Introduction

“Biodiversity” refers to the variety of living organisms from all sources, including terrestrial, marine, and other aquatic ecosystems as well as the ecological complexes to which they belong. At the same time, the presence of various faunal genetic resources located in any region or country is a component of animal biodiversity. Genetic diversity is the basis for a population to evolve and adapt to rapid changes in the environment²⁵. Today’s biodiversity provides the opportunity for sustaining vital environmental services that support life on Earth. Therefore, it is crucial to prioritise species protection and sustainable use. The identification of species paves the way to a deeper understanding of evolutionary processes among varied animal lineages by recording information on patterning and the range of variation.

Various methods have been developed over the decades for the characterization of wild animals which are largely categorized into phenotypic, biochemical and molecular markers. The history of taxonomy and evolutionary research is rooted in the study of morphology. For many years, the classification of species has been most heavily influenced by morphology in its widest meaning, which includes all expressions of structure and form. Morphology was used as a fundamental organising principle to arrange an apparently chaotic variety of living forms into higher (“macrotaxonomic”) levels initially¹⁷. Recently, the DNA barcoding approach based on the cytochrome c oxidase subunit I (COI) gene present in mitochondrial DNA has become adopted as a global biological identification method for all species due to its accuracy when compared to existing classical taxonomic methods²¹. This approach uses DNA extraction, sequencing and barcoding for genomic characterisation⁸, and it has become as an efficient and reliable tool for identifying, confirming and resolving closely related taxa¹⁹. Molecular techniques are increasingly employed in conservation of biological diversity in response to the alarming extinction of different species. Therefore, many conservation geneticists study genetic markers to make decisions for conservation of endangered wild animals. Here, choosing of effective profiling technique is a very critical step, as incorrect data on molecular information may result in incorrect conservation actions²⁹. In this regard, the application of both karyotypes and molecular genetic markers to conserve wild animals has been put into practice²⁴. Identification and characterization of species are two important steps towards crafting sustainable conservation strategies for wild animals. Studying genetic diversity within and between populations, estimation of effective population size and assessment of possible bottlenecks are practices carried out for characterization of wild animal populations.

Molecular characterization is based on studies of both mitochondrial DNA (mtDNA) and nuclear DNA¹. In recent years, advances in molecular techniques have encouraged the application of simple and precise DNA analysis in the taxonomic field. Among the existing genome-based approaches, DNA barcoding stands as a robust strategy to identify existing species and to discover unknown species through comparative analysis of sequence variation^2,18. Extensive research has shown that DNA barcoding can identify a wide range of animal species, including mammals, reptiles, birds, fishes, amphibians and crustaceans^12,28,30. The classification and identification of various life forms, particularly insects have been a major challenge to the scientific community especially with the dwindling interest and funding for taxonomy¹⁶. DNA barcoding provides an alternative approach, allowing expedited examination of species richness of all animal lineages at comparatively low cost. Its capacity to enhance progress depends on the fact that members of most species form a distinct barcode cluster^7,22,23; this relationship has now been operationalized for large-scale surveys by allotting a barcode index number (BIN)^9,20.

There has never been a molecular marker-based study of native faunal species of Tuensang areas of northeast India. Hence, our study aimed to characterize the diversity of animals from these areas using the DNA barcoding technique. Further, the inherent phylogenetic relationships present among those organisms were analyzed. The main objective of this study was to develop a reference library with generated barcodes.

Materials and methods

Study area

One of Nagaland’s largest and most eastern districts is Tuensang, Northeast India. The district of Tuensang covers 4,228 square kilometres and can be found at latitude of 26º 14ʹ 8.67ʺN and longitude of 94º 48ʹ 47.47ʺE, with elevations ranging from 800 to 3500m above mean sea level.          

Figure 1: Map of Tuensang District, Nagaland, India.       Click here to view Figure

Species collection

For molecular characterization, animal species were collected from selected areas of Tuensang district, Nagaland, viz., Tuensang town, Tuensang village, Hakchang village, Helipong village, Ngangpong village, Sangchen compound village, Chendang village, Chingmei village and Momching village. Field research was conducted in this study area to collect the species from forest and agricultural field habitats using different techniques, such as hand collection, pitfall trap, sweep netting, snares, catapults, and air guns. The collected animal tissues were immediately stored in 100% ethanol. For carrying out this research work, a consent letter was obtained from the community leader of the district. The study was conducted from 2019 to 2022.

DNA isolation

The extraction of DNA from collected animal tissue was performed using the Qiagen Tissue Kit following the manufacturer’s protocol. 100 mg of tissue sample was mixed with 180μl of ATL buffer, 20μl of Proteinase K, vortexed, and incubated at 60°C in a dry bath for 4 hours. 200μl of AL buffer was added and mixed in, followed by a 20-minute incubation at 60°C. 200μl of ethanol was poured and put to a DNA silica column for 1 minute of spinning at 12000 rpm. 700 μl of AW1 washed twice, with the AW2 buffer spinning at 12000 rpm for 1 minute. By adding elution buffer to 20 μl of DNA, DNA was eluted and measured using the 1% agarose gel electrophoresis technique.

PCR Amplification and Gel purification

PCR amplification of the isolated DNA samples was done using universal COI primers²⁶. The amplification reaction was performed in 25 μl reaction mixtures with DNA template of 40-80ng. The reaction mixture included 1μl of both forward and reverse primers and 25μl of the master mix containing 1μl dNTPs, 0.5μl Taq DNA polymerase (Barcode Biosciences), 5μl 10x buffer and 14.5μl distilled water. The primers were standard primers available for COI gene amplification, as below:

COIF-    GGTCAACAAATCATAAAGATATTGG-    Tm   51⁰C  and

COIR- TAAACTTCAGGGTGACCAAAAAATCA- Tm 53⁰C. The PCR amplification conditions included an initial denaturation step at 94ºC for 3.5 minutes, 60ºC for 30 seconds, and 72ºC for 1 minute for 35 cycles. On a 1% agarose gel electrophoresis stained with ethidium bromide, PCR product quality was examined.

Sanger Sequencing

An Applied Biosystems 3130xl Genetic Analyzer was used to carry out bidirectional sequencing of PCR products at BioEdge Solutions, Bangalore, India. The data from the sequencing machine was collected and processed in Finch TV (https://finchtv.software.informer.com/1.4/). The obtained electropherogram files were analysed for base-calling peaks in ABI format, which was further converted to pdf and fasta files using Sequence Scanner 2 Software (https://sequence-scanner-software.software.informer.com/2.0/). The sequence data obtained during this study were subjected to NCBI-BLAST in the nucleotide database of GenBank (http://blast.ncbi.nlm.nih.gov/) to identify closely related species.

Construction of phylogenetic tree

To comprehend the link between unknown sequences and relevant top species found by NCBI-BLAST, all the sequences were aligned using the Clustal Omega software ((https://www.ebi.ac.uk/Tools/msa/clustalo/). In this manner, phylogenetic trees were created informing us of the evolutionary relationships among closely related species.

Results and Discussion

DNA barcodes provided a quick, accurate and affordable way to identify species, even those that are challenging to differentiate based on morphology or life stage. A total of 62 species, which included animals belonging to different classes, such as Insecta, Arachnida, Reptilia, Aves and Mammalia, were identified using DNA barcoding technique for the first time from the study areas of Tuensang, Nagaland. The sequences obtained from samples were submitted to GenBank, and their accession numbers and sequence lengths are reported in Table 1. Among 62 sequences, 8 COI sequences that were previously unknown were added to the GenBank database. These sequences species were Cettia brunnifrons (Hodgson, 1845), Psilopogon virens (Boddaert, 1789), Megalaima franklinii (Blyth, 1842), Turdus boulboul (Latham, 1790), Hypsipetes leucocephalus (Gmelin, JF, 1789), Sitta formosa (Blyth, 1843), Oriolus traillii (Vigors, 1832) and Hypsipetes thompsoni (Bingham, 1900).  These accession numbers include: OQ920217, OQ920214, OQ920218, OQ920219, OQ780819, OQ920213, OQ920220 and OQ780820, respectively.

Table 1: Details of accession numbers and amplicon sequence length of insects, spiders, lizards, birds and mammals taken for molecular study.

The species identified included, 20 birds, 11 mammals, 5 spiders, 2 lizards and 24 insects. From the present studied locations, Tragopan blythii (Jerdon, 1870), Sitta formosa (Blyth, 1843) and Rusa unicolor (Kerr, 1792) were reported and declared as Vulnerable species by IUCN.3.1 (Red List, 1964). These species are declining due to overexploitation, traditional Jhum shifting cultivation and hunting activities.

Figure 2: Insects species from study areas identified using molecular markers.   Click here to view Figure

Figure 3: Spiders species from study areas identified using molecular markers.                Click here to view Figure

Figure 4: Lizards and birds species from study areas identified using molecular markers.   Click here to view Figure

Figure 5: Mammals species from study areas identified using molecular markers.                           Click here to view Figure

Figure 6: The evolutionary relationships of: a, insects and spiders; b, birds; and c, mammals and lizards taken for molecular study.                              Click here to view Figure

All the DNA barcoding sequences were aligned and used to create phylogenetic trees informing us of the evolutionary relationships among closely related species, as shown in Figure 5. Extensive research has shown that DNA barcoding can identify a wide range of animal species such as mammals, reptiles, birds, fishes, amphibians and crustaceans^12,28,30. One of the most effective and advanced methods for identifying insects is DNA barcoding, which uses the mitochondrial gene cytochrome c oxidase I (COI) sequence as a DNA identifier¹⁰. The sequences discovered will help to clarify the evolutionary history, for example, of spider mites and will enable quick species identification using molecular methods¹¹. DNA barcodes cannot replace the need for taxonomists, but DNA barcodes provide an additional suite of characters that can be used in taxonomy, assisting identification of species by non-specialists with accurate identification of their specimens. When it comes to saving animals under difficult conditions, genetic information is extremely valuable¹³.

A newly detailed understanding of species diversity can illuminate processes important in speciation, as suggested by the discovery that the most diverse lineages of animals. By using various molecular markers, it is possible to detect unique genetic variation in endangered population or species⁴. Knowledge of genetic diversity directs us to develop breeding programs minimizing inbreeding and safeguarding the loss of genetic variation. It is evident that the combination of morphological and molecular data sets permits a clearer recognition of evolutionary diversity among organisms. Further study is necessary to reveal the diversity among family members¹⁴. Applications of the mitochondrial gene-based species identification techniques cover a wide spectrum, and offer a fascinating perspective for future animal study^6,15.

The mitochondrial gene cytochrome c oxidase I (COI) barcode reference library for animals has become a standard resource for DNA metabarcoding applications recommending as standard metabarcode for metazoans³. These advances have opened up numerous new applications in aquatic sciences and ecological monitoring²⁷. Still, further work is needed to optimize use of COI barcodes for these applications⁵. 

Conclusion

Molecular markers are very useful, providing information on genetic variability among and within animal populations, helping us to develop appropriate conservation strategies. Choosing an efficient profiling technique is important, as selection of an inappropriate technique may lead to incorrect conservation actions. It is important to highlight, however, that DNA barcoding should not replace traditional taxonomy, as integrating both methods can provide a more thorough knowledge of species diversity. The current study improved our knowledge of the faunal diversity of selected areas of Tuensang district, Nagaland. Non-taxonomists, researchers, biodiversity managers and policymakers will use the information provided to strengthen their efforts to develop efficient protective measures for this faunal biodiversity.

Acknowledgment 

The author would like to thank the Head, Department of Zoology, St.Joseph University, Dimapur, Nagaland for her guidance and support.

Conflict of Interest 

The authors declare no conflict of interest.

Funding Source 

There are no funding sources.

Authors’ Contribution        

Chaueichongla Phom (Research scholar)  and Dr. Jeyaparvarthi Somasundaram (Supervisor)

Data Availability Statement

Not applicable

Ethics Approval Statement

The study does not involves an experiment on humans and animals. No Ethical approval was conducted. 

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