Using phylogenetic methods, our research investigates the mechanisms that led to the functional diversity of plants. We combine analyses of gene sequences, genomes, transcriptomes, ecological, physiological and morphological traits to address questions of importance for evolutionary biology in general:
These questions are addressed using comparative approaches, working both at large taxonomic scales and within some selected species. As a study system, we use mainly the evolution of C4 and CAM photosynthesis in grasses and other groups of plants. While transcriptomes and genomes are compared across distantly related families, the origins and consequences of variation in photosynthetic traits are also investigated within the grass species Alloteropsis semialata, the only taxon known to include both C3 and C4 individuals.
C4 photosynthesis is an adaptation that boosts productivity in tropical conditions [1,12]. A number of anatomical and biochemical components must act together to generate a C4 physiology, yet this complex trait is a textbook example of convergent evolution, having evolved more than 60 times independently in flowering plants . We are interested in understanding the factors that eased C4 evolution in some lineages, as well as the effects of the transition from C3 to C4 on the evolutionary trajectories of the plants and their ecological sorting.
The multiple origins of C4 photosynthesis all happened in the last 30 million years [5,6], which supports the hypothesis that declines of atmospheric CO2 during the Oligocene promoted C4 evolution . Phylogenetic models have shown that C4 origins were more likely in those groups possessing leaf anatomies that already fitted some of the C4 requirements [8,15]. In addition, independent C4 origins have recurrently used those genes that were ancestrally highly expressed, so that possessing such genes might have facilitated C4 evolution [9,10,23].
The effects of C4 evolution depend on the organisms. While C4 boosts growth , it also affects the ecological sorting. Across angiosperms, the ecological niche of C4 taxa is correlated to that of their C3 ancestors, indicating significant phylogenetic effects . Despite this effect of evolutionary history, C4 lineages tend to occur in warmer climates than their non-C4 relatives . Within grasses, phylogenetic analyses confirmed that C4 originated mainly in tropical conditions, but surprisingly, the C4 trait increased the rate of migration to colder climates . Therefore, C4 evolution can be said to broaden the ecological niche [12,19], in addition to increasing diversification rates .
Lateral gene transfer (LGT) represents the spread of genetic information among distantly related organisms. While the phenomenon has been widely reported among bacteria, until recently, its relevance for eukaryote evolution remained debated. Some years ago, we have inadvertently discovered that genes used by Alloteropsis semialata for C4 photosynthesis had been laterally acquired from distantly related C4 grasses . Using comparative transcriptomics, we have later shown that these LGT are used by some populations of A. semialata [13,15], while our investigations of the species genome biogeography have shown that these LGT were recently spread among established populations of A. semialata . Bringing our research into the genomics era, we have generated a reference genome for one Australian accession of A. semialata, and compare it to 146 other grass species . We then developed a new pipeline to demonstrate that the genome of A. semialata contains a minimum of 59 LGT acquired from at least nine distinct species of grasses . The majority of these LGT are used by the recipient species, showing that LGT can contribute to the functional diversification of grasses . We are now expanding our investigations to other groups of grasses to identify the conditions that promote LGT among plants.
Relationships among species can be depicted using phylogenetic trees, but reconstructing the history of transitions among character states can be complicated when relying solely on species trees. To gain further insights into the history of modifications underlying adaptive transitions, we decompose complex traits into their constituents and track each of them independently using combinations of species and gene trees. This innovative approach revealed that the complex C4 trait emerged repeatedly within the small grass genus Alloteropsis via a combination of recurrent co-option of pre-extisting components and hybridization . Expanding this approach to other groups, we further showed that genetic modifications associated to some C4 taxa evolved after the C4 physiology itself, so that the number of origins differs among C4 components [3,13].
Reticulate evolution underlying multiple photosynthetic transitions in Alloteropsis; from Dunning et al. 
The origins of C4 photosynthesis are traditionally investigated at large evolutionary scales, through comparisons of species differing in their photosynthetic type. Such research cannot elucidate the microevolutionary processes underlying photosynthetic transitions, and we therefore conduct intraspecific analyses, using as a study system the grass Alloteropsis semialata. This species contains C3 and C4 populations, as well as intermediates . Phylogenetic analyses of chloroplast genomes indicate that the species originated in Central/Eastern Africa . While the intermediates remained ecologically and geographically restricted, the C4 lineage rapidly spread through geographical and ecological spaces, confirming that C4 acts as a niche broadener .
Analyses of nuclear genomes showed that Alloteropsis semialata underwent recurrent events of hybridization during its history . This study system allowed us to track the spread of adaptive loci, showing that C4 components can be acquired independently in isolated populations, and then combined through secondary gene flow . We are currently expanding these investigations to a large sample of populations from across the species range, to reconstruct the history of C4 mutations, and the mechanisms driving their fixation and subsquent spread.
The Alloteropsis semialata system allows comparing recently diverged C4 and non-C4 accessions, as well as C4 populations that evolved independently after the transition to C4. Capitalizing on this system, we recently showed that gene duplication facilitated physiological innovation, by providing rapid increases in gene expression levels . We also use the A. semialata system to identify the anatomical, physiological and genomic traits that differ systematically among photosynthetic types. Sampling the diversity within each photosynthetic type helps differentiate the changes that are responsible for the shifts in photosynthetic type from those that preceded or followed the transition. Focusing on leaf anatomy, we have shown that the only trait differentiating C4 and non-C4 accessions of A. semialata is the proliferation of minor veins, which generated a C4-compatible leaf anatomy in this species and other grasses . In terms of leaf transcriptomes, relatively few genes changed in expression during the transition between photosynthetic types . Interestingly, other changes happened once the plants were already using C4 photosynthesis , confirming that many of the characters traditionally associated with C4 photosynthesis represent secondary adaptations and were not involved in the initial evolutionary transitions [12,17].
Nuclear history of Alloteropsis semialata, showing the distribution of laterally acquired genes (LGT), from Olofsson et al. 
The advent of next generation sequencing allows generating genome data for large numbers of species. While initially used for model species, high-throughput sequencing can also be applied to samples stored in historical collections, such as those found in herbarium and museums. In collaboration with colleagues from Toulouse, we use high-throughput sequencing of historical samples to explore the genomic diversity stored in historical collections. We are able to reconstruct relationships among numerous taxa based on both organellar and nuclear genomes, but can also perform comparative genomics on rare species that would be difficult to collect in the wild [2,3,14,24,25].
Colin Osborne, Sheffield Link
Guillaume Besnard, Toulouse Link
1. Atkinson RRL, Mockford EJ, Bennett C, Christin PA, Spriggs EL, Freckleton RP, Thompson K, Rees M, Osborne CP. 2016. C4 photosynthesis boosts growth by altering physiology, allocation and size. Nature Plants 2: 16038 Link
2. Besnard G, Christin PA, Male PJG, Lhuillier E, Lauzeral C, Coissac E, Vorontsova M. 2014. From museums to genomics: old herbarium specimen sheds light on a C3 to C4 transition. Journal of Experimental Botany 65:6711-6721 Link
3. Besnard G, Bianconi ME, Hackel J, Manzi S, Vorontsova MS, Christin PA. 2018. Herbarium genomics retraces the origins of C4-specific carbonic anhydrase in grasses. Botany Letters
4. Bianconi ME, Dunning LT, Moreno-Villena JJ, Osborne CP, Christin PA. 2018. Gene duplication and dosage effects during the early emergence of C4 photosynthesis. Journal of Experimental Botany 8:1967-1980 Link
5. Christin PA, Besnard G, Samaritani E, Duvall MR, Hodkinson TR, Savolainen V, Salamin N. 2008. Oligocene CO2 decline promoted C4 photosynthesis in grasses. Current Biology 18: 37-43 Link
6. Christin PA, Osborne CP, Sage RF, Arakaki M, Edwards EJ. 2011. C4 eudicots are not younger than C4 monocots. Journal of Experimental Botany 62: 3171-3181 Link
7. Christin PA, Edwards EJ, Besnard G, Boxall SF, Gregory R, Kellogg EA, Hartwell J, Osborne CP. 2012. Adaptive evolution of C4 photosynthesis through recurrent lateral gene transfer. Current Biology 22: 445-449 Link
8. Christin PA, Osborne CP, Chatelet DS, Columbus JT, Besnard G, Hodkinson TR, Garrison LM, Vorontsova MS, Edwards EJ. 2013. Anatomical enablers and the evolution of C4 photosynthesis in grasses. Proceedings of the National Academy of Sciences (PNAS) 110: 1381-1386 Link
9. Christin PA, Boxall SF, Gregory R, Edwards EJ, Hartwell J, Osborne CP. 2013. Parallel recruitment of multiple genes into C4 photosynthesis. Genome Biology and Evolution 5: 2174-2187 Link
10. Christin PA, Arakaki M, Osborne CP, Edwards EJ. 2015. Genomic enablers underlying the clustered evolutionary origins of C4 photosynthesis in angiosperms. Molecular Biology and Evolution 32:846-858 Link
11. Christin PA, Osborne CP. 2013. The recurrent assembly of C4 photosynthesis, an evolutionary tale. Photosynthesis Research 117: 163-175 Link
12. Christin PA, Osborne CP. 2014. The evolutionary ecology of C4 plants. New Phytologist 204: 765-781 Link
13. Dunning LT, Lundgren MR, Moreno-Villena JJ, Namaganda M, Edwards EJ, Nosil P, Osborne CP, Christin PA. 2017. Introgression and repeated co-option facilitated the recurrent emergence of C4 photosynthesis among close relatives. Evolution 71:1541-1555 Link
14. Dunning LT, Liabot AL, Olofsson JK, Smith EK, Vorontsova MS, Besnard G, Simpson KJ, Lundgren MR, Addicott E, Handasyde T, Gallagher RV, Chu Y, Pennington RT, Christin PA, Lehmann CER. 2017. The recent and rapid spread of Themeda triandra. Botany Letters 164:327-337 Link
15. Dunning LT*, Moreno-Villena JJ*, Lundgren MR, Dionora J, Salazar P, Adams C, Nyirenda F, Olofsson JK, Mapaura A, Grundy IM, Kayombo CJ, Dunning LA, Kentatchime F, Ariyarathne M, Yakandawala D, Besnard G, Quick PW, Bräutigam A, Osborne CP, Christin PA. 2019. Key changes in gene expression identified for different stages of C4 evolution in Alloteropsis semialata. Journal of Experimental Botany doi:10.1093/jxb/erz149 Link
16. Dunning LT, Olofsson JK, Parisod C, Choudhury RR, Moreno-Villena JJ, Yang Y, Dionora J, Quick WP, Park M, Bennetzen JL, Besnard G, Nosil P, Osborne CP, Christin PA. 2019. Lateral transfers of large DNA fragments spread functional genes among grasses. Proceedings of the National Academy of Sciences (PNAS) 116:4416-4425. Link
17. Heyduk K, Moreno-Villena JJ, Gilman I, Christin PA, Edwards EJ. 2019. The genetics of convergent evolution: insights from plant photosynthesis. Nature Reviews Genetics doi:10.1038/s41576-019-0107-5 Link
18. Lundgren MR, Osborne CP, Christin PA. 2014. Deconstructing Kranz anatomy to understand C4 evolution. Journal of Experimental Botany 3357-3369 Link
19. Lundgren MR, Besnard G, Ripley BS, Lehmann CER, Chatelet DS, Kynast RG, Namaganda M, Vorontsova MS, Hall RC, Elia J, Osborne CP, Christin PA. 2015. Photosynthetic innovation broadens the niche within a single species. Ecology Letters 18:1021-1029 Link
20. Lundgren MR, Christin PA, Gonzalez Escobar E, Ripley BS, Besnard G, Long CM, Hattersley PW, Ellis RP, Leegood RC, Osborne CP. 2016. Evolutionary implications of C3-C4 intermediates in the grass Alloteropsis semialata. Plant, Cell and Environment 39: 1874-1885 Link
21. Lundgren MR, Christin PA. 2017. Despite phylogenetic effects, C3-C4 lineages bridge the ecological gap to C4 photosynthesis. Journal Experimental Botany 68:241-254 Link
22. Lundgren MR, Dunning LT, Olofsson JK, Moreno-Villena JJ, Bouvier JW, Sage T, Khoshravesh R, Sultmanis S, Stata M, Ripley B, Vorontsova MS, Besnard G, Adams C, Cuff N, Mapaura A, Bianconi M, Long CM, Christin PA, Osborne CP. C4 anatomy can evolve via a single developmental change. Ecology Letters 22: 302-312. Link
23. Moreno-Villena JJ, Dunning LT, Osborne CP, Christin PA. 2018. Highly expressed genes are preferentially co-opted for C4 photosynthesis. Molecular Biology and Evolution 35:94-102 Link
24. Olofsson JK, Bianconi M, Besnard G, Dunning LT, Lundgren MR, Holota H, Vorontsova MS, Hidalgo O, Leitch IJ, Nosil P, Osborne CP, Christin PA. 2016. Genome biogeography reveals the intraspecific spread of adaptive mutations for a complex trait. Molecular Ecology 25: 6107-6123 Link
25. Olofsson JK, Cantera I, Van de Paer C, Hong-Wa C, Zedane L, Dunning LT, Alberti A, Christin PA, Besnard G. 2019. Resolving the phylogeny of the olive tribe (Oleeae) based on low-depth whole genome sequencing. Molecular Ecology Resources doi:10.1111/1755-0998.13016 Link
26. Sage RF, Christin PA, Edwards EJ. 2011. The C4 plant lineages of planet Earth. Journal of Experimental Botany 62: 3155-3169 Link
27. Spriggs E, Christin PA, Edwards EJ. 2014. C4 photosynthesis promoted species diversification during the Miocene grassland expansion. Plos One 9: e97722 Link
28. Watcharamonkol T, Christin PA, Osborne CP. 2018. C4 photosynthesis evolved in warm climates but promoted migration to colder ones. Ecology Letters 21:376-383 Link