GEL - Objective 2

To determine intra- and inter-population genetic diversity based on microsatellite, mitochondrial DNA and allozyme markers in samples from throughout the range of the European lobster

Sampling protocols were established for biopsy collection of the distal segment of the walking leg (pereiopod) in ethanol for DNA analysis, frozen tissue for allozyme analysis, and fertilised eggs for paternity analysis. Data collection and sample to genotype authentication procedures were elaborated and form a model for subsequent studies. Sample collection involved 46 localities covering the range of the European lobster, from northern Norway to the Aegean Sea. In a few localities samples were obtained in several years to test for temporal stability. Most samples were of 100 individuals although only smaller numbers were feasible from several Atlantic localities and from the Mediterranean region (except northern Aegean). A sample of 49 American lobsters was obtained as an outgroup and because of the possible introduction of this species into the wild in Europe. In all specimens were obtained from 4782 individuals, making this one of the largest DNA-based population-genetic studies to-date for any marine organism.

Map illustrating geographic locations for European Lobster samples surveyed during this investigation

Good data quality was ensured through a rigorous microsatellite screening procedure. Since allelic diversity increases with sample size, screening of a sample of 200 lobsters demonstrated that a minimum sample size of 80 individuals should be used. It is recommended, for all future microsatellite population studies, that minimum sample size should be determined empirically, and that two persons should independently verify genotyping. Microsatellite genotypic data at six loci were obtained for 4189 individual European lobsters, representing 51 population samples (including temporal and pooled samples). Genotypic data were obtained for an additional 14 loci for a subset of 12 samples comprising 1182 individuals. One locus in this latter set was subsequently excluded from data analyses due to family data indicating the occurrence of null alleles. At the six loci, three to 13 alleles were found within samples, with an average of 6.9 alleles. The additional 13 loci increased the range to 23 alleles with a mean of 10.8 alleles. The number of alleles per sample, summed over six loci, ranged from 23 (Norway North abbreviated subsequently as NorwayN) to 51 (ScotlandW) and an overall total of 74. For 20 loci, the number of alleles per sample ranged from 94 (NorwayN) to 182 (EnglandSW) and an overall total of 216. There was no significant temporal variation in allele frequencies in the six available sample sets collected from one to 11 years apart.

Screening of the 3kb mtDNA segment I was carried out with five of the enzymes (AseI, AvaII, HincII, HinfI and TaqI) revealing most RFLPs, for 3283 individuals representing 44 population samples. Ninety composite haplotypes were found in the European lobster samples and 36 of these were present in only one sample. The number of haplotypes in individual samples ranged from four (NorwayN) to 31 (ScotlandN). The 2kb segment III was also screened, with two enzymes (DdeI and HinfI) revealing RFLPs, in 447 individuals from ten Mediterranean samples and 87 individuals from 12 Atlantic samples. Four different composite haplotypes were found in the Mediterranean samples with Aegean samples having the highest diversity. Only a single haplotype was found in the Atlantic samples. Haplotype frequencies were not significantly different in three different temporal sample pairs, collected up to five years apart. A major problem of the mtDNA analysis was the presence of additional fragments in the restriction fragment patterns, which frustrated attempts to analyse these patterns on the basis of site changes. Population analysis had therefore to be based on overall haplotype patterns. Various attempts to try to establish if these additional fragments in the European lobster were the result of nuclear copies of mtDNA were unsuccessful. Independent researchers found identical patterns. The high correlation between pair-wise FST values, derived from microsatellites and mtDNA, further validates the composite pattern approach taken to mtDNA data analysis.

Screening of 15 samples (1564 individuals), including two temporal pairs, for six polymorphic allozyme loci (GPI-1*, GPI-2*, IDHP-1*, sMEP*, PGM-1*, PGM-2*), showed a very low level of variation. The samples from NorwayN & NW were only polymorphic at IDHP-1* with only two heterozygotes for this locus being detected in NorwayNW. A previously unreported allele GPI-1* allele was found in AegeanN & NE and IrelandENE.

All three molecular approaches gave concordant results with pairwise Mantel comparisons of allozyme, mtDNA, and microsatellite derived Nei's DSTD and Reynolds' FST matrices showing highly significant positive correlations. The overall level of genetic differentiation found among the European lobster samples was relatively low. Differences among samples were in the form of allele (including haplotype seqq) frequency variation with private alleles being at low frequency only. Allele frequency and FST tests showed significant overall geographical heterogeneity, with FST values being greater for mtDNA (0.078) than for microsatellites (0.018) or allozymes (0.016). These tests, together with bootstrapped UPGMA dendrograms, based on DSTD and FST, showed four major distinct groups: Mediterranean; northern Norway; Netherlands; remaining Atlantic samples. The first three groups differentiate from the main Atlantic group due to reduced gene diversity and not due to unique alleles. Luikart's qualitative graphical method for detecting distortions in the distribution of microsatellite allele frequencies indicates that these populations have suffered genetic bottlenecks. Comparison of gene diversity in samples, as represented by the number of microsatellite alleles or mtDNA haplotypes, shows a highly significant Spearman Rank correlation, with mtDNA, as expected from its reduced effective population size, showing a greater reduction in the bottlenecked populations. Overall there is a significant correlation between geographical distance and genetic differentiation but this results mainly from the geographically extreme (NorwayN and Aegean) samples. No such correlation is found in the main Atlantic group. Microsatellite six-locus composite genotype assignment tests assign 75% of individuals correctly to the NorwayN sample, 62% to the Netherlands sample, 45% to the NorwayNW sample and 15% to the AegeanNE sample. The majority of individuals from remaining samples were randomly assigned to many other samples. As expected, use of 19 loci increased the discriminatory power of the assignment test with, for example, 99% of individuals from NorwayN being assigned to this sample. However, the higher assignment rate is in part due to only 12 samples being involved.

The Mediterranean is further differentiated into three sub-groups: Aegean; Adriatic; MediterraneanW (SpainE + Tyrrhenian). No differentiation was found among the six Aegean samples. High exploitation appears to have been responsible for current low numbers and lowered gene diversity throughout the Mediterranean. Northern Norway consists of two significantly different sub-groups, NorwayN and NorwayNW even though there is only 142km shoreline between the two fjords. Hydrographical barriers appear to be responsible for this reproductive isolation. Both populations have similarly low gene diversity. The populations live north of the Arctic Circle and probably undergo periodic population reductions as the result of extreme environmental conditions at the periphery of the species range.

Both genetic analysis and information from local fisheries biologists indicate a recent bottleneck in the Netherlands (Oosterschelde) population. Low salinity and temperature in the winter of 1962-63 killed virtually all the lobsters in the area, which is also now virtually isolated from the North Sea, due to damming, preventing immigration.

Only microsatellites reveal heterogeneity in the main Atlantic group. In the pair-wise comparisons some samples obtained from geographically adjacent areas (<100km) are significantly different, while some samples from distant areas (>1000km) are not significantly different. There was a 50% increase in the number of significant pair-wise comparisons in the main Atlantic group when 19 loci were used instead of six. Microsatellite analysis indicates significantly different groups on the west (Atlantic) and east (Irish + Celtic Seas) coasts of Ireland. Also, within each of these groups there are significantly different samples and there is no correlation of geographical and genetic distances.

Differences between European and American lobsters were found for mtDNA RFLPs and microsatellites. All individuals were correctly assigned to species by six-locus microsatellite assignment tests. However, examination of further American lobster geographical samples is required before it can be stated definitively that the two species can be identified on the basis of RFLPs or multilocus microsatellite assignment. There was no microsatellite evidence of hybridisation or introgression as a result of the introduction of American lobsters into Europe. However, microsatellites are too variable, and with too many shared alleles, for detection of low levels of introgression. MtDNA, being maternally inherited, is not suitable for identifying hybrids although in can be used to identify the female parental species in an otherwise identified hybrid. Clearly, given the possibility of hybrids occurring in European waters, species-specific nuclear markers should be developed to allow positive hybrid and backcross identification.