DNA Barcode Analysis of Thrips (Thysanoptera) Diversity in Pakistan Reveals Cryptic Species Complexes

RESEARCH ARTICLE DNA Barcode Analysis of Thrips (Thysanoptera) Diversity in Pakistan Reveals Cryptic Species Complexes Romana Iftikhar1, Muhammad Ashfaq1,2*, Akhtar Rasool1, Paul D. N. Hebert2 1 National Institute for Biotechnology and Genetic Engineering (NIBGE), Faisalabad, Pakistan, 2 Centre for Biodiversity Genomics, Biodiversity Institute of Ontario, University of Guelph, Guelph, Ontario, Canada * Abstract OPEN ACCESS Citation: Iftikhar R, Ashfaq M, Rasool A, Hebert PDN (2016) DNA Barcode Analysis of Thrips (Thysanoptera) Diversity in Pakistan Reveals Cryptic Species Complexes. PLoS ONE 11(1): e0146014. doi:10.1371/journal.pone.0146014 Editor: Owain Rhys Edwards, CSIRO, AUSTRALIA Received: June 26, 2015 Accepted: December 12, 2015 Published: January 7, 2016 Copyright: © 2016 Iftikhar et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All data are available on the Barcode of Life Data Systems (www. boldsystems.org) under the dataset DS-MATHR. Funding: This study was partly supported by the Higher Education Commission Pakistan by fellowship grant to RI and research grant (20-1403/R&D/09) to MA. PDNH was supported by International Development Research Centre (grant no. 106106001), and a grant from the Government of Canada through Genome Canada and the Ontario Genomics Institute in support of the International Barcode of Life (iBOL) project. Although thrips are globally important crop pests and vectors of viral disease, species identifications are difficult because of their small size and inconspicuous morphological differences. Sequence variation in the mitochondrial COI-5ʹ (DNA barcode) region has proven effective for the identification of species in many groups of insect pests. We analyzed barcode sequence variation among 471 thrips from various plant hosts in north-central Pakistan. The Barcode Index Number (BIN) system assigned these sequences to 55 BINs, while the Automatic Barcode Gap Discovery detected 56 partitions, a count that coincided with the number of monophyletic lineages recognized by Neighbor-Joining analysis and Bayesian inference. Congeneric species showed an average of 19% sequence divergence (range = 5.6% - 27%) at COI, while intraspecific distances averaged 0.6% (range = 0.0% 7.6%). BIN analysis suggested that all intraspecific divergence >3.0% actually involved a species complex. In fact, sequences for three major pest species (Haplothrips reuteri, Thrips palmi, Thrips tabaci), and one predatory thrips (Aeolothrips intermedius) showed deep intraspecific divergences, providing evidence that each is a cryptic species complex. The study compiles the first barcode reference library for the thrips of Pakistan, and examines global haplotype diversity in four important pest thrips. Introduction Thrips (Thysanoptera) are serious pests and disease vectors on many economically important crops throughout the world [1,2]. Identification of most thrips to a species level is difficult because of their small size, subtle morphological differentiation [3], intraspecific polymorphisms [4], and sexual dimorphisms [5]. Molecular identification of thrips has major advantages to morphology-based analysis because it overcomes the complexities introduced by morphological variation among life stages and the inconspicuous morphological differences among species [3,6]. Several gene regions have been employed for species discrimination [7, 8] and phylogenetic analysis [9]. Crespi et al. [10] employed the nuclear 18S and mitochondrial gene cytochrome c oxidase I (COI) genes to examine phylogenetic relationships between two PLOS ONE | DOI:10.1371/journal.pone.0146014 January 7, 2016 1 / 21 DNA Barcode Analysis of Thrips Diversity in Pakistan Competing Interests: The authors have declared that no competing interests exist. suborders Terebrantia and Tubulifera, while Buckman et al. [9] coupled four nuclear loci (18S & 28S ribosomal DNA (rDNA), Histone 3, Tubulin-alpha 1) with COI to ascertain the phylogenetic relationships of 99 species of thrips from seven families. COI has also been recognized as a particularly suitable marker for thrips identification because it exhibits more consistent interspecific variation [11] than other markers [12,13]. Its analysis has, for example, helped to reveal the number of thrips species inhabiting particular cropping systems [14]. The capacity of the COI-5ʹ (DNA barcode) region to discriminate cryptic species of insects has been well validated [15–18] including for thrips [3,12]. Levels of COI sequence divergence often are helpful in deciding if two sequences derive from different species [19] as most conspecifics show <2% divergence in the barcode region [15,20]. The Barcode Index Number (BIN) system now provides an interim taxonomic system based on COI sequence clusters [21] for all animals and most BINs are congruent with morphological species [22,23]. Barcode data has been used to advance species-level taxonomy in various animal groups [24], often revealing new species [25,26]. Researchers have also employed DNA barcoding to identify pest thrips for quarantine [27,28]. Some pest thrips species are thought to be a complex of multiple cryptic species [29]. For example, COI analysis revealed three lineages of Thrips tabaci [6,30], while Thrips palmi has two clades [27,13]. Likewise, western flower thrips, Frankliniella occidentalis, is now viewed as two species [29]. Members of species complexes have been discriminated by sequence matches [31] or by PCR analysis with species-specific primers, enabling a non-specialist to identify the target species at any life stage [12]. The rapid increase in global trade warrants the development of a universal, anticipatory system with the capacity to identify taxa that are newly encountered in a region because invasive species can reduce local biodiversity and often cause serious economic damage to crops [32]. The accurate identification of pest and invasive species is critical for both control [33,34] and quarantine [35] as misidentifications may lead to ineffective control measures [33]. The varying capacity of thrips species to transmit viral diseases [36] provides an additional incentive to explore their genetic diversity. Prior taxonomic studies on the Thysanoptera of Pakistan [37– 43] have been limited in scope, but 76 species have been reported including members of three families (Aeolothripidae, Phlaeothripidae, Thripidae) (Appendix 1). The present study analyzes patterns of COI sequence diversity among thrips from Pakistan, initiating the development of a regional barcode reference library for Thysanoptera. Furthermore, the study examines the broader geographic patterning of haplotype frequencies in four particularly important pest species. Materials and Methods E (...truncated)