Spatial Distribution And Genetic Diversity Of Odonates And Their Associated Single-stranded DNA Viruses In China-Pakistan

Order Odonata,consisting of more than 6000 species,are widely distributed all over the world and commonly named as dragonflies or damselflies.Odonates are predators,feeding on small insects such as mosquitoes in confined habitats.The augmentative release of dragonflies during immature life stages has shown potential results of biological control.In addition,several recent researches reported dozens of single stranded DNA(ssDNA)viruses associating with odonates.It was proposed that odonates may serve as a good sampling tool for ssDNA viruses because they are top insect predators.Ecology and evolution of Odonata has been studied extensively in 21st century due to their diverse phylogenetic position,ecological and phenotypic diversity and complex life cycle,although few studies have been done to characterize odonates at the molecular level.In this study,a large-scale sampling of odonates was conducted during 2014-2017 from China(central and south zones)and Pakistan(south-western).Mitochondrial cytochrome oxidase subunit(COI)gene sequencing was used to determine odonates genetically.Rolling circle amplification(RCA)was used to enrich and clone ssDNA viruses from several dragonflies collected from Fuzhou(China).The COI sequence alignment was executed using Clustal-W in BioEdit v6.The phylogenetic tree was constructed through Neighbor-Joining method by using Jukes-Cantor model,and genetic divergence was calculated via Kimura 2-Parameter using MEGA7,while Genetic diversity was calculated by DnaSP v5.Moreover,nucleotide mismatch distribution was done to confirm the genetic diversity of all sequences.Similarly,Pairwise distance analysis was conducted to identify the novel viruses.Samples collected from central China(Wuhan)and Pakistan(five locations)revealed 67 diverse sequences of odonates which further revealed the presence of 21 dragonfly species belonging to 15 distinct genera,two families,Gomphidae and Libellulidae,respectively.Maximum genetic divergence was observed for Crocothemis servilia,at 24.96%,followed by Libellulidae sp.with 22.30%while minimum divergence(0.82%)was observed for Melligomphus ardens.Likewise,a significant genetic diversity(341 mutations,)was observed for all species.However,C.servilia species presented maximum value(176 mutations)followed by Libellulidae spp.(150 mutations),whereas minimum value(3 mutations)was observed by Orthetrum testaceum.Interestingly,the diversity of C.servilia,all of which are collected from a single location(Wuhan)of China,is much higher than those from Pakistan,which were collected from 5 different places with a spatial distance exceeding 500 Kms.Our results are useful in understanding the global diversity of odonates and the development of conservation measures of this insect group.Moreover,the identification of cycloviruses from odonates may help us in understanding the diversity and evolution of these viruses.Similarly,the 61 sequences obtained from South China samples revealed the existence of 16 dragonfly species,belonging to 11 genera and three families i.e.Aeshnidae,Gomphidae and Libellulidae,respectively.Maximum divergence was observed by Trithemis genus(18.69%),followed Orthetrum genus(18.16%),whereas minimum value of divergence was noted by Pantala genus(0.31%).On the other hand,maximum genetic diversity was recorded for Orthetrum genus with up to 142 mutations,followed by Trithemis genus(126 mutations),while the smallest value(two mutations)was observed for Pantala genus.Genetic diversity for overall and Libellulidae family sequences was much higher up to 404 mutations and 344 mutations,respectively.Moreover,10 damselfly species,belonging to seven genera and four families(Calopterygidae,Chlorocyphidae,Coenagrionidae,and Devadattidae)were identified from 51 samples collected from South China.Maximum genetic divergence(9.31%),was found in genus Rhinocypha,followed by genus Hetaerina with 8.23%,while minimum divergence was observed for genus Ceriagrion(0.32%)followed by genus Ischnura(0.48%).However,significant genetic divergence,as confirmed by two tests,Tajima's D and Fu's Fs,was observed for damselfly families.Genetic diversity among genera was also investigated,which showed that genus Hetaerina has maximum genetic diversity at 181 mutations,followed by genus Rhinocypha(68 mutations)while minimum genetic diversity was observed for genus Ceriagrion(2 mutations)followed by Ischnura(6 mutations).Three novel species of Cycloviruses were identified from dragonflies.These viruses,named Dragonfly associated cyclovirus-9,-10 and-11(DfCyV-9,-10,-11),respectively,show 56.1-79.6%genome-wide identity with approved species of cyclovirus and 61.6-65.1%among themselves.Thus,according to the current species demarcation criteria,DfCyV-9,-10,and-11 represent three novel cycloviruses.Notably,DfCyV-10 has a predicted Rep protein that is most similar to that of Bat associated cyclovirus 2(BatACyV-2),a cyclovirus discovered from China,with an amino acid sequence identity of 79.4%,but a Cp protein most similar to that of BatACyV-10,a cyclovirus discovered from Brazil,with an 71.7%amino acid sequence identity.Despite great progress in the molecular and morphological odonatology,several ecological zones of the world were still unexplored regarding genetic identification of odonates.This study revealed high genetic diversity of odonates in China and Pakistan,as we identified more than 30 different species which indicate the better quality of ecosystem in the targeted zones.Moreover the identification of cycloviruses from the dragonflies may lead us to find the primary host of these ssDNA viruses.The data presented here may be useful in understanding ecology and evolution of odonates and their vector capabilities.