Towards the conservation of the crucian carp in Europe: Prolific hybridization but no evidence for introgression between native and non-native taxa.

Hybridization plays a pivotal role in evolution, influencing local adaptation and speciation. However, it can also reduce biodiversity, which is especially damaging when native and non-native species meet. Hybridization can threaten native species via competition (with vigorous hybrids), reproductive resource wastage and gene introgression. The latter, in particular, could result in increased fitness in invasive species, decreased fitness of natives and compromise reintroduction or recovery conservation practices. In this study, we use a combination of RAD sequencing and microsatellites for a range-wide sample set of 1366 fish to evaluate the potential for hybridization and introgression between native crucian carp (Carassius carassius) and three non-native taxa (Carassius auratus auratus, Carassius auratus gibelio and Cyprinus carpio) in European water bodies. We found hybridization between native and non-native taxa in 82% of populations with non-natives present, highlighting the potential for substantial ecological impacts from hybrids on crucian carp populations. However, despite such high rates of hybridization, we could find no evidence of introgression between these taxa. The presence of triploid backcrosses in at least two populations suggests that the lack of introgression among these taxa is likely due to meiotic dysfunction in hybrids, leading to the production of polyploid offspring which are unable to reproduce sexually. This result is promising for crucian reintroduction programs, as it implies limited risk to the genetic integrity of source populations. Future research should investigate the reproductive potential of triploid hybrids and the ecological pressures hybrids impose on C. carassius.

The haplotype-resolved Prymnesium parvum (type B) microalga genome reveals the genetic basis of its fish-killing toxins.

The catastrophic loss of aquatic life in the Central European Oder River in 2022, caused by a toxic bloom of the haptophyte microalga Prymnesium parvum (in a wide sense, s.l.), underscores the need to improve our understanding of the genomic basis of the toxin. Previous morphological, phylogenetic, and genomic studies have revealed cryptic diversity within P. parvum s.l. and uncovered three clade-specific (types A, B, and C) prymnesin toxins. Here, we used state-of-the-art long-read sequencing and assembled the first haplotype-resolved diploid genome of a P. parvum type B from the strain responsible for the Oder disaster. Comparative analyses with type A genomes uncovered a genome-size expansion driven by repetitive elements in type B. We also found conserved synteny but divergent evolution in several polyketide synthase (PKS) genes, which are known to underlie toxin production in combination with environmental cues. We identified an approximately 20-kbp deletion in the largest PKS gene of type B that we link to differences in the chemical structure of types A and B prymnesins. Flow cytometry and electron microscopy analyses confirmed diploidy in the Oder River strain and revealed differences to closely related strains in both ploidy and morphology. Our results provide unprecedented resolution of strain diversity in P. parvum s.l. and a better understanding of the genomic basis of toxin variability in haptophytes. The reference-quality genome will enable us to better understand changes in microbial diversity in the face of increasing environmental pressures and provides a basis for strain-level monitoring of invasive Prymnesium in the future.

Massive expansion of sex-specific SNPs, transposon-related elements, and neocentromere formation shape the young W-chromosome from the mosquitofish Gambusia affinis.

BACKGROUND: The Western mosquitofish, Gambusia affinis, is a model for sex chromosome organization and evolution of female heterogamety. We previously identified a G. affinis female-specific marker, orthologous to the aminomethyl transferase (amt) gene of the related platyfish (Xiphophorus maculatus). Here, we have analyzed the structure and differentiation of the G. affinis W-chromosome, using a cytogenomics and bioinformatics approach. RESULTS: The long arm of the G. affinis W-chromosome (Wq) is highly enriched in dispersed repetitive sequences, but neither heterochromatic nor epigenetically silenced by hypermethylation. In line with this, Wq sequences are highly transcribed, including an active nucleolus organizing region (NOR). Female-specific SNPs and evolutionary young transposable elements were highly enriched and dispersed along the W-chromosome long arm, suggesting constrained recombination. Wq copy number expanded elements also include female-specific transcribed sequences from the amt locus with homology to TE. Collectively, the G. affinis W-chromosome is actively differentiating by sex-specific copy number expansion of transcribed TE-related elements, but not (yet) by extensive sequence divergence or gene decay. CONCLUSIONS: The G. affinis W-chromosome exhibits characteristic genomic properties of an evolutionary young sex chromosome. Strikingly, the observed sex-specific changes in the genomic landscape are confined to the W long arm, which is separated from the rest of the W-chromosome by a neocentromere acquired during sex chromosome evolution and may thus have become functionally insulated. In contrast, W short arm sequences were apparently shielded from repeat-driven differentiation, retained Z-chromosome like genomic features, and may have preserved pseudo-autosomal properties.

Sticky steps and the gender gap: how thoughtful practices could help keep caregivers in science.

Many fewer women than men hold senior academic positions, a widely recognized and increasing problem. Our goal is to identify effective and feasible solutions. We begin by providing an in-depth assessment of the drivers of this gender inequity. In our synthesis of existing data, we provide many lines of evidence highlighting caregiving as a primary main factor. This is not a 'new' insight per se, but a point worth repeating that we back up by a strong and synthetic body of recent data. We also believe that our analysis provides a step forward in tackling a complex issue. We then develop a more detailed understanding of the challenges academic caregivers face and discuss whether and why it is important to keep caregivers in science. We find that the attrition due to caregiving should not be seen as a factor but rather as a process with multiple 'sticky steps' that eventually drive caregivers out of science-which, as we argue, is partly also good news. Indeed, it is here that we believe actions could be taken that would have a real impact: for example, one could effectively increase and expand upon current funding practices that focus on caregiver career advancement.

Equilibrated evolution of the mixed auto-/allopolyploid haplotype-resolved genome of the invasive hexaploid Prussian carp.

Understanding genome evolution of polyploids requires dissection of their often highly similar subgenomes and haplotypes. Polyploid animal genome assemblies so far restricted homologous chromosomes to a 'collapsed' representation. Here, we sequenced the genome of the asexual Prussian carp, which is a close relative of the goldfish, and present a haplotype-resolved chromosome-scale assembly of a hexaploid animal. Genome-wide comparisons of the 150 chromosomes with those of two ancestral diploid cyprinids and the allotetraploid goldfish and common carp revealed the genomic structure, phylogeny and genome duplication history of its genome. It consists of 25 syntenic, homeologous chromosome groups and evolved by a recent autoploid addition to an allotetraploid ancestor. We show that de-polyploidization of the alloploid subgenomes on the individual gene level occurred in an equilibrated fashion. Analysis of the highly conserved actinopterygian gene set uncovered a subgenome dominance in duplicate gene loss of one ancestral chromosome set.

DNA Transposon Expansion is Associated with Genome Size Increase in Mudminnows.

Genome sizes of eukaryotic organisms vary substantially, with whole-genome duplications (WGD) and transposable element expansion acting as main drivers for rapid genome size increase. The two North American mudminnows, Umbra limi and Umbra pygmaea, feature genomes about twice the size of their sister lineage Esocidae (e.g., pikes and pickerels). However, it is unknown whether all Umbra species share this genome expansion and which causal mechanisms drive this expansion. Using flow cytometry, we find that the genome of the European mudminnow is expanded similarly to both North American species, ranging between 4.5 and 5.4 pg per diploid nucleus. Observed blocks of interstitially located telomeric repeats in U. limi suggest frequent Robertsonian rearrangements in its history. Comparative analyses of transcriptome and genome assemblies show that the genome expansion in Umbra is driven by the expansion of DNA transposon and unclassified repeat sequences without WGD. Furthermore, we find a substantial ongoing expansion of repeat sequences in the Alaska blackfish Dallia pectoralis, the closest relative to the family Umbridae, which might mark the beginning of a similar genome expansion. Our study suggests that the genome expansion in mudminnows, driven mainly by transposon expansion, but not WGD, occurred before the separation into the American and European lineage.

Sex chromosomes in meiotic, hemiclonal, clonal and polyploid hybrid vertebrates: along the 'extended speciation continuum'.

We review knowledge about the roles of sex chromosomes in vertebrate hybridization and speciation, exploring a gradient of divergences with increasing reproductive isolation (speciation continuum). Under early divergence, well-differentiated sex chromosomes in meiotic hybrids may cause Haldane-effects and introgress less easily than autosomes. Undifferentiated sex chromosomes are more susceptible to introgression and form multiple (or new) sex chromosome systems with hardly predictable dominance hierarchies. Under increased divergence, most vertebrates reach complete intrinsic reproductive isolation. Slightly earlier, some hybrids (linked in 'the extended speciation continuum') exhibit aberrant gametogenesis, leading towards female clonality. This facilitates the evolution of various allodiploid and allopolyploid clonal ('asexual') hybrid vertebrates, where 'asexuality' might be a form of intrinsic reproductive isolation. A comprehensive list of 'asexual' hybrid vertebrates shows that they all evolved from parents with divergences that were greater than at the intraspecific level (K2P-distances of greater than 5-22% based on mtDNA). These 'asexual' taxa inherited genetic sex determination by mostly undifferentiated sex chromosomes. Among the few known sex-determining systems in hybrid 'asexuals', female heterogamety (ZW) occurred about twice as often as male heterogamety (XY). We hypothesize that pre-/meiotic aberrations in all-female ZW-hybrids present Haldane-effects promoting their evolution. Understanding the preconditions to produce various clonal or meiotic allopolyploids appears crucial for insights into the evolution of sex, 'asexuality' and polyploidy. This article is part of the theme issue 'Challenging the paradigm in sex chromosome evolution: empirical and theoretical insights with a focus on vertebrates (Part II)'.

A 180 Myr-old female-specific genome region in sturgeon reveals the oldest known vertebrate sex determining system with undifferentiated sex chromosomes.

Several hypotheses explain the prevalence of undifferentiated sex chromosomes in poikilothermic vertebrates. Turnovers change the master sex determination gene, the sex chromosome or the sex determination system (e.g. XY to WZ). Jumping master genes stay main triggers but translocate to other chromosomes. Occasional recombination (e.g. in sex-reversed females) prevents sex chromosome degeneration. Recent research has uncovered conserved heteromorphic or even homomorphic sex chromosomes in several clades of non-avian and non-mammalian vertebrates. Sex determination in sturgeons (Acipenseridae) has been a long-standing basic biological question, linked to economical demands by the caviar-producing aquaculture. Here, we report the discovery of a sex-specific sequence from sterlet (Acipenser ruthenus). Using chromosome-scale assemblies and pool-sequencing, we first identified an approximately 16 kb female-specific region. We developed a PCR-genotyping test, yielding female-specific products in six species, spanning the entire phylogeny with the most divergent extant lineages (A. sturio, A. oxyrinchus versus A. ruthenus, Huso huso), stemming from an ancient tetraploidization. Similar results were obtained in two octoploid species (A. gueldenstaedtii, A. baerii). Conservation of a female-specific sequence for a long period, representing 180 Myr of sturgeon evolution, and across at least one polyploidization event, raises many interesting biological questions. We discuss a conserved undifferentiated sex chromosome system with a ZZ/ZW-mode of sex determination and potential alternatives. This article is part of the theme issue 'Challenging the paradigm in sex chromosome evolution: empirical and theoretical insights with a focus on vertebrates (Part I)'.

Asexuality Associated with Marked Genomic Expansion of Tandemly Repeated rRNA and Histone Genes.

How does asexual reproduction influence genome evolution? Although is it clear that genomic structural variation is common and important in natural populations, we know very little about how one of the most fundamental of eukaryotic traits-mode of genomic inheritance-influences genome structure. We address this question with the New Zealand freshwater snail Potamopyrgus antipodarum, which features multiple separately derived obligately asexual lineages that coexist and compete with otherwise similar sexual lineages. We used whole-genome sequencing reads from a diverse set of sexual and asexual individuals to analyze genomic abundance of a critically important gene family, rDNA (the genes encoding rRNAs), that is notable for dynamic and variable copy number. Our genomic survey of rDNA in P. antipodarum revealed two striking results. First, the core histone and 5S rRNA genes occur between tandem copies of the 18S-5.8S-28S gene cluster, a unique architecture for these crucial gene families. Second, asexual P. antipodarum harbor dramatically more rDNA-histone copies than sexuals, which we validated through molecular and cytogenetic analysis. The repeated expansion of this genomic region in asexual P. antipodarum lineages following distinct transitions to asexuality represents a dramatic genome structural change associated with asexual reproduction-with potential functional consequences related to the loss of sexual reproduction.

Independent Origin of XY and ZW Sex Determination Mechanisms in Mosquitofish Sister Species.

Fish are known for the outstanding variety of their sex determination mechanisms and sex chromosome systems. The western (Gambusia affinis) and eastern mosquitofish (G. holbrooki) are sister species for which different sex determination mechanisms have been described: ZZ/ZW for G. affinis and XX/XY for G. holbrooki Here, we carried out restriction-site associated DNA (RAD-) and pool sequencing (Pool-seq) to characterize the sex chromosomes of both species. We found that the ZW chromosomes of G. affinis females and the XY chromosomes of G. holbrooki males correspond to different linkage groups, and thus evolved independently from separate autosomes. In interspecific hybrids, the Y chromosome is dominant over the W chromosome, and X is dominant over Z. In G. holbrooki, we identified a candidate region for the Y-linked melanic pigmentation locus, a rare male phenotype that constitutes a potentially sexually antagonistic trait and is associated with other such characteristics, e.g., large body size and aggressive behavior. We developed a SNP-based marker in the Y-linked allele of GIPC PDZ domain containing family member 1 (gipc1), which was linked to melanism in all tested G. holbrooki populations. This locus represents an example for a color locus that is located in close proximity to a putative sex determiner, and most likely substantially contributed to the evolution of the Y.

Zebrafish larvae show negative phototaxis to near-infrared light.

Zebrafish larvae (Danio rerio) are among the most used model species to test biological effects of different substances in biomedical research, neuroscience and ecotoxicology. Most tests are based on changes in swimming activity of zebrafish larvae by using commercially available high-throughput screening systems. These systems record and analyse behaviour patterns using visible (VIS) and near-infrared (NIR) light sources, to simulate day (VIS) and night (NIR) phases, which allow continuous recording of the behaviour using a NIR sensitive camera. So far, however, the sensitivity of zebrafish larvae to NIR has never been tested experimentally, although being a critical piece of information for interpreting their behaviour under experimental conditions. Here, we investigated the swimming activity of 96 hpf (hours post fertilization) and 120 hpf zebrafish larvae under light sources of NIR at 860 nm and at 960 nm wavelength and under VIS light. A thermal source was simultaneously presented opposite to one of the light sources as control. We found that zebrafish larvae of both larval stages showed a clear negative phototactic response towards 860 nm NIR light and to VIS light, but not to 960 nm NIR light. Our results demonstrated that zebrafish larvae are able to perceive NIR at 860 nm, which is almost identical to the most commonly used light source in commercial screening systems (NIR at 850 nm) to create a dark environment. These tests, however, are not performed in the dark from the zebrafish´s point of view. We recommend testing sensitivity of the used test organism before assuming no interaction with the applied light source of commonly used biosensor test systems. Previous studies on biological effects of substances to zebrafish larvae should be interpreted with caution.

Karyotype Variability and Inter-Population Genomic Differences in Freshwater Ostracods (Crustacea) Showing Geographical Parthenogenesis.

Transitions from sexual to asexual reproduction are often associated with polyploidy and increased chromosomal plasticity in asexuals. We investigated chromosomes in the freshwater ostracod species Eucypris virens (Jurine, 1820), where sexual, asexual and mixed populations can be found. Our initial karyotyping of multiple populations from Europe and North Africa, both sexual and asexual, revealed a striking variability in chromosome numbers. This would suggest that chromosomal changes are likely to be accelerated in asexuals because the constraints of meiosis are removed. Hence, we employed comparative genomic hybridization (CGH) within and among sexual and asexual populations to get insights into E. virens genome arrangements. CGH disclosed substantial genomic imbalances among the populations analyzed, and three patterns of genome arrangement between these populations: 1. Only putative ribosomal DNA (rDNA)-bearing regions were conserved in the two populations compared indicating a high sequence divergence between these populations. This pattern is comparable with our findings at the interspecies level of comparison; 2. Chromosomal regions were shared by both populations to a varying extent with a distinct copy number variation in pericentromeric and presumable rDNA-bearing regions. This indicates a different rate of evolution in repetitive sequences; 3. A mosaic pattern of distribution of genomic material that can be explained as non-reciprocal genetic introgression and evidence of a hybrid origin of these individuals. We show an overall increased chromosomal dynamics in E. virens that is complementary with available phylogenetic and population genetic data reporting highly differentiated diploid sexual and asexual lineages with a wide variety of genetic backgrounds.

A transcriptome derived female-specific marker from the invasive Western mosquitofish (Gambusia affinis).

Sex-specific markers are a prerequisite for understanding reproductive biology, genetic factors involved in sex differences, mechanisms of sex determination, and ultimately the evolution of sex chromosomes. The Western mosquitofish, Gambusia affinis, may be considered a model species for sex-chromosome evolution, as it displays female heterogamety (ZW/ZZ), and is also ecologically interesting as a worldwide invasive species. Here, de novo RNA-sequencing on the gonads of sexually mature G. affinis was used to identify contigs that were highly transcribed in females but not in males (i.e., transcripts with ovary-specific expression). Subsequently, 129 primer pairs spanning 79 contigs were tested by PCR to identify sex-specific transcripts. Of those primer pairs, one female-specific DNA marker was identified, Sanger sequenced and subsequently validated in 115 fish. Sequence analyses revealed a high similarity between the identified sex-specific marker and the 3´ UTR of the aminomethyl transferase (amt) gene of the closely related platyfish (Xiphophorus maculatus). This is the first time that RNA-seq has been used to successfully characterize a sex-specific marker in a fish species in the absence of a genome map. Additionally, the identified sex-specific marker represents one of only a handful of such markers in fishes.

A vertebrate reproductive system involving three ploidy levels: hybrid origin of triploids in a contact zone of diploid and tetraploid palearctic green toads (Bufo viridis subgroup).

The rise and consequences of polyploidy in vertebrates, whose origin was associated with genome duplications, may be best studied in natural diploid and polyploid populations. In a diploid/tetraploid (2n/4n) geographic contact zone of Palearctic green toads in northern Kyrgyzstan, we examine 4ns and triploids (3n) of unknown genetic composition and origins. Using mitochondrial and nuclear sequence, and nuclear microsatellite markers in 84 individuals, we show that 4n (Bufo pewzowi) are allopolyploids, with a geographically proximate 2n species (B. turanensis) being their maternal ancestor and their paternal ancestor as yet unidentified. Local 3n forms arise through hybridization. Adult 3n mature males (B. turanensis mtDNA) have 2n mothers and 4n fathers, but seem distinguishable by nuclear profiles from partly aneuploid 3n tadpoles (with B. pewzowi mtDNA). These observations suggest multiple pathways to the formation of triploids in the contact zone, involving both reciprocal origins. To explain the phenomena in the system, we favor a hypothesis where 3n males (with B. turanensis mtDNA) backcross with 4n and 2n females. Together with previous studies of a separately evolved, sexually reproducing 3n lineage, these observations reveal complex reproductive interactions among toads of different ploidy levels and multiple pathways to the evolution of polyploid lineages.

Evaluation of elevated ploidy and asexual reproduction as alternative explanations for geographic parthenogenesis in Eucypris virens ostracods.

Transitions from sexual to asexual reproduction are often coupled with elevations in ploidy. As a consequence, the importance of ploidy per se for the maintenance and spread of asexual populations is unclear. To examine the effects of ploidy and asexual reproduction as independent determinants of the success of asexual lineages, we sampled diploid sexual, diploid asexual, and triploid asexual Eucypris virens ostracods across a European wide range. Applying nuclear and mitochondrial markers, we found that E. virens consists of genetically highly differentiated diploid sexual populations, to the extent that these sexual clades could be considered as cryptic species. All sexual populations were found in southern Europe and North Africa and we found that both diploid asexual and triploid asexual lineages have originated multiple times from several sexual lineages. Therefore, the asexual lineages show a wide variety of genetic backgrounds and very strong population genetic structure across the wide geographic range. Finally, we found that triploid, but not diploid, asexual clones dominate habitats in northern Europe. The limited distribution of diploid asexual lineages, despite their shared ancestry with triploid asexual lineages, strongly suggests that the wider geographic distribution of triploids is due to elevated ploidy rather than to asexuality.

Quantifying the threat of extinction from Muller's ratchet in the diploid Amazon molly (Poecilia formosa).

BACKGROUND: The Amazon molly (Poecilia formosa) is a small unisexual fish that has been suspected of being threatened by extinction from the stochastic accumulation of slightly deleterious mutations that is caused by Muller's ratchet in non-recombining populations. However, no detailed quantification of the extent of this threat is available. RESULTS: Here we quantify genomic decay in this fish by using a simple model of Muller's ratchet with the most realistic parameter combinations available employing the evolution@home global computing system. We also describe simple extensions of the standard model of Muller's ratchet that allow us to deal with selfing diploids, triploids and mitotic recombination. We show that Muller's ratchet creates a threat of extinction for the Amazon molly for many biologically realistic parameter combinations. In most cases, extinction is expected to occur within a time frame that is less than previous estimates of the age of the species, leading to a genomic decay paradox. CONCLUSION: How then does the Amazon molly survive? Several biological processes could individually or in combination solve this genomic decay paradox, including paternal leakage of undamaged DNA from sexual sister species, compensatory mutations and many others. More research is needed to quantify the contribution of these potential solutions towards the survival of the Amazon molly and other (ancient) asexual species.

A tetraploid Amazon molly, Poecilia formosa.

Polyploidization is thought to be an important driving force in evolution as it increases the genetic material on which mutation and selection can act. In the Amazon molly, Poecilia formosa, triploid genotypes can be found in the field and frequently arise from diploid breeding stocks, a tetraploid individual, however, was so far never documented. Here, we report the first tetraploid Amazon molly. Flow cytometry clearly showed the tetraploid DNA content, whereas microsatellite analysis not only confirmed the tetraploidy but also pointed to allotetraploidy. Most likely the fourth genome was received through paternal leakage, namely, by fertilization of a triploid egg with a haploid sperm. The existence of tetraploid individuals offers new explanations for the enormous clonal diversity observed in wild populations of P. formosa.

Automictic reproduction in interspecific hybrids of poeciliid fish.

Automixis, the process whereby the fusion of meiotic products restores the diploid state of the egg, is a common mode of reproduction in plants but has also been described in invertebrate animals. In vertebrates, however, automixis has so far only been discussed as one of several explanations for isolated cases of facultative parthenogenesis. Analyzing oocyte formation in F1 hybrids derived from Poecilia mexicana limantouri and P. latipinna crosses (the cross that led to the formation of the gynogenetic Poecilia formosa), we found molecular evidence for automictic oocyte production. The mechanism involves the random fusion of meiotic products after the second meiotic division. The fertilization of diploid oocytes gives rise to fully viable triploid offspring. Although the automictic production of diploid oocytes as seen in these F1 hybrids clearly represents a preadaptation to parthenogenetic reproduction, it is also a powerful intrinsic postzygotic isolation mechanism because the resulting next generation triploids were always sterile. The mechanism described here can explain facultative parthenogenesis, as well as varying ploidy levels reported in different animal groups. Most importantly, at least some of the reported cases of triploidy in humans can now be traced back to automixis.