Genetics of Local Adaptation in theThree-spined Stickleback

Spatial differentiation in phenotypic traits is commonly observed in the wild, but both the proximate (cf. environmental vs. genetic) and ultimate (cf. adaptive vs. stochastic) causes underlying this differentiation often remain obscure. Studies focussed on the genetic basis of this differentiation can inform us about these issues, especially if the genetic variants under investigation can be linked with information about their functional role(s) and/or gauged against expectations derived from evolutionary null models. However, due to the difficulties in deciphering and studying the genetic basis of phenotypic variability and differentiation in quantitative traits especially in marine vertebrates the occurrence and scale of local adaptation in them is still poorly understood. Yet, identifying patterns of adaptive divergence and the ecological factors that have contributed to them is essential for understanding how natural selection can maintain local adaptation in the face of gene flow. In this thesis I used a genome-wide set of candidate gene-based microsatellite markers, in combination with quantitative genetic approaches, to explore the patterns of adaptive diversity and divergence among stickleback populations from a variety of habitats ranging from global to local geographic scales. Through comparisons of several independent, isolated pairs of marine and freshwater populations, I found that selection is acting on many genomic regions harbouring genes whose putative functions are related to a wide variety of physiological processes. I also found indications that adaptation to freshwater environments may have been achieved through different genetic pathways in different populations. Importantly, the design of my study was such that alternative demographic explanations for observed patterns could be excluded. Focussing on populations within the physically continuous, yet environmentally heterogeneous marine habitat, I further investigated whether selection is acting strongly enough to promote adaptive population structuring despite high gene flow. Signatures of selection were detected in several candidate genes, along with clear evidence for adaptive differentiation in a phenotypic trait (lateral plate number). Analysis of population structure with only these outlier loci uncovered a higher degree of differentiation than was evident in neutral loci, and in some cases, patterns of adaptive differentiation were correlated with environmental variables likely to act as selective agents in the marine environment (viz. salinity and temperature). Evidence for local adaptation among Baltic Sea sticklebacks was confirmed in a common garden experiment, which demonstrated a loss of fitness in populations native to low salinity regions when exposed to high salinity treatments. Overall, the results from this thesis point to the conclusion that adaptive genetic and phenotypic differentiation is common, even in continuous marine habitats lacking obvious physical barriers to dispersal and gene flow. These results are particularly noteworthy, firstly from the perspective that earlier studies conducted using neutral marker genes have largely overlooked the patterns and magnitude of divergence, and secondly due to the comprehensive geographic coverage of the investigations.Maantieteellinen muuntelu eliöiden ilmiasullisissa ominaisuuksissa on tavallista. Ymmärryksemme tämän muuntelun proksimaattisista ja ultimaattista syistä on kuitenkin vajavaista: useinkaan ei ole selvää missä määrin ilmiasulliset erot johtuvat geneettisistä erilaistumisesta ja missä määrin ilmiasullisesta plastisuudesta. Myös luonnonvalinnan ja geneettisen satunnaisajautumisen suhteellinen merkitys geneettisten erojen selittäjänä on yleensä tuntematon, samoin kuin se, jos ja kuinka luonnonvalinta kykenee ylläpitämään paikallisia sopeumia geenivirrasta huolimatta. Tutkimukset populaatioiden välisen ilmiasullisen erilaistumisen perinnöllisestä taustasta yhdessä merkkigeenitutkimusten kanssa tarjoavatkin mahdollisuuksia lisätä ymmärrystämme näistä seikoista. Tarve tämän ymmärryksen kartoittamiselle korostuu erityisesti merikalojen kohdalla, joiden biologiset ominaisuudet ovat rajoittaneet niiden perinnöllisyyden ja paikallisten sopeumien tutkimusta. Käytin tässä väitöskirjatyössä suurta määrää kandidaattigeeneihin ankkuroituja mikrosatelliitti ja SNP merkkigeenejä yhdessä kvantitatiivisen genetiikan menetelmien kanssa selvittääkseni kolmipiikin (Gasterosteus aculeatus) geneettistä erilaistumista ja sen evoluutiivisiä syitä. Tutkimusten maantieteellinen mittakaava vaihteli Itämeren piirin kattavista tutkimuksista maailmanlaajuiseen tarkasteluun sekä meri- että makean veden populaatioissa. Parittaisten meri- ja makean veden populaatioiden vertailu maailmanlaajuisessa mittakaavassa paljasti ison joukon luonnonvalinnan alla olleita fysiologisesti merkittäviä geenejä, jotka ovat todennäköisesti mahdollistaneet kolmipiikkien sopeutumisen makeaan veteen. Valinnan alla olleet geenit eivät olleet aina samoja populaatioparista toiseen, mikä on tulkittavissa osoitukseksi siitä, että sopeutuminen makeaan veteen on voinut tapahtua eri populaatioissa vaihtoehtoisten geneettisten mekanismien avulla. Tutkimukset meripopulaatioiden geneettisestä erilaistumisesta paljastivat, että voimakkaasta geenivirrasta huolimatta, myös meripopulaatiot olivat voimakkaasti toisistaan erilaistuneita. Erilaistuminen ilmeni sekä kandidaattigeeneihin sitoutuneissa merkkigeeneissä, että myös ilmiasullisessa ominaisuudessa (kylkikilpien määrä), jonka ilmenemistä säätelevä geeni on tunnettu. Nämä tulokset tukevat tulkintaa, jonka mukaan kolmipiikin meripopulaatiot ovat paikallisesti sopeutuneita. Tätä tulkintaa tukivat myös valinnan alla olleiden lokusten alleelifrekvenssien korrelaatiot ympäristön suolapitoisuuden kanssa, sekä myös laboratoriossa tehdyt kasvatuskokeet, joissa eri alkuperää olevien kolmipiikkien kelpoisuutta testattiin eri suolapitoisuuksissa mittavien faktorikokeiden avulla. Yleisesti ottaen voidaan sanoa että tähän väitöskirjaan sisältyvien tutkimusten tulokset tukevat näkemystä, jonka mukaan kolmipiikkipopulaatiot niin meressä kuin makeissakin vesissä ovat sekä ilmiasullisesti että perinnöllisesti sopeutuneet paikallisiin olosuhteisiin. Sopeutumista on tapahtunut myös valtamerissä, joista geenivirtaa rajoittavat esteet puuttuvat. Nämä havainnot ovat merkille pantavia siinä perspektiivissä, että aiemmat neutraaleja merkkigeenejä hyödyntäneet tutkimukset eivät ole tätä erilaistumista kyenneet todentamaan. Lisäarvoa havainnoille antavat käytettyjen aineistojen laajuus ja maantieteellinen kattavuus

Temporal stability of genetic diversity and differentiation in the three-spined stickleback (Gasterosteus aculeatus)

Temporal variation in allele frequencies, whether caused by deterministic or stochastic forces, can inform us about interesting demographic and evolutionary phenomena occurring in wild populations. In spite of the continued surge of interest in the genetics of three-spined stickleback (Gasterosteus aculeatus) populations, little attention has been paid towards the temporal stability of allele frequency distributions, and whether there are consistent differences in effective size (Ne) of local populations. We investigated temporal stability of genetic variability and differentiation in 15 microsatellite loci within and among eight collection sites of varying habitat type, surveyed twice over a six-year time period. In addition, Nes were estimated with the expectation that they would be lowest in isolated ponds, intermediate in larger lakes and largest in open marine sites. In spite of the marked differences in genetic variability and differentiation among the study sites, the temporal differences in allele frequencies, as well as measures of genetic diversity and differentiation, were negligible. Accordingly, the Ne estimates were temporally stable, but tended to be lower in ponds than in lake or marine habitats. Hence, we conclude that allele frequencies in putatively neutral markers in three-spined sticklebacks seem to be temporally stable -at least over periods of few generations - across a wide range of habitat types differing markedly in levels of genetic variability, effective population size and gene flow.Peer reviewe

Development and validation of a real-time quantitative PCR assay for the detection and quantification of Perkinsus marinus in the Eastern oyster, Crassostrea virginica

Author Posting. © National Shellfisheries Association, 2009. This article is posted here by permission of National Shellfisheries Association for personal use, not for redistribution. The definitive version was published in Journal of Shellfish Research 28 (2009): 459-464, doi:10.2983/035.028.0306.Perkinus marinus causes a devastating disease, known as Dermo, in the Eastern oyster Crassostrea virginica. Routine detection of the disease is traditionally accomplished by the use of the Ray/Makin assay, using Fluid Thioglycollate Medium (RFTM). A simple real-time quantitative PCR assay was developed as a diagnostic tool to detect and quantify P. marinus, to complement and serve as an alternate to the RFTM method. Using a dual-labeled probe approach, a sensitive assay was designed to accurately detect a range of one to several thousand P. marinus organisms present in oyster tissues. A simple extraction method was used to increase throughput of the assay. Cultured P. marinus cells were quantified prior to DNA extraction, generating a standard curve and allowing cell counts to be derived from PCR cycle threshold values. Direct comparison of the RFTM and real-time PCR methods was accomplished by using tissue samples from the same oyster for both tests. Plotting cycle threshold values against the known Mackin index value generated a standard curve with a coefficient of regression of 0.9. Our results indicate that correlations could be made between this molecular based approach and traditional methods, allowing results generated from the PCR assay to be easily translated into the understood Mackin scale

Variation in sexual brain size dimorphism over the breeding cycle in the three-spined stickleback

Snapshot analyses have demonstrated dramatic intraspecific variation in the degree of brain sexual size dimorphism (SSD). Although brain SSDis believed to be generated by the sex-specific cognitive demands of reproduction, the relative roles of developmental and population-specific contributions to variation in brain SSD remain little studied. Using a common garden experiment, we tested for sex-specific changes in brain anatomy over the breeding cycle in three-spined stickleback (Gasterosteus aculeatus) sampled from four locations in northern Europe. We found that the male brain increased in size (ca. 24%) significantly more than the female brain towards breeding, and that the resulting brain SSD was similar (ca. 20%) for all populations over the breeding cycle. Our findings support the notion that the stickleback brain is highly plastic and changes over the breeding cycle, especially in males, likely as an adaptive response to the cognitive demands of reproduction (e.g. nest construction and parental care). The results also provide evidence to suggest that breeding-related changes in brain size may be the reason for the widely varying estimates of brain SSD across studies of this species, cautioning against interpreting brain size measurements from a single time point as fixed/static.Peer reviewe

Heterochronic development of lateral plates in the three-spined stickleback induced by thyroid hormone level alterations

The three-spined stickleback Gasterosteus aculeatus is an important model for studying microevolution and parallel adaptation to freshwater environments. Marine and freshwater forms differ markedly in their phenotype, especially in the number of lateral plates, which are serially repeated elements of the exoskeleton. In fishes, thyroid hormones are involved in adaptation to salinity, as well as the developmental regulation of serially repeated elements. To study how thyroid hormones influence lateral plate development, we manipulated levels of triiodothyronine and thiourea during early ontogeny in a marine and freshwater population with complete and low plate phenotypes, respectively. The development of lateral plates along the body and keel was heterochronic among experimental groups. Fish with a low dosage of exogenous triiodothyronine and those treated with thiourea exhibited retarded development of bony plates compared to both control fish and those treated with higher a triiodothyronine dosage. Several triiodothyronine-treated individuals of the marine form expressed the partial lateral plate phenotype. Some individuals with delayed development of lateral plates manifested 1-2 extra bony plates located above the main row of lateral plates.Peer reviewe

De Novo Sequencing, Assembly, and Annotation of Four Threespine Stickleback Genomes Based on Microfluidic Partitioned DNA Libraries

The threespine stickleback is a geographically widespread and ecologically highly diverse fish that has emerged as a powerful model system for evolutionary genomics and developmental biology. Investigations in this species currently rely on a single high-quality reference genome, but would benefit from the availability of additional, independently sequenced and assembled genomes. We present here the assembly of four new stickleback genomes, based on the sequencing of microfluidic partitioned DNA libraries. The base pair lengths of the four genomes reach 92–101% of the standard reference genome length. Together with their de novo gene annotation, these assemblies offer a resource enhancing genomic investigations in stickleback. The genomes and their annotations are available from the Dryad Digital Repository (https://doi.org/10.5061/dryad.113j3h7)

Data from: Variation in age and size in Fennoscandian three-spined sticklebacks (Gasterosteus aculeatus)

Average age and maximum life span of breeding adult three-spined sticklebacks (Gasterosteus aculeatus) were determined in eight Fennoscandian localities with the aid of skeletochronology. The average age varied from 1.8 to 3.6 years, and maximum life span from three to six years depending on the locality. On average, fish from marine populations were significantly older than those from freshwater populations, but variation within habitat types was large. We also found significant differences in mean body size among different habitat types and populations, but only the population differences remained significant after accounting for variation due to age effects. These results show that generation length and longevity in three-spined sticklebacks can vary significantly from one locality to another, and that population differences in mean body size cannot be explained as a simple consequence of differences in population age structure. We also describe a nanistic population from northern Finland exhibiting long life spans and small body size

Data from: Local adaptation to salinity in the three-spined stickleback?

Different lines of evidence suggest that the occurrence and extent of local adaptation in high gene flow marine environments – even in mobile and long-lived vertebrates with complex life cycles – may be more widespread than earlier thought. We conducted a common garden experiment to test for local adaptation to salinity in Baltic Sea sticklebacks (Gasterosteus aculeatus). Fish from three different native salinity regimes (high, mid and low) were subjected to three salinity treatments (high, mid and low) in a full-factorial experimental design. Irrespective of their origin, fish subjected to low (and mid) salinity treatments exhibited higher juvenile survival, grew to largest sizes and were in better condition than fish subjected to the high salinity treatment. However, a significant interaction between native and treatment salinities – resulting mainly from the poor performance of fish native to low salinity in the high salinity treatment – provided clear cut evidence for adaptation to local variation in salinity. Additional support for this inference was provided by the fact that the results concur with an earlier demonstration of significant differentiation in a number of genes with osmoregulatory functions across the same populations and that the population-specific responses to salinity treatments exceeded that to be expected by random genetic drift

Archive_JEB

Plate counts and genotypes for STN381. Population codes are as follows in the publication

Data from: Temporal stability of genetic variability and differentiation in the three-spined stickleback (Gasterosteus aculeatus)

Temporal variation in allele frequencies, whether caused by deterministic or stochastic forces, can inform us about interesting demographic and evolutionary phenomena occurring in wild populations. In spite of the continued surge of interest in the genetics of three-spined stickleback (Gasterosteus aculeatus) populations, little attention has been paid towards the temporal stability of allele frequency distributions, and whether there are consistent differences in effective size (Ne) of local populations. We investigated temporal stability of genetic variability and differentiation in 15 microsatellite loci within and among eight collection sites of varying habitat type, surveyed twice over a six-year time period. In addition, Nes were estimated with the expectation that they would be lowest in isolated ponds, intermediate in larger lakes and largest in open marine sites. In spite of the marked differences in genetic variability and differentiation among the study sites, the temporal differences in allele frequencies, as well as measures of genetic diversity and differentiation, were negligible. Accordingly, the Ne estimates were temporally stable, but tended to be lower in ponds than in lake or marine habitats. Hence, we conclude that allele frequencies in putatively neutral markers in three-spined sticklebacks seem to be temporally stable – at least over periods of few generations – across a wide range of habitat types differing markedly in levels of genetic variability, effective population size and gene flow