ProSeq4: A user-friendly multiplatform program for preparation and analysis of large-scale DNA polymorphism datasets.

Preparation of DNA polymorphism datasets for analysis is an important step in evolutionary genetic and molecular ecology studies. Ever-growing dataset sizes make this step time consuming, but few convenient software tools are available to facilitate processing of large-scale datasets including thousands of sequence alignments. Here I report "processor of sequences v4" (proSeq4)-a user-friendly multiplatform software for preparation and evolutionary genetic analyses of genome- or transcriptome-scale sequence polymorphism datasets. The program has an easy-to-use graphic user interface and is designed to process and analyse many thousands of datasets. It supports over two dozen file formats, includes a flexible sequence editor and various tools for data visualization, quality control and most commonly used evolutionary genetic analyses, such as NJ-phylogeny reconstruction, DNA polymorphism analyses and coalescent simulations. Command line tools (e.g. vcf2fasta) are also provided for easier integration into bioinformatic pipelines. Apart of molecular ecology and evolution research, proSeq4 may be useful for teaching, e.g. for visual illustration of different shapes of phylogenies generated with coalescent simulations in different scenarios. ProSeq4 source code and binaries for Windows, MacOS and Ubuntu are available from https://sourceforge.net/projects/proseq/.

How does evolution work in superabundant microbes?

Marine phytoplankton play crucial roles in the Earth's ecological, chemical, and geological processes. They are responsible for about half of global primary production and drive the ocean biological carbon pump. Understanding how plankton species may adapt to the Earth's rapidly changing environments is evidently an urgent priority. This problem requires evolutionary genetic approaches as evolution occurs at the level of allele frequency change within populations driven by genetic drift and natural selection (microevolution). Plankters such as the coccolithophore Gephyrocapsa huxleyi and the cyanobacterium Prochlorococcus 'marinus' are among Earth's most abundant organisms. In this opinion paper we discuss how evolution in astronomically large populations of superabundant microbes (SAMs) may act fundamentally differently than it does in the populations of more modest size found in well-studied organisms. This offers exciting opportunities to study evolution in the conditions that have yet to be explored and also leads to unique challenges. Exploring these opportunities and challenges is the goal of this article.

Evolution of a plant sex chromosome driven by expanding pericentromeric recombination suppression.

Recombination suppression around sex-determining gene(s) is a key step in evolution of sex chromosomes, but it is not well understood how it evolves. Recently evolved sex-linked regions offer an opportunity to understand the mechanisms of recombination cessation. This paper analyses such a region on Silene latifolia (Caryophyllaceae) sex chromosomes, where recombination was suppressed in the last 120 thousand years ("stratum 3"). Locating the boundaries of the stratum 3 in S. latifolia genome sequence revealed that this region is far larger than assumed previously-it is about 14 Mb long and includes 202 annotated genes. A gradient of X:Y divergence detected in the stratum 3, with divergence increasing proximally, indicates gradual recombination cessation, possibly caused by expansion of pericentromeric recombination suppression (PRS) into the pseudoautosomal region. Expansion of PRS was also the likely cause for the formation of the older stratum 2 on S. latifolia sex chromosomes. The role of PRS in sex chromosome evolution has been underappreciated, but it may be a significant factor, especially in the species with large chromosomes where PRS is often extensive.

Pericentromeric recombination suppression and the 'large X effect' in plants.

X chromosome was reported to be a major contributor to isolation between closely related species-the 'large X' effect (LXE). The causes of LXE are not clear, but the leading theory is that it is caused by recessive species incompatibilities exposed in the phenotype due to the hemizygosity of X-linked genes in the heterogametic sex. However, the LXE was also reported in species with relatively recently evolved sex chromosomes where Y chromosome is not completely degenerate and X-linked genes are not hemizygous, such as the plant Silene latifolia. Recent genome sequencing and detailed genetic mapping in this species revealed a massive (> 330 Mb) non- or rarely-recombining pericentromeric region on the X chromosome (Xpr) that comprises ~ 90% of the chromosome and over 13% of the entire genome. If any of the Xpr genes are involved in species incompatibilities, this would oppose interspecific gene flow for other genes tightly linked in the Xpr. Here we test the hypothesis that the previously reported LXE in S. latifolia is caused by the lack of recombination on most of the X chromosome. Based on genome-wide analysis of DNA polymorphism and gene expression in S. latifolia and its close cross-compatible relative S. dioica, we report that the rarely-recombining regions represent a significant barrier for interspecific gene flow. We found little evidence for any additional factors contributing to the LXE, suggesting that extensive pericentromeric recombination suppression on the X-chromosome is the major if not the only cause of the LXE in S. latifolia and S. dioica.

Evolution of sex-determination in dioecious plants: From active Y to X/A balance?

Sex chromosomes in plants have been known for a century, but only recently have we begun to understand the mechanisms behind sex determination in dioecious plants. Here, we discuss evolution of sex determination, focusing on Silene latifolia, where evolution of separate sexes is consistent with the classic "two mutations" model-a loss of function male sterility mutation and a gain of function gynoecium suppression mutation, which turned an ancestral hermaphroditic population into separate males and females. Interestingly, the gynoecium suppression function in S. latifolia evolved via loss of function in at least two sex-linked genes and works via gene dosage balance between sex-linked, and autosomal genes. This system resembles X/A-ratio-based sex determination systems in Drosophila and Rumex, and could represent a steppingstone in the evolution of X/A-ratio-based sex determination from an active Y system.

The role of recombination landscape in species hybridisation and speciation.

It is now well recognised that closely related species can hybridize and exchange genetic material, which may promote or oppose adaptation and speciation. In some cases, interspecific hybridisation is very common, making it surprising that species identity is preserved despite active gene exchange. The genomes of most eukaryotic species are highly heterogeneous with regard to gene density, abundance of repetitive DNA, chromatin compactisation etc, which can make certain genomic regions more prone or more resistant to introgression of genetic material from other species. Heterogeneity in local recombination rate underpins many of the observed patterns across the genome (e.g. actively recombining regions are typically gene rich and depleted for repetitive DNA) and it can strongly affect the permeability of genomic regions to interspecific introgression. The larger the region lacking recombination, the higher the chance for the presence of species incompatibility gene(s) in that region, making the entire non- or rarely recombining block impermeable to interspecific introgression. Large plant genomes tend to have highly heterogeneous recombination landscape, with recombination frequently occurring at the ends of the chromosomes and central regions lacking recombination. In this paper we review the relationship between recombination and introgression in plants and argue that large rarely recombining regions likely play a major role in preserving species identity in actively hybridising plant species.

Mechanisms of prezygotic post-pollination reproductive barriers in plants.

Hybridisation between individuals of different species can lead to maladapted or inviable progeny due to genetic incompatibilities between diverging species. On the other hand, mating with close relatives, or self-fertilisation may lead to inbreeding depression. Thus, both too much or too little divergence may lead to problems and the organisms have to carefully choose mating partners to avoid both of these pitfalls. In plants this choice occurs at many stages during reproduction, but pollen-pistil interactions play a particularly important role in avoiding inbreeding and hybridisation with other species. Interestingly, the mechanisms involved in avoidance of selfing and interspecific hybridisation may work via shared molecular pathways, as self-incompatible species tend to be more 'choosy' with heterospecific pollen compared to self-compatible ones. This review discusses various prezygotic post-pollination barriers to interspecific hybridisation, with a focus on the mechanisms of pollen-pistil interactions and their role in the maintenance of species integrity.

How does speciation in marine plankton work?

Marine plankton species are ecologically important, yet, it remains unclear how they originate in the ocean, where few barriers are apparent to cause the most common type of speciation - divergence in isolation. Here I discuss the use of modern evolutionary genetic approaches to shed light on longstanding questions regarding their evolution.

Pan-European study of genotypes and phenotypes in the Arabidopsis relative Cardamine hirsuta reveals how adaptation, demography, and development shape diversity patterns.

We study natural DNA polymorphisms and associated phenotypes in the Arabidopsis relative Cardamine hirsuta. We observed strong genetic differentiation among several ancestry groups and broader distribution of Iberian relict strains in European C. hirsuta compared to Arabidopsis. We found synchronization between vegetative and reproductive development and a pervasive role for heterochronic pathways in shaping C. hirsuta natural variation. A single, fast-cycling ChFRIGIDA allele evolved adaptively allowing range expansion from glacial refugia, unlike Arabidopsis where multiple FRIGIDA haplotypes were involved. The Azores islands, where Arabidopsis is scarce, are a hotspot for C. hirsuta diversity. We identified a quantitative trait locus (QTL) in the heterochronic SPL9 transcription factor as a determinant of an Azorean morphotype. This QTL shows evidence for positive selection, and its distribution mirrors a climate gradient that broadly shaped the Azorean flora. Overall, we establish a framework to explore how the interplay of adaptation, demography, and development shaped diversity patterns of 2 related plant species.

The origin and evolution of sex chromosomes, revealed by sequencing of the Silene latifolia female genome.

White campion (Silene latifolia, Caryophyllaceae) was the first vascular plant where sex chromosomes were discovered. This species is a classic model for studies on plant sex chromosomes due to presence of large, clearly distinguishable X and Y chromosomes that originated de novo about 11 million years ago (mya), but lack of genomic resources for this relatively large genome (∼2.8 Gb) remains a significant hurdle. Here we report S. latifolia female genome assembly integrated with sex-specific genetic maps of this species, focusing on sex chromosomes and their evolution. The analysis reveals a highly heterogeneous recombination landscape with strong reduction in recombination rate in the central parts of all chromosomes. Recombination on the X chromosome in female meiosis primarily occurs at the very ends, and over 85% of the X chromosome length is located in a massive (∼330 Mb) gene-poor, rarely recombining pericentromeric region (Xpr). The results indicate that the non-recombining region on the Y chromosome (NRY) initially evolved in a relatively small (∼15 Mb), actively recombining region at the end of the q-arm, possibly as a result of inversion on the nascent X chromosome. The NRY expanded about 6 mya via linkage between the Xpr and the sex-determining region, which may have been caused by expanding pericentromeric recombination suppression on the X chromosome. These findings shed light on the origin of sex chromosomes in S. latifolia and yield genomic resources to assist ongoing and future investigations into sex chromosome evolution.

Heterochiasmy and Sex Chromosome Evolution in Silene.

The evolution of a non-recombining sex-specific region is a key step in sex chromosome evolution. Suppression of recombination between the (proto-) X- and Y-chromosomes in male meiosis creates a non-recombining Y-linked region (NRY), while the X-chromosome continues to recombine in females. Lack of recombination in the NRY defines its main properties-genetic degeneration and accumulation of repetitive DNA, making X and Y chromosomes very different from each other. How and why recombination suppression on sex chromosomes evolves remains controversial. A strong difference in recombination rates between the sexes (heterochiasmy) can facilitate or even cause recombination suppression. In the extreme case-complete lack of recombination in the heterogametic sex (achiasmy)-the entire sex-specific chromosome is automatically non-recombining. In this study, I analyse sex-specific recombination rates in a dioecious plant Silene latifolia (Caryophyllaceae), which evolved separate sexes and sex chromosomes ~11 million years ago. I reconstruct high-density RNAseq-based genetic maps including over five thousand genic markers for the two sexes separately. The comparison of the male and female maps reveals only modest heterochiasmy across the genome, with the exception of the sex chromosomes, where recombination is suppressed in males. This indicates that heterochiasmy likely played only a minor, if any, role in NRY evolution in S. latifolia, as recombination suppression is specific to NRY rather than to the entire genome in males. Other mechanisms such as structural rearrangements and/or epigenetic modifications were likely involved, and comparative genome analysis and genetic mapping in multiple Silene species will help to shed light on the mechanism(s) of recombination suppression that led to the evolution of sex chromosomes.

Rapid evolution of hybrid breakdown following recent divergence with gene flow in Senecio species on Mount Etna, Sicily.

How do nascent species evolve reproductive isolation during speciation with on-going gene flow? How do hybrid lineages become stabilised hybrid species? While commonly used genomic approaches provide an indirect way to identify species incompatibility factors, synthetic hybrids generated from interspecific crosses allow direct pinpointing of phenotypic traits involved in incompatibilities and the traits that are potentially adaptive in hybrid species. Here we report the analysis of phenotypic variation and hybrid breakdown in crosses between closely-related Senecio aethnensis and S. chrysanthemifolius, and their homoploid hybrid species, S. squalidus. The two former species represent a likely case of recent (<200 ky) speciation with gene flow driven by adaptation to contrasting conditions of high- and low-elevations on Mount Etna, Sicily. As these species form viable and fertile hybrids, it remains unclear whether they have started to evolve reproductive incompatibility. Our analysis represents the first study of phenotypic variation and hybrid breakdown involving multiple Senecio hybrid families. It revealed wide range of variation in multiple traits, including the traits previously unrecorded in synthetic hybrids. Leaf shape, highly distinct between S. aethnensis and S. chrysanthemifolius, was extremely variable in F2 hybrids, but more consistent in S. squalidus. Our study demonstrates that interspecific incompatibilities can evolve rapidly despite on-going gene flow between the species. Further work is necessary to understand the genetic bases of these incompatibilities and their role in speciation with gene flow.

A CLAVATA3-like Gene Acts as a Gynoecium Suppression Function in White Campion.

How do separate sexes originate and evolve? Plants provide many opportunities to address this question as they have diverse mating systems and separate sexes (dioecy) that evolved many times independently. The classic "two-factor" model for evolution of separate sexes proposes that males and females can evolve from hermaphrodites via the spread of male and female sterility mutations that turn hermaphrodites into females and males, respectively. This widely accepted model was inspired by early genetic work in dioecious white campion (Silene latifolia) that revealed the presence of two sex-determining factors on the Y-chromosome, though the actual genes remained unknown. Here, we report identification and functional analysis of the putative sex-determining gene in S. latifolia, corresponding to the gynoecium suppression factor (GSF). We demonstrate that GSF likely corresponds to a Y-linked CLV3-like gene that is specifically expressed in early male flower buds and encodes the protein that suppresses gynoecium development in S. latifolia. Interestingly, GSFY has a dysfunctional X-linked homolog (GSFX) and their synonymous divergence (dS = 17.9%) is consistent with the age of sex chromosomes in this species. We propose that female development in S. latifolia is controlled via the WUSCHEL-CLAVATA feedback loop, with the X-linked WUSCHEL-like and Y-linked CLV3-like genes, respectively. Evolution of dioecy in the S. latifolia ancestor likely involved inclusion of ancestral GSFY into the nonrecombining region on the nascent Y-chromosome and GSFX loss of function, which resulted in disbalance of the WUSCHEL-CLAVATA feedback loop between the sexes and ensured gynoecium suppression in males.

The Role of Interspecific Hybridisation in Adaptation and Speciation: Insights From Studies in Senecio.

Hybridisation is well documented in many species, especially plants. Although hybrid populations might be short-lived and do not evolve into new lineages, hybridisaiton could lead to evolutionary novelty, promoting adaptation and speciation. The genus Senecio (Asteraceae) has been actively used to unravel the role of hybridisation in adaptation and speciation. In this article, we first briefly describe the process of hybridisation and the state of hybridisation research over the years. We then discuss various roles of hybridisation in plant adaptation and speciation illustrated with examples from different Senecio species, but also mention other groups of organisms whenever necessary. In particular, we focus on the genomic and transcriptomic consequences of hybridisation, as well as the ecological and physiological aspects from the hybrids' point of view. Overall, this article aims to showcase the roles of hybridisation in speciation and adaptation, and the research potential of Senecio, which is part of the ecologically and economically important family, Asteraceae.

Recent expansion of the non-recombining sex-linked region on Silene latifolia sex chromosomes.

Evolution of a non-recombining sex-specific region on the Y (or W) chromosome (NRY) is a key step in sex chromosome evolution, but how recombination suppression evolves is not well understood. Studies in many different organisms indicated that NRY evolution often involves several expansion steps. Why such NRY expansions occur remains unclear, although it is though that they are likely driven by sexually antagonistic selection. This paper describes a recent NRY expansion due to shift of the pseudoautosomal boundary on the sex chromosomes of a dioecious plant Silene latifolia. The shift resulted in inclusion of at least 16 pseudoautosomal genes into the NRY. This region is pseudoautosomal in closely related Silene dioica and Silene diclinis, indicating that the NRY expansion occurred in S. latifolia after it speciated from the other species ~120 thousand years ago. As S. latifolia and S. dioica actively hybridise across Europe, interspecific gene flow could blur the PAR boundary in these species. The pseudoautosomal genes have significantly elevated genetic diversity (π ~ 3% at synonymous sites), which is consistent with balancing selection maintaining diversity in this region. The recent shift of the PAR boundary in S. latifolia offers an opportunity to study the process of on-going NRY expansion.

Evolution of the sex-determining region in Ginkgo biloba.

Sex chromosomes or sex-determining regions (SDR) have been discovered in many dioecious plant species, including the iconic 'living fossil' Ginkgo biloba, though the location and size of the SDR in G. biloba remain contradictory. Here we resolve these controversies and analyse the evolution of the SDR in this species. Based on transcriptome sequencing data from four genetic crosses we reconstruct male- and female-specific genetic maps and locate the SDR to the middle of chromosome 2. Integration of the genetic maps with the genome sequence reveals that recombination in and around the SDR is suppressed in a region of about 50 Mb in both males and females. However, occasional recombination does occur except a small, less than 5 Mb long region that does not recombine in males. Based on synonymous divergence between homologous X- and Y-linked genes in this region, we infer that the Ginkgo SDR is fairly old-at least of Cretaceous origin. The analysis of substitution rates and gene expression reveals only slight Y-degeneration. These results are consistent with findings in other dioecious plants with homomorphic sex chromosomes, where the SDR is typically small and evolves in a region with pre-existing reduced recombination, surrounded by long actively recombining pseudoautosomal regions. This article is part of the theme issue 'Sex determination and sex chromosome evolution in land plants'.

The mode of speciation during a recent radiation in open-ocean phytoplankton.

Despite the enormous ecological importance of marine phytoplankton, surprisingly little is known about how new phytoplankton species originate and evolve in the open ocean, in the absence of apparent geographic barriers that typically act as isolation mechanisms in speciation. To investigate the mechanism of open-ocean speciation, we combined fossil and climatic records from the late Quaternary with genome-wide evolutionary genetic analyses of speciation in the ubiquitous and abundant pelagic coccolithophore genus Gephyrocapsa (including G. huxleyi, formerly known as Emiliania huxleyi). Based on the analysis of 43 sequenced genomes, we report that the best-fitting scenario for all speciation events analyzed included an extended period of complete isolation followed by recent (Holocene) secondary contact, supporting the role of geographic or oceanographic barriers in population divergence and speciation. Consistent with this, fossil data reveal considerable diachroneity of species first occurrence. The timing of all speciation events coincided with glacial phases of glacial-interglacial cycles, suggesting that stronger isolation between the ocean basins and increased segregation of ecological niches during glaciations are important drivers of speciation in marine phytoplankton. The similarity across multiple speciation events implies the generality of this inferred speciation scenario for marine phytoplankton.

Microphase separation in the melts of diblock copolymers with amphiphilic blocks.

Self-assembly of graft diblock copolymers is an actual topic in the development of materials with desirable properties. In the paper, microphase separation in a melt of the diblock copolymer with amphiphilic and non-amphiphilic blocks is investigated using the analytical theory in the strong segregation approximation. Non-amphiphilic blocks are strongly immiscible with the backbone chains of amphiphilic ones but miscible with their side chains. In the theory, the amphiphilic units are considered as dimers, which can easily orient at interfaces. In the case of weakly amphiphilic dimers, the interfacial tension at a flat interface is calculated using density-functional theory. The amphiphilicity effect leads to a decrease in the surface tension and, hence, to weakening of the block stretching and decrease of the spatial period of the structure. In the case of strongly amphiphilic dimers, the phase diagrams are calculated taking into account basic morphological types (spheres and inverse spheres of amphiphilic blocks, cylinders and inverse cylinders, and lamellae). If the amphiphilicity effects dominate, the characteristic size of the amphiphilic block domain is equal to the side chain length, spherical and cylindrical micelles are formed only at very low fractions of the amphiphilic blocks, the lamellae are formed at slightly larger factions, and the micelles from non-amphiphilic blocks are separated by thin interconnected layers from amphiphilic blocks in the broad range of compositions.

Evolution of Mutation Rate in Astronomically Large Phytoplankton Populations.

Genetic diversity is expected to be proportional to population size, yet, there is a well-known, but unexplained lack of genetic diversity in large populations-the "Lewontin's paradox." Larger populations are expected to evolve lower mutation rates, which may help to explain this paradox. Here, we test this conjecture by measuring the spontaneous mutation rate in a ubiquitous unicellular marine phytoplankton species Emiliania huxleyi (Haptophyta) that has modest genetic diversity despite an astronomically large population size. Genome sequencing of E. huxleyi mutation accumulation lines revealed 455 mutations, with an unusual GC-biased mutation spectrum. This yielded an estimate of the per site mutation rate µ = 5.55×10-10 (CI 95%: 5.05×10-10 - 6.09×10-10), which corresponds to an effective population size Ne ∼ 2.7×106. Such a modest Ne is surprising for a ubiquitous and abundant species that accounts for up to 10% of global primary productivity in the oceans. Our results indicate that even exceptionally large populations do not evolve mutation rates lower than ∼10-10 per nucleotide per cell division. Consequently, the extreme disparity between modest genetic diversity and astronomically large population size in the plankton species cannot be explained by an unusually low mutation rate.

The Location of the Pseudoautosomal Boundary in Silene latifolia.

Y-chromosomes contain a non-recombining region (NRY), and in many organisms it was shown that the NRY expanded over time. How and why the NRY expands remains unclear. Young sex chromosomes, where NRY expansion occurred recently or is on-going, offer an opportunity to study the causes of this process. Here, we used the plant Silene latifolia, where sex chromosomes evolved ~11 million years ago, to study the location of the boundary between the NRY and the recombining pseudoautosomal region (PAR). The previous work devoted to the NRY/PAR boundary in S. latifolia was based on a handful of genes with locations approximately known from the genetic map. Here, we report the analysis of 86 pseudoautosomal and sex-linked genes adjacent to the S. latifolia NRY/PAR boundary to establish the location of the boundary more precisely. We take advantage of the dense genetic map and polymorphism data from wild populations to identify 20 partially sex-linked genes located in the "fuzzy boundary", that rarely recombines in male meiosis. Genes proximal to this fuzzy boundary show no evidence of recombination in males, while the genes distal to this partially-sex-linked region are actively recombining in males. Our results provide a more accurate location for the PAR boundary in S. latifolia, which will help to elucidate the causes of PAR boundary shifts leading to NRY expansion over time.