Phylogeny Of Rhus Gall Aphids,and Its Primary Host Plants And Endosymbionts,and Their Coevolutionary Implication

Rhus gall aphids(Insecta: Hemiptera: Aphididae: Eriosomatinae: Fordini)live on the primary host plants of Rhus(Anacardiaceae)and induce galls that are often known as the Chinese galls or Rhus galls,which have been widely used in many fields with rich tannin.The classification was different at genus and species level by different researchers.Rhus gall aphids switch between the primary hosts and secondary hosts to finish their life cycle,and most of them are unique for their primary hosts.They are distributed in eastern Asian and North American to form a disjunct distribution pattern.Also,they have an obligate mutualistic relationship with the endosymbiont-Buchnera inhabiting specialized cells called bacteriocytes in body.The endosymbiont provides essential substances for the normal development of aphids.The coevolutionary pattern of Rhus gall aphids,its primary host plants and endosymbiont Buchnera is special and complex,which has been a good model system for the coevolution study.The primary host plant and gall shape are characteristics for the aphid identification.Herein,we collected Rhus galls with obvious variation in the gall shape,and observed the morphological characteristics of alate viviparous female,combined the gall shape and its primary host plant to identify the aphid species.Then,we assembled the complete mitochondrial genome and universal single-copy nuclear genes of Rhus gall aphids and complete genome of endosymbiont Buchnera with genome skimming method.Combined with the complete chloroplast genome of primary host plants from GenBank,we reconstructed the phylogenetic relationships of Rhus gall aphids,its primary host plants and endosymbiont Buchnera,respectively.We estimated the divergence time of Rhus gall aphids and its primary host plant,respectively,and compared phylogeny of Rhus gall aphids and its primary host plant to investigate co-evolutionary origin and intercontinental biogeographic disjunction.We also compared phylogenetic framework of Rhus gall aphids and endosymbiont Buchnera to investigate their coevolutionary pattern.The main results are as followings:(1)Morphological characteristicsMorphological observation of alate viviparous females of 15 species in six genera showed that Nurudea and Schlechtendalia have both antennae with five antennomeres,and Floraphis,Kaburagia,Meitanaphis and Melaphis have both antennae with six antennomeres.Several samples of the two species Melaphis rhois and M.asafitchi have one five-antennomere antenna.Morphological characters of secondary sensoria and veins of fore wing are more variable across species.Based on morphological characters of alate viviparous females,shape of galls and primary host plants,we found the variable gall shapes in our samples,which formed by Floraphis meitanensis,Schlechtendalia chinensis,Kaburagia rhusicola ovatirhusicola and K.r.ovogallis.And K.r.ovatirhusicola and K.r.ovogallis on Rhus potaninii and R.punjabensis.Furthermore,both of K.r.ovatirhusicola and K.r.ovogallis can live on two primary host plants(Rhus potaninii and R.punjabensis).(2)Mitochondrial genome characteristics,phylogeny and evolution of Rhus gall aphidsWe sequenced the genome of the 93 samples representing 13 species and four subspecies in six genera of Rhus gall aphids,and assembled their mitochondrial genome sequences,which was a double-stranded circular structure,and contained 13protein-coding genes,two rRNA genes,22 tRNA genes and one control region.Some aphid genomes also contained repeat regions.The mitochondrial genome sequence of Rhus gall aphids had a strong bias towards A + T.The start codon of all protein-coding genes is ATN and the stop codons are TAA,TAG or single base T.All the tRNAs could be folded as a classical clover-leaf secondary structure except for trnS1,which lost the dihydrouridine(DHU)arm.The inferred secondary structure of rRNA had many helices and domains.The control region included seven parts: two conserved sequence blocks,two A + T rich region,one T + C rich region,one poly-T region and one G + C rich region,while and the species(Kaburagia,Meitanaphis,Melaphis,Schlechtendalia peitan)had an extra tandem repeat region.Comparative analysis with the mitochondrial genome of other aphids from GenBank showed that in contrast to the trnI-trnQ-trnM in most Aphididae,a trnI-trnM-trnQ order was found in all lineages of Rhus gall aphid mitochondrial genomes.Selection pressure analysis showed that atp8 genes of the most aphids was experiencing strongly positive selection,and the other protein-coding genes were experiencing purified selection.We resolved the phylogenetic relationship among genera and species based on two different types of molecular markers(mitochondrial genome and single-copy nuclear gene).The result showed a consistent phylogenetic relationship among species or subspecies,but there are large differences in the relationships among genera.The six genera of Rhus gall aphids composed six main clades with high support,while only Floraphis and Melaphis is monophyletic.Nurudea shiraii and N.yanoniella formed a monophyletic group that was sister to the remaining Rhus gall aphids,while N.ibofushi was sister to Schlechtendalia chinensis.However,their alate viviparous females have obviously different morphological characters.Meitanaphis flavogallis forms a clade with Kaburagia.Combined with the morphological characters,we suggested that Meitanaphis flavogallis should might be merged into the genus Kaburagia.The conflict analysis also showed that the conflict nodes with a high degree are all inconsistent nodes in the topology of phylogeny.The divergence time results showed that the origin of the ancestors of Rhus gall aphids and the divergence time between genera of Rhus gall aphids was estimated in the Cretaceous,which was the main period of the plate movement of North America and Eurasia.(3)Coevolution between Rhus gall aphids and its primary host plantsWe reconstructed the phylogenetic relationship,and estimated the divergence times of five primary host plants of Rhus gall aphids based on the complete chloroplast genome from GenBank.The results showed that the divergence time of the five species was concentrated in the Eocene to Pleistocene.Compared the correlation of the phylogenetic relationship and divergence time between Rhus gall aphids and its primary host plants,the result showed that the divergence period of the host plants is obviously later than Rhus gall aphids.There is no possibility of Rhus gall aphids shift from other distant plants to present plants based on the strict parasitism with their host plants.However,the divergence time and relationships among some Rhus gall aphids with relatively late diverging time had the correlation and consistence with their corresponding primary host plants,which implied that parallel evolution between these Rhus gall aphids and its primary host plants.(4)Genome characteristics and phylogeny of the primary endosymbiont Buchnera in aphidsHerein,we assembled the complete genome representing 11 species and two subspecies in six genera of the endosymbiont B.aphidicola in Rhus gall aphids.The Buchnera genome is a single circular genome.The genomes ranged from 605,233 to626,129 bp,and contained 532 to 550 protein-coding genes,31 to 33 tRNAs,one ncRNA,one tm RNA and three rRNAs.Comparative genomic analysis showed four rearrangements for protein-coding genes in the entire sampled radiation to date: one inversion and three rearrangements.These genomes are relatively stable in terms of gene order,in contrast to the high levels of nucleotide divergence.There has been substantial gene-loss in the Buchnera lineage.We reconstructed the phylogenetic relationship of the primary endosymbiont Buchnera in aphids,which showed three major clades with high support corresponding to the subfamilies of aphids: “Aphidinae”,“Eriosomatinae” and other six subfamily clades,respectively.Aphidinae is relatively far from the other clades.(5)Coevolution of Rhus gall aphids and its primary endosymbiont BuchneraCophylogenetic analyses showed the complete consistence of phylogenetic relationship between Rhus gall aphids based on mitochondrial genes and its Buchnera based on 144 genes,which had the stronger coevolutionary signal than the cophylogenetic analyses between single-copy nuclear genes of Rhus gall aphids and Buchnera.This result indicated that the evolution of Buchnera was in accordance with mitochondrial genome of their Rhus gall aphid hosts.The phylogenetic relationship among genera of Rhus gall aphids based on single-copy nuclear genes had obvious disagreement with its relationship of Buchnera.The host switch events of the endosymbiont Buchnera between genera of Rhus gall aphids proved that the hybridization might occur in the ancestors of some genera in Rhus gall aphids.In summary,we examined the relationship of Rhus gall aphids,and analyzed the coevolution patterns between Rhus gall aphids,and its primary host plant and primary endosymbionts.These studies not only obtain the genomic information of Rhus gall aphids and its host plant and endosymbionts,but also provide molecular biological evidence for the protection and rational development of this special natural resource.