Phylogenetic relationships ofSphaerophoraceae (Ascomycetes ...

P1. Syst. Evol. 209:75-83 (1998)

Plant Systematics and Evolution © Springer-Verlag1998 Printed in Austria

Phylogenetic relationships of Sphaerophoraceae (Ascomycetes) inferred from SSU rDNA sequences MATS WEDIN, ANDZRS TEHLER, and ANDREA GARGAS

Received June 3, 1996, in revised version September 26, 1996

Key words: Fungi, Ascomycetes, Caliciales, Lecanorales, Sphaerophoraceae, Stereocaulaceae. - Lichens, molecular evolution, phylogeny, small subunit, ribosomal DNA, 18S rDNA. Abstract: SSU rDNA was sequenced from the lichenized fungi Bunodophoron scrobiculatum and Leifidium tenerum (Sphaerophoraceae), and Stereocaulon ramulosum and Pilophorus acicularis (Stereocaulaceae) and analysed by maximum parsimony with 44 homologous ascomycete sequences in a cladistic study. A small insertion (c. 60 nt.) was found in the sequence of Leifidium tenerum. Sphaerophoraceae constitutes a strongly supported monophyletic group which groups together with Lecanora dispersa and the Stereocaulaceae. Together with Porpidia crustulata, this larger group is a sistergroup to the Peltigerineae. This analysis thus supports the Lecanorales as monophyletic, including Sphaerophoraceae and the Peltigerineae, but does not provide strong support for this monophyly. The analysis also suggests that the prototunicate ascus in the Sphaerophoraceae is a reversion to the plesiomorphic state. Based on morphological, anatomical and chemical reasons, Sphaerophoraceae is proposed to belong to one of the groups presently included in the paraphyletic suborder Cladoniineae within the Lecanorales.

Most fungi in the ascomycete order Caliciales s. 1. share the features of prototunicate asci and mazaedia, large masses of spores which have been released from the asci at an early stage. Based on the presence of these characters, the lichen family Sphaerophoraceae has traditionally been classified in the Caliciales. Caliciales was for a long time seen as a model case of a monophyletic group in the ascomycetes. In contrast to this view of monophyly, Tm~LL (1984) suggested that the calicialean families Caliciaceae, Sphinctrinaceae and Mycocaliciaceae formed a monophyletic group, and the rest of Caliciales were a polyphyletic assemblage of families most of which have derived mazaedia and the resulting passive spore dispersal independently. TIBELL'S hypothesis was based on morphological, anatomical and secondary me.tabolite studies. GARGAS & TAYLOR (1992a, 1995) and GARGAS & al. (1995a) presented hypotheses on the phylogeny of the ascomycetes based on sequences of the small subunit of the ribosomal DNA (SSU rDNA) from both lichenized and non-lichenized ascomycetes, including two representatives from different families of Caliciales s. 1.: Mycocalicium albonigrum

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(NYL.) TIBELL (Mycocaliciaceae) and Sphaerophorus globosus (HUDS.) VAIN (Sphaerophoraceae). The two Caliciales taxa did not form a monophyletic group in the cladograms presented, thus supporting TIBELL'S view of the polyphyly of Caliciales. Furthermore, Sphaerophorus was grouped together with Porpidia crustulata (AcH.) HERTEL • KNOPH and Lecanora dispersa (PERS.) SOMMERF., suggesting a closer relationship between it and other Lecanorales. TEHLZR (1990) presented an outline phylogeny of the Ascomycetes with Caliciales as sister group to the rest of the euascomycetes. The basis for that placement was that the Caliciales shared the derived features of hamathecia with paraphyses but lacked other features of the euascomycetes such as apical pore apparatus and bitunicate ascus wall (sporangium wall). Using the Eurotiales as outgroup both the unitunicate ascus and the evanescent, passive spore dispersal (prototunicate) ascus were regarded as plesiomorphic. Hence, taxa which were traditionally thought of as caliciaceous such as Mycocaliciaceae with its apical pore apparatus were not included in Caliciales but were implicitly lumped in the phylogenetically unresolved assemblage of taxa with paraphyses and unitunicate asci with apical pore apparatus (T~HLER 1990). The terminal taxon Caliciales was not diagnosed by TEHLER, and neither did any autapomorphies characterise the group, and it was considered paraphyletic. As evident from the cladogram presented by (TEmpER 1990) only taxa that were unitunicate, prototunicate a n d ascohymenial could be included in the terminal Caliciales. Important to realise is that the prototunicate ascus, which is structurally unitunicate, is to be homologised with spore dispersal and not ascus structure. On the one hand we can conclude that two-walled, bitunicate asci do not appear outside the euascomycetes sensu T~HLZR(TEHLZR 1988, 1990). On the other hand it is well known that the thin-walled, unitunicate ascus is present inside as well as outside the euascomycetes thus clearly indicating the plesiomorphic status of the structure. Consequently the bitunicate ascus is apomorphic. Outside the bitunicate group, the unitunicate ascus or meiosporangium is a feature of the rest of the Eumycota, occurring not only in the chytrid and zygomycete groups but also in the Hemiascomycetes and Taphrinomycetes as well as in the Basidiomycetes (TEHLER 1988). This is in accordance with ERIKSSON(1981) who argues that a primitive type of unitunicate ascus with apical pore apparatus evolved from a true prototunicate type and that this unitunicate type then evolved into semifissitunicate (= bitunicate) ascus. As a result of recent studies on the family Sphaerophoraceae, a reassessment of the genetic classification was made (WEDIN 1993). The genera in the Sphaerophoraceae differ in morphology and chemistry from most other Caliciales, which mainly comprises lichenized and non-lichenized microfungi. The suggested placement of Sphaerophoraceae in Lecanorales (by GARGAS& al. 1995a) was in principle agreed upon by WED~r (1995), who, however, kept the family in Caliciales for the time being, albeit without presenting much detailed discussion on the subject. This discussion will be provided here. The present investigation reports the first two sequences from a larger study on the relationship between different representatives of Caliciales s. 1. and other ascomycetes using SSU rDNA data. The study aims to test the monophyly and relationships of the family Sphaerophoraceae and to discuss relevant morpholog-

Phylogenetic relationships of Sphaerophoraceae

77

ical, anatomical and chemical characteristics. In addition, two new sequences of representatives of the Stereocaulaceae (Lecanorales-Cladoniineae) are included to broaden the sampling within Lecanorales, and help resolve the placement of the

Sphaerophoraceae. Materials and methods Specimens and DNA extractions. Bunodophorom scrobiculatum, Leifidium tenerum, Stereocaulon ramulosum and Pilophorus acicularis from the following collections (with GenBank/EMBL accession numbers) were used for DNA extraction and sequencing: U70958 Bunodophoron scrobiculatum (C. BAB.) WEDtN, New Zealand, WZDIN 5002 (UPS). U70959 Leifidium tenerum (LAuR.) WZDIN, New Zealand, WEDrN4999 (UPS). U70060 Stereocaulon ramulosum R~USCHEL,New Zealand, WED~N4998 (UPS). U70961 Pilophorus acicularis (AcH.) Tn. FR. Canada, WDIN 4829 (UPS). Total DNA was extracted from specimens collected within the last six months by standard fungal miniprep protocols (LEz & al. 1988, LEz & TAYLOR1990). PCR amplifications. Dilutions (10 -1) of the total DNA extraction were used for specific amplification of the fnngal nuclear SSU rDNA using the primers nu-SSU-0819-5', nu-SSU- 0852-3', nu-SSU-1203-5', nu-SSU-1750-3', (GARGAS& TAYLOR1992b, following the nomenclature of GARGAS& DEPRIEST 1996), ITS4 (WmvE & al. 1990) and nu-SSU0021-5' (GARcAS & DEPRIEST 1996). The PCR amplification ran for 30 cycles (1 rain at 94°C) 1 rain at 50°C, 2 rain at 72°C with a 4 sec/cycle extension at 72°C), using Dynazyme II polymerase (Finnzymes Oy, Espoo, Finland) at the final enzyme concentration of c. 0.03 U/#I. Sequencing. Cycle sequencing reactions were carried out with the following primers: nu-SSU-0497-3' (Wr~[TE & al. 1990, following the nomenclature of GARCAS& DEPRIEST 1996), nu-SSU-0402-5 r, nu-SSU-1203-5' (GARGAS& TAYLOR1992b, GARGAS& DEPRIEST 1996), nu-SSU-1580-3' (D~PP,IEST 1992, GARGAS& DEPRIEST1996). The sequencing PCR amplification ran for 25 cycles (30 s at 95°C, 15 s at 50°C, 4 min at 60°C) for sequencing by the DyeDeoxy labelling method (PRISM Ready Reaction DyeDeoxy kit; Perkin-Elmer). The sequences were obtained using an ABI 373 (Perkin-Elmer) automatic sequencer. More than 95 % of the gene was sequenced, and most of the gene was sequenced in both directions. Sequence alignment. The four new sequences were manually aligned with 37 ascomycete sequences obtained from Genbank and three sequences (Nephroma, Peltigera, Solorina) kindly provided by Dr O. ERIKSSON(Table 1). Parsimony analysis. The data matrix was analysed with the computer package PAUP 3.1.1 (SwoFZORD1993). Ten replicates of a heuristic search were performed with the TBR option specified. The polarity of the characters was assessed with outgroup comparison, using representatives of Saccharomycetales as outgroup. This choice of outgroup is based on the relationships of the fungi suggested by GARGAS• al. (1995a). Tree and branch stability measurements. Branch support ("Bremer support") was calculated according to the successively relaxed parsimony approach of BRZMZR(1988, 1994) using the converse constraints approach with PAUP, where 10 replicates of heuristic TBR swapping were preformed to identify the minimum length of pre-defined trees lacking a specific node. Total support is given following KALLERSJO& al. (1992) and the total support index for the most parsimonious tree was calculated according to BRZMER(1994). Jackknifing for rapid identification of well-supported monophyletic groups (FAR~S & al. 1996) was performed using FARR~S' "Parsimony Jackknifing" program (FARRIS 1995), where the cutting point was set to 50% and the number of replicates made was 10000.

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Table 1. Additional sequences (with GenBank/EMBL accession numbers, current family and ordinal classification) used in the analysis U05194 U23537 X53497 M83257 Z30240 U23538 M83258 U00970 Z49753 Z30238 X58572 Z30241 U23539 L37535 U04202 M83259 L28066 L37537 L37538 X89219 X04971 M85054 M83261 X89218 Z27408 U05201 U00975 X54864 L37540 M27607 U23538 L37541 U04236 X89220 Z30239 L37532 M83263 Z49755 Z49754 X58057

Alternaria alternata Arthonia radiata Candida albicans Chaetomium elatum Cudonia confusa Dendrographa leucophaea Eremascus albus Eurotium rubrum Glaziella aurantiaca Gyromitra esculenta Histoplasma capsulatum Inermisia aggregata Lecanacis abietina Lecanora dispersa Leptosphaeria bicolor Leucostoma persoonii Malbranchea gypsea Morchella elata Mycocalicium albonigrum Nephroma arcticum Neurospora crassa Ophiostoma stenoceras Ophiostoma ulmi Peltigera neopolydactyla Plectania nigrella Pleospora herbarum Pleospora rudis Podospora anserina Porpidia crustulata Saccharomyces cerevisiae Schismatomma pericleum Sclerotinia sclerotiorum Septoria nodorum Solorina crocea Spathularia flavida Sphaerophorus globosus Thermoascus crustaceus Tuber rapaeodorum Urnula hiemalis Zygosaccharomyces rouxii

Pleosporaceae Arthoniaceae Saccharomycetaceae Chaetomiaceae ?Geoglossaceae Roccellaceae Eremascaceae Trichocomaceae Glaziellaceae Helvellaceae Onygenaceae Otideaceae Opegraphaceae Lecanoraceae Leptosphaeriaceae Valsaceae Onygenaceae Helvellaceae Mycocaliciaceae Nephromataceae Sordariaceae Ophiostomataceae Ophiostomataceae Peltigeraceae Sarcosomataceae Pleosporaceae Pleosporaceae Lasiosphaeriaceae Porpidiaceae Saccharomycetaceae ?Roccellaceae Sclerotiniaceae Dothideaceae Peltigeraceae Geoglossaceae Sphaerophoraceae Trichocomaceae Tuberaceae Sarcosomataceae Saccharomycetaceae

Dothideales Arthoniales Saccharomycetales Sordariales Leotiales Arthoniales Eurotiales Eurotiales Pezizales Pezizales Onygenales Pezizales Arthoniales Lecanorales Dothideales Diaporthales Onygenales Pezizales Caliciales s. 1. Lecanorales Sordariales Ophiostomatales Ophiostomatales Lecanorales Pezizales Pleosporales Pleosporales Sordariales Lecanorales Saccharomycetales Arthoniales Leotiales Dothideales Lecanorales Leotiales Lecanorales Eurotiales Pezizales Pezizales Saccharomycetales

Results We obtained SSU rDNA sequences for the lichenized fungi Bunodophoron scrobiculatum, Leifidium tenerum, Stereocaulon ramulosum, and Pilophorus acicularis between nucleotides 0004 and 1723 (Bunodophoron) and 1769 (Pilophorus) rela-


79

five to Saccharomyces cerevisiae standard (MANKIN & al. 1986; RUBTSOV & al. 1980). A small insertion (c. 60 nt.) was found at position 1046 (relative to the SSU rDNA sequence of Escherichia coli) in the Leifidium tenerum sequence. This position is a known site for insertions in lichens (GARGAS& al. 1995b) and the insertion may well be a degenerate group I intron (GRUBE & al. 1996). Ten replicates of a heuristic search resulted in a single most parsimonious tree (Fig. 1) of 1544 steps, with a consistency index of 0.56 and retention index of 0.68. The most parsimonious tree found by the heuristic search has a total support of 387 and a total support index of 0.25. Branch lengths (Fig. 1) correspond to number of character changes along the branches, figures above the branches represent branch support ("Bremer support") values (b) and figures below, where present, jackknife values above 50% (j). The analysis strongly supports the selected Sphaerophoraceae as a monophyletic group (b = 14, i.e. 14 extra steps needed to lose the group in the strict consensus tree; j = 99.5%), although the support for a monophyletic Lecanorales s. 1. (highlighted in Fig. 1) is low (b = 1, j < 50%). The resolution within Lecanorales has low branch support values (b = 1, j < 50%) for the group including Porpidia, Sphaerophoraceae, Lecanora, and Stereocaulaceae, b = 2 for this group excluding Porpidia, j < 50%. This may well be attributed to the limited sampling of representatives of the very diverse Lecanorales s. 1. at present, and better support for this group, or a part of it, can be expected with more taxa. The groupings with low branch support, values have as a rule also low jackknife values, and most of these groups are not supported at all by this procedure. Some groups with relatively high branch support values have, however, not particularly high jackknife values, such as the group consisting of Stereocaulaceae and Lecanora dispersa (b = 4 and j = 73%). It is interesting in this context also to note that the Peltigerineae clade has a comparatively high branch support value in this analysis (b = 5), this is contradictory to the bootstrap values presented by E~KSSON & STRAND(1995). The jackknife value for this group, however, is only 57%, pointing out the dilemma with support values derived from different techniques. B~MER'S (1988, 1994) branch support values have an advantage in that the theory behind the procedure is so readily understandable. However, the values may be misleading in that they are based on all characters, not just those characters with a high consistency index. A branch may receive a high branch support value simply because of homoplasious characters. Such effects can be reduced by using the resampling method of parsimony jackknifing. Mycocalicium and members of the orders Eurotiales and Onygenales form the sistergroup to the Lecanorales clade. The Mycocalicium-Eurotialean-Onygenalian clade has a relatively high branch support (b = 4) but a jackknife percentage below 50% in this analysis. Similarly the branch support for the clade containing Lecanotales and the Mycocalicium-Eurotialean-Onygenalian clade is relatively high in this analysis (b = 3) but the jackknife percentage is below 50%. Calicium tricolor was sequenced by GARGAS & TAYLOR(1995) but excluded from their analyses because of supposed long-branch attraction causing spurious placement. When using the resulting tree as a constraint to find the most parsimonious placement of Calicium, it was placed as sister taxon to Leotia lubrica (Leotiales), and not to Mycocalicium, as would have been expected following the

80

M. WEDIN & al.: Neurospora crassa Podospora anserina Chaetomium elatum 13 Ophiostoma ulmi 12 100 I Ophiostomastenoceras 91 Leucostomapersoonii ~ Dendrographaleucophaea 6 Schismatommapericleum radiata 87 I Lecanactis abietina ~ Septorianodorum Pleospora rudis 37 [- Pleospora herbarum 12 100 Alternaria alternata 100 Leptosphaeria bicolor [--- Spathulariafiavida 14 Cudonia confusa 100 Sclerotinia sclerotiorum Pilophorus acicularis 3 12 94

10 changes

3

5r

14 100

~

Stereocaulonraraulosum Lecanora dispersa Bunodophoronscrobiculatum

dia crustulata

Leifutium tenerum Sphaerophorus globosus 66 100

Nephroma arcticum 3~1~

49

Solorina crocea Peltigera neopolydactyla

Eremascus albus Malbranchea gypsea Histoplasma capsulatum Eurotium rubrum 100 1-.-.- Thermoascuscrustaceus 100 Mycocalicium albonigrum Urnula hiemialis Plectania nigrella 2~ Glaziella aurantiaca I'~ ~ lnermisia aggregata 9, I , Morchellaelata V 100 IT,,h,Gnyromitraesculenta Tuber"rapaeodorum 11 Saccharomyces cerevisiae 100 I Zygosaccharomyces rouxii Candida albieans 14

I

3 67

I-I

Fig. 1. Single most parsimonious tree (1544 steps) obtained by ten replicates of a heuristic search using TBR by PAUR Names in bold type represents new sequences, and Lecanorales s. 1. is highlighted. Branch lengths correspond to number of character changes along the branches, figures above the branches represent branch support ("Bremer support") values (b), and figures below represent jackknife values above 50% (j)

hypothesis to TIBELL (1984). We have also found both Calicium tricolor and Leotia lubrica to cause homoplasy in the analyses due to long branch attraction and subsequently excluded these sequences from the present analysis. Discussion

We propose that Sphaerophoraceae be classified in Lecanorales (including Peltigerales, here accepted as suborder Peltigerineae, following HENSSEN& JAHNS 1973, POELT 1974, RAMBOLD & TmEBEL 1992). The circumscription of Lecanorales is currently based mainly on the presence of the bitunicate lecanoralean ascus-type (HAFELLNER 1994), a feature lacking and probably secondarily lost in the Sphaerophoraceae, where the asci must be regarded as having reverted to the


81

plesiomorphic, unitunicate and prototunicate, stage. Lecanorales is subdivided into several suborders (HENSSEN& JAHNS 1973, POELT 1974, RAMBOLD& TmEBZL 1992, HA~LLNZR & al. 1994), several of which are often treated as orders in their own right. In the present analysis, the suborders Peltigerineae (Peltigera, Solorina and Nephroma), Cladoniineae (Porpidia, Stereocaulon and Pilophorus) and Lecanorineae (Lecanora) are represented. Cladoniineae is not supported as monophyletic in the present analysis. Among the suborders in Lecanorales, and on the basis of morphological, anatomical and chemical evidence, we propose that the Sphaerophoraceae belongs to one of the groups presently accommodated in the paraphyletic Cladoniineae. Many members of this paraphyletic group share the general type of thallus organisation (large fruticose to fruticose-foliose thalli) with the members of the Sphaerophoraceae. A number of Cladoniineae have cephalodia, and corticate cephalodia are present in at least one species of the Sphaerophoraceae (Sphaerophorus stereocauloides). Cephalodia are also reported from "Sphaerophorus yangii" (WANG-YANG• LAI 1976), this species was not available for study but it possibly a Bunodophoron species. The development of the pycnidia in the Sphaerophoraceae is of the Umbilicaria-type of VoBIs (Voms 1980; WEDS, unpubl.), and type of conidiophores is most similar to Voms' type VI (TIBELL1984, WEDIN 1993), thus corresponding to types widespread in Lecanoralean fungi, but rarely found outside this order. HENSSEN & JAHNS(1973) briefly compared the ascoma ontogeny between four representatives of Caliciales s. 1.; Calicium, Chaenotheca, Tholurna and Sphaerophorus. They found similarities between Sphaerophorus and Calicium regarding the carbonisation of the exciple and the possession of ascogenous cells with large lumen, and they also noted similarities between Chaenotheca and Tholurna, which however were said to differ considerably in their ontogeny from Sphaerophorus and Calicium. As Calicium tricolor could not be included in this analysis, other representatives of the Caliciaceae, including other species of Calicium, will be added with subsequent studies to evaluate the placement of this family and its possible relationships with the Sphaerophoraceae and the

Mycocaliciaceae. WEDIN (1990) investigated ascomatal ontogeny of Sphaerophorus and Leifidium tenerum, emphasising the later stages of the development, and the results from this study were later used in the separation of the genus Leifidium (WED1N 1993). Further studies of ascoma ontogeny, in combination with additional molecular characters, are needed to fully clarify the placement of Sphaerophoraceae in Lecanorales and for the identification of monophyletic groups in the paraphyletic Cladoniineae. The chemistry of the Sphaerophoraceae is rich and varied, and the groups of lichen substances present include anthraquinones, usnic acids, orcinol depsides,/3orcinol depsides, /3-orcinol depsidones and dibenzofuranes all of which are also present in Lecanorales and provides no contradiction on basis of the chemistry with the suggested placement in the Cladoniineae. Cladoniineae is, however, in the analysis presented here, found to be a paraphyletic grade group (Fig. 1) which probably includes several distinct lines. With the availability of a number of sequences from other representatives of

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Lecanorales s. 1., more detailed hypotheses on relationships can then be investigated in these and other ascomycetes. Portions of this study were undertaken during a visit of MW to AT's lab in Stockholm, and the main part during MW's post-doctoral stay at Karl-Franzens-Universit/it, Graz (Austria). MW is indebted to his host, PAULBLANZ,for generously providing working space and access to the molecular lab, and to MARTINGRUBEfor technical support. PAULADEPRIEST is gratefully acknowledged for helpful discussions of lecanoralean relationships and technical advice and support and OvE ERIKSSONiS thanked for access to the sequences of Solorina crocea, Nephroma arcticum and Peltigera neopolydactyla before their release. KA~ BREMERgave helpful advice regarding analyses of branch support, and PAXWOLSELEY and WmI~IAMPURVIShave kindly commented on the manuscript. MW acknowledges support by grants from the Swedish Royal Academy of Sciences (KVA) and Uppsala University, and by a generous postdoctoral grant from the Swedish Institute.

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