Conus lischkeanus specimen was collected from Moonseom, Jeju Island, Korea, preserved in 95% ethanol solution, and deposited in the Marine Mollusk Resource Bank of Korea (MMRBK; voucher specimen no. MMRBK6746) in Seoul, Korea. The specimen was morphologically identified based on shell characters, which include a conical last whorl covered with yellow-brown periostracum and an angular shoulder. Total genomic DNA was extracted from the foot tissue using an E.Z.N.A. mollusc DNA kit (Omega Bio-tek, Norcross, GA, USA) following the manufacturer’s instructions.

Whole-genome sequencing libraries were prepared using the MGIEasy DNA library prep kit (BGI, Shenzhen, China) according to the manufacturer’s instructions and quantified using the QuantiFluor ssDNA System (Promega Corporation, Madison, WI, USA). Sequencing was conducted on the MGISEQ-2000 system with 150 base-pair reads. A total of 48,608,637 raw reads were obtained, and adapter-trimmed using a skewer program (Jiang et al. 2014) with a mean quality threshold of 20. The mitochondrial genome was assembled from trimmed reads using MITObim v. 1.9.1 (Hahn et al. 2013). Mitochondrial gene annotation was performed using MITOS websever (Bernt et al. 2013) and confirmed through sequence comparison with mitochondrial genomes of other Conus species previously reported (Bandyopadhyay et al. 2008; Cunha et al. 2009; Brauer et al. 2012; Barghi et al. 2016; Chen et al. 2016a, 2016b, 2016c; Gao et al. 2018; Uribe et al. 2018) using Geneious v. 9.1.8 (Kearse et al. 2012). The nucleotide composition, amino acid composition, and relative synonymous codon usage (RSCU) were analyzed using the MEGA X program (Kumar et al. 2018). Nucleotide composition skew was calculated using the following formula: AT-skew = [A – T] / [A + T] and GC-skew = [G – C] / [G + C] (Perna and Kocher 1995).

To determine the relationship between C. lischkeanus and other Conus species, phylogenetic analyses were performed for the nucleotide sequences of 13 protein-coding genes (PCGs) and two rRNA genes from 39 complete or nearly complete mitochondrial genomes of the family Conidae (Table 1). Tomopleura sp., belonging to the family Borsoniidae, was also included as an outgroup in the analysis. A concatenated nucleotide sequence dataset (13,870 bp long) of the 13 PCGs and two rRNA genes was prepared for phylogenetic analysis. The best substitution model for each gene was estimated using jModelTest v. 2.1.10 (Darriba et al. 2012) with the Akaike information criterion (AIC) for the nucleotide dataset. Phylogenetic analyses were conducted using maximum likelihood (ML) and Bayesian inference (BI) methods. ML analysis was performed using RAxML v. 8.2.9 (Stamatakis 2014) with a heuristic search and 10,000 bootstrap replicates. The BI tree was generated using the Markov chain Monte Carlo method, with two independent runs of 1 × 10⁶ generations with four chains, sampling every 100 generations and discarding the first 25% generations as burn-in. Both ML and BI programs were conducted using the CIPRES portal (Miller et al. 2010).

Conus lischkeanus is widely distributed from East Africa to the western Pacific (Röckel et al. 1995), extending to Taiwan, Japan, and Korea (Jeju Island). This species shows a wide range of shell morph and color variations, depending on geographic origin, which were previously classified as a few separate subspecies (Coomans and Filmer 1985) but are now treated as local variations of C. lischkeanus (Röckel et al. 1995). In this study, we determine the complete mitochondrial genome of C. lischkeanus and compare it with other cone snail species to infer the evolutionary diversification of different dietary types. The complete mitochondrial genome of C. lischkeanus (GenBank accession number: OL632021) is 16,120 bp in size, encoding 13 PCGs, 22 tRNA genes, two rRNA genes, and one control region (Fig. 1, Table 2). The overall nucleotide base composition is 29% A, 37.1% T, 17.6% G, and 16.3% C (Table 3). All 13 PCGs, 14 tRNAs, and two rRNA genes are encoded on the heavy strand, whereas eight tRNA genes (trnT, trnM, trnY, trnC, trnW, trnQ, trnG, and trnE) are encoded on the light strand. The gene order is identical to that of other cone snail species, suggesting that the mitochondrial gene order of this genus is highly conserved (Bandyopadhyay et al. 2008; Cunha et al. 2009; Brauer et al. 2012; Barghi et al. 2016; Chen et al. 2016a, 2016b, 2016c; Gao et al. 2018; Uribe et al. 2018). The AT and GC-skew values of the entire genome sequences, which represent the measures of compositional asymmetry, were negative (−0.1233) and positive (0.0390), respectively, similar to those of cone snails (Gao et al. 2018).

Figure 1. 

Mitochondrial genome structure of Conus lischkeanus.

The lengths of 13 PCGs of C. lischkeanus mitochondria range from 162 bp (atp8) to 1,716 bp (nad5) and contain 3,751 codons, excluding termination codons. The base composition of PCGs is 26.3% A, 39.2% T, 17.5% G, and 17.0% C, and the overall AT content was 65.5%, which is very similar to that of the entire mitochondrial genome sequence (AT content of 66.1%; Table 3). All PCGs have ATG as the initiation codon. With the exception of three PCGs (nad1, nad4L, and nad4) with TAG as a termination codon, all PCGs have TAA as a termination codon, which is consistent with complete mitochondrial genomes previously reported (Bandyopadhyay et al. 2008; Cunha et al. 2009; Brauer et al. 2012; Barghi et al. 2016; Chen et al. 2016a, 2016b, 2016c; Gao et al. 2018; Uribe et al. 2018). Fig. 2 shows the RSCU of C. lischkeanus, wherein the five most frequently used codons are UUA (Leu1), UCU (Ser2), CGA (Arg), CCU (Pro), and GUU (Val). In addition, codons with an A or U in the third position are the most frequently used, which is consistent with observations made in other mollusk species (Rawlings et al. 2010; Ren et al. 2010; Lee et al. 2019).

Figure 2. 

The relative synonymous codon usage (RSCU) frequency of the mitochondrial genome of Conus lischkeanus.

Twenty-two tRNA genes were found in the mitochondrial genome of C. lischkeanus. The length of tRNA genes range from 65 bp (trnC, trnE, trnS2, and trnF) to 70 bp (trnL1) (Table 2). All tRNA genes formed typical clover-leaf secondary structures, except for trnS1 and trnS2 which lack or had an imperfect D-arm (Fig. 3), which is common to other mollusk species (Boore 2006; Feng et al. 2020). Meanwhile, two ribosomal RNA genes with a total length of 2,327 bp consisting of small rRNA (rrnS; 952 bp) and large rRNA (rrnL; 1,375 bp) are located between trnE and trnV, and between trnV and trnL1, respectively (Fig. 2, Table 2). The D-loop is 587 bp in length and is located between trnF and cox3, with a short, inverted repeat (IR1; 20 bp), a typical feature of the mitochondrial genome of cone snail species. In contrast, the AT tandem repeat stretch found in C. consors G. B. Sowerby I, 1833 and C. quercinus [Lightfoot], 1786 was not identified in the C. lischkeanus mitochondrial genome (Brauer et al. 2012; Gao et al. 2018).

Figure 3. 

Predicted tRNA structures of Conus lischkeanus.

Phylogenetic analysis using ML and BI methods yield similar results with respect to the tree topology, as shown in Fig. 4. All subgenera, except Kalloconus da Motta, 1991, were monophyletic. A group of three Conus species, (C. capitaneus+(C. imperialis+C. genuanus)) was positioned at the most basal, but the branch reflected relatively weak supporting values (< 70% bootstrap values). Instead, the next monophyletic group consisting of (C. tabidus+(C. lenavati+C. tribblei)) was strongly supported (100% in ML and 1.0 BPP). Moreover, three species belonging to the subgenus Lividoconus Wils, 1970 (including C. lischkeanus) were grouped together with the subgenus Virgiconus Cotton, 1945 species C. virgo Linnaeus, 1758, a sister to a large assemblage of the remaining Conus species that is composed of two well-supported groupings differing in their feeding type: vermivorous species and a mixture of three diet types. The “vermivorous only” clade is composed of three monophyletic groups of the subgenera Virroconus Iredale, 1930, Kalloconus da Motta, 1991, and Lautoconus Monterosato, 1923, with the latter two more closely related to each other than to Virroconus. Within the “mixed diet” clade, aside from a well-supported molluscivorous species (100% BP in ML and 1.0 BPP in BI), all vermivorous species are grouped either with piscivorous or vermivorous species. It is evident that vermivorous species are not monophyletic and are split into four branches, each forming sister relationships with other molluscivorous and/or piscivorous species. Given the mitochondrial genome phylogeny with vermivorous species positioned at the basal, the tree topology coincides with earlier hypothesis that worm-hunting was the ancestral diet type. Meanwhile, the other two diet types such as molluscivorous and piscivorous were secondarily derived (Duda Jr et al. 2001; Puillandre et al. 2014; Gao et al. 2018; Abalde et al. 2019). Notably, piscivorous species in our phylogenetic tree are not monophyletic and split into three branches, which is not consistent with previous mitochondrial genome phylogeny where fish-hunting species formed a monophyletic group (Gao et al. 2018). The polyphyly of piscivorous species in the current study implies that the fish-hunting species have evolved independently from worm-hunting groups multiple times. The complete mitochondrial genome information of the worm-hunting Conus species (C. lischkeanus) in the present study provides valuable insights into the mitochondrial genome diversity and molecular phylogeny of Conus species.

Figure 4. 

Phylogenetic relationships of the genus Conus based on concatenated nucleotide sequences (13 protein coding genes plus two rRNA genes). Numbers above branches are statistical support values for ML (bootstrap values, > 70)/BI (posterior probability values, > 0.7). *: determined in this study.