Meta-analysis of Indonesian Octopus laqueus; Kaneko and Kubodera 2005 (Cephalopoda: Octopodidae) using Mt-DNA COI as Genetic Marker

Octopus laqueus was first discovered not long ago in 2005 in the Ryuku Islands, Japan. Its geographical distribution and molecular identification are therefore still rarely. Nucleotide sequences based on mt-DNA COI for O. laqueus that have been uploaded in the GenBank until before this study was carried out were only six sequences. Since DNA barcoding of mt-DNA COI has some advantageous characteristics, this study aimed to analyse the genetic difference of Indonesian O. laqueus to the data available in the GenBank. Samples were collected in 2019 2020 from Karimunjawa (n=16) and Bangka-Belitung (n=2). The mt-DNA COI was extracted using 10% chelex methods, PCR amplified using Folmer’s primer and sequenced in Sanger methods. Pairwise alignment and genetic distance were carried out in MEGA-X, whereas the phylogenetic tree was reconstructed using Bayesian methods. BLAST identification resulted in 685 bp with a range of 92,07-99,24 percentages of identity. The genetic mean pair-wise distances within-clade were 0,002 and 0,006, whilst the distance between the clade was 0.0883. Combining the suggestion with the ITF current, it is concluded that O. laqueus taken from Karimunjawa raised from the same species as those in Malaysia (MN711655) and Japan (AB302176). Specimens from Bangka-Belitung were suggested came from different species, as they were separated into the second clade by 8.83%. One single sample from Japan (AB430543) which laid outside the two clades by 11.63%-11.38% was also suggested to represent a different species. Overall, this study opens to various further studies on O. laqueus using other loci of genetic markers.


INTRODUCTION
As one of the newly discovered macrobenthic species, O. laqueus is a tropical organism that lives on sand and coral reef substrates of shallow waters of 0 to 18 m, the size may reach up to 48 mm mantle length. Commonly found scattered in Okinawa Island, Japan and northern Great Barrier Reef, Australia (Kaneko and Kubodera, 2005), it is confirmed by Ikeda (2009) that this nocturnal species is widespread in the Indo-Pacific (Ikeda, 2009). Research on O. laqueus has not been done much when compared to its congeneric. Data on nucleotide sequences that have been collected in GenBank (ncbi.nlm.nih.gov) for O. laqueus until before this study was carried out only 6 sequences consisting of 3 sequences of Cytochrom Oxidase sub-unit I (COI) locus (Kaneko et al., 2011;Rosli et al., Unpublished;Fukui and Furuya, unpublished), 1 sequence of COIII locus (Kaneko et al., 2011) Mitochondrial DNA (mt-DNA) is a widely used marker in genetic studies since it possesses several advantageous characteristics and present in high numbers in the cell, also thereby easy to isolate (Zhang and Hewitt, 1996;Kaneko et al., 2011). Aside of it, DNA barcoding of mt-DNA COI is useful for species identification and phylogeographic studies within a single species (Hui et al., 2016). This study therefore, aimed to analyse Indonesian O. laqueus using mt-DNA COI, compare it with available data from the GenBank and register its presence geographically in Indonesia as part of the Indo-Pacific malacological region.

MATERIALS AND METHODS
This study using samples of O. laqueus collected from 2019 to 2020 by local fishermen from Karimunjawa (n=16) and Bangka-Belitung (n=2) Indonesia ( Figure 1). The fishing ground location was confirmed by the local fishermen to ensure that samples were taken within the area of Karimunjawa and Bangka-Belitung. Samples in the form of tentacles tip were preserved in 96% ethanol, sent to the laboratory, renewed with fresh ethanol upon arrival and stored at 4 0 C.
The mt-DNA COI was extracted using 10% chelex (Walsh et al., 1991). The universal primer (forward) LCO1490: 5'-GGT CAA CAA ATC ATA AAG ATA TTG G -3' and (reverse) HCO2198: 3'-TAA ACT TCA GGG TGA CCA AAA AAT CA -5' describe by Folmer et al.1(994) were used for the amplification of the mt-DNA COI fragment. PCR amplification was performed in 25 µl reaction mixture containing 1 µl DNA template, 0.5 µl of each primer at 10 mM, 12.5 µl of Bioline master mix and 10.5 µl ddH2O. The following temperature profile was used: denaturation 94 0 C for 5 min, 35 cycles of denaturation 94 0 C for 1 min, annealing 56 0 C for 1 min, extension 72 0 C for 1.5 min and final extension was conducted at 72 0 C for 1.5 min (Kholilah et al., 2021). PCR products were visualised using 1% agarose gel stained with Biotium® gel red stain. The successfully PCR products were sent to a DNA Sequencing facility to get sequenced using Sanger methods.
The sequences were edited using MEGAX (Kumar et al., 2018) i.e. correct representation of nucleotides was checked based on the chromatogram peaks. Species identification was performed using the identification system for mt-DNA COI from NCBI (www.ncbi.nlm.nih.gov) with BLAST (Basic Local Alignment Search Tools). The identified samples were then compared with another samples of O. laqueus were retrieved from GenBank (BOLD Systems, http://v3.bold systems. org/), i.e., from Japan (n=2, accession no. AB302176 and AB430543, Kaneko et al., 2011) and Malaysia (n=1, accession no. MN711655, Rosli et al., unpublish).  (Kholilah et al.,202 Multiple and pairwise alignments was carried out using Muscle in the software MEGAX (Kumar et al., 2018). The phylogenetic tree was reconstructed using Bayesian method with BEAST2 package programme (Drummond et al., 2007). The best fit model of molecular evolution for each portioning data was determined using jModeltest program (Darriba et al., 2012) and HKY (Hasegawa Kishino & Yano) was selected as the best model. The Markov Chain Monte Carlo (MCMC) analysis with a random starting tree was run for 10 million generations. The genetic pairwise distance also calculated using MEGAX (Kumar et al., 2018).

RESULTS AND DISCUSSION
Samples from Karimunjawa (n= 16) and Bangka-Belitung (n= 2) were obtained from local fishermen using a spear-gun looking for octopus as fishing bait (Table 1). This type of octopus is not an export commodity, so it is not caught in large quantities. All samples can be well amplified (Figure 2). The resulting band be in the range of 700 bp, which is in accordance with the study of Carlini & Graves (1999) targeting the mt-DNA COI locus carried out in the Cephalopod class.
Each sample of O. laqueus from Karimunjawa (n= 16)) and Bangka-Belitung (n= 2) resulted in 685 bp sequences. BLAST identification revealed a range of 92,07-99,24 percentages of identity (Table 1). Of all, 16 specimens in Table 1 have been registered on GenBank with accession number LC552307-LC552342 and verified as O. laqueus, whilst the last two specimens from Bangka-Belitung is yet with accession number (MW559642 and MW559643). Leray et al. (2013) stated that the minimum percentage of identity needed to declare species similarity is 97 %, in other words 3% dissimilarity is a different species. In this case, samples from Bangka-Belitung performed a similarity below 97%, suggesting that they were a different species. However, these low percentages for Bangka-Belitung specimens probably happened due to the limited comparison sequences available at the GenBank.
Research by Hwang et al. (2016), which was carried out using morphological and phylogenetic examinations on well-known cephalopods in Southeast Asia including Thailand, Indonesia, Vietnam and China have been identified three orders, four families and nine genera. Out of those, for the Octopoda order they only found out two species, i.e., O. vulgaris and O. minor. Although O. laqueus was previously reported by Kaneko and Kubodera (2005) only in Japan and Australia, it turns out that this species is also found in Malaysia (Rosli et al., Unpublished) as well as in Indonesia, to be precise in Karimunjawa and Bangka-Belitung.
The phylogenetic tree O. laqueus produced 2 clades with the first clade being the samples originated from Bangka-Belitung and the second clade being those from Karimunjawa, Japan and Malaysia. Meanwhile, one single sample which was also from Japan laid separately from the two formed clades (Figure 3).  (Figure 4), the Indonesian seas, with their complex coastline geometry and narrow passages, provide the only pathway for low latitude Pacific water to flow into the Indian Ocean, this is known as the Indonesian ThroughFlow (ITF). ITF may cause O. laqueus larvae to be distributed by the current flew from Japanese waters to the Indonesian waters. This was also suggested to the larvae of O. cyanea which were planktonic and have been carried away in the water column for 1-2 months before settling (Reberinary & Benbow, 2012). This causes very wide geographical distribution, reaching hundreds of kilometers with the help of currents (Murphy et al., 2002;Casu et al., 2002). Moreover, specimens from Bangka-Belitung made up the second clade suggesting that they were originated from different species. It is however difficult to simply explain that although Bangka-Belitung laid within an area that was passed by the main ITF (Figure 3), they developed a different population, or even a different species. Furthermore, a single sample with accession number AB430543 originating from Japan is also separated from the two clades, confirming that it may be a completely different species. Veron et al. (2009) stated that the main barrier genetic connectivity pathway in marine organisms is currents.
The genetic distance between O. laqueus individuals ranged from 0.000-0.124 ( Table 2). The lowest distance is in the sample from Karimunjawa (0.000), while the highest distance is in the sample from Japan (AB430543-Japan; 0.124). This is inconsistent with the phylogenetic tree (Figure 3), in which that particular sample from Japan creates its own line apart from the two clades.