Molecular phylogeny of the Bacidiaceae (Lecanorales, lichenized ...

783

Stefan EKMAN

Department of Botany, University of Bergen, AlleUgaten 41, N-5007 Bergen, Norway.

E-mail : stefan.ekman!bot.uib.no

Received 28 September 2000 ; accepted 25 January 2001.

The phylogeny of the family Bacidiaceae (Lecanorales, Ascomycota) was investigated using 65 nuclear ITS1-5±8S-ITS2 ribosomal DNA

sequences, 63 of which were newly determined. After exclusion of ambiguous alignment, the data set contained 285 variable

characters, 212 of which were parsimony-informative. Phylogenetic estimations were performed with maximum parsimony

(unweighted and weighted) and maximum likelihood optimality criteria. Four different phylogenetic hypotheses were tested using a

parametric bootstrap approach to simulate the expected null distribution of the difference between the globally optimal tree and the

best (constrained) tree agreeing with the null hypotheses under unweighted and weighted parsimony, and maximum likelihood : (1)

the genus Bacidia is monophyletic ; (2) the genus Bacidina is monophyletic ; (3) the genus Toninia is monophyletic ; and (4) the family

Ramalinaceae is monophyletic and distinct from a monophyletic Bacidiaceae. The monophyly of Bacidia, Toninia, and the Ramalinaceae

was rejected under all circumstances. Hence, Bacidiaceae is likely to be a younger synonym of Ramalinaceae. The monophyly of

Bacidina was not rejected under any optimality criterion. Furthermore, the data set suggests that the Bacidia beckhausii and B.

sabuletorum groups are unrelated to Bacidia s. str., that Megalaria is monophyletic, and that Lecania auct. is polyphyletic.

INTRODUCTION

The Bacidiaceae belongs in the Lecanorales, the largest of the

orders among the ascomycetes. Its presently most widely

used circumscription was established by Eriksson & Hawks-

worth (1987, 1993, 1998), who included 28 genera. In their

sense, the family included crustose and some squamulose,

foliose, and subfruticose taxa with a chlorococcoid photobiont,

biatorine (or sometimes lecanorine) apothecia, a more or less

well-developed annular proper exciple of hyphae dissimilar to

the paraphyses, a well-developed hypothecium, a hymenium

of sparsely branched paraphyses and asci with an amyloid

apex of the ‘Bacidia-type ’ or ‘Biatora-type ’ (Hafellner (1984),

non- or transversely septate (rarely muriform) ascospores, uni-

or plurilocular pycnidia with conidiophores of types II, III,

and VI (Vobis 1980), and ellipsoid, bacilliform or filiform

conidia. Historically, these lichens were included in the

gigantic Lecideaceae (Zahlbruckner (1905, 1921–40), which

included most crustose lichens with biatorine or lecideine

apothecia and a non-Trentepohlia photobiont. They were

treated as such until the comprehensive reclassification of the

Lecanoraceae and Lecideaceae by Hafellner (1984). In that work,

the taxa belonging to the family now known as Bacidiaceae (as

circumscribed by Eriksson & Hawksworth, loc. cit.) were

dispersed over the Bacidiaceae, Biatoraceae, Catinariaceae,

Lecaniaceae, Phyllopsoraceae, Tephromelataceae, Schadoniaceae,

and Squamarinaceae. Later, Hafellner (1988 : 45) reduced the

Biatoraceae and Lecaniaceae into synonymy with the Bacidiaceae.

Even though Eriksson & Hawksworth (1987, 1993, 1998)

amalgamated several of Hafellner’s families, the delimitation

of the family has been questioned. It was suggested by

Rambold (1989 : 21, 73), Rambold & Triebel (1992 : 52, 61),

and Hertel & Rambold (1995) that the Lecanoraceae and

Bacidiaceae as used by Eriksson & Hawksworth were

insufficiently delimited and should be treated as one family,

the name of which would then be Lecanoraceae. This view was

tentatively supported by Ekman (1996a). Based on SSU

rDNA data, however, Ekman & Wedin (2000), demonstrated

that the Bacidiaceae and Lecanoraceae are likely to represent

distinct families, although the genera Tephromela and Scolicio-

sporum probably do not belong in either of the families.

A factor that may have contributed to the confusion around

the distinction between the Lecanoraceae and Bacidiaceae is the

different interpretation of single genera. Bacidina was first

treated in the Lecanoraceae (e.g. Eriksson & Hawksworth 1987,

1993), and subsequently in the Bacidiaceae (Ekman 1996a,

Eriksson & Hawksworth 1998). The genera Megalaria and

Tylothallia have been treated in the Lecanoraceae (Eriksson &

Hawksworth 1998), but Ekman & Tønsberg (1996) presented

data speaking in favour of including Megalaria in the

Bacidiaceae, and Ekman (1997) considered Tylothallia to be

closely related to Cliostomum, a member of the Bacidiaceae

(Eriksson & Hawksworth 1987, 1993, 1998, Ekman & Wedin

2000). Although not a matter of much debate, the delimitation

of the Bacidiaceae relative to the Catillariaceae as used by

Eriksson & Hawksworth is also troubled by misclassified

Mycol. Res. 105 (7) : 783–797 (July 2001). Printed in the United Kingdom.

Molecular phylogeny of the Bacidiaceae (Lecanorales, lichenizedAscomycota)

Molecular phylogeny of the Bacidiaceae 784

genera. Classified in the Catillariaceae by Eriksson &

Hawksworth (1993, 1998), Arthrosporum and Toninia were

considered closely related to, possibly even indistinctly

delimited from, Bacidia, the largest of the genera in the

Bacidiaceae, by Ekman (1996a) and Timdal (1991). For a more

detailed discussion on the relationships between genera of the

Bacidiaceae, see Timdal (1991), Printzen (1995), and Ekman

(1996a).

A further family with unclear relationships to the Bacidiaceae

is the Ramalinaceae, containing the well-known fruticose

lichens Ramalina and Niebla. The Ramalinaceae possess an

amyloid ascus apex that is virtually identical to the one in the

Bacidiaceae, transversely septate spores are common in both

families, and a rare group of secondary metabolites, the

orcinol meta-depsides, are present in both families. With the

inclusion of the newly described crustose genus Ramalinora

(Lumbsch, Rambold & Elix 1995) in the Ramalinaceae, it has

become difficult to draw a clear-cut line between the families.

Although different in important characters, e.g. apothecium

ontogeny, the similarities between Ramalinora, with its only

known species R. glaucolivida (Lumbsch et al. 1995), and

Cliostomum, particularly C. tenerum (Ekman 1997), are striking.

The relationships between the Bacidiaceae and the Ramalinaceae

are definitively in need of scrutiny.

The relationships between the genera within the Bacidiaceae,

whatever circumscription is used, and between species and

species groups within its genera is largely unknown. There is

so far not a single published work dealing with the phylogeny

of the Bacidiaceae or any of its constituent genera (apart from

the outline by Ekman & Wedin 2000). As already mentioned,

Timdal (1991 : 23) pointed to problems with delimiting

Toninia from Bacidia. The genus Bacidina in the sense of Ekman

(1996a) contains a high degree of variation, and its monophyly

can be questioned. Lecania in its traditional sense appears

heterogeneous (Ekman 1996a). Hafellner (1984) questioned

the distinctness of the subfruticose genus Thamnolecania

relative to the crustose Lecania. Ekman (1996a : 45–46)

excluded the ‘Bacidia beckhausii group ’, the ‘Bacidia lutescens

group ’, and the ‘Bacidia sabuletorum group ’ from Bacidia s. str.

but was unable to refer them to any other described genus.

Several of the genera accepted in the Bacidiaceae by Eriksson

& Hawksworth (1998) include only a few known species (e.g.

Adelolecia, Boreoplaca, Herteliana, Rolfidium, Schadonia, Speer-

schneidera, Squamacidia, and Waynea), and their relationships

with the variable, species-rich genera of the family (Bacidia,

Biatora, Lecania, and Toninia) are poorly understood.

In addition to the taxonomic considerations, nomenclature

has been in dispute. The name Bacidiaceae was validly

described by Watson (1929). Both Biatoraceae (Stizenberger

1862 : 163) and Phyllopsoraceae (Zahlbruckner 1905) are older

synonyms, but the validity of the former needs to be

investigated further (Art. 33±7 ; Greuter et al. 2000). However,

the use of Bacidiaceae is maintained here, as it is a relatively

well-established name for the group in question (Eriksson &

Hawksworth 1987, 1993, 1998).

The problems involved in understanding familial and

generic boundaries in this group of lichens stem mainly from

the hazards of interpreting and homologizing morphological

characters. The aim of this investigation is to better understand

the Biatoraceae and its phylogeny using molecular data, in this

case DNA sequences from the ITS1-5±8S-ITS2 ribosomal

DNA. Particular attention is paid to the testing of four null

hypotheses : (1) Bacidia in the sense of Ekman (1996a) forms

a monophyletic group ; (2) Bacidina in the sense of Ekman

(1996a) forms a monophyletic group ; (3) Toninia in the sense

of Timdal (1991) forms a monophyletic group ; and (4) the

Ramalinaceae in the sense of many authors (e.g. Eriksson &

Hawksworth 1998) form a monophyletic group, which is

distinct from a monophyletic Bacidiaceae.

MATERIALS AND METHODS

Specimens

New sequences were obtained from 66 species listed in Table

1. The vast majority, 63 species, belong to the Bacidiaceae as

circumscribed by Eriksson & Hawksworth (1998) with the

corrections suggested by Ekman (1996a, 1997) and Ekman &

Wedin (2000) (Table 1). The remaining three species,

Scoliciosporum umbrinum, Tephromela atra, and T. aglaea, were

included as potential outgroup taxa.

DNA extraction, PCR amplification, sequencing, and

editing

DNA was extracted using the DNeasy Plant Mini Kit4(Qiagen). Complete PCR amplification of the nuclear ITS1-

5±8S-ITS2 ribosomal DNA region was performed using the

primers ITS1F (Gardes & Bruns 1993) and ITS4 (White et al.

1990). The PCR cocktail, the total volume of which was 50 µl,

contained, in addition to extracted DNA, 2±5 mM MgCl#,

200 µM of each of the four dNTPs, 0±7 µM of each primer,

1±5 U of a DNA polymerase (either AmpliTaq or AmpliTaq

Gold, PE Biosystems), together with a Mg#+ free buffer in the

concentration recommended by the manufacturer. The fol-

lowing PCR cycling parameters were used : a two-minute

(AmpliTaq) or a nine-minute hold (AmpliTaq Gold) at 94 °Cfollowed by six cycles including denaturation at 94 °(AmpliTaq) or 95 ° (AmpliTaq Gold) for 60 s, annealing at

62 ° (decreasing 1 ° each cycle) for 60 s, and extension at 72 °for 105 s, then 34 cycles with denaturation at 94 ° (AmpliTaq)

or 95 ° (AmpliTaq Gold) for 30 s, annealing at 56 ° for 30 s,

and extension at 72 ° for 105 s plus an addition of three

seconds each cycle, and finally a 10-min hold at 72 °, after

which the reaction was cooled to a constant 4 °. PCR products

were electrophorized in a 1% agarose gel and visualized using

ethidium bromide. They were subsequently cleaned using the

QiaQuick Spin kit (Qiagen) or, in case of an impure product,

the QiaQuick Gel Extraction kit (Qiagen). The cleaned PCR

product was sequenced with the ITS1F and ITS4 primers,

sometimes also the ITS2 and ITS3 primers (White et al. 1990)

when a long PCR product was obtained due to the presence

of group I introns at the very end of the SSU (Gargas,

DePriest & Taylor 1995). The Big Dye Terminator kit (PE

Biosystems) was used according to the manufacturer’s

instructions except that half-size instead of full-size reactions

were used. The final extension product was cleaned using a

NaAc precipitation protocol according to the manufacturer’s

S. Ekman 785

Table 1. Species from which new ITS1-5±8S-ITS2 nuclear ribosomal DNA sequences were obtained.

Species Generic affiliation CollectionGenBank}EMBLaccession no.

Arthrosporum populorum Haugan 4430 (O) AF282106Bacidia absistens Ekman 3223 (BG) AF282085B. arceutina Ekman 3110 (BG) AF282083B. auerswaldii P. Johansson 20 (UPS) AF282122B. bagliettoana Ekman 3137 (BG) AF282123B. beckhausii Not Bacidia s. str. Holien 6744 (TRH) AF282071B. biatorina Knutsson 94-148 (hb. Knutsson) AF282079B. caligans Bacidina P. Johansson 21 (UPS) AF282096B. circumspecta Ekman L1330 (LD) AF282124B. diffracta Wetmore 26401 (MIN) AF282090B. fraxinea T. Johansson 1620 (BG) AF282088B. hemipolia Not Bacidia s. str. Tønsberg 25091 (BG) AF282072B. hostheleoides 1996, Seaward (hb. Seaward 108121) AF282081B. incompta Ekman 3144 (BG) AF282092B. laurocerasi subsp. laurocerasi Wetmore 74318 (MIN) AF282078B. lutescens ? Ekman L1161 (LD) AF282082B. medialis Ekman L1193 (LD) AF282102B. polychroa Knutsson 91-215 (hb. Knutsson) AF282089B. rosella Ekman 3117 (BG) AF282086B. rubella auct. Ekman 3021 (BG) AF282087B. sabuletorum Not Bacidia s. str. Ekman 3091 (BG) AF282069B. schweinitzii Wetmore 72619 (MIN) AF282080B. scopulicola Ekman 3106 (BG) AF282084B. subincompta auct. Ekman 3413 (BG) AF282125B. suffusa Wetmore 74771 (MIN) AF282091B. vermifera T. Johansson 1619 (BG) AF282109Bacidina arnoldiana Ekman 3157 (BG) AF282093B. chloroticula Tønsberg 18642 (BG) AF282098B. delicata 1996, Fritz (BG) AF282097B. egenula Ekman 3003 (BG) AF282095B. inundata Ekman 3187 (BG) AF282094B. phacodes Ekman 3414 (BG) AF282100Biatora sphaeroides Mycobilimbia Ekman 3454 (BG) AF282068B. vernalis Tønsberg 23757 (BG) AF282070Catillaria globulosa Not Catillaria s. str. Ekman 3142 (BG) AF282073Cliostomum griffithii Ekman 3022 (BG) AF282076Lecania cyrtella Ekman 3017 (BG) AF282067L. naegelii Ekman 3401 (BG) AF282101Megalaria grossa Ekman 3466 (BG) AF282074M. laureri Ekman 3119 (BG) AF282075Thamnolecania brialmontii Convey 121 (AAS) AF282066Toninia alutacea Haugan & Timdal 4824 (O) AF282116T. aromatica Haugan & Timdal 4819 (O) AF282126T. candida Bratli & Timdal 8733 (O) AF282117T. cinereovirens Haugan & Timdal 7953 (O) AF282104T. coelestina Haugan 5985 (O) AF282127T. lutosa Timdal SON28}08 (O) AF282114T. nordlandica Haugan & Timdal 8129 (O) AF282113T. opuntioides Haugan & Timdal 8057 (O) AF282119T. pennina Haugan & Timdal 8122 (O) AF282111T. philippea Haugan & Timdal H3750 (O) AF282112T. plumbina Haugan 4352 (O) AF282107T. rosulata Timdal 8640 (O) AF282121T. sculpturata Haugan & Timdal 7829 (O) AF282110T. sedifolia Knutsson 97-407 (BG) AF282120T. squalida Haugan 4970 (O) AF282103T. talparum Timdal SON120}01 (O) AF282108T. taurica Haugan & Timdal 8060 (O) AF282118T. toniniana TuX rk 20721 (O) AF282115T. tristis subsp. tristis Haugan & Timdal 8109 (O) AF282105T. verrucarioides Bratli & Timdal 8709 (O) AF282128Tylothallia biformigera Ekman 3096 (BG) AF282077Waynea californica Tønsberg 21048 (BG) AF282099

Generic affiliations follow Ekman (1996), Printzen (1995), or Timdal (1991) if other than the one indicated by the generic name of the taxon (‘ ? ’ indicates

uncertain affinity different from the generic name). Abbreviations of public herbaria in which source collections are deposited follow Holmgren, Holmgren &

Barnett (1990). Private herbaria are denoted ‘hb ’.


recommendations. Extension products were subjected to

automatic sequencing on an ABI 377 with the XL upgrade (PE

Biosystems). Sequence fragments were assembled and edited

using Sequencher 3±0 and 3±1.1 (Gene Codes). Partial SSU and

LSU rDNA sequences, sometimes including an intron, at the

beginning and the end of the resulting sequence were

removed before alignment. The start of ITS1 and end of ITS2

were defined using the nuclear rDNA part of the Saccharomyces

cerevisiae chromosome XII sequence Z73326 obtained from

GenBank.

Sequence alignment

The 66 new sequences were aligned together with four

additional sequences : Leifidium tenerum (GenBank accession

number AF117998), Ramalina fastigiata (U84583), Ramalina

siliquosa (U84587), and Sphaerophorus globosus (AF282129).

First, a preliminary alignment including all taxa was performed

using SAM 3±0 (Sequence Alignment and Modeling Software

System) (Krogh et al. 1994, Hughey & Krogh 1996, Hughey,

Karplus & Krogh 1999, Durbin et al. 1998), which is available

on-line at Institut Pasteur (Paris ; http :}}bioweb.pasteur.fr}-

seqanal}motif}sam-uk.html). From the resulting preliminary

alignment it was evident that Scoliciosporum umbrinum,

Tephromela aglaea, and T. atra were dubiously alignable with

the other taxa, and they were hence excluded from the further

analysis. The SAM analysis was reiterated with the remaining

67 taxa. Finally, the alignment was manually optimized.

However, a series of gap-rich regions in the ITS1 and ITS2,

clearly remained ambiguously aligned. These sites were

consequently excluded and the remaining ones were used in

the subsequent phylogenetic analyses and analysis of

phylogenetic signal. The final alignment was submitted

to TreeBASE (Harvard ; http :}}www.herbaria.harvard.edu}treebase) where it is filed under matrix accession number

M849.

Phylogenetic signal

For an a priori estimation of the amount of phylogenetic

signal, the Relative Apparent Synapomorphy Analysis (RASA)

technique was applied (Lyons-Weiler, Hoelzer & Tausch

1996), as implemented in the software RASA 2±3 (Lyons-

Weiler 1999). Unlike other measures of phylogenetic signal,

RASA is tree-independent and is not based on the assumptions

of a tree-building optimality criterion. A rooted test was

performed, and the inferred signal content was compared to

an analytical null model. Here and in the subsequent

phylogenetic analyses, Sphaerophorus globosus and Leifidium

tenerum (Sphaerophoraceae) were used as outgroup taxa

following a suggestion by Ekman & Wedin (2000) that the

Sphaerophoraceae constitute a possible sister-group to the

Bacidiaceae.

Phylogenetic analyses

Phylogenetic analyses were performed on an Apple Power-

Macintosh G3}266 using maximum (unweighted) parsimony

(MP), maximum weighted parsimony (MWP), and maximum

likelihood (ML) optimality criteria as implemented in the

computer programme PAUP* 4±0b2a, except for the analyses

of simulated data, which were analysed using PAUP* 4±0b3aand 4±0b4a (Swofford 1999).

Unweighted parsimony

Gaps were treated as missing data. A heuristic search with

1000 random-addition sequence replicates was performed

using tree bisection-reconnection (TBR) branch-swapping, and

with the MulTrees option on and the steepest descent and

collapse zero-length branches options off. Multiple character

states were interpreted as uncertainties. Branch lengths were

assigned using ACCTRAN character state optimization.

Branch support was estimated using a jackknife analysis

with 10000 replicates with the same search parameters as

above, except that 100 random-addition replicates were tested

in each replicate and that MulTrees was off. The nominal

exclusion of characters was 36±79% (Farris et al. 1996) and

JAC resampling was emulated using PAUP*.

Weighted parsimony

Settings were identical to the unweighted analysis, except that

character state transformations were weighted unequally

according to a symmetric step matrix. Weights were obtained

by charting the absolute average number of changes on 100

equiprobable random trees using MacClade 3±08 (Maddison

& Maddison 1992, Lutzoni 1997). Reciprocal substitutions

were summed, and the relative frequency of each substitution

type was calculated. Frequencies were converted to costs of

changes using the negative natural logarithm of the frequency

and rounded to nearest single-decimal number. The resulting

step matrix was used in PAUP* by applying user-defined

character types. Violations against the triangle inequality were

not present. Branch support was estimated using a jackknife

analysis with 300 replicates. All other search parameters were

identical to the unweighted parsimony jackknife.

Maximum likelihood

In order to investigate what likelihood model best fitted the

data, a likelihood ratio test (Huelsenbeck & Crandall 1997 ;

Huelsenbeck & Rannala 1997) was performed as implemented

in the computer programme MODELTEST 2±1 (Posada &

Crandall 1998). The critical value of rejection was set to 0±008in order to maintain an overall type I error rate of 0±05. The

results favoured a model with unequal nucleotide frequencies

and six different time-reversible substitution types. This

corresponds to the GTR model (e.g. Rodrı!guez et al. 1990,

Yang 1994a). Furthermore, the test favoured the assumption

of invariability (I) in a fraction of sites, pinv

(Gu et al. 1995), and

substitution rate heterogeneity among nucleotide sites

according to a gamma model (Yang 1993).

A χ# test of homogeneity of base frequencies across taxa

was performed, as implemented in PAUP*, since the GTR

likelihood model employed here assumes stationary base

frequencies.

Initial values of the six relative substitution rates (the R

matrix), the proportion of invariable sites (pinv

), and the

S. Ekman 787

gamma curve shape parameter (α) were estimated from one of

the most parsimonious trees using maximum likelihood, and

set constant during the search. The likelihood of this tree was

calculated with four, six, eight, and ten discrete gamma

categories (Yang 1994b), and it was found that the likelihood

was maximized with six categories. Hence, a discrete gamma

model with six categories was applied (dΓ6), and the average

rates of the categories were represented by their means. A tree

search in three parts was conducted : (1) A heuristic search

with 50 random-addition sequence replicates was performed

using nearest-neighbour interchanges (NNI) branch-swapping ;

(2) the most likely trees found in the first search were input

into a second round of branch-swapping with subtree pruning-

regrafting (SPR) ; and (3) the most likely tree(s) found in the

second search were input into a third round of branch-

swapping with tree bisection-reconnection (TBR). After the

search was completed, likelihood parameters were reestimated

according to the most likely tree found, and the heuristic

search was reiterated until tree topology and likelihood

parameters had converged and continued iteration was

unnecessary. Convergence occurred after the third search.

During all searches, the MulTrees option and collapse

effectively zero-length branches options were on, and the

steepest descent option was off.

Each branch in the resulting tree was subjected to a

likelihood ratio test (as implemented in PAUP*) of the null

hypothesis that the branch has zero length. This test was

performed with full reoptimization after forcing a branch

length to zero.

Branch support was estimated using a jackknife analysis

with 100 replicates. Starting trees were obtained using

neighbour-joining (with maximum likelihood distances), which

were subjected to nearest-neighbour interchanges (NNI)

branch-swapping. MulTrees was on, and nchuck and chuck-

score was set to 10 and 1, respectively.

Hypothesis testing

First, the optimal trees agreeing with each of the constraints

inherent in the four null hypotheses were searched for under

unweighted and weighted parsimony, and maximum like-

lihood. The following constraints were used :

Null hypothesis 1 : Bacidia is monophyletic, i.e. B. absistens,

B. arceutina, B. auerswaldii, B. bagliettoana, B. biatorina, B.

circumspecta, B. diffracta, B. fraxinea, B. hostheleoides, B. incompta,

B. laurocerasi, B. medialis, B. polychroa, B. rosella, B. rubella, B.

schweinitzii, B. scopulicola, B. subincompta, B. suffusa, and B.

vermifera form a monophyletic group.

Null hypothesis 2 : Bacidina is monophyletic, i.e. B.

arnoldiana, B. caligans, B. chloroticula, B. delicata, B. egenula, B.

inundata, and B. phacodes form a monophyletic group.

Null hypothesis 3 : Toninia is monophyletic, i.e. T. alutacea,

T. aromatica, T. candida, T. cinereovirens, T. coelestina, T. lutosa,

T. nordlandica, T. opuntioides, T. pennina, T. philippea, T.

plumbina, T. rosulata, T. sculpturata, T. squalida, T. talparum, T.

taurica, T. toniniana, T. tristis, T. sedifolia, and T. verrucarioides

form a monophyletic group.

Null hypothesis 4 : Ramalinaceae is distinct from Bacidiaceae,

i.e. Ramalina fastigiata and R. siliquosa form a monophyletic

group, and all other ingroup taxa form another monophyletic

group.

Search parameters under MP, MWP, and ML were identi-

cal to the searches for the unconstrained trees, except

that six discrete gamma categories (dΓ6) were assumed

rather than tested for in the ML analysis. Different trees

may have different optimal likelihood functions and hence

likelihood parameters in the ML analysis were optimized

for the particular tree topology, rather than, e.g., kept

identical to the parameters of the globally most likely

tree.

When a search yielded more than a single optimal tree

(unweighted and weighted parsimony), the one with the best

likelihood was chosen to represent the null hypothesis. The

likelihood of all the most parsimonious, unweighted and

weighted, trees was calculated using a GTRIdΓ6 model

with the R matrix, pinv

, and α either estimated from the tree

topology (weighted parsimony) or, due to computational

effort, kept constant at the parameters of the globally most

likely tree. Polytomies occurring in the most likely constrained

trees (since effectively zero-length branches were collapsed in

the constrained ML searches) were randomly dichotomized

using MacClade 3±08 (Maddison & Maddison 1992). Branch

lengths of all trees (MP, MWP, ML) selected to represent the

null hypotheses were optimized under a GTRIdΓ6

likelihood model with the R matrix, pinv

, and α estimated.

Branch lengths and likelihood parameters were saved for later

use in the parametric bootstrap. None of the trees representing

the same null hypothesis were identical in topology, which

means that each hypothesis test involved a unique pair of

trees being compared.

The four phylogenetic null hypotheses were tested using a

parametric bootstrap approach (Efron 1985, Felsenstein 1988,

Huelsenbeck, Hillis & Jones 1996, Swofford et al. 1996 : 506,

Hillis, Mable & Moritz 1996 : 523–526, Huelsenbeck, Hillis &

Nielsen 1996). Under a parametric bootstrap, a null distribution

of the null hypothesis is generated by recording the difference

in length}likelihood between the optimal topology and the

best topology agreeing with a null hypothesis for each of

many data sets that were simulated along the best null

hypothesis tree from the original data set. The difference in

length or likelihood should be zero in the ideal case, but due

to stochastic variation or systematic error of the reconstruction

method this is not necessarily so. The difference in

length}likelihood obtained from the original data set (δ) can

then be compared to this null distribution, and the probability

of the null hypothesis being true can be calculated. The

popular Kishino–Hasegawa (Kishino & Hasegawa 1989) and

Templeton tests (Templeton 1983, Felsenstein 1985) were not

employed, since the null distributions that these tests rely on

apply only when the trees being compared can be considered

drawn at random from the population of all possible trees, i.e.

when the trees are fully specified a priori (Goldman, Anderson

& Rodrigo 2000). Consequently, these tests are inapplicable

when one or both of the trees being compared originate from

the analysis of the data set at hand. In fact, most published

applications of these tests are invalid.

The parametric bootstrap was performed in the following

way : 249 data sets per null hypothesis (1–4) per optimality


Fig. 1. One out of four optimal trees under the maximum unweighted parsimony criterion. Tree length¯ 1677 steps. Numbers are

jackknife percentage values above 50% (n¯ 10000). Branches that are collapsed in the strict consensus of all most parsimonious trees

are denoted by an asterisk (*).

criterion (MP, MWP, and ML) were simulated using the

software SEQ-GEN 1±1 (Rambaut & Grassly 1997). The

optimal trees agreeing with the null hypotheses from the

original data set, including their branch lengths and estimated

likelihood parameters (the R matrix, pinv

, and α), were used to

guide the simulation. The ‘REV ’ model (identical to the GTR

model), combined with empirical nucleotide frequencies and

substitution rate heterogeneity among nucleotide sites

S. Ekman 789

Fig. 2. One out of three optimal trees under the maximum weighted parsimony criterion. Tree length¯ 2644±1 steps. A symmetric

step matrix was used, which included weights obtained by charting the absolute average number of changes on 100 equiprobable

random trees (see text). Numbers are jackknife percentage values above 50% (n¯ 300). Branches that are collapsed in the strict

consensus of all most parsimonious trees are denoted by an asterisk (*).


Fig. 3. The single optimal tree under the maximum likelihood criterion. –Ln likelihood¯ 8166.1434. A GTRIdΓ6 model was used,

since it was shown to best explain the observed data according to a likelihood ratio test. Numbers are jackknife percentage values

above 50% (n¯ 100). An asterisk (*) denotes a branch that is not significantly longer than zero under a likelihood ratio test with full

reoptimization after forcing a branch length to zero. The nomenclaturally correct generic names of some clades discussed in the text and

supported by this tree (sometimes also the unweighted and weighted parsimony trees) have been marked. Light grey (as opposed to

dark grey) shading refers to optional inclusion. Informal names of species groups refer to the discussion of Ekman (1996a : 45–46).

S. Ekman 791

according to a continuous gamma model, was used. The

length of the simulated sequences corresponded to the

number of potentially variable sites in the original matrix, i.e.

505¬(1®pinv

) (invariable sites were not added to the

matrices, since only parsimony was used for the analysis ; see

below). A total of (four null hypotheses¬three optimality

criteria¬249¯) 2988 new matrices were simulated.

Each of the (4 null hypotheses¬249¯) 996 data sets

simulated along the optimal constrained MP trees was

analysed in an unconstrained analysis (100 random-addition

sequence replicates, TBR branch-swapping, MulTrees option

on, steepest descent, collapse zero-length branches options

off, nchuck¯ 500, and chuckscore¯ 1), and in a constrained

analysis (same search parameters, the constraint being the

same as the one used to obtain the tree used for simulation).

To facilitate analysis, it was executed in PAUP* as a

continuous series of 996 DATA blocks, each followed by a

PAUP block containing the desired settings and search

commands. For each of the 249 data sets representing a null

hypothesis, the difference between the optimal unconstrained

and the optimal constrained tree was recorded and plotted in

a histogram.

Each of the 996 data sets simulated along the optimal

constrained MWP trees was analysed in a similar way, except

that character state changes were weighted using the same

step matrix as in the initial search for the globally most

optimal MWP tree.

Ideally, the 996 data sets simulated along the optimal

constrained ML trees should be analyzed under maximum

likelihood, since the difference in likelihood between the

optimal unconstrained and constrained trees obtained from

the original data matrix was calculated under this optimality

criterion. However, such an analysis is unrealistic and would

take years to complete even with very moderately aggressive

search parameters. Therefore, differences in parsimony scores

were used in place of differences in likelihood scores for

testing the null hypotheses obtained under the ML optimality

criterion. Although a switch in the optimality criterion can be

motivated, a slight increase in the stochastic variation of the

test can be anticipated, since the difference in parsimony score

will sometimes underestimate and sometimes overestimate

the difference in likelihood scores.

The probability of a null hypothesis being correct is

r}(m1), where r is the rank order of δ among all score

differences and m is the number of bootstrap replicates

(Goldman 1993). Raw probability values were Dunn–S) ida! kcorrected for multiple comparisons (Sokal & Rohlf 1995) to

maintain a total type I error rate of 0±05.The complete analysis as described here, including some

mistakes in need of correction, required approximately four

and a half months of dedicated CPU time.

RESULTS

The final alignment of the 67 taxa was 752 positions in length.

Altogether 247 positions representing 25 blocks of contiguous

alignment sites were excluded due to alignment problems.

The number of included alignment positions was 505. In this

Table 2. A comparison of optimal trees obtained under maximum

parsimony (MPa-d), maximum weighted parsimony (WMPa-c), and

maximum likelihood (MLa).

Tree

Tree length (un-

weighted parsimony)

Tree length

(weighted parsimony) -Ln likelihood

MPa 1677 2654±2 8189±2804MPb 1677 2655±4 8189±4138MPc 1677 2656±6 8189±5331MPd 1677 2657±8 8189±6683MWPa 1690 2644±1 8174±7531MWPb 1690 2644±1 8175±4004MWPc 1690 2644±1 8175±4004MLa 1701 2673±5 8166±1434

The likelihood of each tree was calculated using a GTRIdΓ6 model

with empirical base frequencies, and the R matrix, proportion of invariable

sites pinv

, and the gamma curve shape parameter α estimated.

alignment, 285 characters were variable, and 212 were

parsimony-informative.

The test for phylogenetic signal was highly significant

(βnull

¯ 17±7, βobs

¯ 28±0, ..¯ 2012, rooted tRASA

¯ 38±5,P' 0±001), and it was concluded that the matrix contained a

significant amount of hierarchical signal potentially useful for

phylogenetic analysis.

The heuristic search under the maximum unweighted

parsimony optimality criterion yielded four equally par-

simonious trees of 1677 steps, all with consistency index (CI)

¯ 0±26, retention index (RI)¯ 0±50, and rescaled consistency

index (RC)¯ 0±13 (indices calculated with uninformative

characters excluded). One of the most parsimonious trees,

with jackknife branch support, is shown in Fig. 1. The heuristic

search under the maximum weighted parsimony optimality

criterion yielded three equally parsimonius trees of 2644±1steps, all with consistency index (CI)¯ 0±26, retention index

(RI)¯ 0±52, and rescaled consistency index (RC)¯ 0±14. One

of these trees, with jackknife branch support, is shown in Fig.

2. The heuristic search under the maximum likelihood

optimality criterion yielded a single most likely tree with ln

likelihood¯®8166±1434. Likelihood parameters were rAC

¯2±66, r

AG¯ 3±93, r

AT¯ 1±93, r

CG¯ 0±76, r

CT¯ 7±29 (as-

suming rGT

¯ 1), pinv

¯ 0±19, and α¯ 0±58. This tree, with

jackknife support values, is shown in Fig. 3. The chi-square test

of homogeneity of base frequencies across taxa revealed no

significant differences (χ#¯ 68±3, ..¯ 138, P¯ 1±00). The

optimal trees obtained under the three optimality criteria are

being compared in Table 2. Figs 4–7 demonstrate the outcome

of the parametric bootstrap analyses of the four null

hypotheses under maximum unweighted parsimony, maxi-

mum weighted parsimony, and maximum likelihood. Dunn–

S) ida! k corrected probabilities of the null hypothesis being

correct are provided in Table 3. The parametric bootstrap

rejected the monophyly of Bacidia, Toninia, and the Rama-

linaceae no matter what optimality criterion had been used to

generate the tree representing the null hypotheses. The

monophyly of Bacidina, on the other hand, was not rejected

under any optimality criterion.

DISCUSSION

Common to the optimal trees obtained under maximum

unweighted and weighted parsimony as well as maximum


Fig. 4. The result of parametric bootstraps performed under the null

hypothesis that the genus Bacidia in the sense of Ekman (1996a) is

monophyletic. The null hypothesis was simulated under three

different optimality criteria and the difference in length between the

optimal tree and the best tree agreeing with the null hypothesis was

graphed. Abbreviations : ∆P¯ difference in tree length under

unweighted parsimony, ∆WP¯ difference in tree length under

weighted parsimony, and δ¯ difference in length between optimal

tree and best tree agreeing with the null hypothesis obtained from

the original data.

likelihood is that Lecania, Thamnolecania, Biatora s. l., Tylothallia,

Megalaria, Cliostomum, Ramalina, members of the Bacidia

sabuletorum and B. beckhausii groups (Ekman 1996a), and B.


hypothesis that the genus Bacidina in the sense of Ekman (1996a) is




graphed. Abbreviations are as Fig. 4.

incompta appear at the base of the trees, and that the ‘crown ’

of the trees is composed of two clades, one containing Bacidia

s. str., the other Toninia, Bacidina, and related taxa. However,

the optimal trees obtained under all optimality criteria contain

many ‘weak ’ branches and rather few ‘strong ’ internal

branches as estimated by the jackknife branch support. In

addition, the optimal trees differ conspicuously in which order

S. Ekman 793


hypothesis that the genus Toninia in the sense of Timdal (1991) is




graphed. Abbreviations are as Fig. 4.

some clades appear in the tree. The position of a number of

species is notoriously unstable, e.g. Bacidia bagliettoana, B.

incompta, Thamnolecania brialmontii, and Waynea californica.

Presumably, the rather different trees obtained under the

different optimality criteria is a result of substantial homoplasy

in the matrix (as indicated by, e.g. the low CI, RI, and RC), by


hypothesis that the family Ramalinaceae auct. is monophyletic and

distinct from a monophyletic Bacidiaceae. The null hypothesis was

simulated under three different optimality criteria and the difference

in length between the optimal tree and the best tree agreeing with

the null hypothesis was graphed. Abbreviations are as Fig. 4.

substantially different nucleotide substitution rates (as indi-

cated by the R matrix of the most likely tree ; CT substitutions

are approximately ten timesmore likely thanCG substitutions),

and by substantial rate heterogeneity among nucleotide sites

(as indicated by the rather low value of α, 0±58, of the most

likely tree). The two latter factors are known to create more


Table 3. Probabilities of null hypotheses 1–4 being correct under unweighted and weighted parsimony, and maximum likelihood. ∆P and ∆WP refer to

differences in unweighted or weighted parsimony scores between constrained and optimal trees obtained from simulated data matrices. Raw probabilities

(calculated according to the formula of Goldman 1993) were Dunn-S) ida! k corrected for multiple comparisons (Sokal & Rohlf 1995) to maintain a total

type I error rate of 0±05. The actual null hypothesis at test was that the best tree agreeing with the null hypothesis (the constrained tree) does not differ

significantly from the globally best tree (given an optimality criterion). Hypotheses were rejected if p% 0±05. When the rank order of δ is an interval, p

was calculated from the worst rank (see text for the calculation of p). The lowest probability possible to obtain in this experiment is p¯ 0±047.

Optimality

criterion

Difference of tree

length}likelihood (δ)

Highest ∆P or ∆WP

recorded in simulation Rank order of δ p

(1) Bacidia in the sense of Ekman (1996a) is monophyletic

MP 41 7 1 0±047*MWP 55±9 9±9 1 0±047*ML 39}97±8947 7 1 0±047*

(2) Bacidina in the sense of Ekman (1996a) is monophyletic

MP 6 6 1–3 0±135MWP 9±3 9±9 2 0±091ML 4}3±5009 7 15–40 0±877

(3) Toninia in the sense of Timdal (1991) is monophyletic

MP 25 7 1 0±047*MWP 37±3 10±6 1 0±047*ML 23}63±7593 8 1 0±047*

(4) Ramalinaceae auct. is monophyletic

MP 15 9 1 0±047*MWP 22±9 10±2 1 0±047*ML 14}22±1206 8 1 0±047*

problems to maximum parsimony analysis than to maximum

likelihood analysis, at least when a correct likelihood model is

used (e.g. Yang 1996a,b, 1998). Although still susceptible to

errors due to rate heterogeneity among nucleotide sites,

weighted parsimony is less adversely affected than unweighted

parsimony by unbalanced nucleotide substitution rates, and it

was considered to ‘often provide a close approximation of the

likelihood solutions ’ by Swofford et al. (1996). A parameter-

rich maximum likelihood model like the one used here is

assumed to be more or less immune to these problems. On the

other hand, the likelihood model used may be a poor predictor

of the actual evolutionary events, and even if a correct model

is used, cases are known where other models may reconstruct

the true phylogeny with greater accuracy (Yang 1997). In

addition, likelihood parameter estimates are just estimates ;

they contain errors that may add up to produce an incorrect

phylogeny. If the maximum likelihood tree obtained in this

study is anywhere near the true phylogeny, with several short

internal branches (18 of which are not significantly longer than

zero), the hope of ever obtaining an accurate and well-

supported estimate of the phylogeny of the Bacidiaceae may

have been thwarted by nature itself.

Despite problems with phylogeny estimation and low

branch support, the data set contains sufficient information to

discriminate between alternative phylogenetic scenarios. The

hypotheses that Bacidia in the sense of Ekman (1996a) and

Toninia in the sense of Timdal (1991) are monophyletic were

emphatically rejected by the parametric bootstraps irrespective

of optimality criterion (hypothesis 1 and 3 of Table 3 ; Figs 4,

6), despite the fact that these monographers excluded

numerous species that were previously referred to the two

genera.

A suitable candidate for a redefined Bacidia s. str. obtained

in this study is the clade containing B. hostheleoides and B.

lutescens at the bottom (Figs 1–3). This clade has a 97–100%

jackknife support depending on optimality criterion. The

overall problem with Bacidia in the sense of Ekman (1996a)

seems to be that too many species have been included. In

particular, species with blue-green pigmentation in the

epithecium and}or with fusiform or bacilliform spores (as

opposed to acicular, i.e. with one blunt end and one tapering

end) appear not to be closely related to Bacidia s. str. as

suggested here, but rather to species groups referred to

Toninia as circumscribed by Timdal (1991). Unlike most other

species in need of exclusion from Bacidia s. str., B. medialis has

usually pale apothecia with little pigment. It was considered to

belong to Bacidia s. str. and being closely related to B.

hostheleoides by Ekman (1996a), although a difference in

structure of the proper exciple between the two species was

noted. In the present study, however, it becomes apparent

that B. medialis has little to do with B. hostheleoides, the former

being more closely allied with Bacidina phacodes and Lecania

naegelii. In addition, the affinities of Bacidia incompta to Bacidia

s. str. remain doubtful. As suggested by Ekman (1996a), the B.

beckhausii group appears to be distantly related to Bacidia s. str.

and possibly represents an as yet undescribed genus (although

poorly supported as revealed by jackknife values ; Figs 1–3).

However, Catillaria globulosa seems to belong in this group as

well. This species bears an overall striking similarity with the

other species of the group, except that the spores are 1-septate

instead of 3-septate. Catillaria globulosa was referred to Bacidia

by Wirth (1987), and this treatment has been followed by a

number of authors. The data also lends support to the

exclusion of B. sabuletorum and its relatives from Bacidia s. str.

(Ekman 1996a). Instead, the B. sabuletorum group seems to be

more closely related to Thamnolecania and Lecania (assuming

that L. cyrtella is a member of Lecania s. str.). Unlike the

prediction by Ekman (1996a), however, the B. lutescens group

S. Ekman 795

appears not to bear any close relationship with the B.

beckhausii group, but instead seems to be a part of Bacidia s. str.

Toninia as understood by Timdal (1991) appears to be a

polyphyletic assemblage of species. Apparently, some species

groups are more closely related to taxa traditionally referred

to Arthrosporum, Bacidia, Bacidina, or Waynea than they are to

each other. Unfortunately, the data does not contain resolving

power enough to detail any alternative taxonomic framework

for this group of taxa. The data suggests, however, that some

of the generic names treated as synonyms of Toninia (Timdal

1991 : 30) may have to be resurrected in the future, e.g.

Thalloidima (Fig. 3). A few well-supported groups (here taken

to mean a jackknife support of ca 75% or more under all

optimality criteria) can be discerned (Figs 1–3) : (1) Arthro-

sporum populorum, Toninia plumbina, and T. talparum ; (2)

Toninia aromatica and T. verrucarioides ; (3) Toninia nordlandica,

T. pennina, and T. philippea ; and (4) Toninia toniniana, T.

alutacea, T. candida, T. taurica, T. opuntioides, T. sedifolia, and T.

rosulata. The first group would constitute a slightly expanded

version of the genus Arthrosporum, A. populorum being the

type species, whereas the last group would constitute the

genus Thalloidima, the type species of which is T. candida. The

circumscription of Toninia s. str. is very uncertain. The optimal

trees obtained in this study suggest that Toninia would

include T. cinereovirens (the type species), T. squalida, T. tristis,

and possibly also one or a few species previously referred to

Bacidia, viz. B. bagliettoana, B. auerswaldii, and B. circumspecta.

Great care should be taken in the interpretation, however. If

the position of Bacidina}Woessia}Lecania naegelii as nested

within clades dominated by species of Toninia turns out to be

artefactual, Toninia can be conveniently redefined and become

monophyletic by including in it Arthrosporum, the majority of

misclassified Bacidia species with blue-green pigmentation in

the epithecium and}or with fusiform or bacilliform spores, and

possibly also Waynea.

The case for or against a monophyletic genus Bacidina is

more uncertain. The parametric bootstrap test does not reject

the null hypothesis of a monophyletic Bacidina under any

optimality criterion (hypothesis 2 of Table 3 ; Fig. 5). This

should not be taken to mean that Bacidina is necessarily

monophyletic, just that the data set at hand does not contain

the necessary resolving power to tell whether the genus is

monophyletic or not. A ‘core group ’ of species, Bacidina

chloroticula, B. delicata, B. arnoldiana, B. inundata, B. egenula, and

Bacidia caligans constitute a well-supported group (with

98–100% jackknife support depending on optimality criterion ;

Figs 1–3). This group of species is sometimes referred to

Woessia, the type species being W. fusarioides, a synonym of

Bacidina arnoldiana or W. arnoldiana (see e.g. Diederich &

Se! rusiaux 2000). B. phacodes, the type species of Bacidina, may

or may not be associated with this group. It is interesting to

note that in all of the optimal trees, B. phacodes is more or less

closely associated with Lecania naegelii, since these species are

the only known representatives of the Bacidiaceae that possess

straight macroconidia with multiple transverse septa. Bacidina

may or may not be synonymous with Woessia, and one or

both of Bacidia medialis and Lecania naegelii may have to be

included in Bacidina. It has been proposed to conserve Bacidina

against Woessia if these names are congeneric (Ekman 1996b).

The null hypothesis that the Ramalinaceae is monophyletic

and distinct from a monophyletic Bacidiaceae was rejected by

the parametric bootstraps under all optimality criteria

(hypothesis 4 of Table 3 ; Fig. 7). The jackknife support for

Cliostomum being the sistergroup of Ramalina is very high in

this study, 86–97% depending on optimality criterion (Figs

1–3). However, this should not be immediately interpreted to

mean that Cliostomum is the closest living crustose relative of

the Ramalinaceae, since Ramalinora glaucolivida (Lumbsch et al.

1995) was not part of the study. Accomodating the fruticose

Ramalinaceae and the basically crustose Bacidiaceae in the same

family may seem surprising, but disregarding growth habit,

other morphological characters are similar (see ‘ Introduction ’).

Other families in which the presence of several growth forms

have been confirmed by molecular evidence include the

Roccellaceae (Myllys et al. 1999) and Physciaceae (Lohtander

2000). If the status of the Bacidiaceae and Ramalinaceae as

synonyms is confirmed by further testing, this has implications

for nomenclature, since Ramalinaceae (described by Agardh

1821 : 93) is much older than Bacidiaceae as well as

Phyllopsoraceae and Biatoraceae. Interestingly, the best tree

under weighted parsimony would theoretically allow the

delimitation of a Ramalinaceae in a new, wider sense, and

the Bacidiaceae in a narrower sense without impairing the

monophyly of any of the families (as long as Bacidia incompta

is excluded from both families). The Ramalinaceae in this sense

would consequently have to include Tylothallia, Megalaria,

Cliostomum, Thamnolecania, Biatora, the Bacidia sabuletorum

and B. beckhausii groups, and at least parts of Lecania in

addition to Ramalina and possibly other fruticose genera now

referred to the Ramalinaceae that were not part of this

investigation. However, in such a classification it would be

exceedingly difficult to tell the families apart on morphology.

Synonymizing Ramalinaceae and Bacidiaceae then seems like a

more attractive alternative.

A few additional observations emanate from this data set,

but without having been properly treated in a framework of

phylogenetic hypothesis testing. First of all, the data does not

lend support to the notion that Thamnolecania may be

congeneric with Lecania (Hafellner 1984). Biatora vernalis, the

type species of Biatora, forms a monophyletic group together

with the Bacidia beckhausii group (including Catillaria globulosa)

with high jackknife support (74–88% depending on optimality

criterion ; Figs 1–3). On the other hand, B. sphaeroides, which

is sometimes treated as a member of Biatora (e.g. Coppins

1992), is not immediately associated with B. vernalis. This is

congruent with the idea laid out by Printzen (1995) that B.

sphaeroides, along with a few others (i.e. B. carneoalbida, B.

epixanthoides, and B. tetramera), belong in a separate genus,

Mycobilimbia. Later, Printzen & Lumbsch (2000) informally re-

included Mycobilimbia in Biatora. However, there is no real

incongruence between the tree topologies presented in that

work and the ones presented here, since the studies differ

significantly in taxon sampling. Megalaria grossa, the type

species of the genus, and M. laureri form a monophyletic

group with high jackknife support (74–94% depending on

optimality criterion ; Figs 1–3), which lends some support to

the generic circumscription of Megalaria proposed by Ekman

& Tønsberg (1996). Finally, the genus Lecania as treated, for


example, by Ekman (1996a) and Diederich & Se! rusiaux (2000),

appears to be polyphyletic. Unfortunately, material of the

type species of the genus, L. fuscella, was not available for

sequencing. All of these observations, however, need to be

properly tested with better taxon representation than was

possible here.

The most common use for the ITS rDNA region in

phylogenetics is at the interspecific level (Bridge & Hawks-

worth 1998). However, a study by Hershkovitz & Lewis

(1996) has demonstrated that ITS variation is non-random at

very deep phylogenetic levels, and that it contains conserved

motifs that can resolve even interkingdom relationships. The

study presented here demonstrates that the ITS region can aid

in resolving phylogeny at the intergeneric level within a

family of ascomycetes. There is a price attached to this goal,

however : a fairly large amount of ambiguous alignment had

to be excluded. In addition, phylogenetic reconstruction is

complicated by difficult conditions (unbalanced nucleotide

substitution rates, rate heterogeneity among nucleotide sites)

and rather poor resolution (many short internal branches, low

branch support). Furthermore, taxon sampling is still rather

uneven. The representation of taxa in this study that have

been referred to Bacidia, Bacidina, and Toninia is fairly good.

Other groups are still poorly sampled, particularly members of

potentially ‘basal ’ taxa, (e.g. Lecania, Biatora, Phyllopsora, and

the Ramalinaceae auct.). Before any definite conclusions on the

phylogeny of the Bacidiaceae can be drawn, and the

accompanying nomenclatural novelties can be proposed, more

DNA sequence data from more taxa is needed.

ACKNOWLEDGEMENTS

I am indebted to Christian Printzen for providing me with valuable comments

on the manuscript, to O$ rjan Fritz, Ha/ kon Holien, Per Johansson, Thomas

Johansson, Tommy Knutsson, Mark Seaward, and the curators of AAS, BG,

MIN, and O for providing me with freshly collected herbarium material for

DNA sequencing, and to Mats Wedin for letting me use his Sphaerophoraceae

sequences before they were published in GenBank. I gratefully acknowledge

the staff at the Sequencing Facility at the University of Bergen. I thank the

Research Council of Norway for financial support, and Dag E. Helland for

providing me with space and facilities in his laboratory.

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