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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., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Annals of Botany Company.)

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., (© 2020 The Authors. Evolution © 2020 The Society for the Study of Evolution.)

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.

Aspidistra subrotata Y. Wan & C.C. Huang, 1987 is considered for the first time as a widespread polyploidy complex in the genus Aspidistra Ker Gawler, 1823 from limestone areas of Asia. The chromosome number of the tetraploid is 2n = 76 and the karyotype is formulated as 2n = 44 m + 8 sm + 24 st, while the chromosome number of the diploid is 2n = 38 and the karyotype formula 2n = 22 m + 4 sm + 12 st. In our studies, diploids occupy broader geographical and environmental niche spaces than tetraploids. Although the leaf-shape of Aspidistra subrotata varies quantitatively between and within diploid and/or tetraploid population(s), no obvious discontinuity in the width of leaf has been observed. The tetraploid plants may be distinguished from the diploid plants by their rigid petioles as well as thick deep green lamina. Aspidistra subrotata is therefore an interesting material to explore the formation and the evolutionary dynamics of a natural polyploid complex from limestone areas of the tropical regions.

Whole genome duplication is considered to be a significant contributor to angiosperm speciation due to accumulation of rapid, strong interploid reproductive isolation. However, recent work suggests that interploid reproductive isolation may not be complete, especially among higher order cytotypes. This study evaluates postzygotic reproductive isolation among three cytotypes within a polyploid complex. We conducted reciprocal crosses using two diploid and two hexaploid populations each crossed to tetraploid populations spanning the geographic and phylogenetic range of the Campanula rotundifolia polyploid complex. Interploid and intrapopulation crosses were scored for fruit set, seed number, germination proportion and pollen viability. Postzygotic isolation was calculated for each cross as the product of these fitness components. A subset of offspring was cytotyped via flow cytometry. Postzygotic isolation was significantly lower in tetraploid-hexaploid crosses than diploid-tetraploid crosses, mostly due to substantially higher germination among tetraploid-hexaploid crosses. Tetraploid-hexaploid crosses produced pentaploids exclusively, whereas diploid-tetraploid crosses produced both triploids and tetraploids in high frequencies. Postzygotic isolation was weaker among higher order polyploids than between diploids and tetraploids, and unreduced gametes may facilitate diploid-tetraploid reproduction. This incomplete postzygotic isolation could allow ongoing interploid gene flow, especially among higher order polyploids, which may slow divergence and speciation in polyploid complexes., (© 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.)

Premise of the Study: Microsatellite primers were developed in the perennial herbs of the diploid-polyploid complex Veronica subsect. Pentasepalae (Plantaginaceae) to investigate the role that hybridization has played in the evolution of the group, which includes several endangered species., Methods and Results: Twelve pairs of primers leading to polymorphic and readable markers were identified and optimized from V. jacquinii and V. orbiculata using a microsatellite-enriched library method and 454 GS-FLX technique. The set of primers amplified dinucleotide to pentanucleotide repeats, and the number of alleles per locus ranged from one to six, one to 11, and one to nine for V. orsiniana, V. javalambrensis, and V. rosea, respectively. Transferability analyses were performed in 20 species corresponding to 10 different subgenera., Conclusions: These results indicate the utility of the newly developed microsatellites across Veronica subsect. Pentasepalae, which will help in the study of gene flow patterns and genetic structure.

• The semi-aquatic legumes belonging to the genus Aeschynomene constitute a premium system for investigating the origin and evolution of unusual symbiotic features such as stem nodulation and the presence of a Nod-independent infection process. This latter apparently arose in a single Aeschynomene lineage. But how this unique Nod-independent group then radiated is not yet known. • We have investigated the role of polyploidy in Aeschynomene speciation via a case study of the pantropical A. indica and then extended the analysis to the other Nod-independent species. For this, we combined SSR genotyping, genome characterization through flow cytometry, chromosome counting, FISH and GISH experiments, molecular phylogenies using ITS and single nuclear gene sequences, and artificial hybridizations. • These analyses demonstrate the existence of an A. indica polyploid species complex comprising A. evenia (C. Wright) (2n = 2x = 20), A. indica L. s.s. (2n = 4x = 40) and a new hexaploid form (2n = 6x = 60). This latter contains the two genomes present in the tetraploid (A. evenia and A. scabra) and another unidentified genome. Two other species, A. pratensis and A. virginica, are also shown to be of allopolyploid origin. • This work reveals multiple hybridization/polyploidization events, thus highlighting a prominent role of allopolyploidy in the radiation of the Nod-independent Aeschynomene., (© 2013 The Authors. New Phytologist © 2013 New Phytologist Trust.)

Ploidy has been well studied and used extensively in the genus Opuntia to determine species boundaries, detect evidence of hybridization, and infer evolutionary patterns. We carried out chromosome counts for all members of the Humifusa clade to ascertain whether geographic patterns are associated with differences in ploidy. We then related chromosomal data to observed morphological variability, polyploid formation, and consequently the evolutionary history of the clade. We counted chromosomes of 277 individuals from throughout the ranges of taxa included within the Humifusa clade, with emphasis placed on the widely distributed species, Opuntia humifusa (Raf.) Raf., 1820 s.l. and Opuntia macrorhiza Engelm., 1850 s.l. We also compiled previous counts made for species in the clade along with our new counts to plot geographic distributions of the polyploid and diploid taxa. A phylogeny using nuclear ribosomal ITS sequence data was reconstructed to determine whether ploidal variation is consistent with cladogenesis. We discovered that diploids of the Humifusa clade are restricted to the southeastern United States (U.S.), eastern Texas, and southeastern New Mexico. Polyploid members of the clade, however, are much more widely distributed, occurring as far north as the upper midwestern U.S. (e.g., Michigan, Minnesota, Wisconsin). Morphological differentiation, although sometimes cryptic, is commonly observed among diploid and polyploid cytotypes, and such morphological distinctions may be useful in diagnosing possible cryptic species. Certain polyploid populations of Opuntia humifusa s.l. and Opuntia macrorhiza s.l., however, exhibit introgressive morphological characters, complicating species delineations. Phylogenetically, the Humifusa clade forms two subclades that are distributed, respectively, in the southeastern U.S. (including all southeastern U.S. diploids, polyploid Opuntia abjecta Small, 1923, and polyploid Opuntia pusilla (Haw.) Haw., 1812) and the southwestern U.S. (including all southwestern U.S. diploids and polyploids). In addition, tetraploid Opuntia humifusa s.l., which occurs primarily in the eastern U.S., is resolved in the southwestern diploid clade instead of with the southeastern diploid clade that includes diploid Opuntia humifusa s.l. Our results not only provide evidence for the polyphyletic nature of Opuntia humifusa and Opuntia macrorhiza, suggesting that each of these represents more than one species, but also demonstrate the high frequency of polyploidy in the Humifusa clade and the major role that genome duplication has played in the diversification of this lineage of Opuntia s.s. Our data also suggest that the southeastern and southwestern U.S. may represent glacial refugia for diploid members of this clade and that the clade as a whole should be considered a mature polyploid species complex. Widespread polyploids are likely derivatives of secondary contact among southeastern and southwestern diploid taxa as a result of the expansion and contraction of suitable habitat during the Pleistocene following glacial and interglacial events.