Malika Ainouche

Genetic and epigenetic consequences of recent hybridization and polyploidy in Spartina (Poaceae).

To study the consequences of hybridization and genome duplication on polyploid genome evolution and adaptation, we used independently formed hybrids (Spartina × townsendii and Spartina × neyrautii) that originated from natural crosses between Spartina alterniflora, an American introduced species, and the European native Spartina maritima. The hybrid from England, S. × townsendii, gave rise to the invasive allopolyploid, salt‐marsh species, Spartina anglica. Recent studies indicated that allopolyploid speciation may be associated with rapid genetic and epigenetic changes. To assess this in Spartina, we performed AFLP (amplified fragment length polymorphism) and MSAP (methylation sensitive amplification polymorphism) on young hybrids and the allopolyploid. By comparing the subgenomes in the hybrids and the allopolyploid to the parental species, we inferred structural changes that arose repeatedly in the two independently formed hybrids. Surprisingly, 30% of the parental methylation patterns are altered in the hybrids and the allopolyploid. This high level of epigenetic regulation might explain the morphological plasticity of Spartina anglica and its larger ecological amplitude. Hybridization rather than genome doubling seems to have triggered most of the methylation changes observed in Spartina anglica.

Impact of transposable elements on the organization and function of allopolyploid genomes

Transposable elements (TEs) represent an important fraction of plant genomes and are likely to play a pivotal role in fuelling genome reorganization and functional changes following allopolyploidization. Various processes associated with allopolyploidy (i.e. genetic redundancy, bottlenecks during the formation of allopolyploids or genome shock following genome merging) may allow accumulation of TE insertions. Our objective in carrying out a survey of the literature and a comparative analysis across different allopolyploid systems is to shed light on the structural, epigenetic and functional modifications driven by TEs during allopolyploidization and subsequent diploidization. The available evidence indicates that TE proliferation in the short or the long term after allopolyploidization may be restricted to a few TEs, in specific polyploid systems. By contrast, data indicate major structural changes in the TE genome fraction immediately after allopolyploidization, mainly through losses of TE sequences as a result of recombination. Emerging evidence also suggests that TEs are targeted by substantial epigenetic changes, which may impact gene expression and genome stability. Furthermore, TEs may directly or indirectly support the evolution of new functionalities in allopolyploids during diploidization. All data stress allopolyploidization as a shock associated with drastic genome reorganization. Mechanisms controlling TEs during allopolyploidization as well as their impact on diploidization are discussed.

Rapid structural and epigenetic reorganization near transposable elements in hybrid and allopolyploid genomes in Spartina

*Transposable elements (TE) induce structural and epigenetic alterations in their host genome, with major evolutionary implications. These alterations are examined here in the context of allopolyploid speciation, on the recently formed invasive species Spartina anglica, which represents an excellent model to contrast plant genome dynamics following hybridization and genome doubling in natural conditions. *Methyl-sensitive transposon display was used to investigate the structural and epigenetic dynamics of TE insertion sites for several elements, and to contrast it with comparable genome-wide methyl-sensitive amplified polymorphism analyses. *While no transposition burst was detected, we found evidence of major structural and CpG methylation changes in the vicinity of TE insertions accompanying hybridization, and to a lesser extent, genome doubling. Genomic alteration appeared preferentially in the maternal subgenome, and the environment of TEs was specifically affected by large maternal-specific methylation changes, demonstrating that TEs fuel epigenetic alterations at the merging of diverged genomes. *Such genome changes indicate that nuclear incompatibilities in Spartina trigger immediate alterations, which are TE-specific with an important epigenetic component. Since most of this reorganization is conserved after genome doubling that produced a fertile invasive species, TEs certainly play a central role in the shock-induced dynamics of the genome during allopolyploid speciation.

Tracking the evolutionary history of polyploidy in Fragaria L. (strawberry): New insights from phylogenetic analyses of low-copy nuclear genes

Phylogenetic utility of two nuclear genes (GBSSI-2 and DHAR) was explored in genus Fragaria in order to clarify phylogenetic relationships among taxa and to elucidate the origin of the polyploid species. Orthology of the amplified products was assessed by several methods. Our results strongly suggest the loss of one GBSSI duplicated copy (GBSSI-1) in the Fragariinae subtribe. Phylogenetic analyses provided new insights into the evolutionary history of Fragaria, such as evidence supporting the presence of three main diploid genomic pools in the genus and demonstrating the occurrence of independent events of polyploidisation. In addition, the data provide evidence supporting an allopolyploid origin of the hexaploid F. moschata, and the octoploids F. chiloensis, F. iturupensis and F. virginiana. Accordingly, a new pattern summarizing our present knowledge on the Fragaria evolutionary history is proposed. Additionally, sequence analyses also revealed relaxed constraints on homoeologous copies at high ploidy level, as demonstrated by deletion events within DHAR coding sequences of some allo-octoploid haplotypes.

Molecular investigations in populations of Spartina anglica C.E. Hubbard (Poaceae) invading coastal Brittany (France).

Spartina anglica is a classical example of recent alloploid speciation. It arose during the end of the nineteenth century in England by hybridization between the indigenous Spartina maritima and the introduced East‐American Spartina alterniflora. Duplication of the hybrid genome (Spartina × townsendii) gave rise to a vigorous allopolyploid involved in natural and artificial invasions on different continents. Spartina anglica was first recorded in France in 1906, and since then, it has spread all along the western French coast. Earlier studies revealed that native British populations display consistent morphological plasticity and lack of isozyme variation. In this paper, we use different molecular markers (randomly amplified polymorphic DNA, intersimple sequence repeats and restriction patterns from nuclear and chloroplast DNA sequences) to analyse the genetic patterns of the French populations of S. anglica. Our results show that French populations are mainly composed of one ‘major’ multilocus genotype. This genotype is identical to the first‐generation hybrid S. × townsendii from England. Losses of few markers from this genotype are observed but are restricted to a few populations from Brittany; it is likely that they appeared independently, subsequent to their introduction. In southern Brittany, no hybrids between S. anglica and S. maritima have been found where the two species co‐occur. All French populations of S. anglica display the same chloroplast DNA sequences as S. alterniflora, the maternal genome donor. These findings are consistent with a severe genetic bottleneck at the time of the species formation, as a consequence of a unique origin of the species. Both parental nuclear sequences are present in the allopolyploid populations, revealing that for the markers investigated, no extensive changes have occurred in this young species.

Genetic evidence for hybridization between the native Spartina maritima and the introduced Spartina alterniflora (Poaceae) in South-West France: Spartina × neyrautii re-examined

Abstract. Spartina alterniflora, a perennial grass native to the North American Atlantic coast, was introduced during the 19th century in western Europe (Southern England and western France) where it hybridized with the native Spartina maritima. In England, the sterile hybrid S. × townsendii gave rise by chromosome doubling to the highly fertile allopolyploid Spartina anglica, which has now invaded many salt marshes and estuaries in western Europe, and has been introduced in several continents. In South-West France, another sterile hybrid was discovered in 1892 in the Bidassoa Estuary, and named Spartina × neyrautii. According to their morphology, some authors suggested that S. × neyrautii and S. × townsendii result from reciprocal crosses. During the 20th century, the hybridization site was severely disturbed, and surviving of S. × neyrautii was questioned. In this paper, various Spartina populations are investigated in the Basque region (France and Spain), and compared to the hybrid taxa formed in England (S. × townsendii and S. anglica). The samples were analyzed using molecular fingerprinting (RAPD and ISSR) and Chloroplast DNA sequence (trnL-trnT spacer, trnL intron and trnL-trnF spacer). In the Bidassoa estuary, a hybrid isolated clone has been found, that displays additive species-specific nuclear markers of S. maritima and S. alterniflora, and that is subsequently considered as a surviving clone of S. × neyrautii. The molecular analyses indicate that S. × neyrautii and S. × townsendii share the same maternal (S. alterniflora), and paternal (S. maritima) parental species, but also that the two independent hybridization events have involved different parental (nuclear) genotypes in England and in South-West France.

Making a functional diploid: from polysomic to disomic inheritance

One little understood feature of polyploid speciation is the transition from polysomic to disomic inheritance, and much recent attention has focused on the role of pairing genes in this process. Using computer simulations we studied the effects of mutations, chromosomal inversions, chiasma, neofunctionalization, subfunctionalization and selection on the evolution of disomic inheritance in a polyploid over 10 000 generations. We show that: the evolution of pairing genes is not essential for the establishment of disomic inheritance, as genetic drift, coupled with a threshold for homologue pairing fidelity, is sufficient to explain the transition from polysomic to disomic inheritance; high rates of recombination increase the number of generations required for disomic inheritance to become established; both neofunctionalization and subfunctionalization speed up the transition to disomic inheritance. The data suggest that during polyploid species establishment, selection will favour reduced chiasma number and/or more focused distribution. The data also suggest a new role for subfunctionalization in that it can drive disomic inheritance. The evolution of subfunctionalization in genes across the genome will then act to maintain genes in syntenic blocks and may explain why such regions are so highly conserved.

The enigmatic invasive Spartina densiflora: A history of hybridizations in a polyploidy context

The aim of this study was to explore the origin of the invasive Spartina densiflora by analysing samples from the native region (South America) and from a recently colonized area (California). A combination of various molecular data (chloroplast and nuclear sequences, molecular fingerprint) and ploidy level estimations was used to answer the question whether the reticulate phylogenetic pattern previously detected in S. densiflora was restricted to California, or alternatively, whether a more ancient hybrid origin preceded formation of this species in its native area. We found that this species is heptaploid in both its native and introduced range. Identification of nuclear homeologous sequences indicate that this species has a reticulate origin in its native range, involving a lineage related to the hexaploid clade formed by S. alterniflora, S. foliosa, and S. maritima, and another lineage related to the sub‐Antarctic endemic S. arundinacea that provided the chloroplast genome. The samples from California displayed similar multilocus patterns to the samples from Chile, supporting the hypothesis that this species originated on the southeast American coast (Argentina), from where it eventually spread to the west coast of South America (Chile) first and to the Northern Hemisphere (California) later.

Transcriptome de novo assembly from next-generation sequencing and comparative analyses in the hexaploid salt marsh species Spartina maritima and Spartina alterniflora (Poaceae)

Spartina species have a critical ecological role in salt marshes and represent an excellent system to investigate recurrent polyploid speciation. Using the 454 GS-FLX pyrosequencer, we assembled and annotated the first reference transcriptome (from roots and leaves) for two related hexaploid Spartina species that hybridize in Western Europe, the East American invasive Spartina alterniflora and the Euro-African S. maritima. The de novo read assembly generated 38 478 consensus sequences and 99% found an annotation using Poaceae databases, representing a total of 16 753 non-redundant genes. Spartina expressed sequence tags were mapped onto the Sorghum bicolor genome, where they were distributed among the subtelomeric arms of the 10 S. bicolor chromosomes, with high gene density correlation. Normalization of the complementary DNA library improved the number of annotated genes. Ecologically relevant genes were identified among GO biological function categories in salt and heavy metal stress response, C4 photosynthesis and in lignin and cellulose metabolism. Expression of some of these genes had been found to be altered by hybridization and genome duplication in a previous microarray-based study in Spartina. As these species are hexaploid, up to three duplicated homoeologs may be expected per locus. When analyzing sequence polymorphism at four different loci in S. maritima and S. alterniflora, we found up to four haplotypes per locus, suggesting the presence of two expressed homoeologous sequences with one or two allelic variants each. This reference transcriptome will allow analysis of specific Spartina genes of ecological or evolutionary interest, estimation of homoeologous gene expression variation using RNA-seq and further gene expression evolution analyses in natural populations.

Molecular phylogeny and reticulate origins of the polyploid Bromus species from section Genea (Poaceae)

The origin of polyploid Bromus species of section Genea was investigated using molecular data. This group of annual species native from the Old-World is composed of three diploids, two tetraploids, one hexaploid, and one octoploid. Molecular cloning, sequencing, and phylogenetic analyses were performed on several accessions per species. We used the low copy nuclear gene Waxy, repeated rDNA spacers ITS1 and ITS2 and chloroplast spacers trnT-trnL and trnL-trnF. Our analyses revealed four different lineages involved in the parentage of the polyploids and confirmed their reticulate origin. Three of these lineages are closely related to the diploid species B. sterilis, B. tectorum, and B. fasciculatus. The fourth lineage could not be related to any diploid according to the available data. Our data gave insights on the origin of all the polyploids of section Genea, and chloroplast data allowed us to identify the maternal lineages. The Waxy gene was the most informative regarding origin of the polyploids. The Waxy copies duplicated by polyploidy appear selectively maintained in the polyploid species. No sequence heterogeneity was encountered in the ITS region, where concerted evolution seems to have occurred toward either maternal or paternal repeats. These results provide new information about the origin and molecular evolution of these polyploids and will allow a more accurate taxonomic treatment of the concerned species, based on their evolutionary history.