Species Identification And Phylogeny Analysis Of Cropland Spiders Based On DNA Barcodes And Mitogenomes

As one of important predators of insect pests,cropland spiders play important roles in the integrated pest management.Population protection and reconstruction of natural enemiescan effectively control many agricultural pests,such as planthoppers,leafhoppers,aphids and borers.Rapid and accurate identification of species is theprerequisite and basis for spiders protection and utilization.However,the traditional identification methods based on morphology not only time-consuming,but also requiringexcellent professional knowledge and experience,and hence oftenlead to false identification results.Additionally,for the species-rich spiders,to study thephylogeny and molecular evolutionis also important for their protection and utilization.In the presentstudy,DNA barcodingand phylogenetic resconstraction for the cropland spiders was performed based on both mitochondrial genes and the complete mitogenome sequences.1.Two candidate mitochondrialmarkers,COI(Cytochrome oxidase subunit I,5'658bp)and 16S(16S ribosomal RNA,5' 492bp),were successfully amplified and sequencedby using thebarcode universal primers.Subsequently,289 barcode sequences were generatedfor a total of 56 cropland spider species from36 genera in 14 different familiesfor the first time in China.In order to assess their effectiveness in the discrimination of spiders,the genetic distances of bothcandidatemarkers were estimated and the Neighbor-joining(NJ)trees based on Kimura-2-parameter(K2P)model were constructed by MEGA 5.0 software.As a result,the average divergence values of COI within and between species were 0.0096 and 0.2005,respectively.The average intraspecific andinterspecificgeneticdistances for 16 S were 0.0155 and 0.3107,respectively.The mean value of interspecific K2 P was more than 10 times higher than the average value of the intraspecific for COI and 16 S.Nevertheless,both theCOI and 16 Sgenesoverlapped in intra-and inter-specific genetic distances.The results showed that COI and 16 S could not completely identify the 56 kinds of cropland spiders.When ignoring the 3% thresholds,97% spider species can be identified both COI and 16 Sgene.Meanwhile,NJ trees of K2 P distances based on COI showed that all the individuals belonging to the same species formed single monophyletic clades.However,the NJ tree based on 16 S did not clearly distinguish some taxa at both the species and family level.Individuals of 10 species(17.9%)were not clustered together with their respective groups and two out of 14 families,Araneidae and Linyphiidae,could not form single monophyletic clades.In conclusion,our results highly suggest that the standard barcoding marker COI can be used to distinguish the cropland spiders both in species and family level by NJ tree-based method.In addition,the results from the initial partition and recursive division based on COI and 16 S genes through the ABGD web showed that all the samples(including the outgroup)are divided into 58 groups,which is very close to the actual 57 groups.2.The complete mitogenomes of four spiders,C.xanthogramma(Salticidae),A.angulatus(Araneidae),D.angustivirgatus(Pisauridae)and E.tricuspidata(Thomisidae),wereamplified bylong-PCR method,and then sequenced and anonated.The full length of the mitogenomes are14,563 bp for C.xanthogramma,14,204 bp for A.angulatus,14,783 bp for D.angustivirgatusand 14,352 bp for E.tricuspidata,respectively.The nucleotide composition of fourspider mitogenomes are significantly A+T biased.The overall A+T content of the J-strands accounts for 75.1% inC.xanthogramma,77.0% in D.angustivirgatus,75.4% in A.angulatus and 77.3% in E.tricuspidata,respectively.Additionally,a negative AT-skew and a positive GC-skew are observed in the four spider mitogenomes,except anegative GC-skewis found in C.xanthogramma.The AT-skews are-0.089,-0.121,-0.062 and-0.097 in the mitogenomes of C.xanthogramma,D.angustivirgatus,A.angulatus and E.tricuspidata,respectively.While the values of GC-skew are-0.261,0.262,0.244 and 0.220,respectively.Thirty-seven genes(13 PCGs,22 tRNA genes,and 2 rRNA genes)and a large conrol region were identified in each mitogenome,which is typical of presently available in metazoan mitogenomes.All four mitogenomes share overlapping genes,the number of gene overlaps is 192bp(ranging in size from 1 to 29bp)in C.xanthogramma,155 bp(from 1 to 19 bp)in D.angustivirgatus,210 bp(from 1 to 21 bp)in A.angulatus and 149 bp(from 1 to 15 bp)in E.tricuspidata,respectively.In the mitogenomes ofC.xanthogramma,D.angustivirgatus and A.angulatus,five tRNAs(trnI,trnN,trnQ,trnS1 and trnL2)are translocated and two interchanged their positions(between trnY and trnC),when compared with that of L.polyphemus,which is considered a putative ground pattern for chelicerates.Notably,novel gene order rearrangement of trnL1 was observed in E.tricuspidata mitogenome,which has not been found in those of other sequenced Araneae species yet.The trnL1 is translocated to the new position between trn W and trnY,instead of between ND1 and 12 S rRNA gene that usually presented in Opisthothelae spiders.Phylogenetic analysis using BI and ML methods yielded identical tree topologies based on a concatenated nucleotide dataset of 13 PCGs from 29 Araneae species.Phylogenetic analysis indicates that the species with same genus and family could be well clustered,and the spiders with higher taxonomic level could also formed single monophyletic clades,such as the suborder and superfamily.In summary,the main results of this study include two aspects.Firstly,we screened and assessed the DNA barcode genesin the discrimination ofthe Chinese croplandspiders,and the standard DNA barcodes of the cropland spiderswere obtained.Secondly,the complete mitogenomes of four spiders were sequenced and annotated.The nucleotide composition,secondary structures of tRNAs and gene rearrangement were analyzed,and its application in spiderphylogenetic analysis was also discussed.Our study significantly increases the barcode reference sequence library for Chinese cropland spiders,and will be very useful in pest management,eco-environmental protection and spider evolutionary study.