Barcoding aphids (Hemiptera: Aphididae) of the Korean Peninsula: updating the global data set

DNA barcode (mitochondrial COI) sequences are provided for species identification of aphids from the Korean Peninsula. Most (98%) of the 154 species had distinct COI sequences (average 0.05% intraspecific pairwise divergence) relative to the degree of sequence divergence among species (average value 5.84%). For species in common with other regions, barcodes for Korean samples fell near or within known levels of variation. Based on these results, we conclude that DNA barcodes can provide an effective tool for identifying aphid species in such applications as pest management, monitoring and plant quarantine.     

Unexpected problem in aphid DNA barcoding by universal primers

DNA barcode (mitochondrial COI) sequences have allowed for species identification of aphids. In this study, we newly found a DNA barcoding problem in a part of the DNA sequences for Sitobion avenae. Five S. avenae individuals showed differences of, on average, 32.60% in the DNA sequences from other conspecific individuals, and a BLAST search revealed that the five sequences are similar to those of aphid parasitoids such as Aphidius, Ephedrus and Praon spp. (Hymenoptera: Braconidae). Based on these results, we concluded that the universal primers used in aphid DNA barcodes can amplify barcode sequences from parasitoid species within host aphids.     

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.     

Assessing Nuclear and Mitochondrial DNA Sequence Variation Within Steinernema (Rhabditida: Steinernematidae)

DNA sequence analysis was used to characterize the nuclear ribosomal DNA ITS1 region and a portion of the COII and 16S rDNA genes of the mitochondrial genome from Steinernema entomopathogenic nematodes. Nuclear ITS1 nucleotide divergence among seven Steinernema spp. ranged from 6 to 22%, and mtDNA divergence among five species ranged from 12 to 20%. No intraspecific variation was observed among three S. feltiae strains. Phylogenetic analysis of both nuclear and mitochondrial DNA sequences confirms the existing morphological relationships of several Steinernema species. Both the rDNA ITS1 and mtDNA sequences were useful for resolving relationships among Steinernema taxa.     

Establishment of a mitochondrial DNA sequence database for the identification of fish species commercially available in South Africa

The limitations intrinsic to morphology-based identification systems have created an urgent need for reliable genetic methods that enable the unequivocal recognition of fish species, particularly those that are prone to overexploitation and/or market substitution. The aim of this study was to develop a comprehensive reference library of DNA sequence data to allow the explicit identification of 53 commercially available fish species in South Africa, most of which were locally caught marine species. Sequences of approximately 655 base pairs were generated for all species from the cytochrome c oxidase I (COI) gene, the region widely adopted for DNA barcoding. Specimens of the genus Thunnus were examined in further detail, employing additional mitochondrial DNA control region sequencing. Cumulative analysis of the sequences from the COI region revealed mean conspecific, congeneric and confamilial Kimura 2-parameter distances of 0.10%, 4.58% and 15.43%, respectively. The results showed that the vast majority (98%) of fish species examined could be readily differentiated by their COI barcodes, but that supplementary control region sequencing was more useful for the discrimination of three Thunnus species. Additionally, the analysis of COI data raised the prospect that Thyrsites atun (snoek) could constitute a species pair. The present study has established the necessary genetic information to permit the unambiguous identification of 53 commonly marketed fish species in South Africa, the applications of which hold a plethora of benefits relating to ecology research, fisheries management and control of commercial practices.     

5-bromodeoxyuridine labeling of monomeric and catenated circular mitochondrial DNA in HeLa cells

Bromouracil labeling of the mitochondrial DNA in exponentially growing HeLa cells produces two hybrid mitochondrial DNA species, with density shifts of 41.9 and 54.0 mg/ml relative to unlabeled mitochondrial DNA, as well as heavy mitochondrial DNA, with a shift of 95.3 mg/ml. The two hybrid species result from the difference in thymine composition of the complementary strands of mitochondrial DNA. In addition, mitochondrial DNA with a density intermediate between the hybrid and unlabeled species was found. This quarter heavy mitochondrial DNA represents 25% (w/w) of the total DNA after eight hours of labeling, and forms two peaks with shifts of 20.6 and 27.0 mg/ml relative to unlabeled mitochondrial DNA. 70% (w/w) of the quarter heavy mitochondrial DNA is in catenated forms, while 30% (w/w) is monomeric. Degradation of the catenanes by shearing of purified quarter heavy mitochondrial DNA results in the appearance of hybrid and unlabeled mitochondrial DNA bands, demonstrating that the quarter heavy catenanes contain both hybrid and unlabeled submolecules. The implications of the structure of the quarter heavy catenanes on the mechanism of formation of catenanes are discussed.     

Molecular data support a rapid radiation of aphids in the Cretaceous and multiple origins of host alternation

Many aphids display a remarkably complex life cycle of host alternation, in which cyclical parthenogenesis is combined with the obligate use of two unrelated host plants. We used mitochondrial ribosomal DNA (partial 12S and 16S) sequences to reconstruct the phylogeny of aphids, to determine how many origins of host alternation and correlated major host-plant shifts have occurred. Our results agreed with previous morphological studies in that species clustered with good support at the level of tribes. There was little well-supported phylogenetic structure at levels deeper than tribes, however, except for the monophyly of two subfamilies, Aphidinae and Lachninae. We argue that aphids experienced a rapid radiation at the tribal level, after host shifting from gymnosperms to angiosperms. A rapid radiation is consistent with aphid fossils, which record the presence of few subfamilies in the late Cretaceous, but most extant tribes by the early Tertiary. Plant fossils also record host plants of aphid tribes diversifying during this time. A hypothesized mechanism by which host alternation has evolved (fundatrix specialization), coupled with the rapid radiation, implies that this life cycle may have originated as often as in the ancestor of each tribe that displays it. We also consider, however, an alternative hypothesis of fewer origins. The basal radiation of Aphididae was dated from molecular sequences to have occurred at approximately 80–150 Mya.     

基于三个分子标记的粉毛蚜亚科DNA条形码

【目的】粉毛蚜亚科昆虫是重要的林业害虫,但是由于蚜虫体型较小,形态特征趋于简化,可用于物种鉴定的有效特征非常有限,因此一般基于外部形态特征难以对蚜虫物种实现快速准确的鉴定。本研究获取该亚科2属10种的DNA条形码标准序列,解决部分物种的分类问题,同时比较了3种标记对粉毛蚜亚科（Pterocommatinae）物种快速鉴定的效率。【方法】基于蚜虫的线粒体细胞色素氧化酶C亚基I（cytochrome oxidase subunit I, COI）基因、细胞色素b（cytochrome b, Cytb）基因和蚜虫初级内共生菌Buchnera 6-磷酸葡萄糖酸脱氢酶（gluconate-6-phosphate dehydrogenase, gnd）基因,对2属10种共197号样品进行NJ分析、遗传距离的计算以及基于相似性的物种鉴定分析。【结果】与K-2P模型相比,基于p-distance模型计算得到的遗传距离更小,序列差异频次图上种内距离与种间距离的重叠区域也小于前者;COI序列的物种鉴定成功率最高。获取了粉毛蚜亚科近200条DNA条形码标准序列,并建立了基于3个标记的该亚科物种DNA条形码序列库。【结论】在粉毛蚜亚科DNA条形码研究中,p-distance模型要优于K-2P模型;COI序列具有最高的条形码分析效率;增毛卷粉毛蚜Plocamaphis assetacea可能为蜡卷粉毛蚜Plocamaphis flocculosa的同物异名。     

DNA barcoding of stylommatophoran land snails: a test of existing sequences

DNA barcoding has attracted attention because it is a potentially simple and universal method for taxonomic assignment. One anticipated problem in applying the method to stylommatophoran land snails is that they frequently exhibit extreme divergence of mitochondrial DNA sequences, sometimes reaching 30% within species. We therefore trialled the utility of barcodes in identifying land snails, by analysing the stylommatophoran cytochrome oxidase subunit I sequences from GenBank. Two alignments of 381 and 228 base pairs were used to determine potential error rates among a test data set of 97 or 127 species, respectively. Identification success rates using neighbour‐joining phylogenies were 92% for the longer sequence and 82% for the shorter sequence, indicating that a high degree of mitochondrial variation may actually be an advantage when using phylogeny‐based methods for barcoding. There was, however, a large overlap between intra‐ and interspecific variation, with assignment failure (per cent of samples not placed with correct species) particularly associated with a low degree of mitochondrial variation (Kimura 2‐parameter distance < 0.05) and a small GenBank sample size (< 25 per species). Thus, while the optimum intra/interspecific threshold value was 4%, this was associated with an overall error of 32% for the longer sequences and 44% for the shorter sequences. The high error rate necessitates that barcoding of land snails is a potentially useful method to discriminate species of land snail, but only when a baseline has first been established using conventional taxonomy and sample DNA sequences. There is no evidence for a barcoding gap, ruling out species discovery based on a threshold value alone.     

Comprehensive DNA barcodes for species identification and discovery of cryptic diversity in mayfly larvae from South Korea: Implications for freshwater ecosystem biomonitoring

DNA barcoding of aquatic macroinvertebrates holds much promise as a tool for taxonomic research and for providing baseline reference for phylogenetic analysis and aquatic ecosystem biomonitoring. We obtained 112 novel sequences of the barcode region of the mitochondrial DNA cytochrome c oxidase subunit I gene representing 11 families, 25 genera, and 43 species of mayfly (Insecta: Ephemeroptera) from South Korea. No species shared barcode sequences and all can be identified with barcodes with a possible exception of some species. Minimum levels of interspecific genetic distances ranged from 6.7 to 32.9% (mean: 23.7%), whereas average levels of intraspecific divergence was 3.7%. The latter value was inflated by the presence of very high divergences within some taxa. In fact, approximately 33.3% (15/45) of the species included two or more haplotype clusters showing greater than 5.0% sequence divergence and some values were as high as 32.9%. Many of the species with high intraspecific divergences are para‐ or polyphyletic and represent the possibility of species complexes. Our study suggests that type or topotype specimens should be sequenced to identify accurate barcoding clusters with morphological species concepts and also to determine the status of currently synonymized species.     

Introduced ants reduce interaction diversity in a multi‐species,ant–aphid mutualism

Mutualisms contribute in fundamental ways to the origin, maintenance and organization of biological diversity. Introduced species commonly participate in mutualisms, but how this phenomenon affects patterns of interactions among native mutualists remains incompletely understood. Here we examine how networks of interactions among aphid‐tending ants, ant‐tended aphids, and aphid‐attacking parasitoid wasps differ between 12 spatially paired riparian study sites with and without the introduced Argentine ant Linepithema humile in southern California. To resolve challenges in species identification, we used DNA barcoding to identify aphids and screen for parasitoid wasps (developing inside their aphid hosts) from 170 aphid aggregations sampled on arroyo willow Salix lasiolepis. Compared to uninvaded sites, invaded sites supported significantly fewer species of aphid‐tending ants and ant‐tended aphids. At invaded sites, for example, we found only two species of ant‐tended aphids, which were exclusively tended by L. humile, whereas at uninvaded sites we found 20 unique ant–aphid interactions involving eight species of ant‐tended aphids and nine species of aphid‐tending ants. Ant–aphid linkage density was thus significantly lower at invaded sites compared to uninvaded sites. We detected aphid parasitoids in 14% (28/198) of all aphid aggregations. Although the level of parasitism did not differ between invaded and uninvaded sites, more species of wasps were detected within uninvaded sites compared to invaded sites. These results provide a striking example of how the assimilation of introduced species into multi‐species mutualisms can reduce interaction diversity with potential consequences for species persistence.     

Development of primers to amplify mitochondrial DNA control region of Old World porcupines (subgenus Hystrix)

Eight primers were developed for the amplification of mitochondrial DNA control region of Old world porcupines (subgenus Hystrix). Successful amplifications of low‐quality DNA extracted from old (12 years old) and recent quills were performed, thus facilitating field sampling. Successful cross‐species amplifications were obtained for Hystrix africaeaustralis, H. cristata and H. indica. Length and structure of mitochondrial DNA control region were analysed and its usefulness as genetic marker for interspecific and population investigation was discussed.     

Real‐time PCR for identification of the soybean aphid,Aphis glycines Matsumura

The soybean aphid (Aphis glycines Matsumura) is an economically significant pest in North America, causing extensive damage to soybean crops through direct feeding damage and disease transmission. If unchecked, this pest could cause billions of dollars of damage to soybean crops. Identification of the soybean aphid can be difficult due to its small size, complex life cycle and morphological plasticity. Generally, an expert is required to identify a specimen. Additionally, identification of some life stages, such as eggs, is impossible. DNA barcoding has been successfully used to differentiate aphid species, including A. glycines, based on sequencing of a standardized gene region. Although this method represents an important step towards accurate identification, samples must still be sent to specialized facilities for analysis. Using existing DNA barcode sequences in the publically accessible Barcode of Life Data System (BOLD; www.boldsystems.org ), species‐specific differences were identified and used to develop a real‐time PCR assay to identify soybean aphids. This assay can be run on portable systems for rapid, accurate and simple identification in the field. The use of a non‐destructive DNA extraction protocol allows the original insect to be vouchered and therefore available for further study if necessary. This work represents an important step in soybean aphid management.     

中国蛛缘蝽科物种DNA条形码研究(英文)

【目的】本研究旨在探讨DNA条形码对中国蛛缘蝽科(半翅目：缘蝽总科)物种界定的适用性。【方法】对中国蛛缘蝽科13属23种207个样本的线粒体COI基因DNA条形码序列进行扩增,并扩增稻缘蝽属Leptocorisa 3个物种的31条内转录间隔区1(ITS-1)序列作为辅助标记。使用MEGA 11软件计算种间和种内遗传距离(Kimura 2-parameter, K2P);采用邻接法(neighbor-joining, NJ)进行物种聚类分析;利用中介邻接网络算法构建单倍型网络图。【结果】基于线粒体COI DNA条形码序列得出测试的中国蛛缘蝽科所有23个种的种内平均K2P距离在2%以下,种间K2P距离在0.98%~23.98%之间(平均17.50%)。多数物种彼此能够被较好地分开,且支持率较高。其中,中稻缘蝽Leptocorisa chinensis和大稻缘蝽L. oratoria共享部分COI单倍型,造成COI条形码无法区分二者,可通过ITS-1序列在单倍型网络分析中将二者区分。【结论】本研究得出的中国蛛缘蝽科中绝大部分物种的DNA条形码数据分析结果与基于形态特征的分类单元一致。然而,对于其中亲缘关系极近的物种,单靠线粒体数据尤其是COI条形码序列无法进行准确界定,需引入其他DNA序列或其他类型数据进行区分。     

Faecal genetic analysis to determine the presence and distribution of elusive carnivores: design and feasibility for the Iberian lynx

Noninvasive methods using genetic markers have been suggested as ways to overcome difficulties associated with documenting the presence of elusive species. We present and assess a novel, reliable and effective molecular genetic technique for the unequivocal genetic identification of faeces from the endangered Iberian lynx (Lynx pardinus). From mitochondrial DNA (mtDNA) cytochrome b and D-loop region sequences, we designed four species-specific primers (for products 130-161 bp long) that were considered to be likely to amplify degraded DNA. We compared two DNA extraction methods, various DNA amplification conditions and the robustness and specificity of the primer pairs with 87 lynx samples from 5 potentially different lynx populations and with 328 samples of other carnivore species. The utility of the identification technique was tested with faeces of different ages, with faeces from controlled field experiments, and with faeces collected from locales with possible lynx populations from throughout the state of Andalusia, Spain (8052 km2). Faecal mtDNA extraction was more efficient using PBS wash of the faeces instead of a faeces homogenate. Our assay increased from 92.6 to 99% efficiency with a second amplification and a reduction in template concentration to overcome polymerase chain reaction (PCR) inhibition. Our assay never produced false positives, and correctly identified all lynx faeces. Of 252 faeces samples of unknown species collected throughout Andalusia, 26.6% (from three different areas) were classified as Iberian lynx, 1.4% showed evidence of PCR inhibition and 1.2% were of uncertain origin. This method has proven to be a reliable technique that can be incorporated into large-scale surveys of Iberian lynx populations and exemplifies an approach that can easily be extended to other species.     

Improved COI barcoding primers for Southeast Asian perching birds (Aves: Passeriformes)

The All Birds Barcoding Initiative aims to assemble a DNA barcode database for all bird species, but the 648-bp 'barcoding' region of cytochrome c oxidase subunit I (COI) can be difficult to amplify in Southeast Asian perching birds (Aves: Passeriformes). Using COI sequences from complete mitochondrial genomes, we designed a primer pair that more reliably amplifies and sequences the COI barcoding region of Southeast Asian passerine birds. The 655-bp region amplified with these primers overlaps the COI region amplified with other barcoding primer pairs, enabling direct comparison of sequences with previously published DNA barcodes.     

Identifying gastropod spawn from DNA barcodes: possible but not yet practicable

Identifying life stages of species with complex life histories is problematic as species are often only known and/or described from a single stage. DNA barcoding has been touted as an important tool for linking life-history stages of the same species. To test the current efficacy of DNA barcodes for identifying unknown mollusk life stages, 24 marine gastropod egg capsules were collected off the Philippines in deep water and sequenced for partial fragments of the COI, 16S and 12S mitochondrial genes. Two egg capsules of known shallow-water Mediterranean species were used to calibrate the method. These sequences were compared to those available in GenBank and the Barcode of Life Database (BOLD). Using COI sequences alone, only a single Mediterranean egg capsule was identified to species, and a single Philippine egg capsule was identified tentatively to genus; all other COI sequences recovered matches between 76% and 90% with sequences from BOLD and GenBank. Similarity-based identification using all three markers confirmed the Mediterranean specimens' identifications. A phylogenetic approach was also implemented to confirm similarity-based identifications and provide a higher-taxonomic identification when species-level identifications were not possible. Comparison of available GenBank sequences to the diversity curve of a well-sampled coral reef habitat in New Caledonia highlights the poor taxonomic coverage achieved at present in existing genetic databases, emphasizing the need to develop DNA barcoding projects for megadiverse and often taxonomically challenging groups such as mollusks, to fully realize its potential as an identification and discovery tool.     

Character-based DNA barcoding allows discrimination of genera, species and populations in Odonata

DNA barcoding has become a promising means for identifying organisms of all life stages. Currently, phenetic approaches and tree-building methods have been used to define species boundaries and discover 'cryptic species'. However, a universal threshold of genetic distance values to distinguish taxonomic groups cannot be determined. As an alternative, DNA barcoding approaches can be 'character based', whereby species are identified through the presence or absence of discrete nucleotide substitutions (character states) within a DNA sequence. We demonstrate the potential of character-based DNA barcodes by analysing 833 odonate specimens from 103 localities belonging to 64 species. A total of 54 species and 22 genera could be discriminated reliably through unique combinations of character states within only one mitochondrial gene region (NADH dehydrogenase 1). Character-based DNA barcodes were further successfully established at a population level discriminating seven population-specific entities out of a total of 19 populations belonging to three species. Thus, for the first time, DNA barcodes have been found to identify entities below the species level that may constitute separate conservation units or even species units. Our findings suggest that character-based DNA barcoding can be a rapid and reliable means for (i) the assignment of unknown specimens to a taxonomic group, (ii) the exploration of diagnosability of conservation units, and (iii) complementing taxonomic identification systems.     

Sequence analysis of the mitochondrial DNA control region of ciscoes (genus Coregonus): taxonomic implications for the Great Lakes species flock

Sequence variation in the control region (D-loop) of the mitochondrial DNA (mtDNA) was examined to assess the genetic distinctiveness of the shortjaw cisco ( Coregonus zenithicus ). Individuals from within the Great Lakes Basin as well as inland lakes outside the basin were sampled. DNA fragments containing the entire D-loop were amplified by PCR from specimens of C. zenithicus and the related species C. artedi , C. hoyi , C. kiyi , and C. clupeaformis . DNA sequence analysis revealed high similarity within and among species and shared polymorphism for length variants. Based on this analysis, the shortjaw cisco is not genetically distinct from other cisco species.