Part 5 Genetics and Fisheries Management Genetic variation in fish stocks; Use of molecular tools Estimation of effective population size and population dynamics' parameters; MASTERS IN AQUACULTURE AND FISHERIES Genetics and Selection

1 - Overview of the applications of population genetics to marine resources management Fisheries Management = the application of scientific knowledge to the problems of providing the optimum yield of commercial fisheries products or angling pleasure (Everhart & Youngs 1981) MASTERS IN AQUACULTURE AND FISHERIES Genetics and Selection

Historical Perspective Vast majority of fish stock harvested through fisheries is wild – a conservation problem Until recently there was very limited application of basic genetics principles to fisheries management: Field dominated by taxonomists that care little about differences between individuals Fish are more difficult to observe and so the study of the relationships between phenotype and genotype is more difficult Fish are the ‘last’ major food source captured from wild stocks, with hard to define boundaries The amounts of phenotypic variation found in fish are very wide when compared with other food source animals First genetic results often contradicted ecological and ethological understanding of species So, the long-term perspective that genetic analysis provides has been missing from Fisheries Management theory The result is that many fisheries stocks were the victim of over-exploitation MASTERS IN AQUACULTURE AND FISHERIES Genetics and Selection

Genetic and Phenotypic Variation in Fish Species Include table 1.2 and 1.2 of Pop Gen and Fish. Man. 1 - It appears that fish are phenotypically more variable than other vertebrates; 2 - Specifically, economically important traits such as growth rate vary much between as well as within populations; 3 – Variation between con-specific populations is also very large, artic char 4000%, whereas Darwin's’ finches 15 x less MASTERS IN AQUACULTURE AND FISHERIES Genetics and Selection

Heritability in Fish Species Include table 1.3 of Pop Gen & Fish. Man. Heritability – proportion of variation that is of genetic origin Ranges from 0 (no genetic component) to 1 (no environmental component) Usually lower in fishes than in other vertebrates Fish appear more susceptible to environmental factor: Indeterminate growth – permits greater adjustment to env. Factors Poikilothermic – more sensitive to temperature variations than birds and mammals plasticity in age and size at maturity MASTERS IN AQUACULTURE AND FISHERIES Genetics and Selection

Genetic Divergence Between Populations Classic problem in Fisheries Management: FROM the identification of a fishing stock BECOMES the identification of genetically meaningful management units Many ecological and behavioral differences are due to environmental differences (freshwater/seawater, different spawning times and places, etc.) A considerable part of the difference between populations is also due to environmental differences - similar environmental conditions mold variation within populations and so the differences between populations tend to be due to differences in the environment of each one – The role of genetics in the differentiation has been unclear until recently Markers not available until the mid 70’s MASTERS IN AQUACULTURE AND FISHERIES Genetics and Selection

First Biochemical Studies Identification of previously unrecognized systematic groups: Population units of Pacific herring in the Alaskan peninsula (Grant & Utter, 1984) Reproductively isolated sympatric populations of brown trout (Ferguson & Mason, 1981) Identification of new species of rockfish (Seeb, 1986) Inconsistencies with previous assumptions of genetic divergence Conspecificity of anadromous and landlocked forms of char in North America (Kornfield et al., 1981) No apparent genetic divergence between Fall and Spring spawning Atlantic herring (Ryman et al., 1984) Conspecificity (and local random breeding) of distinct morphological types of Ilyodon previously considered separate species (Turne & Grosse, 1980) Conspecificity of sympatric but trofically specialized forms of Mexican cichlids (Kornfield et al., 1982) MASTERS IN AQUACULTURE AND FISHERIES Genetics and Selection

Management Goals Conservation of genetic variation between and within natural populations Maintenance of the genetic characteristics of stocks that are artificially propagated in hatcheries (e.g. Pacific salmon species in the Northwest USA) Stock enhancement Selective breeding for production traits MASTERS IN AQUACULTURE AND FISHERIES Genetics and Selection

The Role of Genetics in Fisheries Management Differential Harvesting Among Populations Differential Harvesting Within Populations Hatchery Populations Release of Hatchery Fish MASTERS IN AQUACULTURE AND FISHERIES Genetics and Selection

2 - Basic concepts in population and molecular genetics MASTERS IN AQUACULTURE AND FISHERIES Genetics and Selection

Gene Frequencies and Hardy-Weinberg Equilibrium Simplified model for character determination; Applicable to simple traits, such as blood groups or disease resistance; We will assume diploidy; MASTERS IN AQUACULTURE AND FISHERIES Genetics and Selection

Phenotypes and Genotypes Phenotypes are the appearances of characters (visible/measurable); Genotypes are the genetic compositions that cause the phenotypes; Often one phenotype is the product of many genes – For example FCR depends on digestive enzymes in the gut, on hundreds of metabolic enzymes in cells, etc., in total it is estimated that hundreds or thousands of genes will determine FCR (a phenotype we can measure); MASTERS IN AQUACULTURE AND FISHERIES Genetics and Selection

Recessive, Dominant and Codominant Alleles In diploid organisms each gene (locus) is present in two copies (alleles); If both are equal we say the locus is homozygous, otherwise it is termed heterozygous; Recessive alleles do not express themselves in the presence of a dominant one. Difficult to detect… the problem with disease ‘carriers’; Codominance means that to a degree the phenotype of the heterozygote is the product of the expression of both alleles; MASTERS IN AQUACULTURE AND FISHERIES Genetics and Selection

Describing the Genetic Make-up of a Population Genotype Frequencies When we know the relationship between phenotypes and genotypes we can estimate the genotype frequencies in a population; E.g. blood group typing, eye color in fruit flies, etc. Blood group Number of individuals M MN N Frequency (%) Greenland 83.5 15.6 0.9 569 Iceland 31.2 51.5 17.3 747 Mourant(1954) in Falconer 1989 MASTERS IN AQUACULTURE AND FISHERIES Genetics and Selection

Describing the Genetic Make-up of a Population Gene Frequencies Frequency (%) Greenland 91.3 8.7 Iceland 57.0 43.0 MASTERS IN AQUACULTURE AND FISHERIES Genetics and Selection

MN blood Group Genotypes MASTERS IN AQUACULTURE AND FISHERIES Genetics and Selection

Hardy-Weinberg Equilibrium Assumes: Large population; Random mating; No selection; No migration; No mutation; Predicts: Stable gene frequencies from generation to generation; Simple relationship between gene frequencies (allele frequencies) and genotype frequencies. MASTERS IN AQUACULTURE AND FISHERIES Genetics and Selection

H-W Equilibrium Genes in parents Genes in progeny A1 A2 A1A1 A1A2 A2A2 Frequencies p q p2 2pq q2 MASTERS IN AQUACULTURE AND FISHERIES Genetics and Selection

Applications of the H-W Law Determination of the gene frequency of a recessive allele; Frequency of ‘carriers’; Test of H-W equilibrium; MASTERS IN AQUACULTURE AND FISHERIES Genetics and Selection

Changes in gene frequency Random drift; Migration; Mutation; Selection; Assortative Mating; Computer simulation - PopG MASTERS IN AQUACULTURE AND FISHERIES Genetics and Selection

Assortative Mating When mated pairs are of the same phenotype more often than expected by chance (common in humans, e.g. stature, intelligence, etc.); The opposite is called disassortative mating (common in plants with self-sterility systems); Increases frequency of homozygotes (although most characters are multiple loci coded); MASTERS IN AQUACULTURE AND FISHERIES Genetics and Selection

Migration q1 = m qm + (1 – m) q0 = m (qm – q0) + q0 Δq = q1 – q0 Thus teh rate of change depends on: Immigration rate Difference of gene frequencies between immigrants and natives q1 = m qm + (1 – m) q0 = m (qm – q0) + q0 Δq = q1 – q0 = m (qm – q0) MASTERS IN AQUACULTURE AND FISHERIES Genetics and Selection

Mutation Non-recurrent mutation – low chances of mutant allele survival Recurrent mutation – more relevant If u is mutation rate from A1 to A2 and v is mutation rate for reciprocal mutation (A2 to A1) and p0 and q0 are frequencies of A1 and A2 then The change in gene frequency in one generation is Δq = up0 – vq0 And at equilibrium q = u / (u + v) MASTERS IN AQUACULTURE AND FISHERIES Genetics and Selection

Selection Occurs when some genotypes are more fit than others Degrees of dominance with respect to fitness: No dominance; Partial dominance; Complete dominance; Overdominance. MASTERS IN AQUACULTURE AND FISHERIES Genetics and Selection

Changes in Gene Frequency Under Selection If s is the selective advantage of the A1 dominant allele (p), then the frequency of A2 after one generation (q1) is: So, the change of gene frequency from one generation of selection Δq = q1 – q is and substituting p = (1 – q) MASTERS IN AQUACULTURE AND FISHERIES Genetics and Selection

Polymorphism – Possible Causes Heterozygote advantage; Frequency-dependent selection; Heterogeneous environment; Transition stages in evolution (due to environment changes, for example); Neutral mutation (allele); Heterozygosity – a measure of the amount of polymorphism: Proportion of polymorphic loci; Frequency of heterozygotes averaged over all loci tested = average Heterozygosity; MASTERS IN AQUACULTURE AND FISHERIES Genetics and Selection

Small Populations Random drift Sampling effects: Variance in the change of gene frequency Variance of gene frequency among lines MASTERS IN AQUACULTURE AND FISHERIES Genetics and Selection

Random Drift and N MASTERS IN AQUACULTURE AND FISHERIES Genetics and Selection

Inbreeding The mating together of individuals that are related to each other by ancestry. In a bisexual population the number of ancestors of a individual t generations ago is 2t So, in a small population the relatedness of individuals will be greater than in a large population; MASTERS IN AQUACULTURE AND FISHERIES Genetics and Selection

Measuring Inbreeding The coefficient of Inbreeding is F F1 is the inbreeding coefficient of generation 1 and F2 that of generation 2 Probability that a gamete pairs with another of different sort Probability that a gamete pairs with another of the same sort MASTERS IN AQUACULTURE AND FISHERIES Genetics and Selection

} New Inbreeding or the rate of inbreeding General Inbreeding Coefficient for individuals in generation t } New Inbreeding or the rate of inbreeding MASTERS IN AQUACULTURE AND FISHERIES Genetics and Selection

Effective Population Size Ne Real-life populations are not ideal: Structured; Assortative / disassortative mating; Breeding structure; Etc. Ne is the number of individuals that in an ideal population would give rise to the calculated sample variance or rate of inbreeding. So, in an ideal population Ne=1/(2 ΔF) MASTERS IN AQUACULTURE AND FISHERIES Genetics and Selection

Formulas for Ne Self-feritilization Sib-mating Diferent numbers of males and females Unequal numbers in successive generations Non-random distribution of family size MASTERS IN AQUACULTURE AND FISHERIES Genetics and Selection

Complicating Matters In real-life populations populations are far from ideal. Small effective sizes, various mating structures, mutation, selection and migration, all act together to produce the phenotypes and genotypes we see or measure. MASTERS IN AQUACULTURE AND FISHERIES Genetics and Selection

Main Tools Available Allozymes Mitochondrial DNA (mtDNA) Nuclear DNA (nDNA) Microsatellites and minisatellites MASTERS IN AQUACULTURE AND FISHERIES Genetics and Selection

Allozymes Allozymes are electrophoretically distinguishable protein variants First used in fish stocks in late 60’s DISADVANTAGES Needs relatively large amounts of tissue in order to yield enough proteins for visualization Many enzyme systems available (>75) although for each study usually only a small fraction shows polymorphism Potentially subject to selection pressures ADVANTAGES Simple to use and applicable to all species (just needs a source of soluble proteins) Standard protocols that require only minor adjustments from species to species MASTERS IN AQUACULTURE AND FISHERIES Genetics and Selection

MASTERS IN AQUACULTURE AND FISHERIES Genetics and Selection

Mitochondrial DNA First studies (80’s) revealed high levels of sequence diversity Useful for inter and intra-specific analysis Occurs in multiple copies per cell (>1000) Uniparental transmission, no recombination Transmitted via the maternal line – useful in the analysis of sex-specific gene-flow patterns Evolves faster than coding regions of nDNA MASTERS IN AQUACULTURE AND FISHERIES Genetics and Selection

Techniques to Analyze mtDNA RFLPs Restriction fragment length polymorphisms Uses restriction enzymes to cut mtDNA in pieces and the separates them using agarose or acrylamide gel electrophoresis PCR Polymerase Chain Reaction Amplification of the number of copies of a target sequence defined by two flanking primers DNA sequencing MASTERS IN AQUACULTURE AND FISHERIES Genetics and Selection

Nuclear DNA RFLPs nDNA sequences Tandemly repeated DNA RAPDs AFLPs VNTRs -DNA fingerprints (minisatellite sequences) STRs - Microsatellites RAPDs Randomly amplified polymorphic DNA AFLPs Amplified fragment length polymorphisms QTLs Quantitative trait loci MASTERS IN AQUACULTURE AND FISHERIES Genetics and Selection

Summary of the Common Genetic Markers Used for Fisheries Management Studies MASTERS IN AQUACULTURE AND FISHERIES Genetics and Selection

Information Provided by Molecular Tools Population structure Deviations from H-W Law Inbreeding, migration, selection, mating structure, demographic history, phylogeographic history, etc. Genealogical and phylogenetic relationships MASTERS IN AQUACULTURE AND FISHERIES Genetics and Selection

Statistical Methods Used Allozymes, RFLPs, AFLPs, DNA sequences, mini and microsatellites all provide us with allele frequency data (with different levels of polymorphism) Most statistical analysis are based on the analysis of genetic variation and its partition into the various hierarchical levels of structure, from families to demes, to sub-populations and the population as a whole. Most are base in Wright’s F-statistics (FST), adapted by Nei for molecular data (GST) and further adapted to newer markers. MASTERS IN AQUACULTURE AND FISHERIES Genetics and Selection

Variation Usually measured by polymorphism (P, the proportion of polymorphic loci) and heterozygosity (H, the proportion of heterozygous individuals) If the population is in H-W equilibrium then H can be calculated as Where NAa is the number of heterozygote individuals in the sample and N is the sample size and n is number of loci, including the monomorphic ones Where xi is the frequency of the ith allele MASTERS IN AQUACULTURE AND FISHERIES Genetics and Selection

Genetic Distance It is a measure of the gene diversity between populations expressed as a function of genotype diversity According to Nei, For 2 populations X and Y, the probability of identity of 2 randomly chosen genes at a single locus (jk) is The probability of identity of a gene at the same locus in populations X and Y is The normalized identity between populations X and Y with respect to all loci is: The Genetic Distance between populations is then: MASTERS IN AQUACULTURE AND FISHERIES Genetics and Selection

Inter-Population Diversity Determine jk for each population and then the gene identity within all populations (JS) The average gene diversity within populations is The gene identity for the total population is And the inter-population gene diversity is The coefficient of differentiation can be defined as: MASTERS IN AQUACULTURE AND FISHERIES Genetics and Selection

Demographic History Aims at uncovering past population bottlenecks or booms Uses DNA sequence data and plots histograms of pair-wise differences between sequences Needs estimate of molecular clock in order to time events MASTERS IN AQUACULTURE AND FISHERIES Genetics and Selection

Genealogy Theory Uses genealogy data, mainly from DNA sequences but also from microsatellites, RFLPs etc., to reconstruct the genealogies of alleles. Can provide information about population structure as well as past demographic events MASTERS IN AQUACULTURE AND FISHERIES Genetics and Selection

Analysis of Phylogeny Using different data types but preferably DNA sequences together with specific algorithms for grouping the data (maximum likelihood, parsimony, etc.), it estimates the relationships between samples or alleles Software: PAUP – Phylogeny Analysis Using Parsimony PHYLIP MacClade TreeView CAIC - Comparative Analysis of Independent Contrasts Etc… (http://evolution.genetics.washington.edu/phylip/software.html) MASTERS IN AQUACULTURE AND FISHERIES Genetics and Selection

Some Applications MASTERS IN AQUACULTURE AND FISHERIES Genetics and Selection

Conservation MASTERS IN AQUACULTURE AND FISHERIES Genetics and Selection

Determining Family Relationships and Ne Herbinger, C.M., R.W. Doyle, C.T. Taggart, S. Lochmann, A.L. Brooker, J.M. Wright and D. Cook. 1997. Family relationships and effective population size in a natural cohort of cod larvae. Can. J. Fish. Aquat. Sci. 54 (Suppl-1):11-18. Abstract Sibship relationships within a naturally spawned cohort of Atlantic cod (Gadus morhua) larvae on the Western Bank of the Scotian Shelf were investigated by a likelihood ratio method that estimates relationships is among individuals using microsatellite (DNA fingerprint) information. We found no evidence of any temporal or spatial family structure among the larvae from seven different sample collections taken at sequential time intervals during a 21-d period of sampling the larval cohort. There was no evidence that larvae were more related within sample collections than across sample collections. Within each sample collection, there was no evidence of a family structure within or among the depths sampled. Similarly, there was no apparent change in the potential occurrence of sibship with time (successive sample collections), or in association with the passage of a storm during the sampling period. This cohort of cod larvae appears to have been a fairly homogeneous mixture of larvae that were not siblings and came from a large genetic pool. The minimum estimate of the inbreeding effective population size is 2800 individual spawners. MASTERS IN AQUACULTURE AND FISHERIES Genetics and Selection

Mixed Stock Management To determine what impact the winter fishery that takes place in the 3Pn and 3Ps regions has on migratory groups (4T,4R,4S,4Vn and 4Vs) we are looking at the genetic makeup of fish from 3Pn and 3Ps areas during the mixed period and comparing this to the genetic make up of the stocks that are thought to migrate here. From these comparisons it will be possible to determine the level of exploitation of the migratory stocks and to have some estimate of the impact the winter fishery has on the migratory stocks and how this will effect their ability of these stocks to recover. The graphs below illustrates two possible mixing profiles as they may exist. In Figure A the outcome of the fishery would be a harvest which would be mostly focused on fish which come from the inner Gulf region (4T,4R and 4S); Figure B would be a fishery which was mostly focused on resident populations from 3Pn and 3Ps. With either case, consideration of the winter fishery impact must be taken and adjustments made as to how the affected stocks are managed. MASTERS IN AQUACULTURE AND FISHERIES Genetics and Selection

Assigning Individuals to Populations - Forensics Enormous expense is currently incurred by enforcement officers while patrolling the fishing grounds with boats and aircraft. This method of enforcement is inefficient in terms of time, money, and the amount of catch actually monitored. With MGPL's DNA Fingerprinting capabilities, catch restrictions can be enforced at the dock instead of at sea on the basis of forensic analysis of the catch. The genetic information carried by the animals themselves will identify the stock from which they were taken. MASTERS IN AQUACULTURE AND FISHERIES Genetics and Selection

Estimating changes in population size Use of coalescent theory and of pair wise sequence comparisons in order to study and date demographic events; Use of gene diversity estimates in order to detect past bottlenecks in populations; Etc. MASTERS IN AQUACULTURE AND FISHERIES Genetics and Selection

Estimating gene flow among populations MASTERS IN AQUACULTURE AND FISHERIES Genetics and Selection

Software MASTERS IN AQUACULTURE AND FISHERIES Genetics and Selection

Brief summary of computer programs and data analysis approaches http://www.cf.adfg.state.ak.us/geninfo/research/genetics/software/anlink.php LAMARC - Likelihood Analysis with Metropolis Algorithm using Random Coalescence Lamarc is a program for doing Likelihood Analysis with Metropolis Algorithm using Random Coalescence. Lamarc estimates effective population sizes, population exponential growth rates, a recombination rate, and past migration rates for one to n populations assuming a migration matrix model with asymmetric migration rates and different subpopulation sizes. This version can use DNA or RNA sequence data, SNPs, microsatellites, or electrophoretic data. The program can produce: estimates of recombination rate, migration rates between each population pair, population sizes (assuming constant mutation rates among loci), population exponential growth rates, profile likelihood tables, and percentiles.If you know that there is no recombination in your data (for example in mtDNA) you might look also at the other programs: Fluctuate or Migrate. Whichrun 4.1 A computer program for population assignment of individuals based on multilocus genotype data. Microsatellite DNA provides essentially limitless, highly varied information within species.  That this provides a means for distinguishing not only among populations but also individuals has not escaped current theoretic interest (Smouse and Chevillon 1998, Waser and Strobeck 1998).  Here, we present a C++ computer program named WHICHRUN that uses multilocus genotypic data to allocate individuals to their most likely source population. Genetic Mixture Analysis (GMA) Software for estimating the stock proportions within mixed stock fisheries BOTTLENECK version 1.2.02  (16.II.1999) Bottleneck is a program for detecting recent effective population size reductions from allele data frequencies. MASTERS IN AQUACULTURE AND FISHERIES Genetics and Selection

Structure The program structure is a free software package for using multi-locus genotype data to investigate population structure. Its uses include inferring the presence of distinct populations, assigning individuals to populations, studying hybrid zones, identifying migrants and admixed individuals, and estimating population allele frequencies in situations where many individuals are migrants or admixed. It can be applied to most of the commonly-used genetic markers, including microsatellites, RFLPs and SNPs. This method was described in an article by Pritchard, Stephens & Donnelly (2000). Extensions to the method were published by Falush, Stephens and Pritchard (2003). An interesting example from the original paper is shown here.                                                                                                                                             - RST Calc – A program to calculate unbiased estimates of Slatkin's RST and Goldstein et al's (delta-mu)^2 distance for microsatellite data GENEPOP is a population genetics software package originally designed by Michel Raymond (Raymond@isem.univ-montp2.fr) and Francois Rousset (Rousset@isem.univ-montp2.fr), at the Laboratiore de Genetique et Environment, Montpellier, France. MASTERS IN AQUACULTURE AND FISHERIES Genetics and Selection

Presentations Duration 15’, groups of 2-3 (all should participate) When – Friday 17th, 14.00 h What: Results of selection programs in: Salmon Trout Nile Tilapia Catfish Oyster Shrimps Structure: The species and its farming Important traits Heritabilities Structure of selection program Performance (response) of program MASTERS IN AQUACULTURE AND FISHERIES Genetics and Selection