DNA Barcoding

MSU's blog | Created 9 years ago

Traditionally, identifying which species a specimen belonged to required observing its morphological features, and referring to species descriptions and “keys”. However, this method is limited in some circumstances. What if only a feather of a bird was available, like the recent rediscovery of the Night Parrot? Or, for many spiders, only adult males have diagnostic features, so females and juveniles cannot be identified.

DNA barcoding has emerged as a complementary method to help with some of these difficulties. The DNA barcode refers to a short segment of DNA that can be easily sequenced and is unique in every species. This enables researchers to extract DNA from tiny fragments of tissue, sequence the DNA, and compare it to an existing database of known sequences. Because DNA is found in every cell of an organism, identifications can be based on scats, hair, larvae, eggs, or even ancient remains.

For invertebrate fauna, the barcoding region is approximately 650 nucleotides from the Cytochrome Oxidase 1 (CO1) gene of the mitochondrial genome. This region is useful because it accumulates mutations quickly enough for species to show differences, but not so quickly as to show too many differences within a species. This is important, because if there are too many unique barcodes within a single species, it can be difficult to assign an unknown barcode.

In a recent paper published in Invertebrate Systematics, led by WAM researcher Dr. Mark Castalanelli, DNA barcodes were produced for 2,090 Western Australian Mygalomorphae specimens. Mygalomorphs are a highly diverse infraorder of spiders, commonly referred to as tarantulas or trapdoor spiders. They include the iconic funnel-web spider, and are typically heavy bodied, ground dwelling species. One of the truly remarkable features of these spiders is their very small distributions, which is linked to their limited dispersal ability. In the Pilbara, many mygalomorphs qualify as “short range endemics” (SREs), which means they are only found in a limited area, and may need special conservation and management status.

Led by Dr. Mark Harvey, researchers at the Western Australian Museum have been studying the taxonomy of this group, potentially identifying new species in WA. The difficulty with this research is that when using morphology, only adult males have the features needed to differentiate among species. This means that many specimens held in the WAM collection cannot be identified to species with certainty, and are therefore unable to help us map distributions.

Enter DNA barcoding!

By sequencing CO1 for adult males we have a library of known barcodes. So if we also sequence COI from juveniles and females, and compare them to those known males, we can more confidently assign identifications. This allows us to learn more about the distribution of a species, which is especially important for SREs. It also helps us to identify preferred habitats, because females and juveniles associate more strongly with particular habitats, and males are more often caught while wandering about.

In some instances, females and juvenile sequences were totally unique from known adult males, and are assumed to belong to a new species. For these species, a male is now required to identify morphological features and confirm that it is a unique species, before researchers are able to name the new species.

Some males that were thought to belong to a single, morphologically uniform species revealed high genetic variation within the species. For example, Aname mellosa was comprised of 10 distinct lineages, which may represent different species. University of Western Australia PhD candidate, Karl Gruber is currently investigating this ‘species’ in more detail, using more genes to determine how biology and landscape features in the Pilbara are driving genetic differentiation.

DNA barcoding is not a replacement for traditional taxonomy. Rather, it is a complementary tool that is available to people who study the evolutionary history of species. DNA barcoding suffers from its own difficulties. Although DNA does exist in every cell of the body, it is an unstable molecule and can degrade quickly if it is stored inappropriately. As such, the WAM maintains a large ultrafrozen tissue collection, where tissue is kept at -80°C. Other tissues are kept in 100% ethanol at sub-zero temperatures.

As noted above, while highly divergent barcodes might indicate new species, it is also clear that a single species can harbor many divergent lineages. So, good DNA barcoding projects require comprehensive libraries of known barcodes to compare against. This might mean the easiest groups to identify through barcoding, are those where at least one life stage can already be identified through morphology.

Creating the link between a named species and the barcode can also be problematic. For example, one of the first species of Mygalomorphae ever described from Western Australia is Aganippe occidentalis. The original specimen (the holotype), an adult male, was collected from Roebourne (the type location) and the species description and name was published in 1903. The holotype is too old and degraded to be suitable for barcoding, and although we have specimens identified as that species, it would be more reliable if the barcode was based on a specimen from the type location. However, a recent field trip to Roebourne was unable to recover a specimen, complicating barcoding work on this species. Recollecting specimens from type localities has become a focus of many museums across the globe, as molecular methods become a reliable method in the systematist’s toolbox.