Genetic Insights into the Giant Keyhole Limpet (Megathura crenulata), an Eastern Pacific Coastal Endemic: Complete Mitogenome, Phylogenetics, Phylogeography, and Historical Demography

Abstract

Background: The giant keyhole limpet Megathura crenulata is a gastropod mollusk (Fissurella superfamily) that is endemic to the eastern Pacific coast from southern California, USA, to Baja California Sur, Mexico. M. crenulata is socioeconomically important as it produces a potent immune-stimulating protein, called Keyhole Limpet Hemocyanin, which is extracted in vivo and utilized for vaccine development. However, ecological studies are scarce and genetic knowledge of the species needs to be improved. Our objectives were to assemble and annotate the mitogenome of M. crenulata, and to assess its phylogenetic relationships with other marine gastropods and to evaluate its population genetic diversity and structure.

Methods: Samples were collected for mitogenome assembly (n = 3) spanning its geographic range, Puerto Canoas (PCA) and Punta Eugenia (PEU), Mexico, and California (CAL), USA. Total DNA was extracted from gills sequenced using Illumina paired-end 150-bp-read sequencing. Reads were cleaned, trimmed, assembled de novo, and annotated. In addition, 125 samples from eight locations were analyzed for genetic diversity and structure analysis at the 16s rRNA and COX1 genes.

Results: The M. crenulata mitogenomes had lengths of 16,788 bp (PCA) and 16,787 bp (PEU) and were composed of 13 protein-coding regions, 22 tRNAs, two rRNAs, and the D-Loop region. In terms of phylogeographic diversity and structure, we found a panmictic population that has experienced recent demographic expansion with low nucleotide diversity (0.002), high haplotypic diversity (0.915), and low φ_ST (0.047).

Conclusions: Genetic insights into the giant keyhole limpet provides tools for its management and conservation by delimiting fishing regions with low genetic diversity and/or genetically discrete units.

Keywords: Fissurella; genetic diversity; mitogenome; population structure.

Figure 1

Map of the mitogenome of M. crenulata from the PCA sample. Arrows indicate the direction of transcription. Protein-coding genes (PCGs) are in purple, ribosomal RNA in red, transfer RNAs in yellow, D-Loop in dark purple, and the origin of replication in blue.

Figure 2

Inferred Bayesian phylogenetic relationships among eight species of gastropods. (A) Complete mitogenome analysis. (B) Analysis with three mitochondrial genes ATP8, ND6, and COX3. Node support values are from Bayesian bootstrap proportions.

Figure 3

Haplotype frequency and diversity of the 16S rRNA and COX1 genes in eight locations of M. crenulata. California (CAL), Ensenada (ENS), San Quintín (SQT), Isla San Jerónimo (SJO), Puerto Canoas (PCA), Punta Eugenia (PEU), Bahía Asunción (BAS), and Isla Guadalupe (IGP). The circular diagrams indicate the diversity of haplotypes, colors indicate individual haplotypes. n: sample size per locality.

Figure 4

Historical demographic analyses based on 16S rRNA-COX1 concatenated haplotypes of the giant limpet M. crenulata along its geographical distribution. (A) Mismatch distribution. The bars represent the observed values, and the line represents the expected values under a constant population size model. (B) Bayesian skyline plot (BSP) approach. The y-axis is on a logarithmic scale. The x-axis indicates time (years) and starts at zero, corresponding to the present day. The solid blue line shows the median effective population size over time (Ne). The upper and bottom dashed lines represent the 95% confidence interval. The shaded grey area denotes the period during the last glacial maximum (LGM), and the vertical dashed line indicates the last interglacial (LIG) ending. A substitution rate of 0.0157 substitutions/site/million years, reported for marine invertebrates, was applied [40].

Figure 5

The minimum spanning network illustrates haplotypes from eight localities of M. crenulata. The circle sizes represent haplotype frequencies, while colors indicate the respective locality: California (CAL, red), Ensenada (ENS, pink), San Quintín (SQT, orange), Isla San Jerónimo (SJO, white), Puerto Canoas (PCA, blue), Punta Eugenia (PEU, green), Bahía Asunción (BAS, brown), and Isla Guadalupe (IGP, purple). Background circles in shaded color indicate haplogroups. Black circles represent mutational steps.