Studies in the Lasiosphaeriaceae. Monographs of two key genera and a family-level phylogeny
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Table of Contents: (Each of the major characters)
1) Ascoma
2) Asci
3) Hamathecium
4) Ascospores
5) Anamorph
6) Molecular characters
Morphological and developmental characters will be scored for all taxa
studied. The initial list of characters with multiple character states is
derived and amended from characters used in previous studies (Crane et al,
1992; Huhndorf, 1992c; Huhndorf et al 1990; Shearer et al 1993), to be refined
during the course of the project. Character by taxon matrixes will be made
available electronically on the Field Museum gopher and mosaic servers as
well as in the monographs.
1. Ascoma--The ascoma (fruiting body of the teleomorph reproductive phase, containing asci) (Fig. 4a,b) provides a number of characters that are useful in delimiting taxa at the family and genus level. Useful characters include the shape, size, position and wall structure of ascomata. Ascomata may possess a preformed, apical opening (ostiole) for spore dispersal (perithecioid ascoma) or may be entirely closed, opening by weathering or wall disintegration (cleistothecioid ascoma). In the Lasiosphaeriaceae the ascomata are mostly superficial on the substrate and they may be perithecioid or cleistothecioid. The wall may be composed of several layers of different types of cells of varying ontogeny and complexity with a two-layered wall of pseudoparenchymatous cells being at one extreme and a complex multi-layered wall structure composed of carbonaceous, fibrous, cartilaginous and pseudoparenchymatous layers at the other extreme . Both Chaetosphaeria and Lasiosphaeria have been described as having a two-layered wall structure but in most of the species this character has not been well documented.
Ascomatal vestiture (surface hairs, setae, etc.) has historically been used as a genus level character in Chaetosphaeria and Lasiosphaeria, however over the years species with various character states have been included. The development of the centrum (structures within the ascoma, i.e., asci and surrounding sterile elements) has been an important character at the ordinal level. The Sordariales is characterized by a certain centrum type consisting of pseudoparenchyma giving way to true paraphyses and asci produced in a basal layer even though the development of the centrum has been described for very few taxa in the group. Centrum development and wall structures will be elucidated using techniques established by the PI (Huhndorf and Glawe, 1990; Huhndorf, 1991; Crane et al, 1992).
2. Asci--The ascus, the cell where meiosis and ascospore production takes place, has been used as the diagnostic character for Subdivision Ascomycotina (Fig. 4a,c). Variation in ascus function and morphology is recognized from the class to the generic levels. In some well-defined families and genera the morphology of the ascus is very uniform (Xylaria, Hypoxylon, Xylariaceae) but the precise ascus morphology remain to be determined for many others, including the Lasiosphaeriaceae and especially Chaetosphaeria and Lasiosphaeria.
Asci vary in shape, in wall structure and in the structure of the apex and stipe. The ascus apex may contain a ring-like apparatus of varying complexity, a subapical globule beneath the ring characteristic for some members of the Lasiosphaeriaceae, or it may be without discernable structures. For many members of the family the morphology of the ascus apex is not known and the correlation of these structures with generic limits is incomplete.
In general, species of Chaetosphaeria have asci that are short stipitate and have an apical ring. In species described in Lasiosphaeria, Cercophora and Bombardia an apical ring and subapical globule may be present or not, and the asci may have a stipe or not. There has been no thorough study of the patterns of ascus morphology correlated with other morphological characters in these genera.
3. Hamathecium--The hamathecium (sterile tissues within the ascomal centrum interspersed between the asci) in this group has been generalized as being filiform and simple paraphyses, a reduced mass of interascal cells or lacking in some genera or species. The lack of paraphyses in the Nitschkiaceae is one character separating that family from the Lasiosphaeriaceae. In general these structures have been overlooked or not well documented in Chaetosphaeria and Lasiosphaeria with only their presence or absence being noted. Recent classification systems in the ascomycetes (Barr, 1987; Barr, 1990) have utilized this character at various taxonomic levels so that its thorough description for a species is warranted.
4. Ascospores--Ascospore (or meiospore) morphology (Fig. 4a,c) has historically been an important character in ascomycete classification and it still provides a number of criteria that are used in classification at the genus and species level. Ascospore color, shape and septation are diagnostic characters in general. In the Lasiosphaeriaceae, the types of gelatinous appendages and coverings are also important.
In Chaetosphaeria, species with either hyaline or brown, one or multi-septate ascospores have been included depending upon which interpretation of the genus is followed. In species described in Lasiosphaeria, the ascospore type varies from what could be considered typical: hyaline, cylindrical, geniculate, with or without appendages; to a long, filiform, multiseptate spore on one hand; and to a broad, allantoid, nonseptate spore with a gelatinous covering on the other (segregated as Lasiosphaeriella species in the latter extreme). In addition, the spore can delimit an apical cell which becomes pigmented (some species segregated as Cercophora) or the apical portion can swell but not become pigmented.
5. Anamorph--The anamorph is the reproductive phase in which the conidia (or mitospores) result from mitotic division (Fig. 4d,e,f). In this state the fungus is capable of producing enormous numbers of conidia rapidly. Characters of the anamorph useful in classification are the ontogeny and morphology of the conidium, the morphology of the spore-bearing structure, conidiophore, and the sporulating structure, conidioma. In practice, the anamorph is more readily obtained in colonies derived from cultures of the teleomorph than vice versa. Anamorphs of different morphologies can be connected to seemingly identical teleomorphs (Gams and Holubova-Jechova, 1976) or a single teleomorph may produce multiple anamorphs (synanamorphs) (Huhndorf, 1992a; Müller and Samuels, 1982a,b; Samuels pers. comm.). Pure-culture studies are essential to the study of the life cycles of ascomycetes. Cultural studies give additional characters, such as characteristics of colony mats formed in culture dishes, that can be diagnostic in themselves.
Anamorphs are poorly known in the Lasiosphaeriaceae, but most are reported to be hyphomycetous, forming their conidiophores on the substrate surface, never under the shelter of a protective integument. Species of Chaetosphaeria produce phialides (a type of conidiophore forming a succession of conidia) on masses of hyphae that grow out from germinating ascospores (Fig. 4d,f). Species of Lasiosphaeria and Cercophora have ascospores that germinate directly by forming phialides (Fig. 4e).
All of these characters must be observed and taken into account when delimiting taxa in these groups. Although taxa have been segregated from Lasiosphaeria based on single characters, the correlation to other characters and the implication in terms of relationships has often not been discussed. Increasingly, the characteristics of the anamorph are being correlated with the morphology of the teleomorphs, with ascomycetes having similar ascomatal morphology tending to have morphologically similar anamorphs. Thus, one can begin to extrapolate on the kind of anamorph likely to belong to an ascomycete, and also about the relationship of a conidial fungus to other anamorphs whose teleomorphs are known (Rogerson and Samuels, 1985) .
Generation of molecular characters
The DNA sequencing portion of the project is aimed at collecting data for
two levels of phylogenetic analyses; a) resolving phylogenetic relationships
among genera and families of the Sordariales with special emphasis on taxa
in the Lasiosphaeriaceae, and b) developing species level phylogenies for
Lasiosphaeria and Chaetosphaeria.
Complete or partial sequences from the nuclear small-subunit ribosomal DNA
gene (= ssrDNA) have proved appropriate for family and ordinal level analyses
within the Ascomycetes (e.g., White et al., 1990; Bruns et al., 1991;
Kohn, 1992; Spatafora and Blackwell, 1994; Berbee and Taylor, 1994). Spatafora
and Blackwell (1994) were able to resolve the four genera representing four
families in the Sordariales included in their broad survey of ascomycete
phylogeny using a 900bp fragment of ssrDNA (Fig. 3). We plan to sequence
the same portion of the ssrDNA used by Spatafora and Blackwell (1994) (also
Spatafora et al, in prep.). Because this gene has been used extensively
for phylogenetic studies, sequences are available in Genbank for many of
the outgroup taxa that we intend to employ in our analyses (see below).
If this gene proves too conservative or inconclusive, additional data will
be obtained from the less conserved nuclear large-subunit ribosomal DNA
(= lsrDNA) gene (e.g., White et al., 1990; Bruns et al., 1991; Kohn, 1992;
Hopple and Vilgalys, 1994). We would sequence 1000bp of this gene starting
at the 5' end. This region of the gene contains three divergent domains
(relatively fast evolving regions) and there would be ample sequence divergence
between taxa in these regions.
Species level comparisons will be made using sequence data from the internal
transcribed spacer region (= ITS) of nuclear rDNA and, if warranted, the
divergent domain regions of the lsrDNA. The ITS region contains two spacer
regions each ± 300 bp long and the 5.8S rDNA that is subtended by
the spacers. Except for the non-transcribed spacer region that occurs between
the lsrDNA and ssrDNA repeats, the ITS is the most variable part of the
rDNA tandem repeat. The non-transcribed spacer has not proved useful for
species level studies because of numerous insertions and deletions, and
high intraspecific variation (Hibbett, 1992). Most species level phylogenies
within the fungi have been based on ITS and/or partial lsrDNA (e.g., Bruns
et al., 1991; Kohn, 1992; Bunyard et al., 1993; Carbone and Kohn, 1993;
Moncalvo et al., 1993; Zambino and Szabo, 1993; Chapela et al., 1994; Hopple
and Vilgalys, 1994; Mueller and Pine, 1994 and in prep.; Lutzoni and Vilgalys,
1995 and in prep. ).
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