MOL2NET'22, Conference on Molecular, Biomedical & Computational Sciences and Engineering, 8th ed. Conservation Status of Globally Testudines Terrapins Based on COI Mitochondrial Markers

Abstract. Terrapins inhabit brackish water or coastal salt marshes. Terrapins are adapted to intermediate salinities but frequently face saltwater-inundated marsh habitats. To date, 12 species of terrapin have been reported worldwide. The present study aims to determine the global utility of terrapin


Introduction
DNA barcoding is a complementary technology in classical taxonomy and systematics study that has enabled a more precise understanding of the current flora and fauna worldwide [1].This technology utilises the use of a specific part of the DNA, often from the mitochondrion genome that has a simple genetic structure and relatively rapid rate of evolution, to distinguish closely related taxa.Research attempts have been made at the global and local levels to identify freshwater turtles using DNA barcoding [2].The DNA barcoding procedure is summarised in Figure 1.
In this study, we collected four samples of Batagur affinis ssp.from Peninsular Malaysia.The genetic profiles of these samples were evaluated with the cytochrome oxidase subunit I (COI) marker gene and compared to the DNA sequences of other 11 species of terrapins retrieved from the public database portal.Furthermore, B. affinis sequences from this study were submitted to the GenBank database portal for the first time, adding valuable genetic resources for global conservation research.All in all, this study aimed to define the global utility of terrapin DNA barcoding via novel COI sequences and compare them to other COI sequences available in BOLD systems and GenBank.https://mol2net-08.sciforum.net/

Study sites
In this study, four Batagur affinis ssp.individuals from two different population regions on the east and west coasts of the Malaysia Peninsula were selected, and sampling was conducted in 2020 (Figure 2).The blood samples of B. affinis affinis (N = 1) were collected from a captive hatchling population at the Bota Kanan head-starting facility (BK; GPS coordinate: 4.3489° N and 100.8802°E) located at Perak, Malaysia.The blood samples of wild hatchlings B. affinis edwardmolli (N = 3) were collected from a population in the Bukit Paloh area of Kuala Berang (KB; GPS coordinate: 5.0939° N, 102.7821°E) at Terengganu, Malaysia.Venipuncture techniques were used to draw blood from the species through the subcarapacial venous plexus (SVP) and internal jugular vein.Before storing at -20 °C, a total of 1.5 mL of blood was preserved with 0.5 mL of EDTA in a 2 mL microcentrifuge tube at a 1:3 ratio.The research and field permit approval number is B-00335-16-20, issued by the Department of Wildlife and Parks, Peninsular Malaysia.https://mol2net-08.sciforum.net/For PCR, Tuntong set primers 5'-CGCGGAATTAAGCCAACCAG-3' (forward sequence) and 5'-TTGGTACAG-GATTGGGTCGC-3' (reverse sequence) targeting the COI marker gene was used [3].The PCR amplification of COI gene fragment was performed in a Go Taq Flexi PCR (Promega, Madison, USA) reaction mixture containing 2 µL of DNA template, 0.4 µL of primers, 4 µL of 5x PCR buffer, 1.6 µL of 25 mM MgCl2, 0.4 µL of dNTPs, 0.2 µL of Taq DNA polymerase, and 11 µL of distilled water (ddH2O).After an initial denaturation at 94°C for 4 minutes, 35 cycles consisting of denaturation at 94°C for 45 seconds, annealing at 55°C for 35 seconds, and extension at 72°C for 1 minute were performed, and lastly a final extension step for 10 minutes at 72°C.Finally, the PCR products were purified and sent to a local company (First BASE Sdn Bhd) to sequence the COI gene of mitochondrial DNA (mtDNA-COI) using Sanger sequencing technology.Additionally, 17 COI sequences of B. affinis were data mined and downloaded from GenBank, while five COI sequences of B. affinis were mined from BOLD Systems, yielding a total of 26 sequences for this work (Tables 1).https://mol2net-08.sciforum.net/Table 1.List of terrapins species studied through DNA barcoding with the BOLD IDs of their respective COI sequences and the GenBank accession of each species.

DNA barcode sequence quality control measures and analysis
For each sample, chromatograms that show the nucleotide sequences of both DNA strands were generated.Some low-quality noisy sequences on both ends and chromatograms with more than 2% ambiguous bases were trimmed.The bidirectional reads were removed by benchmarking against a quality value of more than 40.A computer programme that is known as SeqScape, version 2.7 (Applied Biosystems) was used to view and combine the forward and reverse chromatograms to get the consensus sequences.The sequences were checked against the GenBank and the BOLD Systems database for accession numbers and BOLD sequence identifiers (Table 1).MEGAX [11] was used to align all the sequences and generate multiple sequence alignments of all sequences with the same length and starting point.Phylogenetic analysis was performed using MEGAX with 1000 bootstrap replicates and the Unweighted Pair Group Method with Arithmetic Mean (UPGMA) [12].Microsoft Excel 2016 was used to make the pie charts that show all terrapins' different family groups and conservation statuses.

Results and Discussion
Twenty-six COI sequences from B. affinis were analysed in this study, in which all of them were from the order Testudines.The species are classified into three families: Geoemydidae (18%), Emydidae (6%) and Pelomedusidae (2%) (Figure 3).All species studied in this study are worldwide terrapins that live in freshwater and brackish water.Notably, [13] identified 13 species of terrapins worldwide but disregarded a Seychelles black terrapin species (Pelusios seychellensis) that was believed to be extinct.However, genetic analysis of the lectotype revealed that this terrapin has not been extinct and has been referred to as Pelusios castaneus.Specimens were possibly mislabeled or mixed up in a private collection before being acquired by the Zoological Museum Hamburg in 1901 [14].https://mol2net-08.sciforum.net/With 100 bootstrap supports, the UPGMA phylogeny of the studied dataset demonstrated cohesive monophyletic clustering of all studied species (Figure 4).Cohesive clustering was also observed between the database reference sequences for the representative species and the produced sequences in the phylogenetic tree.The species were grouped according to their family, with Geoemydidae the most numerous.According to the evolutionary tree, B. baska was originated in India and was closely linked to B. affinis from Malaysia.Additionally, Melanochelys trijuga is closely related to Mauremys caspica from the Persian Gulf, whilst the Malaclemys terrapin is strongly related to Trachemys scripta elegans is native to North America.

Conservation Status
The International Union for the Conservation of Nature (IUCN) Red List is a critical health indicator for biodiversity.The IUCN is far more than a list of species and their states; it is a crucial instrument for informing and catalysing conservation and policy change activity.These steps are critical to preserving the natural resources humans rely on for survival [15,16].The IUCN Red List Categories and Criteria are intended to provide a simple framework for identifying species on the verge of extinction on a global scale.It assigns species to one of the following categories: Not Evaluated (NE), Data Deficient (DD), Least Concern (LC), Near Threatened (NT), Vulnerable (VU), Endangered (EN), Critically Endangered (CR), Extinct in the Wild (EW), or Extinct (EX) [15].Accordingly, 33% of the 12 species of terrapins in this study were classified under the LC category, followed by 25% of the samples belonging to the CR category, 8 % of the samples belonging to the VU category and the remaining samples were classified under EN and NT categories at 17% respectively (Figure 5).
Almost every country with indigenous species has a captive breeding programme.Based on Table 1, three Batagur sp. of terrapins are in CR status, including B. affinis, B. baska, and B. borneoensis.However, in Southeast Asia countries such as Indonesia, Singapore, Thailand, and Vietnam, B. affinis is assumed to be classified under EW [17,18].Furthermore, B. baska could possibly be regionally extinct in Myanmar and Thailand [19].Moreover, B. borneoensis was distributed in Indonesia, Malaysia, and Brunei but it was virtually extinct in Thailand [20].

Conclusions
In conclusion, the COI marker is an effective DNA barcode marker for terrapin species, providing vital evidence that it may be utilised to distinguish and recognise genera and species of these Testudines organisms.Nonetheless, in the future study, other molecular markers and additional samples from new sampling sites should be included to assess terrapin populations extensively.The genomic and bioinformatics analyses of terrapins taxonomy reported here may serve as a foundation for future research on this species throughout the world, allowing for more practical conservation work for this threatened species.

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DNA isolation, PCR, and sequencing Each 200 µL of EDTA whole blood sample was used for nucleic acids extraction.Following cell lysis and protein denaturation, DNA extraction was performed using ReliaPrep TM Blood gDNA Miniprep System with Binding Column technology (Promega, Madison, USA) by following the manufacturer's instructions.According to the input volume of the EDTA whole blood sample, the final extracted volume was adjusted to 200 µL.The quantity and purity of extracted DNA samples were assessed using Thermo Scientific TM NanoDrop 2000c spectrophotometer model ND-2000 (Thermo Fisher Scientific, Waltham, USA).Following the quantification of the isolated nucleic acids, the DNA samples were loaded into 1% (w/v) agarose gel with molecular markers and electrophoresis was performed to evaluate the integrity and intactness of the high molecular weight DNA band.

Figure 3 .
Figure 3.The family of sampled terrapins.

Figure 4 .
Figure 4. UPGMA tree constructed with MEGAX based on COI sequences belonging to order Testudines.

Figure 5 .
Figure 5.The conservation status of the terrapins is based on the IUCN Red List.