Ploidy-specific symbiotic interactions: divergence of mycorrhizal fungi between cytotypes of the Gymnadenia conopsea group (Orchidaceae).

Polyploidy is widely recognized as a major mechanism of sympatric speciation in plants, yet little is known about its effects on interactions with other organisms. Mycorrhizal fungi are among the most common plant symbionts and play an important role in plant nutrient supply. It remains to be understood whether mycorrhizal associations of ploidy-variable plants can be ploidy-specific. We examined mycorrhizal associations in three cytotypes (2x, 3x, 4x) of the Gymnadenia conopsea group (Orchidaceae), involving G. conopsea s.s. and G. densiflora, at different spatial scales and during different ontogenetic stages. We analysed: adults from mixed- and single-ploidy populations at a regional scale; closely spaced adults within a mixed-ploidy site; and mycorrhizal seedlings. All Gymnadenia cytotypes associated mainly with saprotrophic Tulasnellaceae (Basidiomycota). Nonetheless, both adults and seedlings of diploids and their autotetraploid derivatives significantly differed in the identity of their mycorrhizal symbionts. Interploidy segregation of mycorrhizal symbionts was most pronounced within a site with closely spaced adults. This study provides the first evidence that polyploidization of a plant species can be associated with a shift in mycorrhizal symbionts. This divergence may contribute to niche partitioning and facilitate establishment and co-existence of different cytotypes.

Minority cytotypes in European populations of the Gymnadenia conopsea complex (Orchidaceae) greatly increase intraspecific and intrapopulation diversity.

BACKGROUND AND AIMS: Patterns of ploidy variation among and within populations can provide valuable insights into the evolutionary mechanisms shaping the dynamics of plant systems showing ploidy diversity. Whereas data on majority ploidies are, by definition, often sufficiently extensive, much less is known about the incidence and evolutionary role of minority cytotypes. METHODS: Ploidy and proportions of endoreplicated genome were determined using DAPI (4',6-diamidino-2-phenylindole) flow cytometry in 6150 Gymnadenia plants (fragrant orchids) collected from 141 populations in 17 European countries. All widely recognized European species, and several taxa of less certain taxonomic status were sampled within Gymnadenia conopsea sensu lato. KEY RESULTS: Most Gymnadenia populations were taxonomically and/or ploidy heterogeneous. Two majority (2x and 4x) and three minority (3x, 5x and 6x) cytotypes were identified. Evolution largely proceeded at the diploid level, whereas tetraploids were much more geographically and taxonomically restricted. Although minority ploidies constituted <2 % of the individuals sampled, they were found in 35 % of populations across the entire area investigated. The amount of nuclear DNA, together with the level of progressively partial endoreplication, separated all Gymnadenia species currently widely recognized in Europe. CONCLUSIONS: Despite their low frequency, minority cytotypes substantially increase intraspecific and intrapopulation ploidy diversity estimates for fragrant orchids. The cytogenetic structure of Gymnadenia populations is remarkably dynamic and shaped by multiple evolutionary mechanisms, including both the ongoing production of unreduced gametes and heteroploid hybridization. Overall, it is likely that the level of ploidy heterogeneity experienced by most plant species/populations is currently underestimated; intensive sampling is necessary to obtain a holistic picture.

Bridging global and microregional scales: ploidy distribution in Pilosella echioides (Asteraceae) in central Europe.

BACKGROUND AND AIMS: A detailed knowledge of cytotype distribution can provide important insights into the evolutionary history of polyploid systems. This study aims to explore the spatial distribution of different cytotypes in Pilosella echioides at various spatial scales (from the whole distributional range to the population level) and to outline possible evolutionary scenarios for the observed geographic pattern. METHODS: DNA-ploidy levels were estimated using DAPI flow cytometry in 4410 individuals of P. echioides from 46 populations spread over the entire distribution range in central Europe. Special attention was paid to the cytotype structure in the most ploidy-diverse population in south-west Moravia. KEY RESULTS: Five different cytotypes (2x, 3x, 4x, 5x and 6x) were found, the last being recorded for the first time. Although ploidy-uniform (di- or tetraploid) sites clearly prevailed, nearly one-quarter of the populations investigated harboured more (up to all five) cytotypes. Whereas penta- and hexaploids constituted only a minority of the samples, a striking predominance of the triploid cytotype was observed in several populations. CONCLUSIONS: The representative sampling confirmed previous data on cytotype distribution, i.e. the spatial aggregation of mixed-ploidy populations in south-west Moravia and Lower Austria and the predominance of ploidy-uniform populations in other parts of the area investigated. Recurrent origin of polyploids from diploid progenitors via unreduced gametes and their successful establishment are considered the key factors promoting intrapopulational ploidy mixture ('primary hybrid zones'). As an alternative to the generally accepted theory of cytotype co-existence based on the development of different means of inter-ploidy reproductive isolation, it is suggested that a long-term ploidy mixture can also be maintained in free-mating populations provided that the polyploids originate with a sufficient frequency. In addition, the prevalence (or subdominance) of the triploid cytotype in several mixed-ploidy populations represents the first evidence of such a phenomenon in plant systems with exclusively sexual reproduction.

Remarkable coexistence of multiple cytotypes of the Gymnadenia conopsea aggregate (the fragrant orchid): evidence from flow cytometry.

BACKGROUND AND AIMS: One of the prerequisites for polyploid research in natural systems is knowledge of the geographical distribution of cytotypes. Here inter- and intrapopulational ploidy diversity was examined in the Gymnadenia conopsea aggregate in central Europe and potential explanations and evolutionary consequences of the observed spatial patterns investigated. METHODS: DAPI flow cytometry supplemented by confirmatory chromosome counts was used to determine ploidy in 3581 samples of the G. conopsea aggregate from 43 populations. The fine-scale spatial pattern of cytotype distribution (intra- and interploidy associations) was analysed with univariate and bivariate K-functions. KEY RESULTS: Gymnadenia tissues undergo a progressively partial endoreplication, which accounts for about 60 % and 75 % of the total genome in G. conopsea and G. densiflora, respectively. Flow cytometric profiles are therefore species-specific and can be used as a marker for rapid and reliable species recognition. Two majority (4x, 8x) and three minority (6x, 10x, 12x) cytotypes were found, often in mixed-ploidy populations (harbouring up to all five different ploidy levels). The scarcity of the minority cytotypes (about 2·7 %) suggests the existence of strong pre- or postzygotic mating barriers. Spatial structure was observed in plots of populations with the highest cytotype variation, including clumping of individuals of the same ploidy and negative association between tetra- and octoploids. CONCLUSIONS: The remarkable ploidy coexistence in the G. conopsea aggregate has reshaped our perception of intrapopulational ploidy diversity under natural conditions. This system offers unique opportunities for studying processes governing the formation and establishment of polyploids and assessing the evolutionary significance of the various pre- and postzygotic mating barriers that maintain this ploidy mixture.

Morphology and genetics of Lythrum salicaria from latitudinal gradients of the Northern Hemisphere grown in cold and hot common gardens.

The aim of this project was to compare the phenotypic responses of global populations of Lythrum salicaria in cold/dry and hot/humid environments to determine if phenotypic plasticity varied between the native and invasive ranges, and secondarily if this variation was linked to genetic diversity. Common garden studies were conducted in Trebon, Czech Republic, and Lafayette, Louisiana, USA (cold/dry vs. hot/humid garden, respectively), using populations from latitudinal gradients in Eurasia and North America. Lythrum salicaria seeds collected from the same maternal plants across these latitudinal gradients were germinated and grown in Trebon and Lafayette. Tissue masses (above-, below-ground, inflorescence and total) of these individuals were assessed at the end of each growing season (2006-2008). Worldwide field measurements of L. salicaria height were made by volunteers from 2004-2016. Biomass and height data were analyzed using the General Linear Model framework and multivariate techniques. Molecular markers (amplified fragment length polymorphisms) of individuals used in the common garden study were analyzed using traditional genetic diversity metrics and Bayesian clustering algorithms in STRUCTURE. Reaction norms were developed from differences in maternal plant responses in Trebon versus Lafayette. In the common garden studies, stem/leaf, root and total biomass generally were highest for individuals grown from seeds collected in the southern part of the range in the cold garden, particularly by the third year of the study. In contrast, inflorescence biomass in the cold garden was higher by the third year in individuals from mid-latitude populations. As measured by volunteers, plants were taller in Eurasia than in North America moving from north to south with the pattern switching southward of 40°N latitude. Genetic diversity was similar between native and non-native invasive populations regardless of geographical origin of the seed and was not significantly different in the GLM Select model (p > 0.05). Reaction norm slopes showed that Eurasia had larger values than North America for reaction norms for above-ground and total biomass. Plants from the seeds of mother plants from Turkey had wide variation in total biomass when grown in Trebon versus Lafayette; this variation in response within certain populations may have contributed to the lack of population-level differences in plasticity. These results indicate no loss of genetic diversity for L. salicaria during its North American invasion, nor reduction in plastic tissue allocation responses to a varying environment, which may help explain some of its invasive qualities and which could be of adaptive value under changing future environments.

The Enigma of Progressively Partial Endoreplication: New Insights Provided by Flow Cytometry and Next-Generation Sequencing.

In many plant species, somatic cell differentiation is accompanied by endoreduplication, a process during which cells undergo one or more rounds of DNA replication cycles in the absence of mitosis, resulting in nuclei with multiples of 2C DNA amounts (4C, 8C, 16C, etc.). In some orchids, a disproportionate increase in nuclear DNA contents has been observed, where successive endoreduplication cycles result in DNA amounts 2C + P, 2C + 3P, 2C + 7P, etc., where P is the DNA content of the replicated part of the 2C nuclear genome. This unique phenomenon was termed "progressively partial endoreplication" (PPE). We investigated processes behind the PPE in Ludisia discolor using flow cytometry (FCM) and Illumina sequencing. In particular, we wanted to determine whether chromatin elimination or incomplete genome duplication was involved, and to identify types of DNA sequences that were affected. Cell cycle analysis of root tip cell nuclei pulse-labeled with EdU revealed two cell cycles, one ending above the population of nuclei with 2C + P content, and the other with a typical "horseshoe" pattern of S-phase nuclei ranging from 2C to 4C DNA contents. The process leading to nuclei with 2C + P amounts therefore involves incomplete genome replication. Subsequent Illumina sequencing of flow-sorted 2C and 2C + P nuclei showed that all types of repetitive DNA sequences were affected during PPE; a complete elimination of any specific type of repetitive DNA was not observed. We hypothesize that PPE is part of a highly controlled transition mechanism from proliferation phase to differentiation phase of plant tissue development.

Species-specific SSR alleles for studies of hybrid cattails (Typha latifolia x T. angustifolia; Typhaceae) in North America.

PREMISE: Studies of hybridizing species are facilitated by the availability of species-specific molecular markers for identifying early- and later-generation hybrids. Cattails are a dominant feature of wetland communities, and a better understanding of the prevalence of hybrids is needed to assess the ecological and evolutionary effects of hybridization. Hybridization between Typha angustifolia and T. latifolia produce long-lived clones, known as Typha ×glauca, which are considered to be invasive. Although morphological variation in cattails makes it difficult to recognize early- and later-generation hybrids, several dominant, species-specific RAPD markers are available. Our goal was to find codominant, species-specific markers with greater polymorphism than RAPDs, to identify later-generation hybrids more efficiently. • METHODS: We screened nine SSR (simple sequence repeat) loci that were described from populations in Ukraine, and we surveyed 31 cattail populations from the upper Midwest and eastern USA. • KEY RESULTS: Seven SSR loci distinguished the parent taxa and were consistent with known species-specific RAPD markers, allowing easier detection of backcrossing. We used linear discriminant analysis to show that F(1) hybrid phenotypes were intermediate between the parent taxa, while those of backcrossed plants overlapped with the hybrids and their parents. Log(leaf length/leaf width), spike gap length, spike length, and stem diameter explained much of the variation among groups. • CONCLUSIONS: We provide the first documentation of backcrossed plants in hybridizing cattail populations in Michigan. The diagnostic SSR loci we identified should be extremely useful for examining the evolutionary and ecology interactions of hybridizing cattails in North America.