Differentiation and reproductive isolation between diploids and tetraploids
The clear and almost complete segregation of diploids and tetraploids based on the PCA of the nuclear SSRs and the cpDNA network suggests the presence of a strong reproductive barrier among cytotypes in Salicornia, in agreement with several observations of complete reproductive barriers among sympatric cytotypes (Hardy et al., 2000; Husband & Sabara, 2003; Kloda et al., 2008). SSR and cpDNA alleles typical for tetraploids were, however, occasionally found in diploids Allele sharing between diploids and tetraploids at SSRs loci may be because of homoplastic mutations or null alleles. The sharing of SSR alleles or cpDNA haplotypes by diploid and tetraploid lineages may also be explained by the retention of ancestral polymorphisms from their diploid ancestors within the tetraploid lineages. Such an interpretation is fully compatible with the very recent origin of the genus, dated to 1.8–1.4 myrs (Kadereit et al., 2006). However, the sharing of alleles among sympatric individuals from different cytotypes, along with the occurrence of specimens with a typical diploid morphology and nuclear SSR patterns but with a cpDNA haplotype characteristic for tetraploids, is rather indicative of instances of inter-cytotypic gene flow. This hypothesis is favoured by Kaligaric et al. (2008) to explain the sharing of cpDNA haplotypes between Mediterranean diploids and tetraploids and in fact, although there are few examples of the process in the wild, the existence of gene flow among cytotypes has long been recognized (see Chapman & Abbott, 2010, for review).
The high correlation between cytotypes and genotypes observed here confirms previous assessments on the reliability of flower morphology to distinguish among diploid and tetraploid Salicornia (e.g. Lahondère, 2004; Kaligaric et al., 2008). Those cpDNA and SSR markers may therefore prove useful for the determination of the ploidy level of herbarium material, juvenile specimens or specimens with an ambiguous flower morphology. This is especially the case of S. obscura, whose lateral and median flowers are subequal in size, rendering the confusion with both diploid and tetraploid species possible (see Lambinon & Vanderpoorten, 2009, for review). In fact, the molecular analysis of the specimens attributed to S. obscura in the present study revealed that about one-third of them were tetraploids. This suggests that S. obscura has served as a convenient taxonomic repository for morphologically ill-characterized diploid or tetraploid specimens.
Origin and evolution of the tetraploids
The tetraploids exhibit high levels of heterozygosity. Heterozygosity is fixed in the vast majority of populations at loci S2 and S19, with > 95% of heterozygous profiles. More than 50% of specimens are heterozygous at loci S5, S7 and S8. These patterns are consistent with previous isozyme analyses, wherein diploids were strictly homozygous and tetraploids showed either a homozygous or a fixed heterozygous profile (Wolff & Jefferies, 1987). Although the chances of producing an autopolyploid from two unreduced gametes fusing are greatly increased in strong selfers like Salicornia (Shepherd & Yan, 2003), the most parsimonious explanation is that the tetraploids are of allopolyploid origin. Indeed, although the lack of fixed heterozygosity may strengthen the evidence for autopolyploidy (Soltis & Rieseberg, 1986), fixed heterozygosity at many loci in a polyploid is commonly used as evidence for allopolyploidy (Arft & Ranker, 1998; Såstad et al., 2001; Nyberg Berglund et al., 2006). In allopolyploids, indeed, parental genomes may be different enough for chromosome pairing to occur only between chromosomes that originate from the same parental genome. Alleles of each parental genome segregate as if they were from a diploid with disomic inheritance. If the parental genomes are homozygous for different alleles, all gametes will be heteroallelic and all offspring will be heterozygous, i.e. heterozygosity will be fixed. The increased heterozygosity resulting from hybridization in tetraploid Salicornia may, ultimately, result in the formation of new gene combinations and generation of new forms of enzymes, and be critical for the successful establishment in unstable environments with recurrent flooding periods where they typically occur.
At the population level, given the strong reproductive barrier among cytotypes, and provided that the diploids did not suffer more substantially than the tetraploids in the course of the last glaciations, the comparable levels of diversity observed in tetraploids and diploids either suggest an ancient and/or multiple origin of polyploids. Indeed, polyploids are expected to harbour less genetic diversity if polyploid formation is a rare and/or recent event. This is because, although allotetraploids potentially accumulate genetic variation at a faster rate than diploids, newly formed polyploids start out with limited genetic diversity because of founding effects. It therefore takes a considerable amount of time to reach equilibrium between mutation and drift, and ultimately higher levels of genetic diversity (Luttikhuizen et al., 2007). An ancient origin of allotetraploid Salicornia would be consistent with the idea that fast mutation rates at the microsatellite loci would have regenerated the loss of diversity following speciation. The only marginally higher differentiation among than within cytotypes with the SSRs, and the actually higher differentiation within diploids than between diploids and tetraploids in the chloroplast, contrast with the ancient origin hypothesis. The latter is further weakened by the recent origin of the genus (Kadereit et al., 2006) and the lack of phylogenetic resolution within cytotypes, which has been interpreted in terms of a recent and rapid expansion (Kadereit et al., 2007; Murakeözy et al., 2007).
Thus, an alternative explanation for the observed patterns of diversity in tetraploids is that the latter evolved recurrently from diploids. Although a monophyletic origin of European tetraploids was resolved from the phylogenetic analysis of nrDNA ETS sequences (Kadereit et al., 2007), which is consistent with the clear differentiation among cytotypes observed here using nuclear SSRs, a monophyletic origin of the cpDNA lineages observed among tetraploids was statistically rejected. This suggests that allopolyploidization has happened several times from a common gene pool, adding to the mounting evidence for a recurrent origin of polyploids (see Albach, 2007, for review).
Geographic and taxonomic partitioning of genetic variation within cytotypes
Although none of the species, as circumscribed by the most complex taxonomic treatments of, e.g. Lahondère (2004) and Stace (2010), are defined by monophyletic chloroplast lineage, partitioning of genetic variation among species is weak, but significant. The strong underlying phylogeographic structure, however, largely contributes to this differentiation. Levels of differentiation between populations of different species within the same region are indeed very low when compared to those among conspecific populations from different regions. A significant phylogeographic signal is in fact present in the cpDNA data between the Mediterranean and the Atlantic, suggesting that these two regions were colonized anciently and accumulated mutations at a faster rate than migration events. These findings are consistent with the eastern/western pattern of differentiation found by Kadereit et al. (2007).
At the local scale, the results further indicate that taxonomy cannot be retrieved from analyses of genetic relationships. In fact, mean cpDNA Nij kinship coefficients are equal to 1 within and among species. Similarly, although conspecific individuals tend to be significantly more related than individuals from different species at the population scale (see below), kinship coefficients derived from SSR variation among individuals from different species are not significantly lower than those within species as soon as individuals from different populations are considered. The poor relation between taxonomy and genetic variation documented here is consistent with previous analyses using AFLPs (Le Goff, 1999) and DNA sequences (Murakeözy et al., 2007; Kadereit et al., 2007), which failed to resolve monophyletic species groups. This is, however, counter-intuitive given the apparently clear circumscription of at least some species, like S. pusilla, which is readily recognized by its solitary flowers.
In Salicornia, phenotypic plasticity has been suggested as the main factor accounting for the incongruence observed between traditional species concepts and patterns of genetic differentiation (Murakeözy et al., 2007). Owing to their extremely reduced morphology, Salicornia species are defined based on global branching architecture and shape; colour; and size of the lateral vs. central flowers, i.e. a suite of quantitative characters that are indeed arguably more prone to plasticity than complex flower characters found in other groups. The hypothesis that plasticity accounts for the incongruence between species concepts and patterns of genetic differentiation is, however, at odds with the fact that mean Fij among pairs of conspecific individuals in the SSR data tend to be significantly higher than those between pairs of individuals from different species at the population scale, thereby suggesting that the morphological differentiation has a genetical basis. Transplantation experiments indeed revealed that individuals tend to retain their specific morphology (Kadereit et al., 2007).
Another interpretation of the incongruence between taxonomy and genetic variation in Salicornia is the lack of inter-specific reproductive barriers. Salicornia species have, however, traditionally been considered as strong if not complete selfers (Dalby, 1962; Ferguson, 1964). A 100% inbreeding rate was, for instance, reported based upon the genetic identity between 38 maternal plants and 2112 F1 progenies (Noble et al., 1992). In diploid species indeed, the anthers usually dehisce before they are exserted and ripe dehiscing anthers may be seen in contact with presumably receptive stigmas, their pollen spilling onto the stigmatic papillae. Cleistogamy has, in addition, been reported in many instances (see Kadereit et al., 2007, for review). Pistillate flowers can, however, be observed in S. ramosisima (Kadereit et al., 2007), suggesting that outbreeding by wind pollination cannot be completely ruled out. In addition, heterozygosity was recurrently observed in diploids, and the mean level of inbreeding observed within diploid populations (Fis = 0.70) indicates an outcrossing rate of 18% if only selfing accounts for the heterozygote deficit. Such an interpretation is consistent with the existence of fertile inter-specific hybrids such as S. × marshallii between S. pusilla and S. ramosissima, which is readily distinguished by a combination of inflorescences with a single flower, as in S. pusilla, two flowers, and three flowers, as in S. ramosissima. The hybrid nature of such specimens with an intermediate morphology was confirmed by use of the present SSR markers because they display heterozygous genotypes at loci with different homozygous genotypes within sympatric S. pusilla and S. ramosissima. The absence of interspecifric reproductive barriers is also suggested by the partial incongruence reported among nuclear and cpDNA sequences (Murakeözy et al., 2007; Kaligaric et al., 2008). Salicornia is comparable, in this respect, to Quercus, wherein species are not well differentiated genetically owing to the sharing of common cpDNA haplotypes among sympatric species, suggesting appreciable localized cytoplasmic gene flow and high levels of cpDNA fixation within populations (Whittemore & Schaal, 1991), whilst haplotypes sampled among allopatric conspecific specimens are highly divergent (Petit et al., 1993; Manos et al., 1999).
A third and not mutually exclusive interpretation is that the observed morphological differences are not the result of divergent genomes, but are based on single or few point mutations, or even to changes in the mechanisms of gene regulation affecting when and where a gene is expressed. In the beach mouse, for example, a single amino acid mutation contributes to adaptive colour pattern (Hoekstra et al., 2006). In other taxa with reduced morphologies like mosses, Hedenäs & Eldenäs (2008) and Sotiaux et al. (2009) similarly evoked the possibility that a single or a few genes may be responsible for dramatic morphological modifications, whereas the remaining of the genome had no time to sort out. This interpretation is in line with Kadereit’s et al. (2007) hypothesis of recurrent evolution of a species such as S. pusilla from S. ramosissima. The observed association between morphology and genetic variation within populations would thus result from the selfing mating system generating substantial linkage within the genome, linkage that would quickly disappear among unrelated individuals from different populations.
Kinship coefficients derived from both the nuclear and cpDNA markers significantly decreased as soon as individuals from different populations were considered, indicating an extremely low mobility of both seeds and pollen. In fact, Salicornia seeds lack specialized devices for dispersal. Fifty per cent of the seeds can be found within a distance of 10 cm from the mother plant, and most are trapped in sediments, algae or marsh vegetation (see Kadereit et al., 2007, for review). Whilst rare events of long-distance dispersal, probably aided by zoochory, might explain some trans-oceanic phylogenetic patterns (Kadereit et al., 2007), this suggests that routine dispersal at the regional scale is extremely limited in the genus.
The existence of strongly inbred, disconnected populations might explain why, although a significant geographic partitioning of genetic variation is evidenced by the F statistics described above, the log-likelihood values of the models implemented by Instruct steadily increased with the value of K. Under this hypothesis, distinctive traits among species of a same cytotype would represent genetic variation within a single gene pool displaying discontinuous variation because of the coexistence of inbred lineages.
Whatever the evolutionary mechanisms behind it, our results thus strongly suggest that the observed range of morphological variation in Salicornia is unparalleled by genetic differentiation. The results presented here do not support the most complex taxonomic treatments of Lahondère (2004) and Stace (2010) but rather fit with the broad species aggregate concept of, e.g. Valdés & Castroviejo (1990) and Piirainen (2001). Given the strong geographic signal in the data, one possibility would be to recognize, within the study area, one Mediterranean and one Atlantic species within each of the diploid and tetraploid lineages. Such species would be ‘cryptic’ in the sense that they would not be characterized morphologically. ‘Cryptic’ species have increasingly been recognized in other organisms with reduced morphologies like bryophytes (see Vanderpoorten et al., 2010b, for review). Recognition of such lineages solely characterized by their geographic range would, however, dramatically increase the number of ‘species’ in taxa with a substantial geographic structure across their distribution range. Definite taxonomic conclusions will, therefore, be presented elsewhere based upon a comparative analysis of all source of information available, including morphology, cytology and various genetic markers collected for a representative numbers of all Salicornia species across their entire distribution range.