Morphological and DNA barcode species identifications of leafhoppers, planthoppers and treehoppers (Hemiptera: Auchenorrhyncha) at Barrow Island

Abstract

-The hemipteran suborder Auchenorrhyncha comprises a rich assemblage of plant feeding species, many of which are widespread in distribution and act as vectors of viral and fungal diseases affecting plants. Species level identifications in this group generally are possible only by examination of male specimens; prior DNA barcode analyses of a limited range of Auchenorrhyncha indicate that this approach may provide an expedient means to identify species within this diverse group. In this study we explored the utility of DNA barcoding for identification of a wider range of Auchenorrhyncha species than has been examined previously. Diverse fulgoroid (planthopper) and membracoid (leafhopper and allies) Auchenorrhyncha were sampled from Barrow Island, Western Australia, and identified to the least inclusive taxonomic units using morphology. DNA barcodes from 546 adult specimens were obtained and analysed using a General mixed Yule -Coalescent (GMYC) modelling approach to genetically delimit putative species, as a comparison to the morphospecies identifications. Additional DNA barcodes (N = 106) were obtained from nymphs and these were compared to adult DNA barcodes to identify species present among immature specimens.Among adult specimens, 73 species were congruently delimited by morphology and genetic analyses when modelled using a single threshold GMYC. Congruence between morphological and molecular species assignments was greatly reduced when the Yule -Coalescent transition was allowed to vary across genetic lineages. In a separate DNA barcode analysis of all specimens using neighbour joining distance metrics, nymphs and physically degraded specimens were in most cases genetically linked to adult conspecifics. Ten genetic clades detected among the nymphs were not observed among adults and did not match pre-existing sequence accessions in GenBank or DNA barcode records in BOLD.Of the 73 adult Auchenorrhyncha species congruently identified by DNA barcoding and morphology, most were Cicadellidae (N = 53 morphospecies), the remaining 20 morphospecies were sparsely representative of ten other families. Formal identifications to species level were available for only 36% of these 73 morphospecies, owing mainly to an absence of diagnostic male specimens within many of the delimited species. Indeterminate species detected among adults and nymphs are designated with interim species codes.The work presented here demonstrates that DNA barcoding is likely to be a powerful investigative tool for identifying and understanding species limits in the Auchenorrhyncha, particularly if it is used within an integrative taxonomic framework.

A revision of the genusOrosiusDistant (Hemiptera: Cicadellidae) based on male genitalia and DNA barcoding

“…PCR preparations, thermal profiles and quality checking followed that reported in Gopurenko et al . (). We also used PCR procedures reported by Mitchell () to amplify short (<400 bp) overlapping portions of the DNA barcode region.…”

“…The abdomen was removed from adult specimens and digested using a non‐destructive (or semi‐destructive) technique (Gopurenko et al . ) which clears the abdomen for subsequent genitalia examination and provides a DNA sample. The abdomens were then washed and transferred to glycerine for examination and, eventually, storage in vials attached to the pin of the specimens from which they had been removed.…”

“…, ; Gopurenko et al . ), of grapevines at Loxton, South Australia and of lucerne in New South Wales (Pilkington et al . ) have revealed that undescribed species of Orosius occur in all three States.…”

Discovery of a new species ofStromatiumAudinet‐Serville, 1834 (Coleoptera: Cerambycidae) native to Australia, based on morphology and DNA barcoding

“…Using the extracted DNA as a template, a 421 bp portion of the COI gene was amplified by the primer set Scar3AF (5′‐ GCHCCHGAYATAGCNTTYCCNCG ‐3′; Gopurenko et al . ) and Scar3RD (5′‐ AAAATRTAWACTTCDGGRTGNCC ‐3′; Mitchell & Maddox ) as these were found effective for dry preserved museum specimens. PCR amplifications were performed using the iProof polymerase kit (Bio‐Rad, Hercules, CA, USA) in a 20 reaction volume with the following cycling conditions: an initial denaturing step at 98°C for 30 s; 35 cycles of denaturing at 98°C for 10 s, annealing at 57°C for 20 s and extending at 72°C for 1.5 min, and a final extending step of 72°C for 10 min.…”

Morphostructural analysis of the male reproductive system and DNA barcoding in Balclutha brevis Lindberg 1954 (Homoptera, Cicadellidae)

“…Since, moreover, the morphological characterization of numerous species has been frequently integrated, in the last years, by the sequencing data, as taxonomic tool for species identification, this issue manifestly appears to be important also for numerous Auchenorrhyncha species, because of their importance to the global agricultural plants-biosecurity (Gopurenko et al 2013). Recently, the DNA barcoding method has become an increasingly useful and promising tool for ensuring the rapid and accurate identification of species: a partial sequence of the mitochondrial Cytochrome c Oxidase I (COI) gene is currently being used to differentiate between animal taxa (Hebert et al, 2003a(Hebert et al, , 2003b.…”