Molecular Ecology Resources (2009) 9 (Suppl. 1), 99–113 doi: 10.1111/j.1755-0998.2009.02637.

BARCODING FUNGI
Blackwell Publishing Ltd

Evaluation of mitochondrial genes as DNA barcode for

Basidiomycota
A G AT H E V I A L L E ,1*† N I C O L A S F E A U ,1† M AT H I E U A L L A I R E ,† M A RY N A D I D U K H ,‡
F R A N C I S M A RT I N ,¶ J E A N - M A R C M O N C A LV O § and R I C H A R D C . H A M E L I N *†
*Centre d'étude de la forêt, Université Laval, QC, Canada G1 K 7P4, †Laurentian Forestry Centre, Canadian Forest Service, Natural
Resources Canada, 1055 du PEPS, PO Box 10380, Stn Sainte-Foy, QC, Canada G1V 4C7, ‡Department of Ecology and Evolutionary
Biology, University of Toronto, ON, Canada M5S 3B2, §Department of Natural History, Royal Ontario Museum, and Department of
Ecology and Evolutionary Biology, University of Toronto, ON, Canada M5S 2C6, ¶Unité Mixte de Recherche INRA/UHP 1136
‘Interactions Arbres/Microorganismes’, Institut National de la Recherche Agronomique, Centre de Recherches de Nancy, 54280
Champenoux, France

Abstract
Our study evaluated in silico the potential of 14 mitochondrial genes encoding the subunits
of the respiratory chain complexes, including cytochrome c oxidase I (CO1), as Basidiomycota
DNA barcode. Fifteen complete and partial mitochondrial genomes were recovered and char-
acterized in this study. Mitochondrial genes showed high values of molecular divergence,
indicating a potential for the resolution of lower-level relationships. However, numerous
introns occurred in CO1 as well as in six other genes, potentially interfering with polymerase
chain reaction amplification. Considering these results and given the minimal length of 600-bp
that is optimal for a fungal barcode, the genes encoding for the ATPase subunit 6, the cyto-
chrome oxidase subunit 3 and the NADH dehydrogenase subunit 6 have the most promising
characteristics for DNA barcoding among the mitochondrial genes studied. However, biolog-
ical validation on two fungal data sets indicated that no single mitochondrial gene gave a
better taxonomic resolution than the ITS, the region already widely used in fungal taxonomy.
Keywords: Basidiomycota, CO1, DNA barcoding, ITS, mitochondrial genes

Received 31 October 2008; revision received 26 January 2009; accepted 30 January 2009

DNA barcodes are short and standardized sequences of

Introduction
Basidiomycota is one of the major fungal phyla with more
than 30 000 species described and encompasses a broad range
of taxa, morphologies, ecologies and life-history strategies
(Kirk et al. 2001). Many recent molecular phylogenetic studies
using nuclear genes have significantly improved our under-
standing of higher taxonomic level evolutionary relationships
in this phylum (Lutzoni et al. 2004; James et al. 2006; Hibbet
et al. 2007). However, species delimitation and identification
remain problematic in many groups, particularly among
morphologically similar taxa, for example in the Puccinio-
mycotina and Ustilaginomycotina (Stoll et al. 2003; Bridge
et al. 2005; Aime et al. 2006).
Correspondence: Richard C. Hamelin, Fax: 001 418648 5849;
E-mail: Richard.hamelin@ubc.ca
1
Equal contributors
nucleotides from a genomic region universally present in
target lineages and exhibiting sufficient sequence variation
to distinguish species (Hebert et al. 2003; Waugh 2007). DNA
barcoding promises fast, economic and easy identification
of species (Hebert et al. 2003; Hajibabaei et al. 2005). The
selection of a barcode locus is a compromise between the
possibility to design universal DNA primers for polymerase
chain reaction (PCR) amplification and the need for maximal
molecular evolution rates for sufficient discrimination
between closely related taxa. Fungal molecular systematics
has relied heavily on analysis of the nuclear ribosomal RNA
(rRNA) cluster that comprises the small (18S) and large (28S)
ribosomal subunits (Bridge et al. 2005). In general terms,
sequence divergence levels in the 18S and 28S usually allow
differentiation of higher taxonomic levels such as families
and genera while polymorphisms in the internal transcribed
spacer regions (ITS) generally differentiate between species

© 2009 Blackwell Publishing Ltd and Crown in the right of Canada

100 B A R C O D I N G F U N G I
(Bruns et al. 1991; Bridge et al. 2005). The ITS can easily be
amplified from many fungal taxa using a limited set of
primers (White et al. 1990; Gardes & Bruns 1993). Further-
more, based on the finding that differences among species
are generally consistently larger than those within species,
ITS sequences have been frequently utilized for fungal taxa
delimitation and identification (Bruns et al. 1991; Guarro et al.
1999; Bruns 2001; Katsu et al. 2004). This level of polymorph-
ism makes the ITS a logical candidate for DNA barcoding of
fungi (Zeng & De Hoog 2008).
However, utilization of this region for taxonomic and phylo-
genetic purposes in fungi can present some limitations.
On the one hand, a lack of variation among closely related
species has been observed even when the species’ borders
were well defined and supported by other molecular, mor-
phological and/or biological data. For example, molecular
taxonomic studies have demonstrated the inefficiency of the
ITS to resolve well-characterized species in Heterobasidion,
Armillaria, Fusarium and Penicillium genera (Chillali et al.
1998; Skouboe et al. 1999; Bruns 2001; Seifert et al. 2007).
Furthermore, in some cases, the ITS regions could not reveal
known cryptic species in fungal species complexes (Tian
et al. 2004; Seifert et al. 2007); the resolution of these taxo-
nomically challenging and economically important groups
should be one of the most important attributes of a fungal
DNA barcode. On the other hand, intraspecific and even
intra-individual variations have been found in some fungal
groups for these loci (O’Donnell & Cigelnik 1997; Aanen
et al. 2001; Okabe & Matsumoto 2003; Lim et al. 2008). Such
polymorphisms in the ITS can either result from differences
within nuclei (heterogeneity among repeats) or from differ-
ences between nuclei (dikaryotic and multinucleate fungi)
(Aanen et al. 2001; Okabe & Matsumoto 2003). Heterogeneity
among repeats creates complications for direct sequencing
of ITS- PCR products because of the occurrence of multiple
different ITS copies in a single fungal isolate (Aanen et al.
2001; Matheny et al. 2007) and could makes it difficult to
accurately define taxonomic groups at the species level
(Aanen et al. 2001; Smith et al. 2007).
Mitochondrial DNA (mtDNA) has a simple genetic struc-
ture, a limited exposure to genetic recombination, and rapid
rates of evolution compared with nuclear DNA (Xu & Singh
2005; Waugh 2007) and is thus well suited to design mole-
cular markers for the study of closely related taxa (Hebert et al.
2003). A 648-bp region at the 5′-end of the mitochondrial
gene encoding cytochrome c oxidase I (CO1-5′) has been
proposed as the core of global bio-identification systems for
eukaryotes (Hebert et al. 2003; Hebert & Gregory 2005;
Waugh 2007). CO1-5′ appears to possess a greater potential
as species-specific marker than any other mitochondrial gene
as confirmed by taxonomic resolution (> 95%) in most
Metazoa groups, including Lepidoptera and birds (Hebert
et al. 2003, 2004a, b; Cywinska et al. 2006). Promising results
have recently been published using mitochondrial coding
© 2009 Blackwell Publishing Ltd and Crown in the right of Canada
gene regions, alone or concatenated, for fungal taxonomy
and systematics (Paquin et al. 1997; Grasso et al. 2006; Seifert
et al. 2007). The CO1 gene was suitable as a barcode for dis-
criminating fungal species in Penicillium, a taxonomically
challenging genus within ascomycetous fungi (Min & Hickey
2007; Seifert et al. 2007). Nevertheless, despite this specific
case study, the use of CO1-5′ as a DNA barcoding region for
delimiting closely related fungal species has not been exten-
sively studied. Some characteristics of the fungal mito-
chondrial genome represent significant obstacles for the
elaboration of a barcode. Copies of mtDNA genes (or gene
regions) can occur in the nuclear genome [the so-called
nuclear mitochondrial DNA (NUMT)] (Burger et al. 2003;
Richly & Leister 2004; Song et al. 2008) and in the mitochon-
drial genome (Martin et al. 2007). Group I or II introns are
present in the mitochondrial genes with variable fre-
quencies depending on the species sampled, with a strong
preference for protein-coding genes (Paquin et al. 1997;
Burger et al. 2003; Yan & Xu 2005). Furthermore, functional
constraints, unequal substitution patterns and hetero-
geneous evolutionary rates found in mtDNA genes can
potentially affect its usefulness for species delimitation
(Paquin et al. 1997; Roe & Sperling 2007).
In this study, we present a bioinformatics approach that
aims to evaluate the potential of 14 mitochondrial genes
commonly found in fungal mitochondrial genomes as DNA
barcodes for the Basidiomycota. These genes encode hydro-
phobic subunits of the respiratory chain complexes I, III and
IV [including apocytochrome b (cob); cytochrome oxidase
subunits 1, 2 and 3 (CO1 to CO3); NADH dehydrogenase
subunits 1, 2, 3, 4, 4L, 5 and 6 (nad1 to nad6, nad4L), and
ATPase subunits 6, 8 and 9 (atp6, atp8 and atp9)]. Our strat-
egy first consisted in carrying out an inventory of the pub-
licly available mitochondrial sequence data for this group
of fungi to recover a maximum of complete mitochondrial
genomes. We evaluated the structure of each coding gene
to assess (i) intron position and size; (ii) the occurrence of
partial or full length copies of the gene in both nuclear and
mtDNA; and (iii) the information provided in the context
of barcoding. We then assessed and compared the poten-
tial for species level taxonomic resolution with the resolu-
tion obtained for the ITS and 28S loci by sequencing 38
strains within the Chrysomyxa and Melampsora genera
which represent taxonomically challenging groups in
Pucciniomycotina.
Materials and methods
Recovery of mtDNA genome sequences and protein coding
genes for Basidiomycota
Three strategies were designed to recover the entire mito-
chondrial genome of 17 Basidiomycota species from various
public databases (Fig. 1). In the first strategy (Fig. 1-1), six

complete and annotated mtDNA genomes [Cryptococcus
neoformans var. grubii (strain H99), Moniliophthora perniciosa,
Pleurotus ostreatus, Schizophyllum commune, Tilletia indica and
Ustilago maydis] were obtained from the National Center
for Biotechnology Information (NCBI) Genomes databases
[http://www.ncbi.nlm.nih.gov/genomes/ORGANELLES/
fu.html], and two complete but non-annotated genomes
[C. neoformans var. neoformans (strain JEC21) and Puccinia
graminis f. sp. tritici] were obtained from the Stanford Genome
Technology Center and the Broad Institute websites, respec-
tively. The second strategy (Fig. 1-2) was aimed at recovering
sequence contigs that correspond to the mitochondrial gen-
ome of Coprinus cinereus and Cryptococcus neoformans var.
gattii (strain R265) within assembled contigs from whole
genomes. The available expressed sequence tags (ESTs) for
C. cinereus (15 715 ESTs) were screened (tblastx, E-value
threshold at 1e20) for the presence of mitochondrial ESTs
using a set of mitochondrial protein coding genes as well as
rRNA and tRNA of C. neoformans var. grubii (strain H99),
M. perniciosa, S. commune and U. maydis as queries. The
identification of the corresponding mitochondrial contig
was performed by searching the C. cinereus whole-genome
sequence using the tblastx program (E-value threshold
at 1e20) and submitting each EST recovered in the last
search. Likewise, the corresponding mitochondrial contig of
C. neoformans var. gattii (strain R265) was identified using
C. neoformans var. grubii (strain H99) mitochondrial genes as
© 2009 Blackwell Publishing Ltd and Crown in the right of Canada
B A R C O D I N G F U N G I 101
Fig. 1 Bioinformatic process showing three
strategies designed to recover fungal mitoch-
ondrial genomes. Grey boxes correspond to
available data at the origin of each strategy.
Plain line boxes present the tools (query
genes, blast algorithm, program) used to
recover mitochondrial genomes in the
different strategies. Dotted line boxes show
possible output of the different tool boxes
during the mitochondrial genome assembly.
query. The third strategy (Fig. 1-3) consisted in the construc-
tion of a mitochondrial genome assembly for seven other
species (Laccaria bicolor (Martin et al. 2008), Phanerochaete
chrysosporium, Phakopsora pachyrhizi, Postia placenta, Sporidio-
bolus salmonicolor, Sporobolomyces roseus and Ustilago hordei)
using available whole-genome shotgun sequences (WGS)
recovered from Trace Archive databases at the NCBI website
(http://www.ncbi.nlm.nih.gov/Traces/trace.cgi?). For the
latter fungi, all traces were assembled using the Pcap.Rep
program (Huang et al. 2006) with default parameters. Each
whole-genome assembly obtained was screened for potential
mitochondrial contig(s) using the method described above
(strategy 2). If more than one single contig was retrieved, a
new assembly was performed with the Cap3 program imple-
mented with default parameters (Huang & Madan 1999).
In order to improve the comprehensiveness of this last mito-
chondrial genome assembly, an additional blastn search
(E-value threshold at 1e20) was performed against the Trace
Archive database using the 14 genes encoding subunits of
the respiratory chain complexes of each complete and
annotated mtDNA genomes [C. neoformans (strain H99),
M. perniciosa, P. ostreatus, S. commune and U. maydis] as queries.
All similar sequences retrieved by this search were appended
to the previous assembly with one additional run of Cap3
contig assembly (Fig. 1, step 4).
In addition, 24 mitochondrial-protein encoding sequences
(complete CDS features) for nine strains of eight other
102 B A R C O D I N G F U N G I
Basidiomycota species [Agaricus bitorquis, Agrocybe aegerita,
Agrocybe chaxingu, Cryptococcus neoformans var. neoformans
(strain IFM5844), Cryptococcus neoformans var. grubii (strain
IFO410), Rhodotorula glutinis, Suillus luteus, Suillus sinus-
paulianus and Trimorphomyces papilionaceus] were directly
downloaded from the NCBI website. All the sequence data
considered in this study are detailed in Table 1. The
sequence alignments obtained for the 14 mitochondrial
genes encoding subunits of the respiratory chain com-
plexes are available in Fig. S1, Supporting information.
Characterization of 14 genes encoding the subunits of the
respiratory chain complexes
Fourteen genes encoding subunits of the respiratory chain
complexes that are typically present in the mitochondrion
of Basidiomycota were analysed. Each mitochondrial gene
was individually edited in BioEdit version 7.0.0 (Hall 1999)
and characterized as follows. First, the protein-coding,
intronic and intergenic regions were localized based on
multiple comparisons with both the available annotations
of the S. commune (NC003049) and U. maydis (NC008368)
mitochondrial genomes. These multiple comparisons were
performed using the Shuffle-LAGAN program (Brudno
et al. 2003) accessible on the mVISTA website (http://
genome.lbl.gov/vista/mvista/submit.shtml) (Mayor et al.
2000). Each intronic region localized was characterized by
specific protein domain identification and intron secondary
structure modelling. The conserved sequence domains
found in group-I intron endonucleases [e.g. LAGLIDADG
1 and 2 (Dalgaard et al. 1997; Heath et al. 1997) and GIY-YIG
(Tian et al. 1991; Paquin et al. 1995)] and group-II intron
maturases [domain X (Mohr et al. 1993)] were identified by
HMM searches (Hmmer version 2.32; E-value ≤ 1e-02) against
the Pfam database (Eddy 1998; Bateman et al. 2004). Then,
the secondary structures of groups I and II introns were
predicted using the ERPIN search algorithm implemented
in the RNAweasel prediction tool (http://megasun.bch.
umontreal.ca/RNAweasel/) (Lang et al. 2007). Second, tRNA
contents were predicted with the tRNAscan-SE version 1.21
program (http://lowelab.ucsc.edu/tRNAscan-SE/) using
the default search mode and mitochondrial models as source
(Lowe & Eddy 1997).
Presence of mtDNA copies in nuclear and mitochondrial
genomes
mtDNA copies longer than 20 nt were inferred from blastn
hits (E-value < 0.01). For each fungus, each of the 14 genes
encoding subunits of the respiratory chain complexes (com-
plete gene sequence including both exonic and intronic
regions) was used as the query sequence to search against
its own mitochondrial genome and (when available) its own
complete genome assembly.
© 2009 Blackwell Publishing Ltd and Crown in the right of Canada
Pattern of evolution of 14 mitochondrial genes
The 14 genes encoding subunits of the respiratory chain
complexes were initially considered. For comparison, align-
ments of 18S (1699 nucleotides) and 28S (593 nucleotides
located at the 5′-end of the gene) sequences obtained from
the public databases for the same Basidiomycota taxa were
included (Table 1). Orthologous sequences were confirmed
with the Aspergillus niger (strain N909) mitochondrion com-
plete genome sequence (DQ207726) to serve as outgroup.
Inserts were excluded and short gaps were coded as missing
data. For each gene alignment, Kimura 2-parameter distances
(K2P; Kimura 1980) between sequences were computed
pairwise using paup version 4.0b10 for Unix (Swofford 2003).
Statistical significance of differences between distance distri-
butions obtained for the 14 genes was determined by one-
way anova followed by a Tukey test using the r statistical
package (Ihako & Gentleman 1996).
The best-fit maximum-likelihood (ML) model of sequence
evolution was identified for each of the 14 gene alignments
using the likelihood ratio test implemented in the Model-
Test version 3.7 program for Unix (Posada & Crandall 1998).
The parameters allowed to vary in model fitting were base
composition, substitution rates [variation in transition/
transversion (ti/tv) ratio] and rate of heterogeneity across
sites (by both the invariable-sites model and the gamma-
distributed rates model).
Efficiency of the mitochondrial DNA barcode candidates
To investigate the efficiency of the mitochondrial DNA
barcode candidates, we concentrated on the resolution of
species of the Melampsora and Chrysomyxa genera that
cause rust diseases on plants. Specimens were obtained
from fresh collections and from three national Canadian
herbaria. We considered 15 Melampsora specimens representing
five Melampsora species (one to five specimens per species)
collected on aspen and white poplars, and 23 specimens of
Chrysomyxa collected on spruce and Ericaceae (10 species,
1 to 3 specimens per species). Species identification was
based on morphological traits and specificity to the plant
host.
Total genomic DNA was extracted using a modified
protocol of the DNeasy Plant Mini kit (QIAGEN). For each
specimen, a single sorus (uredinium or aecium) was excised
from the infected host tissues. Infected plant material was
then incubated for 2 h at 55 °C in 500 µL of lysis buffer with
10 µL of Proteinase K before using the manufacturer’s protocol.
Full length ITS sequences (~620 bp) were amplified using
primers ITS1F (Gardes & Bruns 1993) and ITS4BR (5′-
TCAACAGACTTGTACATGGTCC-3′) for Melampsora. A
730-bp ITS sequence of the Chrysomyxa specimen was ampli-
fied using two primer pairs, ITS1F (Gardes & Bruns 1993)/
ITS2R2 (5′-GACACTCAAACAGGTGTACCTT-3′) and
© 2009 Blackwell Publishing Ltd and Crown in the right of Canada

Table 1 Available data for the Basidiomycota considered in this study

Publicly available data

Whole genome Complete Mitochondrial Trace

sequences genome genome archive Source and/or GenBank Accession no.
Taxa shotgun assembly assembly file 28S 18S (in parentheses)

Available genome sequences

Coprinus cinereus
Cryptococcus neoformans
var. neoformans (strain JEC21)
Cryptococcus neoformans
var. grubii (strain H99)
Cryptococcus neoformans
var. gattii (strain R265)
Laccaria bicolor
Moniliophthora perniciosa
Phakopsora pachyrhizi
Phanerochaete chrysosporium
Pleurotus ostreatus
Postia placenta
Puccinia graminis
Schizophyllum commune
Sporidiobolus salmonicolor
Sporobolomyces roseus
Tilletia indica
Ustilago hordei
Ustilago maydis
X X X Broad Institute/(AF041494)/(M92991)
X X (not annotated) X X NCBI/Stanford Genome Technology Center (SGTC)
X X X X X Broad Institute/(NC004336)/(AJ560308)
X X X X Broad Institute
X X X X Join Genome Institute (JGI)/NCBI (WGS)/(DQ071702)
X X X (NC005927)/(AY916742)/(AY916739)
X X X NCBI (WGS)/(DQ354537)
X X JGI/NCBI (WGS)
X X X (EF204913)/(DQ071722)/(AY657015)
X X (incomplete) X X X JGI/NCBI (WGS)/(AF139970)
X X X (not annotated) X X Broad Institute/(DQ417387)/(AY125409)
X X X (NC003049)/(DQ071725)/(X54865)
X NCBI (WGS)
X X JGI/NCBI (WGS)
X X (DQ993184)/(AY818977)
X NCBI (WGS)
X X X X Broad Institute/(NC008368)/(AF453938)/(X62396)

Additional corresponding mitochondrial gene sequences

Agaricus bitorquis atp6 (U60235)
Agrocybe aegerita CO1 (AF010257)
cob (AY781064)
Agrocybe chaxingu cob (AY772389)
Cryptococcus neoformans var. atp6 (AY560609)
neoformans (strain IFM 5844) atp9 (AY560609)
CO1 (AY560609)
CO2 (AY138989)
nad6 (AF533432)
Cryptococcus neoformans var. atp6 (AY560610)
grubii (strain IFO410) atp9 (AY560610)
CO1 (AY560610)

B A R C O D I N G F U N G I 103
CO2 (AF534130)
cob (AY560608)
nad4 L (AF538353)
Rhodotorula glutinis atp8 (AB248915)
atp9 (AB248915)
CO3 (AB248915)
nad5 (AB248915)
nad6 (AB248915)
Suillus luteus atp6 (AF002135)
Suillus sinuspaulianus CO3 (AF002136)
Trimorphomyces papilionaceus cob (X85236)
nad1 (X73821)
104 B A R C O D I N G F U N G I
ITS3R2 (5′-AAGGTACACCTGTTTGAGTGTC-3′)/ITS4BR
(5′-TCAACAGACTTGTACATGGTCC-3′). A 740-bp of 28S
sequence was amplified using the ITS4-BRf (5′-GGACCA
TGTACAAGTCTGTTGA-3′) and LR5 primers (Vilgalys
& Hester 1990) for both Melampsora and Chrysomyxa. PCRs
were carried out in a 25-µL reaction volume consisting of
1 µl of undiluted DNA template, 0.35 µm of each primer,
0.2 mm of each dNTPs (GE Healthcare), 1.6 mm MgCl2,
1 µg/µL of BSA, and 1 U of Platinum Taq DNA polymerase
(Invitrogen) in a 1× Taq DNA polymerase buffer (20 mm
Tris-HCl, pH 8, 50 mm KCl), with thermocycling conditions
as follows: denaturation for 3 min at 94 °C, 35 cycles at
94 °C for 30 s, 30 s at 50 °C, and 70 s at 72 °C with a final
extension of 10 min at 72 °C.
Primer pairs were designed for CO1-5′, CO3, atp6 and
nad6. For Melampsora spp., two primer pairs, Cox1MlpAF
(5′-TAAGATGACTTTATAGTACCAA-3′)/Cox1MlpAR (5′-
GCTCCTACCATTACMG-3′) and Cox1MlpB2F (5′-CTGCT
ATGCCCAAGTCTAA-3′)/Cox1MlpCR (5′-ATGTGATGAC
TTCAAACCAC-3′), were used for the amplification of two
composite non-overlapping regions located at the 5′-end of
the CO1 gene. One primer pair, Nad6MLP1F (5′-ATGAAT
TGAGCTCTAAATACCATCT-3′) and Nad6MLP1R (5′-TTG
TCACTTGTCATTACAATAGG-3′), was used for the ampli-
fication of 500 bp of the nad6 gene. Around 660 bp of the
CO3 gene was amplified with CO3_F1(5′-TCAGTATGTT
ATTTTAACGATGTAG-3′) and CO3_R1(5′-TCCTCATCAG
TAAACACTAATA-3′), and 650 bp of the atp6 gene was
amplified with the atp6_F1(5′-TAGAGCAATTTGAAGTTC
AGAATCT-3′)/atp6_R1(5′-GATGAATGATACTGCGATC
TCT-3′) primer pair.
For the Chrysomyxa specimens, Cox1MlpAF/Cox1MlpAR
and Cox1MlpB2F/Cox1MlpCR primers pairs have been tested
but only a short fragment of the CO1 gene was amplified
with the C30 (5′-GCARTTCTRTATTTTGTATTTGG-3′)/
C346CR (5′-CGCWCCTACTAYTASHGG-3′) primer pair. A
450-bp fragment from the mitochondrial nad6 was amplified
using the 23 N6f (5′-CCAGTAACGTCWGTAGTRTATC-3′)
and 504 N6 r (5′-GCAGRAATACRAAAGAGGC-3′) primer
pair. The CO3_F1/CO3_R1 and the atp6_F1/atp6_R1 primer
pairs were used to amplify 660 bp of the CO3 gene and
650 bp of the atp6 gene respectively.
All PCRs of mitochondrial genes were carried out in a
20-µL reaction volume consisting of 1 µL of undiluted DNA
template, 0.2 µm of each primer, 0.15 mm of each dNTPs
(GE Healthcare), 1.5 mm MgCl2, and 1 U of Platinum Taq
DNA polymerase (Invitrogen) in a 1× Taq DNA polymerase
buffer (20 mm Tris-HCl, pH 8, 50 mm KCl), with conditions
as follows: denaturation for 3 min at 95 °C, 36 cycles at
95 °C for 45 s, 47 °C for 30 s with primer pair Cox1MlpAF/
Cox1MlpAR, Nad6MLP1F/Nad6MLP1R, CO3_F1/CO3_R,
atp6_F1/atp6_R1 and C30/C346 or 50 °C for 30 s with
Cox1MlpB2F/Cox1MlpCR and 23 N6f/504 N6 r, and 72 °C
for 70 s. PCR products were visualized by ultraviolet
© 2009 Blackwell Publishing Ltd and Crown in the right of Canada
fluorescence following 1.5% agarose gel eletrophoresis in
1× TAE buffer and ethidium bromide staining. Sequencing
was then performed in both directions with the appropriate
amplification primers using the Big Dye Terminator Cycle
Sequencing Kit version 1.1 on an ABI 3730xl sequencer
(Applied Biosystems) at the CHUL Research Centre
(CRCHUL) Sequencing and Genotyping Platform, Quebec
City, QC, Canada.
Sequences were manually edited using BioEdit version
7.0.9 (Hall 1999) to remove ambiguous base calls and primer
sequences, and were aligned using the ClustalW software
(Thompson et al. 1994). For each data set obtained, we used
the methods previously described to determine K2P intra-
and interspecific genetic distances between pairwise sequ-
ences among each data set using paup. Dendrograms were
constructed using the neighbour-joining algorithm based
on the K2P-distance matrices, as described in Hebert et al.
(2003) and Seifert et al. (2007). A barcode species is defined
if all of its constituent sequences formed a monophyletic
cluster corresponding with morphological and host affinity
traits. Specimen information, including sequence GenBank
Accession numbers, are presented in Table S1, Supporting
information, and are available in the Barcode of Life Data-
base (http://www.barcodinglife.org). Dendrograms obtained
for this study are presented in Fig. S2, Supporting information.
Results and discussion
Characterization of mitochondrial genomes of
Basidiomycota
A total of 15 mitochondrial genomes were retrieved (Table 2),
among which seven [Coprinus cinereus, Cryptococcus neofor-
mans var. gattii (strain R265), Laccaria bicolor, Phanerochaete
chrysosporium, Phakopsora pachyrhizi, Postia placenta and Sporo-
bolomyces roseus] were recovered using the reconstruction
strategies 2, 3 and 4 of our bioinformatic process (Fig. 1).
However, we were unable to construct comprehensive mito-
chondrial contigs from the traces available for Sporidiobolus
salmonicolor (24 879 traces) and Ustilago hordei (1607 traces).
These mitochondrial sequences were consequently excluded
from the assembly by the Pcap.Rep program.
Specific features common to mtDNA were observed in
the mitochondrial genomes (Klich & Mullaney 1992; Yan &
Xu 2005). First, the overall G + C content found in the mito-
chondrial contigs ranged between 21.9% and 37.78%, with
a mean of 31.08%, whereas the G + C content in nuclear
contigs oscillated around 50% (see Fig. S3, Supporting infor-
mation). Second, mitochondrial genome size ranged from
24.8 kb for C. neoformans var. grubii (strain H99) to more than
100 kb for Moniliophthora perniciosa and P. placenta (Table 2).
Such length differences can be explained by the high vari-
ation in gene and intron numbers in mitochondrial genomes.
For example, the ribosomal protein of the small ribosomal
 B A R C O D I N G F U N G I 105

Table 2 General features of mitochondrial genomes for the Basidiomycota considered in this study

Other features* Insert

No. of No of
Final putative putative
assembly No. of G+C No. of group-I group-II
Species size (nt) contigs content % rns§ rnl§ tRNA Rps3§ Total intron intron

C. cinereus‡ 42 448 1 28.30 √ √ 24 √ 1 1 0

C. neoformans var. neoformans 33 199 1 34.80 √ √ 21 √ 9 9 0
(strain JEC21)
C. neoformans var. grubii 24 874 1 34.98 √ √ 21 — 2 2 0
(strain H99)
C. neoformans var. gattii 34 790 1 33.86 √ √ 21 √ 7 7 0
(strain R265)‡
L. bicolor‡ 95 303 1 28.32 √ √ 25 √ 7 7 0
M. perniciosa 109 103 1 31.89 √ √ 28 √ 13 13 0
P. pachyrhizi‡† 35 070 1 34.72 √ √ 24 — 5 5 0
P. chrysosporium‡† 40 093 12 28.76 √ — 3 — ... ... ...
P. ostreatus 73 242 1 26.35 √ √ 26 √ 9 9 0
P. placenta‡† 114 196 4 27.34 √ √ 23 — 17 14 1
P. graminis 79 448 1 37.17 √ √ 22 √ 13 12 1
S. commune 49 704 1 21.86 √ √ 27 √ 0 0 0
T. indica 65 147 1 28.86 √ √ 24 — 9 7 0
S. roseus‡† 45 385 26 37.78 √ √ 19 — 1 1 0
U. maydis 56 814 1 31.20 √ √ 23 √ 10 10 0

*in addition to the 14 genes encoding the hydrophobic subunits of the respiratory chain complexes common to fungal mitochondrial
genomes CO1–2, cob, nad1–6, atp6–9
†incomplete mitochondrial genome sequence, some mitochondrial protein-coding genes not retrieved
‡genome recovered using the reconstruction strategies 2, 3 and 4 of the bioinformatic process described in this study (Fig. 1)
(...) missing data
§target gene (√) retrieved or (—) not retrieved

subunit (rps3) was identified in only 9 out of the 15 mito-
chondrial genomes screened. The set of mitochondrial tRNAs
ranged from 3 tRNA genes for P. chrysosporium to 28 in the
M. perniciosa mitochondrial genome. All 14 genes encoding
the hydrophobic subunits of the respiratory chain complexes
could be localized in all studied genomes, except atp8 in
P. chrysosporium, CO2 in P. placenta and nad6 in P. pachyrhizi
and S. roseus. Similarly, genes encoding the 23S and 16S
ribosomal RNAs of the large and small subunits of the ribo-
some (rnl and rns, respectively) were present in all the mtDNA
genomes investigated, except rnl absent in P. chrysosporium.
Such differences in gene patterns among different mito-
chondrial genomes reflected either the absence of one specific
gene in the mitochondrial genome, as previously reported
for the atp9 gene in Podospora anserina (Yan & Xu 2005), or the
limitation of the data-mining and reconstruction methods
applied to incomplete Trace Archive data sets. The mitochon-
drial genomes of P. placenta (CO2 and rps3 missing), S. roseus
(nad6 and rps3 missing, atp9 incomplete) and P. chrysosporium
(atp8, rnl and rps3 missing, CO1, CO2, CO3, cob, nad1 and
nad5 incomplete) are most likely incomplete since they
resulted from the concatenation of 4, 26 and 12 non-overlapping
contigs, for a total size of 114.1, 45.4 and 40.1 kb, respec-
tively (Table 2).
Characterization of 14 genes encoding the subunits of the
respiratory chain complexes
Half of the 14 genes encoding the subunits of the respiratory
chain complexes (CO1 and CO2, cob, nad1, nad2, nad4 and
nad5) contained insert locations, ranging from one (in nad2)
to 18 (in CO1), for a total of 42 (Fig. 2). One hundred and
twelve of these inserts were identified as probable group I
introns, among which 97 contained putative open reading
frames (ORFs) encoding LAGLIDADG or GIY-YIG endonu-
clease proteins. Two group II introns were also found in this
data set. The insert retrieved in the nad4 gene of P. graminis
contained one ORF which encodes a group II intron maturase.
Likewise, based on secondary structure prediction, the 11th
CO1 insert found in P. placenta was identified as a group-II
intron. Interestingly, this last putative group-II intron poss-
esses an ORF which encodes a LAGLIDADG endonuclease
identical to those found in group I introns. Such a family of
group-II introns, e.g. encoding LAGLIDADG ORFs typical

© 2009 Blackwell Publishing Ltd and Crown in the right of Canada

 106 B A R C O D I N G F U N G I
© 2009 Blackwell Publishing Ltd and Crown in the right of Canada

Fig. 2 Number, position and characterization of inserts in seven mitochondrial genes encoding subunits of the respiratory chain complexes.
 B A R C O D I N G F U N G I 107

Table 3 Properties of the 14 genes encoding subunits of the respiratory chain complexes genes

No. of No. of Maximal

fungal taxon No. of Length of insert(s) + No. of significant
strain with insert(s) continuous exons length copy(ies) [length No. of complete
Gene considered insert(s) location(s) exons (species) variation(nt)] copy(ies) (species)

atp6 18 0 0– 718–865 Mit: 6 (20–760) 1 (S. roseus)

CGA: 5 (32–724) 1 (P. graminis)
atp8 15 0 0 144–62 Mit: 4 (20–162)
CGA: 4 (49–146) 1 (P. graminis)
1 (C. neoformans H99)
atp9 16 0 0 205–222 Mit: 3 (23–57)
CGA: 2 (43–125)
CO1 17 11 18 11–1583 16 446 (P. placenta) Mit: 222* (20–3910)
CGA: 55 (28–1579) 1 (C. neoformans H99)
CO2 15 3 3 68–759 2800 (M. perniciosa) Mit: 162*(20–708) 3 (S. roseus)
CGA: 8 (25–1481) 1 (P. graminis)
CO3 16 0 0 796–839 Mit: 1 (47)
CGA: 10 (28–104)
cob 18 17 9 10–1192 9158 (P. placenta) Mit: 279* (20–920)
CGA: 32 (28–2157) 1 (C. neoformans H99)
nad1 15 6 4 110–997 2106 (C. neoformans H99) Mit: 156* (20–133)
CGA: 9 (23–1509) 1 (C. neoformans H99)
nad2 15 1 1 711–1962 3087 (P. placenta) Mit: 29 (20–1283)
CGA: 14 (30–1502) 1 (C. neoformans H99)
nad3 15 0 0 326–375 Mit: 11 (20–375) 1 (P. graminis) 2 (S. roseus)
CGA: 2 (42–82)
nad4L 17 0 0 262–270 Mit: 6 (21–263) 2 (S. roseus)
CGA: 6 (30–183)
nad4 15 2 2 173–1467 3778 (P. graminis) Mit: 8 (23–1110)
CGA: 12 (29–173)
nad5 12 5 4 169–2064 5449 (P. placenta) Mit: 98*(20–1114)
CGA: 31 (21–1028)
nad6 15 0 0 579–757 885 (T. indica) Mit: 7 (21–24)
CGA: 4 (40–622) 1 (C. neoformans H99)

*Multiple copies of a short DNA fragment; CGA complete genome assembly.

of group-I introns has been observed in the rnl and rns genes
of Agrocybe aegerita and Trimorphomyces papilionaceus, respec-
tively (Toor & Zimmerly 2002). Patterns of presence/absence
for these ORFs resulted in significant length variations for
the different inserts found in those genes (from 200 bp for
the S. roseus insert in cob to 4635 bp for the third C. neoformans
(strain R265) insert in CO1).
As observed in other fungal mtDNA studies (Vaughn et al.
1995; Paquin et al. 1997), we confirmed the prevalence of
introns in the CO1 and cob genes. We furthermore report on
the wide distribution of large putative introns in other genes
such as nad1, nad5 and CO2 (Fig. 2). This contrasts with an
earlier report that CO2, along with atp9 and nad6, rarely
contained introns in fungal mtDNA (Paquin et al. 1997).
Analysis of mtDNA copies of the 14 mitochondrial genes
Over 1180 putative copies of the 14 genes encoding subunits
of the respiratory chain complexes considered in this study
were localized following our fungal genome searches
(Table 3). Size of these copies ranged from 20 nt to the full
length of the query sequence. Two classes of copies were
distinguishable. The first class comprised entire (mostly
perfect) copies that matched almost 90% of the query sequ-
ence length and exhibited up to 95% identity (blastn E-
value of 0.0). Members of this category were rare and confined
to three species: C. neoformans (strain H99) (one complete
copy of atp8, CO1, cob, nad1, nad2 and nad6), P. graminis (one
complete copy of atp6, atp8, CO2 and nad3) and S. roseus (one
complete copy of atp6, three of CO2 and two of nad3 and
nad4L; Table 3). The six complete copies found in C. neofor-
mans (strain H99) were distributed across four supercontigs
(supercontig 1.124 for CO1 and atp8, 1.80 for nad1 and cob,
1.97 for nad2 and 1.76 for nad6). Strong similarities (99%)
were observed between these supercontigs and homologous
regions in the mitochondrial genome of C. neoformans strain
H99. Similarly, all the copies found in P. graminis, except
nad3, were clustered in one single supercontig (2.15), but in

© 2009 Blackwell Publishing Ltd and Crown in the right of Canada

108 B A R C O D I N G F U N G I
this case, only a small part of this supercontig (0.2% of the
total supercontig length) showed a strong homology (98%)
with the P. graminis mitochondrial genome. Given the strong
identities observed in these fungi between these supercontigs
and homologous regions in their respective mitochondrial
genomes, two hypotheses can be advocated. First, the pre-
sence of copies identical to mtDNA sequences in nuclear
DNA can be attributed to recent DNA duplications and
transfers from mitochondria to chromosomes (NUMTs).
Such invasion of nuclear DNA by mtDNA appears as a
continuous evolutionary process that results in the occurrence
of complete rearranged and/or fragmented copies of variable
in sizes and evenly distributed within and among chromo-
somes (Richly & Leister 2004; Bullerwell & Lang 2005; Pamilo
et al. 2007). Examples of recent transfers have been observed
in the ascomycete yeast Saccharomyces cerevisiae (Ricchetti
et al. 1999) and in other nuclear genomes from eukaryotes,
such as plants and human (Mourrier et al. 2001; Stupar et al.
2001; IRGSP 2005). Second, mis- or unassembled parts of the
mitochondrial genome sequence can be retrieved in the com-
plete genome assembly. These technical problems might have
arisen during the assembly of a whole genome sequence,
either from insufficient read quality or from the presence of
nucleotide repeats. In fact, alignment errors (computational
or clone-induced from chimera) resulting in genome region
misassemblies and producing these copies cannot be
completely excluded. Similar computational errors should
have been generated in the S. roseus mitochondrial genome
assembly since perfect copies of the entire CO2, nad3 and
nad4L genes were retrieved in this genome.
The second class of copies included short repetitive ele-
ments scattered throughout mitochondrial genomes. The
mitochondrial genomes of C. cinereus, L. bicolor, M. perniciosa
and P. placenta contained such elements that originated from
the CO1 and 2, cob and nad1 and nad5 genes (Table 3). Most
of these sequences (six in P. placenta, 34 in M. perniciosa and
62 in L. bicolor) of 20–92 nt were repeated from 15 to more
than 30 times and exhibited short hairpin structures. Such
secondary structures have previously been reported in mito-
chondrial fungal genomes and are described as double-
hairpin elements (DHE) (Paquin et al. 1997).
Patterns of evolution
To better understand the pattern and rate of evolution of
the mitochondrial loci, we assessed sequence divergence
levels between 14 ingroup taxa (C. cinereus, C. neoformans
(strain JEC21), C. neoformans (strain H99), C. neoformans (strain
R265), L. bicolor, M. perniciosa, P. pachyrhizi, P. ostreatus, P.
placenta, P. graminis, S. commune, S. roseus, T. indica, and U.
maydis) for each of the 14 mitochondrial genes encoding the
subunits of the respiratory chain complexes. We found high
interspecific mutational variation in each gene as measured
by K2P, with distances ranging from 0.36 (± 0.11) to 0.68
© 2009 Blackwell Publishing Ltd and Crown in the right of Canada
(± 0.23), and by percentage of variable sites (62.6% for atp9
and 77.4% for nad3; Fig. 3a). The substantial divergence
values observed at higher taxonomic levels (e.g. between
genera within Basidiomycota) suggest that these 14 mito-
chondrial genes may be potentially useful markers for the
resolution of lower-level relationships (e.g. between species).
Such high divergence levels were expected for mitochondrial
protein-coding genes (Moriyama & Powell 1997; Hebert
et al. 2003). As a comparison, at similar taxonomic levels
(between genera within Basidiomycota), overall means of
K2P distances observed for the ribosomal nuclear RNA
genes (18S and 28S) were 0.12 (± 0.05) and 0.25 (± 0.05),
respectively. In contrast, numerous insertion/deletion in
the ITS sequences obtained for the same taxa prevented
accurate nucleotidic alignment and determination of the
molecular evolution pattern.
We used likelihood-ratio tests to find the best evolution-
ary model fitting each of the genes under examination. For
each gene, evolutionary models that accounted for unequal
base frequencies provided a significantly better fit to the
data. Base compositional bias was lower for atp9 and CO1
(observed G + C content of 40% and 37%, respectively) rela-
tive to the 12 other mitochondrial genes examined, which
exhibited a mean G + C content of 30% (± 3%). At this level,
such A-T bias may result in an increase of homoplasy
(Lockhart et al. 1994; Foster & Hickey 1999; Rokas et al. 2002).
Limitations in the type of changes at several nucleotidic sites
induced by homoplasy could result in asymmetrical patterns
of among-base substitution rates (Collins et al. 1994). Only
the 18S, 28S and atp8 genes fitted relatively simple models
assuming two [Tamura–Nei (TrN) model for the 18S and
28S genes] or three substitution rates [transitional model
(TIM) for atp8]. The 13 remaining genes fitted transversional
(TVM) and general time reversible (GTR) models, which com-
prise five and six classes of substitution types, respectively
(Fig. 3b). The complexity of these models was furthermore
emphasized by a parameter accounting for the rate of
heterogeneity across sites. In the five cases in which no
invariable site parameter (I) was added to the ML model
(atp8, apt9, nad3, nad4L and 28S; Fig. 3b), the shape parameter
(alpha) of the gamma-distributed rates component was low,
denoting a strong rate of heterogeneity among sites (e.g. a
more uneven distribution of rates among sites). We noted
a significant correlation between the alpha and I parameters
(r2 = 0.74; P < 0.05), which likely resulted from the fact that
more sites were allocated to the invariant site category, the
remaining sites showed a lesser rate of heterogeneity. Low
alpha values correspond to genes with a few sites evolving
at a very high rate, with the remaining sites changing at a
very slow rate. Thus, given these model parameter tend-
encies (e.g. base composition heterogeneity, substitution bias
and low alpha values), the substantial variation observed
in these genes appears to be concentrated at a few sites.
These sites are likely to have multiple substitutions with a
 B A R C O D I N G F U N G I 109

Fig. 3 Attributes of 14 genes encoding subunits of the mitochondrial respiratory chain complexes. (a) Kimura-2-parameter (K2P)
interspecific distances between pairwise sequences with the standard deviation plotted against the percentage of variable sites found for
each gene set; lines below K2P values indicate that the means were not significantly differentiated by a Tukey test. (b) Gamma-shape
parameters, proportion of invariable sites, and among-bases substitution rates components of the best-fitting models for the 14 genes
considered. The classes of substitution types varied according to the colours depicted in the bottom right box. The upper right box contains
the lengths of the different data sets.

resulting reduction in the number of variable and/or
alternative character states available. This translates into
an increased sensitivity to homoplasy (Cummings et al.
1995; Ballard & Whitlock 2004). Such mutational saturation
tendency raised the possibility that convergence in base
composition between unrelated taxa could lead to incorrect
species delimitations (Roe & Sperling 2007).
Efficiency of the mitochondrial DNA barcode candidates
Considering the absence of intron in the in silico analysis
and the potential for high divergence levels, seven fungal
mitochondrial genes, atp6, atp8, atp9, CO3, nad3, nad4L and
nad6, had potential as DNA barcodes. With an optimal length
for a barcode of approximately 600 nt (Min & Hickey 2007),
the set is reduced to three genes: atp6, CO3 and nad6. Atp6
was successfully used for systematics in the Boletales
(Agaricomycotina, Basidiomycota) (Kretzer & Bruns 1999)
and is promising for other groups of Agaricomycotina (J.M.
Moncalvo, unpublished). However, the use of this gene in
Ascomycota proved to be problematic and was abandoned
in the Assembling the Fungal Tree of Life (AFTOL) initiative
(V. Hofstetter, personal communication to J.M.M.). Multiple
divergent atp6 sequences were recovered from several lichen
strains. These divergent atp6 sequences were hypothesized
to originate from autonomously replicating plasmid-like
DNA containing the atp6 gene, as observed in the maize
pathogen Cochliobolus heterosporus (Lin et al. 1988; Hofstetter
et al. 2004). The use of CO3 in Boletales was abandoned since
it contained introns that interfered with PCR amplification
(Kretzer & Bruns 1999). The NADH dehydrogenase subunit
genes are absent from the mitochondrion of several yeasts
(Ascomycota) (Bullerwell et al. 2003). In filamentous fungi,
few studies have considered nad6 as a tool for fungal system-
atics and taxonomy. These studies emphasized the inade-
quacy of individual mitochondrial genes to resolve species
phylogenies (Kouvelis et al. 2004; Pantou et al. 2006). Mito-
chondrial DNA (especially the intergenic sequences of the

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110 B A R C O D I N G F U N G I

Table 4 Efficiency of the ITS, 28S and four mitochondrial loci as DNA barcodes

DNA barcode amplification DNA barcode efficiency

No. of Total length K2P distance K2P distance

primer % amplification used for K2P intraspecific interspecific Species
Marker pair success distan(ce pb) comparisons (range) comparisons (range) resolution *%

Chrysomyxa data set (10 species/23 isolates)

28S 1 100 702 0.0001 0.04 90
(± 0.0003) (± 0.07)
ITS 2 100 859 0.001 0.08 90
(± 0.001) (± 0.1)
CO1 2 29 292 0.001 0.08 50
(± 0.000) (± 0.07)
nad6 1 100 451 0.000 0.06 90
(± 0.01) (± 0.04)
CO3 1 100 517 0.001 0.04 90
(± 0.001) (± 0.02)
atp6 1 91 583 0.001 0.04 70
(± 0.001) (± 0.03)
Melampsora data set (5 species/15 isolates)
28S 1 100 738 0 0.034 80
(± 0) (± 0.01)
ITS 1 100 629 0.0004 0.07 80
(± 0.0008) (± 0.03)
CO1 2 100 744 0.0006 0.003 60
(± 0.001) (± 0.002)
nad6 1 100 500 0 0.005 60
(± 0) (± 0.004)
CO3 1 100 665 0 0.003 20
(± 0) (± 0.003)
atp6 1 100 652 0 0.0006 20
(± 0) (± 0.0009)

*A species is considered resolved if all of its constituent sequences form a monophyletic cluster and are distinct from other sequences.

NADH dehydrogenase subunit genes) was nevertheless
considered as a valuable tool for the discrimination of
closely related species within Ascomycota (Kouvelis et al.
2004; Pantou et al. 2006).
Following our in silico results, we tested the efficiency of
four of the 14 mitochondrial genes for DNA barcode. First,
we tried to amplify the CO1-5′ region by PCR since this locus
was initially proposed as the universal barcode system for
eukaryotes. Then, we compared the efficiency of the nad6,
CO3 and atp6 genes with the 28S, ITS and CO1-5′ loci. To
assess the potential of these loci as DNA barcodes, we
generated a data set for two fungal genera with taxonomic
difficulties (Table S1). The species complex Chrysomyxa ledi
de Bary includes several cryptic species. At least six of them
are distinguishable by their spore morphometry and/or
uredinial host specificity (Crane 2001). A collection of
Melampsora species sampled on aspen and white poplars
was also included in this study. This data set includes the
M. populnea species complex composed of at least four species
distinguished through aecial host specificity, but morpho-
logically similar (Pei & Shang 2005).
The number of primer pairs required for successful PCR
amplifications and sequencing varied according to the locus
and the data set (either Melampsora spp. or Chrysomyxa spp.)
targeted (Table 4). In general, PCR amplification results
obtained from these two rust genera were congruent with
the results obtained in the in silico analyses. The PCR amplifi-
cation of a ~600-bp product from the 5′-end of the CO1 gene
generally required more than a single primer pair due to the
occurrence of numerous large introns. In contrast, no intronic
regions were found in the nad6 and CO3 genes among the
15 fungi considered in the preliminarily in silico analysis,
and successful PCR amplifications of these genes were
obtained using only one single primer pair for the Melamp-
sora and Chrysomyxa strains considered here. The amplifi-
cation and sequencing of the ITS, CO1 and atp6 loci in the
Chrysomyxa data set was particularly complicated. Two pri-
mer pairs were required to obtain readable ITS sequences.
Even using multiple primer pairs in different amplification
reactions, a maximum of 292 bp was obtained for the CO1
gene for 29% of the specimens tested (Table 4). We ampli-
fied more than 650 bp of the atp6 gene in the Chrysomyxa

© 2009 Blackwell Publishing Ltd and Crown in the right of Canada


data set except for two species: C. ledi and C. rhododendri. This
failure might have occurred for these two species because
of (i) the occurrence of intron; (ii) the presence of poly-
morphisms at the primer sites, although these had been
designed in a conserved part of the atp6 gene.
Neither the nuclear (ITS and 28S) nor the mitochondrial
loci fully resolved the different rust taxa under study. Despite
this, ITS and 28S provided greater taxonomic resolution
than the mitochondrial genes (Table 4). Although nad6 and
CO3 provided the same taxonomic resolution as ITS and
28S loci in the Chrysomyxa dataset (90% of the species
resolved; Table 4), these mitochondrial loci resulted in
lower taxonomic resolution than the ribosomal loci in the
Melampsora data set (from 20 to 60%).
Our work demonstrates that the sequences currently
available in public databases are useful to conduct in silico
molecular studies for a large taxonomic group such as Basid-
iomycota. We initially postulated that such in silico analyses
could constitute a helpful resource for facilitating the choice
of genes with sufficient degree of divergence at the appro-
priate taxonomic scale. This approach allowed us to anti-
cipate difficulties for in vivo PCR amplification of mitochondrial
genes in this group of fungi. We predicted that numerous
sporadic introns should occur in mitochondrial genes across
several genera in the Basidiomycota and could compromise
the usefulness of these genes for DNA barcoding. Further-
more, we demonstrated that several fungal mitochondrial
genes, including CO1 that had been proposed for DNA
barcoding, exhibit a range of substantial interspecific diver-
gence which constitutes one of the fundamental require-
ments for a species-level DNA identification system. Despite
such potential for high divergence levels, the taxonomical
resolution observed in mitochondrial genes varies depend-
ing on the combination locus/group of taxa considered.
Finally, our comparison of four of these genes, CO1, atp6,
CO3 and nad6, with nuclear ribosomal regions (ITS and
28S) in two rust data sets (including closely related
species), revealed that ITS and 28S offer a better taxonomic
resolution than the mitochondrial loci in spite of the lower
potential we initially observed for the 28S locus in our in
silico analyses.
Acknowledgements
The authors acknowledge David L. Joly for help with bioinformatics
and Franck Orsupetru Stefani and Philippe Tanguay for comments
on the manuscript. This work was supported by the Natural Sciences
and Engineering Research Council of Canada (NSERC) and Genome
Canada for funding the Canadian Barcode of Life Network and the
Fungal DNA Barcoding Initiative.
Conflict of interest statement
The authors have no conflict of interest to declare and note that
the funders of this research had no role in study design, data
© 2009 Blackwell Publishing Ltd and Crown in the right of Canada
B A R C O D I N G F U N G I 111
collection and analysis, decision to publish, or preparation of
the manuscript.
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Supporting information
Additional supporting information may be found in the online
version of this article:
Fig. S1 Nucleotidic alignments obtained for 14 mitochondrial genes
encoding subunits of the respiratory chain complexes. The sequences
were obtained from available Basidiomycete genomic resources as
detailed in the Material and methods section.
Fig. S2 Dendrograms constructed with the neighbour-joining
algorithm based on the K2P distance matrices of the ITS, 28S, CO1,
atp6, CO3 and nad6 nucleotidic-sequence alignment for the Mela-
mpsora and the Chrysomyxa data sets.
Fig. S3 G + C content comparisons between the nuclear and mito-
chondrial contigs considered in this study. (A) Plot of the G + C
content of each contig (nuclear and mitochondrial) considered in
the genome assemblies; (B) Box plot of G + C content in mitochon-
drial and nuclear contigs considered in the genome assemblies.
Table S1 Information about Chrysomyxa and Melampsora speci-
mens used in this study
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