c Indian Academy of Sciences

RESEARCH ARTICLE

The mitochondrial genome of the quiet-calling katydids, Xizicus fascipes (: : Meconematinae)

MING RU YANG, ZHI JUN ZHOU∗, YAN LIN CHANG and LE HONG ZHAO

College of Life Sciences, Hebei University, Baoding 071002, People’s Republic of China

Abstract To help determine whether the typical arrangement was a synapomorphy for the whole Tettigoniidae, we sequenced the mitochondrial genome (mitogenome) of the quiet-calling katydids, Xizicus fascipes (Orthoptera: Tettigoniidae: Mecone- matinae). The 16,166-bp nucleotide sequences of X. fascipes mitogenome contains the typical gene content, gene order, base composition, and codon usage found in arthropod mitogenomes. As a whole, the X. fascipes mitogenome contains a lower A+T content (70.2%) found in the complete orthopteran mitogenomes determined to date. All protein-coding genes started with a typical ATN codon. Ten of the 13 protein-coding genes have a complete termination codon, but the remaining three genes (COIII, ND5 and ND4) terminate with incomplete T. All tRNAs have the typical clover-leaf structure of mitogenome tRNA, except for tRNASer(AGN), in which lengthened anticodon stem (9 bp) with a bulged nuleotide in the middle, an unusual T-stem (6 bp in constrast to the normal 5 bp), a mini DHU arm (2 bp) and no connector nucleotides. In the A+T-rich region, two (TA)n conserved blocks that were previously described in and two 150-bp tandem repeats plus a partial copy of the composed at 61 bp of the beginning were present. Phylogenetic analysis found: i) the monophyly of Conocephalinae was interrupted by Elimaea cheni from ; and ii) Meconematinae was the most basal group among these five subfamilies.

[Yang M. R., Zhou Z. J., Chang Y. L. and Zhao L. H. 2012 The mitochondrial genome of the quite-calling katydids, Xizicus fascipes (Orthoptera: Tettigoniidae: Meconematinae). J. Genet. 91, 141–153]

Introduction Ensifera , except for Teleogryllus emma (Grylloidea: ) (Ye et al. 2008), and Ellipes minuta (Caelifera: Metazoan mitochondrial DNA (mtDNA) occurs as a small Tridactyloidea) (Sheffield et al. 2010). The gene arrange- ∼ ( 16 kb), circular, double-strand molecules that encodes 13 ment of the fruit fly Drosophila yakuba mitogenome (Clary protein-coding genes, 2 rRNA genes, and 22 tRNA genes and Wolstenholme 1985) was inferred to be the typical (Wolstenholme 1992; Boore 1999). Additionally, it contains arthropod arrangement, which was also found in several a major noncoding region related to the control of replica- other orders such as Coleoptera, Hemiptera, Thysa- tion and transcription (Clayton 1992; Fernandez-Silva et al. nura and Plecoptera; (ii) the diagnostic tRNA rearrangement 2003), and is therefore referred to as the mitochondrial con- found in Acridoidea and Pyrgomorphidae, both of which trol region. In , this region is also known as the share tRNA rearrangement from tRNALys–tRNAAsp to the A+T-rich region because of the high content of these two derived tRNAAsp–tRNALys between COXII and ATP8 genes. nucleotides in the earliest investigated species (Simon et al. This rearrangement was first discovered in the migratory 1994). locust, Locusta migratoria (Flook et al. 1995). Sheffield et al. To date, 38 mitochondrial genome sequences from (2010) subsequently confirmed that this rearrangement was Orthoptera have been deposited in GenBank. All studied not a synapomorphy for the whole Caelifera; (iii) the trans- mitogenomes have an identical arrangement of all protein- location of tRNAAsn–tRNASer (AGN)–tRNAGlu to tRNAGlu– coding genes and rRNA genes and mostly tRNA genes. tRNASer (AGN)–tRNAAsn (genes underlined are encoded by the Three kinds of tRNAs arrangement have been discovered: N strand) (Ye et al. 2008) found only in T. emma (Grylloidea: (i) the typical arthropod arrangement mostly found in Gryllidae). This rearrangement appears to be unique within Orthoptera and has not been observed in another Gryllidae species, Myrmecophilus manni (Fenn et al. 2008), therefore, ∗ For correspondence. E-mail: [email protected]. it is not a synapomorphy for Gryllidae. Keywords. mitochondrial genome; Meconematinae; Orthoptera; phylogeny; Xizicus fascipes.

Journal of Genetics, Vol. 91, No. 2, August 2012 141 Ming Ru Yang et al.

The quiet-calling katydids which represent a disparate Sub-PCR amplification and sequencing assemblage of tettigoniid genera, was composed of 550 Sub-PCR amplifications were performed with L-PCR prod- described species from 78 genera and occur mainly in the ucts as templates. In primer pair of sub-PCR amplifica- Oriental realm and Ethiopian realm. This group includes tions, one was designed using the already sequenced por- the smallest katydids distributed mainly in tropical forests. tions of the mitogenome, and the other was designed accord- Recent investigations show that this group is very numer- ing to aligned mitogenome sequences of Orthoptera species ous, diverse, and represented by species and genera with very through the NCBI nucleotide database and universal primer narrow ranges (Gorochov 2008). In this work, we present (Folmer et al. 1994;Simonet al. 2006; Zhang and Huang the complete mitogenome sequence of Xizicus fascipes 2008). These primer pairs were then used within sub-PCR (Orthoptera: Tettigoniidae: Meconematinae), which is the amplifications to obtain adjacent gene sequences, which first complete mitogenome sequence from the subfamily overlap with known sequences. In the same way, the cycle Meconematinae. was repeated to ‘walk’ around the rest of the genome. This was continued untill the entire mitogenome sequence had Materials and methods been determined. In this study, we designed five rounds of sub-PCR primers to complete the entire genome sequence Specimen collection and DNA extraction amplification (table 1). The cycling protocol consisted of a primary denaturation at 94◦Cfor2min,followedby30 Specimens of X. fascipes were collected at Mount Emei in cycles of denaturation at 94◦C for 10 s, 42–58◦C anneal- Sichuan, China and stored in 100% ethanol at −4◦C. ing for 30 s, and 72◦C elongation for 1–2 min followed by Total genomic DNA was extracted from the leg muscle incubation at 72◦Cfor7min. tissue of a single adult female specimen using the TIANamp The sub-PCR fragments were directly sequenced, whereas Genomic DNA kit (Tiangen Biotech, Beijing, China), and a few fragments that were difficult to sequence directly stored at −20◦C. were cloned into the plasmid vector PMD-19 T (TaKaRa Biotech, Dalian, China) and sequenced. All fragments were L-PCR of the mitochondrial genome sequenced from both strands on ABI 3730 XL DNA Ana- lyzer (Biosune Biotech, Shanghai, China). Initially, three short gene regions (COI, COII and Cytb) were amplified and sequenced with modified conserved insect primers (Simon et al. 1994, 2006) using aligned Sequence assembly, annotation and analysis orthopteran mitogenome sequences obtained from previous studies. Based on these sequences, two L-PCR primer pairs To prepare the mitogenome sequences for uploading into (Liu et al. 2006) were modified to amplify the entire X. MOSAS, we first proofread and assembled raw sequence fascipes mitogenome. The first long PCR product (about data into contigs using the Staden sequence analysis pack- 9 kb) amplified the fragment from the middle of COI to age (Staden et al. 2000), and then uploaded the entire downstream of Cytb, with the primer pair LPA-J (5-CAC mitogenome sequence files into MOSAS for annotation TTA TTT TGA TTY TTT GGW CAC CCT GAA GT-3) (Sheffield et al. 2010). The nucleotide composition of and LPA-N (5-AAG ATA GCA TAW GCA AAT AAR AAG mitogenome different regions and codon usage of protein- TAT CAT TC-3), using the following PCR conditions: a pri- coding genes were analysed using MEGA 4.0 software mary step denaturation at 94◦C for 1 min, followed by 30 (Tamura et al. 2007). Tandem repeats finder was used to cycles of denaturation at 98◦C for 10 s, annealing at 48◦Cfor search for repeats in A+T-rich region (Benson 1999). 30 s, and 68◦C elongation for 10 min, followed by incubation at 68◦C for 10 min. The second product (about 7.5 kb), corre- Phylogenetic analysis sponding to the sequence from upstream of Cytb to the mid- dle of COII, with the primer pair LPB-J (5-AGT TAC YCC Phylogenetic analysis was performed to determine the TGT YCA TAT TCA ACC WGA ATG AT-3) and LPB-N relationships among five katydid subfamilies. For this, (5-TGA TTG GCC CCA CAA ATT TCT GAA CAT TGA the nucleotide sequences of all mitochondrial genes from CC-3), using the following PCR conditions: a primary step X. fascipes (Meconematinae), plus gratiosa denaturation at 94◦C for 1 min, followed by 30 cycles of EU527333 (), simplex EF373911 denaturation at 98◦C for 10 s, annealing at 48◦C for 30 s, and (Tettigoniinae), Deracantha onos EU137664 (Bradypori- 68◦C elongation for 10 min, followed by incubation at 68◦C nae), Ruspolia dubia EF583824 (Conocephalinae), Cono- for 10 min. PCR reactions were performed in a MyCyler Per- cephalus maculatus HQ711931 (Conocephalinae), E. cheni sonal Thermal Cycler (Bio-Rad, Hercules, USA) in 25 μL GU323362 (Phaneropterinae) and Troglophilus neglectus reaction volume set up as follows: 12.5 μLPremixLATaq EU938374 (Rhaphidophoridae, outgroup) were individually Hot Start (TaKaRa Biotech, Dalian, China), 1 μL of each aligned using Clustal W in BioEdit v7.0 (Hall 1999)and primer (10 μM), 3 μL of genomic DNA, 7.5 μL of sterilized concatenated. The nucleotide sequences of protein-coding distilled water. genes were aligned based on reading frame information with

142 Journal of Genetics, Vol. 91, No. 2, August 2012 Mitogenome of quiet-calling katydids )  3 →  Designed in this study. e ). TAAACTTCAGGGTGACCAAAAAATCA TGATTGGCCCCACAAATTTCTGAACATTGACC TATCTACGGCGAATCCYCCTCA GTTGCTCAAACTATTTCTTATGA ATAGGCGRTARACTGTAAAT TAGGGTCCCTGGCCGAATTA CCGATATTTCATCATGCTGA GATTGCGACCTCGATGTTGG ATCCTATTTAYGGGGTATGARCC ATGTCCWGCAATYATATTAGC TGTGAAGGGGCCTTRGGATT CGCCTGTTTAACAAAAACAT GTGCCAGCHGCCGCGGTTAKAC TCAACAAAGTGTCAGTATCA CAGTAATATACCTCTYTTTGG ATTGCTTAYTCRTCTGTTG ACATGYACAYATCGCCCGTC AAGGATTCTCAGGATATTCG TTAGGTTGAGATGGATTAGG 2008 d d d d c a a a a a a b a a a a a a d Modified from Zhang and Huang ( d ). 2006 ( et al. mitogenome. ) Primer R Sequence (5  3 Xizicus fascipes Modified from Liu → c  ). 1994 ( GTTAANCAAACTAATARCYTTCAAACACTTATTTTGATTYTTTGGWCACCCTGAAGTACCCAAAGCACCCTCACAAACAGTTACYCCTGTYCATATTCAACCWGAATGAT LPB-N3651 TAAAGGATTAYYGTGATAGATTGAYGCAACACCTGGCCG N1-N12242 CGTTCAGGTTGATAACCTCA HCO-2198 GGAGTTCGATTAGTTTCAGCATAATMGGGTATCWAATCCTAGTTCATCAGATGACTGAAAGTAAGGAWGAACATAAMCCATGACC CB-N11010 GCTCACGCCGGTYTGAACTCTTRATAAACCCTGATACAMAAGCACCTAGTCCCTCAAGGAAC N2N-1406 AATGTTATTCGGCCAGGAAC A02-R04-4483 GGAATTTGTGCTCTCTTWGT TM-N200 CB-N10608 CCGGTCTGAACTCAGATCATGTA B02-R12-12845 TTTGAYCCTAAGAGWTCCGC A6-N4552 TAACATCTTCAGTGTYATRCTCT N4L-N9629 SR-N14745 B03-R15-13290 C3-N5460 A16-R05-6062 SR-N14220 N4-N8722 N5-N7793 R06-7061 et al. Folmer d d b d ). c d a c a a a a a a a a e a e a 2006 ( et al. Primer F Sequence (5 LPA-J2123 N4L-J9648 LBP-J11184 A02-F03-3563 N4-J9172 B02-F10-11982 TK-J-3790 N4-J8641 B03-F11-12755 A6-J4463 A6-J4500 N5-J7572 LR-J12888 TF-J6400 Primer pairs used in sub-PCR amplification of the Modified from Simon a Table 1. First round TW-J1301 Second round B21-F32-36 Third round SR-J14610 Fourth round LR-J13900 Fifth round N3-J5762

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Toggle Translation in BioEdit v7.0 (Hall 1999). Maximum- et al. 2010). It has a typical gene content found in metazoan likelihood (ML) analysis was performed with PhyML v3.0 mitogenomes (figure 1): 13 protein-coding genes, 22 tRNA (Guindon et al. 2010) online in Montpellier bioinformat- genes, two rRNA genes, and the A+T-rich region. The ics platform (www.atgc-montpellier.fr/phyml). A BioNJ tree gene order and orientation (figure 1;table2)ofX. fascipes was used as a starting tree to search for the ML tree mtDNA is identical to those of D. yakuba (Clary and with the GTR+I+ model. Robustness of the phyloge- Wolstenholme 1985). netic results was tested by bootstrap analysis with 1000 Without considering the A+T-rich region, the X. fascipes replicates. mitochondrial genes are separated by a total of 61 bp of inter- genic spacer sequences, which are spread over 10 regions and range in size from 1 to 17 bp. The largest spacer (17 bp) Results and discussion is present between tRNASer(UCN) and ND1. Summarized all the orthopteran mitogenomes available on GenBank to date, Genome structure, organization and composition the size of the spacer of most species at this location ranges The sequence of the entire X. fascipes mtDNA has been from 16 bp to 34 bp, and a few reach to 59 bp (Kim et al. deposited in the GenBank database under accession number 2005), even 259 bp (Sheffield et al. 2010), except for Gam- JQ326212. The X. fascipes mtDNA is 16,166 bp long, larger phocerus marmoratus (0 bp) (Ma et al. 2009), Mekongiana than all other orthopteran mitogenome sequenced in their xiangchengensis (2 bp) (Zhao et al. 2010). The alignment of entirety except for 16,259 bp of Oedaleus asiaticus (Ma et al. the end of ND1 across orthopterans suggests that, for G. mar- 2009) and 17,004 bp of Physemacris variolosa (Sheffield moratus, it should end 21 bp before the supposed stop codon.

Figure 1. Circular visualization of the mitogenome of Xizicus fascipes. Two pairs of L-PCR primers, LPA-J + LPA-N and LPB-J + LPB-N, were used to amplify the complete mitogenome into two overlapping fragments (from COI to CYTB and from CYTB to COII).

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Table 2. Organization of the Xizicus fascipes mitogenome.

Noncoding Overlapping Gene/region Position Size Direction region region Initiation/termination tRNAIle 1–64 64 F 3 tRNAGln 62–130 69 R 7 tRNAMet 138–200 63 F ND2 201–1226 1026 F 2 ATT/TAA tRNATrp 1225–1290 66 F 8 tRNACys 1283–1349 67 R tRNATyr 1350–1413 64 R 8 COI 1406–2950 1545 F 5 ATT/TAA tRNALeu(UUR) 2946–3010 65 F 2 COII 3013–3696 684 F 2 ATG/TAA tRNALys 3699–3768 70 F 1 tRNAAsp 3768–3834 67 F ATP8 3835–3999 165 F 7 ATC/TAA ATP6 3993–4670 678 F 1 ATG/TAA COIII 4670–5456 787 F ATG/T tRNAGly 5457–5521 65 F ND3 5522–5875 354 F 4 ATT/TAA tRNAAla 5880–5943 64 F 1 tRNAArg 5943–6005 63 F 16 tRNAAsn 6022–6088 67 F 2 tRNASer(AGN) 6091–6157 67 F tRNAGlu 6158–6224 67 F 9 tRNAPhe 6234–6296 63 R ND5 6297–8028 1732 R ATT/T tRNAHis 8029–8094 66 R ND4 8095–9433 1339 R 7 ATG/T ND4L 9427–9723 297 R 1 ATG/TAA tRNAThr 9725–9795 71 F 1 tRNAPro 9795–9859 65 R 1 ND6 9861–10388 528 F 1 ATA/TAA Cytb 10388–11524 1137 F 2 ATG/TAG tRNASer(UCN) 11523–11591 69 F 17 ND1 11609–12562 954 R 6 ATA/TAG tRNALeu(CUN) 12557–12620 64 R lrRNA 12621–13927 1307 R tRNAVal 13928–13998 71 R srRNA 13999–14788 790 R A+T-rich region 14789–16166 1378 F

F, forward (5 → 3); R, reverse (3 → 5).

Fenn et al. (2007) reported, this intergenic spacer sequence aligned the overlap sequence context between ATP8 and was not conserved (figure 2). ATP6, and between ND4 and ND4L, and suggested that both Another common feature of mitogenomes is overlapping overlap by conserved 7 bp nucleotides (figures 3 and 4). genes. The X. fascipes mitogenome contains 14 pairs of There, the initiation codons for ATP6 of G. gratiosa (Zhou genes that overlap between 1 and 8 bp (53 bp in total). As has et al. 2008)andforND4 of T. neglectus appear to be GTG, often been found across the hexapods, the ATP8/ATP6 and which is rare but has been designated as an initiator of G. ori- ND4/ND4L gene pairs appear to overlap 7 bp nucleotides. entalis and Tricholepidion gertschi ND2 (Nardi et al. 2003; In all 38 known orthopteran mitogenomes available on Gen- Kim et al. 2005). The previous analysis of transcription map Bank, there exist 7 bp or 4 bp overlap between ATP8 and across some species suggests that there exists a polycistron ATP6; and 7 bp or 13 bp overlap between ND4 and ND4L in the transcription process of ATP8/ATP6 and ND4/ND4L, except for Troglophilus neglectus, which was spaced 11 bp and it proves that they are both transcribed together (Berthier so that the length of ND4 is obviously shorter than in other et al. 1986). Taanman (1999) deems that monocistronic orthopterans (Fenn et al. 2008). To pursue this further, we transcripts of ATP8 and ND4L are possibly too short to

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Figure 2. Alignment of the end of ND1 and the noncoding sequence between tRNASer(UCN) and ND1 across Orthoptera indicates that ND1 of Gastrimargus marmoratus should end 21 bp before the stop codon they annotated. *Stop codons. interact effectively with the small (28S) ribosomal subunit for orthopteran mitogenomes (65.3%–76.2%, mean 73.2%) (Taanman 1999). (Fenn et al. 2008; Zhou et al. 2008). The nucleotide composition of the X. fascipes mitogenome is biased towards adenine and thymine as in most other Protein-coding genes insects. The A+T content of the entire genome (J-strand) is 70.2%, and this value is relatively lower than that of The initiation and termination codons were identified using most orthopterans, but well within the observed range the ORF finder and by comparison with the situation in

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Figure 3. Alignments of the overlap context between ATP8 and ATP6 of insects sequenced in their entire mitogenome. The box indicates a conserved 7 bp overlap region. Underlined nucleotides indicate the rare initiation codon. *Stop codons. other orthopterans. The typical ATN codon apparently ini- have incomplete termination codons of a single thymine tiates translation of protein-coding genes: ATT for ND2, (T) base (table 2). The presence of incomplete stop codon COI, ND3 and ND5,ATAforND6 and ND1, and ATG for is common in metazoan mitochondrial genomes, and these the other seven genes. Similarly, the conventional termi- truncated stop codons are presumed to be completed via nation codons (TAA and TAG) could be assigned to most posttranscriptional polyadenylation (Ojala et al. 1981). of the protein-coding sequences (eight using TAA and two The A+T content of protein-coding genes, excluding stop using TAG) except that three genes (COIII, ND5 and ND4) codons, is 69.5% in X. fascipes, and around the same value

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Figure 3 (contd)..

in many sequenced orthopterans, such as G. orientalis 69.4% genes. The codon usage and the relative synonymous codon (Kim et al. 2005), M. manni 69.0% (Fenn et al. 2008), E. usage (RSCU) values for the X. fascipes protein-coding cheni 71.4% (Zhou et al. 2010), Anabrus simplex 68.8% genes are shown in table 3. The bias of synonymous (Fenn et al. 2007)andRuspolia dubia 69.8% (Zhou et al. codon usage is correlated with the higher adenine and 2007). The analysis of A+T content at each codon position thymine content of the entire mitogenome. Codon usage shows that the third codon position (79.5%) is higher than the shows a clear bias towards T in the second and T/A in first (64.3%) and the second (64.6%) codon positions. How- the third codon position. Leucine (16.05%), serine (9.06%), ever, the higher content of T-base in the second position, as in isoleucine (8.95%), and phenylalanine (8.68%) are the most other orthopterans, might be related to a preference for non- frequently used amino acids as well as those with AT-rich polar and hydrophobic amino acids in membrane associated codons. proteins. The AT bias of the third codon position is probably introduced by mutational pressure as has been observed in the mitochondrial genome (Clayton 1982; Foster et al. 1997). Transfer RNA coding genes All codons are present in the protein-coding genes of this mitogenome. Excluding termination codons, there are The complete set of 22 tRNA genes is present in the X. a total of 3731 codons in the X. fascipes protein-coding fascipes mitogenome, and the length ranges from 63 to 71 bp.

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Figure 4. Alignments of the overlap context between ND4 and ND4L of insects sequenced in their entire mitogenome. The box indicates a conserved 7 bp overlap region. Underlined nucleotides indicate the rare initiation codon. *Stop codons.

The relative locations for each tRNA gene are shown in Twenty-four nonWatson–Crick pairings (G–U) and six figure 1. All tRNA genes have the typical cloverleaf struc- mismatches were identified in the X. fascipes mitogenome tures except for tRNASer(AGN).ThetRNASer(AGN) was found tRNA genes. Two U–U were proposed in the acceptor stem to have a lengthened anticodon stem (9 bp) with a bulged of tRNAAla; one U–U in the anticodon stem of tRNASer(AGN); nuleotide in the middle, an unusual T-stem (6 bp in constrast two A–A in the anticodon stem of tRNAIle and in the TψC to the normal 5 bp), a mini DHU arm (2 bp) and no connector stem of tRNASer(AGN), respectively; and one C–A in the nucleotides (figure 5) (Steinberg et al. 1997). acceptor stem of tRNALys.

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Figure 4 (contd)..

Ribosomal RNA coding genes (Fenn et al. 2007) to 76.6% in Oxya chinensis (Zhang and Huang 2008), respectively. As in most insect mitogenomes, the two ribosomal genes (lrRNA and srRNA)ofX. fascipes mitogenome were located A+T-rich region between tRNALeu(CUN) and A+T-rich region, and separated by tRNAVal (figure 1;table2). The lengths of lrRNA and srRNA The 1378 bp X. fascipes A+T-rich region was observed in were respectively determined to be 1307 bp and 790 bp, and the conserved location between srRNA and tRNAIle,andwas the length of the two ribosomal genes in other orthopter- composed of 65.5% A and T nucleotides (figure 1;table2). ans range from 1236 bp in Gryllotalpa pluvialis (Fenn et al. The size of X. fascipes A+T-rich region is well within the 2008) to 1342 bp in T. neglectus (Fenn et al. 2008), and range found in other orthopterans, from 70 bp in R. dubia 718 bp in Acrida willemsei (Fenn et al. 2008) to 882 bp (Zhou et al. 2007) to 2277 bp in P. variolosa (Sheffield et al. in R. dubia (Zhou et al. 2007), respectively. The A+T con- 2010). The size differences of A+T-rich region occur not only tent of the lrRNA and srRNA was 74.5% and 73.0%, respec- because of its high rates of nucleotide substitution, insertion tively. These percentages correspond to those found in other or deletion, but also due to the length of tandem repeat unit orthopterans, in which the A+T content of lrRNA and srRNA and the number of tandem repetitions. The sequence analy- ranges from 72.3% in D. onos (Zhou et al. 2009) to 78.9% sis reveals two 150 bp tandem repeats plus a partial copy of in L. migratoria (Flook et al. 1995), and 68.9% in A. simplex 61 bp at the beginning in the A+T-rich region, contributing

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Table 3. Codon usage of protein-coding genes in the Xizicus fascipes mitogenome.

Amino acid Codon n (RSCU) Amino acid Codon n (RSCU)

Phe UUU 265.0 (1.64) Tyr UAU 125.0 (1.59) UUC 59.0 (0.36) UAC 32.0 (0.41) Leu UUA 321.0 (3.22) Ala GCU 84.0 (1.64) UUG 103.0 (1.03) GCC 32.0 (0.62) CUU 74.0 (0.74) GCA 82.0 (1.60) CUC 16.0 (0.16) GCG 7.0 (0.14) CUA 75.0 (0.75) Ter UAA 0.0 (0.00) CUG 10.0 (0.10) UAG 0.0 (0.00) Ile AUU 276.0 (1.65) His CAU 53.0 (1.29) AUC 58.0 (0.35) CAC 29.0 (0.71) Met AUA 174.0 (1.51) Gln CAA 65.0 (1.73) AUG 56.0 (0.49) CAG 10.0 (0.27) Val GUU 75.0 (1.44) Asn AAU 132.0 (1.58) GUC 16.0 (0.31) AAC 35.0 (0.42) GUA 94.0 (1.81) Lys AAA 49.0 (1.32) GUG 23.0 (0.44) AAG 25.0 (0.68) Ser UCU 93.0 (2.20) Asp GAU 59.0 (1.55) UCC 30.0 (0.71) GAC 17.0 (0.45) UCA 89.0 (2.11) Glu GAA 73.0 (1.76) UCG 11.0 (0.26) GAG 10.0 (0.24) AGU 35.0 (0.83) Cys UGU 34.0 (1.74) AGC 16.0 (0.38) UGC 5.0 (0.26) AGA 60.0 (1.42) Trp UGA 100.0 (1.85) AGG 4.0 (0.09) UGG 8.0 (0.15) Pro CCU 76.0 (2.14) Arg CGU 24.0 (1.63) CCC 26.0 (0.73) CGC 5.0 (0.34) CCA 32.0 (0.90) CGA 23.0 (1.56) CCG 8.0 (0.23) CGG 7.0 (0.47) Thr ACU 79.0 (1.54) Gly GGU 46.0 (0.81) ACC 23.0 (0.45) GGC 19.0 (0.34) ACA 90.0 (1.76) GGA 111.0 (1.96) ACG 13.0 (0.25) GGG 50.0 (0.88)

A total of 3731 codons were analysed, excluding termination codon. n, frequency of each codon; RSCU, relative synonymous codon usage. Ter, stop codons.

361 bp to the length of the region. The 361-bp sequence has an unusually lower A+T content (50.1%), which may reduces the A+T content of the entire A+T-rich region. Removing this repeat region, it would reach to 70.7%, which is well within the observed range of orthopterans. Moreover, the A+T-rich region contains two (TA)n conserved blocks ((TA)6 and (TA)7) as in mosquito (Rondan Dueñas et al. 2006),

Figure 6. A phylogeny of five katydid subfamilies was obtained Figure 5. Inferred secondary structure for Xizicus fascipes from phyml analysis. Numbers on each node indicate the num- tRNASer(AGN). The tRNA is labelled with the abbreviations of its ber of times the nodes were supported in an analysis of a 1000 corresponding amino acids (Ser). Dashes (–) indicate Watson–Crick bootstrap replicate dataset. The tree was rooted by T. neglectus base pairing and asterisks (*) indicate G–U base pairing. (Rhaphidophoridae).

Journal of Genetics, Vol. 91, No. 2, August 2012 151 Ming Ru Yang et al. which is also consistent with our previous research (Zhang Fernandez-Silva P., Enriquez J. A. and Montoya J. 2003 Replica- et al. 2011). May be the conserved motif possesses functions tion and transcription of mammalian mitochondrial DNA. Exp. similar to the (TA(A)) motif (Zhang et al. 1995; Zhang and Physiol. 88, 41–56. n Flook P. K., Rowell C. H. and Gellissen G. 1995 The sequence, Hewitt 1997). organization, and evolution of the Locusta migratoria mitochon- drial genome. J. Mol. Evol. 41, 928–941. Folmer O., Black M., Hoeh W., Lutz R. and Vrijenhoek R. 1994 Phylogenetic relationships among katydid subfamilies DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertibrates. Mol. Mar. To understand the phylogenetic relationships among five Biol. Biotechnol. 3, 294–299. katydid subfamilies, a dataset containing the nucleotide Foster P. G., Jermiin L. 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Received 2 January 2012, in final revised form 16 February 2012; accepted 28 February 2012 Published on the Web: 28 June 2012

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