Comparative Morphology Of Larvae And Phylogenetic Analysis Of The Spilomelinae (Lepidoptera:Crambidae) In China

Spilomelinae（Lepidoptera:Pyraloidea:Crambidae）is the largest subfamily in the Cramidae,with 4,097 species belonging to 338 genera known in the world and 450 species belonging to 100 genera recorded in China.Many species in this subfamily are important economic pests in agriculture and forestry.In particular,the larvae feed on economic crops in various ways,including leaf roller,leaf filler,stem or fruit borer.They cause significant economic yield-losses in crop production.However,the tribe classification of many genera and phylogeny of the subfamily has been controversial for a long time,especially the systematic study on the larvae is heavily lacking,which leading to species identification for the larvae in the field is very difficult.Therefore,it is urgent to make progress on the larva classification and employ new features and molecular evidence to reconstruct the phylogeny of the subfamily.In this study,general morphology,chaetotaxy,sensilla on the larval head,maxillary and labial palpi,spinneret,thoracic leg,proleg,crochets were investigated for the first instar larvae of 12 species in 12 genera in Spilomelinae sampled in China using Scanning Electron Microscopy（SEM）in order to illuminate the morphological characteristics and differentiations of the first instar larvae of the subfamily.Meanwhile,mitochondrial genomes were sequenced for some representative species of the subfamily.Phylogenetic analysis was performed based on mitogenomic data to explore the relationships among tribes and the status of some unplaced genera.The main results are as follows:1.Comparative morphological analysis of the first instar larvae of 12 species（Lamprosema sibirialis Millière,1879,Cydalima perspectalis Walker,1859,Pseudebulea fentoni Butler,1881,Haritalodes derogate Fabricius,1775,Pleuroptya balteata Fabricius,1798,Nagiella quadrimaculalis Kollar,1844,Sinomphisa plagialis Wileman,1911,Palpita nigropunctalsi Bremer,1864,Botyodes diniasalis Walker,1859,Patania expictalis Christoph,1881,Tylostega tylostegalis Hampson,1900,Glyphodes pyloalis Walker,1859）of Spilomelinae was investigated.Our results showed that the first instar larvae of these 12species in Spilomelinae in China are polypod type,with classical chewing mouthparts and six slightly protuberant stemmata on both sides of the head.In addition,the chaetotaxy on the head of each species are similar.However,the most remarkable differences for larval morphology among different species were found in the type and the number of sensilla on the head including antennae,maxillary and labial palpi.The number of setae on the thoracic legs and prolegs,characters of crochets of prolegs were recognized as diagnostic differentiations among distinct species.2.Comparative mitogenomic analyses were conducted based on 11 species mitogenomes sequenced in this study,including Glyphodes duplicalis Inoue,Munroe&Mutuura,1981,Syllepte taiwannalis Shibuya,1928,Haritalodes derogate Fabricius,1775,Patania balteata Fabricius,1798,Tylostega tylostegalis Hampson,1900,Pleuroptya chlorophanta Butler,1878,Herpetogramma luctuosalis Guenée,1854,Pseudebulea fentoni Butler,1881,Pycnarmon rdiata Fabricius,1794,Gydalima perspectalis Walker,1859,Notarcha quaternalis Zeller,1832,and published mitogenomes of other 19 species of Spilomelinae in Gen Bank.The results showed that:（1）Mitogenomes sequenced for 11 species in this study were characterized by a circular DNA double-stranded structure,with the whole mitogenomic length ranging from 15,215 bp to15,614 bp.The mitogenome of Pycnarmon rdiata has the longest length（15,614 bp）,while the mitogenome of Notarcha quaternalis has the shortest length（15,215 bp）.The genes and their arrangement in each complete mitogenome of all 11 species were basically similar to those other species in Lepidoptera.（2）Mitogenomes of 30 species were characterized by a circular DNA double-stranded structure,with the length ranging from 14,960 bp to 15,614 bp.The mitogenome of Pycnarmon rdiata has the longest sequence length（15,614 bp）,while Glyphodes Pyloalis has the shortest length（14,960 bp）.The differences of length were mainly caused by the length of AT-rich regions.All species showed different AT skew and GC skew.（3）The mitogenomes contain 13 protein-coding genes except for the mitogenome of Spoladea recurvalis only includes 12 protein-coding genes due to the absence of cytb gene.The start codon of most of protein-coding genes was AT-except for CGA as the start codon for cox1 gene.The cox1,cox2 and nad5 use single T as the stop codon,while other protein-coding genes use TAA or TA as the stop codon.Relative synonymous codon usage analysis showed that the frequency of amino acids Pro,Thr,Arg,Ala,Ser2,Leu2,Val and Gly was used significantly higher than other amino acids.The commonly used codons were CCU,ACU,CGA,GCU,UCU,UUA,GUA（GUU used in few species）and GGA.（4）The tRNA genes and r RNA genes of mitogenomes for 30 species include 22 genes and two genes,respectively.The number of overlap region and intergenic region were different for these species.In terms of t RNA secondary structure,t RNA^Leu（UUR）and t RNA^Leu（CUN）of all species were found to transport Leu,t RNA^Ser（AGN）and t RNA^Ser（UCN）of all species were found to transport Ser,while other amino acids were transported by only one t RNA gene.With the exception of t RNA^Ser（AGN）,which lacks the DHU arm instead of a single loop,the others have the same secondary structure with the typical clover-leaf structure as most t RNAs in Lepidoptera.（5）The phylogeny of the subfamily Spilomelinae was constructed based on four datasets（PCG123,PCG123RT,PCG12 and PCG12RT）.The results of BI and ML analyses yielded similar topological structure with high support values.The phylogenetic relationships among tribes in the subfamily are as follows:（Agroterini+Wurthiini）+（（Spilomelini+Margaroniini）+（（Herpetogrammatini+Nomophilini）+Hymeniini））.Our results supported the placement of genus of Syllepte to the tribe Spilomelini.However,the status of Pycnarmon remains uncertain due to it was recovered as a polyphyletic clade.In addition,the status of Tylostega remains unclear.