Gamete sex and elevation affect genetically based variation for unreduced gamete production in a mixed-ploidy plant.

PREMISE: Unreduced gametes are the primary mechanism of neopolyploid formation. Their production in diploid populations is arguably maladaptive, but the magnitude and patterns of genetically based variation maintained in natural populations are poorly understood. METHODS: We examined variation in male and female unreduced gamete production among plants from different elevations in fireweed, Chamerion angustifolium, grown in a common environment. Using seeds from three high-elevation and three low-elevation diploid populations in one study, and a single diploid population in another, we estimated realized rates of unreduced male (sperm) and female (egg) gamete production by reciprocally pollinating diploid and tetraploid plants and estimating the incidence of tetraploid seeds using flow cytometry. RESULTS: Unreduced gamete frequencies per plant were similar in the two studies (0.12% vs. 0.08%). High-elevation populations had a greater percentage of fruit with seeds from unreduced gametes, but a lower percentage of seeds per fruit than low-elevation populations. Female unreduced gamete frequencies differed among elevations, but male frequencies did not, and the gamete sexes were not correlated at the plant level. CONCLUSIONS: We conclude that genetically based variation for unreduced gametes is maintained within and among natural populations, despite their fitness disadvantages, suggesting that local selection may be ineffective at purging them under some conditions.

Polyploidy increases storage but decreases structural stability in Arabidopsis thaliana.

Whole-genome duplication, leading to polyploidy and endopolyploidy, is widespread throughout the tree of life.1-3 Both polyploidy and endopolyploidy can increase cell size via nucleotypic effects, but the phenotypic consequences of increased cell size at the tissue and whole-organism levels are less well understood.1-4 We quantified the consequences of autopolyploidy and endopolyploidy in nine diploid accessions of Arabidopsis thaliana, representing a gradient in endopolyploidy, to their corresponding experimentally synthesized neo-tetraploid and neo-octoploid cytotypes. The increase in cell size following genome duplication increased plant storage capacity, which increased tolerance of resource limitation, but also incurred biomechanical costs because of a reduction in the amount of cell wall per unit tissue volume. Our findings also show that the functional consequences of autopolyploidy can vary with accession identity, and the presence of this variation suggests that there is potential for adaptation following whole-genome duplication.

Corrigendum: Different Patterns of Ecological Divergence Between Two Tetraploids and Their Diploid Counterpart in a Parapatric Linear Coastal Distribution Polyploid Complex.

[This corrects the article DOI: 10.3389/fpls.2020.00315.].

The role of multiple reproductive barriers: strong post-pollination interactions govern cytotype isolation in a tetraploid-octoploid contact zone.

BACKGROUND AND AIMS: Polyploidy is an important contributor to sympatric speciation and assortative mating is a key mechanism driving cytotype interactions in contact zones. While strong reproductive barriers can mediate the coexistence of different cytotypes in sympatry, positive frequency-dependent mating disadvantage ultimately drives the transition to single-ploidy populations. However, comprehensive estimates of reproductive isolation among cytotypes and across multiple barriers are rare. We quantify the strength of isolation across multiple reproductive stages in a tetraploid-octoploid contact zone to understand the potential for coexistence. METHODS: Assortative mating due to flowering asynchrony, pollinator behaviour, morphological overlap, self-fertilization and gametic competition between tetraploid and octoploid Gladiolus communis in a contact zone in the Western Iberian Peninsula were assessed in natural and experimental populations to quantify reproductive isolation (RI) between cytotypes. KEY RESULTS: Tetraploids and octoploids have a high degree of overlap in flowering time and similar floral morphology, and are visited by generalist insects without cytotype foraging preferences, resulting in weak pre-pollination RI (from 0.00 to 0.21). In contrast, post-pollination isolation resulting from gametic selection was a strong barrier to inter-cytotype mating, with ploidy composition in stigmatic pollen loads determining the levels of RI (from 0.54 to 1.00). Between-cytotype cross-incompatibility was relatively high (RI from 0.54 to 0.63) as was isolation acquired through self-pollination (RI of 0.59 in tetraploids and 0.39 in octoploids). CONCLUSIONS: Total RI was high for both tetraploids (from 0.90 to 1.00) and octoploids (from 0.78 to 0.98). Such high rates of assortative mating will enable cytotype coexistence in mixed-ploidy populations by weakening the impacts of minority cytotype exclusion. This study reveals the key role of gametic selection in cytotype siring success and highlights the importance of comprehensive estimates across multiple reproductive barriers to understand cytotype interactions at contact zones.

Different Patterns of Ecological Divergence Between Two Tetraploids and Their Diploid Counterpart in a Parapatric Linear Coastal Distribution Polyploid Complex.

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.

The influence of experimentally induced polyploidy on the relationships between endopolyploidy and plant function in Arabidopsis thaliana.

Whole genome duplication, leading to polyploidy and endopolyploidy, occurs in all domains and kingdoms and is especially prevalent in vascular plants. Both polyploidy and endopolyploidy increase cell size, but it is unclear whether both processes have similar effects on plant morphology and function, or whether polyploidy influences the magnitude of endopolyploidy. To address these gaps in knowledge, fifty-five geographically separated diploid accessions of Arabidopsis thaliana that span a gradient of endopolyploidy were experimentally manipulated to induce polyploidy. Both the diploids and artificially induced tetraploids were grown in a common greenhouse environment and evaluated with respect to nine reproductive and vegetative characteristics. Induced polyploidy decreased leaf endopolyploidy and stem endopolyploidy along with specific leaf area and stem height, but increased days to bolting, leaf size, leaf dry mass, and leaf water content. Phenotypic responses to induced polyploidy varied significantly among accessions but this did not affect the relationship between phenotypic traits and endopolyploidy. Our results provide experimental support for a trade-off between induced polyploidy and endopolyploidy, which caused induced polyploids to have lower endopolyploidy than diploids. Though polyploidy did not influence the relationship between endopolyploidy and plant traits, phenotypic responses to experimental genome duplication could not be easily predicted because of strong cytotype by accession interactions.

Evolutionary associations between polyploidy, clonal reproduction, and perenniality in the angiosperms.

Clonal reproduction is thought to facilitate polyploid establishment in the angiosperms, but the evolutionary relationship between polyploidy and clonality has not been thoroughly tested. A perennial life history may confer many of the same advantages, and the relative importance of clonality versus perenniality is unknown. We used phylogenetic comparative analyses of 1751 species to examine associations between polyploidy, clonality, and life history. We test hypotheses of co-evolution by determining the sequence of trait development. Polyploidy is associated with both clonality and perenniality across species, and analyses show that clonality can be an important predictor of polyploidy beyond perenniality. Tests of directionality on our full dataset suggest that polyploidy is more likely to promote clonality or perenniality than vice versa, although there are significant differences in patterns of co-evolution among major angiosperm groups. Our results suggest that polyploidy and clonal reproduction are evolutionarily associated across the angiosperms, even when perenniality is considered, but we find little evidence at the whole-angiosperm level for the hypothesis that clonality promotes polyploidy. However, variation among different clades indicates that polyploidy and clonality are interacting in diverse ways, likely to be due to the variable roles of clonality in their evolutionary histories.

Whole-genome duplication decreases clonal stolon production and genet size in the wild strawberry Fragaria vesca.

PREMISE OF THE STUDY: Clonal reproduction is often associated with polyploidy and is expected to influence polyploid establishment success, but the immediate effects of whole-genome duplication (WGD) on clonal reproduction in autopolyploids are unknown. METHODS: We used synthesized neopolyploids to assess the direct effects of WGD on stolon and plantlet production in the wild strawberry Fragaria vesca by (1) comparing absolute clonal investment between diploids and neotetraploids under high and low resource conditions in the greenhouse and (2) determining realized clonal plantlet establishment and genet spatial structure using artificial field populations comprising both cytotypes. KEY RESULTS: Neotetraploids produced fewer stolons and plantlets than diploids at slower weekly rates in the greenhouse when resources were high, resulting in lower total investment in clonal reproduction. Low resources led to smaller reductions in clonal biomass for neotetraploids and less pronounced differences between cytotypes. Comparisons between neotetraploids representing 13 independent WGD events and close diploid relatives revealed considerable variation in the response to polyploidization for some clonal traits. Field populations corroborated greenhouse results; neotetraploid genets were smaller than diploid genets, containing 28% fewer stolons and 46% fewer rooted plantlets. CONCLUSIONS: WGD significantly decreases the clonal output of neotetraploid F. vesca, which is likely attributable to slower whole-plant growth of the neotetraploids than the diploids. In natural populations, smaller neotetraploid genets could decrease the probability of polyploid establishment in this species. However, variation between separate neopolyploid lines emphasizes that the response of clonal investment to WGD may not be uniform across polyploid origins.

Immediate vs. evolutionary consequences of polyploidy on clonal reproduction in an autopolyploid plant.

Background and Aims: Clonal reproduction in polyploids is expected to exceed that in diploids, due to either the immediate direct effects of whole-genome duplication (WGD) or selection during establishment. The timing of polyploidy effects on clonality are largely unknown despite its hypothesized influence on polyploid success. This study tests the direction and timing of divergence in clonal traits in diploid and polyploid Chamerion angustifolium. Methods: Root bud production and biomass allocation patterns were compared between diploids and synthesized tetraploids (neotetraploids), and between neotetraploids and naturally occurring tetraploids grown in a common environment. Key Results: Neotetraploids produced more root buds and fewer sexual structures than diploids and natural tetraploids; diploids and natural tetraploids had similar root bud numbers and sexual investment. The root bud:inflorescence biomass ratio was 71 % higher in neotetraploids than in natural tetraploids. Root bud location suggests that ramet density in neotetraploid genets could be higher than in diploid genets. Conclusions: WGD immediately increases investment in asexual vs. sexual reproduction in C. angustifolium, potentially promoting within-cytotype mating and establishment for neopolyploids. However, evolutionary change after the polyploidization event negates the direct effects of WGD. Natural polyploids and diploids have similar root bud production and biomass allocation patterns, probably resulting from habitat- and ploidy-mediated selection on polyploids to become more like diploids. These results highlight the value of studying the effects of polyploidization in young vs. established polyploids.

Mixed-Ploidy Species: Progress and Opportunities in Polyploid Research.

Mixed-ploidy species harbor a unique form of genomic and phenotypic variation that influences ecological interactions, facilitates genetic divergence, and offers insights into the mechanisms of polyploid evolution. However, there have been few attempts to synthesize this literature. We review here research on the cytotype distribution, diversity, and dynamics of intensively studied mixed-ploidy species and consider the implications for understanding mechanisms of polyploidization such as cytotype formation, establishment, coexistence, and post-polyploid divergence. In general, mixed-ploidy species are unevenly represented among families: they exhibit high cytotype diversity, often within populations, and frequently comprise rare and odd-numbered ploidies. Odd-ploidies often occur in association with asexuality. We highlight research hypotheses and opportunities that take advantage of the unique properties of ploidy variation.

Evolutionary Dynamics of Unreduced Gametes.

Unreduced gametes, which have the somatic (2n) chromosome number, are an important precursor to polyploid formation and apomixis. The product of irregularities in meiosis, 2n gametes are expected to be rare and deleterious in most natural populations, contrary to their wide taxonomic distribution and the prevalence of polyploidy. To better understand this discrepancy, we review contemporary evidence related to four aspects of 2n gamete dynamics in natural populations: (i) estimates of their frequency; (ii) their environmental and genetic determinants; (iii) adaptive and nonadaptive processes regulating their evolution; and (iv) factors regulating their union and production of polyploids in diploid populations. Aided by high-throughput methods of detection, these foci will advance our understanding of variation in 2n gametes within and among species, and their role in polyploid evolution.

Frequency and maintenance of unreduced gametes in natural plant populations: associations with reproductive mode, life history and genome size.

Fertilization involving unreduced (2n) gametes is considered the dominant mechanism of polyploid formation in angiosperms; however, our knowledge of the prevalence of and evolutionary mechanisms maintaining 2n gametes in natural populations is limited. We hypothesize that 2n gametes are deleterious consequences of meiotic errors maintained by mutation-selection balance and should increase in species with relaxed opportunities for selection on sexual processes (asexuality), reduced efficacy of selection (asexuality, selfing) and increased genome instability (high chromosome number). We used flow cytometry to estimate male 2n gamete production in 60 populations from 24 species of Brassicaceae. We quantified variation in 2n gamete production within and among species, and examined associations with life history, reproductive mode, genome size and chromosomal number while accounting for phylogeny. Most individuals produced < 2% 2n male gametes, whereas a small number had > 5% (up to 85%) production. Variation in 2n gamete production was significant among species and related to reproductive system; asexual species produced significantly more 2n gametes than mixed-mating and outcrossing species. Our results, unique in their multi-species perspective, are consistent with 2n gametes being deleterious but maintained when opportunities for selection are limited. Rare individuals with elevated 2n gamete production may be key contributors to polyploid formation.

Direct vs. indirect effects of whole-genome duplication on prezygotic isolation in Chamerion angustifolium: Implications for rapid speciation.

PREMISE OF THE STUDY: The depiction of polyploid speciation as instantaneous implies that strong prezygotic and postzygotic isolation form as a direct result of whole-genome duplication. However, the direct vs. indirect contributions of genome duplication to phenotypic divergence and prezygotic isolation are rarely quantified across multiple reproductive barriers. METHODS: We compared the phenotypic differences between diploid and both naturally occurring and synthesized tetraploids (neotetraploids) of the plant Chamerion angustifolium. Using this information and additional published values for this species, we compared the magnitude of isolation (ecological, flowering, pollinator, and gametic) between diploids and natural-occurring tetraploids to that between diploids and neotetraploids. KEY RESULTS: Differences among ploidy cytotypes were observed for eight of 12 vegetative and reproductive traits measured. Neotetraploids resembled diploids but differed from natural tetraploids with respect to four traits, including flowering time and plant height. Diploid-neotetraploid (2x-4xneo) experimental arrays exhibited lower pollinator fidelity to cytotype and seed set compared with 2x-4xnat arrays. Based on these results and published evidence, reproductive isolation between diploids and neotetraploids across all four life stages averaged 0.48 and deviated significantly from that between diploids and natural tetraploids (RI = 0.96). CONCLUSIONS: Genome duplication causes phenotypic shifts and contributes directly to prezygotic isolation for some barriers (gametic isolation) but cannot account for the cumulative isolation from diploids observed in natural tetraploids. Therefore, conditions for species formation through genome duplication are not necessarily instantaneous and selection to strengthen prezygotic barriers in young polyploids is critical for the establishment of polyploid species in sympatry.

Effect of inbreeding on pollen tube growth in diploid and tetraploid Chamerion angustifolium: Do polyploids mask mutational load in pollen?

PREMISE OF THE STUDY: Deleterious recessive mutations are an important determinant of fitness (mutational load) in the sporophytic phase of plants and a major cause of inbreeding depression; however, their role in gametophyte function is less well documented but may account for variation in pollen tube growth and siring ability, especially between diploid and polyploid plants, which can mask the load. METHODS: We investigated the role of mutational load in pollen performance using the perennial polyploid Chamerion angustifolium by comparing tube growth of pollen, in styles and in growth medium, from inbred (selfed) and outbred diploids to that of inbred and outbred tetraploids. Pollen from tetraploids is expected to mask deleterious mutations more effectively in the outbred condition but reveal them after inbreeding. In contrast, gametophytes from diploids should express the same genetic load in inbred or outbred plants. KEY RESULTS: Pollen tube growth measured in growth medium was highest in outbred tetraploids and generally lower in inbred than outbred plants. The effect of selfing was significant in pollen from tetraploids but not diploids. The differential effect of selfing was also evident in the proportion of pollen reaching the base of styles, but the ploidy × pollination interaction was not significant. Selfing also had a negative effect on sporophyte fitness but was greater in diploids than tetraploids. CONCLUSIONS: Pollen performance is influenced by the expression of mutational load, which is masked in polyploids. This effect may partly explain strong siring success of tetraploids in this species.

Correlated polymorphism in cytotype and sexual system within a monophyletic species, Lycium californicum.

BACKGROUND AND AIMS: Polyploidy has important effects on reproductive systems in plants and has been implicated in the evolution of dimorphic sexual systems. In particular, higher ploidy is associated with gender dimorphism across Lycium species (Solanaceae) and across populations within the species Lycium californicum. Previous research on the association of cytotype and sexual system within L. californicum sampled a limited portion of the species range, and did not investigate evolutionary transitions between sexual systems. Lycium californicum occurs in arid regions on offshore islands and mainland regions in the south-western United States and Mexico, motivating a more comprehensive analysis of intraspecific variation in sexual system and cytotype across the full range of this species. METHODS: Sexual system (dimorphic vs. cosexual) was determined for 34 populations across the geographical range of L. californicum using field observations of pollen production, and was confirmed using morphological measurements and among-plant correlations of primary sexual traits. Ploidy was inferred using flow cytometry in 28 populations. DNA sequence data from four plastid and two nuclear regions were used to reconstruct relationships among populations and to map transitions in sexual system and ploidy. KEY RESULTS: Lycium californicum is monophyletic, ancestrally diploid and cosexual, and the association of gender dimorphism and polyploidy appears to have two evolutionary origins in this species. Compared with cosexual populations, dimorphic populations had bimodal anther size distributions, negative correlations between male and female floral traits, and larger coefficients of variation for primary sexual traits. Flow cytometry confirmed tetraploidy in dimorphic populations, whereas cosexual populations were diploid. CONCLUSIONS: Tetraploidy and gender dimorphism are perfectly correlated in L. californicum, and the distribution of tetraploid-dimorphic populations is restricted to populations in Arizona and the Baja California peninsula. The analysis suggests that tetraploidy and dimorphism likely established in Baja California and may have evolved multiple times.

Distinguishing 2N gamete nuclei from doublets in pollen using flow cytometry and pulse analysis.

The value of flow cytometry for quantifying unreduced (2N) pollen production in plants is well recognized; however, the approach has been limited by technical obstacles to obtaining high quality nuclei fluorescence histograms and the difficulty in distinguishing 2N nuclei from 1N doublets. Here, we use mathematical arguments and observations of fluorescence properties of angiosperm pollen nuclei to generate guidelines for applying pulse analysis to correct for doublets in pollen nuclei data. We show that the theoretical requirements for applying pulse analysis for doublet correction are met when nucleus fluorescence height and/or width measures in the unreduced gamete (2C DNA content) region exhibit bimodality (reflecting singlets and doublets) in combination with unimodal distributions of the same parameters in the reduced gamete (1C) region. These conditions are regularly met in the family Brassicaceae but not in the Asteraceae and Poaceae. We further show that when these requirements are met, pulse analysis estimates of doublet proportions are well correlated with estimates obtained with microscopy. We propose guidelines for doublet correction when estimating frequencies of unreduced male gametes.

Climatic niche differences between diploid and tetraploid cytotypes of Chamerion angustifolium (Onagraceae).

PREMISE OF THE STUDY: Polyploidy-the possession of more than two copies of each chromosome in the nucleus-is common in flowering plants. Polyploid plants can occupy different geographic ranges than their diploid progenitors, but the factors responsible for maintaining these range differences are poorly understood. Polyploidy can have significant physiological consequences, and the present study aims to determine whether previously described physiological differences between cytotypes are correlated with climatic niches and geographic distributions. METHODS: Prior research indicates that tetraploid plants of Chamerion angustifolium (Onagraceae) are more tolerant of drought and less tolerant of freezing than diploids, which suggests that they should occupy a niche that is warmer and drier than that of diploids. We extracted climate data for 134 populations of C. angustifolium classified as pure diploid, pure tetraploid, or mixed-ploidy. We compared climatic conditions between these population categories and generated ecological niche models to compare their geographic distribution with prior qualitative estimates. KEY RESULTS: Pure tetraploid populations occupy habitats that are warmer and drier than those of pure diploid populations. Mixed-ploidy populations occur in habitats that are not strictly intermediate between pure diploid and pure tetraploid populations, but are as cold as pure diploid populations and have intermediate soil moisture deficits. Our niche models were similar to previous qualitative estimates of cytotype geographic distribution. CONCLUSIONS: The correspondence between the physiological tolerances of cytotypes, their climatic niches, and their geographic distributions suggests that physiological traits are at least partially responsible for differences in the realized climatic niches of diploid and tetraploid C. angustifolium.

Pathways of introduction of the invasive aquatic plant Cabomba caroliniana.

The pathway and frequency of species' introductions can affect the extent, impact, and management of biological invasions. Here, we examine the pathway of introduction of the aquatic plant Cabomba caroliniana (fanwort) into Canada and the northern United States using plastid DNA sequence (intergenic spacers atpF-atpH, trnH-psbA, and trnL-trnF) and DNA content analyses. We test the hypothesis that the spread of fanwort is a result of commercial trade by comparing a Canadian population (Kasshabog Lake, ON) to native populations from southern U.S., introduced populations in northern U.S., and plants from commercial retailers. Thirteen plastid haplotypes were identified throughout North America, including one dominant haplotype, which was present in all C. caroliniana populations. Several rare haplotypes were used to infer shared colonization history. In particular, the Canadian population shared two rare alleles with a population from Massachusetts, suggesting range expansion of C. caroliniana from the northern U.S. However, the possibility of a commercial introduction cannot be excluded, as common alleles were shared between the Canadian population and both commercial and southern U.S. sources. Variation in C. caroliniana genome size was bimodal and populations were classified into "high" and "low" categories. The Canadian population had DNA contents similar to several northern U.S. populations (low DNA content). This may provide additional support for range expansion from these introduced populations rather than from commercial sources or populations in the southern U.S., which had high DNA content.

Adaptation of diploid and tetraploid chamerion angustifolium to elevation but not local environment.

Polyploid organisms often have different geographic ranges than their diploid relatives. However, it is unclear whether this divergence is maintained by adaptation or results from historical differences in colonization. Here, we conducted a reciprocal transplant experiment with diploid and autotetraploid Chamerion angustifolium to test for adaptation at the ploidy and population level. In the Rocky Mountains, pure diploid populations occur at high elevations and pure autotetraploid populations occur at low elevations with mixed ploidy populations between. We planted 3134 seedlings in 2004 and 3890 juveniles (bolting) in 2005 among nine plots, three in each of the diploid, mixed ploidy, and tetraploid zones, and monitored survival until 2008. For both seedlings and juvenile plants, elevation significantly influenced survival. The juvenile plants also showed a significant ploidy by elevation interaction, indicating that diploids and tetraploids survived best at their native elevations. In contrast, we found no evidence of local adaptation to plot within elevation. This suggests that the current distribution of diploids and tetraploids across elevations is the result of adaptation and that genome duplication may have facilitated the invasion of lower elevation habitats by limiting the movement of maladapted alleles from diploid populations at higher elevations.