Mitochondrial Genome of Phlebia radiata Is the Second

Largest (156 kbp) among Fungi and Features Signs of

Genome Flexibility and Recent Recombination Events
Heikki Salavirta1, Ilona Oksanen1, Jaana Kuuskeri1, Miia Mäkelä1, Pia Laine2, Lars Paulin2,
Taina Lundell1*
1 Microbiology and Biotechnology, Department of Food and Environmental Sciences, University of Helsinki, Helsinki, Finland, 2 Institute of Biotechnology, DNA
Sequencing and Genomics Laboratory, University of Helsinki, Helsinki, Finland

Abstract
Mitochondria are eukaryotic organelles supporting individual life-style via generation of proton motive force and cellular
energy, and indispensable metabolic pathways. As part of genome sequencing of the white rot Basidiomycota species
Phlebia radiata, we first assembled its mitochondrial genome (mtDNA). So far, the 156 348 bp mtDNA is the second largest
described for fungi, and of considerable size among eukaryotes. The P. radiata mtDNA assembled as single circular dsDNA
molecule containing genes for the large and small ribosomal RNAs, 28 transfer RNAs, and over 100 open reading frames
encoding the 14 fungal conserved protein subunits of the mitochondrial complexes I, III, IV, and V. Two genes (atp6 and
tRNA-IleGAU) were duplicated within 6.1 kbp inverted region, which is a unique feature of the genome. The large mtDNA
size, however, is explained by the dominance of intronic and intergenic regions (sum 80% of mtDNA sequence). The
intergenic DNA stretches harness short (#200 nt) repetitive, dispersed and overlapping sequence elements in abundance.
Long self-splicing introns of types I and II interrupt eleven of the conserved genes (cox1,2,3; cob; nad1,2,4,4L,5; rnl; rns). The
introns embrace a total of 57 homing endonucleases with LAGLIDADGD and GYI-YIG core motifs, which makes P. radiata
mtDNA to one of the largest known reservoirs of intron-homing endonucleases. The inverted duplication, intergenic
stretches, and intronic features are indications of dynamics and genetic flexibility of the mtDNA, not fully recognized to this
extent in fungal mitochondrial genomes previously, thus giving new insights for the evolution of organelle genomes in
eukaryotes.

Citation: Salavirta H, Oksanen I, Kuuskeri J, Mäkelä M, Laine P, et al. (2014) Mitochondrial Genome of Phlebia radiata Is the Second Largest (156 kbp) among
Fungi and Features Signs of Genome Flexibility and Recent Recombination Events. PLoS ONE 9(5): e97141. doi:10.1371/journal.pone.0097141
Editor: Jae-Hyuk Yu, University of Wisconsin - Madison, United States of America
Received November 13, 2013; Accepted April 15, 2014; Published May 13, 2014
Copyright: ß 2014 Salavirta et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study was supported by the Academy of Finland research project grants AF-138331 (Ox-Red) and AF-129869, and a Master’s thesis grant from the
Department of Food and Environmental Sciences, University of Helsinki. The funders had no role in study design, data collection and analysis, decision to publish,
or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: [email protected]

Introduction indispensable cellular processes such as calcium homeostasis, cell

aging and apoptosis, iron metabolism, and synthesis of iron-
Phlebia radiata Fr. is a saprobic, wood-colonizing and white-rot sulphur clusters for oxidoreductive enzymes [9–12].
type of wood decay causing polypore fungal species of the class Essence of mitochondria is accepted to arise from endosymbiosis
Agaricomycetes, phylum Basidiomycota, and is encountered in [13,14], most reliably of the SAR11 clade ancestor marine
Eurasian and North-American forests generally on dead angio- bacterium (pelagibacteria) [15]. Adaptation to the host organism
sperm wood [1,2]. We initiated de novo whole genome sequencing has resulted with co-evolution of the mitochondrial genome and
of P. radiata due to its notable biotechnological abilities in gene flow to the host genome [7,8,16,17]. It was previously
decomposition of wood components and lignocelluloses, and in considered that mitochondrial genomes are small and compact,
oxidation and conversion of synthetic and milled wood lignin, and according to information mostly achieved from metazoa, such as
lignin model compounds [3–5]. The fungus is also efficient in
the only 16 kbp-size human mitochondrial genome [9]. This
degradation of xenobiotics and production of lignin-converting
notion has, together with the retarded mtDNA sizes, previously led
oxidoreductases like lignin peroxidases and manganese peroxidas-
to the proposal of the ‘‘vanishing mitochondria’’, especially in
es, and laccase [3–6].
fungi [8].
The draft assembly of 454-sequenced P. radiata genome resulted
Complete genome sequencing on eukaryotic micro- and macro-
first with ca. 300x coverage of a single scaffold and circular dsDNA
organisms has, however, demonstrated a higher degree of
molecule of over 156 kbp in size, which turned out to be the
mitochondrial genome structural complexity, and variation in
mitochondrial genome. Mitochondria are cellular organelles of
the mtDNA size than was previously realized. Complicated
eukaryotes which support individual life-style and generate proton
network of mini-circle mtDNAs are present in the basal body
motive force for production of ATP and energy via respiration [7–
9]. Mitochondria are also known to participate in many other mitochondrion of the Kinetoplastida protozoa [18], when the

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 Mitochondrial Genome of the Basidiomycota Species Phlebia radiata

largest mt genomes are described for Embryophyta and Char- as well as at molecular level on ribosomal 18S rRNA gene and
ophyta [19,20], i.e. for land plants and green algae. In angiosperm ITS1-5.8S-ITS2 bar coding sequences [33]. For isolation of total
flowering plants, the mtDNA varies highly in size (200 kbp to DNA, the fungus was cultivated in liquid 2% (wt/vol) malt extract
11 Mbp) and may be organized to multiple chromosomes [20–22]. broth for 10 days at 28uC in the dark. After cultivation, the
So far, the largest plant mt genomes were recently sequenced for mycelial mats were harvested and washed with cold ultrapure
Silene species as complex entities with up to 128 circular-mapping water, frozen to 220uC, and lyophilized.
chromosomes [22].
Currently, 162 fully sequenced and annotated fungal mtDNA DNA Isolation
sequences are publicly available. The overwhelming majority (124) Dry mycelium was quickly ground in acid-washed and
of these belong to Ascomycota [19]. Basidiomycota are the second autoclaved mortar. DNA was isolated using a modified version
best represented fungal phylum with 21 complete mt genomes of the hot-CTAB extraction at 65uC [34], followed by phenol-
[19,23–28]. The other publicly available fungal mtDNAs include a chloroform and 3x chloroform-isoamyl alcohol extractions, and
few genomes from species of Blastocladiomycota, Chytridiomy- incubation with 0.1 mg/ml Proteinase K (Fermentas) for 30 min
cota, Glomeromycota, Monoblepharidomycota, one Cryptomy- at 55uC. Total DNA was precipitated overnight with isopropanol
cota (Rozella allomycis), and three previous Zygomycota, now incertae at 4uC, centrifuged at 6500 g 30 min at 4uC, washed twice with
sedis species [19]. Exceptionally, the Microsporidia and the 70% ethanol, and subjected to 50 U/ml of RNAseA (Fermentas)
anaerobic fungi of Neocallimastigomycotina lack traditional treatment at 37uC overnight. After chloroform-isoamyl alcohol
mitochondria, which were modified to other cellular organelles extraction, and re-precipitation with ice-cold 94% ethanol
such as hydrogenosomes [8]. Most fungal mt genomes are overnight at 220uC, DNA was dissolved in sterile TE (10 mM
characterized as single circular dsDNA molecules [7,8,23–29], Tris-HCl buffer with 1 mM EDTA, pH 7.5) solution. Integrity an
when linear or transiently linear chromosome organization was d amount of the isolated total DNA was examined by 1.5% (wt/
reported for a few species [7,8,30–32]. vol) agarose gel electrophoresis, and using the NanoDrop 1000
Fungal mtDNA generally encloses 14 essential protein-coding Spectrophotometer (Thermo Scientific).
genes (atp6,8,9; cob, cox1-3, nad1-6, and nad4L) for protein subunits
of the mitochondrial complexes I, III, IV, and V required for
electron transfer and oxidative phosphorylation. Another com-
454 Sequencing and mt Genome Assembly
Single-stranded template DNA (sstDNA) was sequenced using
mon, but more randomly distributed fungal mtDNA-contained
the 454 sequencing technology with GS FLX Titanium chemistry
gene is rps3, which encodes the small ribosomal subunit protein S3.
Other typical genes to fungal mtDNA are the small (rns) and large (Roche, 454 Life Sciences). Number of obtained reads was 1 876
(rnl) subunit mitochondrial rRNAs, and a tRNA set - generally at 081 containing 752 Mbp of both genomic DNA (gDNA) and
least 23 unique anticodons - sufficient to translate the mtDNA- mitochondrial DNA (mtDNA). All reads were assembled using
encoded proteome [7,8,10,23–28]. However, exceptions are not Newbler (Roche, 454 Life Sciences) software. Mitochondrial
unusual. For example, in the Ascomycota budding yeast Saccha- contigs containing high average sequence coverage (approximately
romyces cerevisiae, the 85.8 kbp mtDNA includes over 40 genes 300x) were placed in proper order, resulting with single scaffold,
encoding e.g. 24 tRNAs and the two rRNAs, but lacks two of the and a finished mtDNA circular genome was defined being 156
14 conserved protein-coding genes (those for Complex I subunits) 348 bp in length. Circularity and sequence orientation, in
[29]. particular for the large duplicated region, was verified with
Together with our study, recent genome sequencing reports genome-walking PCR.
indicate that fungal mitochondrial genomes have a much higher
degree of variation in size, gene content, genomic organization Gene Annotation and Bioinformatic Analyses
and gene order, and gene intron-exon construction than has been The Mold, Protozoan, and Coelenterate Mitochondrial Code
realized previously. We acknowledge that the high number of and the Mycoplasma/Spiroplasma Code (NCBI translation
intron-homing endonucleases (HEs) recognized in the P. radiata table 4) was at first assumed for ORF detection. Protein-coding
mtDNA may play an editing role, both in genome replication and and rRNA genes were annotated by blastp and blastn queries
gene transcription, as well as an integrating role for intron and against non-redundant NCBI databases [35–37], and localised
gene transposition in the mtDNA. Another unique feature is the and annotated in the mtDNA sequence using Artemis [38]
duplicated ‘‘mirror’’ region in the genome, which together with software. Intron-exon boundaries of the conserved genes were
the repetitive-element dense sections may promote both DNA adjusted manually on the basis of ClustalX [39] multiple
recombination and gene transcription. We also discuss mtDNA- Basidiomycota mt coding sequence alignments. Transfer-RNAs
encoded proteome phylogeny in relation to tRNA evolution and were identified with tRNAscan-SE [40]. HEs were recognized
ORF codon usage, in regard to the currently accepted concept of by Pfam 26.0 database [41] queries. Protein domain images
fungal systematics. were generated with ExPASy PROSITE MyDomains Image
Creator (http://prosite.expasy.org/mydomains/) and edited in
Materials and Methods Inkscape version 0.48.2 (http://inkscape.org/). Intron types
were determined with RNAweasel algorithm [42]. Nucleotide
Fungal Isolate and Cultivation sequence repeat elements were identified and analysed with the
Phlebia radiata Fr. strain 79 (FBCC0043) was originally isolated EMBOSS package Nucleic repeats group tools [43], and by
from a distinguishable fruiting body found in South Finland on performing a local blastn [35] query of the complete mtDNA
white-rot decayed alder (Alnus incana), and maintained in the sequence against itself. The hits were clustered as a function of
Fungal Biotechnology Culture Collection at the Department of similarity in CD-HIT Suite [44], and the h-cd-hit-est algorithm
Food and Environmental Sciences, University of Helsinki, as living was run with consecutive 0.75, 0.80, and 0.90 cut-off values,
mycelium on 2% (wt/vol) malt extract, 1.5% (wt/vol) agar slants using the sequence set that returned ,0.001 blastn E-values in
under paraffin oil at 12uC. Species identification is based on both the 1 vs. 1 search.
macroscopic features of the original fruiting body and mycelium,

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 Mitochondrial Genome of the Basidiomycota Species Phlebia radiata

Phylogenetic Analyses Table 1). The highest number of introns (13) was in the cox1 gene,
Genome accessions of completely sequenced fungal mtDNAs which covered ca. 21 kbp (14%) of the genome.
were retrieved from NCBI Organelle Genome Resources website Notably, the genes encoding atp6, tRNA-IleGAU and tRNA-
[19], and linked to corresponding proteomes through GenBank PheGAA were present in two identical copies. The duplicate atp6
[45] queries. Subsequently, super alignments were generated from and tRNA-IleGAU genes were identified in the ‘‘mirror’’ region,
USEARCH [46] de-replicated proteomes with the core of Hal which comprised an inverted and almost identical 6.1 kbp region
pipeline [47], allowing 50% of missing data. Phylogenetic trees in the genome (Figure 1). On the basis of multiple sequence
were constructed from 44 fungal taxa and 2 019 aa remgaps super alignments, cob and nad6 ORFs had C-terminal fused extensions.
alignment first with RAxML 8.0.0 [48] with 100 rapid bootstrap Moreover, alternative 39-ends were found for atp6 and cox2
repetitions and automatic model selection (-f a -d -m PROT- (Figure 3 A, B).
GAMMAAUTO) using Blastocladiomycota as outgroup (best-
scoring aa model was MTZOA), and with PhyloBayes 3.3f [49] Open Reading Frames with Unknown or Non-conserved
using default options, 2 parallel chains were run until maxdiff was Function
,0.1, first 100 trees were discarded as burn-in, and one in ten In total, 108 ORFs in addition to the conserved genes met our
remaining trees were sampled for posterior consensus. The tree initial search criteria (Table S1). From these, 39 produced
was rooted from mid-point. Nodes receiving #0.8 posterior significant (E-value #0.001) blastp hits against the nr database,
consensus (Bayesian) or #80 bootstrap support (ML) were with a Codon Adaptation Index (CAI) range of 0.299–0.800 in
collapsed to polytomies with TreeCollapseCL4 (http:// reference to the conserved protein-coding genes. The majority of
emmahodcroft.com/TreeCollapseCL.html). The trees were edited these ORFs were intronic and were associated with HE domains
in FigTree (http://tree.bio.ed.ac.uk/software/figtree/). (Table 3). A notable exception was ORF793 within the long group
II intron in the middle of cob gene (Figure 1). This intronic ORF
Correlation Analyses was associated with identified RNA-dependent DNA polymerase
Sequence similarity of the core domain aa-sequences from 57 domain (annotated locus PRA_mt0165, reverse transcriptase) and
had particularly low CAI-value of 0.299 (Table S1), which
HEs in the P. radiata mtDNA were analyzed by generating aa-
indicates relatively recent horizontal gene transfer from a
sequence pairwise distance matrix of the LAGLIDADG 1 and 2,
genetically distant source, most probably of viral origin.
and GIY-YIG catalytic ORFs using Geneious 5.5.5 software. In
Due to annotated genome sequence submission requirements,
addition, pairwise distance matrices of the HE domain loci were
ORFs that continued from undisrupted exon reading frames into
calculated using the R environment 2.14.1 package for Windows
putative intronic regions were 59-truncated to their first Met
(http://www.r-project.org/) in order to test correlation of the locus
codons, which shortened eight annotated ORFs, and excluded five
distance to the sequence-similarity based (evolutionary) distance.
ORFs that returned significant E-values (Table S2). These ORFs
The data matrices were tested for being parametric or non-
may represent inteins (‘‘protein introns’’).
parametric. LAGLIDADG 1 aa-sequence similarity scores were Freestanding P. radiata mtDNA ORFs that returned significant
normalized with logarithmic transformation. Parametric Pearson blastp hits were ORF588, ORF319, ORF314, ORF273 and
correlation in PASW Statistics 18, release 18.0.0 (SPSS Inc., ORF90, with respective CAI values of 0.624, 0.577, 0.747, 0.633,
Chicago, IL, USA) was used for LAGLIDADG 1, and GIY-YIG and 0.515 (Table S1), indicative of fungal mitochondrial origin.
type HE domains, when the non-parametric Spearman’s correla- Two of these, ORF588 (PRA_mt0150) and ORF273
tion test was applied to LAGLIDADG 2. (PRA_mt0076), were the most similar to putative DNA polymer-
ases of Pleurotus ostreatus mt genome (Table S1). Two coding
Results sequences, ORF319 and ORF314, were the most similar to
hypothetical proteins annotated in Moniliophthora roreri mtDNA as
P. radiata mtDNA Genome Structure and Conserved orf2 and hyp11, respectively. Notably, the 59-end of ORF319 is
Genes similar to that of the P. radiata mt nad6 gene (36/37 nt identities), as
The mitochondrial genome (mtDNA) of Phlebia radiata isolate 79 it is located at the edge of the mirror region (Figure 1). The best hit
was achieved by de novo 454 sequencing of total DNA using for ORF90 in turn was a hypothetical protein annotated in the
Titanium chemistry, and the final assembly resulted in a single 156 mtDNA of the Ascomycota species Ajellomyces dermatitidis.
348 bp scaffold with a sequence coverage of over 300x,
representing one circular dsDNA molecule (Figure 1) with a GC Transfer RNAs and Codon Usage
percentage of 31.1. The genome contains the 14 protein-coding The tRNAscan-SE algorithm identified 28 tRNAs (Table 2).
genes typical to fungal mtDNA, which are related to the This tRNA set is likely able to sense all the codons of the P. radiata
mitochondrial inner membrane Complexes I, III, IV and V of mtDNA-encoded proteome. With the exception of anticodons of
the respiratory chain, i.e. cox1, atp6, cox2, cox3, nad4L, nad5, atp8, Trp and Ile tRNAs, where possible, U was always the anticodon
nad2, nad3, atp9, cob, nad4, nad6, nad1, in clockwise order of the wobble position base. In the remaining tRNAs, G was always used
mtDNA (Figure 1). Additionally, 31 conserved genes related to over A at the anticodon wobble position. For the tRNA-Cys, the
information transfer (28 tRNAs, rnl, rns, and rps3) were identified tRNAscan-SE Cove algorithm predicted probability for a gene
(Tables 1, 2). match score below the threshold value of 20.0. However, Cove
Identified protein (sum 68 953 bp), rRNA (sum 13 606 bp), and scores were low as well for other Basidiomycota tRNA-Cys genes,
tRNA (sum 2 070 bp) genes including introns cover 55% of the P. e.g. in Phakopsora meibomiae (cove: 19.75), P. ostreatus (cove: 19.67),
radiata mtDNA. However, only about 15% (25 045 bp) of the and Schizophyllum commune (cove: 22.36). This indicates that the
genome refers to conserved coding sequences, when intergenic P.radiata mtDNA tRNA-Cys gene is real despite the low Cove
regions (in total 44% of the genome) and coding-sequence splicing score obtained.
introns (sum 59 584 bp, 38%) dominate the sequence space The GC-content of P. radiata mtDNA ORFs was 26.83% (1st
(Figure 2). The majority of the conserved protein-coding genes letter GC: 34.14%, 2nd letter GC: 33.33%, 3rd letter GC:
were split by long introns into multiple short exons (Figure 1, 13.00%), with no obvious bias observed in codon usage between

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 Mitochondrial Genome of the Basidiomycota Species Phlebia radiata

Figure 1. Gene map of Phlebia radiata mtDNA. Colour of the scale (kb) bar indicates orientation of transcription: clockwise (CW, white), counter-
clockwise (CCW, grey). Bars mark protein-coding (blue) and RNA (red) genes, and alternative C-termini in atp6 and cox2 are depicted (orange). Intron
type is indicated in colour: group I (light grey), group II (yellow), and uncertain (white). Within introns, the hypothetical and identified ORFs are
indicated: over .200 amino-acid long ORFs (turquoise), and homing endonuclease domains GIY-YIG (black) and LAGLIDADG (dark grey). The
transparent ribbon illustrates location of the 6,076 bp inversion-duplication. Asterisk indicates putative tRNA-IleCAU. The inner circle (scale at 12
o’clock in linear units) plots nucleotide bias (G/C skew) up to hexamers along each mtDNA position; G/C (red), A/T (blue), total strand bias (black),
placing oriC around 11:30 o’clock as the largest bias of G over C.
doi:10.1371/journal.pone.0097141.g001

the leading and the lagging strand encoded ORFs. With the mainly in the putative non-translated C-terminal fused extensions
exception of Trp, W-base (A or T/U) ending codons were of cob and nad6.
preferred over S-ending (C or G) codons across all codon families.
Cys codons showed the smallest bias with 72% UGU over 28% Phylogeny
UGC. For Ala, Phe, His, Ile, Asn, Pro and Tyr the same The first phylogenetic analysis was established on the similarity
percentage was $80%, and for the rest $90%. Some codons and variations of codon usage in fungal mtDNA-protein coding
UGA (1), AAG (9), CGC (1), AGG (5), and CGG (2) may be sequence ORFs (Figure 4). The restricted amount of species
unassigned, as they were only present in non-conserved regions, included in the analysis, however, already grouped P. radiata

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 Table 1. Annotated conserved protein-coding and rRNA genes, their characteristics, location and intron types in P. radiata mtDNA.

Coding sequence
Gene Start End Strand Length bp length (bp) Protein length (aa) Introns Average intron length (bp) Coding sequence density Stop codon
group I group II un-certain

cox1 1 21743 + 21743 1590 529 12 1 1550 7.3% TAA

rnl 23389 34087 + 10699 3624 2 2 1769 33.9%

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atp6 36924 37700 + 777 777 258 100.0% TAA
cox2 45480 52022 + 6543 756 251 3 1929 11.6% TAA
cox3 58610 61439 + 2830 813 270 1 2017 28.7% TAA
nad4L 64304 66152 + 1849 273 90 1 1576 14.8% TAA
nad5[1] 66153 76178 + 10026 2007 668 4 1 1604 20.0% TAA
atp8 77128 77286 + 159 159 52 100.0% TAA
nad2 81572 87656 + 6085 1812 603 2 2137 29.8% TAA
nad3[2] 87656 88030 + 375 375 124 100.0% TAA
atp9 103782 104003 2 222 222 73 100.0% TAA
rns 109435 112341 2 2907 1711 3 399 58.9%
[3]
cob 119244 126306 2 7063 1272 423 2 1 1930 18.0% TAA
nad4 127498 130848 2 3351 1473 490 1 1878 44.0% TAA

5
nad6[4] 133402 134382 2 981 981 326 100.0% TAA
atp6 139006 139782 2 777 777 258 100.0% TAA
nad1[5] 140738 143807 2 3070 1017 338 1 2053 33.1% TAG
rps3 150069 151403 + 1335 1335 444 100.0% TAA

[1]
nad5 starts from the adjacent in frame codon to nad4L stop.
[2]
nad3 uses the last A of nad2 stop codon for initiation Met’s first nt.
[3]
Based on multiple sequence alignment of Basidiomycota cob genes the last conserved aa of P. radiata cob is 43 aa before the stop codon.
[4]
Based on multiple sequence alignment of Basidiomycota nad6 genes the last conserved aa of P. radiata nad6 is 122 aa before the stop codon.
[5]
The codon after the putative TAG stop codon is TAA.
Empty cell, not present or observed, or unable to calculate.
doi:10.1371/journal.pone.0097141.t001
Mitochondrial Genome of the Basidiomycota Species Phlebia radiata

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 Mitochondrial Genome of the Basidiomycota Species Phlebia radiata

Table 2. Transfer RNA genes in P. radiata mtDNA.

Transfer RNA[1] Anticodon Start End Strand Length (bp)

Ile GAU 39443 39514 + 72

Ala UGC 55927 55999 + 73
Trp CCA 78296 78369 + 74
Asp GUC 79686 79758 + 73
Phe GAA 89697 89767 + 71
Thr UGU 91722 91793 + 72
Gln UUG 92238 92311 + 74
Lys UUU 93159 93231 + 73
Tyr GUA 95474 95557 + 84
Phe GAA 96533 96603 + 71
Ile[2] CAU 96632 96704 + 73
Ser UGA 97243 97328 + 86
Val UAC 103081 103151 2 71
Ser GCU 107528 107609 2 82
His GUG 107979 108050 2 72
Val UAC 108850 108920 2 71
Met CAU 113144 113216 2 73
Arg UCG 114191 114261 2 71
Leu UAG 114743 114816 2 74
Gly UCC 117118 117188 2 71
Ile GAU 137192 137263 2 72
Arg UCU 146748 146818 2 71
Cys[3] GCA 148390 148461 2 72
Pro UGG 153357 153429 + 73
Asn GUU 153900 153972 + 73
Leu UAA 154617 154701 + 85
Met CAU 154736 154807 + 72
Glu UUC 154832 154902 + 71

[1]
tRNAscan predicts the tRNA type from the anticodon.
[2]
This tRNA was determined to be Ile through comparative means (see below).
[3]
The bit score of tRNA-Cys was below 20, which is a typical cut-off value for a pseudogene. The gene was predicted with exceptionally low score from all Basidiomycota
mtDNAs.
doi:10.1371/journal.pone.0097141.t002

mtDNA-proteome with Agaricomycotina. Exceptional was the polytomies, placing Agaricomycotina as a sister lineage to the
positioning of C. neoformans far out from the other Agaricomycotina Pucciniomycotina/Ustilaginomycotina group. P. radiata positioned
species. nearest to Trametes cingulata and two Ganoderma species (Figure 5).
Our protein phylogenetic approaches, the maximum-likelihood
based RAxML, and the Bayesian Monte Carlo Markov Chain Introns and Intron Homing Endonucleases
sampler PhyloBayes, reconstructed the recognized fungal phyla as Nine of the 16 fungal mitochondrial conserved genes in P.
monophyletic clades. However, in RAxML the branching of radiata mtDNA were interrupted by over 1 000 bp long introns
Chytridiomycota, Glomeromycota, and Dikarya (Ascomycota and (Table 3). RNAweasel [42] algorithm detected 29 group I and two
Basidiomycota) was polytomic, whereas in the PhyloBayes derived group II intron structures, out of which all but one were located
tree, Chytridiomycota and Monoblepharidomycota were a sister within regions that were determined to be intronic also by our
lineage to Glomeromycota and Dikarya, and Glomeromycota manual approach (blastp, blastn, Pfam queries, ClustalX align-
were a sister lineage to Dikarya (Figure 5). Further, in the Bayesian ments). Our semi-manual approach (blastp, blastn, Pfam queries,
phylogeny, the incertae sedis species (previous Zygomycota) R. oryzae ClustalX alignments) predicted seven additional introns. Four of
and M. verticillata were within the Glomeromycota/Dikarya these were associated with core catalytic HE domains within the
branch. nad1, nad2 and rnl gene regions. Despite the lack of sequence
Basidiomycota subphyla were resolved by both methods to homology, three shorter introns were inferred to reside within the
current fungal taxonomy with the single exception of the rns gene (Table 3). In total, 29 introns were associated with HE
Agaricomycotina classified C. neofarmans node (two strains, domains. Introns varied in length from 201 bp (intron 2 in rns) to 3
Figure 5). As with higher-level taxonomy, PhyloBayes seemingly 420 bp (intron 5 in cox1), with an average length of about 1.5 kbp
solved Basidiomycota subphylum level phylogeny with less for the protein-coding gene splicing introns (Table 3).

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 Mitochondrial Genome of the Basidiomycota Species Phlebia radiata

Figure 2. Contribution of the various features of P. radiata mtDNA to genome size. Conserved coding sequence refers to the conserved
fungal mitochondrial proteome ORFs, rRNAs and tRNAs. Significant ORFs refer to additional identified and hypothetical protein coding sequences
with E-value,0.001 obtained by BLASTp queries. Freestanding refers to intronless. Intronic significant ORFs include homing endonucleases, also
exon-to-exon-fused ORFs, excluding intergenic ORF HEGs (see text).
doi:10.1371/journal.pone.0097141.g002

Based on Pfam queries, the P. radiata mtDNA contained 57 Eight of the HE catalytic domains were exceptional in
characteristic protein domains encoded by HE genes (HEGs, Pfam appearing as free-standing after the last putative coding sequence
family PF05204; Table 4) belonging to three different structural exon and stop codon in the genes atp6 and cox2. Notably,
families: LAGLIDADG (47) with subtypes 1 (28) and 2 (19), and alternative C-termini were annotated for both of these genes
GIY-YIG (10). The catalytic HE domains expanded from 21 to (Figure 3A, B). The parametric Pearson’s correlation test of
181 aa (59 to 542 bp), and their aa pairwise sequence similarities pairwise aa-similarity and locus distance for the identified HE
varied from 3.2% to 32% for LAGLIDADG 1, from no identity to domain types (Table S1, S2, S5) resulted in correlation value of
67% similarity for LAGLIDADG 2, and from 12% to 52% for 20,166 with a statistically significant p-value (0.031) for
GIY-YIG type of domains (Table 4, Table S5). The HE motifs LAGLIDADG type 1, and correlation value of 20.256 with,
were predominantly (45/57) situated within group I type introns. however, a statistically insignificant p-value (0.089) for GIY-YIG
One single GIY-YIG domain located within a group II intron, and type. The non-parametric Spearman’s correlation test for
10 motifs located in regions of unrecognized intron type (Table 3). LAGLIDADG type 2 (19) resulted in a test value of 20.040 but
with statistically insignificant p-value (0.607). These results infer

Figure 3. Schematic view of the C-termini regions of P. radiata mtDNA atp6 and cox2 genes. Green lines denote atp6 coding sequence
region and the last exon of cox2. Spheres/ovals represent LAGLIDADG 1 (green) and GIY-YIG (grey) homing endonuclease domains. A) Region of
201 bp (orange) with high 1 vs. 1 sequence similarity, corresponding to the 39-end of the atp6 gene. Codon Adaptation Index (CAI) values are shown
for the atp6 N-terminal region (blue) and for the regions of high sequence similarity. Reference codon usage is from: 1) atp6 N-terminus, 2) atp6 N-
terminus and atp8-9, 3) atp6 N-terminus, atp8-9, cox1-3, nad1-5 and nad4L. B) Separated by three LAGLIDADG 1 domains and a GIY-YIG domain, two
regions of high 1 vs. 1 sequence similarity (orange) exist for the last 66 bp of the cox2 gene. Image was generated with ExPASy PROSITE MyDomains
Image Creator (http://prosite.expasy.org/mydomains/) and edited in Inkscape version 0.48.2 (http://inkscape.org/).
doi:10.1371/journal.pone.0097141.g003

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 Mitochondrial Genome of the Basidiomycota Species Phlebia radiata

Average length (bp)

1604
1769
1929

2137
2017
1576
1878
2053
1550

1930
399
Total length (bp)

20153
8019
7075
5787
1196
5791
4273
2017
1576
1878
2053
Intron 13

1532 G
Intron 12

2004 G

Figure 4. Codon usage of the P. radiata mitochondrial genome

Intron 11

protein-coding gene open reading frames in comparison to

1076 L1

selected Basidiomycota and Ascomycota species. Neighbor-

joining tree with topology derived from codon on codon root mean
squared difference distance matrix (Table S3, Table S4). Agaricomyco-
Intron 1 Intron 2 Intron 3 Intron 4 Intron 5 Intron 6 Intron 7 Intron 8 Intron 9 Intron 10

tina, blue; Ustilaginomycotina, red; Pucciniomycotina, black; Ascomy-

cota species Gibberella zeae = Fusarium graminearum, green, as out-
1649 G

Homing endonuclease associations: G, GIY-YIG; L1, LAGLIDADG 1; L2, LAGLIDADG 2; R, reverse-transcriptase ORF including.

group. Scale bar indicates nucleotide changes per site.

Table 3. Introns, their lengths and types in the conserved P. radiata mtDNA-encoded genes.

doi:10.1371/journal.pone.0097141.g004
1410 L2

that genome (intron) location is to some extent related to the

degree of HE sequence similarity, at least for the LAGLIDADG
1 HEs. However, it may also be concluded that HE motif
1144 L2

transposition to more distant locations are equally allowed, as is

observed for LAGLIDADG 2 and GIY-YG domains.

Inverted Duplication and Other Repeated Elements

432

A distinguishing feature was the inverted duplication region of 6

075 bp in size that accounted for 3.9% of the P. radiata mtDNA.
1447 L1

One region (ID1) expanded from nt position 140 421 to 134 346
and the other (ID2) from nt position 36 285 to 42 360 (Figure 1),
which is named ‘‘mirror region’’ in our EMBL submitted and
1737 G

annotated P. radiata mt genome. Both regions harboured the two

3420

genes atp6 and tRNA-IleGAU, and differed only by 3 nt –in the

plus DNA strand at nt position 40 066 with an additional A, at
1356 L2
1186 L2

nucleotide position 41 338 with absence of T, and at position 42

1732 G

353, T instead of G.
A major difference between the ID1 and ID2 regions was the
1680 L1
1405 L1
1756 L2

1475 L2

start of the coding sequence of the single-copy gene nad6 within the
1786 G

39 end of the ID1 region (Figure 1). Another difference was the
291

occurrence of a single-copy, functionally unknown ORF319

doi:10.1371/journal.pone.0097141.t003

(PRA_mt0074) that was only recognized in ID2. However, the

1540 L1
2278 L2

1152 L2
1912 G

2452 R

Underlined, group II intron type.

ORF was only 1-nt different in the 59 end sequence (first 37 nt)
2967
201

compared to nad6 in ID1. In addition to the mirror region, the P.

radiata mtDNA is frequent with short (#200 nt), dispersed, and
1413 L1
1675 L2

1864 L1
1306 L2
2017 L2
1576 L1

2053 L1
1463 G

2849 G

partially overlapping tandem repeat sequence motifs (Figure 6A,

1878
704

B), in particular between positions 85 000 to 100 000 nt, where

also tRNA encoding genes were clustered (Figure 1). The most
abundant repeat sequence types were dispersed and inverted
repeat sequences, which were almost exclusively localized into
Gene

nad4L
nad5

nad2

nad4
nad1
cox1

cox2

cox3

intronic, and especially into intergenic regions (Figure 6A). These

cob
rns
rnl

repeats were often overlapping, and covered as much as 15% of

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 Mitochondrial Genome of the Basidiomycota Species Phlebia radiata

Figure 5. Phylogeny of fungal mitochondrial proteomes. The statistically most likely tree was derived by Bayesian inference from a multi-gene
superalignment of mtDNA-encoded proteins (2 019 aa positions, 44 taxa). Posterior consensus support values are depicted for branching, and nodes
receiving #0.8 support were collapsed into polytomies. The tree was rooted from mid-point. Colours refer to phyla or sub-phyla: turquoise,
Chytridiomycota; yellow, Monoblepharidomycota; orange brown, Glomeromycota; dark blue, Ascomycota; pink, Ustilaginomycotina (Basidiomycota);
red, Pucciniomycotina (Basidiomycota); bright green, Agaricomycotina (Basidiomycota); Blastocladiomycota node as outgroup. Scale bar indicates
amino-acid substitutions per site. For species and mtDNA accession, see Table 5.
doi:10.1371/journal.pone.0097141.g005

the P. radiata mtDNA. Subsets of these sequences shared high Discussion

sequence similarity (Figure 6B).
mtDNA Size and Genome Organization
Origin of Replication To our knowledge, at 156 348 bp, the mitochondrial genome of
According to G/C skew analysis, origin of replication (oriC) of P. radiata described in our current study is the second largest
the P. radiata mtDNA may be located around 11:30 to 00:30 completely sequenced, gene annotated and located mtDNA
o’clock (position 153 000 to position 7 nt) regarding to the largest among fungi, and presents specific features as signs of genetic
bias of G over C (Figure 1). The lowest G/C skew ratio in turn is flexibility, recombination history and active editing process of the
located around 7:00 to 8:30 o’clock (positions 88 000 to 109 genome. Our findings on the size and original features of the P.
000 nt), in the about 21 kb size intergenic region indicative of a radiata mtDNA, together with other recent Basidiomycota
putative mtDNA replication termination site, which is supported mitochondrial genome studies are thereby not explicitly support-
by switching of the coding strand (orientation of transcription) at ing the previous conclusions for the rather small sized and
this site, around position 103 000 nt. disappearing fungal mitochondrial genomes.
On the contrary, fungal mt genomes apparently vary greatly in
Sequence Accession size, from ca. 12 kb kbp of the Cryptomycota parasite species
The complete and annotated Phlebia radiata 79 mitochondrial Rozella allomycis [19] to over 235 kbp (165 kbp main mtDNA [50])
genome sequence is available under the accession codes [EMBL: of the Basidiomycota Agaricomycotina species Rhizoctonia solani
HE613568] and [NCBI: NC_020148]. strain AG3 Rhs1AP (Table 5). Evidence of large variations in the

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 Mitochondrial Genome of the Basidiomycota Species Phlebia radiata

genome size and a high degree of mtDNA structural complexity

between eukaryotic organism lineages and species is currently
accumulating through sequencing projects. For fungi, such

56 490
34 171
26 106
Mean
extreme variations in the mtDNA size or multi-chromosomal
organization have not, however, been noticed than is updated for
plant (from 0.2 to 10 Mbp mtDNAs), algae and protozoa
mitochondria [18–22]. Fungal mitochondrial genomes are usually
mapped as single circular dsDNA molecules [7,8,23–29]. We
Locus distance (bp)

141 226
114 830
73 172 likewise assume a similar configuration for the P. radiata mtDNA
Max

on the basis of our sequence assembly, bioinformatic analyses and

PCR. Reports on more linear than circular chromosomal structure
of the mtDNA in the Chytridiomycota species Hyaloraphidium
2 214

curvatum [30], in the Ascomycota yeasts Candida albicans [31] and

Min
408
106

Saccharomyces cerevisiae [32,51,52] indicate that the possibility for a

partial linear or linear-circular chromosomal organization for the
P. radiata mtDNA cannot be completely ruled out, despite the
convincing circular assembly which was obtained from the careful
study of our sequence data.
With fungal mt genomes of less than 30 kb in size, usually all the
Mean

14 fungal mtDNA-conserved, mitochondrial inner-membrane

11
16
26

protein complex I, III, IV and V protein subunit-coding genes

are present. Examples of these compact mt genomes within
Basidiomycota are species of the animal-pathogenic genus
Cryptococcus, with some variation of the mtDNA size (24–
Similarity (aa% identity)

34.7 kb), gene order and intronic and ORF coding sequence
Max

between species and variants [53,54]. The large fungal mitochon-

32
67
52

drial genomes, alike P. radiata mtDNA, expand over 100 kb in size

and may include over 50 protein-coding ORFs (Table 5). The
tendency for larger fungal mt genomes (over 90 kbp) in the
Basidiomycota subphylum Agaricomycotina is pinpointed by
Table 4. Intron-homing endonuclease domains and their location in the P. radiata mtDNA.

Min

recent reports on Moniliopthora perniciosa and M. roreri [24,28], T.

3.2

12
0

cingulata (over 90 kb) [26], A. bisporus (135 kbp) [23] and R. solani
(165 kbp/235 kbp) [50]. This tendency is furthermore confirmed
by our study on the 156 kbp P. radiata mtDNA showing up to 126
Mean

102
69

80
84

predicted protein-coding ORFs and 30 RNA genes, which are the

second highest numbers reported for the fungal mitochondrial
genomes.
Max
Length (aa)

115
181
112
136

The largest mtDNAs within Ascomycota are those of the

filamentous species Podospora anserina (over 100 kbp) and Chaeto-
mium thermophilum var. thermophilum (over 120 kbp), similar to many
Min
29
34
21
28

of the Agaricomycotina basidiomycete mtDNAs, when the

smallest (from Ascomycota yeasts) mt genomes are reduced to
about 20 kbp in size (Table 5) with only 10 protein-coding genes
Sum
28
19
10
57

[19]. Among Ascomycota fungi, however, there are yet large

variations at the genus level – between species - of both mtDNA
size and gene content. In the yeast S. cerevisiae, the 85.8 kbp
mtDNA harnesses 19 protein-coding genes when only the
mitochondrial Complex I NADH dehydrogenase subunit encod-
CL0324
CL0324
CL0418
Pfam

ing genes are absent [19,29,51]. In another species of Saccharomyces,

S. castellii, the mtDNA is reduced to 1/3 in size (25.7 kb) and
contains only 9 protein-coding genes [51].
Only slight differences, however, in mtDNA size, gene number
doi:10.1371/journal.pone.0097141.t004

and organisation have been observed in the Basidiomycota genera

Moniliophthora (Agaricomycotina) [24,28], Tilletia (Ustilaginomyco-
tina) [19], Phakopsora (Pucciniomycotina) [27], and Cryptococcus
(Agaricomycotina) [53,54], thus indicating genus-level conserva-
Homing endonuclease

tion of the mitochondrial genomes in the phylum Basidiomycota.

The large differences in gene order and location (loss of synteny)
Catalytic domain
LAGLIDADG-1
LAGLIDADG-2

between the Basidiomycota mitochondrial genomes at higher

taxon levels, as was reported for Ganoderma lucidum (Agaricomyco-
GIY-YIG

tina) mtDNA [55], and is observed in this study for P. radiata

mtDNA, may thus indicate frequent recombination events and
All

flexibility of fungal organelle genomes.

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 Mitochondrial Genome of the Basidiomycota Species Phlebia radiata

Figure 6. Dispersed and inverted repeat sequences in P. radiata mtDNA. Colors: black, conserved protein-coding, rRNA and tRNA genes;
grey, introns; white, intergenic regions. The large 6.2 kb duplication-inversion is excluded from the figures. A) Red ribbons connect regions of
significant (E-value ,161027) nucleotide sequence similarity. B) Coloured ribbons connect similar sequence regions. Only clusters with at least 6
similar repeat members are shown. The sequences were clustered as a function of similarity in CD-HIT Suite [44] (h-cd-hit-est run with consecutive
0.75, 0.80, and 0.90 cut-off values) from a sequence set that returned ,0.001 1 vs. 1 blastn values.
doi:10.1371/journal.pone.0097141.g006

Intergenic and Repetitive Sequences Introns and Homing Endonucleases

In the large angiosperm plant mt genomes, the genome size is Together with the high portion of the intergenic regions,
mainly due to long intergenic regions and non-coding sequence notable in P. radiata mtDNA is the degree of invasion by mobile
(gene spacers, introns, and pseudogenes) [20,22]. Accordingly in DNA-elements, which were recognized as long type I and II self-
the large P. radiata mtDNA, over 80% of the mt genome is either splicing introns (in total over 30 long introns), and including up to
intergenic or intronic sequence (Figure 2). Our analyses show that 57 HE domain-encoding ORFs. Nine of the 15 conserved protein-
most of the intergenic sequence space in the P. radiata mtDNA is coding genes, and the rnl gene in P. radiata mtDNA are invaded by
filled with repeated sequences, in particular between genome nt long introns carrying HE motifs of LAGLIDADG types I and II,
positions from 90 000 to 110 000 surrounding several tRNA- when the ten recognized GIY-YIG motifs correspond a minority
coding gene loci. Accumulation of polymorphic microsatellite of the HE domain types.
repeated elements (1–6 nt in length) were reported for species of Homing endonuclease genes were previously identified within
the Agaricomycotina genus Agrocybe [56], and already in S. cerevisiae mtDNAs of other Basidiomycota species, such as M. perniciosa and
mtDNA, long AT rich stretches were identified [29]. Putative roles M. roreri [24,28] and A. bisporus [23]. However, only four of the
in splicing of mitochondrial polycistronic transcripts may be protein-coding genes (cob, cox1,2, and nad5), and the rns and rnl
proposed for the intergenic regions, as well as action as potential genes were previously reported to contain self-splicing introns with
promoters with regulative element motifs. HE motifs in these fungal mtDNAs, when in the P. radiata mtDNA,
Surprisingly, the mtDNA of P. radiata contains a large (6.1 kbp) nine of the 11 intron-containing conserved genes contained
inverted duplication segment. This is another similar feature to the intronic HE domains. In the elongated cox1 gene of P. radiata,
angiosperm plant mitochondrial genomes, where large duplicated eleven LAGLIDADG and four GIY-YIG motifs were recognized
sequences in the mtDNA apparently function as co-linear in 13 long, self-splicing type I (12) and type II (1) introns. In regard
recombination sites to aid genome organization [21,22]. In the to this, even up to 18 introns, both type I and II, were recognized
Ascomycota species C. albicans, the very similar in size (7 kbp) in the almost 30 kb-size cox1 gene of A. bisporus [57].
inverted repeat regions in the apparently linear mtDNA are A few reports enlighten the enzymatic and molecular functions
directing genome replication via homologous recombination at the of intronic HEs in the fungal mtDNAs. In gene transcription, the
site [31]. Interestingly, the C. albicans mtDNA inverted repeat HE domains are apparently removed from the transcribed pre-
harnesses duplication of the gene cox3, whereas in the P. radiata mRNA resulting in a contiguous RNA transcript [58–60]. It is
mtDNA mirror site, we recognized duplication of the genes atp6 likely that existence of intron-homing endonucleases within fungal
and tRNA-IleGAU. Two inverted repeats of over 4 kbp in size were mtDNA genes is one apparatus for promoting genetic diversity
reported for another Agaricomycotina species, A. bisporus, devoid and adaptive response for the mitochondrial genome, when the
of protein-coding ORFs but containing duplicated sets of tRNA allelic recombination events may be impossible or rare due to the
genes [23]. At the moment, we may suggest a replication-directing mainly uniparental nature of mtDNA inheritance in fungi [10,17].
recombination function for the inverted duplication in the P. In the Ascomycota species Aspergillus nidulans, C-terminal
radiata mt genome, similar to that observed in C. albicans mtDNA fragment of the mtDNA cob gene intron-homing translated
[31]. LAGLIDADG type endonuclease (I-AniI) is involved in splicing

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 Mitochondrial Genome of the Basidiomycota Species Phlebia radiata

Table 5. Fungal mitochondrial genomes, representatives of Basidiomycota and other phyla. Phlebia radiata mtDNA, this study;
*R. solani 162 751 kbp [50], 235 849 kbp [19].

Fungal phylum Species Subphylum or class mtDNA sequence accession mtDNA size (bp)

Basidiomycota
Rhizoctonia solani Agaricomycotina NC_021436 *162 751
Phlebia radiata Agaricomycotina NC_020148 156 348
Agaricus bisporus Agaricomycotina JX271275 135 005
Lentinula edodes Agaricomycotina NC_018365 121 394
Moniliophthora perniciosa Agaricomycotina NC_005927 109 103
Moniliophthora roreri Agaricomycotina NC_015400 93 722
Trametes cingulata Agaricomycotina NC_013933 91 500
Flammulina velutipes Agaricomycotina NC_021373 88 508
Ganoderma sinense Agaricomycotina NC_022933 86 451
Pleurotus ostreatus Agaricomycotina NC_009905 73 242
Ganoderma lucidum Agaricomycotina NC_021750 60 630
Cantharellus cibarius Agaricomycotina NC_020368 58 656
Schizophyllum commune Agaricomycotina NC_003049 49 704
Cryptococcus neoformans var. grubii H99 Agaricomycotina NC_018792 24 919
Cryptococcus neoformans var. grubii Agaricomycotina NC_004336 24 874
Tilletia indica Ustilaginomycotina NC_009880 65 147
Tilletia walkeri Ustilaginomycotina NC_010651 59 352
Ustilago maydis Ustilaginomycotina NC_008368 56 814
Microbotryum lychnidis-dioicae Pucciniomycotina NC_020353 107 808
Microbotryum cf. violaceum Pucciniomycotina NC_020354 92 107
Phakopsora meibomiae Pucciniomycotina NC_014352 32 520
Phakopsora pachyrhizi Pucciniomycotina NC_014344 31 825
Ascomycota
Chaetomium thermophilum var. thermophilum Pezizomycotina NC_015893 127 206
Podospora anserina Pezizomycotina NC_001329 100 314
Fusarium graminearum (Gibberella zeae) Pezizomycotina NC_009493 95 676
Peltigera malacea Pezizomycotina NC_016955 63 363
Fusarium solani Pezizomycotina NC_016680 62 978
Phialocephala subalpina Pezizomycotina NC_015789 43 742
Nakaseomyces bacillisporus Saccharomycotina NC_012621 107 123
Saccharomyces cerevisiae Saccharomycotina NC_001224 85 779
Komagataella (Pichia) pastoris Saccharomycotina NC_015384 35 683
Glomeromycota
Gigaspora rosea Glomeromycetes NC_016985 97 350
Gigaspora margarita Glomeromycetes NC_016684 96 998
Glomus irregulare Glomeromycetes NC_014489 70 800
Glomus (Rhizophagus) intraradices Glomeromycetes NC_012056 70 606
Glomus cerebriforme Glomeromycetes NC_022144 59 633
Chytridiomycota
Spizellomyces punctatus Chytridiomycetes NC_003052 61 347
Rhizophydium sp. 136 Chytridiomycetes NC_003053 68 834
Monoblepharidomycota
Monoblepharella sp. Monoblepharidomycetes NC_004624 60 432
Hyaloraphidium curvatum Monoblepharidomycetes NC_003048 29 593
Harpochytrium sp. Monoblepharidomycetes NC_004623 24 169
Harpochytrium sp. Monoblepharidomycetes NC_004760 19 473
Blastocladiomycota
Allomyces macrogynus Blastocladiomycetes NC_001715 57 473

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 Mitochondrial Genome of the Basidiomycota Species Phlebia radiata

Table 5. Cont.

Fungal phylum Species Subphylum or class mtDNA sequence accession mtDNA size (bp)

Blastocladiella emersonii Blastocladiomycetes NC_011360 36 503

Cryptomycota
Rozella allomycis NC_021611 12 055

doi:10.1371/journal.pone.0097141.t005

of the intronic pre-mRNA, while the second, N-terminal 28,57]. In P. radiata mtDNA, cox1 is apparently the first replicated
endonuclease motif was not essential for intron splicing [58]. and transcribed gene in sense (leading strand) orientation
Instead, the N-terminal motif functioned in cleavage of the DNA (Figure 1). Whether regulation of P. radiata mtDNA gene
target site to initiate the HE intron mobilization. These reports on expression is mediated by the multiple introns and their identified
well-ordered and bi-functional effects of the intronic HEs imply HEs will be of our future concern.
their active involvement in supporting genetic flexibility in the
fungal mitochondrial genomes. Plasmid-originating Features
The HEs recognize longer DNA target regions (14–40 bp) than Although mitochondrial plasmids have been sequenced, and
common DNA-endonucleases and tolerate more sequence varia- plasmid-originating genes are identified in fungal mtDNAs [8,23–
tion, which assists in interrupting and introducing new genetic 28,50,63,64], our sequence analyses supported no individual
elements (usually introns) and ORFs (mainly intronic HE domains) plasmid dsDNA features in the P. radiata mtDNA. We were also
in their target sites [59,60]. In P. radiata mtDNA, two potential unable to detect integrated-plasmid like sequence regions with
examples (genes atp6 and cox2) of HE-transmitted introns and inverted repeat ends as has been reported for other Agaricomycota
alternative coding sequences, both C-terminal, are observed species, i.e. A. bisporus [23], M. perniciosa [24] and A. aegerita [64].
(Figure 3A, B). Unusual splicing of the mtDNA atp6 C-terminus However, putative and degenerative plasmid and viral-originating
was early on reported for the Blastocladiomycota species Allomyces features, such as the cob gene intron-located reverse transcriptase
macrogynus [61]. Additional intron with HE was found as an (RT, RNA-directed DNA polymerase), and DNA polymerase B
insertion including foreign C-terminal atp6 sequence fused in- encoding dpob genes were identified in the P. radiata mtDNA,
frame, which was explained by horizontal gene transfer [61]. thereby possibly indicating previous plasmid-transmitted DNA
However, a more likely explanation is that intronic HEs may have integration events. The mitochondrial type II intron-homing and
transposed to a new site downstream of the stop codon, as has retrovirus-related reverse transcriptase [65,66] may function in
apparently occurred in P. radiata atp6 leading to alternative C- plasmid integration to the mtDNA, and due to template-switching
termini, but in the case of A. macrogynus atp6, introducing somewhat capacity, intron loss and gain to the mitochondrial genomes may
altered homolog of the new C-terminus. occur.
Additional C-terminal coding sequence was likewise inserted
into the mitochondrial intronic rps3 gene of the Ascomycota tRNA Assembly, Codon Usage and Phylogeny
Ophiostoma novo-ulmi subsp. americana [62]. In the latter case, the HE Fungal and animal mitochondrial genomes generally have a
insertion apparently shifted a small portion of the rps3 coding single tRNA gene for each synonymous protein-coding codon
region downstream and disrupted the ORF with a premature stop [67]. This also applies to the mitochondria of Basidiomycota, and
codon. Accordingly, in the P. radiata cox2 gene, the duplicated C- implies extensive codon third nucleotide (wobble) pairing, similar
terminus is intervened by a 2.5 kb sequence containing catalytic to that observed for the tRNAs of the bacterium Mycoplasma
HE domains, and is fused after a premature stop codon in the first capricolum [68], i.e. the tRNA anticodon first nucleotide (anticodon
C-terminus. wobble) U pairs with all four third position nucleotides, and the
The group I and II type self-splicing introns and HEs are first anticodon G pairs with third codon position C or U
interlinked since the self-splicing introns need endonuclease nucleotides.
activity to assist splicing of the transcribed intronic RNA [58– Assuming that these codon/anticodon recognition rules apply,
60]. If the numerous HE motifs in the P. radiata mtDNA are active, the predicted tRNA set of P. radiata mtDNA is sufficient to
the homing processes may modify their target genes, and even the translate its conserved mitochondrial proteome, except for the
genome size and structure considerably - in the course of either a codons Ile AUA (274) and Trp UGA (1), which would require
long or short evolutionary time period - as is seen in the mt unusual ANG and ANC wobble-pairing to their respected tRNA
genomes of the animal-pathogenic Cryptococcus spp. [53,54]. anticodons. However, we infer from Basidiomycota tRNA-Met
Another function for the multiple HE domains could be regulation and tRNA-Ile multiple sequence alignments that one of the P.
of transcription of their target genes, which is observed for radiata mtDNA tRNAs with CAU anticodon is in fact a tRNA-Ile
bacterial viruses [60]. gene, and the predicted anticodon is likely edited. Likewise, based
Considering the density of group I type introns (26) and their on the lack of UGA codons in P. radiata mtDNA conserved ORFs,
high frequency of HE domains in the core genes of P. radiata presence of the canonical CCA anticodon in tRNA-Trp, and the
mtDNA, it is well established to expect that if functional, the HEs high bias towards low GC-content, we infer that P. radiata
could assist in intronic RNA splicing and thereby affect mitochondrial genome does not utilize the Mold, Protozoan, and
transcription of their target genes, with a possibility for alternative Coelenterate Mitochondrial Code (NCBI translation table 4) in
intron splicing. The mitochondrial Complex IV cytochrome which UGA encodes Trp.
oxidase subunit 1 encoding cox1 gene of P. radiata is particularly From the frequency of UGA codons in fungal mtDNA-encoded
interrupted by long self-splicing introns (13) containing multiple proteomes, we infer that UGA has been assigned to Trp multiple
HEs, as seemingly is general in Agaricomycota mtDNAs [24– times in the evolution of Basidiomycota mitochondrial genomes,

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 Mitochondrial Genome of the Basidiomycota Species Phlebia radiata

i.e. in the lineage leading to the Pucciniomycotina genus frequency of repetitive sequence motifs, and especially the
Phakopsora, and in the lineage leading to the Agaricomycotina abundance of intron-homing endonucleases support these conclu-
genus Moniliopthora, but not in the lineage leading to Phlebia. sions. Surprisingly, these features of P. radiata mtDNA, together
Likewise, we infer from sequence alignments of Basidiomycota with the large genome size, are shared with fungal, plant and algae
cox3 genes that in C. neoformans, UGA induces a +1 nt frameshift, mtDNAs.
which restores sequence homology for the last 16 codons of the Accurately characterized reference genomes including the
gene in reference to other Basidiomycota cox3 genes. We conclude mtDNAs are currently needed to aid in de novo sequencing and
that with this repertoire of mtDNA-encoded tRNAs, the evolutionary studies of fungi. The novel P. radiata mtDNA features
mitochondria of P. radiata do not require tRNA import from the observed in our research indicate a general phenomenon for
cytosol. evolutionary pressure and genome diversity in mitochondrial
genomes, not being as stable and compact integrities as previously
mtDNA Proteome Phylogeny considered. The fungal mtDNAs could thus serve as sources for
Maximum-likelihood and Bayesian inference approaches of the evolutionary and biochemical studies of genetic mobile elements,
Basidiomycota mtDNA-encoded proteomes resulted with well- intron loss and gain, virulence and adaptation, and targeted
supported and systematically consistent evolutionary trees in line genetic engineering by the use of homing endonucleases.
with current multigene-based fungal taxonomies [69,70], both in
respect to fungal phyla (-mycota) and within subphyla (-mycotina) Supporting Information
(Figure 5). P. radiata mt proteome grouped together with other
Agaricomycotina species, nearest to Ganoderma spp. and T. Table S1 Intronic and additional ORFs annotated in the
cingulata, which also belong to the same taxonomic class P. radiata mtDNA.
(Agaricomycetes) and share similar, wood-decaying white-rot (XLSX)
saprobic lifestyle with P. radiata. The opportunistic pathogen C. Table S2 ORFs continuing from coding sequence exons
neoformans was the only exception in protein phylogeny by falling into putative intronic regions.
outside the subphylum Agaricomycotina, which is consistent with (XLSX)
our mtDNA proteome ORF codon usage evolutionary analysis
(Figure 4). Multi-protein Bayesian evolutionary analysis positioned Table S3 Root mean squared difference distance ma-
the yet insertae sedis subphyla Kixcellomycotina (Zancudomyces trix of Basidiomycota conserved codons in the protein-
(Smittium) culisetae) nearest to Glomeromycota, and Mucoromyco- coding ORFs.
tina (Rhizopus oryzae) and Mortierellomycotina (Mortierella verticillata) (XLSX)
together between Glomeromycota and Dikarya (Figure 5). Oth- Table S4 Sum squared difference distance matrix of
erwise the relationships between extant taxons were well resolved, Basidiomycota conserved codons in the protein-coding
thus indicating a strong signal for a single common origin of the ORFs.
Basidiomycota and fungal mt genomes. (XLSX)
Table S5 Intronic homing endonuclease (HE) domains
Conclusions
in the P. radiata mtDNA.
Mitochondria are numerous in eukaryotic cells and thereby, (XLSX)
mitochondrial genomes as well have high cellular copy numbers.
Our study confirms the high degree of variety of fungal mtDNAs Acknowledgments
in genome structure and size, gene order and location, and exon-
intron structure of the protein-coding genes. This indicates that for The help of Ms Elina Kokkonen with statistical analyses is warmly thanked.
mtDNA, continuous and adaptive modifications are allowed,
including mobile genetic elements and signs of recombination Author Contributions
events. Several features in the P. radiata mtDNA support such Conceived and designed the experiments: TL LP. Performed the
genetic flexibility and repair mechanisms, and regulation of experiments: PL JK MM. Analyzed the data: HS IO PL. Contributed
transcription. Existence of the long inverted-duplicated region, reagents/materials/analysis tools: TL LP. Wrote the paper: TL HS.

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