Introduction

Molecular phylogenetic analyses provided a different approach compared with traditional morphological methods to estimate the relationships among species based on the topological hypotheses1,2,3. The mitochondrial DNA has a high rate of base substitution and lacks of recombination during inheritance; besides it possesses an unique transmission mode named doubly uniparental inheritance (DUI) in molluscs4,5. Hence mitochondrial genomic analyses was proved as a valid molecular tool in constructing phylogenies, and has been used for phylogenetic analyses in various taxa6,7.

The derived phylogenetic relationships based on molecular data may disagree with the evolutionary hypothesis proposed using morphological data2,3. Neogastropoda was widely accepted as a monophyletic group based on morphological characters; however, Tonnoidea was placed into Neogastropoda based on the molecular analyses8,9,10,11, which contradicted the monophyletic status of Neogastropoda. Among Littorinimorpha, Vermetidae is a peculiar snail family that shows a high rate of gene rearrangement12. Based on the molecular phylogenetic analyses, Vermetidae were regarded as the sister group of the other species in Caenogastropoda,; however, this is opposite to the morphological evidence13. Hence, the relative position of Vermetidae in the mitochondrial phylogenetic analyses has been considered spurious, although the relationship was highly supported in previous molecular phylogenetic analyses14,15.

Strombidae species are important molluscs in shallow water of tropical and subtropical areas from past time until now16,17,18,19. Species in Stromboidae varied greatly in shell shapes, which results in high morphological diversity19,20. Strombus (Linné,1758) and Lambis (Röding, 1798) are the two most abundant genera in Strombidae and were once regarded as the only two genera in strombids21. However, based on the fossil record and molecular phylogenetic analyses, the genus Strombus is justified to be subdivided into several separate genera20,21.

Based on the paleontological studies, Strombidae probably originated from Aporrhaidae during Cenomanian-Turonian, and evolved at low diversities during the rest of the Cretaceous19. During the course of evolution, whereas Aporrhaidae species underwent K/T mass extinction in late Cretaceous, a major genera and species radiation in Strombidae occurred during the early Cenozoic and continued to the Pliocene19,22,23.

In the present study, we determined the complete mitochondrial genomes of H. chiragra and L. lambis, and analyzed the genomic features of the two species, including their structural characters and nucleotide composition. In early taxonomical studies, H. chiragra shared close relationships with L. lambis in Strombidae, yet this was mainly based on their similar tissue anatomies (e.g. egg masses, and radulae)18, regardless of the great morphological difference in adults. To valid the taxonomy relation between H. chiragra and L. lambis, we attempt to determine their phylogenetic relationships based on the mitochondrial genomes. Thus, a robust phylogeny based on the concatenated 13 protein coding genes of 15 Littorinimorpha species was constructed. These data provides a framework for further evolutionary studies among Littorinimorpha.

Materials and Methods

Specimen and mitochondrial DNA extraction

Individuals of H. chiragra and L. lambis were collected from the coastal waters of Quanfu Island, South China Sea. Total genomic DNA was extracted from the foot muscle using a modified standard phenol-chloroform procedure24 and then stored at −20 °C.

Determination of partial sequences

Short fragments of cox1 gene were PCR amplified using the universal primers LCO-1490/HCO-219825. Based on the reference genome of S. gigas26, primers were designed using Primer Premier 527 to amplify short fragments of atp6, cox3, and cytb (Supplementary Table S1). Long PCR primers were designed to amplify the regions between the genes based on the partial sequences obtained.

PCR amplification and sequencing

PCR was performed in a 30 μL reaction mixture containing 3 μL of dNTPs (2.5 mM each), 3 μL of 10 × LA buffer (Mg2+), 1 μL of template DNA (100 ng/μL), 1 μL of each forward and reverse primer, and 0.5 μL of TaKaRa LA-Taq DNA polymerase. The thermal cycling conditions are: 94 °C for 3 min followed by 35 cycles of denaturing at 94 °C for 30 s, annealing at 62 °C for 30 s, and extension at 68 °C for 5 min, with a final extension step of 72 °C for 10 min. PCR products were purified using an EZ-10 Spin Column DNA Gel Extraction Kit (Sangon Biotech), and then directly sequenced using the primer walking method. DNA sequencing was performed on an ABI PRISM 3730 (Applied Biosystems) automatic sequencer.

Genome annotation and sequence analysis

Sequence assembly was performed using the Seqman program, DNASTAR (http//www.DNASTAR.com). The annotations of protein-coding genes were conducted using ORF Finder (https://www.ncbi.nlm.nih.gov/orffinder/) with invertebrate mitochondrial genetic code. The transfer RNA (tRNA) genes were identified using ARWEN (http://130.235.46.10/ARWEN/) and MITOS web servers (http://mitos.bioinf.uni-leipzig.de/index.py) using the mitochloroplast or invertebrate gene code and default search code.

The gene annotation of rrnL and rrnS were conducted using BLAST searches (https://blast.ncbi.nlm.nih.gov/Blast.cgi) by identifying their similarity to gene sequences of S. gigas and Conomurex luhuanus. The A + T content values were computed using MEGA 6.0628 and GC and AT skews were calculated according to the formulae described before29, AT skew = (A − T)/(A + T); GC skew = (G − C)/(G + C), where A, T, G, and C are the occurrences of the four nucleotides. The relative synonymous codon usage (RSCU) values of each protein coding gene were calculated using MEGA 6.0628. The number of base substitutions per site between H. chiragra L. lambis, S. gigas, and C. luhuanus were calculated in MEGA 6.0628 using the Kimura 2-parameter model30.

Phylogenetic analyses

The phylogenetic analyses were based on the concatenated nucleotide and amino acid alignments of thirteen protein-coding genes in seventeen complete mitochondrial genomes, including H. chiragra, L. lambis and 13 other available mitochondrial genomes of Littorinimorpha (Supplementary Table S2). Besides, Tegula lividomaculata and Tegula brunnea from the order Trochida served as outgroup. The thirteen-partitioned nucleotide and amino acid sequences of the protein-coding genes were aligned using MAFFT31 with automatic selection of alignment algorithm. Then the alignments were treated with Gblocks32,33 using default parameters, and the ambiguously aligned regions were removed from the analyses. Multiple gene alignments were concatenated using PhyloSuite34. Then we evaluated the saturation in the codon-based data sets of thirteen protein coding genes in DAMBE735, and the results showed that the DNA sequences were unsaturated in 1st-2nd-3rd and 3rd codon sites. The best-fit partition schemes of amino acid and nucleotide sequences were selected using PatitionFinder 2.1.136. Two methods were used to perform the phylogenetic analyses: Maximum Likelihood (ML) and Bayesian inference (BI). ML analysis was conducted using RAxML37 web server on the CIPRES Science Gateway V.3.3 (http://www.phylo.org/index.php/) based on the partitioned nucleotide alignments, with GTR + G substitution model and 1,000 bootstraps for node reliability estimation. Bayesian analyses were conducted in MrBayes38 for 200 million generations (sampling every 1000 generations) based on the partitioned nucleotide and amino acid alignments. All parameters were checked using Tracer v1.539. The first 50,000 trees were discarded as burnin, and the remaining sampled trees were used to estimate the Bayesian posterior probabilities.

Estimate of divergence time

The estimation of divergence time of the major Littorinimorpha lineages were conducted using BEAST v.1.7.539 based on the partitioned amino acid sequences of 13 protein coding genes. A lognormal relaxed-clock model was selected as the molecular clock model. A Yule process of speciation was chosen for the tree prior. The final Markov chain was set to 100 million generations, sampling every 10,000 generations. The effective sample size of all parameters was above 200. The convergence of the chains was checked with Tracer v.1.539, and the first 1,000 generations sampled were discarded as part of the burn-in process.

The posterior distribution of the estimated divergence times was specified based on the prior fossil knowledge. Two calibration points were selected, using a normal distribution of prior probability: 342.8 Mya was used as prior divergence time for Vermetoidea based on the Paleocene fossil collection in Belgium and the United Kingdom40, and the prior divergence time of Truncatellidae was set as 66.04 Mya according to the oldest fossil record of Paleocene in Belgium41,42,43. Besides, the divergence time of Tegula was 85 Mya based on the Cretaceous fossil record in United States41, and this point was used to cross-validate the accuracy of the dated tree.

Results and Discussion

Genome organization of H. chiragra and L. lambis

The complete mitochondrial genome sequences of H. chiragra and L. lambis are 15,460 bp and 15,481 bp, respectively (Tables 1, 2), and both contain 13 protein coding genes (PCGs), 22 tRNAs and two rRNAs (Fig. 1). This (+) strand encodes for trnD, trnV, trnL, trnL, trnP, trnS, trnH, trnF and the cluster KARNI (trnK, trnA, trnR, trnN, and trnI) and trnS. The (−) strand encodes for the cluster MYCWQGE (trnM, trnY, trnC, trnW, trnQ, trnG, and trnE) and trnT (Fig. 1). Four overlaps between adjacent genes were detected in H. chiragra and L. lambis, in addition, another region between atp8 and trnV was found only in H. chiragra, but not in L. lambis (Fig. 1). The lengths of genes (including PCGs, tRNAs and rRNAs) and intergenic nucleotides are 15129 bp, 331 bp for H. chiragra and 15146 bp, 335 bp for L. lambis, respectively (Tables 1, 2), in which the gene length of the overlapping nucleotides was counted once.

Table 1 Organization of the mitochondrial genome of Lambis lambis (15,481 bp).
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Table 2 Organization of the mitochondrial genome of Harpago chiragra (15,460 bp).
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Figure 1
figure 1

Linear comparison of the gene organization of H. chiragra and L. lambis mitochondrial genomes. The blue lines indicated genes coded by the minor strand. Positive numbers mean the length in bp of non-coding regions between genes and negative numbers represent overlapping nucleotides between genes.

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The organization of the H. chiragra and L. lambis mitochondrial genomes was compared with that of other sepcies in Littorinimorpha (Fig. 2). Among the gastropod species, mitochondrial genomes are estimated to show high rates of gene rearrangement between major lineages44. However, the gene orders of the two newly sequenced mitochondrial genomes were similar to the consensus gene order shared by most previously published species from Littorinimorpha26,45(Fig. 2).

Figure 2
figure 2

Linear comparisons of the organization of the mitochondrial genomes of Littorinimorpha.

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Nucleotide composition

The overall base compositions of the mitochondrial genomes on the (+) strand were both biased toward A and T. For H. chiragra, the nucleotide content was found to be A = 28.26%, T = 37.6%, C = 16.57%, and G = 17.21%. For L. lambis, the nucleotide content was A = 28.61%, T = 37.49%, C = 16.50%, and G = 17.40% (Table 3). For the entire mitochondrial genomes, the AT and GC-skews on the (+) strand were −0.128 and 0.019 for H. chiragra and −0.134 and 0.026 for L. lambis, respectively (Table 3).

Table 3 AT-content, AT-skew, and GC-skew for mitochondrial genes of H. chiragra and L. lambis.
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The nucleotide composition of the single gene region of H. chiragra and L. lambis were calculated. The A + T content of protein coding genes (PCGs), tRNA, rRNA, and non-coding regions (NCRs) is similar between H. chiragra and L. lambis (Table 3). For single genes, similar A + T content was only detected in cox3 (60%), nad2 (68%) and nad4 (66%).

Among the different types of genes, the tRNA genes and rRNA genes of H. chiragra and L. lambis show positive AT skews, whereas all types of protein coding genes show negative AT skews. Both the tRNA and rRNA genes of H. chiragra and L. lambis show positive GC skews. Some protein coding genes (atp6, cob, nad4, nad5, and nad6 in H. chiragra and L. lambis and atp8 in L. lambis) show negative GC skews, while the other types of protein coding genes and RNA genes (tRNA genes and rrnL, rrnS) show positive GC skews.

Protein coding genes (PCGs)

Excluding the termination codons, the mitochondrial genomes of H. chiragra and L. lambis encode 3,744 and 3,745 amino acids, respectively. Comparison between species of start and termination codons of protein coding genes showed that only 2 PCGs initiate or stop with different codons, which were detected in nad4 gene (initiated with GTG in H. chiragra and ATG in L. lambis) and cox1 gene (stopped with TAG in H. chiragra and TAA in L. lambis). In addition, 12 PCGs contain the same start (nad2: ATC; ATG for the other 11 PCGs) and termination (nad2: TAG; nad1: TAG; nad4L: TAG; TAA for the other 9 PCGs) codons between species (Supplementary Fig. S1).

Pairwise divergence among four Strombidae mitochondrial genomes was calculated based on separate and concatenated protein-coding genes (Supplementary Fig S2). The nucleotide divergence between H. chiragra and L. lambis was 0.151, which was the lowest genetic divergence measured here, confirming the close relationship between H. chiragra and L. lambis. Strombus gigas and C. luhuanus have a nucleotide divergence of 0.351, indicating a relatively distant relationship. Compared with the nucleotide divergence among the four Strombidae mitogenomes, the pairwise divergence values calculated using the amino acid sequences were lower, indicating that synonymous substitutions in protein-coding genes were more frequent than nonsynonymous substitutions.

The codon usage of the mitogenomes of H. chiragra and L. lambis was similar to that of other Strombidae species26,45. All codons were used in the mitogenomes of these two species, however the codon frequencies varied between each other. Amino acids encoded by A + T-rich codons are more common than those encoded by G + C-rich codons. The ratio of A + T/G + C-rich codons was 2.61 in L. lambis, which was lower than what was found for H. chiragra (2.70). The relative synonymous codon usage (RSCU) is different between H. chiragra and L. lambis, implying their larger genetic difference than previously recognized (Fig. 3). The RSCU also reflected the nucleotide composition bias. For Phe (UUY), the RSCU was 1.42/1.44 for UUU, but only 0.58/0.56 for UUC in the L. lambis/ H. chiragra mitochondrial genomes, respectively. Amino acids coded by A + T-rich codons are 2.61/2.70 times higher than G + C-rich codons in the L. lambis/H. chiragra, respectively. The codon usage bias observed in the two snails indicated that the two strands were exposed to different mutational pressures during replication, and it increases the frequency of A + T-rich codons, which is similar to the reports in the vertebrate mitogenomes46,47,48.

Figure 3
figure 3

Relative synonymous codon usage (RSCU) of H. chiragra and L. lambis mitochondrial genomes. The termination codon is not given.

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Non-coding regions

There were 34 non-coding regions distributed in the H. chiragra and L. lambis genomes, 403 bp for H. chiragra and 393 bp for L. lambis (Fig. 1). The non-coding sequences were generally characterized by short nucleotide fragments, ranging from 1 bp to 53 bp in H. chiragra and 1 bp to 54 bp in L. lambis among every non-coding fragment. The largest non-coding region was found between the gene cox3 and trnF (53 bp for H. chiragra and 54 bp for L. lambis). This location was proposed as a candidate to contain the control region in other gastropod mitochondrial genomes49. Among the non-coding regions, there were 20 regions with different lengths between H. chiragra and L. lambis and 14 intervals with same length.

Phylogenetic analyses

The selected partition schemes for phylogenetic analyses were listed in Supplementary Tables S3, S4. The topological structure of the trees inferred by two different methods (ML and BI) was essentially uniform (Fig. 4). All nodes in the BI tree were near 100% supported and the nodes in the ML tree were also highly supported. Within Stromboidea, the phylogenetic tree shows that Strombidae form an independent branch as (S. gigas + (C. luhuanus + (L. lambis and H. chiragra))). L. lambis is the closest extant relative of H. chiragra, and this clade clustered with S. gigas and C. luhuanus. Research derived from combined phylogenetic analyses of molecular and morphological data has revealed that Lambis was monophyletic and Strombus was paraphyletic20. However, when the cladistics analyses of species in Lambis were based solely on morphological characters, the results clustered one Lambis species (L. crocata) into the outgroups of species50, suggesting that Strombus is polyphyletic and the Lambis is paraphyletic. Lambis crocata was not included in the present study since there is no complete mitochondrial genome available for this species. Although lacking a sufficient number of species for a robust phylogenetic analysis, our phylogeny is statistically supported and aims to provide a reasonable framework for further phylogenetic research within Stromboidea.

Figure 4
figure 4

Phylogenetic trees derived from BI and ML analyses based on amino acids and nucleotide sequences of 13 protein coding genes. The first and second numbers at each node are the bootstrap values of posterior probabilities based on aligned nucleotide and amino acids sequences of 13 mitochondrial protein coding genes. The third number is ML bootstrap values based on nucleotide sequences.

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Within Littorinimorpha, Stromboidea and Tonnoidea clustered together in the same clade, which was then clustered with (Littorinoidea + Naticoidea), as derived by the BI method. Stromboidea, Tonnoidea, Littorinoidea, and Naticoidea form a well-supported clade based on both ML and BI, confirming their close relationship within Littorinimorpha. Rissooidea was sister to Truncatelloidea, which together formed the second major clade. Vermetoidea formed the third independent well-supported clade within Littorinimorpha.

Estimate of divergence time

To test the accuracy of the dated tree derived from BEAST, we made a cross-validation using the calibration point of genus Tegula. The oldest fossil record of Tegula was stated as 85 Mya41 and the documented time was coincident with the divergence time of T. lividomaculata and T. brunnea (13–309 Mya) in present study. Research documented that the number of species increased from five species within the genus Strombus during the Eocene (53 Mya) to 40 species till the Late Oligocene and the Miocene (23–36 Mya)16, indicating that the species diversification within Strombidae accelerated in the last 36–53 Mya. According to the present dated tree (Fig. 5) the diversification of Strombidae species occurred around 112 (44–206) Mya, and a radiation pattern (accelerated rates of diversification) is detected to occur between 45–75 Mya, which is in agreement with the fossil record in Strombidae. Besides, the diversification pattern of Strombidae species occur between the late Cretaceous and early Paleocene (65–135 Mya), and this might provide implications to understand the Cenozoic replacement event of Aporrhaidae by Strombidae19,22,23. Furthermore, to better resolve the phylogenetic relationships and understand the replacement event, more Strombidae and Aporrhaidae mitochondrial genomes should be inserted into the phylogenetic analyses.

Figure 5
figure 5

Phylogenetic tree with age estimates of 17 mollusk species based on the mitochondrial dataset and Bayesian relaxed dating methods (BEAST). The numbers next to the nodes are presumably ages. The posterior probability of each node is given above the line. Dates are in millions of years (Mya).

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