Squalius Alburnoides Research Articles

Hybridization and invasive species in a threatened freshwater fish community under environmental pressures: Morphometric and molecular evidence

Abstract Mediterranean freshwater systems are under threat owing to increased drought driven by climate change, intensive human land uses and non‐native species. This is causing increased fish hybridization in isolated watercourses. The genetic and morphological characteristics of hybrids of sympatric native and non‐native fish species were studied in four streams of the Mediterranean Guadalquivir basin (south‐west Spain). Fish morphology was analysed using geometric morphometrics, and molecular determination of parenthood was inferred through one mitochondrial gene (cytb) and one nuclear gene (Beta‐actin) for all hybrids and a subset of pure parental specimens. Molecular analyses confirmed hybrids between the native Squalius alburnoides and non‐native Alburnus alburnus in a stream with continuous flow. Haplotype analyses suggested that they originated from backcrossing of hybrid offspring. Intergeneric crosses between native species S. alburnoides and Pseudochondrostoma willkommii, and S. alburnoides and Iberochondrostoma lemmingii were detected in streams under reduced connectivity scenarios. Morphometrics revealed that hybrid phenotypes were similar to S. alburnoides. In some cases, molecular markers uncovered hybridization events that were neither detected in the field nor by morphometric analyses, potentially supporting a backcrossing/introgression scenario. Hybridization is likely to be increasing in Mediterranean rivers where S. alburnoides are present owing to increased fragmentation caused by summer drought exacerbated by climate change and human land uses and pressures. This can become a problem for these endemic vulnerable species if genetic diversity is lost, morphological homogenization occurs and hybrids cannot be easily detected in the field. The potential risks could be addressed by monitoring and eradication of non‐native species and segregation from natives. To avoid native–native crosses, habitat quality and desiccation risk could be tackled by improved water quality and riparian reforestation to provide shade and reduce evapotranspiration. This would need increased coordination and intervention between the institutions that share conservation responsibilities in the area.

First record of intraspecific cleaning behavior by the threatened Leuciscidae Squalius alburnoides (Steindachner, 1866) at the Guadiana River basin (Portugal).

Squalius alburnoides (Steindachner, 1866) is an endemic threatened species from the Iberian Peninsula. Here, we report the first observations of intraspecific cleaning behavior in isolated summer pools in the Guadiana River Basin (Portugal). We found that focal S. alburnoides solicited cleaning by adopting an immobile tail-stand position known as "posing," which immediately signaled a response to a few conspecifics that approached and inspect them. Our study expands the list of cleanerfish species in freshwaters, giving emphasis to the importance of mutual positive behavior within an endangered species, particularly when facing seasonal disturbance.

External parasites: how anchor worms affect Iberian endemic fish

Lernaea cyprinacea, commonly known as anchor worm, is a cosmopolitan invasive copepod that parasitizes a wide variety of freshwater fish, including several native and endemic species of the Iberian Peninsula (Sánchez-Hernández 2017), like the Squalius alburnoides from the Guadiana River basin depicted in the photograph, with 3 different individuals attached. Anchor worm was introduced to Iberia from Asia in the 1970s (Simon Vicente et al. 1973). While males are free-living, adult females attach their anchors – a modification of the head – to the fish host, usually in the fins, gills or mouth. Infestation by this external parasite is easily recognised by the wounds, ulcers and inflammatory processes that it induces in the fish skin and muscle (Hossain et al. 2018, see photograph). It may also induce anaemia, malformations, reduced growth and increased susceptibility to secondary infections (Sánchez-Hernández 2017). Small fish are specially affected in their swimming ability, respiration and health condition due to infections and mechanical compression of internal organs, both resulting from the ulcerous skin lesions developed at anchoring sites (Guagliardo & Tanzola 2016). Since infestation prevalence are hypothesised to be positively correlated with higher water temperatures (Marcogliese 2001), outbreaks are expected to increase in intermittent southern Iberian rivers under a global climate change scenario (Macedo-Veiga et al. 2019), which will likely contribute to the aggravation of the conservation status of the already imperilled native fish species. Although broader studies are needed, reported prevalences are higher in native Iberian fish than in sympatric alien species (Macedo-Veiga et al. 2019)

Widespread Hybridization between Invasive Bleak (Alburnus alburnus) and Iberian Chub (Squalius spp.): A Neglected Conservation Threat

Hybridization between native and nonnative fish species is a major conservation issue, especially in ecosystems with high levels of endemism, such as Iberian streams. To date, hybridization with the invasive bleak Alburnus alburnus has been reported for the Iberian chub Squalius alburnoides and S. pyrenaicus and in scattered locations only. However, the bleak is spreading in the region, potentially increasing the risks of hybridization with other Squalius species. To gather a more comprehensive picture on the current geography of hybridization, we compiled records on hybrids between bleak and chub in Portugal and conducted genetical assessments of hybrids between bleak and S. carolitertii. We found that hybridization with bleak is widespread throughout Portuguese river basins and involves at least S. alburnoides, S. pyrenaicus and S. carolitertii. Hybridization with bleak may not only cause waste of reproductive effort and damage the genetic integrity of these endemic species but also promote shifts in the reproductive dynamics of the S. alburnoides hybrid complex, which includes individuals with various ploidy levels and combinations of parental genomes, reproducing sexually and asexually. We recommend that future studies characterize the fitness of bleak hybrids and their ecological and genetic interactions with native fish, in order to design effective conservation measures.

Allele-specific expression variation at different ploidy levels in Squalius alburnoides

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.

Structural microhabitat use by endemic cyprinids in a Mediterranean‐type river: Implications for restoration practices

Abstract Endemic freshwater fish from the Mediterranean region are among the most threatened species in the world owing to increasing river degradation. Because of such threats, the number of river restoration projects has greatly increased. However, they are seldom planned with consideration of the species' life history, often resulting in erroneous practices that compromise their success. This study assessed the seasonal and size‐related microhabitat use by three endemic cyprinids (Iberian barbel, Luciobarbus bocagei; Iberian straight‐mouth nase, Pseudochondrostoma polylepis; and calandino, Squalius alburnoides) using a modified point electrofishing procedure in a Mediterranean river. A multivariate approach was then employed to analyse both structural resource use and availability data. All species showed non‐random microhabitat use. The barbel and nase shifted to faster‐flowing positions (>25 cm s−1) with a coarser substratum (>150 mm particle size) during spring and to sheltered positions (50–100% instream cover) during autumn. Calandino selected more covered areas in autumn (>60% cover) and shifted to shallower positions from this season (>40 cm) to summer (<35 cm). Significant size‐related differences in microhabitat use were consistent for the three species, with smaller individuals occupying on average shallower microhabitats. Significant resource‐use overlap between species size classes increased from autumn to summer, occurring mainly between juvenile and small adult barbel and calandino. The present study helped develop recommendations for river restoration practices that may have a wider application, particularly for other Mediterranean‐type rivers. Cover was found to be critical for all species, in particular for calandino, which could be classified as a shelter‐oriented eurytopic species. Restoration scenarios should therefore consider maintaining cover habitats during unfavourable flow conditions for these species. Lithophilic spawners, which were highly responsive to velocity and substratum, could particularly benefit from the creation of gravel bedforms and artificial riffles to enhance depth and flow variability.

Reproductive dynamics shapes genomotype composition in an allopolyploid complex.

Hybrid complexes are composed of organisms with multiple combinations of parental genomes (genomotypes) that interconnect through nets of crosses. Although several such complexes are well established without speciation or extinction, mechanisms shaping their dynamics remain poorly understood. In this study, we quantified the reproductive success of the allopolyploid Iberian fish Squalius alburnoides in experimental free-access and directional crosses involving the most common genomotypes. Specifically, we analysed the paternity of the offspring produced when females had free access to male genomotypes and quantified variations in egg allocation, fertilization rate, and offspring survival among crosses involving each male genomotype. The composition of the offspring produced from free-access crosses varied significantly from that expected from random mating, suggesting that offspring production and viability are not independent of parental male genomotype. Moreover, directional crosses producing the genomotype most commonly found in wild populations appeared to be the most successful, with females laying more eggs, and fertilization rate and offspring survival being the highest. These results suggest that reproductive dynamics plays a relevant role in structuring the genomotype composition of populations and opens a path to future research on the ecology and evolutionary biology of allopolyploids and their multiplicity of possible evolutionary pathways.

Gene copy silencing and DNA methylation in natural and artificially produced allopolyploid fish.

Allelic silencing is an important mechanism for coping with gene dosage changes in polyploid organisms that is well known in allopolyploid plants. Only recently, it was shown in the allotriploid fish Squalius alburnoides that this process also occurs in vertebrates. However, it is still unknown whether this silencing mechanism is common to other allopolyploid fish, and which mechanisms might be responsible for allelic silencing. We addressed these questions in a comparative study between Squalius alburnoides and another allopolyploid complex, the Amazon molly (Poecilia formosa). We examined the allelic expression patterns for three target genes in four somatic tissues of natural allo-anorthoploids and laboratory-produced tri-genomic hybrids of S. alburnoides and P. formosa. Also, for both complexes, we evaluated the correlation between total DNA methylation level and the ploidy status and genomic composition of the individuals. We found that allelic silencing also occurs in other allopolyploid organisms besides the single one that was previously known. We found and discuss disparities within and between the two considered complexes concerning the pattern of allele-specific expression and DNA methylation levels. Disparities might be due to intrinsic characteristics of each genome involved in the hybridization process. Our findings also support the idea that long-term evolutionary processes have an effect on the allele expression patterns and possibly also on DNA methylation levels.

Mate Choice Drives Evolutionary Stability in a Hybrid Complex.

Previous studies have shown that assortative mating acts as a driver of speciation by countering hybridization between two populations of the same species (pre-zygotic isolation) or through mate choice among the hybrids (hybrid speciation). In both speciation types, assortative mating promotes speciation over a transient hybridization stage. We studied mate choice in a hybrid vertebrate complex, the allopolyploid fish Squalius alburnoides. This complex is composed by several genomotypes connected by an intricate reproductive dynamics. We developed a model that predicts the hybrid complex can persist when females exhibit particular mate choice patterns. Our model is able to reproduce the diversity of population dynamic outcomes found in nature, namely the dominance of the triploids and the dominance of the tetraploids, depending on female mate choice patterns and frequency of the parental species. Experimental mate choice trials showed that females exhibit the preferences predicted by the model. Thus, despite the known role of assortative mating in driving speciation, our findings suggest that certain mate choice patterns can instead hinder speciation and support the persistence of hybrids over time without speciation or extinction.

Gene expression dosage regulation in an allopolyploid fish.

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.

Quest for the sex chromosomes in Squalius pyrenaicus (Teleostei, Cyprinidae)

Event Abstract Back to Event Quest for the sex chromosomes in Squalius pyrenaicus (Teleostei, Cyprinidae) Marlon F. Pazian1*, Carla S. Pereira1 and M João Collares-Pereira1 1 Faculty of Sciences of the University of Lisbon, Centre for Ecology, Evolution and Environmental Changes, Portugal In teleosts, evolutionary proximity does not dictate similarity in the sex determining systems. Hybridization, even among closely related species, often results in a huge shock. A good example of such deviation is the cyprinid species complex of Squalius alburnoides with unique characteristics that make this a very interesting model of study regarding the differentiation of sex chromosomes in a hybrid context. This complex endemic to the Iberian Peninsula resulted from the hybridization of Squalius pyrenaicus females (P genome) with males from an unknown species closely related to Anaecypris hispanica (A genome). The complex comprises males and females with different levels of ploidy and genome combinations (genomotypes) intertwined by altered modes of reproduction, ranging from normal meiosis to meiotic hybridogenesis. Sex ratio in nature is clearly biased towards the prevalence of triploid females but the complex also comprises an allegedly all-male lineage. Overall, sex chromosomes are very similar with X and Z having euchromatic regions while Y and W are completely heterochromatic. The accumulation of heterochromatin and the potential loss of genes on Y and W chromosomes are considered convergent traits in the process of sex chromosome differentiation. Bearing in mind S. pyrenaicus as the maternal lineage of S. alburnoides and the possibility of a ZW sex determining system in this complex, GISH (genomic in situ hybridization) experiments were performed in 6 specimens (3 males, 3 females; Cheleiros tributary, Portugal) trying to identify polymorphisms that could be associated with differentiating sex chromosomes in S. pyrenaicus. No differences could be found suggesting that either sex determining genes are scattered throughout the genome of this species or that, if present, sex chromosomes remain in a relatively undifferentiated state (morphological and structural). This investigation requires higher resolution cytogenetic tools like repetitive-free chromosome-specific probes, single-copy gene mapping (sex related genes) and chromosome-specific anchoring probes/markers. Acknowledgements The study was supported by Centro de Ecologia, Evolução e Alterações Ambientais [UID/BIA/00329/2013], Fundação para a Ciência e Tecnologia (FCT) [PhD grant number SFRH/BD/44980/2008 awarded to CP], and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) [Post-Doc grant number 240947/2012-6 awarded to MP].

Fish composition in the Guadiamar River basin after one of the worst mining spills in Europe

On 25 April 1998, the tailing pond of the Los Frailes mine in Aznalcollar (Seville, Spain) ruptured, causing one of the most harmful environmental disasters in Europe in recent decades. Through the crack, 6 hm3 of acidic water and metallic mud were spilt, defaunating a large area of the Guadiamar River. After the spill cleanup and habitat restoration, multiple anthropogenic impacts continued to degrade the affected area. This work aimed to provide the most updated list of fish species in the Guadiamar River basin after the spill. Data were collected between 1999 and 2011 by electrofishing, light-traps, minnow-traps and multimesh gill-nests in 78 sampling sites. Species richness values for both native and exotic species in the Guadiamar River basin were high when compared with values for other right bank tributaries of the Guadalquivir River. This may be due to direct contact with the mouth of the Guadalquivir, which allowed the presence of migratory species. It may also be due to its location in the lower part of the Guadalquivir River basin, where exotic species accumulated. Among the Guadiamar River basin species, Luciobarbus sclateri and Squalius alburnoides have the widest distribution. The former is a generalist species resistant to unfavourable habitat changes, and the latter has a very successful breeding strategy. However, when focused on the affected area, there was a marked increase in exotic species, and both pumpkinseed (Lepomis gibbosus) and carp (Cyprinus carpio) co-dominated together with the native L. sclateri and S. alburnoides. The distribution of species within the river basin suggests that the upper section (except the Agrio reservoir) and middle section tributaries may be acting as native species shelters, while the affected area becomes an exotic species source. This information should be useful for monitoring future changes in the species composition and for management planning measures.

The role of prey abundance and flow regulation in the marking behaviour of Eurasian otters in a Mediterranean catchment

Scent marking is a common behaviour used to delineate territories by carnivores, yet it remains insufficiently understood as there are several alternative hypotheses, one being that sites are marked where resources are abundant, thus guaranteeing their exclusive use. Small Mediterranean rivers are highly variable environments throughout the year, representing an ideal scenario to test this theory, as prey availability changes as a function of the spatial and temporal variability of the flow regime. We studied seasonal variation in the habitat selection of latrine (i.e. scent-marked) sites, prey availability and prey selection of the Eurasian otter, Lutra lutra, between two contrasting fluvial systems, the regulated mainstem of River Bullaque and four unregulated tributaries (River Guadiana basin, central Spain). In 2010, we measured habitat variables, collected otter spraints and used electrofishing to compare the diet with prey abundance. Latrines were more frequent at shallow sites, and otters did not discriminate between fluvial systems across seasons. Also, latrines were associated with sites where the main prey, the calandino, Squalius alburnoides, was more abundant in the tributaries during spring and summer. However, this abundance was not translated into prey selection, with calandino being preyed below its availability in the mainstem. This observation suggests that otters mark places where fishing is especially easy (i.e. shallow sites with proportionally more fish). Calandino was substituted by red swamp crayfish, Procambarus clarkii, which was easier to catch but of lower energetic value, where higher water levels made fishing more difficult (i.e. the regulated mainstem).

Identifying parental chromosomes and genomic rearrangements in animal hybrid complexes of species with small genome size using Genomic In Situ Hybridization (GISH)

Genomic In Situ Hybridization (GISH) is a powerful tool to identify and to quantify genomic constituents in allopolyploids, and is mainly based on hybridization of highly and moderate repetitive sequences. In animals, as opposed to plants, GISH has not been widely used in part because there are technical problems in obtaining informative results. Using the allopolyploid Squalius alburnoides Steindachner, 1866 fish complex as a model system, we succeeded in overcoming methodological constraints when dealing with parental species with a small genome size. This hybridogenetic complex has biotypes with different genome compositions and ploidy levels, but parental chromosomes are small, morphologically very similar and therefore cannot be distinguished by conventional cytogenetic approaches. Specimens have a small genome (C-value1.2 pg) with a low level of highly and moderate repetitive sequences, mainly located at pericentromeric chromosome regions. Since it is well known that probe annealing depends on probe concentration and hybridization time to obtain uniform hybridization signals along the chromosome arms, we progressively increased the amount of labeled probes from 100ng up to 1µg and the incubation time from overnight up to 5 days. We also made other smaller improvements. Results showed a clear enhancement of signals with respect to previous data, allowing an accurate and reproducible assignment of the parental genomes in both diploid and triploid fish.It was thus evidenced that high probes’ concentrations and long incubation time are the key to obtain, without extra image editing, uniform and reliable hybridization signals in metaphase chromosomes of animal hybrids from species with small genome size.

Global Analysis of the Small RNA Transcriptome in Different Ploidies and Genomic Combinations of a Vertebrate Complex – The Squalius alburnoides

The Squalius alburnoides complex (Steindachner) is one of the most intricate hybrid polyploid systems known in vertebrates. In this complex, the constant switch of the genome composition in consecutive generations, very frequently involving a change on the ploidy level, promotes repetitive situations of potential genomic shock. Previously in this complex, it was showed that in response to the increase in genome dosage, triploids hybrids could regulate gene expression to a diploid state. In this work we compared the small RNA profiles in the different genomic compositions interacting in the complex in order to explore the miRNA involvement in gene expression regulation of triploids. Using high-throughput arrays and sequencing technologies we were able to verify that diploid and triploid hybrids shared most of their sequences and their miRNA expression profiles were high correlated. However, an overall view indicates an up-regulation of several miRNAs in triploids and a global miRNA expression in triploids higher than the predicted from an additive model. Those results point to a participation of miRNAs in the cellular functional stability needed when the ploidy change.

Synaptonemal Complexes in the Hybridogenetic Squalius alburnoides Fish Complex: New Insights on the Gametogenesis of Allopolyploids

In the Squalius alburnoides fish complex, allotriploid females (3n = 75) reproduce mostly by meiotic hybridogenesis, producing haploid gametes by means of the elimination of the heterospecific chromosome set and recombination between the 2 homospecific genomes. A synaptonemal complexes (SCs) analysis was performed in specimens from a confined southern population (Quarteira, Portugal) to understand chromosome dynamics during gametogenesis. The comparative study between hybrid females with QAA genome composition and the parental bisexual species Squalius aradensis (2n = 50, QQ genome) evidenced: (i) that allotriploid meiocytes comprise the complete chromosome set (75 chromosomes) in prophase I, proving the heterospecific genome (Q) is only excluded after pachytene stage, and (ii) a 2-phase synaptic process where initially, exclusively homologous SCs form and the unmatched univalents remain in a bouquet conformation, followed by the establishment of extensive non-homologous SCs with multivalent associations among the later. These findings disagree with most literature concerning the meiotic process in allotriploid vertebrates, since the most accountable mechanisms (premeiotic exclusion of the unmatched chromosome set and whole genome endoduplication) were not observed.

Natural hybridization with invasive bleak Alburnus alburnus threatens the survival of Iberian endemic calandino Squalius alburnoides complex and Southern Iberian chub Squalius pyrenaicus

Hybrids of invasive bleak Alburnus alburnus and native endemic calandino Squalius alburnoides complex and Southern Iberian chub Squalius pyrenaicus were found in the River Jarama (Tagus River basin, central Spain). Morphological analyses show that hybrids are intermediate in shape between the parent species. Molecular mitochondrial and nuclear analyses evidence that hybridization was different in terms of the direction of gene introgression, with hybrids ♀ A. alburnus × ♂ S. pyrenaicus and ♀ S. alburnoides complex × ♂ A. alburnus. As far as we know, this is the first evidence of interspecific hybridization between a fish hybrid complex and an invasive exotic fish. The hybridization occurred at least during 3 years in a short time period after the arrival of exotic bleak. The spread of bleak in the Iberian Peninsula can be a serious threat for the survival of endemic Squalius species, as well as other close Leuciscinae such as the endangered jarabugo Anaecypris hispanica.

New distribution data on Spanish autochthonous species of freshwater fish

Over the last two years (2008-2010) we performed a monitoring project to update the Spanish freshwater fish fauna. We have collected in 785 sampling localities homogeneously distributed throughout Spain each year. We find as the most important result of this study the occurrence of several species that constitute new records for some rivers or basins in Spain: Lampetra planeri, Petromyzon marinus, Barbus haasi, Iberochondrostoma lemmingii, Squalius alburnoides, Squalius carolitertii, Squalius malacitanus, Cobitis calderoni, Cobitis paludica , and Salaria fluviatilis . Finally we corroborate the existence of Barbatula barbatula in the Duero basin and the assignation of the northeastern populations of this genus to B. quignardi .

Ploidy mosaicism and allele-specific gene expression differences in the allopolyploid Squalius alburnoides

BackgroundSqualius 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.ResultsTo 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.ConclusionsPloidy 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 polymorphic microsatellite from the Squalius alburnoides complex (Osteichthyes, Cyprinidae) cloned by serendipity can be useful in genetic analysis of polyploids

A new microsatellite locus (SAS1) for Squalius alburnoides was obtained through cloning by serendipity. The possible usefulness of this new species-specific microsatellite in genetic studies of this hybrid-species complex, was explored. The polymorphism exhibited by SAS1 microsatellite is an important addition to the set of microsatellites previously used in genetic studies in S. alburnoides complex, that mostly relied in markers described for other species. Moreover, the SAS1 microsatellite could be used to identify the parental genomes of the complex, complementing other methods recently described for the same purpose..