Phylogenetic reassessment of the Teloschistaceae (lichen-forming Ascomycota, Lecanoromycetes)

Pere  Navarro-Rosinés

The Teloschistaceae is a widespread family with considerable morphological and ecological heterogeneity across genera and species groups. In order to provide a comprehensive molecular phylogeny for this family, phylogenetic analyses were carried out on sequences from the nuclear ribosomal ITS region obtained from 114 individuals that represent virtually all main lineages of Teloschistaceae. Our study confirmed the polyphyly of Caloplaca, Fulgensia and Xanthoria, and revealed that Teloschistes is probably non-monophyletic. We also confirm here that species traditionally included in Caloplaca subgenus Gasparrinia do not form a monophyletic entity. Caloplaca aurantia, C. carphinea and C. saxicola s. str. groups were recovered as monophyletic. The subgenera Caloplaca and Pyrenodesmia were also polyphyletic. In the subgenus Caloplaca, the traditionally recognized C. cerina group was recovered as monophyletic. Because this study is based solely on ITS, to maximize taxon sampling, the inclusion of phylogenetic signal from ambiguously aligned regions in MP (recoded INAASE and arc characters) resulted in the most highly supported phylogenetic reconstruction, compared with Bayesian inference restricted to alignable sites.

mycological research 112 (2008) 528–546 journal homepage: www.elsevier.com/locate/mycres Phylogenetic reassessment of the Teloschistaceae (lichen-forming Ascomycota, Lecanoromycetes) Ester GAYAa,b,*, Pere NAVARRO-ROSINÉSb, Xavier LLIMONAb, Néstor HLADUNb, François LUTZONIa a Department of Biology, Duke University, Box 90338, Durham, NC 27708, USA Departament de Biologia Vegetal (Unitat Botànica), Universitat de Barcelona, Av. Diagonal 645, 08028 Barcelona, Spain b article info abstract Article history: The Teloschistaceae is a widespread family with considerable morphological and ecological Received 20 April 2007 heterogeneity across genera and species groups. In order to provide a comprehensive mo- Received in revised form lecular phylogeny for this family, phylogenetic analyses were carried out on sequences 1 October 2007 from the nuclear ribosomal ITS region obtained from 114 individuals that represent virtu- Accepted 15 November 2007 ally all main lineages of Teloschistaceae. Our study confirmed the polyphyly of Caloplaca, Ful- Corresponding Editor: gensia and Xanthoria, and revealed that Teloschistes is probably non-monophyletic. We also H. Thorsten Lumbsch confirm here that species traditionally included in Caloplaca subgenus Gasparrinia do not form a monophyletic entity. Caloplaca aurantia, C. carphinea and C. saxicola s. str. groups Keywords: were recovered as monophyletic. The subgenera Caloplaca and Pyrenodesmia were also poly- Arc phyletic. In the subgenus Caloplaca, the traditionally recognized C. cerina group was recov- Caloplaca ered as monophyletic. Because this study is based solely on ITS, to maximize taxon Fulgensia sampling, the inclusion of phylogenetic signal from ambiguously aligned regions in MP INAASE (recoded INAASE and arc characters) resulted in the most highly supported phylogenetic ITS reconstruction, compared with Bayesian inference restricted to alignable sites. Letrouitiaceae ª 2007 The British Mycological Society. Published by Elsevier Ltd. All rights reserved. Molecular phylogenetics Teloschistaceae Teloschistes Xanthoria Introduction Suprageneric treatment within the Teloschistales The latest classification of the Teloschistales (Eriksson 2006) includes one large (Teloschistaceae) and two much smaller families (Letrouitiaceae and Megalosporaceae). Caloplaca is the largest genus within the Teloschistaceae, along with 11 other smaller genera (Table 1) according to Eriksson (2006). Fulgensia, Teloschistes, and Xanthoria (with ca. ten, 30, and 30 species, respectively) are the next largest genera, followed by mainly monotypic genera with species segregated from the four main genera within this family (e.g. Cephalophysis, Huea, Ioplaca, Josefpoeltia, Seirophora, Xanthodactylon, Xanthomendoza, Xanthopeltis). The classification of the Teloschistaceae at the ordinal level has been highly debated, especially its placement within the Lecanorales versus Teloschistales. The order Teloschistales was first described by Hawksworth & Eriksson (1986), with a single family (Teloschistaceae). Later, two more families * Corresponding author. Department of Biology, Duke University, Box 90338, Durham, NC 27708, USA E-mail address: eb62@duke.edu 0953-7562/$ – see front matter ª 2007 The British Mycological Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.mycres.2007.11.005 Zahlbruckner (1931, 1940) Ozenda & Clauzade (1970) Eriksson & Hawksworth (1986) Kärnefelt (1989) Hawksworth et al. (1995) Eriksson (1999) Kirk et al. (2001) Eriksson et al. (2003) Eriksson (2006) Blastenia) Bombyliospora) Caloplaca) Lethariopsis)) Protoblastenia) Teloschistes)) Xanthoria)) – Bombyliospora# Caloplaca Fulgensia – Protoblasteniax Teloschistes Xanthoria Apatoplaca – – Caloplaca Cephalophysis Follmannia? Fulgensia Ioplaca Leproplaca – – Seirophora Teloschistes Xanthopeltis Xanthoria Apatoplaca – – Caloplaca Cephalophysis – Fulgensia Ioplaca – – – Seirophora Teloschistes Xanthodactylon Xanthopeltis Xanthoria Apatoplaca – – Caloplaca Cephalophysis – Fulgensia Ioplaca – – – – Teloschistes Xanthodactylon Xanthopeltis Xanthoria Apatoplaca – – Caloplaca Cephalophysis – Fulgensia Ioplaca Josefpoeltia – – – Seirophora Teloschistes Xanthodactylon Xanthomendoza Xanthopeltis Xanthoria Apatoplaca – – Caloplaca Cephalophysis – Fulgensia Ioplaca Josefpoeltia – – – Seirophora Teloschistes Xanthodactylon Xanthomendoza Xanthopeltis Xanthoria – – – Caloplaca Cephalophysis – Fulgensia Ioplaca Josefpoeltia – – – Seirophora Teloschistes Xanthodactylon Xanthomendoza Xanthopeltis Xanthoria – – – Caloplaca Cephalophysis – Fulgensia Huea Ioplaca Josefpoeltia – – – Seirophora Teloschistes Xanthodactylon Xanthomendoza Xanthopeltis Xanthoria Phylogenetic reassessment of the Teloschistaceae Table 1 – Genera accepted within the family Teloschistaceae according to different authors Genus name not included in classification because the classification preceded the protologue of the genus. – Genus name existed at the time of the classification, but was nevertheless excluded from the proposed classification. ) Family Caloplacaceae according to Zahlbruckner (1926). )) Family Teloschistaceae according to Zahlbruckner (1898). # Genus synonym to Megalospora (Megalosporaceae). Most of the species from this family were transferred to Letrouitia. x Currently included in the family Psoraceae. 529 530 were included in this order (Eriksson & Hawksworth 1991; Hafellner 1988): Letrouitiaceae (Hafellner & Bellemère 1981a) and Fuscideaceae (Hafellner 1984), even though Kärnefelt (1989, 1994) never accepted the Fuscideaceae as part of the Teloschistales. Earlier, Mattick (1951) had introduced the order Caloplacales in an attempt to join the taxa with polarilocular ascospores. However, this name was not validly published (Art. 36 of the Code). Later on, the order Teloschistales was recognized as a suborder within the Lecanorales (Hafellner et al. 1994; Rambold et al. 1991; Rambold & Triebel 1992). Tehler (1996) included the Fuscideaceae, Letrouitiaceae, and Teloschistaceae within the Lecanorales suborder Teloschistineae. This classification had already been proposed by Henssen & Jahns (1973) based on ontogenic characters. Poelt (1974) also included the Teloschistaceae within the order Lecanorales, but in a different suborder, Buelliineae, based on morphological and anatomical features of the thallus and apothecia. Eriksson (1999) and Eriksson et al. (2001, 2003, 2004) maintained the classification of the Teloschistaceae within the Lecanorales, which was confirmed by molecular data (e.g. Stenroos & DePriest 1998; see revision by Grube & Winka 2002), but without support. Nevertheless, several authors have maintained that the Teloschistales is a valid order (e.g. Kirk et al. 2001). The inclusion of the family Teloschistaceae in the order Lecanorales was revisited in light of recent higher-level phylogenetic studies. Miadlikowska & Lutzoni (2004) demonstrated that none of their phylogenetic trees revealed the order Lecanorales (sensu Eriksson et al. 2003; or Tehler 1996) as monophyletic. Therefore, they emphasized the need of recognizing a monophyletic order Lecanorales (s. str.) restricted to the core of the Lecanorales apart from the Peltigerales and Teloschistales. Lumbsch et al. (2004) preferred a broader use of the order Lecanorales, to include Teloschistales, Caliciales, and Peltigerales, which was recovered as a well-supported monophyletic group. Peršoh et al. (2004) also accepted the Lecanorales in a broad sense and referred to a clade that included the suborder Teloschistineae, with Caloplaca, Megalospora, and Xanthoria. Wiklund & Wedin (2003) also considered the Lecanorales in a broad sense, including the Teloschistaceae and the Caliciaceae, and accepting the suborder Teloschistineae. However, this broad circumscription of the Lecanorales is redundant with the subclass Lecanoromycetidae and diminishes the number of ranks needed for the classification of this large subclass within the Lecanoromycetes (Miadlikowska & Lutzoni 2004). The recognition of the Lecanorales, Teloschistales, and Peltigerales within the Lecanoromycetidae (sensu Miadlikowska & Lutzoni 2004, and Miadlikowska et al. 2007) is in agreement with the classification adopted by a consortium between Myconet (Eriksson 2006), The Dictionary of the Fungi (Kirk et al. 2001) and GenBank (Hibbett et al. 2007). The Fuscideaceae have recently been excluded from the Teloschistales based on a multilocus phylogenetic study by Reeb et al. (2004). Consequently, this family has a status of incerta sedis within the Lecanoromycetidae in Eriksson’s (2006) classification of the Ascomycota. However, a new study (Miadlikowska et al. 2007) shows that the Fuscideaceae is part of the newly recognized Umbilicariales (Hibbett et al. 2007), and that the Letrouitiaceae remains in the Teloschistales. The Letrouitiaceae is a monotypic family (Letrouitia), with about 15 E. Gaya et al. species. Members of this family are widely distributed in subtropical and tropical regions, and are corticolous. Letrouitia includes species that were classified in the species complex Bombyliospora domingensis (Hafellner 1981; Hafellner & Bellemère 1981a), and that were placed in a separate genus based mainly on the unique structure of asci and ascospores. Finally, the Megalosporaceae was classified within the Teloschistales (Eriksson 2005), based on Helms et al. (2003) and Lutzoni et al. (2004). The family Teloschistaceae was first described by Zahlbruckner (1898) who grouped together foliose and fruticose taxa having polarilocular or 4-locule ascospores (Xanthoria, Teloschistes and Lethariopsis). Later, Zahlbruckner (1926) described a second family, Caloplacaceae, for crustose taxa with ascospores that are polarilocular, or rarely with 3-4 locules or simple. At that time he considered four genera to be part of the Caloplacaceae: Caloplaca, Blastenia, Bombyliospora, and Protoblastenia (Table 1). Bombyliospora species have been transferred to Letrouitia (Hafellner 1981; Hafellner & Bellemère 1981a), and Bombyliospora itself is now considered a synonym of Megalospora (Megalosporaceae; Hafellner & Bellemère 1981b). Currently, Protoblastenia is classified within the Psoraceae, and Blastenia (Massalongo 1852, 1853) is a synonym of Caloplaca. The recognition of the Caloplacaceae as a distinct family from the Teloschistaceae was later rejected. Fink (1910) and Malme (1926) already fused the two families into one dTeloschistaceae. Alternatively, crustose genera were grouped within the family Blasteniaceae (Dodge & Baker 1938), including the Placodiaceae (Räsänen 1943). Dodge (1948) used Blasteniaceae also to refer to the Teloschistaceae, even though this change of name was illegitimate, and Rudolph (1955) concurred. Furthermore, Dodge (1971) tried to introduce another family name, Xanthoriaceae (not validly published; Art. 36 of the Code), to encompass Xanthodactylon, Xanthopeltis and Xanthoria. Kärnefelt (1989) conducted an exhaustive revision of this family and the order Teloschistales, and accepted ten genera within the Teloschistaceae (Table 1). With a few exceptions, this remains the main classification in use. The last taxonomical treatment of the family Teloschistaceae was by Oxner (1993). In his flora of the Ukraine, the family Teloschistaceae includes Caloplaca, Fulgensia, Protoblastenia, Pyrrhospora, Teloschistes, and Xanthoria. Oxner (1993) also accepted Caloplaca elegans instead of Xanthoria elegans, as well as C. australis and C. schistidii instead of Fulgensia australis and F. schistidii, respectively. Members of the Teloschistaceae are usually easily recognized by the frequent presence of anthraquinones, giving them an orange to yellow colour (Kþ purple; Santesson 1970a). They include the full spectrum of thallus forms ranging from fruticose to endolithic crustose. Their photobiont belongs to the green alga Trebouxia or its related genera. Their apothecia usually have well-developed thallin margins. The external layer of the ascus tip is Iþ (blue) and ascospores are discharged through a longitudinal slit (Kärnefelt 1989). As Kärnefelt (1989) pointed out, initially, polarilocular ascospores were thought to be a diagnostic trait for this family, but with the inclusion of other genera, such as Apatoplaca, transferred to Caloplaca by Wetmore (1994), Cephalophysis, Fulgensia, and Xanthopeltis, which have simple or septate spores, the main features defining the family had to be reconsidered. Phylogenetic reassessment of the Teloschistaceae As Søchting & Lutzoni (2003) pointed out, the delimitation among genera included within the Teloschistaceae is highly artificial and in need of revision, especially for the closely related species within Caloplaca, Fulgensia, Teloschistes, and Xanthoria. The distinction between the foliose species (Xanthoria) and lobed crustose or placodioid species (e.g. Caloplaca subgenus Gasparrinia) is especially tenuous (Arup & Grube 1999; Kärnefelt 1989; Poelt & Hafellner 1980; Søchting & Lutzoni 2003; Wetmore & Kärnefelt 1998). Infrageneric treatment of Caloplaca Caloplaca is a large and phenotypically heterogeneous genus. More than 1000 species names have been published for Caloplaca alone (Søchting & Lutzoni 2003). However, Kärnefelt (1989) estimated that the family Teloschistaceae comprises approximately 580 species, and Hawksworth et al. (1995) reduced the number to 525 species. Hence, the number of taxa included in Caloplaca and the Teloschistaceae are unresolved at this time. Caloplaca comprises a group of lichens with hyaline polarilocular ascospores, occasionally plurilocular (3-4 locules) or, rarely, simple with a slight wall thickening at the equatorial region. Thalli are mostly crustose, usually with anthraquinones present in the thallus and apothecium. Several anthraquinone syndromes have been reported for this genus, sometimes together with other lichen metabolites (Santesson 1970b; Søchting 1997, 2001). Asci and ascospores have been thoroughly studied by Bellemère & Letrouit-Galinou (1982) and Honegger (1978). Caloplaca is cosmopolitan and found in most xeric and mesic habitats. There have been several attempts to subdivide Caloplaca into smaller taxonomical units that have been recognized as separate genera over time: Blastenia, Follmania, Gasparrinia, Gyalolechia, Huea, Kuttlingeria, Mawsonia, Meroplacis, Polycauliona, Pyrenodesmia, Triophthalmidium, and Xanthocarpia. Most of these segregated genera were reclassified in Caloplaca as subgenera (e.g. subgenus Gasparrinia) or other ranks. Most of these taxonomical units were based mainly on a single character and have been considered as highly artificial (Kärnefelt 1989). Consequently, the current circumscription of Caloplaca is very similar to what was established more than a century ago (Søchting & Lutzoni 2003). Wade (1965) and Clauzade & Roux (1985) provided the most comprehensive infrageneric treatments for Caloplaca. Wade (1965), in his study of Caloplaca in the British Islands, described four sections. Section Caloplaca comprises species with crustose thalli and apothecia with or without a thalline margin and with a continuous or discontinuous photobiont layer present under the hypothecium. This section includes species with apothecia of various pigments. Section Triophthalmidium includes taxa with crustose thalli, apothecia without a thalline margin and ascospores with four cells. Section Gasparrinia refers to taxa with placodioid or squamulous thalli and apothecia with a thalline margin. Section Leproplaca includes taxa with leprarioid thalli that are usually sterile. In their lichen flora of occidental Europe, Clauzade & Roux (1985) proposed six subgenera. Three of these subgenera (Caloplaca, Gasparrinia, and Leproplaca) were similar to Wade’s (1965) concept. However, subgenus Caloplaca sensu Clauzade & Roux was further divided into three groups (C. citrina, 531 C. cerina, and C. ferruginea groups), and subgenus Gaspirrinia was subdivided into five groups (C. aurantia, C. aurea, C. carphinea, C. persica, and C. saxicola; Table 2). Subgenus Pyrenodesmia includes species with white–grey or nearly blackish thalli that are K or Kþ violet, and dark apothecia with a thin or absent thalline margin and an epithecium Kor Kþ violet. Subgenus Gyalolechia regroups species with ascospores that have a thin equatorial wall thickening (< 3 mm). Subgenus Xanthocarpia encompasses species with thin or endolithic thalli and with four-locular or three-septate ascospores. Subsequently, Hansen et al. (1987) subdivided the Caloplaca species from Greenland into ten groups: Cerinae, Chalybeae, Citrinae, Ferrugineae, Nivales, Pauliae, Pyraceae, Saxicolae, Sinapispermae, and Trachyphyllae; and Poelt & Hinteregger (1993) established 21 groups to accommodate the Himalayan species of Caloplaca. ‘Subgenus Gasparrinia’ In most floristic studies Caloplaca subgenus Gasparrinia refers to a group of species with placodiod, squamulose, effigurated (i.e., lobate) thalli, usually with anthraquinones in the thallus and/or apothecia. This group was first described by Tornabene (1849), who listed eight species. Five of the species were later considered outside this group (Wetmore & Kärnefelt 1998). The three remaining species, which Fries (1871) referred to as Caloplaca section Gasparrinia, were Caloplaca callopisma (syn. C. aurantia), C. cirrochroa, and C. murorum (syn. C. saxicola). According to Poelt (1954), the most successful study made on this group was the one by Weddell (1876). Later on, the use of an infrageneric category including all lobed species was accepted by several authors (e.g. Clauzade & Roux 1985 who recognized the five species groups described below; Poelt 1969; Wade 1965; see also Table 2 and Appendix A for a summary of the systematic treatments of this subgenus according to different authors). Caloplaca aurantia group. Characterized by the presence of citriform ascospores. Recently, Sipman & Raus (2002) described a new species (Caloplaca aegaea) that, based on thallus features and ascospore shape, could fit within this group. Caloplaca aurea group. Species within this group have an equatorial wall thickening < 2 mm. Poelt’s (1965) comparative study of Caloplaca aurea and C. paulii, with Fulgensia species, showed that there were intermediate states between the lobed Caloplaca and the genus Fulgensia s. str. According to Clauzade & Roux (1985), C. scrobiculata (syn. C. anularis) is also included within this group (Table 2). Caloplaca carphinea group. This group encompasses two species (Table 2) that are easily distinguished from other lobed taxa by the light yellowish, more or less greenish colour of the thallus, due to the presence of usnic acid. The group is known mainly from the Mediterranean region and Canary Islands. Although these species have been traditionally classified within Gasparrinia, macroscopically they are similar to Dimelaena oreina, which also contains usnic acid. However, the apothecial disk in the C. carphinea group produces emodine and parietin (Hansen et al. 1987; Santesson 1970b), and the ascospores are polarilocular. Breuss (1989) recognized these two taxa, previously treated as subspecies, as distinct species. Caloplaca persica group. This taxonomic entity consists of three corticolous species (Table 2) that had been previously 532 E. Gaya et al. Table 2 – Species included in the five groups described by Clauzade & Roux (1985) within subgenus Gasparrinia, and in the seven groups described by Poelt (1954) within lobed species of Caloplaca Clauzade & Roux (1985) Poelt (1954) C. aurantia group a C. aegaea C. aurantia C. flavescens C. thallincola C. aurea group C. aurea C. paulii C. scrobiculata C. carphinea group C. carphinea C. scoriophila C. persica group C. lobulata C. persica a C. polycarpoides C. saxicola group C. biatorina ssp. biatorina ssp. gyalolechioides C. cirrochroa ssp. cirrochroa ssp. fulva C. C. C. C. C. C. C. C. C. decipiens gloriae granulosa littorea marina microthallina necator obliterans saxicola ssp. arnoldii ssp. biatorinoides ssp. laceratula ssp. miniata ssp. obliterata ssp. pulvinata ssp. saxicola C. scopularis C. tenuata C. tenuatula ssp. inconnexa ssp. verrucariarum ssp. tenuatula var. athallina var. lithophila var. pertenuis var. pervulgata var. tenuatula C. verruculifera a - ohn. näh. Ansch. C. C. C. C. C. C. C. carphinea microthallina rubelliana squamulosa subsoluta tenuata tominii b - Alpinae C. C. C. C. C. aurea australis paulii pruinosa schistidii c - Aurantiae C. aurantia var. aurantia var. heppiana var. papillata C thallincola d - Soraliferae C. arnoldii var. arnoldii var. fulva C. cirrochroa C. microphyllina C. obliterans C. proteus e - Murales C. C. C. C. alcarum decipiens marina murorum var. laceratula var. murorum C. scopularis f - Granulosae C. granulosa C. verruculifera g - Biatorinae C. biatorina var. baumgartneri var. biatorina var. gyalolechioides var. sympecta a Not considered by Clauzade & Roux (1985). included within the genus Xanthoria, and later, treated as Caloplaca section Xanthoriella because of the lack of either inferior cortex or rhizines (Steiner & Poelt 1982). In the same publication, Steiner & Poelt also suggested that the presence of other characters, such as the slightly stipitate apothecia, with a very lax or empty stipe, supported the inclusion of these three species within the same section (Xanthoriella). Conversely, Clauzade & Roux (1985) did not consider this section, even though they maintained these species as a separate group within Gasparrinia. Caloplaca saxicola group. Represents the core of the lobedeffigurate Caloplaca species. In their key, Clauzade & Roux (1985) included taxa of the C. saxicola group as well as species from other groups and even other genera (e.g. Xanthoria), demonstrating the unclear limits of this broad and heterogeneous group. Phylogenetic reassessment of the Teloschistaceae Poelt (1954) subdivided lobed species of Caloplaca into seven groups (a–g; Table 2). Nordin (1972) studied material from Northern Europe and concluded that section Gasparrinia was a well-delimited group. In his study, 16 species were considered without group affiliations. Verseghy (1970, 1971, 1972), in her monograph of Hungarian species, treated Gasparrinia at the genus level and described 13 species with several forms and varieties. Kärnefelt (1989) assembled lobed species into several groups, but without making any formal classification. In the same study, some lobed species that had not been mentioned in previous studies were also considered, e.g. C. ochraceofulva, C. orthoclada, and C. sublobulata. Finally, Wetmore & Kärnefelt (1998) did not delimit groups for the 19 lobed species studied from North and Central America. They considered again that subgenus Gasparrinia was not a natural group, and could not be treated at any taxonomical level. Apart from the species already mentioned, there are other species with more or less well-developed lobed margins that have never been included within Gasparrinia, e.g. Caloplaca cinnabarina, C. dolomiticola, or C. haematodes. Paradoxically, some species in Gasparrinia have thalli that are not clearly lobate (e.g. C. littorea, C. marina, C. microthallina, or C. necator). Regardless of the different classifications of Caloplaca subgenus Gasparrinia, it is clear that the most accute problem is directly associated with the delimitation of the genus Xanthoria (Søchting & Lutzoni 2003). This delimitation between Caloplaca and Xanthoria is based solely on the presence or absence of a lower cortex. Xanthoria thalli usually show two cortical layers, upper and lower, whereas Caloplaca subgenus Gasparrinia presents only an upper cortical layer. However, the development of a lower cortex has been observed in some lobed species, e.g. C. scopularis (Poelt & Romauch 1977) and C. thallincola (Kärnefelt 1989). Phylogenetic studies within the Teloschistales Previous phylogenetic studies on members of the Teloschistales focused mainly on the genus Caloplaca (Arup & Grube 1999), addressed mostly the monophyly of Fulgensia (Gaya et al. 2003; Kasalicky et al. 2000), discussed more specifically relationships between Caloplaca and Xanthoria (Søchting & Lutzoni 2003), and revised Xanthomendoza (Søchting et al. 2002). None of these studies included the genus Teloschistes. Molecular phylogenetic studies within genera of the Teloschistales were centered on Caloplaca subgenus Pyrenodesmia (Muggia et al. 2008), and the C. aurantia group (Søchting & Arup 2002). The genus Xanthoria was the subject of a systematic study by Franc & Kärnefelt (1998), a population study centered on X. calcicola and X. parietina by Lindblom & Ekman (2005), and a research project focused on the phylogeography of X. elegans by Dyer & Murtagh (2001) and Murtagh et al. (2002). In this context, a phylogenetic study with a broad sampling across the Teloschistaceae was needed. The ITS was shown to provide sufficient phylogenetic confidence across this family when signal from ambiguously aligned regions is accommodated in phylogenetic analyses (Gaya et al. 2003). Therefore, we restricted our sequencing efforts to the ITS to maximize our taxon sampling for this study. Our first aim was to circumscribe the Teloschistaceae using monophyly as a grouping criterion, to establish its 533 relationship to the Letrouitiaceae, and to evaluate the monophyly of generic and sub-generic morpho-groups within the Teloschistaceae. With a taxon sampling biased toward the species belonging to the traditionally called subgenus Gasparrinia, we attempted to confirm the polyphyly of lobed species of Caloplaca within a broad taxon sampling of the Teloschistaceae, including taxa from all main groups of Caloplaca. Material and methods Taxon sampling We selected a total of 114 specimens (Supplementary Material Appendix B) from the Teloschistaceae. This sampling included 82 specimens (56 species, 59 taxa) of Caloplaca, 11 specimens (nine species) of Fulgensia, 11 specimens (seven species) of Teloschistes, one specimen of Xanthomendoza, and nine specimens (six species) of Xanthoria. The remaining genera (mainly having few species) in the Teloschistaceae (Eriksson 2006) have not been included in this study as we could not obtain fresh material or because they are doubtful genera (Table 1). In order to evaluate the infrageneric classification of Caloplaca based on morphological characters, the largest number of species sampled were from this genus, and sequences from at least one species per subgenus or group of species (sensu Clauzade & Roux 1985) were used: subgenus Caloplaca (C. citrina group, C. cerina group, C. ferruginea group), subgenus Gasparrinia (C. carphinea group, C. aurea group, C. aurantia group, C. saxicola group), subgenus Gyalolechia, subgenus Leproplaca, subgenus Pyrenodesmia and subgenus Xanthocarpia. The C. persica group that comprises the corticicolous species of subgenus Gasparrinia was not included due to lack of material. Nevertheless, we widely sampled the lobed taxa included in subgenus Gasparrinia. In addition to these 114 sequences of taxa from the family Teloschistaceae, we sequenced two specimens from the genus Letrouitia (Letrouitiaceae). Unfortunately, we could not include the family Megalosporaceae. In order to compare the results of this study with those from Gaya et al. (2003), we maintained the same outgroup that was used in that study: Letharia columbiana, L. vulpina, Protoparmelia badia, and Usnea arizonica (Parmeliaceae), and added Protoblastenia rupestris (Psoraceae). Molecular data DNA isolation and sequencing. Genomic DNA was obtained from fresh samples and herbarium specimens (BCN, C, DUKE, E, GZU, LEB, MARSSJ, MIN, MUB, SANT, TFC Lich, Aptroot herbarium - ABL, U. Arup personal herbarium and J. Etayo personal herbarium; voucher information is detailed in Supplementary Material Appendix B). DNA was isolated using the Puregene Kit (GENTRA Systems, Minneapolis) following the manufacturer’s protocol for filamentous fungi. DNA concentration was determined by visual comparison with positive control (l 100 ladder, concentration 10, 20, 40 ng) on an ethidium bromidestained Tris–borate–ethylenediamine tetraacetate (TBE) agarose gel. Symmetric PCRs were prepared for a 50 ml final volume containing 31.7 ml sterile double-distilled water, 5 ml of 10 Taq 534 polymerase reaction buffer (Boehringer–Mannheim, Indianapolis), 5 ml of 2.5 mM dNTPs, 0.3 ml Taq DNA polymerase (Boehringer–Mannheim), 2.5 ml for each of the 10 mM primers ITS1F or ITS1 or ITS5 and ITS4 (Gardes & Bruns 1993; White et al. 1990), 1.5 ml of 10 mg ml1 bovine serum albumin (BSA; BioLabs), 0.5 ml of 50 mM MgCl2 and 1 ml of template genomic DNA. PCR was performed on Perkin–Elmer GeneAmp 2400 under the following conditions: one cycle of 1 min at 95 C linked to 40 cycles of 1 min at 95 C, 45 s at 52 C, and 2 min at 72 C with the last step increased by increments of 5 s for the last 15 cycles. A final extension step of 10 min at 72 C was added, after which the samples were kept at 4 C. The PCR products were purified using either Cycle-Pure Kit (E.Z.N.A., Omega Bio-Tek, Doraville, GA), or low-binding regenerated cellulose 30,000 NMWL (nominal molecular weight limit) filter units (Millipore, Billerica), following the manufacturer’s instructions. Both strands of the purified PCR products were sequenced using PCR primers used for the symmetric amplification and additional primers 5.8S and 5.8SR (Vilgalys & Hester 1990) or ITS2 and ITS3 (White et al. 1990). Sequencing reactions were prepared in 10 ml final volume using BigDye Terminator v3.1 (ABI PRISM, Perkin-Elmer Biosystems, Wellesley) and following the manufacturer’s instructions. Sequenced products were precipitated with 26 ml deionized sterile water and 64 ml of 95 % ethanol before they were loaded on an ABI Prism 3730 automated DNA sequencer (Perkin–Elmer, Applied Biosystems). Sequence alignment. Sequence fragments were subjected to BLAST searches for a first verification of their identities. They were assembled using Sequencher version 4.1 (Gene Codes Corporation, Ann Arbor) and Sequencer Navigator 1.0.1 (Applied Biosystems), and aligned manually with MacClade 4.01 (Maddison & Maddison 2001). The delimitation of ambiguous regions was done using the method described by Lutzoni et al. (2000). All DNA sequences have been deposited in GenBank (Supplementary Material Appendix B) and the alignment is available in TreeBASE. Phylogenetic analyses MP analyses were conducted using PAUP) version 4.0b10 for UNIX and Macintosh (Swofford 2002) and Bayesian analyses [MCMC with Metropolis coupling (B-MCMCMC)] were carried out using the program MrBayes 3.0b4 (Huelsenbeck & Ronquist 2001). MP analyses. We performed three weighted MP analyses: a first MP analysis (MP1) was executed using exclusively unambiguously aligned sites. The second MP search (MP2) included also coded (INAASE) characters, and we added coded (arc) characters for the third MP search (MP3). In all analyses symmetric step matrices were created for unambiguous portions as follows. The options ‘Show character status/full details/hide excluded characters’ from the Data menu in PAUP) were implemented. From the resulting table, the column States showing all nucleotide states found at each of the unambiguously aligned and non-constant sites was saved as a separate text file. This file was used as an input file for the program STMatrix 2.1 (François Lutzoni & Stefan Zoller, Department of Biology, Duke University), which generates a step matrix (in Nexus format) by calculating frequencies of reciprocal changes from one state to another and E. Gaya et al. converting them into costs of changes using the negative natural logarithm of the frequencies (Felsenstein 1981; Wheeler 1990). ITS1, ITS2, and 5.8S each were subjected to a specific symmetric step matrix. Gaps were used as a fifth character state for unambiguous portions of the alignment. Ambiguously aligned regions were removed from MP searches. However, some of these ambiguously aligned sites were recoded and subjected to specific step matrices obtained with the program INAASE 2.3b (Lutzoni et al. 2000), incorporating the phylogenetic signal from these regions without violating positional homology. Ambiguous regions that were over 100 bp in length, highly variable (i.e., over 32 character states) or that showed major length variation among sequences of the same ambiguous region were recoded into 23 characters with the aid of the program arc v1.5 (Kauff et al. 2003; Miadlikowska et al. 2003) using the nucleotide option, as outlined in Reeb et al. (2004). Each of the 23 characters obtained with arc-nucleotide were subjected to a specific weight: 1.00 for character 1; 0.25 for characters 2–5; 0.10 for characters 6–15 and 0.50 for characters 16–23. All three MP searches were performed using heuristic searches with 1 K random-addition-sequences (RAS), TBR (tree bisection–reconnection) branch swapping, Multrees option in effect, and collapsing branches with maximum branch length equal to zero. In MP1 and MP2, the high number of equally most parsimonious trees that filled the memory before completing the search required stopping these searches before their completion. For this reason, in MP1 we executed three successive searches progressively incrementing the number of trees saved per RAS. In the first round, we saved only one tree per replicate, in the second we saved 100 trees per replicate, and in the third, 1 K trees per replicate were saved. With this strategy we could detect that even when incrementing the number of trees saved per RAS, the topology of the majority rule consensus tree remained the same. Because the power of resolution was higher in MP2, resulting from the addition of INAASE characters, we could perform a search in two steps. In the first step, we saved only one tree per replicate. In the second step, we searched for all equally parsimonious trees, saving all trees only when swapping a tree equal to or shorter than the shortest tree found in the first step. MP3 was conducted in one step by saving all trees as soon as TBR swapping was initiated for each of the 1 K RAS. Branch support was assessed by BS analyses (Felsenstein 1985) with full heuristic searches, 1 K parsimony BS replicates, using two RAS per BS replicate and by saving no more than ten trees per RAS in MP1 and MP2, and 642 parsimony BS replicates, using 50 RAS per BS replicate and by saving all trees per RAS in MP3. In all BS analyses, the same parameters as in the original MP search were used, and constant sites were excluded from all BS analyses. Bayesian analyses. Bayesian analyses were conducted using MrBayes 3.0b4 (Huelsenbeck & Ronquist 2001). PPs were approximated by sampling trees using a B/MCMCMC method. The model of evolution for Bayesian inference was selected using a hierarchical likelihood ratio test (HLRT) (Huelsenbeck & Crandall 1997) with the program Modeltest 3.06 (Posada & Crandall 1998). HLRT implemented in Modeltest suggested that the TrN þ G þ I (Tamura & Nei 1993) was the model that best fitted the data, with estimation of invariable sites, Phylogenetic reassessment of the Teloschistaceae 535 Table 3 – Synopsis of data sets used in MP and Bayesian analyses MP1 analysis MP2 analysis MP3 analysis Bayesian analysis 1067 1001 153 66 848 0 66 46 1074 1001 153 73 (7) 848 7 (7, 0) 73 53 1304 1001 153 301 (7þ228) 848 17 (7, 10) 301 281 1067 848 153 66 848 0 66 NA Total number of characters Number of sites excluded Number of constant sites Number of variable characters Number of ambiguously aligned sites Number of recoded ambiguously aligned regions (INAASE, arc) Total number of analysed characters Number of parsimony informative characters NA, not applicable. and assuming a Gamma distribution for rate heterogeneity among sites. Since the TrN model cannot be implemented in MrBayes, the GTR þ G þ I model was considered instead. Bayesian analyses were initiated on random trees and run for 8 M generations, sampling the Markov chains at intervals of 100 generations. To ensure that all trees from the burnin were excluded, the majority rule consensus tree was calculated with PAUP) using only the last 60 K out of the 80 K trees sampled. The exclusion was made by plotting the loglikelihood values against the time generation, and by setting the stationarity when log-likelihood values reached a stability equilibrium value (Huelsenbeck & Ronquist 2001). A second, independent, run of 8 M generations was performed as described to confirm the result from the first Bayesian analyses. We also used AWTY, as implemented in MrBayes 3.1.2, to test for convergence as a proxy for confirming that our two independent 8 M generation B/MCMCMC runs had reached stationarity. This analysis stopped at 1 M generations. The results were very similar to the first two runs, but likelihoods were slightly worse. Because topological differences were all in the expected parts of the trees with the highest level of phylogenetic uncertainty, none of these differences were seen as significant and would have changed the conclusions derived from this study. Therefore, we present here the result from one of the longest Bayesian analyses of 8 M generations. Results Alignments The size of the ITS final data matrix for this study was 121 sequences by 1067 sites (Table 3). A total of 19 ambiguously aligned regions were delimited, resulting in the exclusion of 848 nucleotide sites. From the 219 remaining sites, 153 were constant, and 66 were variable. In MP1, from the 66 sites left, 46 were parsimony informative. In MP2, seven INAASE coded characters, which substituted seven ambiguously aligned regions, were combined with the 66 characters for a total of 73 non-constant characters and of those, 53 were parsimony informative. In MP3, apart from the seven INAASE characters, arc recoded characters were included, which substituted ten ambiguously aligned regions for a total of 230 characters (corresponding to 71.4 downweighted characters). From the total of 301 non-constant characters of this analysis, 281 were parsimony informative (Table 3). Comparison of resolution and support among phylogenetic analyses The unequally weighted MP1 search yielded 13 003 equally most parsimonious trees (Table 4) of 312.13 steps, which Table 4 – Synopsis of analytical results and internode support Analysis type Number of resolved internodes (50 %) Number of significantly supported internodes Number of equally most parsimonious trees Number of significantly supported internodes in common with MP3 Number of significantly supported internodes lost Number of significantly supported internodes gained MP1 MP2 MP3 Bayesian UNAMB 18 UNAMB þ INA 38 UNAMB D INA D A 73 UNAMB 34 8 24 57 12 13 003 30 3 – 8 23 – 10 49 34 – 47 0 1 – 2 The MP analysis, including INAASE and arc characters (in bold), was used as a reference for all other analyses. Nodes were considered significant if support values were 70 % with MP BS and 95 % with Bayesian analyses. UNAMB, unambiguously aligned sites; INA, addition of INAASE characters; A, addition of arc characters. 536 E. Gaya et al. 100/-/68 95/96 90/100100/88/100/85 -/70 MP3-BS / MP2-BS MP1-BS / PP 88/93/- 100/95 76/99 92/-/52 100/85 100/100 92/100 83/-/75 95/73 72/- -/96 60/- Lineage 2 Lineage 2 Lineage C 100/82 100/99 55/91 85/99 94/79 59/65 -/65 68/99 87/- 100/82/- 66/100/58 75/- 77/- 86/- 100/82 56/73 57/- 100/81 53/100/- 92/- 99/55 -/74 83/Lineage A 66/- 96/73 80/- 67/61 100/80 Lineage 1 99/63 62/77 61/89/- -/59 100/67 -/85 -/90 93/- 98/86 87/100 ? 51/- 64/99/90 -/82 100/- Lineage 1 Lineage 3 95/85/57 59/84 59/65 59/66 59/100 100/-/84 -/73 51/Lineage B 100/90 100/100 78/92 98/100 100/92 77/53 -/54 90/67 57/94 100/72 82/- 64/97 98/- 91/76 57/86 100/54 53/87 100/83 67/98 57/- 100/96 66/55 95/99 91/53 97/92 Protoblastenia rupestris Letharia columbiana Letharia vulpina Usnea arizonica Protoparmelia badia Letrouitia dominguensis Letrouitia parabola Fulgensia fulgida 1 Fulgensia fulgida 2 Fulgensia fulgens Fulgensia desertorum 1 Fulgensia subbracteata Fulgensia poeltii Fulgensia canariensis Fulgensia desertorum 2 Fulgensia bracteata Fulgensia pruinosa Caloplaca flavovirescens Caloplaca flavorubescens Caloplaca gloriae 3 Caloplaca gloriae 2 Caloplaca gloriae 4 Caloplaca gloriae 1 Caloplaca crenularia Caloplaca chalybaea Caloplaca aetnensis Caloplaca erythrocarpa Caloplaca teicholyta 2 Caloplaca teicholyta 1 Caloplaca variabilis Caloplaca demissa 3 Caloplaca demissa 1 Caloplaca demissa 2 Caloplaca carphinea 1 Caloplaca carphinea 2 Caloplaca scoriophila Caloplaca aegaea1 Caloplaca aegaea 2 Caloplaca flavescens 3 Caloplaca flavescens 4 Caloplaca thallincola 2 Caloplaca thallincola 1 Caloplaca flavescens 1 Caloplaca flavescens 2 Caloplaca aurantia 2 Caloplaca aurantia 1 Caloplaca velana v. placidia Fulgensia australis Caloplaca paulii Caloplaca cerina Caloplaca stillicidiorum Caloplaca chlorina Caloplaca cancaraxiticola Caloplaca alpigena Caloplaca velana v. dolomiticola 1 Caloplaca velana v. dolomiticola 2 Caloplaca cirrochroa 1 Teloschistes contortuplicatus Caloplaca proteus 2 Caloplaca cirrochroa 2 Caloplaca proteus 1 Caloplaca xantholyta Teloschistes lacunosus 1 Teloschistes lacunosus 2 Teloschistes lacunosus 3 Teloschistes villosus Teloschistes scorigenus Xanthomendoza fallax Caloplaca ochracea Caloplaca marmorata Caloplaca ferrarii Caloplaca holocarpa Caloplaca verruculifera * Xanthoria candelaria Caloplaca bolacina Caloplaca marina * Caloplaca marina ssp. americana * Caloplaca microthallina * Caloplaca maritima 3 * Caloplaca maritima 2 * Caloplaca maritima 1 * Caloplaca granulosa Caloplaca lithophila * Caloplaca polycarpa Caloplaca coronata Caloplaca arnoldii 1 Caloplaca arnoldii 2 Caloplaca biatorina 1 Caloplaca biatorina 2 Caloplaca arnoldii 1 s. Poelt Caloplaca arnoldii 2 s. Poelt Caloplaca saxicola Caloplaca decipiens 1 Caloplaca decipiens 2 Caloplaca schistidii Caloplaca pusilla Xanthoria calcicola Xanthoria parietina Xanthoria resendei 1 Xanthoria resendei 2 Xanthoria elegans 2 Xanthoria elegans 1 Xanthoria sorediata 2 Xanthoria sorediata 1 Caloplaca alcarum 1 * Caloplaca alcarum 2 * Caloplaca scopularis * Caloplaca inconnexa Caloplaca pyracea Caloplaca ignea 1 ** Caloplaca ignea 2 ** Caloplaca scrobiculata Caloplaca irrubescens1 Caloplaca irrubescens 2 Caloplaca trachyphylla ** Teloschistes chrysophthalmus 1 Teloschistes chrysophthalmus 2 Teloschistes fasciculatus Teloschistes sieberianus 1 Teloschistes sieberianus 2 Caloplaca texana ** Letrouitiaceae Fulgensia s. str. A2 B4 A3 E A3 E B3 C. carphinea group B1 C. aurantia group A2 B2 A1 C. cerina group B2 A2 B4 Xanthoanaptychia? B4 D Xanthoanaptychia? Teloschistes Seirophora Xanthomendoza Oxneria? F C A3 B4 Xanthoria B4 A2 B4 C. saxicola s. str. group Xanthoria schistidii? Xanthoria Rusavskia? B4 A3 B2 A2 Xanthoanaptychia? Teloschistes Teloschistes Fig 1 – Phylogenetic relationships within the Teloschistales (Caloplaca, Fulgensia, Letrouitia, Teloschistes, and Xanthoria), based on an ITS nrDNA data set for 79 species of Teloschistaceae and two species of Letrouitiaceae; taking as an outgroup four species of the family Parmeliaceae (Letharia columbiana, L. vulpina, Protoparmelia badia, and Usnea arizonica) and a species of the Psoraceae (Protoblastenia rupestris). Strict consensus tree of three equally most parsimonious trees generated with the parsimony analysis MP3 accommodating signal from 17 ambiguously aligned regions (INAASE and arc characters). Internodes with BS values from MP3 analysis (BS) 70 % are highlighted by thicker lines. PP values 95 % are marked in bold. Grey shading delimits genera and species groups previously described that have obtained statistical significance in this study. Teloschistes taxa are delimited by dashed lines showing the putative polyphyly of this genus. An asterisk after names shows taxa with littoral preferences belonging to Caloplaca. Two asterisks highlight lobed species from Caloplaca that had not been previously included within subgenus Gasparrinia. All subgenera of Caloplaca are indicated by capital letters and species Phylogenetic reassessment of the Teloschistaceae were part of 32 islands hit 35 times out of 1 K RAS (CI, excluding uninformative characters ¼ 0.466; RI ¼ 0.858). The unequally weighted MP2 search, with INAASE characters, revealed 30 equally most parsimonious trees of 587.47 steps. These trees were found in 30 islands hit 31 times out of 1 K RAS (CI, excluding uninformative characters ¼ 0.538; RI ¼ 0.845). A total of three equally most parsimonious trees was found in one island that was hit six out of 1 K times with the MP3 search based on combined unambiguously aligned sites, INAASE characters and arc characters (Fig 1). The score of the best trees was 3340.10 steps (CI, excluding uninformative characters ¼ 0.351; RI ¼ 0.648). Comparing just the three parsimony treatments, we observed similar topologies throughout the three MP analyses. All three searches recovered the two main lineages 1 and 2 (Fig 1). The main discrepancies detected were in the degree of resolution within these two lineages, especially in lineage 2. The number of internodes with BS support 70 % went from 8, when the analysis was restricted to unambiguously aligned sites (MP1), to 24 when seven INAASE characters were added to these unambiguous sites (MP2), and to 57 when arc characters were added (MP3; Table 4). Only one significantly supported internode in MP2 (BS ¼ 77 %) was not significant (<70 %) in MP3. The Bayesian consensus tree revealed 12 well-supported internodes (PP 95 %). Forty-seven of 57 internodes with BS 70 % in MP3 received PP < 95 % in Bayesian inference, whereas only two internodes with significant PPs received MP3-BS below 70 %. Regarding the topology reconstructed, the Bayesian analysis did not resolve phylogenetic relationships between species groups in the family Teloschistaceae; even the two main lineages were not recovered. Based on these results, we can assert that MP3 analysis provided the most resolved and supported phylogenetic inference for the family Teloschistaceae. Consequently, we will focus mainly on this topology (Fig 1) in the following sections. Phylogenetic relationships One of the most striking results was the phylogenetic placement of the family Letrouitiaceae within the Teloschistaceae (Fig 1). However, this relationship did not receive a single significant support value. Therefore, we cannot exclude the possibility that this family forms a monophyletic group outside the Teloschistaceae, within the Teloschistales, as implied by current classifications (e.g. Eriksson 2006) or within the Teloschistineae as reported by Miadlikowska et al. (2007) with high phylogenetic confidence but for a small taxon sampling within this suborder. 537 As in previous studies (Arup & Grube 1999; Gaya et al. 2003; Søchting & Lutzoni 2003), the same two main sister lineages were recovered within the Teloschistaceae. In the most phenotypically diverse clade (lineage 2), relationships among genera and species groups remained uncertain. However, several monophyletic clades recovered at genus or species complex level were strongly supported. Lineage 1 seemed to show again a slightly higher phenotypic homogeneity than lineage 2. It included most species with anthraquinones in the thallus and a mainly fruticose, foliose or placodiod habit. Again, in this lineage it was not possible to recover high support at deeper internodes. In this study, genera Caloplaca, Fulgensia, and Xanthoria were again recovered as polyphyletic, confirming previous results. The genus Teloschistes appeared also as polyphyletic, with apparently separate origins in the two main lineages. Regarding the genus Caloplaca, species belonging to subgenus Pyrenodesmia, subgenus Leproplaca, subgenus Gasparrinia (C. aurantia group, C. aurea group, C. carphinea group and C. saxicola group) and subgenus Caloplaca (C. citrina group, C. cerina group and C. ferruginea group) (sensu Clauzade & Roux 1985) were recovered in the most phenotypically diverse clade (lineage 2). The C. cerina group was significantly recovered as monophyletic (BS ¼ 86 %), C. carphinea and C. scoriophila always formed a monophyletic clade with BS ¼ 94 %, as well as the C. aurantia group (BS ¼ 100 %). Caloplaca subgenus Gasparrinia (most species of the C. saxicola group, except C. gloriae and one species from the C. aurea group) and several species from subgenus Caloplaca (C. citrina group and C. ferruginea group) were recovered in lineage 1, together with subgenus Gyalolechia and subgenus Xanthocarpia (sensu Clauzade & Roux 1985), the last two subgenera forming a robust monophyletic entity (BS ¼ 96 %). The phylogenetic afiliation of C. scrobiculata (C. aurea group) could not be established with high confidence in any of the analyses, but was always sister to lineage 3. Lineage 3 was recovered in all analyses, but only Bayesian PPs were statistically significant (PP ¼ 100 %). Taxa with mainly foliose or placodioid thalli, and anthraquinones in the thallus, from the genus Xanthoria and Caloplaca saxicola group, were nested within this lineage. Only a few species with reduced thalli, with or without anthraquinones, e.g. C. holocarpa, C. pyracea, or C. coronata were also included in this clade. Relationships among species groups within this lineage remained unresolved, even though the monophyly of several groups was confirmed. Hence, the C. saxicola s. str. group, whose circumscription will be described in a forthcoming paper, appeared as monophyletic with strong evidence (BS ¼ 100 %). Xanthoria was recovered as polyphyletic (Fig 1), with X. elegans and X. sorediata sharing a most groups within these subgenera are referred by numbers: A: Caloplaca subgenus Caloplaca (A1: C. cerina group, A2: C. citrina group, A3: C. ferruginea group); B: Caloplaca subgenus Gasparrinia (B1: C. aurantia group, B2: C. aurea group, B3: C. carphinea group, B4: C. saxicola s. lat. group); C: Caloplaca subgenus Gyalolechia; D: Caloplaca subgenus Leproplaca; E: Caloplaca subgenus Pyrenodesmia; F: Caloplaca subgenus Xanthocarpia. This classification of subgenera and species groups follows Clauzade & Roux (1985). Names followed by question marks refer to genera proposed by Kondratyuk & Kärnefelt (2003) included in our study. Names surrounded by a dotted line indicate the proposal by Frödén & Lassen (2004) for Teloschistes. Lineages A, B and C from Søchting & Lutzoni (2003) and lineages 1 and 2 from Gaya et al. (2003) are italicized. Lineages 1 and 2 recovered in this study are in bold. 538 recent common ancestor (BS ¼ 77 %), X. calcicola and X. parietina forming also a strongly supported monophyletic group (BS ¼ 100 %), and X. candelaria being sister to C. verruculifera (BS ¼ 80 %). In all analyses, Fulgensia s. str., as defined by Gaya et al. (2003), was monophyletic (BS ¼ 92 %). Fulgensia australis was also part of lineage 2, but appeared related without support to C. paulii. Conversely, C. schistidii (syn. F. schistidii) was well nested within the C. saxicola group (BS ¼ 100 %) in lineage 3. As for Fulgensia, the genus Teloschistes was recovered in three clades. Except for T. contortuplicatus, the species of Teloschistes recovered in lineage 2 formed a monophyletic group (BS ¼ 83 %). Species so far included within this genus with anthraquinones in the thallus were otherwise recovered in lineage 1 forming a weakly supported (BS ¼ 66 %) group. Finally, the genus Xanthomendoza was depicted in our study by only one representative: Xanthomendoza fallax, which was recovered as an early diverging lineage (lineage A), within lineage 1. Discussion Comparison of optimization criteria: MP with recoded INAASE and arc characters versus Bayesian methods and MP considering only unambiguously aligned regions Because this phylogenetic study was based only on ITS to resolve relationships across the Teloschistaceae, the inclusion of phylogenetic signal from ambiguously aligned regions in MP (recoded INAASE and arc characters) has proved advantageous in the reconstruction of the phylogeny. This has been shown by an increase in the number of supported internodes in MP2 and MP3, compared with MP1 analysis and Bayesian inference, both restricted to unambiguously aligned regions (Table 4). Because the great majority of nucleotides could not be aligned unambiguously (848; Table 3), the Bayesian method, considered as more efficient than other phylogenetic methods (Alfaro et al. 2003), has not shown a greater resolving power when restricted to non-ambiguously aligned sites than MP when recovering signal from ambiguously aligned regions of the alignment. Our results agree with Reeb et al. (2004) in their nuclear ribosomal LSU and SSU analyses, where higher phylogenetic confidence was not revealed with Bayesian inference when the analyses were restricted to these two genes. They postulate that LSU and SSU evolved slowly, and without the phylogenetic signal recovered from ambiguously aligned regions there was not enough variation to resolve relationships with a high phylogenetic confidence, even for the Bayesian inference. However, when one gene (RPB2) was added to the SSU and LSU, Bayesian analyses using different models of evolution were more efficient than MP analyses even with the addition of signal from ambiguously aligned regions. In Gaya et al. (2003), we suggested that large data sets of ITS sequences for the Teloschistaceae would greatly benefit from methods like INAASE, that have been designed to obtain phylogenetic signal from ambiguously aligned regions. We also pointed out the possibility that ITS alone could provide sufficient phylogenetic information to completely resolve relationships within the Teloschistaceae, and could generate high E. Gaya et al. support values for most internodes if a new method was able to capture phylogenetic signal from all ambiguously aligned regions, even those with more than 32 character states, more than 100 bp in length, or with a considerable variation in length among sequences from the same ambiguous region. By using arc we have been able to recover phylogenetic signal from an additional ten of the remaining 12 ambiguously aligned regions that could not be recoded using INAASE. Miadlikowska et al. (2003) mentioned that variation among their ITS1-HR sequences (recoded with arc) contributed in a great extent to species delimitation and identification, and stated that it can be useful for population studies. We prove that these characters can also be useful for higher-scale phylogenies, as in the case of the Teloschistaceae. Only when including INAASE and arc characters could we reach the level of resolution and support for the family shown here. Nonetheless, deep internodes of our topology lost significant support that was previously recovered in other phylogenetic studies restricted to fewer taxa. This is a common phenomenon for deep internodes when adding many taxa without adding more characters (see Miadlikowska et al. 2007). From this we deduce that the number of taxa exceeded the resolving power of ITS even when including INAASE and arc characters. Nevertheless, ITS remains an excellent marker to resolve, with high phylogenetic confidence, species complexes if used in combination with genes providing complementary resolution and support, such as the nuLSU (e.g. Miadlikowska et al. 2003). Phylogenetic relationships within the family Teloschistaceae The same two main lineages obtained by Arup & Grube (1999) (BS ¼ 91 % and 99 %), Gaya et al. (2003) (lineage 1, PP ¼ 99 % and lineage 2, BS ¼ 89 %) and Søchting & Lutzoni (2003) (without significance), are consistently recovered within the family Teloschistaceae, even though without or with very low support in our study. The taxon sampling here was much broader and included a higher number of species. Although genera Caloplaca, Fulgensia, Teloschistes, and Xanthoria are recovered as polyphyletic, several species groups have been consistently recovered as monophyletic. Agreeing with previous results, we can confirm that lobed species traditionally included within Caloplaca subgenus (sensu Clauzade & Roux 1985), or section (sensu Poelt 1969) Gasparrinia, do not form a monophyletic entity. Caloplaca aurantia, C. carphinea, and C. saxicola s. str. groups are the only groups recovered as monophyletic (with high phylogenetic confidence) within this subgenus. In this way, delimitation of the C. saxicola s. lat. group (sensu Clauzade & Roux 1985) is not in agreement with our molecular phylogeny, and neither are the C. aurea group and sorediate species of the C. saxicola group. In a cladistic study based on morphological characters, Kärnefelt (1989) also did not provide enough evidence to accept Gasparrinia as a separate group. Kärnefelt (1989) pointed out that the use of infrageneric categories within the lobed species could be justified. The C. aurantia group had previously been shown to be separate from the rest of members of subgenus Gasparrinia based on molecular data by Søchting & Arup (2002). They argued that the C. aurantia group is distinguished by several distinct Phylogenetic reassessment of the Teloschistaceae morphological characters. The recently described species C. aegaea (Sipman & Raus 2002) is reported to be part of this group (Fig 1). All four taxa (C. aegaea, C. aurantia, C. flavescens, and C. thallincola) are characterized by having citriform spores, and differ from each other by the different types of cortex and by the presence or absence of calcium oxalate crystals in the cortex. Interspecific relationships have not been resolved in our study, except for the monophyly of C. aegaea, C. aurantia, and C. thallincola. The C. carphinea group is consistently monophyletic in all analyses. One specimen of C. carphinea shares a most recent common ancestor with C. scoriophila (supported only by PP). Breuss (1989) distinguished these two species mainly by the type of cortex, paraplectenchymatous in C. carphinea and scleroplectenchymatous in C. scoriophila, and by the size of the spores, longer in C. scoriophila. According to this author, C. carphinea can be included in the mediterranean element, whereas C. scoriophila shows a more Atlantic distribution. Based on only three specimens, we cannot make conclusions about the monophyly of these two species. In our analyses, the C. aurea group shows three putative origins. In lineage 2, the placement of C. cancarixiticola and C. paulii remains uncertain. In lineage 1, C. scrobiculata is always recovered without support as sister to lineage 3. Contrary to what several authors had suggested, neither C. cancarixiticola nor C. paulii show a close relationship with Fulgensia s. str. in this study. Kärnefelt (1989) and Poelt (1965) had proposed a morphological proximity of the C. aurea group with Fulgensia subgenus Candelariopsis (sensu Poelt 1965 and Poelt & Vězda 1977). Kärnefelt (1989) and Westberg & Kärnefelt (1998) stated that several taxa included within the C. aurea group (e.g. C. paulii) could be related to Fulgensia canariensis and F. schistidii based on cortex structure. Navarro-Rosinés et al. (2000) proposed morphological affinities between C. aurea and typical Fulgensia (subgenus Fulgensia sensu Poelt 1965), and group D from Westberg & Kärnefelt (1998). Caloplaca cancarixiticola was thought to be related to subgenus Candelariopsis and Westberg & Kärnefelt’s subgroups A, B and C by NavarroRosinés et al. (2000) based on a comparison of C. cancarixiticola specifically to F. australis, F. canariensis, and F. schistidii. None of these relationships are corroborated by our study. Clauzade & Roux (1985) considered four sorediate species within the C. saxicola group: C. cirrochroa, C. decipiens, C. obliterans, and C. proteus. The topology recovered in this study does not support this grouping. Hence, C. cirrochroa and C. proteus are recovered within lineage 2, whereas C. decipiens shares a most recent common ancestor with the taxa included in the C. saxicola s. str. group in lineage 1. Unfortunately, C. obliterans could not be included in our study. Although the two specimens of C. proteus are nested in a group together with one of the specimens of C. cirrochroa, the latter shows a second origin of uncertain position. Based on these results, we cannot verify or deny whether C. cirrochroa and C. proteus have evolved independently from different non-sorediate ancestors as stated by Poelt (1969). The phylogenetic placement within the Teloschistaceae of this small group of sorediate taxa remains unresolved. Caloplaca gloriae (syn. C. gomerana, type lost, synonymization pending to study) was considered a species close to C. saxicola by Llimona & Werner (1975), and was included within the C. saxicola group by Clauzade & Roux (1985). However, C. gloriae 539 differs, among other characters, by containing fragilin and caloploicin (Søchting & Lutzoni 2003), two compounds that are also found in Fulgensia (Søchting & Lutzoni 2003). In lineage 2, C. gloriae appears closely related to C. flavorubescens, C. flavovirescens, Fulgensia s. str., and Letrouitia, even though without support. Gaya et al. (2003) had already shown a significant close relationship among C. gloriae, C. flavorubescens, C. flavovirescens, and Fulgensia s. str. Based on results from Miadlikowska et al. (2007), the non-supported phylogenetic placement of Letrouitia as shown in our Fig 1 is most likely incorrect due to the lack of characters for such a high number of taxa. Most taxa of the C. saxicola s. lat. group (sensu Clauzade & Roux 1985), C. scrobiculata (C. aurea group), and other species that could be considered as closely related to subgenus Gasparrinia by being lobed and showing anthraquinones in the thallus, i.e., C. ignea, C. texana, and C. trachyphylla, are recovered in lineage 1. Considering the C. saxicola s. lat. group, this circumscription shows several potential origins. In this sense, most lobed taxa without maritime affinities constitute what we named the C. saxicola s. str. group. This monophyly was also recovered by Gaya et al. (2003) and Arup & Grube (1999). However, in Søchting & Lutzoni (2003, clade B3) the two species of the C. saxicola group included, shared a common ancestor with C. holocarpa. Within this group, we recovered C. pusilla, also named C. saxicola ssp. pulvinata. We will publish this synonymy in a forthcoming morphological study on the C. saxicola s. str. group. In our study, taxa with littoral or maritime affinities, considered by Clauzade & Roux (1985) as part of the C. saxicola s. lat. group, appear in separate clades, not sharing a most recent common ancestor with the C. saxicola s. str. group. One of these clades, groups C. marina, C. maritima, and C. microthallina, which are species with a highly reduced thallus, having microlobes, areoles, granules or warts, depending on the species, and all showing a paraplechtenchymatous cortex (Arup 1992, 1994, 1997). Arup & Grube (1999) and Gaya et al. (2003) had shown a close phylogenetic relationship between C. marina and C. maritima. Caloplaca granulosa is sister to this group of littoral species. This taxon shows well-developed lobes and abundant isidia. Although it is not a littoral species, it grows on steep surfaces, exposed to heavily eutrophicated rainwater runoffs. Caloplaca alcarum and C. scopularis constitute another clade of littoral taxa. One of the C. alcarum specimens and the C. scopularis specimen are recovered as monophyletic, being sisters to the other specimen of C. alcarum. A thorough morphological study of these two species led us to discover a strong similarity in terms of ascospore shape and cortex structure (Gaya 2008). Based on the morphological characters and the phylogenetics results, we suggest the possibility that these two taxa could be in fact the same species, with a wide range of lobe development. In further studies, additional characters should be used to confirm this hypothesis. Arup (1995a) established similarities between Caloplaca alcarum and C. inconspecta (not included in this study), and C. marina, but C. alcarum was never found to be closely related to C. scopularis. Caloplaca alcarum and C. scopularis can be anatomically distinguished from the other clade of littoral species by having a scleroplectenchymatous or sclero-prosoplectenchymatous cortex, but never paraplectenchymatous, as in C. marina and C. maritima. Caloplaca alcarum and C. scopularis are shown here to share 540 a most recent common ancestor with a species from the C. ferruginea group (C. pyracea) and a species from the C. saxicola s. lat. group, C. inconnexa. Phenotypically, there are no similarities among these taxa, except for their preference for euthrophicated habitats. Caloplaca inconnexa and C. pyracea are part of a group of quite problematic and not well-known Caloplaca species. Their circumscriptions as species vary depending on the authors. Magnusson (1946) divided C. pyracea into two species according to their substrate. Saxicolous specimens were classified as C. lithophila, whereas corticolous specimens were named C. pyracea. Later on, these two taxa were synonymized with C. holocarpa (Santesson 1984; Wade 1965), a lignicolous species of uncertain taxonomy. Arup (1994) stated that, apart from the differences of substrate preferences, C. lithophila and C. pyracea were very similar. Clauzade & Roux (1985) considered C. lithophila as a variety, C. tenuatula ssp. tenuatula var. lithophila, and C. inconnexa as a subspecies, C. tenuatula ssp. inconnexa, within the C. saxicola s. lat. group, whereas C. holocarpa and C. pyracea were placed in the C. ferruginea group. Clauzade & Roux (1985) distinguished C. holocarpa from C. pyracea by the presence on the latter of a grey–yellowish thallin margin and bigger apothecia. In our study, we follow the concept of Clauzade & Roux (1985) regarding the taxonomy of these species, and have included another taxon from the C. tenuatula complex, C. polycarpa (syn. C. tenuatula ssp. verrucariarum, Clauzade & Roux 1985). Despite the morphological affinities among these taxa, our analyses do not support a close relationship. Although all of them appear within lineage 3, C. holocarpa shows an uncertain position, C. inconnexa and C. pyracea form a wellsupported monophyletic group, related to C. alcarum and C. scopularis, and C. lithophila and C. polycarpa form a weakly supported monophyletic entity, within a well-supported clade including C. coronata. Caloplaca verruculifera is another littoral species that have been classified within the C. saxicola s. lat. group (e.g. Clauzade & Roux 1985). This species shows a close relationship with X. candelaria in our study, a grouping that also appeared in the phylogenetic analyses by Søchting et al. (2002). Both C. verruculifera and X. candelaria have few apothecia. Caloplaca verruculifera presents a kind of globose isidia called phyllidia (Wetmore & Kärnefelt 1998), whereas X. candelaria abounds in blastidiate soredia (Lindblom 1997; Poelt & Petutschnig 1992a, b). Caloplaca verruculifera does not form a monophyletic entity with C. granulosa, another isidiate species from the C. saxicola s. lat. complex, with which it has been frequently confused, according to Arup (1994). Poelt & Romauch (1977) distinguished these two species by the anatomy of the thallus and stated that C. verruculifera shows a denser medulla than C. granulosa, with strongly conglutinated hyphae. Søchting & Lutzoni (2003) obtained a closer relationship between C. scopularis and C. verruculifera, indicating morphological similarities between these two species in the layout of the hyphae of the cortex and the ability to produce pseudocyphellae in the upper cortex. According to these authors, these features relate C. scopularis and C. verruculifera to a fruticose species, C. coralloides. Results from Søchting & Lutzoni (2003) support the suggestion that C. coralloides could be related to a taxonomical group including C. alcarum, C. scopularis, and C. verruculifera (Arup 1995b). With the addition of new taxa, we can conclude that E. Gaya et al. this grouping does not agree with the current phylogeny. However, we did not include C. coralloides in our study. Further studies considering fruticose species will be necessary to clarify the relationships among this group of taxa. Caloplaca texana and C. trachyphylla, two species morphologically similar to members of subgenus Gasparrinia, are recovered within lineage 1, outside of lineage 3. Caloplaca texana is known from the United States and Mexico (Wetmore & Kärnefelt 1998), and in our topology it is sister to Teloschistes species within lineage 1, but with low support. Caloplaca trachyphylla, a species from North America, Central Asia and Greenland, has been reported to be related to X. elegans, C. gloriae, C. verruculifera, and even to fruticose taxa (e.g. C. coralloides, C. thamnodes) (e.g. Wetmore & Kärnefelt 1998). Caloplaca trachyphylla occupies an uncertain position in our topology. Within the subgenus Caloplaca (Fig 1), the C. citrina and C. ferruginea groups are revealed to be polyphyletic, with potential independent origins both in lineages 1 and 2. These two groups are distinguished mainly by the presence of yellow to orange thalli and apothecia in the C. citrina group, and by whitish, grey, or black thalli and yellow to nearly black apothecia in the C. ferruginea group (Clauzade & Roux 1985). In their keys, the latter authors often included taxa from other groups or subgenera within the C. citrina and C. ferruginea groups. The C. citrina group is represented in lineage 2 by C. flavorubescens, C. flavovirescens, and C. velana. The latter species includes two infraspecific taxa, var. dolomiticola and var. placidia. Caloplaca flavorubescens and C. flavovirescens, difficult to differentiate based only on morphological characters, were treated as subspecies within C. flavorubescens by Clauzade & Roux (1985). Conversely, Giralt et al. (1992) described two new species (C. aegatica and C. alnetorum) and a variety (quercina) based on the corticolous complex of C. flavorubescens, highlighting the great morphological diversity within this taxon. Considering these different taxonomical treatments, a molecular phylogenetic study will be necessary to reassess the morphological diversity of this complex. Regarding C. velana, in our study the two infraspecific taxa included within this species do not belong to the same clade, but more characters are needed to confirm this result. In further studies, it will also be necessary to include all infraspecific taxa of C. velana considered by Clauzade & Roux (1985). These taxa have often been synonymized and included within a broader concept of the species. In lineage 1, the C. citrina group is represented by C. irrubescens, a species with slightly lobed orange to ocher areoles, which shows an uncertain phylogenetic position outside lineage 3, and by C. coronata, a blastidiate taxon that shares a most recent common ancestor with C. lithophila and C. polycarpa, two species from the C. saxicola s. lat. group nested within lineage 3. These two species do not form blastidia, but share the same ecology with C. coronata, mainly coniophilous and ornithocoprophilous, and in the case of C. polycarpa, by sometimes parasitizing other crustose lichens. Recently, Arup (2006) studied the relationships within the C. citrina group in the Nordic countries. In his study, Arup (2006) showed that there are at least five species within what has been called C. citrina, of which four are closely related to one another and to several non-sorediate species (e.g. C. maritima). In our study, we did Phylogenetic reassessment of the Teloschistaceae not include these species, but the position of C. coronata and C. irrubescens (syn. C. subsoluta) does not contradict Arup’s results. In lineage 2, several taxa in the C. ferruginea group with whitish to light grey thalli, lacking anthraquinones, and with ferruginous apothecia, are recovered, i.e., C. aetnensis, C. crenularia, C. erythrocarpa, and C. teicholyta. Caloplaca erythrocarpa and C. teicholyta share a most recent common ancestor with strong support, although the monophyletic delimitation of these species cannot be confirmed nor denied in our analyses. Each have whitish thalli, but in C. teicholyta apothecia are rare and the thallus is more or less lobed and covered by soredia, which gives a pulverulent aspect, whilst in C. erythrocarpa apothecia are abundant and the thallus has no lobes or soredia. C. aetnensis shows similar features, also having a whitish thallus, but in this case areoles and warts form the thallus. The morphological affinities of these three species are translated into a well-supported clade that in turn is nested with high confidence with the two species of the subgenus Pyrenodesmia, C. chalybaea and C. variabilis. Conversely, the phylogenetic placement of C. crenularia, another species of the C. ferruginea group, remains unsupported. This species has a dark brownish thallus and orange–ferruginous to blackish apothecia, with a sinuous margin. C. chalybaea has been considered here as a separate species from C. variabilis based on the information provided by M. Tretiach (pers. comm.) regarding a study on the phylogeny of subgenus Pyrenodesmia. Muggia et al. (2008) presented preliminary results of this study, where several distinct lineages within this group of endolithic lichens were recovered. Based on the taxa included in our study, we can state that the C. cerina group (subgenus Caloplaca) is monophyletic with strong support. This group is recovered in lineage 2 and includes three closely related species, one of them, C. stillicidiorum, has often been considered a variety of C. cerina, the type species of the genus Caloplaca. The subgenus Leproplaca groups leprarioid species without cortex, and is depicted in our study by only one specimen of C. xantholyta, recovered in lineage 2 with an uncertain phylogenetic placement. Conversely, subgenus Xanthocarpia, represented by C. ochracea, is recovered in lineage 1, forming a statistically significant monophyletic entity with C. ferrarii and C. marmorata, two taxa from subgenus Gyalolechia (C. lactea group sensu Navarro-Rosinés & Hladun 1996). These three species are characterized by extremely reduced and often endolithic thalli. Regarding the genus Fulgensia, Westberg & Kärnefelt (1998), in their morphological study, suggested that the circumscription of Fulgensia sensu Poelt was probably polyphyletic. Later, Kasalicky et al. (2000) and Gaya et al. (2003) confirmed this polyphyly with molecular data. In this study, including new species, the polyphyly of Fulgensia is again confirmed. The affiliations of the three independent origins already shown in Gaya et al. (2003) are still uncertain, except for the high support in favor of the inclusion of C. schistidii (syn. F. schistidii) within C. saxicola s. str. The relationships within the C. saxicola s. str. group will be discussed in a forthcoming paper. In our analyses, the group with the highest number of taxa, Fulgensia s. str. (including the type species of the genus, F. fulgens) confirms Fulgensia sensu Kasalicky et al. (2000). All 541 molecular studies carried out thus far (Kasalicky et al. 2000; Gaya et al. 2003; this study) support in part the classification of the subgenera proposed by Poelt (1965). What we call Fulgensia s. str., in our study, fits well with what Poelt (1965) called subgenus Fulgensia. Conversely, subgenus Candelariopsis (F. australis, F. schistidii, and F. pruinosa) sensu Poelt (1965) is not monophyletic (Fig 1), as previously reported by Gaya et al. (2003), Kasalicky et al. (2000) and Westberg & Kärnefelt (1998). In our study, two species described subsequently to the work of Poelt (1965), F. canariensis and F. poeltii, are also included and nested within Fulgensia s. str. Fulgensia s. str. used to group terricolous species with yellow thalli, covered with abundant pruina, cortex darkened by the presence of crystals and spores mostly without septum. The heterogeneity of this group has increased in our analyses by the inclusion of species with different thalli and occasionally septate spores, such as F. canariensis. The new phylogenetic circumscription of Fulgensia s. str. does not correspond exactly to group D from Westberg & Kärnefelt (1998), as it includes F. canariensis, a species that these authors thought belonged to another group (group B). Species included in group D by Westberg & Kärnefelt (1998) were characterized mainly by having a crustose or squamulous yellow thallus, covered by abundant pruina. Spores in this group are simple or uniseptate, sometimes with a visible internal thickening (F. pruinosa). Conversely, F. canariensis shows an areolate thallus, without pruina, and the colour is waxy yellowish-orange. Spores in F. canariensis have a slight equatorial wall thickening. According to Westberg & Kärnefelt (1998) spore shape brings this species nearer to F. fulgida, but septum ontogeny might indicate certain affinity to some species of Caloplaca with polarilocular spores. Based on this character, Breuss (2001) combined F. canariensis into Caloplaca, and stated that this species, apart from being terricolous, can also grow on basaltic rocks. In our results, morphological or ecological differences do not determine a different origin for this species, and we have kept it within Fulgensia. The two specimens of F. desertorum are not recovered as monophyletic. The specimen from Norway forms a robust clade with F. bracteata, whereas the specimen collected in northern Spain shares a most recent common ancestor with F. fulgens. As pointed out in Gaya et al. (2003), it might be possible that the identification of specimens from northern Europe was erroneous. Conversely, the grouping of F. desertorum and F. fulgens does not clarify the identity of these two species. Fulgensia fulgida is the only species that is revealed as monophyletic. The sister relationship between F. fulgida and F. fulgens was already shown by Kasalicky et al. (2000). However, F. pruinosa appeared as sister to F. bracteata with high support, whereas in our topology it is sister to the rest of the Fulgensia s. str. The polyphyly of the genus Teloschistes is here reported for the first time based on molecular data. In our analyses, Teloschistes might have up to three origins, two of them in lineage 2; T. contortuplicatus and a monophyletic group comprising T. lacunosus, T. scorigenus, and T. villosus. These three species do not contain anthraquinones in the thallus, whereas in T. contortuplicatus the thallus is slightly pigmented, yellowish to grey coloured. Søchting & Frödén (2002) grouped Teloschistes species according to their anthraquinone pigmentation into 542 four groups and included the three species without anthraquinones mentioned above within a group of species without, or rarely with, spots of anthraquinones in the thallus. T. contortuplicatus was included within another group characterized by having a thallus fully or partially pigmented, rarely without anthraquinones, which is shared by two of the three Teloschistes species recovered in lineage 1. Consequently, the group without anthraquinones in the thallus is recovered as monophyletic, whereas the species usually pigmented do not correspond to a monophyletic entity (Fig 1). Nevertheless, the four taxa included in lineage 2 are part of a group of species that have been transferred to the genus Seirophora by Frödén & Lassen (2004), who stated that the separation between Seirophora and Teloschistes was supported by molecular data (P. Frödén, unpubl.). According to our results the delimitation of Seirophora is not yet confirmed. The third potential origin of Teloschistes is recovered in lineage 1, and includes species only with yellow-orangish thalli: T. chrysophthalmus, T. fasciculatus, and T. sieberianus. Søchting & Frödén (2002) indicated that even though the production of anthraquinones has an environmental component, as these pigments are produced only under well-lighted conditions, in some species these pigments are environmentally independent, and thalli may not contain anthraquinones even though they are well exposed to sunlight. Some of these unpigmented species are T. lacunosus, T. scorigenus, and T. villosus. It is possible that the phylogenetic relationships of these groups of species, morphologically so different, will be resolved with phylogenetic confidence with the inclusion of more characters. Additionally, it will be necessary to include the type species of the genus, T. flavicans that, according to Frödén & Lassen (2004), may belong to the group of Teloschistes from lineage 1, to complete a taxonomic revision of these fruticose lichens. When comparing results to Gaya et al. (2003) and Søchting & Lutzoni (2003), the polyphyly of the genus Xanthoria is maintained. In our topology, Xanthoria species are recovered in lineage 3, and show a close relationship with taxa from subgenus Gasparrinia included in this lineage. Based on our taxon sampling, lineage B from Søchting & Lutzoni (2003), including Xanthoria and Caloplaca species with crustose thalli, is difficult to interpret. Species in lineage B are characterized by the presence of ellipsoidal conidia and by having parietin as a dominant anthraquinone, together with small amounts of fallacinal, teloschistin, parietinic acid, and emodin (table 4 in Søchting & Lutzoni 2003; chemosyndrome A, Søchting 1997). Comparing lineage B to the clade sister to Xanthomendoza fallax in our study (Fig 1), the morphological homogeneity described by Søchting & Lutzoni (2003) lacks consistency. The C. holocarpa specimen used by Søchting & Lutzoni (2003), and recovered together with taxa from the C. saxicola s. str. group in their clade B3, may not correspond to our understanding of C. holocarpa. Actually, they confirmed that their specimen could correspond to C. tenuatula ssp. inconnexa sensu Clauzade & Roux (1985). According to Claude Roux (pers. comm.) the identity of this specimen is C. polycarpa var. athallina. In our analyses, we do not recover lineage B1 from Søchting & Lutzoni (2003) that grouped X. elegans and X. parietina. Instead, X. elegans appears more closely related to X. sorediata, and X. parietina is nested with X. calcicola. The monophyletic entity constituted by X. parietina and X. calcicola was also E. Gaya et al. revealed in Arup & Grube (1999). Recently, in a population study by Lindblom & Ekman (2005), the separation between X. parietina and X. calcicola has been confirmed. Søchting & Lutzoni (2003) characterized species from their clade B1 by the presence of a foliose thallus, by a paraplectenchymatous cortex, and by fixation to the substrate by means of a lower cortex or hapteria (sensu Kondratyuk & Poelt 1997). After an anatomical study on Xanthoria species we verified that X. elegans and X. sorediata show the same type of cortex structures, proso-scleroplectenchymatous, and non-paraplectenchymatous. The relationship between these two species had already been mentioned (Kondratyuk & Poelt 1997), even though it had been established based on the primary and secondary species concept of Poelt (1963, 1970, 1972). Poelt & Petutschnig (1992a, b) classified X. candelaria within the X. fallax group based on the presence of soredia. In our analyses, we do not recover this relationship. According to Søchting & Lutzoni (2003), phylogenetic separation of X. candelaria, outside the X. fallax group, can be morphologically explained by the presence of ellipsoidal conidia and by the absence of rhizines in X. candelaria. In this study we accepted the combination of the X. fallax group into the genus Xanthomendoza. Just as for Arup & Grube (1999) and Gaya et al. (2003), X. fallax is derived from an early divergence within lineage 1; however, unlike these previous studies, C. texana and the Teloschistes group are revealed as the first divergence within lineage 1 rather than X. fallax. This branch could correspond to group A from Søchting & Lutzoni (2003), which, instead of X. fallax, included X. borealis and X. poeltii, two species also transferred to the genus Xanthomendoza. The X. fallax (or X. ulophyllodes) group was first established by Poelt & Petutschnig (1992a, b) and later recombined as an independent genus (Xanthomendoza) by Søchting et al. (2002). Species of Xanthomendoza are characterized by having true rhizines, by the slightly different cortex structure, and by having narrow, oblong, or bacilliform conidia (Lindblom 1997). Moreover, most species have chemosyndrome A3, described by Søchting (1997). Persistence of Xanthomendoza in presenting a different origin from the rest of Xanthoria species supports its delimitation anticipated by morphological characters. Recently, Kondratyuk & Kärnefelt (2003) have described three new genera in the Teloschistaceae: Oxneria, Rusavskia, and Xanthoanaptychia, respectively segregated from genera Xanthomendoza, Xanthoria, and Teloschistes. Oxneria corresponds to the Xanthomendoza ullophyllodes group, Rusavskia would be equivalent to the presumed natural group of Xanthoria elegans, and Xanthoanaptychia is composed of the Teloschistes villosus group. In Khodosovtsev et al. (2004), the differentiating characters of these newly described genera, from those traditionally accepted, are based mainly on the thallus habit, conidial form, presence/absence of rhizines, and upper and lower cortex structure. We do not recognize these new genera in this study because phylogenetic relationships within the Teloschistaceae need further resolution and support before new generic names can be ascribed to stable monophyletic groups within the Teloschistaceae in a contructive manner. Based on our sampling, Oxneria would be represented by one species (Xanthomendoza fallax); Rusavskia by Xanthoria elegans, X. resendei and X. sorediata; and Xanthoanaptychia by Teloschistes contortuplicatus, T. lacunosus and T. villosus (see Fig 1). Phylogenetic reassessment of the Teloschistaceae Regarding Oxneria, we cannot accept segregating this genus from Xanthomendoza based only on the type of cortex structure. Cortex structure is quite a variable character and it would be risky to use it to discriminate supraspecific entities. As an example, Kondratyuk & Kärnefelt (2003) transferred Xanthomendoza incavata into Oxneria, a species that, according to Søchting et al. (2002), has a cortex structure very similar to X. mendozae, the only species that then remained within genus Xanthomendoza. Moreover, considering the phylogeny obtained by Søchting et al. (2002), Oxneria would be paraphyletic. Genera Rusavskia and Xanthoanaptychia were also differentiated based on the cortex structure and by the presence/ absence of a lower cortex. Species included in Rusavskia revealed several origins both in our topology and in Gaya et al. (2003), just as for Xanthoria species. Xanthoanaptychia is also a polyphyletic genus and the species considered within Xanthoanaptychia correspond to some of those transferred to genus Seirophora by Frödén & Lassen (2004). Until publication of the phylogenetic and morphological study on the genera Seirophora and Teloschistes by P. Frödén (pers. comm.), we believe it more appropriate to maintain the traditional taxonomical categories. Kondratyuk & Zelenko (2002) combined C. schistidii into Xanthoria schistidii. In our study, this species proves to be nested within the C. saxicola s. str. group. Therefore, we do not consider this transfer appropriate at this time. Taxonomic conclusions In our study, we observe that lineage 2 continues to show a high level of phenotypic heterogeneity, and includes several type species: Caloplaca cerina, Fulgensia fulgens, and Letrouitia domingensis. Several species groups are revealed with high confidence (C. aurantia group, C. carphinea group and C. cerina group). Because of the lack of support for intermediate and basal internodes we have decided to make no nomenclatural changes and to wait until new data help to build a more comprehensive phylogenetic classification. Nevertheless, we can make some suggestions based on our results. Accepting the generic entity of the Fulgensia s. str. group could lead to accepting species groups mentioned above at the genus level. These groups have several morphological features that would also support their separation. However, we need to be aware that if C. cerina is the type species of genus Caloplaca, segregation of this species group will necessitate considerable nomenclatural changes for the rest of species currently within Caloplaca. Regarding Teloschistes species included within lineage 2, the proposal by Frödén & Lassen (2004) to use Seirophora would be valid if the monophyly of this group could be confirmed in future studies. Lineage 1 appears more diverse than previously thought. A possible taxonomic solution for this lineage would be to consider it as a part of the redefined genus Xanthoria as proposed by Gaya et al. (2003) and Søchting & Lutzoni (2003). Other possibilities would be to restrict Xanthoria to a subclade within lineage 1, leaving Teloschistes and Xanthomendoza as independent genera. Species of very different morphology, such as those in subgenera Gyalolechia and Xanthocarpia, could be maintained as a subgenus within Xanthoria. Finally, another option would 543 be to consider Xanthoria based solely on lineage 3 and establish independent genera for the rest of the excluded clades. Acknowledgements We thank Claude Roux for critical comments on the manuscript, Valérie Reeb for her invaluable support in the DNA isolation and sequencing, and Ulf Arup, Cécile Gueidan, Jolanta Miadlikowska, and Ulrik Søchting for their useful discussions and help. The authors are also grateful to Ulf Arup, André Aptroot, Javier Etayo, Cécile Gueidan, and Ulrik Søchting, as well as the curators of the herbaria of BCN, C, DUKE, E, GZU, LEB, MARSSJ, MIN, MUB, SANT and TFC Lich for providing material used in this work; Frank Kauff and Stefan Zoller for writing computer programs useful for this study and Molly McMullen for the English revision. This work was carried out within the projects (PB 96-1115-C04-02, BOS 20010869-C04-02, CGL 2005-04322), Spanish government, and CIRIT (2001 SGR 00095, 2005 SGR 01047), Catalonian government. We gratefully acknowledge support from a PhD scholarship (FIprograma propi, Universitat de Barcelona) and a postdoctoral grant from the Fulbright Scholar Program to EG. Supplementary data Supplementary data associated with this article can be found in the online version, at doi: 10.1016/j.mycres.2007.11.005. references Alfaro ME, Zoller S, Lutzoni F, 2003. Bayes or bootstrap? 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