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Unitat de Botànica, Departament de Biologia Animal, de Biologia Vegetal i d’Ecologia, Facultat de Ciències, Universitat Autònoma de Barcelona, 08193-Bellaterra (Barcelona), Spain
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The presence of zygospores in the genus Orphella is newly described. We found zygospores in three species of the genus, O. catalaunica, O. coronata and O. helicospora, which are all the species of the genus known from the Iberian territory. Zygospores are associated with a heterothallic conjugating sexual process in O. coronata, whereas in O. catalaunica and O. helicospora, they form homothallically. In all instances, zygospores are consistently associated with an organized pattern of sterile cells, forming structures comparable to those present with asexual trichospores. We compare the ontogeny of Orphella zygospores with that found in the harpellid Genistellospora homothallica and discuss the possible close relationship of Orphella with Kickxellales (Zygomycetes). We report O. coronata in Spain for the first time, replacing all previous records of O. haysii. Results are supported with line drawings and photographs.
Key words: conjugation, Harpellales, Kickxellales, Plecoptera, systematics
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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, is placed within the Harpellales (Trichomycetes) and, probably, is the most atypical within the order because of the morphology of the thalli and the ontogeny and dispersal of trichospores. Currently, this genus includes six species (Lichtwardt et al 2001), which can be essentially separated by morphology and size of trichospores and accompanying cells, branching pattern of the thallus and basal cell features. Orphella is most comparable to Pteromaktron, an unusual genus described by Whisler (1963), sharing characteristics such as the placement of trichospores on terminal branches (FIG. 38b, c, d), overall thallial structure, and anal protrusion of the apical fertile zone.
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). We provide morphological evidence to support the molecular results, including: (1) morphology and ontogeny of reproductive structures, (2) spore dispersal mechanisms, and (3) sexuality and zygospore production. We concentrate on the third line of evidence because it represents the most interesting novelty of the genus, if not the entire order.
The Harpellales include 180 species in 37 genera, of which 77 (in 29 genera) form zygospores [calculated from Lichtwardt et al (2001), and personal unpublished information]. All the zygospores reported from these species of Harpellales are more or less conical or biconical, with or without a collar and adapted to the particular environment of their host digestive tract (Lichtwardt et al 2001). The zygospores of Orphella species, as we expose subsequently, do not follow this morphological pattern.
Recently, White and Lichtwardt (2004) reported allantoid-spored Orphella specimens identified as O. coronata L. Léger & M. Gauthier from Europe. The authors also suggested the possible conspecificity of the Iberian specimens designated as O. haysii (Santamaria and Girbal 1998) and O. coronata. In agreement with White and Lichtwardt’s proposal and after comparison with type material of O. haysii (FIG. 18), we have decided to classify our specimens with allantoid trichospores as O. coronata.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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), in the context of a wider project for the study and cataloguing of the Iberian Fungi ("Flora Micológica Ibérica" project). Plecopteran hosts were captured and dissected following the methods described by Lichtwardt et al (2001). We measured allantoid trichospores of O. coronata considering the maximum cord distance of the curve. Microscopic slides were mounted as described by Valle and Santamaria (2002), and are kept in the BCB-Mycotheca herbarium (at the institutional address of authors). The slide numbers for photographs and drawings are listed in the corresponding figure caption. For describing and labeling cells and structures in text and figures, especially those associated with trichospores and zygospores, we follow terms and abbreviations used by Santamaria and Girbal (1998) for trichospores with new codes for zygospores which are designated as follows: bc, basal cell of the sporulating head; ch, conjugating hypha; fb, fertile branches; gc, generative cell; h, holdfast cell; ic, intermediate cell; sb, subsidiary branches; sc, supporting cell; sgc, supporting generative cell; sh, secreted holdfast material; tc, terminal cell; tc1, terminal cell formed from intermediate cell; tc2, terminal cell formed from zygosporophore; tc2' and tc2'', ramifications of tc2; tr, trichospore; z, zygospore; zp, zygosporophore. We include precise description of these terms in the paragraphs below. All drawings are based on lactophenol-cotton-blue fixed specimens, using a camera lucida adapted to a Zeiss Universal microscope.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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, 7, 11–12, 34–36).
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–7, 12). The zygosporophore appears inflated just under where the zygospore is attached (zp, FIGS. 1, 3, 5, 7, 9–12), is clavate, bears a terminal cell that is cylindrical and slender initially (tc2, FIGS. 3, 5, 10–12), but broadens and supports two very thin, filiform, prolongations at maturity (tc2', tc2'', FIGS. 5, 7). The intermediate cell is variable, fusiform, slightly curved, even sygmoid (ic, FIGS. 3, 5, 7, 9–12), and bears a slender terminal cell (tc1, FIGS. 2–3, 5, 7, 10–12). The supporting cell, which separates the intermediate cell from the basal cell, is variable but always small and inconspicuous (sc, FIGS. 5, 7, 9–12).
In Orphella coronata, zygosporesare produced heterothallically (FIGS. 13, 22), appearing associated with hyphal conjugations (ch, FIGS. 14, 23) and are helicoidal, measuring 30–35 x 5.5–7 µm (largest outer diameter of helix x breadth of coiled spores) (z, FIGS. 14–16, 22–24). The zygosporophore is cylindrical, more or less curved (zp, FIGS. 15, 23–24), attenuated toward the distal end where a rather elongated (up to 600 µm) and curved terminal cell is attached (tc2, FIGS. 14, 16, 23). The intermediate cell is irregular in shape (ic, FIGS. 14, 23–24) and, replacing the terminal cell, a thin and long specialized hypha arises to establish conjugation bridges (ch, FIGS. 14, 23), forming an irregular and very intricate net of filaments, with anastomozed and inflated zones (FIG. 22). A supporting cell was not observed.
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–31, 36), similar to trichospores in shape, but larger (z, tr, FIGS. 29, 31), measuring 25–28 x 5.5–7.5 µm (compared to 11–17 x 3.5–4.5 µm for trichospores) (largest outer diameter of helix x breadth). The zygosporophore appears inflated just under where the zygospore is attached (zp, FIGS. 27, 30, 34–36), is fusiform and somewhat sinuous and bears a long and filiform terminal cell (up to 150 µm) (tc2, FIGS. 27, 29, 34–36), which later often bifurcates (tc2', tc2'', FIGS. 28, 31). The intermediate cell is curved to sigmoid (ic, FIGS. 27, 30, 34–36) and bears a short, stout prolongation, which we believe to be a terminal cell, although the septum of separation cannot be clearly seen (tc1, FIGS. 34–36). The supporting cell, which separates the intermediate cell from the basal cell, is small and inconspicuous (sc, FIGS. 34–36).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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, 19–20, 25, 33, 38c, d), as a set of cells called a dispersal unit (Williams and Lichtwardt 1987). Some of these dispersal units are located in variable numbers above one basal cell (bc, FIGS. 8, 21, 26, 32, 38c, d), and several basal cells are grouped on the fertile branches in a sort of umbellate, cyme-like structures (FIGS. 21, 26, 32, 38c, d). Williams and Lichtwardt (1987) described the dispersal unit as a three-celled structure because they failed to distinguish the fourth cell, the diminutive supporting generative cell (Santamaria and Girbal 1998). This kind of dispersal unit was first described on Orphella haysii Lichtw. & M.C. Williams (Williams and Lichtwardt 1987), and later for other species of the genus (Santamaria and Girbal 1998, Lichtwardt et al 1991a).
Dissemination mechanisms of the spores are poorly understood because of a lack of evidence for the processes involved with ingestion by the host and posterior trichospore germination within the host digestive tract. However, after examination of numerous specimens, we consider two possibilities of spore dispersion. The first is a consequence of direct observation (in O. coronata), and involves the extrusion of the trichospore content (FIG. 17). This process can occur when the dispersal unit is still attached to the thallus, or when it has been released to the outer or inner host digestive tract environment, depending on whether or not the thallus protrudes from the anus. The second possibility for trichospore dissemination is by the way of the whole dispersal unit, which detaches from the mature and generally protruding thalli (Williams and Lichtwardt 1987); however, this explains only the migrating procedure of the dispersal unit as a whole and leaves unresolved whether the entire unit is ingested by the host, or whether just the trichospore travels along the digestive tract of the Plecoptera.
Regarding the external release of the dispersal unit, the terminal cell seems to function in the same way as trichospore appendages in other Harpellales, i.e. to minimize the water flow effect (Lichtwardt 1986). We have observed the sticky nature of terminal cells in most specimens of Orphella studied, in those accompanying trichospores and, especially, in those related to zygospores, where two long, filiform terminal cells can develop (tc2', tc2'', FIG. 28). This character has been particularly noticed during the manipulation of the specimens for the slide preparation. The presence of these sticking slender terminal cells may be a clue to the capacity of the dispersal unit for setting on the substrate. The extrusion of the trichospore content could be considered a fast way to reproduce in situ, with the purpose of increasing the infestation within the same host gut. This phenomenon was previously reported for other genera such as Ejectosporus Peterson et al (Lichtwardt et al 1991b, Strongman 2005). We have also observed (unpublished observation) a similar spore behavior within Spartiella barbata Tuzet & Manier.
Evidence of sexuality and zygospore production in Orphella species.— –
In some slides containing O. catalaunica we were able to observe two kinds of "trichospores", those typically straight and those, called "anomalous", with a coiled base (FIG. 6). In the beginning, we thought that the "anomalous" spores were purely abnormal trichospores. Nevertheless, the lasting presence of these spores, with a coiled proximal end and a straight apex (FIGS. 5–7, 12), made us think about retracting the thesis of a simple abnormality. We studied in detail the "anomalous" spores and initially called them 
(delta) spores for the similarity of their shape to the Greek letter, and concluded that their ontogeny was clearly different from that of trichospores.
The accompanying cells of the " spores" are more numerous than those in the analogue of trichospore dispersal units (see above in the results section). The presence of " spores" associated with this cell-complex was immediately noticed in O. helicospora and in O. coronata, with the peculiarity that, in the latter, conjugation tubes were also observed (ch, FIGS. 14, 23). Moreover, in O. coronata, the supporting generative cell and the intermediate cell are not clearly identifiable because they can produce projections that may contact other hyphal filaments to conjugate (ch, FIG. 23). The presence of conjugation tubes is enough to accept these " spores" as zygospores in O. coronata, but also to accept the sexuality of both O. catalaunica and O. helicospora because of their similar structure. We have detected the released zygospore dispersal unit in both O. catalaunica and O. helicospora including the zygosporophore and terminal cell, in a structure comparable to the trichospore dispersal units (FIGS. 15, 28).
Other support for our belief in the zygosporic nature of " spores" is the parallelism between the homothallic zygospore development structure in Genistellospora homothallica Lichtw. and the Orphella zygospore complex (FIG. 37). The cells involved in both genera are clearly comparable. Only the terminal cell from the zygosporophore in Orphella does not have a counterpart in G. homothallica. We cannot conclude whether this affinity between reproductive elements is due to a homologous or to an analogous process, although homology might be preferred because of their relative phylogenetic proximity.
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, Lichtwardt 1973, Moss and Young 1978, Gottlieb and Lichtwardt 2001). These works, together with septal studies on Kickxellales and Harpellales (Farr 1965, Manier 1973, Moss 1972, Moss and Lichtwardt 1976, Saikawa et al 1997) pointed to the Kickxellid origin of Harpellales (as well as Asellariales), as was initially proposed by Moss and Young (1978) and Benjamin (1979).
Moss and Young (1978) carried out a comparative study based on asexual spores of Coemansia (Kickxellales) and Smittium (Harpellales). Now, we are trying to reestablish this idea by adding Orphella elements in the argument. Currently, we have more information about other recently described Kickxellales, which may add strength to the morphological affinities between Orphella and Kickxellales. Within Kickxellales species, the sporangiola [monosporous sporangia or merosporangia (Benjamin 1979)], which resemble trichospores of Harpellales, develop in a variable number from a sporocladium [a simple or branched mononuclear cell (Moss and Young 1978)]. According to these authors, the collar area of the trichospores in Harpellales would have evolved from the pseudophialides (short supporting cells placed at the sporangiola base in the Kickxellales) by a progressive degeneration of the lowest septum delimiting this cell (Moss and Young 1978) (FIG. 38). In the genus Orphella, the origin of trichospore associated cells, could be interpreted according to different hypotheses. The most satisfactory, in our opinion, is that which relates the supporting generative cell of Orphella with the pseudophialides in the Kickxellales, because of their similar morphology and relative position on the fertile head. The evolutionary origin of trichospores is not directly comparable to that of sporangiola, because of different position and ontogeny (FIG. 38). We consider that Kickxellales sporangiola and Orphella generative cells might be related by similar ontogeny. Furthermore, when young generative cells of Orphella fertile heads are observed, the similarity with Kickxellales sporangiola becomes apparent. The trichospores and the terminal cells in Orphella would have evolved secondarily from the generative cell, that is, from the original sporangiola of the Kickxellales. This process of cell differentiation within Orphella should obviously be linked to a parallel nuclear cell behavior that could explain all this sequence. In some species of Kickxellales, sporangiola with more than one nucleus (two or three) have been observed (Benjamin 1979). The nuclei of the sporangiola derive from the division of the pseudophialide cell nucleus, one of them remaining within this cell (Benjamin 1979). In sporangiola having three nuclei, two successive mitoses in the pseudophialide cell could have occurred, giving rise to four nuclei, three of them migrating to the sporangiola. It is reasonable that new cells (trichospore and terminal cell) could have evolved from an original sporangiola-like cell by nuclear division, migration, and later septum development.
Initial studies demonstrated molecular evidence of Orphella-Kickxellales affinities (White et al 2003). Additionally, it is interesting to consider Pteromaktron protrudens in all this argument, because it shows, even more evidently than Orphella species, an important resemblance to Kickxellales. Indeed, Moss and Young (1978) suggested the close relationship between Kickxellales and Pteromaktron. In this case, the correspondence of Kickxellales sporangiola with Pteromaktron trichospores seems direct, both by their location and morphology (FIG. 38). Hence, Pteromaktron, or a Pteromaktron-like ancestral taxon, could be the intermediate link between Kickxellales and gut fungi, from where the Harpellales radiated, while Orphella derived directly from species of Kickxellales, probably from some kickxellid resembling Ramicandelaber brevisporus Kurihara, Degawa & Tokumasu (2004), and then being even closer to the Kickxellales than Pteromaktron.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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———.1979. Zygomycetes and their spores. In: B Kendrick, ed. The whole fungus. Vol. 2. National Museum: Ottawa. pp. 573–621.
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Kurihara Y, Degawa Y, Tokumasu S. 2004. Two novel Kickxellalean fungi, Mycöemilia scoparia gen. nov. and Ramicandelaber brevisporus sp. nov. Mycol Res 108:1143–1152.[CrossRef][Medline]
Léger L, Gauthier M. 1931. Orphella coronata n. g., n. sp. Entophyte parasite des larves de Némurides. Trav Lab Hydrobiol Piscic Univ Grenoble 23:67–72.
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———.1986. The Trichomycetes: Fungal Associates of Arthropods. New York: Springer-Verlag. 343 p.
———, Peterson SW, Huss MJ. 1991a. Orphella hiemalis: a new and rare trichomycete occurring in winter-emerging stoneflies (Plecoptera, Capniidae). Mycologia 83:214–219.[CrossRef]
———, ———, Williams MC. 1991b. Ejectosporus, an unusual new genus of Harpellales in winter-emerging stonefly nymphs (Capniidae) and a new species of Paramoebidium (Amoebidiales). Mycologia 83:389–396.[CrossRef]
———, White MM, Colbo MH. 2001. Harpellales in Newfoundland aquatic insect larvae. Mycologia 93:764–7873.[CrossRef]
Manier J-F. 1973. L’ultrastructure de la trichospore de Genistella ramosa Léger et Gauthier, Trichomycète Harpellale parasite du rectum des larves de Baetis rhodani Pict. Compt Rend Acad Sci Paris 276:2159–2162.
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———, Lichtwardt RW. 1976. Development of trichospores and their appendages in Genistellospora homothallica and other Harpellales and fine-structural evidence for the sporangial nature of trichospores. Canad J Bot 54:2346–2364.
———, Young TWK. 1978. Phyletic considerations of the Harpellales and Asellariales (Trichomycetes, Zygomycotina) and the Kickxellales (Zygomycetes, Zygomycotina). Mycologia 70:944–963.[CrossRef]
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Strongman DB. 2005. Synonymy of Ejectosporus magnus and Simuliomyces spica, and a new species, Ejectosporus trisporus, from winter-emerging stoneflies. Mycologia 97:552–561.
Valle LG, Santamaria S. 2002. Baetimyces, a new genus of Harpellales, and first report of Legeriomyces ramosus from the northeastern Iberian Peninsula. Mycologia 94:321–326.
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———, ———, Valle LG, Strongman D. 2003. Orphella: an unusual fungus associated with stoneflies. Abstracts of the Mycological Society of America (MSA) Annual Meeting, Carmel, California. Inoculum 54(3):50.
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