Genetic Diversity And Divergence Time Estimation Of The Rare Butterfly Genus Of Teinopalpus Based On Noninvasive Sampling

The butterfly genus of Teinopalpus,endemic to Asia,has only two sister speciess,T.aureus and T.imperialis.,They are typical mountain butterflies with special habitat requirements,showing rarity in the wild,and high conservation and research value.For example,T.aureus is known as one of the world’s "Three Incredible Butterflies",and both sister species were listed on the IUCN Red List of Protected Species earlier(1985),and both are also listed on the Chinese List of Key Protected Wildlife(Class I or II).The origin of the two sister species from the same ancestral lineage has been confirmed by morphological and molecular evidence,but there are still some doubts about their interspecific relationship.For example,the two sister species have overlapping geospatial occurrence areas(e.g.,in Guangxi and Yunnan)and co-occurrence sites from southeastern Yunnan to the mountains of northern Vietnam,and heterozygous individuals with morphology in between have been recorded in the field.In addition,there are also problems and controversies about "over-subspeciation" in the classification of the two sister species.Therefore,there is an urgent need to obtain more extensive genetic material evidence to systematically verify the interspecific relationships(e.g.,taxonomic status,material differentiation,etc.)and infraspecific relationships(subspecies status,kinship geography,etc.)of the two sister species of Teinopalpus,and to reveal the genetic structure,diversity level,It lays the foundation for future conservation genetics research and also provides a theoretical basis for butterfly species conservation and management.Like other rare and protected species,the genetic analysis study of rare Teinopalpus faces the problem of difficult sampling.For this reason,we firstly tried to extract DNA from non-damaged materials such as feces excreted by larvae,spatula from the foliage of feeding carvings,head shells left by larvae molting,molts and pupal shells left by pupae after fledging,and compared the quality of DNA extracted from muscle tissues of naturally dead individuals reared in protected areas,and mitochondrial molecular marker method to verify the feasibility of the non-damaged method.After that,based on this method,DNA was obtained from the undamaged materials of the two sister species of Teinopalpus,such as feces,food carcasses and pupal shells,collected in recent years in various places,and then PCR amplification technique was applied to determine the mitochondrial DNA sequences of different geographical populations using Teinopalpus-specific primers.The sampling area covered the occurrence area of T.aureus(Jiu Lian Shan,Jing Gang Shan,Wuyi Shan,etc.),the coexistence area of the two sister species(Ma An Di,Yunnan,etc.),and the occurrence area of T.imperialis(Ya’an,Sichuan,Cen Wang Lao Shan,Guangxi,etc.),involving 12 geographic occurrence sites.In combination with the data from six existing occurrence sites(from Prof.Shanyi Zhou’s research team,NCBI database,etc.),we applied mitochondrial molecular markers to analyze the interspecific and infraspecific genetic material differences and variation of rare Teinopalpus more systematically;explore the genetic differentiation and kinship geography;construct a phylogenetic tree and explore the molecular evidence of the origin and dispersal of rare Teinopalpus in combination with the molecular clock model.The main findings and conclusions of the study are as follows:(1)We developed a non-invasive DNA sampling technique for Teinopalpus.By improving the DNA extraction method,DNA was successfully obtained from larval feces,larval cephalic shells and molts,filamentous gland secretions left by larvae on food carvings,and pupae shells after plumage,respectively.although the DNA concentration of nondamaged material was lower compared with that of muscle material of newly dead larvae at rearing,it could meet the requirements of mitochondrial molecular markers and splice to obtain complete cyclic sequences for interspecific and infraspecific genetic relationship analysis.This method can be extended to other insects.(2)The mitochondrial molecular structure of the two sister species of Teinopalpus was characterized.The mitochondrial genomic sequences of T.aureus and T.imperialis were measured to be between 15228 and 15240 bp in length,and the mitochondrial genomic sequences of T.imperialis were between 15226 and 15256 bp in length,both of which are closed-loop molecular structures.The mitochondrial genome has roughly the same gene structure and arrangement order as other butterfly insects,with 37 genes,containing 13 protein-coding genes(COI-III,ND1-6,ATP6,ATP8,Cytb,ND4L),2 r RNA genes(12s r RNA,16 s r RNA),22 t RNA genes and a non-coding control region(D loop).Comparing the intraspecific variation in gene fragment structure of the two sister species,it was found that T.aureus showed more pronounced expression than T.imperialis,especially in the DYS and HN populations.It was found that ND4,ND5,16 Sr RNA,12 Sr RNA,and D-loop showed more obvious structural variation between species,which may be more suitable for interspecific genetic relationship analysis,while COI,COII,ND1,ND3,ND6,Cytb,and ND4 L showed more structural variation within species,which may be more suitable for intraspecific genetic analysis.(3)To reveal the genetic diversity of the two sister species of Teinopalpus based on mitochondrial molecular markers and to infer the historical population dynamics.The results showed that the nucleotide diversity of rare Teinopalpus species was generally low(π < 0.005),especially for T.imperialis(π = 0.0022)compared to T.aureus(π = 0.0047).However,the two sister species(0.993 for T.aureus and 0.995 for T.imperialis)had high haplotype diversity indices(>0.5),and the combination of the two diversity indices suggests that both butterfly populations may have experienced a bottleneck effect,followed by rapid population expansion and variation accumulation.The neutral test(Tajima,s D,Fu,s Fs)of both sister species showed negative results and did not reach the significant level,and combined with their multi-peaked mismatch distribution,it was hypothesized that the two sister species populations were generally stable,but there might be excessive low-frequency polymorphism or purifying selection.(4)The genetic variation and differentiation of the two sister species of Teinopalpus and their gene exchange between geographical populations were revealed based on mitochondrial molecular markers.The results of AMOVA of combined gene sequences showed that the intrapopulation variation rate of T.aureus(63.19%)was significantly higher than the interpopulation variation rate(36.81%),while the variation rates of both T.imperialis were comparable and the interpopulation variation rate(53.3%)was slightly higher than the intrapopulation variation rate(46.7%),implying that the interpopulation variation rate of T.aureus was relatively stronger and reduced the genetic differences between populations.This implies that the relatively stronger gene exchange between populations of T.aureus reduced the genetic variation between populations but increased the genetic variation within populations,while the opposite was true for T.imperialis.The mean differentiation coefficient(Fst)and mean gene flow index(Nm)of each paired geographic population showed that T.aureus populations were strongly genetically differentiated(Fst > 0.25)but with some gene flow,except for the NL population(Fst =0.069),which was weakly differentiated and had strong gene flow(Nm =-3.14);however,the HN(Fst= 0.615),JGS(Fst = 0.53)populations were the most differentiated and had low levels of gene exchange(Nm < 1),and it is speculated that both populations may be driven by genetic drift.In contrast,all geographic populations of T.imperialis were very strongly differentiated(Fst > 0.5),while MAD populations(Fst = 0.82)and CWLS populations(Fst = 0.73)were the most differentiated and had the weakest gene flow(MAD: Nm = 0.11;CWLS: Nm = 0.2).Analysis of geographic distance and genetic distance revealed a strong correlation between T.imperialis(Pearson’s r = 0.73)and a weak correlation between T.aureus(Pearson’s r = 0.31),suggesting that genetic differentiation in T.imperialis populations This suggests that the genetic differentiation of T.imperialis populations may be mainly driven by geographical isolation,while T.aureus is also strongly driven by other factors(e.g.,host plants)in addition to geographical isolation.(5)Phylogeny of mitochondrial genes based on 18 geographical populations of the two sister species.The results show that the two sister species are clearly related generically,with T.aureus: BZS populations diverging first and forming a separate branch;JGS populations clustering with NL populations as a sister group and with WYL as a branch,indicating that JGS is more closely related to NL populations;HN clustering with YN populations as a branch,but with low confidence;WYS clustering with PS and MHS populations as a branch with high confidence,indicating that WYS may be more closely related to MHS and PS populations than other populations;DMS and DYS populations clustered as sister groups with high confidence and then clustered as one with JLS populations,indicating that DMS,DYS,and JLS are more closely related.In T.imperialis: CWLS and MYA populations were separately branched,while four populations,LCS,JJS,GGS and MAD,were clustered into one group,indicating that the other four geographic populations were more closely related to each other except for CWLS and MYA populations.(6)The view that T.aureus is more primitive is based on the divergence time estimates,which is different from the previous view that T.imperialis is more primitive.Since the BZS population where T.aureus first diverged and the CWLS population where T.imperialis first diverged are both located in the mountainous area at the border of Guangxi and Guizhou and extend to Yunnan,it is inferred that the divergence center of Teinopalpus species is at the border of Guangxi and Guizhou,rather than the "Yunnan and Sichuan origin center" or "Nanling origin center" as inferred in the previous study.The original ancestors of Teinopalpus probably diverged into two different populations in this region during the Great Plateau Ice Age and spread and evolved in all directions.