The use of DNA barcodes to estimate phylogenetic diversity in forest communities of southern China

To elucidate potential ecological and evolutionary processes associated with the assembly of plant communities, there is now widespread use of estimates of phylogenetic diversity that are based on a variety of DNA barcode regions and phylogenetic construction methods. However, relatively few studies consider how estimates of phylogenetic diversity may be influenced by single DNA barcodes incorporated into a sequence matrix (conservative regions vs. hypervariable regions) and the use of a backbone family‐level phylogeny. Here, we use general linear mixed‐effects models to examine the influence of different combinations of core DNA barcodes (rbcL, matK, ITS, and ITS2) and phylogeny construction methods on a series of estimates of community phylogenetic diversity for two subtropical forest plots in Guangdong, southern China. We ask: (a) What are the relative influences of single DNA barcodes on estimates phylogenetic diversity metrics? and (b) What is the effect of using a backbone family‐level phylogeny to estimate topology‐based phylogenetic diversity metrics? The combination of more than one barcode (i.e., rbcL + matK + ITS) and the use of a backbone family‐level phylogeny provided the most parsimonious explanation of variation in estimates of phylogenetic diversity. The use of a backbone family‐level phylogeny showed a stronger effect on phylogenetic diversity metrics that are based on tree topology compared to those that are based on branch lengths. In addition, the variation in the estimates of phylogenetic diversity that was explained by the top‐rank models ranged from 0.1% to 31% and was dependent on the type of phylogenetic community structure metric. Our study underscores the importance of incorporating a multilocus DNA barcode and the use of a backbone family‐level phylogeny to infer phylogenetic diversity, where the type of DNA barcode employed and the phylogenetic construction method used can serve as a significant source of variation in estimates of phylogenetic community structure.     

DNA barcoding of flowering plants in Sumatra,Indonesia

The rapid conversion of Southeast Asian lowland rainforests into monocultures calls for the development of rapid methods for species identification to support ecological research and sustainable land‐use management. Here, we investigated the utilization of DNA barcodes for identifying flowering plants from Sumatra, Indonesia. A total of 1,207 matK barcodes (441 species) and 2,376 rbcL barcodes (750 species) were successfully generated. The barcode effectiveness is assessed using four approaches: (a) comparison between morphological and molecular identification results, (b) best‐close match analysis with TaxonDNA, (c) barcoding gap analysis, and (d) formation of monophyletic groups. Results show that rbcL has a much higher level of sequence recoverability than matK (95% and 66%). The comparison between morphological and molecular identifications revealed that matK and rbcL worked best assigning a plant specimen to the genus level. Estimates of identification success using best‐close match analysis showed that >70% of the investigated species were correctly identified when using single barcode. The use of two‐loci barcodes was able to increase the identification success up to 80%. The barcoding gap analysis revealed that neither matK nor rbcL succeeded to create a clear gap between the intraspecific and interspecific divergences. However, these two barcodes were able to discriminate at least 70% of the species from each other. Fifteen genera and twenty‐one species were found to be nonmonophyletic with both markers. The two‐loci barcodes were sufficient to reconstruct evolutionary relationships among the plant taxa in the study area that are congruent with the broadly accepted APG III phylogeny.     

DNA barcoding of invasive plants in China: A resource for identifying invasive plants

Invasive plants have aroused attention globally for causing ecological damage and having a negative impact on the economy and human health. However, it can be extremely challenging to rapidly and accurately identify invasive plants based on morphology because they are an assemblage of many different families and many plant materials lack sufficient diagnostic characteristics during border inspections. It is therefore urgent to evaluate candidate loci and build a reliable genetic library to prevent invasive plants from entering China. In this study, five common single markers (ITS, ITS2, matK, rbcL and trnH‐psbA) were evaluated using 634 species (including 469 invasive plant species in China, 10 new records to China, 16 potentially invasive plant species around the world but not introduced into China yet and 139 plant species native to China) based on three different methods. Our results indicated that ITS2 displayed largest intra‐ and interspecific divergence (1.72% and 91.46%). Based on NJ tree method, ITS2, ITS, matK, rbcL and trnH‐psbA provided 76.84%, 76.5%, 63.21%, 52.86% and 50.68% discrimination rates, respectively. The combination of ITS + matK performed best and provided 91.03% discriminatory power, followed by ITS2 + matK (85.78%). For identifying unknown individuals, ITS + matK had 100% correct identification rate based on our database, followed by ITS/ITS2 (both 93.33%) and ITS2 + matK (91.67%). Thus, we propose ITS/ITS2 + matK as the most suitable barcode for invasive plants in China. This study also demonstrated that DNA barcoding is an efficient tool for identifying invasive species.     

DNA barcoding of Orchidaceae in Korea

Species of Orchidaceae are under severe threat of extinction mainly due to overcollection and habitat destruction; accurate identification of orchid species is critical in conservation biology and sustainable utilization of orchids as plant resources. We examined 647 sequences of the cpDNA regions rbcL, matK, atpF‐atpH IGS, psbK‐psbI IGS and trnH‐psbA IGS from 89 orchid species (95 taxa) and four outgroup taxa to develop an efficient DNA barcode for Orchidaceae in Korea. The five cpDNA barcode regions were successfully amplified and sequenced for all chlorophyllous taxa, but the amplification and sequencing of the same regions in achlorophyllous taxa produced variable results. psbK‐psbI IGS showed the highest mean interspecific K2P distance (0.1192), followed by matK (0.0803), atpF‐atpH IGS (0.0648), trnH‐psbA IGS (0.0460) and rbcL (0.0248). The degree of species resolution for individual barcode regions ranged from 60.5% (rbcL) to 83.5% (trnH‐psbA IGS). The degree of species resolution was significantly enhanced in multiregion combinations of the five barcode regions. Of the 26 possible combinations of the five regions, six provided the highest degree of species resolution (98.8%). Among these, a combination of atpF‐atpH IGS, psbK‐psbI IGS and trnH‐psbA IGS, which comprises the least number of DNA regions, is the best option for barcoding of the Korean orchid species.     

DNA barcoding of Corydalis,the most taxonomically complicated genus of Papaveraceae

The genus Corydalis is recognized as one of the most taxonomically challenging plant taxa. It is mainly distributed in the Himalaya–Hengduan Mountains, a global biodiversity hotspot. To date, no effective solution for species discrimination and taxonomic assignment in Corydalis has been developed. In this study, five nuclear and chloroplast DNA regions, ITS, ITS2, matK, rbcL, and psbA‐trnH, were preliminarily assessed based on their ability to discriminate Corydalis to eliminate inefficient regions, and the three regions showing good performance (ITS, ITS2 and matK) were then evaluated in 131 samples representing 28 species of 11 sections of four subgenera in Corydalis using three analytical methods (NJ, ML, MP tree; K2P‐distance and BLAST). The results showed that the various approaches exhibit different species identification power and that BLAST shows the best performance among the tested approaches. A comparison of different barcodes indicated that among the single barcodes, ITS (65.2%) exhibited the highest identification success rate and that the combination of ITS + matK (69.6%) provided the highest species resolution among all single barcodes and their combinations. Three Pharmacopoeia‐recorded medicinal plants and their materia medica were identified successfully based on the ITS and ITS2 regions. In the phylogenetic analysis, the sections Thalictrifoliae, Sophorocapnos, Racemosae, Aulacostigma, and Corydalis formed well‐supported separate lineages. We thus hypothesize that the five sections should be classified as an independent subgenus and that the genus should be divided into three subgenera. In this study, DNA barcoding provided relatively high species discrimination power, indicating that it can be used for species discrimination in this taxonomically complicated genus and as a potential tool for the authentication of materia medica belonging to Corydalis.     

Evaluating the capacity of plant DNA barcodes to discriminate species of cotton (Gossypium: Malvaceae)

Although two plastid regions have been adopted as the standard markers for plant DNA barcoding, their limited resolution has provoked the consideration of other gene regions, especially in taxonomically diverse genera. The genus Gossypium (cotton) includes eight diploid genome groups (A–G, and K) and five allotetraploid species which are difficult to discriminate morphologically. In this study, we tested the effectiveness of three widely used markers (matK, rbcL, and ITS2) in the discrimination of 20 diploid and five tetraploid species of cotton. Sequences were analysed locus‐wise and in combinations to determine the most effective strategy for species identification. Sequence recovery was high, ranging from 92% to 100% with mean pairwise interspecific distance highest for ITS2 (3.68%) and lowest for rbcL (0.43%). At a 0.5% threshold, the combination of matK+ITS2 produced the greatest number of species clusters. Based on ‘best match’ analysis, the combination of matK+ITS2 was best, while based on ‘all species barcodes’ analysis, ITS2 gave the highest percentage of correct species identifications (98.93%). The combination of sequences for all three markers produced the best resolved tree. The disparity index test based on matK+rbcL+ITS2 was significant (P < 0.05) for a higher number of species pairs than the individual gene sequences. Although all three barcodes separated the species with respect to their genome type, no single combination of barcodes could differentiate all the Gossypium species, and tetraploid species were particularly difficult.     

DNA barcoding as a complementary tool for conservation and valorisation of forest resources

Since the pre-historic era, humans have been using forests as a food, drugs and handcraft reservoir. Today, the use of botanical raw material to produce pharmaceuticals, herbal remedies, teas, spirits, cosmetics, sweets, dietary supplements, special industrial compounds and crude materials constitute an important global resource in terms of healthcare and economy. In recent years, DNA barcoding has been suggested as a useful molecular technique to complement traditional taxonomic expertise for fast species identification and biodiversity inventories. In this study, in situ application of DNA barcodes was tested on a selected group of forest tree species with the aim of contributing to the identification, conservation and trade control of these valuable plant resources.The “core barcode” for land plants (rbcL, matK, and trnH-psbA) was tested on 68 tree specimens (24 taxa). Universality of the method, ease of data retrieval and correct species assignment using sequence character states, presence of DNA barcoding gaps and GenBank discrimination assessment were evaluated. The markers showed different prospects of reliable applicability. RbcL and trnH-psbA displayed 100% amplification and sequencing success, while matK did not amplify in some plant groups. The majority of species had a single haplotype. The trnH-psbA region showed the highest genetic variability, but in most cases the high intraspecific sequence divergence revealed the absence of a clear DNA barcoding gap. We also faced an important limitation because the taxonomic coverage of the public reference database is incomplete. Overall, species identification success was 66.7%.This work illustrates current limitations in the applicability of DNA barcoding to taxonomic forest surveys. These difficulties urge for an improvement of technical protocols and an increase of the number of sequences and taxa in public databases.     

DNA barcoding of Bradysia (Diptera: Sciaridae) for detection of the immature stages on agricultural crops

We investigated the usefulness of mitochondrial cytochrome c oxidase (COI) DNA barcoding of the genus Bradysia for the detection of immature stages and cryptic species complex. Although the larvae of some species in this genus are agricultural pests, immature stages are rarely identified due to the lack of key morphological characteristics. We constructed partial sequences of the COI gene for 25 species of Bradysia as a first step towards a DNA barcode. Using these data, Bradysia impatiens, B. procera and B. peraffinis were identified from larval specimens collected, respectively, from paprika, ginseng and oak sawdust beds used for cultivating shiitake. Our findings reveal a complex of three species within the B. tilicola group. These species were all identified as important pest B. ocellaris based on the morphology of male genital structures; however, the interspecific genetic divergence of the COI region was significantly greater (16.1–19.4%) than the intraspecific variation in each species. Therefore, B. ocellaris may consist of at least three species. The results demonstrate that COI DNA barcodes are useful for Bradysia species identification.     

DNA barcoding provides support for a cryptic species complex within the globally distributed and fishery important opah (Lampris guttatus)

The cornerstone of fisheries management relies on a solid taxonomic base and an understanding of how animals can be grouped into coherent management units. Surprisingly, little is known about the basic biology and ecology of opah (Lampris guttatus), a globally distributed species that is commercially exploited and regionally common in the North Pacific. Recent efforts to collect life history data on this species uncovered evidence of two North Pacific morphotypes. Sequencing of the mitochondrial cytochrome c oxidase I gene (655 bp) for these morphotypes and other specimens collected worldwide (n = 480) produced five strongly diverged and well‐supported clades. Additional sequence data from the cytochrome b gene (1141 bp) as well as the nuclear recombination activating gene 1 (1323 bp) corroborated these results, suggesting these five clades probably represent separate species. Our conclusion that opah is a complex of five separate species has implications for management and indicates a need to gather additional data on these poorly understood fishes.     

DNA barcoding of Murinae (Rodentia: Muridae) and Arvicolinae (Rodentia: Cricetidae) distributed in China

Identification of rodents is very difficult mainly due to high similarities in morphology and controversial taxonomy. In this study, mitochondrial cytochrome oxidase subunit I (COI) was used as DNA barcode to identify the Murinae and Arvicolinae species distributed in China and to facilitate the systematics studies of Rodentia. In total, 242 sequences (31 species, 11 genera) from Murinae and 130 sequences (23 species, 6 genera) from Arvicolinae were investigated, of which 90 individuals were novel. Genetic distance, threshold method, tree‐based method, online BLAST and BLOG were employed to analyse the data sets. There was no obvious barcode gap. The average K2P distance within species and genera was 2.10% and 12.61% in Murinae, and 2.86% and 11.80% in Arvicolinae, respectively. The optimal threshold was 5.62% for Murinae and 3.34% for Arvicolinae. All phylogenetic trees exhibited similar topology and could distinguish 90.32% of surveyed species in Murinae and 82.60% in Arvicolinae with high support values. BLAST analyses yielded similar results with identification success rates of 92.15% and 93.85% for Murinae and Arvicolinae, respectively. BLOG successfully authenticated 100% of detected species except Leopoldamys edwardsi based on the latest taxonomic revision. Our results support the species status of recently recognized Micromys erythrotis, Eothenomys tarquinius and E. hintoni and confirm the important roles of comprehensive taxonomy and accurate morphological identification in DNA barcoding studies. We believe that, when proper analytic methods are applied or combined, DNA barcoding could serve as an accurate and effective species identification approach for Murinae and Arvicolinae based on a proper taxonomic framework.     

DNA barcoding in a biodiversity hot spot: potential value for the identification of Malagasy Euphorbia L. listed in CITES Appendices I and II

The island of Madagascar is a key hot spot for the genus Euphorbia, with at least 170 native species, almost all endemic. Threatened by habitat loss and illegal collection of wild plants, nearly all Malagasy Euphorbia are listed in CITES Appendices I and II. The absence of a reliable taxonomic revision makes it particularly difficult to identify these plants, even when fertile, and thereby compromises the application of CITES regulations. DNA barcoding, which can facilitate species‐level identification irrespective of developmental stage and the presence of flowers or fruits, may be a promising tool for monitoring and controlling trade involving threatened species. In this study, we test the potential value of barcoding on 41 Euphorbia species representative of the genus in Madagascar, using the two widely adopted core barcode markers (matK and rbcL), along with two additional DNA regions, nuclear internal transcribed spacer (ITS) and the chloroplastic intergenic spacer psbA‐trnH. For each marker and for selected marker combinations, inter‐ and intraspecific distance estimates and species discrimination rates are calculated. Results using just the ‘official’ barcoding markers yield overlapping inter‐ and intraspecific ranges and species discrimination rates below 60%. When ITS is used, whether alone or in combination with the core markers, species discrimination increases to nearly 100%, whereas the addition of psbA‐trnH produces less satisfactory results. This study, the first ever to test barcoding on the large, commercially important genus Euphorbia shows that this method could be developed into a powerful identification tool and thereby contribute to more effective application of CITES regulations.     

DNA barcoding in medicinal plants: Testing the potential of a proposed barcoding marker for identification of Uncaria species from China

DNA barcoding, an increasingly popular mean of species identification, has been widely used for global species identification despite a consensus not being reached regarding which DNA sequences can be used as the best plant barcodes. In this study, we tested the feasibility of five candidate DNA barcodes (nrITS, nrITS2, matk, rbcL and trnH-psbA) for identifying Uncaria species. We collected a total of 54 specimens of 10 Uncaria species across its distributional range. BLAST, barcoding gaps, tree-based methods and TAXONDNA analysis were used to investigate the molecular identification capability of the candidate DNA barcodes. The results showed that the ITS2 is most suitable as a candidate DNA barcode for identification of medicinal plants of the genus Uncaria.     

Restoration of tree lines in an agricultural landscape: their effectiveness as a conservation management tool

As remnant vegetation covers <15% of the Australian sheep‐wheat belt, it is important to identify conservation strategies suitable for use in agricultural landscapes. Tree lines are widespread ecological structures in rural areas, and are now the subject of government subsidy schemes in New South Wales. However, the contribution of tree lines to biodiversity conservation is poorly understood. To identify the conservation value of tree lines, the bird communities in 36 tree lines in Cowra Shire, New South Wales, were surveyed 4 times each. The results demonstrated that tree lines were used by a large number of species, six of which were threatened. Different taxa were associated with different physical tree line attributes, with tree line age an important predictor of species occurrence. While mature tree lines tended to support more species, as was reflected in higher Shannon Diversity Index scores, they also harboured relatively more introduced species and nest predators, while young tree lines provided the best habitat for threatened species. However, these tree lines will mature, and as they do so they will provide increasingly suitable habitat for the hyperaggressive native honeyeater, the Noisy Miner (Manorina melanocephala), and introduced species. Therefore, tree lines in agricultural landscapes may only be serving an important role for conservation on short time scales, and the suite of threatened species young tree lines currently support appear likely to decline further in the future.     

Carbon isotope discrimination in leaves of the broad‐leaved paperbark tree,Melaleuca quinquenervia,as a tool for quantifying past tropical and subtropical rainfall

Quantitative reconstructions of terrestrial climate are highly sought after but rare, particularly in Australia. Carbon isotope discrimination in plant leaves (Δ_leaf) is an established indicator of past hydroclimate because the fractionation of carbon isotopes during photosynthesis is strongly influenced by water stress. Leaves of the evergreen tree Melaleuca quinquenervia have been recovered from the sediments of some perched lakes on North Stradbroke and Fraser Islands, south‐east Queensland, eastern Australia. Here, we examine the potential for using M. quinquenervia ?_leaf as a tracer of past rainfall by analysing carbon isotope ratios (δ¹³C) of modern leaves. We firstly assess Δ_leaf variation at the leaf and stand scale and find no systematic pattern within leaves or between leaves due to their position on the tree. We then examine the relationships between climate and Δ_leaf for a 11‐year time series of leaves collected in a litter tray. M. quinquenervia retains its leaves for 1–4 years; thus, cumulative average climate data are used. There is a significant relationship between annual mean ?_leaf and mean annual rainfall of the hydrological year for 1–4 years (i.e. 365–1460 days) prior to leaf fall (r² = 0.64, P = 0.003, n = 11). This relationship is marginally improved by accounting for the effect of pCO₂ on discrimination (r² = 0.67, P = 0.002, n = 11). The correlation between rainfall and Δ_leaf, and the natural distribution of Melaleuca quinquenervia around wetlands of eastern Australia, Papua New Guinea and New Caledonia offers significant potential to infer past rainfall on a wide range of spatial and temporal scales.     

Agrilus mali Matsumara (Coleoptera: Buprestidae), a new invasive pest of wild apple in western China: DNA barcoding and life cycle

Agrilus mali Matsumara (Coleoptera: Buprestidae) is a wood‐boring beetle distributed to eastern China that occasionally injures apple species. However, this wood‐boring beetle is new to the wild apple forests (Malus sieversii) of the Tianshan Mountains (western China) and has caused extensive tree mortality. The development of a biological control program for these wild apple forests is a high priority that requires exploration of the life cycle, DNA barcoding and taxonomic status of A. mali. In this study, to determine the diversity of invasive beetles, a fragment of the mitochondrial cytochrome oxidase gene was analyzed. Based on the results, beetles from Gongliu and Xinyuan counties of Xinjiang were identical but differed from those in the apple nursery of Gongliu by a single‐nucleotide substitution. We summarize the taxonomic status, relationships, and genetic distances of A. mali among other Agrilus species using the Tajima‐Nei model in maximum likelihood phylogeny. Analysis revealed that A. mali was closely related to Agrilus mendax and both belong to the Sinuatiagrulus subgenus. The life cycle of A. mali was investigated based on a monthly regular inspection in the wild apple forests of Tianshan. Similar to congeneric species, hosts are injured by larvae of A. mali feeding on phloem tissue, resulting in serpentine galleries constructed between bark and xylem that prevent nutrient transport and leading to tree mortality. Future studies will focus on plant physiological responses to the invasive beetles and include surveys of natural enemies for a potential classical biological control program.     

The distribution,diversity, and conservation status of Cycas in China

As ancient gymnosperm and woody plants, cycads have survived through dramatic tectonic activities, climate fluctuation, and environmental variations making them of great significance in studying the origin and evolution of flora biodiversity. However, they are among the most threatened plant groups in the world. The principal aim of this review is to outline the distribution, diversity, and conservation status of Cycas in China and provide suggestions for conservation practices. In this review, we describe the taxonomy, distribution, and conservation status of Cycas in China. By comparing Chinese Cycas species with its relatives worldwide, we then discuss the current genetic diversity, genetic differentiation of Cycas, and try to disentangle the potential effects of Quaternary climate changes and topographical events on Cycas. We review conservation practices from both researchers and practitioners for these rare and endangered species. High genetic diversity at the species level and strong genetic differentiation within Cycas have been observed. Most Cycas species in southwest China have experienced population retreats in contrast to the coastal Cycas's expansion during the Quaternary glaciation. Additionally, human activities and habitat fragmentation have pushed these endangered taxa to the brink of extinction. Although numerous efforts have been made to mitigate threats to Cycas survival, implementation and compliance monitoring in protection zones are currently inadequate. We outline six proposals to strengthen conservation measures for Cycas in China and anticipate that these measures will provide guidelines for further research on population genetics as well as conservation biology of not only cycads but also other endangered species worldwide.