Phylogenetic Reconstruction and DNA Barcoding for Closely Related Pine Moth Species (Dendrolimus) in China with Multiple Gene Markers

et al. (2012) Phylogenetic Reconstruction and DNA Barcoding for Closely Related Pine Moth Species (Dendrolimus) in China with Multiple Gene Markers. PLoS ONE 7(4): e32544. doi:10.1371/journal.pone.0032544 Phylogenetic Reconstruction and DNA Barcoding for Closely Related Pine Moth Species (Dendrolimus ) in China with Multiple Gene Markers Qing-Yan Dai 0 Qiang Gao 0 Chun-Sheng Wu 0 Douglas Chesters 0 Chao-Dong Zhu 0 Ai-Bing Zhang 0 Rongling Wu, Pennsylvania State University, United States of America 0 1 College of Life Sciences, Capital Normal University , Beijing , People's Republic of China, 2 Key Laboratory of Zoological Systematics and Evolution, Institute of Zoology, Chinese Academy of Sciences , Beijing , People's Republic of China Unlike distinct species, closely related species offer a great challenge for phylogeny reconstruction and species identification with DNA barcoding due to their often overlapping genetic variation. We tested a sibling species group of pine moth pests in China with a standard cytochrome c oxidase subunit I (COI) gene and two alternative internal transcribed spacer (ITS) genes (ITS1 and ITS2). Five different phylogenetic/DNA barcoding analysis methods (Maximum likelihood (ML)/Neighborjoining (NJ), ''best close match'' (BCM), Minimum distance (MD), and BP-based method (BP)), representing commonly used methodology (tree-based and non-tree based) in the field, were applied to both single-gene and multiple-gene analyses. Our results demonstrated clear reciprocal species monophyly for three relatively distant related species, Dendrolimus superans, D. houi, D. kikuchii, as recovered by both single and multiple genes while the phylogenetic relationship of three closely related species, D. punctatus, D. tabulaeformis, D. spectabilis, could not be resolved with the traditional tree-building methods. Additionally, we find the standard COI barcode outperforms two nuclear ITS genes, whatever the methods used. On average, the COI barcode achieved a success rate of 94.10-97.40%, while ITS1 and ITS2 obtained a success rate of 64.7081.60%, indicating ITS genes are less suitable for species identification in this case. We propose the use of an overall success rate of species identification that takes both sequencing success and assignation success into account, since species identification success rates with multiple-gene barcoding system were generally overestimated, especially by tree-based methods, where only successfully sequenced DNA sequences were used to construct a phylogenetic tree. Non-tree based methods, such as MD, BCM, and BP approaches, presented advantages over tree-based methods by reporting the overall success rates with statistical significance. In addition, our results indicate that the most closely related species D. punctatus, D. tabulaeformis, and D. spectabilis, may be still in the process of incomplete lineage sorting, with occasional hybridizations occurring among them. - Funding: This study was supported by Beijing Municipal Natural Science Foundation Key Projects (No.KZ201010028028 to ZHANG) and by Natural Science Foundation of China (to ZHANG, Grant No. 31071963), by Funding Project for Academic Human Resources Development in Institutions of Higher Learning Under the Jurisdiction of Beijing Municipality (to ZHANG, Grant No. PHR201107120), by The Research Fund for the Doctoral Program of Higher Education (to ZHANG, Grant No. 20101108120002), by Grant from Chinese Academy of Sciences (Grant No. 13914G1001) and a Grant from Public Welfare Project from the Ministry of Agriculture, China (Grant No. 201103024) to ZHU. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. DNA barcoding (http://www.barcodinglife.org) has gained widespread prominence during the past eight years as part of the worldwide campaign to develop a global biodiversity inventory [114]. On 23 Aug. 2011, there were 1,348,985 barcode records from 110,892 species in the Barcode of Life Database (BOLD) (www.barcodinglife.org). However, some reservations still remain about the utility of DNA barcoding [1524]. Two main issues, the choice of barcoding gene and methods for species assignments, have been the central problems. The choice of barcoding gene is one of the primary issues. The 5 prime segment of the mitochondrial (mt) cytochrome c oxidase subunit I (COI) gene (648 bp) was initially proposed to serve as DNA barcode [12], and proved to be of great success in many animal groups [12,25]. Currently, COI has been selected as a standard barcode gene for animal groups. However, the rationale of selection of COI as standard barcode is subject to debate, and with the increase in barcoded taxa, from algae, fungi, bacteria and plants to invertebrates and vertebrates, scientists have found its less effective in some taxon groups [2,20,2628]. The search for the most suitable gene for species identification is not over, with several recent studies testing the efficiencies of different genes, using part of, or the whole of mtDNA genome to look for the optimal DNA barcode gene [2930]. On the other hand, empiricists have also proposed other gene segments as candidate DNA barcode loci, such as the nuclear ITS regions (ITS1, ITS2) [3132]. ITS - Internal Transcribed Spacer (ribosomal DNA repeating unit), which is a commonly used DNA biomarker, was suggested and examined in several plant groups [3132], and fungi (http://www.boldsystems.org/views/projectmenu.php?&). This widely used genetic marker might be suitable as a DNA barcode due to its highly variability. This is especially the case for groups composed of closely related species, where the rate of successful species identification with COI is relatively low (less than 70%) (e.g., fly, [20]). Unlike groups of distantly related species, where the existence of large genetic divergence between species makes discrimination easy, groups of closely related species offer greater challenges for phylogenetic reconstruction and clear species identification. Pine moth species (caterpillar) are one of the most serious pest insect group in China [3339], with outbreaks of the pest regularly causing extensive forest damage [3435,3739]. This pest species group consists of six commonly occurring, closely related species, between which discrimination is very challenging. Taxonomically, three of them (Dendrolims punctatus [40], D. tabulaeformis [41], D. spectabilis [42]) have a very uncertain species status. For instance, the latter two had been suggested as a subspecies of D. punctatus [3435]. However, these species were treated as three different species in several other studies [3739]. Therefore, this species group provides a good model for investigating the efficiency of DNA barcode species identification for closely related species groups. In addition to the selection of barcoding (...truncated)