Comparative Characterization of the Complete Mitochondrial Genomes of the Three Apple Snails (Gastropoda: Ampullariidae) and the Phylogenetic Analyses

Abstract

The apple snails Pomacea canaliculata, Pomacea diffusa and Pomacea maculate (Gastropoda: Caenogastropoda: Ampullariidae) are invasive pests causing massive economic losses and ecological damage. We sequenced and characterized the complete mitochondrial genomes of these snails to conduct phylogenetic analyses based on comparisons with the mitochondrial protein coding sequences of 47 Caenogastropoda species. The gene arrangements, distribution and content were canonically identical and consistent with typical Mollusca except for the tRNA-Gln absent in P. diffusa. An identifiable control region (d-loop) was absent. Bayesian phylogenetic analysis indicated that all the Ampullariidae species clustered on the same branch. The genus Pomacea clustered together and then with the genus Marisa. The orders Architaenioglossa and Sorbeoconcha clustered together and then with the order Hypsogastropoda. Furthermore, the intergenic and interspecific taxonomic positions were defined. Unexpectedly, Ceraesignum maximum, Dendropoma gregarium, Eualetes tulipa and Thylacodes squamigerus, traditionally classified in order Hypsogastropoda, were isolated from the order Hypsogastropoda in the most external branch of the Bayesian inference tree. The divergence times of the Caenogastropoda indicated that their evolutionary process covered four geological epochs that included the Quaternary, Neogene, Paleogene and Cretaceous periods. This study will facilitate further investigation of species identification to aid in the implementation of effective management and control strategies of these invasive species.

Keywords: Ampullariidae; Pomacea canaliculata; Pomacea diffusa; Pomacea maculate; comparative characterization; mitochondrial genome; phylogenetic analysis.

Figure 1

Gene map of the complete mt genomes for P. canaliculata (GenBank accession No. KU052865), P. diffusa (KY008698) and P. maculate (KY008699). The larger ring indicates gene arrangement and distribution. The smaller ring indicates the GC content. ND1-6: NADH dehydrogenase subunits 1–6; COXI-III: cytochrome c oxidase subunits 1–3; ATP6 and ATP8: ATPase subunits 6 and 8; Cytb: cytochrome b; rrn: ribosomal RNA genes; tRNA: transfer RNA gene.

Figure 2

Graphical illustration showing the AT- and GC-skew in the PCGs of the mitochondrial genome of P. canaliculata (PC), P. diffusa (PD) and P. maculate (PM).

Figure 3

Evolutionary rates of the mitochondrial genome of P. canaliculata (PC), P. diffusa (PD) and P. maculate (PM). The rates of non-synonymous substitutions to the rate of synonymous substitutions (Ka/Ks) for each PCG are indicated.

Figure 4

Overall mean p-genetic distances between P. canaliculata, P. diffusa and P. maculate for each of 13 PCG. Values were calculated using the first and second nucleotide positions, the total codon and over the full sequence.

Figure 5

Codon Frequency (A) and RSCU (B) in the mitochondrial genomes of P. canaliculata (PC), P. diffusa (PD) and P. maculate (PM). PC-A indicated the Codon Frequency of P. canaliculata; PD-A indicated the Codon Frequency of P. diffusa; PM-A indicated the Codon Frequency of P. maculate. PC-B indicated the RSCU of P. canaliculata; PD-B indicated the RSCU of P. diffusa; PM-B indicated the RSCU of P. maculate.

Figure 5

Codon Frequency (A) and RSCU (B) in the mitochondrial genomes of P. canaliculata (PC), P. diffusa (PD) and P. maculate (PM). PC-A indicated the Codon Frequency of P. canaliculata; PD-A indicated the Codon Frequency of P. diffusa; PM-A indicated the Codon Frequency of P. maculate. PC-B indicated the RSCU of P. canaliculata; PD-B indicated the RSCU of P. diffusa; PM-B indicated the RSCU of P. maculate.

Figure 6

Graphical map of the BLAST results showing nucleotide identity between the complete mitochondrial genomes of P. canaliculata and species from the orders Sorbeoconcha and Architaenioglossa. BLAST results are presented in order with the sequence most similar to the reference (P. canaliculata) placed closer to the outer edge of the map. The gene regions, Blast identities and AT skew are depicted from outside to inside on the circle. The species from outside to inside are, respectively, P. canaliculata (KU052865), P. maculata (KY008699), P. diffusa (KY008698), M. cornuarietis (NC_025334), T. bacillum (NC_029717), S. libertine (NC_023364), T. sarasinorum (NC_030263) and C. cathayensis (NC_025577).

Figure 7

Graphical map of the BLAST results of mitochondrial CDS identities between P. canaliculata (NC_024586) and Sorbeoconcha and Architaenioglossa species. The sequences most similar to the reference (P. canaliculata) are placed closer to the outer edge of the map. COG (Clusters of Orthologous Groups of proteins), gene region, Blast identity and AT skew are shown from outside to inside. The species from outside to inside are named as in Figure 6.

Figure 8

Bayesian inference tree inferred from the amino acid sequences of 13 PCGs of 47 species of Caenogastropoda. Helix aspersa, NC_021747, was used as the outgroup. The numbers at the nodes represent the Bayesian posterior probabilities. Species in red indicate sequences generated in this study.

Figure 9

Chronogram for the 47 species of Caenogastropoda with a single out-group (Helix aspersa, NC_021747) based on the Bayesian topology calculated from analysis of the 13 PCGs. Divergence times were estimated using three calibrations. Numbers near the nodes indicated the average estimated divergence time estimated in Mya.