High Variability of Mitochondrial Gene Order Among Fungi

GBE
High Variability of Mitochondrial Gene Order among Fungi

Gabriela Aguileta1,2,3,*, Damien M. de Vienne1,2,10, Oliver N. Ross4,5, Michael E. Hood6, Tatiana Giraud7,8,
Elsa Petit6, and Toni Gabaldón1,2,9,*
1
Bioinformatics and Genomics Programme, Centre for Genomic Regulation (CRG), Barcelona, Spain
2
Universitat Pompeu Fabra (UPF), Barcelona, Spain
3
INRA, UR1052 GAFL, Montfavet, France
4
Mediterranean Centre for Marine and Environmental Research (ICM-CSIC), Barcelona, Spain
5
Mediterranean Institute of Oceanography (MIO) Aix-Marseille University, CNRS/INSU, IRD, Marseille, France
6
Biology Department, Amherst College
Downloaded from https://academic.oup.com/gbe/article/6/2/451/537818 by guest on 27 July 2024

7
CNRS, UMR 8079 Ecologie, Systématique et Evolution, Orsay, France
8
UMR 8079 Université Paris Sud, Ecologie, Systématique et Evolution, Orsay, France
9
Institució Catalana de Recerca i Estudis Avancats, Barcelona, Spain
10
Present address: Université Lyon 1, CNRS, UMR 5558, Laboratoire de Biométrie et Biologie Evolutive
*Corresponding author: E-mail: [email protected]; [email protected].
Accepted: January 31, 2014
Abstract
From their origin as an early alpha proteobacterial endosymbiont to their current state as cellular organelles, large-scale genomic
reorganization has taken place in the mitochondria of all main eukaryotic lineages. So far, most studies have focused on plant and
animal mitochondrial (mt) genomes (mtDNA), but fungi provide new opportunities to study highly differentiated mtDNAs. Here, we
analyzed 38 complete fungal mt genomes to investigate the evolution of mtDNA gene order among fungi. In particular, we looked for
evidence of nonhomologous intrachromosomal recombination and investigated the dynamics of gene rearrangements. We inves-
tigated the effect that introns, intronic open reading frames (ORFs), and repeats may have on gene order. Additionally, we asked
whether the distribution of transfer RNAs (tRNAs) evolves independently to that of mt protein-coding genes. We found that fungal mt
genomes display remarkable variation between and within the major fungal phyla in terms of gene order, genome size, composition
of intergenic regions, and presence of repeats, introns, and associated ORFs. Our results support previous evidence for the presence of
mt recombination in all fungal phyla, a process conspicuously lacking in most Metazoa. Overall, the patterns of rearrangements may
be explained by the combined influences of recombination (i.e., most likely nonhomologous and intrachromosomal), accumulated
repeats, especially at intergenic regions, and to a lesser extent, mobile element dynamics.
Key words: Basidiomycota, sordariomycetes, basal fungi, fungal phylogeny, rearrangement rates, genome size reduction,
endosymbiosis.
Introduction origin of mitochondria, a high percentage of the ancestral

Mitochondria play various essential roles in eukaryotic cells, alpha-proteobacterial protein-coding genes were lost and
particularly with respect to their primary functions in respira- replaced by proteins of different origins (Gabaldon and
tory metabolism and energy production. From its origin as a Huynen 2003; Gabaldon and Huynen 2007). The loss of an-
proto-mitochondrion derived from an alpha-proteobacterium cestral bacterial genes resulted in significant genome size re-
to its current state as a cellular organelle, major changes have duction (Bullerwell and Lang 2005).
occurred not only in terms of protein repertoire but also in mt genome evolution differs remarkably among the major
the organization of the mitochondrial (mt) genome (Adams groups of eukaryotes. Comprehensive reviews are available
and Palmer 2003; Gabaldon and Huynen 2004). Previous about mt genome evolution in animals (Boore 1999), plants
studies have shown that subsequent to the endosymbiotic (Levings and Brown 1989; Palmer et al. 2000; Kitazaki and
ß The Author(s) 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted reuse,
distribution, and reproduction in any medium, provided the original work is properly cited.
Genome Biol. Evol. 6(2):451–465. doi:10.1093/gbe/evu028 Advance Access publication February 6, 2014 451
Aguileta et al. GBE
Kubo 2010), protists (Gray et al. 1998; Burger et al. 2003), and most ascomycetes, genes are typically encoded on the same
fungi (Paquin et al. 1997), as well as comparison among these mtDNA strand, whereas in basidiomycetes, they can be
lineages (Burger et al. 2003; Bullerwell and Gray 2004). encoded on both mtDNA strands (references in table 1).
Animal mt genomes are generally gene rich, practically Another remarkable characteristic is that, although fungi are
intron less, and they have a high rate of DNA sequence evo- a lineage more closely allied with animals, mtDNAs in these
lution. Gene order tends to be conserved, especially within organisms show signals of recombination, a characteristic that
major phyla although they can be variable between them is more similar to plant mtDNAs. Also similar to plants, fungal
(Boore 1999). In the last few years, however, examples mt genomes can have large intergenic regions including se-
from different animal groups, in particular molluscs (Boore quence repeats and introns. Interestingly, fungi have mostly
and Brown 1994; Yamazaki et al. 1997; Boore 1999; group I introns, whereas plant mitochondria tend to possess
Kurabayashi and Ueshima 2000; Rawlings et al. 2001), have preferentially group II introns (Lang et al. 2007). Intron num-
challenged this view showing that rearrangements can occur bers are highly variable in fungal mtDNA; for instance,

within animal mt genomes (Perseke et al. 2008; Bernt and although the mitochondrion of the ascomycete Podospora
Middendorf 2011). An important feature is that animal anserina has 15 group I introns and 1 group II intron, that of
mtDNAs typically do not engage in recombination (but see the basidiomycete Schizophyllum commune shows no introns
Rocha 2003; Rokas et al. 2003; Barr et al. 2005). In contrast, at all (Specht et al. 1992; Paquin et al. 1997). In fact, mt
plant mt genomes have sequence characteristics that are as- genome size variation can be explained in large part by differ-
sociated with more frequent recombination, including large ences in the number and length of introns: intron length can
intergenic regions that can house repeated DNA sequences range from a few bp up to 5 kb (Lang et al. 2007). Such fungal
and a variable number of introns and their associated intronic introns often display autonomous proliferation in mt genomes
open reading frames (ORFs) (Palmer et al. 2000). Such repet- via homing endonucleases (HEs), proteins with DNA endonu-
itive genomic elements contribute to the significant increase clease activity that allows them to move easily in the genome
of mt genome size in some plants (e.g., in the Silene genus, by intron transfer, and site-specific integration or “homing”
Sloan et al. 2012). Also, plant mt genomes have experienced (Lazowska et al. 1980; Lambowitz and Perlman 1990; Pellenz
frequent rearrangements, particularly in vascular plants, and et al. 2002).
they have higher gene order variability relative to animal mt The presence of repetitive DNA within mt genomes in the
genomes (Palmer et al. 2000; Kitazaki and Kubo 2010; Galtier form of introns and their associated traits of self-splicing and
2011; Liu et al. 2011 and references therein). Interestingly, insertion endonuclease activity may contribute to the struc-
algal mt genomes do not show many rearrangements and tural dynamics of fungal mt genomes, eliciting changes
are thus a group of plants retaining many characteristics of in gene order, overdispersal of repetitive elements, and the
the ancestral eukaryotic mitochondria (Liu et al. 2011). Plant introduction of new genes through horizontal gene transfer
mt genomes tend to have rates of DNA sequence evolution (HGT) (Vaughn et al. 1995; Ferandon et al. 2010). Moreover,
that are lower than in animals (Palmer et al. 2000; Kitazaki and the repeats accumulated in mt introns have been associated
Kubo 2010). They can also perform extensive RNA editing, with increased recombination and deletions (Rocha 2006), a
although this capability is variable between plant lineages process that is frequently invoked to explain differences in mt
(Hecht et al. 2011; Liu et al. 2011). Other, less-well-studied eu- gene order in fungi but that is remarkably absent in Metazoa
karyotes include the phylogenetically diverse and nonmono- (Saccone et al. 2002). Finally, another factor potentially con-
phyletic protists, whose mt genomes can display the most tributing to gene order variation is the distribution of transfer
variation in organizations (Burger et al. 2003; Bullerwell and RNAs (tRNAs), which display editing, excision, and integration
Gray 2004). Protist mt genomes can be either linear or circular, capabilities, that allow them to change location within the
have multiple linear chromosomes transcribed separately, and genome and participate in HGT events (Tuller et al. 2011).
vary dramatically in size (Burger et al. 2003; Vlcek et al. 2011). Because changes in tRNA location are relatively rare events,
There does not seem to be a generalized tendency in terms of tRNA location within fungal mt genomes has been used to
rate of mt evolution or capacity to recombine among protists study fungal evolution and phylogenetic signal (Cedergren
and they exhibit variability in terms of gene order (Gray et al. and Lang 1985).
1998; Burger et al. 2003). To date, a number of studies have provided insights into
Fungal mt genomes have been less studied than their fungal mt genomes (see references in table 1); however, to
animal or plant counterparts, yet they hold great potential our knowledge, there has not been a large-scale comparative
for illuminating the evolution of organellar genomes. The analysis providing a broader picture of the evolution of fungal
most evident feature is that, although gene content is largely mt genomes, especially of the remarkable variability in gene
conserved, their relative gene order is highly variable, both be- order. Here, we therefore set out to investigate variation in
tween and within the major fungal phyla (Paquin et al. 1997 gene order among fungal mt genomes, including basidiomy-
and references in table 1). One difference between the largest cetes, ascomycetes, and fungi from early diverging lineages.
two fungal phyla, Ascomycota and Basidiomycota, is that in Our species sampling provided the taxonomic depth and
452 Genome Biol. Evol. 6(2):451–465. doi:10.1093/gbe/evu028 Advance Access publication February 6, 2014
Fungal Mitochondrial Genomics GBE
Table 1
List of the Species Analyzed, Accessions, and References
Species Taxonomya GenBank Accession Reference
Allomyces macrogynus Ur NC_001715 Paquin and Lang (1996)
Arthroderma obtusum E NC_012830 Wu et al. (2009)
Beauveria bassiana S NC_017842 Ghikas et al. (2010)
Candida albicans S1 NC_018046 Bartelli et al. (2013)
Candida glabrata S2 NC_004691 Koszul et al. (2003)
Chaetomium thermophilum S NC_015893 Amlacher et al. (2011)
Cordyceps bassiana S NC_013145 Ghikas et al. (2010)
Cryptococcus neoformans B NC_004336 Litter et al. (2005)
Debaryomyces hansenii S1 NC_010166 Sacerdot et al. (2008)

Dekkera bruxellensis S1 NC_013147 Prochazka et al. (2010)
Fusarium oxysporum S NC_017930 Pantou et al. (2008); Al-Reedy et al. (2012)
Gibberella zeae S NC_009493 Herring et al. (unpublished)
Kluyveromyces lactis S2 NC_006077 Zivanovic et al. (2005)
Lecanicillium muscarium S NC_004514 Kouvelis et al. (2004)
Metarhizium anisopliae S NC_008068 Ghikas et al. (2006)
Mycosphaerella graminicola D NC_010222 Torriani et al. (2008)
Microsporum canis E NC_012832 Wu et al. (2009)
Millerozyma farinosa S1 NC_013255 Jung et al. (2010)
Moniliophthora perniciosa B NC_005927 Formighieri et al. (2008)
Microbotryum violaceum-Sl B NC_020353 Lang (unpublished)
Nakaseomyces bacillisporus S2 NC_012621 Bouchier et al. (2009)
Ogataea angusta S1 NC_014805 Eldarov et al. (2011)
Phakopsora pachyrhizi B NC_014344 Stone et al. (2010)
Paracoccidioides brasiliensis E NC_007935 Cardoso et al. (2007)
Peltigera malacea D NC_016955 Xavier et al. (2012)
Penicillium marneffei E NC_005256 Woo et al. (2003)
Phaeosphaeria nodorum D NC_009746 Hane et al. (2007)
Pichia pastoris S1 NC_015384 Kueberl et al. (2011)
Pleurotus ostreatus B NC_009905 Wang et al. (2008)
Podospora anserina S NC_001329 Bullerwell et al. (2000)
Rhizophydium sp.136 Ur NC_003053 Forget et al. (2002)
Schizosaccharomyces japonicus X NC_004332 Bullerwell et al. (2003)
Schizophyllum commune B NC_003049 Forget et al. (2002)
Tilletia indica B NC_010651 Yi et al. (unpublished)
Trametes cingulata B NC_013933 Haridas and Gantt (2010)
Ustilago maydis B NC_008368 Kennell and Bohmer (unpublished)
Vanderwaltozyma polyspora S2 NC_009638 Scanell et al. (unpublished)
Yarrowia lipolytica X NC_002659 Kerscher et al. (2001)
a
Taxonomy: B, basidiomycetes; S1, saccharomycetes1; D, dothideomycetes; E, eurotiomycetes; S, sordariomycetes; Ur, early diverging or basal;
X, other; S2, saccharomycetes2.
balance and established the context for a comprehensive anal- gene order and tRNA distribution among fungal
ysis of gene order evolution in fungi. Basal fungal taxa, being mitochondria.
highly divergent with respect to our sampled ascomycetes and
basidiomycetes, were analyzed separately to obtain reliable Materials and Methods
alignments. We investigated possible causes of gene order
variability, specifically, we assessed 1) evidence of recombina- Data Sets
tion and the dynamics of gene rearrangements and 2) the role To study the evolution of gene order in a representative data
played by intergenic regions and their associated repeats, the set of the major fungal group, the dikarya (constituted by the
number of introns, intronic ORFs, and tRNA distribution. basidiomycetes and the ascomycetes), we obtained the com-
Finally, we discuss how the combined roles of recombination, plete mt genomes and proteomes of 38 species available
chromosomal rearrangements, insertion of introns, and asso- in GenBank (table 1). The complete list of species analyzed
ciated mobile elements can contribute to the high variability of in our main data set (hereafter referred to as the
Aguileta et al. GBE
dikarya data set) includes nine basidiomycetes: Tilletia of gaps (0.1) and the consistency across aligners (>0.16)
indica (NC_010651), Phakopsora pachyrhizi (NC_014344), before they were concatenated. Subsequent phylogenetic re-
Pleurotus ostreatus (NC_009905), Cryptococcus neoformans construction combined neighbor joining and maximum likeli-
(NC_004336), Microbotryum lychnidis-dioicae (NC_020353), hood (ML), using PhyML (Guindon et al. 2009) and RAxML
Moniliophthora perniciosa (NC_005927), S. commune v.7.2.6 (Stamatakis 2006). For the ML analyses, four substitu-
(NC_003049), Trametes cingulata (NC_013933), Ustilago tion rate categories were used, estimating the gamma param-
maydis (NC_008368); 27 ascomycetes: Arthroderma obtusum eter and the fraction of invariable sites from the data. Support
(NC_012830), Beauveria bassiana (NC_017842), Cordyceps values were computed using an approximate likelihood ratio
bassiana (NC_013145), Candida albicans (NC_018046), test. Bootstrap analysis was conducted with 100 resampling
Candida glabrata (NC_004691), Chaetomium thermophilum iterations. Once we inferred the tree, we estimated evolution-
(NC_015893), Debaryomyces hansenii (NC_010166), ary rates with the r8s software v. 1.8 (Sanderson 2003). We
Dekkera bruxellensis (NC_013147), Fusarium oxysporum used the global Langley and Fitch (LF) model, which estimates

(NC_017930), Gibberella zeae (NC_009493), Kluyveromyces a single evolutionary rate across the whole tree (i.e., assuming
lactis (NC_006077), Lecanicillium muscarium (NC_004514), a molecular clock), and the local LF models allowing for the
Metarhizium anisopliae (NC_008068), Microsporum estimation of local rates for groups of clades within the tree.
canis (NC_012832), Millerozyma farinosa (NC_013255), The approximate ages for internal nodes were obtained using
Mycosphaerella graminicola (NC_010222), Nakaseomyces the divergence of basidiomycetes and ascomycetes (Taylor
bacillisporus (NC_012621), Ogataea angusta (NC_014805), and Berbee 2006; Lucking et al. 2009), conservatively set at
Paracoccidioides brasiliensis (NC_007935), Peltigera malacea 500 Ma, and the whole-genome duplication event within the
(NC_016955), Penicillium marneffei (Talaromyces marneffei) Saccharomyces clade (Wolfe and Shields 1997), set at 100 Ma.
(NC_005256), Phaeospheria nodorum (Stagonospora These two dates were used as calibration points. The evolu-
nodorum) (NC_009746), Pichia pastoris (NC_015384), tionary rates and estimated node ages were subsequently
P. anserina (NC_001329), Schizosaccharomyces japonicus used to infer an approximate rate of rearrangements per
(NC_004332), Vanderwaltozyma polyspora (NC_009638) clade.
and Yarrowia lipolytica (NC_002659); and 2 early diverging
fungi as outgroups, Allomyces macrogynus (NC_001715) Whole-Genome Alignments, Recombination, and
and Rhizophydium sp. 136 (NC_003053). Rearrangement Events
The basal fungi data set included representatives of the
main basal clades: 1) Blastocladiomycota: Al. macrogynus Because whole-genome alignment methods produce better
(used as outgroup in the dikarya data set: NC_001715), results, the more similar the genomes are, we decided
Blastocladiella emersonii (NC_011360); 2) Chytridiomycota: to align groups of mt genomes that are not too distant in
Rhizophydium sp. (used as outgroup in the dikarya data terms of sequence identity. To identify which genomes
set: NC_003053); 3) Cryptomycota: Rozella allomycis could be aligned together, we built a composite likelihood
(NC_021611); 4) Glomeromycota: Gigaspora margarita distance matrix based on the concatenated alignment of pro-
(NC_016684), Glomus intraradices (NC_012056); and 5) tein-coding genes cox1, cox2, cox3, atp6, atp8, and atp9. We
Monoblepharidomycota: Harpochytrium sp. JEL105 determined the Euclidian phylogenetic distance and used the
(NC_004623), Hyaloraphydium curvatum (NC_003048), and hierarchical agglomerative clustering method available in R
Monoblepharella sp. JEL15 (NC_004624). (R Development Core Team 2011), with h ¼ 0.4 to determine
the groups of most closely related genomes that could be
used for whole-genome alignment. With the dikarya data
Phylogenetic Inference set, we obtained the nine following groups (hereafter referred
To analyze the evolution of gene order through time and to as fungal clusters): 1) “basidios1,” including Tr. cingulata,
across the sampled species, we first reconstructed a phyloge- Mo. perniciosa, S. commune, and Pl. ostreatus; 2) “basidios2,”
netic tree to map the different gene orders in an evolutionary including T. indica, U. maydis, and C. neoformans; 3)
context. The two data sets defined in this study, the dikarya “basidios3,” including M. violaceum-Sl and Ph. pachyrhizi;
and the basal fungi data sets, were analyzed independently 4) “sordariomycetes,” including B. bassiana, Co. bassiana,
using the same methods. First, protein sequences of the ortho- Ch. thermophilum, P. anserina, F. oxysporum, G. zeae, L. mus-
logs shared by all sampled species, including protein-coding carium, and Me. anisopliae; 5) “saccharomycetes1,” including
genes cox1, cox2, cox3, atp6, atp8, atp9, nad1, nad2 nad3, Ca. albicans, D. bruxellensis, De. hansenii, Mil. farinosa,
nad4, nad5, nad4L, and nad6, were aligned using a combi- O. angusta, and Pi. pastoris; 6) “saccharomycetes2,” including
nation of six different alignment strategies (Muscle, Mafft, and Ca. glabrata, K. lactis, N. bacillisporus, and V. polyspora; 7)
dialignTX, in forward and reverse). These alignments were “dothideomycetes,” including My. graminicola, Pel. membra-
automatically trimmed with trimAl (Capella-Gutierrez et al. nacea, and Ph. nodorum; 8) “eurotiomycetes,” including A.
2009) to remove poorly aligned regions based on the fraction obtusum, Mi. canis, Pa. brasiliensis, and Pen. marneffei; and 9)
“basals,” including Al. macrogynus and Rhizophydium sp. as 1-GOC. As described in Rocha (2006), the empirical models
Neither Schizos. japonicus nor Y. lipolytica could be reliably defined in that study attempt to fit the loss of GOC with
aligned with the other clusters so they were excluded from respect to time. Model 0, proposed by Tamames (2001), is
further analysis. The complete mt genomes in each cluster the simplest model that approximates GOC to a sigmoidal
were aligned with Mauve v.2.3.1 (Darling et al. 2010) using curve described by GOC ¼ 2/1 + eat, where parameter a is
the full alignment option. This general-purpose multiple se- adjusted by regression. Model 1 fits time dependence with a
quence aligner is able to handle whole-genome alignments square root dependence, thus GOC ¼ 1 – ˇat. Model 2 con-
and has the advantage that it identifies syntenic blocks despite siders that GOC decreases with time in a negative proportion
rearrangements and reversals. We further refined the align- to the square of the GOC at time t, hence 1/GOC ¼ at + 1.
ments of the syntenic blocks using t-coffee (Notredame et al. Finally, Rocha (2006) proposes a probabilistic approach where
2000) and analyzed them with GRIMM v. 1.04 (Tesler 2002) the probability (P) of two genes staying together after t con-
and UniMoG (Hilker et al. 2012) to infer a minimal history of secutive generations is given by P ¼ pt. Thus, in Model 3:

rearrangements among the aligned genomes. We assumed GOC ¼ pt. Note that this expression assumes that P is the
the Double-Cut and Join (DCJ), restricted DCJ, Hannenhalli same for all genes, which is thus somewhat unrealistic. We
and Pevzner (HP), inversion, and translocation models. These decided to also use the approach described in Fischer et al.
methods predict optimized rearrangement scenarios between (2006), where a measure of GOL for each branch in the tree is
pairs of gene order lists. GRIMM infers inversions and takes obtained and is thus more specific than the previously de-
only lists of gene orders including the same number of genes, scribed empirical models. Branch-specific GOL (bsGOL) scores
in other words, it does not take into account gene losses, are obtained by minimizing the sum, over all the possible
whereas UniMoG does. The latter has the advantage that it pairwise comparisons at hand, of the squared differences be-
extends the DCJ to include the HP, inversion, and translocation tween the frequency of the observed GOL events and the sum
models. Finally, the syntenic blocks, the intergenic regions, of the predicted branch-specific values. The following expres-
and the individual one-to-one orthologs of all genomes sion is minimized to obtain the bsGOL scores:
were tested for recombination, the most likely mechanism !2
explaining the observed gene order variability. X66 X23
L¼ bij xj GOLi
There are several methods available to detect different i¼1 j¼1
types and signals of recombination (Martin et al. 2011). In
our case, we needed methods that could identify incongruent where bi,j is a Boolean variable that specifies the branches that
blocks of sequence along the alignments. We chose methods are relevant for the estimation of a particular bsGOL (i.e., 0 if it
that look for incongruence in terms of patterns of sites, includ- is not relevant and 1 if it is), xj is obtained by minimizing L and
ing RDP3 v.4.16 (Martin et al. 2010), PhiPack (Bruen et al. is the actual bsGOL value, and GOLi are the estimated values
2006), and Recco (Maydt and Lengauer 2006). However, as from the pairwise comparisons, in other words, GOLi ¼
far as we know, there is no specific software for the detection 1 GOCi (Fischer et al. 2006).
of nonhomologous intrachromosomal recombination, so it is We approximated the GOC and bsGOL models described
possible that the methods available do not perform optimally above to determine which of these models best fitted the data.
for identifying this type of event. Nevertheless, looking for ev- In an attempt to better understand the process of gene order
idence of intrachromosomal recombination is often coincident shuffling, we conducted a test to verify whether the changes
with identifying breakpoints, reversals, and translocations, so observed in gene order occur randomly or whether they sug-
the rearrangements we inferred using GRIMM (Tesler 2002) gest other forces at work: Briefly, for each genome, we listed
and UniMoG (Hilker et al. 2012) were used as a proxy for the the order of genes, made all possible pairwise comparisons of
particular case of intrachromosomal recombination. these lists, estimated the GOC score (Rocha 2006), shuffled
randomly the gene order, and estimated a new GOC value.
We repeated this procedure 100,000 times and compared the
Gene Order Variability: Modeling Gene Order Evolution
original GOC score to the distribution of the GOCs after shuf-
Gene order can be studied directly by the comparison of the fling. We applied the Bonferroni correction for multiple testing
sequential order of mt genes described in their respective ar- and determined the significance (P values) of the comparisons.
ticles and/or genetic maps (see references in table 1). We used This test would indicate whether GOC between a pair of ge-
these data to investigate the most likely evolutionary scenar- nomes is significantly different from random.
ios: We estimated the gene order conservation (GOC) index as
described in Rocha (2006) and the branch-specific GOC de-
scribed in Fischer et al. (2006). GOC is simply defined as the Influence of tRNA Distribution, Intergenic, and Intronic
number of contiguous ortholog pairs that are in common Elements on Gene Order
between compared genomes, normalized by the number of To determine which genomic elements play a significant role
shared orthologs. Conversely, gene order loss (GOL) is defined in shaping mt gene order evolution, if any, we first obtained
Aguileta et al. GBE
the number of mt protein-coding genes, introns, intronic commune, and U. maydis. To our knowledge, rnpB has not
ORFs, and repeats in all our sampled genomes. We also as- been described in other basidiomycete mt genomes; it has so
sessed the distribution of tRNAs, which is variable across far only been recognized in mtDNAs of a few zygomycete and
fungal mt genomes (i.e., they can be dispersed across the ascomycete fungi, two protists, and never in animals and
genome, as in the case of Schizos. japonicus, or present in a plants (Seif et al. 2003, 2005).
few interspersed clusters, as is often the case in sordariomy- The inferred phylogenetic tree including all 38 species in the
cetes), relative to mt protein-coding genes. The number of pro- dikarya data set (fig. 1) is in agreement with previously pub-
tein-coding genes, introns, and their associated intronic ORFs, lished fungal phylogenies (Marcet-Houben and Gabaldon
as well as the number and location of tRNAs, were obtained 2009; Ebersberger et al. 2012), and most internal nodes are
from the annotations available in GenBank. Subsequently, we well supported (i.e., >90%). The global LF model that esti-
looked for simple repeats using RepeatMasker (Smit AFA, mates a single evolutionary rate across the whole tree, that is,
Hubley R, Green P. RepeatMasker Open-3.0.1996-2010; assuming a molecular clock, predicted 1.65 103 substitu-

http://www.repeatmasker.org, last accessed February 18, tions per site per time unit (Myr) and the local LF model, that is,
2014) and mreps (Kolpakov et al. 2003) for detecting without assuming a molecular clock, estimated 1.51 103
tandem repeats across the whole genomes. We focused on for the basal group (Al. macrogynus and Rhizophydium sp.
finding repeats located within intergenic regions because we 136), 2.1 103 for the ascomycetes, and 1.35 103 for
hypothesize that they may be more likely to affect gene order. the basidiomycetes, suggesting a faster evolutionary rate in
Additionally, we asked whether tRNAs are significantly more ascomycetes relative to the basidiomycetes and the
clustered in genomes with high GOC (i.e., where gene order is basal fungi sampled in this study. This rate is lower than
conserved) compared with genomes with low GOC. For every the reported average rates for mammals (i.e., about
taxon, we listed the mt protein-coding genes and tRNAs in 33.88 109/Y, that is approximately 3.4 102/Myr;
order; for each ordered list, we counted the number of Nabholz et al. 2009) but higher than that of plant mt genomes
noncontiguous tRNAs, performed 100,000 random permuta- (i.e., 0.34 109/Y, that is 3.4 104/Myr; Wolfe et al.
tions and recounted the number of noncontiguous tRNAs 1987). These are only approximate comparisons, as we did
each time; we compared the count in the original ordered not analyze population data.
list with the distribution obtained by the permutations; we
chose a 5% threshold for the significance of tRNA clustering.
Rearrangements and Recombination in the Dikarya
Finally, we investigated the influence that the amount of in-
trons, intronic ORFs, intergenic repeats, and the number of Despite the overall conservation of the standard set of mt
predicted rearrangements may have on gene order variability. genes, we found striking variation in gene order among
To this end, we employed Pearson’s w2 test, Fisher’s exact fungal species in the dikarya data set. Noteworthy exceptions
tests, and randomization tests of independence to determine to this trend are the nad4L/nad5 and nad2/nad3 genes, which
the correlation between the different genomic elements (i.e., tend to be next to each other in most species. The overlap of
number of introns, intronic ORFs, and repeats) and the the stop and initiation codons between the particular genes in
number of rearrangement events predicted per fungal cluster. these two pairs is the most likely cause for their contiguity, as it
occurs in Pleurotus mtDNA (Wang et al. 2008).
Because nonhomologous, intrachromosomal recombina-
Results
tion is known to cause chromosomal rearrangements
Our sampling in the dikarya data set provided the necessary (Gordenin et al. 1993; Bi and Liu 1996; Lobachev et al.
taxonomic context and depth for a comprehensive analysis of 1998; Rocha et al. 1999; Waldman et al. 1999; Rocha
gene order evolution in a phylogenetic context. The mtDNA 2003, 2006; Phadnis et al. 2005; Odahara et al. 2009;
of basal fungi was analyzed separately to obtain reliable Lavrov 2010), it could potentially explain the high gene
alignments. order variability we observe in fungal mitochondria. We
thus set out to detect recombination among the syntenic
Phylogenetic Analysis in the Dikarya regions and whole-genome alignments in all the fungal clus-
All the 38 species analyzed in the dikarya data set have the ters. We did find signals of recombination in most alignments
standard core set of mt protein-coding genes (atp6, atp8, but not unequivocal evidence of nonhomologous, intrachro-
atp9, cox1, cox2, cox3, nad1, nad2, nad3, nad4, nad4L, mosomal recombination, as other types of processes may
nad5, nad6, cob, rnl, and a variable number of tRNAs). In have generated similar signals. To be conservative, we
addition to these genes, we found the atypical presence of decided to keep only those results supported with high
rsp3 (encoding the ribosomal protein S3) in S. commune, confidence (P value < 0.05), but in general, most signals
Mo. perniciosa, Pl. ostreatus, Tr. cingulata, and M. lychnidis- did not have a high support. The average recombination
dioicae. We also found rnpB (encoding the RNA subunit of mt rate was estimated as the number of predicted recombina-
RNase P) in the mt genomes of M. lychnidis-dioicae, S. tion events normalized by the average substitution rate
Fungal Mitochondrial Genomics
FIG. 1.—ML phylogeny of our sampled taxa including the 38 species in the dikarya data set. The gene tree was inferred from a concatenated alignment of 14 single-copy orthologous genes (atp6, atp8,
Genome Biol. Evol. 6(2):451–465. doi:10.1093/gbe/evu028 Advance Access publication February 6, 2014
atp9, nad1–nad6, nad4L, cob, and cox1–cox3). RAxML v.7.2.6 (Stamatakis 2006) was used assuming the LG substitution matrix and default parameters. On the right side of each taxon name is a series of
colored boxes representing the mt gene order according to GenBank annotation. Bootstrap support appears next to each node. bsGOL values are shown next to each species name, and they are estimated
PP
by minimizing the following expression: L ¼ ( bi,jxj – GOLi)2, where bi,j is a Boolean variable that specifies the branches that are relevant for the estimation of a particular bsGOL (i.e., 0 if it is not relevant
and 1 if it is), xj is obtained by minimizing L and is the actual bsGOL value, and GOLi are the estimatedvalues from the pairwise comparisons, in other words, GOLi ¼ 1 GOCi (see Fischer et al. 2006 for more
details). Significant tRNA clustering was found in species marked with a spiral. This figure was made using the ETE python environment for tree exploration (Huerta-Cepas et al. 2010).
457
GBE

Aguileta et al. GBE
obtained from r8s for each clade, per fungal cluster. The
recombination events per site per time unit (Myr) were ba-
sidiomycetes (all 3 basidiomycete clusters): 0.11; sordariomy-
cetes: 0.26; saccharomycetes1: 0.02; eurotiomycetes:
0.09; dothideomycetes: 0.06; and saccharomycetes2: 0.15.
Recombination was not detected for the basal fungal cluster
with high confidence. It is noteworthy that most recombina-
tion detection programs lack power when looking for spo-
radic traces of recombination, as it is the case in mt genomes
(Posada and Crandall 2001; Barr et al. 2005; Neiman and
Taylor 2009).
Arguably, a better approach for investigating the evolution

of gene order due to nonhomologous, intrachromosomal re-
combination is to use estimates of gene rearrangements as a
proxy, as both involve identifying breakpoints, inversions and
translocations. We, therefore, compared the gene order lists
to infer the rearrangements that occurred between all pairs of FIG. 2.—GOC between pairs of genomes of the dikarya data set as a
species within each of the fungal clusters in the dikarya data function of their phylogenetic (patristic) distance. Distances were esti-
set. The average minimal rearrangement rates, estimated as mated using the estimated branch lengths in figure 3, listed in table 2.
the number of predicted rearrangement events normalized by Models are fitted by nonlinear regression. Model 0: GOC ¼ 2/1+eat.
the average substitution rate for each clade (per fungal cluster) Model 1: GOC ¼ 1 – ˇat. Model 2: 1/GOC ¼ at + 1. Model 3:
were: 0.03 for basidiomycetes (all three basidiomycete clus- GOC ¼ pt, where parameter a is adjusted by regression and t is the patristic
distance between the two compared taxa.
ters); 0.01 for sordariomycetes; 0.04 for saccharomycetes1;
0.02 for eurotiomycetes; 0.05 for dothideomycetes; 0.02 for
saccharomycetes2; and 0.03 for basals. These results are con-
sistent with the overall higher gene order variability observed eurotiomycetes (A. obtusum, Mi. canis, Pa. brasiliensis, and
in basidiomycetes, saccharomycetes2, followed by the sac- Pen. marneffei) at 0.14 and the sordariomycetes (C. globo-
charomycetes1, and in contrast to what is observed in sordar- sum, P. anserina, G. zeae, F. oxysporum, L. muscarium, Co.
iomycetes, dothideomycetes, and eurotiomycetes. bassiana, B. bassiana, and Me. anisopliae) at 0.1. Also, bsGOL
values show a moderate correlation with branch length values
Gene Order Variability in the Dikarya (R ¼ 0.7, P value < 0.0005, fig. 3). This is also consistent with
older clades, with deeper ancestral nodes, having more rear-
In the dikarya data set, GOC and bsGOL scores, estimated by ranged genes (e.g., basidiomycetes have a higher average
the methods of Rocha (2006) and Fischer et al. (2006), did not bsGOL value than sordariomycetes).
exhibit good fits to the patristic (phylogenetic) pairwise dis-
tance with tested empirical models (supplementary table S1,
Supplementary Material online; fig. 2). The goodness-of-fit Influence of tRNA Distribution, Intergenic, and Intronic
scores obtained were Model 0 ¼ 27.25, Model 1 ¼ 23.25, Elements on Gene Order in the Dikarya
Model 2 ¼ 21.2, and Model 3 ¼ 24.43. Following Fischer’s Rearrangements of the fungal mt genomes were influenced
approach (Fischer et al. 2006) to refine the models with esti- by the sequence characteristics, in particular the amount of
mates of bsGOL, gene order scores were observed to vary repetitive DNA elements at intergenic regions. The average
slightly among fungal clusters (table 2; bsGOL values on the bsGOL value, normalized by the number of fungal species in
right side of each species name in fig. 1). Nevertheless, the each cluster, did not display significant correlation with any of
average bsGOL score per group captures the GOL trend dif- the numbers of genomic elements (i.e., with either the
ferences among fungal clusters: the highest average GOL number of repeats, the number of introns and their associated
score (i.e., where GOL is greatest) is for the basidiomycetes, intronic ORFs, or the number and location of tRNAs, data
with 0.21, followed by the early diverging fungi (Al. macro- not shown). Also, correlations between rearrangements and
gynus and Rhizophydium sp. 136) and the saccharomycetes2 the proportions of introns and intronic ORFs were not signif-
(K. lactis, Ca. glabrata, N. bacillisporus, and V. polyspora) both icant (supplementary table S2, Supplementary Material
at 0.2; at an intermediate average bsGOL level are the sac- online). However, the number of rearrangement events was
charomycetes1 (Mil. farinosa, De. hansenii, Ca. albicans, Pi. significantly correlated with the proportion of repeats at
pastoris, O. angusta, and D. bruxellensis) at 0.18 and the intergenic regions (table 3): Pearson’s w2 test (observed
dothiodeomycetes (My. graminicola, Pel. malacea, and Ph. w2 ¼ 1,158.37, df ¼ 5, P value < 0.0001, alpha ¼ 0.05),
nodorum) at 0.16; at the lowest level of GOL are the Fisher’s exact tests (P value two-tailed < 0.0001,
Table 2
bsGOL, NRPS Rates, and Summary of the Number of Different Genomic Elements per Species
Taxon bsGOLa Branch Lengthsb GOL Ratec Normalized GOL Rated NPRS Ratese tRNAsf Intronic ORFsg Intronsh Repeatsi Intergenic Repeatsj Genome Sizek
Cryptococcus neoformans 3.50E-01 0.7 1.05E+00 2.96E+00 0.002 20 1 2 128 9 24,874
Moniliophthora perniciosa 1.16E-01 0.22 3.36E-01 7.89E-01 0.001 30 11 13 1,648 272 109,103
Microbotryum violaceum-Sl 3.12E-01 0.25 5.62E-01 1.32E+00 0.0007 20 33 22 1,003 380 107,808
Phakopsora pachyrhizi 2.38E-01 0.7 9.38E-01 2.11E+00 0.002 24 2 5 1,898 31 31,825
Pleurotus ostreatus 1.69E-01 0.15 3.19E-01 6.38E-01 0.0009 24 9 12 645 3 73,242
Schizophyllum commune 1.39E-01 0.32 4.59E-01 9.09E-01 0.002 27 0 0 1,718 61 49,704
Tilletia indica 8.97E-02 0.2 2.90E-01 5.74E-01 0.0008 24 0 0 568 52 65,147
Fungal Mitochondrial Genomics
Trametes cingulata 3.00E-01 0.16 4.60E-01 9.11E-01 0.0006 25 26 26 1,196 143 91,500
Ustilago maydis 1.71E-01 0.14 3.11E-01 6.16E-01 0.0005 23 11 12 337 37 56,814
Candida albicans 2.37E-01 0.24 4.77E-01 1.22E+00 0.003 30 0 2 279 40 40,420
Debaryomyces hansenii 1.64E-01 0.19 3.54E-01 8.64E-01 0.004 25 4 4 389 20 29,462
Dekkera bruxellensis 2.15E-01 0.26 4.75E-01 1.16E+00 0.003 25 3 5 3,968 939 76,453
Millerozyma farinosa 2.16E-01 0.2 4.16E-01 9.83E-01 0.004 25 11 14 710 31 39,107
Ogataea angusta 1.41E-01 0.4 5.41E-01 1.54E+00 0.004 26 6 6 944 46 41,719
Pichia pastoris 1.29E-01 0.21 3.39E-01 8.76E-01 0.002 25 3 6 1,080 21 35,683
Mycosphaerella graminicola 1.97E-01 0.16 3.57E-01 8.43E-01 0.0007 27 0 0 218 66 43,964
Peltigera malacea 9.28E-02 0.2 2.93E-01 8.35E-01 0.001 26 17 20 468 31 62,785
Phaeosphaeria nodorum 2.03E-01 0.18 3.83E-01 1.09E+00 0.0009 27 2 4 667 36 49,761
Arthroderma obtusum 7.66E-02 0.02 9.66E-02 2.48E-01 0.0007 25 1 1 325 14 24,105
Microsporum canis 7.66E-02 0.02 9.66E-02 2.38E-01 0.0007 25 1 1 344 10 23,943
Paracoccidioides brasiliensis 1.54E-01 0.24 3.94E-01 9.26E-01 0.001 25 3 13 552 175 71,335
Penicillium marneffei 2.38E-01 0.16 3.98E-01 9.19E-01 0.0007 28 9 11 303 16 35,438
Beauveria bassiana 2.90E-02 0.002 3.10E-02 7.38E-02 0.0002 25 5 5 284 10 29,961
Chaetomium thermophilum 1.45E-01 0.1 2.45E-01 6.29E-01 0.0008 28 3 3 927 113 127,206
Cordyceps bassiana 5.53E-02 0.009 6.43E-02 1.65E-01 0.0005 24 4 5 295 10 32,263
Fusarium oxysporum 5.37E-02 0.01 6.37E-02 1.66E-01 0.0006 25 1 2 298 1 34,477
Gibberella zeae 5.67E-02 0.02 7.67E-02 2.00E-01 0.0006 28 33 35 529 48 95,676
Lecanicillium muscarium 8.50E-02 0.05 1.35E-01 3.54E-01 0.002 25 1 1 241 9 24,499
Metarhizium anisopliae 1.46E-01 0.12 2.66E-01 6.97E-01 0.001 24 1 1 220 2 24,673
Podospora anserina 2.01E-01 0.09 2.91E-01 6.87E-01 0.0008 27 31 34 326 48 100,314
Allomyces macrogynus 1.97E-01 0.3 4.97E-01 7.44E-01 0.0004 25 10 28 219 14 57,473
Rhizophydium sp.136 3.65E-01 1.2 1.56E+00 3.84E+00 0.002 7 1 1 809 13 68,834
Schizosaccharomyces japonicus 5.08E-01 0.87 1.38E+00 3.38E+00 0.002 25 3 2 928 94 80,059
Yarrowia lipolytica 2.00E-01 0.56 7.60E-01 1.76E+00 0.002 27 15 17 792 72 47,916
Candida glabrata 9.56E-02 0.09 1.86E-01 2.78E-01 0.001 23 3 3 797 68 20,063
Kluyveromyces lactis 3.49E-01 0.14 4.89E-01 1.09E+00 0.001 22 3 3 1,190 279 40,291
Nakaseomyces bacillisporus 8.18E-02 0.2 2.82E-01 7.33E-01 0.003 23 0 0 7,896 3,791 107,123
Genome Biol. Evol. 6(2):451–465. doi:10.1093/gbe/evu028 Advance Access publication February 6, 2014
Vanderwaltozyma polyspora 2.62E-01 0.17 4.32E-01 1.06E+00 0.002 23 0 0 1,258 186 21,684
a PP
bsGOL values were estimated by minimizing the following expression: L ¼ ( bi,jxj – GOLi)2, where bi,j is a Boolean variable that specifies the branches that are relevant for the estimation of a particular bsGOL (i.e., 0 if it
is not relevant and 1 if it is), xj is obtained by minimizing L and is the actual bsGOL value, and GOLi are the estimated values from the pairwise comparisons, in other words, GOLi ¼ 1 GOCi (see Fischer et al. [2006] for more
details).
b
These branch lengths were obtained by ML phylogenetic reconstruction with RaxML (Stamatakis 2006).
c
bsGOL normalized by the branch length.
d
bsGOL rates normalized relative to the mean GOL rate value.
e
Rates obtained with r8s with the nonparametric method minimizing local transformations (NPRS) and optimization via Powell’s method (Sanderson 2003).
f
Number of tRNAs (GenBank).
g
Number of intronic ORFs (GenBank).
h
Number of introns (GenBank).
i
Number of repeats (whole genome) detected with Repeatmasker (Smit AFA, Hubley R, Green P. RepeatMasker Open-3.0.1996-2010; http://www.repeatmasker.org, last accessed February 18, 2014).
j
Number of intergenic repeats detected with mreps (Kolpakov et al. 2003).
459
GBE
k
Genome size in bp (GenBank).

Aguileta et al. GBE
Table 3
Number of Intergenic Repeats Normalized by the Number of Species
in Each Fungal Cluster, with and without Outliers, and Rearrangement
Events per Fungal Cluster
Fungal Cluster Intergenic Intergenic Rearrangement
Repeats Repeats Events
(without
Outliers)a
Basidiomycetes 988 (109.78) 336 (37.33) 414
Sordariomycetes 241 (30.13) 32 (4) 42
Dothideomycetes 133 (44.33) 133 (44.33) 24
Eurotiomycetes 215 (53.75) 40 (10) 22
Saccharomycetes1 1,097 (182.83) 158 (26.33) 156

Saccharomycetes2 4,324 (1,081) 254 (63.5) 22
Basals 27 (13.5) 27 (13.5) 14
a
Outliers are defined as the species that have higher than average repeat
content relative to their cluster: Dekkera bruxellensis, Paracoccidioides brasiliensis,
FIG. 3.—Pearson’s correlation between bsGOC values and branch Microbotryum violaceum-Sl, Moniliophthora perniciosa, Chaetomium thermophi-
lengths (R ¼ 0.7, P value < 0.0005) for the dikarya data set. lum, Gibberella zeae, Podospora anserina, Nakaseomyces bacillisporus, and
Kluyveromyces lactis.
alpha ¼ 0.05), Wilk’sG2 test of independence (observed

Wilk’s G2 value ¼ 1,156.383, df ¼ 5, P value < 0.0001), and species with conserved gene order to have significantly clus-
a randomization test of independence with 5,000 Monte tered tRNAs and species with low conservation order to have
Carlo simulations (observed w2: 1,158.37, df ¼ 5, P more scattered tRNAs.
value < 0.0001, alpha ¼ 0.05). Together, these results are
consistent with the hypothesis that repeats favor recombina- Gene Order Variability in Basal Fungi
tion events, thereby promoting rearrangements that change
Basal fungal taxa, being highly divergent with respect to as-
gene order (Gordenin et al. 1993; Bi and Liu 1996; Lobachev
comycetes and basidiomycetes, were analyzed separately to
et al. 1998; Rocha et al. 1999; Waldman et al. 1999; Rocha
obtain reliable alignments. On the basis of the similarity matrix
2003, 2006; Phadnis et al. 2005; Odahara et al. 2009; Lavrov
obtained for the basal data set, we performed a clustering
2010). In general, the more intergenic repeats in fungal mt
analysis (previously described for the dikarya data set)
genomes, the more likely it was to observe rearrangements
that resulted in three clusters of basal species that could
and, therefore, gene order variability.
be reliably aligned together. We thus aligned the
The randomization test assessing pairwise GOC distribu-
Blastocladiomycetes: Al. macrogynus with Bl. emersonii, the
tions and shuffled distributions relative to the patristic (phylo-
Glomeromycetes: Gi. margarita with Gl. intraradices, and the
genetic) distance showed that the pairs of species whose gene
Monoblepharidomycetes: Harpochytrium sp. together with
order has evolved significantly differently from random corre- H. curvatum and Monoblepharella sp.
spond to the well-conserved gene order sets of the sordario- No recombination events were reliably detected in any of
mycetes (figs. 1 and 4). According to our randomization test, the alignments of basal fungi. The evolutionary rates (substi-
the other pairs of species have seen their mt DNA gene order tutions per site per Myr) predicted by r8s assuming the
change more or less randomly. The random permutation test NPRS model were: 8 104,1 for the Blastocladiales,
implemented showed that the groups of species with highly 6 104 for the Monoblepharidales, and 1 103 for the
conserved gene order, such as sordariomycetes, also showed Glomeromycota. The rearrangement rate per clade per Myr,
tRNAs significantly grouped together in a few separate clus- as predicted by UniMoG and r8s were: 0.007 for the
ters along the mt chromosome (shown with a spiral on the Blastocladiales, 0.02 for the Monoblepharidales, and 0.06
right side in fig. 1). On the contrary, in species with high gene for the Glomeromycota. Supplementary figure S1,
order variability (e.g., basidiomycetes), tRNAs tended to be Supplementary Material online, shows the tree inferred for
scattered along the chromosome, consistent with the idea basal fungi, including bsGOL, branch length, and bootstrap
of tRNAs being associated to transposable elements that con- estimates. Results are summarized in supplementary tables
tribute to the variability in their distribution (Devine and Boeke S3–S5, Supplementary Material online. Pairwise GOC
1996; Hughes and Friedman 2004) and favor the reorganiza- models are shown in supplementary figure S2,
tion in the mt genome. Despite the presence of a few species Supplementary Material online. The goodness of fit scores
with low gene order variability and significantly clustered obtained for these empirical GOC models were Model 0:
tRNAs (Y. lipolytica, S. commune, Pl. ostreatus, Mo. perniciosa, 0.93, Model 1: 1.36, Model 2: 0.95, and Model 3: 0.9.
and Tr. cingulata), we nevertheless detected a trend for most bsGOL showed a moderate correlation with phylogenetic
in organelle genome structure among animals, plants, and

unicellular eukaryotes. Mutation rates for mtDNA vary strik-
ingly between these groups of organisms, with animals at the
highest mutation rate spectrum and plants at the lowest.
According to our results, fungal mtDNAs lie at intermediate
mutation rates between animal and plant mtDNA. Important
features are shared between fungal and plant mtDNA: lower
substitution rates than in animal mitochondria, the presence
of introns and associated mobile elements, higher noncoding
DNA than in animal mt genomes, and the capability of recom-
bination and the presence of recombination-associated DNA
repair mechanisms. Galtier (2011) has suggested that life

cycle, metabolic, and ageing (senescence) differences may ex-
plain these striking differences between animal and plant
mtDNA. We argue that such differences could also explain
the discrepancies with respect to animal mtDNA and the sim-
ilarities with plant mt genomes, although these comparisons
have not been specifically addressed, as far as we know.
FIG. 4.—Pairwise GOC values as a function of the phylogenetic (pa- Here, we have shown that there is high variability in terms
tristic) distance between them, for the dikarya data set. Here, we con- of mt gene order among fungi, both between and within the
ducted a randomization test as follows: for each genome, we listed the major phyla (i.e., basidiomycetes, ascomycetes, and early
order of genes, made all possible pairwise comparisons of these lists, es- diverging fungi). The mt genomes of basidiomycetes are
timated the GOC score (Rocha 2006), shuffled randomly the gene order, in general among the most rearranged groups (average
and estimated a new GOC value. We obtained 100,000 reshufflings and bsGOL ¼ 0.21), but other groups defined in this study, in par-
compared the original GOC to the distribution of the shuffled GOCs. We ticular the saccharomycetes1 and saccharomycetes2, also
applied the Bonferroni correction for multiple testing and determined the
show high variability (bsGOL ¼ 0.2 and 0.18, respectively).
significance (P values) of the comparisons. The red dots represent signifi-
On the contrary, the sordariomycetes and the eurotiomycetes
cant P values, which correspond to the group of sordariomycetes (in fig. 1,
the clade grouping Chaetomium thermophilum, Podospora anserina,
have highly conserved gene arrangements (bsGOL ¼ 0.1 and
Gibberella zeae, F. oxysporum, Lecanicillium muscarium, Cordyceps bassi- 0.14, respectively). Although GOL can occur rapidly within a
ana, Beauveria bassiana, and Metarhizium anisopliae). clade, as seen with the pairwise GOC models, it does not
appear to increase linearly with time. The average bsGOL
scores are somewhat more powerful at detecting trends
distance (R ¼ 0.7, P value ¼ 0.004, supplementary fig. S3,
in GOL than the empirical models for pairwise GOC. This
Supplementary Material online), and none of the sampled var-
means that, even if it is not very strong, there is a phylogenetic
iables including introns, intronic ORFs, tRNAs or repeats ap-
component to gene order variability patterns. Moreover,
peared to have an effect on gene order. However, given the
bsGOL scores show a moderate correlation with branch
low number of data points available for the correlation anal-
lengths. This indicates that time contributes somewhat
ysis, we take these results with caution, as there may be low
to GOL although there could be other confounding factors
detection power. We therefore suggest that additional basal
(e.g., gene loss in the saccharomycetes2 and Schizos.
fungi need to be available before stronger conclusions can be
japonicus).
drawn about the proximal causes of gene order variability.
GOC/GOL models measure gene order variability through
Overall, these results suggest that the mitochondria in basal time but do not offer a mechanistic explanation of this pro-
fungi have evolved with a faster evolutionary rate relative to cess. We used a simple nonparametric randomization test to
ascomycetes and basidiomycetes. On the other hand, mtDNA try to identify the propensity of particular fungal groups to
in basal fungi show comparable rates of rearrangements (an have greater change in gene orders. What our test showed
average of 0.029 events/Myr) with respect to ascomycetes is that, except for the sordariomycetes, which have remarkably
and basidiomyctes, with the notable exception of conserved gene order within the group, all other fungal clus-
Blastocladiomycetes (which exhibit a lower rate by one order ters seem to have genes rearranged more or less randomly.
of magnitude). One could think that sordariomycetes display a highly con-
served gene order because they constitute a relatively young
fungal group. Nevertheless, in the same time unit of a million
Discussion years, they have the lowest rearrangement rate compared
Lynch et al. (2006) have pointed out that nonselective forces with the other fungal clusters. Time alone does not explain
such as drift and mutation may account for major differences gene order changes.
Aguileta et al. GBE
Among the genomic elements studied here, repeats at sequences are not divergent enough for software to detect
intergenic regions show the strongest correlation with gene them (at least two phylogenetically informative sites must exist
order. According to theoretical studies, the accumulation of to each side of the recombination breakpoint [Martin et al.
repeats, among other mtDNA structural features, seems to be 2011]). Also, the power to detect recombination depends on
driven mostly by drift and mutation pressure, which are in turn the effective population size, which in the case of mitochon-
largely determined by population size dynamics (e.g., genome dria depends on the bottleneck levels attributable to mt trans-
size reduction or bottlenecks (Lynch and Blanchard 1998; mission (Neiman and Taylor 2009). Finally, although in
Lynch et al. 2006). Although intron-associated ORFs, in par- principle there is one homologous site per base available for
ticular those encoding HEs, have a great potential to insert homologous recombination, there are many more sites avail-
copies in different locations within the genome, thereby able for nonhomologous recombination. This is consistent
changing gene order, we did not observe a strong correlation with the latter type of recombination being more likely to
with gene rearrangements. If they play a role in shaping gene promote gene order changes.

order it appears to be less important than that of simple and Ectopic recombination is often facilitated by the presence
tandem repeats, especially those repeats present at intergenic of repeats in both plant and fungal mtDNAs, and it can
regions. have serious detrimental effects, including disruption of
The distribution of tRNAs contributes to protein-coding coding frames or gene expression alteration (Galtier 2011).
gene order variation among fungi, as they themselves can Different strategies to protect the genome from the negative
change location (Perseke et al. 2008). tRNAs have been asso- effects of ectopic recombination have evolved and they are
ciated with breakpoints involved in nuclear chromosomal rear- remarkably different in plant and animal mtDNAs (Galtier
rangements (Di Rienzi et al. 2009), and our results about 2011): although animal mtDNAs avoid the accumulation of
fungal mtDNAs are consistent with this observation. Species repeats and introns at the cost of a higher mutation rate
showing the highest gene order variability are those showing a (Lynch et al. 2006), plant mtDNAs have selected for efficient
scattered tRNA distribution (e.g., Schizos. japonicus), as op- recombination-mediated DNA repair mechanisms, thus ex-
posed to less variable species, whose tRNAs tended to be plaining the low mutation rate observed in plant mt genomes
clustered (e.g., in sordariomycetes). Another source of gene (Odahara et al. 2009; Davila et al. 2011). Moreover, efficient
order variability is gene loss (e.g., due to transfers to the nu- mismatch repair is often accompanied by gene conversion in
cleus), which could be important in the saccharomycetes2 and plant mtDNA (Davila et al. 2011). In this study, we have not in-
Schizos. japonicus. Finally, although less frequent, HGTs can vestigated recombination-associated DNA repair mechanisms
also contribute to gene order changes (e.g., Bergthorsson in fungal mt genomes; it is nevertheless interesting to specu-
et al. 2004) but we did not investigate it here. late whether fungi have selected for mtDNA repair mecha-
A commonly invoked mechanism to explain gene order nisms similar to those found in plants as defense against
changes is the “tandem-duplication-random-loss” model repeat accumulation. It is known, for instance, that in P. anser-
(Lavrov et al. 2002) that was first proposed to explain gene ina the nuclear-encoded gene grisea protects mtDNA integrity
order in millipedes and suggested that the entire mtDNA was from the deleterious effects of ectopic recombination (Belcour
duplicated with a subsequent loss of gene blocks. In our case, et al. 1991). It would be particularly interesting to test this
this model could partly explain the gene order differences hypothesis in other fungal mtDNAs such as those of the sor-
(only the loss and not the duplication) observed among sac- dariomycetes that show evidence of recombination (Kouvelis
charomycetes2 and in Schizos. japonicus due to the gene loss et al. 2004; Ghikas et al. 2006; Pantou et al. 2008) and
of the NADH gene family (Gabaldon et al. 2005), as these high GOC.
losses necessarily resulted in gene order changes relative to The evolution of gene order in fungal mitochondria, parti-
the ancestral gene arrangement that included the NADH cularly in basidiomycetes, suggests a complex interplay of
genes. The tandem-duplication-random-loss model, however, opposing evolutionary forces. Although mt genes tend to be
cannot account for inversions and transpositions, which may conserved at the sequence level due to their importance in
be better explained by nonhomologous, intrachromosomal cellular metabolism, here we have shown that in fungal
recombination. We suggest that most of the observed gene mtDNA gene order is relatively free to vary, and that this var-
rearrangements among fungal mtDNAs are very likely caused iation is probably largely due to recombination (most likely
by this or a similar recombinational mechanism. Notably, re- nonhomologous, intramolecular). Indeed, in most studies,
combination has been reported in vitro and in natural popu- the diversity of gene order in mitochondria is taken as
lations for fungal mt genomes (van Diepeningen et al. 2010). evidence of effective recombination. Furthermore, some
Difficulties in detecting recombination based on sequence mtDNA sequence characteristics appear to contribute to
data can result from multiple factors, including the scale of the gene order variability. In particular, repeats at intergenic se-
genomic regions involved, where analysis of adjacent nucleo- quences tend to increase the probability of recombination,
tides may fail to detect recombination occurring across large both homologous and nonhomologous, thereby facilitating
physical distances (Neiman and Taylor 2009) or where rearrangement events, in agreement with numerous previous
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FEMS Yeast Res. 9:1283–1292.
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High Variability of Mitochondrial Gene Order among Fungi

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Introduction origin of mitochondria, a high percentage of the ancestral

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alpha ¼ 0.05), Wilk’sG2 test of independence (observed

in organelle genome structure among animals, plants, and

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