The genetic linkage maps established for YCT-RBT hybrids provide novel insights into the genomic consequences of introgression between RBT and YCT. Hybrid and RBT linkage groups were syntenic and had similar marker order, suggesting that RBT and YCT share chromosome arms. In addition, hybrids and RBT shared linkage groups, with one exception where a hybrid linkage group involved a fusion between a bi-armed metacentric and a single-armed acrocentric RBT chromosome. This result was confirmed by additional mapping in YCT. Comparison between hybrid and RBT female maps indicated that introgression suppressed recombination across several large chromosome segments. Two hybrid linkage groups showing recombination suppression (RYHyb20 and RYHyb25_28) contained different chromosome arrangements between YCT and RBT. Segregation distortion was generally limited and distortion patterns varied among parents and families, suggesting that few incompatibilities exist between RBT and YCT genomes. Taken together, these results suggest that RBT and YCT genomes freely introgress, with the exception that differences in chromosome rearrangements between the species could impede introgression across large portions of specific linkage groups.
Chromosome rearrangements between RBT and YCT
The hybrid map is a product of differences in chromosome rearrangements between RBT and YCT, as well as mixed ancestry of the RBT source stock. Using data from karyotypes
[27, 28] and the hybrid map, we suggest that RBT and YCT differ by at least five species-specific chromosome rearrangements. We first consider the fusion between Omy28 and Omy20q in YCT. The most parsimonious explanation for the fusion between the metacentric Omy20 and the acrocentric Omy28 involves two rearrangements. The first rearrangement would have occurred prior to the Omy20-Omy28 fusion as a pericentric inversion of the entire q-arm of Omy20. This inversion would have resulted in Omy20 becoming an acrocentric chromosome and a reduction in the expected 52 haploid chromosome arms
[27, 28] to the observed 51 haploid arms in the hybrid map. The possibility that Omy20q could be inverted in YCT relative to RBT is supported in Chinook salmon (O. tshawytscha), as the Omy20 homologue in Chinook is an acrocentric chromosome and the segment homologous to Omy20q appears inverted compared to RBT (unpublished observations, K. Naish). The second rearrangement would have been a Robertsonian type involving centric fusion between the acrocentric Omy20 homologue and Omy28, conserving chromosome arm number. The three remaining differences may be explained by comparing acrocentric chromosome numbers between RBT (seven in the 60 chromosome race) and YCT (twelve). The seven RBT acrocentric chromosomes have been identified as such in the hybrid map, suggesting that they have an acrocentric YCT homologue. The remaining five YCT acrocentric chromosomes likely have homologs with RBT metacentric chromosomes. The third and fourth rearrangement differences probably represent centric fusion/fission events between two RBT metacentric and four of the five remaining YCT acrocentric chromosomes, which would not change the chromosome arm number. The fifth rearrangement difference may have involved a fission event within a RBT metacentric chromosome arm, generating the fifth acrocentric and a submetacentric chromosome in YCT. Fission within a RBT metacentric arm would gain one YCT chromosome arm and restore the 52 haploid chromosome arm number, countering the loss of a chromosome arm by the pericentric inversion.
The variable number of linkage groups identified in the hybrid maps (28 and 29) is likely due to mixed ancestry of the source RBT stock. Linkage analysis in YCT indicated that the Omy25 and Omy29 homologues each represent acrocentric linkage groups in YCT, but these chromosomes are known to be a Robertsonian polymorphism in RBT
[28, 34, 35]. The Kamloops stock at Hayspur Hatchery appears to include ancestry from inland RBT (2 N = 58; Omy25 and Omy29 are fused as a metacentric chromosomes) as well as the common hatchery RBT derived from coastal California (2 N = 60; Omy25 and Omy29 are acrocentric chromosomes) (R. F. Leary, Montana Fish, Wildlife and Parks, personal communication). Admixture between the two RBT stocks would generate Robertsonian metacentric (2 N = 58) and acrocentric (2 N = 60) polymorphs, as well as Robertsonian heterozygotes (2 N = 59). Subsequent hybridization between the polymorphic Kamloops stock and YCT would produce F1 hybrids that were Robertsonian heterozygotes (for example, Female 2 and Male 1) comprised of 28 linkage groups, containing the RBT metacentric fusion Omy25_Omy29 and the YCT acrocentric homologues to Omy25 and Omy29, as well as Robertsonian acrocentric homozygotes (for example, Female 1 and Male 2) comprised of 29 linkage groups, containing RBT acrocentric chromosomes Omy25 and Omy28 and their YCT homologues (Figure
Conclusions based on recombination differences between the hybrid and RBT maps have limitations, as rates can vary between related species
, as well as among individuals within species
[18, 19, 24]. Indeed, we found significant differences in pairwise recombination rates between the two RBT maps
[21, 33] used to construct the female consensus map across Omy5, Omy8, Omy14, Omy19, Omy20, and Omy22. Furthermore, differences in marker density among hybrid and RBT maps could also account for recombination differences by affecting map distance estimates. However, our objective was to perform a comparative analysis to identify recombination suppression broadly across the genome. Because recombination frequency appears correlated with chromosome arm number
 and broad-scale recombination rates tend to be conserved between closely related species
, we might expect similar recombination rates between RBT and YCT. Therefore, although absolute differences between map distances can be affected by number of markers and number of individuals mapped, the trend revealed by the comparative analyses yields interesting insight into recombination suppression in the hybrids, which could indicate the presence of chromosome rearrangements or genic incompatibilities.
Chromosome rearrangements can generate recombination suppression in heterokaryotypes (chromosomal hybrids) through unbalanced gametes which results in non-recombinants being the only viable gametes
, or by restricting recombination between rearrangements
. Although unbalanced gametes cannot be ruled out, we consider them an unlikely cause of suppression in the F1 hybrids. First, conservation of synteny and marker order between the hybrid and RBT maps
[21, 33] suggests that YCT and RBT chromosomes are highly collinear, which would facilitate normal pairing between homologues and alternate disjunction in F1 hybrids. Second, because RBT and YCT contain the same number of chromosome arms, Robertsonian type rearrangements are considered to have played a significant role in generating the chromosome number differences between the species
. Meiotic pairing between homologues that differ by a Robertsonian rearrangement (i.e., pairing between a metacentric chromosome and the two acrocentric homologues) would produce a trivalent in the F1 hybrids, which would not necessarily cause malsegregation and unbalanced gametes. For example, the rate of nondisjunction was not different in individuals that were heterozygous for Robertsonian rearrangements compared to homozygotes in pink salmon (O. gorbuscha)
, house mouse (Mus musculus domesticus)
, and Eurasian common shrew (Sorex araneus)
, suggesting this type of rearrangement produces balanced gametes.
Recombination was restricted in the F1 hybrids across several loci spanning chromosome fusion and fission differences between RBT and YCT; the Robertsonian fusion/fission within RYHyb25_29 and RYHyb20. This suggests that chromosome rearrangements did indeed suppress recombination. Rearrangements generate extensive linkage disequilibrium in heterokarotypic hybrids
 and suppress recombination across genomic regions that extend beyond rearrangements
[11, 30, 43]. Therefore, broad-scale recombination suppression across other linkage groups in the F1 hybrids could indicate the presence of rearrangements. We suggest that three other metacentric hybrid linkage groups, RYHyb11, RYHyb14, and RYHyb15, contained chromosomes that differed by arrangement between RBT and YCT. These linkage groups might represent Robertsonian rearrangements because recombination was suppressed across putative centromeres which could indicate that F1 hybrids were heterozygous for centric fusions/fissions. However, we found marker order differences within the suppressed regions in each of these three linkage groups compared to RBT linkage maps
[21, 33], which could possibly indicate inversions or translocations, or, alternatively, be due to reduced mapping power in regions with low recombination or the number of progeny used to construct the various maps.
Significant differences in recombination rates between several hybrid and RBT linkage groups could indicate the presence of inter-specific, genic incompatibilities. However, our broad-scale analysis hinders inference across smaller genomic scales where incompatibilities have been reported
[44–46]. Nevertheless, we found numerous instances where two adjacent markers did not recombine in the female-merged hybrid map, but these same markers recombined in the consensus female RBT map. This might suggest that recombination between particular YCT and RBT genomic regions is maladaptive, or that the difference in adjacent recombination is an artifact of the number of offspring genotyped. Finer scale mapping across these regions that differ in recombination rate could be fruitful for identifying the presence of incompatibilities. Inter-specific incompatibilities may also be inferred from segregation distortion, as genotypes that occur less often than expected may be incompatible
[3, 14]. In addition, we expected that inter-specific incompatibilities would show consistent distortion among maps. For example, consistent allelic distortion within both RYHyb01 female hybrid maps and both RYHyb09 male hybrid maps could indicate the presence of incompatibilities. Although segregation distortion was present in several other linkage groups, a lack of consistency across hybrid maps suggests these distorted loci do not reflect genic incompatibilities between RBT and YCT.
Recombination suppression within Robertsonian rearrangements
The recombination suppression pattern across the Robertsonian rearrangement RYHyb25_29 and the presumed Robertsonian rearrangement RYHyb15 differed from patterns reported between chromosome races in the house mouse
[47, 48] and common shrew
. Recombination in mice and shrews that were homozygous for Robertsonian fusions (i.e., homozygous for the metacentric polymorphism) appeared suppressed near centromeres and elevated toward telomeres in comparison to Robertsonian heterozygotes
[39, 47]. However, we observed the opposite; recombination was suppressed in Robertsonian heterozygotes (i.e., hybrids) across the putative centromere and elevated toward telomeres in comparison to fusion homozygotes (i.e., the female RBT consensus map). The mechanisms causing the difference between our results and the house mouse and common shrew are unclear. Given the approximate 3 million year divergence time between RBT and YCT
 and the absence of historical secondary contact, genomic differences could have evolved near the centromeres of rearranged chromosomes that would restrict crossover events in Robertsonian heterozygotes, such as para- or pericentric inversions or genic incompatibilities. Alternatively, mechanistic processes governing meiotic crossover could differ among taxa. Determining how recombination suppression in the Omy25-Omy29 Robertsonian rearrangement differs between intra-specific (RBT chromosome races) and inter-specific hybrids could indicate the efficiency of this type of rearrangement as a barrier to gene flow in salmonids.
Rearrangements protect genomic regions from recombination
Our results suggest that chromosome rearrangement is the main genomic obstacle for gene exchange between RBT and YCT. Rearrangements have been observed to reduce gene flow between several species pairs, including Drosophila pseudoobscura and D. persimilis, Helianthus petiolaris and H. annuus, and S. araneus and S. antinorii[49, 50]. Rearrangements protect genomic regions from recombination, enabling genes within or closely linked to the rearranged genome to differentiate between heterokaryotypes while unrestricted gene flow occurs between regions with similar composition
[45, 51]. As a result, fitness related genes could accumulate within regions that are protected from recombination and diverge in the face of hybridization
[45, 51]. Indeed, genes involved with reproductive isolation have mapped to chromosome rearrangements
[31, 32]. The effectiveness of rearrangements as recombination suppressors may be dependent on how chromosomes are reorganized; rearrangements that change gene order (e.g., inversions or translocations) may be more effective in protecting the genome from being disrupted than rearrangements that do not change gene order (e.g., fusions or fissions). However, several studies indicate that Robertsonian type rearrangements restrict gene flow
[11, 49, 50, 52], but with extreme interbreeding this type of rearrangement may be an ineffective barrier
Given the hybrid linkage map results, we predict that, within introgressed populations, inter-specific recombination will be restricted in particular genomic regions where chromosome arrangement differs between YCT and RBT. This prediction is supported by studies that have reported reduced gene flow across chromosome rearrangements within stable hybrid zones
[10, 11, 50, 52]. Because the majority of hybrid linkage groups appeared to have similar marker order and arrangement with RBT, few chromosomes would be expected to contain regions with restricted recombination, although suppression could potentially extend broadly across these chromosomes
[11, 30, 43]. Within a long standing hybrid zone between house mouse chromosome races, linkage disequilibrium persisted among loci mapping near Robertsonian rearrangements
, indicating that these types of rearrangements can generate and maintain linkage disequilibrium. Given that RBT-YCT introgression is recent and rearrangements produce extensive linkage disequilibrium
, large genomic regions that flank centric fusion/fission differences could be expected to remain intact within some YCT chromosomes. As a consequence, recombination suppression between heterokaryotypes could protect genomic regions from being disrupted and enable co-adapted gene complexes and/or local adaptations linked to the rearrangements to persist within YCT admixed with RBT.
Segregation distortion mechanisms
The low level of segregation distortion observed in the RBT-YCT hybrid maps was unexpected. Several studies have reported high levels of segregation distortion, greater than 15% of loci at P < 0.05, within hybrid maps across a variety of taxa, including interspecific crosses between Mimulus guttatus and M. nastutus, Lepomis cyanellus and L. megalotis, and Nasonia giraulti and N. vitripennis and between intraspecific crosses of Salvelinus alpinus, M. guttatus, Ceratodon purpureus, Coregonus clupeaformis, and Tigriopus californicus. Divergence time estimates between parental lineages used for several of these maps ranged from 0.1-0.2 MYA to 13–16 MYA
[60–63]. We would expect comparable distortion levels to these studies following the estimated 3 million year divergence time between RBT and YCT. Our findings are probably not due to a lack of power for detecting distortion, because we mapped a similar number of markers per linkage group as the studies above, although the number of individuals per mapping panel was less than these studies, except Woram et al.. Nevertheless, it is intriguing that segregation distortion was limited and few consistent distortion patterns emerged between sexes and families. The low level of distortion suggests that few incompatibilities exist between RBT and YCT, and may partially explain why these species readily form hybrid swarms.
Although segregation distortion was limited, we found a few noteworthy cases. A variety of mechanisms may cause distortion
[14, 64, 65]. Understanding the causes of distortion typically requires in-depth study, but we discuss two mechanisms. First, pseudolinkage likely caused allelic distortion within two homeologous linkage groups in Male 2 (RYHyb07/RYHyb18 and RYHyb06/RYHyb27). Pseudolinkage may be implicated when homeologous linkage groups both show allelic segregation distortion
, and we observed this distortion in YCT allele frequencies. Second, meiotic drive could have caused distortion. Meiosis is asymmetric in females and results in one functional haploid gamete per germ cell, compared to males where symmetric meiosis results in four functional gametes per germ cell
. Therefore, any process that results in non-random segregation of chromosomes during oogenesis may lead to distortion in females. Female meiotic drive is characterized by competition among centromeres for spindle fiber attachment during meiosis and oogenesis
. Because the centromere on one chromosome may outcompete the centromere on the homologue for deposition into the oocyte, allele frequencies in female maps may be distorted at loci mapping near centromeres
. Male meiotic drive is characterized by competition between alleles during sperm development and results in differential gamete success. Genomic divergence between species could escalate competition between meiotic drive elements, causing segregation distortion in their hybrid progeny
. Consistent distortion patterns within RYHyb01 between female hybrid maps and within RYHyb09 between male hybrid maps suggest that meiotic drive could be acting within the hybrids.
Conservation of synteny with the RBT sex chromosome, Omy1, suggests that RYHyb28 is the sex-linkage group. Indeed, sex has been established as mapping to homologs in YCT and RBT
. Conservation of sex-linkage groups is uncommon across several salmonid species
[20, 69–71]. Two alternative mechanisms have been proposed to account for the lack of conservation among sex-linkage groups in salmonids: either the sex-determining gene is the same among species but has moved to different chromosomes in different lineages, or sex determination has evolved independently in different lineages
. Regardless of the mechanism, the lack of homology among sex chromosomes could be an important factor for restricting inter-specific hybridization, and could explain why hybridization is not commonly observed between many sympatric salmonid species and, conversely, why two species pairs identified as having homologous sex-linkage regions, YCT and RBT
 and Arctic charr (S. alpinus) and brook charr (S. fontinalis)
, introgress in the wild
[73, 74]. The relationship between conservation of sex chromosomes and introgression is confounded in brown trout (Salmo trutta) and Atlantic salmon (S. salar) because their sex chromosomes lack homology
, yet the species hybridize in the wild
. However, brown trout and Atlantic salmon hybrids suffer reduced viability
 and introgression is rare
. Nevertheless, homology between sex-linkage groups could possibly facilitate introgression between RBT and YCT.