Identification of the sex-determining locus of Atlantic salmon (Salmo salar) on chromosome 2

We have integrated data from linkage mapping, physical mapping and karyotyping to gain a better understanding of the sex-determining locus, SEX, in Atlantic salmon (Salmo salar). SEX has been mapped to Atlantic salmon linkage group 1 (ASL1) and is associated with several microsatellite markers. We have used probes designed from the flanking regions of these sex-linked microsatellite markers to screen a bacterial artificial chromosome (BAC) library, representing an 11.7x coverage of the Atlantic salmon genome, which has been HindIII fingerprinted and assembled into contigs. BACs containing sex-linked microsatellites and their related contigs have been identified and representative BACs have been placed on the Atlantic salmon chromosomes by fluorescent in situ hybridization (FISH). This identified chromosome 2, a large metacentric, as the sex chromosome. By positioning several BACs on this chromosome by FISH, it was possible to orient ASL1 with respect to chromosome 2. The region containing SEX appears to lie on the long arm between marker Ssa202DU and a region of heterochromatin identified by DAPI staining. BAC end-sequencing of clones within sex-linked contigs revealed five hitherto unmapped genes along the sex chromosome. We are using an in silico approach coupled with physical probing of the BAC library to extend the BAC contigs to provide a physical map of ASL1, with a view to sequencing chromosome 2 and, in the process, identifying the sex-determining gene.     

Recombination is suppressed over a large region of the rainbow trout Y chromosome

The previous genetic mapping data have suggested that most of the rainbow trout sex chromosome pair is pseudoautosomal, with very small X-specific and Y-specific regions. We have prepared an updated genetic and cytogenetic map of the male rainbow trout sex linkage group. Selected sex-linked markers spanning the X chromosome of the female genetic map have been mapped cytogenetically in normal males and genetically in crosses between the OSU female clonal line and four different male clonal lines as well as in outcrosses involving outbred OSU and hybrids between the OSU line and the male clonal lines. The cytogenetic maps of the X and Y chromosomes were very similar to the female genetic map for the X chromosome. Five markers on the male maps are genetically very close to the sex determination locus ( SEX ), but more widely spaced on the female genetic map and on the cytogenetic map, indicating a large region of suppressed recombination on the Y chromosome surrounding the SEX locus. The male map is greatly extended at the telomere. A BAC clone containing the SCAR (sequence characterized amplified region) Omy - 163 marker, which maps close to SEX , was subjected to shotgun sequencing. Two carbonyl reductase genes and a gene homologous to the vertebrate skeletal ryanodine receptor were identified. Carbonyl reductase is a key enzyme involved in production of trout ovarian maturation hormone. This brings the number of type I genes mapped to the sex chromosome to six and has allowed us to identify a region on zebrafish chromosome 10 and medaka chromosome 13 which may be homologous to the distal portion of the long arm of the rainbow trout Y chromosome.     

Sex chromosome polymorphisms in Arctic charr and their evolutionary origins

Current data on the Y-specific sex-determining region of salmonid fishes from genera Salvelinus, Salmo, and Oncorhynchus indicate variable polymorphisms in the homologous chromosomal locations of the sex-specific determining region. In the majority of the Atlantic lineage Arctic charr, including populations from the Fraser River, in Labrador Canada, as well as Swedish and Norwegian strains, the sex-determining locus maps to linkage group AC-4. Previously, sex-linked polymorphisms (i.e., variation in the associated sex-linked markers on AC-4) have been described in Arctic charr. Here, we report further evidence for intraspecific sex linkage group polymorphisms in Arctic charr (i.e., the detection of the SEX locus on either the AC-1 or AC-21 linkage group) and a possible conservation of a sex linkage arrangement in Icelandic Arctic charr and Atlantic salmon, involving sex-linked markers on the AC-1/21 homeologs and the European AS-1/6 homeologous linkage groups in Atlantic salmon. The evolutionary origins for the multiple sex-determining regions within the salmonid family are discussed. We also relate the variable sex-determining regions in salmonids to their ancestral proto-teleost karyotypic origins and compare these findings with what has been observed in other teleost species in general.     

Characterization of a chromosomal rearrangement responsible for producing "apparent" XY-female fall-run Chinook salmon in California

Fluorescence in situ hybridization (FISH) was used to identify the X and Y chromosomes of offspring produced by normal and "apparent" XY-female fall-run Chinook salmon (Oncorhynchus tshawytscha) from California. FISH experiments were performed using probes to 2 sex-linked loci, growth hormone pseudogene (GH-Psi), and OtY1, as well as a probe to a sex-linked microsatellite (Omy7INRA). Comparison of FISH staining patterns between the offspring produced by normal and apparent XY-females revealed that the apparent XY-female examined transmitted a "Y-like" chromosome with an attenuated OtY1 and GH-Psi signal to half of its offspring. Segregation analysis of microsatellites derived from rainbow trout (Oncorhynchus mykiss) with respect to phenotypic sex was carried out for 2 normal and 2 apparent XY-female Chinook salmon families. Inheritance patterns of Omy7INRA were consistent with this locus being closely linked to GH-Psi in males and in apparent XY-females carrying the Y-like chromosome. Another microsatellite locus (Omm1077) was closely linked to the primary sex-determining locus (SEX) in males but not to GH-Psi/OtY1 in apparent XY-females. The FISH analyses suggest that apparent XY-female fall-run Chinook salmon in California are not the product of a Y chromosome to autosome translocation. Despite the combined FISH and inheritance analyses, we were unable to differentiate between 2 alternative explanations for apparent XY-females, namely, recombination of markers between the sex chromosomes, or a Y chromosome with a dysfunctional or missing sex-determining region.     

Determination of Genetic Sex in Chinook Salmon (<Emphasis Type="Italic">Oncorhynchus tshawytscha</Emphasis>) Using the Male-Linked Growth Hormone Pseudogene by Real-Time PCR

This study used a real-time quantitative polymerase chain reaction (qPCR) method based on the growth hormone pseudogene (GHp) in chinook salmon (Oncorhynchus tshawytscha) to determine genetic sex. The GHp is present as a single copy in the genome of the male chinook salmon but is absent in the female, providing a means of using this real-time qPCR method to discriminate genetic sex. Comparisons between genomic DNA samples from 2 geographically distinct populations of chinook salmon (Columbia River, Washington, and Yukon River, Alaska) showed, within each population examined, that the males were clearly differentiated from the females. There were no interpopulation differences between males or females. The advantages of this real-time qPCR method are that it is rapid, is amenable to high sample throughput, and provides an accurate numerical value that allows comparisons between samples by statistical methods.     

Effects of a small X-ray exposure to Drosophila melanogaster females on the recovery of genetic damage from X-irradiated males.

Wild-type (Oregon-K) Drosophila melanogaster males were X-irradiated and mated to Oster females (y sc^s1 In49sc⁸; bw; st p^p) that had received a 20 R X-ray exposure (Group MF) or no irradiation (group M). Mature spermatozoa of the irradiated males were sampled and the frequencies of dominant lethals, sex-linked recessive lethals and 2–3 translocations were measured. In the group in which the irradiated males were mated to irradiated females, the survival of eggs was significantly higher than in the group in which only the males were irradiated. However, there was no consistent and detectable difference between the two groups with respect to the frequencies of recessive lethals and translocations.The relatively higher egg survival in the MF group is amenable to an interpretation based on an inducible repair process in females that acts on radiation damage induced in spermatozoa but, such an explanation is inadequate to explain the other results. It is concluded that the observations considered together preclude a general and unifying interpretation based on a low-dose-X-ray-inducible genetic repair process in females (acting on damage in spermatozoa). Possible reasons for the discrepancy between the expectation of differences in response between the MF and M groups (in sex-linked lethal and translocation frequencies) and the observation of no consistent differences between them are discussed.     

Transition in sexual system and sex chromosome evolution in the tadpole shrimp Triops cancriformis

Transitions in sexual system and reproductive mode may affect the course of sex chromosome evolution, for instance by altering the strength of sexually antagonistic selection. However, there have been few studies of sex chromosomes in systems where such transitions have been documented. The European tadpole shrimp, Triops cancriformis, has undergone a transition from dioecy to androdioecy (a sexual system where hermaphrodites and males coexist), offering an excellent opportunity to test the impact of this transition on the evolution of sex chromosomes. To identify sex-linked markers, to understand mechanisms of sex determination and to investigate differences between sexual systems, we carried out a genome-wide association study using restriction site-associated DNA sequencing (RAD-seq) of 47 males, females and hermaphrodites from one dioecious and one androdioecious population. We analysed 22.9 Gb of paired-end sequences and identified and scored >3000 high coverage novel genomic RAD markers. Presence–absence of markers, single-nucleotide polymorphism association and read depth identified 52 candidate sex-linked markers. We show that sex is genetically determined in T. cancriformis, with a ZW system conserved across dioecious and androdioecious populations and that hermaphrodites have likely evolved from females. We also show that the structure of the sex chromosomes differs strikingly, with a larger sex-linked region in the dioecious population compared with the androdioecious population.     

New microsatellite markers for examining genetic variation in peripheral and core populations of the Coastal Giant Salamander (Dicamptodon tenebrosus)

The Coastal Giant Salamander (Dicamptodon tenebrosus) is classified as threatened at the northern periphery of its range in British Columbia (BC), Canada, primarily due to forestry practices and habitat fragmentation. Characterising dispersal behaviour and population connectivity is therefore a priority for this region, while genetic differentiation in core versus peripheral locations remains unstudied in this wide-ranging species. We present seven new polymorphic microsatellite markers for use in population genetic analyses of D. tenebrosus. We examine locus characteristics and genetic variation in 12 streams at the species' northern range limit in BC, and within two regions representing sub-peripheral (North Cascades) and core localities (South Cascades) in Washington State, United States. In BC, the number of alleles per locus ranged from 2-5 and observed heterozygosity ranged from 0.044-0.825. Genetic differentiation was highest between BC and the South Cascades, and intermediate between BC and the North Cascades. Across loci, mean allelic richness was similar across regions, while private allelic richness was highest in the core locality (corrected for sample size). These new microsatellite loci will be a valuable addition to existing markers for detailed landscape and population genetic analyses of D. tenebrosus across its range.     

Sex-linked control of sex pheromone behavioral responses in European corn-borer moths (Ostrinia nubilalis) confirmed with TPI marker gene

In two races of European corn-borer moths (ECB), the E-race females emit and males respond to 99:1 sex pheromone blend of (E)/(Z)-11-tetradecenyl acetates, whereas the Z-race females and males produce and respond to the opposite 3:97 pheromone blend of (E)/(Z)-11-tetradecenyl acetates, respectively. We previously have shown that female production of the final blend ratio is under control of a major autosomal locus but that the sequence of male upwind flight responses to the blend is controlled by a sex-linked (Z-linked) locus. This sex-linked control of behavioral responses in crosses of E and Z ECB now is confirmed by use of sex-linked TPI (triose phosphate isomerase) allozyme phenotypes to determine the origin of the sex chromosomes in F₂ populations. F₁ males from reciprocal E × Z crosses generate similar behavioral-response profiles in wind-tunnel studies, with moderate numbers responding to the Z pheromone and intermediate blends (35%–65% Z), but very few responding to the E pheromone. The F₂ behavioral-response profiles indicate that they are composed of 1:1 mixtures of hybrids and paternal profiles. Analysis of TPI allozyme differences allowed us to separate male F₂ populations into individuals whose Z chromosomes both originated from their grandfathers, and individuals who had one Z chromosome originating from each grandparent. With these partitioned F₂s, the TPI homozygotes exhibited behavioral-response profiles very much like their grandfathers, whereas the TPI hybrids produced response profiles similar to their heterozygous F₁ fathers. These results demonstrate incontrovertibly that the response to sex pheromone in male ECB is controlled by a sex-linked gene that is tightly linked to the TPI locus and therefore is independent of the locus controlling pheromone blend production in females.     

Genetic mapping of sex-linked markers in Salix viminalis L

A total of 88 selective primer combinations were screened using bulked males and females sampled from four families of Salix viminalis. A total of more than 1000 polymorphic fragments was obtained, of which only four cosegregated with sex. These four sex-linked markers were subsequently scored in individuals that were used for bulked sample preparation in additional individuals of the same families, and in individuals in other families. A pair of primers that amplified the sex-linked fragments was constructed from one of the sex-linked amplified fragment length polymorphism (AFLP) fragments. In hybridization of Southern blot filters with the sex-linked DNA fragments, the band was present in females and absent in males, but the opposite pattern of band segregation (a band found in males and no band in females) was never observed in either the AFLP or RFLP experiments. Two of the sex-linked markers were placed on a linkage map. They both map at the same location in a linkage group comprising other markers not segregating with sex. Our data suggest that a single locus governs the sex determination and that nonrecombining sex chromosomes are absent in S. viminalis. A close association was found between skewed sex ratio and segregation distortion at this locus.     

Population Structure and Stock Identification of Steelhead Trout (Oncorhynchus mykiss) in British Columbia and the Columbia River Based on Microsatellite Variation

Variation at 13 microsatellite loci was surveyed from ～3 800 steelhead trout, Oncorhynchus mykiss, from 51 populations in British Columbia, Washington, and the Columbia River drainage. Mean F_ST over all 13 loci and 51 populations was 0.066. Regional structuring of populations was apparent, with Thompson River, upper Fraser River, and Columbia River populations forming distinct groups. In the Nass River, winter-run populations were distinct from the summer-run populations. Significant differences in allele frequencies were observed among regional stock groups at all loci. Analysis of variance components indicated that 5.7% of the total observed variation was distributed among 11 regions, and 2.3% of the variation was among populations within regions. Analysis of simulated mixed-stock samples suggested that variation at the microsatellite loci provided relatively accurate and precise estimates of stock composition for fishery management applications, and this was confirmed by application to actual fishery samples of known origin. Within the Fraser River drainage, individual steelhead trout can be identified to one of the three regions of origin with an accuracy of 94–97%. Microsatellites provided an effective way to determine population structure, and provided reliable estimates of stock composition in mixed-stock fisheries.     

Development and linkage mapping of novel sex-linked markers for marker-assisted cultivar breeding in pistachio (<Emphasis Type="Italic">Pistacia vera</Emphasis> L.)

The dioecious character of Pistacia vera L (the pistachio tree) limits its breeding capacity. Thus, early stage selection of males can save time, labor, and land. This study aimed to develop sex-linked single nucleotide polymorphism (SNP) markers, together with expressed sequence tag-derived simple sequence repeats (EST-SSRs), to determine position of the sex locus in pistachio by constructing a linkage map of its sex chromosome for the first time. Nine novel sex-linked SNP markers were successfully identified by SNaPshot minisequencing analysis of 25 SNP loci from 17 restriction site-associated DNA (RAD) reads in 309 individuals. All nine markers were heterozygous in females and homozygous in males supporting a ZW/ZZ sex determination system in pistachio. A total of 105 segregating SSRs and sex-linked markers were used to identify the sex chromosome and the position of the sex locus through analysis of a Siirt × Ba?yolu F₁ population with 122 progenies. Of these 105 markers, four common and four paternal SSRs were mapped onto the sex chromosome, along with the phenotypic sex locus and sex-linked markers. The resulting consensus map had a total length of 65.19 cM. The sex locus and sex-linked SNP markers were located in the center of the chromosome at a distance of 31.86 and 31.92 cM, respectively. This study presents valuable information about the sex chromosome and sex locus position as well as novel polymorphic EST-SSRs and nine sex-linked SNP markers in pistachio.     

Forensic identification of endangered Chinook Salmon (Oncorhynchus tshawytscha) using a multilocus SNP assay

Upper Columbia River spring-run Chinook salmon are listed as endangered under the Endangered Species Act (ESA). Forensic genetic analyses are needed to assist the National Oceanic and Atmospheric Administration Fisheries Office of Law Enforcement (NOAA Fisheries OLE) during criminal investigations of ESA take violations. Previous genetic studies using allozyme markers have demonstrated that the spring-run and summer-run of Chinook salmon in the Upper Columbia River are genetically differentiated. Because many of the carcasses collected as forensic evidence are of compromised quality, we have developed a PCR based assay to distinguish between the spring-run and the summer-run Chinook salmon in the upper Columbia River Basin. A total of 347 Chinook salmon samples from the upper Columbia River in Washington State were surveyed for single nucleotide polymorphisms (SNPs) using PCR-RFLP at two nuclear genetic loci (Somatolactin and Cytochrome p450A), and a mitochondrial locus (Cytochrome Oxidase III/ND3). We found near fixed differences in our SNP baseline between the summer-run and the spring-run Chinook salmon in the upper Columbia River at these loci enabling us to assign individuals to the most likely population of origin with a high degree of accuracy.     

Short tandem repeat polymorphism in the flanking region of the human phosphoglycerate kinase gene in a Japanese population

The human phosphoglycerate kinase (PGK1) gene is located within Xqll-Xql3 and is closely linked to the androgen receptor gene within a region implicated in a number of X-chromosome-linked urologic disorders. A polymorphism of a TATC short tandem repeat (STR) is present downstream from the PGK1 3' nuclease-sensitive site. We present the PGK1 flanking STR sequence and population genetic data for 190 Japanese males and 83 Japanese females. Ten STR alleles and 29 genotypes were identified in the population. Five alleles--*10, *11, *12, *13, and *14--were common in the Japanese with frequencies greater than 10%. No significant deviations from Hardy-Weinberg equilibrium were established. The power of discrimination was 0.993 for females and 0.819 for males; heterozygosity was 0.759 for females; and the polymorphic information content was 0.936. These data indicate that this STR locus shows a high degree of polymorphism in this Japanese population and may prove to be a useful genetic marker in forensic medicine, in determining the clonality of neoplasms, and potentially in studying predisposition to prostate cancer and other urologic diseases.     

The effective number of a population that varies cyclically in size. I. Discrete generations

We consider a dioecious population having numbers of males and females that vary over time in cycles of length k. It is shown that if k is small in comparison with the numbers of males and females in any generation of the cycle, the effective population number (or size), N(e), is approximately equal to the harmonic mean of the effective population sizes during any given cycle. This result holds whether the locus under consideration is autosomal or sex-linked and whether inbreeding effective population numbers or variance effective population numbers are involved in the calculation of N(e). If, however, only two successive generations in the cycle are considered and the population changes in size between these generations, the inbreeding effective population number, N(eI), differs from the variance effective population number, N(eV). The mutation effective population number turns out to be the same as the number derived using calculations involving probabilities of identity by descent. It is also shown that, at least in one special case, the eigenvalue effective population number is the same as N(eV).     

Electrophoretic polymorphism and sexual dimorphism in the freshwater and anadromous threespine sticklebacks (Gasterosteus aculeatus) of the Little Campbell River,British Columbia

We used single-family crosses to confirm the Mendelian interpretation of allozyme variation and to examine linkage relationships at five polymorphic loci in freshwater and anadromous threespine sticklebacks (Gasterosteus aculeatus) from the Little Campbell River, British Columbia. The electrophoretic pattern of a sixth locus, mitochondrial NADP-dependent isocitrate dehydrogenase (IDH), was found to be sexually dimorphic but otherwise invariant in sticklebacks. This raised the possibility of a sex-linked Idh locus. No differences in IDH quantity or substrate affinities (apparent Km values) were detected between male and female sticklebacks. The electrophoretic pattern of IDH expression was not changed in sticklebacks in which sexual development was altered by hormonal treatment.     

A sex-associated sequence identified by RAPD screening in gynogenetic individuals of turbot (Scophthalmus maximus)

Understanding the genetic basis of sex determination mechanisms is essential for improving the productivity of farmed aquaculture fish species like turbot (Scophthalmus maximus). In culture conditions turbot males grow slower than females starting from eight months post-hatch, and this differential growth rate is maintained until sexual maturation is reached, being mature females almost twice as big as males of the same age. The goal of this study was to identify sex-specific DNA markers in turbot using comparative random amplified polymorphism DNA (RAPD) profiles in males and females to get new insights of the genetic architecture related to sex determination. In order to do this, we analyzed 540 commercial 10-mer RAPD primers in male and female pools of a gynogenetic family because of its higher inbreeding, which facilitates the detection of associations across the genome. Two sex-linked RAPD markers were identified in the female pool and one in the male pool. After the analysis of the three markers on individual samples of each pool and also in unrelated individuals, only one RAPD showed significant association with females. This marker was isolated, cloned and sequenced, containing two sequences, a microsatellite (SEX01) and a minisatellite (SEX02), which were mapped in the turbot reference map. From this map position, through a comparative mapping approach, we identified Foxl2, a relevant gene related to initial steps of sex differentiation, and Wnt4, a gene related with ovarian development, close to the microsatellite and minisatellite markers, respectively. The position of Foxl2 and Wnt4 was confirmed by linkage mapping in the reference turbot map.     

Sex determination in the androdioecious plant Datisca glomerata and its dioecious sister species D. cannabina.

Datisca glomerata is an androdioecious plant species containing male and hermaphroditic individuals. Molecular markers and crossing data suggest that, in both D. glomerata and its dioecious sister species D. cannabina, sex is determined by a single nuclear locus, at which maleness is dominant. Supporting this conclusion, an amplified fragment length polymorphism (AFLP) is heterozygous in males and homozygous recessive in hermaphrodites in three populations of the androdioecious species. Additionally, hermaphrodite x male crosses produced 1:1 sex ratios, while hermaphrodite x hermaphrodite crosses produced almost entirely hermaphroditic offspring. No perfectly sex-linked marker was found in the dioecious species, but all markers associated with sex mapped to a single linkage group and were heterozygous in the male parent. There was no sex-ratio heterogeneity among crosses within D. cannabina collections, but males from one collection produced highly biased sex ratios (94% females), suggesting that there may be sex-linked meiotic drive or a cytoplasmic sex-ratio factor. Interspecific crosses produced only male and female offspring, but no hermaphrodites, suggesting that hermaphroditism is recessive to femaleness. This comparative approach suggests that the hermaphrodite form arose in a dioecious population from a recessive mutation that allowed females to produce pollen.     

No gender differences in the frequencies of HLA-DRB3/B4/B5 heterozygotes in newborns and adults in Koreans

HLA class II haplotypes often contain a second expressed HLA-DRB locus tightly linked to the classical HLA-DRB1 locus on the haplotype, which can be either HLA-DRB3, -DRB4 or -DRB5. These encode the HLA-DR51, -DR52 or -DR53 supertypic specificities and mark the ancestral lineages. HLA-DRB3/B4/B5 heterozygote excess in Welsh male newborns has been reported, suggesting a possibility of male-specific major histocompatibility complex (MHC)-mediated prenatal selection. However, it has not been confirmed in newborns of other ethnic groups or in adult populations. We analyzed the HLA-DRB1 and HLA-DRB3/B4/B5 genes in Korean newborns and healthy adults to examine whether MHC-mediated prenatal or postnatal selection exists. A total of 1,038 newborns (cord blood registry, 516 males and 522 females) and 2,082 healthy adults (hematopoietic stem cell donor registry, 1,111 males and 971 females) were HLA typed. HLA-DRB1/B3/B4/B5 DNA typing was performed using Dynal RELI HLA-DRB SSO Kit (Dyanl Biotech, Wirral, U.K.). Genotype frequencies and homozygosity and heterozygosity rates for DRB3/B4/B5 supertypic loci were compared between males and females in newborns and adults. There were no significant differences in the HLA-DRB3/B4/B5 homozygosity and heterozygosity rates between males and females in both newborns and adults. In the comparison between newborns and adults, homozygosity rate was significantly higher in newborn females than in adult females (31.0% vs 25.0%, p=0.01). Whether there is an age-related change from newborns toward adults has not been well studied in other populations, and further studies are warranted. In conclusion, male-specific heterozygosity excess reported in Welsh newborns has not been observed in Korean population, and there might be some ethnic differences in the gender-specific prenatal selection events.     

Rapid evolutionary change in a sexual signal: genetic control of the mutation 'flatwing' that renders male field crickets (Teleogryllus oceanicus) mute

Colonizing events may expose organisms to physical and ecological environments found nowhere else in their range. Novel selection pressures can then influence subsequent rapid evolutionary changes. Here, I investigate the genetics of one such rapid change in the sexual signal of Polynesian field crickets, Teleogryllus oceanicus, that recently colonized the Hawaiian Islands. In Hawaii, T. oceanicus encounter a deadly parasitoid fly found nowhere else in their range. In <20 generations, a wing mutation, flatwing, that eliminates the crickets' song, an important sexual signal, but protects them from the fly, spread to >90% of males on the island of Kauai. I show, using crosses between flatwing males and females from a population that has never contained flatwings, that the song-suppressing mutation is due to a change in a single sex-linked locus. Contemporary evolution of secondary sexual characteristics has only rarely been identified as the result of single-gene changes and never before as a single sex-linked locus, but sex-linked inheritance is thought to facilitate the rapid evolution of these types of traits. Because divergence of sexual signals can influence reproductive isolation, understanding how colonization events and subsequent selection affect signals, and the genetic mechanisms of such change, can shed light on processes likely to play a role in speciation.