Mitochondrial DNA
COI
One hundred COI sequences were obtained (626 bp), which were composed of 19% guanine, 22% adenine, 29% thymine, and 30% cytosine. A total of 46 polymorphic sites and 39 parsimony informative sites were found, and 17 unique haplotypes were identified (Table 2).
Phylogenetic analysis and genetic structure - Phylogenetic reconstruction with BI showed two monophyletic lineages, hereafter referred to as GOM and MP. GOM included the Gulf of Mexico locations from Florida to Yucatan, while MP included the three locations from the Mexican Pacific (Fig. 2). This analysis also showed two lineages within the MP (A and B) which coincided with the haplotypes in the network (Fig. 3). The average genetic distance between GOM and MP was 4.5%, while the genetic distances between locations within groups ranged from 0–0.2% in GOM and 2.1–3% in MP. The highest intra-location genetic distance was in the Hu locality (3.3%). The general AMOVA detected genetic structure between GOM and MP (FST=0.698, P <0.001; Table 3). Within each group, AMOVA detected significant internal genetic structure (FST=0.224 GOM P <0.001; FST=0.167 MP P=0.040; Table 3). The haplotype network showed two haplogroups—GOM (12 haplotypes) and MP (five haplotypes)—with 27 mutational steps between them. Only two haplotypes were shared between GOM and MP. In addition, two groups, separated by nine mutational steps, were identified in MP. The star-shaped network in GOM could reflect expanding populations, while the haplotype pattern in the MP may suggest a more stable populations without clear differentiation among locations (Fig. 3).
Genetic diversity and demography – The overall genetic diversity for COI was h=0.686 and π=0.020. Genetic diversity within the GOM was h=0.460 and π=0.001 with a total of ten polymorphic sites and two parsimony informative sites. The values for MP were h=0.761 and π=0.023, with a total of 39 polymorphic sites and 39 parsimony informative sites (Table 2). In the GOM, the neutrality test indicated a significant deviation from mutation-drift equilibrium, evidenced by significant negative Tajima’s D and Fu’s Fs’ values, which indicate recent demographic expansion events. In addition, the unimodal mismatch distribution had significant values of R2 and low, non-significant r values (Fig. 4a; Table 2). On the contrary, for the MP, Tajima’s D and Fu’s Fs had positive, non-significant values. The mismatch distribution was bimodal with low and non-significant R2 and r values; therefore, the hypothesis of demographic or population expansion was not supported in MP (Fig. 4b; Table 2). The haplotype network also showed evidence of demographic stability (Fig. 3).
Migration - Migration analyses showed asymmetric migration from GOM to MP, with average Nm=93, with a 95% confidence interval of 12–407 from GOM to MP. In the opposite migration direction (MP to GOM), the average Nm value was less than one, with a confidence interval of 0–75. Within each of these groups, the Nm between locations ranged from 0 to 89,700 for the GOM (Table 4) and from 0 to 16,000 for the MP (Table 5).
Control region
One hundred twenty-one sequences were obtained for the GOM and MP (683 bp), composed of 16% guanine, 29% adenine, 33% thymine and 22% cytosine. 173 polymorphic sites and 130 parsimony informative sites were detected. A total of 97 haplotypes were identified (Table 2).
Genetic structure - The average genetic distance between GOM and MP was 11.7%, while the genetic distances between the locations within GOM ranged from 0.3–3.2% and for the two locations of MP was 15.6%. The highest intrapopulation genetic distance was in the Hu locality (average 6.3%). The general AMOVA detected genetic structure between GOM and MP (FST=0.698, P <0.001; Table 6). Within each group, the AMOVA detected significant internal genetic structure (FST=0.314 GOM P <0.001; FST=0.724 MP P <0.001), but the genetic structure was stronger in MP than GOM. The haplotype network confirmed the existence of two groups—GOM (79 haplotypes) and MP (18 haplotypes)—separated by 69 mutational steps, with only three shared haplotypes. Within the MP, two subgroups were detected, separated by 20 mutational steps: the first with Cu haplotypes and the second with Hu haplotypes. The network also highlighted the presence of a Cu haplotype, which was separated from the rest of the haplotypes by 40 mutational steps (Fig. 5).
Genetic diversity and demographic analyses – The overall genetic diversity for the control region was h=0.993 and π=0.051. Genetic diversity within the GOM was h=0.992 and π=0.015 with a total of 93 polymorphic sites and 50 parsimony informative sites, while for the MP was h=0.987 and π=0.078 with a total of 130 polymorphic sites and 116 parsimony informative sites (Table 2). GOM showed a higher genetic diversity, especially in the Si locality (Fig. 5).
In the GOM, the neutrality test Tajima’s D and Fu’s Fs had significant negative values, indicating a possible recent demographic expansion. A bimodal mismatch distribution was found, with significant values of R2 and low and non-significant r values (Fig. 4c; Table 2). For the MP, Tajima’s D was positive and Fu’s Fs was negative, both with non-significant values. The mismatch distribution was multimodal with low and non-significant R2 and r values (Fig. 4d; Table 2).
Migration - Migration analyses showed asymmetric migration between the groups; from GOM to MP there was an average Nm=31, with the 95% confidence interval from 5–208, while there was no migration in the opposite direction. Within each group, Nm values ranged from 0 to 3,910 for the GOM (Table 7), while in the MP, no migrants were detected from Cu to Hu and only 34 from Hu to Cu.
Microsatellites
Most of the locations were in H-W equilibrium, except Ta, Ca and Al in the GOM, due to the presence of null alleles. All loci were in linkage equilibrium. Null alleles were detected in loci Mce8, Mce11 and Mce25. However, the ENA test determined that including these loci in the analysis did not affect the genetic structure values (FST=0.426 with ENA, FST=0.412 without ENA).
Genetic structure - In the general analysis, which included all of the samples, the average genetic distance between GOM and MP was 66.3%. When analyzing only males, the mean distance was 66.1%, and for females it was 68.3%. In the overall AMOVA, 20.4% of the variation was between groups, 58.7% within groups, and 20.9% among locations within groups, which indicated significant heterogeneity among locations (FST=0.412, P <0.001; Table 8). The STRUCTURE results supported the existence of two genetic groups: GOM and MP (K=2, Ln (K)=-10,893.6; Fig. 6a). However, running this analysis separately by sex resulted in five genetic groups for males (K=5, Ln (K)=-2,598.7, Fig. 6b; FST=0.417 P <0.001), whereas for females we identified the same two genetic groups found in the overall analysis (K=2, Ln (K)=-7,393.2, Fig. 6c; FST=0.416 P <0.001). Significant FST values were detected for each sex (Table 8). Given the finding of different genetic structure between the sexes, we ran the STRUCTURE analysis separately within each sex-specific genetic group. In addition, since the Hu locality presented a mixture of genotypes from both coasts, it was included in the separate analyses for both coasts. The results for each coast are described below. In the GOM, AMOVA showed significant FST values in all cases (Table 8). With STRUCTURE, five genetic groups were detected when considering all of the individuals (K=5, Ln (K)=-5,467.4, Fig. 7a; FST=0.294 P <0.001). We also detected that males presented three genetic groups while the females had only two (K=3, Ln (K)=-1,900.1, Fig. 7b, FST=0.348 P <0.001 for ♂; K=2, Ln (K)=-4,760.5, Fig. 7c, FST=0.275 P <0.001 for ♀). In the MP, AMOVA showed significant FST values in all cases (Table 8). With STRUCTURE, there were three genetic groups when analyzing all the individuals and when analyzing separately by sex (K=3, Ln (K)=-2,973.5, Fig. 8a, FST=0.133 P <0.001 both sexes; K=3, Ln (K)=-1,003.5, Fig. 8b, FST=0.233 P <0.001 for ♂; K=3, Ln (K)=-2,008.83, Fig. 8c, FST=0.166 P <0.001 for ♀).
Genetic diversity – The genetic diversity for each locality was on average Ho=0.438 (range: 0.363–0.530) and average HE=0.520 (range: 0.418–0.643). The number of alleles per location ranged from three to nine, with an average of five alleles. The heterozygosity of the GOM was on average HO=0.447 (range: 0.363–0.530) and average HE=0.486 (range: 0.418–0.554), and each location had between three and four alleles, with an average of four. In the MP, heterozygosity was on average HO=0.472 (range: 0.450–0.513) and average HE=0.614 (range: 0.584–0.643), and each location exhibited between five and nine alleles, with an average of six. Heterozygosity in males was on average of HO=0.443 (range: 0.338-0.642) and average HE=0.580 (range: 0.402–0.634). The number of alleles per locality ranged from two to five, with an average of three alleles. Heterozygosity in females was on average Ho=0.435 (range 0.368–0.544) and average HE=0.522 (range: 0.411–0.642). The number of alleles per locality varied between two and eight, with an average of four alleles.
Migration and population sizes – The Nm was estimated within each of the STRUCTURE genetic groups within each sex, but since the Hu locality presented genotypes from both coasts, the Nm between coasts was estimated including Hu in both the GOM and MP groups. In the GOM, the number of male migrants (Nm♂) between genetic groups ranged from 7–227 individuals per generation, and migration was asymmetric in all cases. The locations that received the highest Nm were Sa and Hu, receiving 22–227 individuals from the other groups, while the lowest exchange was between the locations La, Ta and Ca with Me, with Nm=7 in both directions. In the MP, Nm♂ between locations ranged from 5–36 individuals per generation, and there was evidence of asymmetric gene flow in all cases except between Cu and Ba. The lowest gene flow was from Hu to Ba. The number of female migrants (Nm♀) presented a similar pattern, but with a lower Nm (range 5–16). The highest exchange of individuals occurred between Ba and Cu, while the lowest Nm was from Hu to Cu. Finally, the Ne for the GOM locations present the same ranged for each sex between 7,457 and 14,126 individuals, while the Ne for the MP ranged between 18,345 and 36,161 males and between 12,236-21,948 females.