Background

The bigfin reef squid, scientifically known as Sepioteuthis lessoniana d’Orbigny, is one of the members of the Loliginidae family. This species is considered to be the most widely distributed loliginid squid in the Indo-West Pacific region because besides its largest distribution range from Japan to northern Australia, it also occurs in many other places in the Pacific and Indian Oceans such as New Zealand, the Hawaiian Islands, South India, West Africa, the Madagascar island, Red Sea, and Western Mediterranean [1]. In Vietnam, bigfin reef squid occurs in the waters of the Gulf of Tonkin, the Southern Central, the Southern East, and the Southern West [2,3,4] and has been considered as one of the most economically important seafood resources.

In its distribution areas, this species usually occurs in water from the surface to a depth of about 100 m, in coastal habitats with the bottom layer usually covered by seagrass beds and coral reefs [1]. Such habitats are relatively common in the Southwestern and Southeastern seas of Vietnam, which are characterized by the coastal habitats of the two large islands, Phu Quoc and Con Dao. In fact, bigfin reef squid used to be fairly abundant and has been considered as a famous seafood in these two islands compared to other distribution areas in Vietnam. These two islands are separated by about 345 km as the crow flies and the shortest sea distance between them is about 410 km. Sharing characteristics with other loliginid squids, the planktonic paralarvae of S. lessoniana are widely distributed after hatching by coastal and ocean currents [5, 6] while juveniles and adults live in coastal and consistently move inshore to start mating and spawning [1]. These indicated that adult squid individuals from Con Dao and from Phu Quoc are almost impossible to mate together directly, and therefore bigfin reef squid individuals from Con Dao island and from Phu Quoc island must belong to two separate populations.

Bigfin reef squid in particular and squid in general are crucial components in the food chains and webs as well as the biodiversity of marine ecosystems and are seafood of high economic value for fisheries. Despite playing such important role, the species S. lessoniana has faced to two risks, including overexploitation and loss of suitable habitat as a result of climate change [7, 8] which can lead to species extinction and loss of valuable marine resources without effective strategies for conservation and sustainable exploitation.

Such strategies are often established based on a fundamental conception of the importance of intraspecific genetic diversity, which is determined by the genetic diversity of the constituent populations and the gene flow among them and is measured at markers scattered throughout the genome [9]. Low population genetic diversity is often thought to be a consequence of inbreeding degradation and increased genetic drift and this low diversity itself is the cause to reduce individual vitality, along with a depleted capacity for population growth [10]. On the other hand, high genetic diversity is often considered as the driving force to promote population survival and ensure the ability of populations to adapt to changing environmental pressures [11].

In case of the bigfin reef squid in Vietnam, there were several populations which used to be evaluated for genetic diversity, including the population in the Gulf of Tonkin based on the mitochondrial DNA noncoding region [3] and populations in Nha Trang Bay and Phu Quoc island based on mitochondrial COI and 16S rRNA sequences [4]. In the study by Cheng et al. in 2013, the haplotype diversity and the nucleotide diversity were respectively determined at 0.73 and 0.004 for the S. lessoniana population in Phu Quoc; however, this data was only based on 17 samples. Thus, population genetic data of bigfin reef squid in Con Dao and Phu Quoc islands have been still lacking and need to be supplemented to create the scientific basis for establishment of conservation and exploitation strategies [4].

For bigfin reef squid, previous studies on population genetic diversity were conducted by two groups of methods, including using mitochondrial DNA sequence data [3, 4] and using DNA fingerprinting data generated through the allozyme marker [12] and microsatellite marker [13, 14].

In general, DNA fingerprinting data can be generated using many markers such as simple sequence repeat (SSR), inter simple sequence repeat (ISSR) [15, 16], random amplified polymorphic DNA (RAPD), and amplified fragment length polymorphism (AFLP) [17]. Although not direct reflecting the allele status of the locus, dominant markers still have been widely used in population genetic diversity assessment due to their high sensitivity in expressing the differences among the surveyed individuals. With the development of molecular biology, many dominant markers have been developed recently, such as start codon targeted polymorphism (SCoT) [18] and CAAT box–derived polymorphism (CBDP) [19] markers. SCoT and CBDP markers target the start codon and CAAT box of functional genes, respectively. Both of these markers are widely used in studies of population genetics in plants; while SCoT markers have also been used in studies of population genetics in animals [20,21,22], there are almost no found publications on the use of CBDP markers in this field. However, in the gene structure in eukaryotes, the CAAT box is located upstream of the transcription initiation site and signals the binding site for the RNA transcription factor, namely, NF-Y subunits; the sequence of 5′-GGTTA-3′ is conserved for the CAAT box [23].

With the aim of determining the genetic diversity and variation of the two bigfin reef squid populations in Con Dao and Phu Quoc islands and the genetic differentiation between them, in this study, in addition to mitochondrial COI gene sequence data, DNA fingerprinting data generated using two techniques of SCoT and CBDP were also used for analysis.

Methods

Materials

Samples were collected in October 2020 by night fishing with lure-hooks in the same way to exploit form local fishermen. In a total of collected 59 juvenile and adult samples of S. lessoniana, 28 samples belonged to the Con Dao population were caught in the waters around Con Dao island, and 31 samples belonged to the Phu Quoc population were caught in the waters around Phu Quoc island. Fishing localities of these samples were not more than 10 km offshore. Collected specimens were tentacular tissues of captured individuals, individually labeled and preserved in 95% ethanol at −20 °C until DNA extraction. The field trip sample notation was done continuously for all the caught samples, but only samples belonging to the S. lessoniana species were used in this study.

Twenty-eight representative samples of the Con Dao population were assigned as Sles036, Sles037, Sles039–Sles049, Sles052–Sles055, Sles057, Sles058, Sles060–Sles065, Sles067, Sles068, and Sles070. Thirty-one representative samples of the Phu Quoc population were assigned as Sles072–Sles091 and Sles093–Sles103. Samples were not numbered consecutively as above due to exclusion of different species individuals during fishing.

DNA extraction

Total genomic DNA was extracted from ethanol-preserved tentacular tissue using cetyltrimethylammonium bromide (CTAB) method [24] which modified by Adamkewicz and Harasewych [25]. Three DNA samples were extracted from each individual sample. The DNA concentration and quality were measured using spectrophotometry method [26] using a NanoScan2 system (Analytik Jena). The DNA samples with OD260/OD280 values between 1.8 and 2.0 were kept at −20 °C for the subsequent PCRs.

DNA fingerprinting

In this study, DNA fingerprinting data was obtained by using SCoT and CBDP techniques. PCRs were performed in 50 μL reactions containing 25 μL My Red HS Taq mix (Bioline), 0.2 μM primer, and approximately 30 ng DNA templates. The PCRs were performed using an Eppendorf Mastercycler Pro S thermal cycler (Germany). The thermal program used in SCoT technique was as follows: initial denaturation at 94 °C for 5 min; 36 cycles of 94 °C for 15 s, 50 °C for 15 s, 72 °C for 45 s; final extension at 72 °C for 10 min [18]. The thermal program used in CBDP technique: initial denaturation at 94 °C for 5 min; 6 cycles of 94 °C for 45 s, 35 °C for 45 s, 72 °C for 90 s; 30 cycles of 94 °C for 45 s, 51 °C for 45 s, 72 °C for 90 s; final extension at 72 °C for 10 min [19].

After screening, 10 primers for each technique were chosen for PCR; the sequences and amplification features of these primers are shown in Table 1.

Table 1 Primers used in the study and their amplification features in total 59 samples of whole species in surveyed region
Full size table

To obtain DNA fingerprints for investigated samples, PCR products were separated in 2% agarose gel, electrophoresis was performed at 60 V for 3 h using TBE buffer, the gel then was stained with ethidium bromide (0.5 μg/mL), and photographed under 254/312 nm wavelength lights using UVP GelStudio Plus System (Analytik Jena, Germany).

COI amplification and sequencing

The DNA sequence of the mitochondrial COI gene was isolated amplified using the primer pairs LCO1490 (5′-GGTCAACAAATCATAAAGATATTGG-3′) and HCO2198 (5′-TAAACTTCAGGGTGACCAAAAAATCA-3′) [27]. PCRs were performed in 60 μL reactions containing 30 μL My Red HS Taq mix (Bioline), 0.4 μM each primer, and approximately 40 ng DNA templates using the Eppendorf Mastercycler Pro S thermal cycler (Germany). The thermal program used to amplify COI gene was as follows: initial denaturation at 95 °C for 5 min; 30 cycles of 95 °C for 15 s, 46 °C for 15 s, 72 °C for 30 s; final extension at 72 °C for 10 min. This program was modified from Folmer et al. [27] and Kim et al. [28] by gradient testing for primer annealing temperature.

PCR products were confirmed by electrophoresis on 1.0% agarose gel electrophoresis and then were purified using AccuPrep® Gel Purification Kits (Bioneer, Korea). Purified DNA samples together with two primers LCO1490 and HCO2198 were preserved in icepack during sending to 1st BASE DNA Sequencing Services (Singapore) for sequencing. DNA sequencing was performed by Sanger method using ABI 3730 XL sequencer.

Data analysis

The parameters of genetic diversity and variation were calculated for each of two populations and for the whole species in the surveyed region. DNA fingerprint data and mitochondrial COI sequence data were analyzed using different specialized software to calculate the diversity and variation parameters and construct the dendrograms for genetic relationship among studied samples.

Based on DNA fingerprinting data

Since both CBDP and SCoT markers are dominant, each observed band was assumed to represent the genotype at a single biallelic locus [29] and DNA fingerprinting data from them can be combined together for genetic analysis (SCoT and CBDP data sets possessed the same overall utility when being applied in samples of whole species in surveyed region, which were analyzed using iMEC—an online marker efficiency calculator, data not shown). The bands were scored as presence (1) or absence (0) characters to construct the binary data matrix. In studies on population genetic diversity and variation, it is generally recognized that increasing the number of investigated loci gives more reliable results [30]. Accordingly, in this study, the analysis was performed with all bands generated using both SCoT and CBDP techniques.

POPGENE 32 software was used to calculate genetic diversity and variation parameters: the percentage of polymorphic bands (PPB), the expected heterozygosity (He), Shannon index (I), the gene differentiation (GST), the genetic distance between investigated populations (D), and gene flow between them (Nm) [38].

DNA fingerprinting data, which showed close grou** of individuals by population, again better reflects the genetic differentiation and the genetic variation between the two populations compared to mitochondrial COI sequence data. This could be explained by the fact that the SCoT and CBDP data reflect the genetic variation of many genes across different regions of the genome while the mitochondrial COI sequence data only reflect the genetic variation of a single gene.

Analysis of molecular variance (AMOVA) revealed high genetic variation within populations and low genetic differentiation among two populations in investigated regions. Based on the mitochondrial DNA non-coding region, Aoki et al. showed that pairwise gene differentiations among 7 bigfin reef squid populations in Japan, Taiwan, and Gulf of Tonkin of Vietnam were in range of 0.0041 to 0.8593 with the average of 0.3695 [3]. Comparing to this study and the interpretation of gene differentiation values in previous studies [39, 40], the gene differentiation between Con Dao and Phu Quoc populations based on DNA fingerprinting data was at moderate level and based on mitochondrial COI sequence data was at relatively low level.

Based on DNA fingerprinting data, the genetic distance between Con Dao and Phu Quoc populations in current study (D = 0.0361) was significantly higher than between Nagasaki and Rayon populations (D = 0.003, using allozyme technique) [12], although the geographical distance between the Nagasaki and Rayon populations is much farther than between the Con Dao and Phu Quoc populations.

Using mitochondrial COI sequence to investigate 11 populations of bigfin reef squid in Southeast Asia, Cheng et al. showed that the significantly different genetic variation distribution among populations (77.51% or 23.24%) and within populations (22.49% or 76.76%) depended on the investigate lineages [4]. Compared with the results from this study, genetic variation within populations in current study was much higher. This may due to in the presentative sample sets for Con Dao and Phu Quoc populations included different lineages of bigfin reef squid which inherently identified by Cheng et al. and the surveyed region in current study was also smaller.

As mentioned above, the information on the genetic diversity, differentiation, and gene flow among the populations is necessary to establish the strategies for conservation and sustainable exploitation of the species. The findings from the current study indicate that the Con Dao population had a higher genetic diversity than the Phu Quoc population. The main reason for that difference may be that Con Dao island is exposed to strong ocean currents from two different directions, while there has been only a weak ocean current bringing planktonic paralarvae to Phu Quoc island. Accordingly, to maintain the long-term genetic diversity of the populations in surveyed regions, several fishery management strategies including the promotion of programs of artificial propagation and local as well as translocational releasing to increase the size of local populations and to overcome barriers to gene flows among populations; establishing regulations to limit fishing in the breeding season; and enhancing the protection and restoration of coral reefs and seagrass beds to ensure the quality of such habitats, especially at spawning grounds for the species should be fully considered.

Conclusions

For the bigfin reef squid species in the surveyed region, the Con Dao population had the higher genetic diversity than the Phu Quoc population; however, the levels of genetic diversity based on COI data of these populations were at relatively higher compared to other populations in Southeast Asia. Between the investigated populations existed a low to moderate genetic differentiation and a genetic exchange via gene flow.

DNA fingerprinting data better showed the gene differentiation between the two investigated populations than the mitochondrial COI gene sequence data; the COI gene sequence data reflects the phylogenetic relationships of the individuals investigated in the current study with bigfin reef squid in some other sea areas in Southeast Asia.

From this study, several fishery management strategies for conservation and sustainable exploitation of bigfin reef squid are proposed.