Systematic and taxonomic approaches to entomophthoralean species

June 6, 2017 | Autor: Richard Humber | Categoria: Biotechnology, Taxonomy, Classification
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Proceedings and Abstracts, 5th International Colloquium on Invertebrate Pathology and Microbial Control, Adelaide, Australia, 20-24 August 1990. ISBN 0 646 00549 9 pp. 133-137

SYSTEMATIC AND TAXONOMIC APPROACHES TO ENTOMOPHTHORALEAN SPECIES Richard A. Humber USDA-ARS Plant Protection Research Unit US Plant, Soil, and Nutrition Laboratory Tower Road, Ithaca, New York 14853-0331, USA Recent revisions of the higher level taxonomy and systematics of the Entomophthorales have underscored many latent problems In the taxonomy of species within this order. The descriptions of most species must be augmented to include consideration of the states of newly recognized familial and generic characters. Many species having wide geographical distributions and/or very diverse host specificities are being recognized as species complexes resolvable into distinct taxa characterized by unique combinations of morphological, ecological, pathological, biochemical, and geographical factors. Some of the new taxonomic techniques and concepts which both support and drive this revision of species and species complexes are briefly discussed. A guide is presented to working taxonomic characters that can or should be used to clarify the taxonomy of species in the Entornophthorales.

Throughout the 1980's, the fungi of the order Entomophthorales (Zygomycotina) were subjected to a series of taxonomic and systematic examinations and revisions. This rather intense activity in a number of laboratories in several countries had some mixed results: The most positive outcome was the universal discontinuance of the use of Entomophthora Fres. in the broad sense for a biologically and morphologically diverse array of entomopathogenic fungi that could not be included in a single genus whose circumscription has any predictive value about biological characters. The first serious multigeneric classification was proposed in the mid-1960's by Batko (see Humber 1981) but remained unaccepted (in print but not in discussions) for 15 years. The more recent classifications all built upon the Batkoan classification and tried to correct its errors although sharp conflict erupted over the criteria upon which to construct a post-Batkoan classification: Variant classifications based on phenetics (Remaudière & Hennebert, 1980; Remaudière & Keller, 1980) and on perceived phylogenetic relationships (Humber 1981, 1982, 1984a-b; BenZc'ev & Kenneth 1982a-b; BenZe’ev et al., 1987) were proposed. The systematics principles underlying these two classifications were fundamentally less compatible than their proposed arrangements of taxa (Humber 1981, 1982). These methodological conflicts may still exist, but

a more uniform classification has emerged. Entomophaga Batko is now universally accepted as an entomophthoraceous genus distinct from the ancylistaceous genus Conidiobolus Bref.; there is wide agreement that Strongwellsea Batko & Weiser is distinct from the other erynioid genera (Humber 1982). Some taxonomic discord may remain over the disposition of the Batkoan subgenera of Zoophthora Batko (1966). Humber (1989) noted that Erynia Nowak.—as used by either Remaudière & Hennebert (1980) or by Humber & Ben-Ze’ev (1982) and Ben-Ze’ev & Kenneth (1982b)— was invalidly published, and, after reanalyzing the criteria used to delimit families and genera, raised Batko’s (1966) subgenera to generic status. CHANGING SPECIES CONCEPTS Ben-Ze’ev & Kenneth (1982a) proposed the following criteria to separate species: (1) shapes and sizes of all spore forms; (2) sizes and natures of distinctive cells (hyphal bodies, conidiophores and conidiogenous cells, rhizoids and cystidia); (3) biochemical characters, and (4) host range and key aspects of pathobiology (the host-pathogen association). Whether Balazy (1986) overemphasized morphology in his study of Zoophthora spp. will remain uncertain until these taxa can be studies by molecular systematics techniques.

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The re-evaluation of familial and generic concepts has real implications for the taxonomy of species in this order. A vital task for the “new” systematics is to verify the states of newly recognized characters omitted from previously published descriptions. Empusa apiculata Thax., and E. major Thax. illustrate how changing taxonomic concepts led to some unexpected results: Specimens of these taxa must now be divided into pairs of conver-gent species (Keller 1987; IHumber 1989) whose conidia and rhizoids are virtually indistinguishable but whose nuclear cytology places them in separate families as species of Batkoa Humber (Ento-mophthoraccae) or Conidiobolus (Ancylistaccae). In a different example, Entomphthora helvetica BenZe’ev et al. (1985) was described as differing from the similar E. erupta Dustan largely in (1) wholly divergent pathobiologies (sporulating on dead vs. living hosts, respectively) and (2) nuclear dimensions. Pathobiological characters must be regarded as taxonomically significant at both the generic and specific levels (Humber 1982, 1989; Ben-Ze’ev et al. 1985).

other correlated significant differences (Humber, unpublished). The taxonomic value of electrophoretic analysis of enzyme systems is well accepted for separating species, and has also proven useful for studying infraspecific variation (Latgé & Boucias 1984). The enzyme profiles of related species may be wholly distinct (with no common banding positions for strains of two taxa for any enzyme system studied) or a mixture of common and divergent enzymes. The latter state requires especially careful evaluation of a broad range of diverse criteria before making taxonomic conclusions about the identity or difference of the strains in question. Fatty acid analyses: The value of fatty acid analyses for specific taxonomy in the Entomophthorales species still remains unproven even though its methods are relatively simple. Studies on this subject indicate that some species do have fairly distinctive fatty acid profiles (Tyrrell 1967, 1968; 1971; Miura et al. 1983) . Direct genomic comparisons: Relatively little use has been made of the powerful techniques of molecular genetics to solve problems of entomophthoralean systematics. Programs using such techniques are under way at the University of South Florida (Tampa) to study species of Basidiobolus Eidam (Cochrane et al. 1989), and at the University of Toronto (Scarborough Campus) to study the Entomophaga aulicae and E. grylli (Fres.) Batko species complexes (Walsh et al. 1989). It is reassuring that studies of restriction fragment length polymorphisms with two ribosomal DNA probes and several restriction endonucleases for a wide range of Entomophaga species (Hajek et al. 1990; Walsh et al. 1989) supported the identical taxonomic conclusions that had been generated by e1ectrophoretic analysis of enzyme systems on starch gels (Soper et al. 1983; Humber 1984a

Enzyme polymorphisms: If any molecular technique could be said to have been widely accepted into systematics of the Entomophthorales, it is the comparison of polymorphic enzyme systems by starch gel electrophoresis. Observed differences in isozyme migration patterns among Australian strains of aphid pathogens originally identified as Erynia neoaphidis Rem. & Henn. [=Pandora neoaphidis (Rem. & Henn.) Humber] led Milner et al. (1983) to describe Pandora kondoiensis (Milner et al.) Humber. Similarly, Soper et al. (1983) distinguished two North American members of the E. grylli species complex in North America by electrophoretic analysis of isoenzyme polymorphisms; these differences were perfectly correlated with the host specificities and types of spores produced by these taxa. Humber (1989) recognized the pathogen of Melanoplus spp. that produces resting spores (but no usual conidial phase) as Entomophaga calopteni (Bessey) Humber; four other members of this species complex (the second North American fungus, one from Japan, and one from Australia) are distinguished by their isozyme profiles and

THE ROLE OF BIOGEOGRAPHY Geographical data seem to be increasingly critical for correct identifications. This should be expected in view of the increasing tendency to regard widely distributed species as species complexes and of the greater effort now being expended to study entomophthoraleans from

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ecosystems whose fungal flora has remained inadequately explored. Few floristic studies have been undertaken on entomophthoralean fungi, but it is a familiar idea that many of this order's species are globally distributed. As some of these species are recognized to be species complexes, however, the importance of the geographical source of a collection increases. For example, serious studies of Conidiobolus species in the eastern United States by Drechsler and, later, by King, and in India by Srinivasan, Thirumalachar, and Narasimhan are summarized by King (1976a-b, 1977); many species occur in both the US and India, but many others may be assumed to have restricted, regional distributions. Many new and indigenous species will probably be described as a result of floristic studies in previously unexplored regions. Morphological, biochemical, and genetic studies of the taxonomy of Basidiobolus have indicated strong tendencies for isolated populations to become genetically distinct (Cochrane et al. 1989) although the taxonomic implications of the genetic data remain unappraised. A 1989 panzootic of gypsy moth larvae, Lymantria dispar, in the northeastern United States was caused by Entomophaga maimaiga Soper et al. (Hajek et al. 1990). The identification of this taxon (whose presence was previously confirmed only for Japan) was based on comparisons of enzyme systems and restriction fragment length polymorphisms for numerous strains of the E. aulicae and E. grylli species complexes from Japan, North America, and elsewhere. Without proof of the complete biochemical identity of American strains with those from Japanese populations of L. dispar, the causative fungus might have been identified wrongly as a new species or as E. aulicae. This evidence for the Japanese origin of the causative fungus, also strongly suggested that this panzootic derived from intentional introductions in 1910-11 of a fungus imported from Japan (Speare & Colley 1912). Small but consistent differences in taxonomic characters among collections assignable to a species complexes or poorly defined species can be hard to assess. If, however, such differences are correlated with widely separated collections (e.g., from differing continents), geographical data do offer significant support for taxonomic decisions. The realization that Zoopbtbora aphidis (Hoffm. in Fres.) Batko is a rare species limited to Europe led to the

description of the globally distributed Pandora neoapbldis of the globally distributed Pandora neoapbldis (Rem. & Henn.) Humber (Remaudière & Hennebert 1980) and, directly, to the last decade’s systematic upheavals. RESOLUTION OF SPECIES COMPLEXES Taxonomists and systematists working with the Entornophthorales have increasingly recognized many common and long-recognized taxa to be species complexes, groups of morphologically similar but taxonomically distinct species. While there is wide agreement that taxa such as Entomophthora muscae (Keller 1984), Entomophaga grylli (Fres.) Batko (Soper et al. 1983), and E. aulicae (Soper et al. 1988) are species complexes, there are no established standards about either what the exact limits of species should be, or how best to resolve species complexes. Studies on the E. muscae species complex led to the recognition of nuclear diameter as a useful taxonomic character (Ben-Ze'ev & Zelig 1984; Keller & Wilding 1985); Humber (1981, 1984b) originally stressed the taxonomic value of chromosomal organization and mitotic mechanisms. Balazy (1986) regarded the wide host range of Zoophthora radicans (Bref.) Batko to indicate that this taxon is a species complex. Careful comparisons of fungal morphology and cultural characters for extensive collections of Zoophthora led Balazy (1986) to recognize five new Zoophthora species among his Polish collections. Balazy acknowledged the desirability of including biochemical and physiological studies in this taxonomic study but was unable to incorporate such studies in his analyses. The criteria used to delimit species must also be applied when resolving species complexes. The resolution of a species complex depends on correlating sometimes small but consistent differences in morphology (if any exist), host ranges, pathobiological characters, geographical distributions, cultural characters, and, ideally, one or more sets of molecular traits (fatty acid profiles or polymorphisms in enzyme systems or restriction fragment lengths). The precise taxonomy needed to distinguish species within complexes serves as a significant model for updating circumscriptions of existing species or describing new species.

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HOW, THEN, CAN WE IDENTIFY SPECIES?

(plus complete morphological characterization) or rhizoids and holdfasts.

Once upon a time, all entomopathogens in this order were assigned to Entomophthora, Tarichium Cohn, or Massospora Peck. Species of Entomophthora in its broadest (and now abandoned sense) could often be identified merely by knowing the identity of the host. Such a simple procedure— albeit overlaid with geographical data for the fungi in question—is still useful as a convenient (if fallible) shortcut to identify a surprisingly large proportion of taxa. This may be noteworthy but is not a suggestion to return to a taxonomy recognizing separate species for morphologically similar fungi occurring on differing hosts. There is no agreement about precise limits for species definitions in the Entomophthorales. No rigid guidelines to delimit species can be so easily defined as for genera and families. Because the definition of any taxon is augmented by each new study of that organism, it may never be possible to codify exactly what a species concept must include or exclude. A broad agreement on an evolving set of “working criteria” to guide the circumscription of species in the Entomophthorales does seem possible. The following lists suggest many diverse characters from which a broad and judicious selection of data can support a responsible taxonomic decision process. These lists are intended to be suggestive rather than comprehensive:

Pathobiological characters: Host specificity (field and experimental data may be of equal value, but common sense is vital when using lab infection studies); details of infection processes; means by which host mortality occurs; relative timing of host death and/or sporulation with respect to lighVdark cycles, etc. Ecological characters: Geographical occurrence; seasonal occurrence; special environmental conditions (cold or heat; wetness or dryness; etc.); restriction to particular habitats (riparian, lotic, etc.). Molecular characters: Enzyme banding positions for particular enzyme systems; fatty acid spectra; genomic comparisons (DNA hybridization or thermal renaturation curves; restriction fragment length polymorphisms; direct genomic sequencing).

The adoption of molecular systematics techniques additions will assist greatly in the resolution of many problems in entomophthoralean systematics. Although such techniques will lead to more accurately predictive classifications, very few laboratories now have access to them. No matter what sorts of new techniques may be adopted in systematics laboratories, it is important to remember that the only way to assure that a classification remains readily useable by the vast majority of its users (who are not taxonomists) is to avoid basing the identifications of taxa primarily on molecular characters; taxonomic decisions based on molecular evidence should always be correlated with other (more traditional and more accessible) sorts of characters. A good classification, no matter what criteria may be used to construct it, must provide bases to identify species with high (if not absolute) accuracy from characters observable with no fancier equipment than dissecting and compound microscopes.

Conidial characters: Size and shape; number and size of (primary) conidial nuclei; mode of formation and discharge; and other consistent characters (e.g., the presence and number of oil droplets, color, surface decoration, presence of any distinctive adhesive device, etc.). All primary and secondary conidial forms should be characterized. Special care should be given to characterize any special “aquatic” spores (formed when conidiophores and/or conidia are submerged in water), and, if possible, germ conidia. Resting spore characters: Size and shape; color and/or surface decoration; mode development as zygospores, azygospores, or chiamydospores; number (and nature) of nuclei in mature propagules; mode of germination (direct or indirect; see Humber 1989).

REFERENCES Balazy, S. 1986. Taxonomic criteria for inter- and intra-specific differentiation in the Entomophthoraceae, exemplified by the subgenus Zoophthora. in Fundamental and applied aspects of invertebrate pathology (R.A. Samson, J. M. Vlak, and D. Peters, eds.), p. 201-205. Foundation 4th Internat. Colloq. Invertebr. Pathol., Wageningen.

Vegetative cell characters: Size, shape, walled or protoplastic state; the absence or presence (plus morphology) of cystidia; the absence or presence

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Batko, A. 1966. On the subgenera of the fungus genus Zoophthora Batko 1964 (Entomophthoraceae). Acta Mycol. 2: 15-21.

—, and N. Wilding. 1985. Entomophthora brevinucleata sp. nov. (Zygomycctcs, Entomophthoraceae), a pathogen of gall midges (Diptera, Cecidomyiidae). Entomophaga 30: 55-63.

Ben-Ze’ev, I.S., S. Keller, and A.B. McEwen. 1985. Entomophthora erupta and Entomophthora helvetica sp. nov. (Zygomycctes: Entomophthorales), two pathogens of Miridae (Heteroptera) distinguished by pathobiological and nuclear features. Canad. J. Bot. 63: 1469-1475.

King, D.S. 1976a. Systematics of Conidiobolus (Entomophthorales) using numerical taxonomy. I. Biology and cluster analysis. Can. J. Bot. 54: 45-65. —. 1976b. Systematics of Conidiobolus (Entomophthorales) using numerical taxonomy. II. Taxonomic considerations. Can. J. Bot. 54: 1285-1296.

—, and R.G. Kenneth. 1982a. Features-criteria of taxonomic value in the Entomophthorales: I. A revision of the Batkoan classification. Mycotaxon 14: 393-455.

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—, and —. 1982b. Features-criteria of taxonomic value in the Entomophthorales: 11. A revision of the genus Erynia Nowakowski 1881 (= Zoopbthora Batko 1964). Mycotaxon 14: 456-475.

Latgé, J.P., and D.G. Boucias. 1984. Intraspecific variability in Conidiobolus obscurus. J. Gen. Appl. Microbiol. 30: 135-150. Milner, R.J., R.J. Mahon, and W.V. Brown. 1983. A taxonomic study of the Erynia neoaphidis Remaudière & Hennebert (Zygomycctcs: Entomophthoraceae) group of insect pathogenic fungi, together with a description of the new species Erynia kondoiensis. Austral. J. Bot. 31: 173-188.

—, —, and A. Uziel. 1987. A reclassification of Entomophthora turbinata in Thaxterosporium gen. nov., Neozygitaceae fam. nov. (Zygomycetcs: Entomophthorales). Mycotaxon 28: 313-326. —, and Y. Zelig. 1984. Entomophthora israelensis sp. nov. (Zygomycetes: Entomophthoralcs), a fungal pathogen of gall midges (Diptera: Cccidomyiidae). Mycotaxon 21: 463-474.

Miura, Y., T. Nagai, and I. Ishiyama. 1983. Fatty acid and lipid compositions of Conidiobolus. J. Appl. Bacteriol. 54: 85-90.

Cochrane, B.J., J.K. Brown, R.P. Wain, B.G. Yangco, and D. Te Strake. 1989. Genetic studies in the genus Basidiobolus. I. Isozyme variation among isolates of human and natural populations. Mycologia 81: 504-513.

Remaudière, G., and G.L. Hennebert. 1980. Révision systématique de Entomophthora aphidis Hoffm. in Fres. Description de deux nouveaux pathogènes d'aphides. Mycotaxon 11: 269-321.

Hajek, A.E., R.A. Humber, J.S. Elkinton, B. May, S.R.A Walsh, and J.C. Silver. 1990. Panzootic of Japanese Entomophaga maimaiga in North American gypsy moth populations. [submitted to Science).

—, and S. Keller. 1980. Révision systématique des genres d'Entomophthoraceae à potentialité entomopathogène. Mycotaxon 11: 323-338. Soper, R.S., B. May, and B.J. Martinell. 1983. Entomophaga grylli enzyme polymorphism as a technique for pathotype identification. Envir. Entomol. 12: 720-723.

Humber, R.A. 1981. An alternative view of certain taxonomic criteria used in the Entornophthorales (Zygomycetes). Mycotaxon 13: 191-240.

—, M. Shimazu, R.A. Humber, M.E. Ramos, and A.E. Hajek. 1988. Isolation and characterization of Entomophaga maimaiga sp. nov., a fungal pathogen of gypsy moth, Lymantria dispar, from Japan. J. Invertebr. Pathol. 51: 229-241.

—. 1982. Strongwellsea vs. Erynia: the case for a phylogenetic classification of the Entomophthorales (Zygomycetes). Mycotaxon 15: 167-184. —. 1984a. The identity of Entomophaga species (Entomophthorales: Entomophthoraceae) attacking Lepidoptera. Mycotaxon 21: 265-272.

Speare, A.T., and R.H. Colley. 1912. The artificial use of the brown-tail fungus in Massachusetts, with practical suggestions for private experiment, and a brief note on a fungous disease of the gypsy caterpillar. Wright and Potter, Boston.

—. 1984b. Foundations for an evolutionary classification of the Entomophthorales (Zygomycetes). In Fungus/ Insect Relationships: Perspectives in Ecology and Evolution (Q. Wheeler and M. Blackwell, eds.), pp. 166-183. Columbia University Press, New York.

Tyrrell, D. 1967. The fatty acid compositions of 17 Entomophthora isolates. Can. J. Microbiol. 13: 75576o. —. 1968, The fatty acid compositon of some Entomophthoraceae. II. The occurrence of branchcd-chain fatty acids in Conidiobolus denaesporus Drechsl. Lipids 3: 368-372.

—. 1989. Synopsis of a revised classification for the Entomophthorales (Zygomycotina). Mycotaxon 34: 441-460.

—. 1971. The fatty acid composition of some Entomophthoraceae. III. Can. J. Microbiol. 17: 1115-1118.

—, and I. Ben-Ze’ev. 1981. Erynia (Zygomycetcs: Entomophthorales): emendation, synonymy, and transfers. Mycotaxon 13: 506-516.

Walsh, S.R.A, D. Tyrrell, R.A. Humber, and J.C. Silver. 1989. Use and development of DNA probes for the identification of Entomophaga isolates: Proc. Ann. Mtg. Mycol. Soc. Amer., Univ. of Toronto, Ontario. [abstr.]

Keller, S. 1984. Entomophthora muscae als Artenkomplex. Mitt. Schweiz. Entomol. Ges. 57: 131-132. —. 1987. Arthropod-pathogenic Entomophthorales of Switzerland. I. Conidiobolus, Entomophaga, and Entomophthora. Sydowia 40: 122-167.

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