Polyploidy and hybridization in the Mediterranean: unravelling the evolutionary history of Centaurium (Gentianaceae).

BACKGROUND AND AIMS: Polyploidy is considered one of the main mechanisms of plant evolution and speciation. In the Mediterranean basin, polyploidy has contributed to making this region a biodiversity hotspot, along with its geological and climatic history and other ecological and biogeographic factors. The Mediterranean genus Centaurium Hill (Gentianaceae) comprises ca. 25 species, of which 60% are polyploids, including tetraploids and hexaploids. To date, the evolutionary history of centauries has been studied using Sanger sequencing phylogenies, which have been insufficient to fully understand the phylogenetic relationships in this lineage. The goal of this study is to gain a better understanding of the evolutionary history of Centaurium by exploring the mechanisms that have driven its diversification, specifically hybridization and polyploidy. We aim at identifying the parentage of hybrid species, at the species or clade level, as well as assessing whether morphological traits are associated with particular ploidy levels. METHODS: We sequenced RADseq markers from 42 samples of 28 Centaurium taxa, and performed phylogenomic analyses using maximum likelihood, summary coalescent SVDquartets and NeighborNet approaches. To identify hybrid taxa, we used Phylonetworks and the fastStructure algorithm. To infer the putative parental species of the allopolyploids, we employed genomic analyses (SNIPloid). The association between different traits and particular ploidy levels was explored with NMDS. KEY RESULTS: Our phylogenetic analyses confirmed the long-suspected occurrence of recurrent hybridization. The allopolyploid origin of the tetraploid C. serpentinicola and the hexaploids C. mairei, C. malzacianum and C. centaurioides were also confirmed, unlike that of C. discolor. We inferred additional signatures of hybridization events within the genus and identified morphological traits differentially distributed in different ploidy levels. CONCLUSIONS: This study highlights the important role that hybridization has played in the evolution of a Mediterranean genus such as Centaurium, leading to a polyploid complex, which facilitated its diversification and may exemplify that of other Mediterranean groups.

The puzzle of plant hybridisation: a high propensity to hybridise but few hybrid zones reported.

An interesting conundrum was recently revealed by R. Abbott when he found that the number of hybrid zones reported in the literature for plants is very low, given the propensity of plants to hybridise. In another literature survey on hybrid zones performed over the period 1970-2022, we found that the number of hybrid zones reported for vertebrates was 2.3 times greater than that reported for vascular plants, even though there are about six times more vascular plant species than vertebrates. Looking at the number of papers reporting hybrid zones, there are 4.9 times more on vertebrates than on vascular plants. These figures support the relevance of this conundrum. In this paper we aim to shed light on this question by providing a structured discussion of the causes that may underlie this conundrum. We propose six non-mutually exclusive factors, namely lack or deficit of spatial structure, lack or deficit of genetic structure, effects of hybridisation between non-closely related species, lability of plant hybrid zones over time, botanists' perception of hybridisation, and deficit of population genetic data. There does not appear to be a single factor that explains our puzzle, which applies to all cases of plants where hybridisation is detected but no hybrid zone is reported. It is argued that some plant features suggest that the puzzle is not, at least entirely, due to insufficient knowledge of the specific cases, a hypothesis that should be addressed with a wider range of empirical data across different taxonomic groups.

Demography-driven and adaptive introgression in a hybrid zone of the Armeria syngameon.

Syngameons represent networks of otherwise distinct species connected by limited gene exchange. Although most studies have focused on how species maintain their cohesiveness despite gene flow, there are additional relevant questions regarding the evolutionary dynamics of syngameons and their drivers, as well as the success of their members and the network as a whole. Using a ddRADseq approach, we analysed the genetic structure, genomic clines and demographic history of a coastal hybrid zone involving two species of the Armeria (Plumbaginaceae) syngameon in southern Spain. We inferred that a peripheral population of the sand dune-adapted A. pungens diverged from the rest of the conspecific populations and subsequently hybridized with a locally more abundant pinewood congener, A. macrophylla. Both species display extensive plastid DNA haplotype sharing. Genomic cline analysis identified bidirectional introgression, but more outlier loci with excess A. pungens than A. macrophylla ancestry, suggesting the possibility of selection for A. pungens alleles. This is consistent with the finding that the A. pungens phenotype is selected for in open habitats, and with the strong correlation found between ancestry and phenotype. Taken together, our analyses suggest an intriguing scenario in which bidirectional introgression may, on the one hand, help to avoid reduced levels of genetic diversity due to the small size and isolated location of the A. pungens range-edge population, thereby minimizing demographic risks of stochastic extinction. On the other hand, the data also suggest that introgression into A. macrophylla may allow individuals to grow in open, highly irradiated, deep sandy, salt-exposed habitats.

Genome-wide footprints in the carob tree (Ceratonia siliqua) unveil a new domestication pattern of a fruit tree in the Mediterranean.

Intense research efforts over the last two decades have renewed our understanding of plant phylogeography and domestication in the Mediterranean basin. Here we aim to investigate the evolutionary history and the origin of domestication of the carob tree (Ceratonia siliqua), which has been cultivated for millennia for food and fodder. We used >1000 microsatellite genotypes to delimit seven carob evolutionary units (CEUs). We investigated genome-wide diversity and evolutionary patterns of the CEUs with 3557 single nucleotide polymorphisms generated by restriction-site associated DNA sequencing (RADseq). To address the complex wild vs. cultivated status of sampled trees, we classified 56 sampled populations across the Mediterranean basin as wild, seminatural or cultivated. Nuclear and cytoplasmic loci were identified from RADseq data and separated for analyses. Phylogenetic analyses of these genomic-wide data allowed us to resolve west-to-east expansions from a single long-term refugium probably located in the foothills of the High Atlas Mountains near the Atlantic coast. Our findings support multiple origins of domestication with a low impact on the genetic diversity at range-wide level. The carob was mostly domesticated from locally selected wild genotypes and scattered long-distance westward dispersals of domesticated varieties by humans, concomitant with major historical migrations by Romans, Greeks and Arabs. Ex situ efforts to preserve carob genetic resources should prioritize accessions from both western and eastern populations, with emphasis on the most differentiated CEUs situated in southwest Morocco, south Spain and eastern Mediterranean. Our study highlights the relevance of wild and seminatural habitats in the conservation of genetic resources for cultivated trees.

Interstitial Arabidopsis-Type Telomeric Repeats in Asteraceae.

Tandem repeats of telomeric-like motifs at intra-chromosomal regions, known as interstitial telomeric repeats (ITR), have drawn attention as potential markers of structural changes, which might convey information about evolutionary relationships if preserved through time. Building on our previous work that reported outstanding ITR polymorphisms in the genus Anacyclus, we undertook a survey across 132 Asteraceae species, focusing on the six most speciose subfamilies and considering all the ITR data published to date. The goal was to assess whether the presence, site number, and chromosomal location of ITRs convey any phylogenetic signal. We conducted fluorescent in situ hybridization (FISH) using an Arabidopsis-type telomeric sequence as a probe on karyotypes obtained from mitotic chromosomes. FISH signals of ITR sites were detected in species of subfamilies Asteroideae, Carduoideae, Cichorioideae, Gymnarhenoideae, and Mutisioideae, but not in Barnadesioideae. Although six small subfamilies have not yet been sampled, altogether, our results suggest that the dynamics of ITR formation in Asteraceae cannot accurately trace the complex karyological evolution that occurred since the early diversification of this family. Thus, ITRs do not convey a reliable signal at deep or shallow phylogenetic levels and cannot help to delimitate taxonomic categories, a conclusion that might also hold for other important families such as Fabaceae.

Hybridization and cryptic speciation in the Iberian endemic plant genus Phalacrocarpum (Asteraceae-Anthemideae).

Understanding the role and impact of reticulation in phylogenetic inquiry has improved with extended use of high throughput sequencing data. Yet, due to the dynamism of genomes over evolutionary time, disentangling old hybridization events remains a serious challenge. Phalacrocarpum (DC.) Willk. is one of the 27 Iberian endemic plant genera, currently considered monotypic but including three subspecies. Its uncertain phylogenetic relationships within tribe Anthemideae (Asteraceae) point to an Early Miocene divergence from its sister group, and its persistent taxonomic instability has been proposed to be due to hybridization. We aim at understanding the evolutionary history of this genus using SNPs called from a genotyping-by-sequencing (GBS) analysis, Sanger sequences-from three plastid DNA regions (psbJ-petA, petB-petD, trnH-psbA) and the nuclear ribosomal ITS regions (cloned)-as well as leaf morphometric multivariate analysis. SNP data and Sanger sequences strongly support the unforeseen existence of a cryptic species in the eastern populations of P. oppositifolium subsp. anomalum. Broad molecular and morphometric patterns of variation found in conflictive populations from the Sanabria Valley region convincingly identify a recent previously undocumented hybrid zone. By contrast, evidence is less conclusive on relationships between subspecies hoffmannseggii, oppositifolium and a second conflictive group distributed along the Galician-Portuguese border (Orense massifs). Although genetic clustering analysis of SNP data suggests that the former subspecies was the maternal progenitor in hybridization events that gave rise to the other two groups, we found considerable uniqueness of ITS ribotypes and plastid haplotypes in them. This result, in the context of Pleistocene climatically-driven range shifts in NW Iberian Peninsula, can be due to periods of isolation, genetic bottlenecks and drift superimposed on old hybridization events. Our study confirms the idea that unravelling old hybridization events may be compromised by the suite of evolutionary processes accumulated subsequently, particularly in areas with a history of climatic instability.

Genomics of Evolutionary Novelty in Hybrids and Polyploids.

It has long been recognized that hybridization and polyploidy are prominent processes in plant evolution. Although classically recognized as significant in speciation and adaptation, recognition of the importance of interspecific gene flow has dramatically increased during the genomics era, concomitant with an unending flood of empirical examples, with or without genome doubling. Interspecific gene flow is thus increasingly thought to lead to evolutionary innovation and diversification, via adaptive introgression, homoploid hybrid speciation and allopolyploid speciation. Less well understood, however, are the suite of genetic and genomic mechanisms set in motion by the merger of differentiated genomes, and the temporal scale over which recombinational complexity mediated by gene flow might be expressed and exposed to natural selection. We focus on these issues here, considering the types of molecular genetic and genomic processes that might be set in motion by the saltational event of genome merger between two diverged species, either with or without genome doubling, and how these various processes can contribute to novel phenotypes. Genetic mechanisms include the infusion of new alleles and the genesis of novel structural variation including translocations and inversions, homoeologous exchanges, transposable element mobilization and novel insertional effects, presence-absence variation and copy number variation. Polyploidy generates massive transcriptomic and regulatory alteration, presumably set in motion by disrupted stoichiometries of regulatory factors, small RNAs and other genome interactions that cascade from single-gene expression change up through entire networks of transformed regulatory modules. We highlight both these novel combinatorial possibilities and the range of temporal scales over which such complexity might be generated, and thus exposed to natural selection and drift.

Genome size variation at constant chromosome number is not correlated with repetitive DNA dynamism in Anacyclus (Asteraceae).

BACKGROUND AND AIMS: Changes in the amount of repetitive DNA (dispersed and tandem repeats) are considered the main contributors to genome size variation across plant species in the absence of polyploidy. However, the study of repeatome dynamism in groups showing contrasting genomic features and complex evolutionary histories is needed to determine whether other processes underlying genome size variation may have been overlooked. The main aim here was to elucidate which mechanism best explains genome size evolution in Anacyclus (Asteraceae). METHODS: Using data from Illumina sequencing, we analysed the repetitive DNA in all species of Anacyclus, a genus with a reticulate evolutionary history, which displays significant genome size and karyotype diversity albeit presenting a stable chromosome number. KEY RESULTS: By reconstructing ancestral genome size values, we inferred independent episodes of genome size expansions and contractions during the evolution of the genus. However, analysis of the repeatome revealed a similar DNA repeat composition across species, both qualitative and quantitative. Using comparative methods to study repeatome dynamics in the genus, we found no evidence for repeat activity causing genome size variation among species. CONCLUSIONS: Our results, combined with previous cytogenetic data, suggest that genome size differences in Anacyclus are probably related to chromosome rearrangements involving losses or gains of chromosome fragments, possibly associated with homoploid hybridization. These could represent balanced rearrangements that do not disrupt gene dosage in merged genomes, for example via chromosome segment exchanges.

Advances in genotyping microsatellite markers through sequencing and consequences of scoring methods for Ceratonia siliqua (Leguminosae).

PREMISE OF THE STUDY: Simple sequence repeat (SSR) or microsatellite markers have been used in a broad range of studies mostly scoring alleles on the basis of amplicon size as a proxy for the number of repeat units of an SSR motif. However, additional sources of variation within the SSR or in the flanking regions have largely remained undetected. METHODS: In this study, we implemented a next-generation sequencing-based genotyping approach in a newly characterized set of 18 nuclear SSR markers for the carob tree, Ceratonia siliqua. Our aim was to evaluate the effect of three different methods of scoring molecular variation present within microsatellite markers on the genetic diversity and structure results. RESULTS: The analysis of the sequences of 77 multilocus genotypes from four populations revealed SSR variation and additional sources of polymorphism in 87% of the loci analyzed (42 single-nucleotide polymorphisms and five insertion/deletion polymorphisms), as well as divergent paralog copies in two loci. Ignoring sequence variation under standard amplicon size genotyping resulted in incorrect identification of 69% of the alleles, with important effects on the genetic diversity and structure estimates. DISCUSSION: Next-generation sequencing allows the detection and scoring of SSRs, single-nucleotide polymorphisms, and insertion/deletion polymorphisms to increase the resolution of population genetic studies.

Morphological Innovations and Vast Extensions of Mountain Habitats Triggered Rapid Diversification Within the Species-Rich Irano-Turanian Genus Acantholimon (Plumbaginaceae).

The Irano-Turanian floristic region spans a topographically complex and climatically continental territory, which has served as a source of xerophytic taxa for neighboring regions and is represented by a high percent of endemics. Yet, a comprehensive picture of the abiotic and biotic factors that have driven diversification within this biota remains to be established due to the scarcity of phylogenetic studies. Acantholimon is an important component of the subalpine steppe flora of the Irano-Turanian region, containing c. 200 cushion-forming sub-shrubby pungent-leaved species. Our recent molecular phylogenetic study has led to enlarging the circumscription of this genus to include eight mono- or oligospecific genera lacking the characteristic life-form and leaves. Using the same molecular phylogeny, here we investigate the tempo and mode of diversification as well as the biogeographic patterns in this genus, to test the hypothesis that a combination of key morphological innovations and abiotic factors is behind Acantholimon high species diversity. Molecular dating analysis indicates that Acantholimon s.l. started to diversify between the Late Miocene and the Pliocene and the biogeographic analysis points to an Eastern Iran-Afghanistan origin. Macroevolutionary models support the hypothesis that the high diversity of the genus is explained by accelerated diversification rates in two clades associated with the appearance of morphological key innovations such as a cushion life-form and pungent leaves; this would have favored the colonization of water-stressed, substrate-poor mountainous habitats along the newly uplifted IT mountains during the Mio-Pliocene. Given the apparent similarity of mountain habitats for most species of Acantholimon, we hypothesize that its current high species diversity responds to a scenario of non-adaptive radiation fueled by allopatric speciation rather than evolutionary radiation driven by ecological opportunity. Similar scenarios might underlie the high diversity of other speciose genera in the topographically complex Irano-Turanian landscape, though this remains to be tested with fine-grained distribution and climatic data.

High and uneven levels of 45S rDNA site-number variation across wild populations of a diploid plant genus (Anacyclus, Asteraceae).

The nuclear genome harbours hundreds to several thousand copies of ribosomal DNA. Despite their essential role in cellular ribogenesis few studies have addressed intrapopulation, interpopulation and interspecific levels of rDNA variability in wild plants. Some studies have assessed the extent of rDNA variation at the sequence and copy-number level with large sampling in several species. However, comparable studies on rDNA site number variation in plants, assessed with extensive hierarchical sampling at several levels (individuals, populations, species) are lacking. In exploring the possible causes for ribosomal loci dynamism, we have used the diploid genus Anacyclus (Asteraceae) as a suitable system to examine the evolution of ribosomal loci. To this end, the number and chromosomal position of 45S rDNA sites have been determined in 196 individuals from 47 populations in all Anacyclus species using FISH. The 45S rDNA site-number has been assessed in a significant sample of seed plants, which usually exhibit rather consistent features, except for polyploid plants. In contrast, the level of rDNA site-number variation detected in Anacyclus is outstanding in the context of angiosperms particularly regarding populations of the same species. The number of 45S rDNA sites ranged from four to 11, accounting for 14 karyological ribosomal phenotypes. Our results are not even across species and geographical areas, and show that there is no clear association between the number of 45S rDNA loci and the life cycle in Anacyclus. A single rDNA phenotype was detected in several species, but a more complex pattern that included intra-specific and intra-population polymorphisms was recorded in A. homogamos, A. clavatus and A. valentinus, three weedy species showing large and overlapping distribution ranges. It is likely that part of the cytogenetic changes and inferred dynamism found in these species have been triggered by genomic rearrangements resulting from contemporary hybridisation.

Genome size and base composition variation in natural and experimental Narcissus (Amaryllidaceae) hybrids.

BACKGROUND AND AIMS: Although there is evidence that both allopolyploid and homoploid hybridization lead to rapid genomic changes, much less is known about hybrids from parents with different basic numbers without further chromosome doubling. Two natural hybrids, Narcissus × alentejanus (2n = 19) and N. × perezlarae (2n = 29), originated by one progenitor (N. cavanillesii, 2n = 28) and two others (N. serotinus, 2n = 10 and N. miniatus, 2n = 30, respectively) allow us to study how DNA content and composition varies in such hybrids. METHODS: Flow cytometry measurements with two staining techniques, PI and DAPI, were used to estimate 2C values and base composition (AT/GC ratio) in 390 samples from 54 wild populations of the two natural hybrids and their parental species. In addition, 20 synthetic F(1) hybrid individuals were also studied for comparison. KEY RESULTS: Natural hybrids presented 2C values intermediate between those found in their parental species, although intra-population variance was very high in both hybrids, particularly for PI. Genome size estimated from DAPI was higher in synthetic hybrids than in hybrids from natural populations. In addition, differences for PI 2C values were detected between synthetic reciprocal crosses, attributable to maternal effects, as well as between natural hybrids and those synthetic F(1) hybrids in which N. cavanillesii acted as a mother. CONCLUSIONS: Our results suggest that natural hybrid populations are composed of a mixture of markedly different hybrid genotypes produced either by structural chromosome changes, consistent with classic cytogenetic studies in Narcissus, or by transposon-mediated events.

Five molecular markers reveal extensive morphological homoplasy and reticulate evolution in the Malva alliance (Malvaceae).

The Malva alliance is a well-defined group with extensive morphological homoplasy. As a result, the relationships among the taxa as well as the evolution of morphological traits have remained elusive and the traditional classifications are highly artificial. Using five molecular markers (nuclear ITS, plastid matK plus trnK, ndhF, trnL-trnF, psbA-trnH), we arrived at a phylogenetic hypothesis of this group, the genera Alcea, Althaea and Malvalthaea being studied here for the first time with molecular data. Althaea and, in particular, Lavatera and Malva are highly polyphyletic as currently circumscribed, because their diagnostic characters, the number and degree of fusion of the epicalyx bracts, evolve in a highly homoplasious manner. In contrast, fruit morphology largely agrees with the molecularly delimited groups. Hybrid origins confirmed for the genus Malvalthaea and for Lavatera mauritanica and hybridization in the group of ruderal small-flowered mallows underline the importance of reticulate evolution in shaping the history of this group and complicating the interpretation of morphological evolution.

Ecology matters: Atlantic-Mediterranean disjunction in the sand-dune shrub Armeria pungens (Plumbaginaceae).

Inferring the evolutionary history of Mediterranean plant lineages from current genetic, distributional and taxonomic patterns is complex because of a number of palaeoclimatic and geological interconnected factors together with landscape heterogeneity and human influence. Therefore, choosing spatially simplified systems as study groups is a suitable approach. An amplified fragment length polymorphism (AFLP) study using two restriction enzyme combinations (EcoRI/MseI and KpnI/MseI) was carried out to estimate the structure of genetic variation throughout the range of Armeria pungens. This species has a West Iberian-Corso/Sardinian disjunct distribution on coastal sand-dune ecosystems. Bayesian, amova and genetic distance analyses of the AFLP data revealed the same distinguishable genetic groups, which do not match the main geographical disjunction. Corso-Sardinian populations were found to be genetically closer to southwest Portuguese than to those from the Gulf of Cadiz (the closest geographically). Eastwards long-distance dispersal is therefore invoked to explain this geographical disjunction. A GIS analysis based on bioclimatic envelope modelling aiming to characterize the current locations of A. pungens found strong similarities between the Portugal and Corsica-Sardinia sites and less so between these areas and the Gulf of Cadiz. This coincident pattern between AFLP and climatic data suggests that the geographical disjunction is better explained by climatic factors than by the likeliness of a stochastic dispersal event. Such a combined phylogeographical-GIS modelling approach proves to be enlightening in reconstructing the evolutionary history of plant species.

Better the devil you know? Guidelines for insightful utilization of nrDNA ITS in species-level evolutionary studies in plants.

The internal transcribed spacers (ITS) of the nuclear ribosomal 18S-5.8S-26S cistron continue to be the most popular non-plastid region for species-level phylogenetic studies of plant groups despite the early warnings about their potential flaws, which may ultimately result in incorrect assumptions of orthology. It has been gradually realized that the alternative target regions in the nuclear genome (low-copy nuclear genes, LCNG) are burdened with similar problems. The consequence is that, to date, developing useful LCNG for non-model organisms requires an investment in time and effort that hinders its use as a real practical alternative for many labs. It is here argued that ITS sequences, despite drawbacks, can still produce insightful results in species-level phylogenetic studies or when non-anonymous nuclear markers are required, provided that a thoughtful use of them is made. To facilitate this, two series of guidelines are proposed. One helps to circumvent problems of ITS amplification from the target organism, including spurious results from contaminants, paralogs and pseudogenes, as well as detection of sequencing artifacts. The other series helps to find out causes for unresolved clades in phylogenetic reconstruction, to integrate gene phylogenies, to distinguish horizontal transfer from lineage sorting, and to reveal if ITS phylogeny is not a good estimate of organism phylogeny.

Fine-scale geographical structure, intra-individual polymorphism and recombination in nuclear ribosomal internal transcribed spacers in Armeria (Plumbaginaceae).

BACKGROUND AND AIMS: Isolation and drift are the main causes for geographic structure of molecular variation. In contrast, the one found in a previous survey in Armeria (Plumbaginaceae) for nuclear ribosomal ITS multicopy regions was species-independent and has been hypothesized to be due to extensive gene-flow and biased concerted evolution. Since this was inferred from a genus-level phylogenetic analysis, the aim of this study was to check for the occurrence of such structure and the validity of the proposed model at a local scale, in a southern Spanish massif (Sierra Nevada), as well as to examine the evolutionary implications at the organism level. METHODS: In addition to 117 sequences of direct PCR products from genomic DNA, 50 sequences of PCR products from cloned DNA were obtained to analyse cases of intragenomic polymorphisms for the ITS regions. KEY RESULTS: Sequence data confirm the occurrence of a species-independent structure at a local scale and reveal insights through the analysis of contact areas between different ITS copies (ribotypes). A comparison between cloned and direct sequences (a) confirms that, within these contact areas, ITS copies co-occur both in different individuals and within single genomes; and (b) reveals recombination between different copies. CONCLUSIONS: This study supports the utility of direct sequences for detecting intra-individual polymorphism and for partially inferring the ITS copies involved, given previous knowledge of the variability. The main evolutionary implication at the organism level is that gene-flow and concerted evolution shape the geographic structure of ITS variation.

Origin of the disjunct tetraploid Cardamine amporitana (Brassicaceae) assessed with nuclear and chloroplast DNA sequence data.

Seventy-four nucleotide sequences from the ITS regions of nuclear ribosomal DNA and 76 from the trnL-trnF spacer of chloroplast DNA were used to address the origin of tetraploid Cardamine amporitana, the conspecifity of central Italian and northeastern Spanish populations, and the possible cause for such geographic disjunction. Because of the complex lineage relationships in Cardamine, the sampling included 22 taxa. In the results, both data sets are highly congruent in supporting a close relationship of C. amporitana to the widespread Eurasian C. amara. Low genetic variability in northeastern Spanish populations of C. amporitana suggests long-distance dispersal from central Italy. The interior position of the single northeastern Spanish haplotype in a statistical parsimony network of trnL-trnF haplotypes however does not support this scenario and invokes other plausible phylogeographic explanations. The disappearance of geographically intermediate populations and genetic impoverishment by migration and isolation, both probably associated with Quaternary climatic oscillations, appears as an alternative hypothesis to explain the phylogeographic pattern. A recent hybridization event is reported between C. amporitana and a diploid from the C. pratensis group in central Italy on the basis of additive polymorphisms in ITS for all the 22 distinguishing nucleotides.

Additive polymorphisms and reticulation in an ITS phylogeny of thrifts (Armeria, Plumbaginaceae).

A parsimony analysis of 133 sequences of the nuclear ribosomal DNA ITS1+5.8S+ITS2 region from 71 taxa in Armeria was carried out. The presence of additive polymorphic sites (APS; occurring in 14 accessions) fits the reticulate scenario proposed in previous work for explaining the ITS pattern of variation on a much smaller scale and is based mainly on the geographical structure of the data, irrespective of taxonomic boundaries. Despite the relatively low bootstrap values and large polytomies, part of which are likely due to disruptive effects of reticulation and concerted evolution in these multicopy sequences, the ITS analysis has phylogenetic and biogeographic implications. APS detected in this study are consistent with hypothesized hybridization events, although biased concerted evolution, previously documented in the genus, needs to be invoked for specific cases and may be responsible for a possible "sink" effect in terminals from a large clade. The causes for sequences of the same species appearing in different clades (here termed transclade) are discussed.