BACKGROUND
the nematophagous fungus pochonia chlamydosporia  zare & gams  is a widespread soil fungus distributed worldwide in nematode suppressive soils.
pochonia chlamydosporia can infect the females and eggs of plant endoparasitic nematodes as a facultative parasite  <cit> .
it has already demonstrated its efficacy as a bca against both root-knot nematodes  and cyst nematodes   <cit> , major agricultural pests that cause tremendous economic losses worldwide estimated at  <dig> billion dollars annually  <cit> .
the fungus p. chlamydosporia belongs to the family clavicipitaceae .
based on spore morphology and polymorphisms of the nuclear ribosomal internal transcribed spacer  region, this species has been recognized as having at least two distinct varieties, p. chlamydosporia var.
chlamydosporia and p. chlamydosporia var.
catenulate  <cit> .
many members of clavicipitaceae are invertebrate-pathogenic fungi.
it was previously suggested that the host relatedness and host habitat hypotheses could explain the evolution of parasite-host relationships  <cit> .
therefore, exploring the phylogenetic relationships and evolutionary trajectories of the invertebrate-pathogenic fungi in clavicipitaceae is a topic of interest.
multi-gene phylogenetic analyses showed that clavicipitaceae is paraphyletic and consists of three well-defined clades, at least one of which is shared with the members of another fungal family   <cit> .
the phylogenetic placement of the genus pochonia in the family clavicipitaceae was inferred from six nuclear genes  and one mt gene  using phylogenetic analysis, and the phylogenetic trees inferred from different genes produced different topological structures  <cit> .
therefore, a comparison of larger scale genome sequences is necessary because it can provide deep insights into the phylogenetic relationships and evolution of the fungi in clavicipitaceae.
recently, the complete nuclear genome sequence of p. chlamydosporia was published.
based on a phylogenetic analysis inferred from nuclear genome sequences, p. chlamydosporia appeared to be most closely related to the metarhizium species  <cit> .
mitochondrial markers can be successfully applied in evolutionary biology and systematics because mt genomes often evolve faster than nuclear genomes and allow for robust phylogenetic analyses based on the conserved proteins of the oxidative phosphorylation system  <cit> .
the continuously increasing number of recent fungal mt genome studies – more than  <dig> complete fungal mt genomes are available today at ncbi - provide powerful tools for comparative studies to reveal the patterns and mechanisms of mitogenome evolution  <cit> .
thus far, fungal mitogenomes vary considerably in size, the largest being  <dig>  bp long in rhizoctonia solani ag <dig> ia   <cit>  and the smallest being  <dig>  bp long in hanseniaspora uvarum   <cit> .
researchers have demonstrated that different lengths of intronic sequences and intergenic regions result in different sizes of mitogenomes  <cit> , particularly for the mitogenome of moniliophthora perniciosa in basidiomycota, which has a length of  <dig>  bp and encodes several hypothetical orfs in the larger sized intergenic regions and introns  <cit> .
however, a depiction of the size variation and related mechanisms in ascomycota has not yet been reported.
moreover, the nearly ubiquitous presence of rps <dig> genes, which are encoded in the group i introns within rnl genes, has been attributed to a vertical mode of inheritance rather than horizontal inheritance during evolution  <cit> .
in one study, the molecular evolution of the rps <dig> gene in filamentous ascomycetes fungi  was analysed, and the group i intron-encoded version of rps <dig> appeared to have a rather complex evolutionary history in ascomycetes fungi  <cit> .
however, only a few fungi in hypocreales were included in that study.
to better understand the evolution of the fungal mitogenome in hypocreales, a phylogenetic analysis of the rps <dig> genes is required.
furthermore, it was reported that trn genes are able to change their location in genomes and participate in horizontal gene transfer  events, as they have editing, excision and integration capabilities  <cit> .
because changes in trn location are relatively rare events, the locations of trn genes in fungal mt genomes have been used to study fungal evolution and phylogenetic signals  <cit> .
to date,  <dig> different types of aminoacyl-trnas  have been identified in fungal mitogenomes, and conserved trn clusters are found in pezizomycotina by employing comparative genomic approaches  <cit> .
the distribution of trn genes may contribute to gene order variation in fungal mitogenomes  <cit> .
six operations have been used to explain rearrangements in mt gene order: inversion, transposition, reverse transposition, tandem-duplication-random-loss  <cit> , deletion and replication slippage  <cit> .
however, the evolution of gene orders in pezizomycotina mitogenomes is suggested to be mainly driven by transpositions  <cit> .
as in animal mt genomes, trn in fungal mitogenomes might have played a role in gene shuffling as they intervene between genes and can act like mobile elements  <cit> .
taking advantage of published fungal mitogenomes and the novel p. chlamydosporia mitogenome, we investigated the trn rearrangements in pezizomycotina, and addressed the role of rearrangements in evolution.
in this paper, the p. chlamydosporia strain  <dig> isolated from a rkn in china was sequenced using illumina sequencing, and a complete mt genome of  <dig>  bp was obtained.
we aim to  present a complete and annotated mt genome sequence of p. chlamydosporia,  compare the mt genome of p. chlamydosporia with the genomes of other fungi in pezizomycotina to identify the common and specific characteristics of the mt genomes of invertebrate-pathogenic fungi, and  provide powerful insights into the evolution and phylogenetic relationships of the invertebrate-pathogenic fungi in hypocreales.
RESULTS
general characteristics of the mt genome in p. chlamydosporia
the complete mt genome of p. chlamydosporia is a circular dna molecule with a length of  <dig>  bp.
the low g + c content  is similar to other fungal mitogenomes in clavicipitaceae .
the mitogenome encodes an essential set of conserved genes including three cytochrome c oxidase subunits , apocytochrome b , three atp synthase subunits , seven subunits of nadh dehydrogenase , the small and large ribosomal rna subunits , an intronic orf coding for a putative rps <dig>  and  <dig> trn.
all of the  <dig> genes are encoded on the same dna strand .
the set of trn genes found  is the smallest set currently known for pezizomycotina fungi , but the trn genes can recognize the codons for all  <dig> amino acids.
in total, the coding regions , of which the  <dig> typical protein-coding genes related to oxidative phosphorylation account for  <dig>  bp, and together with the rps <dig>  within the rnl group i intron , cover  <dig> % of the total mt genome, and the rest are trn, rrna genes and intergenic regions.table  <dig>
general features of the mitogenomes
species
length
gc
coding genes
trn
rrnas
accession
pochonia chlamydosporia
metarhizium anisopliae
beauveria bassiana
beauveria pseudobassiana
cordyceps brongniartii
lecanicillium muscarium
cordyceps militaris
hypocrea jecorina
fusarium solani
fusarium graminearum
fusarium verticillioides
fusarium oxysporum
verticillium dahliae
neurospora crassa
aspergillus fumigatus
aspergillus niger
aspergillus tubingensis
trichophyton rubrum
epidermophyton floccosum
paracoccidioides brasiliensis
candida parapsilosisfunctional map of the complete mitogenome of
p. chlamydosporia
.
all genes are transcribed on the same strand.
there are  <dig> encoding genes,  <dig> transfer rna genes and  <dig> ribosomal rna genes .
the circular map was generated using organellargenomedraw  <cit> .
using the abundant sequenced resources available , the sequence variance of the p. chlamydosporia mitogenome was investigated, but no single nucleotide polymorphism  was found.
this indicates that a high quality mitogenome was assembled.
moreover, we compared the published mt gene sequences of p. chlamydosporia with our genome sequence.
in total,  <dig> mt gene sequences from ncbi are available, including six coding segments in the imi  <dig> strain , four genes in the imi  <dig> strain , one gene in the cbs  <dig> strain  and one gene in the cbs  <dig>  strain .
our analysis showed that  <dig> sequences were matched to our genome sequence with identities greater than or equal to 95% , suggesting that these gene sequences are highly conserved in p. chlamydosporia strains.
phylogenetic relationships of p. chlamydosporia to other fungi in pezizomycotina
utilizing published fungal mitogenomes in addition to the novel p. chlamydosporia mitogenome, the phylogenetic relationships in  <dig> species in pezizomycotina were inferred, using candida parapsilosis as an outgroup .
phylogenetic trees were constructed based on the  <dig> conserved protein-coding genes associated with the oxidative phosphorylation system  using a maximum likelihood  approach.
the topological structures based on both nucleotide and amino acid sequences were identical, and a clear genealogical relationship was shown .
p. chlamydosporia is most closely related to the entomopathogenic fungus m. anisopliae, with a bootstrap value of 100%, which is similar to the result of phylogenetic analysis based on genome-encoded orthologous proteins  <cit> .
the two fungi form a cluster with five additional entomopathogenic fungi, namely, cordyceps brongniartii, beauveria pseudobassiana, b. bassiana, lecanicillium muscarium and c. militaris.
interestingly, although both p. chlamydosporia and l. muscarium were once put into the same genus , the two species are assigned to different sub-clades in the phylogenetic tree.
however, all of the invertebrate-pathogenic fungi cluster into a clade  clearly separated from the plant-parasitic fungi, including four fusarium plant pathogens , which form clade b with the fungus hypocrea jecorina that specializes in colonizing pre-degraded wood.
both invertebrate-pathogenic and plant-pathogenic fungi shared a common ancestor in clade c represented by a soil born plant pathogen, verticillium dahliae, suggesting that hypocreales is a monophyletic group .
the phylogeny shows that plant pathogenic and invertebrate-pathogenic fungi in hypocreales form independent clusters and have evolved separately.
a third group comprises fungi that infect animals or humans, including several members of eurotiomycetes, i.e., aspergillus fumigates, paracoccidioides brasiliensis, epidermophyton floccosum and trichophyton rubrum; this group is distant from p. chlamydosporia and the entomopathogenic fungi.
the phylogenetic relationships inferred from mt proteins are principally consistent with the current pezizomycotina taxonomic system .figure  <dig>
the mitogenome phylogenetic analysis and comparative analysis.
a).
phylogenetic placement of p. chlamydosporia using  <dig> fungal mitogenomes.
the phylogeny was generated based on  <dig> conserved encoded proteins  using a ml approach.
c. parapsilosis was used as an outgroup.
p. chlamydosporia is closely related to m. anisopliae with a bootstrap support value of 100%.
b).
synteny comparisons between p. chlamydosporia and m. anisopliae, p. chlamydosporia and l. muscarium.
syntenic regions were identified by performing blastn with a threshold of 1e- <dig>  and alignment blocks of less than  <dig> bp are not shown.
comparison of mitogenome organization in p. chlamydosporia and related species
we first compared the mitogenomes of p. chlamydosporia, m. anisopliae and l. muscarium , and found that they are highly similar in both size  and g + c content  .
the total length of the gene regions  in p. chlamydosporia mt genome  is  <dig> bp less than that of l. muscarium and  <dig> bp less than that of m. anisopliae.
however, the entire length of the p. chlamydosporia mitogenome is  <dig>  bp and  <dig> bp larger than l. muscarium and m. anisopliae respectively, mainly due to longer intergenic regions in p. chlamydosporia.
we also compared the contents of the simple repeat motifs in each of the genomes, including low complexity sequences , simple repeats  n), inverted repeats and palindromes, which have been previously suggested to be putative elements for recombination or regulation  <cit> .
it was found that ~ <dig> %  of the p. chlamydosporia mitogenome consists of repeat sequences compared with only ~ <dig> %  of the l. muscarium mitogenome and ~ <dig> %  of the m. anisopliae mitogenome.
the distributions of repeat elements are also different .
in l. muscarium, there are no repeat sequences located in the regions between nad <dig> and rnl, nad <dig> and cob, rns and cox <dig>  however, in m. anisopliae and p. chlamydosporia, there are no repeat sequences in the regions between rnl and nad <dig>  cox <dig> and nad <dig>  there are repeat sequences residing in the regions between rns and cox <dig> in p. chlamydosporia, but only one at rich motif is found in this region in m. anisopliae .
repeat sequences in this region display the unique mitogenomic features of p. chlamydosporia.
in addition, sequence similarity studies and syntenic gene analyses were performed comparing p. chlamydosporia with five other mitogenomes, including m. anisopliae, l. muscarium, b. bassiana, v. dahliae and a. fumigatus.
the analyses show identical gene order in m. anisopliae, l. muscarium, b. bassiana and p. chlamydosporia mt genomes.
v. dahliae, as shown in previous work  <cit> , displayed differences at the regions nad6-rnl, nad3-atp <dig> and cox1-nad <dig> with a gene order of nad6-atp <dig>  nad3-nad <dig> and cox1-rnl instead .
a larger difference of gene order is observed between the mitogenomes of p. chlamydosporia and a. fumigatus .
comparing the two mitogenomes of p. chlamydosporia and m. anisopliae,  <dig>  bp  of m. anisopliae mitogenome sequences match to  <dig>  bp  of p. chlamydosporia mitogenome sequences with  <dig> % identity determined by blastn .
a long-range synteny between the p. chlamydosporia and m. anisopliae mt genomes is observed.
the majority of the unmatched sequences reside within the intergenic regions between nad <dig> and rnl.
however, comparing the p. chlamydosporia and l. muscarium mitogenomes,  <dig>  bp  of the l. muscarium mitogenome sequences match to  <dig> % of the p. chlamydosporia mitogenome sequences with  <dig> % identity .
comparisons of p. chlamydosporia mt genomes with the other three species  showed that only  <dig>  bp  of b. bassiana,  <dig>  bp  of v. dahliae and  <dig>  bp  of a. fumigatus mitogenomes matched to p. chlamydosporia mt sequences with  <dig> %,  <dig> % and  <dig> % identities, respectively .
although the sequences of most of the mt genes in v. dahliae matched to homologous genes in p. chlamydosporia mitogenome, the alignments of some genes  between the two species present multiple blocks, and no alignments are found in the intergenic regions in the p. chlamydosporia and v. dahliae mitogenomes.
based on the above syntenic analysis results, an evolutionary relationship between p. chlamydosporia and the other fungi is also indicated, that is, that p. chlamydosporia is most closely related to m. anisopliae.
the result is similar to that obtained from the genomic dna analysis  <cit> .
analysis of mitogenome size variation among the fungi in sordariomycetes
the sizes of the mitogenome in the fungi in sordariomycetes vary significantly, from  <dig>  bp in m. anisopliae to  <dig>  bp in f. graminearum .
to identify the causes of this mitogenome size variation in sordariomycetes, five mitogenomes  of different sizes were used for a comparative analysis .
despite the size variance ranging from  <dig>  bp in m. anisopliae to  <dig>  bp in n. crassa,  <dig> common mt protein-coding gene sets, including cox1- <dig>  cob, nad1- <dig>  nad4l, atp <dig>  atp8- <dig> and rsp <dig>  are encoded in all of the five genomes.
we calculated the size of the crs, trn regions, rrna regions, introns and intergenic regions in each mitogenome separately .
in p. chlamydosporia mitogenome, the lengths of the crs, trn regions, rrna regions, introns and intergenic regions are  <dig>  bp ,  <dig>  bp ,  <dig>  bp ,  <dig> bp  and  <dig>  bp  of the genome sequences, respectively.
a comparison of the five mitogenomes showed that, in general, the length of each region  increases with genome size, as well as the proportions of introns and intergenic regions, with the exception of h. jecorina, which has the smallest length of rrna genes but the largest length of introns of the five mitogenomes.
in addition, the length of its intergenic regions is less than that of f. oxysporum, although the genome size of h. jecorina is larger than that of f. oxysporum .
the results indicate that, in sordariomycetes, crs, intergenic regions and introns are the three main contributors to mitogenome size variation, with the length of intergenic regions or introns being the primary contributor in most of the mitogenomes.
the length of the crs in n. crassa and h. jecorina is the second highest contributor because several intron-encoded orfs and unidentified reading frames exist in their intergenic regions .figure  <dig>
a comparison of the mitogenomes of five pezizomycotina fungi with considerable size differences.
there are  <dig>   <dig>   <dig>   <dig>  and  <dig> protein-coding genes in p. chlamydosporia, m. anisopliae, f. oxysporum, h. jecorina, and n. crassa, respectively.
size comparisons indicate that the larger the mitogenomes are primarily due to larger intergenic regions, secondarily to introns, and to a lesser extent to longer coding regions.
codon usage in the fungal mitogenomes of pezizomycotina
we analysed codon usage bias in the mitogenome of p. chlamydosporia, and compared it with other fungal mitogenomes in pezizomycotina.
genetic code four  <cit>  was used to transcribe the coding gene sequences in the p. chlamydosporia mt genome.
a total of  <dig> codons were found, including the high-frequency codons tta , ata , gta  and ttc  , which are amino acids with hydrophobic side chains.
six codons  were absent in p. chlamydosporia but present in the closely related species m. anisopliae.
however, two codons  were absent in m. anisopliae but present in p. chlamydosporia.
clearly, nearly in all of the fungal mitogenomes, codon usage is biased strongly towards codons ending in a or t, and more than 84% of the codons in p. chlamydosporia mitogenomes end in a or t , possibly due to the high at content found in fungal mitogenomes.
similar results were obtained from m. anisopliae and l. muscarium mitogenomes  <cit> .
molecular evolution of rps <dig> in pezizomycotina
we identified and annotated the rps <dig> genes in the  <dig> fungal mitogenomes.
the length of the rps <dig> sequences ranged from  <dig>  bp in the t. rubrum and e. floccosum mitogenomes to  <dig>  bp in the f. verticillioides mitogenome .
when the  <dig> rps proteins were aligned by blastp the similarity levels were greater than or equal to 30%.
however, the c-terminal domains of these proteins obtained from the pfam database indicated a common function .
the addition of  <dig> more fungal rps <dig> gene sequences showed matches with e-value cutoffs of less than 2e- <dig>  hence they were omitted from comparisons.
e-value cutoffs of  <dig> were obtained for the rps <dig> genes of f. oxysporum, m. anisopliae, h. jecorina, f. solani, f. graminearum and f. verticillioides .
the unique evolutionary relationships among these species can be elucidated from phylogenetic analysis based on the sequences of the rps <dig> genes .
a phylogenetic tree shows that p. chlamydosporia rps <dig> is more closely related to f. oxysporum and m. anisopliae rps <dig>  they then group together with h. jecorina and other three fusarium species to form clade a. clade a separates distinctly from clade b, which contains the other five entomopathogenic fungi  .
evidently, the phylogenetic relationships among the species of hypocreales inferred from the rps <dig> genes are different from those inferred from the other  <dig> protein-coding genes .
to measure the rate and selective pressure of the rps <dig> genes, we calculated nonsynonymous/synonymous substitution rate ratios  by adopting different models .
the dn/ds value for the  <dig> rps <dig> genes is  <dig>  using the codeml program with a jtt model in paml  <cit> .
because every functional protein contains amino acid sites under selective constraints, averaging the evolutionary rates across sites leads to a lower capacity to detect positive selection.
thus, we adopted the site models m <dig> , m <dig> , m <dig>  and m <dig>  for the ω ratio analysis  <cit> .
because the m7-m <dig> comparison is a very stringent test of positive selection  <cit> , we calculated the value of the likelihood ratio test  statistic , and the significant results of this comparison clearly indicates signals of positive selection in the rps <dig> genes.
to further confirm whether the hypocreales rps <dig> genes are evolving under positive selection or not, we took the rps <dig> sequence of t. rubrum as a reference to calculate the dn and ds of each rps <dig> sequence with the reference sequence using dnasp  <cit>  and codeml  <cit> .
similar results were obtained, that is, dn/ds >  <dig> in hypocreales .
the above results indicate that the fungal rps <dig> protein is not only under functional constraints but also under positive selection in hypocreales.
trn gene distribution and putative gene rearrangements in pezizomycotina
the distribution of trn genes on mt genomes is considered as a factor that potentially contributes to the gene order variation in pezizomycotina  <cit> .
firstly, we analyzed the distribution of  <dig> trn genes in the p. chlamydosporia mitogenome and found that they cluster into three groups , with the exception of four trn genes  that scatter as a single gene throughout the mt genome .
then, we compared the  <dig> trn genes  in the  <dig> fungal mitogenomes of pezizomycotina and found that they are presented in all mitogenomes whereas some of the other seven trn genes  are often missing from a fungal mitogenome  as reported in previous studies  <cit> .
some trn genes have multiple copies, such as trnm, which has at least two copies in each mitogenome.
a sequence alignment showed that the trn gene sequences display high similarities among these fungal species, such as for trnn, where  <dig> out of the 71/ <dig> bp  are in consensus in the  <dig> mitogenomes, and completely identical  in b. bassiana, b. pseudobassiana and c. brongniartii .
a comparative analysis showed that the distribution of trn genes is variable in pezizomycotina, but identical in the order hypocreales .
the trn clusters are conspicuously observed in these mitogenomes, as reports in previous studies  <cit> .
excepting the largest trn cluster  conserved in all of the pezizomycotina mitogenomes, the others are generally conserved at the order level, such as  and  in hypocreales,  in sordariales,  in eurotiales and  in onygenales, although minor differences exist owing to the lack of a trn or a transposition event .
the clustering of trn is suggested to be a unique characteristic of all pezizomycotina  <cit> .figure  <dig>
the schematic representation of
trn
clusters.
a).
analysis of the trn genes residing in the complete mitogenomes of  <dig> fungi that belong to nine families.
the nine families are clavicipitaceae, hypocreaceae, nectriaceae, plectosphaerellaceae, sordariaceae, lasiosphaeriaceae, trichocomaceae, ajellomycetaceae and arthrodermataceae.
b).
an example of high sequence similarities in trn genes .
the consensus bases are marked in boldface.
based on a display of the orthologous protein-coding genes, trn genes and rrnas in the  <dig> mitogenomes , a similar gene order can be observed in the hypocreales mitogenomes, with the exception of trng, which changed position in fusarium species.
the gene order is essentially identical in the seven invertebrate-parasitic fungi  and h. jecorina, as well as the four fusarium fungi, as found in previous studies  <cit> .
the same is also observed in eurotiales and onygenales.
however, marked differences in gene order are observed between sordariomycetes and eurotiomycetes .
this indicates that in pezizomycotina, which has closer phylogenetic relationships among its fungi, a greater number of identical gene orders are displayed, supporting the conclusion that the greater the identical gene orders the closer relationship they share  <cit> .
using crex analysis  <cit> , possible gene rearrangements in the  <dig> mitogenomes were predicted.
a total of  <dig> transposition events were identified,  <dig> of which involve changes in trn position .
among these, there are some rearrangement events that only occur in certain fungi , but no transposition event was found in clavicipitaceae.
several events occur at the order level, for example, the trng location in hypocreales involves at least three events .
most of the rearrangements occur in pezizomycotina, such as trnh , trni and trns , between sordariomycetes and eurotiomycetes .
notably, many transposition events  occur in the region near nad <dig> , perhaps due to historical changes in gene order in this region.
although four types of rearrangement operations  were considered in the program crex  <cit> , only transposition events were predicted in our study, supporting the suggestion that the evolution of gene orders within pezizomycotina is mostly driven by transpositions  <cit> .
even though the mechanisms of rearrangements are not fully understood, these rearrangement events may provide clues about the phylogeny and evolution of the fungal species in pezizomycotina.
discussion
the mt genome represents a major model system in studies on evolutionary genomics, such as in animals  <cit>  and fungi  <cit> .
here, the organization of the complete mt genome of p. chlamydosporia that is a promising bca parasitizing nematode eggs is provided and its phylogenetic relationships to other invertebrate-pathogenic fungi are investigated.
synteny and phylogenetic analyses show that p. chlamydosporia is more closely related to m. anisopliae than to l. muscarium and other entomopathogenic fungi , even though both p. chlamydosporia and l. muscarium were once assigned to the same genus, verticillium.
our results are consistent with previous results on the nuclear genome, in which the genome of p. chlamydosporia was found to be most closely related to entomopathogenic fungi metarhizium spp.
<cit> , as well as on the phylogenetic analyses of the p. chlamydosporia serine proteases  <cit> .
our phylogenetic results also show that fungi attributed to the same genus group together at first, such as the three species of beauveria , and the two species of lecanicillium .
these five species then cluster into a clade because all of their teleomorphic forms are attributed to the same genus, cordyceps  <cit> .
similarly, the four fusarium species and the three aspergillus species form two separate clades.
according to our results, p. chlamydosporia and m. anisopliae also group together with a bootstrap value of 100%.
the teleomorph of p. chlamydosporia is metacordyceps chlamydosporia, but the teleomorph of m. anisopliae is unknown, therefore, we hypothesize that the teleomorphic form of m. anisopliae may also belong to the genus metacordyceps.
our phylogenetic tree showed that, in hypocreales, all of the invertebrate-pathogenic fungi cluster together to form a monophyletic group, which is noticeably distinguished from a cluster comprising plant pathogens.
this suggests that these invertebrate-pathogenic fungi have a most recent common ancestor .
notably, in our phylogenetic tree, the fungus h. jecorina  groups together with the four fusarium species, and is obviously outside the group entomopathogens.
this is in agreement with some previous studies  <cit> , but distinctly different from that in others  <cit> , in which h. jecorina was within entomophagous group and more closely related to m. anisopliae.
because the results were obtained from both the total genomic data  <cit>  and the mitogenomic data  <cit> , we believe this may reflect to the different evolutionary rates of the nuclear and mt genomes or the much bigger data  provided by the nuclear genome in comparison with mt genes.
considering h. jecorina that is attributed to the family hypocreaceae in fungal taxonomy, we think it is more reasonable that h. jecorina is outside the group entomopathogens, which belong to the family clavicipitaceae.
based on the phylogenetic relationship in our study, the evolution of fungi in hypocreales may be speculated to have evolved from being parasitic on plants towards being parasitic on invertebrates.
we think that a host habitat hypothesis can be used to explain the host shift in hypocreales fungi, which suggests that host shifts tend to follow the host’s microhabitat or feeding habitat, resulting in a group of related endoparasites that exploit distantly related organisms at higher taxonomic levels  <cit> .
nikoh and fukatsu reported that the entomoparasitic fungi of cordyceps  have an interkingdom host jumping from animalia to fungi by overlapping the ecological niches of the unrelated hosts  <cit> .
because most hypocreales fungi grow naturally in soil, interkingdom host jumping events in hypocreales might occur in an underground environment.
it is thus worthwhile to study the evolution of hypocreales fungi in depth in the future.
among the ascomycete fungi, it was reported that the group i intron encoded version of rps <dig> appears to have a rather complex evolutionary history  <cit> .
however, the evolution of rps <dig> in hypocreales fungi has not been reported so far.
in this study, our phylogenetic analysis shows that mitogenome rps <dig> genes display an evolutionary pattern distinctly different from those inferred from the  <dig> mt protein-coding genes in hypocreales fungi .
a jtt model showed a dn/ds value less than  <dig>  indicating that the encoded protein is under functional constraints, i.e., natural selection is operating to minimize the number of amino acid changes, thereby maintaining the activity of the protein; a result similar to that found in a study with ophiostomatoid fungi  <cit> .
however, a site model analysis showed a clear signal of positive selection in these rps <dig> sequences.
moreover, the dn/ds values obtained for all of the rps <dig> sequences in hypocreales - in relation to the reference sequence of t. rubrum - are also greater than  <dig>  suggesting that positive selection acts on rps <dig> in hypocreales fungi.
such a positive selection on rps <dig> was also observed previously in gymnosperms  <cit> .
although the special function of rps <dig> in ascomycetes fungi is unclear, it is known that rps <dig> plays a critical role in ribosome biogenesis and dna repair in other eukaryotes  <cit> .
therefore, further investigation is needed to fully understand the evolution of fungal rps <dig> genes.
as shown recently by the comparison of  <dig> complete fungal mt genomes from all the major phyla, remarkable variation is observed in genome size, gene order, composition of intergenic regions, repeats and introns  <cit> .
in our study, we compared the sizes of the mt genomes from fungal genera belonging to sordariomycetes and showed that, crs, intronic and intergenic regions all contributed to genome size variation.
however, the major contributor was the length of the intergenic regions or the intronic regions in the majority of the fungal mitogenomes .
our result indicates that, in sordariomycetes, the length of intergenic regions is also an important contributor to fungal mitogenome size variation, the same as the length of introns.
a remarkable characteristic of fungal mitogenomes is that, similar to plant mitogenomes, fungal mitogenomes show signals of recombination.
a comparison of  <dig> complete fungal mitogenomes  showed that the patterns of rearrangements may be explained by the combined influences of recombination, accumulated repeats, especially at intergenic regions, and to a lesser extent, mobile element dynamics  <cit> .
in this study, we compared the trn in the  <dig> mitogenomes of pezizomycotina fungi, and found that the gene order for all the protein- and rrna-coding genes were principally conserved in sordariomycetes, but trn clusters were conserved at the order level .
obvious differences of gene order and trn clusters were observed between sordariomycetes and eurotiomycetes.
our results support the previous reports that the sordariomycetes and the eurotiomycetes have highly conserved gene arrangements  <cit> .
moreover, several gene order rearrangement events in the  <dig> pezizomycotina fungi mitogenomes were estimated by crex analysis, and  <dig> transposition events in the mitogenomes were identified .
out of these,  <dig> events displayed changes in trn order.
our results support the view that the evolution of gene order in pezizomycotina is mostly driven by transpositions  <cit> , although four operations of inversion, transposition, reverse-transposition and tandem-duplication-random-loss were considered in crex program  <cit> .
the observed transpositions also support the previous suggestion that trn genes should be considered as mobile elements involved in gene rearrangement  <cit> .
our results may provide an illustration of trn genes location changes in gene rearrangement in pezizomycotina fungi mitogenomes.
CONCLUSIONS
in this study, the complete and annotated mt genome sequence of p. chlamydosporia is provided.
the relationships among the invertebrate pathogenic fungi in hypocreales are determined.
according to different model predictions, our results indicate that the rps <dig> gene has experienced positive selection leading to a unique evolutionary pattern in hypocreales.
a comparison of the mitogenome sizes in sordariomycetes shows that intergenic regions are as important as introns contributing to mitogenome size variation in sordariomycete.
gene rearrangement analysis shows that the operation of transposition drives the rearrangement events of gene order in pezizomycotina, and most of them display changes in trn order.

 the nematophagous fungus pochonia chlamydosporia zare & gams is a widespread soil fungus distributed worldwide in nematode suppressive soils. pochonia chlamydosporia can infect the females and eggs of plant endoparasitic nematodes as a facultative parasite. it has already demonstrated its efficacy as a bca against both root-knot nematodes and cyst nematodes, major agricultural pests that cause tremendous economic losses worldwide estimated at billion dollars annually. the fungus p. chlamydosporia belongs to the family clavicipitaceae. based on spore morphology and polymorphisms of the nuclear ribosomal internal transcribed spacer region, this species has been recognized as having at least two distinct varieties, p. chlamydosporia var. chlamydosporia and p. chlamydosporia var. catenulate. many members of clavicipitaceae are invertebrate-pathogenic fungi. it was previously suggested that the host relatedness and host habitat hypotheses could explain the evolution of parasite-host relationships. therefore, exploring the phylogenetic relationships and evolutionary trajectories of the invertebrate-pathogenic fungi in clavicipitaceae is a topic of interest. multi-gene phylogenetic analyses showed that clavicipitaceae is paraphyletic and consists of three well-defined clades, at least one of which is shared with the members of another fungal family. the phylogenetic placement of the genus pochonia in the family clavicipitaceae was inferred from six nuclear genes and one mt gene using phylogenetic analysis, and the phylogenetic trees inferred from different genes produced different topological structures. therefore, a comparison of larger scale genome sequences is necessary because it can provide deep insights into the phylogenetic relationships and evolution of the fungi in clavicipitaceae. recently, the complete nuclear genome sequence of p. chlamydosporia was published. based on a phylogenetic analysis inferred from nuclear genome sequences, p. chlamydosporia appeared to be most closely related to the metarhizium species. mitochondrial markers can be successfully applied in evolutionary biology and systematics because mt genomes often evolve faster than nuclear genomes and allow for robust phylogenetic analyses based on the conserved proteins of the oxidative phosphorylation system. the continuously increasing number of recent fungal mt genome studies – more than complete fungal mt genomes are available today at ncbi - provide powerful tools for comparative studies to reveal the patterns and mechanisms of mitogenome evolution. thus far, fungal mitogenomes vary considerably in size, the largest being bp long in rhizoctonia solani ag ia and the smallest being bp long in hanseniaspora uvarum. researchers have demonstrated that different lengths of intronic sequences and intergenic regions result in different sizes of mitogenomes, particularly for the mitogenome of moniliophthora perniciosa in basidiomycota, which has a length of bp and encodes several hypothetical orfs in the larger sized intergenic regions and introns. however, a depiction of the size variation and related mechanisms in ascomycota has not yet been reported. moreover, the nearly ubiquitous presence of rps genes, which are encoded in the group i introns within rnl genes, has been attributed to a vertical mode of inheritance rather than horizontal inheritance during evolution. in one study, the molecular evolution of the rps gene in filamentous ascomycetes fungi was analysed, and the group i intron-encoded version of rps appeared to have a rather complex evolutionary history in ascomycetes fungi. however, only a few fungi in hypocreales were included in that study. to better understand the evolution of the fungal mitogenome in hypocreales, a phylogenetic analysis of the rps genes is required. furthermore, it was reported that trn genes are able to change their location in genomes and participate in horizontal gene transfer events, as they have editing, excision and integration capabilities. because changes in trn location are relatively rare events, the locations of trn genes in fungal mt genomes have been used to study fungal evolution and phylogenetic signals. to date, different types of aminoacyl-trnas have been identified in fungal mitogenomes, and conserved trn clusters are found in pezizomycotina by employing comparative genomic approaches. the distribution of trn genes may contribute to gene order variation in fungal mitogenomes. six operations have been used to explain rearrangements in mt gene order: inversion, transposition, reverse transposition, tandem-duplication-random-loss, deletion and replication slippage. however, the evolution of gene orders in pezizomycotina mitogenomes is suggested to be mainly driven by transpositions. as in animal mt genomes, trn in fungal mitogenomes might have played a role in gene shuffling as they intervene between genes and can act like mobile elements. taking advantage of published fungal mitogenomes and the novel p. chlamydosporia mitogenome, we investigated the trn rearrangements in pezizomycotina, and addressed the role of rearrangements in evolution. in this paper, the p. chlamydosporia strain isolated from a rkn in china was sequenced using illumina sequencing, and a complete mt genome of bp was obtained. we aim to present a complete and annotated mt genome sequence of p. chlamydosporia, compare the mt genome of p. chlamydosporia with the genomes of other fungi in pezizomycotina to identify the common and specific characteristics of the mt genomes of invertebrate-pathogenic fungi, and provide powerful insights into the evolution and phylogenetic relationships of the invertebrate-pathogenic fungi in hypocreales.  general characteristics of the mt genome in p. chlamydosporia the complete mt genome of p. chlamydosporia is a circular dna molecule with a length of bp. the low g + c content is similar to other fungal mitogenomes in clavicipitaceae. the mitogenome encodes an essential set of conserved genes including three cytochrome c oxidase subunits, apocytochrome b, three atp synthase subunits, seven subunits of nadh dehydrogenase, the small and large ribosomal rna subunits, an intronic orf coding for a putative rps and trn. all of the genes are encoded on the same dna strand. the set of trn genes found is the smallest set currently known for pezizomycotina fungi, but the trn genes can recognize the codons for all amino acids. in total, the coding regions, of which the typical protein-coding genes related to oxidative phosphorylation account for bp, and together with the rps within the rnl group i intron, cover % of the total mt genome, and the rest are trn, rrna genes and intergenic regions.table general features of the mitogenomes species length gc coding genes trn rrnas accession pochonia chlamydosporia metarhizium anisopliae beauveria bassiana beauveria pseudobassiana cordyceps brongniartii lecanicillium muscarium cordyceps militaris hypocrea jecorina fusarium solani fusarium graminearum fusarium verticillioides fusarium oxysporum verticillium dahliae neurospora crassa aspergillus fumigatus aspergillus niger aspergillus tubingensis trichophyton rubrum epidermophyton floccosum paracoccidioides brasiliensis candida parapsilosisfunctional map of the complete mitogenome of p. chlamydosporia. all genes are transcribed on the same strand. there are encoding genes, transfer rna genes and ribosomal rna genes. the circular map was generated using organellargenomedraw. using the abundant sequenced resources available, the sequence variance of the p. chlamydosporia mitogenome was investigated, but no single nucleotide polymorphism was found. this indicates that a high quality mitogenome was assembled. moreover, we compared the published mt gene sequences of p. chlamydosporia with our genome sequence. in total, mt gene sequences from ncbi are available, including six coding segments in the imi strain, four genes in the imi strain, one gene in the cbs strain and one gene in the cbs strain. our analysis showed that sequences were matched to our genome sequence with identities greater than or equal to 95%, suggesting that these gene sequences are highly conserved in p. chlamydosporia strains. phylogenetic relationships of p. chlamydosporia to other fungi in pezizomycotina utilizing published fungal mitogenomes in addition to the novel p. chlamydosporia mitogenome, the phylogenetic relationships in species in pezizomycotina were inferred, using candida parapsilosis as an outgroup. phylogenetic trees were constructed based on the conserved protein-coding genes associated with the oxidative phosphorylation system using a maximum likelihood approach. the topological structures based on both nucleotide and amino acid sequences were identical, and a clear genealogical relationship was shown. p. chlamydosporia is most closely related to the entomopathogenic fungus m. anisopliae, with a bootstrap value of 100%, which is similar to the result of phylogenetic analysis based on genome-encoded orthologous proteins. the two fungi form a cluster with five additional entomopathogenic fungi, namely, cordyceps brongniartii, beauveria pseudobassiana, b. bassiana, lecanicillium muscarium and c. militaris. interestingly, although both p. chlamydosporia and l. muscarium were once put into the same genus, the two species are assigned to different sub-clades in the phylogenetic tree. however, all of the invertebrate-pathogenic fungi cluster into a clade clearly separated from the plant-parasitic fungi, including four fusarium plant pathogens, which form clade b with the fungus hypocrea jecorina that specializes in colonizing pre-degraded wood. both invertebrate-pathogenic and plant-pathogenic fungi shared a common ancestor in clade c represented by a soil born plant pathogen, verticillium dahliae, suggesting that hypocreales is a monophyletic group. the phylogeny shows that plant pathogenic and invertebrate-pathogenic fungi in hypocreales form independent clusters and have evolved separately. a third group comprises fungi that infect animals or humans, including several members of eurotiomycetes, i.e., aspergillus fumigates, paracoccidioides brasiliensis, epidermophyton floccosum and trichophyton rubrum; this group is distant from p. chlamydosporia and the entomopathogenic fungi. the phylogenetic relationships inferred from mt proteins are principally consistent with the current pezizomycotina taxonomic system.figure the mitogenome phylogenetic analysis and comparative analysis. a). phylogenetic placement of p. chlamydosporia using fungal mitogenomes. the phylogeny was generated based on conserved encoded proteins using a ml approach. c. parapsilosis was used as an outgroup. p. chlamydosporia is closely related to m. anisopliae with a bootstrap support value of 100%. b). synteny comparisons between p. chlamydosporia and m. anisopliae, p. chlamydosporia and l. muscarium. syntenic regions were identified by performing blastn with a threshold of 1e- and alignment blocks of less than bp are not shown. comparison of mitogenome organization in p. chlamydosporia and related species we first compared the mitogenomes of p. chlamydosporia, m. anisopliae and l. muscarium, and found that they are highly similar in both size and g + c content. the total length of the gene regions in p. chlamydosporia mt genome is bp less than that of l. muscarium and bp less than that of m. anisopliae. however, the entire length of the p. chlamydosporia mitogenome is bp and bp larger than l. muscarium and m. anisopliae respectively, mainly due to longer intergenic regions in p. chlamydosporia. we also compared the contents of the simple repeat motifs in each of the genomes, including low complexity sequences, simple repeats n), inverted repeats and palindromes, which have been previously suggested to be putative elements for recombination or regulation. it was found that ~ % of the p. chlamydosporia mitogenome consists of repeat sequences compared with only ~ % of the l. muscarium mitogenome and ~ % of the m. anisopliae mitogenome. the distributions of repeat elements are also different. in l. muscarium, there are no repeat sequences located in the regions between nad and rnl, nad and cob, rns and cox however, in m. anisopliae and p. chlamydosporia, there are no repeat sequences in the regions between rnl and nad cox and nad there are repeat sequences residing in the regions between rns and cox in p. chlamydosporia, but only one at rich motif is found in this region in m. anisopliae. repeat sequences in this region display the unique mitogenomic features of p. chlamydosporia. in addition, sequence similarity studies and syntenic gene analyses were performed comparing p. chlamydosporia with five other mitogenomes, including m. anisopliae, l. muscarium, b. bassiana, v. dahliae and a. fumigatus. the analyses show identical gene order in m. anisopliae, l. muscarium, b. bassiana and p. chlamydosporia mt genomes. v. dahliae, as shown in previous work, displayed differences at the regions nad6-rnl, nad3-atp and cox1-nad with a gene order of nad6-atp nad3-nad and cox1-rnl instead. a larger difference of gene order is observed between the mitogenomes of p. chlamydosporia and a. fumigatus. comparing the two mitogenomes of p. chlamydosporia and m. anisopliae, bp of m. anisopliae mitogenome sequences match to bp of p. chlamydosporia mitogenome sequences with % identity determined by blastn. a long-range synteny between the p. chlamydosporia and m. anisopliae mt genomes is observed. the majority of the unmatched sequences reside within the intergenic regions between nad and rnl. however, comparing the p. chlamydosporia and l. muscarium mitogenomes, bp of the l. muscarium mitogenome sequences match to % of the p. chlamydosporia mitogenome sequences with % identity. comparisons of p. chlamydosporia mt genomes with the other three species showed that only bp of b. bassiana, bp of v. dahliae and bp of a. fumigatus mitogenomes matched to p. chlamydosporia mt sequences with %, % and % identities, respectively. although the sequences of most of the mt genes in v. dahliae matched to homologous genes in p. chlamydosporia mitogenome, the alignments of some genes between the two species present multiple blocks, and no alignments are found in the intergenic regions in the p. chlamydosporia and v. dahliae mitogenomes. based on the above syntenic analysis results, an evolutionary relationship between p. chlamydosporia and the other fungi is also indicated, that is, that p. chlamydosporia is most closely related to m. anisopliae. the result is similar to that obtained from the genomic dna analysis. analysis of mitogenome size variation among the fungi in sordariomycetes the sizes of the mitogenome in the fungi in sordariomycetes vary significantly, from bp in m. anisopliae to bp in f. graminearum. to identify the causes of this mitogenome size variation in sordariomycetes, five mitogenomes of different sizes were used for a comparative analysis. despite the size variance ranging from bp in m. anisopliae to bp in n. crassa, common mt protein-coding gene sets, including cox1- cob, nad1- nad4l, atp atp8- and rsp are encoded in all of the five genomes. we calculated the size of the crs, trn regions, rrna regions, introns and intergenic regions in each mitogenome separately. in p. chlamydosporia mitogenome, the lengths of the crs, trn regions, rrna regions, introns and intergenic regions are bp, bp, bp, bp and bp of the genome sequences, respectively. a comparison of the five mitogenomes showed that, in general, the length of each region increases with genome size, as well as the proportions of introns and intergenic regions, with the exception of h. jecorina, which has the smallest length of rrna genes but the largest length of introns of the five mitogenomes. in addition, the length of its intergenic regions is less than that of f. oxysporum, although the genome size of h. jecorina is larger than that of f. oxysporum. the results indicate that, in sordariomycetes, crs, intergenic regions and introns are the three main contributors to mitogenome size variation, with the length of intergenic regions or introns being the primary contributor in most of the mitogenomes. the length of the crs in n. crassa and h. jecorina is the second highest contributor because several intron-encoded orfs and unidentified reading frames exist in their intergenic regions.figure a comparison of the mitogenomes of five pezizomycotina fungi with considerable size differences. there are and protein-coding genes in p. chlamydosporia, m. anisopliae, f. oxysporum, h. jecorina, and n. crassa, respectively. size comparisons indicate that the larger the mitogenomes are primarily due to larger intergenic regions, secondarily to introns, and to a lesser extent to longer coding regions. codon usage in the fungal mitogenomes of pezizomycotina we analysed codon usage bias in the mitogenome of p. chlamydosporia, and compared it with other fungal mitogenomes in pezizomycotina. genetic code four was used to transcribe the coding gene sequences in the p. chlamydosporia mt genome. a total of codons were found, including the high-frequency codons tta, ata, gta and ttc, which are amino acids with hydrophobic side chains. six codons were absent in p. chlamydosporia but present in the closely related species m. anisopliae. however, two codons were absent in m. anisopliae but present in p. chlamydosporia. clearly, nearly in all of the fungal mitogenomes, codon usage is biased strongly towards codons ending in a or t, and more than 84% of the codons in p. chlamydosporia mitogenomes end in a or t, possibly due to the high at content found in fungal mitogenomes. similar results were obtained from m. anisopliae and l. muscarium mitogenomes. molecular evolution of rps in pezizomycotina we identified and annotated the rps genes in the fungal mitogenomes. the length of the rps sequences ranged from bp in the t. rubrum and e. floccosum mitogenomes to bp in the f. verticillioides mitogenome. when the rps proteins were aligned by blastp the similarity levels were greater than or equal to 30%. however, the c-terminal domains of these proteins obtained from the pfam database indicated a common function. the addition of more fungal rps gene sequences showed matches with e-value cutoffs of less than 2e- hence they were omitted from comparisons. e-value cutoffs of were obtained for the rps genes of f. oxysporum, m. anisopliae, h. jecorina, f. solani, f. graminearum and f. verticillioides. the unique evolutionary relationships among these species can be elucidated from phylogenetic analysis based on the sequences of the rps genes. a phylogenetic tree shows that p. chlamydosporia rps is more closely related to f. oxysporum and m. anisopliae rps they then group together with h. jecorina and other three fusarium species to form clade a. clade a separates distinctly from clade b, which contains the other five entomopathogenic fungi. evidently, the phylogenetic relationships among the species of hypocreales inferred from the rps genes are different from those inferred from the other protein-coding genes. to measure the rate and selective pressure of the rps genes, we calculated nonsynonymous/synonymous substitution rate ratios by adopting different models. the dn/ds value for the rps genes is using the codeml program with a jtt model in paml. because every functional protein contains amino acid sites under selective constraints, averaging the evolutionary rates across sites leads to a lower capacity to detect positive selection. thus, we adopted the site models m, m, m and m for the ω ratio analysis. because the m7-m comparison is a very stringent test of positive selection, we calculated the value of the likelihood ratio test statistic, and the significant results of this comparison clearly indicates signals of positive selection in the rps genes. to further confirm whether the hypocreales rps genes are evolving under positive selection or not, we took the rps sequence of t. rubrum as a reference to calculate the dn and ds of each rps sequence with the reference sequence using dnasp and codeml. similar results were obtained, that is, dn/ds > in hypocreales. the above results indicate that the fungal rps protein is not only under functional constraints but also under positive selection in hypocreales. trn gene distribution and putative gene rearrangements in pezizomycotina the distribution of trn genes on mt genomes is considered as a factor that potentially contributes to the gene order variation in pezizomycotina. firstly, we analyzed the distribution of trn genes in the p. chlamydosporia mitogenome and found that they cluster into three groups, with the exception of four trn genes that scatter as a single gene throughout the mt genome. then, we compared the trn genes in the fungal mitogenomes of pezizomycotina and found that they are presented in all mitogenomes whereas some of the other seven trn genes are often missing from a fungal mitogenome as reported in previous studies. some trn genes have multiple copies, such as trnm, which has at least two copies in each mitogenome. a sequence alignment showed that the trn gene sequences display high similarities among these fungal species, such as for trnn, where out of the 71/ bp are in consensus in the mitogenomes, and completely identical in b. bassiana, b. pseudobassiana and c. brongniartii. a comparative analysis showed that the distribution of trn genes is variable in pezizomycotina, but identical in the order hypocreales. the trn clusters are conspicuously observed in these mitogenomes, as reports in previous studies. excepting the largest trn cluster conserved in all of the pezizomycotina mitogenomes, the others are generally conserved at the order level, such as and in hypocreales, in sordariales, in eurotiales and in onygenales, although minor differences exist owing to the lack of a trn or a transposition event. the clustering of trn is suggested to be a unique characteristic of all pezizomycotina.figure the schematic representation of trn clusters. a). analysis of the trn genes residing in the complete mitogenomes of fungi that belong to nine families. the nine families are clavicipitaceae, hypocreaceae, nectriaceae, plectosphaerellaceae, sordariaceae, lasiosphaeriaceae, trichocomaceae, ajellomycetaceae and arthrodermataceae. b). an example of high sequence similarities in trn genes. the consensus bases are marked in boldface. based on a display of the orthologous protein-coding genes, trn genes and rrnas in the mitogenomes, a similar gene order can be observed in the hypocreales mitogenomes, with the exception of trng, which changed position in fusarium species. the gene order is essentially identical in the seven invertebrate-parasitic fungi and h. jecorina, as well as the four fusarium fungi, as found in previous studies. the same is also observed in eurotiales and onygenales. however, marked differences in gene order are observed between sordariomycetes and eurotiomycetes. this indicates that in pezizomycotina, which has closer phylogenetic relationships among its fungi, a greater number of identical gene orders are displayed, supporting the conclusion that the greater the identical gene orders the closer relationship they share. using crex analysis, possible gene rearrangements in the mitogenomes were predicted. a total of transposition events were identified, of which involve changes in trn position. among these, there are some rearrangement events that only occur in certain fungi, but no transposition event was found in clavicipitaceae. several events occur at the order level, for example, the trng location in hypocreales involves at least three events. most of the rearrangements occur in pezizomycotina, such as trnh, trni and trns, between sordariomycetes and eurotiomycetes. notably, many transposition events occur in the region near nad, perhaps due to historical changes in gene order in this region. although four types of rearrangement operations were considered in the program crex, only transposition events were predicted in our study, supporting the suggestion that the evolution of gene orders within pezizomycotina is mostly driven by transpositions. even though the mechanisms of rearrangements are not fully understood, these rearrangement events may provide clues about the phylogeny and evolution of the fungal species in pezizomycotina. discussion the mt genome represents a major model system in studies on evolutionary genomics, such as in animals and fungi. here, the organization of the complete mt genome of p. chlamydosporia that is a promising bca parasitizing nematode eggs is provided and its phylogenetic relationships to other invertebrate-pathogenic fungi are investigated. synteny and phylogenetic analyses show that p. chlamydosporia is more closely related to m. anisopliae than to l. muscarium and other entomopathogenic fungi, even though both p. chlamydosporia and l. muscarium were once assigned to the same genus, verticillium. our results are consistent with previous results on the nuclear genome, in which the genome of p. chlamydosporia was found to be most closely related to entomopathogenic fungi metarhizium spp., as well as on the phylogenetic analyses of the p. chlamydosporia serine proteases. our phylogenetic results also show that fungi attributed to the same genus group together at first, such as the three species of beauveria, and the two species of lecanicillium. these five species then cluster into a clade because all of their teleomorphic forms are attributed to the same genus, cordyceps. similarly, the four fusarium species and the three aspergillus species form two separate clades. according to our results, p. chlamydosporia and m. anisopliae also group together with a bootstrap value of 100%. the teleomorph of p. chlamydosporia is metacordyceps chlamydosporia, but the teleomorph of m. anisopliae is unknown, therefore, we hypothesize that the teleomorphic form of m. anisopliae may also belong to the genus metacordyceps. our phylogenetic tree showed that, in hypocreales, all of the invertebrate-pathogenic fungi cluster together to form a monophyletic group, which is noticeably distinguished from a cluster comprising plant pathogens. this suggests that these invertebrate-pathogenic fungi have a most recent common ancestor. notably, in our phylogenetic tree, the fungus h. jecorina groups together with the four fusarium species, and is obviously outside the group entomopathogens. this is in agreement with some previous studies, but distinctly different from that in others, in which h. jecorina was within entomophagous group and more closely related to m. anisopliae. because the results were obtained from both the total genomic data and the mitogenomic data, we believe this may reflect to the different evolutionary rates of the nuclear and mt genomes or the much bigger data provided by the nuclear genome in comparison with mt genes. considering h. jecorina that is attributed to the family hypocreaceae in fungal taxonomy, we think it is more reasonable that h. jecorina is outside the group entomopathogens, which belong to the family clavicipitaceae. based on the phylogenetic relationship in our study, the evolution of fungi in hypocreales may be speculated to have evolved from being parasitic on plants towards being parasitic on invertebrates. we think that a host habitat hypothesis can be used to explain the host shift in hypocreales fungi, which suggests that host shifts tend to follow the host’s microhabitat or feeding habitat, resulting in a group of related endoparasites that exploit distantly related organisms at higher taxonomic levels. nikoh and fukatsu reported that the entomoparasitic fungi of cordyceps have an interkingdom host jumping from animalia to fungi by overlapping the ecological niches of the unrelated hosts. because most hypocreales fungi grow naturally in soil, interkingdom host jumping events in hypocreales might occur in an underground environment. it is thus worthwhile to study the evolution of hypocreales fungi in depth in the future. among the ascomycete fungi, it was reported that the group i intron encoded version of rps appears to have a rather complex evolutionary history. however, the evolution of rps in hypocreales fungi has not been reported so far. in this study, our phylogenetic analysis shows that mitogenome rps genes display an evolutionary pattern distinctly different from those inferred from the mt protein-coding genes in hypocreales fungi. a jtt model showed a dn/ds value less than indicating that the encoded protein is under functional constraints, i.e., natural selection is operating to minimize the number of amino acid changes, thereby maintaining the activity of the protein; a result similar to that found in a study with ophiostomatoid fungi. however, a site model analysis showed a clear signal of positive selection in these rps sequences. moreover, the dn/ds values obtained for all of the rps sequences in hypocreales - in relation to the reference sequence of t. rubrum - are also greater than suggesting that positive selection acts on rps in hypocreales fungi. such a positive selection on rps was also observed previously in gymnosperms. although the special function of rps in ascomycetes fungi is unclear, it is known that rps plays a critical role in ribosome biogenesis and dna repair in other eukaryotes. therefore, further investigation is needed to fully understand the evolution of fungal rps genes. as shown recently by the comparison of complete fungal mt genomes from all the major phyla, remarkable variation is observed in genome size, gene order, composition of intergenic regions, repeats and introns. in our study, we compared the sizes of the mt genomes from fungal genera belonging to sordariomycetes and showed that, crs, intronic and intergenic regions all contributed to genome size variation. however, the major contributor was the length of the intergenic regions or the intronic regions in the majority of the fungal mitogenomes. our result indicates that, in sordariomycetes, the length of intergenic regions is also an important contributor to fungal mitogenome size variation, the same as the length of introns. a remarkable characteristic of fungal mitogenomes is that, similar to plant mitogenomes, fungal mitogenomes show signals of recombination. a comparison of complete fungal mitogenomes showed that the patterns of rearrangements may be explained by the combined influences of recombination, accumulated repeats, especially at intergenic regions, and to a lesser extent, mobile element dynamics. in this study, we compared the trn in the mitogenomes of pezizomycotina fungi, and found that the gene order for all the protein- and rrna-coding genes were principally conserved in sordariomycetes, but trn clusters were conserved at the order level. obvious differences of gene order and trn clusters were observed between sordariomycetes and eurotiomycetes. our results support the previous reports that the sordariomycetes and the eurotiomycetes have highly conserved gene arrangements. moreover, several gene order rearrangement events in the pezizomycotina fungi mitogenomes were estimated by crex analysis, and transposition events in the mitogenomes were identified. out of these, events displayed changes in trn order. our results support the view that the evolution of gene order in pezizomycotina is mostly driven by transpositions, although four operations of inversion, transposition, reverse-transposition and tandem-duplication-random-loss were considered in crex program. the observed transpositions also support the previous suggestion that trn genes should be considered as mobile elements involved in gene rearrangement. our results may provide an illustration of trn genes location changes in gene rearrangement in pezizomycotina fungi mitogenomes.  in this study, the complete and annotated mt genome sequence of p. chlamydosporia is provided. the relationships among the invertebrate pathogenic fungi in hypocreales are determined. according to different model predictions, our results indicate that the rps gene has experienced positive selection leading to a unique evolutionary pattern in hypocreales. a comparison of the mitogenome sizes in sordariomycetes shows that intergenic regions are as important as introns contributing to mitogenome size variation in sordariomycete. gene rearrangement analysis shows that the operation of transposition drives the rearrangement events of gene order in pezizomycotina, and most of them display changes in trn order. 
a phylogenetic tree inferred from mt proteins in pezizomycotina fungi supports that p. chlamydosporia is most closely related to the entomopathogenic fungus m. anisopliae. a comparison of mitogenome sizes shows that the length of the intergenic regions or the intronic regions is the major size contributor in most of mitogenomes in sordariomycetes. our rps and trn gene order results also establish a foundation for further exploration of the evolutionary trajectory of the fungi in hypocreales. the invertebrate-pathogenic fungi in hypocreales cluster together and clearly separate from a cluster comprising plant-pathogenic fungi and hypocrea jecorina. the invertebrate-pathogenic fungi in hypocreales referred to in this paper form a monophyletic group sharing a most recent common ancestor. gene rearrangement analysis shows that operation of transposition drives the rearrangement events in pezizomycotina, and most events involve in trn position changes, but no rearrangement was found in clavicipitaceae. the complete mitogenome sequence of p. chlamydosporia is bp in size, containing the typical protein-coding genes, two ribosomal rna genes, an intronic orf coding for a putative ribosomal protein and a set of transfer rna genes which recognize codons for all amino acids. sequence similarity studies and syntenic gene analyses show that % and % of p. chlamydosporia mitogenome sequences match % of metarhizium anisopliae sequences and % of lecanicillium muscarium sequences with % and % identities, respectively. the complete mitochondrial genome is expected to open new avenues for understanding the phylogenetic relationships and evolution of the invertebrate-pathogenic fungi in hypocreales. we present the complete annotated mitogenome sequence of p. chlamydosporia. based on evolutionary and phylogenetic analyses, we have determined the relationships between the invertebrate-pathogenic fungi in hypocreales. the fungus pochonia chlamydosporia parasitizes nematode eggs and has become one of the most promising biological control agents for plant-parasitic nematodes, which are major agricultural pests that cause tremendous economic losses worldwide. evolutionary analysis shows that rps is under positive selection, leading to the display of unique evolutionary characteristics in hypocreales. moreover, the variability of trn distribution has a clear impact on gene order in mitogenomes.
 the fungus pochonia chlamydosporia parasitizes nematode eggs and has become one of the most promising biological control agents for plant-parasitic nematodes, which are major agricultural pests that cause tremendous economic losses worldwide. the complete mitochondrial genome is expected to open new avenues for understanding the phylogenetic relationships and evolution of the invertebrate-pathogenic fungi in hypocreales.  the complete mitogenome sequence of p. chlamydosporia is bp in size, containing the typical protein-coding genes, two ribosomal rna genes, an intronic orf coding for a putative ribosomal protein and a set of transfer rna genes which recognize codons for all amino acids. sequence similarity studies and syntenic gene analyses show that % and % of p. chlamydosporia mitogenome sequences match % of metarhizium anisopliae sequences and % of lecanicillium muscarium sequences with % and % identities, respectively. a phylogenetic tree inferred from mt proteins in pezizomycotina fungi supports that p. chlamydosporia is most closely related to the entomopathogenic fungus m. anisopliae. the invertebrate-pathogenic fungi in hypocreales cluster together and clearly separate from a cluster comprising plant-pathogenic fungi and hypocrea jecorina. a comparison of mitogenome sizes shows that the length of the intergenic regions or the intronic regions is the major size contributor in most of mitogenomes in sordariomycetes. evolutionary analysis shows that rps is under positive selection, leading to the display of unique evolutionary characteristics in hypocreales. moreover, the variability of trn distribution has a clear impact on gene order in mitogenomes. gene rearrangement analysis shows that operation of transposition drives the rearrangement events in pezizomycotina, and most events involve in trn position changes, but no rearrangement was found in clavicipitaceae.  we present the complete annotated mitogenome sequence of p. chlamydosporia. based on evolutionary and phylogenetic analyses, we have determined the relationships between the invertebrate-pathogenic fungi in hypocreales. the invertebrate-pathogenic fungi in hypocreales referred to in this paper form a monophyletic group sharing a most recent common ancestor. our rps and trn gene order results also establish a foundation for further exploration of the evolutionary trajectory of the fungi in hypocreales. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords nematode egg-parasite funguspochonia chlamydosporiabiological control agentmitochondrial genomerps3rearrangementevolutionphylogenyissue-copyright-statement© the author 2015 