Patrik Mráz

Anthropogenic disturbance as a driver of microspatial and microhabitat segregation of cytotypes of Centaurea stoebe and cytotype interactions in secondary contact zones

BACKGROUND AND AIMS In a mixed-ploidy population, strong frequency-dependent mating will lead to the elimination of the less common cytotype, unless prezygotic barriers enhance assortative mating. However, such barriers favouring cytotype coexistence have only rarely been explored. Here, an assessment is made of the mechanisms involved in formation of mixed-ploidy populations and coexistence of diploid plants and their closely related allotetraploid derivates from the Centaurea stoebe complex (Asteraceae). METHODS An investigation was made of microspatial and microhabitat distribution, life-history and fitness traits, flowering phenology, genetic relatedness of cytotypes and intercytotype gene flow (cpDNA and microsatellites) in six mixed-ploidy populations in Central Europe. KEY RESULTS Diploids and tetraploids were genetically differentiated, thus corroborating the secondary origin of contact zones. The cytotypes were spatially segregated at all sites studied, with tetraploids colonizing preferentially drier and open microhabitats created by human-induced disturbances. Conversely, they were rare in more natural microsites and microsites with denser vegetation despite their superior persistence ability (polycarpic life cycle). The seed set of tetraploid plants was strongly influenced by their frequency in mixed-ploidy populations. Triploid hybrids originated from bidirectional hybridizations were extremely rare and almost completely sterile, indicating a strong postzygotic barrier between cytotypes. CONCLUSIONS The findings suggest that tetraploids are later immigrants into already established diploid populations and that anthropogenic activities creating open niches favouring propagule introductions were the major factor shaping the non-random distribution and habitat segregation of cytotypes at fine spatial scale. Establishment and spread of tetraploids was further facilitated by their superior persistence through the perennial life cycle. The results highlight the importance of non-adaptive spatio-temporal processes in explaining microhabitat and microspatial segregation of cytotypes.

Polyploidy in Phenotypic Space and Invasion Context: A Morphometric Study of Centaurea stoebe s.l.

The taxonomy of the Centaurea stoebe complex is controversial. Diploid and tetraploid plants occur in its native European range, but to date only tetraploids have been recorded from its introduced range in North America. We examined morphological differentiation of C. stoebe using multivariate and univariate approaches to clarify the taxonomic status of the known cytotypes. We measured more than 40 morphological traits on plants originating from 78 populations, grown from seed under uniform glasshouse conditions. The ploidy of almost 300 plants from 2 native and 20 introduced populations from Canada was assessed to test for the absence of diploids from North America. Finally, we explored whether postintroduction processes have resulted in phenotypic changes in introduced plants which may have contributed to the invasion success of C. stoebe. Morphometric analyses showed a clear separation of 2x and 4x plants and thus supported recognition of both cytotypes as separate taxa. Differences in the life cycle, the number of florets, the shape of capitula, and the shape of young rosette leaves were the best discriminant characters. Only minor differences were found between native and introduced tetraploids. All plants from the introduced range except for one hexaploid were found to be tetraploid. Rare diploids from Canada were identified as Centaurea diffusa or Centaurea psamogenna.

Allopolyploid origin of highly invasive Centaurea stoebe s.l. (Asteraceae)

Spotted knapweed (Centaurea stoebe) occurs from Western Asia to Western Europe both as diploid and tetraploid cytotypes, predominantly in single-cytotype populations with higher frequency of diploid populations. Interestingly, only tetraploids have been recorded so far from its introduced range in North America where they became highly invasive. We performed phylogenetic and network analyses of more than 40 accessions of the C. stoebe and C. paniculata groups and other related taxa using cloned internal transcribed spacer (ITS) and sequences of the chloroplast trnT-trnL and atpBrbcL regions to (i) assess the evolutionary origin of tetraploid C. stoebe s.l., and (ii) uncover the phylogeny of the C. stoebe group. Both issues have not been studied so far and thus remained controversial. Cloned ITS sequences showed the presence of two slightly divergent ribotypes occurring in tetraploid cytotype, while only one major ribotype was present in diploid C. stoebe s.str. This pattern suggests an allopolyploid origin of tetraploids with contribution of the diploid C. stoebe s.str. genome. Although we were not able to detect the second parental taxon, we hypothesize that hybridization might have triggered important changes in morphology and life history traits, which in turn may explain the colonization success of the tetraploid taxon. Bayesian relaxed clock estimations indicate a relatively recent--Pleistocene origin of the tetraploid C. stoebe s.l. Furthermore, our analyses showed a deep split between the C. paniculata and C. stoebe groups, and a young diversification of the taxa within the C. stoebe group. In contrast to nrDNA analyses, the observed pattern based on two cpDNA regions was inconclusive with respect to the origin and phylogeny of the studied taxa, most likely due to shared ancient polymorphism and frequent homoplasies.

Rare recent natural hybridization in Hieracium s. str. – evidence from morphology, allozymes and chloroplast DNA

The first proven data on natural hybridization in the genus Hieracium s. str. are presented. Plants with intermediate morphological characters between the diploids H. alpinum and H. transsilvanicum were found in the Muntii Rodnei (Romanian Eastern Carpathians) in 2001 and in the Chornohora Mts (Ukrainian Eastern Carpathians) in 2003. While plants of intermediate morphology between usually so called basic species are usually tri- or tetraploid in Hieracium s. str., these plants were diploid (2n=18) like both parental species in this region. The Romanian plant did not produce fertile achenes in free pollination and in control backcrosses with H. transsilvanicum, two hybrids from Ukraine were completly seed sterile in free pollination and reciprocal crosses. Pollen stainability as an indirect measure of male fertility was quite high in the studied Ukrainian hybrid plants and similar to the parental taxa. Evidence from allozyme analysis also confirmed the hybrid origin of the studied plants. Sequencing and PCR-RFLP analyses of the trnT-trnL intergenic spacer revealed that all hybrid plants had the H. transsilvanicum chloroplast DNA haplotype. Maternal inheritance of chloroplast DNA in this particular cross was proved with artificial hybrids from reciprocal experimental crosses between H. alpinum and H. transsilvanicum. In both localities, the natural hybrid plants were found in disturbed habitats, exceptionally allowing contact of the otherwise ecologically vicariate parental species. Morphologically, the hybrid plants belong to H. × krasani Woł.

Trichomes in the tribe Lactuceae (Asteraceae) — taxonomic implications

Although the infratribal classification of the Lactuceae has been the focus of several studies, relationships between the genera are not yet fully resolved. While trichome characters have been shown to be an important tool in taxa delimitation in many plant families, a detailed study on trichomes within the Lactuceae was still lacking. We assess here how trichome types can contribute to the clarification of generic relationships in this tribe. Trichome diversity was investigated on stems and leaves of 135 representatives of 53 genera by light and scanning electron microscopy. According to their morphology and anatomy, trichomes were classified into eight types and several subtypes. Phenetic analysis based on the presence or absence of particular trichome types was performed. The previously supposed monophyly of exclusively American genera and affinities between several closely related genera were confirmed. A new circumscription of the subtribe Hieraciinae is proposed, in accordance with recent molecular data. According to the present study, this subtribe is characterized by a unique combination of two trichome types and comprises the following genera: Andryala, Hieracium, Hispidella, Pilosella, Stenotheca. The previously included genera Arnoseris, Hololeion and Tolpis do not belong to this assemblage and their removal from the Hieraciinae should be reconsidered.

Drought tolerance and plasticity in the invasive knapweed Centaurea stoebe s.l. (Asteraceae): effect of populations stronger than those of cytotype and range

BACKGROUND AND AIMS Spotted knapweed (Centaurea stoebe s.l., Asteraceae) is native to Europe, where it occurs as a diploid (2xEU) and tetraploid cytotype (4xEU), but so far only the tetraploid has been reported in the introduced range in North America (4xNA). In previous studies, significant range shifts have been found towards drier climates in 4xEU compared with 2xEU, and in 4xNA when compared with the native range. In addition, 4x plants showed thicker leaves and reduced specific leaf area compared with 2x plants, suggesting higher drought tolerance in 4x plants. It is thus hypothesized that the 4x cytotype might be better pre-adapted to drought than the 2x, and the 4xNA better adapted than the 4xEU due to post-introduction selection. METHODS Plants of the three geocytotypes (2xEU, 4xEU and 4xNA ), each represented by six populations, were subjected to three water treatments over 6 weeks in a greenhouse experiment. Plasticity and reaction norms of above- and below-ground biomasses and their ratio, survival rate, stomatal conductance and carbon isotope discrimination were analysed using linear and generalized linear mixed effect models. KEY RESULTS AND CONCLUSIONS Above-ground and total biomasses of European tetraploids were slightly less affected by drought than those of European diploids, and 4xEU plants maintained higher levels of stomatal conductance under moderate drought than 4xNA plants, thus supporting the pre-adaptation but not the post-introduction evolution hypothesis. Plasticity indexes for most of the traits were generally higher in 2xEU and 4xNA than in 4xEU plants, but these differences were not or were only marginally significant. Interestingly, the effect of population origin and its interaction with treatment was more important than the effects of geocytotype and range. Population means for the control treatment showed several significant associations either with latitude or some aspect of climatic data, suggesting evolution of local adaptations, especially within the 2xEU and 4xEU geocytotypes.

Chemotaxonomic significance of flavonoids and phenolic acids in the Hieracium rohacsense group (Hieracium sect. Alpina; Lactuceae, Compositae)

Five apomictic taxa from the Hieracium rohacsense group were studied for their phenolic constituent composition. The following substances represent dominant compounds in the leaves: chlorogenic acid, 3,5-dicaffeoylquinic acid, luteolin 7-O-β-d-glucopyranoside, luteolin 4′-O-β-d-glucuronopyranoside and apigenin 4′-O-β-d-glucuronopyranoside. Within the group only quantitative differences were found, luteolin 7-O-glucoside being the most important chemotaxonomic marker. Each taxon has its own specific quantitative pattern, invariable within the taxon. Based on these characteristic profiles, H. rohacsense can be distinguished from a closely related and still undescribed taxon from Mt. Pip Ivan. The proportion of luteolin 7-O-glucoside to apigenin 4′-O-glucuronoside also clearly separates the individuals of two morphologically close species—H. ratezaticum and H. pseudocaesium, which corresponds to a few slight but recognisable morphological and phenological characteristics. The ontogenetic stage of leaf development and seasonal variation are also important factors, which must be taken into consideration, as the quantity of the substances changes during leaf ontogeny and with season.

Invasion success in polyploids: the role of inbreeding in the contrasting colonization abilities of diploid versus tetraploid populations of Centaurea stoebe s.l.

Summary 1.As a consequence of founder effects, inbreeding can hamper colonization success: Firstly, in species with self-incompatibility controlled by an S-locus, inbreeding may decrease cross-compatibility, mainly due to the sharing of identical S-alleles between closely related mating partners. Secondly, inbreeding can reduce fitness of inbred relative to outbred offspring (i.e. inbreeding depression). Polyploids often show reduced inbreeding depression compared to diploids, which may contribute to the overrepresentation of polyploids among invasive species. This is the first study that tests how the effects of inbreeding differ between geo-cytotypes (i.e. ploidy levels within a given range). 2.Our model organism, Centaurea stoebe, is strictly self-incompatible and comprises three geo-cytotypes: diploids are more frequent than tetraploids in the native range, while only tetraploids occur in the invasive range. We conducted a breeding experiment (sib-mating vs. outcrossing) with 14 native diploid, 13 native tetraploid and 15 invasive tetraploid populations. We recorded cross-compatibility and estimated a cumulative index for offspring fitness. Since frequent inbreeding can result in purging of genetic load responsible for inbreeding depression, our analyses included a metric for within-population relatedness, based on eight microsatellite markers, to assess the effect of purging. 3. Inbreeding was found to reduce cross-compatibility, which was similarly pronounced in diploids and tetraploids. It also caused inbreeding depression in cumulative fitness, which was significant in diploids but not in tetraploids. No evidence of purging was observed as inbred fitness was not affected by within-population relatedness. 4.Synthesis. Our results provide new insights into the contrasting invasion success of the cytotypes of C. stoebe. As the effects of cross-compatibility and purging were comparable between cytotypes, both processes can be ruled out to affect the colonization success of diploids vs. tetraploids. Our findings are consistent with the hypothesis that polyploidy increases the masking of recessive mutations, which maintains high fitness in inbred tetraploids and may thus facilitate colonization of new ranges. We highlight that reduced inbreeding depression may add to previously acknowledged advantages of polyploids in range expansions, a mechanism that may hitherto have been underestimated due to a lack of data on variation in inbreeding depression across geo-cytotypes. This article is protected by copyright. All rights reserved.

The population genetics of the fundamental cytotype-shift in invasive Centaurea stoebe s.l.: genetic diversity, genetic differentiation and small-scale genetic structure differ between cytotypes but not between ranges

Polyploids are overrepresented in invasive species. Yet, the role of genetic diversity and drift in colonization success of polyploids remains unclear. Here, we investigate genetic diversity, genetic differentiation and small-scale genetic structure in our model system, the three geo-cytotypes of Centaurea stoebe: monocarpic diploids and polycarpic (allo)tetraploids coexist in the native range (Eurasia), but only tetraploids are reported from the invasive range (North America). For each geo-cytotype, we investigated 18–20 populations varying in size and habitat type (natural vs. ruderal). Population genetic analyses were conducted at eight microsatellite loci. Compared to diploids, tetraploids revealed higher genetic diversity and lower genetic differentiation, whereas both were comparable in tetraploids between both ranges. Within spatial distances of a few meters, diploid individuals were more strongly related to one another than tetraploids. In addition, expected heterozygosity in diploids increased with population size and was higher in natural than in ruderal habitats. However, neither relationship was found for tetraploids. The higher genetic diversity of tetraploid C. stoebe may have enhanced its colonization abilities, if genetic diversity is correlated with fitness and adaptive capabilities. Furthermore, the inheritance of a duplicated chromosome set as well as longevity and frequent gene flow reduces drift in tetraploids. This counteracts genetic depletion during initial introductions and in subsequent phases of small or fluctuating population sizes in ruderal habitats. Our findings advocate the importance of studying colonization genetic processes to gain a more mechanistic understanding of the role of polyploidy in invasion dynamics.

Hieracium × grofae — a rediscovered diploid hybrid from the Ukrainian Carpathians

Diploid hybrid plants (2n = 18) between sexual diploid cytotypes of Hieracium alpinum and H. umbellatum were found in the Ukrainian Eastern Carpathians. They were identified with H. × grofaeWoł., originally used for the combination H. decipiens × H. umbellatum var. lactaris. As H. decipiens sensu Woł. (non Tausch) does not produce viable pollen grains and is most probably a polyploid apomict, it is unlikely to produce diploid hybrid plants with diploid H. umbellatum. Both parent species, Hieracium alpinum and H. umbellatum are also given by Wołoszczak from the original locality. Thus we conclude that H. × grofae is result of hybridization between H. alpinum and high mountain form of H. umbellatum. Hybrid plants are morphologically intermediate between the parent species, and moreover resemble closely or they are identical with the experimental hybrids of the same parent combination. Hybrids produce rather high amount of homogeneously sized pollen grains (values of standard deviation and coefficient of variation lower then upper limits for diploids — 3μm or 7.5%, respectively), and might probably serve as male parents in further crosses; on the other hand, they are fully seed-sterile. A lectotype of H. × grofae, a second proved nothotaxon in the genus Hieracium s.str., is designated. Localities of H. × grofae are located in subalpine belt of the Marmarosh Mts, the Svydovets’ Mts and the Horhany Mts (all in the Ukrainian Eastern Carpathians). Biotopes of hybrid plants usually represent secondary formed and disturbed pastures allowing close contact of altitudinally vicariant parent taxa.