DNA based analysis of thrips diversity and thrips-borne Iris yellow spot virus (Tospovirus: Bunyaviridae) from Pakistan

Thrips (Thysanoptera) are one of the most economically important groups of crop pests at a global scale which damage a wide range of field and horticultural crops. Some thrips species also serve as vectors of plant viruses. Despite the importance of this tiny insect as pests, predators, fungal feeders, gall formers, pollinators and virus vector, scant work was carried out on their systematics in Pakistan. Currently thrips taxonomy in Pakistan is solely based on morphological identification. Present study focused on thrips species identification based on the morphological characters, and developing a database of thrips fauna and their characterization based on DNA barcoding. Thrips were collected from multiple plants during 2009-2012 at 158 sites in three climatic regions of Pakistan. Twelve species from five genera of the suborder Tubulifera and twenty nine species from seventeen genera of the suborder Terebrantia were identified following standard taxonomic keys. A checklist of species reported in Pakistan since 1947 including thrips from the current survey was compiled. A comparison of our species with those previously reported from this region showed that one species (Apterygothrips pellucidus Ananthakrishnan) from Tubulifera and seven species (Chaetanaphothrips orchidii Moulton, Chirothrips meridionalis Bagnall, Megalurothrips distalis Karny, M. usitatus Bagnall, Neohydatothrips samayunkur Kudo, Taeniothrips major, Thrips trehernei Priesner) from Terebrantia and four genera (Apterygothrips, Chaetanaphothrips, Neohydatothrips, Taeniothrips) were the first reports from Pakistan. Mitochondrial COI sequences were used for discriminating 471 thrips that represented 55 species in the current survey. Sequence analysis revealed that the intraspecific and interspecific distances ranged from 0.0% to 7.5% and 2.3% to 22.3%, respectively. In addition, the study showed that four of the major thrips species in the region, Aeolothrips intermedius, Haplothrips reuteri, Thrips palmi and Thrips tabaci were cryptic species complexes. The study showed that DNA barcoding successfully discriminated regional thrips species including those which were morphologically cryptic. A barcode reference library for thrips from Pakistan was compiled and regional lineages of four important virus-vector thrips were connected with those from other countries by haplotype networks. A survey to determine the \ud incidence of selected tospoviruses was carried out in onion-growing regions of the Punjab province of Pakistan during February-May and September-October 2012 in thirteen administrative districts. Plants with symptoms suggestive of Iris yellow spot virus (IYSV) infection were collected and tested for the presence of the virus by ELISA and RT-PCR. Sequence analysis of RT-PCR amplified nucleocapsid (N) gene confirmed IYSV infection of onion in Pakistan. This was the first report of IYSV infecting onion in Pakistan. A global analysis of more than 100 IYSV N gene sequences was carried out to determine the comparative population structure, spatial and temporal dynamics with reference to its genetic diversity and evolution. Global IYSV population could be grouped into two genotypes, IYSVBR and IYSVNL and the analysis showed that the two genotypes were almost equally distributed. A temporal shift was observed from IYSVNL to IYSVBR genotype over a period of 15 years (1997 to 2013). The diversity in IYSV population and temporal shift in IYSVBR genotype is attributable to genetic recombination, abundance of purifying selection, insignificant positive selection and population expansion. Restricted gene flow between the two major IYSV genotypes (IYSVBR and IYSVNL) further emphasizes the role of genetic drift in modeling the population architecture, evolutionary lineages and epidemiology of IYSV