Allozymatic diagnosis of four economically important Liriomyza species (Diptera, Agromyzidae)

Liriomyza bryoniae, L. huidobrensis, L. sativae and L. trifolii are leafminers of great economic importance; morphologically they are difficult to separate, many being inseparable at the pre-adult stages. Horizontal starch gel electrophoresis of larvae, pupae and adults was carried out in order to search for diagnostic allozymic characters. Analysis of the variation patterns at 15 genetic loci reveals that each species can be identified at all three stages of the life cycle. A biochemical key is presented.     

Identification of forensically important blow fly species (Diptera: Calliphoridae) in China by mitochondrial cytochrome oxidase I gene differentiation

Abstract Unambiguous and rapid sarcosaphagous insect species identification is an essential requirement for forensic investigations. Although some insect species are difficult to classify morphologically, they can be effectively identified using molecular methods based on similarity with abundant authenticated reference DNA sequences in local databases. However, local databases are still relatively incomplete in China because of the large land area with distinct regional conditions. In this study, 75 forensically important blow flies were collected from 23 locations in 16 Chinese provinces, and a 278‐bp segment of the cytochrome oxidase subunit I gene of all specimens was successfully sequenced. Phylogenetic analysis of the sequenced segments showed that all Calliphorid specimens were properly assigned into nine species with relatively strong supporting values, thus indicating that the 278‐bp cytochrome oxidase subunit one region is suitable for identification of Calliphorid species. The clear difference between intraspecific threshold and interspecific divergence confirmed the potential of this region for Calliphorid species identification, especially for distinguishing between morphologically similar species. Intraspecific geographic variations were observed in Lucilia sericata (Meigen, 1826) and Lucilia caesar (Linnaeus, 1758).     

The complete mitochondrial genome of the leafminer Liriomyza sativae (Diptera: Agromyzidae): great difference in the A+T-rich region compared to Liriomyza trifolii

The complete mitochondrial genome sequence of Liriomyza sativae Blanchard (15,551 bp) was determined and analyzed in this study. The circular genome contained 37 genes including 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes and an A + T-rich region. The initiation codons of COI and ND1 were ‘ATCA’ and ‘GTG’, respectively. ND2 gene used the truncated termination codon ‘T’. All the tRNA genes had the typical cloverleaf secondary structures except for tRNA^Ser(AGN) gene, which was found with the absence of a DHU arm. In addition, a tRNA-like secondary structure (tRNA^Met) was found in the A + T-rich region. The great difference was that the length of L. sativae A + T-rich region was 597 bp shorter than that of Liriomyza trifolii (Burgess). Meanwhile, some minor differences such as ‘TATA’ block were also observed in L. sativae in contrast to ‘TACA’ block in L. trifolii. There were also some essential structure elements such as ‘TATA’ block, ‘G(A)_nT’ block, poly-T stretch and stem-and-loop structure in the A + T-rich region of L. sativae mitochondrial genome.     

Influence of Liriomyza species (Diptera: Agromyzidae) and their host plants, on oviposition by Opius dissitus females (Hymenoptera: Braconidae)

Two factors, among others, influence the oviposition of Opius dissitus Muesebeck. The first was the host species, the second the plant upon which the pest host larvae develop. O. dissitus females prefer Liriomyza trifolii (Burgess) larvae over those of L. huidobrensis (Blanchard). O. dissitus females were more attracted to the larvae of L. trifolii or L. huidobrensis when these were present on courgette rather than tomato or lettuce.     

Brazilian populations of Drosophila maculifrons (Diptera: Drosophilidae): low diversity levels and signals of a population expansion after the Last Glacial Maximum

The Quaternary period was marked by considerable changes in climate. Such palaeoclimatic changes affected the population dynamics of many species, both in the Northern and in the Southern Hemisphere. However, the extent of these impacts on the demographic patterns of Neotropical species presenting different ecological requirements remains unclear. Drosophila maculifrons DUDA 1947 belongs to the guaramunu group of Drosophila and represents a potential indicator of the genetic consequences caused by the climatic fluctuations of the Quaternary, because it seems to be sensitive to temperature and humidity shifts. The aim of this study was to evaluate the evolutionary processes subjacent to the patterns of intraspecific diversity and structure of different populations of D. maculifrons. In total, 152 individuals were collected in the south and south‐east Brazil. Phylogenetic and phylogeographical analyses were performed based on sequences of COI and COII mitochondrial genes. In general, the results pointed to Brazilian populations of D. maculifrons being extremely impoverished in terms of mitochondrial diversity and population structure, which could be explained by a recent population expansion event dated to approximately 12 000 years ago. In fact, with the assistance of species palaeo‐distribution modelling strategies, it was possible to infer that most of the sampled region did not present the D. maculifrons environmental suitability requirements at least during the period of the Last Glacial Maximum. © 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 112 , 55–66.     

Integrative taxonomy of the Clavus canalicularis species complex (Drilliidae,Conoidea, Gastropoda) with description of four new species

ABSTRACT

The conoidean family Drilliidae Olsson, 1964 is a species-rich lineage of marine gastropods, showing a high degree of diversification in comparison to other families of Conoidea. Despite intensive molecular phylogenetic studies during the last decade that have led to notable rearrangements of conoidean systematics, the genus- and species-level taxonomy of Drilliidae has not thus far been affected and remains entirely based on shell features. In the current study we revisit species delimitation in a morphological cluster of species from the Indo-Pacific referred to as the Clavus canalicularis complex, using an integrative taxonomy approach. The species in the complex possess robust thick-walled shells typically over 15?mm in height with sculpture of prominent rounded nodules located at the whorl’s shoulder, sometimes sharp and squamiform, or producing long spines. We find that in addition to five known species, the complex comprises four new species. These are described as Clavus brianmayi n. sp. (New Caledonia), Clavus davidgilmouri n. sp. (the Philippines), Clavus andreolbrichi n. sp. (Vanuatu and New Ireland) and Clavus kirkhammetti n. sp. (Madagascar). Clavus exasperatus (Reeve, 1843), which was previously considered widely distributed in Indo-Pacific, is shown to be confined to the western Indian Ocean.     

DNA barcoding of the genus Lepidion (Gadiformes: Moridae) with recognition of Lepidion eques as a junior synonym of Lepidion lepidion

DNA sequences of cytochrome c oxidase I gene (COI) from Lepidion spp. were employed to test the efficiency of species identification. A sample of 32 individuals from five Lepidion species was sequenced and combined with 26 sequences from other BOLD projects. As a result, 58 Lepidion DNA sequences of the COI gene belonging to eight of the nine recognized Lepidion species were analysed. Sequences were aligned and formed seven clades in a Bayesian phylogenetic tree, where Lepidion lepidion and Lepidion eques grouped jointly. The Kimura 2‐parameter genetic distances, among congeners were, on average, 4.28%, 16 times greater than among conspecifics (0.27%). The main diagnostic meristic data of Lepidion spp. were compiled and a detailed morphological revision of the congeneric species L. eques and L. lepidion was made. The eye diameter was significantly different between L. eques and L. lepidion (P < 0.001). The number of anal fin rays ranged from 45 to 51 in L. lepidion and from 47 to 54 in L. eques, but no significant differences were obtained in the mean values of this variable (P = 0.07). According to the morphological and genetic analyses, the results strongly suggest that the Mediterranean codling L. lepidion and the North Atlantic codling L. eques are conspecific, making L. eques a junior synonym of L. lepidion.     

Phylogeography of Simulium siamense Takaoka and Suzuki complex (Diptera: Simuliidae) in Thailand

The phylogeography of Simulium siamense complex was inferred from mitochondrial DNA sequences. A 586‐bp fragment of the cytochrome oxidase I was sequenced for 92 individuals from 13 populations throughout Thailand, representing five cytoforms (A, B, C, F and G). The cytoforms are not genetically different at the molecular level except for cytoform B, which is genetically distinct from the others. This might indicate that cytoform B is a distinct species. Further morphological and molecular work using other genes is needed to clarify this. Our results also argue for the need of integrated approach, both cytological and molecular studies to understanding biodiversity of black flies. The star‐like shape of the mtDNA genealogy is consistent with the sudden population expansion of the mismatch distribution analysis and large negative values of Fu's F_s and Tajima's D‐tests, indicating a population demographic expansion. The expansion time is estimated to be in the late Pleistocene (about 120 000 years ago). Therefore, the overall low level of genetic structure could be due to sharing a recent history. The ancestral haplotype was found in the mountainous area in northeastern Thailand, suggesting that this area could have been the refugium of the species complex during the Pleistocene glaciations. Our results are consistent with previous findings about population expansion in response to the Pleistocene climatic change, thus revealing the importance of this historical event in shaping the genetic structure and diversity of Southeast Asian mainland species.     

Evaluation of sequential sampling plans for estimation of leafmines density of Liriomyza sativae Blanchard (Diptera: Agromyzidae) in cucumber greenhouses

This study was conducted to develop sequential sampling plans to estimate leafmine density by Liriomyza sativae (Blanchard) at two fixed-precision levels in a cucumber greenhouse. The within-greenhouse spatial patterns of leafmines were aggregated. The slopes and intercepts of Taylor’s power law did not differ between years. A fixed-precision level sampling plan was developed using the parameters of Taylor’s power law generated from total number of leafmines in a cucumber leaf at two precision levels (D) of 0.1 and 0.25. The resulting sampling plans were tested with sequential bootstrap simulations (n = 500) using 10 independent data sets for validation. Bootstrap simulation within a wide range of densities demonstrated that actual D′ values at desired D = 0.25 averaged less than or equal to 0.25 in all cases. Even at the lowest density of leafmine (0.27 mine per leaf), the actual mean D′ was 0.24 at D = 0.25. This result shows that the sampling plan developed in this study is effective and reliable for estimating the mine densities in cucumber greenhouses.     

The feasibility of using the sterile insect technique against Liriomyza trifolii (Diptera: Agromyzidae) infesting greenhouse chrysanthemum

Liriomyza trifolii is a serious pest of chrysanthemum greenhouses and other crops around the world. The larvae feed within the leaves of the host plants and create serpentine mines. The Sterile Insect Technique (SIT) has been successfully used against some Dipteran species in the field, and it is especially efficient against isolated populations like those on islands. Therefore, SIT against pest populations in confined environments such as greenhouses should have high potential for success. The objectives of this study were to determine the optimum gamma‐irradiation dosage required for the sterilisation of L. trifolii adults, and to determine the quality (emergence percentage, flight ability, longevity, copulatory success, and sperm transfer) of the irradiated males compared with that of non‐irradiated (normal) males. We found that sterility (< 0.7 mine per female) was achieved with the dose of 170 ± 5% Gy. The copulatory success and sperm transfer during copulation of the sterile males were not significantly different from those of normal males. Moreover, the longevity, emergence percentage and flight ability of irradiated males were also not significantly different from normal males. The SIT experiments suggest that the release of sterile L. trifolii can significantly reduce the reproductive capacity of a wild leafminer population. Our studies indicate that sterilisation of L. trifolii flies is feasible and that sterile males are of high quality and competitive with normal males. Based on these data, research on the use of SIT against L. trifolii populations in greenhouses is ongoing.     

Redesign of PCR primers for mitochondrial cytochrome c oxidase subunit I for marine invertebrates and application in all‐taxa biotic surveys

DNA barcoding is a powerful tool for species detection, identification and discovery. Metazoan DNA barcoding is primarily based upon a specific region of the cytochrome c oxidase subunit I gene that is PCR amplified by primers HCO2198 and LCO1490 (‘Folmer primers’) designed by Folmer et al. (Molecular Marine Biology and Biotechnology, 3 , 1994, 294). Analysis of sequences published since 1994 has revealed mismatches in the Folmer primers to many metazoans. These sequences also show that an extremely high level of degeneracy would be necessary in updated Folmer primers to maintain broad taxonomic utility. In primers jgHCO2198 and jgLCO1490, we replaced most fully degenerated sites with inosine nucleotides that complement all four natural nucleotides and modified other sites to better match major marine invertebrate groups. The modified primers were used to amplify and sequence cytochrome c oxidase subunit I from 9105 specimens from Moorea, French Polynesia and San Francisco Bay, California, USA representing 23 phyla, 42 classes and 121 orders. The new primers, jgHCO2198 and jgLCO1490, are well suited for routine DNA barcoding, all‐taxon surveys and metazoan metagenomics.     

叶螨线粒体COI基因中央区段的PCR扩增

根据二点叶螨线粒体ＣＯＩ基因序列设计１对ＰＣＲ引物,对叶螨科不同种属的线粒体ＣＯＩ基因中央区段进行了ＰＣＲ扩增,结果表明该对引物能成功扩增叶螨科７属９种叶螨的约３４０ｂｐ的同源片段,由于叶螨ＤＮＡ序列资料非常有限,扩大引物的使用范围成为快速获得物特定基因序列的有效途径,研究结果使根据叶螨线粒体ＣＯＩ基因序列信息探索其系统演化成为可能,另外,还对模板ＤＮＡ分离方法进行了优化。     

Rapid polymerase chain reaction-restriction fragment length polymorphism method for discrimination of the two Atlantic cryptic deep-sea species of scabbardfish

The present investigation provides an efficient diagnostic method based on polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) analysis to discriminate between two cryptic species of scabbardfish, Aphanopus carbo and A. intermedius, with commercial relevance in several European fish markets. Two DNA fragments from the mtDNA, including control region and partial cytochrome oxidase subunit I genes of about 1100 bp and 700 bp, respectively, were isolated by PCR amplification. Digestion of the amplicon including the control region with HaeII and the amplicon including the COI gene with Sau3AI restriction enzymes allowed an unequivocal discrimination between the two scabbardfish species. This PCR–RFLP method allowed a clear and rapid discrimination of the trichiurid species studied.     

Incomplete estimates of genetic diversity within species: Implications for DNA barcoding

DNA barcoding has greatly accelerated the pace of specimen identification to the species level, as well as species delineation. Whereas the application of DNA barcoding to the matching of unknown specimens to known species is straightforward, its use for species delimitation is more controversial, as species discovery hinges critically on present levels of haplotype diversity, as well as patterning of standing genetic variation that exists within and between species. Typical sample sizes for molecular biodiversity assessment using DNA barcodes range from 5 to 10 individuals per species. However, required levels that are necessary to fully gauge haplotype variation at the species level are presumed to be strongly taxon‐specific. Importantly, little attention has been paid to determining appropriate specimen sample sizes that are necessary to reveal the majority of intraspecific haplotype variation within any one species. In this paper, we present a brief outline of the current literature and methods on intraspecific sample size estimation for the assessment of COI DNA barcode haplotype sampling completeness. The importance of adequate sample sizes for studies of molecular biodiversity is stressed, with application to a variety of metazoan taxa, through reviewing foundational statistical and population genetic models, with specific application to ray‐finned fishes (Chordata: Actinopterygii). Finally, promising avenues for further research in this area are highlighted.     

Resurrection of Rabdophaga salicivora Shinji (Diptera: Cecidomyiidae), a Japanese gall midge formerly misidentified as a North American species, Rabdophaga rigidae (Osten Sacken), with observations on the phylogenetic relationships of its populations in Japan and the Russian Far East

Intercontinental biotic connections between Eurasia and North America are common in many gall midge genera (Diptera: Cecidomyiidae), but only a few species have been recorded from both continents. In Japan, four gall midge species had been previously considered to be identical to North American species, but three of these cases have already been disproved. We examined the remaining species, Rabdophaga rigidae, which had been originally described from Japan as Rabdophaga salicivora in 1938, later recorded from the Russian Far East in 1967, and synonymized with a North American species, R. rigidae, in 1982. Morphological features and partial sequence data of the mtDNA cytochrome oxidase subunit I (COI) region suggested that the Japanese species is a distinct species and is identical to the species recorded from the Russian Far East. We therefore apply the original name, R. salicivora, to the Japanese and the Russian species. In addition, on the basis of a molecular phylogenetic analysis, we conclude that R. salicivora possibly came to the Japanese Archipelago through the Korean Peninsula and established itself first in the southern parts of Japan. Then, it expanded its distribution range to northern parts of Honshu, but could not reach Hokkaido, probably because of the Tsugaru Strait between Honshu and Hokkaido.     

Different phylogeographic patterns in two Japanese Silpha species (Coleoptera: Silphidae) affected by climatic gradients and topography

To reveal differences in phylogeographic patterns of flightless insect species occurring in different regions of Japan, we studied the phylogeography and demographic history of Silpha beetles occurring in cool-temperate habitats of two major islands, Honshu and Hokkaido, using sequences of the mitochondrial cytochrome oxidase subunit I (COI) gene. Honshu has a more mountainous topography, and cool-temperate habitats occur discontinuously, whereas Hokkaido, located to the north of Honshu, has more continuous cool-temperate habitats. A species endemic to Honshu, S. longicornis occurs on Honshu, whereas S. perforata occurs on Hokkaido and the East Asian continent. Our results indicate that the ancestors of S. longicornis colonized Honshu via a south-west route c . 0.7 Mya and the species has highly divergent populations in isolated mountainous areas of Honshu, whereas S. perforata colonized Hokkaido via a northern route less than 90 000 years ago and has less divergent geographic populations. During the last glacial period, S. perforata was probably restricted to refugia in southern Hokkaido and later expanded into northern Hokkaido, whereas S. longicornis populations existed in many isolated refugia, probably because of the complex topography of Honshu. Thus, our study demonstrates that, even between closely related species, interactions among biology, latitudinal climatic gradients and topography can produce different phylogeographic patterns.  © 2009 The Linnean Society of London, Biological Journal of the Linnean Society , 2009, 98 , 452–467.     

The origin of the Tibetan Mastiff and species identification of Canis based on mitochondrial cytochrome c oxidase subunit I (COI) gene and COI barcoding

DNA barcoding is an effective technique to identify species and analyze phylogenesis and evolution. However, research on and application of DNA barcoding in Canis have not been carried out. In this study, we analyzed two species of Canis, Canis lupus (n = 115) and Canis latrans (n = 4), using the cytochrome c oxidase subunit I (COI) gene (1545 bp) and COI barcoding (648 bp DNA sequence of the COI gene). The results showed that the COI gene, as the moderate variant sequence, applied to the analysis of the phylogenesis of Canis members, and COI barcoding applied to species identification of Canis members. Phylogenetic trees and networks showed that domestic dogs had four maternal origins (A to D) and that the Tibetan Mastiff originated from Clade A; this result supports the theory of an East Asian origin of domestic dogs. Clustering analysis and networking revealed the presence of a closer relative between the Tibetan Mastiff and the Old English sheepdog, Newfoundland, Rottweiler and Saint Bernard, which confirms that many well-known large breed dogs in the world, such as the Old English sheepdog, may have the same blood lineage as that of the Tibetan Mastiff.     

Revision of the lygaeid genus Nysius (Heteroptera: Lygaeidae: Orsillinae) of Japan,with description of a new species

Japanese species of the lygaeid–orsilline genus Nysius were revised, and five species including a new species, N. hidakai, were recognized. Male and female genitalia of all the species were illustrated, and a key to species of Japanese Nysius was provided. The DNA barcoding information of each species except N. expressus were recorded.     

Spatial expansion and population structure of the neotropical malaria vector, Anopheles darlingi (Diptera: Culicidae)

Extensive population structuring is known to occur in Anopheles darlingi , the primary malaria vector of the Neotropics. We analysed the phylogeographic structure of the species using the mitochondrial cytochrome oxidase I marker. Diversity is divided into six main population groups in South America: Colombia, central Amazonia, southern Brazil, south-eastern Brazil, and two groups in north-east Brazil. The ancestral distribution of the taxon is hypothesized to be central Amazonia, and there is evidence of expansion from this region during the late Pleistocene. The expansion was not a homogeneous front, however, with at least four subgroups being formed due to geographic barriers. As the species spread, populations became isolated from each other by the Amazon River and the coastal mountain ranges of south-eastern Brazil and the Andes. Analyses incorporating distances around these barriers suggest that the entire South American range of An. darlingi is at mutation–dispersal–drift equilibrium. Because the species is distributed throughout such a broad area, the limited dispersal across some landscape types promotes differentiation between otherwise proximate populations. Moreover, samples from the An. darlingi holotype location in Rio de Janeiro State are substantially derived from all other populations, implying that there may be additional genetic differences of epidemiological relevance. The results obtained contribute to our understanding of gene flow in this species and allow the formulation of human mosquito health protocols in light of the potential population differences in vector capacity or tolerance to control strategies.  © 2009 The Linnean Society of London, Biological Journal of the Linnean Society , 2009, 97 , 854–866.