| The subfamily Pyraustinae is one of the largest subfamilies in the Crambidae.The larvae of many species are economically important pests in agricultural and forestry crops.It currently contains more than 1,413 recognized species placed in 190 genera.The phylogeny and classification of the subfamily Pyraustinae has long become very difficult based on morphological characters which is mainly due to that there are many undescribed sibling species and close-related genera,even without clearly interspecific and intraspecific diagnostic characteristics in the subfamily.DNA barcoding is a molecular-based tool for biological identification which employs a short standardized gene region as a useful technique for species assignment and for the discovery of cryptic diversity.It also has great potential to assist in clarifying morphology-based taxonomic investigations and inferring the evolutionary relationships among living organisms.In this thesis,the research status of DNA barcoding technique is briefly introduced and the taxonomic study on the subfamily Pyraustinae is reviewed.However,the general aim of the thesis is to build a comprehensive reference DNA barcode library and investigate cryptic species diversity in the subfamily Pyraustinae from China and North America,and attempt to deal with the systematics and phylogeny of the subfamily combining both morphological characteristics and mitochondrial COI gene sequence.1.A comprehensive reference library of DNA barcode sequences and its application for species identification to the Pyraustinae from China and North America.In total,922 COI sequences,including 665 standard barcodes and 251 mini-barcodes were recovered from 253 species in 52 genera with a success rate of 79%.These taxa included representatives of 101 species in 31 genera and 154 species in 27 genera from China and North America respectively.More than three COI barcodes were obtained from 111 species,but 142 species had only oneor two COI barcodes.Of the 54 genera sampled in the Pyraustinae,only two genera Munroeodes,Torulisquama were not successfully barcoded.We also attempted to test and confirm the utility of DNA barcoding for species identification in the Pyraustinae.Our results indicated that the intraspecific genetic distance ranged from 0-7.75%(mean 0.39%),whereas interspecific variation ranged from 0-18.57%(mean 9.12%)for standard barcodes.A clear gap exists between intraspecific and interspecific divergences was observed at a 2.0%threshold which made it possible to identify and distinguish species in the Pyaustinae.For mini-barcodes,the intraspecific genetic distance ranged from 0-33.19%(mean 1.16%),whereas interspecific variation ranged from 0-34.49%(mean 11.36%).No clear gap exists between intraspecific and interspecific divergences at 2.0%threshold was observed.The analysis showed that there is bigger overlap in the range of intra-and interspecific COI sequence divergence.The neighbour-joining analysis of standard barcodes resulted in a TaxonID tree with 165 morphospecies forming distinct,cohesive units displaying greater species resolution at 86.4%.By contrast,TaxonID tree of mini-barcode showed weaker resolution(63.6%)with undifferentiated or overlapping barcodes.2.DNA barcoding and morphology reveal cryptic species diversity in Pyraustinae from China and North America.The standard barcodes analysis indicated that twelve species displayed high intra-specific divergence(>2%).These deep divergences and discrete clusters may indicate the presence of cryptic species.In neighbor-joining(NJ)and minimum-evolution(ME)analyses,haplotypes for six species,Anania coronata,A.hortulata,Perispasta caeculalis,Pyrausta fodinalis,P.perrubralis,P.subsequalis had distinct geographic lineages formed non-overlapping monophyletic clusters and mean divergences above 2.0%.Further analyses of genital structures revealed diagnostic differences that correspond to the barcode lineages of these complexes of species.On the basis of both molecular and morphological evidence,we proposed that fourspecies which are considered new to science,and three subspecies that should be raised to species,as follows:Anania aduncata sp.nov.,Anania tennesseensis sp.nov.,Perispasta brevis sp.nov.,Pyrausta chiapaslis sp.nov.,Pyrausta septentrionicola stat.nov.,Pyrausta monticola stat.nov.,Pyrausta shastanalis stat.nov..3.Phylogeny of the subfamily Pyraustinae based on material from China and North America,and morphological and molecular analysisThe topology of the three phylogenetic trees constructed by maximum parsimony(MP),maximum likelihood(ML)and Bayesian inference(BI)based on mitochondrial COI gene weredifferent.The topology of the consensus phylogram generated by MP revealeddeeper-level splits within the Pyraustinae,which were broadly congruent with those in the ML consensus tree.In contrast,Bayesian inference yielded a single tree with topology in terms of shallow relationships among the exemplar taxa and provided unsupported polytomy.Overall,molecular phylogenetic analyses did not produce a well-supported monophyletic ingroup,including all 45 exemplars of the subfamily Pyraustinae.However,tree topologies generated by three methods were in general agreement,with all our analyses suggesting that 36 exemplars among the ingroup should be placed in the subfamily Pyraustinae,which agrees with taxonomical treatments based on morphology.Four exemplars within the ingroup,including Charitoprepes,Lamprophaia,Mabra and Mimetebulea appear to be more closely related to the Spilomelinae in contrast to the other members of Pyraustinae,while two exemplars within the outgroup,Aetholix,Udea appear to be more closely related to Pyraustinae in contrast to the other members of Spilomelinae.Conversely,conflicting results of different analyses led to the uncertain systematic relationship of Carminibotys,Euclasta,Heterocnephes,Pseudebulea,Triunicidia.The topology of the two phylogenetic trees constructed by maximum parsimony(MP),and Bayesian inference(BI)based on the combined morphological characters and mitochondrial COI gene were different.The topology of the consensus phylogram generated by MP provided deeper-level splits within the Pyraustinae.In contrast,Bayesian inference yielded a single tree with topology in terms of shallow relationships among the exemplar taxa and provided unsupported polytomy.Overall,the combined analyses did not produce a well-supported monophyletic ingroup,including all 22 exemplars of the subfamily Pyraustinae from China.However,tree topologies generated by two methods were in general agreement,with both analyses suggesting that 19 exemplars among ingroup should be placed in the subfamily Pyraustinae,which is in concordance with taxonomical treatment based on morphology.The genus Mimetebulea appear to be more closely related to Spilomelinae in contrast to the other members of Pyraustinae,whereas conflicting results of different analyses led to the uncertain systematic relationships of two exemplars within ingroup,including Carminibotys and Pseudebulea. |
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