Phylogenomics and adaptive evolution in stickleback fishes

How predictable is evolution? There is no fully satisfactory answer to this 100-year old question yet. However, within the past two decades, much progress has been made towards unravelling various factors that influence the predictability of evolution. Much of this work has focused on the similarity of evolutionary responses in replicate populations of a given taxon that have independently colonised similar environments – a phenomenon known as parallel evolution. The fish species in the family Gasterosteidae (sticklebacks) have become popular models to study the repeatability of evolution. This thesis focuses on evolutionary history and parallel evolution in two ecologically similar and geographically co-distributed species in the family Gasterosteidae, the three-spined stickleback (Gasterosteus aculeatus) and the nine-spined stickleback (Pungitius pungitius). Freshwater populations of both species evolved similar phenotypic traits after marine ancestors independently colonised freshwater environments. A highly resolved phylogeny is a prerequisite for untangling the processes that have shaped the underlying genomic divergence, including natural selection and population demographic history. Therefore, my thesis begins by resolving the worldwide phylogenetic relationships and demographic history of both focal species, using state of the art phylogenomic analyses. The results indicate that extant three-spined stickleback populations originated from the Eastern Pacific in the late Pleistocene, and the Atlantic populations were colonised from the Pacific ancestors via the Arctic Ocean. In contrast, nine-spined sticklebacks have a more ancient history, diversifying in the late Pliocene, and their current distribution is the result of multiple waves of trans-Arctic colonisation from the Far East, with several divergent lineages having evolved across their geographic range. The thesis then moves on to investigate the genetic basis of parallel freshwater adaptation in each of the two species, using the information gained in the previous chapter to set up specific hypotheses and define simulation parameters. For three-spined sticklebacks, the level of parallel evolution at the genotype level was 10 times higher among the freshwater populations in the ancestral Eastern Pacific region than anywhere else in the world. Empirical data and simulations demonstrate that these patterns are determined by a reduction in standing genetic variation outside the ancestral Eastern Pacific region, a result that can be explained by the demographic history of the species. A comparison of the two species revealed fundamental differences in the way standing genetic variation – the raw material upon which selection acts – is distributed among populations. This was exemplified by 2-fold higher degree of genetic structuring and 23-fold stronger isolation-by-distance in ninethan in three-spined sticklebacks. Conversely, the proportion of genetic parallelism in three-spined stickleback is 123.4 times greater than the nine-spined stickleback. Taken together, the thesis resolved the phylogenetic affinities and demographic history of stickleback fishes using state-of-art methods and a global sampling strategy. Based on this knowledge, the thesis further uncovered profound heterogeneity in the repeatability of evolution within and between the two model species in response to freshwater colonisation. Hence, the two stickleback species with their contrasting demographic and evolutionary histories constitute a model system to study how differences in the distribution of standing genetic variation can influence the predictability of evolution.Voiko evoluutiota ennustaa? Tähän lähes sata vuotta vanhaan kysymykseen ei edelleenkään osata vastata, mutta kuluvan vuostituhannen aikana on opittu ymmärtämään evoluution ennustettavuuteen vaikuttavia tekijöitä. Viimeaikainen tutkimus on keskittynyt keskenään samanlaisen kolonisaatiohistorian omaavien populaatioiden evoluution samankaltaisuuteen eli paralleeliin evoluutioon, ja piikkikaloista (Gasterosteidae) on tullut alan käytetyimpiä mallilajeja. Tässä väitöskirjassa tarkastellaan kahden ekologialtaan ja levinneisyydeltään samankaltaisen piikkikalalajin, kolmipiikin (Gasterosteus aculeatus) ja kymmenpiikin (Pungitius pungitius), evoluutiohistoriaa ja paralleelia evoluutiota. Molemmilla lajeilla esiintyy samankaltaisia makean veden muotoja, jotka ovat kehittyneet meressä eläneiden yksilöiden levittäydyttyä järviin ja jokiin. Tutkimuksen edellytys on tarkka fylogenia, joka osoittaa tutkittavien lajien ja populaatioiden polveutumishistorian. Väitöskirjani alkaa molempien tutkimuslajien fylogenian ja demografisen historian kuvaamisella. Tulosten mukaan kolmipiikin itäisen Tyynenmeren populaatiot syntyivät myöhäisellä pleistoseenkaudella ja Atlantin populaatiot syntyivät Tyynenmeren yksilöiden levittäydyttyä Atlantille Pohjoisen jäämeren kautta. Kymmenpiikkipopulaatiot ovat sen sijaan ovat eriytyneet jo myöhäisellä plioseenikaudella ja lajin nykyinen levinneisyys on seurausta useista itäisestä Aasiasta lähteneistä levittäytymisistä. Väitöskirjan seuraavissa osissa tutkitaan paralleelin makeaan veteen sopeutumisen geneettistä perustaa. Kolmipiikin itäisen Tyynenmeren populaatioissa paralleelin evoluution aste oli kymmenkertainen maailman muihin populaatioihin verrattuna. Sekä aineisto että simulaatiot tukevat tulkintaa, jonka mukaan ero selittyy itäisen Tyynenmeren alueen korkeammalla muuntelun määrällä, mikä puolestaan johtuu demografisesta historiasta. Lajien välinen vertailu osoittaa, että geneettisen muuntelun määrä, joka mahdollistaa evoluution, eroaa populaatioiden välillä huomattavasti. Esimerkiksi kymmepiikkipopulaaitoiden geneettinen rakenne ja populaatioiden erityminen oli huomattavasti voimakkaampaa kuin kolmipiikillä Tässä väitöskirjassa esitetään tarkat, viimeisimpiin menetelmiin ja maailmanlaajuiseen otantaan perustuvat kolmi- ja kymmenpiikin fylogeniat. Fylogenioiden perusteella makeanveden kolonisaatioiden ja niitä seuraavien sopeutumisten voidaan osoittaa olevan geneettisesti erilaisia eri lajeilla. Erilaiset demografiset ja evolutiiviset historiat omaavat piikkikalalajit toimivat hyvänä mallina geneettisen muuntelun vaikutukseen evoluution ennustettavuudelle

Allopatric origin of sympatric whitefish morphs with insights on the genetic basis of their reproductive isolation

The European whitefish (Coregonus lavaretus) species complex is a classic example of recent adaptive radiation. Here, we examine a whitefish population introduced to northern Finnish Lake Tsahkal in the late 1960s, where three divergent morphs (viz. littoral, pelagic, and profundal feeders) were found 10 generations after. Using demographic modeling based on genomic data, we show that whitefish morphs evolved during a phase of strict isolation, refuting a rapid sympatric divergence scenario. The lake is now an artificial hybrid zone between morphs originated in allopatry. Despite their current syntopy, clear genetic differentiation remains between two of the three morphs. Using admixture mapping, we identify five SNPs associated with gonad weight variation, a proxy for sexual maturity and spawning time. We suggest that ecological adaptations in spawning time evolved in allopatry are currently maintaining partial reproductive isolation in the absence of other barriers to gene flow.Peer reviewe

Worldwide phylogeny of three-spined sticklebacks

Stickleback fishes in the family Gasterosteidae have become model organisms in ecology and evolutionary biology. However, even in the case of the most widely studied species in this family – the three-spined stickleback (Gasterosteus aculeatus) – the worldwide phylogenetic relationships and colonization history of the different populations and lineages remain poorly resolved. Using a large collection of samples covering most parts of the species distribution range, we subjected thousands of single nucleotide polymorphisms to coalescent analyses in order to reconstruct a robust worldwide phylogeny of extant G. aculeatus populations, as well as their ancestral geographic distributions using Statistical-Dispersal Vicariance and Bayesian Binary MCMC analyses. The results suggest that contemporary populations originated from the Pacific Ocean in the Late Pleistocene, and the Atlantic was colonized through the Arctic Ocean by a lineage that diverged from Pacific sticklebacks ca 44.6 Kya. This lineage contains two branches: one that is distributed in the Mediterranean area, from the Iberian Peninsula to the Black Sea (‘Southern European Clade’), and another that is comprised of populations from northern Europe and the east coast of North America (‘Trans-Atlantic Clade’). Hence, the results suggest that the North American East Coast was colonized by trans-Atlantic migration. Coalescence-based divergence time estimates suggest that divergence among major clades is much more recent than previously estimated.Peer reviewe

Estimating uncertainty in divergence times among three-spined stickleback clades using the multispecies coalescent

Incomplete lineage sorting (ILS) can lead to biased divergence time estimates. To explore if and how ILS has influenced the results of a recent study of worldwide phylogeny of three-spined sticklebacks (Gasterosteus aculeatus), we estimated divergence times among major clades by applying both a concatenation approach and the multispecies coalescent (MSC) model to single-nucleotide polymorphisms. To further test the influence of different calibration strategies, we applied different calibrations to the root and to younger nodes in addition to the ones used in the previous study. Both the updated calibrations and the application of the MSC model influenced divergence time estimates, sometimes significantly. The new divergence time estimates were more ancient than in the previous study for older nodes, whereas the estimates of younger nodes were not strongly affected by the re-analyses. However, given the applied MSC method employs a simple substitution model and cannot account for changes in population size, we suggest that different analytical approaches and calibration strategies should be used in order to explore uncertainty in divergence time estimates. This study provides a valuable reference timeline for the ages of worldwide three-spined stickleback populations and emphasizes the need to embrace, rather than obscure, uncertainties around divergence time estimates.Peer reviewe

Knowledge, Attitudes, and Social Responsiveness Toward Corona Virus Disease 2019 (COVID-19) Among Chinese Medical Students—Thoughts on Medical Education

Purpose: To assess knowledge, attitudes, and social responsiveness toward COVID-19 among Chinese medical students.Methods: Self-administered questionnaires were used to collect data from 889 medical students in three well-known Chinese medical universities. The questionnaire was comprised of three domains which consisted of demographic characteristic collection, seven items for knowledge, and eight items for attitudes and social responsiveness toward COVID-19. Data from different universities were lumped together and were divided into different groups to compare the differences, including (1) students at the clinical learning stage (Group A) or those at the basic-medicine stage (Group B) and (2) students who have graduated and worked (Group C) or those newly enrolled (Group D).Results: Medical students at group B had a weaker knowledge toward COVID-19 than did students at group A, especially in the question of clinical manifestations (p < 0.001). The percentage of totally correct answers of COVID-19 knowledge in group C was higher than that in Group D (p < 0.001). There were significant differences between groups C and D in the attitudes and social responsiveness toward COVID-19. Surprisingly, we found that the idea of newly enrolled medical students could be easily affected by interventions.Conclusions: In light of this information, medical education should pay attention not only to the cultivation of professional knowledge and clinical skills but also to the positive interventions to better the comprehensive qualities including communicative abilities and empathy

Chromosome-level genome assembly of a high-altitude-adapted frog (Rana kukunoris) from the Tibetan plateau provides insight into amphibian genome evolution and adaptation

Background The high-altitude-adapted frog Rana kukunoris, occurring on the Tibetan plateau, is an excellent model to study life history evolution and adaptation to harsh high-altitude environments. However, genomic resources for this species are still underdeveloped constraining attempts to investigate the underpinnings of adaptation. Results The R. kukunoris genome was assembled to a size of 4.83 Gb and the contig N50 was 1.80 Mb. The 6555 contigs were clustered and ordered into 12 pseudo-chromosomes covering similar to 93.07% of the assembled genome. In total, 32,304 genes were functionally annotated. Synteny analysis between the genomes of R. kukunoris and a low latitude species Rana temporaria showed a high degree of chromosome level synteny with one fusion event between chr11 and chr13 forming pseudo-chromosome 11 in R. kukunoris. Characterization of features of the R. kukunoris genome identified that 61.5% consisted of transposable elements and expansions of gene families related to cell nucleus structure and taste sense were identified. Ninety-five single-copy orthologous genes were identified as being under positive selection and had functions associated with the positive regulation of proteins in the catabolic process and negative regulation of developmental growth. These gene family expansions and positively selected genes indicate regions for further interrogation to understand adaptation to high altitude. Conclusions Here, we reported a high-quality chromosome-level genome assembly of a high-altitude amphibian species using a combination of Illumina, PacBio and Hi-C sequencing technologies. This genome assembly provides a valuable resource for subsequent research on R. kukunoris genomics and amphibian genome evolution in general.Peer reviewe

Population Structure Limits Parallel Evolution in Sticklebacks

Population genetic theory predicts that small effective population sizes (N-e) and restricted gene flow limit the potential for local adaptation. In particular, the probability of evolving similar phenotypes based on shared genetic mechanisms (i.e., parallel evolution), is expected to be reduced. We tested these predictions in a comparative genomic study of two ecologically similar and geographically codistributed stickleback species (viz. Gasterosteus aculeatus and Pungitius pungitius). We found that P. pungitius harbors less genetic diversity and exhibits higher levels of genetic differentiation and isolation-by-distance than G. aculeatus. Conversely, G. aculeatus exhibits a stronger degree of genetic parallelism across freshwater populations than P. pungitius: 2,996 versus 379 single nucleotide polymorphisms located within 26 versus 9 genomic regions show evidence of selection in multiple freshwater populations of G. aculeatus and P. pungitius, respectively. Most regions involved in parallel evolution in G. aculeatus showed increased levels of divergence, suggestive of selection on ancient haplotypes. In contrast, haplotypes involved in freshwater adaptation in P. pungitius were younger. In accordance with theory, the results suggest that connectivity and genetic drift play crucial roles in determining the levels and geographic distribution of standing genetic variation, providing evidence that population subdivision limits local adaptation and therefore also the likelihood of parallel evolution.Peer reviewe

On the causes of geographically heterogeneous parallel evolution in sticklebacks

The three-spined stickleback (Gasterosteus aculeatus) is an important model system for the study of parallel evolution in the wild, having repeatedly colonized and adapted to freshwater from the sea throughout the northern hemisphere. Previous studies identified numerous genomic regions showing consistent genetic differentiation between freshwater and marine ecotypes but these had typically limited geographic sampling and mostly focused on the Eastern Pacific region. We analysed population genomic data from global samples of the three-spined stickleback marine and freshwater ecotypes to detect loci involved in parallel evolution at different geographic scales. Most signatures of parallel evolution were unique to the Eastern Pacific and trans-oceanic marine-freshwater differentiation was restricted to a limited number of shared genomic regions, including three chromosomal inversions. On the basis of simulations and empirical data, we demonstrate that this could result from the stochastic loss of freshwater-adapted alleles during the invasion of the Atlantic basin and selection against freshwater-adapted variants in the sea, both of which can reduce standing genetic variation available for freshwater adaptation outside the Eastern Pacific region. Moreover, the elevated linkage disequilibrium associated with marine-freshwater differentiation in the Eastern Pacific is consistent with secondary contact between marine and freshwater populations that evolved in isolation from each other during past glacial periods. Thus, contrary to what earlier studies from the Eastern Pacific region have led us to believe, parallel marine-freshwater differentiation in sticklebacks is far less prevalent and pronounced in all other parts of the species global distribution range. Population genomic data from a global dataset of three-spined sticklebacks show that parallel signatures of marine to freshwater differentiation are less common than previously thought.Peer reviewe

Worldwide phylogenetic tree of Gasterosteus aculeatus

The time-calibrated worldwide phylogenetic tree of Gasterosteus aculeatus

Genetic population structure constrains local adaptation in sticklebacks

Repeated and independent adaptation to specific environmental conditions from standing genetic variation is common. However, if genetic variation is limited, the evolution of similar locally adapted traits may be restricted to genetically different and potentially less optimal solutions or prevented from happening altogether. Using a quantitative trait locus (QTL) mapping approach, we identified the genomic regions responsible for the repeated pelvic reduction (PR) in three crosses between nine-spined stickleback populations expressing full and reduced pelvic structures. In one cross, PR mapped to linkage group 7 (LG7) containing the gene Pitx1, known to control pelvic reduction also in the three-spined stickleback. In the two other crosses, PR was polygenic and attributed to 10 novel QTL, of which 90% were unique to specific crosses. When screening the genomes from 27 different populations for deletions in the Pitx1 regulatory element, these were only found in the population in which PR mapped to LG7, even though the morphological data indicated large-effect QTL for PR in several other populations as well. Consistent with the available theory and simulations parameterized on empirical data, we hypothesize that the observed variability in genetic architecture of PR is due to heterogeneity in the spatial distribution of standing genetic variation caused by >2x stronger population structuring among freshwater populations and >10x stronger genetic isolation by distance in the sea in nine-spined sticklebacks as compared to three-spined sticklebacks.Peer reviewe