Complete mitochondrial genome of Zeugodacus tau (Insecta: Tephritidae) and differentiation of Z. tau species complex by mitochondrial cytochrome c oxidase subunit I gene

The tephritid fruit fly Zeugodacus tau (Walker) is a polyphagous fruit pest of economic importance in Asia. Studies based on genetic markers indicate that it forms a species complex. We report here (1) the complete mitogenome of Z. tau from Malaysia and comparison with that of China as well as the mitogenome of other congeners, and (2) the relationship of Z. tau taxa from different geographical regions based on sequences of cytochrome c oxidase subunit I gene. The complete mitogenome of Z. tau had a total length of 15631 bp for the Malaysian specimen (ZT3) and 15835 bp for the China specimen (ZT1), with similar gene order comprising 37 genes (13 protein-coding genes—PCGs, 2 rRNA genes, and 22 tRNA genes) and a non-coding A + T-rich control region (D-loop). Based on 13 PCGs and 15 mt-genes, Z. tau NC_027290 (China) and Z. tau ZT1 (China) formed a sister group in the lineage containing also Z. tau ZT3 (Malaysia). Phylogenetic analysis based on partial sequences of cox1 gene indicates that the taxa from China, Japan, Laos, Malaysia, Bangladesh, India, Sri Lanka, and Z. tau sp. A from Thailand belong to Z. tau sensu stricto. A complete cox1 gene (or 13 PCGs or 15 mt-genes) instead of partial sequence is more appropriate for determining phylogenetic relationship.


Introduction
Zeugodacus tau (Walker) is the most common tephritid fruit fly species of the genus Zeugodacus found in Southeast Asia [1]. It is among the economically important species belonging to the Dacinae subfamily, occurring from Pakistan to Philippines and south to Indonesia [2]. It is a polyphagous fruit pest, infesting host fruits of the families Anacardiaceae, Cucurbitaceae, Elaeocarpaceae, Moraceae, Myrtaceae, Oxalidaceae, Rutaceae, Sapotaceae, and Solanaceae [3][4][5][6][7]. The adult male flies are attracted to Cue lure.
Studies based on cytogenetics, partial sequences of mitochondrial cytochrome c oxidase subunit I (cox1) gene and allozymes have revealed that Z. tau (previously referred to as a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 Bactrocera tau (Walker)) is a species complex comprising eight species (or morphs) in Thailand, with species A designated as Z. tau sensu stricto [8][9][10]. Z. tau A may be reliably separated from Z. tau B, C, D, E, F, G, and I by the heat shock protein 70 cognate gene Bthsc1 [11].
Phylogenetic analysis using mitochondrial cox1 gene sequences revealed that the Z. tau population in Himachal Pradesh (India) is closely related to Z. tau sp. A from Thailand [12]. The overall genetic variability in this Indian taxon is substantial, with 10 different haplotypes detected in 16 individuals. A study of 23 Z. tau populations (Myanmar and western Yunnan; Laos and southern Yunnan; central Yunnan; Thailand; southern China, central China and northern Vietnam; and southwestern China), based on mitochondrial NADH dehydrogenase gene (nad1), revealed six genetic groups corresponding to geographical characteristics, and strong genetic structure for the populations in western China, Thailand, and Laos [13]. Z. tau in China has also been reported to exhibit seven cytochrome b haplotypes (NCBI GenBank 26-JUL-2016: AY953491-AY953497).
To date, there is only a single report on the complete mitochondrial genome (mitogenome) of Z. tau [14]. The taxon is from Shenzhen, China. We report here the complete mitogenome of Z. tau from Malaysia and compare it to that of China as well as the mitogenome of other congeners. We also carry out phylogenetic analysis using cox1 gene to determine the relationship of Z. tau taxa from different geographical regions.

Specimen collection and mitochondrial DNA extraction
Male fruit flies of Z. tau were collected in Malaysia (Kuala Lumpur-3.1390˚N, 101.6869˚E) and China (Zhuhai, Guangdong-22.2710˚N, 113.5767˚E) by means of Cue lure according to the method of Yong et al. [15]. The specimens were preserved in absolute ethanol and stored in -20˚C freezer until use. Z. tau is an insect pest. It is not endangered or protected by law. No permits are required to study this fruit fly. The extraction of mitochondrial DNA was according to the method of Yong et al. [16].

Mitogenomes and cytochrome c oxidase subunit I sequences from GenBank
The complete mitogenomes of Tephritidae available from GenBank (Table 1)

Phylogenetic analysis
Alignment of nucleotide sequences and reconstruction of phylograms based on 15 mt-genes and cytochrome c oxidase subunit I gene sequences followed that described in Yong et al. [26].
The complete mitogenome of Z. tau had a total length of 15631 bp for the Malaysian specimen (ZT3) and 15835 bp for the China specimen (ZT1), with similar gene order comprising 37 genes (13 protein-coding genes-PCGs, 2 rRNA genes, and 22 tRNA genes) and a non-coding A + T-rich control region (D-loop) ( Table 2, Fig 1, S1 and S2 Tables). The control region was flanked by rrnS and trnI genes respectively, with 745 bp in Z. tau ZT3 and 946 bp in Z. tau ZT1. It contained a long polyT-stretch of 14 bp in Z. tau ZT3 and 19 bp in Z. tau ZT1. It also contained in both taxa a long poly A-stretch (20 bp) after 'ATAGA' motif.
There were 16 intergenic regions with spacing sequence and 9 regions with overlaps in both Z. tau ZT3 and Z. tau ZT1. The region between trnR and trnN genes in both taxa was separated by the largest sequence of 34 bp. This sequence had clear stem-loop structures.

Phylogenetic relationship and genetic divergence
The phylogenetic relationship of some of the component taxa of genus Bactrocera was not congruent between ML and BI analyses (Fig 2). For example, ML analysis indicated B. melastomatos to be a member of the B. dorsalis complex, but in BI analysis it was basal to the other taxa of subgenus Bactrocera. Nonetheless, the genus Bactrocera was monophyletic.
Phylogenetic analysis based on partial cox1 sequences from bp 50-700 indicated that the Z. tau taxa from China, Bangladesh, India (Meghalaya, north of Bangladesh) and Malaysia formed a clade with several haplotypes (Fig 3). The uncorrected genetic distance ranged from 'p' = 0 to 'p' = 0.72% (S5 Table).
Based on the partial cox1 sequence from bp 900-1500, the Z. tau taxa from India, Sri Lanka, Malaysia, Laos, China and Japan formed a clade with Z. tau sp. A from Thailand (Fig 4), with uncorrected genetic distance ranging from 'p' = 0% to 'p' = 1.39% (S6 Table). This clade was distinctly different from Z. tau sp. B, C, D, E, F, G, and I from Thailand, with uncorrected genetic distance ranging from 'p' = 9.03% to 'p' = 14.06% (S6 Table).

Haplotype diversity and nucleotide diversity
Twelve haplotypes were revealed in the present 18 cox1 sequences (from bp 50-700) of Z. tau from four geographical regions (China, Malaysia, Bangladesh and India) (Fig 5). A common haplotype was found in China (3 sequences), Bangladesh (2 sequences) and India (1 sequence). The haplotype/gene diversity was 0.8954 ± 0.0653, and the nucleotide diversity was 0.0033 ± 0.0022.
Sixteen haplotypes were revealed in the 22 cox1 sequences (from bp 900-1500) of Z. tau sensu stricto from six geographical regions (China, Laos, Malaysia, India, Sri Lanka, and Thailand sp. A) (Fig 6). A common haplotype was found in China (2 sequences), Japan (1 sequence), India (1 sequence) and Sri Lanka (1 sequence). Another haplotype was common to Malaysia (2 sequences) and India (1 sequence). The haplotype/gene diversity was 0.9437 ± 0.0372, and the nucleotide diversity was 0.0056 ± 0.0034. Z. tau sp. B, C, D, E, F, G and I from Thailand formed a distinct cluster from Z. tau sensu stricto, and each was represented by a distinct haplotype. The haplotypes of Z. tau F and Z. tau B had a small difference of 4 bp.
The gene order of Z. tau mitogenome conforms to other Zeugodacus and other tephritid mitogenomes [15,20,24,25,[28][29][30]. The mitogenome of Z. tau ZT3 (Malaysia) is shorter than that of Z. tau ZT1 (China) and Z. tau NC_027290 (China), while Z. tau ZT1 is longer than Z. tau NC_027290 ( Table 2). The difference in the total size of the mitogenome is due mainly to the length of the control region-745 bp for Z. tau ZT3, 946 bp for Z. tau ZT1 and 801 bp for Z. tau NC_027290 (Table 2).
There are differences in the spacing/overlap sequence in some intergenic regions among Z. tau mitogenomes: -1 bp in Z. tau NC_027290 between trnC and trnY versus 1 bp in Z. tau ZT3 and Z. tau ZT1 ( Table 2).
The difference in size of the nad1 gene (1020 bp in Z. tau NC_027290, and 940 bp in Z. tau ZT1 and Z. tau ZT3) and the stop codon (TAA in NC_027290 and incomplete T in ZT1 and ZT3) can be attributed to annotation of the intergenic space between trnS2 and nad1 genes (overlap of 65 bp in NC_027290 and spacing sequence of 15 bp in ZT3 and ZT1); this intergenic space is 15 bp in most of the Zeugodacus taxa. Incomplete stop codons have been reported in other taxa of tephritid fruit flies [15,20,24]. The incomplete stop codons can be converted to TAA by post-translational polyadenylation [33].
A long poly-A stretch of 20 bp is present in the control region after 'ATAGA' motif in the Malaysian and China taxa of Z. tau. In addition, a long poly-T stretch is present in the control In both T. tau ZT3 and Z. tau ZT1, the TCC-loop was absent in trnF while trnS1 lacked the DHU-loop (S1 and S2 Figs). The TCC-loop and DHU-loop of tRNA act as special recognition site during protein biosynthesis or translation [34][35][36]. It has been reported that misacylation of tRNA can affect the survivability of an organism [36]. However, deviant tRNA secondary structures are frequent in Arthropoda [37].
The mitochondrial cox1 gene has been commonly used for differentiation of various taxa of Z. tau [9,12,[38][39][40][41][42][43][44][45][46]. In the present study based on partial sequences of cox1 gene (Figs 3-6), the Z. tau taxa showed several haplotypes. The taxa from China, Japan, Laos, Malaysia, Bangladesh, Inida, and Sri Lanka were genetically similar to Z. tau sp. A from Thailand, with 'p' = 0-1.39% (S5 and S6 Tables). As Fuzhou (Foochow), Fujian, China is the type locality of Z. tau, the taxa from various geographical regions that grouped with those from China can be designated as Z. tau sensu stricto. Although many taxa had been included for comparison, none were similar to any of the Z. tau sp. B, C, D, E, F, G, and I reported from Thailand. Among the Z. tau taxa from Thailand, the genetic distance bewteen Z. tau F and Z. tau B was 'p' = 0.69% (S6 Table) with haplotype difference of 4 bp (Fig 6), indicating that these two taxa may be conspecific.
In the present study based on partial cox1 sequences, Z. tau ZT1 (China) and Z. tau NC_027290 (China) were not closely related to each other compared to Z. tau ZT3 (Malaysia) (Figs 3 and 4). This differs from their closer relationship based on complete cox1 gene (S3 Fig)  and 15 mt-genes (Fig 2). A complete cox1 gene (or 13 PCGs or 15 mt-genes) instead of partial sequence is therefore more appropriate for determining phylogenetic relationship.
At the higher-level phylogeny, the phylogenetic analysis indicated that Anastrepha fraterculus (Tribe Toxotrypanini) of subfamily Trypetinae was grouped with Tribe Dacini of Dacinae, while Procecidochares utilis (Tribe Ceceidocharini) of subfamily Trypetinae was basal to the clade containing Dacinae and A. fraterculus (Fig 2). This discrepancy may be due to insufficient taxon sampling. A broader taxa sampling, particularly Trypetinae, is needed to better elucidate the higher-level phylogeny of the tribes and subfamilies of Tephritidae.
In summary, we have successfully sequenced the complete mitogenome of Z.tau from Malaysia and China and confirmed that they were conspecific. Based on partial cox1 sequences, the taxa from China, Japan, Laos, Malaysia, Bangladesh, India, Sri Lanka, and Z. tau sp. A from Thailand are conspecific and belong to Z. tau sensu stricto. The mitogenome will prove useful for studies on phylogenetics and systematics of fruit flies of the Z. tau species complex and other taxa of Tephritidae.