t i l e DNA Bar-Coding Based Identification of Neverita didyma (Roding 1798) and its Phylogeny

The Phylum Mollusca’s class Gastropoda contains a diverse range of organisms that live in several environments. Neverita didyma is a commercially important snail that is also known as the moon shell or bladder moon snail. Its molecular identification as N. didyma was confirmed in this investigation. The current study’s sequence has been submitted to NCBI under the accession number ON358159. It showed 91.36% similarity with the sequence under accession number NC046594. Neighbour- joining tree revealed that N.didyma from Pakistani coastal waters is distinctly clustered with other species of N. didyma. The present molecular-based confirmation of this species will be helpful for taxonomists, and biodiversity monitors.

The Phylum Mollusca's class Gastropoda contains a diverse range of organisms that live in several environments. Neverita didyma is a commercially important snail that is also known as the moon shell or bladder moon snail. Its molecular identification as N. didyma was confirmed in this investigation. The current study's sequence has been submitted to NCBI under the accession number ON358159. It showed 91.36% similarity with the sequence under accession number NC046594. Neighbour-joining tree revealed that N.didyma from Pakistani coastal waters is distinctly clustered with other species of N. didyma. The present molecular-based confirmation of this species will be helpful for taxonomists, and biodiversity monitors. G astropods live in several environments, including terrestrial, freshwater, and marine environments (Loker, 2010). During low tide, a variety of gastropods can be found along the coast. The gastropods in the marine environment are Euryhaline and have a diverse colour pattern (Haynes, 2005;Tan and Clements, 2008).
Neverita didyma belongs to the family of Naticidae commonly called moon shell or bladder moon snail. It is a carnivore animal and has sexual reproduction (Lee, 1999;Liu and Sun, 2009). It inhabits in the marine benthic environment. The species is widely distributed along the China coast, Indian Ocean, Madagascar, Mozambique, and South Africa. In China, this species has a significant economic and nutritional value (Liu et al., 2013;Zhao et al., 2018), yet no commercial use of this species has been reported in Pakistan and its research is also restricted to the work of (Khan and Dastgir, 1971;Tirmizi and Zehra, 1983;Barkati and Rehman, 2005;Kazmi et al., 2018;Ghani et al., 2018Ghani et al., , 2019Aslam et al., 2020). Khan and Dastgir (1971) described it as N. didyma, and Barkati and Rehman reported it as N. lamarkii in 2005. Until now, no molecular-based identification of this species was reported from this region.
It is significantly important to determine the potential natural resources of the region with an accurate taxonomic approach to understand the diversity of species in the area (Ran et al., 2020). Many species are very complex morphologically, therefore, the DNA bar code-based identifications are being appreciated worldwide. The molecular-based taxonomic investigations on Mollusca species from Pakistani coastal waters are limited to the work of (Zafar et al., 2016;Humayun et al., 2019;George et al., 2021).
The cytochrome oxidase subunit 1 (COX1) is considered suitable for investigations of genetic differentiation, taxonomy, and evolution of species. In the present investigation, we focused to confirm its identification and understanding of phylogenetic relationships. The molecular-based confirmation of this species will be helpful for bio-diversity monitors.

Materials and methods
N. didyma individuals were hand-picked at random and transported to the laboratory. The morphological identification was done using the available literature (Bosch et al., 1995;Khan and Dastagir, 1971). The tissues O n l i n e

F i r s t A r t i c l e
were taken and maintained at -20 0C for further study. For PCR amplification of COX1 gene the phenolchloroform procedure was used to isolate total genomic DNA from muscle tissue (Sambrook, 1989). A 100mg DNA template, 2.5ul dNtp (2.5mM each), 2.5ul10 X buffer, 2ul Mgcl2, (20mM), 1μl primers for cytochrome oxidase 1 (COX1) gene (10M each), and 0.25μlof Taq polymerase (5U Ml*1) were used for amplification. The thermocycler conditions were denaturation at 94 o C for 5 min; 35 cycles of 94 o C for 30 sec., annealing at 50 o C for 30 sec., extension at 72 o C for 30 sec.; and a final extension at 72 o C for 7 min (Table I). Gel electrophoresis (1 percent agarose gel with ethidium bromide) was done to confirm successful amplification.
The successful PCR product was sequenced. Softwares (BIOEDIT and MEGA 6) were used to do the necessary insertion and deletion (Tamura et al., 2013). A neighbour joining tree (NJ) was constructed using the Kimura 2 parameter (K2P) model in MEGA 6 (Tamura et al., 2013).

Results
Initially, the individuals of N. didyma were identified using morphological characteristics (Fig. 1).
After species confirmation as N. didyma using NCBI's Nucleotide BLAST TOOL, a 671 bp fragment of the COX1 gene was submitted to the National Center for Biotechnology Information under accession number (ON358159). The sequence of the present study showed significant alignment with the mitochondrial complete genomes of N. didyma accession numbers as NC046594 and MK548644, the query coverage was 100 percent and the percent identity was 91.36 %. The difference within the Naticidae family was calculated to be 0.109, the pairwise differentiation among the specie of N. didyma depicted that the sequence under accession number (NC046594) is nearest to the Pakistani specimen (Table II).
Nine COX1 sequences of related gastropods were downloaded from NCBI and used to build the phylogenetic tree. The accession number of each is presented in Figure  2. The phylogenetic tree based on neighbour joining indicated significant distinction among the Naticidae species. The N. didyma from Pakistan showed withingroup distinction with 97% bootstrap support (Fig. 2).

Discussion
The family Naticidae of order Littorinimorpha has 260 to 270 species worldwide (Huelsken et al., 2008) and over 200 species are inhabiting in Indo-Pacific region (Kabat, 1990). The members of the family Naticidae has immense commercial importance as food along the coast of the western Pacific and Southeast Asia (Zhang, 2016;Poutiers, 1998). However, in Pakistan the consumption of seafood is much lesser than in Southeast Asian countries, therefore, less attention was given to certain species of commercial importance. In scientific investigations, the exact identification is difficult based on morphological features (Fontanilla et al., 2014). It is said that traditional taxonomy sometimes fails to identify species (Packer et al., 2009). The N. didyma was first time reported from Pakistan by Khan and Dastgir (1971) later on reported by Barkati and Rehman (2005) as N. lamarkii. However, Ghani et al. (2018Ghani et al. ( , 2019 and Kazmi (2018) reported this species as N. didyma. To our knowledge no early bar-codebased identification was carried out in this species from South Asia. Nevertheless, Wang et al. (2019) published the full mitochondrial genome of N. didyma, while Zhao et al. (2018) analyzed the species' cryptic genetic diversity. The NJ tree of the present study revealed its distinction from the similar species found other than Pakistan.

Conclusion
The COXI gene has been a valuable method of identifying organisms. N. didyma, a member of the Naticidae family is evaluated morphologically and genetically in present research. The CO1 result of this investigation confirmed the N. didyma species. To our knowledge, this is the first molecular-based identification of N. didyma from the Pakistani marine environment. The outcome of the present investigation will be useful for taxonomists and biodiversity monitors.

Acknowledgment
We deeply thank Petroleum Marine Development Committee Malir District, Karachi for support of this study.

Ethics statement
No mandatory ethical approval was needed for the animals studied; however, all the methods were carried out in line with international norms for invertebrates.

Declaration
We declare no conflict of interest.

Data availability statement
The sequences which support this study are openly available in GenBank of NCBI at (https://www.ncbi.nlm. nih.gov/nucleotide).

Statement of conflict of interest
The authors have declared no conflict of interest.