Mitochondrial genomes from the lichenized fungi Peltigera membranacea and Peltigera malacea: Features and phylogeny
Mitochondrial genomes from the fungal partners of two terricolous foliose lichen symbioses, Peltigera membranacea and Peltigera malacea, have been determined using metagenomic approaches, including RNA-seq. The roughly 63 kb genomes show all the major features found in other Pezizomycotina, such as unidirectional transcription, 14 conserved protein genes, genes for the two subunit rRNAs and for a set of 26 tRNAs used in translating the 62 amino acid codons. In one of the tRNAs a CAU anticodon is proposed to be modified, via the action of the nuclear-encoded enzyme, tRNA Ile lysidine synthase, so that it recognizes the codon AUA (Ile) instead of AUG (Met). The overall arrangements and sequences of the two circular genomes are similar, the major difference being the inversion and deterioration of a gene encoding a type B DNA polymerase. Both genomes encode the RNA component of RNAse P, a feature seldom found in ascomycetes. The difference in genome size from the minimal ascomycete mitochondrial genomes is largely due to 17 and 20 group I introns, respectively, most associated with homing endonucleases and all found within protein-coding genes and the gene encoding the large subunit rRNA. One new intron insertion point was found, and an unusually small exon of seven nucleotides (nt) was identified and verified by RNA sequencing. Comparative analysis of mitochondrion-encoded proteins places the Peltigera spp., representatives of the class Lecanoromycetes, close to Leotiomycetes, Dothidiomycetes, and Sordariomycetes, in contrast to phylogenies found using nuclear genes.
Mitochondrial genomes from the lichenized fungi Peltigera
membranacea and Peltigera malacea: Features and phylogeny
Basil Britto XAVIER
, Vivian P. W. MIAO
ıas O. J
Olafur S. ANDR
Department of Life and Environmental Sciences, University of Iceland, 101 Reykjav
Department of Microbiology and Immunology, University of British Columbia, Vancouver, Canada
Received 14 October 2011
Received in revised form
20 April 2012
Accepted 21 April 2012
Available online 5 May 2012
Mitochondrial genomes from the fungal partners of two terricolous foliose lichen symbio-
ses, Peltigera membranacea and Peltigera malacea, have been determined using metagenomic
approaches, including RNA-seq. The roughly 63 kb genomes show all the major features
found in other Pezizomycotina, such as unidirectional transcription, 14 conserved protein
genes, genes for the two subunit rRNAs and for a set of 26 tRNAs used in translating the
62 amino acid codons. In one of the tRNAs a CAU anticodon is proposed to be modiﬁed,
via the action of the nuclear-encoded enzyme, tRNA Ile lysidine synthase, so that it recog-
nizes the codon AUA (Ile) instead of AUG (Met). The overall arrangements and sequences of
the two circular genomes are similar, the major difference being the inversion and deteri-
oration of a gene encoding a type B DNA polymerase. Both genomes encode the RNA com-
ponent of RNAse P, a feature seldom found in ascomycetes. The difference in genome size
from the minimal ascomycete mitochondrial genomes is largely due to 17 and 20 group I
introns, respectively, most associated with homing endonucleases and all found within
protein-coding genes and the gene encoding the large subunit rRNA. One new intron inser-
tion point was found, and an unusually small exon of seven nucleotides (nt) was identiﬁed
and veriﬁed by RNA sequencing. Comparative analysis of mitochondrion-encoded proteins
places the Peltigera spp., representatives of the class Lecanoromycetes, close to Leotiomycetes,
Dothidiomycetes, and Sordariomycetes, in contrast to phylogenies found using nuclear genes.
ª 2012 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.
Mitochondrial (mt) DNA has been used extensively in evolu-
tionary and population studies of all types of eukaryotes and
is usually inherited in a uniparental manner (Galtier et al.
2009). Analysis of mt DNA has proven useful in determining
population structures and phylogenies of fungi, ﬁrst by using
length polymorphisms of particular sections, later by se-
quencing sections between highly conserved PCR priming
sites, and more recently by sequencing of whole mt genomes
(Anderson & Kohn 1998). Mt DNA accumulates mutations
faster than nuclear DNA, making it more suitable for differen-
tiating closely related organisms, and part of the mt cox1 gene
has been suggested as a universal ‘barcode’ for rapid identiﬁ-
cation of eukaryotic organisms (Hebert et al. 2003). Informa-
tion from mt DNA can be compared to that from nuclear
DNA from the same fungal cells, and in the case of lichenized
fungi (mycobionts) that are found in nature only as symbionts
with green algae or cyanobacteria, both types of fungal phy-
logenies can be compared to those of their symbionts.
* Corresponding author. Tel.: þ354 525 4627; fax: þ354 525 4069.
E-mail address: email@example.com
journal homepage: www.elsevier.com/locate/funbio
fungal biology 116 (2012) 802e814
1878-6146/$ e see front matt er ª 2012 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.
Comparisons of mt genomes may be particularly valuable in
understanding the evolution and gene function in this large
and diverse group of organisms: Lichen mycobionts, thought
to include w40 % of all ascomycetes (Nash 2008) form deeply
branching groups in the fungal kingdom (Gargas et al. 1995;
Lutzoni et al. 2001) with the greatest numbers of lichenized
species within the ascomycete class Lecanoromycetes, in the
subphylum Pezizomycotina. Currently there are mt genomes
from over 90 fungal species in GenBank, mostly from ascomy-
cetes and basidiomycetes. Although lichens have been stud-
ied using mt DNA markers (Nelsen et al. 2009; Werth 2010),
no lecanoromycete mt genome has been published so far.
The genus Peltigera includes about 90 species of mainly ter-
ricolous foliose lichens with widespread distribution. They
have been the subject of many studies ranging from biogeog-
raphy, evolution, and taxonomy, ecophysiology to symbiosis
and population biology. In addition to the mycobiont, all Pelti-
gera have a cyanobacterial symbiont (Nostoc sp.) and some
species also have a chlorophyte alga as a major photobiont;
in such cases the Nostoc is conﬁned to small spots (cephalo-
dia). Besides photosynthesis, the Nostoc symbiont can carry
out nitrogen ﬁxation and thus Peltigera lichens channel both
carbon and nitrogen into the ecosystem.
Here we p resent an analysis and comparative study of
the fungal mt genomes from Peltigera membranacea and Pel-
tigera malacea of the family Peltigeraceae in the class Leca-
noromycetes. P. membranacea is ubiquitous in mixed
birchwood communities, the climax community of preva-
lent lowland lavaﬁelds and glacioﬂuvial plains in Iceland.
For comparison and stronger inference of common results
we used a g eographically distant sample of another spe-
cies, P. malacea, which has similar major characters, but is
distant from P. membranacea within the genus when rRNA
sequences are compared (Miadlikowska & Lutzoni 2004).
Since Peltigera mycobionts have proven r efractory to culti-
vation att empts (E. Stocker and M. Grube, pers. comm.),
the mt genomes were assembled from data generated by
massive parallel sequencing of natural metagenomic i so-
lates represented by whole thalli from ﬁeld collections.
Transcriptomics (RNA-seq) data were also used in add ition
to the DNA sequence data for annotation and e xpress ion
These ﬁrst complete mt genomes from lichens, including
coding sequences for 15 conserved proteins, now makes it
possible to apply more complete molecular information to
questions such as the phylogeny of the Pezizomycotina, which
has proven difﬁcult to resolve (James et al. 2006). These mt ge-
nome sequences and further delineation of the component
genomes of the symbionts, including that of associated micro-
biota, the Nostoc photobiont, and the mycobiont nucleus, will
provide a natural genetic framework for extending research
on these lichen species.
Methods and materials
Source of lichens and DNA extraction
Peltigera membranacea (accession XBB013, LA-31632 (IINH)) was
collected at Keldnagil, Reykjavik, Iceland within a 12 m span
W). Peltigera malacea (acces-
sion DB3992, UBC) was collected near Little Fort, British Co-
W). Samples were extensively
washed with water and processed using a typical procedure
for recovery of fungal genomic DNA (Sinnemann et al. 2000).
In brief, lyophilized lichen was ground into a powder under
liquid nitrogen, and lysed in 1 % lauryl sulfate or lauryl sarco-
sine; cell debris and proteins were removed by ammonium ac-
etate precipitation and by partitioning with chloroform and
RNA was digested with RNAase. Genomic DNA (>50 kb as sub-
sequently determined by agarose gel electrophoresis) was re-
covered by isopropanol precipitation and quantiﬁed on
a NanoDrop spectrophotometer (Thermo Scientiﬁc), and pro-
cessed for sequencing at commercial facilities via the Roche
454 and the Illumina/Solexa methodology. Approximately
1.76 and 1.64 gigabases of 454 data, and 1.4 and 0.3 gigabases
of Illumina data, were obtained for P. membranacea and P. mal-
Mt genome assembly and annotation, nuclear genes
After an initial Newbler assembly of the 454 reads, contigs
having high homology with fungal mt genomes and high cov-
erage were sorted out and their component reads reassembled
using the MIRA assembler (Chevreux et al. 1999). Subsequent
assembly and annotation were performed using CLC Geno-
mics Workbench software (CLC bio, Denmark), an integrated
platform for analyzing and visualizing next generation se-
quencing data. Illumina reads were used to resolve uncer-
tainties due to homopolymer runs in 454 reads. The average
fold coverage with 454 reads was 267 and 372, and with Illu-
mina reads 47 and 5, for Peltigera membranacea and Peltigera
The genome assemblies were validated in two ways. Illu-
mina mate-pair sequences with mean insert sizes of 1950 bp
and 3600 bp were mapped onto the mt genome. The sequence
pairs were distributed evenly throughout the genome and no
discrepancies were found. The TAR2 region in P. membranacea
and the cox2ernpB regions in both mt genomes were veriﬁed
by PCR and Sanger sequencing.
For annotation of the mt DNA, the BLAST s uite of tools
(Altschul et al. 1990) was used for searches i n GenBank and
comparisons to known fungal mt sequences and identiﬁca-
tion of repeats. RNA weasel (Lang et al. 2007)wasusedfor
identiﬁcation of tRNA genes, group I introns, rns,andrnpB
(including new annotations of rnpB and rps3 in several pub-
lished mt genomes, see Table 1). The large subunit ribo-
somal RNA gene (rnl ) was ann otated based on homology
to the closest rela tives in GenBank and f rom RNA-seq reads
in P. membranacea (below). Single nucleotide polymorphisms
(SNPs) were determined in the Illumina d ata with CLC
Genomics Workbench using the standard quality ﬁlter and
a minimum setting of 3 reads for e ach minor allele. Compar-
ative physical maps of the two genomes were constructed
using the CG View online server (Fig 1)(Grant & St othard
The nuclear genes, rpb1 and tilS, were found by BLAST
searches of the draft assemblies of P. membranacea and P. mal-
acea metagenomes (unpublished), and annotated using RNA-
Mitochondrial genomes from the lichenized fungi Peltigera membranacea and Peltigera malacea 803
Table 1 e Fungal species, genes, mt genomes, GenBank accession numbers.
Species Class mt features Nuclear proteins
GC % Protein
ORF tRNAs dpoB rnpB rps3 cox1 (bp) Cox1
TilS accession Rpb1 accession
Peltigera membranacea Lecanoromycetes 62.8 27 14 1 26 1 1 1 10198 631 6 JN088165 JN032401 JN596954
Peltigera malacea Lecanoromycetes 63.4 27 14 1 26 1 1 1 12690 627 9 JN088164 JN032402 JN639885
Aspergillus niger Eurotiomycetes 31.1 26 14 2 25 0 0 1
5059 566 3 NC_007445 XP_001398711 XP_001389676
Aspergillus tubingensis Eurotiomycetes 33.6 26 14 2 25 0 0 1
5073 565 4 NC_007597
Aspergillus nidulans Eurotiomycetes 33.2
25 14 0 28 0 1
4973 567 3
Epidermophyton ﬂoccossum Eurotiomycetes 30.9 23 14 4 25 0 0 1 2703 528 1 NC_007394
Paracoccidioides brasiliensis Eurotiomycetes 71.3 20 14 0 25 0 0 1 12428 710 8 NC_007935
Penicillium marneffei Eurotiomycetes 35.5 24 14 10 28 0 0 1 10027 561 7 NC_005256 XP_002144809 XP_002146907
Penicillium digitatum Eurotiomycetes 28.9 25 14 0 27 0 0 1 1775 591 0 NC_015080
Trichophyton rubrum Eurotiomycetes XP_003231777
Fusarium oxysporum Sordariomycetes 34.4 31 14 0 25 0 0 1 1593 530 0 AY945289 EGU79617
Gibberella zeae Sordariomycetes 95.5 31 14 1 28 0 0 1 16914 530 12 NC_009493 XP_384534 XP_381092
Glomerella lindemuthiana Sordariomycetes 1380 460 2 HM450146
Neurospora crassa Sordariomycetes 64.8 36 14 6 28 0 0 1 7972 557 4
Podospora anserina Sordariomycetes 100 30 13 37 27 0 0 1 24506 541 14 NC_001329 XP_001912461
Verticillium dahliae Sordariomycetes 27.2 27 14 0 25 0 0 1 1623 540 0 NC_008248
Verticillium albo-atrum Sordariomycetes XP_003002835
Mycosphaerella graminicola Dothideomycetes 43.9 31 14 8 27 0 1
0 2010 669 0 NC_010222 EGP84297 EGP87916
Phaeosphaeria nodorum Dothideomycetes 49.7 29 12 3 27 0 0 1 1668 556 0 NC_009746 XP_001801796
Phialocephala subalpina Leotiomycetes 43.7 27 15 4 28 0 0 1 1722 573 0 JN031566
Sclerotinia sclerotiorum Leotiomycetes XP_001589873 XP_001593006
Coprinopsis cinerea Agaricomycetes XP_001828577
Moniliophthora perniciosa Agaricomycetes 109 31 14 54 26 1 0 1 8929 533 6 NC_005927
Schizophyllum commune Agaricomycetes 49.7 21 14 5 27 0 0 1 1583 527 0 NC_003049 XP_003037684
Mortierella verticillata Zygomycetes 58.7 27 14 8 26 0 1 1 5811 534 3 NC_006838
Rhizopus oryzae Zygomycetes 54.1 26 14 9 24 0 1 0 4994 505 3 NC_006836
Glomus intraradices Glomeromycetes 70.6 37 14 0 26 0 0 0 7990 529 12 NC_012056
c Identiﬁed in this study using the deposited genomes: M. graminicola rnpB (mt24998-24821), A. tubingensis rps3 (mt16536-17753), A. niger rps3 (mt16403-17620).
d Identiﬁed in this study using A. nidulans mt genome: rnpB (mt20776-20962), rps3 (mt2957-4189).
804 B. B. Xavier et al.
RNA analysis of Peltigera membranacea
Total RNA was isolated using the Totally RNA kit (Ambion)
from thalli, apothecia, and rhizines from naturally moist,
fresh material collected directly into RNAlater solution
(Ambion) to inactivate RNAases, and polyA mRNA was iso-
lated, fragmented, and reverse transcribed according to
Illumina protocols (see e.g. http://www.illumina.com/applica-
tions/sequencing/rna.ilmn). After ligating adaptors the size
range 250e300 bp was selected via agarose gel electrophoresis
and sequenced on a Genome Analyzer IIx (Illumina). RNA se-
quences from different tissues and seasons were combined
and mapped to the P. membranacea mt genome using CLC Ge-
nomics Workbench. RNA sequence mapping served two pur-
poses: Conﬁrmation of gene candidate annotation, and
identiﬁcation of extragenic, transcriptionally active regions
(TAR), deﬁned as any tract of >150 nucleotides (nt) with a min-
imum coverage of at least four reads/nt, at least 150 nt away
from an annotated gene (Bruno et al. 2010). The RNA structures
and their minimum free energy were predicted using the
RNAfold program (Zuker & Stiegler 1981; McCaskill 1990;
Hofacker et al. 1994).
For phylogenetic analysis, protein sequences from Peltigera
membranacea, Peltigera malacea and other fungi were aligned
using StatAlign (Nov
ak et al. 2008) and regions with unambig-
uous alignments were considered for further analysis. Trees
were built and assessed using three different methods, maxi-
mum parsimony (Swofford 1993; Tamura et al. 2011), maxi-
mum likelihood (PhyML 3.0) (Guindon & Gascuel 2003;
Guindon et al. 2010) and Bayesian approaches (MrBayes v3.2)
(Huelsenbeck & Ronquist 2001). A model generator was used
to determine the optimal substitution model WAG (Whelan
& Goldman 2001) using the Akaike Information Criterion, the
Bayesian Information Criterion and hierarchical likelihood-
ratio tests, with a g-distribution of four categories (Keane
et al. 2006). Maximum parsimony bootstrap was performed
with 10000 replicates where the initial trees were obtained
Fig 1 e Peltigera mt genomes. Circular gene map representing the mt DNA of P. membranacea. All genes are transcribed
clockwise (feature labels are afﬁxed to the 5
end), dpoB* is a pseudogene. Genes encoding proteins and ribosomal RNA are
indicated by red and yellow boxes, respectively; tRNA genes, in green, are speciﬁed by standard one-letter amino acid code
and anticodon. Introns and HEGs are shown in blue and light blue, and extragenic, TARs and an unidentiﬁed ORF are in dark
green and sky blue, respectively. The pink inner ring shows regions of identity with the genome of P. malacea; dpoB is only
found in P. malacea. Numbers inside the map are in kb.
Mitochondrial genomes from the lichenized fungi Peltigera membranacea and Peltigera malacea 805
by random addition of sequences. Maximum likelihood boot-
strap (Felsenstein 1985) support was calculated with 10000
replicates and branch support for trees was also examined
by statistical analysis using the approximate likelihood-ratio
test (aLRT) (Anisimova & Gascuel 2006). The Bayesian analysis
was replicated up to 5 million generations sampling trees ev-
ery 1000 generations, with a burn-in value of 20 000 to improve
effective sample sizes to >800. Stability of the log-likelihood
values was assessed by visualization in Tracer v1.5
(Rambaut & Drummond 2009).
Results and discussion
General features of the mt genomes
The mt genomes of Peltigera membranacea and Peltigera malacea
can be represented as circles with nearly the same number of
basepairs, 62 785 and 63 363, respectively (Fig 1). The genome
sizes, low G þ C content (w27 %) and numbers of genes and
open reading frames (ORFs) are within the range commonly
observed among fungi (Table 1). Each of the Peltigera mt ge-
nomes has 15 predicted ORFs for known protein coding genes:
three for ATP synthase F
subunits (encoded by atp6, atp8, and
atp9), three for cytochrome c oxidase subunits (cox1, cox2, and
cox3), seven for nicotinamide adenine dinucleotide ubiqui-
none oxidoreductase subunits (nad1, nad2, nad3, nad4, nad4L,
nad5, nad6) and one each for cytochrome oxidase b (cytb),
and ribosomal protein subunit 3 (rps3). The genomes share
98 % identity in the coding regions, and 77 % overall, with di-
vergence associated primarily with intronic regions ( Fig 1).
Both genomes have a full set of tRNA genes, as well as genes
for the large (rnl ) and small (rns) ribosomal subunit RNAs,
the RNA component of mt RNAse P (rnpB), an ORF with no sig-
niﬁcant match in GenBank, and a gene or pseudogene for
a B-type DNA polymerase (dpoB)(Table 1). As in most ascomy-
cetes, the Peltigera mt genes are all oriented in the same direc-
tion. Since the genomic DNA was not clonal, considerable
single nucleotide sequence polymorphism was detected,
5 % at 77 sites in P. membranacea, and 935 sites in P. malacea.
These polymorphisms are the subject of a separate study. The
mt genome accessions in GenBank are JN088165 for P. membra-
nacea and JN088164 for P. malacea.
The mt genes of both Peltigera membranacea and P. malacea con-
tain 4480 codons (not counting dpoB and rps3). Codon usage is
similar in both species and, consistent with the transmem-
brane localization of most mt proteins, codons for the nonpo-
lar amino acids Leu, Phe, and Ile are the most abundant
(Supplementary Table 1). As in other mt genomes, there is
a strong bias for the use of A and U in synonymous codons:
84 % of third positions are A or U, and 62 % of the Leu codons
are UUA. The genes cytb, cox2, and nad6 appear to use GUG,
AUU, and AUA, respectively, as initiation codons. Stipulation
of an AUG codon in these cases would call for subsequent
RNA editing, deletion of highly conserved amino acids, or
overlap with preceding genes, resulting in a long N-terminal
extension. These alternatives are not supported by transcript
mapping (Fig 2). GUG is well established as an alternative ini-
tiation codon in bacteria and the ﬁrst position anticodon C in
mt initiator tRNA appears to allow ﬂexible pairing, permitting
binding to AUA and AUU codons in addition to AUG
(Bullerwell et al. 2003). All genes have a UAA stop codon, ex-
cept for cox2, cox3, and dpoB which have UAG.
Fig 2 e P. membranacea mt transcriptome. Mapped reads are shown below the annotated mt genome (see text). Discontin-
uous line indicates RNA sequence coverage. The 35 base RNA-seq reads give a qualitative picture of what genome sequences
are transcribed and found in RNA molecules in the mitochondrion and a semi-quantitative indication of the RNA levels. Two
regions with no identiﬁed protein or tRNA coding produce deﬁned RNA products present at a high level (TAR1 and TAR2).
Intronic sequences are absent or at low levels. rRNAs are found at very high levels but the method does not detect RNA
molecules smaller than w150 bases (e.g. tRNAs). RNA sequences spanning splice joints are detected but not presented in this
806 B. B. Xavier et al.
The Peltigera membranacea and Peltigera malacea mt genomes
each encode 26 tRNAs that specify all 20 common amino
acids. There are two distinct tRNAs for amino acids that
have six codons (Arg, Leu, Ser), and two distinct tRNAs for
Tyr in each species. Both Peltigera species also have three dif-
ferent tRNA genes with CAU anticodons, normally specifying
Met: One encodes the elongator tRNA
, another the initiator
, but the third encodes a tRNA
. The Peltigera mito-
chondria use all three Ile codons (AUU, AUC, and AUA), and
a tRNA with a GAU anticodon that should pair with the Ile co-
dons AUC and AUU by wobble pairing (Crick 1966) was identi-
ﬁed, but no tRNA gene with a UAU anticodon was detected for
pairing to AUA (an unmodiﬁed version of this anticodon
should also pair with the Met codon AUG). The third tRNA
gene with a CAU anticodon therefore appeared a likely candi-
date for the role of decoding AUA, as it conforms to the deter-
minants of a tRNA
described in bacterial genomes (Silva et al.
2006), and presumably could be restricted to decoding AUA by
a lysidine modiﬁcation (Takemoto et al. 2009) as has been
demonstrated in potato mitochondria (Weber et al. 1990). A
similar situation seems to exist in other fungal mt genomes
where there appears to be an excess of tRNA genes with
CAU anticodons and none with UAU anticodons. Phylogenetic
analysis places these four tRNA types on distinct branches
(Fig 3A). Consistent with this model, nuclear homologs of
tilS, encoding tRNAIle lysidine synthetase necessary for the
lysidine modiﬁcation (Suzuki & Miyauchi 2010), can be found
in P. membranacea and P. malacea, as well as in representatives
from major fungal groups (Fig 3B; Table 1). Their deduced pro-
tein sequences have clear homology to bacterial TilS and con-
servation of key residues (Soma et al. 2003). In contrast to the
mitochondrion, the cytoplasm of eukaryotes makes use of
with inosine in the ﬁrst position of the anticodon,
allowing decoding of all three Ile codons.
The Peltigera mt rnl genes contain a 2621 bp intron with an em-
bedded ORF of 473 amino acids with conserved residues of the
Rps3 protein (Supplementary Fig 1). The group I intron splicing
) were veriﬁed
by RNA sequencing and are identical to those previously
reported among ﬁlamentous ascomycetes. Although it has
not always been speciﬁcally annotated ( e.g. Aspergillus tubin-
gensis, Aspergillus niger, Table 1), the rps3 gene appears to be
common and strongly conserved in all major fungal groups
(Bullerwell et al. 2000) including the Pezizomycotina, supporting
the hypothesis that this arrangement is ancestral to the
Fig 3 e Fungal mt Met and Ile tRNAs and TilS. A) The phylogenetic tree of mt Met and Ile tRNAs was constructed using the
maximum likelihood method implemented in the PhyML program (v3.0 aLRT) using the substitution model HKY85. Reli-
ability of internal branching was assessed using the aLRT test and the likelihood ratios are shown above the branches (>0.90
indicates signiﬁcant likelihood). Sequences marked as trnI CAU were originally annotated as trnM in GenBank. B) Selected
regions from the N-terminal half of tRNA
lysidine synthetase containing recognition and catalytic functions were aligned.
Conserved amino acids are highlighted and residues putatively involved in the recognition of ATP, tRNA, and
(Nakanishi et al. 2005) are marked with ﬁlled stars, open stars and triangles respectively. For full names of fungi and ac-
cession numbers see Table 1. E. coli TilS accession: BAA77863.
Mitochondrial genomes from the lichenized fungi Peltigera membranacea and Peltigera malacea 807
lineage. The placement of rps3 within the rnl gene and their
co-transcription has been suggested to enhance expression
of rps3 (Sethuraman et al. 2009), but as gauged by RNA se-
quencing, the transcript level of rps3 in Peltigera membranacea
is lower than that of the other protein coding genes in the mi-
tochondrion (Fig 2).
Respiratory chain genes (atp, cyt, cox, and nad)
The majority of the proteins encoded by mt genes have a role
in the respiratory chain. ATP synthase is a large protein com-
plex that transforms the energy of the electromotive force into
high energy bonds in ATP. Most of its subunits are encoded by
nuclear genes, but several subunits are usually encoded by the
mitochondria of fungi and other eukaryotes lineages, e.g. atp9
is present in most fungal mitochondria but absent from Podo-
spora anserina (Cummings et al. 1990). The atp9 gene, as well as
the atp6 and atp8 genes, are present in both Peltigera spp. RNA
sequencing shows that the atp9 gene is highly expressed in
Peltigera membranacea (Fig 2), with a 74 base 5
UTR and 146
UTR that presumably contribute to stability and trans-
lational efﬁciency (Chen & Dieckmann 1997).
The cox1, cox2, and cox3 genes encode three subunits of cy-
tochrome c oxidase (‘complex IV’), the last enzyme in the re-
spiratory electron transport chain of mitochondria, while
cytb encodes a subunit of respiratory chain complex III (Yang
et al. 2011). The most conserved genes, cox1 and cytb, are often
riddled with introns in ascomycetes (Paquin et al. 1997). In Pel-
tigera, the cox genes and cytb together host 50 % of all of the
mt introns, with cox1 bearing the greatest number: in Peltigera
malacea only 16 % of this gene is coding. Fungal Cox1 proteins
vary in length from 460 to 710 residues (Table 1), but are gen-
erally in the range of 525e550. The predicted Cox1 protein of P.
membranacea is 631 residues. The larger than average size
arises from a 3
extension that encodes the C-terminal 77
amino acids, and is consistent with transcriptomic data
(Fig 2). P. malacea similarly has an additional 73 amino acids.
The extensions are 93 % identical in amino acid sequence, in-
dicating common ancestry, but neither sequence is conserved
in other known Cox1 proteins. The extensions do not have as
strong a codon usage bias as observed in other mt genes
encoding proteins, possibly reﬂecting a recent addition. The
longest Cox1 extensions are found in Mycosphaerella gramini-
cola (141 residues) and Paracoccidioides brasiliensis (156 residues)
(Table 1), but neither have similarity to each other or to
In contrast to the plethora of non-coding intervening se-
quences and group I introns in the mt DNA that separate pro-
tein coding genes into many fragments, a gene fusion is
proposed between cytb and cox2. No canonical stop codon is
detected for cytb, no canonical start for cox2, and both coding
sequences are in the same frame. These observations suggest
the possibility of a fusion of the two genes, with the single stop
codon at the end of cox2 serving both. A preponderance of fu-
sion mRNAs (Fig 2) among transcripts for this region supports
this model in P. membranacea, although it is also consistent
with the possibility of a single message with an exceptionally
long, untranslated extension, or a polycistronic message, as
found in bacteria. Fusion proteins between cytb and cox2
have not been reported, but other fusion transcripts have
been reported from the mt genomes of eukaryotic microbes.
Analyses of proteins in Acanthamoeba castellanii revealed that
a fusion transcript (cox1 and cox2) gave rise to two products
upon translation (Lonergan & Gray 1996; Gawryluk & Gray
2010), while the possibility of a new multidomain protein
from a fusion transcript (cytb and cox3) in the dinoﬂagellate
Oxyrrhis marina remains open (Slamovits et al. 2007).
dpoB and rnpB
The dpoB gene, encoding a type B2 DNA polymerase (Dpo), is
most often found in free linear plasmids that may populate
fungal mitochondria (Grifﬁths 1995), but integrated
forms have occasionally been reported in mt genomes
(Mouhamadou et al. 2004; Formighieri et al. 2008). Dpo includes
an N-terminal domain of variable size that in some cases
codes for the terminal protein for priming plasmid replication
(Rohe et al. 1992; Laday et al. 2008), a 3
(with three conserved regions ExoI, ExoII, and ExoIII) and a C-
terminal polymerase domain (with a series of conserved ‘Pol’
regions) (Blanco et al. 1991; Rohe et al. 1992). Integrated ortho-
logs of dpoB were observed at the same locus, between nad4
and nad1, in both Peltigera spp. In Peltigera malacea, the 697
amino acid predicted Dpo contains all three conserved regions
of the exonuclease domain and most of the polymerase
domain (Pol1, Pol2a, Pol2b, Pol3, and Pol4 regions)
(Supplementary Fig 2). The translation ends shortly after the
Pol4 region, much like that of the integrated Aa-polB gene of
Agrocybe aegerita (Bois et al. 1999), without a clear Pol5 region
that may be more typical of other classes of Dpo proteins. In
Peltigera membranacea, dpoB is represented as a pseudogene
(dpoB*) on the opposite strand. The main 1461 nt section of
dpoB* is disrupted by three stop codons. Its hypothetical trans-
lation (reading through the stops) includes the three Exo mo-
tifs, Pol1 and Pol2a regions. No relict of the Pol2b region is
evident in the adjacent sequence, but an ORF immediately
downstream on the same strand contains another fragment
of dpoB*, with a 262-residue predicted translation that includes
Pol3 and Pol4 regions. There are no clear footprints (e.g. re-
peats) in the regions surrounding the dpoB genes to indicate
recent plasmid integration events, as have been deduced in
other fungi (Formighieri et al. 2008). Analyses of geographically
well-separated samples of P. malacea and P. membranacea by
PCR mapping indicate that the rearrangements in this region
of the mt DNA are species-speciﬁc (unpublished), even if their
origins are unclear. Interestingly, the closest known relative
of the Peltigera dpoB gene resides in a basidiomycete,
Moniliophthora roreri, (55 % identity to GenBank accession
YP004347432, and Supplementary Fig 3). This suggests an inci-
dence of horizontal gene transfer in a common ancestor, fol-
lowed by rearrangement in one lineage during or after
divergence of the two Peltigera spp.
Mitochondrially-encoded rnpB genes are irregularly distrib-
uted among fungi (Seif et al. 2003) and not always annotated
(e.g. Mycosphaerella graminicola and Aspergillus nidulans,
Table 1). They have been reported in a few ﬁlamentous and
yeast-form ascomycetes, zygomycetes (Seif et al. 2005) and
most recently, possibly in the mycorrhizal symbiont, Glomus
intraradices (Lee & Young 2009). The rnpB genes in Peltigera
are AT-rich, similar in size (75.2 % and 75.8 % AT, and 245
808 B. B. Xavier et al.
and 249 nt, respectively), and carry sequences corresponding
to conserved regions CR1 and CRV (Chen & Pace 1997). They
are ﬂanked downstream by a tRNA gene as in other fungi,
but are ﬂanked upstream not by tRNA genes, as is typical,
but by cox2 (Fig 1), an arrangement described previously only
in Schizosaccharomyces ﬁbuligera (Seif et al. 2003).
Group I introns and homing endonucleases
Group I introns are mobile genetic elements expressing self-
splicing RNAs (ribozymes) varying from 142 bp to >3000 bp.
They interrupt a wide range of organelle genes and nuclear
rDNA genes in fungi, algae and many other unicellular eukary-
otes. There are ﬁve main subgroups and several subdivisions
of group I introns, based on their RNA secondary structure
and, in contrast to introns with homing endonucleases, they
recognize and insert into their target sequences sites through
base pairing with short (4-6 nt) internal guide sequences, ribo-
zyme mediated reverse splicing and reverse transcription into
DNA (Adams et al. 2004; Haugen et al. 2005; Lang et al. 2007).
The mt introns are useful markers for population and evolu-
tion studies as they target conserved sequences (Mullineux
et al. 2010) but are less subject to strong selection and there-
fore rapidly accumulate mutations (degenerate) or may be
eliminated by precise deletion.
Homing endonucleases recognize and cleave speciﬁc DNA
sites (generally 20e40 bp) inserting sequences that include the
reading frame encoding the corresponding endonuclease.
Thus, when DNA with a homing endonuclease gene (HEG) in-
sertion enters a cell compartment containing DNA with the cor-
responding recognition site, but no HEG insertion, the DNA is
cleaved and subsequently repaired by homologous recombina-
tion to the homologous chromosome containing the HEG
(Stoddard 2011). Homing endonucleases form at least ﬁve fam-
ilies, and two of them, characterized by the amino acid se-
quences in their core nuclease domains as LAGLIDADG and
GIY-YIG types, are commonly found in mitochondria, most of-
ten associated with group I introns(Haugenet al. 2005).Position-
ing HEGs within self splicing group I introns prevents their
disrupting functional proteins, and placement of the group I in-
trone HEG combinationin highlyconserved genesequences fa-
cilitates their spread to virgin populations, even to other
species. During evolution, endonuclease function may be lost
with corresponding sequence degeneration although selection
may act to enlist the remaining protein for enhancing intron
RNA folding and splicing (Edgell et al. 2011; Stoddard 2011).
In Peltigera species, as in other fungi, the majority of mt
group I introns carry HEGs, most often for the LAGLIDADG
type enzymes. Of the 17 group I introns in Peltigera membrana-
cea, nine have LAGLIDADG sequences (two degenerate), four
have GIY-YIG sequences and four, including the intron in rnl
that carries rps3 (discussed above), lack HEGs. Similarly, of
the 20 group I introns in Peltigera malacea, 12 have LAGLIDADG
sequences, ﬁve have GIY-YIG (two of each type degenerate),
and three, including the one carrying rps3, lack HEGs. In
P. membranacea, RNAs corresponding to many of the HEGs
can clearly be detected, but other intronic RNAs are generally
found at lower levels (Fig 2).
The cox1 gene is the most common target for insertion by
group I introns but the number of integrated elements varies
widely (e.g. from none in Verticillium dahliae to 14 in Podospora
anserina among the ascomycetes, Table 1). Because insertion
of the LAGLIDADG HEGs into this gene has been observed in
diverse taxonomic groups, the positions of insertion can be
used to examine the evolutionary dynamics of intron mobility
erandon et al. 2010). There are six and nine introns with
HEGs in the cox1 genes of P. membranacea and P. malacea
at a total of ten insertion sites (Fig 4) of which all
but one, ‘AJ’ (designated in this study), are considered widely
erandon et al. 2010). Presence of a Peltigera intron
at a site also occupied by LAGLIDADG HEG orthologs in other
ascomycetes and basidiomycetes suggests that the intron-
containing state at that position is likely ancestral, e.g. orthol-
ogous HEGs from both Peltigera spp. and other fungi occur at
site ‘K’, suggesting it is ancestral among dikaryomycota
(Supplementary Fig 4). Since introns at sites ‘G’, ‘Y’, and ‘AE’
are often found in ascomycete mitochondria but absent
from P. membranacea, this is interpreted as losses from P. mem-
branacea, rather than independent gains in P. malacea. A loss
from P. malacea is likewise inferred at site ‘AD’. Insertion at
the site ‘AJ’, after the S554 codon of the cox1 gene in P. malacea
has not been reported before and is not found in P. membrana-
cea. In this case, rather than a loss, the intron is predicted to be
a recent gain, possibly of a particularly active HEG-intron com-
bination. Putative orthologs of the HEG at ‘AJ’ in P. malacea oc-
cur not only at a variety of other locations within cox1, e.g.
intron 7 (39 % identity, 60 % similarity) at position ‘U’ in G.
zeae (YP_001249330) or orf 441 in intron 5 (43 % identity, 63 %
similarity) at ‘AK’, another novel position in P. anserina
(YP_004376358.1) (see also Fig 4), but also within other mt
genes, e.g. P. anserina nad3 intronic orf403 (NP_074914.1) and
Moniliophthora roreri nad5 intron 1 (YP_004376358.1). The over-
all impression remains, however, that intron loss is more
common in P. membranacea than in the P. malacea lineage.
The largest protein-coding gene in Peltigera membranacea,
nad5, is interrupted by three introns that fragment the gene
into four parts. The second and third introns are so close
that an exon of only seven nt is predicted (Supplementary
Fig 5). Small exons are not uncommon; in Peltigera, exons of
28 bp and 33 bp are also observed in nad4L and cox1 respec-
tively. Nonetheless, small exons, such as the 3 bp exons in
cox1 in Podospora anserina (Cummings et al. 1989)orAgaricus
erandon et al. 2010), should be annotated with cau-
tion and ideally be supported by transcriptomic data, as alter-
native splicing options may also be possible. The 7 bp exon in
P. membranacea nad5, in a highly conserved region and with
the predicted splicing veriﬁed by RNA-seq data, shows that
conservation of only a few nt is necessary for splicing, as
has been inferred from structural analysis (Adams et al. 2004).
Non-coding regions: repeats and TARs
Intergenic regions or non-coding regions of the mt DNA vary
greatly among species, but may play important roles in gene
and genome function. The intergenic regions amount to
30 % and 27 % in Peltigera membranacea and Peltigera malacea re-
spectively and are rich in simple direct, inverted and tandem
Mitochondrial genomes from the lichenized fungi Peltigera membranacea and Peltigera malacea 809
repeats which can mediate genome evolution, e.g. by errors in
recombination producing duplications, inversions and dele-
tions (Alexander et al. 2010; Stone et al. 2010).
In P. membranacea and P. malacea there are ﬁve and four
pairs of direct, perfect or near-perfect repeats from 76 bp to
205 bp (Supplementary Table 2). Two of these pairs are at
equivalent positions in the two mt genomes. The ﬁrst pair is
w124 bp in both species, and is in an rnl intron, as well as
1250 bp downstream in the highly conserved rnl sequence.
The partners of the other pair, 82 bp in P. membranacea and
129 bp in P. malacea, are in the intergenic region between
nad6 and trnK, and 10 kb away in the intergenic region be-
tween cox2 and rnpB. Although the repeat pairs (other than
in rnl ) have >80 % identity within each species, they show
no signiﬁcant homology between the species. Thus there ap-
pears to be selection for maintaining these repeats, but not
their sequence per se. Repeats can be found in many other as-
comycete mt genomes but they do not appear to be a generally
conserved feature (unpublished observations).
It has recently been shown that fungal mitochondria pro-
duce a variety of RNA molecules derived from intergenic re-
gions, and that the concentration of these RNAs is similar to
that of the protein coding RNAs (Bruno et al. 2010). Two such
transcriptionally active regions (TARs) were detected by
RNA-seq analysis (Fig 2). Each produces prominent RNAs
that are predicted to form energetically stable structures sim-
ilar to those in prokaryotes (Weinberg et al. 2009). The 800 nt
TAR1 RNA (DG ¼193 kcal mol
) appears highly conserved
(91 % identity) between the two Peltigera spp., but the 1284 nt
TAR2 RNA (D ¼392 kcal mol
) with a triple w50 nt tandem
repeat embedded, does not have a close homolog in P. malacea.
The TAR RNAs are in many ways similar to short stable RNAs
that have been found in bacteria in recent years, most without
functional correlation, but some are thought to be involved in
regulation or RNA processing (Storz et al. 2011).
The two Peltigera mt genomes are identical in the number and
order of genes, except for the inversion of dpoB, and differ little
in their coding sequences (Fig 1). The arrangement of mt genes
in the two Peltigera spp. was compared to that of representa-
tives with the same basic gene set from four fungal phyla
(Supplementary Fig 6). Approximately the same number of
rearrangements separates the Peltigera mt genomes from
those of other Pezizomycotina classes, with more differences
observed as the species are further apart in the phylogenetic
tree, as observed in other studies (Pantou et al. 2008). Two fea-
tures common to the Pezizomycotina mt genomes, rps3 embed-
ded in an intron within rnl and a one base overlap of the nad4L
and nad5 reading frames are also found in other phyla and
may therefore be ancestral.
A phylogenetic analysis of 14 concatenated mt protein se-
quences from the two Peltigera spp. and 12 representative fun-
gal species (Table 1) using maximum likelihood, maximum
parsimony and Bayesian methods returned the same well-
supported tree topology with robust bootstrap and posterior
probability values (Fig 5a). The same tree topology was obtained
using individual proteins Nad5 and Cox1 (data not shown). The
genus Peltigera is a coherent group according to analysis of nu-
clear ribosomal RNA genes (Miadlikowska & Lutzoni 2004)and
the two representative mt genomes described here show this
group separating deeply from those of other published se-
quences. Using the estimate of approximately 200 myr for the
separation of Eurotiomycetes and Sordariomycetes (Taylor &
Berbee 2006), and an even rate of amino acid substitution, the
two Peltigera species may have diverged roughly 10 myrs ago.
The analysis based on 14 mt proteins, as well as another
analysis based on the 12 mt proteins common to a larger set
of taxa (Supplementary Fig 7), show a clear divergence of Leca-
noromycetes from Eurotiomycetes, in contrast to studies based
Fig 4 e Group I introns in cox1. Insertion sites for introns carrying a LAGLIDADG HEG (black triangles) or a GIY-YIG HEG (white
triangle) in P. malacea and P. membranacea (above and below the sequence, respectively) are shown relative to the derived
Cox1 amino acid sequence of P. membranacea. Reference amino acid positions are numbered above each tract, and insertion
sites are labeled as in F
erandon et al. 2010. Insertion site AJ is novel. Insertion site AK (black arrow) is novel and not found in
the Peltigera mt genomes, but the equivalent position in P. anserina is used by a presumptive ortholog of the intron at AJ in
810 B. B. Xavier et al.
on other nuclear and mt markers in which Lecanoromycetes ap-
pear as a sister group to Eurotiomycetes (Miadlikowska &
Lutzoni 2004; James et al. 2006; Nelsen et al. 2009). Further-
more, Lecanoromycetes appears closer to Leotiomycetes and
Dothidiomycetes than to Sordariomycetes. The mt protein results
partly agree with a more extensive phylogeny of Pezizomyco-
tina made with a set of ﬁve nuclear genes (Spatafora et al.
2006), but differ in the placement of the single representatives
from Dothidiomycetes and Leotiomycetes. When the full se-
quence of the nuclear encoded RNA polymerase large subunit
(Rpb1) is used for analysis, a well-supported tree topology
(Fig 5B) similar to that previously found with nuclear encoded
proteins is generated (James et al. 2006). This incongruence,
found using maximum likelihood, maximum parsimony and
Bayesian methods, may reﬂect biological processes such as
horizontal gene transfer, introgression or incomplete lineage
sorting leading to inconsistencies in tree-building (Galtier &
Daubin 2008; Wiens et al. 2010). Interestingly, a similar incon-
gruence between nuclear and mt phylogenies has been de-
scribed for the Enterographa group of lichens within the
Arthoniales (Ertz et al. 2009).
Metagenomic and next generation sequencing platforms are
increasingly practical tools for a variety of detailed investiga-
tions of microorganisms in natural ecosystems, including li-
chen symbionts that are generally recalcitrant to pure
culture in vitro (Magain et al. 2010; Bates et al. 2011; Schneider
et al. 2011). The mt genomes of the mycobionts of Peltigera
membranacea and Peltigera malacea, selectively assembled after
respective whole thallus DNA sequencing and annotated with
support of transcriptomic data from thalli of P. membranacea,
were similar to those of related non-symbiotic fungi, and dif-
fered from each other primarily in their population of group I
introns and the condition of a plasmid-derived dpoB gene.
These results suggest that there are few options for speciﬁc
adaptations to a symbiotic state of existence at the level of
organellar genome structure in terms of the basic suite of
genes, but that the mt genomes of lichenized fungi are none-
theless dynamic in other respects. The mt genome of P. mem-
branacea seems to have been more effective at eliminating
invasive mobile elements: intron loss is more common in
this lineage, and the integrated dpoB sequence has been
eroded. The Peltigera HEG at ‘AJ’ as well as the DpoB sequences
show considerable similarity to equivalent elements in the ba-
sidiomycete, Moniliophthora roreri, raising questions about hor-
izontal movement of genes between ascomycete and
basidiomycete mitochondria that potentially complicate phy-
logenetic analysis (Brown 2003; Marcet-Houben & Gabald
2010). Concatenated whole mt genome protein sequences
were used to establish a well-supported phylogeny within
the Pezizomycotina, but a contrasting phylogeny was also con-
structed from a conserved nuclear protein. It will be interest-
ing to determine, with the growing number of fungal mt
genomes available through various sequencing projects,
whether this disparity reﬂects fundamental differences in
the evolution of mt and nuclear protein coding genes.
This research was funded by the Icelandic Research Fund. The
authors are grateful to Dr. B.F. Lang for reviewing the rnpB
data, Dr. M. Grube for useful discussion and an anonymous re-
viewer for helpful comments. We also thank deCODE Genetics
and Droplaug N. Magn
Olafur Þ. Magn
usson for technical
Appendix A. Supplementary material
Supplementary material related to this article can be found
online at doi:10.1016/j.funbio.2012.04.013.
Fig 5 e Phylogenetic trees based on mitochondrion encoded proteins and Rpb1, a nuclear encoded protein. A) Fourteen proteins
encoded by the mitochondria of P. membranacea and P. malacea were concatenated and aligned to corresponding sequences
from representatives of other classes of fungi (Table 1). Branch support value, in percent, indicates Bayesian posterior proba-
bility, followed by maximum likelihood bootstrap value if different. Branches are drawn to scale, with the bar indicating 0.2
substitutions per site. B) The complete protein sequences of Rpb1 from the species in A) or their closest relatives available in
GenBank, plus Phaeosphaeria nodorum (Table 1), were aligned and used for building a maximum likelihood tree. Branch support
values as in A) with the bar indicating 0.4 substitutions per site. B [ Basidiomycotina;E[ Eurotiomycetes;Lec[ Lecanoromycetes;
L [ Leotiomycetes;D[ Dothidiomycetes;S[ Sordariomycetes. For full names and accession numbers see Table 1.
Mitochondrial genomes from the lichenized fungi Peltigera membranacea and Peltigera malacea 811
Adams PL, Stahley MR, Gill ML, Kosek AB, Wang J, Strobel SA,
2004. Crystal structure of a group I intron splicing intermedi-
ate. RNA 10: 1867e1887.
Alexander RP, Fang G, Rozowsky J, Snyder M, Gerstein MB, 2010.
Annotating non-coding regions of the genome. Nature Reviews
Genetics 11: 559e571.
Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ, 1990. Basic
local alignment search tool. Journal of Molecular Biology 215:
Anderson JB, Kohn LM, 1998. Genotyping, gene genealogies and
genomics bring fungal population genetics above ground.
Trends in Ecology and Evolution 13: 444e449.
Anisimova M, Gascuel O, 2006. Approximate likelihood-ratio test
for branches: a fast, accurate, and powerful alternative. Sys-
tematic Biology 55: 539e552.
Bates ST, Cropsey GWG, Caporaso JG, Knight R, Fierer N, 2011.
Bacterial communities associated with the lichen symbiosis.
Applied and Environmental Microbiology 77: 1309e1314.
Blanco L, Bernad A, Blasco MA, Salas M, 1991. A general structure
for DNA-dependent DNA-polymerases. Gene 100:27e38.
Bois F, Barroso G, Gonzalez P, Labarere J, 1999. Molecular cloning,
sequence and expression of Aa-polB, a mitochondrial gene
encoding a family B DNA polymerase from the edible basid-
iomycete Agrocybe aegerita. Molecular and General Genetics 261:
Brown, 2003. Ancient horizontal gene transfer. Nature Reviews
Genetics 4: 121.
Bruno VM, Wang Z, Marjani SL, Euskirchen GM, Martin J,
Sherlock G, Snyder M, 2010. Comprehensive annotation of the
transcriptome of the human fungal pathogen Candida albicans
using RNA-seq. Genome Research 20: 1451e1458.
Bullerwell CE, Burger G, Lang BF, 2000. A novel motif for identi-
fying Rps3 homologs in fungal mitochondrial genomes. Trends
in Biochemical Sciences 25: 363e365.
Bullerwell CE, Leigh J, Seif E, Longcore JE, Lang BF, 2003. Evolution
of the fungi and their mitochondrial genomes. In: Dilip KA,
George GK (eds), Applied Mycology and Biotechnology. Elsevier,
Amsterdam, pp. 133e159.
Chen JL, Pace NR, 1997. Identiﬁcation of the universally conserved
core of ribonuclease P RNA. RNA 3: 557e560.
Chen W, Dieckmann C, 1997. Genetic evidence for interaction
and speciﬁc nucleotides in the 5’ untranslated
region of mitochondrial cytochrome b mRNA in Saccharomy-
ces cerevisiae. Molecular and Cellular Biology 17: 6203e6211.
Chevreux TP, Wetter T, Suhai S, 1999. Genome sequence assembly
using trace signals and additional sequence information. Paper
presented. Computer Science and Biology: proceedings of the German
conference on bioinformatics (GCB),Germany,99,pp.45e56.
Crick FH, 1966. Codon-anticodon pairing: the wobble hypothesis.
Journal of Molecular Biology 19: 548e 555.
Cummings DJ, Michel F, McNall KL, 1989. DNA sequence analysis
of the 24.5 kilobase pair cytochrome oxidase subunit I mito-
chondrial gene from Podospora anserina: a gene with sixteen
introns. Current Genetics 16: 381e406.
Cummings DJ, McNally KL, Domenico JM, Matsuura ET, 1990. The
complete DNA sequence of the mitochondrial genome of Po-
dospora anserina. Current Genetics 17: 375e402.
Edgell D, Chalamcharla V, Belfort M, 2011. Learning to live to-
gether: mutualism between self-splicing introns and their
hosts. BMC Biology 9: 22.
Ertz D, Miadlikowska J, Lutzoni F, Dessein S, Rasp
e O, Vigneron N,
Hofstetter V, Diederich P, 2009. Towards a new classiﬁcation
of the Arthoniales (Ascomycota) based on a three-gene phylog-
eny focussing on the genus Opegrapha. Mycological Research
Felsenstein J, 1985. Conﬁdence limits on phylogenies - an ap-
proach using the bootstrap. Evolution 39: 783e791.
erandon C, Moukha S, Callac P, Benedetto J-P, Castroviejo M,
Barroso G, 2010. The Agaricus bisporus cox1 gene: the longest
mitochondrial gene and the largest reservoir of mitochondrial
group I introns. PLoS One 5: e14048.
Formighieri EF, T iburcio RA, Armas ED, Medrano FJ, Shimo H,
Carels N, Goes-Neto A, Cotomacci C, Carazzolle MF, Sardi-
nha-Pinto N, Thomazella DP, Rincones J, Digiampietri L,
Carraro DM, Azeredo-Espin AM, Reis SF, Deckmann AC,
Gramacho K, Goncalves MS, Moura Neto JP, Barbosa LV,
Meinhardt LW, Cascardo JC, Pereira GA, 2008. The m ito-
chondrial genome of the p hytopathogenic basidiomycete
Moniliophthora perniciosa is 109 kb in size and contains
a stable integrated plasmid. Mycological Research 112:
Galtier N, Daubin V, 2008. Dealing with incongruence in phylo-
genomic analyses. Philosophical Transactions: Biological Sciences
Galtier N, Nabholz B, Glemin S, Hurst GDD, 2009. Mitochondr ial
DNA as a marker of molecular diversity: a reappraisal. Molec-
ular Ecology 18:
s A, DePriest P, Grube M, Tehler A, 1995. Multiple origins of
lichen symbioses in fungi suggested by SSU rDNA phylogeny.
Science 268: 1492e1495.
Gawryluk RM, Gray MW, 2010. An ancient ﬁssion of mitochon-
drial Cox1. Molecular Biology and Evolution 27:7e10.
Grant JR, Stothard P, 2008. The CGView Server: a comparative
genomics tool for circular genomes. Nucleic Acids Research 36:
Grifﬁths AJF, 1995. Natural plasmids of ﬁlamentous fungi. Micro-
biology and Molecular Biology Reviews 59: 673e685.
Guindon S, Gascuel O, 2003. A simple, fast, and accurate algo-
rithm to estimate large phylogenies by maximum likelihood.
Systematic Biology 52: 696e704.
Guindon S, Dufayard J-F, Lefort V, Anisimova M, Hordijk W,
Gascuel O, 2010. New algorithms and methods to estimate
maximum likelihood phylogenies: assessing the performance
of PhyML 3.0. Systematic Biology 59: 307e 321.
Haugen P, Simon DM, Bhattacharya D, 2005. The natural history
of group I introns. Trends in Genetics 21: 111e119.
Hebert PDN, Cywinska A, Ball SL, deWaard JR, 2003. Biological
identiﬁcations through DNA barcodes. Proceedings of the Royal
Society of London Series B: Biological Sciences 270: 313e321.
Hofacker IL, Fontana W, Stadler PF, Bonhoeffer LS, Tacker M,
Schuster P, 1994. Fast folding and comparison of RNA secondary
structures. Monatshefte F
ur Chemie/Chemical Monthly 125:
Huelsenbeck JP, Ronquist F, 2001. MRBAYES: Bayesian inference
of phylogenetic trees. Bioinformatics 17: 754e755.
James TY, Kauff F, Schoch CL, Matheny PB, Hofstetter V, Cox CJ,
Celio G, Gueidan C, Fraker E, Miadlikowska J, Lumb sch HT,
Rauhut A, Reeb V, Arnold AE, Amtoft A, Stajich JE, Hosaka K,
Sung G-H, Johnson D, O/’Rourke B, Crockett M, Binder M,
Curtis JM, Slot JC, Wang Z, Wilson AW, Schuszler A,
Longcore JE, O/’Donnell K, Mozley-Standridge S, Porter D,
Letcher PM, Powell MJ, Taylor JW, White MM, Grifﬁth GW,
Davies DR, Humber RA, Morton JB, Sugiyama J, Rossman AY,
Rogers JD, Pﬁster DH, Hewitt D, Hansen K, Hambleton S,
Shoemaker RA, Kohlmeyer J, Volkmann-Kohlmeyer B,
Spotts RA, Serdani M, Crous PW, Hughes KW, Matsuura K,
Langer E, Langer G, Untereiner WA, Lucking R, Budel B,
Geiser DM, Aptroot A, Diederich P, Schmitt I, Schultz M,
Yahr R, Hibbett DS, Lutzoni F, McLaughlin DJ, Spatafora JW,
Vilgalys R, 2006. Reconstructing the early evolution of Fungi
using a six-gene phylogeny. Nature 443: 818e822.
Keane T, Creevey C, Pentony M, Naughton T, McInerney J, 2006.
Assessment of methods for amino acid matrix selection and
812 B. B. Xavier et al.
their use on empirical data shows that ad hoc assumptions for
choice of matrix are not justiﬁed. BMC Evolutionary Biology 6: 29.
Laday M, Stubnya V, Hamari Z, Hornok L, 2008. Characterization
of a new mitochondrial plasmid from Fusarium proliferatum.
Plasmid 59: 127e133.
Lang BF, Laforest MJ, Burger G, 2007. Mitochondrial introns:
a critical view. Trends in Genetics 23: 119e125.
Lee J, Young JP, 2009. The mitochondrial genome sequence of the
arbuscular mycorrhizal fungus Glomus intraradices isolate 494
and implications for the phylogenetic placement of Glomus.
New Phytologist 183: 200e211.
Lonergan KM, Gray MW, 1996. Expression of a continuous open
reading frame encoding subunits 1 and 2 of cytochrome c
oxidase in the mitochondrial DNA of Acanthamoeba castellanii.
Journal of Molecular Biology 257: 1019e1030.
Lutzoni F, Pagel M, Reeb V, 2001. Major fungal lineages are derived
from lichen symbiotic ancestors. Nature 411: 937e940.
Magain N, Forrest LL, S
erusiaux E, Gofﬁnet B, 2010. Microsatellite
primers in the Peltigera dolichorhiza c omplex (lichenized as-
comycete, Peltigerales). American Journal of Botany 97:
Marcet-Houben M, Gabald
on T, 2010. Acquisition of prokaryotic
genes by fungal genomes. Trends in Genetics 26:5e8.
McCaskill JS, 1990. The equilibrium partition function and base
pair binding probabilities for RNA secondary structure. Bio-
polymers 29: 1105e1119.
Miadlikowska J, Lutzoni F, 2004. Phylogenetic classiﬁcation of
peltigeralean fungi (Peltigerales, Ascomycota) based on ribo-
somal RNA small and large subunits. America n Journal of Botany
Mouhamadou B, Barroso G, Labarere J, 2004. Molecular evolution
of a mitochondrial polB gene, encoding a family B DNA poly-
merase, towards the elimination from Agrocybe mitochondrial
and Genomics 272: 257e263.
Mullineux ST, Costa M, Bassi GS, Michel F, Hausner G, 2010. A
group II intron encodes a functional LAGLIDADG homing en-
donuclease and self-splices under moderate temperature and
ionic conditions. RNA 16: 1818e1831.
Nash TH, 2008. Lichen Biology, 2nd edn. Cambridge University
Press, New York.
Nakanishi K, Ogiso Y, Nakama T, Fukai S, Nureki O, 2005.
Structural basis for anticodon recognition by methionyl-
tRNA synthetase. Nature Structural & Molecular Biology 12:
Nelsen MP, L
ucking R, Grube M, Mbatchou JS, Muggia L, Plata ER,
Lumbsch HT, 2009. Unravellingthe phylogeneticrelationships of
lichenised fungi in Dothideomyceta. Studies in Mycology 64:
os I, Lyngsø R, Hein J, 2008 . StatAlign: an extend-
able software package for joint Bayesian estimation of
alignments and evo lutionary trees. Bioinformatics 24:
Pantou MP, Kouvelis VN, Typas MA, 2008. The complete mito-
chondrial genome of Fusarium oxysporum: insights into fungal
mitochondrial evolution. Gene 419:7e15.
Paquin B, Laforest M-J, Forget L, Roewer I, Wang Z, Longcore J,
Lang BF, 1997. The fungal mitochondrial genome project:
evolution of fungal mitochondrial genomes and their gene
expression. Current Genetics 31: 380e395.
Rambaut A, Drummond A, 2009. Tracer v1.5. Available from http:
Rohe M, Schrunder J, Tudzynski P, Meinhardt F, 1992. Phyloge-
netic relationships of linear, protein-primed replicating ge-
nomes. Current Genetics 21: 173e176.
Schneider T, Schmid E, de Castro JV, Cardinale M, Eberl L, Grube M,
Berg G, Riedel K, 2011. Structure and function of the symbiosis
partners of the lung lichen (Lobaria pulmonaria L. Hoffm.) ana-
lyzed by metaproteomics. Proteomics 11: 2752e2756.
Seif E, Forget L, Martin NC, Lang BF, 2003. Mitochondrial RNase P
RNAs in ascomycete fungi: lineage-speciﬁc variations in RNA
secondary structure. RNA 9: 1073e1083.
Seif E, Leigh J, Liu Y, Roewer I, Forget L, Lang BF, 2005. Compara-
genomics in zygomycetes: bacteria-like
RNase P RNAs, mobile elements and a close source of the
group I intron invasion in angiosperms. Nucleic Acids Research
Sethuraman J, Majer A, Friedrich NC, Edgell DR, Hausner G, 2009.
Genes within genes: multiple LAGLIDADG homing endonu-
cleases target the ribosomal protein S3 gene encoded within
an rnl group I intron of Ophiostoma and related taxa. Molecular
Biology and Evolution 26: 2299e2315.
Silva FJ, Belda E, Talens SE, 2006. Differential annotation of tRNA
genes with anticodon CAT in bacterial genomes. Nucleic Acids
Research 34: 6015e6022.
Sinnemann SJ, Andresson OS, Brown DW, Miao VP, 2000. Cloning
and heterologous expression of Solorina crocea pyrG. Current
Genetics 37: 333e338.
Slamovits CH, Saida rri aga JF, Larocque A, Keeling PJ, 2007. T he
highly reduced a nd fragmented mitochondrial genome of
the early-branching dinoﬂagellate Oxyrrhis marina shares
characteristics with both apicomplexan and dinoﬂagellate
mitochondrial g enomes. Journal of Molecular Biology 372:
Soma A, Ikeuchi Y, Kanemasa S, Kobayashi K, Ogasawara N,
Ote T, J-i Kato, Watanabe K, Sekine Y, Suzuki T, 2003. An
RNA-modifying enzyme that governs both the codon and
amino acid speciﬁcities of isoleucine tRNA. Molecular Cell 12:
Spatafora JW, Sung G-H, Johnson D, Hesse C, O’Rourke B,
Serdani M, Spotts R, Lutzoni F, Hofstetter V, Miadlikowska J,
Reeb V, Gueidan C, Fraker E, Lumbsch T, Lucking R, Schmitt I,
Hosaka K, Aptroot A, Roux C, Miller AN, Geiser DM, Hafellner J,
Hestmark G, Arnold AE, Budel B, Rauhut A, Hewitt D,
Untereiner WA, Cole MS, Scheidegger C, Schultz M, Sipman H,
Schoch CL, 2006. A ﬁve-gene phylogeny of Pezizomycotina.
Mycologia 98: 1018e1028.
Stoddard BL, 2011. Homing endonucleases: from microbial ge-
netic invaders to reagents for targeted DNA modiﬁcation.
Stone CL, Buitrago MLP, Boore JL, Frederick RD, 2010. Analysis of
the complete mitochondrial genome sequences of the soy-
bean rust pathogens Phakopsora pachyrhizi and P. meibomiae.
Mycologia 102: 887e897.
Storz G, Voge l J, Wassarman KM, 2011. Regulation by small
RNAs in bacteria: expanding frontiers. Molecular Cell 43:
T, Miyauchi K, 2010. Discovery and characterization of
tRNAIle lysidine synthetase (TilS). FEBS Letters 58 4:
Swofford, 1993. PAUP: a computer program for phyl ogenetic in-
ference using maximum parsimony. Journal of General
Physiology 102: A9.
Takemoto C, Spremulli LL, Benkowski L A, Ueda T,
Yokogawa T, Watanabe K, 2009. Unconventional decoding
of the AUA codon as methionine by mitochondrial tRNA-
Met with the anticodon f5CAU as revealed with a mito-
chondrial in vitro translation system. Nucleic Acids Resear ch
Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S, 2011.
MEGA5: Molecular evolutionary genetics analysis using maxi-
mum likelihood, evolutionary distance, and maximum parsi-
mony methods. Molecular Biology and Evolution 28:2731e2739.
Taylor JW, Berbee ML, 2006. Dating divergences in the fungal tree
of life: review and new analyses. Mycologia 98: 838e849.
Weber F, Dietrich A, Weil JH, Marechal-Drouard L, 1990. A potato
mitochondrial isoleucine tRNA is coded for by a mitochondrial
Mitochondrial genomes from the lichenized fungi Peltigera membranacea and Peltigera malacea 813
gene possessing a methionine anticodon. Nucleic Acids Re-
search 18: 5027e5030.
Weinberg Z, Perreault J, Meyer MM, Breaker RR, 2009. Exceptional
structured noncoding RNAs revealed by bacterial metage-
nome analysis. Nature 462: 656e659.
Werth S, 2010. Population genetics of lichen-forming fungi e
a review. Lichenologist 42: 499e519.
Whelan S, Goldman N, 2001. A general empirical model of protein
evolution derived from multiple protein families using
a maximum-likelihood approach. Molecular Biology and Evolu-
tion 18: 691e699.
Wiens JJ, Kuczynski CA, Stephens PR, 2010. Discordant mito-
chondrial and nuclear gene phylogenies in emydid turtles:
implications for speciation and conservation. Biological Journal
of the Linnean Society 99: 445e461.
Yang M, Ge Y, Wu J, Xiao J, Yu J, 2011. Coevolution study of mi-
tochondria respiratory chain proteins: Toward the under-
standing of protein-protein interaction. Journal of Genetics and
Genomics 38: 201e207.
Zuker M, Stiegler P, 1981. Optimal computer folding of large RNA
sequences using thermodynamics and auxiliary information.
Nucleic Acids Research 9: 133e148.
814 B. B. Xavier et al.