A review of over a decade of DNA barcoding in South Africa: a faunal perspective

For over a decade, molecular short standardised DNA fragments, termed DNA barcodes, have been developed for species discrimination around the world. As of 2010, the vast majority of barcoding research was biased toward particular taxonomic groups and geographic regions largely because researchers in developed countries were the ones with the resources and capacity to carry out such work. To rectify this, the International Barcode of Life Project was launched with the intent to extend the geographic and taxonomic coverage of the barcode reference library. South Africa committed to this mission in an attempt to catalogue all of its known biodiversity and, possibly, help identify new species. To date, approximately 48 000 South African faunal barcodes are housed in the Barcode of Life Data System (BOLD), which represent only 2.3% of all known South African animal species. Although insects are the best represented in absolute terms, with over 37 000 samples recorded, they are still grossly lacking with just over 1% representation. Much like the global trend, there is a general taxonomic bias, with fish, birds and mammals showing the greatest representation. Moreover, geographic bias is also present, with the Free State province particularly under-represented on BOLD, likely owing to limited human capacity. Although few studies have been published with respect to barcoding, the majority reveal that the cytochrome c oxidase 1 (CO1) gene, used in isolation or in conjunction with other molecular markers, can greatly benefit South African biodiversity research. Several limitations of DNA barcoding are discussed and recommendations specific to South Africa provided.     

Molecular identification of polydorid polychaetes (Annelida: Spionidae): is there a quick way to identify pest and alien species?

The early detection and correct identification of polydorid polychaete species is essential as they are often encountered as invasive alien pests in aquaculture facilities or the intertidal where they may modify the ecosystem. Accurate identification is, however, often hampered by high levels of morphological similarity among species. This taxon will therefore benefit from the development of a library of sequences, such as COI barcodes, to aid identification. However, the universal primers for the cytochrome c oxidase I (COI) DNA barcoding marker has failed to consistently amplify this gene for polydorids, greatly hampering the development of such a library. We describe the development of unique PCR primers for the COI gene that work across four genera and nine species of polydorids. We also compared its efficacy with sequence data for mitochondrial cytochrome b and nuclear 18S rRNA, and a concatenated dataset consisting of all three markers. The nuclear 18S rRNA gene showed the least variation both intra- (0.0–1.2%) and interspecifically (0.6–4.3%), and was the most accurate for species identifications among the three markers. Although COI was characterised by higher intraspecific variation compared with Cyt b (0.0–14.5% and 0.0–4.2%, respectively), Cyt b showed considerably higher levels of interspecific variation (16.6–30.2%) compared with COI (2.2–20.7%). Of the two mitochondrial DNA markers, COI was actually less accurate for species identifications, having suggested two species within Boccardia pseudonatrix that was not supported by the other markers. Overall, the concatenated dataset yielded the most consistent intraspecific groupings, suggesting that this is the most accurate means of identifying polydorids using DNA sequence data. Thus, there may not be a quick and easy way to identify these species accurately using only molecular data.     

国际草田耕作制度研究进展

豆科牧草与其他牧草或作物间套作或轮作是国际牧草耕作制度研发方向。发展有机、可持续的、绿色和生物多样性的农牧业是发达国家牧草耕作制度的基本政策,保护当地植物和生物多样性是非洲国家牧草耕作制度发展前沿,将家畜养殖和农牧业生产相结合、集雨和地膜覆盖种植牧草和新技术应用是亚洲部分国家牧草耕作制度研发方向。     

利用DNA条形编码探讨柞蚕饰腹寄蝇的分类学地位

柞蚕饰腹寄蝇(Blepharipa tibialis Chao)是柞蚕的主要寄生性害虫之一。测定了柞蚕饰腹寄蝇的线粒体细胞色素酶C亚基I(COI)基因5′端的部分片段(657bp,GenBank登录号EU433559),并利用该DNA条形编码探讨了其分类学地位。将GenBank数据库登载的寄蝇科11个属的15个代表种的序列组成数据集,基于Kimura-2-Parameter计算了序列间的遗传距离,采用邻近距离法(Neighbour-Joining)构建了系统树。Blast检索表明这段序列可以作为DNA条形编码用于柞蚕饰腹寄蝇的鉴定。柞蚕饰腹寄蝇与饰腹寄蝇属内5个种间的平均遗传距离(0.122)是饰腹寄蝇属内种间平均遗传距离(0.063)的2倍,而11个形态学属间的平均遗传距离(0.122)则与柞蚕饰腹寄蝇和这11个形态学属之间的平均遗传距离(0.124)基本一致;柞蚕饰腹寄蝇在构建的NJ树中也没有与饰腹寄蝇属的种类聚在一起。基于上述结果,建议将柞蚕饰腹寄蝇从饰腹寄蝇属划分出来,单独作为一个新的属。     

DNA barcoding for identification of cryptic species in the field and existing museum collections: a case study of Aethomys and Micaelamys (Rodentia: Muridae)

DNA barcoding has been proposed as a method for species identification. However, this method has been criticised for its over-reliance on a single mitochondrial gene. In this study, four mitochondrial gene regions and one nuclear gene region were used to investigate their different abilities to identify tissue associated with museum specimens of Aethomys chrysophilus, Aethomys ineptus and Micaelamys namaquensis. Aethomys chrysophilus and the more recently elevated A. ineptus are indistinguishable on morphological grounds; however, their ranges are largely parapatric with only one syntopic locality currently known. All of the mitochondrial gene regions were able to separate M. namaquensis from A. chrysophilus and A. ineptus, but they varied in their abilities to resolve differences between A. chrysophilus and A. ineptus. The sequence results identified a specimen from KwaZulu-Natal that was misclassified and should have been identified as A. ineptus. Seven specimens that had not been reclassified following the elevation of A. ineptus to species level were identified as A. ineptus. Individuals of A. chrysophilus from Malawi could not be classified as either A. chrysophilus or A. ineptus, and may be a hybrid or a new, distinct species. This study indicates that DNA barcoding may be used to separate M. namaquensis from A. chrysophilus and A. ineptus, and although it was not able to separate A. chrysophilus and A. ineptus, it did indicate specimens from Malawi may be a new cryptic species.     

Molecular species identification and population genetics of chondrichthyans in South Africa: current challenges,priorities and progress

Molecular genetic techniques, such as DNA barcoding and genotyping, are increasingly being used to assist with the conservation and management of chondrichthyans worldwide. Southern Africa is a shark biodiversity hotspot, with a large number of endemic species. According to the IUCN Red List, a quarter of South Africa's chondrichthyans are threatened with extinction. South Africa's commercial shark fisheries have increased over the last decade and there is a need to ensure sustainable utilisation and conservation of this fisheries resource. Here, we give an overview of the molecular techniques that are used to assist in the identification of species, cryptic speciation and possible interspecific hybridisation, as well as the assessment of population structure and reproductive behaviour of endemic and commercially important chondrichthyan species of southern Africa. We discuss the potential application of these techniques for management and conservation of several species affected by South African fisheries. Acquiring baseline barcode data of all chondrichthyans in southern African waters and assessing the population structure of exploited species on a local and greater regional scale are recommended as research priorities. Future prospects should also include high-throughput molecular marker development and investigation of intraspecies functional variation using next generation sequencing technology.     

Phylogeography of Buthus Leach, 1815 (Scorpiones: Buthidae): a multigene molecular approach reveals a further complex evolutionary history in the Maghreb

The distribution of the scorpion genus Buthus Leach, 1815 includes southwestern Europe, North and Central Africa and extends east towards the Arabian Peninsula. Phylogenetic relationships within the genus are complex and remain partially unresolved despite several previous assessments. A set of three mitochondrial markers, 12s, 16s and CO1, revealed the presence of five well-supported clades: three clades endemic to Morocco, one clade distributed across the Maghreb region and southwestern Europe and one endemic to Tunisia and Algeria. Morocco presents high levels of endemism and appears to be the centre of diversity for the genus. Further differentiation was found within the clade distributed in Tunisia and Algeria, with the discovery of new phylogenetic patterns. In addition, a phylogeny combining all published CO1 data for the genus emphasized the ongoing complex situation regarding the genus’ taxonomy. Highly similar sequences were attributed to different species by different authors throughout the tree, and no differentiated monophyletic species could be resolved. This lack of congruence between morphologically defined species and genetic lineages is a major hindrance in the study of the highly complex genus Buthus.     

Single-strand conformation polymorphism (SSCP) analysis as a new diagnostic tool to distinguish dorsal-spined larvae of the Elaphostrongylinae (Nematoda: Protostrongylidae) from cervids

Single-strand conformation polymorphism (SSCP) was used to genetically differentiate morphologically indistinguishable first-stage larvae (L(1)) of the six species of elaphostrongyline nematodes. A partial fragment (317-336bp) of the first internal transcribed spacer (pITS-1) plus 5' flanking region (76bp of the 18S gene) of the nuclear ribosomal DNA (rDNA) was amplified from individual L(1) of known identity and subjected to SSCP. The results showed that the four species of elaphostrongylines found in North American cervids, Parelaphostrongylus tenuis, P. andersoni, P. odocoilei and Elaphostrongylus rangiferi, could be distinguished from one another based on their distinct (i.e. species-specific) SSCP profiles. In addition, E. alces, a species that occurs in moose in Fennoscandinavia, also had a distinct SSCP profile with respect to the other species of elaphostrongylines. However, the SSCP profiles of E. cervi could not be distinguished from those of E. rangiferi because of a lack of interspecific sequence differences in this region of the ITS-1. The distinct SSCP profiles for the other species were consistent with the interspecific differences in ITS-1 sequences, which ranged from 2 (between P. tenuis and P. andersoni) to 59bp (between genera). The pITS-1 SSCP approach was also used to identify unknown elaphostrongyline L(1) from different hosts and localities in North America. The ability to distinguish between L(1) of the four elaphostrongyline species that occur in North American cervids has important diagnostic and epidemiological implications.     

The sclerodactylid holothurians of southern Africa,with the erection of one new subfamily and two new genera (Echinodermata: Holothuroidea)

The sclerodactylid subfamily Sclerodactylinae is restricted to include only those forms with a short, compact, tubular calcareous ring and a new subfamily Sclerothyoninae is diagnosed to accommodate two new southern African monotypic genera Sclerothyone and Temparena, with compact but non-tubular calcareous rings. The new genera are erected respectively for Ludwig and Heding's (1935) Cucumaria? velligera and C.l chuni, formerly classified in Pentamera. This brings the total number of sclerodactylid holothurians known from southern Africa to six genera and seven species. All taxa are diagnosed and/or described, a key is provided for all southern African species and their local distributions are mapped.     

Identification of cryptic species of Culicoides (Diptera: Ceratopogonidae) in the subgenus Culicoides and development of species-specific PCR assays based on barcode regions

Culicoides biting midges (Diptera: Ceratopogonidae) are vectors of important diseases affecting wild and domestic animals. During the last decade they have played a major role in the epidemiology of the largest bluetongue epizootic ever recorded in Europe, the disease is transmitted between hosts almost exclusively by bites of Culicoides midges and affects both domestic and wild ruminants however severe disease usually occurs in certain breeds of sheep and some species of deer. An accurate vector identification is of major importance in arthropod borne diseases surveillance, as great differences in vectorial capacity are found even between close species. Unfortunately, specialized taxonomic knowledge of Culicoides identification is rarely available in routine surveillance, mainly based on wing morphology. Recently, some European species of Culicoides belonging to the subgenus Avaritia Fox, 1955 and Culicoides Latreille, 1809 have been described as new bluetongue virus vectors.In the present study, by using a fragment of the barcode region (COI gene) we report the presence of up to 11 species within the subgenus Culicoides in Catalonia (NE Spain), a region recently affected by a bluetongue epizootic. The molecular analysis revealed new non-described cryptic species which were grouped in three complexes of morphologically similar species, two in the Pulicaris complex resembling Culicoides pulicaris, two in the Fagineus complex resembling Culicoides fagineus and three in the Newsteadi complex resembling Culicoides newsteadi. The phylogenetic relationships among them showed that cryptic species detected in both Pulicaris and Fagineus complexes were closely related, whereas those in the Newsteadi complex were more distant. Accurate analysis of all species using morphological and molecular approaches resulted in the detection of diagnostic metric traits for cryptic species and the design of several new species-specific single and multiplex PCR assays to identify unambiguously all the species, most of them still lacking a specific molecular diagnosis.     

A morphological analysis of the skull size and shape of Kerivoulinae (Chiroptera: Vespertilionidae) from Vietnam

Southeast Asia is a region of high biodiversity, containing species of plants and animals that are yet to be discovered. In this region, bats of the subfamily Kerivoulinae are widespread and diverse with six species recorded in Vietnam. However, the taxonomy of the Kerivoulinae in Asia is complicated. In our study, we used diagnostic characters and multivariate analysis to determine morphological differences between the genera Kerivoula and Phoniscus. We showed that the two genera are distinguishable by the size of second upper incisors, the shape of skull, nasal sinus, canines, second upper and lower premolars. In addition, the two genera can be osteometrically separated by measurements of the braincase height, interorbital width and shape of anterior palatal emargination. Our data clearly revealed the morphological variations in the skull shape of Kerivoula hardwickii in Vietnam. This suggests a possible separation into three morphotypes, representing cryptic species supported by statistical differences with wide variation in skull shape, size and teeth. These results demonstrated Kerivoula hardwickii can be separated three subspecies, and the result will serve as the basis for the future assessment and classification of this group in Southeast Asia.     

First record of freshwater fish on the Cape Verdean archipelago

The Cape Verdean islands form a distinct aquatic freshwater ecoregion characterized mainly by temporal water bodies with an adapted invertebrate community. Freshwater fish were not previously recorded from the archipelago. During a non-exhaustive survey of freshwater bodies on five islands of the archipelago, the first presence of a freshwater fish was recorded. Using barcoding sequences, the species was identified as the guppy (Poecilia reticulata), a highly invasive species alien to the Cape Verdean Islands.     

Comparative Morphology of Three Native Lernaeids (Copepoda: Cyclopoida) from Amazonian Fishes and Descriptions of Two New Genera

Abstract

Morphological studies were carried out on adult postmetamorphic females of three native South American genera of Lernaeidae (Crustacea: Copepoda: Cyclopoida) and these were compared with the imported species Lernaea cyprinacea. Two of the native species represent new genera, which are described as Amazolernaea sannerae gen. et sp. nov. and Bedsylernaea collaris gen. et sp. nov. The third native species had already been described as Perulernaea gamitanae Thatcher and Paredes, 1985. Amazolernaea sannerae from the cichlids Cichla monoculus and speckled pavon C. temensis was characterized as having four similar petal-like anchors, a long slender neck, thoracopods 1 and 2 close together, and a genital pore located equatorially in the well-developed hindbody. Bedsylernaea collaris differs from all known lernaeids in having a large inflated head region with one bulbous anchor on either side, a slender neck, a stout hindbody with a large pregenital prominence, a genital pore that is anterior to the midpoint on the hindbody, and collar-like extensions on the anterior extremity of the hindbody. Perulernaea gamitanae has two large and two small unbranched anchors, a long neck with prominent glands, and an elongate hindbody with a genital pore that is posterior to the midpoint. In contrast, all valid species of Lernaea have four frequently branching anchors, a neck region and hindbody that are not well defined, a short abdomen, and a genital pore at or near the posterior extremity.     

祁连山高山草地毒杂草侵入对蝗虫相对多度的影响

为研究祁连山高山草地毒杂草侵入对蝗虫群落组成、相对多度以及群落数量的影响,2008-2009年6-9月在野外调查基础上,对毒杂草(狼毒、甘肃臭草和披针叶黄华)侵入型草地和天然草地植被群落、土壤理化特性以及蝗虫多样性及群落数量进行调查分析。结果表明,毒杂草侵入显著降低草地物种丰富度、Shannon-Wiener指数和均匀度指数(P<0.05),植被群落高度和生物量明显增加(P<0.05);甘肃臭草侵入型草地土壤有机质和含水量最高,狼毒侵入型草地土壤紧实度和天然草地全N含量显著大于其他样地(P<0.05);毒杂草侵入影响蝗虫群落组成、降低物种多样性、改变物种相对多度。但蝗虫群落数量因杂草种类呈现较大差异,蝗虫群落数量因甘肃臭草侵入而降低、随狼毒和披针叶黄华侵入而增加。高山草地毒杂草侵入通过改变植被结构和营养价值、影响蝗虫土壤理化特性和栖息生境,而草地蝗虫群落组成、数量和多样性对此做出积极响应。该研究不仅为研究高山草地毒杂草型退化对昆虫以及无脊椎动物的影响提供参考,而且也为高山草地生物多样性维持和保育提供有益依据。     

Diet analysis of small mammal pests: A comparison of molecular and microhistological methods

Knowledge of what pest species are eating is important to determine their impact on stored food products and to plan management strategies accordingly. In this study, we investigated the food habits of 2 rodents, Rattus rattus (ship rat) and Mus musculus castaneus (house mouse) as well as an insectivore, Suncus murinus (shrew), present in human dwellings. Both a microhistological approach and a DNA barcoding approach were used in the present study. Following DNA extraction, amplification was performed using group‐specific primers targeting birds, plants and invertebrates. Resulting polymerase chain reaction products were sequenced and analyzed to identify the different prey species present in the gut contents. The findings from the application of both techniques were in agreement, but the detection of prey type with each technique was different. The DNA barcoding approach gave greater species‐level identification when compared to the microhistological method, especially for the invertebrate and avian prey. Overall, with both techniques, 23 prey taxa were identified in the gut contents of the 3 species, including 15 plants, 7 insects and a single bird species. We conclude that with a selection of suitable “barcode genes” and optimization of polymerase chain reaction protocols, DNA barcoding can provide more accurate and faster results. Prey detection from either technique alone can bias the dietary information. Hence, combining prey information of both microhistological analysis and DNA barcoding is recommended to study pest diet, especially if the pest is an omnivore or insectivore species.     

新疆草地药用及毒害植物的霜霉菌研究

对新疆草地药用植物及有毒有害植物的霜霉进行了系统的分类研究，采自新疆天山及平原绿洲的９７份草地药用及毒害植物霜霉菌标本，共鉴定出分别寄生于１５科３６属４４种寄生植物上的５属３４种霜霉菌，包括２个新种，７个全国新记录种有５个新寄主记录，其中寄生于２６属３４种草地药用植物上的霜霉菌５属２５种，寄生于１０属１１种草地毒害植物上的霜霉菌２属１０种，从生物多样性的保护和持续利用及生态系统的角度出发，在挖掘我     

甘肃多儿国家级自然保护区维管植物区系分析

基于野外实地考察、植物标本的采集与鉴定以及结合文献研究的方法,分析了多儿保护区维管植物区系的物种组成和分布区类型。结果表明,该区维管植物共115科469属1323种,包括蕨类植物14科25属61种,裸子植物4科8属25种,被子植物97科436属1237种。多儿保护区维管植物区系优势现象明显,≥20种的优势科共879种,占该区维管植物总种数的66.44%;小属和单种属成分比例较大,占该区维管植物总属数的95.31%,总种数的75.51%。科级水平上,热带科（30.43%）稍多于温带科（26.96%）;属级水平上,温带科（72.49%）明显高于热带科（13.01%）;种级水平上,中国特有种有545种,其中538种与周边省份所共有,体现了该区系明显的过渡性和交汇性。该保护区维管植物区系地理成分复杂多样,属的分布有15个分布型和5个变型,温带地理成分占绝对优势,尤其以北温带分布为主。该区珍稀濒危野生植物较多,被列入保护名录有37种;一些古老、孑遗植物在该保护区具有良好的原始类群分布。本研究进一步完善了多儿保护区维管植物区系特征,为以后了解该保护区生物多样性提供可靠依据。     

Partial sequence of the DNA-dependent DNA polymerase gene of fowl adenoviruses: a reference panel for a general diagnostic PCR in poultry

Adenoviruses are frequent infectious agents in different poultry species. The traditional, serological typing of new isolates by virus neutralisation tests is now in transition to be replaced by PCR and sequencing. The first PCRs, recommended for the detection of adenoviruses, had been designed to target the gene of the major capsid protein, the hexon. In birds, members of three different genera of the family Adenoviridae may occur. Accordingly, three specific hexon PCRs had to be elaborated for the detection of adenoviruses in poultry. A significantly more sensitive PCR, targeting the viral DNA-dependent DNA polymerase gene, has been described recently. This method proved to be an efficient alternative for the general detection of adenoviruses irrespective of their genus affiliation. Fowl adenoviruses (FAdVs), isolated from chicken to date, comprise twelve serotypes classified into five virus species (FAdV-A to E). The polymerase gene sequence has been determined yet only from three FAdV types representing three species. In the present work, the panel of polymerase gene sequences was completed with those of the rest of FAdVs. The newly determined sequences will facilitate the identification of new FAdV isolates as an existing species or as a putative new FAdV. Once the polymerase sequence is known, more specific PCRs for the amplification of the hexon and other genes can be designed and performed according to the preliminary species classification.     

Mixed‐method study on medicinal herb collection in relation to wildlife conservation: the case of giant pandas in China

Medicinal herb collection has historical and cultural roots in many rural communities in developing countries. Areas where herb collection occurs may overlap with biodiversity hotspots and crucial habitat of endangered and threatened species. However, impacts of such practices on wildlife are unknown and possibly underestimated, perhaps due to the elusive nature of such activities. We examined this phenomenon in Wolong Nature Reserve, China, a protected area in the South‐Central China biodiversity hotspot that also supports a community of Tibetan, Qiang and Han people who use herb collection as a supplementary source of livelihood. We adopted a participatory approach in which we engaged local people in outlining spatial and temporal dynamics of medicinal herb collection practices. We found that the overall spatial extent of herb collection increased in the past two decades. We then overlaid herb collection maps with localities of giant panda (Ailuropoda melanoleuca) feces collected over two time points in the reserve. Using a Bayesian parameter estimation, we found evidence for declined giant panda occurrence in the areas most recently impacted by emerging medicinal herb collection. Our methodology demonstrates the potential power of integrating participatory approaches with quantitative methods for processes like herb collection that may be difficult to examine empirically. We discuss future directions for improving explanatory power and addressing uncertainty in this type of mixed‐method, interdisciplinary research. This work has implications for future attempts to understand whether and how prevalent but subtle human activities may affect wildlife conservation.