Phylogeography Of Four Cicada Species From China And Adaptive Evolution Of Subpsaltria Yangi

Cicadas are excellent subjects for speciation and bio-geographical studies because of their low dispersal ability caused by the generally long,subterranean pre-adult stage and short,adult lifespan compared with other insects.The acoustic signals produced by male cicadas can be used in studies of cryptic species,sibling species,and elucidation of conflicting gene trees and speciation,and rapid assessment of local cicada biodiversity.Although extensive phylogeographic studies have been conducted on cicadas,they were mainly focused on cicadas in Australia,New Zealand,the Mediterranean and America,etc.Traditional morphological,phylogenetic,bioacoustic and behavioral methods have been applied to cicadas in China,but few studies were focused on the phylogeography Chinese cicadas.Therefore,the origin,dispersal and formation of distribution pattern and evolutionary history of some representative cicada species in China were studied in the present dissertation based on morphological,molecular,bioacoustical,behavioral and ecological data.In addition,cosidering that the distribution range of the Chinese endemic cicada Subpsaltria yangi is narrow but the habitat differentiation of this species is significant,we applied a Genotyping-by-sequencing(GBS)approach combined with molecular,bioacoustic and ecological data,to investigate the genetic composition and genomic divergence of this species.We also conducted dispersal-vicariance and phylogeographic analyses,and compared the genotype and calling-song phenotype of different populations of this species,aiming to elucidate the potential impact of geological isolation and host shift on the evolutionary history of this species.The main results are as follows:1)Integrative taxonomy and population differentiation of Hyalessa maculaticollis.This species is widely distributed in East Asia,and is noted for its great morphological variability.The variation in this species and its allies Hyalessa fuscata has been a long-standing controversy.The population differentiation,genetic structure and phylogeography of this species were explored based on morphological observations,mitochondrial and nuclear DNA analyses,and comparison of the calling song structure of males.The results revealed that the abundant intraspecific morphological variations are consistent with the high levels of genetic divergence in this species.Phylogenetic and network analyses indicated that H.maculaticollis was composed of two major lineages,i.e.,teh Chinese and Japan lineages.The East China Sea(ECS)land bridge acted as a dispersal corridor for H.maculaticollis during the glacial period.The climatic oscillations in the Pleistocene and the terrain structure of East Asia influenced the population differentiation.The divergence time between the two sides of the East China Sea was estimated to be ?1.05(95% CI = 0.80–1.30)Ma,which was about the same period that the sea level increased rapidly during the ?Ryukyu Coral Sea Stage?(0.2–1.3 Ma).Populations of H.maculaticollis were structured phylogeographically,with the China populations differentiated into more haplogroups and highly structured.Qinling Mountains and the mountainous regions around the Sichuan Basin were presumed to have been major refugia for H.maculaticollis in glacial periods,and a recent population expansion has been detected for populations distributed in the area to the north of Qinling Mountains.The high degree of haplotype and nucleotide diversity showed in East China populations suggested that the flat terrains with low-altitude hills were suitable for the survival of H.maculaticollis.The species H.fuscata,formerly treated as an independent species from H.maculaticollis by some researchers based on acoustic analyses of the calling song structure,was confirmed to be a junior synonym of H.maculaticollis by our results of analyses based on morphological variation,the calling song structure and acoustic playback experiments.2)Phylogeography of Platypleura hilpa.Climate change and geographical events played key roles in driving population genetic structure of organisms.Different scenarios were suggested for species occurring in tropical and subtropical areas,where have never been covered by ice sheets.We used an integrative approach based on mitochondrial genes,nuclear genes and microsatellite markers to study the population genetic structure,diversity and demographic history of the cicada P.hilpa occurring in coastal areas of southern China and northeastern Indo-Burma,aiming to elucidate the potential impact of climate change and geological events on its evolutionary history.Platypleura hilpa comprises five main lineages with nearly unique sets of haplotypes and distinct geographic distributions.Ecological niche modeling(ENM)and diversification analyses revealed that several climatic factors were associated with the divergence.Combined with molecular data,the ancestral range reconstructions indicated a major split occurred in the Pleistocene.The potential range during the Last Glacial Maximum(LGM)had apparently increased in South China,remained more or less stable on the Leizhou Peninsula,and expanded on the exposed South China Sea Shelf.The highest genetic diversity in the South China lineage suggested this region has been climatically stable.The percentages of total,marginally,moderately and highly suitable habitats were predicted to decrease by 2050.The Pleistocene sea-level fluctuations had profound effects on regional genetic structure of P.hilpa.The Beibu Gulf represented a more important geographic barrier than the Qiongzhou Strait in blocking gene flow among the populations.These results contributed to a better understanding of the pressure climatic change imposes on insects in coastal areas of East Asia.3)Distribution pattern and evolutionary history of Karenia caelatata.The Hengduan Mountains(HDM)are a biodiversity hotspot and had a complex and unique geological structure.Karenia caelatata produced calling songs using their forewings clapping the lateral margins of opercula,which is an endemic species distributed in the HDM,the Qinling-Bashan Mountains,the Yunnan-Guizhou Plateau and the Nanling region of China.Morphological variations and differentation of acoustical signals among geographical populations of K.caelatata made it an ideal model to investigate the mechanism of speciation and diversification of montane insects in eastern Asia.The population differentiation,genetic structure and phylogeography of this species were explored based on morphological,acoustic and molecular data.The results revealed that the abundant intraspecific morphological variations were consistent with the high levels of genetic divergence in this species.The calling song structure of HDM population was significantly different from that of other populations.Phylogenetic,BPPS and network analyses indicated that K.caelatata is composed of six major lineages with almost unique sets of haplotypes and distinct geographic distributions.In conjunction with molecular dating,the ancestral range reconstructions indicated that a major split occurred in the Pleistocene(?3.0 Ma),which is approximately consistent with the uplift of the HDM.The results indicated that historical events such as orogeny and climate change played key roles in promoting the diversification of this species.Based on gene flow analysis,we speculated that the species originated from the HDM,and spread eastward to Dalou Mountains(Guizhou Plateau),northward to Qionglai Mountains,and then to Qin-Ba Mountains.The population in the HDM had a high differentiation rate according to the results of analyses of diversity and population differentiation,which verified the uplift-driven diversification hypothesis.Ecological niche modeling(ENM)analyses revealed that the potential range during the LGM was basically the same as the present distribution.The HDM,Qin-Ba Mountains and Nanling regions might have provided glacial refugia for this species.These results provided a good model for understanding how the complex geological events in East Asian continent shaped the diversity of species.4)Population differentiation and phylogeography of S.yangi.Geographical isolation has been identified as major factor that contribute to the formation of new species.To determine whether geographical barriers contributed to population divergence or speciation in the rare cicada S.yangi which is endemic to the Loess Plateau and adjacent areas of northwestern China,the population differentiation,genetic structure and phylogeography of this species were investigated by analysing mitochondrial and nuclear DNA and comparing the calling song structure among different populations.The results revealed a low level of genetic differentiation and relatively simple phylogeographic structure for this species,but two independent clades corresponding to geographically isolated populations were recognized.Genetic and geographical distances were significantly correlated among lineages.Results of divergence-time estimation are consistent with a scenario of isolation due to glacial refugia and interglacial climate oscillation in northwestern China.Significant genetic divergence was found between the population occurring in the Helan Mountains and other populations,and during the LGM population expansion has occurred in the Helan Mountains and/or adjacent areas.This population was also significantly different in calling song structure from other populations.Geographical barriers(i.e.,the deserts and semi-deserts surrounding the Helan Mountains),possibly coupled with related ecological differences,may have driven population divergence and allopatric speciation.5)Adaptive evolution in S.yangi based on Genotyping-by-sequencing(GBS).A draft genomic sequence of S.yangi was reported based on a Genotyping-by-sequencing(GBS)data and mitochondrial genes,to which a set of 7,216 single nucleotide polymorphisms(SNPs)were aligned.This species comprised four main lineages with nearly unique sets of phylogenetic and calling song structure,which were distinctly related with the host plants and the ecological environment of habitats.Results of pairwise comparisons of the genotypic and phenotypic variations of different populations indicated that only geographic distance between pairs of populations can well predict the genome divergence.Analysis of spatial autocorrelation of phenotypic variables and geographic distance revealed that acoustical signal and geographic distance together explain over 60% of the genome divergence among populations.Our results suggested that geomorphic barriers and climate oscillations associated with Pleistocene glaciation serve as the primary factors in shaping the population genetic structure of this species,and highlighted the importance of host shift in driving population divergence and speciation of herbivorous insects.The association of genotype and calling-song phenotype among different populations of S.yangi improved our understanding of the process of speciation and adaptive micro-evolution of this species.This provides a possible example of incipient speciation in Cicadidae,and informs future studies on population differentiation,speciation and phylogeography of other insects with a high degree of endemism in the Helan Mountains and adjacent areas.This study may also inform future studies on the molecular mechanisms underlying the relationship between genetic/phenotypic changes and adaptation of herbivorous insects to novel host plants and new niches.In conclusion,our results indicate that climate change and geological historical events are important factors leading to the diversification,speciation and the formation of phylogeography of cicadas in East Asia.Our results suggest that the integration of morphology,molecular biology,bioacoustics and behavioral ecology,besides solving some controversies related to the systematics of cicadas,may also provide new information for further studies on population differentation,distribution pattern and genealogy of other insects occurring in East Asia.