C4 photosynthesis provided an immediate demographic advantage to populations of the grass Alloteropsis semialata.

C4 photosynthesis is a key innovation in land plant evolution, but its immediate effects on population demography are unclear. We explore the early impact of the C4 trait on the trajectories of C4 and non-C4 populations of the grass Alloteropsis semialata. We combine niche models projected into paleoclimate layers for the last 5 million years with demographic models based on genomic data. The initial split between C4 and non-C4 populations was followed by a larger expansion of the ancestral C4 population, and further diversification led to the unparalleled expansion of descendant C4 populations. Overall, C4 populations spread over three continents and achieved the highest population growth, in agreement with a broader climatic niche that rendered a large potential range over time. The C4 populations that remained in the region of origin, however, experienced lower population growth, rather consistent with local geographic constraints. Moreover, the posterior transfer of some C4-related characters to non-C4 counterparts might have facilitated the recent expansion of non-C4 populations in the region of origin. Altogether, our findings support that C4 photosynthesis provided an immediate demographic advantage to A. semialata populations, but its effect might be masked by geographic contingencies.

Leaf anatomy explains the strength of C4 activity within the grass species Alloteropsis semialata.

C4 photosynthesis results from anatomical and biochemical characteristics that together concentrate CO2 around ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), increasing productivity in warm conditions. This complex trait evolved through the gradual accumulation of components, and particular species possess only some of these, resulting in weak C4 activity. The consequences of adding C4 components have been modelled and investigated through comparative approaches, but the intraspecific dynamics responsible for strengthening the C4 pathway remain largely unexplored. Here, we evaluate the link between anatomical variation and C4 activity, focusing on populations of the photosynthetically diverse grass Alloteropsis semialata that fix various proportions of carbon via the C4 cycle. The carbon isotope ratios in these populations range from values typical of C3 to those typical of C4 plants. This variation is statistically explained by a combination of leaf anatomical traits linked to the preponderance of bundle sheath tissue. We hypothesize that increased investment in bundle sheath boosts the strength of the intercellular C4 pump and shifts the balance of carbon acquisition towards the C4 cycle. Carbon isotope ratios indicating a stronger C4 pathway are associated with warmer, drier environments, suggesting that incremental anatomical alterations can lead to the emergence of C4 physiology during local adaptation within metapopulations.

Upregulation of C4 characteristics does not consistently improve photosynthetic performance in intraspecific hybrids of a grass.

C4 photosynthesis is thought to have evolved via intermediate stages, with changes towards the C4 phenotype gradually enhancing photosynthetic performance. This hypothesis is widely supported by modelling studies, but experimental tests are missing. Mixing of C4 components to generate artificial intermediates can be achieved via crossing, and the grass Alloteropsis semialata represents an outstanding study system since it includes C4 and non-C4 populations. Here, we analyse F1 hybrids between C3 and C4 , and C3 +C4 and C4 genotypes to determine whether the acquisition of C4 characteristics increases photosynthetic performance. The hybrids have leaf anatomical characters and C4 gene expression profiles that are largely intermediate between those of their parents. Carbon isotope ratios are similarly intermediate, which suggests that a partial C4 cycle coexists with C3 carbon fixation in the hybrids. This partial C4 phenotype is associated with C4 -like photosynthetic efficiency in C3 +C4 × C4 , but not in C3 × C4 hybrids, which are overall less efficient than both parents. Our results support the hypothesis that the photosynthetic gains from the upregulation of C4 characteristics depend on coordinated changes in anatomy and biochemistry. The order of acquisition of C4 components is thus constrained, with C3 +C4 species providing an essential step for C4 evolution.

Hybridization boosts dispersal of two contrasted ecotypes in a grass species.

Genetic exchanges between closely related groups of organisms with different adaptations have well-documented beneficial and detrimental consequences. In plants, pollen-mediated exchanges affect the sorting of alleles across physical landscapes and influence rates of hybridization. How these dynamics affect the emergence and spread of novel phenotypes remains only partially understood. Here, we use phylogenomics and population genomics to retrace the origin and spread of two geographically overlapping ecotypes of the African grass Alloteropsis angusta. In addition to an ecotype inhabiting wetlands, we report the existence of a previously undescribed ecotype inhabiting Miombo woodlands and grasslands. The two ecotypes are consistently associated with different nuclear groups, which represent an advanced stage of divergence with secondary low-level gene flow. However, the seed-transported chloroplast genomes are consistently shared by distinct ecotypes inhabiting the same region. These patterns suggest that the nuclear genome of one ecotype can enter the seeds of the other via occasional pollen movements with sorting of nuclear groups in subsequent generations. The contrasting ecotypes of A. angusta can thus use each other as a gateway to new locations across a large part of Africa, showing that hybridization can facilitate the geographical dispersal of distinct ecotypes of the same grass species.

Low dispersal and ploidy differences in a grass maintain photosynthetic diversity despite gene flow and habitat overlap.

Geographical isolation facilitates the emergence of distinct phenotypes within a single species, but reproductive barriers or selection are needed to maintain the polymorphism after secondary contact. Here, we explore the processes that maintain intraspecific variation of C4 photosynthesis, a complex trait that results from the combined action of multiple genes. The grass Alloteropsis semialata includes C4 and non-C4 populations, which have coexisted as a polyploid series for more than 1 million years in the miombo woodlands of Africa. Using population genomics, we show that there is genome-wide divergence for the photosynthetic types, but the current geographical distribution does not reflect a simple habitat displacement scenario as the genetic clusters overlap, being occasionally mixed within a given habitat. Despite evidence of recurrent introgression between non-C4 and C4 groups, in both diploids and polyploids, the distinct genetic lineages retain their identity, potentially because of selection against hybrids. Coupled with strong isolation by distance within each genetic group, this selection created a geographical mosaic of photosynthetic types. Diploid C4 and non-C4 types never grew together, and the C4 type from mixed populations constantly belonged to the hexaploid lineage. By limiting reproductive interactions between photosynthetic types, the ploidy difference probably allows their co-occurrence, reinforcing the functional diversity within this species. Together, these factors enabled the persistence of divergent physiological traits of ecological importance within a single species despite gene flow and habitat overlap.

Genetic and morphological divergence between Littorina fabalis ecotypes in Northern Europe.

Low dispersal marine intertidal species facing strong divergent selective pressures associated with steep environmental gradients have a great potential to inform us about local adaptation and reproductive isolation. Among these, gastropods of the genus Littorina offer a unique system to study parallel phenotypic divergence resulting from adaptation to different habitats related with wave exposure. In this study, we focused on two Littorina fabalis ecotypes from Northern European shores and compared patterns of habitat-related phenotypic and genetic divergence across three different geographic levels (local, regional and global). Geometric morphometric analyses revealed that individuals from habitats moderately exposed to waves usually present a larger shell size with a wider aperture than those from sheltered habitats. The phenotypic clustering of L. fabalis by habitat across most locations (mainly in terms of shell size) support an important role of ecology in morphological divergence. A genome scan based on amplified fragment length polymorphisms (AFLPs) revealed a heterogeneous pattern of differentiation across the genome between populations from the two different habitats, suggesting ecotype divergence in the presence of gene flow. The contrasting patterns of genetic structure between nonoutlier and outlier loci, and the decreased sharing of outlier loci with geographic distance among locations are compatible with parallel evolution of phenotypic divergence, with an important contribution of gene flow and/or ancestral variation. In the future, model-based inference studies based on sequence data across the entire genome will help unravelling these evolutionary hypotheses, improving our knowledge about adaptation and its influence on diversification within the marine realm.

Transcriptomic resources for evolutionary studies in flat periwinkles and related species.

The flat periwinkles, Littorina fabalis and L. obtusata, comprise two sister gastropod species that have an enormous potential to elucidate the mechanisms involved in ecological speciation in the marine realm. However, the molecular resources currently available for these species are still scarce. In order to circumvent this limitation, we used RNA-seq data to characterize the transcriptome of four individuals from each species sampled in different locations across the Iberian Peninsula. Four de novo transcriptome assemblies were generated, as well as a pseudo-reference using the L. saxatilis reference transcriptome as backbone. After transcripts' annotation, variant calling resulted in the identification of 19,072 to 45,340 putatively species-diagnostic SNPs. The discriminatory power of a subset of these SNPs was validated by implementing an independent genotyping assay to characterize reference populations, resulting in an accurate classification of individuals into each species and in the identification of hybrids between the two. These data comprise valuable genomic resources for a wide range of evolutionary and conservation studies in flat periwinkles and related taxa.

Evolutionary history of two cryptic species of northern African jerboas.

BACKGROUND: Climatic variation and geologic change both play significant roles in shaping species distributions, thus affecting their evolutionary history. In Sahara-Sahel, climatic oscillations shifted the desert extent during the Pliocene-Pleistocene interval, triggering the diversification of several species. Here, we investigated how these biogeographical and ecological events have shaped patterns of genetic diversity and divergence in African Jerboas, desert specialist rodents. We focused on two sister and cryptic species, Jaculus jaculus and J. hirtipes, where we (1) evaluated their genetic differentiation, (2) reconstructed their evolutionary and demographic history; (3) tested the level of gene flow between them, and (4) assessed their ecological niche divergence. RESULTS: The analyses based on 231 individuals sampled throughout North Africa, 8 sequence fragments (one mitochondrial and seven single copy nuclear DNA, including two candidate genes for fur coloration: MC1R and Agouti), 6 microsatellite markers and ecological modelling revealed: (1) two distinct genetic lineages with overlapping distributions, in agreement with their classification as different species, J. jaculus and J. hirtipes, with (2) low levels of gene flow and strong species divergence, (3) high haplotypic diversity without evident geographic structure within species, and (4) a low level of large-scale ecological divergence between the two taxa, suggesting species micro-habitat specialization. CONCLUSIONS: Overall, our results suggest a speciation event that occurred during the Pliocene-Pleistocene transition. The contemporary distribution of genetic variation suggests ongoing population expansions. Despite the largely overlapping distributions at a macrogeographic scale, our genetic results suggest that the two species remain reproductively isolated, as only negligible levels of gene flow were observed. The overlapping ecological preferences at a macro-geographic scale and the ecological divergence at the micro-habitat scale suggest that local adaptation may have played a crucial role in the speciation process of these species.

Hybridization patterns between two marine snails, Littorina fabalis and L. obtusata.

Characterizing the patterns of hybridization between closely related species is crucial to understand the role of gene flow in speciation. In particular, systems comprising multiple contacts between sister species offer an outstanding opportunity to investigate how reproductive isolation varies with environmental conditions, demography and geographic contexts of divergence. The flat periwinkles, Littorina obtusata and L. fabalis (Gastropoda), are two intertidal sister species with marked ecological differences compatible with late stages of speciation. Although hybridization between the two was previously suggested, its extent across the Atlantic shores of Europe remained largely unknown. Here, we combined genetic (microsatellites and mtDNA) and morphological data (shell and male genital morphology) from multiple populations of flat periwinkles in north-western Iberia to assess the extent of current and past hybridization between L. obtusata and L. fabalis under two contrasting geographic settings of divergence (sympatry and allopatry). Hybridization signatures based on both mtDNA and microsatellites were stronger in sympatric sites, although evidence for recent extensive admixture was found in a single location. Misidentification of individuals into species based on shell morphology was higher in sympatric than in allopatric sites. However, despite hybridization, species distinctiveness based on this phenotypic trait together with male genital morphology remained relatively high. The observed variation in the extent of hybridization among locations provides a rare opportunity for future studies on the consequences of different levels of gene flow for reinforcement, thus informing about the mechanisms underlying the completion of speciation.

Phylogeographic history of flat periwinkles, Littorina fabalis and L. obtusata.

BACKGROUND: The flat periwinkles, Littorina fabalis and L. obtusata, are two sister species widely distributed throughout the Northern Atlantic shores with high potential to inform us about the process of ecological speciation in the intertidal. However, whether gene flow has occurred during their divergence is still a matter of debate. A comprehensive assessment of the genetic diversity of these species is also lacking and their main glacial refugia and dispersal barriers remain largely unknown. In order to fill these gaps, we sequenced two mitochondrial genes and two nuclear fragments to perform a phylogeographic analysis of flat periwinkles across their distribution range. RESULTS: We identified two main clades largely composed by species-specific haplotypes corresponding to L. obtusata and L. fabalis, with moderate to strong support, respectively. Importantly, a model of divergence with gene flow between the two species (from L. obtusata to L. fabalis) was better supported, both in Iberia and in northern-central Europe. Three mitochondrial clades were detected within L. fabalis and two within L. obtusata, with strong divergence between Iberia and the remaining populations. The largest component of the genetic variance within each species was explained by differences between geographic regions associated with these clades. Our data suggests that overall intraspecific genetic diversity is similar between the two flat periwinkle species and that populations from Iberia tend to be less diverse than populations from northern-central Europe. CONCLUSIONS: The phylogeographic analysis of this sister-species pair supports divergence with gene flow. This system thus provides us with the opportunity to study the contribution of gene flow and natural selection during diversification. The distribution of the different clades suggests the existence of glacial refugia in Iberia and northern-central Europe for both species, with a main phylogeographic break between these regions. Although the genetic diversity results are not fully conclusive, the lower diversity observed in Iberia could reflect marginal conditions at the southern limit of their distribution range during the current interglacial period.

Population-Specific Selection on Standing Variation Generated by Lateral Gene Transfers in a Grass.

Evidence of eukaryote-to-eukaryote lateral gene transfer (LGT) has accumulated in recent years [1-14], but the selective pressures governing the evolutionary fate of these genes within recipient species remain largely unexplored [15, 16]. Among non-parasitic plants, successful LGT has been reported between different grass species [5, 8, 11, 16-19]. Here, we use the grass Alloteropsis semialata, a species that possesses multigene LGT fragments that were acquired recently from distantly related grass species [5, 11, 16], to test the hypothesis that the successful LGT conferred an advantage and were thus rapidly swept into the recipient species. Combining whole-genome and population-level RAD sequencing, we show that the multigene LGT fragments were rapidly integrated in the recipient genome, likely due to positive selection for genes encoding proteins that added novel functions. These fragments also contained physically linked hitchhiking protein-coding genes, and subsequent genomic erosion has generated gene presence-absence polymorphisms that persist in multiple geographic locations, becoming part of the standing genetic variation. Importantly, one of the hitchhiking genes underwent a secondary rapid spread in some populations. This shows that eukaryotic LGT can have a delayed impact, contributing to local adaptation and intraspecific ecological diversification. Therefore, while short-term LGT integration is mediated by positive selection on some of the transferred genes, physically linked hitchhikers can remain functional and augment the standing genetic variation with delayed adaptive consequences.

Comparative mitogenomic analysis of three species of periwinkles: Littorina fabalis, L. obtusata and L. saxatilis.

The flat periwinkles, Littorina fabalis and L. obtusata, offer an interesting system for local adaptation and ecological speciation studies. In order to provide genomic resources for these species, we sequenced their mitogenomes together with that of the rough periwinkle L. saxatilis by means of next-generation sequencing technologies. The three mitogenomes present the typical repertoire of 13 protein-coding genes, 22 transfer RNA genes, two ribosomal RNA genes and a putative control region. Although the latter could not be fully recovered in flat periwinkles using short-reads due to a highly repetitive fragment, in L. saxatilis this problem was overcome with additional long-reads and we were able to assemble the complete mitogenome. Both gene order and nucleotide composition are similar between the three species as well as compared to other Littorinimorpha. A large variance in divergence was observed across mitochondrial regions, with six- to ten-fold difference between the highest and the lowest divergence rates. Based on nucleotide changes on the whole molecule and assuming a molecular clock, L. fabalis and L. obtusata started to diverge around 0.8 Mya (0.4-1.1 Mya). The evolution of the mitochondrial protein-coding genes in the three Littorina species appears mainly influenced by purifying selection as revealed by phylogenetic tests based on dN/dS ratios that did not detect any evidence for positive selection, although some caution is required given the limited power of the dataset and the implemented approaches.

Population genomic evidence for adaptive differentiation in Baltic Sea three-spined sticklebacks.

BACKGROUND: The degree of genetic differentiation among populations experiencing high levels of gene flow is expected to be low for neutral genomic sites, but substantial divergence can occur in sites subject to directional selection. Studies of highly mobile marine fish populations provide an opportunity to investigate this kind of heterogeneous genomic differentiation, but most studies to this effect have focused on a relatively low number of genetic markers and/or few populations. Hence, the patterns and extent of genomic divergence in high-gene flow marine fish populations remain poorly understood. RESULTS: We here investigated genome-wide patterns of genetic variability and differentiation in 10 marine populations of three-spined stickleback (Gasterosteus aculeatus) distributed across a steep salinity and temperature gradient in the Baltic Sea, by utilizing > 30,000 single nucleotide polymorphisms obtained with a pooled RAD-seq approach. We found that genetic diversity and differentiation varied widely across the genome, and identified numerous fairly narrow genomic regions exhibiting signatures of both divergent and balancing selection. Evidence was uncovered for substantial genetic differentiation associated with both salinity and temperature gradients, and many candidate genes associated with local adaptation in the Baltic Sea were identified. CONCLUSIONS: The patterns of genetic diversity and differentiation, as well as candidate genes associated with adaptation in Baltic Sea sticklebacks were similar to those observed in earlier comparisons between marine and freshwater populations, suggesting that similar processes may be driving adaptation to brackish and freshwater environments. Taken together, our results provide strong evidence for heterogenic genomic divergence driven by local adaptation in the face of gene flow along an environmental gradient in the post-glacially formed Baltic Sea.

Multiplex single-nucleotide polymorphism typing of the human Y chromosome using TaqMan probes.

BACKGROUND: The analysis of human Y-chromosome variation in the context of population genetics and forensics requires the genotyping of dozens to hundreds of selected single-nucleotide polymorphisms (SNPs). In the present study, we developed a 121-plex (121 SNPs in a single array) TaqMan array capable of distinguishing most haplogroups and subhaplogroups on the Y-chromosome human phylogeny in Europe. RESULTS: We present data from 264 samples from several European areas and ethnic groups. The array developed in this study shows >99% accuracy of assignation to the Y human phylogeny (with an average call rate of genotypes >96%). CONCLUSIONS: We have created and evaluated a robust and accurate Y-chromosome multiplex which minimises the possible errors due to mixup when typing the same sample in several independent reactions.

Molecular phylogeny and biogeographic history of the European Maja spider crabs (Decapoda, Majidae).

We have assessed for the first time the phylogenetic relationships and biogeographic history of the crabs of the genus Maja that inhabit European coasts: M. brachydactyla, M. crispata, M. goltziana and M. squinado. Using mitochondrial markers, we have recovered a well-resolved phylogenetic tree that supports a single origin for the European species, most likely from an Indo-West Pacific ancestor during the Early Miocene. In this phylogeny, M. goltziana appears as the basal European species, with a sister lineage bifurcating into an Eastern Atlantic (M. brachydactyla) and a Mediterranean (M. crispata and M. squinado) clade. We propose the Tethyan Seaway as the initial colonization route, although an entrance through South Africa cannot be discounted. The Eastern Atlantic/Mediterranean split seems to predate the Messinian salinity crisis, which, in turn, could have promoted the recent divergence within the Mediterranean. In addition, Pleistocene glaciations could explain the current diversity in the Eastern Atlantic Ocean, where a unique mitochondrial lineage is found. According to this, the genetic profile of South African crabs appears to belong to M. brachydactyla, questioning the validity of the putative species M. capensis.