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Phylogenetic analysis of ambrosial species in the genus Raffaelea based on 18S rDNA sequences
Mycol. Res. 102 (6) : 661–665 (1998)
661
Printed in Great Britain
Phylogenetic analysis of ambrosial species in the genus Raffaelea based on 18S rDNA sequences
K E V I N G. J O N E S A N D M E R E D I T H B L A C K W E L L Department of Plant Biology, Louisiana State University, Baton Rouge, LA 70803
Cladistic analysis of characters derived from nuclear-encoded 18S rDNA sequences was used to infer the phylogenetic placement of ambrosial species of the anamorph genus Raffaelea among perithecial ascomycetes. Of eight species in the genus investigated, seven resolve as a monophyletic lineage that forms a sister group to the sexual genus Ophiostoma. Raffaelea hennebertii, the single species excluded from this lineage, appears to be allied phylogenetically with species of Melanospora. These data are discussed in relation to the monophyly of Raffaelea and its relationship with other sexual and asexual fungal associates of wood-boring Coleoptera. In addition, we report the presence of significant length variation in PCR-amplified 18S rDNA fragments for three of the Raffaelea species studied.
The anamorph genus Raffaelea was introduced by Arx & Hennebert (1965) to accommodate the primary ambrosia fungus of the pinhole borer Platypus cylindricus Fab. Subsequent to the description of the type species, R. ambrosiae v. Arx & Henneb., nine additional species have been ascribed to the genus, seven of which are similarly ambrosial symbionts of mycetophagous wood-boring Coleoptera (Batra, 1967 ; Scott & Du Toit, 1970 ; Guerrero, 1966 ; Funk, 1970). Raffaelea is characterized by the formation of sporodochia in which the conidia develop singly at the tip of each conidiophore and successively at the tip of each growing point that arises immediately below the cicatricial scar of a fallen conidium (Arx & Hennebert, 1965 ; Batra, 1967). Beyond this common mode of conidium production, individual species vary considerably in pigment production (Batra, 1967), conidium shape and size (Sutton, 1975), yeast-like budding of conidia (Funk, 1970), production of rhizoidal hyphae at the base of the conidiophore (Scott & Du Toit, 1970), and production of aleuriospores (Arx & Hennebert, 1965 ; Funk, 1970). In addition, evidence for indeterminate growth of conidiophores has been noted for R. sulcati Funk (Funk, 1970), and catenate conidia have been recorded in R. santoroi Guerrero (Guerrero, 1966). This latter deviation led Sutton (1975) to suggest that R. santoroi may be better placed within the genus Sporothrix Hekt. & Perkins ex Nicot & Mariat. In the absence of known teleomorph stages, the monophyly of Raffaelea and its placement among sexual ascomycete taxa remain unclear. In these respects, Raffaelea presents analogous difficulties to those encountered previously with Ambrosiella Brader ex v. Arx & Henneb., an asexual genus whose component species were nominally grouped by their ambrosial association with coleopterans and a relatively plastic monilioid pattern of conidium production (Batra, 1967). Recently,
phylogenetic analysis of partial 18S rDNA sequences has demonstrated Ambrosiella to be polyphyletic with species related either to Ophiostoma H. & P. Sydow or to Ceratocystis Ellis & Halst. (Cassar & Blackwell, 1996). The efficacy of molecular data in defining monophyletic groups within Ambrosiella has prompted the present comparable study to address taxonomic questions relating to ambrosial species of Raffaelea. MATERIALS AND METHODS The type isolates of eight species of Raffaelea were included in this study (Table 1). Collectively, these taxa represent all described species within the genus isolated as mutualists of scolytid or platypodid beetles. Fungi were cultured in 2 % malt extract medium, harvested on filter paper, and snap-frozen in liquid nitrogen prior to DNA isolation. Total nucleic acids were extracted from frozen homogenized material following the procedure of Lee & Taylor (1990). For R. ambrosiae and R. sulcati, an additional purification step using caesium chloride (Yoon, Glawe & Shaw, 1991) proved necessary to obtain Table 1. Isolates of Raffaelea included in the present study Taxon Raffaelea Raffaelea Raffaelea Raffaelea Raffaelea Raffaelea Raffaelea Raffaelea
Source albimanens Scott & Du Toit ambrosiae v. Arx & Henneb. arxii Scott & Du Toit canadensis Batra hennebertii Scott & Du Toit santoroi Guerrero sulcati Funk tritirachium Batra
CBS 271.70 CBS 185.64 CBS 273.70 CBS 168.66 CBS 272.70 CBS 399.67 CBS 806.70 CBS 726.69
Phylogeny of Raffaelea based on 18S rDNA
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Table 2. List of taxa and GenBank Accession numbers used in this study Taxon
GenBank locus
Ambroziozyma platypodis (Baker & Kreger-van Rij) v. d. Walt Aspergillus fumigatus Fres. Balansia sclerotica (Pat.) Ho$ hnel Candida albicans (C. P. Robin) Berkhout Ceratocystis fimbriata Ellis & Halst. Ceratocystis virescens (Davidson) Mor. Chaetomium globosum Kunze : Fr. Claviceps paspali Stevens & Hall Daldinia concentrica (Bull. : Fres.) Ces. & de Not. Diaporthe phaseolarum (Cooke & Ellis) Sacc. Hypocrea schweinitzii (Fres.) Sacc. Hypomyces polyporinus Peck Leucostoma persoonii (Nitschke) Ho$ hnel Melanospora fallax Zukal. Melanospora zamiae Corda Microascus trigonosporus Emm. & Dodge Neurospora crassa Shear & Dodge Ophiostoma piliferum (Fr.) H. & P. Sydow Ophiostoma ulmi (Buisman) Nannf. Petriella setifera (Schmidt) Curzi Protomyces inouyei Henn. Talaromyces flavus (Klocker) Stolk & Samson Taphrina deformans (Berk.) Tul. Xylaria curta Fr.
L36984 M55626 U32399 X53497 U36987 U32419 U20355 U32401 U32402 L36985 L36986 U32410 M83259 U47842 U78356 L36987 X04971 U20377 U83261 U32421 D11377 M83262 L36988 U32417
template DNA suitable for use in polymerase chain reactions (PCRs). Using dilute aliquots of genomic DNA, approximately 1500 base pairs (bp) from the 5« end of the 18S rDNA were amplified by PCR (Saiki et al., 1988). Amplification products were verified by electrophoresis through 1 % agarose-TAE gels (Sambrook, Fritsch & Maniatis, 1989). The rate of migration relative to a 1 Kb DNA ladder (Gibco BRL) was used to estimate the sizes of amplification products (Sambrook et al., 1989). All taxa were initially amplified using primers NS1 and NS6 (White et al., 1990). Three isolates produced NS1–NS6 amplification products of anomalous sizes (see Results). Genomic DNA from these taxa was subsequently amplified using primes NS1 and NS4, and DNA from R. tritirachium and R. albimanens was additionally amplified using primes NS5 and NS6 (White et al., 1990). Predicted sizes of amplification products were based on the 18S rDNA sequence of Saccharomyces cerevisiae Meyen ex Hansen (Rubtsov et al., 1980). Direct sequencing of double-stranded PCR products using P$$ end-labelled primers NS1, NS2, NS3 and NS4 was performed as described previously ( Jones & Blackwell, 1996). About 900 nucleotide positions were obtained for each taxon. Raffaelea DNA sequences were aligned with those from the taxa listed in Table 2. The ingroup consists of 18 perithecial ascomycetes, Aspergillus fumigatus and Talaromyces flavus from the eurotialean plectomycetes, and two species of saccharomycetalean yeasts (Candida albicans and Ambroziozyma platypodis) ; the outgroup comprises the archiascomycetes Taphrina deformans and Protomyces inouyei. Maximum parsimony analysis was performed via heuristic search options in PAUP 3\1\1. (Swofford, 1993) using stepwise addition (random sequence addition) and branch swapping (tree bisection-reconnection) algorithms. Support for the inferred
clades within resulting trees was obtained by bootstrap analysis (Felsenstein, 1985) from 500 resamplings of the data set. Decay indices (Bremer, 1988) were estimated by examining the collapse of branches in consensus trees up to 10 steps longer than the most parsimonious. Tree stability in terms of supported resolution (¯ total support index, ti) was calculated as the sum of all branch decay indices (t) divided by the length of the most parsimonious trees, (s) (Bremer, 1994). Theoretical ti values can range from 0 in totally unresolved trees (no branch support, t ¯ 0) to 1 where there is a single most parsimonious tree, supported by all characters in the data (t ¯ s). Raffaelea DNA sequences generated during the present study have been submitted to GenBank (Accession Nos. U44474–U44481, inclusive). RESULTS 18S rDNA size variation Amplification of genomic DNAs with primers NS1 and NS6 at an annealing temperature of 53 °C revealed length variation in 18S rDNA fragments from the Raffaelea species (Fig. 1, lanes 7–14). Five taxa yielded DNA products of the predicted size of 1±46 Kb (Fig. 1, lanes 10–14). An additional smaller fragment amplified from R. ambrosiae and R. canadensis (Fig. 1, lanes 12 and 14) was abolished when amplifications were performed with primer annealing at 56°. For R. tritirachium, the NS1–NS6 product was approximately 600 bp larger than expected (Fig. 1, lane 9). Comparison of the NS1–NS4 (Fig. 1, lanes 3 and 4) and NS5–NS6 (Fig. 1, lanes 1 and 2) amplification products from R. albimanens and R. tritirachium demonstrated that the additional DNA sequence in the former taxon is located within the distal (3«) 300 bp of the NS1–NS6 18S rDNA fragment, and thus lies outside the region considered in the phylogenetic study. Two species, R. sulcati
M 1
2
3
4
5
6
7
8
9 10 11 12 13 14
M
Fig. 1. Agarose gel electrophoresis of PCR products derived by amplification of partial 18S rDNA sequences from R. albimanens (lanes 1, 3 and 13), R. ambrosiae (lane 12), R. arxii (lane 11), R. canadensis (lane 14), R. hennebertii (lane 10), R. tritirachium (lanes 2, 4 and 9), R. santoroi (lanes 6 and 8) and R. sulcati (lanes 5 and 7). Genomic DNA was amplified using primers NS5 and NS6 (lanes 1 and 2), NS1 and NS4 (lanes 3–6) or NS1 and NS6 (lanes 7–14). Fragment sizes were assessed relative to a 1 Kb DNA ladder marker (Lanes M).
K. G. Jones and M. Blackwell
663 73 Raffaelea sulcati Raffaelea tritirachium 1 73 2 Raffaelea albimanens
Raffaelea santoroi Raffaelea ambrosiae Raffaelea arxii 3 79 Raffaelea canadensis 2 100 Ophiostoma piliferum 56 >10 Ophiostoma ulmi 1 100 Diaporthe phaseolarum 9 Leucostoma persoonii 2 Chaetomium globosum 97 61 9 Neurospora crassa 1 53 Daldinia concentrica 2 Xylaria curta Balansia sclerotica 87 100 Claviceps paspali 2 >10 1 89 Hypomyces polyporinus Hypocrea schweinitzii 5 2 Melanospora zamiae 60 72 1 Raffaelea hennebertii 99 3 Melanospora fallax 95 7 8 100 Ceratocystis fimbriata 9 Ceratocystis virescens 100 Microascus trigonosporus 10 100 Petriella setifera 9 Aspergillus fumigatus 100 >10 Talaromyces flavus Ambrosiozyma platypodis 100 >10 Candida albicans Taphrina deformans Protomyces inouyei 56 93 1
Fig. 2. Strict consensus of 4 most parsimonious cladograms containing 32 taxa. T. deformans and P. inouyei are the designated outgroup taxa. Bootstrap replication frequencies above 50 % and decay indices are given above and below the nodes, respectively.
and R. santoroi yielded anomalous NS1–NS6 amplification products c. 400 bp smaller than anticipated (Fig. 1, lanes 7 and 8). Comparable amplifications at increased primer stringency (55° annealing) produced no detectable products. Both taxa revealed NS1–NS4 derived fragments of expected size (Fig. 1, lanes 5 and 6) ; these products were used as sequencing templates for subsequent phylogenetic analyses. Phylogenetic analyses The rDNA data set constructed for this study comprises 32 taxa and approximately 900 bp of DNA sequence for each isolate. Of the aligned nucleotide positions sampled, 209 are phylogenetically informative and were used to infer tree topologies. An heuristic search of the data produced four equally parsimonious trees of 509 steps with consistency (CI), retention (RI) and rescaled consistency (RC) indices of 0±552, 0±721 and 0±398, respectively. Because of the excessive computation time required to find all trees ten steps larger than the next most parsimonious, decay indices were estimated from a 9000 tree subgroup of all trees up to 10 steps greater that the most parsimonious solution. The undecayed nodes remaining in the consensus cladogram of these trees (denoted by the value " 10 in Fig. 2) were given a score of 10 for the summation of decay indices and calculation of the total support index (ti). Although the computed ti value of 0±257
will slightly underestimate the true total support, this figure still lies in the median range of values reported for molecularbased phylogenies (Bremer, 1994). In the strict consensus tree (Fig. 2) seven of the Raffaelea species resolve as a monophyletic group that received a bootstrap value of 93 % and a decay index of three steps. Within this clade, a number of infra-generic lineages of the species were discerned, although the support for these divisions is low based on both bootstrap and decay index values. This probably reflects the paucity of phylogenetic signal available from the use of a conserved genomic region in resolving relationships between closely allied taxa. The Raffaelea clade forms a sister group to the clade containing species of Ophiostoma, which thus represent the sexual taxa closest to Raffaelea of the species analysed. Both the bootstrap value (79 %) and the decay index (2 steps) lend only moderate support to this anamorph-teleomorph connection. A single species, R. hennebertii was excluded from the main group of Raffaelea species. Rather, this taxon resided in a clade containing two species of Melanospora (Fig. 2). The inclusion of R. hennebertii in the data set renders the genus Melanospora paraphyletic. When the two Melanospora species are excluded from the analysis, R. hennebertii remains as a sister taxon to the clade containing members of the Hypocreaceae and Clavicipitaceae (data not shown). The putative Ophiostomatalean clade, along with representatives from the Diaporthales (D. phaseolarum, L. persoonii), Sordariales (C. globosum, N. crassa) and Xylariales (D. concentrica, X. curta), forms one of two major clades which together contain all perithecial ascomycetes included in the analysis (Fig. 2). Ordinal-level relationships within this first group of perithecial taxa are poorly resolved in comparison to those of the second perithecial clade which comprises members of the Microascales, Melanosporales and Hypocreales (Fig. 2). Robust support was obtained for a monophyletic lineage of perithecial taxa distinct from the eurotialean plectomycetes and saccharomycetalean yeasts. The monophyly of the ingroup was maintained with respect to the outgroup (Fig. 2). DISCUSSION Amplification of partial 18S rDNA sequences from ambrosial species of Raffaelea revealed significant length variation in 18S ribosomal genes within the genus. For R. tritirachium this variation is manifested by the insertion of an additional 600 bp towards the 3« end of the 18S rDNA. Sequencing of the NS5-NS6 amplification product which harbours the extra sequences has revealed the presence of two insertions with characteristics of group I introns (Cech, 1988 ; unpublished data). Introns of this nature have previously been reported to punctuate the 18S rDNA of a number of ascomycete taxa (DePriest & Been, 1992 ; Gargas, DePriest & Taylor, 1995). For R. sulcati and R. santoroi the length variation is more cryptic since the apparent loss of 400 bp from the 18S rDNA would render the genes non-functional. One possibility is that the truncated products are due to primer-template mismatch coupled with disruption of the normal annealing site by an intron-type insertion event. The observed lack of amplification at higher annealing temperature and the clustering of R.
Phylogeny of Raffaelea based on 18S rDNA santoroi and R. sulcati with R. tritirachium in the phylogenetic analysis (Fig. 2) indirectly support this contention. It is uncertain whether the normal NS1-NS4 derived rDNA fragments from R. sulcati and R. santoroi are amplified from the same genomic templates as the NS1-NS6 PCR products. Thus the possibility remains that the latter products arise from DNA pseudogenes that effectively compete with full-length sequences for primer binding during amplification. The full alignment of nucleotide positions from the NS1-NS4 products of R. sulcati and R. santoroi with the comparable sequences from other Raffaelea species would validate the use of these rDNA fragments for subsequent phylogenetic analyses. Cladistic analysis of partial sequences of the 18S rDNA has shown ambrosial species of Raffaelea to be polyphyletic. The non-monopoly of the genus is due solely to the placement of R. hennebertii in a clade separate from the other seven species analysed. Although the present study has considered ambrosial species of Raffaelea, the available evidence does not suggest a close phylogenetic affinity between R. hennebertii and either of the two non-ambrosial taxa ascribed to the genus : preliminary 18S rDNA sequence data from the saprobic species R. variabilis Sutton (Sutton, 1975) supports placement of this taxon amongst the major group of ambrosial species (unpublished data). The taxonomic status of R. castellanii (Pinoy in Castell.) de Hoog, a causal agent of human sporocladiosis, has been questioned (Sutton, 1975). This species was considered by de Hoog (1974) to be a variety of Sporothrix schenckii Hekt. & Perkins, although the author also noted similarities with the conidial states of Pseudallescheria boydii (Shear) McGinnes et al., a member of the Microascales (Berbee & Taylor, 1992 b). The culture of R. hennebertii examined (CBS 272\70) fits the original description of the isolate (Scott & Du Toit, 1970) in gross morphological appearance, but we have not been able to observe sporulation in the culture. Verification of the polyphyly of Raffaelea will require that further isolates of R. hennebertii be collected and scrutinized. When R. hennebertii is excluded from consideration, Raffaelea resolves as a monophyletic lineage which forms a sister group to species of the sexual genus Ophiostoma. A proposed link between these genera is not without precedence ; in a synopsis of the Ophiostomatales, Arx & van der Walt (1987) placed Ophiostoma and Raffaelea within the family Ophiostomataceae on the basis of conidial characteristics. Raffaelea was included by Weijman & de Hoog (1975) in a rather homogenous group of genera with Graphium Corda-like conidial states, the members of which have been frequently (although far from exclusively) associated with species of Ophiostoma (Seifert & Okada, 1994). Similarly, the proposed accommodation of R. santoroi in Sporothrix (Sutton, 1975) also suggests a possible link with Ophiostoma (Berbee & Taylor, 1992 a ; de Hoog, 1994). The significance of wood-boring Coleoptera in the dispersal biology of Ophiostoma has long been recognized (reviewed in Malloch & Blackwell, 1994), and the evolution of the ambrosial habit from less intimate symbioses with species of Ophiostoma probably has occurred in the genus Ambrosiella (Cassar & Blackwell, 1996). The Ophiostoma-Raffaelea clade was present in all of the equally parsimonious trees generated from the data analysis,
664 and ordinal-level placement of Raffaelea within the Ophiostomatales would appear tenable from a molecular perspective in addition to the morphological and life-history considerations cited above. In the present analysis, the branch supporting the ophiostomatalean clade is comparatively short in relation to the branches which subtend the component genera (Fig. 2). This suggests that the Ophiostomatales have undergone rapid phyletic radiation, and a capacity for accelerated speciation within the order has indeed been documented (Brasier, 1987, 1991). Rapid radiation may make it difficult to collect the relevant synapomorphies that originally leant cohesion to the order, and this paucity of phylogenetic signal may contribute to the lack of robust support for the Raffaelea-Ophiostoma clade. Another consideration in this regard is the problem of taxon sampling. Previous phylogenetic studies of genera containing some strictly asexual species have shown that asexual taxa are dispersed among their sexual counterparts (LoBuglio, Pitt & Taylor, 1993 ; Geiser, Timberlake & Arnaud, 1996). Thus the resolution of distinct Raffaelea and Ophiostoma lineages in Fig. 2 may be interpreted as the absence in the data set of close sexual progenitors to the Raffaelea anamorphs. Incomplete taxon sampling is understandable given that Ophiostoma alone contains over eighty currently accepted species (Hausner, Reid & Klassen, 1993 ; Seifert, Wingfield & Kendrick, 1994), and we cannot preclude that some Raffaelea species are derived from alternative sexual ophiostomatalean genera. A more inclusive sampling from the Ophiostomatales, coupled with sequencing of a more variable gene region than the 18S rDNA should further resolve the evolutionary position of Raffaelea within the order. Although Raffaelea does not show the marked polyphyly seen in the ambrosial genus Ambrosiella (Cassar & Blackwell, 1996), some species within the latter genus resolve in an identical position to that observed here for the main Raffaelea lineage. The present study and that of Cassar and Blackwell (1996) utilise similar taxon sampling, but the nucleotide sites sampled are not entirely congruent for the two ambrosial genera, and conclusions as to their relationship would be premature. However, the patent morphological differences between Raffaelea and Ambrosiella may mask their close phylogenetic relationship and raise interesting questions about host beetle-mediated effects on patterns of conidium development. The authors thank Brandye Sawyer for technical assistance. This work was funded by a National Science Foundation Award (DEB-9208027) to M. Blackwell. REFERENCES Arx, J. A. von & Hennebert, G. . (1965). Deux champignons ambrosiae. Mycopathologia et Mycologia Applicata 25, 309–315. Arx, J. A. von & Van der Walt, J. P. (1987). Ophiostomatales and Endomycetales. In The Expanding Realm of Yeast-like Fungi (ed. G. S. de Hoog, M. T. Smith & A. C. M. Weijman), pp. 167–176. Elsevier : Amsterdam. Batra, L. R. (1967). Ambrosia fungi : a taxonomic revision and nutritional status of some species. Mycologia 59, 979–1017. Berbee, M. L. & Taylor, J. W. (1992 a). 18S ribosomal RNA gene sequence characters place the human pathogen Sporothrix schenckii in the genus Ophiostoma. Experimental Mycology 16, 87–91.
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661
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Phylogenetic analysis of ambrosial species in the genus Raffaelea based on 18S rDNA sequences
K E V I N G. J O N E S A N D M E R E D I T H B L A C K W E L L Department of Plant Biology, Louisiana State University, Baton Rouge, LA 70803
Cladistic analysis of characters derived from nuclear-encoded 18S rDNA sequences was used to infer the phylogenetic placement of ambrosial species of the anamorph genus Raffaelea among perithecial ascomycetes. Of eight species in the genus investigated, seven resolve as a monophyletic lineage that forms a sister group to the sexual genus Ophiostoma. Raffaelea hennebertii, the single species excluded from this lineage, appears to be allied phylogenetically with species of Melanospora. These data are discussed in relation to the monophyly of Raffaelea and its relationship with other sexual and asexual fungal associates of wood-boring Coleoptera. In addition, we report the presence of significant length variation in PCR-amplified 18S rDNA fragments for three of the Raffaelea species studied.
The anamorph genus Raffaelea was introduced by Arx & Hennebert (1965) to accommodate the primary ambrosia fungus of the pinhole borer Platypus cylindricus Fab. Subsequent to the description of the type species, R. ambrosiae v. Arx & Henneb., nine additional species have been ascribed to the genus, seven of which are similarly ambrosial symbionts of mycetophagous wood-boring Coleoptera (Batra, 1967 ; Scott & Du Toit, 1970 ; Guerrero, 1966 ; Funk, 1970). Raffaelea is characterized by the formation of sporodochia in which the conidia develop singly at the tip of each conidiophore and successively at the tip of each growing point that arises immediately below the cicatricial scar of a fallen conidium (Arx & Hennebert, 1965 ; Batra, 1967). Beyond this common mode of conidium production, individual species vary considerably in pigment production (Batra, 1967), conidium shape and size (Sutton, 1975), yeast-like budding of conidia (Funk, 1970), production of rhizoidal hyphae at the base of the conidiophore (Scott & Du Toit, 1970), and production of aleuriospores (Arx & Hennebert, 1965 ; Funk, 1970). In addition, evidence for indeterminate growth of conidiophores has been noted for R. sulcati Funk (Funk, 1970), and catenate conidia have been recorded in R. santoroi Guerrero (Guerrero, 1966). This latter deviation led Sutton (1975) to suggest that R. santoroi may be better placed within the genus Sporothrix Hekt. & Perkins ex Nicot & Mariat. In the absence of known teleomorph stages, the monophyly of Raffaelea and its placement among sexual ascomycete taxa remain unclear. In these respects, Raffaelea presents analogous difficulties to those encountered previously with Ambrosiella Brader ex v. Arx & Henneb., an asexual genus whose component species were nominally grouped by their ambrosial association with coleopterans and a relatively plastic monilioid pattern of conidium production (Batra, 1967). Recently,
phylogenetic analysis of partial 18S rDNA sequences has demonstrated Ambrosiella to be polyphyletic with species related either to Ophiostoma H. & P. Sydow or to Ceratocystis Ellis & Halst. (Cassar & Blackwell, 1996). The efficacy of molecular data in defining monophyletic groups within Ambrosiella has prompted the present comparable study to address taxonomic questions relating to ambrosial species of Raffaelea. MATERIALS AND METHODS The type isolates of eight species of Raffaelea were included in this study (Table 1). Collectively, these taxa represent all described species within the genus isolated as mutualists of scolytid or platypodid beetles. Fungi were cultured in 2 % malt extract medium, harvested on filter paper, and snap-frozen in liquid nitrogen prior to DNA isolation. Total nucleic acids were extracted from frozen homogenized material following the procedure of Lee & Taylor (1990). For R. ambrosiae and R. sulcati, an additional purification step using caesium chloride (Yoon, Glawe & Shaw, 1991) proved necessary to obtain Table 1. Isolates of Raffaelea included in the present study Taxon Raffaelea Raffaelea Raffaelea Raffaelea Raffaelea Raffaelea Raffaelea Raffaelea
Source albimanens Scott & Du Toit ambrosiae v. Arx & Henneb. arxii Scott & Du Toit canadensis Batra hennebertii Scott & Du Toit santoroi Guerrero sulcati Funk tritirachium Batra
CBS 271.70 CBS 185.64 CBS 273.70 CBS 168.66 CBS 272.70 CBS 399.67 CBS 806.70 CBS 726.69
Phylogeny of Raffaelea based on 18S rDNA
662
Table 2. List of taxa and GenBank Accession numbers used in this study Taxon
GenBank locus
Ambroziozyma platypodis (Baker & Kreger-van Rij) v. d. Walt Aspergillus fumigatus Fres. Balansia sclerotica (Pat.) Ho$ hnel Candida albicans (C. P. Robin) Berkhout Ceratocystis fimbriata Ellis & Halst. Ceratocystis virescens (Davidson) Mor. Chaetomium globosum Kunze : Fr. Claviceps paspali Stevens & Hall Daldinia concentrica (Bull. : Fres.) Ces. & de Not. Diaporthe phaseolarum (Cooke & Ellis) Sacc. Hypocrea schweinitzii (Fres.) Sacc. Hypomyces polyporinus Peck Leucostoma persoonii (Nitschke) Ho$ hnel Melanospora fallax Zukal. Melanospora zamiae Corda Microascus trigonosporus Emm. & Dodge Neurospora crassa Shear & Dodge Ophiostoma piliferum (Fr.) H. & P. Sydow Ophiostoma ulmi (Buisman) Nannf. Petriella setifera (Schmidt) Curzi Protomyces inouyei Henn. Talaromyces flavus (Klocker) Stolk & Samson Taphrina deformans (Berk.) Tul. Xylaria curta Fr.
L36984 M55626 U32399 X53497 U36987 U32419 U20355 U32401 U32402 L36985 L36986 U32410 M83259 U47842 U78356 L36987 X04971 U20377 U83261 U32421 D11377 M83262 L36988 U32417
template DNA suitable for use in polymerase chain reactions (PCRs). Using dilute aliquots of genomic DNA, approximately 1500 base pairs (bp) from the 5« end of the 18S rDNA were amplified by PCR (Saiki et al., 1988). Amplification products were verified by electrophoresis through 1 % agarose-TAE gels (Sambrook, Fritsch & Maniatis, 1989). The rate of migration relative to a 1 Kb DNA ladder (Gibco BRL) was used to estimate the sizes of amplification products (Sambrook et al., 1989). All taxa were initially amplified using primers NS1 and NS6 (White et al., 1990). Three isolates produced NS1–NS6 amplification products of anomalous sizes (see Results). Genomic DNA from these taxa was subsequently amplified using primes NS1 and NS4, and DNA from R. tritirachium and R. albimanens was additionally amplified using primes NS5 and NS6 (White et al., 1990). Predicted sizes of amplification products were based on the 18S rDNA sequence of Saccharomyces cerevisiae Meyen ex Hansen (Rubtsov et al., 1980). Direct sequencing of double-stranded PCR products using P$$ end-labelled primers NS1, NS2, NS3 and NS4 was performed as described previously ( Jones & Blackwell, 1996). About 900 nucleotide positions were obtained for each taxon. Raffaelea DNA sequences were aligned with those from the taxa listed in Table 2. The ingroup consists of 18 perithecial ascomycetes, Aspergillus fumigatus and Talaromyces flavus from the eurotialean plectomycetes, and two species of saccharomycetalean yeasts (Candida albicans and Ambroziozyma platypodis) ; the outgroup comprises the archiascomycetes Taphrina deformans and Protomyces inouyei. Maximum parsimony analysis was performed via heuristic search options in PAUP 3\1\1. (Swofford, 1993) using stepwise addition (random sequence addition) and branch swapping (tree bisection-reconnection) algorithms. Support for the inferred
clades within resulting trees was obtained by bootstrap analysis (Felsenstein, 1985) from 500 resamplings of the data set. Decay indices (Bremer, 1988) were estimated by examining the collapse of branches in consensus trees up to 10 steps longer than the most parsimonious. Tree stability in terms of supported resolution (¯ total support index, ti) was calculated as the sum of all branch decay indices (t) divided by the length of the most parsimonious trees, (s) (Bremer, 1994). Theoretical ti values can range from 0 in totally unresolved trees (no branch support, t ¯ 0) to 1 where there is a single most parsimonious tree, supported by all characters in the data (t ¯ s). Raffaelea DNA sequences generated during the present study have been submitted to GenBank (Accession Nos. U44474–U44481, inclusive). RESULTS 18S rDNA size variation Amplification of genomic DNAs with primers NS1 and NS6 at an annealing temperature of 53 °C revealed length variation in 18S rDNA fragments from the Raffaelea species (Fig. 1, lanes 7–14). Five taxa yielded DNA products of the predicted size of 1±46 Kb (Fig. 1, lanes 10–14). An additional smaller fragment amplified from R. ambrosiae and R. canadensis (Fig. 1, lanes 12 and 14) was abolished when amplifications were performed with primer annealing at 56°. For R. tritirachium, the NS1–NS6 product was approximately 600 bp larger than expected (Fig. 1, lane 9). Comparison of the NS1–NS4 (Fig. 1, lanes 3 and 4) and NS5–NS6 (Fig. 1, lanes 1 and 2) amplification products from R. albimanens and R. tritirachium demonstrated that the additional DNA sequence in the former taxon is located within the distal (3«) 300 bp of the NS1–NS6 18S rDNA fragment, and thus lies outside the region considered in the phylogenetic study. Two species, R. sulcati
M 1
2
3
4
5
6
7
8
9 10 11 12 13 14
M
Fig. 1. Agarose gel electrophoresis of PCR products derived by amplification of partial 18S rDNA sequences from R. albimanens (lanes 1, 3 and 13), R. ambrosiae (lane 12), R. arxii (lane 11), R. canadensis (lane 14), R. hennebertii (lane 10), R. tritirachium (lanes 2, 4 and 9), R. santoroi (lanes 6 and 8) and R. sulcati (lanes 5 and 7). Genomic DNA was amplified using primers NS5 and NS6 (lanes 1 and 2), NS1 and NS4 (lanes 3–6) or NS1 and NS6 (lanes 7–14). Fragment sizes were assessed relative to a 1 Kb DNA ladder marker (Lanes M).
K. G. Jones and M. Blackwell
663 73 Raffaelea sulcati Raffaelea tritirachium 1 73 2 Raffaelea albimanens
Raffaelea santoroi Raffaelea ambrosiae Raffaelea arxii 3 79 Raffaelea canadensis 2 100 Ophiostoma piliferum 56 >10 Ophiostoma ulmi 1 100 Diaporthe phaseolarum 9 Leucostoma persoonii 2 Chaetomium globosum 97 61 9 Neurospora crassa 1 53 Daldinia concentrica 2 Xylaria curta Balansia sclerotica 87 100 Claviceps paspali 2 >10 1 89 Hypomyces polyporinus Hypocrea schweinitzii 5 2 Melanospora zamiae 60 72 1 Raffaelea hennebertii 99 3 Melanospora fallax 95 7 8 100 Ceratocystis fimbriata 9 Ceratocystis virescens 100 Microascus trigonosporus 10 100 Petriella setifera 9 Aspergillus fumigatus 100 >10 Talaromyces flavus Ambrosiozyma platypodis 100 >10 Candida albicans Taphrina deformans Protomyces inouyei 56 93 1
Fig. 2. Strict consensus of 4 most parsimonious cladograms containing 32 taxa. T. deformans and P. inouyei are the designated outgroup taxa. Bootstrap replication frequencies above 50 % and decay indices are given above and below the nodes, respectively.
and R. santoroi yielded anomalous NS1–NS6 amplification products c. 400 bp smaller than anticipated (Fig. 1, lanes 7 and 8). Comparable amplifications at increased primer stringency (55° annealing) produced no detectable products. Both taxa revealed NS1–NS4 derived fragments of expected size (Fig. 1, lanes 5 and 6) ; these products were used as sequencing templates for subsequent phylogenetic analyses. Phylogenetic analyses The rDNA data set constructed for this study comprises 32 taxa and approximately 900 bp of DNA sequence for each isolate. Of the aligned nucleotide positions sampled, 209 are phylogenetically informative and were used to infer tree topologies. An heuristic search of the data produced four equally parsimonious trees of 509 steps with consistency (CI), retention (RI) and rescaled consistency (RC) indices of 0±552, 0±721 and 0±398, respectively. Because of the excessive computation time required to find all trees ten steps larger than the next most parsimonious, decay indices were estimated from a 9000 tree subgroup of all trees up to 10 steps greater that the most parsimonious solution. The undecayed nodes remaining in the consensus cladogram of these trees (denoted by the value " 10 in Fig. 2) were given a score of 10 for the summation of decay indices and calculation of the total support index (ti). Although the computed ti value of 0±257
will slightly underestimate the true total support, this figure still lies in the median range of values reported for molecularbased phylogenies (Bremer, 1994). In the strict consensus tree (Fig. 2) seven of the Raffaelea species resolve as a monophyletic group that received a bootstrap value of 93 % and a decay index of three steps. Within this clade, a number of infra-generic lineages of the species were discerned, although the support for these divisions is low based on both bootstrap and decay index values. This probably reflects the paucity of phylogenetic signal available from the use of a conserved genomic region in resolving relationships between closely allied taxa. The Raffaelea clade forms a sister group to the clade containing species of Ophiostoma, which thus represent the sexual taxa closest to Raffaelea of the species analysed. Both the bootstrap value (79 %) and the decay index (2 steps) lend only moderate support to this anamorph-teleomorph connection. A single species, R. hennebertii was excluded from the main group of Raffaelea species. Rather, this taxon resided in a clade containing two species of Melanospora (Fig. 2). The inclusion of R. hennebertii in the data set renders the genus Melanospora paraphyletic. When the two Melanospora species are excluded from the analysis, R. hennebertii remains as a sister taxon to the clade containing members of the Hypocreaceae and Clavicipitaceae (data not shown). The putative Ophiostomatalean clade, along with representatives from the Diaporthales (D. phaseolarum, L. persoonii), Sordariales (C. globosum, N. crassa) and Xylariales (D. concentrica, X. curta), forms one of two major clades which together contain all perithecial ascomycetes included in the analysis (Fig. 2). Ordinal-level relationships within this first group of perithecial taxa are poorly resolved in comparison to those of the second perithecial clade which comprises members of the Microascales, Melanosporales and Hypocreales (Fig. 2). Robust support was obtained for a monophyletic lineage of perithecial taxa distinct from the eurotialean plectomycetes and saccharomycetalean yeasts. The monophyly of the ingroup was maintained with respect to the outgroup (Fig. 2). DISCUSSION Amplification of partial 18S rDNA sequences from ambrosial species of Raffaelea revealed significant length variation in 18S ribosomal genes within the genus. For R. tritirachium this variation is manifested by the insertion of an additional 600 bp towards the 3« end of the 18S rDNA. Sequencing of the NS5-NS6 amplification product which harbours the extra sequences has revealed the presence of two insertions with characteristics of group I introns (Cech, 1988 ; unpublished data). Introns of this nature have previously been reported to punctuate the 18S rDNA of a number of ascomycete taxa (DePriest & Been, 1992 ; Gargas, DePriest & Taylor, 1995). For R. sulcati and R. santoroi the length variation is more cryptic since the apparent loss of 400 bp from the 18S rDNA would render the genes non-functional. One possibility is that the truncated products are due to primer-template mismatch coupled with disruption of the normal annealing site by an intron-type insertion event. The observed lack of amplification at higher annealing temperature and the clustering of R.
Phylogeny of Raffaelea based on 18S rDNA santoroi and R. sulcati with R. tritirachium in the phylogenetic analysis (Fig. 2) indirectly support this contention. It is uncertain whether the normal NS1-NS4 derived rDNA fragments from R. sulcati and R. santoroi are amplified from the same genomic templates as the NS1-NS6 PCR products. Thus the possibility remains that the latter products arise from DNA pseudogenes that effectively compete with full-length sequences for primer binding during amplification. The full alignment of nucleotide positions from the NS1-NS4 products of R. sulcati and R. santoroi with the comparable sequences from other Raffaelea species would validate the use of these rDNA fragments for subsequent phylogenetic analyses. Cladistic analysis of partial sequences of the 18S rDNA has shown ambrosial species of Raffaelea to be polyphyletic. The non-monopoly of the genus is due solely to the placement of R. hennebertii in a clade separate from the other seven species analysed. Although the present study has considered ambrosial species of Raffaelea, the available evidence does not suggest a close phylogenetic affinity between R. hennebertii and either of the two non-ambrosial taxa ascribed to the genus : preliminary 18S rDNA sequence data from the saprobic species R. variabilis Sutton (Sutton, 1975) supports placement of this taxon amongst the major group of ambrosial species (unpublished data). The taxonomic status of R. castellanii (Pinoy in Castell.) de Hoog, a causal agent of human sporocladiosis, has been questioned (Sutton, 1975). This species was considered by de Hoog (1974) to be a variety of Sporothrix schenckii Hekt. & Perkins, although the author also noted similarities with the conidial states of Pseudallescheria boydii (Shear) McGinnes et al., a member of the Microascales (Berbee & Taylor, 1992 b). The culture of R. hennebertii examined (CBS 272\70) fits the original description of the isolate (Scott & Du Toit, 1970) in gross morphological appearance, but we have not been able to observe sporulation in the culture. Verification of the polyphyly of Raffaelea will require that further isolates of R. hennebertii be collected and scrutinized. When R. hennebertii is excluded from consideration, Raffaelea resolves as a monophyletic lineage which forms a sister group to species of the sexual genus Ophiostoma. A proposed link between these genera is not without precedence ; in a synopsis of the Ophiostomatales, Arx & van der Walt (1987) placed Ophiostoma and Raffaelea within the family Ophiostomataceae on the basis of conidial characteristics. Raffaelea was included by Weijman & de Hoog (1975) in a rather homogenous group of genera with Graphium Corda-like conidial states, the members of which have been frequently (although far from exclusively) associated with species of Ophiostoma (Seifert & Okada, 1994). Similarly, the proposed accommodation of R. santoroi in Sporothrix (Sutton, 1975) also suggests a possible link with Ophiostoma (Berbee & Taylor, 1992 a ; de Hoog, 1994). The significance of wood-boring Coleoptera in the dispersal biology of Ophiostoma has long been recognized (reviewed in Malloch & Blackwell, 1994), and the evolution of the ambrosial habit from less intimate symbioses with species of Ophiostoma probably has occurred in the genus Ambrosiella (Cassar & Blackwell, 1996). The Ophiostoma-Raffaelea clade was present in all of the equally parsimonious trees generated from the data analysis,
664 and ordinal-level placement of Raffaelea within the Ophiostomatales would appear tenable from a molecular perspective in addition to the morphological and life-history considerations cited above. In the present analysis, the branch supporting the ophiostomatalean clade is comparatively short in relation to the branches which subtend the component genera (Fig. 2). This suggests that the Ophiostomatales have undergone rapid phyletic radiation, and a capacity for accelerated speciation within the order has indeed been documented (Brasier, 1987, 1991). Rapid radiation may make it difficult to collect the relevant synapomorphies that originally leant cohesion to the order, and this paucity of phylogenetic signal may contribute to the lack of robust support for the Raffaelea-Ophiostoma clade. Another consideration in this regard is the problem of taxon sampling. Previous phylogenetic studies of genera containing some strictly asexual species have shown that asexual taxa are dispersed among their sexual counterparts (LoBuglio, Pitt & Taylor, 1993 ; Geiser, Timberlake & Arnaud, 1996). Thus the resolution of distinct Raffaelea and Ophiostoma lineages in Fig. 2 may be interpreted as the absence in the data set of close sexual progenitors to the Raffaelea anamorphs. Incomplete taxon sampling is understandable given that Ophiostoma alone contains over eighty currently accepted species (Hausner, Reid & Klassen, 1993 ; Seifert, Wingfield & Kendrick, 1994), and we cannot preclude that some Raffaelea species are derived from alternative sexual ophiostomatalean genera. A more inclusive sampling from the Ophiostomatales, coupled with sequencing of a more variable gene region than the 18S rDNA should further resolve the evolutionary position of Raffaelea within the order. Although Raffaelea does not show the marked polyphyly seen in the ambrosial genus Ambrosiella (Cassar & Blackwell, 1996), some species within the latter genus resolve in an identical position to that observed here for the main Raffaelea lineage. The present study and that of Cassar and Blackwell (1996) utilise similar taxon sampling, but the nucleotide sites sampled are not entirely congruent for the two ambrosial genera, and conclusions as to their relationship would be premature. However, the patent morphological differences between Raffaelea and Ambrosiella may mask their close phylogenetic relationship and raise interesting questions about host beetle-mediated effects on patterns of conidium development. The authors thank Brandye Sawyer for technical assistance. This work was funded by a National Science Foundation Award (DEB-9208027) to M. Blackwell. REFERENCES Arx, J. A. von & Hennebert, G. . (1965). Deux champignons ambrosiae. Mycopathologia et Mycologia Applicata 25, 309–315. Arx, J. A. von & Van der Walt, J. P. (1987). Ophiostomatales and Endomycetales. In The Expanding Realm of Yeast-like Fungi (ed. G. S. de Hoog, M. T. Smith & A. C. M. Weijman), pp. 167–176. Elsevier : Amsterdam. Batra, L. R. (1967). Ambrosia fungi : a taxonomic revision and nutritional status of some species. Mycologia 59, 979–1017. Berbee, M. L. & Taylor, J. W. (1992 a). 18S ribosomal RNA gene sequence characters place the human pathogen Sporothrix schenckii in the genus Ophiostoma. Experimental Mycology 16, 87–91.
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