BACKGROUND
insect mitogenomes are generally compact with few intergenic spacers and possess stable gene content and organization.
they are usually about  <dig> kb in size and bear  <dig> protein-coding genes ,  <dig> ribosomal rna genes ,  <dig> transfer rna genes , and one control region  that includes replication and transcription origins
<cit> .
however, extensive studies have revealed that gene order rearrangement and size variation that results from the presence of tandem repeats  and other non-coding regions occur more often than previously expected.
much attention has been paid to studies focusing on unveiling genomic diversities and evolutionary trends.
recently, a mitogenomic investigation of congeneric species has yielded a valuable approach for assessing mtdna evolutionary trends
<cit> .
unfortunately, in spite of the large number of insect species, the limited availability of complete mitogenomic sequence data, including those of congeneric species, impedes a thorough understanding of the insect mitogenomes.
orthoptera, the largest polyneopteran order, contains  <dig> subfamilies and over  <dig>  described species, taxonomically divided into two suborders: caelifera  and ensifera
<cit> .
orthoptera mitogenomes generally possess a relatively stable gene content and organization identical to the insect ancestor
<cit> .
only a trnd-trnk rearrangement in the lineage acridomorpha
<cit> , inversion of the gene cluster trne-trns-trnn in teleogryllus emma <cit> , and occasional inversion of trnw in the migratory locust
<cit>  have been discovered.
a mitogenomic divergence in the at-bias between the two suborders has been demonstrated, i.e., the at-content was generally lower in ensifera than in caelifera
<cit> .
additionally, a possible stem-loop structure has been implicated in mtdna replication initiation in a few caeliferans
<cit> , which contrasts with the recognition of the mtdna replication origin  that involves a long t-stretch in most other insects
<cit> .
however, representative katydid mitogenomes, e.g., anabrus simplex and deracantha onos, revealed the existence of a long t-stretch
<cit> .
furthermore, mitogenomes of  <dig> caelifera and  <dig> ensifera species available from genbank demonstrate a distinct taxon sampling imbalance between the two suborders.
thus, additional ensifera taxon sampling is essential to investigate the mitogenomic genetic diversities and evolutionary trends.
the genus sinochlora tinkham
<cit> , chinese bush katydid, belongs to the subfamily phaneropterinae in the suborder ensifera.
in sinochlora, one species s. longifissa is widespread in east asia including japan, korea, and southern china, and two species are widely distributed and indigenous in southern china
<cit> .
other species are endemic in various large mountains in southern china including low-altitude areas in tibet
<cit> .
investigation of the mitogenomic evolutionary trends of the genus is helpful to unveil the molecular mechanism of the divergence patterns.
herein, we chose two representative species, s. longifissa distributed in east asia, and s. retrolateralis narrowly endemic in southern china, for mitogenomic investigation.
we sequenced the mitogenomes of the two congeners, unveiled novel mitogenomic characteristics, and outlined the possible rearrangement mechanism.
additionally, we also compared the recognition sequences of the or in known orthoptera mitogenomes.
overall, we attempted to provide the molecular basis for understanding diversification of the genus sinochlora and depict molecular diversity and evolutionary trends of orthoptera mitogenomes.
RESULTS
genome organization
we sequenced the complete mitogenome of s. longifissa  and the nearly complete mitogenome of s. retrolateralis  with a partial cr.
the mitogenomes of s. longifissa and s. retrolateralis have been deposited in the genbank database under accession numbers of kc <dig> and kc <dig>  respectively.
they are currently the largest orthoptera mitogenomes on genbank.
their large sizes are due to two large non-coding regions, i.e., the cr and one intergenic spacer  located between trns and nad <dig>  both mitogenomes  resemble the available ensiferan mitogenomes
<cit>  and the proposed ancestral insect mitogenome
<cit>  in gene content and orientation, trna anticodons, and trna/rrna structures .
additionally, there are several gene overlaps, such as the open reading frames  of atp6-atp <dig> and nad4l-nad <dig>  each of which overlaps by seven nucleotides .
a few other igs are also present in the mitogenomes .
sl, s. longifissa; sr, s. retrolateralis.
the number of intergenic nucleotides before the gene starts are shown in parenthesis.
the asterisk indicates that the tandem repeat region in s. retrolateralis was not completely sequenced.
nucleotide composition
like other ensiferans, the two mitogenomes show comparatively low a+t bias  , compared with most caeliferans
<cit> .
the at skew is  <dig>  in s. longifissa and  <dig>  in s. retrolateralis, while the gc skew is - <dig>  in the former and - <dig>  in the latter, indicating weak a skew and strong c skew in the j-strand.
through comparison of nucleotide skew in all sequenced orthoptera, the skew divergence between the two suborders was detected.
within the ensifera, the at skew values of the whole j-strand range from - <dig>  to  <dig>  and gc skew values are lower than - <dig> , showing the same skew patterns as in sinochlora species .
by contrast, within the caelifera except a tridactylid ellipes minuta <cit> , absolute values of at skew and gc skew of the j-strand range from  <dig>  to  <dig> , indicating pronounced a skew and c skew .
the synonymous fourfold degenerate third codon positions  suffer less selective constraints, and thus could indicate background mutational pressures on nucleotide skew
<cit> .
the skew patterns at the p4fd between the two suborders are consistent with those on the j-strand .
it is proposed that nucleotide composition bias and skew are caused by the selection-mutation-drift equilibrium in the molecular evolution
<cit> .
the asystematical directional mutation pressure and corresponding deaminations of cytosine and adenine in the mitogenomes are involved in the mutation processes
<cit> .
start and stop codons
all pcgs except cox <dig> and nad <dig> start with typical atn codons.
a previous study concerning orthoptera mitogenomes suggested that the cox <dig> gene may start with an irregular tetranucleotide codon auga
<cit> .
similar irregular tetranucleotide codons were also proposed as start codons in drosophila <cit> .
however, there is no experimental evidence for the use of a 4-bp start codon in any creature.
recent research on characteristics of mature mrna and rrna genes from d. melanogaster mitochondria showed that ucg serves as the start codon of its cox <dig> gene
<cit> .
the result is consistent with predictions of the start of cox <dig> suggested by comparison of conserved amino acid positions
<cit> .
by comparing amino acid sequences of cox <dig> in all sequenced orthoptera, we observed that the first conserved codon is the tcn serine codon, downstream of which at least three codon positions are also conserved and there are no standard atn bases .
thus, in both sinochlora species, the conserved tct serine codon may also serve as a start codon.
concerning the nad <dig> gene, it has been proposed to start with atn codons in other sequenced orthopterans; however, there are no conserved amino acid positions within the span of over  <dig> amino acids downstream of trnp .
then we propose that the nad <dig> may start with ctg in s. longifissa and ttg in s. retrolateralis, considering previous designation of such start codons
<cit> .
the two start codons, creating no igs or overlap between trnp and nad <dig> genes, also appear to be more plausible in the evolutionary economic perspective
<cit> .
two standard stop codons taa/tag and two incomplete stop codons t/ta are utilized in the pcgs.
for s. longifissa, six pcgs  terminate with taa, one  with tag, one  with ta, and the other four  with t. the stop codons differ between the two congeners in that taa is utilized as the cox <dig> stop codon, and ta is utilized as the atp <dig> and cox <dig> stop codon in s. retrolateralis.
a partial t or ta stop codon, which was proposed to create complete taa stop codons via posttranscriptional polyadenylation
<cit> , is also present in other metazoan mitochondrial genes.
novel gene order rearrangement involving the control region
one of our most significant findings is the novel gene order rearrangement "rrns-trni-trnm-nad2-cr-trnq-trnw" in both sinochlora species .
the two mitogenomes are the first representatives that have a large-scale translocation involving the cr in orthoptera.
such gene order has not been observed in other sequenced insect mitogenomes.
the gene cluster rrns-cr-trni-trnq-trnm-nad <dig> in the proposed ancestral mitogenome has been discovered to function as a hot spot for gene rearrangements in arthropod mitogenomes.
duplicate trni and partial trnq genes have been discovered in the cr close to the rrns gene in some blowflies
<cit> .
in a mantid, a complicate set of repeat units dispersing in both ends of the cr also translocated between trnm and nad <dig> <cit> .
a plague thrips displayed cr duplications that are distant from the rrna genes
<cit> .
various locations of the cr and neighboring trna genes have been exhibited in lice
<cit> .
location variability of the putative cr has also been reported as a product of the trna gene translocation, such as rrns-trnq-cr-trni-trns-trnm-nad <dig> in a springtail
<cit> , or the trna and rrna gene translocation, such as rrnl-trnv-trns-trnc-cr-rrns in a mite
<cit> .
the cr and the neighboring genes have also been found to be duplicated in some ticks
<cit> .
organization of the control region
for both sinochlora species, the putative cr is composed of two major sections with different nucleotide compositions: a highly a+t-biased region  adjacent to nad <dig> and several gc-rich tr units adjacent to trnq.
for the former, the a+t content is  <dig> % in s. longifissa and  <dig> % in s. retrolateralis.
by contrast, the tr region has low bias toward a+t .
the atr is conserved between the two congeners in length, nucleotide composition and secondary structure.
its length is  <dig>  bp in s. longifissa and  <dig>  bp in s. retrolateralis, consistent with the a+t-rich region of other orthopterans
<cit> .
the two species share  <dig> % sequence identity in this region.
a long t-stretch and several conserved potential hairpin structures are thought to play significant roles in initiating and/or regulating the transcription and replication of mtdna
<cit> .
they were also found in the both sinochlora species.
the long t-stretch  is situated in the initial quarter of the atr on the j-strand, and a similar t-stretch  is situated in the initial third of the atr on the minority strand  .
the two species also share  <dig> % sequence identity in the domain located between these two t-stretches.
the domain has seven similar sites capable of forming 8- to 33-bp stem-loop structures .
additionally, there are three similar sites for the formation of 11- to 25-bp stable hairpins, with 7- to 11-bp loops supported by 1– <dig> gc matches in the stems, adjacent to the 5’ end of the t-stretch on the j-strand .
in s. longifissa, the tr region is  <dig>  bp in length, comprising nine full 175-bp copies and a partial 153-bp copy.
in s. retrolateralis, a total of five 175-bp repeat copies have been successfully sequenced from both ends of the tr; however, we failed to sequence through the whole tr region due to the presence of a large number of repeat copies.
we estimate that there are twelve 175-bp tandem copies in s. retrolateralis based on the length  of the tr indicated by gel electrophoresis.
the consensus tr motifs of the two species share  <dig> % sequence identity.
in s. longifissa, four of the nine tr motifs are identical to the consensus tr motif, and the other five have a few mutations and/or deletions.
among the nine tr motifs, all but the fourth possess an orf.
the consensus orf could be translated into  <dig> amino acids, starting with lysine  and ending with valine  .
low shared identities  with nuclear sequences in search of the genbank database exclude the possibility that the orf is transferred from the nuclear genome.
it has  <dig> % identity with the antisense strand of nad <dig>  and  <dig> % identity with the sense strand of cox <dig>  the shared bases are scattered in alignment, among which over 60% are a and t .
this suggests that the orf might not be obtained through horizontal transfer between mitochondria.
in contrast, no orf could be found in the s. retrolateralis tr motif.
the tr motif could be duplicated through slipped-strand mispairing
<cit> .
moreover, potential stem-loop structures in a repeated unit and its flanking part have been demonstrated to cause an increase in slipped-strand mispairing frequency
<cit> .
stem-loop structures were detected in the tr region of the two sinochlora species.
for example, in the first tr unit and its junctions at both ends in s. longifissa, the nucleotide sequence can potentially form seven 8- to 27-bp hairpins with 5- to 10-bp loops supported by 1– <dig> gc matches in the stem .
in the corresponding regions in s. retrolateralis, the nucleotide sequence can potentially form six 10- to 29-bp hairpins with 5- to 15-bp loops supported by 1– <dig> gc matches in the stem .
similar complicated hairpin structures in tr were also detected in the louse bothriometopus <cit>  and termites
<cit> .
however, there are no conserved stem-loops among the trs and at the joints in these insects.
unassigned intergenic spacers and trns pseudogenes
eleven igss with identical locations are present in s. longifissa  and s. retrolateralis ; one additional igs is situated between atp <dig> and cox <dig> in s. longfissa  and between cox <dig> and trnk in s. retrolateralis  .
the largest igs  lies between trns and nad <dig>  by comparing the igs of currently available orthoptera mitogenomes, we found a 7-bp conserved motif  downstream the nad <dig> across orthoptera, with the only exception of mekongiella xizangensis .
the conserved motif has been proposed as a binding site for mitochondrial transcription termination factor  in orthoptera
<cit> .
similar conserved motifs, which were proposed as a binding site of the mtterm, between trns and nad <dig> have also been found in lepidoptera
<cit> , and coleoptera
<cit> .
it has been confirmed that one of the two binding sites of mtterm lies downstream of nad <dig> in drosophila <cit> .
in the igs region between trns and nad <dig>  we detected the vestige of one additional trns gene copy in both sinochlora species.
while both trns pseudogenes possess the same anticodon to the functional trns, they are unable to form stable cloverleaf secondary structures .
relative-rate tests show that both pseudogenes evolved more than twice as fast as their functional paralogs .
this suggests that the redundant trns pseudogenes have experienced more relaxed selective constraints.
in addition, the igs region in s. longifissa could be folded into three 19- to 25-bp stable stem-loop structures .
the anticodon lies in the stem of the second hairpin and the 7-bp conserved motif aforementioned lies adjacent to the 3’ end of the third hairpin .
in contrast, there is no hairpin in the igs region in s. retrolateralis.
results of all possible pairwise contrasts between trns genes and trns pseudogenes are significant .
sd, standard deviation; sr, s. retrolateralis; sl, s. longifissa; mm, myrmecophilus manni; ser, trns; pser, trns pseudogenes.
mechanism of genome rearrangements
the predominant mechanism of mitogenome rearrangements is considered to be partial genome duplication followed by a random
<cit>  or non-random loss of the duplicated gene copies
<cit> .
for the rearrangement involving the cr neighbourhood, we propose that the ancestor of the two sinochlora species possessed the same gene order as ancestral insects
<cit> .
firstly, the gene cluster cr-trni-trnq-trnm-nad <dig> was duplicated, likely promoted by the stem-loop structures detected in the cr .
subsequently, non-random deletions might happen to the entire subset of the duplicated genes, dependent on the gene copy’s transcriptional polarity and location in the genome, because the cr includes a transcriptional control sequence
<cit> .
namely, the redundant genes which possess the same polarity, such as the redundant gene cluster trni-trnm-nad <dig>  were successively deleted during replication .
by contrast, persistence of the trns pseudogenes may have resulted from the tandem duplication of trns followed by a subsequent random loss of the trns paralogs.
apparently, more knowledge of transcription signals in insect mitogenomes and sampling additional closely related taxa will be helpful to understand the rearrangement mechanism.
recognition sequences of replication origins
the t-stretch is involved in the recognition of the or of mtdna at least among holometabolous insects, whereas similar t-stretch was not found in the upstream portion of the or for l. migratoria
<cit> .
however, after a thorough re-investigation, we detected long t-stretches or variants of t-stretch in the putative cr of available orthoptera mitogenomes .
the long t-stretch can be observed in the available sequences of all katydids , except ruspolia dubia, and varies in size from  <dig>  to  <dig> bp .
the t-stretches could participate in the formation of or be positioned adjacent to a possible stem-loop structure.
by contrast, in crickets and grasshoppers, t-stretch variants can be detected within a potential stem-loop structure .
in the t-stretch variants, a few transitions between thymine and cytosine occurred to form ncn sequences.
the t-stretch variants lie proximal to the middle part of the cr in the crickets, varying in size from  <dig> to  <dig> bp .
the t-stretch variants vary in size from  <dig> to  <dig> bp in grasshoppers , with the exception of alulatettix yunnanensis.
in a. yunnanensis, a small t-stretch could be observed in the stem portion of a hairpin structure, similar to that observed in katydids .
human mtdna synthesis is initiated from the sites near the stem base of the secondary structure located around the light strand origin, and the small t-stretch  located in the loop portion participates in the initiation process
<cit> .
the secondary structure in human and other vertebrate mtdna is very similar to the aforementioned stem-loop structure in the orthopterans
<cit> .
therefore, the long t-stretch, which participates in the formation of or is adjacent to a possible stem-loop structure in katydids, and the t-stretch variants within the stem-loop structure of crickets and most grasshoppers, could also play a crucial role in mtdna replication initiation.
notably, the t-stretch in cockroaches and termites also participates in the formation of a certain stem-loop structure .
however, the stem-loop structure around the mtdna or could not be detected in drosophila <cit> .
therefore, the or recognition sequences of mtdna, although generally detected in orthoptera, have diverged not only among orthoptera but also throughout insect evolution.
CONCLUSIONS
the two sinochlora congeners represent the first two orthopterans that have a large-scale translocation involving the cr.
it seems that the present mitogenome rearrangements are a consequence of tandem duplication followed by non-random loss of paralogs.
however, future research including additional taxon sampling is needed to determine rearrangement mechanisms and evolutionary processes.
comparison of the or recognition sequences among orthoptera and other insects will aid in further understanding of mechanisms underlying mtdna replication.
divergence in nucleotide bias and skew of mtdna exists between the two suborders of orthoptera.
future studies on mtdna-based phylogeny of orthoptera should therefore take into consideration the base compositional heterogeneity, which could lead to incorrect phylogenetic inferences
<cit> .

 insect mitogenomes are generally compact with few intergenic spacers and possess stable gene content and organization. they are usually about kb in size and bear protein-coding genes, ribosomal rna genes, transfer rna genes, and one control region that includes replication and transcription origins. however, extensive studies have revealed that gene order rearrangement and size variation that results from the presence of tandem repeats and other non-coding regions occur more often than previously expected. much attention has been paid to studies focusing on unveiling genomic diversities and evolutionary trends. recently, a mitogenomic investigation of congeneric species has yielded a valuable approach for assessing mtdna evolutionary trends. unfortunately, in spite of the large number of insect species, the limited availability of complete mitogenomic sequence data, including those of congeneric species, impedes a thorough understanding of the insect mitogenomes. orthoptera, the largest polyneopteran order, contains subfamilies and over described species, taxonomically divided into two suborders: caelifera and ensifera. orthoptera mitogenomes generally possess a relatively stable gene content and organization identical to the insect ancestor. only a trnd-trnk rearrangement in the lineage acridomorpha, inversion of the gene cluster trne-trns-trnn in teleogryllus emma, and occasional inversion of trnw in the migratory locust have been discovered. a mitogenomic divergence in the at-bias between the two suborders has been demonstrated, i.e., the at-content was generally lower in ensifera than in caelifera. additionally, a possible stem-loop structure has been implicated in mtdna replication initiation in a few caeliferans, which contrasts with the recognition of the mtdna replication origin that involves a long t-stretch in most other insects. however, representative katydid mitogenomes, e.g., anabrus simplex and deracantha onos, revealed the existence of a long t-stretch. furthermore, mitogenomes of caelifera and ensifera species available from genbank demonstrate a distinct taxon sampling imbalance between the two suborders. thus, additional ensifera taxon sampling is essential to investigate the mitogenomic genetic diversities and evolutionary trends. the genus sinochlora tinkham, chinese bush katydid, belongs to the subfamily phaneropterinae in the suborder ensifera. in sinochlora, one species s. longifissa is widespread in east asia including japan, korea, and southern china, and two species are widely distributed and indigenous in southern china. other species are endemic in various large mountains in southern china including low-altitude areas in tibet. investigation of the mitogenomic evolutionary trends of the genus is helpful to unveil the molecular mechanism of the divergence patterns. herein, we chose two representative species, s. longifissa distributed in east asia, and s. retrolateralis narrowly endemic in southern china, for mitogenomic investigation. we sequenced the mitogenomes of the two congeners, unveiled novel mitogenomic characteristics, and outlined the possible rearrangement mechanism. additionally, we also compared the recognition sequences of the or in known orthoptera mitogenomes. overall, we attempted to provide the molecular basis for understanding diversification of the genus sinochlora and depict molecular diversity and evolutionary trends of orthoptera mitogenomes.  genome organization we sequenced the complete mitogenome of s. longifissa and the nearly complete mitogenome of s. retrolateralis with a partial cr. the mitogenomes of s. longifissa and s. retrolateralis have been deposited in the genbank database under accession numbers of kc and kc respectively. they are currently the largest orthoptera mitogenomes on genbank. their large sizes are due to two large non-coding regions, i.e., the cr and one intergenic spacer located between trns and nad both mitogenomes resemble the available ensiferan mitogenomes and the proposed ancestral insect mitogenome in gene content and orientation, trna anticodons, and trna/rrna structures. additionally, there are several gene overlaps, such as the open reading frames of atp6-atp and nad4l-nad each of which overlaps by seven nucleotides. a few other igs are also present in the mitogenomes. sl, s. longifissa; sr, s. retrolateralis. the number of intergenic nucleotides before the gene starts are shown in parenthesis. the asterisk indicates that the tandem repeat region in s. retrolateralis was not completely sequenced. nucleotide composition like other ensiferans, the two mitogenomes show comparatively low a+t bias, compared with most caeliferans. the at skew is in s. longifissa and in s. retrolateralis, while the gc skew is - in the former and - in the latter, indicating weak a skew and strong c skew in the j-strand. through comparison of nucleotide skew in all sequenced orthoptera, the skew divergence between the two suborders was detected. within the ensifera, the at skew values of the whole j-strand range from - to and gc skew values are lower than -, showing the same skew patterns as in sinochlora species. by contrast, within the caelifera except a tridactylid ellipes minuta, absolute values of at skew and gc skew of the j-strand range from to, indicating pronounced a skew and c skew. the synonymous fourfold degenerate third codon positions suffer less selective constraints, and thus could indicate background mutational pressures on nucleotide skew. the skew patterns at the p4fd between the two suborders are consistent with those on the j-strand. it is proposed that nucleotide composition bias and skew are caused by the selection-mutation-drift equilibrium in the molecular evolution. the asystematical directional mutation pressure and corresponding deaminations of cytosine and adenine in the mitogenomes are involved in the mutation processes. start and stop codons all pcgs except cox and nad start with typical atn codons. a previous study concerning orthoptera mitogenomes suggested that the cox gene may start with an irregular tetranucleotide codon auga. similar irregular tetranucleotide codons were also proposed as start codons in drosophila. however, there is no experimental evidence for the use of a 4-bp start codon in any creature. recent research on characteristics of mature mrna and rrna genes from d. melanogaster mitochondria showed that ucg serves as the start codon of its cox gene. the result is consistent with predictions of the start of cox suggested by comparison of conserved amino acid positions. by comparing amino acid sequences of cox in all sequenced orthoptera, we observed that the first conserved codon is the tcn serine codon, downstream of which at least three codon positions are also conserved and there are no standard atn bases. thus, in both sinochlora species, the conserved tct serine codon may also serve as a start codon. concerning the nad gene, it has been proposed to start with atn codons in other sequenced orthopterans; however, there are no conserved amino acid positions within the span of over amino acids downstream of trnp. then we propose that the nad may start with ctg in s. longifissa and ttg in s. retrolateralis, considering previous designation of such start codons. the two start codons, creating no igs or overlap between trnp and nad genes, also appear to be more plausible in the evolutionary economic perspective. two standard stop codons taa/tag and two incomplete stop codons t/ta are utilized in the pcgs. for s. longifissa, six pcgs terminate with taa, one with tag, one with ta, and the other four with t. the stop codons differ between the two congeners in that taa is utilized as the cox stop codon, and ta is utilized as the atp and cox stop codon in s. retrolateralis. a partial t or ta stop codon, which was proposed to create complete taa stop codons via posttranscriptional polyadenylation, is also present in other metazoan mitochondrial genes. novel gene order rearrangement involving the control region one of our most significant findings is the novel gene order rearrangement "rrns-trni-trnm-nad2-cr-trnq-trnw" in both sinochlora species. the two mitogenomes are the first representatives that have a large-scale translocation involving the cr in orthoptera. such gene order has not been observed in other sequenced insect mitogenomes. the gene cluster rrns-cr-trni-trnq-trnm-nad in the proposed ancestral mitogenome has been discovered to function as a hot spot for gene rearrangements in arthropod mitogenomes. duplicate trni and partial trnq genes have been discovered in the cr close to the rrns gene in some blowflies. in a mantid, a complicate set of repeat units dispersing in both ends of the cr also translocated between trnm and nad. a plague thrips displayed cr duplications that are distant from the rrna genes. various locations of the cr and neighboring trna genes have been exhibited in lice. location variability of the putative cr has also been reported as a product of the trna gene translocation, such as rrns-trnq-cr-trni-trns-trnm-nad in a springtail, or the trna and rrna gene translocation, such as rrnl-trnv-trns-trnc-cr-rrns in a mite. the cr and the neighboring genes have also been found to be duplicated in some ticks. organization of the control region for both sinochlora species, the putative cr is composed of two major sections with different nucleotide compositions: a highly a+t-biased region adjacent to nad and several gc-rich tr units adjacent to trnq. for the former, the a+t content is % in s. longifissa and % in s. retrolateralis. by contrast, the tr region has low bias toward a+t. the atr is conserved between the two congeners in length, nucleotide composition and secondary structure. its length is bp in s. longifissa and bp in s. retrolateralis, consistent with the a+t-rich region of other orthopterans. the two species share % sequence identity in this region. a long t-stretch and several conserved potential hairpin structures are thought to play significant roles in initiating and/or regulating the transcription and replication of mtdna. they were also found in the both sinochlora species. the long t-stretch is situated in the initial quarter of the atr on the j-strand, and a similar t-stretch is situated in the initial third of the atr on the minority strand. the two species also share % sequence identity in the domain located between these two t-stretches. the domain has seven similar sites capable of forming 8- to 33-bp stem-loop structures. additionally, there are three similar sites for the formation of 11- to 25-bp stable hairpins, with 7- to 11-bp loops supported by 1– gc matches in the stems, adjacent to the 5’ end of the t-stretch on the j-strand. in s. longifissa, the tr region is bp in length, comprising nine full 175-bp copies and a partial 153-bp copy. in s. retrolateralis, a total of five 175-bp repeat copies have been successfully sequenced from both ends of the tr; however, we failed to sequence through the whole tr region due to the presence of a large number of repeat copies. we estimate that there are twelve 175-bp tandem copies in s. retrolateralis based on the length of the tr indicated by gel electrophoresis. the consensus tr motifs of the two species share % sequence identity. in s. longifissa, four of the nine tr motifs are identical to the consensus tr motif, and the other five have a few mutations and/or deletions. among the nine tr motifs, all but the fourth possess an orf. the consensus orf could be translated into amino acids, starting with lysine and ending with valine. low shared identities with nuclear sequences in search of the genbank database exclude the possibility that the orf is transferred from the nuclear genome. it has % identity with the antisense strand of nad and % identity with the sense strand of cox the shared bases are scattered in alignment, among which over 60% are a and t. this suggests that the orf might not be obtained through horizontal transfer between mitochondria. in contrast, no orf could be found in the s. retrolateralis tr motif. the tr motif could be duplicated through slipped-strand mispairing. moreover, potential stem-loop structures in a repeated unit and its flanking part have been demonstrated to cause an increase in slipped-strand mispairing frequency. stem-loop structures were detected in the tr region of the two sinochlora species. for example, in the first tr unit and its junctions at both ends in s. longifissa, the nucleotide sequence can potentially form seven 8- to 27-bp hairpins with 5- to 10-bp loops supported by 1– gc matches in the stem. in the corresponding regions in s. retrolateralis, the nucleotide sequence can potentially form six 10- to 29-bp hairpins with 5- to 15-bp loops supported by 1– gc matches in the stem. similar complicated hairpin structures in tr were also detected in the louse bothriometopus and termites. however, there are no conserved stem-loops among the trs and at the joints in these insects. unassigned intergenic spacers and trns pseudogenes eleven igss with identical locations are present in s. longifissa and s. retrolateralis; one additional igs is situated between atp and cox in s. longfissa and between cox and trnk in s. retrolateralis. the largest igs lies between trns and nad by comparing the igs of currently available orthoptera mitogenomes, we found a 7-bp conserved motif downstream the nad across orthoptera, with the only exception of mekongiella xizangensis. the conserved motif has been proposed as a binding site for mitochondrial transcription termination factor in orthoptera. similar conserved motifs, which were proposed as a binding site of the mtterm, between trns and nad have also been found in lepidoptera, and coleoptera. it has been confirmed that one of the two binding sites of mtterm lies downstream of nad in drosophila. in the igs region between trns and nad we detected the vestige of one additional trns gene copy in both sinochlora species. while both trns pseudogenes possess the same anticodon to the functional trns, they are unable to form stable cloverleaf secondary structures. relative-rate tests show that both pseudogenes evolved more than twice as fast as their functional paralogs. this suggests that the redundant trns pseudogenes have experienced more relaxed selective constraints. in addition, the igs region in s. longifissa could be folded into three 19- to 25-bp stable stem-loop structures. the anticodon lies in the stem of the second hairpin and the 7-bp conserved motif aforementioned lies adjacent to the 3’ end of the third hairpin. in contrast, there is no hairpin in the igs region in s. retrolateralis. results of all possible pairwise contrasts between trns genes and trns pseudogenes are significant. sd, standard deviation; sr, s. retrolateralis; sl, s. longifissa; mm, myrmecophilus manni; ser, trns; pser, trns pseudogenes. mechanism of genome rearrangements the predominant mechanism of mitogenome rearrangements is considered to be partial genome duplication followed by a random or non-random loss of the duplicated gene copies. for the rearrangement involving the cr neighbourhood, we propose that the ancestor of the two sinochlora species possessed the same gene order as ancestral insects. firstly, the gene cluster cr-trni-trnq-trnm-nad was duplicated, likely promoted by the stem-loop structures detected in the cr. subsequently, non-random deletions might happen to the entire subset of the duplicated genes, dependent on the gene copy’s transcriptional polarity and location in the genome, because the cr includes a transcriptional control sequence. namely, the redundant genes which possess the same polarity, such as the redundant gene cluster trni-trnm-nad were successively deleted during replication. by contrast, persistence of the trns pseudogenes may have resulted from the tandem duplication of trns followed by a subsequent random loss of the trns paralogs. apparently, more knowledge of transcription signals in insect mitogenomes and sampling additional closely related taxa will be helpful to understand the rearrangement mechanism. recognition sequences of replication origins the t-stretch is involved in the recognition of the or of mtdna at least among holometabolous insects, whereas similar t-stretch was not found in the upstream portion of the or for l. migratoria. however, after a thorough re-investigation, we detected long t-stretches or variants of t-stretch in the putative cr of available orthoptera mitogenomes. the long t-stretch can be observed in the available sequences of all katydids, except ruspolia dubia, and varies in size from to bp. the t-stretches could participate in the formation of or be positioned adjacent to a possible stem-loop structure. by contrast, in crickets and grasshoppers, t-stretch variants can be detected within a potential stem-loop structure. in the t-stretch variants, a few transitions between thymine and cytosine occurred to form ncn sequences. the t-stretch variants lie proximal to the middle part of the cr in the crickets, varying in size from to bp. the t-stretch variants vary in size from to bp in grasshoppers, with the exception of alulatettix yunnanensis. in a. yunnanensis, a small t-stretch could be observed in the stem portion of a hairpin structure, similar to that observed in katydids. human mtdna synthesis is initiated from the sites near the stem base of the secondary structure located around the light strand origin, and the small t-stretch located in the loop portion participates in the initiation process. the secondary structure in human and other vertebrate mtdna is very similar to the aforementioned stem-loop structure in the orthopterans. therefore, the long t-stretch, which participates in the formation of or is adjacent to a possible stem-loop structure in katydids, and the t-stretch variants within the stem-loop structure of crickets and most grasshoppers, could also play a crucial role in mtdna replication initiation. notably, the t-stretch in cockroaches and termites also participates in the formation of a certain stem-loop structure. however, the stem-loop structure around the mtdna or could not be detected in drosophila. therefore, the or recognition sequences of mtdna, although generally detected in orthoptera, have diverged not only among orthoptera but also throughout insect evolution.  the two sinochlora congeners represent the first two orthopterans that have a large-scale translocation involving the cr. it seems that the present mitogenome rearrangements are a consequence of tandem duplication followed by non-random loss of paralogs. however, future research including additional taxon sampling is needed to determine rearrangement mechanisms and evolutionary processes. comparison of the or recognition sequences among orthoptera and other insects will aid in further understanding of mechanisms underlying mtdna replication. divergence in nucleotide bias and skew of mtdna exists between the two suborders of orthoptera. future studies on mtdna-based phylogeny of orthoptera should therefore take into consideration the base compositional heterogeneity, which could lead to incorrect phylogenetic inferences. 
the orthoptera mtdna recognition sequence of the replication origin may be one of two kinds: a long t-stretch situated in or adjacent to a possible stem-loop structure or a variant of a long t-stretch located within a potential stem-loop structure. the two kinds of recognition sequences of replication origin suggest that the regulatory sequences involved in the replication initiation process of mtdna have diverged through orthoptera evolution. a question still remains regarding whether a long thymine-nucleotide stretch involved in the recognition of the replication origin exists in the control region of orthoptera mitochondrial dna. orthoptera mitochondrial genomes follow the ancestral insect gene order, with the exception of a trnd-trnk rearrangement in acridomorphs and rare trna inversions. the unique sinochlora mitogenomes reveal that the mtdna architecture within orthoptera is more variable than previously thought, enriching our knowledge on mitogenomic genetic diversities. additionally, we performed comparative mitogenomic analysis to depict evolutionary trends of orthoptera mitogenomes. both mitogenomes are characterized by a large size resulting from multiple copies of an approximately bp gc-rich tandem repeat within cr; a novel gene order, compared to the ancestral order; and redundant trns pseudogenes located between trns and nad multiple independent duplication events followed by random and/or non-random loss occurred during sinochlora mtdna evolution.
 orthoptera, the largest polyneopteran insect order, contains suborders and subfamilies. orthoptera mitochondrial genomes follow the ancestral insect gene order, with the exception of a trnd-trnk rearrangement in acridomorphs and rare trna inversions. a question still remains regarding whether a long thymine-nucleotide stretch involved in the recognition of the replication origin exists in the control region of orthoptera mitochondrial dna. herein, we completed the sequencing of whole mitogenomes of two congeners, which possess overlapping distribution areas. additionally, we performed comparative mitogenomic analysis to depict evolutionary trends of orthoptera mitogenomes.  both sinochlora mitogenomes possess genes and one cr, a common gene orientation, normal structures of transfer rna and ribosomal rna genes, rather low a+t bias, and significant c skew in the majority strand, resembling all the other sequenced ensiferans. both mitogenomes are characterized by a large size resulting from multiple copies of an approximately bp gc-rich tandem repeat within cr; a novel gene order, compared to the ancestral order; and redundant trns pseudogenes located between trns and nad multiple independent duplication events followed by random and/or non-random loss occurred during sinochlora mtdna evolution. the orthoptera mtdna recognition sequence of the replication origin may be one of two kinds: a long t-stretch situated in or adjacent to a possible stem-loop structure or a variant of a long t-stretch located within a potential stem-loop structure.  the unique sinochlora mitogenomes reveal that the mtdna architecture within orthoptera is more variable than previously thought, enriching our knowledge on mitogenomic genetic diversities. the novel genome rearrangements shed light on mtdna evolutionary patterns. the two kinds of recognition sequences of replication origin suggest that the regulatory sequences involved in the replication initiation process of mtdna have diverged through orthoptera evolution. sinochlora longifissasinochlora retrolateralismitochondrial genomegenome rearrangementsrecognition sequence 