New mitochondrial primers for metabarcoding of insects,designed and evaluated using in silico methods

Insect metabarcoding has been mainly based on PCR amplification of short fragments within the “barcoding region” of the gene cytochrome oxidase I (COI). However, because of the variability of this gene, it has been difficult to design good universal PCR primers. Most primers used today are associated with gaps in the taxonomic coverage or amplification biases that make the results less reliable and impede the detection of species that are present in the sample. We identify new primers for insect metabarcoding using computational approaches (ecoprimers and degeprime ) applied to the most comprehensive reference databases of mitochondrial genomes of Hexapoda assembled to date. New primers are evaluated in silico against previously published primers in terms of taxonomic coverage and resolution of the corresponding amplicons. For the latter criterion, we propose a new index, exclusive taxonomic resolution, which is a more biologically meaningful measure than the standard index used today. Our results show that the best markers are found in the ribosomal RNA genes (12S and 16S); they resolve about 90% of the genetically distinct species in the reference database. Some markers in protein‐coding genes provide similar performance but only at much higher levels of primer degeneracy. Combining two of the best individual markers improves the effective taxonomic resolution with up to 10%. The resolution is strongly dependent on insect taxon: COI primers detect 40% of Hymenoptera, while 12S primers detect 12% of Collembola. Our results indicate that amplicon‐based metabarcoding of insect samples can be improved by choosing other primers than those commonly used today.     

Molecular biological study on speciation and phylogeny of the order Entomobryomorpha (Collembola: Hexapoda)

In order to elucidate the phylogenetic relationship among groups of the order Entomobryomorpha (Collembola), the sequences on the ITS 1 to ITS 2 fragments of the rRNA gene were analyzed in 11 species of three families. In order to avoid the potential risks and inconsistencies of a single method or data set, the phylogenetic reconstructions were based on three different approaches: methods of maximum parsimony, maximum likelihood and neighbor joining. The inferred phylogenies supported monophyly of the order Entomobryomorpha. The relationships between families were different, but the orders of branching within each family were the same. Entomobryidae and Isotomidae were paraphyletic, whereas Tomoceridae was monophyletic. Tomoceridae was subdivided into two branches; the molecular analysis provided results distinctive enough to separate the two genera by the high bootstrap value. On the other hand, two different populations of putative Homidia koreana appeared to be different species, although their chaetotaxy is identical. A wide coverage of characters, including not only morphological characters but also genetic data such as allozymes and DNA sequences, will give a more accurate picture of the classification and phylogeny of the studied group.     

16S mitochondrial sequences associate morphologically dissimilar males and females of the family Phoridae (Diptera)

The genus Phora (Diptera, Phoridae) includes more than 50 species widely distributed in temperate regions of Eurasia and Africa. Taxonomic identification of most species is based upon the morphology of the male hypopygium: hence for many species, including the type species of the genus, females are unknown. We used mitochondrial 16S ribosomal RNA sequences to match males with previously unidentified females from Phora atra , P. stictica , and P. holosericea . We then identified morphological characters that allow identification of females of P. atra and P. stictica without recourse to DNA sequencing. Our results show that small scale sequencing can aid in the development of taxonomic characters for use in the field to identify previously cryptic females. This iterative method of identifying populations genetically followed by re-examination of morphology should allow development of better keys for rapid identification of heretofore cryptic populations of insects. We also found that sequences from individual of P. holosericea from Cambridge, England and Malakhovka, Russia, were more similar to each other than to sequences from a conspecific fly also collected in Cambridge. This result suggests that there is previously un-suspected population structuring in this species.  © 2002 The Linnean Society of London, Biological Journal of the Linnean Society , 2002, 77 , 267–273.     

Fossil evidence for key innovations in the evolution of insect diversity

Explaining the taxonomic richness of the insects, comprising over half of all described species, is a major challenge in evolutionary biology. Previously, several evolutionary novelties (key innovations) have been posited to contribute to that richness, including the insect bauplan, wings, wing folding and complete metamorphosis, but evidence over their relative importance and modes of action is sparse and equivocal. Here, a new dataset on the first and last occurrences of fossil hexapod (insects and close relatives) families is used to show that basal families of winged insects (Palaeoptera, e.g. dragonflies) show higher origination and extinction rates in the fossil record than basal wingless groups (Apterygota, e.g. silverfish). Origination and extinction rates were maintained at levels similar to Palaeoptera in the more derived Polyneoptera (e.g. cockroaches) and Paraneoptera (e.g. true bugs), but extinction rates subsequently reduced in the very rich group of insects with complete metamorphosis (Holometabola, e.g. beetles). Holometabola show evidence of a recent slow-down in their high net diversification rate, whereas other winged taxa continue to diversify at constant but low rates. These data suggest that wings and complete metamorphosis have had the most effect on family-level insect macroevolution, and point to specific mechanisms by which they have influenced insect diversity through time.     

Comparative morphology of mandibular structures in lycid larvae and its phylogenetic implications (Polyphaga, Hexapoda)

The morphology of the larval mandibular structures of the family Lycidae (Polyphaga) is characterized by facultative dicondyly, posterior articulation with long mandibular rods, lateral location of the anterior condyle and its articulation with a paired non‐cranial structure. It is compared to that of Eucrustacea, Chilopoda, Entognatha, Microcoryphia and Zygentoma and found to be more reminiscent of the Entognatha. The phylogenetic implications of this conclusion are discussed, with the Pterygota and Dicondylia hypothesized to be non‐monophyletic.     

A forgotten homology supporting the monophyly of Tracheata: The subcoxa of insects and myriapods re-visited

The current discussion about the relationships of higher arthropod taxa has led to a conflict between the traditional Tracheata (=Atelocerata) concept (hexapods united with myriapods), the Tetraconata concept (hexapods united with crustaceans, excluding myriapods), and the Paradoxopoda or Myriochelata concept (myriapods united with cheliceratans), with major contradictions between morphological and molecular data. We have analyzed a character set which apparently has completely vanished from the recent debate, namely the equipment of the trunk pleura of myriapods and insects with a characteristic set of concentric sclerites around the leg base and accompanying muscles. Based on the work of Heymons (1899) these sclerites were thought to be remains of the first appendage article, then denominated “subcoxa”. We have re-visited this old idea and show the arrangement of the much discussed pleural structures by SEM for the first time. Obviously a characteristic pattern of concentric pleural plates around the leg-base is present in all major myriapod taxa, including for the first time evidence for their presence in Progoneata. Because of their equal structure and orientation, the pleural sclerites present in entognathous and ectognathous insects may be derived from the same ground pattern. We conclude that the pleurites of Hexapoda and Myriapoda seem to be homologous structures, and there is evidence that the “subcoxa” of Tracheata is homologous with the coxa of crustaceans. Since no other arthropods show these sclerites, the transformation of the crustacean coxa to the pleural region in myriapods and insects is probably a synapomorphy congruent with the traditional Tracheata-hypothesis. Further investigations of recently published molecular work using the phylogenetic network software SplitsTree V.4 indicate that information content of several data sets is not convincing.     

What is the epipleurite? A contribution to the subcoxal theory as applied to the insect abdomen

The epipleurites were originally described by Hopkins in 1909 on the imago and larva of a beetle. Then this term was widely used in insect morphology, mainly for larvae, to designate certain sclerites of the pleural region. They have recently been interpreted as tergopleural (i.e. pleural but not strictly appendicular) by Deuve in 2001, but a study of embryonic development by Kobayashi et al. in 2013 has shown that they are instead eupleural (i.e. appendicular) and correspond to a dorsal part of the subcoxa. Their presence in the abdominal segments of insects illustrates the fundamental importance of the subcoxa in segmental structure, with a function of anchoring and supporting the appendage when the latter is present. However, the epipleurites are normally separated and functionally dissociated from the coxosternum, which integrates the ventral component of the subcoxa. In females, the epipleurite of segment IX of the abdomen corresponds to the gonangulum, as already pointed out by Deuve in 1994 and 2001, and it is involved in gonopod articulation. At segments VIII and IX of both males and females of holometabolans, the formation process of the genital ducts leads to an internalisation of the whole subcoxosternum (i.e. the coxosternum with the exception of the coxal and telopodal territories), and it is the two flanking epipleurites that ventrally close the abdomen in relation to the rearward displacement of the gonopore. This model may be generalised, in its broad lines, to a large part of the hemimetabolans. The body plan of the insect abdomen underlines the morphological and functional importance of the subcoxa in its fundamental structure, but the study of the Hexapoda in general also indicates the presence of a more proximal segment, the precoxa, which would belong to the groundplan but is more cryptic because it is often closely associated with the subcoxa and/or the paranotal lobe. Its location, which is sometimes on the ventral flank of the paranotal lobe, is in line with the hypothesis of a dual origin of the pterygote wing.     

Ribosomal DNA gene and phylogenetic relationships of Diplura and lower Hexapods

HennigdividedInsectas.lat.(=Hexapoda)intotwowelldefinedtaxonomicgroups:Entog-nathaandEctognatha[1].ThemostdistinctivecharacterofEntognatha(includingProtura,Col-lembolaandDiplura)liesintheenclosedmouthpartscondition,whereasthepresenceofexposedmouthpartsisthemainfeatureofEctognatha(includingMicrocoryphia,Zygentomaandtheptery-goteinsects).ControversiesaboutthephylogeneticrelationshipsofhightaxaEntognathahavekeptgrowinginrecentyears,withthemonophylyofDipluraandthephylogeneticpositionsofDiplur…     

有关节肢动物系统发育若干问题的研究进展

近年来对节肢动物系统发育问题的认识有了相当大的变化,尤其是支序分析研究方法的使用,以及来自分子系统学和分子发育生物学的证据,成为继比较形态学之后有力的研究手段,近年来的研究显示线虫,缓步类和有爪类是节肢动物的近缘类群,而非以前绝大多数学者所认为的软体动物的环节动物,对于节肢动物门的单系性及其内部类群间关系的问题,目前倾向于认为（1）节肢动物为单系群;（2）由甲壳类,六足类和多足类联合形成单系群－具颚类;（3）与六足类分子系统学两方面研究的支持;对第3点的争议虽较大,但就论据而言确为优势观点。     

Ribosomal DNA gene and phylogenetic relationships of Diplura and lower Hexapods

The monophyly of Diplura and its phylogenetic relationship with other hexapods are important for understanding the phylogeny of Hexapoda. The complete 18SrRNAgene and partial 28SrRNA gene (D3-D5 region) from 2 dipluran species (Campodeidae and Japygidae), 2 proturan species, 3 collembolan species, and 1 locust species were sequenced. Combining related sequences in GenBank, phylogenetic trees of Hexapoda were constructed by MP method using a crustaceanArtemia salina as an outgroup. The results indicated that: (i) the integrated data of 18SrDNA and 28SrDNA could provide better phylogenetic information, which well supported the monophyly of Diplura; (ii) Diplura had a close phylogenetic relationship to Protura with high bootstrap support.