Phylogenetic relationships among Bactrocera species (Diptera: Tephritidae) inferred from mitochondrial DNA sequences

Several members of the dipteran family Tephritdae are serious pests because females lay eggs in ripening fruit. The genus Bactrocera is one of the largest within the family with over 500 described species arranged in 28 subgenera. The phylogenetic relationships among the various species and subgenera, and the monophyly of specific groups have not been examined using a rigorous phylogenetic analysis. Therefore, phylogenetic relationships among 24 Bactrocera species belonging to 9 subgenera were inferred from DNA sequence of portions of the mitochondrial 16S rRNA, cytochrome oxidase II, tRNA(Lys), and tRNA(Asp) genes. Two morphological characters that traditionally have been used to define the four groups within the subgenus Bactrocera were evaluated in a phylogenetic context by mapping the character states onto the parsimony tree. In addition, the evolutionary trend in male-lure response was evaluated in a phylogenetic context. Maximum parsimony analyses suggested the following relationships: (1) the genus Bactrocera is monophyletic, (2) the subgenus B. (Zeugodacus) is paraphyletic, (3) the subgenus B. (Daculus) is a sister group to subgenus B. (Bactrocera), and (4) the subgenus B. (Bactrocera) is monophyletic. The mapping analyses suggested that the morphological characters exhibit a simple evolutionary transition from one character state to another. Male-lure response was identified as being a labile behavior that has been lost on multiple occasions. Cue-lure response was plesiomorphic to methyl-eugenol response, and the latter has evolved independently within the Bactrocera and Zeugodacus groups of subgenera. The implications of our results for devising a coherent, consolidated classification for Bactrocera is discussed.     

福州实蝇监测初报

自2005年5月29日至2006年5月28日在福州金山福建农林大学校园内进行了实蝇监测。采用甲基丁香酚(M e)、诱蝇酮(Cue)和地中海实蝇诱芯(T),诱集到橘小实蝇[Bactrocera(Bactrocera)dorsalis]、瓜实蝇[B.(Zeugodacus)cucurbi-tae]、南瓜实蝇[B.(Zeugodacus)tau]和具条实蝇(B.scutellata)4种实蝇,但未诱到地中海实蝇(Ceratitis capitata)。其中,橘小实蝇诱集量最大,8月中下旬达到高峰。本研究为掌握福州地区实蝇发生动态提供了基础资料。     

Multigene Phylogeography of Bactrocera caudata (Insecta: Tephritidae): Distinct Genetic Lineages in Northern and Southern Hemispheres

Bactrocera caudata is a pest of pumpkin flower. Specimens of B. caudata from the northern hemisphere (mainland Asia) and southern hemisphere (Indonesia) were analysed using the partial DNA sequences of the nuclear 28S rRNA and internal transcribed spacer region 2 (ITS-2) genes, and the mitochondrial cytochrome c oxidase subunit I (COI), cytochrome c oxidase subunit II (COII) and 16S rRNA genes. The COI, COII, 16S rDNA and concatenated COI+COII+16S and COI+COII+16S+28S+ITS-2 nucleotide sequences revealed that B. caudata from the northern hemisphere (Peninsular Malaysia, East Malaysia, Thailand) was distinctly different from the southern hemisphere (Indonesia: Java, Bali and Lombok), without common haplotype between them. Phylogenetic analysis revealed two distinct clades (northern and southern hemispheres), indicating distinct genetic lineage. The uncorrected ‘p’ distance for the concatenated COI+COII+16S nucleotide sequences between the taxa from the northern and southern hemispheres (‘p’ = 4.46-4.94%) was several folds higher than the ‘p’ distance for the taxa in the northern hemisphere (‘p’ = 0.00-0.77%) and the southern hemisphere (‘p’ = 0.00%). This distinct difference was also reflected by concatenated COI+COII+16S+28S+ITS-2 nucleotide sequences with an uncorrected ''p'' distance of 2.34-2.69% between the taxa of northern and southern hemispheres. In accordance with the type locality the Indonesian taxa belong to the nominal species. Thus the taxa from the northern hemisphere, if they were to constitute a cryptic species of the B. caudata species complex based on molecular data, need to be formally described as a new species. The Thailand and Malaysian B. caudata populations in the northern hemisphere showed distinct genetic structure and phylogeographic pattern.     

海南寡鬃实蝇亚科分类研究(双翅目: 实蝇科)

根据2000～2004年间海南省实蝇监测和野外调查的部分研究结果,确认海南寡鬃实蝇亚科Dacinae现知下列14种：桔小实蝇 Bactrocera (Bactrocera) dorsalis (Hendel), 辣椒果实蝇 B. (B.) latifrons (Hendel), 锈红果实蝇B. (B.) rubigina (Wang et Zhao), 五指山果实蝇 B. (B.) wuzhishana Lin et Yang, sp. nov., 何氏华实蝇B. (Sinodacus) hochii (Zia), 短条果实蝇 B. (Zeugodacus) abbreviata (Hardy), 普通果实蝇 B. (Z.) caudata (Fabricius), 两带果实蝇B. (Z.) cilifera (Hendel), 瓜实蝇 B. (Z.) cucurbitae (Coquillett), 黑颜果实蝇 B. (Z.) diaphora (Hendel), 宽带果实蝇 B. (Z.) scutellata (Hendel), 南亚果实蝇 B. (Z.) tau (Walker), 海口棍腹实蝇 Dacus (Callantra) haikouensis Wang et Chen, 海南寡鬃实蝇 Dacus (Dacus) hainanus Wang et Zhao。五指山果实蝇 B. (B.) wuzhishana Lin et Yang, sp. nov. 区别于果实蝇属内其他种的鉴别特征如下：1）颜具2个黑色大斑点;2）中胸盾片的缝后黄色侧条短而窄,后端约与缝后翅上鬃处于同一水平;3）翅前缘带较窄,末端稍加宽;4）足的股节除中股节基部的1/2和后股节基部的3/5呈黄色外,余全黑色;腹部完全黑色。除附新种特征图外,还提供海南寡鬃实蝇亚科的种类修订名录和分种检索表。     

基于mtDNA Cytb 的六种果实蝇的分子鉴定

本研究首次对果实蝇属的桔小实蝇Bactrocera dorsalis、瓜实蝇B. cucurbitae、南瓜实蝇B. tau、番石榴实蝇B. correcta、具条实蝇B. scutellata、黑漆实蝇B. scutellaris等6种实蝇mtDNA Cytb基因进行了测序。对这6种实蝇72个个体mtDNA Cytb基因中段420 bp的碱基序列进行分析,得到38种单倍型,发现了116个变异位点,其中30个位点较为稳定。对这6种实蝇与其各自鉴别位点的对应关系研究表明,mtDNA Cytb基因可以作为这6种实蝇种类鉴别的分子标记。     

云南边境地区果实蝇属种类DNA条形码鉴定

【目的】果实蝇属Bactrocera中有国际上重要的检疫性害虫, 基于形态的物种鉴定有一定的局限性。另一方面, 云南边境地区为东南亚地区实蝇入侵我国的重要通道。因此, 对该地区实蝇分子鉴定方法的研究对于该属物种的快速准确鉴定具有重要意义。本研究旨在探讨DNA条形码技术在果实蝇属物种鉴定中的有效性。【方法】使用线粒体基因COI和COII序列的通用引物对果实蝇属20个物种60份样品进行PCR扩增、测序和序列分析; 采取距离方法和建树方法评价2种序列的鉴别能力。【结果】COI和COII序列平均长度分别为682 bp和339 bp, 种内和种间遗传差异较大, 有较明显的遗传距离间隔（barcoding gap）, 鉴定成功率分别为91.2%和90.7%。另外, 分子系统树表明华实蝇亚属Sinodacus不是单系群。【结论】COI和COII序列均能够将绝大多数果实蝇属物种进行准确鉴别, 应用COI或COII序列进行果实蝇属物种鉴定具有一定的可行性。     

Barcoding Queensland Fruit Flies (Bactrocera tryoni): impediments and improvements

Identification of adult fruit flies primarily involves microscopic examination of diagnostic morphological characters, while immature stages, such as larvae, can be more problematic. One of the Australia’s most serious horticultural pests, the Queensland Fruit Fly (Bactrocera tryoni: Tephritidae), is of particular biosecurity/quarantine concern as the immature life stages occur within food produce and can be difficult to identify using morphological characteristics. DNA barcoding of the mitochondrial Cytochrome Oxidase I (COI) gene could be employed to increase the accuracy of fruit fly species identifications. In our study, we tested the utility of standard DNA barcoding techniques and found them to be problematic for Queensland Fruit Flies, which (i) possess a nuclear copy (a numt pseudogene) of the barcoding region of COI that can be co‐amplified; and (ii) as in previous COI phylogenetic analyses closely related B. tryoni complex species appear polyphyletic. We found that the presence of a large deletion in the numt copy of COI allowed an alternative primer to be designed to only amplify the mitochondrial COI locus in tephritid fruit flies. Comparisons of alternative commonly utilized mitochondrial genes, Cytochrome Oxidase II and Cytochrome b, revealed a similar level of variation to COI; however, COI is the most informative for DNA barcoding, given the large number of sequences from other tephritid fruit fly species available for comparison. Adopting DNA barcoding for the identification of problematic fly specimens provides a powerful tool to distinguish serious quarantine fruit fly pests (Tephritidae) from endemic fly species of lesser concern.     

中国果实蝇属一新记录种及云南镞果实蝇亚属记述

Nine species in the subgenus Zeugodacus Hendel were known to occur in Yunnan. We add seven more species to the fauna of Yunnan. Among them, one species, Bactrocera (Zeugodacus) vultus Hardy, 1973, is reported from China for the first time. This species is redescribed in detail and its male genitalia are illustrated and described for the first time. A key to the species of the subgenus Zeugodacus from Yunnan is provided.     

First genetic characterization of the brown dog tick <Emphasis Type="Italic">Rhipicephalus sanguineus</Emphasis> sensu lato in Peninsular Malaysia

The brown dog tick Rhipicephalus sanguineus sensu lato (s.l.) is a species complex comprising three main mitochondrial lineages, namely tropical, temperate and southeast European lineages. Despite its medical and veterinary importance, little attention has been paid to the genetic lineage of this species in Southeast Asia. Rhipicephalus sanguineus s.l. from Malaysia was investigated genetically, for the first time, using the mitochondria-encoded cytochrome c oxidase subunit I (COI) and 16S ribosomal RNA (16S) genes. Specifically, a pair of primers was developed to amplify the COI sequences in the present study. Both genes unambiguously assigned Malaysian material into the tropical lineage of R. sanguineus s.l. The 16S sequences were highly conserved; no variation site was observed. The COI sequences revealed slightly higher variation by recovering four haplotypes, one of which is restricted to the northernmost of Peninsular Malaysia. This finding will be a stepping stone in promoting more biological studies of this species complex in this region.     

The molecular phylogenetic signature of Bali cattle revealed by maternal and paternal markers

Bali cattle is a domestic cattle breed that can be found in Malaysia. It is a domestic cattle that was purely derived from a domestication event in Banteng (Bos javanicus) around 3,500 BC in Indonesia. This research was conducted to portray the phylogenetic relationships of the Bali cattle with other cattle species in Malaysia based on maternal and paternal lineage. We analyzed the cytochrome c oxidase I (COI) mitochondrial gene and SRY of Y chromosome obtained from five species of the Bos genus (B. javanicus, Bos gaurus, Bos indicus, Bos taurus, and Bos grunniens). The water buffalo (Bubalus bubalis) was used as an outgroup. The phylogenetic relationships were observed by employing several algorithms: Neighbor-Joining (PAUP version 4.0), Maximum parsimony (PAUP version 4.0) and Bayesian inference (MrBayes 3.1). Results from the maternal data showed that the Bali cattle formed a monophyletic clade, and together with the B. gaurus clade formed a wild cattle clade. Results were supported by high bootstrap and posterior probability values together with genetic distance data. For the paternal lineage, the sequence variation is low (with parsimony informative characters: 2/660) resulting an unresolved Neighbor-Joining tree. However, Bali cattle and other domestic cattle appear in two monophyletic clades distinct from yak, gaur and selembu. This study expresses the potential of the COI gene in portraying the phylogenetic relationships between several Bos species which is important for conservation efforts especially in decision making since cattle is highly bred and hybrid breeds are often formed. Genetic conservation for this high quality beef cattle breed is important by maintaining its genetic characters to prevent extinction or even decreased the genetic quality.     

A molecular phylogeny for the Tribe Dacini (Diptera: Tephritidae): systematic and biogeographic implications

With well over 700 species, the Tribe Dacini is one of the most species-rich clades within the dipteran family Tephritidae, the true fruit flies. Nearly all Dacini belong to one of two very large genera, Dacus Fabricius and Bactrocera Macquart. The distribution of the genera overlap in or around the Indian subcontinent, but the greatest diversity of Dacus is in Africa and the greatest diversity of Bactrocera is in south-east Asia and the Pacific. The monophyly of these two genera has not been rigorously established, with previous phylogenies only including a small number of species and always heavily biased to one genus over the other. Moreover, the subgeneric taxonomy within both genera is complex and the monophyly of many subgenera has not been explicitly tested. Previous hypotheses about the biogeography of the Dacini based on morphological reviews and current distributions of taxa have invoked an out-of-India hypothesis; however this has not been tested in a phylogenetic framework. We attempted to resolve these issues with a dated, molecular phylogeny of 125 Dacini species generated using 16S, COI, COII and white eye genes. The phylogeny shows that Bactrocera is not monophyletic, but rather consists of two major clades: Bactrocera s.s. and the 'Zeugodacus group of subgenera' (a recognised, but informal taxonomic grouping of 15 Bactrocera subgenera). This 'Zeugodacus' clade is the sister group to Dacus, not Bactrocera and, based on current distributions, split from Dacus before that genus moved into Africa. We recommend that taxonomic consideration be given to raising Zeugodacus to genus level. Supportive of predictions following from the out-of-India hypothesis, the first common ancestor of the Dacini arose in the mid-Cretaceous approximately 80mya. Major divergence events occurred during the Indian rafting period and diversification of Bactrocera apparently did not begin until after India docked with Eurasia (50-35mya). In contrast, diversification in Dacus, at approximately 65mya, apparently began much earlier than predicted by the out-of-India hypothesis, suggesting that, if the Dacini arose on the Indian plate, then ancestral Dacus may have left the plate in the mid to late Cretaceous via the well documented India-Madagascar-Africa migration route. We conclude that the phylogeny does not disprove the predictions of an out-of-India hypothesis for the Dacini, although modification of the original hypothesis is required.     

Floral fragrances in two closely related fruit fly orchids,Bulbophyllum hortorum and B. macranthoides (Orchidaceae): assortments of phenylbutanoids to attract tephritid fruit fly males

Floral chemical components are important cues used by plants to attract pollinators. One outstanding case is “fruit fly orchids” in the genus of Bulbophyllum to attract their pollinators by releasing characteristic fragrances. Dacini fruit flies are main or exclusive pollinators which are strongly attracted to certain natural chemicals, either methyl eugenol (ME: a phenylpropanoid) or raspberry ketone (RK: a phenylbutanoid). Furthermore, zingerone (ZN: a phenylbutanoid) has been characterized as the attractant for both ME- and RK-sensitive fruit fly species. In the present study, we examined chemical profiles of two closely related Bulbophyllum orchids—B. hortorum, and B. macranthoides subsp. tollenoniferum—distributed in Papua New Guinea and the Southeast Asian countries, respectively. We first observed that RK-sensitive flies were attracted to these orchids by ex situ cultivation in Penang, Malaysia. These Bulbophyllum orchids contained RK and/or ZN as their main floral components. Other than these attractants, multiple phenylbutanoids including potential attractants for RK-sensitive species were identified from these orchids. Therefore, we examined attractiveness of potential phenylbutanoid attractants to an RK-sensitive melon fly, Zeugodacus cucurbitae, using laboratory-reared flies. Furthermore, we analyzed molecular phylogenetic relationships among phenylpropanoid- or phenylbutanoid-producing orchids to see a relation between chemical profiles and phylogenetic classification in the related species.     

A multi‐locus approach resolves the phylogenetic relationships of the Simulium asakoae and Simulium ceylonicum species groups in Malaysia: evidence for distinct evolutionary lineages

A multi‐locus approach was used to examine the DNA sequences of 10 nominal species of blackfly in the Simulium subgenus Gomphostilbia (Diptera: Simuliidae) in Malaysia. Molecular data were acquired from partial DNA sequences of the mitochondria‐encoded cytochrome c oxidase subunit I (COI), 12S rRNA and 16S rRNA genes, and the nuclear‐encoded 18S rRNA and 28S rRNA genes. No single gene, nor the concatenated gene set, resolved all species or all relationships. However, all morphologically established species were supported by at least one gene. The multi‐locus sequence analysis revealed two distinct evolutionary lineages, conforming to the morphotaxonomically recognized Simulium asakoae and Simulium ceylonicum species groups.     

Phylogenetic relationships within the genus Jesogammarus (Crustacea, Amphipoda, Anisogammaridae) deduced from mitochondrial COI and 12S sequences

The genus Jesogammarus contains 16 species in two subgenera, Jesogammarus and Annanogammarus. To examine relationships among species in the genus, a molecular phylogenetic study including eight species of the former subgenus and four of the latter was conducted using partial DNA sequences of the mitochondrial COI and 12S rRNA genes. MP, NJ, and ML trees based on the combined COI and 12S data indicated monophyly of the subgenus Annanogammarus, though the monophyly of Jesogammarus was left unresolved. Consistent with few morphological differences, Jesogammarus (A.) naritai and J. (A.) suwaensis showed low genetic differentiation and did not show reciprocal monophyly, which suggests a close affinity of these taxa.     

基于COI基因分析的双齿蚋分子分类研究

为了确定COI基因序列分析在蚋科中的分类地位,本研究测定了双齿蚋(Simulium bidentatum) COI基因序列,分析该基因序列特征;下载与其同源性较高的12个蚋种的COI基因序列15条,进行同源性分析和分子系统发育研究。结果显示,双齿蚋COI基因序列(DQ534946)与S. notiale、S. snowi、S. nyssa等蚋种的COI基因序列同源性最高,在贝叶斯分析所构建的分子系统进化树中,不同蚋组的序列各自聚成一枝,双齿蚋序列(DQ534946)未与其他任何蚋种聚为一枝。研究结果表明,双齿蚋COI基因序列与蚋亚属序列同源性较高,COI基因序列分析能较好建立蚋亚属内组间关系,双齿蚋未与其他蚋组序列聚为一枝,分类至淡足蚋组并不合适,支持分类至阿根蚋组。本研究进一步明确了COI基因序列分析在蚋科分子系统发育研究中的意义,并在一定程度上解决了双齿蚋在蚋科的分类争议。     

云南六种实蝇的RAPD快速鉴定

采用RAPD技术构建了果实蝇属（Bactrocera）的南瓜实蝇(B. tau)、黑漆实蝇(B.scutellaris)、具条实蝇(B. scutellata)、瓜实蝇(B. cucurbitae)、桔小实蝇(B. dorsalis)和番石榴实蝇(B. correcta)6种实蝇的指纹图谱.从131个引物中筛选出5个重复性好、多态性高的引物,共扩增出302条谱带,其中111条谱带具有遗传多态性.引物OPC-01、OPI-17、OPL-07、OPL-08以及OPL-16可以用于6种实蝇的分类鉴定.     

Molecular evolution of tephritid fruit flies in the genus Bactrocera based on the cytochrome oxidase I gene

Fruit flies of the genus Bactrocera (Diptera: Tephritidae) are one of the major economically important insects in Asia and Australia. Little attention has been given to analyses of molecular phylogenetic relationships among Bactrocera subgenera. By using mitochondrial cytochrome oxidase I gene (COI) sequences, the phylogenetic relationships among four subgenera, Asiadacus, Bactrocera, Hemigymnodacus, and Zeugodacus, were investigated. Nucleotide diversity within subgenera ranged from 11.7 to 12.4%, and the net divergence among subgenera ranged from 11.2 to 15.7%. Phylogenetic trees calculated from both maximum parsimony and neighbor-joining phylogenetic analysis methods were highly congruent in terms of tree topologies. Phylogenetic analysis of mitochondrial COI sequences suggests that tephritid fruit fly species, which attack cucurbit plants, that is, Asiadacus, Hemigymnodacus and Zeugodacus, were more closely related to each other than to fruit fly species of the subgenus Bactrocera, which attack plants of numerous families. Our data supports previous classification of Bactrocera based on morphological characters. However, the phylogenetic tree showed the polyphyletic of fruit flies in subgenus Zeugodacus. Possible causes of speciation among fruit flies species in this genus were also discussed.     

Differential abundance and core members of the bacterial community associated with wild male Zeugodacus cucurbitae fruit flies (Insecta: Tephritidae) from three geographical regions of Southeast Asia

Zeugodacus cucurbitae (Coquillet) is one of the most significant and widespread tephritid pest species of agricultural crops. This study reports the bacterial communities associated with Z. cucurbitae from three geographical regions in Southeast Asia (Thailand, Peninsular Malaysia, and Sarawak). The bacterial microbiota were investigated by targeted 16S rRNA gene (V3–V4 region) sequencing using the Illumina Mi-Seq platform. At 97% similarity and filtering at 0.001%, there were seven bacterial phyla and unassigned bacteria, comprising 11 classes, 23 orders, 39 families and 67 genera. The bacterial diversity and richness varied within and among the samples from the three geographical regions. Five phyla were detected for the Sarawak sample, and six each for the Thailand and Peninsular Malaysia samples. Four phyla—Actinobacteria, Bacteroidetes, Firmicutes, and Proteobacteria—were represented in all the fruit fly specimens, forming the core members of the bacterial community. Proteobacteria was the predominant phylum, followed by Bacteroidetes, Firmicutes, and Actinobacteria. Fifty-three genera were represented in the Thailand sample, 56 in the Peninsular Malaysia sample, and 55 in the Sarawak sample. Forty-two genera were present in all the three geographical regions. The predominant core members were order Enterobacteriales (Proeteobacteria), and family Enterobacteriaceae (Enterobacteriales). Klebsiella (Enterobacteriaceae) was the predominant genus and K. oxytoca the predominant species with all specimens having?>?10% relative abundance. The results indicate the presence of a great diversity as well as core members of the bacterial community associated with different populations of Z. cucurbitae.

     

A real‐time PCR toolbox for accurate identification of invasive fruit fly species

Significant plant pests such as fruit flies that travel with fresh produce between countries as eggs or larvae pose a great economic threat to the agriculture and fruit industry worldwide. Time‐limited and expensive quarantine decisions require accurate identification of such pests. Immature stages are often impossible to identify, making them a serious concern for biosecurity agencies. Use of COI barcoding PCR, often the only molecular identification resource, is time‐consuming. We assess the suitability of the COI barcoding region for real‐time PCR assays to identify four pest fruit fly species (Family: Tephritidae), in a diagnostic framework. These species, namely Mediterranean fruit fly (Ceratitis capitata), Queensland fruit fly (Bactrocera tryoni), African invader fly (Bactrocera invadens) and Island fly (Dirioxa pornia) each provide a different set of genetic species delimitation problems. We discuss the benefits and limitations of using a single‐gene TaqMan^? real‐time approach for such species. Our results indicate that COI‐based TaqMan^? real‐time PCR assays, in particular for genetically distinct species, provide an accurate, sensitive and rapid diagnostic tool.     

Anadromy and heterogenous population of a tropical shad Tenualosa ilisha in Malaysia,as revealed by otolith microchemistry and molecular evidence

Tenualosa ilisha was found recently in the Perak River in western Peninsular Malaysia. Molecular phylogenetic and haplotype network analyses suggest that T. ilisha has two genetically distinct populations/groups: (i) Peninsular Malaysia (Malaysia population), and (ii) Peninsular Malaysia, Thailand, India and Bangladesh (Indian Ocean population). The results also suggest that the T ilisha population in Peninsular Malaysia is genetically heterogeneous with a typical anadromous migration pattern.