Gametic Isolation Research Articles

Chromosomal evolution, environmental heterogeneity, and migration drive spatial patterns of species richness in Calochortus (Liliaceae)

We used nuclear genomic data and statistical models to evaluate the ecological and evolutionary processes shaping spatial variation in species richness in Calochortus (Liliaceae, 74 spp.). Calochortus occupies diverse habitats in the western United States and Mexico and has a center of diversity in the California Floristic Province, marked by multiple orogenies, winter rainfall, and highly divergent climates and substrates (including serpentine). We used sequences of 294 low-copy nuclear loci to produce a time-calibrated phylogeny, estimate historical biogeography, and test hypotheses regarding drivers of present-day spatial patterns in species number. Speciation and species coexistence require reproductive isolation and ecological divergence, so we examined the roles of chromosome number, environmental heterogeneity, and migration in shaping local species richness. Six major clades-inhabiting different geographic/climatic areas, and often marked by different base chromosome numbers (n = 6 to 10)-began diverging from each other ~10.3 Mya. As predicted, local species number increased significantly with local heterogeneity in chromosome number, elevation, soil characteristics, and serpentine presence. Species richness is greatest in the Transverse/Peninsular Ranges where clades with different chromosome numbers overlap, topographic complexity provides diverse conditions over short distances, and several physiographic provinces meet allowing immigration by several clades. Recently diverged sister-species pairs generally have peri-patric distributions, and maximum geographic overlap between species increases over the first million years since divergence, suggesting that chromosomal evolution, genetic divergence leading to gametic isolation or hybrid inviability/sterility, and/or ecological divergence over small spatial scales may permit species co-occurrence.

CRISPR-Cas9-based method for isolating microgametes of Eimeria tenella

Eimeria tenella infections are known to cause severe caecal damage and death of the infected chicken. Gamogony is an essential stage in E. tenella life cycle and in the establishment of coccidiosis. Prior research had extensively explored isolation and separation of the parasite gametes - microgamete (male) and macrogamete (female). However, there is little information on the efficient, highly purified and distinctly separated male and female gametes. In this study, we generated a genome editing line expressing mCherry fluorescent protein fused with GCS1 protein in E. tenella by using Toxoplasma gondii CRISPR-Cas9 system, flow cytometry and fluorescence microscopy. This allowed precise separation of E. tenella male and female gametes in the transgenic parasite population. The separation of male and female gametes would not only build on our understanding of E. tenella transmission, but it would also facilitate development of gametocidal compounds as drug targets for E. tenella infection.

Meiotic analysis of induced translocation heterozygotes in Lepidium sativum Linn.

Aim: To assess the cytological effects of Gamma rays on the pollen mother cells and post meiotic products of Lepidium sativum Linn. which resulted in isolation of translocation heterozygote and aneuploid gametes. Methodology: During the experiment, parameters such as Meiotic stages, chromosomal structure, and pollen fertility were studied. Fresh seeds of Garden cress were orderly arranged in plastic and divided into four groups. The fresh seeds of each group were individually irradiated with 60Gy, 150Gy, 300Gy, 450Gy and 600Gy @ 7.247KGyh-1 in the Floriculture Laboratory of National Botanical Research Institute (NBRI), Lucknow (India). The source for gamma ray irradiation was Cobalt-60. The irradiated seeds were sown in triplicates along with their respective controls. Results: Meiotic analysis of pollen mother cells (PMCs) at 300Gy showed translocation heterozygotes, however, in the control sets, the rate was negligible and phenotypically the plants were weak and short heighted. The cytological illustration of chromosomal configuration at diakinesis and metaphase I exhibited the superiority of tetravalents, bivalent and other forms of chromosomal associations such as multivalents. The induced translocation heterozygotes resulted in stickiness at metaphase and anaphase states along with the formation of bridges at anaphase stage. Due to chromosomal anomalies, the pollen fertility was reduced to 37.54±0.25% in contrast to control plants (98.20±0.16%). Interpretation: The current research work would be of paramount interest as translocation heterozygote serves as a source for raising aneuploid offspring having novel genetic combinations. Key words: Chromosomal anomalies, Heterozygotes, Lepidium sativum Linn, Meiosis, Pollen fertility

Extensive introgression among strongylocentrotid sea urchins revealed by phylogenomics.

Gametic isolation is thought to play an important role in the evolution of reproductive isolation in broadcast-spawning marine invertebrates. However, it is unclear whether gametic isolation commonly evolves early in the speciation process or only accumulates after other reproductive barriers are already in place. It is also unknown whether gametic isolation is an effective barrier to introgression following speciation. Here, we used whole-genome sequencing data and multiple complementary phylogenomic approaches to test whether the well-documented gametic incompatibilities among the strongylocentrotid sea urchins have limited introgression. We quantified phylogenetic discordance, inferred reticulate phylogenetic networks, and applied the Δ statistic using gene tree topologies reconstructed from multiple sequence alignments of protein-coding single-copy orthologs. In addition, we conducted ABBA-BABA tests on genome-wide single nucleotide variants and reconstructed a phylogeny of mitochondrial genomes. Our results revealed strong mito-nuclear discordance and considerable nonrandom gene tree discordance that cannot be explained by incomplete lineage sorting alone. Eight of the nine species examined demonstrated a history of introgression with at least one other species or ancestral lineage, indicating that introgression was common during the diversification of the strongylocentrotid urchins. There was strong support for introgression between four extant species pairs (Strongylocentrotus pallidus ⇔ S. droebachiensis, S. intermedius ⇔ S. pallidus, S. purpuratus ⇔ S. fragilis, and Mesocentrotus franciscanus ⇔ Pseudocentrotus depressus) and additional evidence for introgression on internal branches of the phylogeny. Our results suggest that the existing gametic incompatibilities among the strongylocentrotid urchin species have not been a complete barrier to hybridization and introgression following speciation. Their continued divergence in the face of widespread introgression indicates that other reproductive isolating barriers likely exist and may have been more critical in establishing reproductive isolation early in speciation.

Isolation of gametes and zygotes from Setaria viridis.

Setaria viridis, the wild ancestor of foxtail millet (Setaria italica), is an effective model plant for larger C4 crops because S. viridis has several desirable traits, such as short generation time, prolific seed production and a small genome size. These advantages are well suited for investigating molecular mechanisms in angiosperms, especially C4 crop species. Here, we report a procedure for isolating gametes and zygotes from S. viridis flowers. To isolate egg cells, ovaries were harvested from unpollinated mature flowers and cut transversely, which allowed direct access to the embryo sac. Thereafter, an egg cell was released from the cut end of the basal portion of the dissected ovary. To isolate sperm cells, pollen grains released from anthers were immersed in a mannitol solution, resulting in pollen-grain bursting, which released sperm cells. Additionally, S. viridis zygotes were successfully isolated from freshly pollinated flowers. Isolated zygotes cultured in a liquid medium developed into globular-like embryos and cell masses. Thus, isolated S. viridis gametes, zygotes and embryos are attainable for detailed observations and investigations of fertilization and developmental events in angiosperms.

Endocrine disrupting chemicals and their detection in an IVF laboratory

Abstract The endocrine disruptors are chemicals with the capacity to influence physiological processes in the organism, most often through hormonal control. They are present in the environment and in the products of daily use. They are often found in food, released from plastic bottles for water, present in cosmetics or fertilizers. Latest research suggests that they can be released from plastics used in the IVF laboratories and can be even present in the manipulation and cultivation media used for isolation and fertilization of gametes and subsequent cultivation of embryos. Permanent and long-term utilization of these substances has adverse effects in human reproductive health, mainly by the means of interfering with synthesis and action mechanisms of reproductive hormones. Moreover, some endocrine disruptors show a range of adverse effects directly on the gametes or embryos cultured in the in vitro conditions. The article provides an overview on bisphenols detected in plastics and media commonly used in the IVF laboratory and considers their possible impact on effectiveness of the IVF methods in a human laboratory.

Aneuploidy rates and morphokinetic parameters of embryos cultured in distinct culture media: a sibling oocyte study.

Do embryos from sibling oocytes assigned to distinct single-step media culture systems demonstrate differences in early embryo development, morphokinectics or aneuploidy rates? Embryo quality, morphokinetic parameters and aneuploidy rates from trophectoderm biopsy were similar between sibling embryos cultured in distinct media systems from the time of gamete isolation. Studies on the effect of commercially available embryo culture media systems have demonstrated inconsistent impact on human embryonic development, morphokinetics, aneuploidy rates and clinical outcomes. In addition, these studies have been primarily randomized at the level of the embryo or the patient to culture media. Prospective sibling oocyte cohort derived from 200 subjects undergoing IVF at a tertiary academic medical center between February 2018 and November 2019. Sibling oocytes were allocated to Global® or SAGE® media system based upon laterality of ovary from which they were retrieved. All embryos were cultured in a time-lapse incubator. Blastocysts underwent trophectoderm biopsy for preimplantation genetic testing for aneuploidy using next-generation sequencing. One hundred twenty-seven subjects (n = 127) had paired blastocysts for biopsy in each culture media system. There was no difference in top quality blastocyst formation (47.1 ± 31.0 vs 48.1 ± 27.2%; P = 0.87) nor aneuploidy rate (62.3 ± 34.0 vs 56.1 ± 34.4%; P = 0.07) for sibling embryos cultured in Global versus SAGE media system. Embryo morphokinetic parameters including time to each cell division from two cells (t2) to eight cells (t8), time to morula stage (tM), time to blastocele formation (tSB), time to fully formed blastocyst (tB) and time to expansion of the blastocyst (tEB) were similar between paired blastocysts from each culture media system. Pregnancy outcomes and offspring health data were not available for analysis. Commercially available culture media may not have a differential impact on embryo development and blastocyst aneuploidy rate when patient and stimulation-related factors are held constant. There was no external funding for this study. C.H. is owner of a consultancy company, IVF Professionals, Chief Scientific Officer at Apricity, Executive Director at TMRW and co-owner and shareholder of Aria Fertility. She has received speaker fees, consulting fees and travel support from Cooper Surgical and Vitrolife. J.B. is an employee and shareholder of vitrolife. N/A.

P–221 Prospective randomized sibling study on gamete preparation, insemination and subsequent culture of human oocytes in a time-lapse system using media systems with and without antioxidants

Abstract Study question Does the addition of antioxidants for gamete preparation, insemination and embryo culture lead to differences in embryo development and clinical outcome Summary answer Using an antioxidant-containing media system for sperm preparation, insemination and embryo culture imparts significantly higher good-quality blastocyst rates and improved clinical outcome in elderly patients. What is known already A previous study showed that adding combined antioxidants for sequential embryo culture in conventional incubators (interrupted culture) improves embryo viability and clinical outcome, especially for elderly patients. Here we investigated the combined effect of three antioxidants Acetyl-L-Carnitine (10 µM), N-Acetyl-L-Cysteine (10 µM), and α-Lipoic Acid (5 µM) during sperm preparation, insemination, and time-lapse culture in a single step medium on human embryo development and clinical outcome. Study design, size, duration Prospective randomized single center study including 143 couples for IVF/ICSI between August 2018 and December 2019. Inclusion required at least eight cumulus-oocyte-complexes (COCs) after retrieval. Cycles involving PGT, split IVF/ICSI, and surgically retrieved sperm were excluded. Immediately after retrieval oocytes were randomly distributed to a study or control media system with or without antioxidants (Vitrolife). Similarly, ejaculates were split and prepared with and without antioxidants. Participants/materials, setting, methods Sibling oocytes were inseminated in the respective group with accordingly prepared sperm. Single step embryo culture was conducted in medium with (Gx-TL) and without (G-TL) antioxidants in the EmbryoScope+. Embryo quality and clinical outcome were assessed in relation to maternal age (<35/>35 years). Good-quality embryos on day 3 were defined as 8- to 10-cells with even cells and low fragmentation; good-quality blastocysts as > 3BB. Clinical outcome was assessed after single vitrified blastocyst transfer (SVBT). Main results and the role of chance From 143 participants (female age, 34.7±3.2 years), a total of 2424 COCs were collected; 1180 COCs/916 metaphase-II (MII) oocytes were allocated to Gx-TL media and 1244 COCs/981 MII oocytes to G-TL media. Age-related analysis in Gx-TL compared with G-TL in relation to allocated MII oocytes revealed a trend for higher fertilization rates in Gx-TL for both age groups (<35: 72.1% vs. 66.9%; >35: 70.7% vs. 64.9%, P < 0.1). Good-quality day 3 embryo development/MII oocytes was higher, albeit not significant, in the elderly patients in Gx-TL (<35: 35.9% vs. 34.4%; >35: 31.1% vs. 27.9%). Overall day 5/6 blastocyst rate was similar for both media (<35: 48.2% vs. 49.9%; >35: 42.3% vs. 39.5%). Day 5/6 GQB rate was comparable for younger patients (<35: 23.8% for Gx-TL vs. 26.0% for G-TL) but significantly higher in Gx-TL in elderly patients (>35: 20.7% vs. 14.4%; P < 0.05). A total of 200 SVBT were performed; 99 in the Gx-TL- and 101 in the G-TL-arm. We noted almost similar implantation and ongoing pregnancy rates between Gx-TL vs G-TL in the younger (<35) age group (50.0% vs. 55.4%; 50.0% vs. 55.6%) but higher albeit not significant rates for Gx-TL in older (>35) patients (44.1% vs. 33.3%; 44.1% vs. 33.3%). Limitations, reasons for caution In almost 95% of the cycles, oocytes were inseminated by ICSI; thus results may not equally apply for cycles with IVF. The use of a closed time-lapse system may have prevented from some environmental oxidative stress. Therefore results may come out different with a similar study using standard incubation. Wider implications of the findings: Supplementation of antioxidants to media for gamete isolation and preparation, as well as subsequent single step time-lapse culture may improve GQE/B rates and clinical outcomes in certain age groups, plausibly through the reduction of oxidative stress. Further studies in selected sub-groups (severe OAT syndrome / testicular cases) may be indicated. Trial registration number UMIN000034482

Proteins of penial mamilliform glands in closely related <em>Littorina</em> species (Mollusca, Caenogastropoda): variability and possible contribution to reproductive isolation

The forces driving reproductive isolation emergence during the process of sympatric speciation are still intensely debated. Mechanisms of gametic isolation (which are known to form rapidly in several models) take the central place in these debates. Nevertheless, the approximative capacity of a few investigated models to other taxa could be questioned, generating demand for the adoption of additional model organisms to study sympatric speciation. The group of closely related species of the genus Littorina (subgenus Neritrema) sympatrically inhabiting seashores are promising. In this study, we performed comparative proteomic analysis of penial tissues of four Neritrema species to identify potential effectors contributing to gametic isolation. Among 272 analyzed proteins, 13 mamilliform gland-specific proteins (possibly transferred to the female during copulation) were detected, as well as five proteins specifically expressed in the epithelium of the penial basal part. Eight of these proteins were species-specific and may be involved in the maintenance of reproductive barriers.

Isolation of male and female gametes, zygotes and proembryos of leek (Allium tuberosum Roxb).

The isolation of male and female gametes is an effective method to study the fertilization mechanisms of higher plants. An osmotic shock method was used to rupture pollen grains of Allium tuberosum Roxb and release the pollen contents, including generative cells, which were mass collected. The pollinated styles were cut following 3 h of in vivo growth, and cultured in medium for 6-8 h, during which time pollen tubes grew out of the cut end of the style. After pollen tubes were transferred into a solution containing 6% mannitol, tubes burst and released pairs of sperm cells. Ovules of A. tuberosum were incubated in an enzyme solution for 30 min, and then dissected to remove the integuments. Following transfer to a dissecting solution free of enzymes, each nucellus was cut in the middle, and squeezed gently on the micropylar end, resulting in the liberation of the egg, zygote and proembryo from ovules at selected stages. These cells can be used to explore fertilization and embryonic development using molecular biological methods for each cell type and development stage.

Gene Expression and Genome Editing Systems by Direct Delivery of Macromolecules Into Rice Egg Cells and Zygotes.

Polyethylene glycol calcium (PEG-Ca2+)-mediated transfection allows rapid and efficient examination to analyze diverse cellular functions of genes of interest. In plant cells, macromolecules, such as DNA, RNA and protein, are delivered into protoplasts derived from somatic tissues or calli via PEG-Ca2+ transfection. To broaden and develop the scope of investigations using plant gametes and zygotes, a procedure for direct delivery of macromolecules into these cells has recently been established using PEG-Ca2+ transfection. This PEG-Ca2+-mediated delivery into rice egg cells/zygotes consists of four microtechniques, (i) isolation of gametes, (ii) production of zygotes by electrofusion of gametes, (iii) PEG-Ca2+-mediated delivery of macromolecules into isolated egg cells or produced zygotes, and (iv) culture and subsequent analyses of the transfected egg cells/zygotes. Because the full protocol for microtechniques (i) and (ii) have already been reported in Toda et al., 2016 , microtechniques (iii) and (iv) are mainly described in this protocol.

In Vitro Production of Zygotes by Electrofusion of Rice Gametes.

In angiosperms, fertilization and embryogenesis occur in the embryo sac, which is deeply embedded in ovular tissue. In vitro fertilization (IVF) systems using isolated gametes have been utilized to dissect postfertilization events in angiosperms, such as egg activation, zygotic development, and early embryogenesis. In addition, using IVF systems, interspecific zygotes and polyploid zygotes have been artificially produced, and their developmental profiles/mechanisms have been analyzed. Taken together, the IVF system can be considered a powerful technique for investigating the fertilization-induced developmental sequences in zygotes and generating new cultivars with desirable characteristics. Here, we describe the procedures for the isolation of rice gametes, electrofusion of gametes, and the culture of the produced zygotes and embryo.

Step-by-step protocols for rice gamete isolation

A detailed, step-by-step protocol for isolation of rice gametes for transcriptional profiling, with a general workflow that includes controls for RNA contamination from surrounding cells and tissues is presented. Characterization of the transcriptome and other -omics studies of flowering plant gametes are challenging as a consequence of the small sizes and relative inaccessibility of these cells. Collecting such poorly represented cells is also complicated by potential contamination from surrounding sporophytic, adjacent gametophytic tissues and difficulties in extracting high-quality intact cells. Here we present detailed, step-by-step procedures for collecting intact, unfixed rice (Oryza sativa) egg cells and sperm cells without enzymatic treatments. In addition, we also present a general workflow for assessing sample purity by RT-PCR, using primers specific for marker genes preferentially expressed in surrounding cells and tissues. These protocols should facilitate future studies of genome-scale characterization of gametes in this important model crop.

Genetic Contributions to Ectopic Sperm Cell Migration in Caenorhabditis Nematodes.

Reproductive barriers involving gametic incompatibilities can act to enhance population divergence and promote the persistence of species boundaries. Observing gametic interactions in internal fertilizing organisms, however, presents a considerable practical challenge to characterizing mechanisms of such gametic isolation. Here we exploit the transparency of Caenorhabditis nematodes to investigate gametic isolation mediated by sperm that can migrate to ectopic locations, with this sperm invasion capable of inducing female sterility and premature death. As a step toward identifying genetic factors and mechanisms associated with female susceptibility to sperm invasion, we characterized a panel of 25 C. elegans genetic mutants to test for effects on the incidence and severity of sperm invasion in both conspecific and inter-species matings. We found genetic perturbations to contribute to distinct patterns of susceptibility that identify ovulation dynamics and sperm guidance cues as modulators of ectopic sperm migration incidence and severity. Genotypes confer distinctive phenotypic sensitivities to the sperm from conspecific C. elegans males vs. heterospecific C. nigoni males, implicating evolution of functional divergence in the history of these species for components of sperm-reproductive tract interactions. Sexually-antagonistic co-evolution within species that drives divergent trait and molecular evolution between species provides a working model to explain mismatched species-specific gametic interactions that promote or mitigate ectopic sperm migration.

X exceptionalism in Caenorhabditis speciation.

Speciation genetics research in diverse organisms shows the X-chromosome to be exceptional in how it contributes to "rules" of speciation. Until recently, however, the nematode phylum has been nearly silent on this issue, despite the model organism Caenorhabditis elegans having touched most other topics in biology. Studies of speciation with Caenorhabditis accelerated with the recent discovery of species pairs showing partial interfertility. The resulting genetic analyses of reproductive isolation in nematodes demonstrate key roles for the X-chromosome in hybrid male sterility and inviability, opening up new understanding of the genetic causes of Haldane's rule, Darwin's corollary to Haldane's rule, and enabling tests of the large-X effect hypothesis. Studies to date implicate improper chromatin regulation of the X-chromosome by small RNA pathways as integral to hybrid male dysfunction. Sexual transitions in reproductive mode to self-fertilizing hermaphroditism inject distinctive molecular evolutionary features into the speciation process for some species. Caenorhabditis also provides unique opportunities for analysis in a system with XO sex determination that lacks a Y-chromosome, sex chromosome-dependent sperm competition differences and mechanisms of gametic isolation, exceptional accessibility to the development process and rapid experimental evolution. As genetic analysis of reproductive isolation matures with investigation of multiple pairs of Caenorhabditis species and new species discovery, nematodes will provide a powerful complement to more established study organisms for deciphering the genetic basis of and rules to speciation.

Evolutionary Dynamics of Male Reproductive Genes in the Drosophila virilis Subgroup.

Postcopulatory sexual selection (PCSS) is a potent evolutionary force that can drive rapid changes of reproductive genes within species, and thus has the potential to generate reproductive incompatibilities between species. Male seminal fluid proteins (SFPs) are major players in postmating interactions, and are important targets of PCSS in males. The virilis subgroup of Drosophila exhibits strong interspecific gametic incompatibilities, and can serve as a model to study the genetic basis of PCSS and gametic isolation. However, reproductive genes in this group have not been characterized. Here we utilize short-read RNA sequencing of male reproductive organs to examine the evolutionary dynamics of reproductive genes in members of the virilis subgroup: D. americana, D. lummei, D. novamexicana, and D. virilis. We find that the majority of male reproductive transcripts are testes-biased, accounting for ∼15% of all annotated genes. Ejaculatory bulb (EB)-biased transcripts largely code for lipid metabolic enzymes, and contain orthologs of the D. melanogaster EB protein, Peb-me, which is involved in mating-plug formation. In addition, we identify 71 candidate SFPs, and show that this gene set has the highest rate of nonsynonymous codon substitution relative to testes- and EB-biased genes. Furthermore, we identify orthologs of 35 D. melanogaster SFPs that have conserved accessory gland expression in the virilis group. Finally, we show that several of the SFPs that have the highest rate of nonsynonymous codon substitution reside on chromosomal regions, which contributes to paternal gametic incompatibility between species. Our results show that SFPs rapidly diversify in the virilis group, and suggest that they likely play a role in PCSS and/or gametic isolation.

Isolation of GFP-tagged plasma membrane protein from Arabidopsis egg cells.

Angiosperms possess a double fertilization system for sexual reproduction. Double fertilization is regulated by interactions among proteins localized in the plasma membrane of each sex gamete. A few plasma membrane resident proteins regulating double fertilization have been identified in male gametes. In contrast, no fertilization regulators in female gamete plasma membrane have been identified, largely due to difficulties in the isolation and collection of female gametes. We had produced Arabidopsis transgenic plant pDD45::GFP-AtPIP2;1 where the egg cell plasma membrane was specifically labeled with GFP (Igawa et al. 2013). The protein extract derived from approximately 200 pistils, which contained unfertilized and mature egg cells, was subjected to immunoprecipitation using anti-GFP antibody. As a result, both GFP and AtPIP2;1 were specifically detected in immunoprecipitated proteins from pistil tissues of pDD45::GFP-AtPIP2;1 transgenic plant, but not in those of wild type pistils. It was revealed that specific proteins expressed in the egg cells were successfully isolated from pistil cell population. The method described here showed the feasibility of isolating specific egg cell plasma membrane protein without gamete isolation and collection procedures.

Isolation of Fucus serratus Gametes and Cultivation of the Zygotes.

Zygotes of the Fucale species are a powerful model system to study cell polarization and asymmetrical cell division (Bisgrove and Kropf, 2008). The Fucale species of brown algae grow in the intertidal zone where they reproduce by releasing large female eggs and mobile sperm in the surrounding seawater. The gamete release can be induced from sexually mature fronds in the laboratory and thousands of synchronously developing zygotes are easily obtained. In contrast to other eukaryotic models, such as land plants (Brownlee and Berger, 1995), the embryo is free of maternal tissues and therefore readily amenable to pharmacological approaches. The zygotes are relatively large (up to 100 µm in diameter), facilitating manipulations and imaging studies. During the first hours of zygote development, the alignment of the axis to external cues such as light is labile and can be reversed by light gradients from different directions. A few hours before rhizoid emergence, the alignment of the axis and the polarity are fixed and the cells germinate accordingly. At this stage the zygotes are naturally attached to the substratum through the secretion of cell wall adhesive materials ( Kropf et al., 1988 ; Hervé et al., 2016 ). The first cell division occurs about 24 h after fertilisation and the early embryo is composed of only two cell types that differ in size, shape and developmental fates (i.e., thallus cells and rhizoid cells) ( Bouget et al., 1998 ). The embryo can be successfully cultivated in the laboratory for a few more days (4 weeks maximum) and has an invariant division pattern during the early stages, which allows cell lineages to be traced histologically.

Fertilization success of sterlet Acipenser ruthenus and Siberian sturgeon Acipenser baerii gametes under conditions of heterospecific mating

Species may be prevented from interspecific hybridization by a number of different reproductive barriers that operate precopulatory and postcopulatory. In situation, when natural precopulatory reproductive barriers are affected by anthropogenic factors, postcopulatory reproductive barriers may be important for maintaining gametic isolation and hence preventing interspecific hybridization. This is highly topical in sturgeon (order Acipenseriformes) which exhibits remarkable ease of interspecific hybridization. The objectives of the present study were to evaluate the fertilization success of Acipenser ruthenus and Acipenser baerii spermatozoa under the interspecific competitive conditions and assessed, whether their spermatozoa tend to differentially fertilize eggs of conspecifics. We set up several in vitro fertilization experiments: (i) pooled eggs of both species were fertilized by sperm of each species separately; (ii) eggs of each species were fertilized by pooled sperm; (iii) pooled eggs were fertilized by pooled sperm and (iv) purebred and hybrid control groups. Using parental assignment by molecular markers, we found that when these species competed in pooled sperm, 78.9% of progeny were sired by A. ruthenus and 21.1% by A. baerii, demonstrating higher fertilization success for the former, irrespective of conspecificity of fertilized eggs. When pooled eggs were inseminated by A. ruthenus or A. baerii sperm separately, progeny almost equally comprised hybrid and purebred individuals. Hence, neither A. ruthenus nor A. baerii eggs showed a tendency to biased fertilization by spermatozoa of conspecific males. These findings together show that there may not be postcopulatory mechanisms preventing hybridization between A. ruthenus and A. baerii.

Correlated evolution of male and female reproductive traits drive a cascading effect of reinforcement in Drosophila yakuba.

Selection against maladaptive hybridization can drive the evolution of reproductive isolation in a process called reinforcement. While the importance of reinforcement in evolution has been historically debated, many examples now exist. Despite these examples, we typically lack a detailed understanding of the mechanisms limiting the spread of reinforced phenotypes throughout a species' range. Here we address this issue in the fruit fly Drosophila yakuba, a species that hybridizes with its sister species D. santomea and is undergoing reinforcement in a well-defined hybrid zone on the island of São Tomé. Within this region, female D. yakuba show increased postmating-prezygotic (gametic) isolation towards D. santomea when compared with females from allopatric populations. We use a combination of natural collections, fertility assays, and experimental evolution to understand why reinforced gametic isolation in D. yakuba is confined to this hybrid zone. We show that, among other traits, D. yakuba males from sympatric populations sire fewer progeny than allopatric males when mated to allopatric D. yakuba females. Our results provide a novel example of reinforcement acting on a postmating-prezygotic trait in males, resulting in a cascade of reproductive isolation among conspecific populations.