Reproductive isolation in a nascent species pair is associated with aneuploidy in hybrid offspring.

Speciation may occur when the genomes of two populations accumulate genetic incompatibilities and/or chromosomal rearrangements that prevent inter-breeding in nature. Chromosome stability is critical for survival and faithful transmission of the genome, and hybridization can compromise this. However, the role of chromosomal stability on hybrid incompatibilities has rarely been tested in recently diverged populations. Here, we test for chromosomal instability in hybrids between nascent species, the 'dwarf' and 'normal' lake whitefish (Coregonus clupeaformis). We examined chromosomes in pure embryos, and healthy and malformed backcross embryos. While pure individuals displayed chromosome numbers corresponding to the expected diploid number (2n = 80), healthy backcrosses showed evidence of mitotic instability through an increased variance of chromosome numbers within an individual. In malformed backcrosses, extensive aneuploidy corresponding to multiples of the haploid number (1n = 40, 2n = 80, 3n = 120) was found, suggesting meiotic breakdown in their F1 parent. However, no detectable chromosome rearrangements between parental forms were identified. Genomic instability through aneuploidy thus appears to contribute to reproductive isolation between dwarf and normal lake whitefish, despite their very recent divergence (approx. 15-20 000 generations). Our data suggest that genetic incompatibilities may accumulate early during speciation and limit hybridization between nascent species.

RNA-seq reveals transcriptomic shock involving transposable elements reactivation in hybrids of young lake whitefish species.

Identifying the molecular basis of reproductive isolation among diverging lineages represents an essential step toward understanding speciation in natural populations. Postzygotic barriers can lead to hybrid breakdown, a syndrome that has been documented in several systems, potentially involving the reactivation of transposable elements. In northeastern North America, two lake whitefish lineages have repeatedly colonized postglacial lakes ~12,000 years ago, and a dwarf limnetic species has evolved multiple times from the normal benthic species. Reproductive isolation is incomplete between them; viable hybrids can be generated in the laboratory but significant mortality occurs and is associated with a malformed phenotype in backcross embryos, thus revealing a hybrid breakdown syndrome. By means of RNA-seq analyses, the objective of this study was to determine which genes were misregulated in hybrids and rigorously test the hypothesis of transposable element reactivation. We compared the transcriptomic landscape in pure embryos, F1-hybrids, and healthy and malformed backcrosses at the late embryonic stage. Extensive expression differences consistent with previously documented adaptive divergence between pure normal and dwarf embryos were identified for the first time. Pronounced transcriptome-wide deregulation in malformed backcrosses was observed, with over 15% of transcripts differentially expressed in all comparisons, compared with 1.5% between pure parental forms. Convincing evidence of transposable elements and noncoding transcripts reactivation in malformed backcrosses is presented. We propose that hybrid breakdown likely results from extensive genomic incompatibilities, plausibly encompassing transposable elements. Combined with previous studies, these results reveal synergy among many reproductive barriers, thus maintaining divergence between these two young whitefish species.

Internal exposure of whitefish (Coregonus lavaretus) to estrogens.

Gonad malformations have been found in fish all over the world. Particularly in Lake Thun (Switzerland) a high prevalence of gonad deformations in whitefish has been observed. Very often, a link between exposure to endocrine disrupting compounds and altered gonad morphology exists. Hence, we analyzed the estrogenic burden in bile and muscle from whitefish (coregonids) from Lake Thun and linked it to observed gonad malformations. Estrogenicity in bile, measured with the yeast estrogen screen (YES) was exclusively caused by the natural steroids estrone and 17beta-estradiol. Estrogenicity determined in muscle tissue using YES was similar in cases and controls, and between the sexes. Furthermore, endocrine active compounds in the lake water were investigated using passive sampling devices to monitor tributaries and the main outflow of Lake Thun. Here, we found accumulated estrogenicity. With target chemical analysis small amounts of estrone and bisphenol A were determined. We conclude, that the whitefish from Lake Thun are not suffering from (xeno)estrogens. The present study contributed substantially to the search for the cause for gonad malformations in Lake Thun whitefish, even though the cause of the malformations remains yet to be discovered.

Long-term estrogen exposure of whitefish Coregonus lavaretus induces intersex but not Lake Thun-typical gonad malformations.

A high prevalence of gonad morphological variations has been observed in whitefish Coregonus lavaretus from Lake Thun (Switzerland). To clarify the role of endocrine disruption as a possible cause of the gonad alterations, whitefish were reared in a long-term laboratory experiment under exposure to 17 beta-estradiol (E2). Fish were fed from first-feeding until 3 yr of age at a daily rate of 0 (control), 0.5 or 50 microg E2 kg(-1) fish. E2 exposure resulted in a time- and concentration-dependent increase of prevalence and intensity of intersex gonads, i.e. gonads that macroscopically appeared as either testis or ovary but microscopically contained both male and female germ cells. Four types of intersex could be distinguished: Types 1 and 2 were composed of mainly male tissue, with Type 1 containing single oocytes and Type 2 displaying an ovary-like lamellar structure of the tissue. In Type 3, an increased percentage of the tissue was occupied by female germ cells, while in Type 4, the majority of the gonad tissue consisted of female germ cells. Chronic E2 exposure additionally resulted in a concentration-dependent shift of the sex ratio towards females, a reduced condition factor, retarded gonad growth together with delayed maturation of germ cells, and elevated levels of hepatic vitellogenin mRNA. However, Lake Thun-typical alterations of gonad morphology were not induced by chronic E2 exposure. The results provide evidence that estrogen-active compounds unlikely play a role in the etiology of gonad malformations in Lake Thun whitefish.

Unexplained gonad alterations in whitefish (Coregonus spp.) from Lake Thun, Switzerland: levels of persistent organic pollutants in different morphs.

Since 2000, a surprisingly high number of macroscopical gonad alterations has been reported in whitefish (Coregonus spp.) from Lake Thun, Switzerland. This unique phenomenon is still unexplained and has received much public attention. As one possible trigger for these effects, the presence of persistent, bioaccumulative and toxic compounds acting as endocrine disruptors in the lake has been discussed. In this study, concentrations of selected persistent organic pollutants were examined in two morphs of whitefish from Lake Thun and their link to the observed abnormalities was investigated. Analyzed compound classes included polychlorinated biphenyls, polychlorinated dibenzo-p-dioxins and dibenzofurans, polychlorinated naphthalenes, polybrominated diphenyl ethers and hexabromocyclododecanes. The target substances were identified in all samples and concentrations of the analyzed compounds were highly correlated among each other. These correlations show that the analyzed substances have the same distribution pattern throughout the lake and that uptake, accumulation and elimination processes are similar. Significant differences in contaminant levels within the samples existed between the two analyzed morphs of whitefish, most likely due to different age, food patterns and growth rate. No difference in contaminant levels was observed between fish with abnormal gonads and fish with normal gonads, suggesting no causal link between the investigated lipophilic organohalogen compounds present in fish and the observed gonad abnormalities in whitefish from Lake Thun. A comparison to existing data shows that concentrations in Lake Thun whitefish are at the lower bound of contaminant levels in whitefish from Swiss lakes or from European waters.

How normal is abnormal? Discrimination between deformations and natural variation in gonad morphology of European whitefish Coregonus lavaretus.

The gonad morphology of whitefish Coregonus lavaretus collected in Lake Thun, Switzerland, and two neighbouring lakes was assessed in order to differentiate between 'normal' and 'abnormal' character states of gonad morphology, which had been previously described in C. lavaretus from Lake Thun (constrictions, asymmetries, aplasia, compartmentations, fusions and hermaphroditism). In total, 4668 fish were collected and analysed using two complementary sampling schemes: (1) monthly samples of catches by the commercial fishermen and (2) samples of ripe spawners of all known 33 spawning sites of the three lakes. Considerable variation in gonad morphology in C. lavaretus populations of all lakes was found. Notably, all deviation types were observed in fish of all three lakes. Asymmetries and constrictions were frequent in all three lakes and showed systematic differences in frequency between the two sampling strategies. This indicates that asymmetries and constrictions represent to a large extent natural variation in gonad morphology of C. lavaretus and are also prone to considerable measurement error. In contrast, aplasia, fusions, compartmentations and hermaphroditism occurred predominantly in one C. lavaretus form of Lake Thun and in particular in populations spawning at great depths. This suggests that these deviation types are probably reliable indicators for gonad deformations and supports the interpretation that Lake Thun harbours a unique case of deformed gonads in C. lavaretus of yet unknown origin.

Gonadal alterations in male whitefish Coregonus fatioi: no evidence for genetic damage reducing viability in early life stages.

In recent years, numerous cases of morphological gonadal alterations in fish have been recorded throughout the world and across a wide range of species. In the whitefish Coregonus fatioi from the pre-alpine Lake Thun (Switzerland), the frequency of gonadal alterations is particularly high and the variety of alteration types large. Little is known about the proximal causes and the direct consequences of these morphological features on population persistence. In particular, the potential for the observed alterations to be the phenotypic expression of reduced genetic quality has not yet been addressed. In this study, we used offspring survival during embryogenesis as a proximate indicator of male genetic quality and tested whether the presence of gonadal alterations in males is an indicator of reduced quality. Embryos resulted from in vitro fertilizations of gametes from 126 males and females. We found no significant correlation between embryo survival and gonadal alteration in adults. Our findings suggest that in C. fatioi of Lake Thun, alterations in gonad morphology are not a phenotypic expression of variation in genetic quality.

Changes in the expression of genetic characteristics across cohorts in skeletal deformations of farmed salmonids.

Genetic analysis of disorder incidence in farmed animals is challenged by two factors. Disorders in different cohorts and environments could be caused by different factors, leading to changes in heritability and to less than unity genetic correlations across cohorts. Moreover, due to computational limitations, liability scale heritabilities at very low incidence may differ from those estimated at higher incidence. We tested whether these two dilemmas occur in skeletal deformations of farmed salmonids using multigeneration data from the Finnish rainbow trout breeding programme and previous salmonid studies. The results showed that heritability was close to zero in cohorts in which management practices maintained incidence at a low level. When there was a management failure and incidence was unusually high, heritability was elevated. This may be due to computational limitations at very low incidence and/or because deformations are induced by different factors in different cohorts. Most genetic correlations between deformations recorded in different generations were weakly to strongly positive. However, also negative correlations between generations were present, showing that high liability at one time can be genetically connected to low liability at another time. The results emphasise that genetic architecture of binary traits can be influenced by trait expression.

Frequent and unexplained gonadal abnormalities in whitefish (central alpine Coregonus sp.) from an alpine oligotrophic lake in Switzerland.

The occurrence of a large number of whitefish with morphologically altered gonads in Lake Thun (Switzerland) has been reported by commercial fishermen since the year 2000. Macroscopic and microscopic examination of gonads revealed that 35% (281 out of 808) of whitefish from Lake Thun were affected. Frequency of gonadal abnormalities varied significantly with sex, age and ecotype of whitefish. Apparent females (26% [106/408]) showed significantly less gonadal alterations than apparent males (40% [151/225]). Fish with deformed gonads were observed in all age classes from 1 to 6 yr, but were most frequent in the year classes 3+ to 5+. The gonadal alterations showed different frequencies among the 3 ecotypes of whitefish living in the lake: 1 ecotype was less affected (26%) than the other 2 forms (41% and 32%, respectively). The gonadal alterations included adhesions/fusions to the peritoneal wall and the lateral trunk musculature (overall: 5%; in females: 5%; in males: 5%), asymmetry (4%; 6%; 4%), atrophy (4%; 6%; 1%), compartmentations (11%; 4%; 18%), constrictions (3%; 1%; 7%) and hermaphroditism (1.1% of sampled fish, and 10 additional specimens collected by fishermen during the filleting process). In some cases, more than one alteration was observed in the same fish. Hermaphroditism manifested as 3 different types: (1) the lobular type, in which a consecutive sequence of testicular and ovarian lobes are found on the same gonad strand; (2) discrete gonads within one fish, with one gonad strand being ovarian tissue and the other being testicular tissue; and (3) the mosaic type, in which oocytes lie in tissue with normal testicular morphology, or spermatids or sperm are present in ovarian tissue. Several features of the gonadal alterations described in this study of whitefish from Lake Thun have also been reported for whitefish from other areas. However, the Lake Thun situation appears to be unique because of the high prevalence of fish affected and the broad variation of abnormal features. The causes of the frequent appearance of these gonadal alterations in whitefish from Lake Thun remain unclear.