Sequencing And Analysis Of The Complete Mitochondrial Genomes For Five Species Of Phylloscopus And Development Of Their SSR Markers

Approximately 66 species and 112 subspecies of warblers in the genus Phylloscopus(order Passeriformes,superfamily Sylvioidea,family Phylloscopidae)exist worldwide.Oftentimes,several Phylloscopus spp.share the same habitat.There are many barriers to species classification,field identification,and phylogeny that have always been a challenge for research in the field of avian taxonomy.This study is the first to determine the complete mitochondrial genome sequences of five species of Phylloscopus using first generation sequencing technology.These sequences,combined with published mitochondrial genome sequences for Phylloscopus inornatus and Phylloscopus proregulus,were used to analyze the base composition,protein-coding genes(PCGs),and phylogeny of all seven species.Our study was also the first to develop 27 pairs of microsatellite primers for six species of Phylloscopus,which we achieved through the use of restriction site–associated DNA(RAD)technology.The main results of our study are provided below.1.Mitochondrial genome structures and characteristics in five species of PhylloscopusThe complete mitochondrial genome lengths were as follows: Phylloscopus tenellipes,16,904 bp;Phylloscopus coronatus,16,905 bp;Phylloscopus borealis,16,881 bp;Phylloscopus schwarzi,16,920 bp;and Phylloscopus borealoides,16,904 bp.The complete mitochondrial genomes contained two noncoding control regions and 37 typical mitochondrial genes comprising 22 transfer RNA genes,2 ribosomal RNA genes,13 protein-coding genes,and 2control regions.The size and coding direction of genes in the whole mitochondrial genomes for each of the five species of Phylloscopus were very similar to those of Phylloscopus inornatus and Phylloscopus proregulus.Furthermore,the AT content of protein-coding genes in the five species ranged from 50.6% to 52.1%.2.Comparative and phylogenetic analysis of mtDNA from seven species of PhylloscopusThere was a slight A + T bias in the mitochondrial genomes of the seven species of Phylloscopus.Furthermore,the codon usage patterns in those seven species were similar,with two-and four-fold degenerate codons being overused at the third codon position.The total length of 13 PCGs averaged 11,395 bp,accounting for 67.44% of the seven complete mitochondrial genomes.Genetic distances in the 13 PCGs showed low variation in the mitochondrial genomes of the seven species.The nonsynonymous/synonymous substitution rate ratios(Ka/Ks)of all 13 PCGs were less than 0.28.A previous analysis had found that the molecular evolutionary pattern of single PCGs in the seven species of Phylloscopus is similar to that of other vertebrates.In addition,phylogenetic topology studies have shown that the genetic relationship between P.tenellipes and P.borealoides is closer than between P.borealis and P.borealoides.Taken together,the above findings provide a reliable basis for upgrading P.borealoides from subspecies to species.3.Development of general microsatellite markers for PhylloscopusSecond-generation sequencing technology(i.e.,RAD)was used to simplify the genomic sequencing of 4 individuals from three species of Phylloscopus.An average of 6,016 simple sequence repeat loci were detected in 4 samples.Primer 6.24 software was used to design 82 pairs of primers for those loci.Six species(P.tenellipes,P.coronatus,P.borealis,P.schwarzi,P.borealoides,and P.inornatus;one sample from each species)were selected for screening.From the 82 pairs of primers,27 pairs of microsatellite loci with good amplification efficiency were identified.Information on polymorphism at the loci was obtained by high-performance capillary electrophoresis(HPCE)of 48 specimens from the six species.Results showed that the 27 loci comprised 10 low polymorphic loci,9 moderate polymorphic loci,and 8 high polymorphic loci.Thus,the microsatellite primers could be used for detecting intraspecific and interspecific genetic diversity,genetic mapping of quantitative traits,genetic linkage mapping,and identification of families and individuals from the genus Phylloscopus.This study is of theoretical significance,as there are few reports on the genetics of Phylloscopus at home and abroad.The results we obtained herein broaden the understanding of the taxonomic position of Phylloscopus within the Sylviidae family and help elucidate the phylogeny of Phylloscopus spp.Moreover,the microsatellite markers we identified provide reliable information for genetic diversity analysis,genetic mapping of quantitative traits,genetic linkage mapping,and identification of taxonomic families and individual species.