x 1 µm (FIG. 14). Each fertile branch with 2–8 generative cells (FIG. 12). Zygospores not found.

Etymology. – L, gracile = delicate (referring to the general aspect of the thin thallus).

Specimens examined.. SPAIN. BARCELONA: L’Estany, l’Estany stream, prepared from Chironomidae Chironomini (Chironomus spp.) larvae, L.G. Valle, 21-XI-2000, BCB-Tr0414 (HOLOTY PE), Tr0409, Tr0412, Tr0415 (ISO-TY PES); same locality, 19-XII-2000, L.G. Valle, BCB-Tr0438, Tr0440–0444, Tr0446–0448; Moià, Moià stream, prepared from Chironomidae (Chironomus sp.) larvae, 13-V-2002, L.G. Valle, BCB-Tr1352; Palas de Torrella; Cardener river, prepared from Chironomidae Diamesinae larvae, 24-IV-2002, L.G. Valle, BCB-Tr1331.

An outstanding characteristic of this species is the peculiar horseshoe-shaped basal cell (FIGS. 17, 18), similar to that of another species that we consider closely related, S. delicatum Lichtw., described from Australia in Chironomus larvae (Lichtwardt and Williams 1990). We have studied the type of this species (microscope slide AUS-24-7, Holotype [FH]) and both may be distinguished by thallial and sporangial features. In S. delicatum, there is a main hyphal axis clearly continuous with the basal cell and some lateral, basally septate branches (2–4), arising more or less symmetrically from both sides of this axis (FIG. 19). In S. gracilis, there is not a definite main axis (FIGS. 15, 16). Smittium delicatum secondary branches are longer, thinner and more sparsely arranged than those of S. gracilis. The trichospores, although comparable, are quite different, being slightly longer and narrower in S. delicatum.

Smittium gracilis could be related, as well, to species of the genus Furculomyces M.C. Williams & Lichtw. by its thallial characteristics, but we have not found the zygospores, the most important element that defines the genus Furculomyces, with its furcula-like conjugation tubes (Lichtwardt and Williams 1992b). We have examined the type of Furculomyces boomerangus (M.C. Williams & Lichtw.) Lichtw. & M.C. Williams (microscope slide AUS-42-M-2 Holotype [FH]) for comparison with our species.

An unidentified species of Smittium recently has been found in Norway (Lichtwardt, pers comm), sharing many characteristics with S. gracilis, in its horseshoe-shaped basal cell, and trichospore features. We think that those specimens may correspond to the species here proposed.

Smittium hecatei L.G. Valle & Santam., sp. nov. FIGS. 20–27

Thallus a basali cellula ramosus, verticillatus in mediis atque superis areis. Basalis cellula ramificans atque lato pedi sim-ilem structuram formans (usque 12 µm lata) in maturo thallo secreta tenaculi materia tectam. Trichosporae subcylindricae ad elongato-ellipsoidales, cum duabus magnitudinibus: typus , 30–37(–49) x 3.5–5.5 µm, cum collari 3.5–6 x 3.5 µm; typus ß, 14.5–24 x 2.5–3.5 µm, cum collari 1.5–2.5 x 2–2.5 µm, in utroque typo cylindricis ad leviter decrescentibus versus extremum. Unaquaeque fertilis rama cum 8–14 magisve genitalibus cellulis, variabili magnitudine (longiores cum trichosporas formant) atque decrescentibus versus distalia extrema (circa 2.5–3.5 µm diam). Zygosporae ignotae. In Chironomidarum larvarum proctodaeo incoli.

Thalli branched from the basal cell, verticillate in the medial and upper areas. Basal cell not distinguished in young thalli, ramified and differentiated in a kind of broadened-leg (12 µm wide) coated with secreted holdfast material in mature thalli (FIG. 22). Bulbous lateral protuberances often can arise from the basal cell, occasionally slightly perforating the hindgut lining. Trichospores subcylindrical to long-ellipsoidal, with two size ranges (FIGS. 20, 24): type , 30–37(–49) x 3.5–5.5 µm, with a collar of 3.5–6 x 3.5 µm; type ß, 14.5–24 x 2.5–3.5 µm, with a collar of 1.5–2.5 x 2–2.5 µm, cylindrical or slightly narrowing to the end in both types (FIG. 21). Each fertile branch with 8–14 or more generative cells (FIG. 20), variable in size (longer when producing trichospores ), and narrowing to the distal ends (ca. 2.5–3.5 µm diam). Zygospores not found.

Etymology. – Gr., hecate = Greek goddess (who was venerated by different people, among them, by fishers who asked her for a good fishing; we could also ‘‘venerate’’ her to get the best trichomycete hosts).

Specimens examined.. SPAIN. BARCELONA: Moià, Moià stream, prepared from Chironomidae Diamesini (Diamesinae) larvae, 13-III-2002, L.G. Valle, BCB-Tr1348 (HOLO-TY PE), BCB-Tr1349–1350, Tr1361–1362 (ISOTY PES); same locality, 14-III-2002, L.G. Valle and S. Santamaria, BCB-Tr1364–1371, Tr1373–1378, Tr1380–1384; same locality, prepared from axenic cultures (Moià pq7), BCB-Tr1494; Gualba, Gualba de Baix, Gualba stream, prepared from Chironomidae Orthocladiinae (Cricotopus spp.) larvae, 28-III-2001, L.G. Valle, BCB-Tr0656–0659.

This species is characterized by the presence of a wide trichospore size range, which we have considered separable into two forms. We define these two forms as type , the larger, and type ß, the smaller, which measures nearly half the length of the former type. Both types are found randomly on the same thallus but arising from different fertile branches (FIG. 20). The absence of small, ovate trichospores can be use to segregate this species from other dimorphic species as S. dimorphum Lichtw. & M.C. Williams (Lichtwardt and Williams 1983), which also show a longer collar in the larger, subcylindrical trichospores than those present in trichospores of S. hecatei.

Another diagnostic characteristic of S. hecatei is the presence of numerous generative cells per fertile branch, usually longer in those branches giving rise to trichospores of type . The basal cell is also a very peculiar feature to separate this species from others, showing an elephant leg-like morphology (FIG. 22), with a coat of secreted holdfast material covering its perimeter.

The trichospores of S. hecatei are similar to trichospores of S. megazygosporum (Manier and Coste 1971) but differentiated by the slightly flared outward collar in the latter species. Also, the morphology of the basal cell is an important character to be considered for the separation of both species, when trichospores ß are not observed in S. hecatei.

Smittium elongatum Lichtw. also can be compared with the species here described, although the differences can be easily discerned when closely observed because S. elongatum has a markedly verticillate pattern of ramification, with many branches arising from each verticil (observations from the examined type, microscope slide COL-4-15, Holotype [FH]), while in S. hecatei, less ramified, just a few branches are observed per verticil, and these have more or less pinnate secondary ramifications. Otherwise, S. elongatum does not show the long series of generative cells observed in S. hecatei. The presence of the small trichospores of type ß and the peculiar base are other characters to distinguish both species. Smittium elongatum also displays a wide trichospore size range, with larger maximums, (20–)34(–44) x 3–6 µm, and a collar of 2–4 µm (Lichtwardt 1972).

The observations with SEM revealed the cylindrical shape of the trichospore appendage in all its length. Some vesicular bodies can be seen on the appendage surface (FIGS. 26, 27), which probably are related with the presence of adherent materials allowing the trichospore to be attached in the vicinity of the host environment.

Smittium heterosporum L.G. Valle & Santam., sp. nov. FIGS. 28–39

Thallus ramosus, verticillatus in mediis atque superis areis. Basalis cellula cum simplici, disciformi vel campanulato tenaculo. Trichosporae dimorphae: typus , elongato-ellipsoidales, (36–)50–62(–74) x (7.5–)9–13 µm, cum subtiliter granulata pagina et collari leviter decrescenti versus extremum, (10–)12.5–17(–20) x 3–4(–5.5) µm; typus ß, parvae, ovato-ellipsoidales ad pyriformes, 14–27 x 8.5–11 µm, cum subtiliter vel nullimodo granulata pagina et collari 8–9 x 1.5 µm. Zygosporae fusiformes, (160–)170–180(–190) x 15–18 µm; zygosporophorum excentricum, prope unum extremorum polarium locatum. Liberae zygosporae cum collari 17–20(–30) x 10–13 µm. In Chironomidarum larvarum proctodaeo incoli.

Thalli branched, verticillate in the medial and upper areas. Some broadenings and constrictions are observed in the basal segment with hyphal cells highly variable diameter (6–14 µm or more). Basal cell with a simple, discoid or slightly campanulate holdfast (FIG. 30). Trichospores dimorphic: type , long-ellipsoidal, (36–)50–62(–74) x (7.5–)9–13 µm, with a delicately granulated surface (more or less apparent depending on the individual, FIGS. 29, 35 and 39), collar slightly narrowing toward the end, (10–)12.5–17(–20) x 3–4(–5.5) µm, filiform appendage (but ribbon-like under SEM! FIG. 38), measuring up to eight times the trichospore length; type ß, smaller, ovate-ellipsoidal or pyriform, 14–27 x 8.5–11 µm, with the surface slightly granulated or not, collar 8–9 x 1.5 µm, appendage identical to that of type . At the lowermost and medial areas of fertile branches, the generative cells may not arise directly from the main axis but from lateral peduncles, variable in size (18–50 µm). Zygospores fusiform, (160–)170–180(–190) x 15–18 µm; zygosporophore eccentric, located near one of the polar ends (FIG. 32). Once released, the zygospore shows a collar of 17–20(–30) x 10–13 µm, and a large appendage (FIG. 32). Specialized conjugation hyphae giving rise to angulous conjugation bridges, from which a peduncle develops, supporting both zygosporophore and zygospore.

Etymology. – Gr., hetero = different; L., sporum = spore (referring to trichospore).

Specimens examined.. SPAIN. BARCELONA: Sant Llorenç Savall; Vall d’Horta stream, prepared from Orthocladiinae (Chironomidae) larvae, 6-III-2001, L.G. Valle, BCB-Tr0598 (HOLOTY PE), BCB-Tr0601 (ISOTY PE); same locality, prepared from axenic cultures (SmV.H.-pq3), 11-XII-2001, BCB-Tr1175–1177, 5-II-2002, BCB-Tr1242–1243, Tr1249; same locality, 7-II-2002, BCB-Tr1266; same locality, prepared from axenic cultures (SmV.H.-pq2), 28-II-2002, BCB-Tr1298; same locality, prepared from axenic cultures (SmV.H.-pq2), 5-III-2002, BCB-Tr1301–1302; Marganell, Marganell stream, prepared from Chironomidae Orthocla-diinae and Diamesiinae (Sympotthastia spp., Potthastia spp.) larvae, 13-I-2002, L.G. Valle, BCB-Tr1194, Tr1199; same locality, 6-III-2002, L.G. Valle, BCB-Tr1309–1315; same locality, prepared from axenic cultures (SmV.H.-pq1), 13-II-2002, BCB-Tr1288; same locality, prepared from axenic cultures (SmV.H.-pq4), 28-II-2002, BCB-Tr1293, Tr1295–1297. GI-RONA: Boadella d’Empordà, Muga river, prepared from Chironomidae Orthocladiinae (Cricotopus bicinctus Meig.) larvae, 14-VI-2001, L.G. Valle, BCB-Tr0840. TARRAGONA: Horta de St. Joan, Ports de Beseit, Mas de la Franqueta, prepared from Chironomidae Diamesinae larvae, 24-III-2001, L.G. Valle, BCB-Tr0762–0765, Tr0767–0768, Tr0772, Tr0775–0777.

As defined by the specific epithet, this species is characterized by the dimorphic trichospores, here named and ß. Some characters of S. heterosporum are shared with other species, but the composite features were enough to segregate this from others. Smittium macrosporum Kobayasi may be considered as the most closely related species but having somewhat shorter trichospores (extreme size 42 x 7.5 µm, according to Kobayasi et al 1969). Smittium macrosporum trichospores resemble those of S. heterosporum by its finely coarse surface and by the presence of branches (or peduncles) growing intercalated between generative cells. Smittium macrosporum shows few generative cells per branch, usually two, while we have distinguished up to eight cells per branch in S. heterosporum. The typical dimorphism in S. heterosporum, and the presence of trichospores measuring up to 70 µm long are other important characters that can be used to determine this species. Another species with trichospores showing a punctate wall, but different in other trichospore features, is S. incrassatum Kobayasi (Kobayasi et al 1971).

The zygospores of S. heterosporum are the longest ever described in a species of Smittium, and are characterized, as well, by their extremely eccentric collar (FIG. 32). The conjugating filaments are formed from specific hypha that resemble the furcula-like structures of Furculomyces (Lichtwardt and Williams 1992b), a genus described for the previously named Smittium boomerangus M.C. Williams & Lichtw. (Lichtwardt and Williams 1990). For comparison we have examined the type of Furculomyces boomeragus (microscope slide AUS-42-M2, Holotype [FH]).

We have observed an interesting phenomenon, already reported in other species, which is the presence of released sporangiospores initiating their development prematurely in the midgut and sometimes with the young thalli having the ability to perforate the peritrophic matrix. Smittium perforatum M.C. Williams & Lichtw. also reveals this capacity (Williams and Lichtwardt 1987, Lichtwardt et al 1997). The thallial morphology of the initial developmental stages are similar in both species, but the features of mature individuals are entirely different, with smaller trichospores ([33–]38[–45] x [7–]7.9[–8.2] µm) and a shorter collar ([6–]7[–8] x [3–]4.2[–4.6] µm) in Smittium perforatum (type examined!, microscope slide RMBL-28–15 Holotype [FH]). Sweeney (1981a) reported that S. morbosum, along with its pathogenic action, perforate the hindgut lining. Smittium longisporum M.C. Williams, Lichtw. & S.W. Peterson (Williams et al 1982) is another species in which the precocious sporangiospore extrusion has been observed (Lichtwardt et al 1997).

All the collections are highly uniform in trichospore features. The trichospores represent the main type in nearly all the studied samples. It might be of some interest to mention the peculiar holdfast found in some thalli from Tarragona (Tr0765), which show a globose base and a skirt-like membrane partially covering it (FIG. 30).

Sporangiospore extrusion has been frequently observed in axenically cultured material, without additional help (e.g., induced pH changes [Horn 1989]). The sporangial content (sporangiospore) is released through the apical pole (opposite to the collar, FIG. 31). Once empty, the observation of the granulated surface of the outer sporangial (trichospore) wall is easier (FIGS. 29 and 35). In addition, the sporangio-spore extrusion and posterior germination has been viewed in natural conditions inside the chironomid gut lumen. A less frequent episode has been noticed: the extrusion of the zygospore content. The released endospore has been recognized as a fusiform material, ejected through one of the acute poles. We have not detected the whole extrusion because part of the endoplasm remained inside the zygospore.

Observations with SEM revealed a very interesting characteristic, namely the presence of a ribbon-like appendage (FIG. 38), unique among the described species of Smittium. In addition, the observation with electronic microscopy shows the coarse outer surface of trichospores (FIG. 39).

Smittium inexpectans L.G. Valle & Santam., sp. nov. FIGS. 40–44

View larger version (149K): [in this window] [in a new window]   FIGS. 40–55. Smittium inexpectans, S. prostratum and S. pseudodimorphum. 40–44. S. inexpectans (FIG. 40, BCB-Tr1156; FIGS. 41–42, BCB-Tr1095; FIGS. 43–44, BCB-Tr1084). 40. Overall view of the thallus. 41. Free trichospore showing the collar. 42–43. Fertile branches with few trichospores. 44. Aspect of a secondary axis with few trichospores (arrows). 45–48. S. prostratum (BCB-Tr1550). 45. Overall view of a thallus. 46. Free trichospore with its collar. 47. Detail of trichospore collar and appendage (arrow). 48. Lateral view of the basal prostrate area, with holdfast material secreted all along the contact surface (arrowheads). 49–55. S. pseudodimorphum (FIGS. 49–52, 54–55, BCB-Tr0605; FIG. 53, BCB-Tr0623). 49. Detail of trichospore collar and appendage (arrow). 50. Conjugant hyphae with zygospores and trichospores of type , young tri-chospore type ß is focused at the lower part (arrowhead). 51. Aspect of a zygospore with its zygosporophore (arrow) and part of the supporting cell (arrowhead). 52, 54. Fertile branches, some producing trichospores type ß (arrow) and others, from the same thallus, producing trichospores type (arrowhead). 53. Lobulate basal cells (arrows) with holdfasts. 55. Detail of a fertile branch bearing trichospores type ß, showing its long collars (arrow). All FIGS. photographed from lactophenol cotton-blue mounted slides. FIGS. 40–54, by DIC. FIG. 55, by phase contrast. Scale bar FIGS. 40, 50–51, 54 = 50 µm; Scale bar FIGS. 41–44, 46–47, 52–53, 55 = 20 µm; Scale bar FIGS. 45, 48 = 40 µm; Scale bar FIG. 49 = 10 µm.

Thalli pinnate to verticillately branched, with a main and other hyphal axes arising from the basal cell and tapering to the tips. The basal cell is slightly broadened with a simple, disk-like secreted holdfast, located between the lateral basal branches. Trichospores subcylindrical, often with a median bulge, 23–27 x 2–2.5 µm, each showing, after released from generative cells, a cylindrical collar, 5.5–6 x 2 µm (FIG. 41). Each fertile branch with 4–6 generative cells, variable in length. Zygospores not found.

Etymology. – L., inexpectans = not expected.

Specimens examined.. SPAIN. BARCELONA: Fogars de Montclú s; Santa Fe del Montseny, Riera de Santa Fe, prepared from Chironomidae Orthocladiinae larvae, 25-X-2001, L.G. Valle and S. Santamaria, BCB-Tr1156 (HOLO-TY PE); same locality, 7-XI-2001, L.G. Valle and S. Santamaria, BCB-Tr1095, BCB-Tr1089.

Smittium inexpectans is characterized by its relatively long collar (FIG. 41). Thallial characters are somewhat peculiar, because the pattern of basal ramification is not common, with its laterally emerging branches, tapering toward the apex, as wide as the basal cell, without any septa at the base; and the holdfast, placed in the middle of these lateral branches (FIG. 40). Smittium kansense Lichtw. & Grigg has similar trichospores but differs in its shorter collar (2 µm, according to Lichtwardt and Grigg 1998). Other species with similar-size trichospores are S. phytotelmatum Lichtw. (Lichtwardt 1994), S. delicatum (Lichtwardt and Williams 1990), S. typhellum Manier & Coste (Manier and Coste 1971), and S. angustum M.C. Williams & Lichtw. (Lichtwardt and Williams 1992a), all separable from our species by the presence of shorter trichospore collars, as well as by other thallial features.

Smittium prostratum L.G. Valle & Santam., sp. nov. FIGS. 45–48

Thallus postratus super proctodaeo, cum lateralibus ramis verticillatas ramificationes in superis areis ferentibus. Secreta tenaculi materia secus thalli paginam hanc adhaerens cum proctodaeo. Basalis cellula variabilis, plerumque cum lateralibus ramis. Trichosporae subcylindricae ad elongato-ellipsoidales, 26–30 x 3.5–4.5 µm, cum campanulato collari, decrescenti versus extremum, 4.5–5.5 x 2.5–3.5 µm. Quoque fertile ramae 2–4(–6) genitales cellulas. Zygosporae ignotae. In Chironomidarum larvarum proctodaeo in-coli.

Thalli prostrate on the hindgut lining, with lateral branches that form verticillate ramifications at the upper areas. Holdfast material secreted along the thallial surface keeping it in contact with the gut lining, functioning as glue (FIG. 48). Basal cell variable, usually with lateral branches. Trichospores subcylindrical to elongate-ellipsoidal, 26–30 x 3.5–4.5 µm, with a campanulate collar, narrowing toward the end, 4.5–5.5 x 2.5–3.5 µm (FIGS. 46–47). Each fertile branch with 2–4(–6) generative cells. Zygospores not found.

Etymology. – L., prostratum = prostrate (referring to the position of the thallus).

Specimen examined.. SPAIN. HUESCA: Puente de la Reina de Jaca, Asabón river, prepared from Chironomid Ortho-cladiinae larvae, 264-IX-2002, L.G. Valle, BCB-Tr1550 (HO-LOTY PE).

The main diagnostic characteristic of this species undoubtedly is the position of thalli inside the hindgut, being prostrate (i.e., horizontally in relation to the gut lining). The holdfast not only is concentrated in the basal cell but covers the entire thallial axis that remains in contact with the gut lining (FIG. 48). An increase of the basal surface may improve its capacity to develop a larger and more ramified thallus, allowing more trichospore production.

Trichospore morphology in S. prostratum is similar to that of other species, but collar morphology is a good character for its isolation, being wider and more campanulate than in the related species. Trichospores of S. cylindrosporum are somewhat larger ([21–]26–33[–41] x 4–6 µm, according to Lichtwardt and Arenas 1996) and more cylindrical. Smittium typhellum also shows similar trichospore sizes but has shorter and cylindrical collars (Manier and Coste 1971, Manier and Mathiez 1965).

Another species where a lateral direction of the thallial growth has been reported is S. fecundum, although in this example the lateral extension is not so evident and the holdfast material is not continuous but secreted at the tip of small pits laterally produced from the axial cells. Other genera of Harpel-lales also have similar patterns of thallial development (e.g., Lancisporomyces vernalis Santam., where the main axis lies on the gut lining, being fixed to it by numerous discontinuous peg-like holdfasts and profuse lateral branches [Santamaria 1997]). In addition, Baltomyces styrax Cafaro (Cafaro 1999) has a similar layout. In all these cases the increase in the contact surface could be a result of a phenomenon of evolutionary convergence.

Smittium pseudodimorphum L.G. Valle & Santam., sp. nov. FIGS. 49–55

Thallus e basali cellula ramosa, verticillatus in mediis atque superis areis. Basalis cellula simplex vel bilobata, cum secreta tenaculi materia. Trichosporae dimorphae: typus , sub-cylindricae, (45–)50–55(–66) x (4–)5.5(–7) µm, cum cylindrico vel convergentibus marginibus collari 9–11.5 x 3.5 µm; typus ß, late ellipsoidales, (12–)14–16.5 x 5.5–6 µm, cum collari cylindrico, 12.5–18 x 1.5–2 µm. Genitales cellulae longiores in ramis trichosporas typi ferentibus, breviores atque plures in ramis trichosporas typi ß ferentibus. Zygosporae biconicae, 82–97 x (14–)16–18(–20) µm, zygosporophorum in extremo quadrante zygosporae longitudis locata. Liberae zygosporae cum collari, 16.5–20 x .5–7 µm atque conspicua appendice. In Chironomidarum larvarum proctodaeo incoli.

Thalli branched from the basal cell, verticillate at the medial and upper areas. Basal cell simple or bilobulated (FIG. 53), with secreted holdfast material. Trichospores dimorphic: type , subcylindrical, (45–)50–55(–66) x (4–)5.5(–7) µm, with a thickened apex, collar cylindrical or with convergent margins (FIGS. 49–50), 9–11.5 x 3.5 µm; type ß, broadly ellipsoidal, (12–)14–16.5 x 5.5–6 µm, collar cylindrical, 12.5–18 x 1.5–2 µm (FIGS. 52, 54–55). Both trichospore types growing in the same thallus but on different branches, the trichospores of type ß being less common. Generative cells longer in branches producing the trichospores of type , shorter and more numerous in fertile branches producing the trichospores of type ß. Zygospores biconical, 82–97 x (14–)16–18(–20) µm (FIGS. 50–51). Once released, the zygospore shows a collar of 16.5–20 x .5–7 µm, and a visible appendage.

Etymology. – L., pseudo = false, similar to; dimorphum = dimorphic (referring to the similarity with Smittium dimorphum).

Specimens examined.. SPAIN. BARCELONA: Palas de Torrella, Cardener river, prepared from Chironomidae Diamesiinae larvae, 15-III-2001, L.G. Valle, BCB-Tr0605 (HO-LOTY PE). TARRAGONA: Prades; Riudabella, La Milana stream, prepared from Chironomidae Diamesinae larvae, 19-III-2001, L.G. Valle and S. Santamaria, BCB-Tr0622, Tr0624–0625.

As inferred by the specific epithet, this species is similar to S. dimorphum, one of the several dimorphic species described within the genus. Trichospores of the types and ß are similar in size range in both species, but the main difference between them is the presence, in S. pseudodimorphum, of a longer collar in both trichospore types (FIGS. 49, 55), most obvious in trichospores of type ß (2.5–4 µm in S. dimorphum, Lichtwardt and Williams 1983). In S. pseudodimorphum, trichospores are slightly longer (38–50 x 5.5–6.5 µm in S. dimorphum, Lichtwardt and Williams 1983). Zygospores of S. pseudodimorphum are broader and more biconical ([82–97 x [14–]16–18[–20] µm) than those of S. dimorphum (71–96 x 13–14 µm). The zygospore collar is broader and campanulated in the American species (12–17 µm diam, according to Lichtwardt and Williams 1983). The basal cell is different between the two species, being bilobulate in most of the individuals of S. pseudodimorphum (FIG. 53). Smittium pseudodimorphum presents a well-distinguished and differentiated holdfast, while it is inconspicuous in S. dimorphum (Lichtwardt and Williams 1983). For comparison we have examined the type of Smittium dimorphum (microscope slide MBL-62-7 Holotype [FH]).

Trichospores of type ß may be compared to those found on S. alpinum Lichtw. (Lichtwardt 1984), although in S. pseudodimorphum they are somewhat larger ([23–]33[–44] x [10–]12[–14] µm) and the collar is likewise larger ([10–]14[–19] x [2–]3[–4] µm). In both species zygospores are nearly identical.

Smittium esteparum Ferrington, Lichtw. & López Lastra (Lichtwardt et al 1999) is another dimorphic species, although it is distinguished from S. pseudodimorphum by its trichospore and holdfast features, as well as by its smaller zygospores. Smittium orthocladii Manier is similar to the Spanish species, although trichospores are more ellipsoidal and shorter and the ß trichospores are smaller than in S. pseudodimorphum. Zygospores in S. orthocladii were observed to be more fusiform (Manier 1970) when we examined the type (microscope slide COUL 9.63 Holotype [PC]).

	PREVIOUSLY KNOWN SPECIES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS NEW SPECIES PREVIOUSLY KNOWN SPECIES DISCUSSION KEY TO THE IBERIAN... LITERATURE CITED

 
Smittium alpinum Lichtw. FIG. 56

Specimens examined. – SPAIN. TARRAGONA: Horta de Sant Joan; Mas de la Franqueta, prepared from Chironomidae Diamesini larvae, 24-V-2001, L.G. Valle, BCB-Tr0773, Tr0778.

Other collections examined. – USA, Glacier National Park, prepared from Diamesa sp., 11-VIII-1975, Lichtwardt, microscope slide MBL-13-10 (HOLOTY PE, FH).

Species reported from the USA and Europe (Sweden, France and Switzerland; Lichtwardt 1984). Some variations have been noticed between the American and the European collections, with longer trichospores in the former (Lichtwardt 1984). A remarkable characteristic is the presence of a long cylindrical collar (FIG. 56), especially in some of the individuals studied (14–17 x 2 µm). Trichospores measure 30.5–36 x 9–12 µm, being somewhat narrower but mostly coincident with other European thalli of S. alpinum ([23–]33[–44] x [10–]12[–14] µm, according to Lichtwardt 1984). We have not found zygospores, which have been reported only from the American samples. This is the first report of S. alpinum from the Iberian Peninsula.

Smittium chironomi Tuzet & Manier FIG. 57

Specimens examined. – SPAIN. SEGOVIA: Vegas de Matute, Moros river, prepared from Chironomidae Diamesinae larvae, 24-IX-2001, L.G. Valle, BCB-Tr1009, Tr1012–1013, Tr1015.

This species was described from France (Tuzet and Manier 1953) and last reported but not definitely identified from England, where several experimental studies were carried out with cultured material (Moss 1972). This species previously was reported from Spain, where smaller trichospores than typical were described (Santamaria and Girbal 1997). The Span-ish samples have slightly shorter and broader trichospores (13–20 x 3–4 µm) and the collar is slightly longer (2–3 µm) than those of the original description.

Smittium culicis Manier FIGS. 58–63

Specimens examined. – SPAIN. BARCELONA: Viladrau; Collpregon stream, prepared from Chironomidae Orthocladiinae (Eukiefferiella sp.) larvae, 23-I-0, L.G. Valle, BCB-Tr1219, Tr1227–1228, Tr1230; Moià, Moià stream, prepared from Chironomidae Orthocladiinae (Chironomus sp.) larvae, 13-III-2002, L.G. Valle, BCB-Tr1318; same locality, L.G. Valle and S. Santamaria, 14-III-2002, Tr1356, Tr1360, Tr1379. GIRONA: Tor, Llosa river, prepared from Culicidae larvae, 7-VIII-2000, L. Ribas, L.G. Valle and S. Santamaria, BCB-Tr0198.

Other collections examined. – FRANCE. Montpellier, prepared from Culex pipiens, 21-V-1959, Manier, microscope slide TY PE M.17C (HOLOTY PE, PC).

Smittium culicis is a cosmopolitan species, since it has been reported nearly from all continents where Trichomycetes have been searched (France, Tunisia [Manier et al 1964], Canada, New Zealand and USA [Lichtwardt 1986], Chile [Lichtwardt and Arenas 1996], Argentina [Lichtwardt et al 1999]). It comprises thalli and collections with a broad trichospore size range and morphometric variations that have been solved taxonomically creating what often has been designed as a species complex (Lichtwardt et al 2001). Our specimens include individuals at the lower limits of trichospore size range ([17–]20–25.5 x 4–6 µm, with a collar of 4–7.5 µm long).

The typical hosts for S. culicis are the Culicidae larvae, although it also has been reported and isolated in axenic cultures from Chironomidae (Chironomus spp.) in France and from Simuliidae in the USA (Lichtwardt 1986, Lichtwardt et al 2000). Our specimens were found in both Chironomidae and Culicidae, with zygospores observed in the Orthocladiinidae Eukiefferiella sp., an unusual host. No remarkable differences have been observed between the examined populations in spite of host variation.

We report for the first time the presence of zygospores (FIG. 61), which typically are biconical, measuring 47–55 x 11–12 µm, with a collar of 4.5–8 x 3.5–5.5 µm, having cylindrical or slightly out-flared margins. In those thalli where zygospores develop, trichospore size is smaller (14.5–19.5 x 5–6 µm) and the number per fertile branch decreases in comparison with those seen in nonsexual phases. Zygosporophores and zygospores develop at the top of a peduncle formed from one of the conjugants, next to the conjugation tube (FIG. 58).

The observation of a coat-like substance on both trichospores and trichospore collars, when studied with scanning electronic microscopy (FIGS. 62, 63), is remarkable.

Smittium culisetae Lichtw. – Specimens examined.. SPAIN. BARCELONA: Caldes de Montbui, prepared from Culicidae larvae, 13-X-94, J. Girbal, BCB-Tr0049–0056; Cerdanyola del Vallès, Bellaterra, UAB, prepared from Culicidae larvae, 15-VII-2002, L.G. Valle, Tr-BCB-1502.

The species is widely distributed (Lichtwardt 1986). We have studied the material previously reported by Santamaria and Girbal (1997). These specimens perfectly match the trichospore size range proposed for the species ([11–]16[–30] x [3–]4[–7] µm, according to Lichtwardt 1964). Smittium culisetae is distinguished from S. culicis by its characteristic maximum breadth located in the basal section of the trichospore.

Smittium dipterorum Lichtw. FIG. 76

Specimens examined.. SPAIN. BARCELONA: Gualba de dalt (Parc del RACC), Gualba stream, prepared from Chironomidae Orthocladiinae larvae, 7-XI-2001, L.G. Valle, BCB-Tr1122.

Other collections examined.. COSTA RICA. Monteverde, prepared from Simulium sp., 11-XI-1991, Lichtw., CR-260-2 (HOLOTY PE, FH), CR-17-3 (PARATY PE, FH).

This is the second world record of this species, previously known only from Costa Rica (Lichtwardt 1997) in the tract of Simuliidae and Chironomiidae (Orthocladius spp.). Our specimens have almost cylindrical trichospores measuring 15–20 x 2.5–3.5 µm, with a collar of 1.5–3 µm, slightly flared outward ([10–]12–18[–26] x 2–3[–4] µm, in Lichtwardt 1997). The thallus is profusely branched at the base, showing many radiating branches with verticillate ramifications (FIG. 76). Each fertile branch includes 4–6(–8) generative cells. We have not clearly observed the basal cell because the only mature thalli collected were crowded.

Smittium fecundum Lichtw. & M.C. Williams FIGS. 64–68

Specimens examined.. SPAIN. BARCELONA: L’Espunyola; Can Macià, in a little stream proceeding from a pond, prepared from Chironomidae Orthocladiinae larvae, 4-II-2002, L.G. Valle, BCB-Tr1245–1248, Tr1251, Tr1254–1265, Tr1267–1277; same locality, prepared from axenic cultures (SmMacià-pq4 and pq7), same locality, 28 Feb 2002, BCB-Tr1294, Tr1299, Tr1320, Tr1322.

Other collections examined.. USA. COLORADO: Gunnison Country, prepared from Psectrocladius sp., 27-VII-1995, Lichtwardt and Williams, RMBL-61-8 (HOLOTY PE, FH).

This species was known only from the type locality in the USA (Lichtwardt and Williams 1999). The Spanish specimens show trichospores with a narrower collar (5.5–7.5 µm) in comparison to the type, although they absolutely are comparable and the differences are not important. Iberian specimens have trichospores measuring (14–)25–28(–31) x 7–7.5(–9.5) µm, similar to those described for the American specimens (17–20[–27] x 5–8 µm, in Lichtwardt and Williams 1999), although we have observed some trichospores longer and wider. The original description was prepared, in part, on the basis of cultured material, where the formation of long series of generative cells was observed. Likewise, we have seen this arrangement only in cultured specimens. In the natural hosts, the number of generative cells per fertile branch clearly decreases (4–6[–8]). The characteristic peg-like holdfasts have been observed in some but not in all of the examined thalli.

We report for the first time the presence of zygospores, which are biconical, slightly asymmetric, rounded at the medial section, measuring 70–85 x 18–19 µm, with the zygosporophore eccentrically placed at approximately one-third the distance from one apex (FIG. 67). Zygosporophores and zygospores grow at the top of a sterile peduncle measuring 42–54 x 8–10 µm (FIG. 68). Zygospore collar measures 9–12 µm in length. These sexual spores are comparable to those of S. alpinum, which also are biconical with a pronounced rounded or broadened middle section (Lichtwardt 1984), as well as those of S. ouseli M.C. Williams & Lichtw. (Williams and Lichtwardt 1984).

Observations using SEM techniques revealed the presence of a cylindrical appendage, with a micro-granulation on the surfaces of both trichospores and trichospore collars. The margins of the collar are seen clearly, being inwardly folded at the distal end (FIG. 66).

Smittium megazygosporum Manier & Coste FIGS. 69–72

Specimens examined.. SPAIN. BARCELONA: Avià, Clarà stream, prepared from Chironomidae Diamesini (Diamesinae) larvae, 2-IV-2001, L.G. Valle, BCB-Tr0669–0671, Tr0673–0675; Rupit, Rupit stream, prepared from Chironomidae Orthocladiinae (Eukiefferiella gr. minor) larvae, 26-IV-2001, L.G. Valle, BCB-Tr0559–0562; Marganell, Marganell stream, prepared from Chironomidae Orthocladiinae larvae, 13-I-2002, L.G. Valle, BCB-Tr1188–1200; same locality, prepared from axenic cultures (SmMarg_pq4), 28-II-2002, BCB-Tr1293, Tr1295–1297, Tr1324. TARRAGONA: Riudabella, Milana stream, prepared from Chironomidae Orthocladiinae (Cricotopus sp.) larvae, 19-III-2001, L.G. Valle and S. Santamaria, BCB-Tr0623.

Other collections examined.. FRANCE. Herault, prepared from Syncricotopus paquiventris, XI-1965, Manier and Coste, TY PE LIR-11.65 (HOLOTY PE, PC).

Smittium megazygosporum was described in France from the Orthocladiinae Syncricotopus rufiventris (Manier and Coste 1971) and also was reported from Argentina in Cricotopus sp. (Lichtwardt et al 1999). The description of this species is wide enough to include several forms with overlapping trichospore size ranges. Taking this into account, we could consider this as a species complex, comparable to S. culicis. Until now, the descriptions for the specimens found in France and Argentina comprise individuals with extreme trichospore sizes of 36–49 x 3.5–6 µm, and collar of 3.5–7 µm (from Manier and Coste 1971, and Lichtwardt et al 1999). We have found trichospores measuring (35–)40–47(–53) x 3.5–5.5 µm, with collar of (3–)4.5–5.5 x 2.5–4 µm. Some of the collections of S. megazygosporum examined surpass the trichospore length of those previously described. The trichospore size differences seem not to be very important, we consider them as intraspecific variations. We did not found zygospores, an essential feature to fully confirm the identity of the species, however, all the thallial and sporangial characters indicate that these Iberian collections correspond to the description of S. megazygosporum.

We have studied some cultured material (from Marganell collection) with SEM techniques, it is worth mentioning that the cylindrical appendage is helically arranged just after release (FIG. 72). We also mention the smooth trichospore outer wall (FIG. 72). Most of the trichospore collars strangely have been broken and appear incomplete (FIG. 71). These events have been observed in other species (S. culicis and S. hecatei), giving rise to abnormal morphologies (i.e., shorter collars, usually also somewhat outwardly flared).

Cultured material shows trichospores that frequently extrude their content with no external help. These apically released sporangiospores are viable and produce new thallial colonies.

Smittium pusillum Manier & Coste FIGS. 73–74

Specimens examined.. SPAIN. CANTABRIA: Cabezón de Liébana, Bullo river, prepared from Chironomidae Orthocladiinae larvae, 30 Sep 2002, L.G. Valle, BCB-Tr1591.

Other collections examined.. FRANCE. Herault, prepared from Procladius sp., 2-IV-1968, Manier and Coste, TY PE VEND 4.68 (HOLOTY PE, PC).

This is the third record of S. pusillum, which originally was described from France. The second report occurred in the Rocky Mountains (USA), within Chironomidae Orthocladiinae (Cricotopus spp. and Orthocladius spp.) larvae (Williams and Lichtwardt 1987, Lichtwardt and Williams 1988). A peculiar character of this species that we have not observed is the presence of a pseudorhizoidal basal cell, reported in the original description, although without any illustration (Manier and Coste 1971) and not mentioned in the second record of the species (Williams and Lichtwardt 1987). We have observed only some lateral and nonseptate branches arising from the basal cell in some individuals. The spore features of the Spanish specimens are coincident with those described in France (14–22 x 2.5–3 µm, in Manier and Coste 1971), although we have observed some larger and broader trichospores (15–24 x 2.5–3.5 µm) as reported from the American specimens (14–25 x 3.5–4 µm, according to Williams and Lichtwardt 1987). The collar measures 3–4 x 1.5–2 µm in our specimens but reaches 4.5 µm in those described by Williams and Lichtwardt (1987). We have not found zygospores.

Smittium simulii Lichtw. – Specimens examined.. SPAIN. BARCELONA: El Brull, Montseny, La Castanya, la Castanya stream, prepared from Simuliidae larvae, 3-X-2000, L.G. Valle and S. Santamaria, BCB-Tr0326, Tr0328; Fogars de Montclú s, Sta. Fe del Montseny, Riera de Sta Fe, prepared from Simuliidae larvae, 14-IX-94, J. Girbal and S. Santamaria, BCB-Tr0066; Cerdanyola del Vallès, UAB, prepared from Culicidae larvae, 15-VII-2002, L.G. Valle, Tr-BCB-1502–1513; same locality, prepared from axenic cultures, 10-XI-2002, BCB-Tr1682. GIRONA: Setcases; Coma d’Orri, Ter river, prepared from Chironomidae larvae, 12-IX-2000, L.G. Valle and S. Santamaria, BCB-Tr0273.

This is a frequent and cosmopolitan species with a wide host range, from the more typical Simuliidae through the less typical Chironomidae (Lichtwardt et al 1987) to the more infrequent Culicidae (Lichtwardt et al 2001). We also have found this species in all of these hosts, the more remarkable being those individuals found in Culicidae larvae. This species was reported previously from Spain (Girbal and Santamaria 1998