| Liriomyza is a large genus including numerous species in it.Of those species,about 160 species is damaging cultivated crops and ornamental plants.Due to the small size of these insects(1.3-2.1 mm in length),similarity in morphological characteristics,obvious niche overlap,rapid interspecific competition and displacement,as well as the limitations of the status or integrity of insect body,it is difficult to classify and identify these species;and this will cause confusion in practical work like plant quarantine,monitoring or control.In this study,five species of Liriomyza,L.trifolii,L sativae,L.huidobrensis,L.bryoniae and L.chinensis were collected from 38 cities across 11 provinces in mainland of China from 2014 to 2017.The first three species(L trifolii,L.sativae,and L.huidobrensis)are the most destructive invasive species.In order to further investigate the variation of morphological characteristics and genetic differentiation at inter-species and intra-species level of Liriomyza,as well as to provide accurate knowledge for quarantine identification,a comprehensive comparison was made on the morphological characteristics from 4 771 samples of five species of Liriomyza.Furthermore,in order to develop a more stable,rapid and convenient identification method,the whole dorsal features of 285 samples across five species were observed and recorded by pictures(see Appendix Ⅰ).In addition,the mitochondrial Cytochrome Oxidase I(COI)gene was sequenced to validate the identification of species.Moreover,the COI gene fragment sequences from 258 individuals of Liriomyza and the EF-la gene fragment sequences of 255 individuals of Liriomyza were analyzed;284 individuals of Liriomyza were also analyzed based on microsatellite markers to make a thorough investigation of the genetic structure of Liriomyza,the mitochondrial genome of L.chinensis was sequenced and analysed.The main findings are as follows:1.Morphological identification characteristics of five species of LiriomyzaThe male external genitalia of five species of Liriomyza are basically the same as described in Spencer(1973),indicating external genitalia is still the most reliable features for morphological identification of Liriomyza.However,most of the existing literature were based on Spencer’s hand-drawn identification images,and there is a lack of clear physical comparison and identification charts.Therefore,we took clear photos of five species of Liriomyza by actual male genitalia,as a supplement to the hand-drawn identification charts.In addition,the most frequently used identification characteristics of Liriomyza were found to have some limitations:1)The color characteristic of the outer and inner vertical bristles on hind-margin of eye is unstable and the boundary between species is not clear,which could only be used as an auxiliary identification feature;2)The ratio of the end segment length of the forewing M3+4 veins to the second end segment length varied within species and overlaped between species;therefore,there is no obvious absolute boundary of this ratio between species.We can not distinguish L.trifolii and L.sativae based on these characters;therefore it can only be used as an auxiliary identification feature to distinguish other species;3)The abaxial pattern of the abdomen is variable,while the L.trifolii is unique.In order to identify the five species of Liriomyz quickly and accurately without dissecting or DNA sequencing,this paper summarizes the most stable and reliable morphological characteristics of the five species:1)L.trifolii:A yellow longitudinal stripe divides black transverse stripes into two rows on the abdomen in dorsal view,or at least there is a large or small yellow plaque which can be distinguished from other species in the middle of the visible penultimate black transverse stripe.And the color of the outer and inner vertical bristles on hind-margin of eye are mostly yellow;2)L.sativae:The bottom edge of the penultimate black transverse stripe on the abdomen in dorsal view is smooth,without obvious notch,and the color of the outer vertical bristles on hind-margin of eye are black;3)L.bryoniae:The bottom edge of the penultimate black transverse stripe on the abdomen in dorsal view is smooth or a large arc with no notch in the middle,and the color of outer and inner vertical bristles on hind-margin of eye are yellow;4)L.huidobrensis:The leg segment have obvious black patches,and both the inner and outer bristles are black;5)L.chinensis:The small shield is completely black,and the color of the inner bristles are yellow,the location of the outer bristles are circular or irregular black patches which are obviously different from other species of Liriomyza species.2.Genetic structure analyses based on mitochondrial COI gene and nuclear EF-la geneThe results showed that COI gene from five species of Liriomyza populations showed relatively conservative characteristics among populations from different geographical and host plants;therefore COI gene can be used as a molecular marker for Liriomyza species identification.The mutation rate of EF-1a gene is relatively higher;therefore EF-1a gene can be used to study the genetic structure of Liriomyza population.The phylogenetic tree based on COI haplotypes showed that the five species of Liriomyza have obvious interspecific differentiation.L.trifolii and L.sativae have the closest relationship;although their morphology is very similar,there is no gene exchange or hybridization between species.The relationship of L.bryoniae and L.huidobrensis is the closest while the relationship between L.chinensis and other four species of Liriomyza is less close,which is the same as the similarity of the male genitalia.The phylogenetic tree based on EF-la haplotypes is consistent witih the phylogenetic tree based on COI haplotypes,but the interspecific differentiation between the haplotypes of L trifolii and L.sativae is not obvious.The genetic structure analyses results showed that differentiation degree and direction of nuclear gene and mitochondrial gene are not completely consistent.3.Genetic structure analysis based on microsatellite markersEight microsatellite loci of L.trifolii were selected to further analyze the genetic structure of 20 L.trifolii populations and 6 populations of the other four Liriomyza species.The results are consistent with the results based on COI gene and EF-la gene.The genetic distances among the five species of Liriomyza are significantly further than those populations within the species.The highly interspecific differentiation showed the five species(especially between L.trifolii and L.sativae)have high level of genetic differentiation both in mitochondrial and nuclear genes,and the interspecies differentiations are obvious.The observed heterozygosity(Ho)in half of L.trifolii populations are greater than 0.5,indicating that the genetic diversity of L trifolii populations in China is at a high level.The level of genetic diversity of the intra-populations in China is much lower than inter-populations of Liriomyza,indicating that geographic isolation and other factors still have a certain degree of influence on gene exchange among the populations of Liriomyza,influence of host plants on the genetic differentiation were not detected.The results also showed that some populations of L.trifolii in mainland China were in an high or moderate degree of genetic differentiation,and they could be divided into two groups,the populations from coastal areas of south China are clustered into one group and the northern populations are another group,and the invasion and spread trend of L trifolii should be from south to north.The results of bottleneck test analyses show that the population of L trifolii in mainland China is in a steady growth period.4.Mitochondrial genome of L.chinensisThe total length of mitochondrial gene of L.chinensis is 16175 bp(GenBank login number MG252777),including 37 mitochondrial genes(13 protein coding genes,22 transporting tRNA,2 ribosomal rRNA)and one non-coding region(control region).The sequence and structure of L.chinensis are basically consistent as the model organism Drosophila melanogaster.23 genes(9 protein-coding genes and 14 transporting tRNA)are distributed on the J-chain,14 genes on the N-chain(4 protein-coding genes,8 transporting tRNA and 2 ribosomal rRNA),16 gene have 64 bp spacers in total,the shortest was only one bp,and the longest was 19 bp,located in tRNAGlu and tRNAPhe.There are nine overlapping genes in the mitochondrial genome of L.chinensis,and the longest overlapping gene is 8 bp between tRNATrp and tRNACys.The mitochondrial genome of L.chinensis is similar to other species of Liriomyza.In addition,the genomic sequence of L.chinensis is consistent with the ancestral ancestor trnI-trnQ-trnM.Similar to the other five species of Agromyzidae,no rearrangement was found in the whole mitochondrial genomes of L.chinensis,indicating that the mitochondrial genome of Agromyzidae are conservative in length and location of gene spacers.In this paper,the phylogenetic relationships of 5 species of Liriomyza were constructed by analyzing the base sequences of 13 protein coding genes in mitochondrial genes.The consistent results showed that L.chinensis should belong to Liriomyza. |
