Phylogeography and Historical Introgression in Stickleback Fishes

Phylogeography and historical introgression in Stickleback fishes

Cui Wang

Ecological Genetics Research Unit Faculty of Biological and Environmental Sciences University of Helsinki Finland

Academic dissertation

To be presented for public examination with the permission of the Faculty of Biological and Environmental Sciences of the University of Helsinki in Au- ditorium 2, Info Centre Korona (Viikinkaari 11), on 9th of March 2018 at 12.00 o’clock noon.

Helsinki 2018

Supervised by Prof. Juha Merilä University of Helsinki, Finland

Dr. Takahito Shikano University of Helsinki, Finland

Thesis advisory committee Doc. Perttu Seppä University of Helsinki, Finland

Dr. Helena Johansson University of Helsinki, Finland

Pre-examiners Doc. Laura Kvist University of Oulu, Finland

Asst. Prof. Robert Ekblom Uppsala University, Sweden

Opponent Prof. Jacob Höglund Uppsala University, Sweden

Custos Prof. Juha Merilä University of Helsinki, Finland

Layout and cover by: © Cui Wang ISBN 978-951-51-4084-5 (paperback) ISBN 978-951-51-4085-2 (PDF) http://ethesis.helsinki.fi Unigrafia, Helsinki 2018

穷且益坚，不坠青云之志。老当益壮，宁移白首之心？ ——王勃

The more difficult it is, the stronger we need to be. Never give up your dream!

Contents

Abstract ...... 6 1. Introduction ...... 8 1.1 Phylogeography and climate changes...... 8 1.1.1 Population genetics and climate changes ...... 8 1.1.2 Phylogeography of freshwater fishes ...... 9 1.1.3 Molecular markers and phylogeography ...... 9 1.2 Study systems ...... 10 1.2.1 Taxonomy and distribution of Pungitius species ...... 10 1.2.2 Intraspecific genetic divergence of Pungitius species ...... 11 1.2.3 Interspecific introgression among Pungitius species ...... 12 2. Aims of the thesis ...... 12 3. Materials and Methods ...... 13 3.1 Study species and sampling ...... 13 3.2 Molecular markers ...... 15 3.3 Statistical analyses ...... 16 4. Results and discussion ...... 20 4.1 Phylogeography of Pungitius species ...... 20 4.2 Historical introgression among Pungitius species...... 24 4.3 Reproductive isolation between species ...... 27 Conclusions and outlook ...... 29 Acknowledgements ...... 30 References ...... 31

The content of the dissertation is based on the following articles:

I Cui Wang, Takahito Shikano, Henri Persat, Juha Merilä 2015. Mitochondrial phylogeography and cryptic divergence in the stickleback genus Pungitius. Journal of Bi- ogeography 42: 2334-2348. II Cui Wang, Takahito Shikano, Henri Persat, Juha Merilä 2017. Phylogeography and historical introgression in smoothtail nine-spined sticklebacks, Pungitius laevis (Gas- terosteiformes: Gasterosteidae). Biological Journal of the Linnean Society 121: 340-354 III Baocheng Guo, Takahito Shikano, Cui Wang, Alexandra Kravchenko, Juha Merilä. A phylogenomic perspective to diversity, hybridization and evolutionary affinities in the stickleback genus Pungitius. Submitted manuscript.

The following table shows the contributions of authors to the original articles. The authors are referred to by their first initials, and the articles by their roman numer- als.

I II III Original idea TS JM CW TS JM CW JM BG TS Sample collection JM HP TS HP TS JM JM TS AK BG Molecular data collection TS CW TS BG JM CW Data analyses CW TS CW TS BG Manuscript preparation TS CW JM HP CW TS JM HP JM BG CW TS

AK: Alexandra Kravchenko HP: Henri Persat

BG: Baocheng Guo JM: Juha Merilä

CW: Cui Wang TS: Takahito Shikano

Abstract

Pleistocene glaciations have profoundly influenced the genetic diversity of organsims in the Northern Hemisphere. Large ice sheets covered vast areas of the Eurasian continent, driving species southward to different isolated refugia, often resulting in deep divergences within species. Phylogeographic studies carried out on Pungitius species based on mitochondrial DNA (mtDNA) support profound intraspecific genetic divergence in refugia during glaciation cycles. However, compared to species distributed at lower latitudes, those distributed at higher latitudes may have also occurred in cryptic refugia in periglacial areas during glaciations, complicating the inferences of the phylogeographic patterns of the fish species with a circumpolar distribution, such as the Pungitius sticklebacks. Moreover, comprehensive phylogenetic studies of Pungitius species have been lacking in the sense that not all extant species have been included into analyses. In this dissertation, I carried out phylogeographic studies on seven Pungitius species using both mtDNA and genome-wide nuclear SNP markers, with worldwide sampling of populations to shed light on intra- and interspecific divergence in this genus, as well as to study their historical demography and interspecific hybridization.

By sequencing five mtDNA regions, I found six highly divergent Pungitius lineages including those corresponding to P. pungitius, P. platygaster, P. tymensis and P. kaibarae, and two independent monophyletic lineages of P. laevis. I also found a third lineage of P. laevis that clustered together with P. pungitius. To understand whether this clustering of the P. laevis lineage III and P. pungitius mtDNA was a result of convergence or interspecific introgression, I conducted phylogeographic and population genetic analyses using both mtDNA and nuclear gene sequences. The results indicated asymmetric mitochondrial introgression from P. pungitius to P. laevis and genetic admixture of these species. Hence, the results suggest that the P. laevis lineage III has experienced historical hybridization. Deep intraspecific mitochondrial divergence was found within P. laevis in central and southern France, coinciding with major drainages, suggesting that these areas correspond to distinct glacial refugia for the species explaining the observed intraspecific divergence.

To further clarify evolutionary relationships between different Pungitius species and populations, as well as to study the prevalence and extent of introgression among recognized species, phylogenomic datasets were constructed from restriction-site associated DNA in combination with mitochondrial genomes. All divergences in the Western Pale- arctic were estimated to have occurred during the Pleistocene (≤ 2.6 Ma). The phylogenetic patterns suggest a major split in Pungitius genus occurred early in history, resulting in an East Asian group (P. kaibarae, P. tymensis, P. sinensis) and European - North America group (P. hellenicus, P. platygaster, P. laevis and P. pungitius). The genus probably originated from the Western Pacific and spread to Europe and North America through the Arctic Ocean in several waves after the opening of the Bering Strait. Four cases of incongruence between nuclear and mtDNA-based trees revealed evidence for frequent hybridizations and mitogenome capture during the evolutionary history of this genus. Further analyses of these four cases of cytonuclear incongruence also revealed evidence for nuclear introgression, but the estimated levels of autosomal introgression were low.

gent lineages within species. In fact, Qua- 1. Introduction ternary glaciations have been the most in- fluential climatic changes that have affect- 1.1 Phylogeography and climate ed the phylogeographic patterns of terres- changes trial organisms in the past few million years (Hewitt 2004). When ice sheets ac- 1.1.1 Population genetics and climate cumulated on the Northern Hemisphere changes during glaciations, most surviving animal The history of species in space and time populations retreated to southern refugia is a topic of continued interest for evolu- (Hewitt 1996). Several refugia have been tionary biologists. Unlike the limitations of inferred for many species in the Eurasian fossil records, phylogeographic studies continent, including those in Iberian, Ital- allow inferences of the demographics and ian and Balkan peninsulas (Hewitt 2000; evolutionary relationships among species Provan & Bennett 2008). These refugia based on genetic markers, and thus, have were geographically isolated from each become important tools in reconstructing other with no or limited gene flow, result- species’ histories (Avise 2000). Ancient ing in deep divergences and in some cases, climatic and geographic changes have trig- triggering speciation events (Hewitt 1996). gered genetic responses in various species, After the glaciations, species recolonizing either due to random genetic drift or fixa- northern areas often lost genetic diversity tion/elimination of beneficial/detrimental in the leading edge of expansions because alleles, respectively (Hoffmann & Willi of serial founder effects (Hewitt 2004). 2008). Consequently, populations of a giv- Species adapted to high latitude environ- en species may have diverged into several ments may have also had cryptic refugia at genetically and morphologically distinct periglacial regions (instead or in addition species or lineages over time. For example, to southern ones) during the glaciations, in the Cenozoic era, frequent speciations and thus, their colonization history and occurred globally, because of recurrent genetic divergence may differ from those geological activities, such as the opening that recolonized from southern refugia and closing of the Bering Strait, the rise only (Stewart et al. 2010). The post-glacial and fall of sea levels, glaciation cycles, and recolonizations have also lead to repeated changing waterways (Craw et al. 2016; secondary contacts between formerly iso- Romaschenko et al. 2014). These geologi- lated populations and species, leading to cal events likely facilitated species disper- intraspecific mixture or interspecific hy- sal and migration, or restricted them in bridization (Aurelle et al. 2002; Zemlak et certain regions, thus enhancing the diver- al. 2008). Hybridization is especially easy gence of species, and evolution of diver- for young species at the onset of speciation

because of incomplete reproductive barri- past climatic and/or geological events on ers (Gérard et al. 2006; Harrison & Larson phylogeographic patterns. 2014).

1.1.3 Molecular markers and phylogeog- 1.1.2 Phylogeography of freshwater fishes raphy The genetic consequences of climatic The development of phylogeography has and geographic changes during Pleistocene allowed genealogy to be analyzed in con- glaciations can be especially obvious for junction with knowledge of spatial distri- freshwater species, because their occur- bution, and used genetic markers have rence in spatially complex and limited hab- evolved from isozymes to DNA sequences itats restrict their dispersal. Comparative (Avise et al. 1987; Avise 2000). Numerous phylogeographic studies on freshwater and phylogeographic studies based on mtDNA anadromous fish species often show deep have elucidated micro- and macroevolu- divergences in the deglaciated regions, but tionary processes on various spatial and also often lowered genetic diversity at temporal scales, providing insights into higher latitudes (Bernatchez & Wilson species’ history through theory of popula- 1998). This is possibly due to the fact that tion genetics (Avise et al. 1987). separate major drainages in deglaciated regions served as geographic barriers for However, because mtDNA is maternal- gene flow even prior to the Pleistocene ly inherited, it has become clear that infer- glaciations, whereas fishes in the glaciated ring population history on the basis of regions may have expanded into them from mtDNA variability alone can be mislead- the same refugium (Gum et al. 2005). As a ing (Hurst & Jiggins 2005). For example, result, different lineages or species may mitochondrial capture often happens in come into contact in interglacial secondary taxa experiencing interspecific hybridiza- contact zones either through the drainage tion. Although this phenomenon can some- connections perturbed by glaciations, or as times be inferred from polyphyletic phy- a consequence of population expansions logeny, it nevertheless needs to be further from different refugia, as in the case of validated with the aid of nuclear genetic brown trout (Salmo trutta), perch (Perca markers (Moore 1995). Therefore, biparen- fluviatilis), chub (Leuciscus cephalus) and tally inherited nuclear markers need to be many others (Nesbø et al. 1999; Perdices used together with mtDNA to fully under- et al. 2003, Gum et al. 2005; McKeown et stand the history of a given species. Specif- al. 2010). Thus, freshwater fishes with ically, the combination of mitochondrial wide distribution ranges have become use- and nuclear markers can aid in distinguish- ful models for investigating the effects of ing incomplete lineage sorting from inter-

specific hybridization and in identifying 1.2 Study systems hybrids in secondary contact zones. With 1.2.1 Taxonomy and distribution of recent developments in DNA-sequencing Pungitius species technology, several new approaches have become available for genotyping numerous Species in the genus Pungitius are small nuclear genetic markers simultaneously freshwater fishes of the family Gas- (Capella-Gutierrez et al. 2014; Davey & terosteidae, widely distributed in the Blaxter 2010). For instance, restriction-site Northern Hemisphere. The number of rec- associated DNA-markers (RAD-tags) are ognized species in the genus Pungitius is markers generated by randomly cutting not agreed upon, ranging from two to ten DNA at restriction sites throughout the depending on the source (Wootton 1976; genome, yielding a reduced representation Eschmeyer et al. 2017; Keivany & Nelson of genome-wide loci. Hence, thousands of 2000, 2004; Reshetnikov et al. 2003; markers representing the whole genome Kottelat & Freyhof 2007). Here, I follow can be generated without requirement for the classification that is based mainly on specific primers, making this method pos- variation in armor traits, according to sible to be used on any organism (Emerson which ten species are recognized. These et al. 2010). Modern sophisticated se- include: P. platygaster, P. pungitius, P. quencing technologies have made it possi- sinensis, P. bussei, P. hellenicus, P. kaiba- ble to use RAD-sequencing to infer phy- rae, P. laevis, P. polyakovi, P. stenurus logenies of many individuals or species, and P. tymensis (Eschmeyer et al. 2017). with high confidence and low cost (Baird Of these, P. pungitius has the widest cir- et al. 2008; Hohenlohe et al. 2010; Eaton cumpolar distribution across Eurasia and & Ree 2013). Access to a large number of North America, whereas the other species genome-wide markers will increase the all have more localized distributions accuracy of phylogenetic analyses, such as (Kottelat & Freyhof 2007; Bogutskaya et divergence time estimation, distinguishing al. 2008; Aldenhoven et al. 2010). P. genetic introgression from incomplete lin- sinensis, P. tymensis, P. bussei and P. kai- eage sorting, as well as helping in inferring barae are all locked in river drainages in genomic regions responsible for reproduc- East Asia, whereas P. polyakovi occurs on tive barriers (Rubin et al. 2012; Martin et Sakhalin Island and P. stenurus is only al. 2013, 2015). found in Lake Hulun in China (Shedko et al. 2005; Bogutskaya et al. 2008; Eschmeyer et al. 2017). P. laevis is confined to the water systems of Central and Southern Europe, as well as Ireland and Southern Great Britain (Takahashi et al.

2001; Kottelat & Freyhof 2007), whereas 2010; Shikano et al. 2010; Teacher et al. P. platygaster and P. hellenicus occur in 2011). The nine-spined stickleback (P. the Black, Caspian and Aral Sea basins. pungitius) has been the most widely inves- The taxonomy and systematics of this ge- tigated species. Based on analyses of nus are based heavily on the use of body mtDNA and microsatellite markers, it has armor traits - lateral plate numbers and been found to have diverged into three presence or absence of pelvic apparatus - lineages in North America and two line- as diagnostic characters (Takata et al. ages in Europe, probably as a consequence 1987a; Keivany 1996; Keivany & Nelson of repeated isolation into different refugia 2000). However, armor traits are evolu- during glaciation cycles (Aldenhoven et al. tionary very labile (Nelson 1971; 2010; Shikano et al. 2010; Teacher et al. Takahashi et al. 2001), and as such, may 2011). In East Asia, three ecotypes (viz. lead to misidentification of species due to freshwater, brackishwater and Omono convergent evolution (Takahashi et al. type) of P. pungitius have been identified 2001; Shapiro et al. 2009; Wang et al. based on morphological traits and popula- 2015). High-order relationships within tion genetic analyses using allozyme Gasterosteidae are well solved by nuclear markers. Nevertheless, the phylogenetic and mtDNA data, but species number and relationships and evolutionary history relationships within the genus Pungitius among these ecotypes have not been clear- are still poorly resolved (Brooks & ly resolved (Niwa 1987; Takata et al. McLennan 1991; Keivany & Nelson 2004; 1987a, b; Takahashi et al. 2001; Tsuruta & Kawahara et al. 2009). In fact, several phy- Goto 2006; but see Takahashi et al. 2016). logeographic studies have revealed deep Furthermore, Bae & Suk (2015) found that intraspecific genetic divergences within P. kaibarae in the Korean peninsula has Pungitius species (Takahashi & Goto diverged into two highly divergent line- 2001; Aldenhoven et al. 2010; Shikano et ages (Southeast and Northeast) on the basis al. 2010; Teacher et al. 2012), suggesting of mtDNA variability, a divergence which that there might be more species in the was attributed to Pleistocene glaciations. genus than currently recognized. Additional evidence from microsatellite markers has suggested that intraspecific 1.2.2 Intraspecific genetic divergence of divergences within Pungitius species can Pungitius species often be deep (Bae & Suk 2015). Thus far, most of these studies have revealed intra- Several phylogeographic studies carried specific genetic divergence, however they out on Pungitius species have suggested are generally focused on specific species, differentiated lineages in North America, often with limited geographic sampling. In Europe and East Asia (Aldenhoven et al. fact, species such as P. hellenicus have

never been included into phylogenetic among the extant taxa. Unexpectedly, in- analyses based on DNA markers. There- trogression was found between P. pungi- fore, systematic studies including both tius and P. tymensis, and between P. nuclear and mitochondrial genetic markers, pungitius and P. kaibarae, as inferred by as well as sampling of multiple species are incongruent phylogenetic patterns between required to achieve a more complete pic- mtDNA and nuclear markers – in spite of ture of the evolutionary history of Pungi- the fact that these species do not currently tius species. exist in sympatry (Takahashi et al. 2016). Thus, historical introgression before the 1.2.3 Interspecific introgression among species spread to their current ranges Pungitius species seems likely.

All phylogenetic studies conducted from East Asian individuals of P. sinensis have 2. Aims of the thesis shown that the species does not cluster together as a single group in mitochondrial My general aim in this dissertation was genealogy (Takahashi & Goto 2001; Bae to apply sticklebacks as models to explore & Suk 2015; Wang et al. 2015; Takahashi the evolutionary history of fish species, as et al. 2016;). For example, P. sinensis in well as to understand processes behind Japan was found to have diverged into two inter- and intraspecific genetic divergence, lineages; one lineage was a sister species using phylo- and population genomic ap- with P. tymensis and the other with the proaches. I was particularly interested in freshwater type of P. pungitius (Takahashi the occurrence of interspecific introgres- & Goto 2001). Because all these phyloge- sion, and its implications for reconstructing nies were constructed on the basis of the evolutionary history of species in the mtDNA, it is possible that the results are genus Pungitius. By assessing genetic di- caused by introgression between P. sinen- versity and constructing phylogenetic hy- sis and P. pungitius in East Asia potheses with the aid of molecular mark- (Takahashi & Goto 2001; Takahashi et al. ers, I also hoped to shed light on the spe- 2016). Recent studies that have included cies’ demographic history over space and nuclear genetic markers in addition to time. To reconcile the issue of polyphyletic mtDNA have found several cases of genet- topology of previous Pungitius phyloge- ic introgression in the genus Pungitius nies (cf. Takahashi & Goto 2001), I tested (Takahashi et al. 2016; Chapter II, III), several hypotheses regarding the possible suggesting that genomic divergence and evolutionary processes behind this pattern, historical relationships within this genus such as convergent evolution and interspe- are strongly influenced by introgression cific hybridization. Considering that the

divergence between Pungitius species and tained with RAD-sequencing technology. populations appears to have mostly oc- The main aim was to reconstruct robust curred during the Pleistocene glaciation phylogenetic hypotheses for Pungitius spe- cycles (Aldenhoven et al. 2010; Shikano et cies, as well as to estimate the proportion al. 2010; Teacher et al. 2011), this disser- of the genome for each species that has tation should also provide insights into been subjected to introgression. In addi- how historical climatic changes have influ- tion, by estimating divergence times be- enced intraspecific genetic diversity and tween different species and lineages, I interspecific genetic divergence and intro- hoped to shed more light on the biogeo- gression in freshwater fishes. These objec- graphic history of this genus, as well as tives are approached in the following three resolve the long-standing debate about the chapters: likely number of species in this genus.

Chapter I The aim of this chapter was to use the widely distributed Pungitius 3. Materials and Methods species as models to carry out phylogeographic studies using five mitochondrial 3.1 Study species and sampling DNA regions, so as to investigate the evo- Extensive sampling of seven Pungitius lutionary history of stickleback species, species (P. pungitius, P. laevis, P. and how freshwater species diverged and platygaster, P. hellenicus, P. sinensis, P. evolved during major climatic changes, tymensis and P. kaibarae) covering much like the glaciation cycles. of their known distribution areas were in-

cluded in this thesis (Figure 1). Due to the Chapter II Based on the results of wide distribution and ongoing collection of Chapter I, in this Chapter I utilized nu- samples, sample sizes and geographic cov- clear genetic markers and mitochondrial erage of sampling increased as the work DNA sequences for phylogeographic anal- progressed from Chapter I to III. The yses of P. laevis. In this chapter, I also fishes were collected with hand nets, seine aimed to address the deep intraspecific nets or minnow traps, and species were divergences, and in particular, interspecific putatively identified using morphological hybridization as a possible explanation for criteria (Wootton 1976; Kottelat & Freyhof the patterns of divergence in this species. 2007; Eschmeyer et al. 2017).

Chapter III The objective of this chap- The global-scale phylogeographic study ter was to reconstruct the phylogenetic included 193 individuals of four Pungitius history and genetic introgression of Pungi- species; across the five mtDNA regions tius species using genome-wide SNPs ob-

that were sequenced, 524 polymorphic tymensis, and P. kaibarae) by increasing sites were identified (Chapter I). Specifi- the number of informative SNPs. In this cally, this chapter targeted P. pungitius study, two individuals from 34 locations (143 individuals, 35 sites) including line- were analyzed (Figure 1). The three-spined ages from Europe, East Asia and North stickleback (G. aculeatus), the black- America, and P. laevis (35 individuals, 11 spotted stickleback (G. wheatlandi), the sites) from France. Three distinct forms of brook stickleback (C. inconstans) and the Japanese P. pungitius, referred to as four spined stickleback (Apeltes quad- Omono, freshwater and brackish-water racus) were included as outgroups into the types, were also identified according to phylogenetic analyses, and also to aid in their morphological characteristics (Chap- estimating divergence times between dif- ter I). Samples of P. platygaster and P. ferent lineages. The three-spined stickle- tymensis were obtained from Romania and back genomic data used in Chapter III Japan, respectively. In addition, mtDNA of were retrieved from Ensembl database one P. kaibarae individual was obtained (release-76). from GenBank (accession number: EU332749; Chapter I).

The results of Chapter I revealed deep divergences within P. laevis, and signs of genetic introgression between P. pungitius and P. laevis in France. Therefore, a more thorough sampling of P. laevis covering its whole distribution range (114 individuals from 25 sites) – including seven main drainage basins in France (viz. Seine, Loire, Dordogne, Charente, Meuse, Rhine and Rhône basins) and 22 individuals of P. pungitius from five sites in southeastern France – was conducted in Chapter II (Figure 1). In this chapter, I used mitochondrial cytochrome b gene (128 polymorphic sites) and eight nuclear genes (73 SNPs). In Chapter III, I aimed to increase the resolution of the relationships between seven species (P. pungitius, P. laevis, P. platygaster, P. hellenicus, P. sinensis, P.

Figure 1. Sampling locations used in Chapter I, II and III. Coloured shaded patterns show the distribution ranges of the different species; different symbols indicate sampling sites for each species. Filled circle = P. pungitius; diamond = P. tymensis; squares = P. sinensis; half circle = P. kaibarae; triangle = P. platygaster; star = P. hellenicus; black stripe filled square = P. laevis.

3.2 Molecular markers

Nuclear and mitochondrial molecular markers are biparentally and maternally inherited, respectively. Hence, they provide complementary insights into the evo-

lutionary history of stickleback fishes. To al., 2010; Chapter I) were used to amplify study the phylogeography of Pungitius the five mitochondrial regions in Chapter species, I started with five mitochondrial I. The nuclear genes, including exon- genes (in total 3236 bps, including: primed intron-crossing markers ATPase 6 [551 bps], cytochrome b [1104 (04174E20, 19231E4, 36298E1 and bps], control region [558 bps], 12S rRNA 55305E1) and four conserved coding re- [422 bps] and 16S rRNA [601 bps]) for gions (myh6, plagl2, SH3PX3 and sreb2), phylogenetic inference and divergence used in Chapter II were amplified using time estimation (Chapter I). As the results primers designed in earlier studies (Li et of Chapter I indicated that genetic intro- al. 2007; Li et al. 2010). In Chapter III, a gression may have occurred between restriction enzyme (PstI) was used to di- Pungitius species, eight nuclear genes gest the total DNA into fragments, and the (04174E20 [274bps], 19231E4 [452bps], fragments were ligated with P1 adaptor 36298E [467bps], 55305E1 [853bps], composed of a forward amplification pri- myh6 [593bps], plagl2 [703bps], SH3PX3 mer site, Illumina sequencing primer site [731bps] and sreb2 [832bps]) were includ- and a barcode to distinguish each sample. ed in Chapter II, in combination with the The DNA was sheared and ligated to a P2 mitochondrial cytochrome b gene. This adaptor containing the complement of the combined dataset allowed me to investi- reverse amplification primer site to select gate possible interspecific hybridization. In the tags with P1 adaptor (Baird et al. Chapter III, the number of nuclear mark- 2008). The successfully selected RAD-tags ers was further increased by use of RAD- with both P1 and P2 adaptors were ampli- seq, which generated a genome-wide panel fied and sequenced on Illumina HiSeq2000 of SNPs that were used to construct phylo- platform at the Beijing Genome Institute genetic hypotheses about the species rela- (BGI). tionships. This data also allowed me to study the occurrence of genetic introgres- 3.3 Statistical analyses sion on a larger genomic scale. The five mitochondrial gene sequences Total DNA was extracted from fin-clips were aligned and concatenated for further using a silica-based method (Chapter I, analyses in Chapter I. Basic statistics of II), or standard phenol-chloroform method the concatenated fragments, such as num- (Chapter III). Polymerase chain reaction ber of haplotypes, nucleotide diversity (π) (PCR) was performed following Shikano and haplotype diversity (ĥ) were calculated et al. (2010), and nine primer sets (Kocher for each lineage or species using the pro- et al., 1989; Palumbi, 1996; Fu et al., gram DNAsp v. 5 (Librado & Rozas 1999; Takahashi & Goto, 2001; Shikano et 2009). After removing the gaps of the con-

catenated alignment, phylogenetic relation- lotype diversity (ĥ) for mtDNA and num- ships were constructed under Bayesian ber of alleles (Na), expected heterozygosi-

Inference. Divergence time and species ty (HE) and inbreeding coefficient (FIS) for tree estimations were performed by using nuclear SNPs. Phylogenetic analyses were the fossil record of three-spined stickle- conducted under Bayesian Inference for back (Bell et al. 2009) for calibration, uti- both mtDNA and nuclear datasets. To see lizing a coalescent model within and Yule if the evolutionary history of maternally speciation model between species. Sudden inherited genetic markers was different expansion tests (including sum of squared from that of biparentally inherited markers, deviations (SSD) and Harpending’s rag- incongruence between mtDNA and nuclear gedness index (HRI)) were conducted with SNPs phylogenies was tested using Con- the program Arlequin v3.1 (Excoffier & gruence Among Distance Matrices Lischer 2010) and neutrality indecies (in- (CADM) program (Legendre & Lapointe cluding Tajima’s D and Fu’s FS statistics) 2004). I also performed Principal Compo- were calculated to investigate the demo- nent Analysis (PCA) using Eigensoft (Pat- graphic history of each lineage or species. terson et al. 2006) to infer the phylogenetic To investigate whether the morphological relationships among the species, and car- traits used for species identification (lateral ried out population admixture analysis on plate, caudal keel and pelvic apparatus) nuclear SNPs using STRUCTURE 2.3.4 were significantly correlated with phylog- (Pritchard et al. 2000) to get further insight eny, I tested the association between phy- into possible introgression. Finally, the logeny and armor traits using D statistics colonization route of the putative hybrid (Fritz & Purvis 2010) with 1000 permuta- lineage P. laevis lineage III was inferred tions. based on the leading-edge effect of postglacial genetic diversity distribution, under Mitochondrial and nuclear data were the assumption that the genetic diversity analyzed separately in Chapter II. Hardy- declines from the site of origin towards the Weinberg equilibrium and linkage disequi- more recently colonized areas (Hewitt librium were calculated for nuclear SNPs 2000, 2004). To examine if any gene flow with the program Genepop 4.2 (Raymond between species has occurred recently or in & Rousset 2004; Rousset 2008). For both the past, I estimated genetic divergence datasets, basic statistics of each population, between populations of different species, as well as variance analyses (ANOVA) and tested whether these divergences were among lineages/species and hierarchical correlated with the geographic distances analyses of molecular variance (AMOVA; between them (i.e. isolation-by-distance). Excoffier & Lischer 2010) were calculated, If gene flow has occurred recently rather including nucleotide diversity (π) and hap- than historically, one would expect more

genetic divergence between more distant Heterogeneity in Fst and dxy (see BOX 2) populations than among populations situat- measures of divergence between Pungitius ed closer to each other. species was plotted against 21 linkage groups using custom-written functions of In Chapter III, the RAD sequences genomics.py from Dr. Simon Martin were aligned and mapped to the three- (https://github.com/simonhmartin/genomic spined stickleback genome using BWA s_general/blob/master/genomics.py) with 0.7.5a (Li & Durbin 2009) to identify the aid of BCFtools (Li et al. 2009) to SNPs. Firstly, I carried out a multidimen- identify regions across the genome poten- sional scaling (MDS) using PLINK1.9 tially related to reproductive isolation: Fst (Purcell et al. 2007) on the genome-wide or dxy values significantly higher than the SNPs to get a basic idea of the genetic sim- baseline can be indicative of such regions ilarity among species. Secondly, I con- (e.g. dxy ≥ 0.2, Chapter III). structed phylogenetic relationships using 560 149 of the 2 888 502 SNPs and 459 233 orthologous genes using a Maximum Likelihood (ML) method in RAxML 8.1.3 (Stamatakis 2014). Four more individuals were sequenced for the five mitochondrial DNA regions used in Chapter I, to com- pare the phylogenetic patterns between nuclear and mitochondrial genomes in order to study interspecific hybridization. Thirdly, I investigated genetic introgression using orthologous genes by using ABBA-BABA tests (Green et al. 2010; Martin et al. 2013). Given a topology of (((P1, P2), P3), O), the number of alleles shared between P1 and P3 should be the same as the numbers of alleles shared between P2 and P3 on the condition that there is no genetic introgression between P3 and either P1 or P2 (see BOX 1; Green et al. 2010; Durand et al. 2011). To investigate patterns of genomic divergence, species phylogenies were also estimated for each of the 21 linkage groups separately.

BOX 1. Detecting genetic introgression using ABBA - BABA test

Genetic introgression between species can be estimated by calculating the proportion of SNPs showing biased ABBA-BABA pattern. In the given topology (((P1, P2), P3, O) as sketched below, P1, P2 and P3 represent three closely related species, and O represents an outgroup. P1 P2 P3 O

Assume that A is an ancestral allele, and B is a derived allele. If there is no gene flow or incomplete lineage sorting between P1 and P3 or between P2 and P3, the most frequent- ly observed pattern should be BBAA. If we consider that there is no gene flow but only incomplete lineage sorting evenly distributed between taxa, the counts of ABBA or BABA pattern (CABBA or CBABA) should be equal between P1 and P3 or between P2 and P3. In this case, the value of D calculated with the formula

푛 σ푖=1 퐶ABBAሺ푖ሻ−퐶퐵퐴퐵퐴ሺ푖ሻ D(P1, P2, P3, O)= 푛 σ푖=1 퐶퐴퐵퐵퐴ሺ푖ሻ+퐶퐵퐴퐵퐴ሺ푖ሻ would equal 0. When there is gene flow between P1 and P3, the frequency of the AB- BA-pattern would exceed that of BABA, such that the value of D would be significantly positive. In contrast, if there is gene flow between P2 and P3, the value of D would be significantly negative.

BOX 2. Genetic divergence between populations based on Fst and Dxy

In the SNPs dataset, Fst measures the genetic divergence between populations on the basis of allele frequency differences, which are inferred from the formula

퐻푡−퐻푠 Fst = 퐻푡

where Ht represents the expected heterozygosity in the total population, and Hs represents the average expected heterozygosity in the subpopulations.

Dxy measures absolute pairwise genetic distance between populations, calculated from the formula

Dxy=σ푖푗 푋푖푌푗퐷푖푗

Where the Dxy is a sum of the distance (Dij) between the ith individual from the population X and the jth individual from Y.

Speciation genes indicate those genes that are involved in reproductive isolation or ecological adaptation, thus these genomic regions are more resistant to gene flow between species compared to neutral regions (Cruickshank & Hahn 2014). In genomic regions under natural selection (including ‘speciation genes’), the genetic divergence is expected to exceed that in regions evolving neutrally, yielding outlier peaks. Detection and validation of these peaks can help to identify speciation genes.

4. Results and discussion

4.1 Phylogeography of Pungitius species The Bayesian phylogeny tree based on nese Omono type, freshwater-brackish the five mtDNA regions revealed six high- water complex, North American, Western ly divergent Pungitius clades, correspond- European (WE) and Eastern European ing to P. tymensis, P. kaibarae, P. (EE) lineages (Chapter I). Although di- platygaster, P. laevis lineage I, P. laevis vergence within Pungitius species – espe- lineage II, and P. laevis lineage III, the cially in the case of P. pungitius – have latter of which clustered together with P. been investigated before (Takata et al. pungitius (Chapter I). The widely distrib- 1987a; Takahashi et al. 2001; Aldenhoven uted P. pungitius was further identified to et al. 2010; Shikano et al. 2010; Teacher et consist of five lineages including the Japa- al. 2012; Takahashi et al. 2016), this study

found that deep intraspecific divergence vergence by facilitating species dispersal also occurred in its sister species P. laevis, or isolation, and it can be difficult to trace which is split into three lineages in the their footprints because the subsequent mtDNA phylogenetic tree (Chapter I). glaciations may have obscured this signal. Further divergence time estimation based From the mtDNA data, the divergence on both mtDNA (1.95 million years ago between Pungitius species was estimated (Mya)) and nuclear data (0.9 Mya) showed to have been initiated 4.4 Mya when the the P. laevis lineage I diverged from line- ancestor of P. tymensis and P. kaibarae age II during Pleistocene glaciations diverged from other species. This coin- (Chapter I, III), indicating this species cides with the opening of the Bering Strait, may have retreated to different refugia which provided a passage for ancestors to when glaciers accumulated and were hence move out of Asia and colonize Europe and geographically isolated for a long time North America through the Arctic Sea during the glaciations. The distribution of (Figure 2, Chapter III). All divergences in P. laevis lineage I, II and III were confined the Western Palearctic were estimated to to separate river basins: the Loire river basin, the southern region of the Charente river basin, and eastern region of the Seine basin, respectively (Chapter II). Because the permafrost reached the northern parts of France during the glaciation period (Bertran et al. 2014), and many species used central and southwestern France as refugia, it seems likely that the first two regions have provided shelter for P. laevis lineage I and II, respectively (Chapter II). As the P. laevis lineage III showed genetic admixture with P. pungitius, the history of this lineage in the interspecific introgression will be discussed below.

It is widely accepted that Pleistocene glaciations had profound impacts on the phylogeography of fishes in the Nearctic and Palearctic (Bernatchez & Wilson 1998). However, earlier geographic changes may have also affected the genetic di-

Figure 2. Phylogeny of Pungitius species based on single nucleotide polymorphisms. Species G. aculeatus, G. wheatlandi, A. quadracus, and C. inconstans were included as outgroups. Different colors indicate different species/lineages.

have occurred during the Pleistocene (≤ Apart from showing distinguishable 2.6 Mya; Chapter I), which was character- phylogeographic patterns, species experi- ized by a series of glaciation and deglacia- encing retreats and recolonizations during tion cycles starting 2.58 Mya (Ogg et al. the Pleistocene glaciations also often dis- 2004). These inferences are concordant play specific patterns in their levels of ge- with divergence time estimates based on netic diversity. According to the leading- nuclear SNPs (Chapter III), barring a few edge hypothesis (Hewitt 1996, 1999, topological incongruences regarding P. 2000), populations following the retreat of sinensis, P. laevis and P. pungitius, which the ice sheet rapidly expand, colonizing will be discussed later. The RAD-based large ranges and preventing more southern phylogeny revealed a clear pattern of early lineages or populations to advance, result- divergence between two major clades: one ing in lowered genetic diversity in former- including P. tymensis, P. kaibarae and P. ly glaciated areas than in regions that re- sinensis from East Asia, and the other in- mained unglaciated (Hewitt 1996). Com- cluding P. platygaster, P. hellenicus, P. pared to the Western European (WE) line- laevis and P. pungitius, with the latter dis- age, the Eastern European (EE) lineage of tributed across large parts of Eurasia and P. pungitius residing in the northern parts North America (Chapter III). The phylo- of Europe showed reduced genetic diversi- genetic analyses suggest that the genus ty, likely as a result of population expan- originated in the West Pacific, and the sion after glaciation. Genetic diversity of common ancestor of the latter clade P. laevis lineage II in southern France was crossed the Arctic Sea following the open- significantly higher than that of lineages I ing of the Bering Strait, eventually reach- and III in former periglacial regions, sug- ing inland Europe (Chapter III). Howev- gesting that southern France may have er, the several lineages of P. laevis and P. been a refugium for P. laevis lineage II, pungitius were possibly formed in different and that the other lineages have colonized refugia during glaciation cycles. In contrast their current territories later. to more locally distributed species in this clade (e.g. P. hellenicus, P. laevis & P. My results also clarify the taxonomic platygaster), P. pungitius has a global dis- confusion stemming from use of morpho- tribution including Eurasia and North logical traits as diagnostic characters in America. My data suggest that several Pungitius systematics. In contrast to previ- waves of dispersal from the Northern Pa- ous studies that have claimed the Pungitius cific through the Arctic Ocean to Europe individuals in the Omono river (Omono and North America might have been in- type) and Japanese freshwater habitats volved in the establishment of its current (freshwater type) to be considered as distribution range.

different ecotypes of P. pungitius (a hypothesis, I calculated D-statistics sepa- claim also supported by mtDNA phylog- rately using Far East and non - Far East eny in Chapter I; Takata et al. 1987b; populations of P. pungitius at position P2 Takahashi et al. 2001), the SNP-based (BOX 1). Each population of P. sinensis phylogeny classified them as two differ- (including freshwater type of P. pungi- ent species: P. kaibarae and P. sinensis, tius), P. kaibarae (including Omono type respectively (Chapter III). Takahashi et of P. pungitius), and one representative al. (2016) independently reached a simi- population of P. laevis was used at P3, to lar conclusion, suggesting that morpho- distinguish between gene flow and in- logical traits are not reliable diagnostic complete lineage sorting. The results characters in Pungitius systematics. showed significant positive D-statistics between these species pairs, indicating 4.2 Historical introgression interspecific hybridization, rather than incomplete lineage sorting as the cause among Pungitius species of the phylogenetic incongruence (Table Four cases of phylogenetic discord- 1, Chapter III). Genetic admixture anal- ance between mitochondrial and nuclear ysis and PCA using nuclear genes con- genomes were found in the seven Pungi- firmed that interspecific hybridization tius species, suggesting extensive hybrid- occurred between P. laevis and P. pungi- ization in this genus. Although the phy- tius, resulting in a hybrid P. laevis line- logenetic analysis based on SNPs clearly age III (Chapter II). However, MDS distinguished P. sinensis, P. kaibarae, P. analyses based on SNP data showed two laevis and P. pungitius as independent populations of P. tymensis clustering species, some populations of the first together with P. sinensis and P. kaiba- three species carry mtDNA of P. pungi- rae, which may be a result of genetic tius (Chapter III). Specifically, P. introgression. The D-statistics calculated sinensis included the freshwater type of using these species supported the hy- P. pungitius, and P. kaibarae included pothesis that gene flow had occurred the Omono type of P. pungitius. The dis- between P. tymensis and P. sinensis or P. crepancy between mtDNA and nuclear kaibarae. gene phylogenies may be explained by several factors, such as convergent evo- Genetic introgression has been fre- lution, incomplete lineage sorting and quently reported to occur between spe- genetic introgression (van Oppen et al. cies (Echelle & Connor 1989; Largiadèr 2001; Fleischer et al. 2008). As P. sinen- & Scholl 1996; Baack & Rieseberg 2007; sis and P. kaibarae are distributed in East Rheindt & Edwards 2011; Twyford & Asia, I supposed that if introgression has Ennos 2012). However, whether this has taken place, it has occurred between Far occurred during contemporary or histori- East populations of P. pungitius and P. cal events is poorly understood. Alt- sinensis or P. kaibarae. To evaluate this hough the signs of hybridization were

Table 1. Tests of admixture between different Pungitius species. P1, P2 and P3 indicate species/population used in a given topology position

when testing for admixture using Patternson’s D-statistics. Z is the test-statistic and nSig/n gives the number of tests out of all possible tests (for a given set of taxa) that were significant. F = estimated admixture proportion. O = outgroup.

P1 P2 P3 O Z (min – max) nSig/n F (%) P. pungitius – P. sinensis PH, PL, (NA, FE, EU, WU) FE PP PS(PP[FW]) CI 1.6 - 31.38 30/39 0.75 – 5.73 PP PH, PL, (NA, FE, EU, WU) None-FE PP PS CI 0.32 - 2.90 0/18 - PP P. pungitius – P. kaibarae PH, PL, (NA, FE, EU, WU) FE PP PK(PP[OM]) CI 0.61 – 15.52 17/26 0.43 – 4.26 PP PH, PL, (NA, FE, EU, WU) None-FE PP PK CI 0.23 – 2.97 0/12 - PP P. pungitius – P. laevis (lineage III) (NA, FE, EU, WU) PP PP-FR- PL(III) PH 21.57 – 43.98 5/5 2.11 – 7.61 MAR (NA) PP (NA) PP PL(III) PH 1.63 0/1 - P. tymensis – P. sinensis PS(CN) PS(RU) PT(RU) PH 3.65 – 13.14 3/3 3.00 – 4.00 PS(RU) PS(CN&JP) PT(JP) PH 0.08 – 11.59 2/4 2.00 – 3.00 P. tymensis – P. kaibarae PK PP[OM] PT PS 20.8 – 29.74 2/2 8.00 – 12.00 CI: C. inconstans; PH: P. hellenicus; PK: P. kaibarae; PL: P. laevis; PP: P. pungitius; PS: P. sinensis; PT: P. tymensis; FW: freshwater type of P. pungitius; OM Omono type of P. pungitius; EU: East Europe lineage of P. pungitius; FE: Far East lineage of P. pungitius; NA: North Ameri- ca lineage of P. pungitius; WU: East Europe lineage of P. pungitius.

common in the genus Pungitius, D- fluctuations in water levels and connec- statistics and genetic admixture analyses tivity, which may also have influenced of the RAD-data showed that the extent the distribution and secondary contacts at of genetic introgression varies from spe- coastal areas. For example, the rise of ice cies to species, and are mostly negligibly sheets during the last maximum glacia- low. The highest extent of hybridization tions caused the sea levels to drop to occurred between the two youngest spe- nearly 134 meters below the present level cies P. pungitius and P. laevis, with ca (Dutton et al. 2015). The sea level sub- 2.1-7.6% of the genome shared (Chapter sequently rose to 7 m higher than at pre- III). sent during the last interglacial period (Gibson et al. 2007). This likely had a

Gene flow tests based on pairwise Fst- significant impact on river connectivity. estimates between interspecific popula- Hence, different lineages of fish species tions rejected the hypothesis of recent or previously inhabiting separated refugia ongoing gene flow among the two spe- may have met and hybridized during or cies, which, together with the low intro- after these geomorphological events. In gression ratio, suggests that the hybridi- the four cases of detected hybridization zation most likely occurred in the ancient between Pungitius species, secondary past (Chapter II). Similarly, only 0.8– contact seems to have occurred both in 5.7 % of the genome was estimated to be Europe and East Asia as indicated by involved in introgression between P. introgression of mtDNA of P. pungitius pungitius and P. sinensis, and between P. into P. laevis, P. kaibarae, P. sinensis. P. pungitius and P. kaibarae, which, to- laevis lineage III distributed in northern gether with the wide range of mtDNA France was revealed to be a hybrid line- capture, also possibly suggest historical age that was genetically mixed between introgression events. P. laevis and P. pungitius (Figure 3). However, because P. laevis lineages I Natural hybridization is commonly and II are restricted to southwestern and seen in freshwater fishes (Schwartz mid-France, and P. pungitius was land- 1981). Among 139 species pairs of fish locked within the Saône basin, the intro- hybridizing in wild and aquaculture, 89% gression is unlikely to have happened of the cases were related to the removal recently to result in a widely distributed of spatial barriers (Scribner et al. 2000). P. laevis lineage III. Hypothesis testing

This may be due to the low complexity based on comparing Fst and geographic of spawning habitats and susceptibility to distance among lineages suggested the secondary contacts between previously gene flow between P. laevis and P. isolated populations when new water pungitius may have occurred during gla- connections are formed (Scribner et al. ciations when these species occupied a 2000). In addition, historical events and refugium in southern France and a human disturbance could both lead to

P. laevis P. pungitius

Figure 3. Bayesian clustering of P. laevis and P. pungitius individuals at K = 2 to K = 5 (K refers to the number of genetic clusters in the data) based on nuclear genetic data. K=2 was selected using Evanno’s method (Evanno et al. 2005). Blue = P. laevis; green = P. pungitius. Each vertical bar represents an individual (Chapter II). secondary contact formed thereafter (Chapter II). tween parental species, whether due to 4.3 Reproductive isolation between pre- or postzygotic reproductive barriers. species For example, although anadromous and For both terrestrial and marine species, freshwater types of three-spined stickle- it is widely accepted that smaller genetic backs are often morphologically and ge- distances between parental species lead netically divergent, they may hybridize to more successful hybridization because in estuaries, indicating incomplete of reduced chances of genetic incompati- prezygotic isolation (Jones et al. 2006). bilities (Montanari et al. 2014). Cryptic species, which are genetically divergent Comparison of mtDNA and nuclear but morphologically similar, may be es- SNP-based phylogenies indicated that all pecially prone to hybridization (Patel et the species experiencing introgression al. 2015). Hybridization is an indication had obtained their mtDNA from P. of incomplete reproductive isolation be-

pungitius. This asymmetric mitochondri- asymmetric introgression, retaining al introgression may have occurred for mtDNA of only one parental species several reasons, including adaptive intro- (Wirtz 1999). gression of the mtDNA, population size difference among hybridizing species, Several reproductive isolation events sex ratio bias, mating preferences as well have been revealed between Pungitius as artificial transfer of one species to the species. For example, breeding experi- habitat of other species (Wilson & Ber- ments have been carried out between natchez 1998; Wirtz 1999; Crespin et al. freshwater (recognized as P. sinensis in 1999; Telschow et al. 2006; Toews & this thesis, but see also: Takahashi et al. Brelsford 2012; Johnson et al. 2015). For 2016) and brackish-water type of P. example, species with male-killing sym- pungitius in East Asia, revealing sterility bionts will cause a biased sex ratio in the of F1 male hybrids during reciprocal population, resulting in lower intraspecif- hybridization of the parental species, ic mating rate due to the reduced female following Haldane’s rule (Takahashi et choice. Females must mate with males al. 2005). Thus, if female P. pungitius from the immigrant species, in which prefer to mate with male P. sinensis, then case the symbionts may continue to kill the female F1 hybrids would carry male offspring, thus reducing the popula- mtDNA from P. pungitius, and if they tion size of the genetic markers of the backcross to P. sinensis, the mtDNA local species and causing asymmetric would be transferred to the latter species. gene flow from the invading species The same hypotheses could also be ap- (Telschow et al. 2006). In other cases, plied to P. pungitius and P. laevis in Eu- females of species A may have mating rope. Although the geographic distance preferences for males of species B, but between P. kaibarae (Omono type) and females of species B do not prefer to P. pungitius populations is similar to that mate with males of species A. Conse- between P. sinensis (freshwater type) and quently, the hybrids of the next genera- P. pungitius populations, the former pair tions will only carry mtDNA from spe- shares much less nuclear alleles in com- cies A, resulting in asymmetric gene mon than the latter. This could be a result flow of maternal markers (Laurie 1997; of stronger reproductive isolation be- Demuth & Wade 2007). These processes tween P. pungitius and P. kaibarae, but also involve reproductive isolation be- an alternative explanation can be that tween species, thus the species boundary introgressive hybridization first hap- in the process of speciation is often pened between P. pungitius and P. sinen- blurred (Good et al. 2008). When sis, and P. sinensis hybridized with P. postzygotic isolation has evolved, genet- kaibarae later on (Takahashi & Goto ic incompatibility – especially male ste- 2001). Because the SNP phylogeny rility in hybrids or low fitness of off- showed the divergence time between P. spring – will also contribute to the kaibarae and P. sinensis to be 3.0 Mya,

and the divergence time of mtDNA was peaks (Turner et al. 2005; Cruickshank less than 0.66 mya, it is very likely that & Hahn 2014). These “genomic islands” the two species obtained a common are responsible for maintaining the spe- mtDNA during Quaternary glaciations cies boundary, and thus are less likely to and diverged thereafter (Wang et al. be subjected to introgression from other 2015). In nature, introgressive hybridiza- species (Feder et al. 2012). The genomic tion between sympatric species may oc- comparison between Pungitius species cur in conjunction with sharp population showed varied peaks across the 21 link- size declines, when a species loses con- age groups. However, the average diver- specific mating opportunities and hybrid- gence levels among species or popula- izes with other species. This has been tions among linkage groups were not observed in Scandinavian house mice significantly different. Pairwise dXY was (M. domesticus), asp vipers (Vipera as- in general less than 0.2 across the ge- pis) and an antelope (Ferris et al. 1983; nome, except for 203 genomic regions Pinto et al. 2016). During glaciation which had values higher than 0.2 (Chap- periods, the ice sheets and permafrost ter III). Some of these high peaks corre- have covered most parts of the Eurasian sponded to important functional genes in continent and North America, reaching the three-spind stickleback genome, and up to northern France and northern parts have been inferred to be involved in fish of Japan in East Asia (Hewitt 2000; speciation in other studies. Thus, these Sakaguchi et al. 2012; Barr & Solomina genomic regions may contain Pungitius 2014). Because P. pungitius has the wid- speciation genes. i.e. genes that have est distribution and intraspecific genetic contributed to reproductive isolation and diversity in comparison to other conge- local adaptations (Carling & Brumfield neric species (Figure 1, Chapter III), it 2009; Via et al. 2012). However, alt- is possible that the population size of P. hough preliminary genetic studies may pungitius was much larger than in the identify these genes involved with speci- other species before recolonization, and ation, further studies are still needed to the mtDNA of P. pungitius was fixed understand the process of speciation and during introgressive hybridization. role these candidates play (Noor et al. 2001; Rieseberg 2001; Navarro & Barton Genes coding for traits involved with 2003; Kenney & Sweigart 2016). reproductive barriers will be less likely to introgress than neutral loci (Barton & Hewitt 1981; Payseur et al. 2004; Gay et Conclusions and outlook al. 2007). As a result, genetic divergence among the whole genome will not be In conclusion, by using mitochondrial homogenous, and genomic regions relat- DNA fragments, mitochondrial genomes ing to reproductive isolation or natural and nuclear SNPs of seven Pungitius selection tend to give rise to high Fst species, I was able to construct a well-

resolved phylogeny and detect several testing for mating preferences, hybrid ancient hybridization events in this ge- fitness in different crosses, or population nus. The divergence timelines of the spe- demographics censuses can all help to cies were closely related to geographic address the causes of unidirectional in- and/or climatic changes during the late trogression in the genus Pungitius. Pliocene and Pleistocene glaciation cycles. Intraspecific genetic diversity tend- ed to decline from south to north, follow- Acknowledgements ing the leading-edge hypothesis which predicts that postglacial recolonization First, I would like to say thank you to results in a gradient of decreasing genetic my supervisor Professor Juha Merilä for diversity from refugia towards newly offering me such a great chance to study colonized territories. During this process, in this beautiful country Finland. Thank temporary secondary contact zones ap- you Juha for generously providing me pear to have formed when different spe- the spacious office and lab, for offering cies recolonize new territories from dif- me different projects to lead me to the ferent refugia. Hybridization occurs field of science, and for helping me apply commonly in the genus Pungitius, and for scholarships throughout my PhD has resulted in several phylogenetic in- study. Through your lead and guidance, I congruences between mtDNA and nucle- learned to be humble and to learn from ar SNPs. Hypothesis testing regarding other people, and to take an objective the genetic divergence over geographic view with everything in life. I also want distance rejected the possibility of recent to say thank you to my second supervisor gene flow, suggesting that the observed Dr. Takahito Shikano. Thank you, Taka, hybridization events have occurred in the your patient and thorough guidance al- past. Levels of genetic introgression in lowed me to go through all the difficul- the nuclear genomes were low, whereas ties during study, and become an inde- mitochondrial capture from P. pungitius pendent thinker. I still remember when I was commonly found among the hybrid- got stuck with some analysis for months, izing pairs of taxa, showing that the evo- you discussed with me and encouraged lutionary history of maternal genetic me to solve the problem little by little, markers can be very different from bipa- making me feel more and more confident rental markers. Putative reproductive with myself. I want to thank my thesis isolation genes were inferred from ge- committee members Dr. Perttu Seppä nomic divergence comparisons between and Dr. Helena Johansson for offering different species pairs. However, more me so much valuable guidance to com- work is needed to test the function of plete my study in time. I am especially these genes and their possible role in grateful to the pre-examiners of this dis- speciation. Breeding experiments such as sertation, Dr. Laura Kvist and Dr. Robert

Ekblom for their constructive efforts to star. Lastly, many thanks to LUOVA for improve this thesis, and all your advice the travel support to attend conferences are highly appreciated. Also, I want to say and seminars. Thanks to the support from thank you to my collaborator, Henri, who CIMO, the Chinese Scholarship Council, I have never met in person, but has still Research foundation of University of Hel- played an important role in my thesis. sinki and the dissertation completion grant Thank you Baocheng and Zitong, for put- from University of Helsinki, I can accom- ting so much effort into this thesis. Alt- plish my PhD study. hough some of my friends may not have the chance to read this dissertation, I still want to say thank you to all my previous References and current colleagues and friends. Thank you, Marika, Heini, Kirsi, Anni and Aldenhoven JT, Miller MA, Corneli PS, Minttu, you are my first teachers in the Shapiro MD (2010) Phylogeography of University to get me familiar with the ninespine sticklebacks (Pungitius university, group and lab, and I will never Pungitius) in North America: glacial forget your help and humor! Thank you, refugia and the origins of adaptive Jackie, for your kindness and patience to traits. Molecular Ecology 19, 4061- check my language in my every publica- 4076. tion! Thank you, Chris, Abhilash, Profes- Aurelle D, Cattaneo-Berrebi G, Berrebi P sor Liao, Federico, Paolo, Pasi and Ari for (2002) Natural and artificial secondary the helpful discussions when I came contact in brown trout (Salmo trutta, across some confusions in my study. L.) in the French western Pyrenees as- Thank you, Alex and Scott, for your gen- sessed by allozymes and microsatel- erous help with translating references. lites. Heredity 89, 171. Thank you, Sergey, Kristina, Bohao, Avise JC (2000) Phylogeography: the Xueyun, Shichao and Minastiina for your history and formation of species Har- friendship and some relaxing times in the vard university press, Cambridge, office. Thank you, Hui, for your long-term Mass. company at lunch and farmer experience. Avise JC, Arnold J, Ball RM, et al. (1987) Thank you, Antti Korpin Tie dormitory Intraspecific phylogeography: the mi- friends, for those leisure moments we tochondrial DNA bridge between shared, you are all my treasures! population genetics and systematics. Most importantly, I want to say thank Annual Review of Ecology and System- you to everyone in my family, especially atics 18, 489522. my grandparents and my husband Kari for Baack EJ, Rieseberg LH (2007) A ge- your great tolerance. Thank you Armi, nomic view of introgression and hybrid Hannu and Timo for your enormous sup- speciation. Current opinion in genetics port to me. Also, my dear son Samuel, is & development 17, 513-518. bringing happiness to my life like a rising

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