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Allopolyploid plants are long known to be subject to a homoeolog expression bias of varying degree. The same phenomenon was only much later suspected to occur also in animals based on studies of single selected genes in an allopolyploid vertebrate, the Iberian fish Squalius alburnoides. Consequently, this species became a good model for understanding the evolution of gene expression regulation in polyploid vertebrates. Here, we analyzed for the first time genome-wide allele-specific expression data from diploid and triploid hybrids of S. alburnoides and compared homoeolog expression profiles of adult livers and of juveniles. Co-expression of alleles from both parental genomic types was observed for the majority of genes, but with marked homoeolog expression bias, suggesting homoeolog specific reshaping of expression level patterns in hybrids. Complete silencing of one allele was also observed irrespective of ploidy level, but not transcriptome wide as previously speculated. Instead, it was found only in a restricted number of genes, particularly ones with functions related to mitochondria and ribosomes. This leads us to hypothesize that allelic silencing may be a way to overcome intergenomic gene expression interaction conflicts, and that homoeolog expression bias may be an important mechanism in the achievement of sustainable genomic interactions, mandatory to the success of allopolyploid systems, as in S. alburnoides.

Sex determination is a highly variable process that utilizes many different mechanisms to initiate the cascade of differentiation processes. The molecular pathways controlling sexual development are less conserved than previously assumed, and appear to require active maintenance in some species; indeed, the developmental decision of gonad phenotype in gonochoristic species is not fixed at an early developmental stage. Much of the knowledge about sex determination mechanisms was derived from research on gonochoristic, non-seasonal breeders. In this study, the transcriptome of resting adult gonads of a seasonal breeder, the endangered Iberian cyprinid fish Squalius pyrenaicus, was analyzed to assess the expression patterns and evolutionary rates of sex-biased genes that could be involved in maintenance of gonad identity as well as in sex determination. Remarkably, some crucial female genes-such as aromatase cyp19a1a, estrogen receptor esr1a, and foxl2-were expressed more abundantly in S. pyrenaicus testis than in ovaries. Moreover, contrary to the higher evolutionary rate changes observed in male-biased genes, higher d N /d S ratios were observed for female-biased genes than for male-biased genes in S. pyrenaicus. These results help unravel the impact of seasonality in sex determination mechanisms and the evolution of genes, and highlight the need to study fish at different gonadal maturation states to understand the function of sex-biased genes. Mol. Reprod. Dev. 83: 1102-1115, 2016. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

How allopolyploids are able not only to cope but profit from their condition is a question that remains elusive, but is of great importance within the context of successful allopolyploid evolution. One outstanding example of successful allopolyploidy is the endemic Iberian cyprinid Squalius alburnoides. Previously, based on the evaluation of a few genes, it was reported that the transcription levels between diploid and triploid S. alburnoides were similar. If this phenomenon occurs on a full genomic scale, a wide functional ''diploidization'' could be related to the success of these polyploids. We generated RNA-seq data from whole juvenile fish and from adult livers, to perform the first comparative quantitative transcriptomic analysis between diploid and triploid individuals of a vertebrate allopolyploid. Together with an assay to estimate relative expression per cell, it was possible to infer the relative sizes of transcriptomes. This showed that diploid and triploid S. alburnoides hybrids have similar liver transcriptome sizes. This in turn made it valid to directly compare the S. alburnoides RNA-seq transcript data sets and obtain a profile of dosage responses across the S. alburnoides transcriptome. We found that 64% of transcripts in juveniles' samples and 44% in liver samples differed less than twofold between diploid and triploid hybrids (similar expression). Yet, respectively 29% and 15% of transcripts presented accurate dosage compensation (PAA/PA expression ratio of 1 instead of 1.5). Therefore, an exact functional diploidization of the triploid genome does not occur, but a significant down regulation of gene expression in triploids was observed. However, for those genes with similar expression levels between diploids and triploids, expression is not globally strictly proportional to gene dosage nor is it set to a perfect diploid level. This quantitative expression flexibility may be a strong contributor to overcome the genomic shock, and be an immediate evolutionary advantage of allopolyploids.

When comparing the known picture of polyploidy in animals and in plants, it is possible to recognize some similarities, namely: (i) multiple and recurrent origins in several well-established taxonomic groups; (ii) a strong and regular association with hybridization events; (iii) the production of genotypic diversity; (iv) a rapid genomic reshuffling; (v) a very active role of transposable elements in allopolyploids; (vi) a comparatively privileged occurrence in harsher environments when compared with their diploid relatives, and (vii) gene silencing and divergence of duplicated genes without disruption of duplicated loci. Research on polyploidy was highly biased towards plants during the last century because polyploidy in animals was for long time considered rare, occasional and irrelevant from an evolutionary perspective. However, as empirically observed in plants, genome rediploidization starts in polyploid organisms immediately after the polyploid shock. Given the speed and dynamicity of this process, evidence of genome multiplication is completely erased over time, and hence, only the most recent events are likely to be acknowledged. Although varying in expression between and within taxonomic groups, polyploidy and hybridization are ubiquitous in animals and may be recurrent, fostering evolution. Since evolutionary allopolyploid genomes behave as biologically diploid, zoologists have to challenge the old paradigm of an irrelevant evolutionary role in animals using current genomic and cytogenomic tools. These methods are most likely to reveal the role of polyploid mechanisms in producing evolutionary novelties. Nonsexual complexes are the perfect models to bridge the gap between empirical and theoretical research, while the evolutionary process is in action. Such animal complexes represent a transient stage that, in general, moves towards a polyploid stage, where bisexuality might be recovered, ultimately giving rise to a new gonochoric species. These pathways are herein illustrated by the Iberian allopolyploid Squalius alburnoides. Some general aspects on this fish's complex are updated and reviewed, namely the reproductive modes of the distinct genomotypes, since variable ploidies and genomic combinations occur in natural populations. Most recent data on the mechanisms of gene expression regulation and the importance of the genomic context in driving allelic expression are also included. It was first demonstrated that a regulatory mechanism involving dosage compensation by gene-copy silencing exists in allotriploid females and that allelic expression patterns differed either between genomically equivalent individuals or within the same individual (between tissues and genes). Thus, instead of a whole haplome inactivation, a biased silencing towards repression of a specific allele was observed as well as a reduction of the transcript levels to the diploid state. See also sister article focusing on plants by Tayalé and Parisod in this themed issue., (Copyright © 2013 S. Karger AG, Basel.)

Background: Squalius alburnoides is an Iberian cyprinid fish resulting from an interspecific hybridisation between Squalius pyrenaicus females (P genome) and males of an unknown Anaecypris hispanica-like species (A genome). S. alburnoides is an allopolyploid hybridogenetic complex, which makes it a likely candidate for ploidy mosaicism occurrence, and is also an interesting model to address questions about gene expression regulation and genomic interactions. Indeed, it was previously suggested that in S. alburnoides triploids (PAA composition) silencing of one of the three alleles (mainly of the P allele) occurs. However, not a whole haplome is inactivated but a more or less random inactivation of alleles varying between individuals and even between organs of the same fish was seen.In this work we intended to correlate expression differences between individuals and/or between organs to the occurrence of mosaicism, evaluating if mosaics could explain previous observations and its impact on the assessment of gene expression patterns., Results: To achieve our goal, we developed flow cytometry and cell sorting protocols for this system generating more homogenous cellular and transcriptional samples. With this set-up we detected 10% ploidy mosaicism within the S. alburnoides complex, and determined the allelic expression profiles of ubiquitously expressed genes (rpl8; gapdh and β-actin) in cells from liver and kidney of mosaic and non-mosaic individuals coming from different rivers over a wide geographic range., Conclusions: Ploidy mosaicism occurs sporadically within the S. alburnoides complex, but in a frequency significantly higher than reported for other organisms. Moreover, we could exclude the influence of this phenomenon on the detection of variable allelic expression profiles of ubiquitously expressed genes (rpl8; gapdh and β-actin) in cells from liver and kidney of triploid individuals. Finally, we determined that the expression patterns previously detected only in a narrow geographic range is not a local restricted phenomenon but is pervasive in rivers where S. pyrenaicus is sympatric with S. alburnoides.We discuss mechanisms that could lead to the formation of mosaic S. alburnoides and hypothesise about a relaxation of the mechanisms that impose a tight control over mitosis and ploidy control in mixoploids.

A new method for the fast identification of the genomic composition of the cyprinid Squalius alburnoides is presented. The method is based on a length polymorphism detected in the beta-actin gene, which serves as the basis for the development of a semi-quantitative PCR.

A non-intrusive method for individual identification of Iberian cyprinid complex Squalius alburnoides is presented, with possible application to other fish complexes and species. The proposed methodology is based on scale patterns that vary greatly between individuals.