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Whole‐genome duplication is considered an important speciation mechanism in plants. However, its effect on reproductive isolation between higher cytotypes is not well understood. We used backcrosses between different ploidy levels and surveys of mixed‐ploidy contact zones to determine how reproductive barriers differed with cytotype across a polyploid complex. We backcrossed F1 hybrids derived from 2X‐4X and 4X‐6X crosses in the Campanula rotundifolia autopolyploid complex, measured backcross fitness, and estimated backcross DNA cytotype. We then sampled four natural mixed‐ploidy contact zones (two 2X‐4X and two 4X‐6X), estimated ploidy, and genotyped individuals across each contact zone. Reproductive success and capacity for gene flow was markedly lower for 2X‐4X than 4X‐6X hybrids. In fact, 3X hybrids could not backcross; all 2X‐4X backcross progeny resulted from neotetraploid F1 hybrids. Further, no 3X individuals were found in 2X‐4X contact zones, and 2X and 4X individuals were genetically distinct. By contrast, backcrosses of 5X hybrids were relatively successful, particularly when crossed to 6X individuals. In 4X‐6X contact zones, 5X individuals and aneuploids were common and all cytotypes were largely genetically similar and spatially intermixed. Taken together, these results provide strong evidence that reproduction is low between 2X and 4X cytotypes, primarily occurring via unreduced gamete production, but that reproduction and gene flow are ongoing between 4X and 6X cytotypes. Further, it suggests whole‐genome duplication can result in speciation between diploids and polyploids, but is less likely to create reproductive barriers between different polyploid cytotypes, resulting in two fundamentally different potentials for speciation across polyploid complexes., To assess the role of ploidy in determining reproductive isolation and speciation in polyploid contact zones, we used backcrosses between different ploidy levels and surveys of mixed‐ploidy contact zones to determine how reproductive barriers differed with cytotype across a polyploid complex. Reproductive success and capacity for gene flow was markedly higher for 4X‐6X hybrids than 2X‐4X hybrids, which was also seen in natural mixed‐ploidy contact zones. Our results suggest whole‐genome duplication can result in speciation between diploids and polyploids, but is less likely to create reproductive barriers between different polyploid cytotypes, resulting in two fundamentally different potentials for speciation across polyploid complexes.

Lippia alba is a phenotypically variable tropical shrub thought to comprise a young autopolyploid complex. Chromosome numbers in L. alba include 2n = 30, 38, 45, 60, and 90. High levels of chemical and phenotypic variation associated with economic and medicinal importance were reported. However, the genetic background including chromosome composition remains under-explored. Furthermore, the occurrence of at least four ploidal levels in L. alba and the lack of data for polyploid plants in tropical areas also merit further study of L. alba. Here we assessed the chromosome composition using two new satellite repeats (CL98 and CL66) applied as FISH probes to mitotic chromosomes, and we proposed to calculate the degree of homozygosis for CL66 satDNA (named as index h) and to associate it to meiotic instability. The CL98 mapping showed few variations in both number of signals and position. However, the levels of structural homozygosity for a satellite repeat CL66 were very variable. The numbers of CL66-bearing-chromosomes were under-represented in tetraploids relative to diploids implying that CL66 arrays have been lost in tetraploid lineages as a result of increased meiotic instability. High percentage of irregularities was observed in meiotic cells, especially in polyploids. L. alba complex comprised a mixture of homomorphic and heteromorphic chromosomes. Overall, the polyploid complex presents features typical of both young and older stable polyploids. It seems that L. alba genome is still in the process of stabilization.

Species are the basic units of biodiversity and evolution. Nowadays, they are widely considered as ancestor-descendant lineages. Their definition remains a persistent challenge for taxonomists due to lineage evolutionary role and circumscription, i.e., persistence in time and space, ecological niche or a shared phenotype of a lineage. Recognizing and delimiting species is particularly methodically challenging in fast-evolving, evolutionary young species complexes often characterized by low genetic divergence, hybrid origin, introgression and incomplete lineage sorting (ILS). Ranunculus auricomus is a large Eurasian apomictic polyploid complex that probably has arisen from the hybridization of a few sexual progenitor species. However, even delimitation and relationships of diploid sexual progenitors have been unclearly ranging from two to twelve species. Here, we present an innovative workflow combining phylogenomic methods based on 86,782 parameter-optimized RADseq loci and target enrichment of 663 nuclear genes together with geometric morphometrics to delimit sexual species in this evolutionary young complex (

When differentiated lineages come into contact, their fates depend on demographic and reproductive factors. These factors have been well-studied in taxa of the same ploidy, but less is known about sympatric lineages that differ in ploidy, particularly with respect to demographic factors. We assessed prezygotic, postzygotic, and total reproductive isolation in naturally pollinated arrays of diploid-tetraploid and tetraploid-hexaploid population mixes of Campanula rotundifolia by measuring pollinator transitions, seed yield, germination rate, and proportion of hybrid offspring. Four frequencies of each cytotype were tested, and pollinators consistently overvisited rare cytotypes. Seed yield and F1 hybrid production were greater in 4X-6X arrays than 2X-4X arrays, whereas germination rates were similar, creating two distinct patterns of reproductive isolation. In 2X-4X arrays, postzygotic isolation was near complete (3% hybrid offspring), and prezygotic isolation associated with pollinator preference is expected to facilitate the persistence of minority cytotypes. However, in 4X-6X arrays where postzygotic isolation permitted hybrid formation (44% hybrids), pollinator behavior drove patterns of reproductive isolation, with rare cytotypes being more isolated and greater gene flow expected from rare into common cytotypes. In polyploid complexes, both the specific cytotypes in contact and local cytotype frequency, likely reflecting spatial demography, will influence likelihood of gene exchange.

Polyploidy plays a significant role in the evolution and diversification of flowering plants. In several polyploid complexes, high morphological variability and plasticity coupled with cytogenetic diversity make it difficult to disentangle their evolutionary history. The main goal of this study was to gain insights into the role of whole genome duplications as one of the factors shaping the evolution of flowering plants. Linum suffruticosum s.l. has been described as a polyploid complex, with high morphological variability, but nothing is known about current cytogeographical patterns. We investigated cytotype diversity and distribution patterns in 151 populations covering most of the distribution range, in the Iberian Peninsula, south-eastern France, north-western Italy and Morocco, using flow cytometric analyses complemented with chromosome counts. A high cytogenetic diversity was found with five major cytotypes being detected (diploids, tetraploids, hexaploids, octoploids and decaploids) and with new ploidy levels being reported for the first time. The different ploidies were distributed parapatrically, with geographical structure and several contact zones. Most of the populations comprised one cytotype, but a few mixed-ploidy populations were observed. Our results suggest that whole genome duplications are one of the key mechanisms, alone or together with hybridization, governing the diversification of L. suffruticosum s.l. Genome size and/or chromosome counts might be useful tools for identifying specimens of L. suffruticosum s.l. Also, geographical overlap and high cytogenetic diversity suggest multiple origins of the polyploids. The diversity observed here has been mostly neglected to date and should be accounted when studying the biosystematics of this complex.

Hybridization associated with polyploidization and apomixis is a frequent mechanism of speciation.Sorbusis a genus with ongoing hybridization resulting in a polyploid complex with different parental lineage.Triparensis the smallest hybridogenous subgenus ofSorbusso far known to comprise only two taxa,S. intermediaandS. × liljeforsiithat combine the genomes of three taxa (S. ariaagg.,S. aucupariaandS. torminalis). To elucidate the origins ofS. dacica,S. paxianaandS. tauricola, three new trigenomic candidates formerly believed to be of biparental origin with eitherS. ariaagg. × S. aucupariaorS. ariaagg. × S. torminalislineage we combined data from HPLC and chloroplast DNA analysing additional 33 related taxa as well. We concluded that the ‘torminalis-type’ flavonoid profile and the ‘aucuparia-type’ plastid indicate the participation of bothS. torminalisandS. aucupariaresulting in the formation ofS. dacica,S. paxianaandS. tauricola.Sorbus ariaagg. as the third ancestor and as a necessary link to meet genes ofS. torminalisandS. aucupariain one genome is obvious from morphological features (densely tomentose undersides of leaves). The tetraploid cytotypes and obligate pseudogamy ofS. dacicaandS. paxianawere determined by flow cytometry and are published here for the first time. The most probable evolutionary scenario forTriparensspecies is: 1. a diploid sexualS. aucupariaas pollen acceptor hybridized with a tetraploid apomictic taxon from theS. ariaagg. producing a triploid apomictic taxon with ‘aucuparia-type’ plastid inherited maternally; 2. during a second crossing event this subgenusSorariahybrid as maternal progenitor hybridized with the sexual diploidS. torminalis(providing gene(s) of apigenin O-glucuronide synthesis) forming a tetraploidTriparenshybrid with ‘aucuparia-type’ plastid and ‘torminalis-type’ flavonoids.

While it is known that whole genome duplication (WGD) and reticulate evolution play important roles in plant evolution, the origins and evolutionary histories of most polyploid and reticulate groups are still poorly known. The North Temperate haplostemonous (NTH) Ludwigia L. (sections Isnardia (L.) W. L. Wagner & Hoch, Ludwigia, Microcarpium Munz, and Miquelia P. H. Raven) group, characterized by having 4-merous and haplostemonous flowers, pluriseriate and free seeds, glabrous and convex nectaries, and a north-temperate distribution, is a polyploid complex (2×, 4×, 6×, and 8×) of 24 species with frequent reports of inter- and intrasectional hybridization. Although earlier biosystematics studies postulated some evolutionary scenarios and recent molecular phylogenetic studies have partially tested these propositions, the full history of their reticulate evolution remains puzzling. In this study, we sequenced four chloroplast regions (rpL16, rpoB-trnC, trnL-trnF, and ycf6-psbM) and conducted extensive molecular cloning of the biparentally inherited single-copy nuclear PgiC gene (376 clones in total), sampling 23 of the 24 NTH Ludwigia species whose chromosome numbers and ploidy levels were confirmed. Both the chloroplast and PgiC trees include strongly supported sister clades of section Ludwigia (four diploid species) and the “Microcarpium complex” (composed of sections Isnardia, Microcarpium, and Miquelia), which together are sister to the rest of Ludwigia. In the PgiC tree, eight clades are identified within the Microcarpium complex, with four clades including no extant diploid species. Neither sections Isnardia nor Microcarpium are monophyletic, while the monospecific section Miquelia has a hybrid origin. By integrating our phylogenetic trees with previous cytological hypotheses, the reticulate evolution of NTH Ludwigia is disentangled and four to eight extinct diploid species are inferred. Ancestral area reconstruction supports a North American origin of L. ovalis whose current East Asian distribution reflects a relict of the Arcto-Tertiary Geoflora. Based on our results, we propose to synonymize sections Microcarpium and Miquelia under the expanded section Isnardia.

International audience; The economically important Allium ampeloprasum L. represents a polyploid complex, comprising hexa- and octoploid Great Headed Garlic horticultural cultivars (6x-8x GHG) and several traditional varieties of the tetraploid (4x) leeks (Leek, Bulbous leek, Kurrat and Pearl onion). Its wild representatives were indicated as rare in the Mediterranean region. This study aims to explore the diversity and origin of polyploidy in this complex, including its wild relatives A. baeticum Bossier and A. guttatum Steven with particular focus on the poorly investigated North-African region. Natural populations were sampled in Algeria in various bioclimatic conditions, then subjected to karyological and molecular phylogenetic analyses based on nuclear rDNA ITS region and chloroplast trnL-trnF and trnD-trnT intergenic spacers. Comparative analyses included available Genbank accession sequences representing old-world relatives. Chromosome count surveys revealed an unexpected higher occurrence of diploid (2n = 16) than tetraploid (2n = 32) cytotypes. The phylogenetic analyses first allowed positioning the Algerian material within the A. ampeloprasum complex. Interestingly, all the Algerian diploid and tetraploid populations from A. ampeloprasum and A. baeticum form a distinct monophyletic group. The results provide novel and robust evidence demonstrating that the North African diploid A. ampeloprasum genetic pool widely contributed as a source of progenitors not only for the A. ampeloprasum and A. baeticum Algerian tetraploids, but also in the formation of the GHG and Leek cultivated allopolyploids. Therefore, the North African populations emerge as an important reservoir of new wild genetic resources of great interest for tracing the origin of crop domestication and for breeding programs of cultivated varieties.

This study is the first report of a genetic stability analysis of a polyploid complex maintained in vitro for a long-time. Twenty-two accessions of Lippia alba, a medicinal species of economic importance, had been maintained under in vitro culture conditions for 7 years through sprouting of axillary buds. Four clones of each accession were analyzed, being three plants from in vitro bank and one cultivated in the field. We investigated the genetic stability of diploid, aneuploid, triploid, tetraploid, and hexaploid accessions. The investigation was carried out using flow cytometry, inter simple sequence repeat (ISSR) and simple sequence repeat (SSR) markers. No significant variation in nuclear DNA content was observed between the in vitro conserved plants and their respective field plant. Out of 23 ISSR primers screened, 8 primers were found to produce clear reproducible bands resulting in a total of 5456 bands. 86.36% of the analyzed plantlets (19 accessions) showed at least one polymorphic band. The polymorphic rate ranged from 1.61 to 33.87%. The SSR markers were used to confirm the absence or low occurrence of variation in accessions that showed no polymorphism or polymorphism for only one ISSR primer. The genetic instability detected in our study at the molecular level may be attributed to the natural instability of L. alba genome combined with the long-time in vitro maintenance.

Evolutionary processes in plants such as hybridization and polyploidy are relevant to speciation and little studied in subtropical South America. In subtropical South America, the genus Mimosa is h...

The genus Sesleria represents a taxonomically intricate polyploid complex of morphologically highly varied and weakly differentiated taxa. In the wider area of the Western Carpathians, the genus is represented by three tetraploid (S. caerulea, S. heufleriana, and S. uliginosa) and three octoploid (S. hungarica, S. sadleriana, and S. tatrae) species. Although these taxa play a dominant role in a number of plant communities, their identification is often problematic. We examined relative (RGS; 637 individuals, 49 populations) and absolute (AGS; 64 individuals, 19 populations) genome size variation of all six taxa and revealed that genome size could be an effective marker for their identification. Genome size allowed not only separation of the two ploidy levels (4x vs. 8x), but helped to identify at least four of the six studied species. Among tetraploids, RGS appeared to be diagnostic for S. heufleriana (even at individual level), but failed to differentiate S. caerulea from S. uliginosa. RGS appeared to be diagnostic for octoploid species at population level, although the identity of populations at Vrsatec (Slovakia) and Naszaly (Hungary) require further studies. We found that octoploid plants from Slavec (Slovakia), formerly identified as S. heufleriana, actually represent S. hungarica, which is the first reliable record of this species from Slovakia. In the geographically isolated or peripheral populations, an increased or decreased genome size has been detected.

Polyploidy has played an important evolutionary role in the genus Festuca (Poaceae), and several ploidy levels (ranging from 2n = 2x = 14 to 2n = 12x = 84) have been detected to date. This study aimed to estimate the genome size and ploidy level of two subspecies belonging to the F. yvesii polyploid complex by flow cytometry and chromosome counting. The phenotypic variation of the cytotypes was also explored, based on herbarium material. The genome size of F. yvesii subsp. lagascae has been estimated for the first time. Nuclear 2C DNA content of F. yvesii subsp. summilusitana ranged from 21.44 to 31.91 pg, while that of F. yvesii subsp. lagascae was from 13.60 to 22.31 pg. We report the highest ploidy level detected for Festuca (2n = 14x = 98) and previously unknown cytotypes. A positive correlation between holoploid genome size and chromosome number counts shown herein was confirmed. The morphometric approach showed a slight trend towards an increase in the size of some organs consistent with the variation in the ploidy level. Differences in characters were usually significant only among the most extreme cytotypes of each subspecies, but, even in this case, the high overlapping ranges prevent their distinction.

[EN] Centaurea aspera polyploid complex represents a comprehensive model. The aim of this short communication was to study the mating system of the three main species. The results showed that allotetraploid C. seridis was self-compatible (SC), while autotetraploid C. gentilii was self-incompatible (SI) as the diploid parental C. aspera (SI)., This work was supported by Generalitat Valenciana through AICO/2019/227 project.

Background and Aims Polyploidy has played a major role in the origin of new plant species, probably because of the expansion of polyploid populations in the species’ ecological niche, and because reproductive isolation can be established between a new polyploid population and its diploid progenitor species. It is well established that most polyploid species are polyphyletic, with multiple independent origins, and that polyploid genomes may undergo rapid change after their duplication and hybridization associated with their origin. We considered whether multiple independent origins and rapid genomic change might lead to reproductive isolation between polyploid populations of the same ploidy but with potentially different evolutionary histories. Methods We tested our hypothesis by assessing differences in DNA content and morphology, the evolution of reproductive isolation, and the phylogenetic placement of two broadly sympatric hexaploid lineages of the wind-pollinated annual plant Mercurialis annua hitherto regarded as populations of the same species. Key Results The two hexaploid lineages of M. annua have slightly divergent DNA content, and distinct inflorescence morphology. They also fall into largely different clades of a chloroplast phylogeny and are reproductively isolated from one another. Conclusions The distinct evolutionary histories of the two hexaploid lineages of M. annua have contributed to the remarkable reproductive diversity of the species complex. It seems likely that reproductive interference between them will eventually lead to the displacement of one lineage by the other via pollen swamping. Thus, whereas polyploidization can contribute to speciation, diversification might also be compromised by reproductive interference.

Polyploidy plays a paramount role in phytodiversity, but the causes of this evolutionary pathway require further study. Here, we use phylogenetic methods to examine possible polyploidy-promoting factors by comparing diploid representatives of the comprehensive European polyploid complex Leucanthemum with members of its strictly diploid North African counterpart Rhodanthemum. We investigate genetic divergence and gene flow among all diploid lineages of both genera to evaluate the role of genomic differentiation and hybridization for polyploid speciation. To test whether hybridization in Leucanthemum has been triggered by the geological conditions during its diversification, we additionally generate a time-calibrated phylogeny of 46 species of the subtribe Leucantheminae. Leucanthemum shows a significantly higher genetic divergence and hybridization signal among diploid lineages compared with Rhodanthemum, in spite of a similar crown age and diversification pattern during the Quaternary. Our study demonstrates the importance of genetic differentiation among diploid progenitors and their concurrent affinity for natural hybridization for the formation of a polyploid complex. Furthermore, the role of climate-induced range overlaps on hybridization and polyploid speciation during the Quaternary is discussed.

Although plant species originated from Arctic regions commonly grow in alpine habitats at mid-latitudes, some populations of these species exist also in some specific habitats below the treeline. Local populations at lower elevations may have different origins, ploidy levels, mating systems and/or morphological traits from alpine populations, but comparative studies between alpine and low-elevation populations are scarce. We aimed to reveal the ecological and genetic differentiations between higher and lower populations of Vaccinium vitis-idaea in Hokkaido, northern Japan by comparing 22 populations growing in diverse environments. We analysed the ploidy level of individual populations using flow cytometry. Genetic differentiation among populations, and genetic diversity within populations were calculated using microsatellite markers. Fruit and seed production were recorded under natural conditions, and a pollination experiment was conducted to reveal the variations in mating system across populations. Furthermore, we compared shoot growth and leaf characteristics among populations. Most of the low-elevation populations were tetraploid, whereas all but one of the alpine populations were diploid. Tetraploid populations were clearly differentiated from diploid populations. Some tetraploid populations formed huge clonal patches but genetic diversity was higher in tetraploids than in diploids. Alpine diploids were self-incompatible and produced more seeds per fruit than tetraploid populations. In contrast, tetraploids showed high self-compatibility. Leaf size and foliar production were greater in tetraploid populations. Our results indicate that the genetic compositions of low-elevation tetraploid populations are different from those of alpine diploid populations. Most populations at lower elevations contained unique ecotypes suited to persistence in isolated situations. Local, low-elevation populations of typical alpine species maintain ecologically and genetically specific characteristics and could be valuable in terms of evolutionary and conservation biology. The present study demonstrates the biological importance of small and isolated populations at the edges of species distribution., Plant species originated from Arctic regions commonly grow in alpine habitats at mid-latitudes, but some species exist also in specific low-elevation habitats. We investigated ecological and genetic differentiations between alpine and low-elevation populations of Vaccinium vitis-idaea in northern Japan. Most of the alpine populations were diploids as well as northern populations. Unexpectedly, most of the low-elevation populations were tetraploids that were first record in this species. Tetraploids showed unique genetic composition, higher self-compatibility and larger morphological traits compared to diploids. Therefore, alpine and low-elevation populations are composed of different ecotypes suited to persistence in each specific environment.

The aim of this work is to review our knowledge on the evolutionary significance of triploid plants, modes of their origin and mechanisms preventing triploid formation, and to answer the question of whether triploids act rather as mediators or barriers in gene flow between populations possessing different numbers of chromosome sets (diploid and tetraploid). The production of unreduced gametes is considered to be the major pathway of triploid formation in otherwise diploid populations, following the merger of haploid and diploid gamete. In contrast, in mixed di-tetraploid populations, triploids are usually formed by interploidy hybridization. The frequency of formation and fitness of triploids is reduced by a number of reproductive barriers, both pre- and postzygotic, the most important of which is the so-called triploid block caused by an unbalanced ratio of originally maternal and paternal genes in the nourishing tissue endosperm. Although the formation of triploids is accompanied by many barriers, which also further translate to their reduced viability, followed by additional reduction in fertility due to meiotic problems, triploids play a more important role in populations than might be expected solely on the basis of their rare occurrence in nature. Triploids act as mediators of gene flow...

SummaryPolyploidy and hybridization are important processes in the evolution of spore-dispersed plants. Few studies, however, focus these dynamics in heterosporous lycophytes, such as Isoëtes, where polyploid hybrids are common and thought to be important in the generation of their extant diversity. We investigate reticulate evolution in a complex of western North American quillworts (Isoëtes) and provide insights into the evolutionary history of hybrids, and the role of polyploidy in maintaining novel diversity.We utilize low copy nuclear markers, whole plastomes, restriction site-associated DNA sequencing, cytology, and reproductive status (fertile or sterile) to investigate the reticulate evolutionary history of western North American Isoëtes.We reconstruct the reticulate evolutionary history and directionality of hybridization events in this complex. The presence of high level polyploids, plus frequent homoploid and interploid hybridization suggests that there are low prezygotic reproductive barriers in this complex, hybridization is common and bidirectional between similar—but not divergent—cytotypes, and that allopolyploidization is important to restore fertility in some hybrid taxa.Our data provide five lines of evidence suggesting that hybridization and polyploidy can occur with frequency in the genus, and these evolutionary processes may be important in shaping extant Isoëtes diversity.

International audience; We explored diversity, distribution and evolutionary dynamics of Ty1-Copia retrotransposons in the genomes of the Hordeum murinum polyploid complex and related taxa. Phylogenetic and fluorescent in situ hybridization (FISH) analyses of reverse transcriptase sequences identified four Copia families in these genomes the predominant BARE1 (including three groups or subfamilies, A, B and C), and the less represented RIRE1, IKYA and TAR-1. Within the BARE1 family, BARE1-A elements and a subgroup of BARE1-B elements (named B1) have proliferated in the allopolyploid members of the H. murinum complex (H. murinum and H. leporinum), and in their extant diploid progenitor, subsp. glaucum. Moreover, we found a specific amplification of BARE1-B elements within each Hordeum species surveyed. The low occurrence of RIRE1, IKYA and TAR-1 elements in the allopolyploid cytotypes suggests that they are either weakly represented or highly degenerated in their diploid progenitors. The results demonstrate that BARE1-A and BARE1-B1 Copia elements are particularly well represented in the genomes of the H. murinum complex and constitute its genomic hallmark. No BARE1-A and -B1 homologs were detected in the reference barley genome. The similar distribution of RT-Copia probes across chromosomes of diploid, tetraploid and hexaploid taxa of the murinum complex shows no evidence of proliferation following polyploidization.

Polyploidy is widely recognized as a major evolutionary force in plants and has been reported in the genus Lippia (Verbenaceae). Lippia alba, the most studied species, has been documented as a polyploid complex involving at least four ploidal levels. L. alba presents remarkable chemical and genetic variation and represents a model for understanding genome organization. Although the economic and medicinal importance of the species has been widely described, no established polyploid induction protocol has been reported so far. Here, we describe the production of synthetic polyploid plants of L. alba using colchicine. The ploidal levels were estimated by flow cytometry and chromosome counting. In addition, FISH and molecular markers approaches were used to confirm the stability of the synthetic polyploids. The major component of the essential oils was estimated by GCMS to compare with the natural individuals. Tetraploids and triploids were produced providing new opportunities for investigating medicinal, pharmacological, and economic applications as well as addressing intrinsic questions involved in the polyploidization process in tropical plants.

• The Balkan Peninsula is recognized as one of the hotspots of biodiversity in Europe. This area has shown since the Last Glacial Maximum appropriate conditions for species diversification and hybridization, which has led to the existence of numerous taxonomically unresolved entities. Here, we focus on the Western Balkans and explore the genetic structure and relationships among species belonging to the V. austriaca - V. orbiculata diploid-polyploid complex, including populations showing intermediate morphologies. • A combination of nuclear markers (microsatellites), plastid DNA regions (trnH-psbA, ycf6-psbM) and ploidy level estimations using flow cytometry are employed to assess the genetic structure and evolutionary dynamics of this polyploid complex. To reconstruct the evolutionary history, an approximate Bayesian computation approach is combined with projections of the species distribution models onto the climatic scenarios of the Mid-Holocene (6 ka BP) and Last Glacial Maximum (22 ka BP). • Four main groups were found: one well-established entity within the diploid level, V. dalmatica, a second diploid-tetraploid group which corresponds to V. orbiculata, a hexaploid cluster harboring V. austriaca subsp. jacquinii individuals, and an enigmatic tetraploid group. According to the molecular data obtained, this latter cluster represents an allopolyploid cryptic lineage −with V. orbiculata and V. dalmatica as putative parents− morphologically similar to V. orbiculata, but genetically more related to V. austriaca subsp. jacquinii. Veronica dalmatica and this “uncertain tetraploid” group are involved in the formation of the hexaploid taxon V. austriaca subsp. jacquinii, with the possibility of recent gene flow among different cytotypes. • The present study supports a scenario of diversification from a diploid common ancestor leading to two different but interrelated lineages. The first one would correspond with the diploid V. orbiculata plus tetraploid individuals of this species arising through allo- and autopolyploidization, and the second one would involve all ploidy levels with allopolyploidization being prevalent.

Polyploidization is a widespread mechanism of evolutionary divergence in flowering plants. Ecological divergence of polyploid lineages has been proposed as a key process shaping the distribution of cytotypes in nature (niche shift hypothesis); however, evidence for the role of niche separation in replicated diploid-polyploid species pairs is still needed. This study aimed to assess the role of abiotic factors shaping current cytotype distributions. For that, we examined the distribution and environmental niches of two varieties recognized in diploid-tetraploid Jasione maritima across the species range and within a putative contact zone on the Iberian Peninsula. We counted chromosomes, screened for ploidy across Iberian Peninsula and characterized environmental requirements using niche modeling tools. We found that J. maritima var. maritima is composed by diploids with disjunct distribution in the west coast of France and northwest Iberian Peninsula, and by tetraploids in Iberian Peninsula, while var. sabularia is tetraploid. In the Iberian Peninsula, two parapatric contact zones along a linear coastal distribution were detected, one between diploid and tetraploid var. maritima, and the other between tetraploids of the two varieties. Environmental variables of diploid populations from France are distinct from those of southern diploid populations, which are more similar to tetraploids. In general, niche modeling results are congruent with the observed distribution patterns, although the results suggest a wider contact zone between varieties and cytotypes. Tetraploids of both varieties revealed different degrees of environmental divergence in comparison with their diploid counterpart. Tetraploid var. sabularia differed environmentally from diploids suggesting niche divergence. In contrast, tetraploid var. maritima overlapped with diploid environmental niche and currently occupies its entire predicted range, whereas diploids are restricted to northern areas of their suitable environment. Differences in ecological envelopes facilitate the recognition of functional units of biodiversity within polyploid groups, allowing the study of factors related to post-polyploidization divergence. Thus, whereas changes in environmental requirements may have allowed tetraploid var. sabularia to spread in habitats not favorable to diploids, other factors are involved with the distribution of diploid and tetraploid var. maritima.

Polyploidy is a phenomenon that alters the genetic diversity of populations and has been reported as one of the most important evolutionary forces for plant diversification. The Psidium cattleyanum complex comprises a group of wild populations with several ploidy levels reported in the literature. The multiple cytotypes, associated with its wide distribution area, make this species a potential key model for understanding evolutionary processes related to polyploidization. In this study, we isolated and characterized nuclear microsatellite markers of P. cattleyanum and tested their transferability to other nine species of the genus. We performed a preliminary analysis of genetic diversity and population structure in three populations of P. cattleyanum. The three populations analyzed had different chromosome numbers, being polyploid cytotypes (2n = 6x = 66, 2n = 7x = 77 and 2n = 8x = 88). We designed 46 primer pairs and successfully amplified 37 markers, from which the 10 best were selected for analysis. Considering both the PIC and DP values, most of markers were highly informative. The new SSR markers were used to assess the levels of genetic diversity of the populations and detected one population with predominance of sexual reproduction. DAPC analysis pointed the formation of three groups, which corresponded to the populations analyzed. The markers were successfully amplified in related species, with some species presenting 80% transferability. By producing this panel of polymorphic microsatellites, we contribute to the understanding evolution in groups of natural polyploids for future studies.

Species are the basic units of biodiversity and evolution. Nowadays, they are widely considered as ancestor-descendant lineages. Their definition remains a persistent challenge for taxonomists due to lineage evolutionary role and circumscription, i.e., persistence in time and space, ecological niche or a shared phenotype of a lineage. Recognizing and delimiting species is particularly methodically challenging in fast-evolving, evolutionary young species complexes often characterized by low genetic divergence, hybrid origin, introgression and incomplete lineage sorting (ILS). Ranunculus auricomus is a large Eurasian apomictic polyploid complex that probably has arisen from the hybridization of a few sexual progenitor species. However, even delimitation and relationships of diploid sexual progenitors have been unclearly ranging from two to twelve species. Here, we present an innovative workflow combining phylogenomic methods based on 86,782 parameter-optimized RADseq loci and target enrichment of 663 nuclear genes together with geometric morphometrics to delimit sexual species in this evolutionary young complex (< 1 Mya). For the first time, we revealed a fully resolved and well-supported maximum likelihood (ML) tree phylogeny congruent to neighbor-net network and STRUCTURE results based on RADseq data. In a few clades, we found evidence of discordant patterns indicated by quartet sampling (QS) and reticulation events in the neighbor-net network probably caused by introgression and ILS. Together with coalescent-based species delimitation approaches based on target enrichment data, we found five main genetic lineages, with an allopatric distribution in Central and Southern Europe. A concatenated geometric morphometric data set including basal and stem leaves, as well as receptacles, revealed the same five main clusters. We accept those five morphologically differentiated, geographically isolated, genetic main lineages as species: R. cassubicifolius s.l. (incl. R. carpaticola), R. flabellifolius, R. envalirensis s.l. (incl. R. cebennensis), R. marsicus and R. notabilis s.l. (incl. R. austroslovenicus, R. calapius, R. mediocompositus, R. peracris and R. subcarniolicus). Our comprehensive workflow combing phylogenomic methods supported by geometric morphometrics proved to be successful in delimiting closely related sexual taxa and applying an evolutionary species concept, which is also transferable to other evolutionarily young species complexes.

[EN] How polyploids become established is a long-debated question, especially for autopolyploids that seem to have no evolutionary advantage over their progenitors. The Centaurea aspera polyploid complex includes diploid C. aspera and two related tetraploids C. seridis and C. gentilii. Our purpose was to study the mating system among these three taxa and to analyze its influence on polyploid establishment. The distribution and ploidy level of the Moroccan populations, and forced intra- and inter-specific crosses were assessed. Allotetraploid C. seridis produced more cypselae per capitulum in the intra-specific crosses. It is a bigger plant and autogamous, and previous studies indicated that selfing forces the asymmetric formation of sterile hybrids. All these characteristics help C. seridis to avoid the minority-cytotype-exclusion effect and become established. Inter-specific hybridization was possible between C. aspera and C. gentilii, and with the symmetric formation of hybrids. However, 49% of the hybrid cypselae were empty, which probably reveals postzygotic barriers. Autotetraploid C. gentilii produced the same number of cypselae per capitulum as those of the diploid parental, has an indistinguishable field phenotype, is allogamous, and symmetrically produces hybrids. Therefore, C. gentilii does not seem to have the same competitive advantages as those of C. seridis., This research was funded by the Conselleria d'Educacio, Cultura i Esport (Generalitat Valenciana) with project AICO/2019/227.

PREMISE OF THE STUDY Both intrinsic and extrinsic factors contribute to a species distribution. Among plants, the extrinsic effects of glaciation and intrinsic effects of whole genome duplication are powerful drivers of biogeographical patterns, but the interplay of these factors is poorly understood. Here, we investigate the roles glaciation and whole-genome duplication have played in the evolution of the widespread polyploid complex Campanula rotundifolia. METHODS We assessed the cytotype of 37 populations that spanned the geographic and cytotypic range of the C. rotundifolia complex. We constructed a chloroplast phylogeny for these populations and used RAD-seq to create nuclear phylogenies and networks for a subset of 23 populations; and estimated divergence times of major clades using Bayesian estimation of substitution rates. KEY RESULTS Campanula rotundifolia originated in south-central Europe and underwent range expansion throughout much of Europe and North America. Multiple genome duplications have occurred in C. rotundifolia-at least two tetraploid and three hexaploid formations. CONCLUSIONS Nuclear and chloroplast phylogenies are largely congruent with a history of populations surviving glacial maxima in known Pleistocene refugia in Europe and North America. Divergent European clades are consistent with two disjunct glacial refugia within Europe. North America was colonized by hexaploids derived from Western European lineages. A glacial refugium in Midwestern North America likely facilitated post-glacial recolonization of North America and limited genetic divergence. These results implicate both glaciation and whole-genome duplication as contributing factors to the extant biogeography of C. rotundifolia.

International audience; Prospero seisumsianum (Ruksans & Zetterl.) Yıldırım, a species recently described in southeastern Turkey, was recorded for the first time in the flora of Iraq based on samples collected from the Amadya and Duhok areas (northern Iraq). Our contribution here will be to find out more about its description, cytology, and chorology. The specimen collected from the Mesopotamian dry plain differs slightly from those collected from the mountainous Zagros forest area, but converges after a cultivation period. Both are treated here as P. seisumsianum and have the same genome size (2C = 8.77 ± 0.24 pg) corresponding to a diploid level. Three chromosome counts gave the same 2n = 2x = 14 karyotype. We hypothesize here that all the historical mentions of P. ?autumnale? in Iraq actually correspond to P. seisumsianum. Comparative morphological description, genome size, ecological niche, and biogeography of the related taxa, and a revised key for Prospero taxa from the Irano-Anatolian enlarged to its Mediterranean border are given in the present study.

Poa alpina, a widespread alpine grass in the temperate and subarctic zone, is a polyploid complex including many cytotypes with a broad range of chromosome numbers. To assess the occurrence and frequency of cytotypes, and to test for effects of past and current land use on their distribution, we measured DNA content using flow cytometry in 543 individuals of Poa alpina from agriculturally used grassland parcels and from natural sites in 12 local communities from three cultural traditions in the Swiss Alps. We tested whether geography (local community), affiliation to three cultural traditions, current land use, fertilization, and elevation affected DNA-content of populations. DNA content among individuals was highly variable (2C values ranging 100–600 pg) indicating chromosome numbers between 14 and 69. Cultural tradition, geography (local community), and current land use significantly affected mean DNA-content of Poa alpina in populations from agriculturally used parcels. The range of DNA-content and cytotypes was larger in pastures than in meadows, and the distribution of cytotypes differed among cultural traditions. Plants with more chromosomes grew larger in a common garden suggesting higher vigour. Among reproducing plants, an almost equal share was either reproducing by seeds or by bulbils. Seed-producing individuals had a higher DNA-content. Results presented here suggest that human land use is reflected directly in the distribution of cytotypes of different ploidy level and chromosome number. The effect of different cultural traditions is an indication that the current distribution of cytotypes does not only reflect geographic variability and current land use, but also differences in land use in the past that are persisting to the present.

Microsatellite primers were developed in Lippia alba complex to better understanding the origins and evolution of the species. We sought to increase the numbers of available simple sequence repeat (SSR) markers. We performed low-coverage (~ twofold) genomic DNA sequencing of a diploid accession and generated a de novo assembly comprising 175,572 contigs. Sixteen SSR loci were selected and of these 13 SSR loci were successfully amplified in 20 L. alba tetraploid accessions and in 12 other Lippia species. Only one SSR locus was monomorphic, whereas 12 loci were polymorphic, yielding one to nine alleles. The heterozygosity was similar among markers, with values of 0.274–0.485; the polymorphism information content values varied from 0.237 to 0.367. These markers were successfully amplified in related species with 85% of transferability on average. Thus, we demonstrate the utility of including a de novo assembly step to obtain SSR markers from low-coverage genomic datasets.

Background and Aims The distribution of cytotypes and its potential correlation with environmental variables represent a cornerstone to understanding the origin and maintenance of polyploid lineages. Although many studies have addressed this question in single species at a regional scale, only a few have attempted to decipher this enigma in groups of closely related species at a broad intercontinental geographical scale. Here, we consider ca. 20 species of a diploid-polyploid complex (Veronica subsect. Pentasepalae) of recent and rapid diversification represented in Europe and North Africa to study the frequency and distribution of cytotypes and its relationship with environmental variables. Methods A total of 680 individuals (207 populations) were sampled. Ploidy levels were determined using flow cytometry. Ecological differentiation among cytotypes was tested using climatic and environmental variables related to temperature, precipitation, vegetation and biogeographical region, among others, and by performing univariate and multivariate (constrained PCoA) analyses. Key Results Four ploidy levels (2x, 4x, 6x, 8x) were found and genome downsizing was observed to occur within the group. Plants of different ploidy level are ecologically differentiated, with hexaploids and octoploids occurring in wetter and colder habitats with a higher seasonality than diploids. A south-to-north distribution pattern was found, with diploids occupying southern refugial areas and octoploids being more frequent in northern regions of Europe above the permafrost boundary. Conclusions The distribution of cytotypes can be explained by ecological differentiation, the geographical position of refuge areas during the Quaternary climatic oscillations, as well as by ice and permafrost retreat patterns. The Balkan Peninsula constitutes the most important contact zone between cytotypes. This work provides the first comprehensive ploidy screening within V. subsect. Pentasepalae at a broad scale and indicates that polyploidy and genome downsizing might have contributed to the colonization of new habitats in a recently diverged polyploid complex.

Some tropical Bignoniaceae form sporophytic apomictic polyploid complexes are similar to better studied temperate plants. Handroanthus ochraceus is a widely distributed Neotropical savanna tree with both monoembryonic/self-sterile, and polyembryonic/apomictic and self-fertile populations, but lacking chromosome number and morphological comparative studies. We tested if monoembryonic/non-apomictic and polyembryonic/apomictic populations differed in ploidy and morphological features, as a basis to understand evolution and biogeography of these plants. Chromosome number and embryo number per seed were investigated, and uni- and multivariate analyses of flower and leaf morphology were done for five populations of H. ochraceus. We found two pure monoembryonic diploid (2n = 40), and one pure polyembryonic (62–94% of polyembryonic seeds) tetraploid population (2n = 80). One of the diploid populations presented only one individual with 3.2% polyembryonic seeds and was considered a non-apomictic population. Another population showed predominantly polyembryonic (27–66% of polyembryonic seeds) tetraploid individuals, but one diploid individual with 2% of polyembryonic seeds, and was considered a mixed apomictic and non-apomictic population. Morphological analyses confirmed breeding system clusters, and that stigma width, as well as pollen grain area, was consistently larger in polyembryonic populations. Polyploid plants showed larger cells, as well as larger organs and other distinctive features, which will be useful to identify apomictic populations and to future taxonomic discussions. The species can be considered an agamic complex with apomixis associated with neopolyploidy. This trend is also found in other tropical sporophytic apomictics, contrasting with the usual reports linking diploidy or paleopolyploidy to this kind of apomictics.