BACKGROUND
insect mitogenomes are usually small closed-circular molecules  containing  <dig> protein-coding genes ,  <dig> ribosomal rna  genes,  <dig> transfer rna  genes, and a large non-coding element termed the a + t-rich or control region  <cit> .
because of their unique features, including coding content conservation, maternal inheritance, and rapid evolution, mitogenome sequences have been widely used as molecular markers for diverse evolutionary studies  <cit> .
the order of the genes in the drosophila yakuba mitogenome, the first insect mitogenome to have its sequence determined, is shared by the majority of insect species, and this is therefore considered to be the ancestral order for the entire class insecta  <cit> .
various gene rearrangements have been reported in other insect mitogenomes and the most common type of rearrangements involves trna genes  <cit> .
all trna gene rearrangements can be classified as translocation, local inversion, or remote inversion   <cit> .
these rearrangements represent a molecular mitochondrial signature at the order or lower taxonomic levels  <cit> .
therefore, in addition to sequence data, the mitochondrial gene order can provide important evidence to establish evolutionary relationships  <cit> .
with the increasing availability of sequence data, the mitogenome has become a model for investigating the mode and mechanism of genome evolution  <cit> .
lepidoptera , one of the two largest insect orders, has more than  <dig>  described species that have been classified into 45– <dig> superfamilies  <cit> .
based on the information available in the genbank database up to  <dig>  more than  <dig> complete or nearly complete mitogenome sequences have been determined for the lepidoptera species.
all these species are limited to six superfamilies, tortricoidea, bombycoidea, noctuoidea, pyraloidea, geometroidea, and papilionoidea, which belong to the lepidopteran lineage ditrysia.
taxonomic sampling is still poor mainly because of the absence of information about the non-ditrysian lineages.
a better understanding of the lepidoptera mitogenomes requires an expansion of taxon samplings, especially of the non-ditrysian lineages .
all the lepidoptera mitogenomes available in genbank are characterized by the gene order trnmtrnitrnq, revealing a translocation of trnm compared with the ancestral gene order trnitrnqtrnm.
it is not known whether this rearrangement is common for the whole order lepidoptera or whether it occurred after the split of lepidoptera.
mitogenome sequencing of non-ditrysian lineages of lepidoptera will help address this interesting question and shed light on the underlying mechanisms of mitogenome evolution.
the ghost moth genus thitarodes viette  belongs to the family hepialidae  and hepialoidea is the most successful among the non-ditrysian lineages in terms of extant diversity  <cit> .
thitarodes are the only known hosts for the ascomycete ophiocordyceps sinensis , commonly known as the chinese caterpillar fungus, which is a prized traditional chinese medicine that is believed to boost immunity and increase stamina  <cit> .
members of the thitarodes occupy diverse habitats on the alpine meadows in the himalayas and on the tibetan plateau  <cit> .
among them, t. renzhiensis is distributed on the renzhi and baima snow mountains, at altitudes of 3880–4750 m, in northwest yunnan province, china  <cit> .
thitarodes yunnanensis is found only on laojun mountain, at elevations of 3680–3750 m, in northwest yunnan province, china  <cit> .
there are distinct differences in the male genital structure between these two species.
compared with t. yunnanensis, there is a heavily sclerotized curved spine on the end of the valve in t. renzhiensis <cit> .
in the present study, we sequenced the entire mitogenomes of the ghost moths, t. renzhiensis and t. yunnanensis.
to confirm our findings, the  <dig> -bp sequence from rrns to nad <dig> was also determined for a third species in thitarodes.
we compared the sequences with other insect mitogenomes, particularly with those of previously determined lepidoptera species.
RESULTS
genome structure, organization, and composition
the complete mitogenomes of t. renzhiensis and t. yunnanensis are circular molecules with  <dig>  bp and  <dig>  bp, respectively.
the two mitogenome sequences have been deposited in genbank .
because of expansion of the a + t-rich region, the mitogenome sequence of t. renzhiensis is longer than any other complete lepidoptera mitogenome sequenced to date.
the  <dig> -bp mtdna sequence comprising partial rrns, the a + t-rich region, trni, trnq, trnm, and partial nad <dig>  was also determined for thitarodes sp.
.
the t. renzhiensis and t. yunnanensis mitogenomes both include the entire set of  <dig> genes  and the a + t-rich region that is usually present in animal mitogenomes  <cit> .
the order of the genes in the sequences of the three thitarodes species was identical to that of the inferred ancestral insects, but different from the gene order in all other lepidoptera mitogenomes sequenced to date because of the rearrangement of three trna genes between the a + t-region and nad <dig> .
j and n refer to the majority and minority strand, respectively.
position numbers refer to positions on the majority strand.
in addition to the a + t-rich region, a total of 49-bp and 81-bp noncoding sequences are present in the mitogenomes of t. renzhiensis and t. yunnanensis, respectively.
in the t. yunnanensis mitogenome, trni and trnq are separated by a 27-bp intergenic spacer 3ctttttcaacta whereas there is no such intergenic spacer in t. renzhiensis.
there is a 15-bp intergenic spacer between trns and nad <dig> in both mitogenomes.
in this region, a conserved motif atactaa is present in all previously sequenced lepidoptera mitogenomes  <cit> , while the corresponding sequence is atactat in t. renzhiensis and atactac in t. yunnanensis.
like other insect mitogenomes, the two newly sequenced mitogenomes contain overlapping genes.
a total of 16-bp overlapping sequences occupy the same three locations in each of the two mitogenomes.
one 8-bp overlap is located between trnw and trnc oriented on opposite strands; the other two locations are between atp <dig> and atp <dig>  and between trnk and trnd  on the majority strand .
the nucleotide compositions of the two thitarodes mitogenomes are significantly biased toward a and t. the a + t content of the majority strand in t. yunnanensis is  <dig>  %, higher than that of t. renzhiensis .
these values fall within the range of the a + t content for other lepidoptera species; from  <dig>  % in ochrogaster lunifer to  <dig>  % in coreana raphaelis <cit> .
the nucleotide skew statistics for the entire majority strand of t. renzhiensis  and t. yunnanensis  indicate slight a or t skews and a moderate c skew.
a similar trend has been observed in other lepidoptera mitogenomes ; the at-skew ranges from − <dig>   to  <dig>   and the gc-skew is always negative varying from − <dig>   to − <dig>  .
for pcgs, all stop codons were excluded from analysis.
transfer and ribosomal rna genes
the two mitogenomes have the complete set of  <dig> trna genes  that are present in most metazoan mitogenomes.
the predicted cloverleaf structures for the trna genes are presented in figures  <dig> and  <dig>  all trna genes were determined by trnascan-se  <dig>   <cit>  and the program arwen  <cit>  except for trns in t. yunnanensis.
the trns could not form the typical cloverleaf structure, because the dihydrouridine  arm is replaced by an unpaired stretch of  <dig> and  <dig> nucleotides in t. renzhiensis and t. yunnanensis, respectively.
this feature is common to many arthropod mitogenomes  <cit> .
the trns in t. yunnanensis, therefore, was determined by comparison with that of t. renzhiensis and previously determined lepidoptera mitogenomes.
the trnqtrnk, and trnn genes each have an identical sequence between t. yunnanensis and t. renzhiensis.
a total of  <dig> mismatched base pairs and g-u wobble pairs are located in the acceptor , dhu , tψc , and anticodon  stems of the trna secondary structures in the two mitogenomes.
in t. renzhiensis,  <dig> mismatched base pairs  and 10 g-u wobble pairs have been identified in  <dig> trna genes; in t. yunnanensis,  <dig> a-c, 1 c-u, and  <dig> u-u mismatched pairs as well as 11 g-u wobble pairs occur in  <dig> trna genes.
as in the korean hairstreak c. raphaelis <cit> , the anticodon of trns in t. renzhiensis and t. yunnanensis is ucu instead of gcu that is present in other sequenced lepidoptera mitogenomes.
the anticodons of the remaining trnas are each identical to those of all other available lepidoptera mitogenomes.
various gene orders have been found in holometabolous insect mitogenomes, and trna genes have frequently been involved in mitogenome rearrangements  <cit> .
in the ancestral type, the trna gene order between the a + t-rich region and nad <dig> is trnitrnqtrnm <cit> .
in all previously sequenced lepidoptera mitogenomes, the order is trnmtrnitrnq, implying that translocation of trnm has taken place  <cit> .
by contrast, the present study revealed the ancestral gene arrangement in t. renzhiensist.
yunnanensis, and thitarodes sp.. therefore, the translocation of trnm is not a mitochondrial signature for the whole lepidoptera order.
rather, the translocation event likely occurred after hepialoidea diverged from other lepidopteran lineages.
the trna rearrangements are generally considered to be a consequence of tandem duplication of part of mitogenome, followed by random and/or nonrandom loss of duplicated copies  <cit> .
similar non-coding sequences, ranging from 40 bp in parnassius bremeri <cit>  to 87 bp in sasakia charonda , are present at the position originally occupied by the transposed trnm in all previously sequenced lepidoptera species but not in the three currently reported thitarodes species.
because these intergenic sequences have similar lengths to those of typical trna genes, they were presumed to be remnants of the trnm gene and its boundary sequences  <cit> .
if this is the case, the tandem duplication of mtdna sequences including trnm followed by loss could be responsible for the trna rearrangement patterns in previously sequenced lepidoptera mitogenomes.
however, the intergenic sequences have also been considered to be products of a partial duplication of the neighboring nad <dig> because of the high levels of sequence identities  between these intergenic sequences and nad <dig> <cit> .
if so, the duplication of partial nad <dig> should be a relatively recent event considering the high sequence identities.
however, low identities  between the intergenic sequences and nad <dig> were also detected in other sequenced lepidoptera mitogenomes  <cit> .
we should note that the short length  and high a + t content  of the intergenic sequences make it difficult to determine the origin of the intergenic sequences based solely on sequence identities.
therefore, cautions should be taken before definite conclusions regarding the source of the intergenic sequences are drawn.
regardless of the origin of the intergenic sequences, the most likely mechanism for the trna rearrangement in the previously sequenced lepidoptera species is through a tandem duplication of the region that includes trnitrnq, and trnm, followed by losses of the supernumerary genes.
clearly, more work is required in future studies to test this assumption.
the ribosomal rna genes  are located between trnl and trnv, and between trnv and the a + t-rich region, respectively.
the rrnl gene is  <dig>  bp long in t. renzhiensis and  <dig>  bp in t. yunnanensis; their a + t content is  <dig>  % and  <dig>  %, respectively .
in t. renzhiensis, rrns is 779 bp long and the a + t content is  <dig>  %; in t. yunnanensis, it is 777 bp long with an a + t content of  <dig>  % .
protein-coding genes
the mitogenomes of t. renzhiensis and t. yunnanensis contain the  <dig> pcgs that are usually present in animal mitogenomes and they are arranged along the mitogenome in congruence with the standard order in insects  <cit> .
in t. renzhiensis, the start codons for cox <dig> and nad <dig> are cga and ttg, respectively; the other pcgs start with the typical atn codons, three  with att, two  with ata, and the remainder with atg .
compared with t. renzhiensist.
yunnanensis has a different initiation codon ata in nad <dig>  ambiguities always arise when attempting to annotate the initiation codon for cox <dig> in a wide variety of species including lepidoptera and many irregular initiation codons, including attacg  <cit> , ttag  <cit>  and cga  <cit> , have been postulated for cox <dig> in the sequenced lepidoptera species.
a study based on the transcript information of anopheles funestus revealed that the translation initiation codon for the cox <dig> gene is tcg, rather than the atypical, longer codons that had been proposed earlier  <cit> .
recently, expressed sequence tag data from the legume pod borer maruca vitrata have shown that cox <dig> may start with the cga codon for arginine  <cit> .
here, we tentatively designate cga as the cox <dig> start codon, partly because this start codon has been found previously to be well conserved in  <dig> lepidoptera species  <cit> .
further investigations are required to clarify the mechanism of cox <dig> initiation in lepidoptera.
in both t. renzhiensis and t. yunnanensis, the annotated start codon of nad <dig> is ttg, consistent with those in a. funestus <cit>  and m. vitrata <cit> , which were annotated based on transcript information.
the atp8cobcox3nad <dig>  and nad <dig> genes in t. renzhiensis have the conventional stop codon taa; the remaining  <dig> have incomplete stop codons t or ta,  <dig> that are located in the genes that have trna genes at their 3' end, and  <dig> belonging to atp <dig> and nad <dig> upstream of cox <dig> and cob, respectively .
the only difference between the stop codons in the two mitogenomes is for nad <dig> which uses the complete stop codon taa in t. yunnanensis and the incomplete stop codon t in t. renzhiensis.
partial stop codons are common in the mitogenomes of most insects including all sequenced lepidoptera species  <cit> .
the polycistronic transcript molecule is processed into mature rna by precise endonucleolytic cleavages using the recognition signals of trna secondary structures; the truncated stop codons are presumed to be completed via post-transcriptional polyadenylation  <cit> .
the atp <dig> and atp <dig> genes in the two mitogenomes have a 7-bp overlap, a common feature of lepidoptera and many other arthropod mitogenomes  <cit> .
the a + t content of the pcgs, excluding stop codons, is  <dig>  % and  <dig>  % in t. renzhiensis and t. yunnanensis, respectively, which is lower than the a + t content of the mitogenomes as a whole .
in t. renzhiensis and t. yunnanensis, the third codon positions have a considerably higher a + t content  than the first and second codon positions; the strongest bias toward t is in the second codon positions , and the lowest content of g is in the third codon positions .
the codon usage bias has been detected in the currently sequenced thitarodes mitogenomes.
there are a total of  <dig> codons, excluding stop codons, in each of the two thitarodes mitogenomes.
among them, the three most abundant codons in both mitogenomes are uua , auu , and uuu .
as a consequence, leu , ile, and phe are the three most frequently used codon families .
the rarest used codon family is cys.
when pcgs on the majority and minority strands are considered separately, the three most abundant codon families are also leu , ile, and phe .
the usage of both four- and two-fold degenerate codons exhibits a strong a + t-bias in the third codon positions.
g + c-rich codons are less preferred, with ugc absent in t. renzhiensis, and cug, ccg, and acc lost in t. yunnanensis .
a similar codon usage pattern and a + t-bias in the third codon positions are present in previously sequenced lepidoptera mitogenomes, which were analyzed and averaged for each superfamily .
the a + t-rich region
the length and a + t content of the a + t-rich regions are  <dig>  bp and  <dig>  % in t. renzhiensis, 978 bp and  <dig>  % in t. yunnanensis, and 559 bp and  <dig>  % in thitarodes sp.. the a + t-rich region of t. renzhiensis is the longest of all the sequenced lepidoptera mitogenomes; the shortest is 319 bp in o. lunifer <cit> .
the a + t-rich region of t. renzhiensis includes a tandem repeat region consisting of eight 113-bp copies and one partial copy of a 57-bp sequence .
in t. yunnanensis, the a + t-rich region includes four complete repeat units  and one truncated repeat unit .
however, the a + t-rich region of thitarodes sp.
consists entirely of non-repetitive sequences .
the conspicuous macrorepeat units  commonly found in other insects are also present in previously sequenced lepidoptera mitogenomes, for example, bombyx mandarina   <cit> , papilio maraho   <cit> , and spilonota lechriaspis   <cit> .
an explanation for the origin of these repeat sequences is slipped-strand mispairing during mtdna replication  <cit> .
these repeat sequences mainly account for length variations in lepidoptera mitogenomes.
downstream of the rrns gene in the previously sequenced lepidoptera mitogenomes, there is a widely conserved structure that includes the motif ‘ataga’ and a 16–22 bp poly-t stretch.
it has been suggested that this structure might function as a signal for mtdna replication initiation  <cit> .
however, these conserved elements are not found in the mitogenomes of t. yunnanensist.
renzhiensis, and thitarodes sp., indicating that these structural motifs are not conserved in the non-ditrysian lepidoptera species.
the thitarodes mitogenomes may adopt a different strategy for replication initiation.
the absence of the conserved elements in the a + t-rich region, together with above mentioned structures, such as the presence of the ancestral gene arrangement, and the absence of the intergenic spacer upstream of nad <dig>  demonstrates that the thitarodes mitogenomes have unusual features compared with the previously determined lepidoptera mitogenomes.
therefore, in future studies, more attention should be paid to non-ditrysian lineages when comparing lepidoptera mitogenomes.
CONCLUSIONS
thitarodes renzhiensis and t. yunnanensis mitogenomes are the first representatives of non-ditrysian lineages of lepidoptera.
the arrangement of the trna genes between the a + t-region and nad <dig> is trni-trnq-trnm, different from those of previously sequenced lepidoptera mitogenomes but identical to the ancestral gene order.
therefore, the previously identified trna rearrangement is not a synapomorphy for lepidoptera mitogenomes.
this result indicates that the trna rearrangement event likely occurred after hepialoidea diverged from other lepidopteran lineages.
in addition, compared with other lepidoptera mitogenomes, t. renzhiensis and t. yunnanensis mitogenomes have other unique structural characters such as the lack of the intergenic spacer upstream of nad <dig>  therefore, non-ditrysian lineages should be taken into consideration in future comparative mitogenomic studies of lepidoptera.
characterization of the two mitogenomes has contributed to our understanding of lepidoptera mitogenomes and provided insights into mitogenome evolution, especially gene rearrangements.

 insect mitogenomes are usually small closed-circular molecules containing protein-coding genes, ribosomal rna genes, transfer rna genes, and a large non-coding element termed the a + t-rich or control region. because of their unique features, including coding content conservation, maternal inheritance, and rapid evolution, mitogenome sequences have been widely used as molecular markers for diverse evolutionary studies. the order of the genes in the drosophila yakuba mitogenome, the first insect mitogenome to have its sequence determined, is shared by the majority of insect species, and this is therefore considered to be the ancestral order for the entire class insecta. various gene rearrangements have been reported in other insect mitogenomes and the most common type of rearrangements involves trna genes. all trna gene rearrangements can be classified as translocation, local inversion, or remote inversion. these rearrangements represent a molecular mitochondrial signature at the order or lower taxonomic levels. therefore, in addition to sequence data, the mitochondrial gene order can provide important evidence to establish evolutionary relationships. with the increasing availability of sequence data, the mitogenome has become a model for investigating the mode and mechanism of genome evolution. lepidoptera, one of the two largest insect orders, has more than described species that have been classified into 45– superfamilies. based on the information available in the genbank database up to more than complete or nearly complete mitogenome sequences have been determined for the lepidoptera species. all these species are limited to six superfamilies, tortricoidea, bombycoidea, noctuoidea, pyraloidea, geometroidea, and papilionoidea, which belong to the lepidopteran lineage ditrysia. taxonomic sampling is still poor mainly because of the absence of information about the non-ditrysian lineages. a better understanding of the lepidoptera mitogenomes requires an expansion of taxon samplings, especially of the non-ditrysian lineages. all the lepidoptera mitogenomes available in genbank are characterized by the gene order trnmtrnitrnq, revealing a translocation of trnm compared with the ancestral gene order trnitrnqtrnm. it is not known whether this rearrangement is common for the whole order lepidoptera or whether it occurred after the split of lepidoptera. mitogenome sequencing of non-ditrysian lineages of lepidoptera will help address this interesting question and shed light on the underlying mechanisms of mitogenome evolution. the ghost moth genus thitarodes viette belongs to the family hepialidae and hepialoidea is the most successful among the non-ditrysian lineages in terms of extant diversity. thitarodes are the only known hosts for the ascomycete ophiocordyceps sinensis, commonly known as the chinese caterpillar fungus, which is a prized traditional chinese medicine that is believed to boost immunity and increase stamina. members of the thitarodes occupy diverse habitats on the alpine meadows in the himalayas and on the tibetan plateau. among them, t. renzhiensis is distributed on the renzhi and baima snow mountains, at altitudes of 3880–4750 m, in northwest yunnan province, china. thitarodes yunnanensis is found only on laojun mountain, at elevations of 3680–3750 m, in northwest yunnan province, china. there are distinct differences in the male genital structure between these two species. compared with t. yunnanensis, there is a heavily sclerotized curved spine on the end of the valve in t. renzhiensis. in the present study, we sequenced the entire mitogenomes of the ghost moths, t. renzhiensis and t. yunnanensis. to confirm our findings, the -bp sequence from rrns to nad was also determined for a third species in thitarodes. we compared the sequences with other insect mitogenomes, particularly with those of previously determined lepidoptera species.  genome structure, organization, and composition the complete mitogenomes of t. renzhiensis and t. yunnanensis are circular molecules with  bp and  bp, respectively. the two mitogenome sequences have been deposited in genbank. because of expansion of the a + t-rich region, the mitogenome sequence of t. renzhiensis is longer than any other complete lepidoptera mitogenome sequenced to date. the -bp mtdna sequence comprising partial rrns, the a + t-rich region, trni, trnq, trnm, and partial nad was also determined for thitarodes sp.. the t. renzhiensis and t. yunnanensis mitogenomes both include the entire set of genes and the a + t-rich region that is usually present in animal mitogenomes. the order of the genes in the sequences of the three thitarodes species was identical to that of the inferred ancestral insects, but different from the gene order in all other lepidoptera mitogenomes sequenced to date because of the rearrangement of three trna genes between the a + t-region and nad. j and n refer to the majority and minority strand, respectively. position numbers refer to positions on the majority strand. in addition to the a + t-rich region, a total of 49-bp and 81-bp noncoding sequences are present in the mitogenomes of t. renzhiensis and t. yunnanensis, respectively. in the t. yunnanensis mitogenome, trni and trnq are separated by a 27-bp intergenic spacer 3ctttttcaacta whereas there is no such intergenic spacer in t. renzhiensis. there is a 15-bp intergenic spacer between trns and nad in both mitogenomes. in this region, a conserved motif atactaa is present in all previously sequenced lepidoptera mitogenomes, while the corresponding sequence is atactat in t. renzhiensis and atactac in t. yunnanensis. like other insect mitogenomes, the two newly sequenced mitogenomes contain overlapping genes. a total of 16-bp overlapping sequences occupy the same three locations in each of the two mitogenomes. one 8-bp overlap is located between trnw and trnc oriented on opposite strands; the other two locations are between atp and atp and between trnk and trnd on the majority strand. the nucleotide compositions of the two thitarodes mitogenomes are significantly biased toward a and t. the a + t content of the majority strand in t. yunnanensis is  %, higher than that of t. renzhiensis. these values fall within the range of the a + t content for other lepidoptera species; from  % in ochrogaster lunifer to  % in coreana raphaelis. the nucleotide skew statistics for the entire majority strand of t. renzhiensis and t. yunnanensis indicate slight a or t skews and a moderate c skew. a similar trend has been observed in other lepidoptera mitogenomes; the at-skew ranges from − to and the gc-skew is always negative varying from − to −. for pcgs, all stop codons were excluded from analysis. transfer and ribosomal rna genes the two mitogenomes have the complete set of trna genes that are present in most metazoan mitogenomes. the predicted cloverleaf structures for the trna genes are presented in figures  and all trna genes were determined by trnascan-se and the program arwen except for trns in t. yunnanensis. the trns could not form the typical cloverleaf structure, because the dihydrouridine arm is replaced by an unpaired stretch of and nucleotides in t. renzhiensis and t. yunnanensis, respectively. this feature is common to many arthropod mitogenomes. the trns in t. yunnanensis, therefore, was determined by comparison with that of t. renzhiensis and previously determined lepidoptera mitogenomes. the trnqtrnk, and trnn genes each have an identical sequence between t. yunnanensis and t. renzhiensis. a total of mismatched base pairs and g-u wobble pairs are located in the acceptor, dhu, tψc, and anticodon stems of the trna secondary structures in the two mitogenomes. in t. renzhiensis, mismatched base pairs and 10 g-u wobble pairs have been identified in trna genes; in t. yunnanensis, a-c, 1 c-u, and u-u mismatched pairs as well as 11 g-u wobble pairs occur in trna genes. as in the korean hairstreak c. raphaelis, the anticodon of trns in t. renzhiensis and t. yunnanensis is ucu instead of gcu that is present in other sequenced lepidoptera mitogenomes. the anticodons of the remaining trnas are each identical to those of all other available lepidoptera mitogenomes. various gene orders have been found in holometabolous insect mitogenomes, and trna genes have frequently been involved in mitogenome rearrangements. in the ancestral type, the trna gene order between the a + t-rich region and nad is trnitrnqtrnm. in all previously sequenced lepidoptera mitogenomes, the order is trnmtrnitrnq, implying that translocation of trnm has taken place. by contrast, the present study revealed the ancestral gene arrangement in t. renzhiensist. yunnanensis, and thitarodes sp.. therefore, the translocation of trnm is not a mitochondrial signature for the whole lepidoptera order. rather, the translocation event likely occurred after hepialoidea diverged from other lepidopteran lineages. the trna rearrangements are generally considered to be a consequence of tandem duplication of part of mitogenome, followed by random and/or nonrandom loss of duplicated copies. similar non-coding sequences, ranging from 40 bp in parnassius bremeri to 87 bp in sasakia charonda, are present at the position originally occupied by the transposed trnm in all previously sequenced lepidoptera species but not in the three currently reported thitarodes species. because these intergenic sequences have similar lengths to those of typical trna genes, they were presumed to be remnants of the trnm gene and its boundary sequences. if this is the case, the tandem duplication of mtdna sequences including trnm followed by loss could be responsible for the trna rearrangement patterns in previously sequenced lepidoptera mitogenomes. however, the intergenic sequences have also been considered to be products of a partial duplication of the neighboring nad because of the high levels of sequence identities between these intergenic sequences and nad. if so, the duplication of partial nad should be a relatively recent event considering the high sequence identities. however, low identities between the intergenic sequences and nad were also detected in other sequenced lepidoptera mitogenomes. we should note that the short length and high a + t content of the intergenic sequences make it difficult to determine the origin of the intergenic sequences based solely on sequence identities. therefore, cautions should be taken before definite conclusions regarding the source of the intergenic sequences are drawn. regardless of the origin of the intergenic sequences, the most likely mechanism for the trna rearrangement in the previously sequenced lepidoptera species is through a tandem duplication of the region that includes trnitrnq, and trnm, followed by losses of the supernumerary genes. clearly, more work is required in future studies to test this assumption. the ribosomal rna genes are located between trnl and trnv, and between trnv and the a + t-rich region, respectively. the rrnl gene is  bp long in t. renzhiensis and  bp in t. yunnanensis; their a + t content is  % and  %, respectively. in t. renzhiensis, rrns is 779 bp long and the a + t content is  %; in t. yunnanensis, it is 777 bp long with an a + t content of  %. protein-coding genes the mitogenomes of t. renzhiensis and t. yunnanensis contain the pcgs that are usually present in animal mitogenomes and they are arranged along the mitogenome in congruence with the standard order in insects. in t. renzhiensis, the start codons for cox and nad are cga and ttg, respectively; the other pcgs start with the typical atn codons, three with att, two with ata, and the remainder with atg. compared with t. renzhiensist. yunnanensis has a different initiation codon ata in nad ambiguities always arise when attempting to annotate the initiation codon for cox in a wide variety of species including lepidoptera and many irregular initiation codons, including attacg, ttag and cga, have been postulated for cox in the sequenced lepidoptera species. a study based on the transcript information of anopheles funestus revealed that the translation initiation codon for the cox gene is tcg, rather than the atypical, longer codons that had been proposed earlier. recently, expressed sequence tag data from the legume pod borer maruca vitrata have shown that cox may start with the cga codon for arginine. here, we tentatively designate cga as the cox start codon, partly because this start codon has been found previously to be well conserved in lepidoptera species. further investigations are required to clarify the mechanism of cox initiation in lepidoptera. in both t. renzhiensis and t. yunnanensis, the annotated start codon of nad is ttg, consistent with those in a. funestus and m. vitrata, which were annotated based on transcript information. the atp8cobcox3nad and nad genes in t. renzhiensis have the conventional stop codon taa; the remaining have incomplete stop codons t or ta, that are located in the genes that have trna genes at their 3' end, and belonging to atp and nad upstream of cox and cob, respectively. the only difference between the stop codons in the two mitogenomes is for nad which uses the complete stop codon taa in t. yunnanensis and the incomplete stop codon t in t. renzhiensis. partial stop codons are common in the mitogenomes of most insects including all sequenced lepidoptera species. the polycistronic transcript molecule is processed into mature rna by precise endonucleolytic cleavages using the recognition signals of trna secondary structures; the truncated stop codons are presumed to be completed via post-transcriptional polyadenylation. the atp and atp genes in the two mitogenomes have a 7-bp overlap, a common feature of lepidoptera and many other arthropod mitogenomes. the a + t content of the pcgs, excluding stop codons, is  % and  % in t. renzhiensis and t. yunnanensis, respectively, which is lower than the a + t content of the mitogenomes as a whole. in t. renzhiensis and t. yunnanensis, the third codon positions have a considerably higher a + t content than the first and second codon positions; the strongest bias toward t is in the second codon positions, and the lowest content of g is in the third codon positions. the codon usage bias has been detected in the currently sequenced thitarodes mitogenomes. there are a total of codons, excluding stop codons, in each of the two thitarodes mitogenomes. among them, the three most abundant codons in both mitogenomes are uua, auu, and uuu. as a consequence, leu, ile, and phe are the three most frequently used codon families. the rarest used codon family is cys. when pcgs on the majority and minority strands are considered separately, the three most abundant codon families are also leu, ile, and phe. the usage of both four- and two-fold degenerate codons exhibits a strong a + t-bias in the third codon positions. g + c-rich codons are less preferred, with ugc absent in t. renzhiensis, and cug, ccg, and acc lost in t. yunnanensis. a similar codon usage pattern and a + t-bias in the third codon positions are present in previously sequenced lepidoptera mitogenomes, which were analyzed and averaged for each superfamily. the a + t-rich region the length and a + t content of the a + t-rich regions are  bp and  % in t. renzhiensis, 978 bp and  % in t. yunnanensis, and 559 bp and  % in thitarodes sp.. the a + t-rich region of t. renzhiensis is the longest of all the sequenced lepidoptera mitogenomes; the shortest is 319 bp in o. lunifer. the a + t-rich region of t. renzhiensis includes a tandem repeat region consisting of eight 113-bp copies and one partial copy of a 57-bp sequence. in t. yunnanensis, the a + t-rich region includes four complete repeat units and one truncated repeat unit. however, the a + t-rich region of thitarodes sp. consists entirely of non-repetitive sequences. the conspicuous macrorepeat units commonly found in other insects are also present in previously sequenced lepidoptera mitogenomes, for example, bombyx mandarina, papilio maraho, and spilonota lechriaspis. an explanation for the origin of these repeat sequences is slipped-strand mispairing during mtdna replication. these repeat sequences mainly account for length variations in lepidoptera mitogenomes. downstream of the rrns gene in the previously sequenced lepidoptera mitogenomes, there is a widely conserved structure that includes the motif ‘ataga’ and a 16–22 bp poly-t stretch. it has been suggested that this structure might function as a signal for mtdna replication initiation. however, these conserved elements are not found in the mitogenomes of t. yunnanensist. renzhiensis, and thitarodes sp., indicating that these structural motifs are not conserved in the non-ditrysian lepidoptera species. the thitarodes mitogenomes may adopt a different strategy for replication initiation. the absence of the conserved elements in the a + t-rich region, together with above mentioned structures, such as the presence of the ancestral gene arrangement, and the absence of the intergenic spacer upstream of nad demonstrates that the thitarodes mitogenomes have unusual features compared with the previously determined lepidoptera mitogenomes. therefore, in future studies, more attention should be paid to non-ditrysian lineages when comparing lepidoptera mitogenomes.  thitarodes renzhiensis and t. yunnanensis mitogenomes are the first representatives of non-ditrysian lineages of lepidoptera. the arrangement of the trna genes between the a + t-region and nad is trni-trnq-trnm, different from those of previously sequenced lepidoptera mitogenomes but identical to the ancestral gene order. therefore, the previously identified trna rearrangement is not a synapomorphy for lepidoptera mitogenomes. this result indicates that the trna rearrangement event likely occurred after hepialoidea diverged from other lepidopteran lineages. in addition, compared with other lepidoptera mitogenomes, t. renzhiensis and t. yunnanensis mitogenomes have other unique structural characters such as the lack of the intergenic spacer upstream of nad therefore, non-ditrysian lineages should be taken into consideration in future comparative mitogenomic studies of lepidoptera. characterization of the two mitogenomes has contributed to our understanding of lepidoptera mitogenomes and provided insights into mitogenome evolution, especially gene rearrangements. 
their ancestral gene order indicates that the trna rearrangement event likely occurred after hepialoidea diverged from other lepidopteran lineages. all three thitarodes species possess the ancestral gene order with trni-trnq-trnm located between the a + t-rich region and nad which is different from the gene order trnm-trni-trnq in all previously sequenced lepidoptera species. the anticodon of trns in t. renzhiensis and t. yunnanensis is ucu instead of the mostly used gcu in other sequenced lepidoptera mitogenomes. the formerly identified conserved elements of lepidoptera mitogenomes are absent in the thitarodes mitogenomes. the mitogenomes of t. renzhiensis and t. yunnanensis exhibit unusual features compared with the previously determined lepidoptera mitogenomes. characterization of the two ghost moth mitogenomes has enriched our knowledge of lepidoptera mitogenomes and contributed to our understanding of the mechanisms underlying mitogenome evolution, especially gene rearrangements. to gain new insights into lepidoptera mitogenome evolution, we sequenced the mitogenomes of two ghost moths that belong to the non-ditrysian lineage hepialoidea and conducted a comparative mitogenomic analysis across lepidoptera. the mitogenomes of thitarodes renzhiensis and t. yunnanensis are  bp and  bp long with an a + t content of  % and  %, respectively. compared with the ancestral insect gene order, these mitogenomes all contain a trna rearrangement. both mitogenomes include protein-coding genes, transfer rna genes, ribosomal rna genes, and the a + t-rich region.
 lepidoptera encompasses more than described species that have been classified into 45– superfamilies. the previously determined lepidoptera mitochondrial genomes are limited to six superfamilies of the lineage ditrysia. compared with the ancestral insect gene order, these mitogenomes all contain a trna rearrangement. to gain new insights into lepidoptera mitogenome evolution, we sequenced the mitogenomes of two ghost moths that belong to the non-ditrysian lineage hepialoidea and conducted a comparative mitogenomic analysis across lepidoptera.  the mitogenomes of thitarodes renzhiensis and t. yunnanensis are  bp and  bp long with an a + t content of  % and  %, respectively. both mitogenomes include protein-coding genes, transfer rna genes, ribosomal rna genes, and the a + t-rich region. different tandem repeats in the a + t-rich region mainly account for the size difference between the two mitogenomes. all the protein-coding genes start with typical mitochondrial initiation codons, except for cox and nad in both mitogenomes. the anticodon of trns in t. renzhiensis and t. yunnanensis is ucu instead of the mostly used gcu in other sequenced lepidoptera mitogenomes. the -bp sequence from rrns to nad was also determined for an unspecified ghost moth, which has no repetitive sequence in the a + t-rich region. all three thitarodes species possess the ancestral gene order with trni-trnq-trnm located between the a + t-rich region and nad which is different from the gene order trnm-trni-trnq in all previously sequenced lepidoptera species. the formerly identified conserved elements of lepidoptera mitogenomes are absent in the thitarodes mitogenomes.  the mitogenomes of t. renzhiensis and t. yunnanensis exhibit unusual features compared with the previously determined lepidoptera mitogenomes. their ancestral gene order indicates that the trna rearrangement event likely occurred after hepialoidea diverged from other lepidopteran lineages. characterization of the two ghost moth mitogenomes has enriched our knowledge of lepidoptera mitogenomes and contributed to our understanding of the mechanisms underlying mitogenome evolution, especially gene rearrangements. thitarodes renzhiensisthitarodes yunnanensismitochondrial genomegene rearrangement 