Historic DNA for taxonomy and conservation: A case-study of a century-old Hawaiian hawkmoth type (Lepidoptera: Sphingidae)

Abstract

Analysing historic DNA from museum specimens offers the unique opportunity to study the molecular systematics and phylogenetics of rare and possibly extinct taxa. In the Hawaiian fauna, the hawkmoth, Hyles calida calida, occurs on several of the main islands and is quite frequent, whereas Hyles c. hawaiiensis is restricted to the Island of Hawaii where it appears to be very rare. Analysis of mitochondrial DNA sequences shows that Hyles c. hawaiiensis differs from the nominotypical subspecies by an average p-distance of 2.8%, which is of a similar order of magnitude to that found between other species of Hyles, suggesting that Hyles c. hawaiiensis should perhaps be awarded species status, although more data are required for a formal taxonomic revision. Given the rarity of this taxon, these analyses should be undertaken urgently so that conservation measures can be implemented before it becomes extinct.

Advantages of an easy-to-use DNA extraction method for minimal-destructive analysis of collection specimens

“…First, the gene fragments of the cytochrome oxidase subunits COI and COII plus the intermediate tRNA Leu (trnL2) were annotated using MITOS [36]. In a second step sanger sequences of COI, COII and trnL2 sequences of the same or closely related species from an earlier publication [37] were mapped on the mitochondrial reference genome to validate the annotated positions of each gene. These sequences are available on their NCBI accession numbers (see Fig 8).…”

“…Positions in the alignments with zero coverage were masked out with "N". The consensus sequences were used together with sanger sequences from previous publications [37] to enable a multi sequence alignment, which was done using MAFFT [39], allowing adjusting of sequence direction…”

DNA from 33-year-old dried moth specimens help confirm larva as the elusive <i>Wiseana fuliginea</i>

“…Amplification of the DNA from the museum voucher moths with the same primers used by Brown et al (1999a) and Richards et al (2017) was unsuccessful, most likely due to degradation of the DNA by endogenous nuclease activity and hydrolytic damage (Wandeler et al 2007). This was overcome by designing a series of primers that amplified smaller overlapping regions of the porina mitochondrial COI gene (< 400 bp, Table 1), similar to the method used by Hundsdoerfer and Kitching (2017) to successfully retrieve mitochondrial COI and COII sequences from 100-year-old museum hawkmoth specimens. New primers were named using the system described by Folmer et al (1994) where L and H refer to light and heavy DNA strands, CO refers to cytochrome oxidase, and the numbers refer to the position of the Drosophila yakuba 5' nucleotide.…”

Molecular Marker Study for Hyles euphorbiae (Lepidoptera: Sphingidae) Based on Mitochondrial DNA Genes in Erbil Province

“…Classification in the past was done depended on superficially phenotype body characters like pattern and of wing and abdomen in mature stage, color in larval immature stage and genital feature (Hundsdoerfer and Kitching, 2017). Species identification requires data from more sources such behavior phenotype and DNA markers so only a very little variation in molecular level is enough also quite efficient to detect unknown insect (Funk andOmland, 2003, Dayrat, 2005).…”

“…The sequence data comprise about 2300 bp of the mitochondrial genes cytochrome c oxidase subunit I (COX I), cytochrome c oxidase subunit II (COX II), and the gene of the ribosomal transfer RNA for leucine (tRNA-leu). In the mitochondrial genome of Hyles, this ribosomal region lies between the two COX genes (Hundsdoerfer et al, 2005a). The primers were designed depending on the various marker sequence of commonly used mitochondrial genes like cytochrome oxidase I, II and 16S rRNA genes for species level classification (Folmer et al, 1994, Caterino et al, 2000.…”