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Department of Botany, The Field Museum, 1400 S. Lake Shore Drive, Chicago, Illinois 60605
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The phylogenetic relationships of many lichen-forming perithecioid ascomycetes are unknown. We generated nuLSU and mtSSU rDNA sequences of members of seven families of pyrenocarpous lichens and used a Bayesian framework to infer a phylogenetic estimate. Members of the perithecioid Protothelenellaceae, Thelenellaceae and Thrombiaceae surprisingly cluster within the mainly discocarpous Lecanoromycetes, while Strigulaceae, Verrucariaceae and Pyrenulaceae are related to the ascolocular Chaetothyriomycetes. Micromorphological studies of the ascomata showed that the two main groups of pyrenocarpous lichen-forming fungi differ in their ascus types. The Strigulaceae, Verrucariaceae and Pyrenulaceae have apically and laterally thick-walled asci, whereas the Thelenellaceae, Protothelenellaceae and Thrombiaceae have only apically thickened asci. The latter two show ring-shaped amyloid apical structures. Based on morphological and molecular evidence we propose to reduce Thrombiaceae to synonymy with Protothelenellaceae.
Key words: ascus type, Bayesian analysis, Chromatochlamys, molecular phylogeny, mtSSU rDNA, nucleotide composition bias, nuLSU rDNA, Protothelenella, pyrenolichens, Thelenella, Thrombium, systematics
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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), and consequently other characters have been employed, such as ascoma development (Nannfeldt 1932) or ascus structure (Luttrell 1951, 1955). Nannfeldt (1932) distinguished between ascohymenial and ascolocular ascoma development. The ascus may be functionally single-walled (unitunicate or prototunicate), or double-walled (bitunicate) (Luttrell 1955; Reynolds 1981, 1989). However, these single-character classifications have not been supported by molecular evidence: Discomycetes and Pyrenomycetes have been shown to be of polyphyletic origin (Gargas and Taylor 1995, Lutzoni et al 2001). Classifications based on ascus type (e.g., Luttrell 1955) or ascoma development alone (Nannfeldt 1932) also are rejected by molecular data (Berbee 1996, Wedin et al 1998, Lindemuth et al 2001).
Among lichen-forming ascomycetes the discocarpous species outnumber the pyrenocarpous taxa, and most phylogenetic studies so far have concentrated on lichen-forming discomycetes. The classification of pyrenocarpous lichen-forming fungi, especially at higher taxonomic levels, still is largely unsettled. Aptroot (1998) surveyed the historical developments in the classification of pyrenocarpous fungi.
The majority of pyrenocarpous lichens belong to a group with gelatinizing paraphyses, the Verrucariaceae, including genera such as Dermatocarpon, Endocarpon and Verrucaria. This group has been shown to be closely related to the non-lichenized Chaetothyriales (Lutzoni et al 2001) and consequently was placed in the Chaetothyriomycetes (Eriksson et al 2004). However, many other families containing lichenized pyrenocarpous ascomycetes still are listed as "of uncertain position" in the most recent outline of the Ascomycota (Eriksson et al 2004), e.g., Pyrenulales, Trichotheliales, Protothelenellaceae, Thelenellaceae and Thrombiaceae.
A number of these poorly known pyrenocarpous taxa have been classified previously in the genus Microglaena. This genus is a heterogeneous assemblage of lichenized ascomycetes characterized by sessile to immersed perithecia, reticulate, persistent paraphyses, thick-walled asci that generally are interpreted as bitunicate and muriform ascospores (Morgan-Jones and Swinscow 1965, Vezda 1968; Harris 1973:38, 1975:26; Mayrhofer and Poelt 1985). The generic name Microglaena was introduced by Koerber (1855) based on the type species M. wallrothiana. Almost concurrently, Nylander (1855) erected the genus Thelenella based on Thelenella modesta. Lönnroth (1858) recognized the synonymy of Microglaena wallrothiana and Thelenella modesta and thereafter both generic names were used alternately. Eriksson (1981) pointed out that Microglaena Körber is an illegitimate name, being a later homonym of Microglaena Ehrenb. (Chlorophyta), and should be replaced by Thelenella Nyl. During a revision of the European taxa of Microglaena Mayrhofer and Poelt (1985) removed several species from the genus and divided the remaining core group into three genera: Chromatochlamys, Protothelenella and Thelenella (syn. Microglaena). Because of its deviating ascus structures they separated Protothelenella into a new family: Protothelenellaceae. Later, Mayrhofer (1987a) erected the Thellenellaceae to accommodate the genera Chromatochlamys, Julella and Thelenella. The classification of the above-named genera and families into the system of Ascomycota proved difficult. The genus Thelenella/Microglaena was placed in discocarpous Pertusariaceae (Rabenhorst 1870:138, Pabst 1876, Franck 1877), in the pyrenocarpous families Verrucariaceae (e.g., Zahlbruckner 1907, 1926; Clements 1909, Zschacke 1934), Thelidiaceae (Watson 1929), Pyrenulaceae (Harris 1975), Polyblastiaceae (Kopa
evskaja et al 1977), Microglaenaceae (later Thelenellaceae) (V
zda 1968, Henssen and Jahns 1973, Poelt 1973, Barr 1979), and in the ascolocularous Pleosporaceae (Dothideomycetes) or Mycoporaceae (Arx and Müller 1975), due to the putative bitunicate nature of its asci (Morgan-Jones and Swinscow 1965, Hale and Culberson 1966, 1970; Poelt and Vezda 1981). While most recent authors considered Thelenella a pyrenocarpous lichen, Eriksson (1981) had a deviating interpretation. He considered the ascus opening in Thelenella as bilabiate and the ascomata as perithecioid apothecia, supporting the early classification in the Pertusariaceae. Later he emphasized the need for a new family if the ascomata proved to be pseudothecia (Eriksson 1982). The Thelenellaceae as established by Mayrhofer (1987a) are kept incertae sedis in the system of the Ascomycota (Mayrhofer 1987a, Eriksson et al 2004).
The genus Thrombium comprises another group of enigmatic pyrenocarpous lichens. It is characterized by immersed or sessile perithecia, persistent, non or sparsely branched paraphyses, amyloid ascus tips and simple, hyaline ascospores (Zschacke 1934, Swinscow 1964, Breuss 2002). This group formerly was treated within the Verrucariaceae because of superficial similarities in thallus and ascospore morphology (Zahlbruckner 1926). However, Thrombium is characterized by a persistent hamathecium, as members of the Adelococcaceae (Verrucariales), whereas taxa in the Verrucariaceae have gelatinizing paraphyses. Poelt (1973) suggested close affinities to the Protothelenellaceae, but later annulled this statement (Mayrhofer and Poelt 1985). Poelt and Vezda (1981) segregated the genus in its own family, and later David and Hawksworth (1991) validated the name Thrombiaceae. The family’s position within the Ascomycota is unsettled (Eriksson et al 2004).
We use sequence data of the nuclear large subunit and mitochondrial small subunit rDNA to evaluate the phylogenetic position of the enigmatic pyrenocarpous families Protothelenellaceae, Thellenellaceae and Thrombiaceae. The Protothelenellaceae, with two genera, Protothelenella and Mycowinteria, includes 12 species worldwide (Aptroot and van Iperen 1998; Mayrhofer 1987b, 2002a). The Thelenellaceae includes Chromatochlamys (three species) (Mayrhofer and Poelt 1985), which recently was synonymized with Thelenella (Fryday and Coppins 2004), Thelenella (ca. 20 species) (Mayrhofer 2002b) and Julella (ca. four species) (Aptroot 2002). The Thrombiaceae, with the single genus Thrombium, includes five species worldwide (Breuss 2002). The analysis includes sequences of members of the Pertusariaceae, Verrucariaceae, Pyrenulaceae, Strigulaceae and Dothideomycetes, groups that are putatively related to Thrombiaceae, Protothelenellaceae and Thelenellaceae.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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in, S slope of Mt. Huser along small creek S of summit, at forest edge (Picea orientalis, Fagus orientalis), 01 July 2001, A. Guttová, J. Halda, Z. Palice 8634 & C. Printzen (HB Z. Palice). Protothelenella sphinctrinoidella (Nyl.) H. Mayrhofer & Poelt. ANTARCTICA. SOUTH SHETLAND ISLANDS: Livingston Island, Bayers Peninsula, Chester Cone, H.T. Lumbsch 19022b (F). Psoroglaena epiphylla Lücking. MEXICO. VERACRUZ: Sierra de Los Tuxtlas, Volcán San Martín Tuxtla, Feb 2003, Herrera-Campos et al (F). Pyrenula nitida (Weigel) Ach. GERMANY. HESSE: Main-Kinzig Kreis, Steinau an der Straße, Teufelshöhle, beneath parking lot, 25 May 2002, I. Schmitt & S. Pauls (F). Strigula stigmatella (Ach.) R.C. Harris. AUSTRIA. SALZBURG: Hohe Tauern, Glockner Gruppe, Kapruner Tal. Kesselfall, NW facing slopes. 26 Aug 1996, J. Hafellner 47259 (GZU). Thelenella antarctica (I.M. Lamb) O.E. Erikss. ANTARCTICA. SOUTH SHETLAND ISLANDS: Livingston Island, South Bay, Punta Polaca, 22 Jan 2002, H.T. Lumbsch 19006a (F). Thrombium epigaeum (Pers.) Wallr. AUSTRIA, TIROL: Pitztal, hiking path from Jerzens to the Hochzeiger, 8 Sep 1996, H.T. Lumbsch 11179 (F). Light microscopy.— – For LM studies of the asci we prepared squash mounts of the ascomata of selected species, stained with Lugol’s solution/potassium hydroxide (IKI) and/or lactophenol cottonblue.
DNA extraction, amplification and sequencing.— –
Specimens and sequences used for the molecular analyses are compiled (TABLE I). Total DNA was extracted using the Qiagen Plant Mini Kit (Qiagen) following the instructions of the manufacturer. The nuclear LSU rDNA was amplified with the primer pairs AL1R (Döring et al 2000) and LR6 (Vilgalys and Hester 1990), and the mitochondrial SSU rDNA with mrSSU1, mrSSU2, mrSSU2R, mrSSU3R (Zoller et al 1999), and MSU7 (Zhou and Stanosz 2001). Primers were selected to ensure that at least one primer of each pair was fungal specific to avoid amplification of photobiontic DNA. The 25 µL PCR reactions contained 2.5 µL buffer, 2.5 µL dNTP mix, 1 µL of each primer (10 µM), 5 µL BSA, 2 µL Taq, 2 µL genomic DNA extract and 9 µL water. Thermal cycling parameters were: initial 3 min denaturation at 95 C, followed by 30 cycles 1 min at 95 C, 1 min at 52 C (mtSSU primers) or 53 C (AL1R/LR6), 1 min at 73 C and a final elongation 7 min at 73 C. Amplification products were viewed on 1% agarose gels stained with ethidium bromide and subsequently purified using the Nucleo Spin DNA purification kit (Macherey-Nagel). Fragments were sequenced using the Big Dye Terminator reaction kit (ABI PRISM, Applied Biosystems). Sequencing and PCR amplifications were performed using the same sets of primers. Cycle sequencing was executed with the following program: 25 cycles of 95 C for 30 sec, 48 C for 15 sec, 60 C for 4 min. Sequenced products were precipitated with 10 µL of sterile dH2O, 2 µL of 3 M NaOAc, and 50 µL of 95% EtOH before loaded on an ABI 3100 (Applied Biosystems) automatic sequencer. Sequence fragments obtained were assembled with SeqMan 4.03 (DNASTAR) and manually adjusted.
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). Polarity of characters was assessed using four members of the Sordariomycetes as outgroups as in other studies on lichen-forming fungi (e.g., Lumbsch et al 2004).
Phylogenetic analysis.— –
The data sets were analyzed singly and in combination using the programs PAUP* 4.0b (Swofford 2003) and MrBAYES 3.0 (Huelsenbeck and Ronquist 2001). We employed a Bayesian approach (Huelsenbeck et al 2000, Larget and Simon 1999) with Markov Chain Monte Carlo (MCMC) tree sampling to make a phylogenetic estimate. Posterior probabilities of each node were calculated by counting the frequency of trees visited during the course of the MCMC analysis. The program MrBAYES was employed to sample the trees. The analyses were performed assuming the general time reversible model (Rodriguez et al 1990) including estimation of invariant sites and assuming a discrete gamma distribution with six rate categories (GTR + I + 
) for the combined and the single-gene analyses. No molecular clock was assumed. The MCMC process was set so that 12 chains ran simultaneously for 2 000 000 generations. Trees were sampled every 100th generation for a total of 20 000 trees. We plotted the log-likelihood scores of sample points against generation time using TRACER 1.0 (http://evolve.zoo.ox.ac.uk/software.html?id=tracer) and determined that stasis was achieved when the log-likelihood values of the sample points reached a stable equilibrium value (Huelsenbeck and Ronquist 2001). The initial 1000 trees were discarded as burn-in before stasis was reached. Using PAUP*, majority-rule consensus trees were derived from 19 000 trees sampled after reaching likelihood convergence to calculate the posterior probabilities of the tree nodes. Unlike nonparametric bootstrap values (Felsenstein 1985), these are estimated probabilities of the clades under the assumed model (Rannala and Yang 1996) and hence posterior probabilities $95% are considered significant supports. Branch lengths were obtained using the "sumt" option of MrBayes. Phylogenetic trees were drawn using TREEVIEW (Page 1996).
We used a Bayesian approach to examine the heterogeneity in phylogenetic signal between the two data partitions (Buckley et al 2002). For the two genes and the concatenated analyses, the set of topologies reaching 0.95 posterior probability was estimated. The combined analysis topology then was compared for conflict with the 0.95 posterior intervals of the single-gene analyses, and the two single-gene analyses were compared. If no conflict was evident (i.e., branches supported by at least 0.95 posterior probability support in conflict among the three analyses), it was assumed the two data sets were congruent and could be combined. If conflict is evident, the two data sets can be interpreted as incongruent and the concatenated analysis may be potentially misleading (Bull et al 1993).
As discussed below the topology of the backbone of the phylogeny within the Lecanoromycetes differed from that obtained in other recent studies focusing on Lecanoromycetes phylogeny (e.g., Grube et al 2004, Lücking et al 2004, Lumbsch et al 2004). We evaluated the potential influence of deviating GC content in some sequences using the LogDet correction (Lockhart et al 1994). The LogDet method was developed to correct for base composition inequalities. For this analysis, minimum evolution (ME) trees were inferred using the heuristic search command under the distance criterion in Paup*. Constant characters were excluded.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). The nuLSU data produced a matrix of 908 unambiguously aligned nucleotide position characters, 474 of which were variable. The mtSSU alignment was 764 positions long including 521 variable characters. The Bayesian approach for testing data sets for incongruence indicated that the topology of the majority-rule consensus tree from the combined analysis lies within the 0.95 posterior intervals for the two separate data sets (data not shown). This is consistent with the hypothesis that the two partitions have evolved along the same underlying topology and hence a combined analysis was performed. The combined alignment is available in TreeBASE (http://www.treebase.org/treebase/), matrix number SN1868.
The likelihood parameters in the sample had the following average values (± one SD): LnL –3123.000 (± 6.267), base frequencies 
(A) = 0.297 (± 0.004), (C) = 0.179 (± 0.003), (G) = 0.25 (± 0.003), (T) = 0.274 (± 0.003), rate matrix r(AC) = 1.088 (± 0.057), r(AG) = 3.269 (± 0.172), r(AT) = 1.946 (± 0.087), r(CG) = 1.189 (± 0.053), r(CT) = 5.661 
± 0.268), r(GT) = 1.0 (± 0.0), gamma shape parameter = 0.752 (± 0.009) and the proportion of invariable site p(invar) = 0.284 (± 0.004).
The 50% majority-rule consensus tree of 19 000 sampled trees is shown (FIG. 1). Members of the Protothelenellaceae, Thelenellaceae, and Thrombiaceae all cluster within the Lecanoromycetes. Thelenellaceae, including one species each of Chromatochlamys and Thelenella, forms a strongly supported group (pp 1.00), which is a sistergroup to Ostropales s.lat. (pp 1.00). Basal to that are Protothelenella and Thrombium, which form a supported sistergroup (pp 0.97). The basal placement of Protothelenella and Thrombium to Ostropales s.lat. is not supported. Members of the monophyletic Agyriales (pp 1.00) are in a clade with all aforementioned groups (pp 1.00). Sister to this assemblage is the monophyletic Pertusariales (pp 0.98). The posterior probability of the node connecting Pertusariales to the other groups is 0.99. The Lecanorales are strongly supported as monophyletic (pp 1.00), and constitute the most basal lineage within the Lecanoromycetes, which itself is strongly supported as monophyletic class (pp 1.00). Sistergroup to the Lecanoromycetes are Chaetothyriomycetes + Eurotiomycetes. The Chaetotyriomycetes include members of the lichen-forming or lichenicolous pyrenocarpous Verrucariaceae (Agonimia, Norrlinia, Dermatocarpon) and Strigulaceae (Strigula) (pp 1.00), which are next to a clade of non-lichenized fungi with putative ascolocular ascoma development (Capronia, Ceramothyrium, Glyphium) (pp 1.00). Most basal in the Chaetothyriomycetes are the lichen-forming Pyrenulales (Pyrenula) (pp 1.00). The clade bearing Chaetothyriomycetes and Eurotiomycetes is strongly supported (pp 1.00). The Dothideomycetes (pp 1.00) include lichenized and non-lichenized taxa which are characterized by ascolocular development. They are basal to Lecanoromycetes/Chaetothyriomycetes/Eurotiomycetes.
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, Lücking et al 2004, Lumbsch et al 2004), but shows some similarities to a recent phylogeny published by Miadlikowska and Lutzoni (2004). While the present study places Ostropales as a sister group to Agyriales, as in Kauff and Lutzoni (2002) and Miadlikowska and Lutzoni (2004), the other studies show ostropalean fungi as a sister-group to all remaining Lecanoromycetes, and Agyriales as a sister to Pertusariales. When examining the data matrix more thoroghly, we noticed a strong base composition heterogeneity. The GC content ranged between 0.42–0.43 in the two Pyrenula and Thelotrema spp. and 0.49 in Diploschistes thunbergianus and Pertusaria amara (all data available upon request). To evaluate whether the observed differences in base frequencies are responsible for the deviating topologies in the backbone of the Lecanoromycetes phylogeny, we employed the LogDet distance; developed to accommodate for inequalities of base frequencies (Lockhart et al 1994). The minimum evolution tree obtained by employing LogDet corrected distances in PAUP* basically agreed with the majority rule consensus tree obtained from the Bayesian analysis. However, the backbone topology of the Lecanoromycetes phylogeny was different. A simplified tree shows the differences between the phylogenies obtained using the GTR + I + (A) and LogDet (B) models (FIG. 2). The topology of this simplified tree A) corresponds to the B/MCMC tree shown (FIG. 1). In contrast, the tree based on the LogDet algorithm (FIG. 2B) shows the Ostropales s.lat. (with Thelenellaceae as sister-group) as the most basal lineage within the Lecanoromycetes. Protothelenellaceae and Thrombiaceae are a sistergroup to Agyriales, which together are a sister clade to the two lineages of Pertusariales. The Lecanorales are a sistergroup to the clade bearing Agyriales, Protothelenellaceae + Thrombiaceae, and Pertusariales (FIG. 2B).
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). The genera Protothelenella and Thrombium show similar thickening patterns and additionally exhibit ring-like amyloid patterns in the ascus apex, which often seem to fuse with tube-shaped structures in mature asci. (FIG. 3E–G; see also Eriksson 1981, p. 96, FIG. 118 D, a double, amyloid ring). FIG. H shows mature asci of Thrombium epigaeum, which have released some of their ascospores. The picture examplifies the difficulty to determine with LM methods whether the asci are functionally unitunicate or bitunicate. Ascus apices of representatives of the Verrucariaceae (Psoroglaena), Strigulaceae (Strigula) and Pyrenulaceae (Pyrenula) all displayed asci with apically and laterally evenly thickened walls, and no amyloid reactions (FIG. 3I–K).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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demonstrated that the perithecioid Porinaceae also belong here. A close relationship of the Thelenellaceae and the Pertusariales, as suggested previously (Rabenhorst 1870, Pabst 1876, Franck 1877, Eriksson 1981), could not be supported. Instead, the Thelenellaceae form a supported sistergroup with Ostropales s.lat (FIGS. 1, 2). Harris (1989) criticized the placement of Chromatochlamys in Thelenellaceae, but later revised his opinion (Harris 1995) and even suggested to synonymize the genera Chromatochlamys and Thelenella. The present molecular data confirm a close relation-ship of Chromatochlamys and Thelenella as proposed by Mayrhofer and Poelt (1985) and Harris (1995). Micromorphological reexaminations showed remarkable similarities in ascus structure, as both genera have thickened ascus apices, which lack amyloid reactions (FIG. 3A–D). The genus Chromatochlamys recently has been subsumed formally in Thelenella (Fryday and Coppins 2004).
Striking resemblances in ascus structure and ascus staining properties also could be found in the genera Protothelenella and Thrombium (FIG. 3E–G). As mentioned in Mayrhofer and Poelt (1985:25 and FIGS. 21, 22 p 78) these genera display a distinct blue ring pattern in the ascus apex when stained with iodine. In contrast to an earlier suggestion by Poelt (1973), Mayrhofer and Poelt (1985) suspected no affinities between the two genera, because they found several distinctive features such as: periphyses (present in Thrombium, absent in Protothelenella), ascospores (simple in Thrombium, muriform in Protothelenella), hymenial gel (absent in Thrombium, present in Protothelenella) and structural differences in the perithecial walls. For all these differences, the similarities in ascus structure and the molecular data suggest a close relationship of the two genera. Consequently, we propose the following synonyms, merging Protothelenellaceae and Thrombiaceae into one family, Protothelenellaceae:
Protothelenellaceae Vezda, Mayrhofer & Poelt in Herzogia 7:26. 1985.
Thrombiaceae Poelt & Vezda ex J.C. David and D. Hawksw., Syst Ascom 10:15. 1991.
We cannot resolve the placement of the Protothelenellaceae within the Lecanoromycetes with the current data set. They might be related to the Thelenellaceae and Ostropales s.lat. (FIGS. 1, 2A), or to the Agyriales (FIG. 2B). Close connections between Protothelenellaceae (incl. Thrombiaceae) or Thelenellaceae and either Verrucariaceae (e.g., Dermatocarpon), Strigulaceae, Pyrenulaceae, or the ascolocularous Dothideomycetes, as has been suggested before, are rejected by the current analysis. Verrucariaceae, Strigulaceae and Pyrenulaceae are members of the Chaetothyriomycetes. Micromorphological studies showed that the ascus types of lichenized pyrenomyctes from this group differ from those placed in the Lecanoromycetes. Representatives of Verrucariaceae (Psoroglaena), Strigulaceae (Strigula) and Pyrenulaceae (Pyrenula) have ascus walls that are even evenly thickened at the top and at the sides and show no amyloid reactions (FIG. 3I–K).
Formerly, almost all pyrenocarpous lichens were placed in the single class Loculoascomycetes (e.g., Barr 1987). However, our current study shows that pyrenocarpous lichens occur in distantly related groups of Ascomycota. Only few, e.g., the lichen-forming Arthopyreniaceae, belong to strictly ascolocular fungi, which are placed currently in the Dothideomycetes (FIG. 1). Fungi in this group are functionally bitunicate and ascolocular. The bulk of perithecioid lichens belongs to the Chaetothyriomycetes. These include the species-rich families Pyrenulaceae, Verrucariaceae, and Strigulaceae. These taxa typically have functionally bitunicate asci and ascohymenial ascoma development. A few families of lichenized fungi containing only a limited number of species (Thelenellaceae, Protothelenellaceae) belong to the Lecanoromycetes. These taxa have more or less thick-walled asci that are usually interpreted as bitunicate. Our observations on the ascus opening in Thrombium indicate that the asci in this genus may also be interpreted as functionally unitunicate. Interestingly, also Breuss (2002) did not mention bitunicate asci in his description of the genus Thrombium. Thus, ascus types should be interpreted cautiously. It should be noted in this connection that most euascomycetes, with the exception of cleistothecial ascomycetes, usually have a thin, firm ectotunica and a more flexible endotunica. And the unitunicate ascomycetes normally have two main ascal wall layers, which however, do not separate during ascospore release (Reynolds 1971). Asci may consist of two walls that can be separated in a squash preparation, but still may be functionally unitunicate. Such a case occurs in the Gomphillaceae. The asci in this group also were formerly interpreted as bitunicate (Hafellner 1984), but subsequent studies showed them to be functionally unitunicate (Lücking 1997).
The placement of pyrenocarpous lichens in the Lecanoromycetes is surprising at first, because the Lecanoromycetes contain almost exclusively discocarpous taxa. However, this class exhibits an extraordinary variability of ascoma types, some taxa having closed, perithecioid ascomata (e.g., members of the Coccotremataceae, Gyalectaceae, Pertusariaceae Thelotremataceae). Moreover, Grube et al (2004) showed that the "truly" pyrenocarpous Porinaceae are related to ostropalean Lecanoromycetes as well. Our current data add further examples to the immense diversity of ascoma types in the Lecanoromycetes.
Our study also has raised the question of the influence of nucleotide composition bias on the backbone topology of the Lecanoromycetes phylogeny. A thorough investigation of this problem is beyond the scope of this paper, but the different results obtained by employing the GTR + I + or LogDet substitution models show that caution is needed in interpreting higher-level phylogenetic relationships with the poor data and taxon sampling at hand. Known cases of bias caused by nucleotide composition inequalities in well-studied groups (e.g., Xia et al 2003) call for our alertness.
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ACKNOWLEDGMENTS |
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k Palice (Prague) for providing several of the valuable pyrenolichens examined in this study. Special thanks go to Helmut Mayrhofer (Graz) for helpful comments on the manuscript. Many thanks also go to Steffi Kautz (Essen) for assistance with DNA extractions. Sequencing was performed in the Pritzker Lab for Molecular Systematics and Evolution at The Field Museum (Chicago). We are very grateful to Robert Lücking (Chicago) for supplying samples of Psoroglaena and Aspidothelium to study their ascus-types. This study was financially supported by a fellowship within the Postdoc-Program of the German Academic Exchange Service (DAAD) to IS, and a start-up fund from the Field Museum to HTL. |
FOOTNOTES |
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1 Corresponding author, E-mail: ischmitt{at}fieldmuseum.org
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LITERATURE CITED |
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