Major clades of parmelioid lichens (Parmeliaceae, Ascomycota) and the evolution of their morphological and chemical diversity

Molecular Phylogenetics and Evolution 39 (2006) 52–69 www.elsevier.com/locate/ympev

Major clades of parmelioid lichens (Parmeliaceae, Ascomycota) and the evolution of their morphological and chemical diversity Oscar Blanco a, Ana Crespo a, Richard H. Ree b, H. Thorsten Lumbsch b,¤ a

Departamento de Biología Vegetal II, Facultad de Farmacia, Universidad Complutense de Madrid, Madrid 28040, Spain b Department of Botany, The Field Museum, 1400 S. Lake Shore Drive, Chicago, IL 60605, USA Received 31 March 2005; revised 11 November 2005; accepted 28 December 2005 Available online 14 February 2006

Abstract Parmelioid lichens comprise about 1500 species and have a worldwide distribution. Numerous species are widely distributed and well known, including important bioindicators for atmospheric pollution. The phylogeny and classiWcation of parmelioid lichens has been a matter of debate for several decades. Previous studies using molecular data have helped to establish hypotheses of the phylogeny of certain clades within this group. In this study, we infer the phylogeny of major clades of parmelioid lichens using DNA sequence data from two nuclear loci and one mitochondrial locus from 145 specimens (117 species) that represent the morphological and chemical diversity in these taxa. Parmelioid lichens are not monophyletic; however, a core group is strongly supported as monophyletic, excluding Arctoparmelia and Melanelia s. str., and including Parmeliopsis and Parmelaria. Within this group, seven well-supported clades are found, but the relationships among them remain unresolved. Stochastic mapping on a MC/MCMC tree sampling was employed to infer the evolution of two morphological and two chemical traits believed to be important for the evolutionary success of these lichens, and have also been used as major characters for classiWcation. The results suggest that these characters have been gained and lost multiple times during the diversiWcation of parmelioid lichens. © 2006 Elsevier Inc. All rights reserved. Keywords: Parmeliaceae; Ascomycota; Lichens; Stochastic mapping; Character evolution

1. Introduction In the course of co-evolution between ascomycete fungi and their photosynthetic partners in lichen symbioses, numerous morphological structures and secondary metabolites have evolved that are believed to have an important adaptive role for the success of these symbiotic organisms (Lange, 1992; Rikkinen, 1995; Sanders, 2001). The morphological traits occur mostly in foliose lichens and include cortical layers composed of fungal hyphae that protect photosynthetic partners from high insolation (Büdel and Scheidegger, 1996; Jahns, 1973, 1988), and pores of diVerent kinds and sizes that facilitate gas exchange through the cortex—analogues to stoma in bryophytes and kormophytes.

*

Corresponding author. Fax: +1 312 665 7158. E-mail address: [email protected] (H.T. Lumbsch).

1055-7903/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.ympev.2005.12.015

The morphology and physiological eVects of these pores have been studied intensely during the last decades (Beltman, 1978; Green et al., 1981, 1985; Hale, 1973, 1981; Lumbsch and Kothe, 1992; Sancho et al., 2000; Yoshimura and Hurutani, 1987), but their evolutionary history is currently poorly understood. In addition to morphological adaptations, lichens are well known for the number and quantity of secondary metabolites they produce (Culberson, 1969; Huneck and Yoshimura, 1996). Of particular interest is the adaptive value of cortical substances, including UV absorbent compounds or pigments that screen visible light and UV (BeGora and Fahselt, 2001; Bjerke et al., 2002; Rikkinen, 1995; Solhaug and Gauslaa, 1996; Solhaug et al., 2003). These adaptive morphological structures and secondary metabolites were frequently used as taxonomic characters without any clear concept about their evolutionary origins. While alternative hypotheses about the evolutionary origin

O. Blanco et al. / Molecular Phylogenetics and Evolution 39 (2006) 52–69

of lichen symbioses exist (Gargas et al., 1995; Lutzoni et al., 2001) and a high plasticity in the life-style of some ostropalean fungi has been demonstrated (Wedin et al., 2004), the evolution of adaptive morphological and chemical characters in lichens has been examined only in few studies. These include the reconstruction of the evolution of polyspory (Reeb et al., 2004) and the evolution of tri-partite symbioses (Miadlikowska and Lutzoni, 2004). This lack of attention is surprising, given the importance of these characters in lichen symbiosis and their frequent use in taxonomy. In this study, we have used parmelioid lichens (Parmeliaceae) (DePriest, 1999), a group of morphologically complex lichen-forming fungi (Henssen and Jahns, 1974), as a model to study the evolution of these traits focusing in two morphological and two chemical characters. Parmeliaceae is one of the largest families of lichenforming fungi, and has a worldwide distribution. It includes several common and well-known species, including taxa such as Parmelia sulcata, Flavoparmelia caperata, and Punctelia subrudecta that are frequently used as bioindicators for atmospheric pollution (Crespo et al., 2004; Nimis et al., 2002). In general, the family is characterized morphologically by a certain type of ascoma ontogeny and the presence of an ascomatal structure called a cupulate exciple (Henssen and Jahns, 1974). Some authors prefer to segretate some taxa from Parmeliaceae at the family level, such as Alectoriaceae or Hypogymniaceae (Elix, 1979; Eriksson and Hawksworth, 1998; Kärnefelt and Thell, 1992; Krog, 1982; Poelt, 1973), but segregation of these families is not supported by molecular evidence (Mattsson and Wedin, 1999; Mattsson et al., 2004; Thell et al., 2004; Wedin et al., 1999). Traditionally, two large genera, Parmelia and Cetraria, were distinguished within the core group of the family, and these correspond more or less to the parmelioid (DePriest, 1999) and cetrarioid group (Kärnefelt et al., 1992; Randlane and Saag, 1993; Randlane et al., 1997), respectively. Parmelioid lichens encompass approximately 1500 taxa (Hale and DePriest, 1999), which were formerly classiWed within Parmelia sensu lato. This group includes species which are mainly foliose, mostly rhizinate lichens with laminal apothecia and simple, hyaline ascospores (Hale, 1987), and have their centre of diversity in oceanictemperate, tropical, and subtropical ecosystems. Generic concepts in lichen-forming fungi have changed dramatically since the late 1960s (Rambold and Triebel, 1999), with the Parmeliaceae being a prominent example (DePriest, 1999; Hale, 1984; Nimis, 1998). In crustose groups, ascomatal characters (e.g., ascus-type, ascoma and ascospore development) and hamathecial features have often been utilized to circumscribe segregate genera, but these characters have not been as widely used in parmelioid lichens, which frequently occur as sterile species. For this reason, morphological and chemical characters have more often been employed to segregate genera in this group, though acceptance of the new genera has not been uniform (e.g., Clauzade and Roux, 1985; Eriksson and

53

Hawksworth, 1986; Llimona and Hladun, 2001; Purvis et al., 1992). Based on Hale’s concept, DePriest (1999) gave an overview of the historic development of generic concepts in parmelioid lichens and included 36 genera in this group (Table 1). In recent years, some of these segregates have been included within other genera based on morphological and/or molecular evidence, e.g., Rimeliella within Canomaculina (Elix, 1997); Chondropsis, Paraparmelia, and Neofuscelia within Xanthoparmelia (Blanco et al., 2004a; Elix, 2003; Hawksworth and Crespo, 2002); and Canomaculina, Concamerella, and Rimelia within Parmotrema (Blanco et al., 2005). Other groups have been found to be heterogeneous such as Melanelia (Blanco et al., 2004b) (Table 1). Crespo et al. (2001) demonstrated the monophyly of a group of parmelioid lichens using mitochondrial rDNA, a result corroborated by Mattsson et al. (2004), who used four diVerent loci in a higher-level phylogenetic study. However, the relationships among major clades within parmelioids remain poorly understood, and the evolution of morphological and chemical characters within parmelioid lichens has not yet been studied. We chose to study two morphological and two chemical traits that are usually interpreted as being adaptive for these symbiotic fungi (Hale, 1981; Rikkinen, 1995). The morphological characters are two types of perforations of the thallus surface that allow gas exchange of the photosynthetic partner: (a) pores in the epicortex and (b) a cortical structure that reaches the algal layer, called a pseudocyphella (Fig. 1). The epicortex is a polysaccharide layer about 0.6 m thick, lying over the cortex of several, unrelated macrolichens. This layer is analogous to the cuticle in higher plants. Pores are 15–40 m in diam., with a density of 100–400 pores per mm¡2, and may be restricted to the epicortex or include cortical layers, but not the medulla (Beltman, 1978; Hale, 1973, 1981). Pseudocyphellae are ontogenetically derived from pores in the cortex, have a diameter of 200–2000 m and a density of 1–2 per mm¡2. The cortex is dissolved or partially reduced in these structures and medullary hyphae are usually involved (Feuerer and Marth, 1997; Hale, 1973, 1981). The type of perforation is usually believed to be an important character at the generic level and was even regarded as important at the subfamilial rank (Elix, 1993; Hale, 1981; Henssen, 1992). The two chemical characters investigated are the presence of cortical substances that are believed to play an important ecological role in screening UV and visible light: the pigment usnic acid absorbs both UV and visible light, and atranorin, a colourless depside of the -orcinol type, has a strong UV absorption (Huneck and Yoshimura, 1996). All four characters described above have been regarded as key characters distinguishing genera and generic groups within parmelioid lichens (Elix, 1993). Sequences of two nuclear and single mitochondrial ribosomal DNA loci were used for phylogeny reconstruction. Most sequences were used in studies focused on

54

O. Blanco et al. / Molecular Phylogenetics and Evolution 39 (2006) 52–69

Table 1 Genera of Parmelioid lichens as accepted by DePriest (1999) Changes in classiWcations since DePriest (1999) Allantoparmelia Almbornia Arctoparmelia Bulborrhizina Bulbothrix Canomaculina Canoparmelia Cetrariastrum Concamerella Everniastrum Flavoparmelia Flavpunctelia Hypotrachyna Karoowia Melanelia Melanelia s. lat. Melanelia s. lat. Myelochroa Namakwa Neofuscelia Omphalora Paraparmelia Parmelia Parmelina Parmelinella Parmelinopsis Parmotrema Parmotremopsis Placoparmelia Pleurosticta Pseudoparmelia Psiloparmelia Punctelia Relicina Relicinopsis Rimelia Xanthomaculina Xanthoparmelia

Number of species

Genera included in this study

3 2 5 1 52

¡ ¡ + ¡ +

49 5

+ ¡

40 35 7 ca. 190 19 8 11 19 28 2

+ + + + + + + + + ¡ + ¡ + + + + + + ¡ ¡ + ¡ ¡ + + ¡ + + +

Parmotrema

Parmotrema

Melanelixia Melanohalea

Xanthoparmelia 1 Xanthoparmelia ca. 45 ca. 15 5 25 ca. 350 2 1 3 4 12 34 54 5 Parmotrema 2 ca. 650

A

60µm

B

60µm

Fig. 1. SEM picture of the morphology of perforations in the upper surface of parmelioid lichens. (A) Pored epicortex in Parmotrema cetratum. (B) Pseudocyphella in Melanohalea exasperata.

certain generic groups within parmelioid lichens (Blanco et al., 2004a,b, 2005). Here, they are used with 20 new sequences to infer higher-level phylogenetic relationships within this group in Bayesian and maximum parsimony

frameworks. On the basis of the Bayesian tree sampling, histories of morphological and chemical character evolution are mapped and used to evaluate previous hypotheses developed on the basis of classical comparative methods.

O. Blanco et al. / Molecular Phylogenetics and Evolution 39 (2006) 52–69

2. Materials and methods 2.1. Taxon sampling We sampled 109 species of parmelioid lichens, including the major clades and representatives of the morphological and chemical diversity within this group. The sample includes 20 out of 31 parmelioid genera based on the DePriest (1999) classiWcation, considering that some genera were reduced to synonymy (Blanco et al., 2004a, 2005), and the two newly described genera Melanelixia and Melanohalea (Blanco et al., 2004b). Samples of eight other Parmeliaceae were also included. Pseudevernia furfuracea was used as outgroup since it has been shown that it does not belong to parmelioid lichens (Mattsson et al., 2004). The data set includes 406 sequences, the majority of which are from previous publications by us (Blanco et al., 2004a,b, 2005; Crespo et al., 2001), and 20 sequences newly generated for this study. GenBank accession numbers, locality, and voucher information are given in Table 2. 2.2. Molecular methods Samples prepared from freshly collected and frozen herbarium specimens were ground with sterile glass pestles. Total genomic DNA was extracted using the DNeasy Plant Mini Kit (Qiagen) according to the manufacturer’s instructions with slight modiWcations described in Crespo et al. (2001). Dilutions of the total DNA were used for PCR ampliWcations of nuclear ITS and LSU rRNA loci, and the mitochondrial SSU rRNA. Fungal nu ITS rDNA was ampliWed using the primers ITS1F (Gardes and Bruns, 1993) and ITS4 (White et al., 1990); nu LSU rDNA was ampliWed using the primers LR0R (Rehner and Samuels, 1994) and LR5 (Vilgalys and Hester, 1990), and mt SSU rDNA was ampliWed using the primers mrSSU1 and mrSSU3R (Zoller et al., 1999), NMS1 and NMS2 (Li et al., 1994), and MSU1 and MSU7 (Zhou and Stanosz, 2001). AmpliWcations were performed in 50 L volumes containing a reaction mixture of 10 L diluted DNA, 5 L of 10£ DNA polymerase buVer (Biotools) (containing MgCl2 2 mM, 10 mM Tris–HCl, pH 8.0, 50 mM KCl, 1 mM EDTA, and 0.1% Triton X-100), 1 L of dinucleotide triphosphate (dNTPs), containing 10 mM of each base, 2.5 L of each primer (10 M), 1.25 L of DNA polymerase (1 U/L), and 27.5 L dH2O. AmpliWcations for nu ITS and LSU rDNA were carried out in an automatic thermocycler (Techne Progene) and performed using the following programs: initial denaturation at 94 °C for 5 min, and 30 cycles of: 94 °C for 1 min, 54–60 °C (ITS rDNA) and 60 °C (LSU rDNA) for 1 min, 72 °C for 1.5 min, and a Wnal extension at 72 °C for 5 min. PCR ampliWcation for mitochondrial rDNA was carried out in a Hybaid OmniGene thermocycler using the following program: initial denaturation at 94 °C for 5 min and 35 cycles of: 94 °C for 1 min, 57–58 °C for 1 min, and 72 °C for 1.5 min, and a Wnal extension at 72 °C for 5 min.

55

The PCR products were subsequently cleaned using the DNA PuriWcation Column kit (Biotools) according to the manufacturer’s instructions. The cleaned PCR products were sequenced with the same primers used in the ampliWcations. The ABI Prism Dye Terminator Cycle Sequencing Ready reaction kit (Applied Biosystems) was used and the following settings were carried out: denaturation for 3 min at 94 °C and 25 cycles at: 96 °C for 10 s, 50 °C for 5 s, and 60 °C for 4 min. Sequencing reactions were electrophoresed on a 3730 DNA analyser (Applied Biosystems). Sequence fragments obtained were assembled with SeqMan 4.03 (DNAStar) and manually adjusted. 2.3. Sequence alignments The nu ITS and mt SSU rDNA data sets contained highly variable sequence portions in the alignment. Because standard alignment programs such as Clustal (Thompson et al., 1994) become less reliable when highly variable sequences are involved, we used an alignment procedure that employs a linear Hidden Markov Model (HMM) for the alignment, as implemented in the software SAM (Hughey and Krogh, 1996). Sequences of 145 specimens (Table 2) were aligned separately for the three regions. Ninety-nine base pairs in the mt SSU, 61 bp in the nu ITS and 188 in the nu LSU data set that could not be aligned with statistical conWdence were excluded from the phylogenetic analysis. 2.4. Phylogenetic analyses For phylogenetic analysis, we used a Bayesian approach that allows eYcient analysis of complex nucleotide substitution models in a parametric statistical framework (Huelsenbeck et al., 2001; Larget and Simon, 1999) and includes estimation of uncertainty (Huelsenbeck et al., 2000). Posterior probabilities and bootstrap support values from maximum parsimony or maximum likelihood analyses have been demonstrated to diVer, and these diVerences have been interpreted in diVerent ways (Alfaro et al., 2003; Simmons et al., 2004; Suzuki et al., 2002; Wilcox et al., 2002). Bayesian support values appear to be overestimates in certain cases, especially when short branches are involved. In contrast, bootstrap values are commonly underestimates and can be viewed as helpful lower bounds of support values (Douady et al., 2003), but are more sensitive to long-branch attraction. We also performed maximum parsimony analyses, including nonparametric bootstrapping. Here, we adopt a conservative perspective and consider well-supported clades to be those that have a posterior probability of at least 0.95 as well as bootstrap support equal to or above 70%. 2.4.1. Bayesian analyses The Bayesian (B/MCMC) analyses were performed using MrBayes 3.0 (Huelsenbeck and Ronquist, 2001). Posterior probabilities were approximated by sampling trees using a Markov chain Monte Carlo (MCMC) method. For all data sets the general time reversible model of nucleotide substitu-

56

Table 2 Species and specimens of Parmeliaceae analysed Species

Voucher

Locality

mt SSU

nu ITS

nu LSU

Pored

Pseudocyphellae

Atranorin

Usnic acid

Parmelioid (DePriest, 1999)

Arctoparmelia centrifuga Bulbothrix meizospora B. setschwanensis Canoparmelia crozalsiana Everniastrum cirrhatum E. nepalense Flavoparmelia baltimorensis 1 F. baltimorensis 2 F. caperata 1 F. caperata 2 F. soredians Flavopunctelia Xaventior 1 F. Xaventior 2 Hypotrachyna adducta H. ciliata H. costaricensis H. endochlora H. immaculata H. inWrma H. laevigata H. revoluta H. sinuosa H. taylorensis Karoowia saxeti Melanelia disjuncta Melanelia stygia Melanelixia fuliginosa 1 M. fuliginosa 2 M. fuliginosa 3 M. fuliginosa 4 M. fuliginosa 5 M. glabra 1 M. glabra 2 M. subargentifera 1 M. subargentifera 2 M. subaurifera 1 M. subaurifera 2 M. subaurifera 3 M. subaurifera 4 M. elegantula 1 M. elegantula 2 M. elegantula 3 M. elegantula 4 M. exasperata 1 M. exasperata 2 M. aV. exasperata 1

MAF 6879 GPGC 02-000786 MAF 10212 MAF 7658 Trest 149 GPGC 02-000924 MAF 7660 MAF 10174 MAF 6045 MAF 10175 MAF 10176 MAF 6046 DCH-19 MAF 10206 MAF 10185 MAF 10211 MAF 10178 MAF 7462 MAF 10210 MAF 10177 MAF 6047 MAF 10179 MAF 9921 Aptroot 53350 Mayrhofer 13743 Haikonen 20365 MAF 7640 MAF 10222 MAF 10219 MAF 10223 MAF 10229 MAF 7634 MAF 10228 MAF 6049 MAF 9909 MAF 10221 MAF 10217 MAF 10216 MAF 10215 MAF 10226 MAF 10218 MAF 10231 MAF 10224 MAF 7636 MAF 10214 MAF 10227

Sweden India China Spain Costa Rica India USA USA Spain China Spain Spain China China China Costa Rica Great Britain Australia China Great Britain Spain Great Britain Great Britain Taiwan Austria Finland Spain Spain Spain Spain Spain Spain Spain Spain Spain Spain Spain Spain Great Britain Spain Spain Spain Spain Spain Spain Spain

AF351156 AY611127 — AY586594 AY611128 AY611129 AY586583 AY586584 AF351163 AY586585 AY586586 AF351164 AY586587 AY785277 AY785280 AY785276 AY611130 AY611131 AY785278 AY611132 AF351166 AY611133 AY582298 AY582299 AY611134 AY611154 AY611141 AY611142 AY611143 AY611146 AY611145 AY582300 AY611144 AY611155 AY582301 AY611152 AY611157 AY611158 AY611156 AY611136 AY611135 AY611151 AY61137 AY611140 AY611138 AY611139

AY581054 AY611068 AY611069 AY586571 AY611070 AY611071 AY586559 AY586560 AY581059 AY586561 AY586562 AY581060 AY586563 AY785270 AY785273 AY785269 AY611072 AY611073 AY785271 AY611074 AY611075 AY611076 AY581061 AY581063 AY611077 AY611097 AY611084 AY611085 AY611086 AY611089 AY611088 AY581064 AY611087 AY611098 AY581065 AY611096 AY611100 AY611101 AY611099 AY611079 AY611078 AY611094 AY611080 AY611083 AY611081 AY611082

AY578917 AY607780 AY607781 AY584831 AY607782 AY607783 AY584832 AY584833 AY578922 AY584834 AY584835 AY578923 — AY785263 AY785266 AY785262 AY607784 AY607785 AY785264 AY607786 AY607787 AY607788 AY578924 AY578926 AY607789 AY607809 AY607796 AY607797 AY607798 AY607801 AY607800 AY578927 AY607799 AY607810 AY578928 AY607807 AY607812 AY607813 AY607811 AY607791 AY607790 AY607806 AY607792 AY607795 AY607793 AY607794

Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes No No Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes No No Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes No No No No No No No

No No No No No No No No No No No Yes Yes No No No No No No No No No No No Yes Yes No No No No No No No No No No No No No Yes Yes Yes Yes Yes Yes Yes

No Yes Yes Yes Yes Yes No No No No No Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes No Yes No No No No No No No No No No No No No No No No No No No No No No No

Yes No No No No No Yes Yes Yes Yes Yes No No No No No No No No No No Yes No Yes No No No No No No No No No No No No No No No No No No No No No No

O. Blanco et al. / Molecular Phylogenetics and Evolution 39 (2006) 52–69

Group

MAF 10225 MAF 10230 MAF 10213 Vitikainen 16196 Ahti 60893 MAF 10207 MAF 10208 MAF 7272 MAF 6804 MAF 9756 MAF 7281 MAF 6054 MAF 6058 MAF 6057 MAF 6056 LWG 20-77171 MAF 9913 MAF 7639 MAF 10220 MAF 10182 MAF 6878 Osorio 9424 MAF 6061 Osorio 9423 MAF 7657 MAF 7636 Cole 7983 MAF 6965 MAF 7656 MAF 7650 MAF 6067 MAF 10164 MAF 10166 HO 324283 GPGC 02-000696 MAF 10163 MAF 9914 MAF 9919 MAF 6922 MAF 7661 MAF 10162 Elix 42705 MAF 9918 MAF 10184 MAF 10183 MAF 6768 MAF 6744 Elix 30651 MAF 6793

Spain Spain Spain Finland Finland China China Russia Sweden Spain Japan Great Britain Spain Spain Spain India Spain Spain China Australia Australia Uruguay Portugal Uruguay Australia Spain USA Portugal Uruguay Spain Portugal China Portugal Australia India Australia Spain Portugal Australia USA USA Australia Portugal Australia Australia USA USA Australia Spain

AY611149 AY611153 AY611147 AY611148 AY611150 AY611160 AY611159 AY611161 AF351172 AY582319 AY611162 AY582320 AY611163 AY611164 AF351173 AY611165 AY582321 AY582322 AY611168 AY611166 AF351174 AY586598 AY586589 AY582297 AY582295 AY586590 AY586591 AY586580 AY582296 AY586600 AF351184 AY586599 AY586592 AY586581 AY586582 AY586593 AY582323 AY582324 AY586595 AY586596 AY586597 AF351183 AY582325 AY785281 AY785282 AY582328 AY582302 — AF351186

AY611092 AY611095 AY611090 AY611091 AY611093 AY611103 AY611102 AY036988 AF350027 AY295109 AY036975 AY581083 AY611104 AY611105 AY581084 AY611106 AY581085 AY581086 AY611110 AY611107 AY611108 AY586576 AY586565 AY581057 AY581055 AY586567 AY586568 AY586566 AY581056 AY586578 AY586579 AY586577 AY586569 AY586557 AY586558 AY586570 AY581087 AY581088 AY586572 AY586573 AY586574 AY586575 AY581089 AY785274 AY785275 AY581092 AY581066 AY581062 AY581096

AY607804 AY607808 AY607802 AY607803 AY607805 AY607815 AY607814 — AY578947 AY578948 AY607816 AY578949 AY607817 AY607818 AY578950 AY607819 AY578951 AY578952 — AY607820 AY607821 AY584847 AY584837 AY578920 AY578918 AY584839 AY584840 AY584838 AY578919 AY584849 AY584850 AY584848 AY584841 AY584829 AY584830 AY584842 AY578953 AY578954 AY584843 AY584844 AY584845 AY584846 AY578955 AY785267 AY785268 AY578958 AY578929 AY578925 AY578962

No No No No No Yes Yes No No No No No Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes No No No No No No Yes Yes Yes Yes Yes Yes

Yes Yes Yes Yes Yes No No Yes Yes Yes Yes Yes No No No No No No No No No No No No No No No No No No No No No No No No No Yes Yes Yes Yes Yes Yes No No No No No No

No No No No No No No No No No Yes No Yes No Yes No Yes No Yes No Yes No Yes No Yes No Yes No Yes No Yes No Yes No Yes No Yes No Yes No Yes No Yes No Yes No Yes No Yes No Yes No Yes No Yes No Yes No Yes No Yes No Yes No Yes No Yes No Yes No Yes No No No Yes No Yes No Yes No Yes No Yes No Yes No No Yes No Yes No Yes No No No Yes No Yes (continued on next page)

O. Blanco et al. / Molecular Phylogenetics and Evolution 39 (2006) 52–69

M. aV. exasperata 2 M. aV. exasperata 3 M. exasperatula M. olivacea M. septentrionalis Myelochroa irrugans M. metarevoluta Parmelia pinnatiWda P. saxatilis P. serrana P. squarrosa P. sulcata Parmelina pastillifera P. quercina P. tiliacea Parmelinella wallichiana Parmelinopsis horrescens P. minarum 1 P. minarum 2 P. neodamaziana P. subfatiscens Parmotrema cetratum P. crinitum P. Wstulatum P. haitiense P. hypoleucinum P. perforatum P. perlatum P. pilosum P. pseudoreticulatum P. reticulatum 1 P. reticulatum 2 P. robustum P. subcaperatum P. subtinctorium P. tinctorum Pleurosticta acetabulum Punctelia borreri P. pseudocoralloidea P. rudecta 1 P. rudecta 2 P. subXava P. subrudecta Relicina subnigra R. sydneyensis Xanthoparmelia angustiphylla X. atticoides X. brachinaensis X. conspersa

57

58

Table 2 (continued) Group

Voucher

Locality

mt SSU

nu ITS

nu LSU

Pored

Pseudocyphellae

Atranorin

Usnic acid

MAF 7524 MAF 7440 MAF 7659 MAF 7432 MAF 7525 MAF 7665 MAF 9912 GZU 46511 MAF 9956 MAF 9955 MAF 6900 MAF 7471 MAF 7072 MAF 6206 MAF 6802 MAF 9916 MAF 9915 MAF 7532 Elix 42648 MAF 6052 MAF 9908 MAF 6216 MAF 6794 — MAF 6784 Elix 42635 Elix 30650 Elix 30294 MAF 9917 MAF 9910

Australia Australia Spain Australia Australia Australia Spain Namibia Spain USA Australia Australia Spain Spain Spain Spain Australia Australia Australia Spain Spain Spain Spain Poland Spain Australia Australia Australia Spain Spain

AY582332 AY582333 AY582304 AY582303 AY582334 AY582305 AY582308 AY582326 AY582330 AY582338 AF351171 AY582314 AY582313 AY582306 AY582335 AY582336 AY582315 AY582316 AY582337 AY582307 AY582312 AY582339 AF351169 — AY582309 — AY582317 AF351158 AY582329 AY582340

AY581097 AY581098 AY581068 AY581067 AY581099 AY581069 AY581072 AY581090 AY581094 — AY581077 AY581078 AY581076 AY581070 AY037006 AY581100 AY581079 AY581080 AY581101 AY037005 AY581075 AY581104 AY581071 — AY037004 AY581102 AY581081 AY581058 AY581093 AY581105

AY578963 AY578964 AY578931 AY578930 AY578965 AY578932 AY578935 AY578956 AY578960 AY578970 AY578941 AY578942 AY578940 AY578933 AY578966 AY578967 AY578943 AY578944 AY578968 AY578934 AY578939 AY578972 — AJ421433 AY578936 AY578969 AY578945 AY578921 AY578959 AY578973

Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes

No No No No No No No No No No No No No No No No No No No No No No No

No No No No No No No No No No Yes Yes No No No No Yes Yes No No No No No

Yes Yes No No Yes No No Yes Yes Yes No No No No Yes Yes No No Yes No No Yes No

Yes Yes Yes Yes Yes Yes

No No No No No No

No No Yes No No No

No Yes No Yes Yes Yes

MAF 7494 MAF 7460 MAF 7667 MAF 7463 MAF 10209 MAF 7523 MAF 6070 BCN-13861 BCN-13862

Australia Spain Australia Australia Spain Australia Spain Spain Spain

AY582310 AY582341 AY582311 AY582318 AY582327 AY582342 AY582343 AY582344 AY582345

AY581073 AY581106 AY581074 AY581082 AY581091 AY581107 AY581108 AY581109 AY581110

AY578937 AY578974 AY578938 AY578946 AY578957 AY578975 AY578976 AY578977 AY578978

Yes Yes Yes Yes Yes Yes Yes Yes Yes

No No No No No No No No No

No No No Yes No No No No No

No Yes No No Yes Yes Yes Yes Yes

X. subdiZuens X. subincerta X. sublaevis X. subprolixa X. subspodochroa X. sub verrucigera X. tegeta X. tinctina 1 X. tinctina 2 X. tinctina 3

O. Blanco et al. / Molecular Phylogenetics and Evolution 39 (2006) 52–69

Species X. crespoae 1 X. crespoae 2 X. delisei X. aV. delisei X. digitiformis X. glabrans X. aV. glabrans X. hueana X. isidiovagans X. lineola X. lithophila X. lithophiloides X. loxodes 1 X. loxodes 2 X. mougeotii 1 X. mougeotii 2 X. murina X. norcapnodes X. notata X. pokornyi 1 X. pokornyi 2 X. protomatrae X. pulla X. pulla X. pulloides X. reptans X. scotophylla X. semiviridis X. stenophylla

No No Yes New sequences not used in previous studies are in bold. The presence/absence of morphological and chemical characters are shown.

No No No Yes Yes No

Yes Yes No

No No Yes No Yes Yes No No Yes No No Yes No No No No No Yes Yes Yes

AY607824 AY607825 AY785265 AY584836 — — AY607822 AY607823 AY607826 AY607827 — AY611111 AY785272 AY586564 — AF410829 — AY611109 AY611112 AY611113 Spain Spain Australia India Austria — Spain Spain Spain Spain Nonparmelioid

Brodoa atrofusca Cetraria aculeata Pannoparmelia angustata Parmelaria subthomsonii Parmeliopsis ambigua P. ambigua P. ambigua P. hyperopta Pseudevernia furfuracea Vulpicida pinastri

MAF 6780 MAF 6781 Elix 42640 LWG 20-77151 GZU 46209 Tehler 8110 MAF 10186 MAF 10181 MAF 6772 MAF 6783

AY643090 AY643091 AF351170 AY586588 AF351175 — — AY611167 AY611169 AF351185

AY578961 AY578979 AY578980 AY578971 Spain Spain Spain Spain X. transvaalensis X. verrucigera X. vicentei 1 X. vicentei 2

MAF 9841 MAF 9920 MAF 7248 MAF 9954

AY582331 AY582346 AY582347 —

AY581095 AY581111 AY581112 AY581103

Yes Yes Yes Yes

No No No No

No No No No

Yes Yes Yes Yes

O. Blanco et al. / Molecular Phylogenetics and Evolution 39 (2006) 52–69

59

tion (Rodríguez et al., 1990) including estimation of invariant sites and assuming a discrete gamma distribution with six rate categories (GTR +I+G) was used and parameters were calculated for each partition separately as proposed by Nylander et al. (2004). In the separate single-partition analyses, MrBAYES was run with eight simultaneous chains for all data sets. In the combined analysis, 12 simultaneous chains were used. MrBAYES was run for 106 generations on each data set and 2 £ 106 generations for the combined analysis. Trees were sampled every 100 generations yielding 10,000 and 20,000 trees, respectively. The Wrst 1000 trees were discarded as the “burn in” of the chain. We plotted the log-likelihood scores of sample points against generation time using TRACER 1.0 (http://evolve.zoo.ox.ac.uk/software. html?id D tracer) to conWrm probable chain stationarity after the Wrst 100,000 generations. For the remaining trees in each analysis, a majority-rule consensus tree with average branch lengths was calculated using the sumt option of MrBayes. Phylogenetic trees were visualized using the program Treeview (Page, 1996). Congruence between the data sets was assessed by comparing bootstrap support of clades above 70% for each locus. We used this approach instead of comparing posterior probabilities to avoid the potential problem of overestimated support for short branches in Bayesian analyses. 2.4.2. Maximum parsimony analyses The maximum parsimony (MP) analyses were performed employing PAUP* 4.0 (SwoVord, 2003) using the heuristic search option with 200 random sequence additions, TBR branch swapping and MulTrees option in eVect, equally weighted characters and gaps treated as missing data. Nonparametric bootstrap support (Felsenstein, 1985) for each clade was estimated based on 2000 replications, using the heuristic search option with 200 random sequence additions and the MulTrees option in eVect. To assess homoplasy levels, consistency index (CI), retention index (RI), and rescaled consistency (RC) index (Farris, 1989) were calculated from each parsimony search in PAUP*. 2.5. Character evolution To evaluate the evolution of morphological and chemical characters in a Bayesian framework, we have used stochastic mapping and Bayesian Metropolis-coupled Markov Chain Monte Carlo (MC/MCMC) tree sampling. Stochastic mapping was originally developed by Nielsen (2002) to infer the phylogenetic locations of nucleotide substitutions and the method was extended to morphological characters by Huelsenbeck et al. (2003). Stochastic mapping estimates the history of character changes on a phylogeny using simulations that employ an explicit transformation model, thus avoiding the tendency of parsimony to underestimate the actual number of changes that occurred (Felsenstein, 1978). It takes as input character-state data for extant species and a phylogenetic tree of those species, and uses a model parame-

60

O. Blanco et al. / Molecular Phylogenetics and Evolution 39 (2006) 52–69

terized by a matrix of instantaneous rates of change between states (Q), a vector of stationary frequencies for character states (), and tree length, measured as the expected amount of change in the character of interest. Characters were coded as follows: epicortex nonpored (0) or pored (1); pseudocyphellae absent (0) or present (1); usnic acid absent (0) or present (1); and atranorin absent (0) or present (1) (Table 2). Presence and absence of atranorin and usnic acid was studied using standardized TLC and HPLC methods (Lumbsch, 2002). Presence or absence of pores/fenestrations and pseudocyphellae was examined using SEM. Small pieces of thalli ca. 5 mm in diam. were cut from samples, air-dried, Wxed to a metallic stub, and sputtered with gold-palladium in a vacuum. A Jeol (JSM 6400) electron microscope was used for the analysis. Uncertainty in the phylogeny was accommodated by mapping each character over 19,000 trees sampled in the combined analysis. For each character, a mapping resulting in the observed states at terminal nodes was performed for each tree. The simulations were run with branch lengths scaled to yield a total tree length of 1 and the bias parameter 0 was drawn from a uniform prior distribution. Each mapping was recorded as two sets of taxon bipartitions (with a bipartition corresponding to a clade subtended by a character-state change): one set for gains of state 1, and one for losses. Recording mappings in this manner facilitates the summary of results across trees that may diVer in topology. Stochastic mapping was carried out using a software library, stmap, written by RHR and available from http:// www.phylodiversity.net/rree/software.html. 3. Results 3.1. Phylogenetic analyses Ambiguously aligned regions and major insertions, representing spliceosomal and group I introns in the nuclear

ribosomal DNA (Bhattacharya et al., 2000; Cubero et al., 2000; Gargas et al., 1995), were excluded from all analyses (99 bp for mt SSU, 188 for the nu LSU, and 61 for the nu ITS). We produced a matrix of 2237 unambiguously aligned nucleotide position characters in the combined analysis, including 645 of the mt SSU, 1038 of the nu LSU, and 554 of the nu ITS rDNA. Five hundred and forty-six characters were variable in the mt SSU, 850 in the nu LSU, and 493 in the ITS rDNA data set. Congruence in phylogenetic signal between the three loci was high overall, with the majority of clades supported by one single-gene analysis not being contradicted in the others. Areas of conXict, in which strongly supported clades were contradicted in another analysis, were restricted to a very few terminal nodes: (a) in the mt SSU tree Hypotrachyna adducta and H. ciliata clustered together with 93% bootstrap support, while in the nu ITS tree H. adducta is sister group of H. inWrma (100%); (b) Parmotrema haitiense grouped with P. pilosum in the mt SSU tree (82%), whereas P. haitiense is sister to P. subtinctorum in the nu ITS tree (100%); (c) Melanohalea exasperata is monophyletic in the nu ITS tree (93%), while this species is paraphyletic in the mt SSU tree. The combined alignment is available in Treebase (S1400). (http://www.treebase.org/treebase). Summary statistics for the data sets analysed under maximum parsimony and B/MCMC are given in Table 3. The amount of homoplasy diVers between the gene partitions: the mt SSU rDNA exhibits the lowest amount of homoplasy, while the highest amount of homoplasy is present in the nu ITS rDNA gene partition. Base composition varies considerably between the mitochondrial and nuclear data sets. The nu LSU rDNA was the most GC rich (51.9%), while the mitochondrial data set had a much lower GC content (39.4%). The percentage of invariable sites also diVers between the data sets, being lowest in the mt SSU rDNA (13.5%). The gamma shape parameter alpha is similar between the gene partitions. The nu ITS,

Table 3 Comparison of performance of data partitions under parsimony and in a Bayesian framework

No of parsimony informative sites No. steps CI RI RC No. nodes with bootstrap 7 70% Mean likelihood Amean/all Cmean/all Gmean/all Tmean/all r (AC)mean/all r (AG)mean/all r (AT)mean/all r (CG)mean/all r (CT)mean/all mean/all P(invar) mean/all No. of nodes with PP 7 0.95

Nu LSU

Nu ITS

Mt SSU

Combined

164 1054 0.298 0.648 0.193 33 ¡6593 (§3.907) 0.228 (§0.003) 0.225 (§0.003) 0.294 (§0.003) 0.254 (§0.003) 1.445 (§0.211) 4.944 (§0.849) 0.769 (§0.093) 0.644 (§0.073) 9.900 (§0.188) 0.500 (§0.0002) 0.219 (§0.013) 48

231 2172 0.227 0.677 0.153 59 ¡10603 (§1.118) 0.224 (§0.006) 0.287 (§0.005) 0.227 (§0.005) 0.263 (§0.003) 1.061 (§0.133) 3.818 (§0.324) 2.359 (§0.257) 0.776 (§0.078) 9.769 (§0.098) 0.570 (§0.0018) 0.324 (§0.041) 75

141 655 0.394 0.822 0.324 28 ¡4438 (§2.567) 0.327 (§0.003) 0.155 (§0.003) 0.239 (§0.004) 0.280 (§0.004) 0.953 (§0.120) 2.936 (§0.377) 1.353 (§0.172) 0.553(§0.088) 3.374 (§0.480) 0.501 (§0.0002) 0.135 (§0.014) 42

536 3959 0.268 0.696 0.187 76 ¡21656 (§2.530) 0.251 (§0.003) 0.227 (§0.003) 0.264 (§0.004) 0.258 (§0.003) 1.080 (§0.008) 3.192 (§0.208) 1.611 (§0.113) 0.600 (§0.038) 8.756 (§0.676) 0.578 (§0.007) 0.181 (§0.003) 85

Parmelioid lichens

Xanthoparmelia lithophiloides Xanthoparmelia scotophylla Xanthoparmelia subspodochroa Xanthoparmelia lithophila Xanthoparmelia norcapnodes Xanthoparmelia crespoae 1 Xanthoparmelia crespoae 2 Xanthoparmelia murina Xanthoparmelia digitiformis Xanthoparmelia tinctina 2 Xanthoparmelia tinctina 3 Xanthoparmelia tinctina 1 Xanthoparmelia semiviridis Xanthoparmelia reptans Xanthoparmelia vicentei 2 Xanthoparmelia vicentei 1 Xanthoparmelia isidiovagans Xanthoparmelia conspersa Xanthoparmelia atticoides Xanthoparmelia lineola Xanthoparmelia sublaevis Xanthoparmelia stenophylla Xanthoparmelia subdiffluens Xanthoparmelia protomatrae Xanthoparmelia-clade Xanthoparmelia angustiphylla Xanthoparmelia brachinaensis Xanthoparmelia notata Xanthioarmelia subverrucigera Xanthoparmelia verrucigera Xanthoparmelia tranvaalensis Xanthoparmelia delisei Xanthoparmelia loxodes 2 Xanthoparemelia aff. glabrans Xanthoparmelia puloides Xanthoparmelia pokornyi 1 Xanthoparmelia pulla Xanthoparmelia pokornyi 2 Xanthoparmelia loxodes 1 Xanthoparmelia aff. delisei Xanthoparmelia subprolixa Xanthoparmelia subincerta Xanthoparmelia glabrans Xanthoparmelia mougeotii 1 Xanthoparmelia mougeotii 2 Xanthoparmelia tegeta Xanthoparmelia hueana Karoowia saxeti Parmotrema crinitum Parmotrema perlatum Parmotrema robustum Parmotrema pilosum Parmotrema fistulatum Parmotrema tinctorum Parmotrema reticulatum 2 Parmotrema reticulatum 1 Parmotrema pseudoreticulatum Parmotrema cetratum Parmotrema haitiense Parmotrema subtinctorium Parmotrema subcaperatum Parmelaria subthomsonii Parmotrema-clade Parmotrema hypoleucinum Parmotrema perforatum Canoparmelia crozalsiana Flavoparmelia caperata 1 Flavoparmelia caperata 2 Flavoparmelia baltimorensis 1 Flavoparmelia baltimorensis 2 Flavoparmelia soredians Punctelia rudecta 1 Punctelia rudecta 2 Punctelia subrudecta Punctelia borreri Punctelia pseudocoralloidea Punctelia subflava Flavopunctelia flaventior 1 Flavopunctelia flaventior 2 Melanelixia subaurifera 1 Melanelixia subaurifera 3 Melanelixia subaurifera 2 Melanelixia subaurifera 4 Melanelixia fuliginosa 2 Melanelixia fuliginosa 5 Melanelixia-clade Melanelixia fuliginosa 3 Melanelixia fuliginosa 4 Melanelixia fuliginosa 1 Melanelixia subargentifera 1 Melanelixia subargentifera 2 Melanelixia glabra 2 Melanelixia glabra 1 Parmelia pinnatifida Parmelia saxatilis Parmelia-clade Parmelia serrana Parmelia squarrosa Parmelia sulcata Pleurosticta acetabulum Melanohalea exasperata 2 Melanohalea exasperata 1 Melanohalea elegantula 4 Melanohalea exasperatula Melanohalea elegantula 2 Melanohalea-clade Melanohalea elegantula 3 Melanohalea elegantula 1 Melanohalea aff. exasperata 1 Melanohalea aff. exasperata 3 Melanohalea aff. exasperata 2 Melanohalea olivacea Melanohalea septentrionalis Melanelia disjuncta Parmelinopsis minarum 1 Parmelinopsis minarum 2 Parmelinopsis subfatiscens Parmelinopsis horrescens Parmelinopsis neodamaziana Hypotrachyna revoluta Hypotrachyna immaculata Everniastrum cirrhatum Everniastrum nepalense Hypotachyna endochlora Hypotrachyna laevigata Hypotrachyna taylorensis Hypotrachyna sinuosa Hypotrachyna adducta Hypotrachyna infirma Hypotrachyna ciliata Hypotrachyna costaricensis Bulbothrix meizospora Bulbothrix setschwanensis Parmelinella wallichiana Parmelina pastillifera Parmelina tiliacea Parmelina quercina Parmelina-clade Myelochroa metarevoluta Myelochroa irrugans Parmeliopsis ambigua Parmeliopsis hyperopta Relicina subnigra Relicina sydneyensis Cetaria aculeata Vulpicida pinastri Melanelia stygia Pannoparmelia angustata Arctoparmelia centrifuga Brodoa atrofusca Pseudevernia furfuracea

61

Hypotrachyna-clade

O. Blanco et al. / Molecular Phylogenetics and Evolution 39 (2006) 52–69

0.1

Fig. 2. Phylogenetic relationships of parmelioid lichens inferred from a combined analysis of nuclear ITS, LSU and mitochondrial SSU rDNA sequences. 50% majority-rule consensus tree of 19,000 trees sampled using a Bayesian MC/MCMC analysis. Branches with posterior probabilities above 0.94 and also bootstrap support under parsimony equal or above 70% are indicated in bold.

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O. Blanco et al. / Molecular Phylogenetics and Evolution 39 (2006) 52–69

Table 4 Morphological, chemical and geographical characters of main clades of parmelioid lichens Cell wall polysaccharide

Pores/fenestrations

Pseudocyphellae

Centre of distribution

Ecology

Hypotrachyna-clade Melanelixia-clade Melanohalea-clade

Isolichenan Not determined Not determined

Present Present Absent

Subcosmopolitan Northern Hemisphere Northern Hemisphere

Tropical Temperate Temperate

Parmelia-clade Parmelina-clade Parmotrema-clade

Isolichenan Isolichenan Unknown type

Absent Present Present/Absent

Absent Absent On verrucae or isidia tips, circular to slightly elliptic EYgurate, linear Absent Punctiform or absent

Both Hemispheres Both Hemispheres Southern Hemisphere

Xanthoparmelia-clade

Xanthoparmelia-type lichenan

Present

Absent

Southern Hemisphere

Temperate to boreal Temperate Tropical, subtropical to temperate Subtropical, semi-arid

having the highest level of homoplasy, provides the highest level of highly supported internodes (58 with MP, 75 with B/MCMC). In this regard, the contribution of mt SSU is about half of the ITS (28 with MP and 42 with B/ MCMC). Monophyly of a core group of parmelioid lichens is strongly supported (Fig. 2), but parmelioid lichens as circumscribed by DePriest (1999) are polyphyletic. Arctoparmelia and Melanelia s. str. are outside this well-supported clade, and two genera not accepted as parmelioid in DePriest (1999), Parmeliopsis and Parmelaria, fall within it. In all analyses, the backbone of the phylogeny of parmelioid lichens lacks support. However, several well-supported Table 5 Frequencies of gains and losses of a pored epicortex in the phylogeny of parmelioid lichens from stochastic mapping simulations Gains ! 0 losses 0 1 2 3 4 5 6 7 8

1

2

3

4

5

6

7

8

9

10

0.02 0.05 0.06 0.01 0.01 0.01 0.01 0.03 0.03 0.02 0.02 0.01 0.04 0.01 0.01 0.07 0.03 0.01 0.39 0.02 0.09 0.01

Changes are shown up to a cumulative frequency of 0.95.

clades are resolved (Fig. 2), and seven of these are discussed below. The majority of these well-supported clades are characterized by a unique combination of some diagnostic characters (Table 4). 3.1.1. Xanthoparmelia-clade This clade corresponds to the genera Xanthoparmelia and Karoowia in the circumscription accepted by Blanco et al. (2004a). The genera include species that have cell wall Table 7 Frequencies of gains and losses of usnic acid in the phylogeny of parmelioid lichens from stochastic mapping simulations Gains ! losses

0 1

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

2 3 4 5

6

7

8

9

10

11

0.10 0.02 0.01 0.58 0.02 0.004 0.01 0.03 0.004 0.04 0.07 0.01 0.01 0.01 0.01 0.01

0.01 0.01 0.004

Changes are shown up to a cumulative frequency of 0.95. Table 6 Frequencies of gains and losses of pseudocyphellae in the phylogeny of parmelioid lichens from stochastic mapping simulations Gains ! losses 0 1 2 3 4 5 6 7 8

0

1

2

3

4

0.03 0.04

0.01 0.03 0.03 0.01

0.01 0.02

5 0.07 0.13 0.01

0.04 0.03

Changes are shown up to a cumulative frequency of 0.95.

6 0.46 0.01

7 0.03

Table 8 Frequencies of gains and losses of atranorin in the phylogeny of parmelioid lichens from stochastic mapping simulations Gains ! losses 0 1 2 3 4 5 6 7

0

1

2

3

4

5

0.01 0.02 0.01

6

0.05 0.03 0.01

7

0.07 0.05 0.01

8

0.24 0.05 0.01

9

10

11

0.27 0.05 0.01

0.05 0.01

0.01

Changes are shown up to a cumulative frequency of 0.95.

O. Blanco et al. / Molecular Phylogenetics and Evolution 39 (2006) 52–69

polysaccharides with Xanthoparmelia-type lichenan. Most species occur in the Southern Hemisphere in arid or semiarid subtropical areas, and to some extend into temperate regions. The species in this clade lack pseudocyphellae, have a pored epicortex, and show a considerable variation in cortical chemistry. Species lacking phenolic cortical metabolites are placed here, as well as species containing usnic acid or atranorin.

63

3.1.2. Parmotrema-clade This clade includes the large genus Parmotrema and some smaller genera, including Flavoparmelia, Flavopunctelia, Punctelia, Parmelaria, and Canoparmelia crozalsiana. Parmotrema in the classiWcation of Blanco et al. (2005) includes species that have a cell wall polysaccharide of unknown type. Its centre of distribution is in the Southern Hemisphere. It is especially diverse in tropical and

Fig. 3. The four most probable distinct mappings of pored epicortex history on the phylogeny of parmelioid lichens. Thick branches represent the presence of a pored epicortex. Each mapping is shown on a majority-rule consensus of the trees on which individual histories were simulated: i.e., the set of trees over which simulations of character history yielded an identical set of transformations. Identical sets were determined on the basis of the taxon bipartitions deWning the branches on which gains and losses occurred, and their probabilities are derived from frequencies of occurrence in the stochastic mapping analysis (see text).

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O. Blanco et al. / Molecular Phylogenetics and Evolution 39 (2006) 52–69

subtropical areas, with some species extending into temperate regions. In this clade some species have a pored epicortex, or pseudocyphellae, and may contain atranorin or usnic acid as cortical pigment. 3.1.3. Melanelixia-clade This recently described genus (Blanco et al., 2004b) has a cell wall polysaccharide that has not yet been determined. This genus is distributed in the Northern Hemisphere, mainly in temperate regions. It is characterized by having a pored or fenestrate epicortex, lacking pseudocyphellae. Neither atranorin nor usnic acid are present as cortical compounds, but species in this group do contain melanoid substances that are responsible for their brown colour. 3.1.4. Parmelia-clade This clade represents the monophyletic Parmelia s. str. with isolichenan as a cell wall polysaccharide. It has a worldwide distribution. The species have linear to eYgurate pseudocyphellae, lacking pores, and contain atranorin as a cortical substance.

3.1.6. Hypotrachyna-clade This clade includes species of the genera Bulbothrix, Everniastrum, Hypotrachyna, Parmelinella, and Parmelinopsis. The genus Hypotrachyna is not monophyletic in our analysis. All these genera have isolichenan as a polysaccharide cell wall. They are currently poorly known and have their centre of speciation in the tropical and subtropical regions of both hemispheres. All species in this clade have a pored epicortex and lack pseudocyphellae. They may contain atranorin, usnic acid or lichenoxanthone as cortical substances. 3.1.7. Parmelina-clade The Parmelina clade includes species of Myelochroa, a group widely distributed in the Northern Hemisphere, and Parmelina, which is cosmopolitan, but occurs mainly in the Southern Hemisphere. The species in this clade have isolichenan as polysaccharide cell walls, a pored epicortex, lack pseudocyphellae, contain atranorin and lack usnic acid as cortical compound. 3.2. Character evolution

3.1.5. Melanohalea-clade This clade corresponds to the recently described genus Melanohalea (Blanco et al., 2004b). Its cell wall polysaccharide is not determined. It occurs in the Northern Hemisphere mainly in temperate regions, and includes species lacking a pored epicortex, but having pseudocyphellae. This genus, as Melanelixia, has a brown coloration that is due to melanoid compounds and its species also lack atranorin and usnic acid.

Tables 5–8 summarize the posterior probabilities of gains and losses in each of the examined character over the set of trees sampled by MCMC. Figs. 3–6 show the four most probable distinct mappings of each character, where a distinct mapping refers to a unique set of taxon bipartitions corresponding to the clades subtended by changes in character state (see Section 2). All characters exhibit considerable variability in the

Fig. 4. The two most probable distinct mappings of pseudocyphellae history on the phylogeny of parmelioid lichens. Thick branches represent the presence of pseudocyphellae. Each mapping is shown on a majority-rule consensus tree as in Fig. 3.

O. Blanco et al. / Molecular Phylogenetics and Evolution 39 (2006) 52–69

65

3.2.1. Pored epicortex The character mapping suggests that a pored epicortex has been gained 0–10 times and lost 0–8 times over the tree (Table 5, Fig. 3). The three most probable gain–loss pairs account for about 55% of the posterior probability density and suggest no gains and 5–7 losses of the pored epicortex. The proportion of stochastic mappings including more losses than gains is 0.68. 3.2.2. Pseudocyphellae This trait has evolved 1–7 times and been lost 0–8 times (Table 6, Fig. 4). Fifty-nine percent of the posterior probability density is represented by two combinations of gains and losses suggesting 0–1 losses and 5–6 gains of the pseudocyphellae. The proportion of stochastic mappings including more gains than losses is 0.78.

Fig. 5. The most probable distinct mapping of usnic acid history on the phylogeny of parmelioid lichens. Thick branches represent the presence of usnic acid. The mapping is shown on a majority-rule consensus tree as in Fig. 3.

distribution of gains and losses, indicating the phylogenetic uncertainty associated with the inference of character evolution.

3.2.3. Usnic acid For this character the character mapping analysis suggests 1 and 5–11 gains and 3–9 and 13–15 losses (Table 7, Fig. 5). The most probable numbers of gains and losses (9 and 4, respectively) accounts for 58% of the posterior probability density. The proportion of stochastic mappings including more gains than losses is 0.66. 3.2.4. Atranorin Five to 11 gains and 1–7 losses for atranorin are inferred from character mapping (Table 8, Fig. 6). Fiftyone percent of the posterior probability density is represented by the two most probable gain–loss pairs that suggest 8–9 gains and 1–2 losses. The proportion of

Fig. 6. The two most probable distinct mappings of atranorin history on the phylogeny of parmelioid lichens. Thick branches represent the presence of atranorin. Each mapping is shown on a majority-rule consensus tree as in Fig. 3.

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stochastic mappings including more gains than losses is 0.91. 4. Discussion Although this is a phylogenetic study that samples broadly both taxonomically and morphologically parmelioid lichens, and includes most currently accepted genera, the species studied represent less than 10% of the total species diversity in the lineage. Further, some geographic regions that are centres of distribution of parmelioid lichens, such as southern Africa and south-eastern Asia remain poorly sampled. Therefore, the phylogenetic relationships presented here should be regarded as preliminary. The nuclear and mitochondrial gene partitions supported the same overall topology and no substantial intergenomic conXict was found for the major clades. The combined three-region data set provided the most robust support of the parmelioid lichen phylogeny overall, indicating that multi-locus data sets will be necessary to further resolve the phylogenetic relationships in this lineage of fungi. Characteristic diVerences in bootstrap and Bayesian support values are well documented (e.g., Alfaro et al., 2003; Douady et al., 2003; Suzuki et al., 2002; Wilcox et al., 2002). Our results conWrm the common pattern that the Bayesian support is always higher than bootstrap values. Both metrics appear to converge to some extent in the combined analyses. The increase in strongly supported nodes in the combined analyses gives hope that with the addition of further gene partitions, the backbone of the parmelioid lichens phylogeny can be resolved. 4.1. Major clades and intergeneric relationships within parmelioid lichens Although the backbone is not resolved, we can nevertheless draw some conclusions about the phylogeny of parmelioid lichens. The monophyly of a core group of parmelioid lichens is strongly supported. This group includes Parmelaria, a cetrariod genus (Randlane et al., 1997), and Parmeliopsis, a genus not included in the classiWcation of parmelioid lichens by DePriest (1999). Other genera, formely considered as parmelioid, fell outside this monophyletic group, e.g., Arctoparmelia and Melanelia s. str. Parmelaria, a genus of two species with laminal to submarginal apothecia, is very close to Parmotrema in thallus morphology (Culberson, 1962). Our molecular analyses support this relationship (Blanco et al., 2005) with Parmotrema. Parmeliopsis has not been considered as parmelioid (DePriest, 1999), although it Wts well morphologically, since it has a special type of conidiophores (Glück, 1899), which do not occur in other Parmeliaceae. In agreement with previous molecular

studies (Crespo et al., 2001; Mattsson et al., 2004) our data suggest that Parmeliopsis belongs to parmelioid lichens. Arctoparmelia, a genus morphologically similar to Xanthoparmelia (Elix, 1993), falls outside of the core of parmelioid group. However, Arctoparmelia diVers chemically from other parmelioid lichens in having Cetraria-type lichenan as cell wall polysaccharide (Elix, 1993). Our data conWrm previous molecular analyses in which no relationship between Arctoparmelia and Xanthoparmelia or other investigated parmelioid genera has been found (Blanco et al., 2004a; Thell et al., 2004). Melanelia s. str. is also outside of the core of parmelioid lichens. Our results corroborate previous studies (Blanco et al., 2004b; Mattsson et al., 2004; Thell et al., 2004) where Melanelia stygia is distantly related to Melanelixia and Melanohalea, and more closely related with cetrarioid lichens. Thell (1995) transferred the Cetraria hepatizon group to Melanelia based on morphological and chemical characters demonstrating relationships with cetrarioid lichens. However, Melanelia is still polyphyletic (Blanco et al., 2004b; Mattsson et al., 2004), since M. stygia and M. disjuncta do not cluster together. M. disjuncta has been shown as closely related to Pleurosticta acetabulum (Mattsson et al., 2004). Our results do not show this relationship. Additional studies are necessary to clarify the status of M. disjuncta. Within the monophyletic core group of parmelioid group, seven well-supported clades are recovered by the molecular data, and these are also deWned by a combination of morphological, chemical, and geographical characters (Table 3). These clades correspond to genera or generic groups and support recent generic rearrangements, such as the enlargement of Parmotrema (Blanco et al., 2005) and Xanthoparmelia (Blanco et al., 2004a), and the segregation of Melanelixia and Melanohalea from Melanelia (Blanco et al., 2004b). Two of these clades, Parmotrema and Xanthoparmelia, were also shown by Thell et al. (2004). The results further suggest that additional studies are necessary to clarify the monophyly of other currently accepted genera, such as Karoowia, Canoparmelia, and Parmelaria. Generic concepts in the Hypotrachyna-clade clearly need revision, since Hypotrachyna itself is polyphyletic. Larger taxon sampling is needed in some groups, such as the Parmelia- and Parmelina-clades, and more taxa and characters will be required to discover the relationships of other genera, such as Parmeliopsis, Pleurosticta, and Relicina. 4.2. Character evolution Morphological characters, such as the type of cortical perforations and cortical chemistry, have generally been regarded as key characters for classiWcation in parmelioid lichens. The presence of diVerent types of cortical perforations was even believed to be important at subfamilial rank (Elix, 1993; Hale, 1981; Henssen, 1992). However, neither

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those species having pseudocyphellae nor those having a pored epicortex form a monophyletic group, and cortical chemistry does not appear to be important at the suprageneric level, showing variability within some genera (e.g., Xanthoparmelia, Hypotrachyna). It thus appears that the taxonomic value of these characters has been overestimated in classiWcations of parmelioid lichens. Phylogenetic inferences of character evolution suggest that the pored epicortex is a plesiomorphic state within parmelioid lichens and has been lost several times independently within the group. However, some character mappings involving numerous gains from a poreless ancestor are also present, albeit at low probability, in the 0.95% conWdence interval estimated by stochastic mapping. Pseudocyphellae appear to have been gained more often than lost in the phylogeny of parmelioid lichens, evolving independently in the Punctelia-, Flavopunctelia-, Parmelia-, and Melanohalea-clades and also in Melanelia disjuncta. The presence of usnic acid shows less phylogenetic conservation than the two morphological characters examined. This compound appears to have been lost and gained several times, supporting recent views (Blanco et al., 2004a; Elix, 2003) that the taxonomic value of this character has been overestimated in previous classiWcations. Similarly, atranorin also exhibits considerable homoplasy, with a clear trend toward more gains than losses. The distribution of this character suggests that atranorin is of limited taxonomic value at generic or suprageneric level in parmelioid lichens. We currently have no knowledge about the processes that regulate the presence or absence of secondary metabolites in lichen-forming fungi, but further investigation of genes involved in the production of secondary metabolites, such as polyketide synthases (Grube and Blaha, 2003; Schmitt et al., 2005) are likely to improve our understanding of the evolution of such characters. Acknowledgments This project has been supported by the Spanish Ministry of Science and Technology (CGL2004-1848/BOS) from the Ministry of Educación and Science to A.C. and a start up fund of the Field Museum to H.T.L. Sequencing was carried out at the Unidad de Genómica (Parque CientíWco de Madrid) by M. Isabel García and SEM facilities were provided by the CAI de Microscopía Electrónica Luis Bru of the UCM by Eugenio Baldonedo. We are indebted to various colleagues for sending fresh material of several species, oVering some sequences and also for their contributions and critical comments, notably to J.A. Elix, P.K. Divakar, and M.C. Molina. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.ympev. 2005.12.015.

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References Alfaro, M.E., Zoller, S., Lutzoni, F., 2003. Bayes or bootstrap? A simulation study comparing the performance of Bayesian Markov chains Monte Carlo sampling and bootstrapping in assessing phylogenetic conWdence. Mol. Biol. Evol. 20, 255–266. BeGora, M.D., Fahselt, D., 2001. Usnic acid and atranorin concentrations in lichens in relation to bands of UV irradiance. Bryologist 104, 134–140. Beltman, H.A., 1978. Vegetative Strukturen der Parmeliaceae und ihre Entwicklung. Bibl. Lichenol. 11, 1–193. Bhattacharya, D., Lutzoni, F., Reeb, V., Simon, D., Nason, J., Fernández, F., 2000. Widespread occurrence of spliceosomal introns in the rDNA genes of ascomycetes. Mol. Biol. Evol. 17, 1971–1984. Bjerke, J.W., Lerfall, K., Elvebakk, A., 2002. EVects of ultraviolet radiation and PAR on the content of usnic and divaricatic acids in two arcticalpine lichens. Photochem. Photobiol. Sci. 1, 678–685. Blanco, O., Crespo, A., Elix, J.A., Hawksworth, D.L., Lumbsch, H.T., 2004a. A new classiWcation of parmelioid lichens containing Xanthoparmelia-type lichenan (Ascomycota: Lecanorales) based on morphological and molecular evidence. Taxon 53, 959–975. Blanco, O., Crespo, A., Divakar, P.K., Esslinger, T.L., Hawksworth, D.L., Lumbsch, T.H., 2004b. Melanelixia and Melanohalea, two new genera segregated from Melanelia (Parmeliaceae) based on molecular and morphological evidence. Mycol. Res. 108, 873–884. Blanco, O., Crespo, A., Divakar, P.K., Elix, J.A., Lumbsch, H.T., 2005. Phylogeny of parmotremoid lichens (Acomycotina, Lecanorales). Mycologia 97, 150–159. Büdel, B., Scheidegger, C., 1996. Thallus morphology and anatomy. In: Nash, III, T.H. (Ed.), Lichen Biology. Cambridge University Press, Cambridge, pp. 37–64. Clauzade, G., Roux, C., 1985. Likenoj de Okcidenta Europo. Ilustrita Determinlibro. Bull. Soc. Bot. Centre-Ouest, Nouv. Ser., Num. Spec. 7, 1–893. Crespo, A., Blanco, O., Hawksworth, D.L., 2001. The potential of mitochondrial DNA for establishing phylogeny and stabilising generic concepts in the parmelioid lichens. Taxon 50, 807–819. Crespo, A., Divakar, P.K., Argüello, A., Gasca, C., Hawksworth, D.L., 2004. Molecular studies on Punctelia species of the Iberian Peninsula, with an emphasis on specimens newly colonizing Madrid. Lichenologist 36, 299–308. Cubero, O.F., Bridge, P.D., Crespo, A., 2000. Terminal-sequence conservation identiWes spliceosomal introns in ascomycete 18S RNA genes. Mol. Biol. Evol. 17, 751–756. Culberson, W.L., 1962. The systematic position of Platysma thomsonii Stirton. Bryologist 65, 304–307. Culberson, C.F., 1969. Chemical and Botanical Guide to Lichen Products. University of North Carolina Press, Chapel Hill. DePriest, P.T., 1999. Development of Mason E. Hale’s list of epithets in the parmelioid genera (lichen-forming Ascomycotina): a bibliographic review. Bryologist 102, 442–461. Douady, C.J., Delsuc, F., Boucher, Y., Doolittle, W.F., Douzery, E.J., 2003. Comparison of Bayesian and maximum likelihood bootstrap measures of phylogenetic reliability. Mol. Biol. Evol. 20, 248–254. Elix, J.A., 1979. A taxonomic revision of the lichen genus Hypogymnia in Australasia. Brunonia 2, 175–245. Elix, J.A., 1993. Progress in the generic delimitation of Parmelia sensu lato lichens (Ascomycotina: Parmeliaceae) and a synoptic key to the Parmeliaceae. Bryologist 96, 359–383. Elix, J.A., 1997. The lichen genera Canomaculina and Rimeliella (Ascomycotina, Parmeliaceae). Mycotaxon 65, 475–479. Elix, J.A., 2003. The lichen genus Paraparmelia, a synonym of Xanthoparmelia (Ascomycota, Parmeliaceae). Mycotaxon 87, 395–403. Eriksson, O., Hawksworth, D.L., 1986. An alphabetical list of the generic names of ascomycetes—1986. Syst. Ascomycetum 5, 3–111. Eriksson, O., Hawksworth, D.L., 1998. Outline of the ascomycetes. Syst. Ascomycetum 16, 83–301. Farris, J.S., 1989. The retention index and the rescaled consistency index. Cladistics 5, 417–419.

68

O. Blanco et al. / Molecular Phylogenetics and Evolution 39 (2006) 52–69

Felsenstein, J., 1978. Cases in which parsimony or compatibility methods will be positively misleading. Syst. Zool. 27, 401–410. Felsenstein, J., 1985. ConWdence limits on phylogenies: an approach using the bootstrap. Evolution 39, 783–791. Feuerer, T., Marth, C., 1997. Anatomy of pseudocyphaellae and bulbate cilia in Parmeliaceae. Mitt. Inst. Allg. Bot. Hamburg 27, 101–107. Gardes, M., Bruns, T.D., 1993. ITS primers with enhanced speciWcity for basidiomycetes-application to the identiWcation of micorrhizae and rust. Mol. Ecol. 2, 113–118. Gargas, A., DePriest, P.T., Grube, M., Tehler, A., 1995. Multiple origins of lichen symbioses in fungi suggested by SSU rDNA phylogeny. Science 268, 1492–1495. Glück, H., 1899. Entwurf zu einer vergleichenden Morphologie der Flechten- Spermagonien. Verh. Naturhist.-Med. Ver. Heidelberg 6, 81–216. Green, T.G.A., Snelgar, W.P., Brown, D.H., 1981. Carbon dioxide exchange in lichens. Carbon dioxide exchange through the cyphellate lower cortex of Sticta latifrons Rich. New Phytol. 88, 421–426. Green, T.G.A., Snelgar, W.P., Wilkins, A.L., 1985. Photosynthesis, water relations and thallus structure of Stictaceae lichens. In: Brown, D.H. (Ed.), Lichen Physiology and Cell Biology. Plenum Press, New York and London, pp. 57–75. Grube, M., Blaha, J., 2003. On the phylogeny of some polyketide synthase genes in the lichenized genus Lecanora. Mycol. Res. 107, 1419–1426. Hale Jr., M.E., 1973. Fine structure of the cortex in the lichen family Parmeliaceae viewed with the scanning-electron microscope. Smithsonian Contr. Bot. 10, 1–92. Hale Jr., M.E., 1981. Pseudocyphellae and pored epicortex in the Parmeliaceae: their delimitation and evolutionary signiWcance. Lichenologist 13, 1–10. Hale Jr., M.E., 1984. An historical review of the genus concept in lichenology. Beih. Nova Hedwigia 79, 11–23. Hale Jr., M.E., 1987. A monograph of the lichen genus Parmelia Acharius sensu stricto (Ascomycotina: Parmeliaceae). Smithsonian Contr. Bot. 66, 1–54. Hale, B.W., DePriest, P.T., 1999. Mason E. Hale’s list of epithets in the parmelioid genera. Bryologist 102, 462–544. Hawksworth, D.L., Crespo, A., 2002. Proposal to conserve the name Xanthoparmelia against Chondropsis nom. cons. (Parmeliaceae). Taxon 51, 807. Henssen, A., 1992. Placoparmelia patagonica, a new lichen genus and species from Argentina (Parmeliaceae). Lichenologist 24, 133–142. Henssen, A., Jahns, H.M., 1974. Lichenes, Eine Einführung in die Flechtenkunde. Georg Thieme Verlag, Stuttgart. Huelsenbeck, J.P., Ronquist, F., 2001. MRBAYES: Bayesian inference of phylogenetic trees. Bioinformatics 17, 754–755. Huelsenbeck, J.P., Rannala, B., Masly, J.P., 2000. Accommodating phylogenetic uncertainty in evolutionary studies. Science 288, 2349–2350. Huelsenbeck, J.P., Ronquist, F., Nielsen, R., Bollback, J.P., 2001. Bayesian inference of phylogeny and its impact on evolutionary biology. Science 294, 2310–2314. Huelsenbeck, J.P., Nielsen, R., Bollabck, J.P., 2003. Stochastic mapping of morphological characters. Syst. Biol. 52, 131–158. Hughey, R., Krogh, A., 1996. SAM: Sequence alignment and modelling software system. Technical Report UCSC-CRL-96-22, University of California, Santa Cruz, CA. Huneck, S., Yoshimura, I., 1996. IdentiWcation of Lichen Substances. Springer-Verlag, Berlin, Heidelberg. Jahns, H.M., 1973. Anatomy, morphology and development. In: Ahmadjian, V., Hale, M.E. (Eds.), The Lichens. Academic Press, New York and London, pp. 3–58. Jahns, H.M., 1988. The lichen thallus. In: Galun, M. (Ed.), CRC Handbook of Lichenology, vol. I. CRC Press, Boca Raton, FL, pp. 95–143.

Kärnefelt, I., Thell, A., 1992. The evaluation of characters in lichenized families, exempliWed with the alectorioid and some parmelioid genera. Plant Syst. Evol. 180, 181–204. Kärnefelt, I., Mattsson, J.-E., Thell, A., 1992. Evolution and phylogeny of cetrarioid lichens. Plant Syst. Evol. 183, 113–160. Krog, H., 1982. Evolutionary trends in foliose and fruticose lichens of the Parmeliaceae. J. Hattori Bot. Lab. 52, 303–311. Lange, O.L., 1992. PXanzenleben unter Stress. Flechten als Pioniere der Vegetation an Extremstandorten der Erde. Rostra Universitatis Wirceburgensis, Würzburg. Larget, B., Simon, D.L., 1999. Markov chain Monte Carlo algorithms for the Bayesian analysis of phylogenetic trees. Mol. Biol. Evol. 16, 750–759. Li, K.-N., Rouse, D.I., German, T.L., 1994. PCR primers that allow intergeneric diVerentiation of ascomycetes and their application to Verticillium spp. Appl. Environ. Microbiol. 60, 4324–4331. Llimona, X., Hladun, N.L., 2001. Checklist of the lichens and lichenicolous fungi of the Iberian Peninsula and Balearic Islands. Bocconea 14, 1–581. Lumbsch, H.T., 2002. Analysis of phenolic products in lichens for identiWcation and taxonomy. In: Kranner, I., Beckett, R.P., Varma, A.K. (Eds.), Protocols in Lichenology. Culturing, Biochemistry, Ecophysiology and Use in Biomonitoring. Springer-Verlag, Berlin, Heidelberg, pp. 281–295. Lumbsch, H.Y., Kothe, H.W., 1992. Thallus surfaces in Coccocarpiaceae and Pannariaceae (lichenized ascomycetes) viewed with scanning electron microscopy. Mycotaxon 43, 277–282. Lutzoni, F., Pagel, M., Reeb, V., 2001. Major fungal lineages are derived from lichen symbiotic ancestors. Nature 411, 937–940. Mattsson, J.-E., Wedin, M., 1999. A re-assessment of the family Alectoriaceae. Lichenologist 31, 431–440. Mattsson, J.-E., Articus, K., Wiklund, E., Wedin, M., 2004. The monophyletic groups within the Parmeliaceae. In: Articus, K. (Ed.), Phylogenetic Studies in Usnea (Parmeliaceae) and Allied Genera. Acta Univ. Upsal. 931, sine pagin. Miadlikowska, J., Lutzoni, F., 2004. Phylogenetic classiWcation of peltigeralean fungi (Peltigerales, Ascomycota) based on ribosomal RNA small and large subunits. Am. J. Bot. 91, 449–464. Nielsen, R., 2002. Mapping mutations on phylogenies. Syst. Biol. 51, 729–739. Nimis, P.L., 1998. A critical appraisal of modern generic concepts in lichenology. Lichenologist 30, 427–438. Nimis, P.L., Scheidegger, C., Wolseley, P.A. (Eds.), 2002. Monitoring with Lichens Monitoring Lichens. Nato Science Series. IV. Earth and Environmental Sciences. Kluwer Academic Publishers, Dordrecht, The Netherlands. Nylander, J.A.A., Ronquist, F., Huelsenbeck, J.P., Nieves-Aldrey, J.L., 2004. Bayesian phylogenetic analysis of combined data. Syst. Biol. 53, 47–67. Page, R.D.M., 1996. Treeview: an application to display phylogenetic trees on personal computers. Comput. Appl. Biosci. 12, 357–358. Poelt, J., 1973. ClassiWcation. In: Ahmadjian, V., Hale, M.E. (Eds.), The Lichens. Academic Press, New York, pp. 599–632. Purvis, O.W., Coppins, B.J., Hawksworth, D.L., James, P.W., Moore, D.M. (Eds.), 1992. The Lichen Flora of Great Britain and Ireland. Natural History Museum Publications & British Lichen Society, London. Rambold, G., Triebel, D., 1999. Generic concepts in lichenized and lichenicolous ascomycetes since 1950—a historical approach. Symb. Bot. Upsal. 32, 123–164. Randlane, T., Saag, A., 1993. World list of cetrarioid lichens. Mycotaxon 47, 395–403. Randlane, T., Saag, A., Thell, A., 1997. A second updated world list of cetrarioid lichens. Bryologist 100, 109–122. Reeb, V., Lutzoni, F., Roux, C., 2004. Contribution of RPB2 to multilocus phylogenetic studies of the Pezizomycotina (Euascomycetes, Fungi) with special emphasis on the lichen-forming Acarosporaceae and evolution of polyspory. Mol. Phylogenet. Evol. 32, 1036–1060.

O. Blanco et al. / Molecular Phylogenetics and Evolution 39 (2006) 52–69 Rehner, S.A., Samuels, G., 1994. Taxonomy and phylogeny of Gliocladium analyzed from nuclear large subunit ribosomal DNA sequences. Mycol. Res. 98, 625–634. Rikkinen, J., 1995. What’s behind the pretty colours? A study on the photobiology of lichens. Bryobrothera 4, 1–239. Rodríguez, F., Oliver, J.F., Martín, A., Medina, J.R., 1990. The general stochastic model of nucleotide substitution. J. Theor. Biol. 142, 485–501. Sancho, L.G., Schroeter, B., Del Prado, R., 2000. Ecophysiology and morphology of the globular erratic lichen Aspicilia fruticulosa (Eversm.) Flag. from central Spain. Bibl. Lichenol. 75, 137–147. Sanders, W.B., 2001. Lichens: The Interface between Mycology and Plant Morphology. BioScience 51, 1025–1035. Schmitt, I., Martín, M.P., Kautz, S., Lumbsch, H.T., 2005. Diversity of nonreducing polyketide synthase genes in the Pertusariales (lichenized Ascomycota): a phylogenetic perspective. Phytochemistry 66, 1241–1253. Simmons, M.P., Pickett, K.M., Miya, M., 2004. How meaningful are Bayesian support values? Mol. Biol. Evol. 21, 188–199. Solhaug, K.A., Gauslaa, Y., 1996. Parietin, a photoprotective secondary product of the lichen Xanthoria parietina. Oecologia 108, 412–418. Solhaug, K.A., Gauslaa, Y., Nybakken, L., Bilger, W., 2003. UV-induction of sun-screening pigments in lichens. New Phytol. 158, 91–100. Suzuki, Y., Glazko, G.V., Nei, M., 2002. Overcredibility of molecular phylogenies obtained by Bayesian phylogenetics. Proc. Natl. Acad. Sci. USA 99, 16138–16143. SwoVord, D.L., 2003. PAUP*. Phylogenetic analysis using parsimony (*and other methods). Sinauer Associates, Sunderland, MA. Thell, A., 1995. A new position of the Cetraria commixta group in Melanelia (Ascomycotina, Parmeliaceae). Nova Hedwigia 60, 407–422. Thell, A., Feuerer, T., Kärnefelt, I., Myllys, L., Stenroos, S., 2004. Monophyletic groups within the Parmeliaceae identiWed by ITS rDNA, -tubulin and GAPDH sequences. Mycol. Prog. 3, 297–314.

69

Thompson, J.D., Higgins, D.G., Gibson, T.J., 1994. Clustal W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-speciWc gap penalties and weight matrix choice. Nucleic Acids Res. 22, 4673–4680. Vilgalys, R., Hester, M., 1990. Rapid genetic identiWcation and mapping of enzymatically ampliWed ribosomal DNA from several Cryptococcus species. J. Bacteriol. 172, 4238–4246. Wedin, M., Döring, H., Mattsson, J.-E., 1999. A multi-gene study of the phylogenetic relationships of the Parmeliaceae. Mycol. Res. 103, 1185– 1192. Wedin, M., Döring, H., Gilenstam, G., 2004. Saprotrophy and lichenization as options for the same fungal species on diVerent substrata: environmental plasticity and fungal lifestyles in the Stictis-Conotrema complex. New Phytol. 164, 459–465. White, T.J., Bruns, T.D., Lee, S., Taylor, J., 1990. AmpliWcation and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis, M.A., Gelfand, D.H., Sninsky, J.J., White, T.J. (Eds.), PCR Protocols. Academic Press, San Diego, pp. 315–322. Wilcox, T.P., Zwickl, D.J., Heath, T.A., Hillis, D.M., 2002. Phylogenetic relationships of the dwarf boas and a comparison of Bayesian and bootstrapping measures of phylogenetic support. Mol. Phylogenet. Evol. 25, 361–371. Yoshimura, I., Hurutani, R., 1987. Fine structures of cyphellae, pseudocyphellae and allied structures in lichen family Lobariaceae as determined by scanning electron microscopy. Bull. Kochi Gakuen College 18, 345–359. Zhou, S., Stanosz, G.R., 2001. Primers for ampliWcation of mt SSU rDNA, and a phylogenetic study of Botryosphaeria and associated anamorphic fungi. Mycol. Res. 105, 1033–1044. Zoller, S., Scheidegger, C., Sperisen, C., 1999. PCR primers for the ampliWcation of mitochondrial small subunit ribosomal DNA of lichen-forming ascomycetes. Lichenologist 31, 511–516.