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Experimentelle Phykologie und Sammlung für Algenkulturen, Albrecht-von-Haller-Institut, Universität Göttingen, Untere Karspüle 2, 37073 Göttingen, Germany
Gerhard Rambold
Lehrstuhl Pflanzensystematik, Universität Bayreuth, Universitätsstraße 30, D-95448 Bayreuth, Germany
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONTAXONOMYCONCLUSIONSLITERATURE CITED |
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The monophyletic origin of the ascomycete family Physciaceae, its position within the Lecanorales and the phylogenetic structure within the family were investigated using nuclear rDNA sequence analyses. The common origin of the Caliciaceae and Physciaceae as previously shown (Wedin et al 2000) was confirmed. Further it could be shown that the Caliciaceae are nested within the Physciaceae. A unique region in loop 37 of the SSU rRNA secondary structure model was identified, which characterizes the Physciaceae/Caliciaceae. The SSU rDNA sequence data did not support a particular relationship with any other Lecanoralean family. Analyses of ITS rDNA sequences revealed a bifurcation of the Physciaceae/Caliciaceae clade, which was found to be congruent with the distribution of certain morphological characters. The congruence with the ITS phylogeny demonstrated the phylogenetic significance of ascus type, hypothecium pigmentation, ascospore characters and excipulum type. Fine-structure details of ascospores and the structure of excipula were found to be important in the recognition of convergences in these traits. Other previously used characters, i.e., growth habit, certain ascospore types or structure of the upper cortex, were found to be of multiple origins within the Physciaceae. All monophyletic lineages of noncrustose growth habit exhibit uniform ascospore types, indicating a higher evolutionary age of ascospore types than foliose growth habit. The taxonomic segregation of the Physciaceae into the Physciaceae and Caliciaceae is proposed here.
Key words: acetate-polymalonate pathway, ascospore type, ascus type, atranorin, Bayesian analysis, Caliciaceae, excipulum type, growth habit, hypothecium pigmentation, internal transcribed spacer, Lecanorales, lichenized ascomycetes, molecular phylogeny, rRNA, SSU, systematics
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONTAXONOMYCONCLUSIONSLITERATURE CITED |
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, in its currently accepted circumscription, comprises 27 genera of lichenized ascomycetes of various growth forms (Eriksson et al 2003), i.e., crustose, foliose and fruticose lichens. Zahlbruckner placed these taxa into two separate families, the Buelliaceae Zahlbr. (1907) and the Physciaceae Zahlbr. (1898). Poelt (1973) unified both families into the Physciaceae, and this concept essentially has remained unchanged until now. However, Poelt kept Dermatiscum Nyl. in a separate family and placed it together with the Physciaceae, Candelariaceae Hakul. and Teloschistaceae Zahlbr. in the suborder Buelliineae. Henssen and Jahns (1974) established the suborder Physciineae with the Physciaceae as the only family, essentially comprising the same genera as in the Physciaceae of Poelt, however, they included Dermatiscum as well. Hafellner et al (1979) published a survey on the Physciaceae, adding the genus Dermiscellum Hafellner, H. Mayrhofer & Poelt to the family. Rambold and Triebel (1992) regarded asci with an amyloid tholus enclosing a less amyloid axial body as a diagnostic feature for determining suborders and thus placed the Physciaceae in the suborder Lecanorineae. This view is kept until now, as documented in the most recent "Outline of Ascomycota" (Eriksson et al 2003). Molecular support for a common origin of the Physciaceae and the Caliciaceae first was presented by Wedin et al (2000), who included two Physciacean genera of different growth forms and four genera of the Caliciaceae in their survey. This finding was unexpected because the Caliciaceae were considered to belong to a different order of Ascomycetes, the Caliciales. Relationships of the Physciaceae to other ascomycete families previously were considered only within the Lecanorales, e.g., to the Lecanoraceae, to the Teloschistaceae and to the Candelariaceae.
The circumscription of the Physciaceae currently is based on ascus and ascospore types. Asci of the Lecanora-type (
Physcia-type) or the Bacidia-type (Buellia-type Lecidella-type Biatora-type) (Bellemère and Letrouit-Galinou 1981, 1987; Hafellner 1984) and pigmented, septate, thick-walled ascospores, categorized into 20 types (Mayrhofer 1982, 1984; but see Matzer and Mayrhofer 1996) are the essential character states for the recognition of this family.
Before the unification of the families Buelliaceae and Physciaceae by Poelt (1973), these two were distinguished by growth habit, which demonstrates the importance given to this character in the past. The Buelliaceae comprised crustose taxa, while foliose taxa were assigned to the Physciaceae. In addition, the excipulum type was regarded as an important trait and used to separate the genera Buellia and Rinodina (Zahlbruckner 1926), which constituted the Buelliaceae. Species of Rinodina develop an apothecial margin with algal cells (thalline excipulum), but they are absent in proper excipula of Buellia species. When not employing growth form as primary phylogenetic trait (e.g., Poelt 1973), the delimitation of Rinodina becomes uncertain. In addition, the remarkable diversity in ascospore types described in this genus (Mayrhofer 1982, 1984, Matzer and Mayrhofer 1996) might suggest that Rinodina in its current circumscription is not a monophyletic genus. In fact, ITS rDNA studies revealed that Rinodina in its actual concept represents a paraphyletic assemblage comprising the closest relatives of most foliose genera of the Physciaceae (Grube and Arup 2001). Both genera, Buellia and Rinodina, remained large and diverse assemblages even after the segregation of the genera Phaeorrhiza, Rinodinella, Mobergia, Hafellia, Amandinea, Diploicia and Diplotomma s. str. and were subject of recent investigations (e.g., Nordin 2000, Grube and Arup 2001).
In our study, we were able to obtain SSU and/or ITS rDNA sequence data of 23 of the 27 genera as listed in Eriksson et al (2003). These data proved to be particularly useful in the understanding of phylogenetic relationships among these genera as well as in testing the phylogenetic significance of various morphological characters.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONTAXONOMYCONCLUSIONSLITERATURE CITED |
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could not be included in this work because no material suitable for PCR amplification was available.
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. A set of mycobiont specific 5'-PCR primers was used in combination with nonspecific 3'-PCR primers (Table II). PCR was performed with 1 µl of the dissolved total lichen DNA in a 50 µl reaction volume containing a reaction mix of 0.2 mM of each of the four dNTPs, 2 mM MgCl2, 4% DMSO, 0.2 µM of each PCR primer, and 1 unit SilverStar Taq-polymerase with the supplier's buffer (Eurogentec). After an initial denaturing step at 95 C for 5 min, 33 cycles of denaturing at 94 C for 40 s, annealing at 51 C for 30 s with a time increment of 2 s per cycle, and extension at 72 C for 120 s with a time increment of 2 s per cycle, were performed and followed by a final extension at 72 C for 10 min. PCR products were cleaned by an alcohol precipitation and sequenced in both directions with a set of sequencing primers as listed in Table II. However, when PCR yielded double bands, each band was extracted from an agarose gel and cloned with the pGEM-T Vector-System I (Promega) and competent Escherichia coli cells, strain DH5
. The insert subsequently was amplified from transformant colonies with M13 primers and sequenced directly. For cycle sequencing reactions, IR-700 and IR-800 labeled primers were used with the Thermo Sequenase Sequencing kit with 7-deaza-dGTP (Amersham). Reactions were run on a LI-COR NEN Global IR2 sequencer, and sequences were assembled with AlignIR version 1.2 software (LI-COR).
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) and the resulting alignments were refined manually with BioEdit (Hall 1999). Only nearly full-length SSU rDNA sequences (
1600 bp) were included, constituting an alignment of 67 taxa. SSU sequences were aligned to the insertion-free SSU sequence AF088254 from Xanthoria elegans to detect and remove SSU introns. Positions that were conserved or comprised only one base change were excluded before phylogenetic analyses, as were ambiguously aligned positions. The resulting SSU data matrix then contained 293 variable positions of which 273 were parsimony informative. A model of sequence evolution that fit this dataset best was selected with the hierarchical likelihood-ratio test (hLRT) under the Akaike information criterion (AIC) as implemented in the program Modeltest (version 3.06, Posada et al 1998). For the SSU rDNA dataset, the "TVM+G" model (Rodríguez et al 1990) was selected, and likelihood parameters (Lset) were set as follows: Base frequencies (Base) were A = 0.2363, C = 0.2786, G = 0.1902 T = 0.2950, substitution types (Nst) = 6 with substitution values (Rmat) AC = 0.9678, AG = 2.6625, AT = 0.8204, CG = 0.3888, CT = 2.6625, GT = 1.0000; the site-to-site rate heterogeneity was modeled as a gamma distribution (Yang 1994) with a shape parameter = 1.9369, resolved with four rate classes. The proportion of invariable sites was set to zero (Pinvar = 0). This model was employed in a maximum-likelihood search. Tree search was started with a neighbor-joining tree and subsequent heuristic search using the tree bisection-reconnection (TBR) branch-swapping algorithm. All other parameters were as the default settings of PAUP. Further, maximum-parsimony (MP) and distance analyses were conducted. In maximum-parsimony analyses, the sites were weighted (rescaled consistency index [RC] over an interval of 1–1000 (Bhattacharya and Medlin 1995) and then used as input for bootstrap analyses (1000 replications). Introduced gaps were treated as fifth character state in the MP analyses. Heuristic search conditions were with starting trees built stepwise with 10 random additions of taxa, using the TBR algorithm. Best scoring trees were held at each step. For distance analyses, the neighbor-joining method (NJ, Saitou and Nei 1987) and the Jukes-Cantor model was used. Bootstrap analyses were conducted with 1000 replicates. This simple model was employed as a methodical counterbalance to the more complex models and to test the robustness of the analyses. For obtaining likelihood-based branch support values, Bayesian analysis and Markov chain Monte Carlo tree search algorithms as implemented in MrBayes, version 3.01b (Huelsenbeck and Ronquist 2001) were employed. A six-parameter model of evolution was specified, the exact parameter values being estimated during tree search. The rate variation of nucleotide substitution was modeled by a gamma distribution approximated by six rate categories. One million generations were calculated, of which every 100th was saved. Four differently heated chains were run, corresponding to the default setting of MrBayes 3.01b. The first 2000 saved trees were discarded before constructing a majority rule consensus tree from the remaining 8000 trees. Branch lengths were averaged across all 8000 trees, from which the consensus was calculated. Branch supports of the consensus tree, referred to as posterior probabilities, correspond to the fraction of the 8000 trees that contained the supported nodes and were regarded as significant when exceeding 0.95 (Larget and Simon 1999).
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) was selected using Modeltest with Lset parameters Base = equal; Nst = 6; Rmat = (2.1951, 3.9535, 2.3711, 1.3580, 9.5189, 1.0000); Rates = gamma; Shape = 1.4215; Ncat = 4; Pinvar = 0. For the reduced ITS dataset, the same alignment positions as in the extended dataset were analyzed. Again, the "SYM+G" model was selected with Lset parameters Base = equal; Nst = 6; Rmat = (3.0820, 4.8120, 3.3231, 2.0149, 11.9643, 1.0000); Rates = gamma; Shape = 1.5530; Ncat = 4; Pinvar = 0. ITS phylogenies were inferred with the methods as for the SSU sequence data. Alternative tree topologies for the ITS phylogeny corresponding to hypotheses as suggested by phenotypic characters were examined. For this purpose the ML phylogeny of the reduced dataset (as shown in Fig. 2) was manipulated manually in TreeView (version 1.5.3, Page 1996) and tested with the approximately unbiased test and the Kishino-Hasegawa test (Kishino and Hasegawa 1989) as implemented in CONSEL (version 0.1f, Shimodaira and Hasegawa 2001). Both tests carried out in CONSEL were based on the ML criterion, while the KH-test based on the MP criterion was calculated in PAUP.
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) as implemented in the alignment editor Align (Hepperle 2000). When rooting the ingroup with a suitable outgroup, the tRASA value had to increase compared to the unrooted ingroup. In addition, the outgroup should not appear extraneous to the ingroup in an unrooted RASA plot (Lyons-Weiler et al 1998).
To identify morphological characters that correlated with the ITS phylogeny, the obtained topologies were compared with phenotypic data summarized in Nordin and Mattson (2001), Scheidegger et al (2001) and citations therein. Further, Purvis et al (1992) and the genera dataset of LIAS (Rambold and Triebel 1996–2002) were consulted. Descriptive data of species were taken from Awasthi (1975), Esslinger (1986), Giralt (2001), Hale and Vobis (1978), Kalb (1987), Kashiwadani (1975), Matzer et al (1997), Mayrhofer (1984), Mayrhofer, Sheard and Matzer (1992), Moberg (1977, 1987), Moberg and Nash, (1999), Moberg and Purvis (1997), Nimis and Tretiach (1997), Nordin (2000), Scheidegger (1993), Sérusiaux and Wessels (1984), Scheidegger (1993), Sheard (1992), Swinscow and Krog (1976, 1988). Assignment of taxonomic categories followed the nomenclature as given in Myconet (Eriksson et al 2003), except when otherwise noted.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONTAXONOMYCONCLUSIONSLITERATURE CITED |
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Apart from the Caliciaceae-Physciaceae clade, the families Parmeliaceae and Sphaerophoraceae, and the Cladoniaceae-Stereocaulaceae clade were well supported. Support for most other families of the Lecanorales (e.g., the Teloschistaceae) was insignificant. No more than 216 parsimony-informative positions were in the SSU alignment for the Lecanoralean clade. Consistent separation of the Caliciaceae-Physciaceae clade from other members of the Lecanorales was based on only 12 positions (Table IV). Within the Caliciaceae-Physciaceae clade, the resolution of many relationships was ambiguous. Among the sequences from this clade, 193 sites were variable, of which 98 were parsimony informative.
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Two adjacent complementary 4-base stretches (position 1220–1223 and 1260–1263 of the reference sequence AF088254 from Xanthoria elegans) were found to be unique for the Caliciaceae-Physciaceae (Table IV). They form the base of hairpin loop 37 in the secondary structure model of the eukaryotic SSU rDNA of Wuyts et al (2002). These sequence stretches were used to construct PCR primers specific for Caliciaceae and Physciaceae (Phy 1200 F, Phy 1200a R and Phy 1200b R; Table II) to discriminate against all other fungal families, e.g., common contaminant lichenicolous fungi growing on Physciacean specimens. In addition, these primers were especially helpful for crustose taxa that grew in contact with other crustose lichens of different taxonomic groups.
ITS rDNA analyses – In ITS rDNA phylogeny, the monophyly of lineages found in the SSU analyses could be confirmed and further refined. Members of the Physciaceae were clearly separated into two major clades, A and B, and each of these clades was divided further into two subclades (subclades I–IV, Figs. 2, 3). Monophyly of both major clades was well resolved, as was the monophyly of three of the four subclades. Relationships within the subclades, however, were ambiguous in most cases. Because no ITS sequences for Caliciacean taxa were available, the relationship of the Caliciaceae to putative relatives in the Physciaceae could not be tested further. Subclade I (Fig. 2) consisted of the genera Heterodermia, Mobergia, Physcia, Tornabea and Rinodina p.p. Several other species of Rinodina, Buellia lindingeri, and members of the genera Anaptychia, Hyperphyscia, Phaeophyscia, Phaeorrhiza, Physconia and Rinodinella formed Subclade II. Subclade III comprised the genera Diploicia, Diplotomma, Dirinaria and Pyxine. With the exception of B. lindingeri, the polyphyletic genus Buellia was found to be restricted to Subclade IV, which also included the genera Amandinea, Australiaena, Dermatiscum, Dimelaena, Hafellia and Santessonia. Monophyletic origin for this subclade was consistently low in the various analyses (Bayesian: 76%, NJ: 17%, MP: 18%).
To examine the monophyly of certain Physciacean genera, additional taxa not represented in Fig. 2 were included in the ITS rDNA analyses (Tables I, III, Fig. 3). With this enlarged dataset, the monophyly of the genera Physcia, Physconia, Heterodermia, Dirinaria and Pyxine could be supported and the grouping of Physciella chloantha with Phaeophyscia was well resolved (Subclade II, Fig. 3). Anaptychia was found to be potentially polyphyletic. A. runcinata and A. bryorum were placed together with Physconia spp. in one lineage, but A. ciliaris and A. ulotrichoides were distinct from it (Subclade II, Fig. 3). The genera Buellia and Rinodina were clearly polyphyletic in the ITS rDNA analyses.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONTAXONOMYCONCLUSIONSLITERATURE CITED |
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SSU rDNA phylogeny –
The proportion of parsimony-informative positions within the Lecanoralean SSU rDNA alignment was comparatively small. Further, a large proportion of these positions appeared to be quite variable and resulted in a high content of homoplasy, especially when distantly related taxa were compared. In addition, the number of informative positions supporting a single node in a cladogram was in a similar magnitude as that of presumable sequence errors or point mutations that were not deleted by selection. An unfavorable relation of phylogenetic signal and "background noise" (nucleotide substitutions that do not reflect relatedness of taxa), as well as the large number of uninformative sites, might obscure bootstrap analyses, the most widespread method for evaluating reliability of phylogenetic reconstructions (Zharkikh and Li 1992, Hillis and Bull 1993). The effect may be clearly seen in recently published Euascomycete SSU rDNA phylogenies in which basal nodes are all without support (Stenroos et al 1998, Wedin et al 2000, Lutzoni et al 2001). In contrast to bootstrap analyses that ignore a considerable fraction of alignment positions for each bootstrap replicate, Bayesian analysis makes use of all alignment positions during the process of significance measurement. Among other reasons, this increases the support particularly of basal branches. Further, this method revealed phylogenies that corresponded well to maximum-likelihood analyses but needed much less computing time. Therefore, this method of phylogenetic analysis was preferred over all other methods of phylogenetic reconstruction.
In addition to taxon sampling, the choice of outgroup might significantly influence tree topologies. This might explain apparent differences among the Lecanorales phylogeny shown here and phylogenies of other authors (e.g., Lutzoni et al 2001, Stenroos et al 1998, Wedin et al 2000). These authors used taxa outside the Euascomycetes as outgroups, such as Saccharomyces cerevisiae or even more distantly related taxa. However, the focus of this study was to clarify the position of the Physciaceae within the Lecanorales, and therefore S. cerevisiae was suspected to be too distantly related to the taxa under consideration and might have resulted in a too-high degree of homoplasy. Hence, Bulgaria inquinans (Helotiales) was chosen as outgroup for the Lecanoralean phylogeny.
Monophyly of a clade uniting Caliciaceae and Physciaceae –
Despite the lack of resolution for basal nodes in the Lecanoralean phylogeny, the Caliciaceae-Physciaceae clade was highly supported and could be unambiguously recognized by two adjacent complementary 4-base stretches forming the base of hairpin loop 37 of the SSU rDNA (Wuyts et al 2002). The uniqueness of these positions is assumed to originate from an ancient reciprocal strand exchange as can be deduced from the high similarity of the corresponding regions in other Lecanoralean taxa (Table IV). In addition to the monophyly of the Caliciaceae-Physciaceae clade, an affiliation of the Caliciaceae to a particular lineage within the Physciaceae is indicated by the SSU phylogeny. When compared to the ITS phylogeny, the Physciacean taxa closest related to the Caliciaceae are members of Subclade IV (Fig. 2). Both are connected by the presence of a true excipulum, Beltraminia-type ascospores and similar ascospore ontogeny and ornamentation, characters that are considered to be important phylogenetic traits in this lichen group (Nordin 2000, Wedin et al 2000).
According to Hafellner (1984), the coherence of the Caliciaceae-Physciaceae clade might be questioned because different ascus types or subtypes are observed in this clade: the prototunicate ascus in the Caliciaceae, the Bacidia-type ascus in Buellia and allied genera and the Lecanora-type ascus found in the taxa of Clade A. However, the latter two ascus types were shown to be variable (Ekman 1996, Thell et al 1995) and might closely resemble each other at certain ontogenetic stages. Therefore, it is assumed that both ascus types are related. The prototunicate ascus of the Caliciaceae, in fact, is not interpreted as an ascus type of its own but rather a reduction of an ancient nonprototunicate ascus type, as proposed by Wedin et al (2000). Compared to other ascus types within the Lecanorales that are well distinguished by amyloid structures such as tubes or caps (Porpidia-type, resp. Lecidea-type), the occurrence of different but structurally related ascus types is not regarded as a contradiction to the monophyly of the Caliciaceae-Physciaceae clade.
All three ascus types found in species of the Caliciaceae-Physciaceae clade also are found in families included in the sister clade of the Caliciaceae-Physciaceae clade (Fig. 1), which corroborates the sister-group relationship between these two clades. Further, it could be suspected that the Caliciaceae-Physciaceae clade might be more closely related to other Lecanoralean families with the same ascus types than to families with different ascus types. Potential relationships of the Caliciaceae-Physciaceae to other Lecanoralean families then would be to the Lecanoraceae, Parmeliaceae, Candelariaceae (with Lecanora-type asci) or Bacidiaceae and Ramalinaceae (both with Bacidia-type asci). A closer relationship of the Caliciaceae-Physciaceae clade to any of these Lecanoralean families than to families with different ascus types could not be validated or rejected on the basis of the presented Lecanoralean SSU rDNA phylogeny.
ITS-rDNA phylogeny –
To root Physciaceae ITS phylogenies, various taxa already have been used as outgroup (Grube et al 2001, Lohtander et al 2000). As seen in the SSU phylogeny (Fig. 1), the genus Lecidea appeared as the taxon closest to the root of the Physciaceae. When using Lecidea lapicida (AF 282124) as outgroup, the tRASA test statistic increased considerably. This effect was consistent, regardless of the portion of ambiguous positions removed from the alignment before phylogenetic inference. In addition, Lecidea lapicida did not appear as an extraneous element in an unrooted RASA plot.
The distinction of two major clades in the ITS rDNA phylogeny is congruent with the distribution of certain morphological character states (Fig. 2, Table V). The distribution of the ascus type, hymenium pigmentation, excipulum type and various ascospore types correlated significantly to the clades of the ITS rDNA phylogeny. However, no character could be detected that was absolutely free of homoplasy. Taxa that exhibit deviations from the typical character states of their assigned subclade are listed in Table VI. Taxa of Clade A typically are characterized by the Lecanora-type ascus, an unpigmented hypothecium, a thalline excipulum and ascospores with wall thickenings or Rinodinella-type ascospores. Pluriseptate ascospores were the exception in this clade. Members of Clade B typically have Bacidia-type asci, a pigmented hypothecium, a proper excipulum and usually ascospores without distinctive wall thickenings or Dirinaria-type ascospores. Pluriseptate ascospores are more frequent in Clade B than in Clade A and might have evolved several times here (Nordin 2000).
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) and Hafellner (1984) and used to delimit families of lichenized ascomycetes (e.g., Hafellner 1984). With respect to the Physciaceae, ascus structures and types already had been discussed by Rambold et al (1994). Our study revealed the ascus type as one of the most constant phylogenetic traits in the Physciaceae, corroborating the subdivision of this family as seen in the ITS phylogeny. This coincides with a survey in the Pezizaceae (Pezizomycetes), where ascus types also correlated well with rDNA phylogenies (Hansen et al 2001), supporting the phylogenetic significance of this trait. The only critical genus in this study is Australiaena, which inserts in Subclade IV but develops a Lecanora-type ascus. Further, a couple of Rinodina species develop a Bacidia-type ascus (Table VI). It is of interest to note that these Rinodina species exhibit additional character states that assign them to Clade B (Giralt 2001). Therefore, these taxa are not considered exceptions but in need of a revision. All Buellia-like genera established by Marbach (2000) are described with Bacidia-type asci, which indicates their relationship to taxa included in Clade B as described here. In other lichen families, the taxonomic significance of ascus structures and types have been controversially discussed due to the observation of ascus-type variation within families or even genera that were homogenous with respect to other morphological characters (Lumbsch et al 2001, Hafellner 1984, Rambold et al 1998a). In Buellia oidalea and B. oidaliella, both with extremely large spores, an axial body is expressed that closely resembles that of the Lecanora-type ascus (Nordin 2000). However, in most of the investigated Physciaceae, ascus type did not seem to correlate with ascospore size (data not shown) as observed in the Bacidiaceae (Ekman 1996).
Ascospore characters also delimited clades A and B from each other. In contrast to members of Clade B, taxa of Clade A usually develop ascospores with distinct wall thickenings, designated to about 16 different types (Mayrhofer 1982, 1984, Nordin 1997, but see Matzer and Mayrhofer 1996). Rinodinella takes an exceptional position in this aspect, because it develops ascospores with unusually thin ascospore walls without any thickenings (Rinodinella-type). Physcia-, Pachysporaria- and Polyblastidium-types occur together within well separated genera (e.g., Physcia, Phaeophyscia and Heterodermia, Mayrhofer 1982) and regularly form intermediates even within single species (e.g., Swinscow and Krog 1988). They therefore are considered as belonging to one major type in this study. This ascospore type is found in five lineages of Clade A, i.e., Heterodermia, Physcia and Rinodina atrocinerea, Mobergia and Rinodina confragosa (Subclade I), as well as in Buellia lindingeri, Phaeophyscia (including Physciella) and Hyperphyscia (Subclade II). Also the Physconia-ascospore type appears homoplasious. This ascospore type might have developed in Subclade II in the assumed common ancestor of Anaptychia and Physconia and in Subclade I in Tornabea. Phaeorrhiza might be an exception because it develops the Beltraminia ascospore type, which otherwise is typical for Clade B. However, Mayrhofer and Poelt (1978) described occasional thickenings of the ascospore. In Clade B, ascospores with wall thickenings evolved only in few cases (Table VI). Most common are the Buellia- and the Beltraminia-ascospore type and pluriseptate ascospores that do not exhibit distinct wall thickenings. Beltraminia- and Buellia-ascospore types, originally delimited by the presence/absence of a torus (Mayrhofer 1984), were not distinguished in this study, following Matzer and Mayrhofer (1996). The Beltraminia-ascospore type differs from the Rinodinella-type in that its ascospore walls are uniformly thickened. In the assumed common ancestor of Diploicia, Pyxine and Dirinaria the Dirinaria ascospore type with wall thickenings and Type-B ontogeny evolved. This spore type also is found in a group of Rinodina species in Clade A (Fig. 3), represented by the species R. gennarii, R. olea and R. nimisii. Notably, R. pyrina clustered within these species and develops Physconia-type ascospores. Further Rinodina species with type B ascospore ontogeny are known (Giralt 2001, Scheidegger 2001), whose phylogenetic position was not investigated. Marbach (2000) described Cratiria Marbach, Fluctua (Malme in J. Steiner) Marbach and Sculptolumina Marbach with pronounced ascospore wall thickenings. As mentioned above, major phylogenetic traits denote these three genera as members of Clade B. Considering their tropical origin, an affiliation with Subclade III might be suspected. Triseptate, submuriform (Diplotomma-type) or muriform ascospores seem to have evolved several times (Nordin 2000). Especially in the genus Diplotomma, the number of septa seems to be a variable trait.
Hypothecium pigmentation turned out to be a highly conserved phylogenetic trait and proved to be of similar phylogenetic significance as ascus type. Exceptions were recognized in few species of the genera Dimelaena and Rinodina (Giralt 2001, Sheard 1974, Mayrhofer et al 1996). Of note in this connection is Dimelaena oreina, generally described to have a hyaline hypothecium. Hafellner et al (1979) also mention specimens with dark hypothecium. In addition, the closest relative of the genus Dimelaena treated in this study, Dermatiscum thunbergii, retained the hypothecial pigmentation. All genera described by Marbach (2000) have a pigmented hypothecium, again strongly suggesting their relatedness to taxa of Clade B.
The distribution of thalline (Lecanorine) and proper (Lecideine, true) excipulum also was found to correspond to the bipartition of the Physciaceae. However, more exceptions were found in this trait than in the previously discussed characters. In Clade A, which is characterized by a thalline excipulum, Buellia lindingeri exhibits a proper excipulum. In addition, Phaeorrhiza sareptana (Tomin) H. Mayrhofer & Poelt, not included in this study (but see Grube and Arup 2001), develops a lecideine excipulum. For Rinodina species with lecideine excipulum and taxa assigned to Clade B with thalline excipulum, see Table VI. All genera described by Marbach (2000) exhibit true excipula. Excipulum type may be a less conservative character than the presence of hypothecium pigmentation, and therefore the genera Buellia and Rinodina may be more readily distinguished by this latter trait. In addition, hypothecium pigmentation is a character easy to recognize and apparently almost as well conserved as ascus type.
Upper cortex structure turned out to be quite variable and is not indicative of the relatedness of genera, as was initially implied by the former taxonomies, e.g., uniting species of the genera Anaptychia and Heterodermia or Physcia and Physconia (Zahlbruckner 1926). Poelt (1965) realized the insignificance of this trait and published a more refined system, which, in its coarse outline, has remained unchanged. The different structures of the upper cortex (elongated hyphae forming a prosoplectenchymatic cortex versus more or less isodiametric hyphae forming a paraplectenchymatic cortex) do not correlate with the delimitation of clades or subclades. In Clade A, both subclades contain both forms of upper cortex. In Subclade I, Physcia is characterized by a paraplectenchymatic cortex, in Subclade II, Physconia, Phaeophyscia and Hyperphyscia develop this cortex type. A prosoplectenchymatous upper cortex is found in Heterodermia and Tornabea (Subclade I) and Anaptychia (Subclade II). The genera Anaptychia and Physconia are separated by this character but intermix and are not resolved in phenotypic as well as genotypic surveys, as published in Nordin et al (2001) and Lohtander et al (2000). A closer look also reveals that there is still some variability within each cortex type. For instance, Moberg (1977) distinguished Physconia grisea from all other Physconia species, partly due to a different cortex structure. This points to a high intrageneric variability, which eventually might enable the reversal of this character state. Therefore, a separation of A. runcinata from Physconia, based on this character, might not depict natural relationships. In addition to these two genera, variation in the upper cortex structure seems to follow genus delimitations well.
The foliose growth habit seems to have evolved five times in Clade A. Hyperphyscia, Heterodermia, Physcia and Phaeophyscia appear as independent lineages, being separated by crustose Rinodina species. Anaptychia and Physconia form a single foliose lineage, signaling a closer relationship between these two genera than revealed for the other foliose genera in this clade. This assumption is confirmed further by the common ascospore type developed in both genera. The fruticose thallus of Tornabea, often regarded as closely related to Anaptychia (Mayrhofer 1982, Nordin and Mattson 2001), probably evolved independently. Therefore, the fruticose thallus does not represent an enhancement of a foliose growth habit but a developmental line of its own (Poelt 1965). In Clade B more highly organized growth forms evolved at least three times: once in Santessonia (fruticose), once in Dermatiscum (umbilicate) and once leading to the related genera Dirinaria and Pyxine (foliose). Both genera also are characterized by a common ascospore type. In addition, the umbilicate genus Dermiscellum is believed to belong to this clade. Multiple origins of noncrustose growth forms were postulated by Frey (1963) and Poelt (1965). Poelt assumed Rinodina and Buellia species as the closest relatives of each of these foliose/fruticose lineages. After asserting the evolutionary independence of these lineages with molecular data, it became evident that major ascospore types have remained constant during the radiation of these higher organized lineages, resulting in a remarkable uniformity of ascospore types in noncrustose lineages. This might suggest that the evolution of major ascospore types predates the origin of these foliose genera. Therefore, different major ascospore types (no intermediates or intraspecific transitions observed between these major ascospore types) might be considered as indicative of the delimitation of genera.
Secondary compounds derived from the acetate-polymalonate pathway delimit the foliose taxa of Subclade I from those of Subclade II (Nordin and Mattson 2001, Scheidegger et al 2001). Foliose taxa of Subclade I are characterized by the presence of secondary compounds, such as atranorin or norstictic acid, synthesized via the acetate-polymalonate pathway, while taxa of Subclade II lack such substances. Contradicting statements concerning Anaptychia ciliaris were found. Whereas Kurokawa (1973), followed by Hafellner et al (1979) and Nordin and Mattson (2001), found minor amounts of atranorin in this species, Scheidegger et al (2001) noted the absence of this substance. Rinodina oxydata was the only species found in Subclade II that synthesizes atranorin. Two lineages of Rinodina species in Subclade I, annotated with branch support numbers 9 and 14 in Fig. 3, lack secondary compounds. Also in Tornabea scutellifera, no secondary compounds were found. In addition, Tornabea scutellifera develops ascospores of the Physconia-type, which is typical of the Phaeorrhiza/Anaptychia/Physconia cluster in Subclade II. To evaluate these contradicting affiliations of phenotypic traits on one side and ITS sequence data on the other, the phylogenetic positions of R. sophodes and T. scutellifera were examined with the approximately unbiased test and the Kishino-Hasegawa test. Various constrains were set as shown in Table VII A–I. Under ML as well as MP settings, all tested alternative topologies were rejected at the 5% significance level.
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, Grube and Arup (2001) and a morphological survey by Nordin and Mattson (2001). A. runcinata shares adnate lobe tips, a lower cortex and hairs on the upper cortex with Physconia spp. and deviates in these characteristics from A. ciliaris (Nordin and Mattson 2001). In contrast, Anaptychia species, by definition, share the same upper cortex structure. Further, Helms et al (2001) reported photobiont ITS variants from A. ciliaris and A. runcinata that were closely related to each other but distantly related to all the studied Physconia photobionts. Therefore, mycobiont selectivity toward particular photobionts might be another trait that supports a delimitation of Anaptychia from Physconia species. In a ML-based Kishino-Hasegawa test and the approximately unbiased test as implemented in CONSEL (Shimodaira and Hasegawa 2001), the hypothesis that Anaptychia is either monophyletic or paraphyletic but basal to Physconia could not be rejected at the 5% significance level (Table VII J, K). Therefore, uncertainty remains about the phylogenetic relationship between these two genera. The genus Rinodina (type species: R. sophodes) was found to be a polyphyletic assemblage, which confirms the findings of Grube and Arup (2001). Species of Rinodina were found in at least six independent lineages of clade A. Further branches of Rinodina species might be found, because Rinodina is by far the largest genus in Clade A and therefore least well represented. In addition, none of the typically corticolous, lignicolous and blastidate Rinodina species were included in this study. Mayrhofer (1984) described 15 ascospore types in this genus demonstrating high diversity when compared with the foliose genera. Hyperphyscia, Tornabea and Mobergia appear to be small and well-delimited genera in which monophyly seems likely but cannot be discussed here because they were represented by only one species in this study. Monophyly of Phaeorrhiza nimbosa and P. sareptana already was shown by Grube and Arup (2001).
The delimitation of Subclade III and Subclade IV, based on phenotypic traits only, seems problematic. Diploicia, Dirinaria and Pyxine are delimited from all other investigated members of Clade B by their Dirinaria-type ascospores. However, Diplotomma s. str. has triseptate or Diplotomma-type (= submuriform) ascospores that also occur in Subclade IV. Species of Diplotomma s. str. are characterized by calcium oxalate in the thallus, often pruinose apothecia and a thick perispore that distinguishes them from most of the taxa of Subclade IV (Nordin 2000). A pruina, which consists of calcium oxalate, also is found in other members of Subclade III, which supports a closer relationship of these taxa. A close affiliation of Diplotomma species and Diploicia already had been shown by Molina et al (2002). However, both genera are well delimited by their ascospore-types as well as ITS sequences and therefore were confirmed as two distinct genera in this study. The conspecifity of species determined as Diplotomma epipolium and D. venustum could be confirmed here on the basis of ITS sequence comparisons. Both share a seven base-pair deletion in the most variable part of ITS-1, compared to other Diplotomma species. Because of the high variability of this part of ITS-1 no unambiguous alignment with other Physciacean ITS sequences was possible and therefore it could not be used in phylogenetic analyses. This might have caused the poor resolution among the sequences from Diplotomma in Fig. 3. Buellia pulverulenta (Anzi) Jatta was not supported as a member of the Diplotomma cluster (Subclade III), as it was assumed by Nordin and Mattson (2001), but was found to be closely related to Buellia geophila in our study (Fig. 3, Subclade IV).
The taxa of Subclade IV appear as a rather diverse assemblage, with respect to morphological characters as well as ITS rDNA sequence data. This particularly applies to the large genus Buellia even after the segregation of Amandinea, Australiaena, Dimelaena, Diplotomma and Hafellia (Fig. 3). Although weakly supported, Dermatiscum and Dimelaena form a clade. This coincides with their thalline excipulum, which otherwise is rare within Subclade IV. The common origin of these two genera is further corroborated by Nordin's (2001) cladogram in which both genera form a well-supported clade. Ascospore ontogeny and ornamentation are regarded as important phylogenetic traits in this clade (Nordin 2000, Wedin et al 2000), but the phylogenetic relevance of these characters could not be tested further due to the poor resolution among the taxa included in this molecular approach. Compared to the known diversity of this group, the number of available ITS sequences still is low and addition of further taxa might strongly influence cladistic analyses.
The clade affiliation of some taxa was unexpected, referring to older literature, or disagreed with trait assignment to certain subclades as described here. The closer relationship of the genera Dirinaria and Pyxine to Diplotomma (Clade B) than to taxa of Clade A was unexpected. In former taxonomic surveys (Awasthi 1975, Nordin and Mattson 2001, Scheidegger et al 2001), Dirinaria and Pyxine were assumed to be related to the foliose genera Physcia or Heterodermia, or at least to be descendants of an ancestor related to Rinodina (Mayrhofer 1982). This was due to similarities in growth habit, excipulum type and the presence of ascospore wall thickenings, which were thought to indicate a close relationship with these taxa. In contrast, conflicting characters such as ascus type and hypothecium pigmentation were given less weight. However, a closer look at ascospore and excipulum types reveals differences in fine structure that question the synapomorphy of these traits. The lecanorine excipula of Clade A differ in their alga distribution from the lecanorine excipula of Dirinaria (Awasthi 1975). These fine structure differences gain taxonomic weight when correlated with the Physciacean ITS phylogeny. Here the Dirinaria excipulum and stages of the early Pyxinean excipulum development are interpreted as convergent autapomorphies in these genera, where Pyxine might represent a transitory state between Diploicia and Dirinaria. This interpretation also corroborates the finding of Dirinaria to be a fairly derived genus with species represented by long branches in ITS trees (Fig. 3). The interpretation of excipulum characteristics to be homoplastic is supported by further examples of lecanorine excipula within Clade B and lecideine excipula in Clade A as listed in Table VI.
Buellia lindingeri (Clade A) and Amandinea (Rinodina) cacuminum (Clade B) already were recognized to exhibit character states leading to contradicting taxonomic assignments (Rambold et al 1994). Here, ITS sequence data show their phylogenetic position distinct from other species of Rinodina and Buellia, respectively. The ITS phylogeny supports the phylogenetic significance of the morphological markers under discussion, favoring ascus over excipulum characteristics. In the case of Amandinea cacuminum (formerly Rinodina cacuminum), the according recombination was done (Mayrhofer and Sheard 2002). Additional candidates still to be investigated in this respect are R. biloculata (Nyl.) Sheard, R. ericina (Nyl.) Giralt, R. insularis (Arnold) Hafellner and R. kalbii Giralt & Matzer. In these species, the excipulum and ascus types lead to differing assignments and it would be of great interest to see whether the priority of traits as stated above also holds in these cases.
To assess the plesiomorphy and evolutionary constancy of the phenotypic traits that appeared to be most highly conserved in the Physciaceae and Caliciaceae, ascus types and hypothecium pigmentation were compared with taxa of other families that were depicted as basal taxa to the Physciaceae-Caliciaceae clade in the SSU phylogeny. These were the genera Lecidea (Lecideaceae) and Porpidia (Porpidiaceae), standing closer to the root of ingroup than any other investigated taxon. Neither Lecidea nor Porpidia develop asci of a type found in the Physciaceae-Caliciaceae. Asci of Lecidea are characterized by a ILugol + palely stainable tholus, and a ILugol + dark blue cap-like structure. Those of Porpidia develop a ILugol + palely stainable tholus, and a central ILugol + dark blue tube-like structure. Therefore, it is assumed that the Lecanora-type asci of the Physciaceae found in Clade A and the Bacidia-type and prototunicate asci of Clade B (which includes the Caliciaceae) are apomorphies. They are most likely convergent, but not homologous to similar ascus types found in other Lecanoralean families. In addition, ascospores in Lecidea and Porpidia are quite different from those of the ingroup taxa. Both develop elliptic, unicellular, hyaline ascospores, Lecidea without, and Porpidia with a halonate perispore. Connections between outgroup and ingroup are seen in apothecial traits. Lecidea and Porpidia usually exhibit a pigmented hypothecium which, however, is hyaline in some of their species. Both genera develop true excipula. A true excipulum as well as a pigmented hypothecium coincide with Clade B. Therefore, these character states are considered plesiomorphic within the Physciaceae-Caliciaceae. The thalline excipulum as well as an unpigmented hypothecium found throughout Clade A correspondingly appear as autapomorphies of this clade. Naturally, thalline excipula occurring in Clade B are likewise apomorphic.
The different contents of homoplasy in different morphological traits as described here are paralleled by findings in the Pezizomycetes (Hansen et al 2001). In this study, ascus types also turned out as phylogenetically highly conserved characters that defined monophyletic clades. Other characters such as ascospore ornamentation and excipulum structure exhibited a higher degree of variability. The different degrees of phylogenetic conservation in these traits from such distantly related groups as Pezizaceae and Physciaceae is conspicuous and may be interpreted as evidence for the general ranking of "phylogenetic reliability" of these characters.
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showed four members of the Caliciaceae to be paraphyletic with taxa of Clade B. This also is confirmed in this study. However, the phylogenetic positions of those Caliciaceae presented in Wedin et al (2002) receive little support and appear incongruent with the phylogeny obtained from nrSSU data as presented in this study. Therefore, the phylogenetic relationships between the Caliciaceae and Clade B remain uncertain.
Regarding the type species Calicium viride as the anchor to position the family, two alternative scenarios might be deduced from the present data: First, the Caliciaceae might form a sister group to Subclade IV and Subclade III might be the sister group to the previous two groups, Clade A being the sister taxon to all of the previous three groups. Second, the Caliciaceae might be nested within Subclade IV. Therefore, establishing a higher order taxon comprising the taxa of Clade B necessarily would include the Caliciaceae also. For reasons of nomenclatorial priority, the establishment of such a taxon would imply the transfer of these taxa into the Caliciaceae. Wedin and Grube (2002) proposed the conservation of the family name Physciaceae against the name Caliciaceae for the complete Physciaceae/Caliciaceae clade. Although the authors appreciate this attempt, we suggest instead documenting the apparent split in the Physciaceae/Caliciaceae clade by nomenclature. In the event that a transfer of most Physciaceae species (Clade B comprises more species than Clade A) into the Caliciaceae would be difficult for lichenologists to adapt to, we suggest conserving the family name Buelliaceae against Caliciaceae for all taxa in Clade B (including the Caliciaceae) and maintaining the Physciaceae in a narrower circumscription. However, a "nomen conservandum" is not proposed here and the strict rules of nomenclature are followed.
A phenotypic delimitation of the resulting families of the Physciaceae, represented by the taxa of Clade A, and Caliciaceae (including the taxa of Clade B) could be based on a combination of three characters, of which at least two indicate the correct affiliation. Only Australiaena might be problematic under this concept, since it has asci that resemble the Lecanora-type ascus and exhibits fairly variable ascospores that occasionally develop ascospore wall thickenings. All other taxa fit the delimitation as given below. The ascospores for both families constantly being pigmented, septate and, with the exception of Rinodinella, thick-walled (see also Table V).
Caliciaceae Chevall. (1826) emend. G. Helms, G. Rambold & T. Friedl
Asci of Bacidia-type or prototunicate; hypothecium pigmented; ascospores without distinct wall thickenings. (Species usually with proper excipula, exceptional taxa: see Table VI.)
Physciaceae Zahlbr. (1898) emend. G. Helms, G. Rambold & T. Friedl
Asci of Lecanora-type; hypothecium hyaline; ascospores with distinct wall thickenings or of Rinodinella-type. (Species usually with thalline excipula, exceptional taxa: see Table VI.)
The interrelations among the taxa of Clade B, especially Subclade IV and Caliciaceae Pers. await a more thorough exploration. A subsequent division of the newly emended Caliciaceae in families, corresponding to subclades III, IV and the Caliciaceae in their former circumscription, might be appropriate, if further evidence could be found that supports this grouping. The critical taxon that prevents a clear delimitation of the two subclades is Diplotomma. ITS, as well as SSU rDNA sequence data, assign this genus to Subclade III, while it seems problematic to consistently delimit this genus from Subclade IV with phenotypic characters. Accordingly, groupings of taxa assigned to Diplotomma based on nrITS data (this study) and phenotypic traits (Nordin 2000) exhibit incongruencies.
A segregation of the Caliciaceae in their new circumscription into three families, as outlined above, would form an alternative concept. For Subclades III and IV, existing family names, such as Pyxinaceae (E.M. Fries) Stitzenberger (1862) and Buelliaceae Zahlbr. (1907), respectively, could be adopted. Provided further investigations do not unravel the taxa with prototunicate asci as a polyphyletic assemblage, the Caliciacean concept could be re-established in the former circumscription. In case the Caliciaceae and Subclade IV intermix, taxa of both groups could be designated to the Caliciaceae, resulting in a tripartite family concept with Physciaceae, Pyxinaceae and Caliciaceae. In all these scenarios, the new concept of the Physciaceae as proposed above would persist.
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