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      Towards a global DNA barcode reference library for quarantine identifications of lepidopteran stemborers, with an emphasis on sugarcane pests

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          Abstract

          Lepidopteran stemborers are among the most damaging agricultural pests worldwide, able to reduce crop yields by up to 40%. Sugarcane is the world’s most prolific crop, and several stemborer species from the families Noctuidae, Tortricidae, Crambidae and Pyralidae attack sugarcane. Australia is currently free of the most damaging stemborers, but biosecurity efforts are hampered by the difficulty in morphologically distinguishing stemborer species. Here we assess the utility of DNA barcoding in identifying stemborer pest species. We review the current state of the COI barcode sequence library for sugarcane stemborers, assembling a dataset of 1297 sequences from 64 species. Sequences were from specimens collected and identified in this study, downloaded from BOLD or requested from other authors. We performed species delimitation analyses to assess species diversity and the effectiveness of barcoding in this group. Seven species exhibited <0.03 K2P interspecific diversity, indicating that diagnostic barcoding will work well in most of the studied taxa. We identified 24 instances of identification errors in the online database, which has hampered unambiguous stemborer identification using barcodes. Instances of very high within-species diversity indicate that nuclear markers (e.g. 18S, 28S) and additional morphological data (genitalia dissection of all lineages) are needed to confirm species boundaries.

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          DNA barcoding and taxonomy in Diptera: a tale of high intraspecific variability and low identification success.

          DNA barcoding and DNA taxonomy have recently been proposed as solutions to the crisis of taxonomy and received significant attention from scientific journals, grant agencies, natural history museums, and mainstream media. Here, we test two key claims of molecular taxonomy using 1333 mitochondrial COI sequences for 449 species of Diptera. We investigate whether sequences can be used for species identification ("DNA barcoding") and find a relatively low success rate (< 70%) based on tree-based and newly proposed species identification criteria. Misidentifications are due to wide overlap between intra- and interspecific genetic variability, which causes 6.5% of all query sequences to have allospecific or a mixture of allo- and conspecific (3.6%) best-matching barcodes. Even when two COI sequences are identical, there is a 6% chance that they belong to different species. We also find that 21% of all species lack unique barcodes when consensus sequences of all conspecific sequences are used. Lastly, we test whether DNA sequences yield an unambiguous species-level taxonomy when sequence profiles are assembled based on pairwise distance thresholds. We find many sequence triplets for which two of the three pairwise distances remain below the threshold, whereas the third exceeds it; i.e., it is impossible to consistently delimit species based on pairwise distances. Furthermore, for species profiles based on a 3% threshold, only 47% of all profiles are consistent with currently accepted species limits, 20% contain more than one species, and 33% only some sequences from one species; i.e., adopting such a DNA taxonomy would require the redescription of a large proportion of the known species, thus worsening the taxonomic impediment. We conclude with an outlook on the prospects of obtaining complete barcode databases and the future use of DNA sequences in a modern integrative taxonomy.
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            Revisiting the insect mitochondrial molecular clock: the mid-Aegean trench calibration.

            Phylogenetic trees in insects are frequently dated by applying a "standard" mitochondrial DNA (mtDNA) clock estimated at 2.3% My(-1), but despite its wide use reliable calibration points have been lacking. Here, we used a well-established biogeographic barrier, the mid-Aegean trench separating the western and eastern Aegean archipelago, to estimate substitution rates in tenebrionid beetles. Cytochrome oxidase I (cox1) for six codistributed genera across 28 islands (444 individuals) on both sides of the mid-Aegean trench revealed 60 independently coalescing entities delimited with a mixed Yule-coalescent model. One representative per entity was used for phylogenetic analysis of mitochondrial (cox1, 16S rRNA) and nuclear (Mp20, 28S rRNA) genes. Six nodes marked geographically congruent east-west splits whose separation was largely contemporaneous and likely to reflect the formation of the mid-Aegean trench at 9-12 Mya. Based on these "known" dates, a divergence rate of 3.54% My(-1) for the cox1 gene (2.69% when combined with the 16S rRNA gene) was obtained under the preferred partitioning scheme and substitution model selected using Bayes factors. An extensive survey suggests that discrepancies in mtDNA substitution rates in the entomological literature can be attributed to the use of different substitution models, the use of different mitochondrial gene regions, mixing of intraspecific with interspecific data, and not accounting for variance in coalescent times or postseparation gene flow. Different treatments of these factors in the literature confound estimates of mtDNA substitution rates in opposing directions and obscure lineage-specific differences in rates when comparing data from various sources.
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              DNA barcode-based delineation of putative species: efficient start for taxonomic workflows

              The analysis of DNA barcode sequences with varying techniques for cluster recognition provides an efficient approach for recognizing putative species (operational taxonomic units, OTUs). This approach accelerates and improves taxonomic workflows by exposing cryptic species and decreasing the risk of synonymy. This study tested the congruence of OTUs resulting from the application of three analytical methods (ABGD, BIN, GMYC) to sequence data for Australian hypertrophine moths. OTUs supported by all three approaches were viewed as robust, but 20% of the OTUs were only recognized by one or two of the methods. These OTUs were examined for three criteria to clarify their status. Monophyly and diagnostic nucleotides were both uninformative, but information on ranges was useful as sympatric sister OTUs were viewed as distinct, while allopatric OTUs were merged. This approach revealed 124 OTUs of Hypertrophinae, a more than twofold increase from the currently recognized 51 species. Because this analytical protocol is both fast and repeatable, it provides a valuable tool for establishing a basic understanding of species boundaries that can be validated with subsequent studies.
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                Author and article information

                Contributors
                timothy.lee@austmus.gov.au
                Journal
                Sci Rep
                Sci Rep
                Scientific Reports
                Nature Publishing Group UK (London )
                2045-2322
                7 May 2019
                7 May 2019
                2019
                : 9
                : 7039
                Affiliations
                [1 ]ISNI 0000 0004 0470 8815, GRID grid.438303.f, Department of Entomology, , Australian Museum Research Institute, ; 1 William St, Darlinghurst, NSW 2010 Australia
                [2 ]GRID grid.467741.7, Biosecurity Operations, NAQS, , Department of Agriculture and Water Resources, ; 1 Pederson Road, Eaton, NT 0812 Australia
                [3 ]Northern Territory Department of Primary Industry and Resources, GPO Box 3000, Darwin, NT 0801 Australia
                [4 ]Department of Agriculture and Water Resources, 114 Catalina Crescent, Airport Business Park, Cairns Airport, Cairns, QLD 4870 Australia
                [5 ]ISNI 0000 0004 1794 5158, GRID grid.419326.b, African Insect Science for Food and Health (ICIPE), ; PO Box 30772-00100 Nairobi, Kenya
                [6 ]ISNI 0000 0001 2171 2558, GRID grid.5842.b, IRD/CNRS, UMR IRD 247 EGCE, Laboratoire Evolution Génomes Comportement et Ecologie, Avenue de la terrasse, BP1, 91198, Gif-sur-Yvette, France and Université Paris-Sud 11, ; 91405 Orsay, France
                [7 ]ISNI 0000 0001 2214 904X, GRID grid.11956.3a, Department of Conservation Ecology and Entomology, Faculty of AgriSciences, , University of Stellenbosch, Private Bag X1, ; Matieland, Western Cape 7602 South Africa
                [8 ]ISNI 0000 0000 8633 7245, GRID grid.482601.9, South African Sugarcane Research Institute, ; 170 Flanders Drive, Mount Edgecombe, KwaZulu-Natal 4300 South Africa
                [9 ]GRID grid.467576.1, Sugar Research Australia, ; 71378 Bruce Highway, Gordonvale, QLD 4865 Australia
                [10 ]GRID grid.467576.1, Sugar Research Australia, ; 50 Meiers Road, Indooroopilly, QLD 4068 Australia
                Author information
                http://orcid.org/0000-0003-2839-3873
                http://orcid.org/0000-0002-7152-3517
                Article
                42995
                10.1038/s41598-019-42995-0
                6504866
                31065024
                738b3ccf-22bf-4c4f-8c0f-9c1a4518117b
                © The Author(s) 2019

                Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

                History
                : 28 November 2018
                : 5 April 2019
                Funding
                Funded by: FundRef https://doi.org/10.13039/100008709, Sugar Research Australia (Sugar Research Australia Ltd.);
                Award ID: 2016/041
                Award ID: 2016/041
                Award ID: 2016/041
                Award ID: 2016/041
                Award ID: 2016/041
                Award Recipient :
                Funded by: Subcommittee for Plant Health Diagnostic for the development of the National Diagnostic Protocols for exotic moths, Plant Health Australia
                Categories
                Article
                Custom metadata
                © The Author(s) 2019

                Uncategorized
                entomology,biodiversity,phylogenetics
                Uncategorized
                entomology, biodiversity, phylogenetics

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