Pathways to polyploidy: indications of a female triploid bridge in the alpine species <Emphasis Type="Italic">Ranunculus kuepferi</Emphasis> (Ranunculaceae)

Polyploidy is one of the most important evolutionary processes in plants. In natural populations, polyploids usually emerge from unreduced gametes which either fuse with reduced ones, resulting in triploid offspring (triploid bridge), or with other unreduced gametes, resulting in tetraploid embryos. The frequencies of these two pathways, and male versus female gamete contributions, however, are largely unexplored. Ranunculus kuepferi occurs with diploid, triploid and autotetraploid cytotypes in the Alps, whereby diploids are mostly sexual, while tetraploids are facultative apomicts. To test for the occurrence of polyploidization events by triploid bridge, we investigated 551 plants of natural populations via flow cytometric seed screening. We assessed ploidy shifts in the embryo to reconstruct female versus male gamete contributions to polyploid embryo and/or endosperm formation. Seed formation via unreduced egg cells (B_III hybrids) occurred in all three cytotypes, while only in one case both gametes were unreduced. Polyploids further formed seeds with reduced, unfertilized egg cells (polyhaploids and aneuploids). Pollen was highly variable in diameter, but only pollen >27 μm was viable, whereby diploids produced higher proportions of well-developed pollen. Pollen size was not informative for the formation of unreduced pollen. These results suggest that a female triploid bridge via unreduced egg cells is the major pathway toward polyploidization in R. kuepferi, maybe as a consequence of constraints of endosperm development. Triploids resulting from unreduced male gametes were not observed, which explains the lack of obligate sexual tetraploid individuals and populations. Unreduced egg cell formation in diploids represents the first step toward apomixis.     

Effect of triploid fitness on the coexistence of diploids and tetraploids

The conditions for the coexistence of diploids, triploids and tetraploids in a single population were investigated with a deterministic model under the assumptions that diploids might produce 2 n gametes, and that triploids had a lower fitness than other cytotypes and generated equal proportions of haploid and diploid gametes. When diploids produced only haploid gametes, the dynamics of the cytotypes were similar to that of heterozygote disadvantage with two alleles at a single locus, with triploids being equivalent to the heterozygotes. Production of 2 n gametes by diploids increased the pool of diploid gametes and created a stable equilibrium involving a majority of diploids and a minority of polyploids. When the fitness of tetraploids was equal to or higher than that of diploids, increased triploid fitness decreased the threshold of 2 n gametes necessary to deterministically fix tetraploids in the population. Conversely, when tetraploids were less fit than diploids, the rate of 2 n gamete production leading to the exclusion of diploids first decreases and then increased with increasing triploid fitness. Triploids are repeatedly found in diploid-tetraploid hybridizations and are rarely totally sterile. They might play a determinant role in the future of multiple cytotype populations. The effect of triploids depends on the relative fitness of diploids and tetraploids and is also a function of their fitness.     

Production of diploid and triploid offspring by inbreeding of the triploid planarian <Emphasis Type="Italic">Dugesia ryukyuensis</Emphasis>

Triploidy has generally been considered to be an evolutionary dead end due to problems of chromosomal pairing and segregation during meiosis. Thus, the formation of tetraploids and diploids from triploid types is a rare phenomenon. In the present study, we demonstrated that inbreeding of the triploid planarian Dugesia ryukyuensis resulted in both diploid and triploid offspring in nature. In the triploids of D. ryukyuensis, chiasmata between homologous chromosomes were observed in both female and male germ lines. This result suggests that both diploid and triploid offspring of this species are produced bisexually by zygotic fusion between sperm and eggs. Hence, this phenomenon may be a novel mechanism in planarian for escaping the triploid state.     

Meiotic chromosome configurations in oocytes of <Emphasis Type="Italic">Cobitis taenia</Emphasis> and its polyploid hybrids

Diploid and triploid hybrid females of Cobitis as a rule produce unreduced eggs which mainly develop gynogenetically, but some of the eggs incorporate sperm genome and develop into triploids and tetraploids, respectively. Here, we observed for the first time the meiotic chromosomes in the germinal vesicles (GVs) of mature oocytes of three diploid C. taenia (2n = 48) and 20 allopolyploid females of Cobitis (18 triploid 3n = 74 and 2 tetraploid 4n = 99). The majority of GVs in diploid, triploid and tetraploid females contained 24, 74 and 96 or 99 bivalents, respectively. These results directly indicated premeiotic endomitosis as a mechanism underlying the formation of unreduced eggs in allopolyploid females of Cobitis.     

Geographical separation of diploid and triploid cytotypes of <Emphasis Type="Italic">Allium scorodoprasum</Emphasis> in Lithuania

The aim of this study was to determine the geographical and ecological distribution of Allium scorodoprasum cytotypes in Lithuania. 46 populations were studied: environmental variables were recorded and somatic chromosome numbers of 797 individuals from all populations were assessed. Diploids were found in 13 populations in the northern part of the study area, while triploids occurred in 33 populations from the western part of Lithuania. A. scorodoprasum frequently occurred in river valleys, particularly in the rarely flooded high level floodplains or on lower terraces. Only one population was recorded in Alno-Ulmion forest and three on the Glechometalia hederaceae forest edges, while 42 populations occurred in mesophilous or semidry Arrhenatheretalia elatioris, Mesobromion erecti and Trifolio-Geranietea sanguinei grasslands. No distinct differences in relative frequencies of diploid and triploid cytotypes in different plant communities were found whereas the soil chemical composition between the habitats of both cytotypes differed statistically. These differences however were indicative of the geographical separation of habitats rather than ecological differentiation between the two cytotypes. The results suggest that there were at least two separate roads of colonization of diploid and triploid A. scorodoprasum into Lithuania.     

Genetic differentiation in the polyploid complex of <Emphasis Type="Italic">Suaeda corniculata</Emphasis> (C.A. Mey.) Bunge in Eastern Siberia

Data on differentiation of the polyploid complex of Suaeda corniculata (C.A. Mey.) Bunge in East Siberia based on variability of morphological characteristics, karyological analysis, and genetic polymorphism in inter simple sequence repeat (ISSR) markers were given for the first time. It was established that the samples studied belonged to three cytotypes: diploids (2n = 2x = 18), tetraploids (2n = 4x = 36) and hexaploids (2n = 6x = 54). The analysis of variability of morphological characteristics by the method of principal coordinates showed division of the samples into three separate groups. On the basis of morphological and molecular-genetic methods, a genetically differentiated diploid population was revealed in Yakutia.     

Comparison of age and growth performance of diploid and tetraploid loach <Emphasis Type="Italic">Misgurnus anguillicaudatus</Emphasis> in the Yangtze River basin,China

Diploids and tetraploids of the cyprinid loach Misgurnus anguillicaudatus coexist in many natural habitats in Asia. However, little is known about the biological and ecological differences between these two ploidy forms. We examined age, body size and growth rates of fish in a mixed ploidy population of M. anguillicaudatus in the Yangtze River basin in China. The sex ratios of both diploid and tetraploid M. anguillicaudatus were highly skewed toward females, who tended to be larger than males. The age distributions of the collected specimens clearly indicated that tetraploids lived longer than diploids. For example, we found a substantial number of tetraploids that were 5+ years old, while the oldest diploids were 4+ years old. Tetraploids were also longer and heavier than diploids for both sexes. Using the empirical body size and age data, we inferred the growth patterns of these fish with the von Bertalanffy growth function. The estimated asymptotic body lengths (L _∞) indicated that tetraploids could indeed achieve larger body sizes than diploids. This difference was partially due to the increased growth rate, as demonstrated by lower growth coefficient (K) and higher growth performance index (φ’). In sum, we show that tetraploid M. anguillicaudatus exhibited significantly increased longevity and superior growth performance compared to diploids. These differences may contribute to the ecological competitiveness of tetraploid M. anguillicaudatus, thus enabling them to coexist with diploids in certain ecological settings.     

Genetic diversity and structure analyses on the natural populations of diploids and triploids of tiger lily, <Emphasis Type="Italic">Lilium lancifolium</Emphasis> Thunb., from Korea,China, and Japan

Tiger lily, Lilium lancifolium Thumb. is an endemic species in Korea. Genetic structure and diversity of diploid and triploid tiger lily accessions were analyzed using SSRP and IRAP molecular markers. All genetic indices analyzed showed that the diploids hold higher genetic variations compared to the triploids which were collected from Korea, China, and Japan. All variations found in triploids were also found in diploids to support the autotriploidy of L. lancifolium. It was also revealed that the triploid populations have experienced population regrowth from small population after population bottleneck. The population and phylogenetic analyses showed that analyzed tiger lilies were partitioned into three populations, Pop 1 from West Sea of Korean Peninsula, Pop 2 from South Sea of Korean Peninsula and Pop 3 of triploids in which many accessions of West Sea and South Sea were admixed in each partitioned group. Although the level of population differentiation is not extensive, F_ST analysis revealed some level of differentiation among the three populations. The analyses of genetic structure and phylogenetic dendrogram revealed admixture in some accessions among populations. All the results obtained in our analyses imply that autotriploid tiger lilies were originated from a small number of accessions of diploid L. lancifolium.     

Embryological and genetic evidence of amphimixis and apomixis in <Emphasis Type="Italic">Boehmeria tricuspis</Emphasis>

Apomixis is a widespread alternative mode of sexual reproduction resulting in offspring that are genetically identical to the maternal plant. Boehmeria tricuspis (Hance) Makino is a perennial, wind-pollinated, herbaceous plant in the nettle family Urticaceae. The diploid B. tricuspis is monoecious but the triploid B. tricuspis is gynoecious, bearing female inflorescences only. Apomixis in B. tricuspis was first reported 50 years ago, but the mode of apomixis in the species has not been described yet. Here, we provide embryological observations of the embryo sac formation proving that triploid B. tricuspis reproduced apomictically following the Antennaria type of diplospory, and that the diploid individuals were the sexual genotype with the classical Polygonum-type maturation pattern of embryo sac development. A subsequent flow cytometry seed screen (FCSS) showed that the triploids were obligate apomicts with autonomous endosperm development, and the diploids reproduced sexually. In addition, a progeny test by molecular marker assays further demonstrated the above results.     

Autotetraploids of Vicia cracca show a higher allelic richness in natural populations and a higher seed set after artificial selfing than diploids

Background and Aims

Despite the great importance of autopolyploidy in the evolution of angiosperms, relatively little attention has been devoted to autopolyploids in natural polyploid systems. Several hypotheses have been proposed to explain why autopolyploids are so common and successful, for example increased genetic diversity and heterozygosity and the transition towards selfing. However, case studies on patterns of genetic diversity and on mating systems in autopolyploids are scarce. In this study allozymes were employed to investigate the origin, population genetic diversity and mating system in the contact zone between diploid and assumed autotetraploid cytotypes of Vicia cracca in Central Europe.

Methods

Four enzyme systems resolved in six putative loci were investigated in ten diploid, ten tetraploid and five mixed-ploidy populations. Genetic diversity and heterozygosity, partitioning of genetic diversity among populations and cytotypes, spatial genetic structure and fixed heterozygosity were analysed. These studies were supplemented by a pollination experiment and meiotic chromosome observation.

Key Results and Conclusions

Weak evidence of fixed heterozygosity, a low proportion of unique alleles and genetic variation between cytotypes similar to the variation among populations within cytotypes supported the autopolyploid origin of tetraploids, although no multivalent formation was observed. Tetraploids possessed more alleles than diploids and showed higher observed zygotic heterozygosity than diploids, but the observed gametic heterozygosity was similar to the value observed in diploids and smaller than expected under panmixis. Values of the inbreeding coefficient and differentiation among populations (ρ_ST) suggested that the breeding system in both cytotypes of V. cracca is mixed mating with prevailing outcrossing. The reduction in seed production of tetraploids after selfing was less than that in diploids. An absence of correlation between genetic and geographic distances and high differentiation among neighbouring tetraploid populations supports the secondary contact hypothesis with tetraploids of several independent origins in Central Europe. Nevertheless, the possibility of a recent in situ origin of tetraploids through a triploid bridge in some regions is also discussed.     

Patterns of cytotype variation of <Emphasis Type="Italic">Turnera sidoides</Emphasis> subsp. <Emphasis Type="Italic">pinnatifida</Emphasis> (Turneraceae) in mountain ranges of central Argentina

Cytogeographical variability among 564 plants from 26 populations of Turnera sidoides subsp. pinnatifida in mountain ranges of central Argentina was analysed with meiotic chromosome counts and flow cytometry and is described at regional and local scales. Populations were primarily tetraploids (2n = 4x = 28), although diploid (2n = 2x = 14), hexaploid (2n = 2x = 42), and mixed populations of diploids and triploids (2n = 3x = 21) were also found. Diploids, triploids, and hexaploids were fewer in number and restricted to narrow areas, while tetraploids were the most common and geographically widespread cytotype. Diploids grew at higher altitudes and in colder and wet locations; tetraploids had the broadest ecological spectrum, while hexaploids occurred at the lowest altitudes and in drier conditions. The cytotypes were also spatially segregated at a microgeographical scale. Diploids grew in the piedmont, tetraploids were in the adjacent valley, and in the contact zone of both cytotypes, patches of diploids and triploids were found. At a regional scale, the distribution of the cytotypes may be governed by a combination of ecological and historical variables, while segregation in the contact zone may be independent of the selective environment because the cytotypes are unable to coexist as a result of reproductive exclusion. The role of triploids is also discussed.     

Inferring the potential of plastid DNA-based identification of derived ferns: a case study on the <Emphasis Type="Italic">Asplenium trichomanes</Emphasis> aggregate in Europe

The utility of three plastid DNA regions to identify fern species was explored with focus on the European representatives of the Asplenium trichomanes aggregate. The sampling included representatives of the three diploid and the four tetraploid taxa recognized in the European flora plus Macaronesia. Besides European samples, the compiled data set comprised specimens of a putative Hawaiian endemic and one species occurring in Southeast Asia. By combining the sequences of three non-coding plastid regions, 13 haplotypes were recovered of which four were found in more than one taxon. Evidences for four distinct diploid lineages were found that correspond to Asplenium anceps, A. inexpectans, A. trichomanes s.s., and A. tripteropus. The four tetraploids occurring in Europe shared haplotypes with A. inexpectans. Thus, DNA barcoding can successfully identify the diploids, but fail to separate the tetraploids from their diploid ancestors. As a consequence, barcoding analyses of ferns need to take into account the differences of ploidy level measured by evidence independent from the DNA barcode. Evidence for uneven accumulation of intra-species DNA variation was recovered by comparing all species. Furthermore, the study provided evidence that the current taxonomy of these ferns requires to be revised. The two European diploids form well-separated clades and need to be recognized as A. inexpectans and A. trichomanes s.s. To keep name consistency for all European tetraploids, a new name Asplenium jessenii is introduced to replace A. trichomanes subsp. hastatum.     

Natural hybridization among subspecies of <Emphasis Type="Italic">Turnera sidoides</Emphasis> L. (Passifloraceae) revealed by morphological and genetic evidence

Turnera sidoides is a complex of outcrossing, perennial, rhizomatous herbs that is widely distributed in southern South America. Five subspecies are recognized taxonomically based on morphological features and geographical distribution. In certain regions, the areas of distribution of the subspecies overlap partially. In such contact zones, the extent of reproductive barriers among subspecies is still largely unknown, but morphologically intermediate individuals have been found in the field, indicating that hybridization may actually occur between subspecies. Crossability among subspecies of T. sidoides has been shown by experimental studies with cultivated plants, but the mechanisms involved in natural populations are still unknown. To investigate the mechanisms that underlie gene flow within the T. sidoides complex, in this paper we analyze the morphological and genetic variation, as well as the crossability among taxa in a contact zone between subspecies pinnatifida and sidoides, in southeastern Uruguay. Our results constitute the first evidences of ongoing natural hybridization between subspecies of T. sidoides and suggest that, although hybridization may not have been of significance in the early phase of the species differentiation, reticulate evolution is ongoing enhancing the current morphological and genetic variability of the complex.     

Expression and DNA methylation pattern of reproduction-related genes in partially fertile triploid Pacific oysters <Emphasis Type="Italic">Crassostrea gigas</Emphasis>

Partial or complete sterility is an obvious feature in triploid Pacific oyster (Crassostrea gigas) which contributes to improving rearing performances. Despite the significance of sterility, the molecular mechanism behind it remains elusive and related research was limited. This study focused on six reproduction-related genes and compared their different behavior in gene expression and DNA methylation pattern between triploid and diploid oysters in order to provide more molecular information. The gonadal development of triploid oyster was examined by histology before molecular analysis. Gametogenesis disturbance was observed in triploid oysters at different development stages (stage II and III) with more serious impairment in females. QPCR showed significant gene expression difference between diploid and triploid in two genes: putative Vg and cgER. Gene expression of putative Vg was delayed in triploids while for cgER triploid oyster showed higher expression and the difference was significant at stage III. DNA methylation pattern of these two genes were further investigated by bisulfite sequencing. Between diploid and triploid oysters, no difference was observed in total methylation level but some individual loci showed different patterns: significantly high methylation rate of loci 2284 in cgER was observed in triploid oyster which has a higher expression of this gene. This study indicated that putative Vg and cgER might play a role in partial sterile in triploid C. gigas. Gene expression could be regulated by the methylation pattern at specific individual locus, which deserves equivalent attention as well as total DNA methylation level.     

Factors influencing distribution and local coexistence of diploids and tetraploids of <Emphasis Type="Italic">Vicia cracca</Emphasis>: inferences from a common garden experiment

Vicia cracca diploids and autotetraploids are highly parapatric in Europe; tetraploids reside in western and northern part, whereas diploids occupy much drier south-eastern part. They meet together in a Central European contact zone. This distribution pattern raised questions about a transformative effect of polyploidization on plant performance and environmental tolerances. We investigated plant survival, growth, and seed production in two water regimes in a common garden experiment using seeds collected from five localities in the Central European contact zone where diploids and tetraploids occur in sympatry. Obtained data imply that tetraploids of V. cracca are not generally superior in performance to diploids. Significantly larger seeds from tetraploid mother plants collected in the field were not correlated with greater stature of the seedlings. Nonetheless, tetraploids might have a potential to out-compete diploids in the long run due to the tetraploids’ ability of greater growth which manifested in the second year of cultivation. Considering the response of diploids and tetraploids to water supply, drought stressed tetraploids but not diploids produced a higher proportion of aborted seeds than watered ones, which implies that tetraploids are more drought susceptible than diploids. On the other hand, decreased plant height in drought stresses tetraploids, which simultaneously increased total seed production, may suggest that tetraploids have a greater ability to avoid local extinction under unfavourable conditions by enhancing biomass allocation into production of seeds at the cost of lower growth. The significant interaction between ploidy level and locality in several traits suggests possible polyfyletic origin of tetraploids and the necessity to clarify the history of the tetraploids in Europe.     

Cytotype distribution inEmpetrum (Ericaceae) at various spatial scales in the Czech Republic

Ploidy levels inEmpetrum (crowberry) from the Czech Republic and from one adjacent locality in Poland were estimated by flow cytometry to examine cytotype distribution patterns at large (within the country), medium (within mountain ranges) and small (within particular localities) spatial scales. Diploid, triploid, and tetraploid individuals were found. Triploids are reported from Central Europe for the first time; they occurred in the Krkono?e Mts. Exclusively diploid plants were observed in three mountain ranges (the Kru?né hory Mts., Labské pískovce Mts., Adr?pa?sko-Teplické skály Mts.), exclusively tetraploids were observed in the Jeseníky Mts., and both cytotypes were observed in the ?umava Mts., Jizerské hory Mts. and Krkono?e Mts. Except for the latter mountain range, diploids and tetraploids were always found in different habitats. Spatial isolation is supposed to be the main barrier preventing cytotype mating. A mosaic-like sympatric occurrence of different cytotypes was demonstrated in the Krkono?e Mts., where peat bogs and rocky places were not spatially separated. Eight of 11 localities studied there were inhabited by diploids and tetraploids (five localities), diploids and triploids (one locality) or all three ploidy levels (two localities). Diploid and triploid plants occasionally intermingled at 0.3 × 0.3 m. Flower sex in crowberries was strongly associated with ploidy level: diploids usually had unisexual flowers, the tetraploids bore exclusively bisexual flowers. However, a few diploid plants with hermaphrodite flowers occurred in one population in the Krkono?e Mts.     

MicroRNA Alternations in the Testes Related to the Sterility of Triploid Fish

The knowledge of understanding the molecular traits of the sterile triploid fish is sparse. Herein, we analyzed the microRNA (miRNA) alternations in the testes of the sterile triploid fish produced by crossing the tetraploid fish with the diploid fish, compared with those of tetraploids and diploids used as the controls. A total of 136, 134, and 142 conserved miRNAs and 105, 112, and 119 novel miRNAs were identified in the diploid, triploid, and tetraploid fish, respectively. The genes targeted by the differentially expressed miRNAs were identified and were enriched in the GO term cell surface receptor signaling pathway, cellular process, G-protein coupled receptor signaling pathway, and metabolic process. KEGG pathway enrichment was also assessed to evaluate the target genes with differentially expressed miRNAs and these genes were enriched in four pathways (synthesis and degradation of ketone bodies, pentose and glucuronate interconversions, cyanoamino acid metabolic process, and ascorbate and aldarate metabolism). Nine differentially expressed miRNAs were verified by quantitative real-time PCR analysis (qPCR). The upregulated miRNAs in triploids, including miR-101a, miR-199-5p, miR-214, miR-222, and miR-193a, showed the same results with high-throughput sequencing. Among the selected downregulated miRNAs, miR-7b and miR-153b had significantly lower expression levels in triploids. Dnah3 and Tekt1 genes targeted by miR-199-5p showed lower expression in triploids by qPCR. These verified differentially expressed miRNAs may participate in testicular development and sperm activity by targeting functional genes, which were identified with differential expression in the triploid. This evidence provides insights into the epigenetic regulatory mechanisms of sterility in triploid cyprinids.     

Cytogenetic evidences on the evolutionary relationships between the tetraploids of the section <Emphasis Type="Italic">Rhizomatosae</Emphasis> and related diploid species (<Emphasis Type="Italic">Arachis</Emphasis>, Leguminosae)

Rhizomatosae is a taxonomic section of the South American genus Arachis, whose diagnostic character is the presence of rhizomes in all its species. This section is of particular evolutionary interest because it has three polyploid (A. pseudovillosa, A. nitida and A. glabrata, 2n?=?4x?=?40) and only one diploid (A. burkartii, 2n?=?2x?=?20) species. The phylogenetic relationships of these species as well as the polyploidy nature and the origin of the tetraploids are still controversial. The present study provides an exhaustive analysis of the karyotypes of all rhizomatous species and six closely related diploid species of the sections Erectoides and Procumbentes by cytogenetic mapping of DAPI/CMA heterochromatin bands and 5S and 18–26S rDNA loci. Chromosome banding showed variation in the DAPI heterochromatin distribution pattern, which, together with the number and distribution of rDNA loci, allowed the characterization of all species studied here. The bulk of chromosomal markers suggest that the three rhizomatous tetraploid species constitute a natural group and may have at least one common diploid ancestor. The cytogenetic data of the diploid species analyzed evidenced that the only rhizomatous diploid species—A. burkartii—has a karyotype pattern different from those of the rhizomatous tetraploids, showing that it is not likely the genome donor of the tetraploids and the non-monophyletic nature of the section Rhizomatosae. Thus, the tetraploid species should be excluded from the R genome, which should remain exclusively for A. burkartii. Instead, the karyotype features of these tetraploids are compatible with those of different species of the sections Erectoides and Procumbentes (E genome species), suggesting the hypothesis of multiple origins of these tetraploids. In addition, the polyploid nature and the group of diploid species closer to the tetraploids are discussed.