‘Direct PCR’ optimization yields a rapid, cost‐effective, nondestructive and efficient method for obtaining DNA barcodes without DNA extraction

Abstract

Macroinvertebrates that are collected in large numbers pose major problems in basic and applied biodiversity research: identification to species via morphology is often difficult, slow and/or expensive. DNA barcodes are an attractive alternative or complementary source of information. Unfortunately, obtaining DNA barcodes from specimens requires many steps and thus time and money. Here, we promote a short cut to DNA barcoding, that is, a nondestructive PCR method that skips DNA extraction ('direct PCR') and that can be used for a broad range of invertebrate taxa. We demonstrate how direct PCR can be optimized for the larvae and adults of nonbiting midges (Diptera: Chironomidae), a typical invertebrate group that is abundant, contains important bioindicator species, but is difficult to identify based on morphological features. After optimization, direct PCR yields high PCR success rates (>90%), preserves delicate morphological features (e.g. details of genitalia, and larval head capsules) while allowing for the recovery of genomic DNA. We also document that direct PCR can be successfully optimized for a wide range of other invertebrate taxa that need routine barcoding (flies: Culicidae, Drosophilidae, Dolichopodidae, Sepsidae; sea stars: Oreasteridae). Key for obtaining high PCR success rates is optimizing (i) tissue quantity, (ii) body part, (iii) primer pair and (iv) type of Taq polymerase. Unfortunately, not all invertebrates appear suitable because direct PCR has low success rates for other taxa that were tested (e.g. Coleoptera: Dytiscidae, Copepoda, Hymenoptera: Formicidae and Odonata). It appears that the technique is less successful for heavily sclerotized insects and/or those with many exocrine glands.

Remarkable fly (Diptera) diversity in a patch of Costa Rican cloud forest: Why inventory is a vital science

“…A repeated point in the literature is that morphotaxonomy is expensive and DNA barcoding relatively cheap (e.g., Carbayo & Marque 2011;Hebert et al 2016;Smith et al 2008;Wong et al 2014;Yu et al 2012). So too, there is the argument that molecular techniques are fast, while it will take hundreds of years to describe all the species present on our planet.…”

Conochironomus (Diptera: Chironomidae) in Asia: new and redescribed species and vouchering issues

“…The latter stage has high value in systematics and descriptive taxonomy (Brundin 1966, Krosch & Cranston 2012) and biomonitoring (Raunio et al 2007), and it can provide barcode DNA (Krosch & Cranston 2012, Kranzfelder et al 2015. Fortunately, it is likely that the development of quick extraction methods (Kranzfelder et al 2016), Direct PCR (Wong et al 2014), and cheap NGS barcoding (Meier et al 2015) will rapidly increase the number of species with matched life history stages.…”

Cryptic species in the nuisance midge Polypedilum nubifer (Skuse (Diptera: Chironomidae) and the status of Tripedilum Kieffer

“…Live specimens were preserved in 70% ethanol or isopropanol (adults) or in 99% ethanol or isopropanol (larvae). Those recognised on gross morphology as resembling P. nubifer were subjected to DNA extraction and barcoding (Wong et al, 2014). Post-extraction carcasses have been vouchered, slide mounted and preserved in ANIC, the Raffles Museum of Biodiversity Research (RMBR) (now Lee Kong Chian Natural History Museum), University of Singapore, The Natural History Museum (NHM=BMNH), London, England and in the Senckenberg Deutsches Entomologisches Institut (SDE), Müncheberg, Germany (SDEI).…”