Resolving evolutionary relationships in the lichen-forming genus Porpidia and related allies (Porpidiaceae, Ascomycota)

MOLECULAR PHYLOGENETICS AND EVOLUTION Molecular Phylogenetics and Evolution 32 (2004) 66–82 www.elsevier.com/locate/ympev

Resolving evolutionary relationships in the lichen-forming genus Porpidia and related allies (Porpidiaceae, Ascomycota) Jutta Buschboma,b,* and Gregory Muellerb a

University of Chicago, Committee on Evolutionary Biology, Culver Hall, Rm. 402, 1025 E. 57th St., Chicago, IL 60637, USA b Field Museum of Natural History, Botany Department, 1400 S. Lake Shore Drive, Chicago, IL 60605, USA Received 25 February 2003; revised 30 October 2003 Available online 27 February 2004

Abstract The lichen-forming genus Porpidia (Porpidiaceae, Ascomycota) provides excellent opportunities for evolutionary, reproductive, and ecological studies of crustose epilithic lichen symbioses. However, despite the fact that the genus itself seemed to be clearly delimited, the group was thought to be a hopeless case with regard to intrageneric relationships and species delimitations due to apparently rampant homoplasy throughout most character systems. Contrary to the situation for non-molecular data, a robust and generally well-resolved phylogeny was recovered based on DNA-sequence data. Separate and combined analyses of nuclear ribosomal RNA large subunit and nuclear b-tubulin gene fragments were performed using maximum parsimony, maximum likelihood, and Bayesian approaches. Branch support was estimated using non-parametic bootstrapping and posterior probabilities, while monophyly of a priori defined groups was tested using posterior probabilities. The results reveal a highly supported ‘‘Porpidia sensu lato,’’ however, Porpidia itself is not monophyletic. Several smaller genera of the Porpidiaceae and probably the large genus Lecidea (Lecideaceae) are nested within the group. Most taxa analyzed fall into one of four major subgroups within Porpidia s.l., though the basal relationships among these subgroups could not be supported. This phylogeny will make it possible to re-evaluate morphological and chemical characters in the group, and to conduct detailed studies of species delimitations within the monophyletic subgroups. Ó 2003 Elsevier Inc. All rights reserved. Keywords: Ascomycota; Homoplasy; Lecanorales; Molecular phylogenetics; Porpidia; Resolution; Taxon sampling

1. Introduction The approximately 35–40 species of the genus Porpidia K€ orber (1855) are among the over 40% of ascomycete species that live in obligate symbiosis with green algae and cyanobacteria, called ‘‘lichens.’’ Porpidia, as well as the family Porpidiaceae, include exclusively crustose taxa that form colorful thalli on siliceous to slightly calcareous rock surfaces worldwide in association with their unicellular green algal photobionts of the genus Trebouxia (Chlorophyta; Ahmadjian, 1993; Hildreth and Ahmadjian, 1981). Members of Porpidia * Corresponding author. Present address: Institut f€ ur Bioinformatik, Heinrich-Heine-Universit€at, Universit€atsstrasse 1, Geb. 25.02.02, 40225 D€ usseldorf, FRG, Germany. Fax: +49-211/81-15767. E-mail address: [email protected] (J. Buschbom).

1055-7903/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.ympev.2003.11.012

are inhabitants of exposed to shaded, but always humid (micro-) localities in temperate to arctic zones. Porpidia belongs to the largest group of lichen-forming ascomycetes, the Lecanoromycetes, and here to the very diverse and species-rich order Lecanorales. No molecular phylogenetic study that covers the whole diversity of taxa within the Lecanorales exists to date. Suborder- and family-level relationships within the Lecanorales are thus unknown. Phylogenetic studies within the Lecanorales have so far concentrated on particular genera or families (e.g., Crespo and Cubero, 1998; Grube and Arup, 2001; Lumbsch and Schmitt, 2001; Mattsson and Wedin, 1998; Miadlikowska and Lutzoni, 2000). These taxa were often assumed a priori to be monophyletic, thereby necessitating very limited outgroup sampling which limited the studies ability to test and challenge the assumption of monophyly of the investigated group. The present study

J. Buschbom, G. Mueller / Molecular Phylogenetics and Evolution 32 (2004) 66–82

shows that those assumptions should be taken with caution even in taxonomic groups that previously were thought to be clearly circumscribed. In its turbulent taxonomic history Porpidia is closely entwined with the crustose genus Lecidea. During the 150 years following Erik AchariusÕ initial description of Lecidea in 1803, this genus became the ‘‘garbage bin’’ for crustose lichen taxa with generally green algal photobionts, photobiont-free apothecial margins and hyaline, single-celled ascospores. At its height, the genus had grown to include approximately 1600 species (Hertel, 1975). Only in the second half of the last century were efforts started, most significantly by Hertel (1967, 1977, 1984, 1995) to revise the genus based on morphological, chemical, ecological, and biogeographical data. As currently circumscribed Lecidea sensu stricto (Lecideaceae) includes approximately 100 species whose within-genus revision is currently under way (Hertel, 1995). One of the largest genera split from Lecidea is the genus Porpidia. It was recognized early on that species with relatively large, ellipsoidal ascospores that are characterized by the presence of a gelatinous outermost spore wall layer (‘‘halo’’), and with darkly pigmented hypothecia (apothecial base), and exciples (apothecial margins) might form a distinct evolutionary group (e.g., Zahlbruckner in Handel-Mazzetti, 1930; Choisy in Tronchet, 1936; Vezda, 1972). However, the taxonomy of the group was stabilized only in 1984 with the recognition of the potential importance of ascus-tip characteristics (Hafellner, 1984; Hertel, 1984). Hafellner (1984) erected the family Porpidiaceae based on the presence of a tubular structure in the ascus tip that stains dark blue in iodine solution (see also Hertel, 1984). Between 15 and 20 genera are currently accepted in the Porpidiaceae, of which Porpidia is the largest (Eriksson et al., 2001; Hafellner et al., 1993; Hawksworth et al., 1995; Rambold, 1989; Tehler, 1996). Several revisions of the genus Porpidia based on traditionally employed morphological, chemical, ecological, and biogeographical characters exist (Gowan, 1989a,b; Gowan and Ahti, 1993; Hertel and Knoph, 1984; Inoue, 1983; Knoph, 1984; Schwab, 1986; Fryday, 1997). Gowan (1989b) delimited four subgroups within the genus based on combinations of characters, including thallus thickness, spore size, excipular cell diameter, amyloid reaction of the medulla, etc. Still, despite these efforts and despite Porpidia having been thought to be well-delimited and easily identifiable, species delimitations, and subgeneric relationships within Porpidia have remained problematic. Relationships could not be resolved due to substantial intraspecific variability and apparent homoplasy of employed characters. Consequently, the evolutionary significance of different character systems, including the presence of contrasting reproductive modes, secondary compounds, and morphological features, within the genus could not be assessed.

67

Gowan (1988) conducted the first and only cladistic study in Porpidia and the Porpidiaceae. She used morphological, chemical, and ecological data. Her analysis resulted in a tree that showed very little structure. Integrating over all characters available at that time, the resulting star-like topology clearly illustrates the homoplasious nature of these character sets within Porpidia. It also documents the need and importance of a well-supported and robust phylogenetic hypothesis as a prerequisite for our understanding of evolutionary patterns and processes within the group. At the onset of the present project, however, the question remained if molecular (DNA-sequence) data, in contrast to morphological and chemical data, would result in a wellresolved phylogeny or if they would be as ‘‘noisy’’ as the previously used character systems. The present study is the first molecular study to investigate the evolutionary relationships of the genus Porpidia and related allies. The purpose of the present paper is to clarify the evolutionary relationships within the group and thus to provide a robust and well-supported phylogenetic framework as a basis for detailed phylogenetic and population genetic investigations. To this end, the following hypotheses were tested: (1) monophyly of the family Porpidiaceae, (2) monophyly of the genus Porpidia, and (3) monophyly of the four previously proposed subgeneric groups within the genus as defined by Gowan (1989b; see Table 1). DNA-sequence data of LSU and b-tubulin gene fragments were analyzed using a wide range of phylogenetic approaches, including maximum parsimony, maximum likelihood, and Bayesian approaches. This comprehensive approach was taken since it has been proposed that congruence between interspecific phylogenetic hypotheses based on independent data sets and different phylogenetic methods can be ‘‘considered some of the strongest support for phylogenetic relationships’’ (Cunningham, 1997).

2. Material and methods 2.1. Taxon and character sampling Twenty-five taxa in Porpidia, 13 taxa representing other genera within the Porpidiaceae and 30 outgroup taxa were included in the analyses (Table 1). Outgroup sampling was orientated along the systems of Hafellner et al. (1993) and Tehler (1996). Both systems delimit the suborder Cladoniineae from the Lecanorineae for taxa of the Lecanorales with tubular or cap-like structures in the ascus-tips. Thus, potential relatives to the Porpidiaceae occur within the Cladoniineae. Representatives of all four subdivisions within Porpidia (Gowan, 1989b), as well as most families within the Cladoniineae are included in the study. Furthermore, several major groups within the Lecanorales, such as, Parmeliaceae, Telo-

68

Table 1 List of taxa included in the study and voucher information for specimens used for DNA-extraction Taxon

Locality

Depository

LSU

b-Tubulin

59

R. Harris 4.9.1998

Wisconsin, USA

F

AY532938

AY536796

66

J. Buschbom 2911

Ontario, Canada

F

AY532937

AY536795

58, 57

J. Buschbom 3082

Minnesota, USA

F

AY532936

AY536794

189 113 121 127 182 74 153 114 116 120 183 80 108 110 130 141

J. J. J. J. J. J. J. J. J. J. J. J. J. J. J. J.

BC, Canada Qu
ebec, Canada Nunavut, Canada Nunavut, Canada Sweden Greenland Austria Qu
ebec, Canada Nunavut, Canada Nunavut, Canada Sweden Greenland Greenland Nunavut, Canada Greenland Nunavut, Canada

F F F F F F F F F F F F F F F F

AY532940 AY532945 AY532946 AY532947 AY532948 AY532949 AY532950 AY532954 AY532955 AY532956 AY532957 AY532958 AY532960 AY532961 AY532962 AY532963

AY536798 AY536804 AY536805 AY536806 AY536807 AY536808 AY536809 AY536813 AY536814 AY536815 AY536816 AY536817 AY536819 AY536820 AY536821 AY536822

187 155 33, 62 159 190 164 157 39, 64

K. Glew 000810-2 [Glew] J. Buschbom 19.8.2001-2a J. Buschbom 2953 J. Buschbom 30.8.2001-12 J. Buschbom 4.09.2001-1 J. Buschbom 25.08.2001-41 B. Coppins 21.8.2001 J. Buschbom 12.10.1998-5

Washington, USA Sweden New York, USA Finland Sweden Sweden Great Britain North Carolina, USA

K. Glew F F F F F F F

AY532942 AY532943 AY532944 AY532953 AY532964 AY532959 AY532965 AY532973

AY536801 AY536802 AY536803 AY536812 AY536799 AY536818 AY536823 AY536831

166 56

J. Buschbom 30.8.2001-1 J. Buschbom 25.8.1999-15

Finland Poland

F F

AY532941 AY532951

AY536800 AY536810

Lecanoromycetes: Porpidia Suborder Cladoniineae Lecideaceae group Porpidiaceae Porpidia ALBOCAERULESCENS COMPLEX Porpidia albocaerulescens (Wulfen) Hertel & Knoph (sexual) Porpidia albocaerulescens var. polycarpiza (Vain.) Rambold & Hertel (sexual) Porpidia albocaerulescens (Wulfen) Hertel & Knoph (asexual) Porpidia carlottiana Gowan Porpidia flavocoerulescens (Hornem.) Hertel & A.J. Schwab

Porpidia glaucophaea (K€ orb.) Hertel & Knoph Porpidia melinodes (K€ orb.) Gowan & Ahti

Porpidia ochrolemma (Vain.) Brodo & R. Sant.

MACROCARPA COMPLEX Porpidia contraponenda (Arnold) Knoph & Hertel Porpidia crustulata (Ach.) Hertel & Knoph Porpidia diversa Gowan Porpidia macrocarpa (DC.) Hertel & A.J. Schwab Porpidia nigrocruenta (Anzi) Diederich & S
erus. Porpidia soredizodes (Lamy ex Nyl.) J.R. Laundon Porpidia tahawasiana Gowan SPEIREA COMPLEX Porpidia cinereoatra (Ach.) Hertel & Knoph Porpidia grisea Gowan

Buschbom Buschbom Buschbom Buschbom Buschbom Buschbom Buschbom Buschbom Buschbom Buschbom Buschbom Buschbom Buschbom Buschbom Buschbom Buschbom

25.11.2001-1 9.7.00-40 16.7.2000-17 19.7.2000-43 26.8.2001-66 27.8.2000-4 12.9.2001-5 7.7.2000-70 18.7.2000-4 19.7.2000-36 26.8.2001-35 27.8.2000-15 23.8.2000-2 16.7.2000-64 26.8.2000-51 17.7.2000-28

J. Buschbom, G. Mueller / Molecular Phylogenetics and Evolution 32 (2004) 66–82

Extraction Voucher Nos.

Porpidia lowiana Gowan Porpidia speirea (Ach.) Kremp. Porpidia tuberculosa (Sm.) Hertel & Knoph SUPERBA COMPLEX Porpidia superba (K€ orb.) Hertel & Knoph Porpidia zeoroides (Anzi) Knoph & Hertel

Porpidia sp. 3 Lecanoromycetes: Outgroups Suborder Acarosporineae Hymeneliaceae Hymenelia lacustris M.Choisy Tremolecia atrata (Ach.) Hertel Suborder Cladoniineae Micareaceae group Cladoniaceae Cladonia rangiferina (L.) F.H.Wigg. Mycobilimbiaceae Mycobilimbia hypnorum (Lib.) Kalb & Hafellner Psoraceae Protoblastenia rupestris (Scop.) J. Steiner Psora rubiformis (Ach.) Hook. Stereocaulaceae Stereocaulon vesuvianum Pers. Lecideaceae group Catillariaceae Sporastatia testudinea (Ach.) Massal. Lecideaceae Cecidonia umbonella (Nyl.) Triebel & Rambold (on 175) Cecidonia xenophana (K€ orber) Triebel & Rambold (on 179) Melanolecia transitoria (Arnold) Hertel in Poelt & Vezda Lecidea subgenus Lecidea Lecidea fuscoatra (L.) Ach. Lecidea subgenus Rehmiopsis Lecidea atrobrunnea (Ramond ex Lam. & DC.) Schaer. Lecidea confluens (Weber) Ach. Lecidea lapicida var. swartzioidea (Nyl.) Nyl. Lecidea subgenus Cladopycnidium Lecidea tessellata Fl€ orke Porpidiaceae

J. J. J. J.

109 34

Finland Minnesota, USA New York, USA Ontario, Canada

F F F F

AY532952 AY532970 AY532974 AY532975

AY536811 AY536828 AY536832 AY536833

J. Buschbom 16.7.2000-64 V. Reeb VR 9-VII-98/38

Nunavut, Canada France

F F

AY532972 AY532976

AY536830 AY536834

95 98 30 180 181 179

R. Poulsen 547 R. Poulsen 903 J. Buschbom 3161 J. Buschbom 14.9.2001-9 J. Buschbom 16.9.2001-10 J. Buschbom 26.8.2001-9

Kerguelen Islands Kerguelen Islands Minnesota, USA Austria Austria Sweden

C C F F F F

AY532939 AY532971 AY532966 AY532967 AY532968 AY532969

AY536797 AY536829 AY536824 AY536825 AY536826 AY536827

138 178

J. Buschbom 3155 J. Buschbom 1.9.2001-20

North Carolina, USA F Norway F

AY533006 AY533007

AY536776 AY536844

40 174 161 107 103

J. J. J. J. J.

Buschbom Buschbom Buschbom Buschbom Buschbom

Minnesota, USA Sweden Sweden Nunavut, Canada Greenland

F F F F F

AY533000 AY533005 AY533008 AY533009 AY533002

AY536772 AY536787 AY536835 AY536837 AY536843

154 186 188 167

J. J. J. J.

Buschbom 17.7.2000-17 Buschbom 21.08.2001-9b Buschbom 26.08.2001-9 Poelt & M. Grube 93-432

Nunavut, Canada Sweden Sweden Austria

F F F GZU

AY533011 AY532990 AY532991 AY532999

AY536840 AY536768 AY536769 AY536786

169

J. Buschbom 16.8.2001-46

Sweden

F

AY532995

AY536782

50 171 100 175

J. J. J. J.

Minnesota, USA Sweden Nunavut, Canada Sweden

F F F F

AY532993 AY532994 AY532996 AY532997

AY536780 AY536781 AY536783 AY536784

22

J. Buschbom 2979

Ontario, Canada

F

AY532998

AY536785

J. J. J. J. J.

Greenland Sweden Nunavut, Canada Minnesota, USA Greenland

F F F F F

AY532977 AY532978 AY532979 AY532980 AY532982

AY536762 AY536763 AY536764 AY536765 AY536766

Amygdalaria consentiens (Nyl.) Hertel, Brodo & M.Inoue 102 170 Amygdalaria elegantior (H.Magn.) Hertel & Brodo 99 Amygdalaria panaeola (Ach.) Hertel & Brodo 69 Bellemerea alpina (Sommerf.) Clauzade & Roux 101

Buschbom Buschbom Buschbom Buschbom

Buschbom Buschbom Buschbom Buschbom

Buschbom Buschbom Buschbom Buschbom Buschbom

31.8.2001-14 3152 2943 2990

3034c 16.8.2001-25 26.8.2001-71 16.7.2000-11 23.8.2000-41

3123 21.8.2001-44 18.07.2000-14 21.08.2001-9b

22.8.2000-56 26.08.2001-67 16.7.2000-38 3118 23.8.2000-22

J. Buschbom, G. Mueller / Molecular Phylogenetics and Evolution 32 (2004) 66–82

UNKNOWN AFFILIATION Porpidia cf. austroshetlandica Hertel Porpidia stephanodes (Stirt.) Hertel Porpidia sp. 1 Porpidia sp. 2

168 31 25 26, 54

69

70

Table 1 (continued) Extraction Voucher Nos.

Locality

Depository

LSU

b-Tubulin

Bellemerea subsorediza (Lynge) R.Sant. Clauzadea monticola (Ach.) Hafellner & Bellmere Farnoldia jurana (Schaer.) Hertel Immersaria usbekica (Hertel) M.Barbero, Nav.-Ros. & Cl.Roux Koerberiella wimmeriana (K€ orb.) Stein Notolecidea subcontinua (Nyl.) Hertel Pachyphysis ozarkana R.C.Harris & Ladd Stenhammarella turgida (Ach.) Hertel Stephanocyclos henssenianus Hertel

158 67 172 94

J. Buschbom 25.8.2001-38a B. Coppins 20.02.2000 J. Buschbom 15.09.2001-1 C. Roux 1.09.2000-5

Sweden Great Britain Austria Spain

F F F F

AY532983 AY533004 AY532984 AY532985

AY536767 AY536771 AY536774 AY536777

156 96 51 38, 61 97

J. Buschbom 25.8.2001-9 R. Poulsen 737a J. Buschbom 11.10.1997-2 F. Lutzoni FL 96.8.30-1 1/3 R. Poulsen 835

Sweden Kerguelen Islands Missouri, USA Austria Kerguelen Islands

F C F F C

AY532986 AY532987 AY532988 AY532981 AY532989

AY536778 AY536790 AY536791 AY536842 AY536839

53

J. Buschbom 3034c

Minnesota, USA

F

AY533010

AY536838

152

Bratli, H. & Timdal, E. 8694

Norway

O

AY533001

AY536841

Cetraria nivalis (L.) Ach. Lecanora cenisia Ach.

144 52

J. Buschbom 25.8.2000-92 J. Buschbom 3015

Greenland Minnesota, USA

F F

AY533003 AY532992

AY536770 AY536779

Nephroma arcticum (L.) Torss. Nephroma resupinatum (L.) Ach. Peltigera aphthosa (L.) Willd. Peltigera neckeri Hepp ex M€ ull.Arg. Pseudocyphellaria croccata (L.) Vain.

JM, JM, JM, JM, JM,

Sharnoff & Sharnoff 1484.27 USA Sharnoff & Sharnoff 1192.22 USA Lutzoni 97.06.29 Canada Miadlikowska 5240 Poland McCune 23785 USA

CANL CANL F UGDA-L OSU

AF286828a AF286829a AF286759a AF286766a AF286826a

AY536788 AY536789 AY536792 AY536793 AY536836

F

AY533012

AY536846

F

AY533013 AY533015 AY533014 AY533016

AY536773 AY536775 AY536845 AY536761

Rhizocarpaceae group Rhizocarpaceae Rhizocarpon geminatum K€ orb. Squamarinaceae group Squamarinaceae Squamarina gypsacea (J.E. Smith) Poelt Suborder Lecanorineae Parmeliaceae Lecanoraceae Suborder Peltigerineae Peltigeraceae

Lobariaceae

133 134 136 135 137

Suborder Teloschistineae Teloschistaceae Xanthoria elegans (Link) Th. Fr.

105

J. Buschbom 30.8.2000-5

Greenland

Non-lichenized outgroups Leotiales Cudonia circinans (Pers.) Fr. Geoglossum glabrum Pers. Trichoglossum hirsutum (Pers.) Boud. Pezizales Morchella esculenta (L.) Pers.

147 148 149 185

J. Platt 232 ANK 1546 J. Platt 267 P. Leacock

Unknown Unknown Unknown Illinois, USA

Higher-level classification within the Lecanorales is based on Tehler (1996). Subgeneric classifications are following Gowan (1989b) in Porpidia and Rambold (1989) in Lecidea. Nomenclature follows Santesson (1993). Extraction numbers are identifying different specimens of the same taxon (if more than one extraction was done: first number represents extraction used for LSU, second for b-tubulin). The last two columns present GenBank accession numbers of deposited DNA-sequences. a Sequences obtained from J. Miadlikowska/GenBank.

J. Buschbom, G. Mueller / Molecular Phylogenetics and Evolution 32 (2004) 66–82

Taxon

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schistaceae, and Peltigeraceae, are represented in the sample. The phylogeny is rooted by four non-lichenized ascomycetes of the Leotiales and Pezizales. Morchella esculenta (Pezizales) was assigned as outgroup in all analyses. Gene loci selected for DNA-sequence-based phylogenetic analyses were a 1.4 kb fragment at the 50 end of the nuclear large subunit RNA gene (LSU) and a 1 kb fragment at the 30 -end of the nuclear gene coding for b-tubulin. Specimens were identified based on macro- and micro-morphological and chemical characters. Secondary metabolic compounds present in the thalli were identified by thin layer-chromatography (TLC; Culberson, 1972; Culberson and Ammann, 1979; Culberson et al., 1981). For TLC-analyses and DNA-extraction, small thallus fragments were sampled from specimens and thallus parts that were apparently free of parasites and parasymbionts. The haploid thalli of the lichens were sampled for extraction as far as possible. The fruiting bodies of the investigated group often contain high concentrations of phenolic substances which seemed to inhibited PCR amplification. However, apothecia had to be sampled for endolithic and gall-forming species due to the lack of (independent) thallus tissue. 2.2. Lab procedures DNA was extracted from specimens with the Puregene kit (Gentra Systems), following the protocol for filamentose fungi. The LSU region was amplified using primers LROR and LR7 (Vilgalys, www.biology.duke.edu/fungi/mycolab) and the following PCR protocol: 95 °C for 1 min, 35
(95 °C for 1 min, 52 °C for 45 s, 72 °C for 1:30 min), 72 °C for 4 min. Amplification of the b-tubulin fragment was performed using primers BT1843R and BT2870 (Table 2) and a cycle of 95 °C for 1 min, 20
(95 °C for 1 min, 48 °C for 45 s, 72 °C for 2 min), 15
(95 °C for 1 min, 48 °C for 45 s, 72 °C for 2 min + 0.5 s/cycle), 72 °C for 10 min. Amplification products were visualized on agarose gels and purified using Microcon-100 spin columns (Millipore). Amplification products from the gall-forming species of Ceci-

Table 2 Sequences of newly designed primers for amplification and sequencing of b-tubulin gene fragments Primer

Sequence

BT1843R BT1852R BT1269R BT2870 BT1413 BT1269

GGYCAATCYGGWGCYGGNAACAA GGWGCYGGIAACAAYTGGGC CGCAARTTGGCYGTSAAYATGGT TACCAATGYAAGAADGCYTTDCG TCRGAAGCAGCCATCAT ACCATRTTSACRGCCARTTTGCG

Sequences are cited in 50 –30 direction. Degeneracy follows international nomenclature: R ¼ A or G, S ¼ C or G, W ¼ A or T, Y ¼ T or C, D ¼ A or G or T, N ¼ A or C or G or T.

71

donia had to be cloned (TOPO Cloning Kit, Invitrogen) to separate gall and host DNA-copies. Fluorescencelabeled ddNTPs were incorporated into the cleaned PCR products using BIG dye V2 (Perkin–Elmer) during cycle sequencing. The following internal primers were used for sequencing both strands in addition to the PCR primers: LIC15R, LR3R, LR5R, LR17R, LIC2044, LR6, LR5, and LR3 for the LSU (Vilgalys and Hester, 1990; Vilgalys www.biology.duke.edu/fungi/mycolab; Lutzoni unpubl.) and BT1852R, BT1269R, BT1283R, Bt1a, BT1413, BT1283, BT1269 (see Table 2; Glass and Donaldson, 1995; Fernandez, unpubl.; Lutzoni, unpubl.). Automated sequencing of the labeled fragments was performed using ABI 377 and ABI 3100 automated sequencers (Perkin–Elmer). The resulting chromatograms were evaluated using Sequence Analysis 3.4 (Amersham Biosciences). BLAST searches in GenBank were performed for each sequence coming from the sequencer to verify that the sequences were of fungal (ascomycete) origin and did not represent the algal or cyanobacterial photobionts. Additionally, the BLAST results ensured that the intended loci were amplified. Contigs of sequence reads and subsequent alignments were assembled using Sequencher 3.7–4.1 (Gene Codes, 2000). Sequence alignments were subsequently optimized by eye using MacClade 4.0 (Maddison and Maddison, 2000). Ambiguously aligned regions within the LSU were delimited based on secondary structure and excluded from the analyses. Introns had to be removed for both loci because they were not present in all taxa. 2.3. Analyses of evolutionary relationships Phylogenetic relationships were reconstructed using unequally weighted maximum parsimony (MP) and maximum likelihood (ML) methods as implemented in PAUP*4.0b10 (Swofford, 1998) as well as a Bayesian approach (BMC3 ) using MrBayes 3.0b3 (Huelsenbeck and Ronquist, 2001). The debate over whether, and how, to combine (incongruent) data sets of multiple loci is still ongoing (e.g., Ballard et al., 1998; Bull et al., 1993; Kluge, 1989; Huelsenbeck et al., 1996). The present study follows the approaches described by Bull et al. (1993), Mason-Gamer and Kellogg (1996), Cunningham (1997), and OÕDonnell and Cigelnik (1997). Both gene loci were analyzed separately. Then incongruence among datasets was investigated through a procedure suggested by Mason-Gamer and Kellogg (1996) which evaluates if conflict between highly supported relationships in the single-gene-based trees exists. In the present study, internodes with P 75% bootstrap support (MP analyses) or 95% posterior probability (BMC3 analyses) were considered highly supported. In addition, the incongruence length difference test (ILD; Farris et al., 1994),

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implemented in PAUP*4.0 as partition homogeneity test, was performed. Employing MP, the ILD-test randomly resampled the partitions 100 times and analyzed each replicate per heuristic search with settings as in the original searches (see below) but with only 10 random additions per replicate. Heuristic searches with TBR branch swapping were carried out for MP and ML analyses. The following settings were chosen: only the best trees were saved with the maximum number of trees to be saved set to autoincrease; starting tree options were set to stepwise addition and swap on best tree only; during random additions only a single tree was kept at each step; TBR branch swapping was performed on the best tree only with the ÔMULTREESÕ option in effect and the number of rearrangements per replicate limited to 10 million to avoid the search getting stuck in one replicate, a limit that was rarely, if ever reached. One thousand random addition sequence replicates were carried out for MP. Transition and transversion costs between nucleotides as estimated from the aligned sequences were incorporated into the MP analyses through stepmatrices (Maddison and Maddison, 2000). Three different schemes to investigate the extend of homoplasy in different data partitions were applied to the protein-coding b-tubulin gene sequence data: (a) all codon positions were included and equally weighted, (b) third positions were excluded, i.e., they were given a weight of zero, (c) the DNA sequences were translated into amino-acid sequences using the universal code. MP analyses employing heuristic searches were performed as described above under all three schemes. Support for relationships under MP was estimated using non-parametric bootstrapping with 1000 pseudoreplicates. Trees were optimized in each replicate using the original heuristic search setting with the exception of only 10 random sequence additions per pseudoreplicate. For ML tree optimization, 100 random sequence addition replicates were conducted. The parameters of the nucleotide substitution models were iteratively estimated from the data using likelihood ratio tests. Starting values were estimated in PAUP based on the most-parsimonious topologies found under MP. The tree resulting from a search with these parameters was subsequently used to re-estimate the parameters. The iterations were continued until no major changes in topologies occurred between iterations. For all three partitions, the Tamura and Nei (1993) model with a probability of invariant sites and a gamma distribution modeling rate heterogeneity among sites showed the best fit. Nucleotide frequencies in the substitution models were set to equal for the LSU, for the other two partitions they were optimized. A comparison with the parameter values output by BMC3 runs (see below) confirmed the choices for the models. Branch support was investigated using non-parametric bootstrapping

with 80 pseudoreplicates and 2 random sequence additions per pseudoreplicate. Markov Chain Monte Carlo searches in MrBayes were conducted assuming the GTR model with rate heterogeneity between sites and a proportion of sites being invariant (in accordance with the models used for ML). The default settings were used as priors for all other parameters. For the separate analyses of the LSU and b-tubulin datasets, runs of 2 million generations were carried out. For the combined search three 2 million—generation runs and one 10 million—generation run were conducted. Every 100th generation was sampled and after inspection the first 1 million (2 million for the 10 million run) generations were removed as burn-in phase for each chain. This resulted in 10,000 trees per run (80,000 for the 10 million run) on which the results are based. Homoplasious characters can either contribute to resolving ingroup relationships or only introduce random noise into the dataset. Their polarization will be strongly influenced by the specifics of the taxon sampling used. Thus, a taxon-sampling jackknife approach was designed to explore the influence of varying taxon samplings on the polarization of homoplasious characters present in the dataset. One hundred jackknife replicates were constructed so that in each replicate 50% of all taxa were removed from the dataset. The resulting matrices of the combined gene datasets were analyzed using MP as described above, but with 100 random addition sequence replicates per jackknife replicate. Monophyly of taxon groups proposed a priori in the literature was tested using a Bayesian approach (Lewis, 2001; Lumbsch et al., in press; Miller et al., 2002). Constraints were constructed representing each proposed hypothesis using MacClade. The trees saved in the (BMC3 ) plateaus were then filtered for those congruent with a constraint using PAUP.

3. Results 3.1. Gene sequences and alignment A total of 68 taxa, represented by 86 DNA-sequences, was included in the analyses. Alignment of the LSU sequences resulted in a 3138 site long matrix. Two hundred and three ambiguously aligned sites (in 14 regions) and 1836 intron sites were excluded from the analyses. Two hundred and ninety sites in the included regions were variable of which 191 were parsimony-informative. The alignment of the b-tubulin sequences resulted in 1052 sites of which the 188 sites of three introns present had to be excluded. Of 363 variable sites, 338 were found to be parsimony-informative (codon position 1, 49; position 2, 16; and position 3, 273).

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Upon first inspection, both genes and all codon positions are affected by multiple parallel substitutions at individual nucleotide sites (Table 3). Most strongly affected were the third codon positions in b-tubulin with two-thirds of the sites showing multiple substitutions. However, phylogenetic analyses of b-tubulin that excluded third codon positions, or analyzed amino acid sequences, did not result in improved resolution of evolutionary relationships (results not shown). Hundreds (3rd positions excluded) to thousands (amino acid coding) of most parsimonious trees were found in those searches. Strict consensus trees of those trees resulted in extensive polytomies. The amino acid analysis uncovered no resolution within the Lecanorales, while the analysis of the nucleotide sequences with 3rd positions excluded retrieved the internode supporting the ingroup (‘‘Porpidia s.l.,’’ see below) but with little resolution above or below this internode in the strict consensus tree. Thus, in all further analyses 3rd codon positions were included, that is, all codon positions were equally weighted. b-Tubulin is a member of a small gene family for which Landvik et al. (2001) described a gene duplication event involving b-tubulin within the Ascomycota. It remains unclear at which point the duplication occurred, and thus which groups of ascomycetes possess paralogs. The primers used in this study were optimized to produce a single homologous amplification product within the Porpidiaceae. However, on rare occasion a deviating copy was sequenced for taxa within the Porpidiaceae. Those sequences could easily be identified in preliminary phylogenetic analyses due to the extensive outgroup sampling and were subsequently eliminated. These deviating sequences either represent a paralogous copy of b-tubulin or infection of the extracted thalli by fungal parasites or parasymbionts. Outgroup sequences were tested for paralogous copies by including them into a wider taxonomic context. For this, tubulin sequences of ascomycetes, basidiomycetes, and plants (as outgroups) were chosen from GenBank to represent the taxonomic diversity available in the database (Spring, 2002). Included in the sampling were all sequences submitted by Landvik et al. (2001). All mycobiont sequences formed a single lichenized clade in the MP analysis of the aligned

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nucleotide sequences that represents the Lecanoromycetes (results not shown). Paralogous copies thus could only be present if a recent duplication event had occurred within the Lecanoromycetes. No evidence exists to date that would suggest such an event. 3.2. Phylogenetic tree reconstruction In a maximum parsimony search of the LSU gene, 644 most parsimonious trees of 1544.68 steps were found. The consistency index (CI) for these trees is 0.3995. The basal relationships within the strict consensus tree are neither well-resolved, nor do they correspond to traditional taxonomic groupings. The genus Porpidia and the family Porpidiaceae are found throughout the tree with major Lecanoromycetes lineages (e.g., Cladoniaceae, Parmeliaceae, Peltigeraceae, Teloschistaceae, etc.) appearing within Porpidia. However, tip clades suggested in the LSU MP tree are identical or similar to the ones found with b-tubulin, although none of these relationships are supported. Bootstrap support of 75% or higher is only found in the tips of the tree. These results hold true also for the ML and BMC3 approaches. The ML search found only a single most likely tree ()lnL ¼ 6609.28; Fig. 1A). However, small changes in the likelihood model during the iterations brought about changes in the topology of the backbone of the tree. Accordingly, posterior probabilities of the extremely short branches in the backbone are below 95%. The maximum parsimony analysis of the b-tubulin fragment resulted in only two most parsimonious trees of 6416.7 steps with a CI of 0.1793. The two MP trees differ only in the placement of two lineages (P. macrocarpa and Porpidia sp. 2) within one of the tip clades. The ML analyses produced four trees of )lnL ¼ 15962.74 (Fig. 1B). Those trees differ slightly in the resolution of the relationship of Lecidea tessellata and Immersaria usbekica, as well as in the relationships in the Rhizocarpon, Sporastatia, Hymenelia, and Tremolecia group. The ML trees and the 50% majority rule consensus tree of the BMC3 analysis are congruent and differ only in resolution. The recovered relationships based on the b-tubulin dataset are consistent with

Table 3 Number of sites affected by multiple substitutions per partition Gene

LSU b-Tubulin

Combined

Partition

included pos1 pos2 pos3

Total no. of variable sites

Parsimonyinformative sites

Number of sites with 2, 3 or 4 different nucleotides present

Sum of sites showing multiple substitutions

2

4

3+4

(%)

3

290 65 25 273

191 49 16 273

209 57 17 101

61 3 8 35

20 5 0 137

81 8 8 172

0.28 0.12 0.32 0.63

653

529

384

107

162

269

0.41

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Fig. 1. Most-likely trees found in maximum likelihood analyses of the LSU (A) and b-tubulin (B) gene fragments. Wide internodes are supported by P 95% posterior probabilities. Four highly supported subgroups within Porpidia s.l. were found (groups I–IV). Support values for these groups are noted: values below the branches represent posterior probabilities, those above the branches denote non-parametric bootstrap support found in maximum parsimony analyses. Gray-scale indicates taxonomic groups, that is, light gray, Porpidiaceae; medium gray, Lecideaceae; and black, Lecanoromycete outgroups and non-lichenized ascomycetes. MLT, most-likely tree.

morphological and chemical data. Internodes showing monophyly of a ‘‘Porpidia s.l.’’ (see below) group, a Lecideaceae/Porpidiaceae-group, a subgroup within the Lecanorales and the Lecanoromycetes are recovered

with some of the reconstruction methods and show varying support. Under MP, the lichen-forming Lecanoromycetes do not come together as a monophyletic group at the base of the tree. Since paralogy of included

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Fig. 1. (continued)

DNA-sequences was investigated and could be rejected, these unrealistic relationships are probably due to insufficient taxon sampling in that part of the tree, suggesting long-branch-attraction. Correcting for possible long-branch attraction (Huelsenbeck, 1997, 1998), ML and BMC3 analyses present the lichen-forming group to be monophyletic. The separate analyses of the LSU and b-tubulin datasets reveal no highly supported evolutionary rela-

tionships that are in conflict in MP analyses, or in the ML/BMC3 -approaches. In addition, no significant incongruence in phylogenetic signal ðP ¼ 0:44Þ between the two datasets was detected using the ILD-test. Thus, both gene regions were combined for further analyses. The MP analysis of the combined datasets resulted in a single most parsimonious tree (8037.31 steps, CI ¼ 0.2200). ML produced a single most likely tree ()lnL ¼ 23073.63; Fig. 2). The ML tree is slightly more

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Fig. 2. Single most-likely tree found in a maximum likelihood reconstruction of the combined LSU and b-tubulin data set. Wide internodes are supported by P 95% posterior probabilities. Numbers below selected branches are posterior probability values, those above represent MP (left)/ML (right) non-parametric bootstrap values. MLT: most-likely tree. Subgroups and gray-scale are defined as in Fig. 1.

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resolved than the 50% majority rule consensus tree produced by BMC3 , with which it is otherwise congruent. Consensus trees produced from 2 million generation intervals of the 10 million run and of the 3 parallel 2 million runs provided the same result. The topologies resulting from the combined analyses are similar to the ones that were found with b-tubulin alone. However, the addition of the LSU improved resolution and increased branch support. Independent of reconstruction method, the combined dataset resolves the same backbone in all trees. All lichen-forming species included in the present study are classified as belonging to the Lecanoromycetes and group together with high posterior probability (PP). Within the Lecanorales, all four suborders, as defined by Lumbsch et al. (in press), are represented: the Acarosporineae (Hymenelia and Tremolecia) are basal to a highly supported group, including Peltigerineae (Peltigera, Nephroma, and Pseudocyphellaria), Teloschistineae (Xanthoria) and Lecanorineae (including all other lichen taxa, except Rhizocarpon and Sporastatia). Interestingly, Rhizocarpon and Sporastatia, previously classified as Lecanorineae (Cladoniineae), appear outside the supported Lecanorineae, Peltigerineae and Teloschistineae group in all combined analyses. The taxa sampled here from the families Lecideaceae and Porpidiaceae form a single monophyletic group in the MP analysis of the combined dataset, with Clauzadea (formerly Porpidiaceae) and Mycobilimbia hypnorum (i.e., the ‘Lecidea’ hypnorum group; Lecanorineae incertae sedis) being seemingly unrelated to either family. However, neither bootstrap support P75% nor posterior probabilities P95% were obtained for these relationships. A highly supported ingroup could be found (combined data set: 77% MP bootstrap, 87% ML bootstrap, 100% PP) independent of reconstruction method in all combined and b-tubulin analyses. This ingroup, labeled ‘‘Porpidia sensu lato,’’ includes all analyzed species of Porpidia and Lecidea, as well as representatives of several smaller genera within the Porpidiaceae (Amygdalaria, Immersaria, Stenhammarella, Stephanocyclos), and Lecideaceae (Cecidonia). Four highly supported (both by bootstrap and PP) subgroups within Porpidia s.l. are apparent in b-tubulin and combined analyses in all approaches (groups I–IV; Figs. 1B and 2). Support for the relationships within groups I and IV is generally high. Several taxa remain unattached to any of the groups and might represent independent subgroups in an analysis with more complete taxon sampling. Among those taxa are Porpidia stephanodes and Stephanocyclos henssenianus, which in most trees form a monophyletic group at the base of Porpidia s.l. In most analyses Porpidia tahawasiana is the sister lineage to group IV, however, this relationship shows no support. The positions of Immersaria usbekica and Lecidea tessellata are unclear. Lecidea tessellata was never found closely related to groups I and II, while

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Immersaria usbekica sometimes appeared as the sister taxon to group II. 3.3. Tests of monophyly No tree among the 80,000 trees sampled in the plateau of the 10 million—generation run showed the genus Porpidia s.s. as being monophyletic. Thus, monophyly of Porpidia as previously circumscribed is rejected (PP ¼ 0). Monophyly of the family Porpidiaceae can also be rejected at the highest significance level (PP ¼ 0). The same result was found for the four subgeneric groups proposed by Gowan (1989b). All subgroups have a posterior probability of zero. The results of the current phylogenetic analyses open up questions about the significance of the monophyly of the Porpidiaceae excluding obviously outlying genera, as for example, Clauzadea and the ‘Lecidea’ hypnorum group, as well as the monophyly of Porpidia s.l. and Porpidia s.l. minus subgroup II (Lecidea/Cecidonia). However, those are a posteriori hypothesis that should be tested in future studies on the basis of independent data sets. 3.4. Evaluation of conflict and signal in the data Despite the fact that Porpidia s.l. and the four subgroups within it are clearly defined and highly supported, the relationships between groups I–IV remain unresolved. Of special interest is the placement of Lecidea (including Cecidonia). Lecidea is placed within Porpidia s.l. as sister group to group I in MP, ML as well as in BMC3 analyses of the combined data. With over 100 species, Lecidea is the largest and traditionally most distantly classified genus to appear within Porpidia s.l. Due to the lack of bootstrap and PP support, however, it would still be possible for Lecidea to fall outside of Porpidia as traditionally delimited (including or excluding Porpidia stephanodes and S. henssenianus). Thus, we investigated if the lack of resolution at the base of Porpidia s. l. is due to a lack of signal (i.e., informative sites) or to extensive homoplasy, and, furthermore, if signal for the placement of Lecidea (group II) can be found. A reduced matrix, retaining only the ingroup taxa of Porpidia s.l. (with Bellemerea alpina as outgroup), contained 338 parsimony informative sites in the combined dataset if delimitation of ambiguous regions is kept the same as in the total dataset (Table 4). Realignment and subsequent inclusion of previously ambiguously aligned regions in the LSU reduced the number of excluded regions from 14 to 4 (introns still had to be excluded). The realignment resulted in an additional 27 parsimony informative sites. Re-analysis of this matrix (using MP heuristic searches and bootstraps) did not result in increased support for the relationships between the four

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Table 4 Effect of outgroup sampling and character inclusion (re-alignment of previously ambiguous regions) on the number of parsimony informative sites, tree length, and consistency indices Analysis

Gene partition

Number of taxa

Ambiguous regions

Parsimony inform. sites

MPT length

CI

RI

RC

Original

Comb LSU b-Tubulin

86 86 86

14 14 14

529 191 338

8037.31 1599.55 6437.76

0.2200 0.3858 0.1788

0.5640 0.4939 0.5749

0.1241 0.1905 0.1028

Reduced taxa

Comb LSU b-Tubulin

57 57 57

14 14 14

338 60 278

2725.88 381.53 2344.35

0.3752 0.4922 0.3562

0.7319 0.6928 0.7362

0.2746 0.3410 0.2622

Reduced taxa and re-included sites

Comb LSU b-Tubulin

57 57 57

4 4 4

365 87 278

2698.01 521.63 2176.38

0.3833 0.4673 0.3632

0.7323 0.6826 0.7404

0.2807 0.3190 0.2689

MPT, most parsimonious tree; CI, consistency index; RI, retention index; and RC, rescaled consistency index.

subgroups. While the reduction in taxa resulted in an increase in consistency and retention indices (Table 4), the re-inclusion of previously ambiguous sites did not affect the indices. The additional sites therefore did not provide especially clear phylogenetic signal. Rather they revealed considerable levels of homoplasy within Porpidia s.l. In both genes, visual inspection of the parsimony informative sites within the ingroup showed that sites potentially informative for the subgroup relationships are not only in conflict among each other, but are also commonly subject to multiple parallel substitutions. These results suggest that the analyzed gene regions provide enough signal at this level, but that the information available is noisy. We investigated if there is signal that supports a specific topology among the high level of homoplasy or if the homoplasy represents random noise. The 100 jackknife replicates produced 102 most parsimonious trees (Table 5). A first observation is that monophyly was independent of the taxa sampled for each of the four subgroups in the resulting trees. With regard to the placement of Lecidea (including Cecidonia), three trees were uninformative since one of the four groups was missing completely from the replicates. In only two of

Table 5 Results of taxon jackknifing procedure (100 replicates) with regard to the position of subgroup II (Lecidea and Cecidonia) within Porpidia s.l. Tree topology

No. of replicates

Lecidea groups within Porpidia 97 Subgroup II is sister group to subgroup I 54 Subgroup II is part of the lineage to subgroup III 34 Subgroup II takes a paraphyletic position in 9 Porpidia s.l. Lecidea takes a basal position in Porpidia s.l. (i.e., as sister group to Porpidia)

2

One of the four groups is missing (trees excluded)

3

Total number of trees

102

the retained 99 trees did subgroup II (Lecidea and Cecidonia) appear basal to Porpidia and its related genera. Porpidia stephanodes and S. henssenianus are basal to subgroup II in one of those topologies and missing from the other tree. Among the 97 trees in which subgroup II is falling within Porpidia as traditionally circumscribed, it is most often found as sister to subgroup I (54 replicates) or in the same clade as subgroup III (34 replicates). Most relevant taxa that could be added in future studies of the group are anticipated to either fall into one of the four subgroups, which are expected to persist, or to expand existing lineages (e.g., Immersaria). Thus, the jackknifing results suggest that even with a more complete taxon sampling within Porpidia s.l. the present position of Lecidea inside of Porpidia would persist.

4. Discussion 4.1. Phylogenetic resolution The present study is the first to provide a well-resolved and robust phylogeny for the genus Porpidia. The monophyly of the clade Porpidia s.l. is highly supported, as are four main subgroups and many relationships of taxa within them. The main characteristics of the final phylogeny are independent of reconstruction method, loci analyzed (with some exceptions in the LSU-only dataset), and taxa sampled. Despite generally high branch support throughout the ingroup, basal relationships between the four subgroups and several lineages that are not associated with any of the subgroups were not adequately resolved. A large fraction of variable sites in both analyzed gene fragments of LSU and b-tubulin has been subject to multiple parallel substitutions even within the ingroup, introducing a complex pattern of homoplasy within and among sites. This is especially true for the sites that provide information about the basal relationships within

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Porpidia s.l. Thus, the lack of resolution in this part of the tree is not due to a dearth of substitutions, which would be expected if the basal short internodes reflect short time intervals between speciation events, i.e., a rapid radiation. If a rapid radiation occurred, additional evolutionary processes must have altered its signature. The polarization of homoplasy within a dataset will depend on the taxa present in the matrix. A taxon jackknife approach was implemented (Lanyon, 1985) to investigate the influence that taxon sampling has on the relationships within the tree. As pointed out by Felsenstein (1988), the approach cannot assess statistical significance of topologies since taxa do not represent statistically independent sample points. Nevertheless, in the present study the approach was useful as it revealed that neither the monophyly of Porpidia s.l., nor the monophylies of each of the four subgroups are affected by taxon sampling. It thus gives an indication of what might be expected if more taxa were to be included in the future. Furthermore, despite a lack in statistically significant resolution in the backbone of Porpidia s.l., the data suggest that Lecidea might have arisen from within Porpidia as traditionally defined. In contrast to morphological and chemical characters, DNA-sequence data provide information that clarify the evolutionary relationships within Porpidia s.l. The discrepancy between these character systems might be attributed to a dearth of non-molecular characters in the group. However, a survey of phylogenetically informative characters in Porpidia s.l. suggests that this is probably not the case (Fryday and Buschbom, unpublished). It seems instead, that the lack of resolution provided by non-molecular characters is a consequence of the lack of our understanding of the delimitation, development, function, and evolution of those characters within the group and within lichen-forming ascomycetes as a whole. A stable phylogeny for Porpidia s.l. offers an important opportunity for understanding and re-evaluating traditionally employed characters within the group. 4.2. Evolutionary relationships within Porpidia sensu lato The present study lays the foundation for a future revision of Porpidia s.l. based on all character systems available. Such a revision will make it possible to address the question at which taxonomic rank Porpidia s.l. and the observed subgroups should be recognized. Gowan (1989b) already suggested that Porpidia is polyphyletic with smaller genera of the Porpidiaceae arising from within it. Alternatively, the generic status of several genera within the Porpidiaceae has been questioned (cp. Amygdalaria, Stenhammarella), since they are delimited from Porpidia on the basis of characteristics of uncertain significance (e.g., Brodo and Hertel, 1987; Brusse, 1988). The present results not only support these

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notions, but furthermore suggest that the group might also include genera of the Lecideaceae. The importance of ascus characters for classification within the Lecideaceae/Porpidiaceae will need to be reevaluated. Ascus characters play a prominent role in currently widely accepted higher-level classifications of the Lecanorales (e.g., Eriksson et al., 2001; Hafellner, 1984; Hafellner et al., 1993; Hawksworth et al., 1995; Rambold and Triebel, 1992; Tehler, 1996). Accordingly, Lecanorineae genera with predominately crustose taxa and well-developed tubular structures in the ascus-tips that stain blue after application of iodine solutions have been placed in the Porpidiaceae, while taxa that show only darker coloring caps in the ascus-tips have been treated in the Lecideaceae. In the phylogeny presented in this paper, Porpidia s.l. is not delimited along the lines of contrasting ascus-tip characters: taxa with either type of ascus tip are included in, as well as excluded from, Porpidia s.l. Thus, ascus characters do not represent a default character system within the Lecanorales that unconditionally reflects major evolutionary splits. Similar conclusions were drawn in investigations of the Bacidiaceae and Lecanoraceae (Ekman and Wedin, 2000), as well as the Agyriales (Lumbsch et al., 2001). With regard to the present group of species, Hertel (1987) had already pointed out that Porpidia- and Lecidea-type ascus-tips are not as strictly distinct as originally described, but that a continuum exists between the two ascus-tip types. In certain Lecidea species (e.g., L. lygomma Nyl.—subgenus Rehmiopsis, Rambold, 1989) ascus-tips can be regularly found that show two ‘‘teeth’’ reaching down distally from the cap. In addition, transitions between ascus-tip types have occurred at least twice within Porpidia s.l., since Lecidea is not monophyletic: Lecidea tessellata representing the monotypic subgenus Cladopycnidium is never found to be sister group to subgroup II (Lecidea subgenus Lecidea, Lecidea subgenus Rehmiopsis and Cecidonia). Nevertheless, while ascus-tip characters do not delimit Porpidia s.l., they provide one important morphological feature that distinguishes the two Lecidea lineages (subgroup II and L. tessellata) from all other lineages in Porpidia s.l. The subgroups found in the current phylogeny have not been proposed in their present circumscriptions before. The subgeneric divisions proposed by Gowan (1989b) show affinities with subgroups I, III, and IV found here, but are not identical to any of them. The assignment of taxa to these three subgroups is not intuitive. The observed distribution of taxa to the four subgroups suggests that parallelism, convergence, and reversals played an important role in the evolution of many morphological as well as chemical characters within Porpidia s.l. For example, a hyphal exciple structure was identified as a synapomorphy of the albocaerulescens complex by Gowan (1989b). However, this character state can be found in subgroups I

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(Porpidia ochrolemma), III (all species), and IV (Porpidia flavocoerulescens and Porpidia melinodes). Similar situations can be found with regard to chemical characters. For example, the apparently closely related stictic and norstictic acid pathways (Gowan, 1989a) are present in some taxa in all subgroups. It is expected that the subgroups will be identified by specific combinations of character states in the future. More work needs to be done to identify informative character combinations for each subgroup, since most characters need to be reevaluated and character variability within each subgroup needs to be taken into account.

placement that was generally accepted in subsequent classifications (e.g., Eriksson et al., 2001; Tehler, 1996). However, already Hafellner (1995), while excluding Sporastatia from the Acarosporaceae, questioned this relationship (‘‘? Catillariaceae’’). The position of Sporastatia in the present tree suggests that the genus is close to the Acarosporineae, a conclusion that is supported by the hymenial structure and ascus-tip anatomy of its species (T. Lumbsch, pers. comm.).

4.3. Phylogenetic context of the Lecideaceae/Porpidiaceae within the Lecanorales

Overall, a generally well-structured and resolved phylogeny of the taxonomically difficult genus Porpidia was recovered. Most taxa sampled within Porpidia s.l. fall into one of four highly supported subgroups, whose taxonomic rank will need to be determined in a future revision of the group based on molecular and non-molecular characters. The position of Lecidea (i.e., subgroup II) within Porpidia s.l., is still unsupported and requires further investigation, targeted to resolve the backbone of Porpidia s.l. through analyses of additional genetic loci and the inclusion of non-molecular characters. The present phylogeny provides insight into evolutionary processes and patterns within this group of lichen-forming ascomycetes (Buschbom, 2003). It offers the opportunity to investigate the evolution of different character systems throughout the group at the interspecific level. In addition, it presents a phylogenetic framework for population genetic investigations, especially due to the high resolution within the subgroups and tips of the tree.

Starting out with the goal to merely provide insight into evolutionary relationships within the genus Porpidia, the present study shows that a much wider group of related taxa and genera needs to be considered in investigations involving the ‘‘genus.’’ Nevertheless, a highly supported Porpidia s.l. clade was found that restricts the scope of the group to the Porpidiaceae and Lecideaceae for future investigations. With the exception of two groups (Clauzadea and the ‘Lecidea’ hypnorum group), the taxa sampled from the families Lecideaceae/Porpidiaceae form a monophyletic group in analyses of b-tubulin and the MP combined analysis. This relationship, however, lacks support, a situation that probably can be improved with a more inclusive taxon sampling in this part of the tree. Eriksson et al. (2001) and others treated Clauzadea and the ‘Lecidea’ hypnorum group within the Porpidiaceae. Our findings are in agreement with a recent revision of the genus Clauzadea based on morphological, chemical, and ecological characters (Meyer, 2002). This investigation found Clauzadea to be intermediate between the Porpidiaceae and the ‘Lecidea’ hypnorum group. Furthermore, the study concluded that Clauzadea only has a marginal position within the Porpidiaceae, if it is a member of the family at all. The question of the closest sister group to the Lecideaceae/Porpidiaceae remains open, since none of the previously proposed related families within the Cladoniineae included in this study seem to be close. This result is in accordance with studies that questioned the existence of the Cladoniineae (Ott and Lumbsch, 2001; Printzen, 1995). Furthermore, Rhizocarpon geminatum and Sporastatia testudinea, previously placed in families of the Cladoniineae, appear outside a supported Lecanorineae, Peltigerineae, and Teloschistineae group. A basal placement of Rhizocarpon (Rhizocarpaceae) in the Lecanorales was already proposed by Honegger (1980) based on ascus-tip characters. The family-level placement of Sporastatia is still unclear. Rambold and Triebel (1992) assigned the genus to the Catillariaceae, a

5. Conclusions and outlook

Acknowledgments The first author would like to thank I. Brodo (CANL), E.S. Hansen (C), R. Harris (NY), H. Hertel (M), R. Moberg (UPS), F. Oberwinkler (GZU), G. Rambold (M), C. Wetmore (MIN), and especially A. Fryday (MSC) for sharing their expertise of Porpidia, many fruitful discussions on the subject, collection support as well as access to the respective herbaria. B. Coppins, C. Roux, and R. Bettner kindly collected material. R. Poulsen made available collections from the Kerguelen Islands for DNA-extraction; K. Glew a specimen from Mt. Rainier, Washington, USA. T. Lumbsch offered helpful comments on the manuscript. F. Lutzoni provided advice in the early stages of the project. Research stations in Schefferville and Iqaluit (Canada), Qeqertarsuaq (Greenland), Abisko (Sweden), and Kevo (Finland) provided support during fieldwork. The Nunavut Research Institute, Qikiqtani Inuit Association, Danish Polar Center and National Park Service of Canada enabled collection work on Baffin Island,

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Greenland, and Vancouver Island. This study was supported by a NSF DDIG Grant (DEB-0105024), as well as awards and grants from the American Society of Plant Taxonomists, the Botanical Society of America, the Explorers Club, Scott Polar Research Institute and the University of Chicago Graduate Student Hinds Fund. Visits to the herbarium at Michigan State University were supported by NSF Grant DBI-9808735 to A. Pranther. J.B. received fellowships from the German Academic Exchange Service (DAAD), the Field Museum of Natural History and the Mycological Society of America.

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