Lateral gene transfer generates accessory genes that accumulate at different rates within a grass lineage.

Lateral gene transfer (LGT) is the movement of DNA between organisms without sexual reproduction. The acquired genes represent genetic novelties that have independently evolved in the donor's genome. Phylogenetic methods have shown that LGT is widespread across the entire grass family, although we know little about the underlying dynamics. We identify laterally acquired genes in five de novo reference genomes from the same grass genus (four Alloteropsis semialata and one Alloteropsis angusta). Using additional resequencing data for a further 40 Alloteropsis individuals, we place the acquisition of each gene onto a phylogeny using stochastic character mapping, and then infer rates of gains and losses. We detect 168 laterally acquired genes in the five reference genomes (32-100 per genome). Exponential decay models indicate that the rate of LGT acquisitions (6-28 per Ma) and subsequent losses (11-24% per Ma) varied significantly among lineages. Laterally acquired genes were lost at a higher rate than vertically inherited loci (0.02-0.8% per Ma). This high turnover creates intraspecific gene content variation, with a preponderance of them occurring as accessory genes in the Alloteropsis pangenome. This rapid turnover generates standing variation that can ultimately fuel local adaptation.

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.

Alloteropsis semialata as a study system for C4 evolution in grasses.

BACKGROUND: Numerous groups of plants have adapted to CO2 limitations by independently evolving C4 photosynthesis. This trait relies on concerted changes in anatomy and biochemistry to concentrate CO2 within the leaf and thereby boost productivity in tropical conditions. The ecological and economic importance of C4 photosynthesis has motivated intense research, often relying on comparisons between distantly related C4 and non-C4 plants. The photosynthetic type is fixed in most species, with the notable exception of the grass Alloteropsis semialata. This species includes populations exhibiting the ancestral C3 state in southern Africa, intermediate populations in the Zambezian region and C4 populations spread around the palaeotropics. SCOPE: We compile here the knowledge on the distribution and evolutionary history of the Alloteropsis genus as a whole and discuss how this has furthered our understanding of C4 evolution. We then present a chromosome-level reference genome for a C3 individual and compare the genomic architecture with that of a C4 accession of A. semialata. CONCLUSIONS: Alloteropsis semialata is one of the best systems in which to investigate the evolution of C4 photosynthesis because the genetic and phenotypic variation provides a fertile ground for comparative and population-level studies. Preliminary comparative genomic investigations show that the C3 and C4 genomes are highly syntenic and have undergone a modest amount of gene duplication and translocation since the different photosynthetic groups diverged. The background knowledge and publicly available genomic resources make A. semialata a great model for further comparative analyses of photosynthetic diversification.

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.

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.

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.

Continued Adaptation of C4 Photosynthesis After an Initial Burst of Changes in the Andropogoneae Grasses.

C$_{4}$ photosynthesis is a complex trait that sustains fast growth and high productivity in tropical and subtropical conditions and evolved repeatedly in flowering plants. One of the major C$_{4}$ lineages is Andropogoneae, a group of $\sim $1200 grass species that includes some of the world's most important crops and species dominating tropical and some temperate grasslands. Previous efforts to understand C$_{4}$ evolution in the group have compared a few model C$_{4}$ plants to distantly related C$_{3}$ species so that changes directly responsible for the transition to C$_{4}$ could not be distinguished from those that preceded or followed it. In this study, we analyze the genomes of 66 grass species, capturing the earliest diversification within Andropogoneae as well as their C$_{3}$ relatives. Phylogenomics combined with molecular dating and analyses of protein evolution show that many changes linked to the evolution of C$_{4}$ photosynthesis in Andropogoneae happened in the Early Miocene, between 21 and 18 Ma, after the split from its C$_{3}$ sister lineage, and before the diversification of the group. This initial burst of changes was followed by an extended period of modifications to leaf anatomy and biochemistry during the diversification of Andropogoneae, so that a single C$_{4}$ origin gave birth to a diversity of C$_{4}$ phenotypes during 18 million years of speciation events and migration across geographic and ecological spaces. Our comprehensive approach and broad sampling of the diversity in the group reveals that one key transition can lead to a plethora of phenotypes following sustained adaptation of the ancestral state. [Adaptive evolution; complex traits; herbarium genomics; Jansenelleae; leaf anatomy; Poaceae; phylogenomics.].

Contrasted histories of organelle and nuclear genomes underlying physiological diversification in a grass species.

C4 photosynthesis evolved multiple times independently in angiosperms, but most origins are relatively old so that the early events linked to photosynthetic diversification are blurred. The grass Alloteropsis semialata is an exception, as this species encompasses C4 and non-C4 populations. Using phylogenomics and population genomics, we infer the history of dispersal and secondary gene flow before, during and after photosynthetic divergence in A. semialata. We further analyse the genome composition of individuals with varied ploidy levels to establish the origins of polyploids in this species. Detailed organelle phylogenies indicate limited seed dispersal within the mountainous region of origin and the emergence of a C4 lineage after dispersal to warmer areas of lower elevation. Nuclear genome analyses highlight repeated secondary gene flow. In particular, the nuclear genome associated with the C4 phenotype was swept into a distantly related maternal lineage probably via unidirectional pollen flow. Multiple intraspecific allopolyploidy events mediated additional secondary genetic exchanges between photosynthetic types. Overall, our results show that limited dispersal and isolation allowed lineage divergence, with photosynthetic innovation happening after migration to new environments, and pollen-mediated gene flow led to the rapid spread of the derived C4 physiology away from its region of origin.

C4 anatomy can evolve via a single developmental change.

C4 photosynthesis is a complex trait that boosts productivity in warm environments. Paradoxically, it evolved independently in numerous plant lineages, despite requiring specialised leaf anatomy. The anatomical modifications underlying C4 evolution have previously been evaluated through interspecific comparisons, which capture numerous changes besides those needed for C4 functionality. Here, we quantify the anatomical changes accompanying the transition between non-C4 and C4 phenotypes by sampling widely across the continuum of leaf anatomical traits in the grass Alloteropsis semialata. Within this species, the only trait that is shared among and specific to C4 individuals is an increase in vein density, driven specifically by minor vein development that yields multiple secondary effects facilitating C4 function. For species with the necessary anatomical preconditions, developmental proliferation of veins can therefore be sufficient to produce a functional C4 leaf anatomy, creating an evolutionary entry point to complex C4 syndromes that can become more specialised.

Gene duplication and dosage effects during the early emergence of C4 photosynthesis in the grass genus Alloteropsis.

The importance of gene duplication for evolutionary diversification has been mainly discussed in terms of genetic redundancy allowing neofunctionalization. In the case of C4 photosynthesis, which evolved via the co-option of multiple enzymes to boost carbon fixation in tropical conditions, the importance of genetic redundancy has not been consistently supported by genomic studies. Here, we test for a different role for gene duplication in the early evolution of C4 photosynthesis, via dosage effects creating rapid step changes in expression levels. Using genome-wide data for accessions of the grass genus Alloteropsis that recently diversified into different photosynthetic types, we estimate gene copy numbers and demonstrate that recurrent duplications in two important families of C4 genes coincided with increases in transcript abundance along the phylogeny, in some cases via a pure dosage effect. While increased gene copy number during the initial emergence of C4 photosynthesis probably offered a rapid route to enhanced expression, we also find losses of duplicates following the acquisition of genes encoding better-suited isoforms. The dosage effect of gene duplication might therefore act as a transient process during the evolution of a C4 biochemistry, rendered obsolete by the fixation of regulatory mutations increasing expression levels.

Tripartite ATP-Independent Periplasmic (TRAP) Transporters and Tripartite Tricarboxylate Transporters (TTT): From Uptake to Pathogenicity.

The ability to efficiently scavenge nutrients in the host is essential for the viability of any pathogen. All catabolic pathways must begin with the transport of substrate from the environment through the cytoplasmic membrane, a role executed by membrane transporters. Although several classes of cytoplasmic membrane transporters are described, high-affinity uptake of substrates occurs through Solute Binding-Protein (SBP) dependent systems. Three families of SBP dependant transporters are known; the primary ATP-binding cassette (ABC) transporters, and the secondary Tripartite ATP-independent periplasmic (TRAP) transporters and Tripartite Tricarboxylate Transporters (TTT). Far less well understood than the ABC family, the TRAP transporters are found to be abundant among bacteria from marine environments, and the TTT transporters are the most abundant family of proteins in many species of ß-proteobacteria. In this review, recent knowledge about these families is covered, with emphasis on their physiological and structural mechanisms, relating to several examples of relevant uptake systems in pathogenicity and colonization, using the SiaPQM sialic acid uptake system from Haemophilus influenzae and the TctCBA citrate uptake system of Salmonella typhimurium as the prototypes for the TRAP and TTT transporters, respectively. High-throughput analysis of SBPs has recently expanded considerably the range of putative substrates known for TRAP transporters, while the repertoire for the TTT family has yet to be fully explored but both types of systems most commonly transport carboxylates. Specialized spectroscopic techniques and site-directed mutagenesis have enriched our knowledge of the way TRAP binding proteins capture their substrate, while structural comparisons show conserved regions for substrate coordination in both families. Genomic and protein sequence analyses show TTT SBP genes are strikingly overrepresented in some bacteria, especially in the ß-proteobacteria and some α-proteobacteria. The reasons for this are not clear but might be related to a role for these proteins in signaling rather than transport.

Massive over-representation of solute-binding proteins (SBPs) from the tripartite tricarboxylate transporter (TTT) family in the genome of the α-proteobacterium Rhodoplanes sp. Z2-YC6860.

Lineage-specific expansion (LSE) of protein families is a widespread phenomenon in many eukaryotic genomes, but is generally more limited in bacterial genomes. Here, we report the presence of 434 genes encoding solute-binding proteins (SBPs) from the tripartite tricarboxylate transporter (TTT) family, within the 8.2 Mb genome of the α-proteobacterium Rhodoplanes sp. Z2-YC6860, a gene family over-representation of unprecedented abundance in prokaryotes. Representing over 6 % of the total number of coding sequences, the SBP genes are distributed across the whole genome but are found rarely in low-GC islands, where the gene density for this family is much lower. This observation, and the much higher sequence identity between the 434 Rhodoplanes TTT SBPs compared with the average identity between homologues from different species, is indicative of a key role for LSE in the expansion. The TTT SBP genes were found in the vicinity of genes encoding membrane components of transport systems from different families, as well as regulatory proteins such as histidine-kinases and transcription factors, indicating a broad range of functions around the sensing, response and transport of organic compounds. A smaller expansion of TTT SBPs is known in some species of the ß-proteobacteria Bordetella and we observed similar expansions in other ß-proteobacterial lineages, including members of the genus Comamonas and the industrial biotechnology organism Cupriavidus necator, indicating that strong environmental selection can drive SBP duplication and specialisation from multiple evolutionary starting points.