DNA barcoding as a method for species identification: case study in Ahaetulla snake

Species identification using molecular analyses has recently been developed, one of which is DNA barcoding method. The purpose of this study was to apply the DNA barcoding method to identify Ahaetulla snake which previously had not been identified molecularly. The specimens used came from Sumatra, Java, Bali, Lombok and additional samples obtained from Genbank. Cytochrome B gene was extracted and amplified to obtain DNA barcode. Phylogenetic analysis using ML method was used to determine the kinship relationship of the Ahaetulla snake. Morphological analyses were added to confirm the results of molecular identification. The results of molecular analysis using DNA barcoding method showed the presence of two Ahaetulla species in this study, there are A. prasina and A. mycterizans. This difference is indicated by the formation of two separate main clades, clade I contains A. prasina and clade II contains A. mycterizans. The results of the morphological analyses also showed differences in the character of the two different clade species. Thus, molecular analyses result agrees with morphological analyses. It can be concluded that the DNA barcoding method can be used as a valid method to identify Ahaetulla snake species.


Introduction
Traditional method for species determination is by identifying morphological characters compare to the other species. However, this traditional method fails to discriminate some species due to several conditions, for example, species that having various external body colorations especially when specimens are not fresh [1], museum preserved species [2] and samples showing phenotypic plasticity [3].
Nowadays, DNA barcode attracts the taxonomies attention as the latest system in the identification of almost all fauna species, both interspecific and intraspecific, quickly and accurately. DNA barcoding uses small regions of mitochondrial DNA (mtDNA) that used as a barcode to amplify a gene. Various primers set of mitochondrial gene for barcoding are available in published studies [4]. With DNA barcode, identification by taxonomies will be easier. This identification technique fully supports the improvement of animal classification as well as helps to sort out any ambiguity at the species level. However, DNA barcoding with the support of traditional taxonomy has the capability to identify species complexes within populations [5].
The COI gene is a part of mtDNA which is commonly used as a barcode to identify species, however, the cyt b gene is also part of mtDNA which has a high mutation rate, therefore it also can be used as a barcode. The variation in the Cyt B sequence causes this gene to be widely used to compare species in the same genus or family. The uniqueness of the Cyt B gene sequence is that there are parts that are conserved in species level, so it can be used for grouping based on animal species or for determining kinship relationships between animal species [6]. The results of the study by Laopichienpong et al. [7] showed that the use of the Cyt B gene was more effective for the identification of Ahaetulla snakes than the COI gene.
In this study, Cyt B gene was isolated from Ahaetulla snake that widely distributed in Indonesia. There are at least three species distributed in this country, A. prasina, A. mycterizans, and A. fasciolata [8]. Although it is widely distributed with vulnerable population, this snake is also threatened by human killed and traded. Faith and Williams [9] argued that the most significant contribution of DNA barcoding to conservation efforts is its role in improving and speeding up phylogenetic diversity assessments. Previous study by Leo et al. [10] showed that there are no significant morphological differences among A. prasina of Java and Sulawesi. Therefore, it is necessary to do research about identification of Ahaetulla snake using molecular analyses.
This study aimed to hold molecular analyses by using Cyt B gene as barcode in Ahaetulla snake of Indonesia. Morphological analyses also added to confirm the validity of molecular analyses.

Samples collection
All samples were collected from 10 localities along Sumatra, Java, Bali, and Lombok Island ( Figure 1). The total of 10 samples including of 3 samples of Sumatra, 5 samples of Java, a sample of Bali and a sample of Lombok. Four samples were taken from Genbank used as outgroup (www.ncbi.nlm.nih.gov). There are A. prasina, A. mycterizans, Chrysopelea ornata, and Dendrelapis pictus.  Table 1. List of specimens used as in group and outgroup in the phylogenetic analysis.

Phylogenetic analyses
Cyt B sequences were contiged by Sequencher (Gene codes, Ann Arbor, Michigan, USA). The sequences were aligned using MEGA7 by Clustal W method and genetic distance was calculated as uncorrected pairwise distance [15]. Phylogenetic tree was inferred using maximum likelihood (ML) method. For ML, tree was constructed by RAxML using automatically bootstrap option by RAxML. Bootstrap value equal or more than 70% was defined as significant value [16].

DNA barcoding analyses
The results of the genetic distance analysis showed that there were two identified species based on the genetic comparisons with A. prasina and A. mycterizans sequences from Genbank. The genetic distance between A. prasina and A. mycterizans is about >6%. According to Jeong [17], Cyt B sequences in reptiles that have a genetic distance >5% are considered as different species. The results of this genetic distance have identified that A. prasina and A. mycterizans in this study have >6% genetic distance. The next analyses is the construction of phylogenetic trees to determine the relationship among Genus Ahaetulla in Indonesia.

Specimens
Locality Genbank Accession no.  In A. prasina of Indonesia clade, there are 2 subclades formed based on their localities (ML=99). Subclade I consist of A. prasina of Sumatra and West Java, while subclade II consist of A. prasina of Central Java, East Java, and Bali. The clustering joining of A. prasina of Sumatra and West Java is predicted because of the integration of the mainland of Sumatra and Java during glacial period. At that time, it was estimated that there were many species migrations which made it easier for species from Java and Sumatra to move each other [18]. This makes a pattern of adaptation which then affects the genetic structure of the species. Likewise, the Bali island which once joined with the mainland of Java allowed species migration from Java to Bali or vice versa. The island is part of the mainland of Sundaland that was once connected to become a major mainland in the past [19]. Within A. mycterizans clade, 2 subclades were formed (ML=78) consisting of A. mycterizans sequence from Genbank and A. mycterizans of Lombok and Banten. A. mycterizans of Lombok and Banten formed a clade with significant bootstrap (ML=100) although Lombok is located in Lesser Sunda and Banten in Sundaland. This analyse indicates that A. mycterizans has high similarity Cyt B sequence with A. mycterizans of Banten. It can be predicted that A. mycterizans of Lombok could be originating from Sundaland. We refer to Inger [20] which proposed that fauna in Wallace's line, including Lesser Sunda region, originated from Sundaland region.

Source
This result of molecular identification adds evidence that molecular analyses with Cyt-B gene as barcode can be used as alternative method for identifying species

Morphological analyses
To confirm the identification result, we also add morphological identification. The specimens used are representative of A. prasina and A. mycterizans used in this study. The results of morphological identification are shown in Figure 3 and Table 2. As illustrated in Figure 3, the differences between A. mycterizans and A. prasina are: 1) A. mycterizans eyes is bigger than A. prasina, 2) upper surface of snout is convex in A. mycterizans but flat or even depressed in A. prasina. The differences between A. mycterizans and A. prasina not illustrated in Figure 3 and not mentioned in Table 4    Based on the results of morphological identification, our specimen of Lombok agrees with five diagnostic morphological characters given for A. mycterizans by Miralles and David [21]: 1) ventral scale less than 200, 2) Anal entire, 3) Snout convex above, 5) venter with grey longitudinal lines. Moreover, our identification of A. mycterizans also agrees with recent studies that A. mycterizans eyes is wider than A. prasina and the number of ventral scales has a big influence in distinguishing A. prasina and A. mycterizans [22].
The morphological characters of Ahaetulla of East Java agree with diagnostics characters of A. prasina given by De Rooij [23 ] (who mentioned as Dryophis prasinus): 1. number of ventral scales 194-235 (206), 2. anal divided, 3. Snout more than twice the diameter of eye, 4. Sub caudal scales 151-207 (162).Therefore, we agree that there are 2 species found in this study based on morphological characters, A. prasina and A. mycterizans.

Conclusion
From those both analyses, it can be concluded that molecular analysis using Cyt B gene as a barcode can be used to identify species accurately because it shows appropriate result with morphological identification. Among animal taxonomists, the COI gene is the most often used for species identification. Based the results of this study, it can also be added that the Cyt B gene is also an effective gene used as a DNA barcode for species identification. Besides to be used for species identification, DNA barcoding method may also be used to analyse the biogeography of species in the distribution area.