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DNA barcoding is a molecular tool for the identification of species of organisms using a relatively short DNA sequence from a standard position in the genome. The cytochrome c oxidase subunit I (COI) mitochondrial region is fast becoming the standard barcode region. In most groups, the COI is a 648 nucleotide base pair sequence.
DNA barcodes vary among individuals of the same species, but only to a very minor degree. The intraspecies variations in DNA barcode regions are significantly less than interspecies variations.
The barcode of life (BoL) is a catalogue of DNA barcodes compiled for the use of taxonomic identification. DNA barcoding of specimens obtained from natural history museums, herbaria, zoos, aquaria, frozen tissue collections, seed banks, type culture collections and other biological material repositories are exceptional for contributing information to the BoL database (BoLD), as the specimens have been expertly identified.
The BoL method of identification is quickly becoming preferred to the use of traditional dichotomous keys. This is due to four principal flaws in the use of keys. Firstly, phenotypic plasticity and genetic variability in characters necessary for species recognition can result in incorrect identifications. Second, morphologically cryptic taxa are often disregarded in many dichotomous keys, which can significantly influence identifications. Third, some morphological keys are often designed for the identification of organisms at a particular life stage or gender. Finally, dichotomous keys are only useful for the identification of species recognised at the time of printing; therefore newly recognised species cannot be identified. Barcoding protocols created by the Consortium for the Barcode of Life can be followed to obtain DNA barcode sequences acceptable for entry into the BoLD.
When fully developed, the BoL identification system will provide a reliable, cost-effective and accessible solution to the current problems associated with species identification. Research specimens will be able to be identified by referencing the closest matching COI sequences on the BoLD.
The genus Cherax presents a particularly difficult group of organisms to identify into separate species. Currently, novel characteristics, which can be difficult to interpret, are used in dichotomous keys for Cherax identification. The morphological and habitat variations within several species have been found to be as great as that between species. Therefore, caution is advised when interpreting morphology based studies of Cherax (Austin & Knott, 1996). The use of COI to identify Cherax specimens would greatly enhance the reliability of research papers on species of this genus, as correct identification of specimens is almost certain. There is, of course, a margin of doubt which is introduced by human error.
The aim of this study was to determine if COI samples from known species of freshwater Cherax could be combined with information from the BoLD to corroborate correct taxonomic identification. The COI sequences from both laboratory samples and BoLD were used to create phylogenetic trees to facilitate the examination of interspecies relationships.
Materials and Methods
The protocol was followed as per the lab manual (Koenders, 2010). The only significant change that was made to the procedure was not conducting a qualitative test on the DNA by loading and running the gel. Due to complications met while carrying out the procedure, sequences from Cherax specimens obtained prior had to be used. None of these sequences had corresponding information regarding identification by either DNA barcoding or morphological examination.
The phylogenetic tree created using the minimum evolution method (Figure 1.) shows four of the lab specimens form a monophyletic clade. These are B12, C32, B72, and A72. The closest relationship between these specimens is between B12 and C32. The closest branch of database entry specimens to the clade of lab specimens is the C. crassimanus group. It should be recognised that all database entry specimens group into monophyletic clades of specimens with the same name. The only lab specimen that is not included in the large monophyletic clade of lab specimens is A62. The A62 specimen sits within the C. quinquecarinatus clade. Bootstrapping percentages show a high level of confidence in the majority of branches. Some low percentages that should be noted, however, are the 57% confidence between all Cherax specimens and the outgroup E. strictiforns, the 56% confidence between the C. quinquecarinatus clade and the C. tenuimanus clade, and the 60% confidence between the C. crassimanus clade and the C. priessii clade.
Figure 1. - Minimum evolution phylogenetic bootstrap consensus tree constructed using COI sequences of 28 Cherax specimens and 3 different closely related outgroup species. Bootstrap percentages indicating the percent of replicate trees that showed similar branching are shown next to the top of the branches.
The phylogenetic tree created using the neighbour-joining method (Figure 2.) is extremely similar to the minimum evolution tree (Figure 1.). All database entry specimens in figure 2. group into monophyletic clades of specimens with the same name. Figure 2. also shows four of the lab specimens forming a monophyletic clade. These are B12, C32, B72, and A72. The closest relationship between these specimens, however, is between B12 and B72. The C. crassimanus group is still the closest branch of database entry specimens to the clade of lab specimens. A62 sits within the C. quinquecarinatus clade. It is still the only lab specimen that is not included in the large monophyletic clade of lab specimens. Bootstrapping percentages show a high level of confidence in the majority of branches. The same low bootstrapping percentages that were noted for figure 1. are also present in figure 2; however, the values have changed slightly. Figure 2. shows 52% confidence between the C. quinquecarinatus clade and the C. tenuimanus clade, and 60% confidence between the C. crassimanus clade and the C. priessii clade. The 60% confidence between all Cherax specimens and the outgroup E. strictiformus in figure 2 is the same level of confidence as is shown in figure 1.
Figure 2. - Neighbour-joining phylogenetic bootstrap consensus tree constructed using COI sequences of 28 Cherax specimens and 3 different closely related outgroup species. Bootstrap percentages indicating the percent of replicate trees that showed similar branching are shown next to the top of the branches.
The two types of phylogenetic trees used, minimum evolution (Figure 1.) and neighbour-joining (Figure 2.), are representatives of two different strategies for constructing phylogenetic trees. These types of phylogenetic trees were also chosen as they have been found to show a high performance in obtaining the correct tree. The minimum evolution method uses the exhaustive search strategy, which examines all or a large number of possible trees and select the best one depending on certain criteria. The neighbour joining method uses a different strategy known as stepwise clustering, which constructs trees step-by-step after examining local topological relationships of a tree. By examining tree constructed using different strategies, a more comprehensive study of the evolutionary history and relationships of South Western Australian Cherax species can be conducted.
The minimum evolution tree (Figure 1.) shows a considerably low bootstrapping value of 32% between B12 and B72 within the lab specimen clade. Although this casts doubt on the relationship between these two specimens, higher bootstrapping values are given at prior branches. These values support relationships with other lab specimens. There is a relatively low confidence that links C. crassimanus COI(3) and C. crassimanus COI(4) to the other two database C. crassimanus specimens. It is possible that this is caused by the use of sequences from specimens that are geographically distant. The 100% bootstrapping value between the lab specimen clade, including B12, B72, C32, and A72, and the clade containing C. crassimanus COI, and C. crassimanus COI(2) suggests that these lab specimens are members of the species C. crassimanus. The one lab specimen that is not included in this clade sits within the C. quinquecarinatus clade. The positioning of A62 within this clade is supported by high bootstrapping values, suggesting that A62 is of the species C. quinquecarinatus.
The tree constructed using the neighbour-joining method (Figure 2.) is extremely similar to the minimum evolution tree (Figure 1.) in both the orientation of the specimens and the bootstrapping values. This similarity was also found by Saitou and Imanishi (1989) in the neighbour-joining and minimum evolution trees constructed as part of their study. The lab specimens B12, B72, C32, and A72 show the same relationship with the C. crassimanus clade as is presented in the minimum evolution tree (Figure 1.), which ultimately leads to the same conclusion that these lab specimens are all of the species C. crassimanus. It is possible that some, or even all, of these lab specimens belong to the species C. glaber, of which there are currently no database specimens to compare COI sequences with. While this is theoretically possible, it is unlikely that any of the lab specimens within the C. crassimanus clade do belong to C. glaber. This is due to these two species having little genetic similarities between them (Austin & Knott).
The A62 lab specimen sits within the same clade in the neighbour-joining tree as it does in the minimum evolution tree, therefore suggesting that it belongs to C. quinquecarinatus. The strong correlations between the two trees strengthen the conclusions reached concerning the identity of the previously unidentified lab specimens.
Other vital components present in both trees that should be noted are the low bootstrapping values linking the C. tenuimanus clade to the C. quinquecarinatus clade and the value linking the C. priessii clade to the C. crassimanus clade. Although these values are unlikely to impact the identification of the lab specimens, they are still concerning from an evolutionary history perspective, suggesting that there may be less divergence between the species than is currently acknowledged.
As the lab specimens used in the construction of both phylogenetic trees (Figure 1. and Figure 2.) had no corresponding morphological identifications, the effectiveness of using BoL as a means of specimen identification can be scarcely commented on. The positioning of the outgroups (E. strictiforns, E. eungella, and E. fosser) in both trees supports that the specimens belong to the Cherax genus. This is a positive sign that DNA barcoding can be used as a means of determining the genus of a lab sample. Th e outgroups used were chosen because they belong to the same family as Cherax, Parastacidae.
DNA barcoding has the potential of being an invaluable tool to biologists. Highly comprehensive phylogenetic trees can be used to determine the evolutionary history of animal species and suggest when a new species may be present in a data set. As for DNA barcoding being used for identification purposes, until further studies have been conducted on the effectiveness of the BoL, and considerably more COI sequences added to its database, morphological identification should be used in conjunction with BoL to support identifications.