Tholurna dissimilis and generic delimitations in Caliciaceae inferred from nuclear ITS and LSU rDNA phylogenies (Lecanorales, lichenized ascomycetes)

Mycol. Res. 107 (12): 1403–1418 (December 2003). f The British Mycological Society

1403

DOI: 10.1017/S0953756203008694 Printed in the United Kingdom.

Tholurna dissimilis and generic delimitations in Caliciaceae inferred from nuclear ITS and LSU rDNA phylogenies (Lecanorales, lichenized ascomycetes)

Leif TIBELL Department of Systematic Botany, Evolutionary Biology Centre, Uppsala University, Norbyva¨gen 18D, S-75236 Uppsala, Sweden. E-mail : [email protected] Received 3 February 2003; accepted 10 September 2003.

The phylogenetic relationships of Tholurna dissimilis were investigated in relation to a phylogeny of twenty-three species in Caliciaceae and eighteen species from Physciaceae. ITS and LSU regions of the nuclear ribosomal DNA were used for the reconstruction of phylogenies by maximum parsimony methods. Calicium adaequatum was shown to be the closest relative of and possibly congeneric with Tholurna. Calicium is thus not monophyletic unless Tholurna is included. Calicium in the molecular phylogeny contains several distinct clades, which to some extent can be characterized morphologically. Cyphelium in a traditional sense is probably not monophyletic. Cyphelium s. str. has immersed apothecia, large smooth spores and a very thin excipulum throughout. C. inquinans and C. karelicum, which form a distinct and highly supported clade, may be accommodated in Acolium, possibly along with other Cyphelium and Calicium species. The phylogenies presented here do not support the recognition of neither Physciaceae nor Caliciaceae in a narrow sense, but they also do not exclude this. Numerous spliceosomal and unclassified insertions were found in the LSU sequences. They to some extent offered phylogenetic information both with respect to location and by their sequence similarities.

INTRODUCTION Calicioid lichens, colloquially known as ‘pin lichens’ or ‘stubble lichens ’, have received considerable attention during the last few decades. They are often used for bioindication of old-growth forests and many of the species are on Red Lists in different countries. As will be detailed below, calicioids is a historic concept based on a long 19th and 20th century tradition of considering these lichens and non-lichen fungi as forming a taxon and (or) natural group, despite having a variable morphology and ecology. This tradition was based on the conviction that the occurrence of a mazaedium, prototunicate asci, and to some extent stalked apothecia were reliable, cardinal characteristics for circumscription or recognition of the group (Tibell 1998). Tholurna dissimilis was described by Norman (1861) and attracted attention both because of its unusual morphology and habitat ecology. It is a mazaediate species with the apothecia sitting on podetia organized in almost spherical clusters with the podetia projecting in all directions. Tholurna also has a specialized ecology occurring in extremely exposed situations, on decaying twigs in the uppermost parts of low Picea trees growing above the tree-line. These twigs are often used by birds

as perching places and they are well manured. Tholurna was originally thought to be endemic to Scandinavia, but during the 20th century it was reported from British Columbia (Otto 1964) and Central Europe (O¨sthagen 1974). A monotypic family, Tholurnaceae, was suggested by Ra¨sa¨nen (1943). The peculiar pronounced ornamentation of the spores of Tholurna was noted by Sato (1967). Earlier classification of calicioid lichens and fungi Calicium and Sphaerophorus were the first calicioid lichen genera to be described (Persoon 1794). Acharius (1803, 1814, 1815, 1816, 1817) was particularly interested in calicioid lichens. He further described Cyphelium (Acharius 1815) and Coniocybe (Acharius 1816) placing them in ‘ Plantae Calicioidea ’ along with Calicium and Sphaerophorus. ‘ Plantae Calicioidea ’ was subsequently named ‘Coniocarpi ’ by Fries (1817) and considered by him to be one of four major groups of lichens. Fe´e (1824) referred calicioid lichens to two different families, Caliciaceae and Sphaerophoraceae. The Zahlbruckner (1903) classification of calicioid lichens recognized three families, Caliciaceae, Sphaerophoraceae and Cypheliaceae, and had a major impact

Tholurna and generic delimitations in Caliciaceae during the 20th century (Sato 1975). In Zahlbruckner’s classification, Caliciaceae, in addition to Calicium, included Chaenotheca, Coniocybe, Stenocybe, Pyrgidium, and Sphinctrina – none of them considered to belong to Caliciaceae today. The newly described Cypheliaceae included Cyphelium, Farriola, Pyrgillus, Tylophoron, and Tylophorella. Vainio (1927) accepted Caliciaceae, Sphaerophoraceae and Cypheliaceae as tribes, and added the Tholurnae as a separate tribe. Tholurnae included only the monotypic and morphologically aberrant Tholurna dissimilis. In Caliciaceae Vainio also included several newly described, non-lichenized genera such as Chaenothecopsis, Microcalicium, Strongylopsis, and Strongyleuma. Na´dvornı´ k (1942a) included Cypheliaceae in Caliciaceae as an ‘Unterfamilie ’, and the same arrangement was retained by Ra¨sa¨nen (1943), who, however, recognized Tholurnaceae along with Sphaerophoraceae and Caliciaceae in Coniocarpae. Poelt (1974) accepted this arrangement of Caliciales, but did not recognize Cypheliaceae at any level (the genera being included in Caliciaceae) and added the newly described Mycocaliciaceae (Schmidt 1970) for the non-lichenized genera Chaenothecopsis, Mycocalicium, Phaeocalicium, Stenocybe, and Strongyleuma. This group of fungi was included as an ‘ Anhang ’ to the Caliciales by Henssen & Jahns (1973), but these authors accepted Cypheliaceae as a family. The classification of calicioid lichens and fungi was thoroughly revised by Tibell (1984) in a paper where it was concluded that Caliciales as then construed was an heterogeneous assemblage of genera forming a biological but not a natural group. Monophyletic entities were identified and eight families were recognized : Caliciaceae, Calycidiaceae, Coniocybaceae, Microcaliciaceae, Mycocaliciaceae, Sclerophoraceae, Sphaerophoraceae and Sphinctrinaceae. In addition, eight tropical genera were not referred to a family but considered to have an uncertain position. In Caliciaceae, Tibell accepted Acroscyphus, Calicium, Cyphelium, Texosporium, Thelomma and Tholurna, thus rejecting both Cypheliaceae and Tholurnaceae. Caliciaceae was characterized as containing lichenized species with a mazaedium, and dark spores with a thick wall. Based on a cladistic analysis of morphological features, they were thought to be most closely related to Sphinctrinaceae. Acroscyphus is a monotypic genus described by Le´veille´ (1846). A. sphaerophorpoides has a thallus of aggregates of branched, podetia-like structures with apically immersed apothecia and a complex secondary chemistry (Shibata et al. 1968). It has a fragmented distribution in east and south-east Asia, South, Central and North America, and South Africa. It is rare and mainly occurs in high mountains. Calicium Pers. (Persoon 1794 ; lectotype Calicium viride Pers. 1794) contains some 40 species. They are all crustose and have stalked apothecia. They have a

1404 cosmopolitan distribution and mainly occur on bark or wood, more rarely on rocks. Cyphelium Ach. (Acharius 1815; lectotype Lichen tigillaris Ach. 1798=Cyphelium tigillare (Ach.) Ach. 1815) as presently circumscribed contains about 15 species. They are all crustose and have sessile or immersed apothecia. They have a cosmopolitan distribution and mainly occur on bark or wood, more rarely on rocks. Some of the species are commensals on lichens. Texosporium Na´dv. is a monotypic genus described by Na´dvornı´ k (in Tibell & Hofsten 1968). Its only species, T. sancti-jacobi, is crustose with immersed apothecia. It is endemic to south-east North America, where it occurs on soil in arid areas. Thelomma Massal. (Massalongo 1860 ; type Cyphelium mammosum Hepp 1857) contains seven species. They are crustaceous, occur on rocks or wood, and have immersed apothecia. Most species occur in areas with a mediterranean in the northern hemisphere. The genus is most probably heterogeneous. Molecular phylogenies of calicioid lichens and non-lichen fungi When SSU rDNA data became available, it was shown that Caliciaceae belongs to Lecanorales (Wedin & Tibell 1997, Wedin et al. 2000), whereas Sphinctrinaceae and Mycocaliciaceae belong to Mycocaliciales (Wedin & Tibell 1997, Tibell & Wedin 2000). Recently, mtSSU and nITS rDNA sequences were used by Wedin et al. (2002) to investigate the relationships between Physciaceae and Caliciaceae. In Physciaceae/ Caliciaceae two main groups were recognized : the Physcia-group, containing species of Anaptychia, Buellia, Phaeorrhiza, Physcia, and Rinodina, having a Lecanora-type of ascus ; and the Buellia-group, including species of Amandinea, Buellia, Calicium, Cyphelium, Pyxine, Texosporium, and Tholurna. In this phylogeny Tholurna was the sister species to Texosporium, and these two taxa in turn formed the sister group of Cyphelium inquinans. Molecular phylogenies of generic relationships in Lecanorales have primarily been obtained from SSU rDNA data, and only to a limited degree from LSU sequences (Bhattacharya et al. 2002a, Kauff & Lutzoni 2002) and mtSSU sequences (Wedin et al. 2002). There are, however, few LSU sequences of Lecanorales publicly available, particularly in the groups studied here (one in Caliciaceae and none in Physciaceae). Mitochondrial DNA has also been used for investigating generic relationships between parmelioid lichens (Crespo et al. 2001). Insertions have long been known to occur in the rDNA of lichenized fungi (DePriest & Been 1992). Most of these insertions have been identified as group I introns and are located in the SSU (DePriest 1993a, b, Gargas et al. 1995, Stenroos & DePriest 1998, Thell 1999, Thell & Miao 1999). Recently occurrences of

L. Tibell spliceosomal introns in the SSU have also been reported (Zoller et al. 1999, Cubero et al. 2000, Bhattacharya et al. 2002a). Intron sequence potential for reconstructing phylogenies has to some extent been investigated (Stenroos & DePriest 1998, Myllys et al. 1999, 2001, Thell 1999, Thell & Miao 1999, Thell et al. 2000). Introns have thus proved to contribute phylogenetic resolution in phylogenies based on SSU or ITS data. Insertions are however, problematic from some points of view. They evolve rapidly, are often lost (Bhattacharya et al. 2002b) and hence structural homology may be difficult to ascertain. Moreover they may degrade and consequently be difficult to recognize as belonging to a certain type of introns. Further, they seem to be transferred between organisms (Friedl et al. 2000, Coates et al. 2002) and may be transposed within the genome. A common assumption has been that introns at a certain insertion point are homologous, but this is not necessarily so since different introns may have moved to new locations and repeated insertions of non-homologous insertions may have taken place at the same position. Recently introns in the LSU have also received attention since fungal LSU rDNA seems to have been invaded by spliceosomal introns (Bhattacharya et al. 2002a).

1405 Table 1. Sequences downloaded from GenBank. Species

DNA

Accession no.

Amandinea punctata Anaptychia ciliaris A. runcinata B. dijiana B. disciformis B. elegans B. epigaea B. frigida B. schaereri Calicium viride Diplotomma alboatrum Fulgensia bracteata F. fulgens Heterodermia boryi H. speciosa Hyperphysica adglutinata Leifidium tenerum Lempholemma polyanthes Leptogium cyanescens Melanelia stygia M. stygia Phaeophyscia orbicularis Physcia adscendens P. aipolia Rinodina sophodes R. tunicata Texosporium sancti-jacobi

ITS1-5.8S-ITS2 rDNA ITS1-5.8S-ITS2 rDNA ITS1-5.8S-ITS2 rDNA ITS1-5.8S-ITS2 rDNA ITS1-5.8S-ITS2 rDNA ITS1-5.8S-ITS2 rDNA ITS1-5.8S-ITS2 rDNA ITS1-5.8S-ITS2 rDNA ITS1-5.8S-ITS2 rDNA LSU rDNA ITS1-5.8S-ITS2 rDNA LSU rDNA ITS1-5.8S-ITS2 rDNA ITS1-5.8S-ITS2 rDNA ITS1-5.8S-ITS2 rDNA ITS1-5.8S-ITS2 rDNA ITS1-5.8S-ITS2 rDNA LSU rDNA LSU rDNA ITS1-5.8S-ITS2 rDNA LSU rDNA ITS1-5.8S-ITS2 rDNA ITS1-5.8S-ITS2 rDNA ITS1-5.8S-ITS2 rDNA ITS1-5.8S-ITS2 rDNA ITS1-5.8S-ITS2 rDNA ITS1-5.8S-ITS2 rDNA

AF250780 AF250782 AJ42124 AF250788 AF250784 AJ421415 AF250785 AF281307 AF250791 AF355670 AF408677 AF277668 AF278772 AJ421419 AF250794 AF250795 AF117998 AF356691 AF356672 AF115763 AF421434 AF250799 AF250802 AF224390 AF250813 AF250816 AY143396

Aim of the investigation

Sequences downloaded from GenBank are listed in Table 1. New sequences were produced from isolations as indicated in Table 2. Collections identified by UPSC numbers were isolated from cultured material. Lichen sample vouchers of the new sequences are listed in Table 2.

Either 10–30 apothecia or about 0.5 cm2 of an axenic culture of the mycobiont was extracted. PCR amplification was conducted by using the primers ITS1-F (Gardes & Bruns 1993), and ITS 4 (White et al. 1990) to specifically amplify the fungal ITS1-5.8S-ITS2, and by using the primers ITS1-F (Gardes & Bruns 1993) or 5.8Sr and LR7 (both : http:// www.biology.duke.edu/fungi/mycolab/primers.htm) to amplify the LSU. The PCR ran for 35 cycles (1 min at 94 xC, 1 min at 54 x, 45 s at 72 x with a 4 s cyclex1 extension at 72 x) using ABI or Promega Taq. Part of the amplifications were carried out by using Ready To Go PCR Beads (Pharmacia Biotech, Piscataway, NJ) according to the protocol of the producer. Before sequencing the PCR product was purified using the Qiaquick Spin kit and protocol by Qiagen. Sequencing reactions were carried out with the following primers : ITS1-F, ITS2, ITS3, ITS4, 5.8Sr, LR0R, LR3, LR5 and LR7 (White et al. 1990). The sequencing reaction ran for 26 cycles (30 s at 96 x, 15 s at 50 x, 4 min at 60 x) for sequencing by the BigDye labelling method (Perkin–Elmer) and for 30 cycles (20 s at 95 x, 15 s at 50 x, 3 min at 60 x) for sequencing by the MegaBACE labelling method (Pharmacia–Amersham). Contigs were assembled in Bioedit (http://www.mbio.ncsu.edu/RNaseP/info/ programs/BIOEDIT/bioedit.html).

Extractions and PCR amplifications

Sequence alignment and parsimony analyses

For the new sequences total DNA was extracted from the samples using the Qiagen DNeasy Plant Mini Kit.

The LSU sequences were aligned manually. The ITS1-5.8S-ITS2 sequences were aligned using ClustalW

Generic delimitation in Caliciaceae at present rests on morphological features and the object of this investigation is to find the closest relatives of Tholurna in relation to phylogenies based on LSU and ITS rDNA sequences of calicioid species. In a first step a phylogeny was derived from LSU sequences from a selection of calicioid species and other Lecanorales species using Lempholemma polyanthes in the Lichinaceae as outgroup. Then, in a more extensive taxon sampling, the relationships between Tholurna and other members of Caliciaceae and Physciaceae were investigated using analyses of ITS1-5.8S-ITS2 sequences. A further aim is to clarify phylogenetic relationships between genera in the Caliciaceae and Physciaceae, and also to explore the phylogenetic signal in the insertions of the LSU rDNA sequences investigated. MATERIAL AND METHODS Material studied

Tholurna and generic delimitations in Caliciaceae

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Table 2. Newly produced sequences.

Species

Code

Origin

Collection (herbarium location/culture collection)

DNA

GenBank accession no.

Acroscyphus sphaerophoroides Calicium abietinum C. abietinum C. adaequatum C. adaequatum C. adaequatum C. adaequatum C. adspersum C. chlorosporum C. denigratum C. denigratum C. denigratum C. glaucellum C. glaucellum C. glaucellum C. glaucellum C. hyperelloides C. hyperelloides C. lenticulare C. montanum C. montanum C. pinastri C. quercinum C. quercinum C. robustellum C. salicinum C. salicinum C. trabinellum C. tricolor C. tricolor C. viride C. viride C. viride Cyphelium inquinans C. inquinans C. inquinans C. karelicum C. notarisii C. notarisii C. tigillare C. tigillare C. tigillare C. tigillare Diplotomma alboatrum Leifidium tenerum Tholurna dissimilis T. dissimilis

T141 T117 T117 T101 T101 T235 T235 V2299 T120 T102 T102 T236 T139 T139 T208 T209 T210 T210 T283 T147 T147 T284 T170 T170 T211 T122 T212 T173 T214 T214 T136 T136 T174 T138 T149 T149 T271 T126 T126 T140 T140 T269 T269 T183 V2091 T146 T146

Nepal India India Sweden Sweden Sweden Sweden New Zealand India Sweden Sweden Sweden Sweden Sweden Sweden Argentina New Zealand New Zealand Canada Portugal Portugal Czech Republic Sweden Sweden New Zealand Sweden New Zealand Sweden New Zealand New Zealand Sweden Sweden Sweden Sweden Sweden Sweden Sweden Sweden Sweden Sweden Sweden Sweden Sweden Sweden New Zealand Sweden Sweden

Shimizu (TNS) Tibell 21852 (UPS) Tibell 21852 (UPS) Tibell 22182 (UPS) Tibell 22182 (UPS) Tibell 22468 (UPS) Tibell 22468 (UPS) Tibell 16902/UPSC2299a Tibell 21867 (UPS) Tibell 22200 (UPS) Tibell 22200 (UPS) Tibell 22469 (UPS) Tibell 22319 (UPS) Tibell 22319 (UPS) Tibell 17027/UPSC2520 Tibell 18507 (UPS)/culture Tibell 16708/UPSC2152 Tibell 16708/UPSC2152 Articus 732 (UPS) Boom 2344 (hb. v. d. Boom) Boom 2344 (hb. v. d. Boom) Palice 4044 (hb. Palice) Tibell 22287 (UPS) Tibell 22287 (UPS) Tibell 16689/UPSC2149 Tibell 22284 (UPS) Tibell 16581/UPSC2069 Tibell 22350 (UPS) Tibell 16640/UPSC2150 Tibell 16640/UPSC2150 Tibell 22297 (UPS) Tibell 22297 (UPS) Tibell 22351 (UPS) Tibell 22316 (UPS) Tibell 22283 (UPS) Tibell 22283 (UPS) 2001, Tibell (UPS) Hermansson 10058 (hb. Hermansson) Hermansson 10058 (hb. Hermansson) Tibell 22343 (UPS) Tibell 22343 (UPS) Tibell 22719 (UPS) Tibell 22719 (UPS) 2001, Nordin (UPS) Tibell 16587/UPSC2091 Tibell 22346 (UPS) Tibell 22346 (UPS)

ITS1-5.8S-ITS2 rDNA ITS1-5.8S-ITS2 rDNA LSU rDNA ITS1-5.8S-ITS2 rDNA LSU rDNA ITS1-5.8S-ITS2 rDNA LSU rDNA LSU rDNA ITS1-5.8S-ITS2 rDNA ITS1-5.8S-ITS2 rDNA LSU rDNA ITS1-5.8S-ITS2 rDNA LSU rDNA ITS1-5.8S-ITS2 rDNA ITS1-5.8S-ITS2 rDNA ITS1-5.8S-ITS2 rDNA ITS1-5.8S-ITS2 rDNA LSU rDNA ITS1-5.8S-ITS2 rDNA ITS1-5.8S-ITS2 rDNA LSU rDNA ITS1-5.8S-ITS2 rDNA ITS1-5.8S-ITS2 rDNA LSU rDNA ITS1-5.8S-ITS2 rDNA ITS1-5.8S-ITS2 rDNA ITS1-5.8S-ITS2 rDNA ITS1-5.8S-ITS2 rDNA ITS1-5.8S-ITS2 rDNA LSU rDNA ITS1-5.8S-ITS2 rDNA LSU rDNA ITS1-5.8S-ITS2 rDNA ITS1-5.8S-ITS2 rDNA ITS1-5.8S-ITS2 rDNA LSU rDNA ITS1-5.8S-ITS2 rDNA ITS1-5.8S-ITS2 rDNA LSU rDNA ITS1-5.8S-ITS2 rDNA LSU rDNA ITS1-5.8S-ITS2 rDNA LSU rDNA LSU rDNA LSU rDNA ITS1-5.8S-ITS2 rDNA LSU rDNA

AY450562 AY450563 AY452501 AY450564 AY452502 AY450565 AY452503 AY452504 AY450566 AY450567 AY452505 AY450568 AY453646 AY450569 AY450570 AY450571 AY450572 AY453634 AY450573 AY450574 AY453635 AY450575 AY450576 AY453636 AY450577 AY450578 AY453645 AY450579 AY450580 AY453637 AY450581 AY453638 AY450582 AY450583 AY450584 AY453639 AY450585 AY452496 AY453640 AY452497 AY453641 AY452498 AY453642 AY452500 AY453643 AY452499 AY453644

a

UPSC, isolations made from material kept in the Uppsala University Culture Collection of Fungi with culture collection number.

as implemented by the Bioedit software packet. Insertions were excluded from the LSU alignment. The alignments can be obtained from the author on request. The ITS region contained areas which were difficult to align. In one analysis gap-rich and ambiguous parts were deleted, resulting in an alignment including 413 characters, thus being 157 characters (27.5 %) shorter than the original one. The data matrix was analysed by the computer software PAUP* 4.0 beta 10 (Swofford 1998). Analyses of LSU and ITS rDNA applied a heuristic search using 1000 replicates (MaxTrees: increase=auto, collapse branches if maximum branch length=0,

MulTrees=yes, gaps treated as missing data, characters given equal weight). The analyses of the introns used a branch-and-bound search (MaxTrees=100: increase=auto, collapse branches if maximum branch length=0, MulTrees=yes, gaps treated as missing data, characters given equal weight). Jack-knife support values were obtained from an heuristic search using 1000 replicates each with 5 random addition sequence replicates, TBR branch swapping algorithm, steepest descent off, MaxTrees=100: increase=auto, MulTrees on, Hold=1, collapse zero length branches when maximum length is zero, gaps treated as missing data, nominal deletion of characters (36.79 %).

L. Tibell

1407

Fig. 1. Jack-knife consensus tree from an analysis of 23 nLSU rDNA sequences representing Leptogium cyanescens, Tholurna dissimilis, other species in Caliciaceae, Diplotomma alboatrum, and further species in the Lecanorales using Lempholemma polyanthes as outgroup. Numbers above branches refer to jack-knife values, and below the branches clade names and a letter identify the clades indicated. Well-supported clades are formed by the Tholurna-clade (A: Calicium adaequatum, Tholurna dissimilis) ; the Tholurna-clade plus Cyphelium inquinans ; the Calicium viride-clade (D : C. quercinum and C. viride); and the Cyphelium tigillare-clade (E : C. notarisii and C. tigillare).

Lichinaceae was chosen as outgroup in the analysis of the LSU sequences since it was identified by Schultz et al. (2001) and Kauff & Lutzoni (2002) as sister group to a large clade including Lecanorales. The phylogeny of Caliciaceae/Physciaceae obtained from the LSU sequences allowed the localization of a closer outgroup for the ITS phylogeny, and hence a less ambiguous alignment of the ITS-based phylogeny. Thus Melanelia stygia was chosen as outgroup in an analysis of ITS sequences among lecanoralean taxa forming a sistergroup with species of Caliciaceae and Physciaceae in the LSU phylogeny (Fig. 1). Selection of Fulgensia bracteata or Leifidium tenerum as outgroup did not significantly change the topology of the phylogeny and only to a small degree affected the support values of the strongly supported clades. In order to obtain enhanced resolution a phylogeny was also inferred from combined LSU and ITS data for a selection of the species. Branch and bound searches (MaxTrees=100, collapse branches if maximum branch length=0, MulTrees= yes, gaps treated as missing data, characters given

equal weight) of some intron matrices were also undertaken. Jack-knife values were obtained from an heuristic search (5 random additional replicates, TBR branch swapping algorithm, steepest descent off, MulTrees off, collapse zero length branches when maximum length is zero, gaps treated as missing data) using 1000 replicates, resample=normal, with 36.9 % of the characters deleted in each replicate. Spliceosomal introns (Bhattacharya et al. 2002a) were identified by sequence features. The unclassified introns were searched for Group I features, but several of them were short and only few of the characteristic features as described by Cech (1988) were found.

RESULTS Sequences Sixteen new LSU rDNA and 29 new ITS1-5.8S-ITS2 sequences were produced in this study (Table 2).

Tholurna and generic delimitations in Caliciaceae Alignments Alignment of the generally rather conserved LSU sequences excluding the introns was easy and were made by hand. Alignment of some regions of the ITS sequences and the introns was difficult and ClustalW was used to assist the alignment. The alignment parameters gap opening penalty and gap extension penalty were varied for each alignment to obtain an optimal homology as judged from the balance between block stability in conservative regions and homology degree in gap-rich regions. Gap open penalty was varied between 5 and 80 and gap extension penalty between 0.1 and 1. Generally alignments judged as optimal were obtained close to the default values (10 and 0.2), but sometimes other values were preferred. Strongly supported branches in the analyses were retained over a wide range of gap open and gap extension values, whereas the topology in the poorly supported parts of the trees sometimes varied when the alignment parameters were changed.

A phylogeny inferred from LSU sequences The LSU alignment (gap open penalty=20 ; gap extension penalty=0.4) included 23 taxa, and 166 out of the 1179 characters were parsimony informative. The heuristic search yielded 7 most parsimonious trees (CI=0.51, RI=0.59). A jack-knife tree is presented in Fig. 1 indicating jack-knife support. A jack-knife tree was illustrated since it included some weakly supported clades not found in the consensus tree. In this part of the analysis Lempholemma polyanthes (Lichinaceae) was used as outgroup. Lempholemma was part of a trichotomy with Leptogium cyanescens (Collemataceae) and the rest of the Lecanorales forming a monophyletic group (jack=100). This phylogeny identified suitable outgroup candidates – Fulgensia bracteata (Teloschistaceae), Leifidium tenerum (Sphaerophoraceae) and Melanelia stygia (Parmeliaceae) – for further analyses of the monophyletic but rather weakly supported Caliciaceae/Physciaceae (jack=58). Strongly supported clades in the study group included the Tholurna-clade (A : Tholurna dissimilis and Calicium adaequatum, jack=100) ; Cyphelium inquinans, Tholurna dissimilis and Calicium adaequatum ( jack=100); The Cyphelium tigillare-clade (E : Cyphelium notarisii and C. tigillare, jack=99) ; and the Calicium viride-clade (D : represented by Calicium quercinum and C. viride, jack=100). The Calicium glaucellum-clade (B : represented by C. denigratum, C. glaucellum and C. montanum) formed a weakly supported clade ( jack=56), and Diplotomma alboatrum, as the only representative of Physciaceae, formed a polytomy with all these groups in addition to three more Calicium species belonging to the Calicium hyperelloides-clade (C. abietinum, C. hyperelloides, C. tricolor ; Fig. 2).

1408 A phylogeny inferred from ITS1-5.8S-ITS2 sequences The ITS alignment (gap open penalty=10 ; gap extension penalty=0.2) included 51 taxa, and 332 out of the 593 characters were parsimony informative. The heuristic search yielded 29 most parsimonious trees (CI=0.34, RI=0.58). A strict consensus tree of these including jack-knife support (actual deletion level 36.7 %) is presented in Fig. 2. Melanelia stygia was used as outgroup since it was part of a polytomy with Fulgensia bracteata, Leifidium tenerum and the study group in the LSU based analysis. Choosing Fulgensia bracteata or Leifidium tenerum as outgroup did not change the topology of the resulting consensus tree. Caliciaceae and Physciaceae formed a well-supported monophyletic clade (jack=99). The relationships between the species of the study group are in some cases weakly supported and partly unresolved. Species in Caliciaceae and Physciaceae formed a polytomy, which includes some well-supported monophyletic groups. Thus Calicium adaequatum, and Tholurna dissimilis form a strongly supported clade ( jack=100), the Tholurna-clade (A). Cyphelium inquinans and C. karelicum also form a strongly supported clade ( jack=99), the Acolium-clade (F). All these species together with Acroscyphus sphaerophoroides and Texosporium sancti-jacobi form a moderately well-supported clade ( jack=87). Another well-supported clade contains several Calicium species ( jack=95), with two subclades. One of these ( jack=79) is the Calicium glaucellum-clade (B : C. denigratum, C. glaucellum, C. montanum, C. pinastri, and C. trabinellum). The sister group to this clade containing C. abietinum, C. hyperelloides, C. tricolor and C. robustellum, the Calicium hyperelloides-clade (C), has good support ( jack=92). Cyphelium notarisii and C. tigillare form a highly supported clade ( jack=100), the Cyphelium tigillare clade (E), and so do Calicium quercinum, C. salicinum and C. viride, the Calicium viride clade (D, jack=100). Some representatives of Physciaceae also form wellsupported clades. Thus Physcia aipolia and P. adscendens form a well-supported clade ( jack=96), and together with Rinodina sophodes a group with good support is formed ( jack=86). Anaptychia ciliaris and A. runcinata ( jack=99) and Heterodermia boryi and H. speciosa ( jack=100) form strongly supported groups. All these species along with R. tunicata, Phaeophyscia orbicularis and Hyperphyscia adglutinata together form a rather well-supported group ( jack=87), the Physciagroup (Wedin et al. 2002). Another clade includes Buellia dijiana, B. disciforme, B. elegans, and B. epigaea ( jack=91), representing the Buellia-group (Wedin et al. 2002). Diplotomma alboatrum and a clade consisting of Calicium chlorosporum and C. lenticulare remain unresolved in a basal polytomy. The position of Amandinea punctata, Buellia frigida and B. schaereri in the Buellia-group is poorly supported. The ITS region contained areas which were difficult to align. In one analysis gap-rich and ambiguous

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Fig. 2. Strict consensus tree of 19 equally parsimonious trees from an analysis of 51 ITS1-5.8S-ITS2 rDNA sequences representing Tholurna dissimilis with other species in Caliciaceae and Physciaceae using Melanelia stygia as outgroup. Numbers above branches refer to jack-knife values, and below the branches clade names and a letter identify the clades are indicated. Well-supported clades are formed by the Tholurna-clade (A : T. dissimilis and Calicium adaequatum) ; the Calicium viride-clade (D : C. viride, C. salicinum and C. quercinum) ; the Cyphelium tigillare-clade (E : C. tigillare and C. notarisii) ; and the Acolium-clade (F : Cyphelium inquinans and C. karelicum). The Calicium glaucellum-group (B); the Calicium hyperelloides-group (C: C. abietinum, C. hyperelloides, C. robustellum and C. tricolor); and the Physcia-group are moderately supported. Caliciaceae/Physciaceae also receives a high support.

regions were deleted, resulting in an alignment including 413 characters, thus being 157 characters (27.5%) shorter than the original one. An analysis of this tree resulted in 278 most parsimonious trees and a less resolved consensus tree (not shown). The Tholurna-, Calicium viride-, C. glaucellum-, and Cyphelium tigillareclades were also here monophyletic and strongly supported. The Physcia-group, however, was divided into three clades receiving moderate support.

Relationships were also less resolved in the Calicium glaucellum-clade and in the Calicium viride-clade. In the alignment based on the complete sequences the Caliciaceae/Physciaceae comprised a polytomy of eight branches. In the analysis based on the 413 character alignment the corresponding polytomy included 21 branches. Generally the support values for comparable clades were lower. Thus removal of the gap-rich regions seems to have caused loss of phylogenetic signal.

Tholurna and generic delimitations in Caliciaceae

1410

Fig. 3. The single most parsimonious tree from an analysis of 18 ITS1-5.8S-ITS2/LSU rDNA sequences representing Tholurna dissimilis with other species in Caliciaceae and Buellia alboatra using Melanelia stygia as outgroup. Numbers above branches refer to jack-knife values, and below the branches clade names and a letter identify the clades are indicated. Well-supported clades are the Tholurna-clade (A : Calicium adaequatum, Tholurna dissimilis) ; a more inclusive clade formed by the Tholurna-clade and Cyphelium inquinans ; the Calicium viride-clade (D : Calicium quercinum, C. salicinum and C. viride); and the Cyphelium tigillare-clade (E : Cyphelium notarisii and C. tigillare). The Calicium glaucellum-clade (B : C. denigratum, C. glaucellum and C. montanum) and the Calicium hyperelloides-clade (C: C. abietinum, C. hyperelloides and C. tricolor) are moderately supported. All members of Caliciaceae along with Diplotomma alboatrum also form a well-supported clade.

A phylogeny based on combined ITS1-5.8S-ITS2 and LSU sequences The LSU-ITS alignment (gap open penalty=10 ; gap extension penalty=0.2) included 19 taxa, and 383 out of the 1977 characters were parsimony informative. The heuristic search yielded one most parsimonious tree (CI=0.53, RI=0.60), which including jack-knife support (actual deletion level 36.8 %) is presented in Fig. 3. Melanelia stygia was, as in the LSU analysis, used as outgroup. In this analysis, Caliciaceae forms a strongly supported group (jack=100) along with the only

representative of Physciaceae, Diplotomma alboatrum, that was included in the study. Tholurna forms a strongly supported group with Calicium adaequatum (jack=100), the Tholurna-clade (A), and their sister group relationship to Cyphelium inquinans is also strongly supported (jack=100). The Calicium virideclade (D : C. quercinum, C. salicinum and C. viride) is strongly supported (jack=100). The Calicium hyperelloides-clade (C: C. abietinum, C. hyperelloides and C. tricolor) is moderately supported (jack=85), and so is the Calicium glaucellum-clade (B: C. denigratum, C. montanum and C. glaucellum). Again Cyphelium

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Table 3. Insertions in the nuclear rDNA LSU. Location refer to the homologous position before the intron in Saccharomyces cerevisiae GenBank accession no. J013551.

Species

GenBank accession no.

Location

Length

Clade

Flanking sequences

Calicium montanum C. quercinum Cyphelium notarisii C. tigillare C. tigillare C. tigillare C. tigillare Diplotomma alboatrum Calicium adaequatum T101 C. adaequatum T235 C. hyperelloides C. quercinum C. adaequatum T101 C. adaequatum T235 Tholurna dissimilis Calicium glaucellum C. abietinum C. tricolor C. adspersum Tholurna dissimilis Calicium abietinum C. montanum Diplotomma alboatrum Calicium glaucellum C. tricolor C. adaequatum T101 C. adaequatum T235 C. abietinum C. denigratum

Afzx Afzx Afzx Afzx Afzx Afzx Afzx Afzx Afzx Afzx Afzx Afzx Afzx Afzx Afzx Afzx Afzx Afzx Afzx Afzx Afzx Afzx Afzx Afzx Afzx Afzx Afzx Afzx Afzx

921 921 921 921 921 947 947 1021 1021 1021 1021 1021 1044 1044 1044 1044 1147 1147 1318 1318 1318 1318 1377 1377 1377 1383 1383 1383 1383

56 243 59 53 53 50 50 210 54 54 (189) (61) 205 205 353 61 70 56 333 229 267 248 234 314 228 223 (179) 163 (69)

B D E E E E E

CCGAAAG/ATGGTGAA CCGAAAG/ATGATGAA CCGAAAG/ATGGTGAA CCGAAAG/ATGGTGAA CCGAAAG/ATGGTGAA TAGGGTG/AAGCCAGA TAGGGTG/AAGCCAGA TATAGGG/GCGAAAGA TATAGGG/GCGAAAGA TATAGGG/GCGAAAGA CAAAGGG/ TATAGGG/ AACCATCT/ATAGCTGG AACCATCT/ATAGCTGG AACCATCG/AGTAGCTG AATCATCT/ATACCTGG CATCCTTA/ACCTATT CATCCTTA/ACCTATT CACACACG/GTGTTAATT CACACACG/GTGTTAGTT CACAAAAG/GTGTTAATT CACAAAGC/GTGTTAATT CTAAGGAGT/GTGTAA CTAAGGAGT/GTGTAA CTAAGGAGT/GTGTAA GTGTAG/CAACTCACC GTGTAG/ GTGTAA/ GTGTAA/CAACTTACC

A A C D A A A B C C A C B B C A A C B

S1 S2 S3 S4 S5 S6 S7 N1 S8 S9 S10 S11 N2 N3 N4 N5 S12 S13 N6 N7 N8 N9 N10 N11 N12 N13 N14 N15 N16

A, Tholurna-clade (cf. Fig. 2); B, Calicium glaucellum-clade; C, C. hyperelloides-clade; D, C. viride-clade; E, Cyphelium tigillare-clade. S, spliceosomal intron; N, not classified insertion. Intron lengths within parentheses indicate incomplete introns.

notarisii and C. tigillare form a strongly supported clade (E, jack=100). The lower branches in the in-group have low support (jack<50) and that leaves the relationships between the aforementioned groups unresolved. Insertion patterns There are numerous insertions in the LSU sequences located at eight different positions (Table 3). The number of insertions in one species varies from none to three. Some insertions were recognized as spliceosomal introns (Bhattacharya et al. 2002a). They were located at four different positions relative to the sequence of Saccharomyces cerevisiae (921, 947, 1021, 1147) and varied considerably in length (50–243 nucleotides) and donor and acceptor sequences (Table 4). Eight other insertions (Table 3) located at three positions (1044, 1377, 1383) were very variable in length (61–314 nucleotides) and secondary structure and contained one to four of the sequence signatures characteristic of the conserved regions P, Q, R or S (Cech 1988) of Group I introns. Some of these insertions may be akin to what was described as ‘degenerate Group I introns’ from the SSU rDNA of Arthonia lapidicola (Grube et al. 1996) and Lecanorales (Stenroos & De Priest 1998). In the alignment of these introns it

Table 4. Location and sequence features of spliceosomal introns in the rDNA LSU sequences studied. Positions refer to the homologous position before the intron in Saccharomyces cerevisiae GenBank accession no. J013551. Position

Exon end

Donor site

Branch site

Acceptor site

921 767

GAAAG

CTAAC

CAG AAG

947 793 1021 867

GGGTG

GTATG GTACG GTAAG GTACG

TACTAAC

TAG

TAGGG

GTAAGC GTAAGT

TGCTAAC AGCTAAC

ATCCTTA

GTAAGT

AAGGAGT

GTCGGT

TGCTAAT CACTAAT GACTAAA

AGG AGC TCG CAG TAG GGT

1147 993 1377

was clear, however, that some parts were highly conserved, such as the (AC)AGACTA(AA) site, partly corresponding to the R site (Cech 1988). Eight insertions (Table 3) of variable length (163–353) were not recognized as spliceosomal and had no sequence signatures characteristic of Group I introns. They were partly located at the same positions as the previously mentioned group of introns (1044 and 1383), but also at position 1318.

Tholurna and generic delimitations in Caliciaceae

1412 N6-N9 (jack=100) and N10-N12 (jack=91). All random sequences formed weakly supported clades with each other or remained unresolved in the network, the only exception being R5, which formed a clade with N2 (jack=71). It was concluded that non-random phylogenetic signal was indeed obtained by the alignment procedure also in the homoplasy rich intron sequences.

Spliceosomal introns were found in position 921, 947, 1021 and 1147. Spliceosomal introns at position 921 occur in species representing three different clades (B, D and E ; Table 3). Only Cyphelium tigillare had a spliceosomal intron at position 947. Spliceosomal introns at position 1021 are found in species from clades A and D. Calicium abietinum and C. tricolor, both belonging to the C. hyperelloides-clade (C), have spliceosomal introns at position 1147. Unclassified introns occur at position 1044 in C. adaequatum (clade A) and C. glaucellum (clade B). An unclassified long insertion in Tholurna dissimilis (clade A) is also located here. Unclassified introns at position 1377 occur in C. glaucellum (clade B) and C. tricolor (clade C). Few taxa were investigated and the species are represented by one collection only, except for in C. adaequatum and Cyphelium tigillare. In these two species both specimens had identical sets of insertions. Unclassified insertion at position 1318 occur in clades A, B and C, and at position 1383 in clades B and C. Some phylogenetic information may be obtained from insertion patterns. Thus in the Tholurna-clade (A) spliceosomal introns occur at position 1021, unclassified introns at positions 1044, 1318 and 1383. The Calicium glaucellum-clade (B) has spliceosomal introns at positions 921 and unclassified introns at positions 1044, 1318, 1377 and 1383. The Calicium viride-clade (D) has spliceosomal introns at position 921 and 1021. The Cyphelium tigillare-clade (E) has spliceosomal introns at positions 921 and 947. The Calicium hyperelloides-clade (C) has a short spliceosomal intron at position 1147, and unclassified introns at position 1318 and 1383.

Thirteen spliceosomal introns (Tables 3 and 4) were assumed to be homologous irrespective of position. They were aligned (gap open penalty=10 ; gap extension penalty=0.2, minor manual adjustments). A branch-and bound search included 54 out of 190 characters that were parsimony informative resulted in one network (Fig. 4) 198 steps long (CI=0.64, RI=0.62). Spliceosomal introns S4 and S5 from Cyphelium tigillare grouped together and received good support (jack=90, differing in one transition and one transversion). These introns and S3 (from C. notarisii) formed a weakly supported group (jack=60). Intron S1 (from Calicium montanum) in turn is their closest sister intron, although with only moderate support (jack=70). All these introns, like S2 of Calicium quercinum are located at position 921. Introns S6 and S7 from Cyphelium tigillare at position 947 form a strongly supported clade (jack=100). Introns S8 and S9 from Calicium adaequatum at position 1021 are strongly supported (jack=100). Cyphelium tigillare and Calicium quercinum thus both have two different spliceosomal introns which possibly have separate evolutionary histories.

Insertion phylogenies

Unclassified insertions

Homologies between different insertions are difficult to establish and insertions may have been transposed to different locations within the genome. The phylogeny of the insertions was here investigated regardless of their location in the LSU. Under theses conditions it is also very difficult to identify an outgroup. The degree of positional homology between the introns is low and the alignment is unstable with respect to changes in the alignment parameters, and the resulting phylogenies differ apart from in the relationships of a few, strongly supported groups. To find out if there was phylogenetic signal or random pattern causing the structure of the resulting trees an experiment was undertaken. In an alignment fifteen random sequences of similar length and relative base frequency as the fifteen non-spliceosomal introns were created. The fifteen insertion sequences were aligned together with the fifteen random sequences. Default alignment parameters were used and in a heuristic search a consensus tree of 2 equally parsimonious trees was obtained (not shown). Introns N2, N3, N13 and N14 from Calicium adaequatum plus N4 from Tholurna dissimilis (jack=94) formed a strongly supported group, like

To find out if the unclassified insertions would show phylogenetic relationships they were aligned (gap open penalty=20; gap extension penalty=1) using ClustalW. The heuristic search included 235 out of 451 characters that were parsimony informative resulted in one most parsimonious tree 965 steps long (CI=0.60, RI=0.57) presented in Fig. 5. Introns N2 and N3 at position 1044 from Calicium adaequatum formed a strongly supported clade ( jack=100), as did N13 and N14 at position 1383, also from C. adaequatum ( jack=100). Intron N4 from Tholurna dissimilis was sister group to N13 and N14 in a weakly supported clade ( jack=56) including also the four introns from C. adaequatum. All introns from position 1377, N10 from Diplotomma alboatrum, N11 from C. glaucellum and N12 from C. tricolor, formed a distinct clade from all the other introns ( jack=99). It is interesting to note that intron N10 from Buellia alboatra grouped with introns from Calicium (N11 and N12). The insertion N4 and N7 in Tholurna thus seem to have a different recent evolutionary histories. Moreover insertions with different evolutionary background may have also have occurred both in Calicium glaucellum (N5, position

Spliceosomal introns

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Fig. 4. The most parsimonious tree from a branch-and-bound analysis of 13 spliceosomal introns in LSU rDNA sequences from species in Caliciaceae. Number at branches refer to jack-knife values. Well-supported clades are formed by introns S8 and S9 at position 1021 from two different specimens of Calicium adaequatum ; S6 and S7 at position 947 from two different specimens of Cyphelium tigillare ; and S4 and S5 at position 921 from two different specimens of Cyphelium tigillare, differing in one transition and one transversion). Furthermore a moderately supported group is formed by S4 and S5 (from Cyphelium tigillare) and S3 from C. notarisii (all at position 921) and S3, S4 and S5 together with S1 from Calicium montanum, also at position 921 form a moderately supported group.

1044 and N11, position 1377) and C. abietinum (N8, position 1318 and N15, position 1383). Since these insertions were difficult to align a range of alignment parameters were tested. Strongly supported clades, however, did not change when the alignment parameters were altered. Thus if gap open penalty was decreased to 5 and gap extension penalty to 0.2 the N6-N9 clade was still supported ( jack=62), and the N10/N11/N12-clade ( jack=99) was still strongly supported, but N15/N16 received only weak support (jack=61).

DISCUSSION The position of Tholurna The LSU phylogeny (Fig. 1) gives a weak support ( jack=58) to a clade comprising 17 specimens of Caliciaceae together with Diplotomma alboatrum in the Physciaceae. Tholurna forms a strongly supported clade with Calicium adaequatum (jack=100). The sister group relationship of these species with Cyphelium

inquinans is also strongly supported (jack=100). In the ITS based phylogeny (Fig. 2) Tholurna again appears as the sister group of Calicium adaequatum with strong support (jack=100). Consequently also in the combined ITS-LSU phylogeny (Fig. 3), Tholurna forms a strongly supported group with Calicium adaequatum (jack=100). Both Tholurna (N4) and Calicium adaequatum (N2, N3) have insertions at position 1044. Otherwise these two species differ in their insertion profiles, C. adaequatum having a splicesomal intron (S8, S9) at position 1021 and another insertion (N13, N14) at position 1383, whereas Tholurna has a different insertion (N7) at position 1318. In the phylogeny (Fig. 5) insertions N13/N14 from Calicium adaequatum formed the sister group to N4 from Tholurna, although only with weak support. The close relationship between Tholurna and Calicium may seem surprising, particularly since Tholurna in a long 20th century tradition has been considered as quite isolated, and sometimes even referred to a distinct tribe (Vainio 1927) or family (Na´dvornı´ k 1942, Ra¨sa¨nen 1943). Is there any morphological or

Tholurna and generic delimitations in Caliciaceae

1414

Fig. 5. The most parsimonious tree from a branch-and-bound analysis of 14 unclassified insertions in LSU rDNA sequences from species in Caliciaceae along with two unclassified insertions from Diplotomma alboatrum. Numbers at branches refer to jack-knife values. Well-supported clades are formed by N2 and N3 at position 1044 from Calicium adaequatum and N13 and N14 at position 1383 likewise from C. adaequatum. N4 from Tholurna, also at position 1044, joins this clade, although with low support. All these represent the Tholurna-clade. Insertions N6 from Calicium adspersum and N8 from C. abietinum at position 1318 form a strongly supported group, as do N15 from C. abietinum and N16 from C. denigratum at position 1383.

ecological evidence supporting the relationship between Tholurna and C. adaequatum ? Some morphological similarities may actually be found. The capitulum in C. adaequatum is campanulate. This is unique in Calicium, and the apothecia of Tholurna have a very similar shape. Both Tholurna and C. adaequatum also have a very strong surface ornamentation of the ascospores consisting of spirally arranged ridges resulting from the irregular rupturing of the outermost part of the spore wall. Spiral ornamentations formed by a similar ontogentic process are, however, also found in other species of Calicium, but the resulting ornamentation is not quite as distinctive as in these two species. There is also an ecological similarity insofar that C. adaequatum is the only species in Calicium that almost exclusively occurs on twigs, preferably of Alnus and Populus (Tibell 1999) and Tholurna also exclusively occurs on twigs, but on twigs of Picea abies. The phylogenies do not support recognition of the family Tholurnaceae as different from Caliciaceae if Calicium adaequatum is kept in Calicium. These phylogenetic relationships also have consequences for Calicium. C. adaequatum cannot be retained in Calicium unless both Tholurna and probably also the Acolium-clade and Acroscyphus

sphaerophoroides and Texosporium sancti-jacobi are included in Calicium. A more attractive alternative is perhaps to include C. adaequatum in Tholurna, or if retaining Tholurna as a monophyletic genus to describe a new genus for C. adaequatum. The position of Cyphelium Although the taxon sampling has not been designed to reveal the phylogenetic relationships of Cyphelium, a few remarks may be made from the phylogenies presented. C. inquinans and C. karelicum form a strongly supported clade, the Acolium-clade (Fig. 2) in the ITS phylogeny ( jack=99), distinct from the also strongly supported C. tigillare-clade ( jack=100). In the analysis of Wedin et al. (2000) based on SSU rDNA sequences, C. tigillare and C. inquinans also did not form a monophyletic group. In the ITS phylogeny the Acolium-clade is sister group to Acroscyphus sphaerophoroides and Texosporium sancti-jacobi with moderate support ( jack=76). In the LSU and combined ITS-LSU phylogenies, C. inquinans groups with the Tholurna-clade ( jack=100 and 99, respectively). C. notarisii and C. tigillare also form a strongly supported group in the LSU ( jack=100), ITS ( jack=100) and combined ITS-LSU phylogeny ( jack=100). C. notarisii

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and C. tigillare have no unclassified introns, but both have a spliceosomal intron in position 921, and in the phylogeny based on spliceosomal introns (Fig. 4) S4 and S5 from C. tigillare form the sister group to S3 from C. notarisii albeit with rather low support (jack=60). Tibell (1971) remarked that Cyphelium ‘is probably heterogeneous, and it is not at all clearly delimited from Calicium. Thus the present conception of Cyphelium is mainly a practical one, based on a few easily observed characters. Though groups within the genus are undoubtedly very homogenous the relationships between these groups, and consequently the coherence of the genus, is still difficult to establish ’. Now we may have tools to start unravelling these relationships. C. inquinans and C. karelicum belong to ‘Sect. Tympanium Na´dv. ’ (Na´dvornı´ k 1942a, b ; not validly published), characterized by having sessile apothecia with a very thick excipulum at the base and ornamented spores. Cyphelium notarisii and C. tigillare belong to ‘Sect. Scyphulium Na´dv. ’ (Na´dvornı´ k 1942a, 1942b ; not validly published), characterized by having immersed apothecia, a very thin excipulum and smooth spores. ‘Sect. Scyphulium Na´dv. ’, here corresponding to the Cyphelium tigillare-clade, includes the type species of Cyphelium, C. tigillare, and further C. notarisii and most probably C. trachylioides, although this species was not included in the present study. Other species should then be excluded from Cyphelium. Pseudacolium, based on Cyphelium notarisii, becomes a synonym of Cyphelium. Acolium (Ach.) S. Gray (untypified) may be based on Calicium tympanellum Ach. (=Cyphelium inquinans) and the name is thus available for species closely related to Cyphelium inquinans. Acolium also includes C. karelicum and possibly some other Cyphelium s. lat. species, and sequence comparisons should be made with species such as C. lucidum, C. pinicola, and C. lecideinum. It may also include some additional Calicium s. lat. species. These relationships need to be further studied by sequence comparisons.

includes the type species of Calicium, and may form the core of Calicium in a more narrow sense. In the ITS phylogeny C. chlorosporum and C. lenticulare somewhat surprisingly form a strongly supported clade (jack=99). Here C. chlorosporum has spores with a strong spiral ornamentation, whereas the spore ornamentation in C. lenticulare is formed by minute warts. A large and moderately well-supported clade in the ITS phylogeny (jack=79) is the C. glaucellum-clade (B). Three species from this clade only receive low support as a monophyletic group in the LSU phylogeny (jack=56), but the C. glaucellum-clade as represented by the same three species in the combined analysis is moderately well supported (jack=84). The spores in this group have an irregular, areolate or weakly striate surface ornamentation but a strong spiral ornamentation does not occur. These species occur in cool temperate areas and contain depsides, depsidones and the tetronic acid derivative vulpinic acid, but no xanthones. The C. hyperelloides-clade (C), containing C. abietinum, C. hyperelloides, C. tricolor and C. robustellum, has a good support in the ITS phylogeny (jack=92) and moderate support in the combined analysis (jack=85). Spliceosomal introns at position 1147 were found in C. abietinum (S12) and C. tricolor (S13). An insertion (N8) was found at position 1318 in C. abietinum, and another (N12) at position 1377 in C. tricolor, but no insertion was found in C. hyperelloides. This clade contains species with a warm temperate to tropical distribution and all, except for C. abietinum, contain xanthones in the thallus, secondary substances only found in this group in the genus. Texosporium sancti-jacobi was found to be the sister species of Tholurna by Wedin et al. (2002), but this relationship was not supported here. In the ITS phylogeny Acroscyphus was the sister taxon to Texosporium and these two species in turn were most closely related to the Acolium-group, whereas the relationship to Tholurna was more distant.

The phylogeny of Calicium

The phylogeny of Caliciaceae and Physciaceae

Although the present analyses do not resolve the basal relationships in Caliciaceae and do not include a wide enough taxon sampling, some comments may be made on the phylogeny of the Calicium species included. C. quercinum, C. salicinum and C. viride, form a wellsupported group, the C. viride-clade (D, Fig. 2) in the ITS phylogeny (jack=100). C. quercinum and C. viride likewise form a strongly supported group in the LSU phylogeny (jack=100), and in the combined analysis the C. viride-clade is strongly supported (jack=100). In the LSU analysis C. adspersum is the sister group to these species with weak support (jack=52). No insertion was found in C. viride, but a spliceosomal intron occurred in position 1021 in C. quercinum. These four species all have spores with a spiral ornamentation and occur in cool temperate to temperate areas. The group

The phylogenies here are not easy to compare to those of Wedin et al. (2002) insofar that their analysis is based on combined data from nITS rDNA and mtSSU in a different taxon sampling. Nevertheless in the present study a representative selection of the species treated by Wedin et al. (2002) were included in the ITSbased analysis. Wedin et al. (2002) in their molecular phylogeny found two main groups, coinciding with the Buellia-group and the Physcia-group characterized by ascus features. The Buellia-group has a Bacidia-type of ascus, while the Physcia-group has a Lecanora-type of ascus (Rambold et al. 1994). The four representatives of Caliciaceae included in the study of Wedin et al. (Calicium viride, Cyphelium inquinans, Texosporium sancti-jacobi and Tholurna dissimilis) were all members of the Buellia-group, but did not form a monophyletic

Tholurna and generic delimitations in Caliciaceae

1416

group. This also led Wedin et al. to the conclusion that Caliciaceae and Physciaceae should be merged. In the present ITS phylogeny (Fig. 2) the resolution in the lower part of the Caliciaceae and Physciaceae phylogeny was low, which resulted in a comprehensive polytomy of all species of Caliciaceae and Physciaceae with strong support (jack=99). The species of Caliciaceae did not form a monophyletic group and are represented by the clades discussed in previous sections. With respect to Physciaceae, the representatives of the Buellia-group formed a well-supported clade with Buellia dijiana, B. disciformis, B. elegans and B. epigaea (jack=91). Amandinea punctata, B. frigida and B. schaereri also group with the Buellia-clade, although with low support (<50%). The following representatives of the Physcia-group formed a moderately well-supported group : Anaptychia ciliaris, A. runcinata, Heterodermia boryi, H. speciosa, Hyperphyscia adglutinata, Phaeophyscia orbicularis, Physcia aipolia, P. adscendens, Rinodina sophodes and R. tunicata (jack=87). These two Physciaceae clades were part of the unresolved basal polytomy of Caliciaceae and Physciaceae along with Diplotomma alboatrum. This last species in the ITS phylogeny does not group with Buellia s. str. as has earlier been shown by Molina et al. (2000). This ITS phylogeny thus only offers limited information on the relationships between Caliciaceae and Physciaceae. It does not, however, offer evidence neither for each of these families not being monophyletic, nor for a wider concept of Physciaceae as suggested by Wedin et al. (2002). Information from a more extensive taxon sampling and additional genes will hopefully further clarify the phylogeny of and relationships between Physciaceae and Caliciaceae. Pending this no formal nomenclatural changes are suggested here, but some possible consequences for the classification of these families will be summarized below.

Calicium may be monophyletic, but this could not be shown in the present analyses. Calicium contains some well-supported clades. Thus the Calicium viride-clade (Calicium quercinum, C. salicinum and C. viride) is strongly supported and is morphologically characterized by having spores with a distinctive spiral ornamentation. If Calicium turns out to be paraphyletic this group may represent the core genus. The C. glaucellum-clade is moderately supported comprising C. denigratum, C. glaucellum, C. montanum, C. pinastri and C. trabinellum. These species occur in temperate to cool temperate areas, have a varied secondary chemistry but do not contain xanthones. C. glaucellum is morphologically very similar to C. trabinellum. They are sympatric in large areas, but there is a difference insofar that C. trabinellum occurs in cooler areas outside their areas of sympatry and C. glaucellum in warmer. Their ITS sequences are also quite similar, and in the analysis presented here C. glaucellum is paraphyletic versus C. trabinellum. This problem is in need of further study based on more sequences and a wider geographical representation. The sister group to this clade, the Calicium hyperelloides-clade, is well supported, and includes C. abietinum, C. hyperelloides, C. tricolor and C. robustellum. These species occur in temperate to tropical areas and all, except for C. abietinum, contain xanthones. The phylogenies presented here do not support the recognition of neither Physciaceae nor Caliciaceae in a narrow sense. But they also do not exclude this. The recognition of a wider Physciaceae including Caliciaceae as suggested by Wedin et al. (2002) cannot be refuted but is also not supported. Numerous spliceosomal, tentative group I and unclassified insertions were found in the LSU sequences. They to some extent offered phylogenetic information both with respect to insertion position and by their sequence similarities.

CONCLUSIONS

ACKNOWLEDGEMENTS

Tholurna dissimilis in this taxon sampling is most closely related to, and possibly congeneric with Calicium adaequatum. There are also some morphological and ecological features supporting this relationship. Cyphelium in a traditional sense is not monophyletic. Along with the type species, C. tigillare, C. notarisii (and most probably C. trachylioides) form a strongly supported monophyletic group, Cyphelium s. str. It is characterized by having immersed apothecia, a thin excipulum throughout and large, smooth spores. Some other species traditionally included in Cyphelium, could be accommodated in Acolium, based on C. inquinans as type. Here belongs also C. karelicum. These species are characterized by having sessile apothecia, a thick excipulum, particularly at the base and comparatively small, strongly ornamented spores. Calicium as presently circumscribed is not monophyletic. After the exclusion of C. adaequatum,

The fellow members of the lichen group in Uppsala, Kristina Articus, Roland Moberg, Anders Nordin, and Rolf Santesson, have supplied cordial support and taken an interest in the investigations. It is my pleasure to acknowledge the Department of Systematic Botany, Evolutionary Biology Centre, Uppsala University, for providing basic facilities for sequencing, and in particular the technical assistance with the sequencing reactions from Nahid Heidari. M. Vinuesa kindly placed two unpublished sequences at my disposal. Mats Wedin kindly gave me access to a sequence of Texosporium sancti-jacobi before its publication. Pieter v. d. Boom, Janolof Hermansson, Anders Nordin and Zdenek Palice generously supplied material for DNA-isolations. The work was supported by grants from the Olsson-Borg Foundation of Uppsala University, and the Magnus Bergvall Foundation, Stockholm.

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Corresponding Editor: M. Grube