Genome size variation and species relationships in Hieracium sub-genus Pilosella (Asteraceae) as inferred by flow cytometry

BACKGROUND AND AIMS: Hieracium sub-genus Pilosella (hawkweeds) is a taxonomically complicated group of vascular plants, the structure of which is substantially influenced by frequent interspecific hybridization and polyploidization. Two kinds of species, 'basic' and 'intermediate' (i.e. hybridogenous), are usually recognized. In this study, genome size variation was investigated in a representative set of Central European hawkweeds in order to assess the value of such a data set for species delineation and inference of evolutionary relationships. METHODS: Holoploid and monoploid genome sizes (C- and Cx-values) were determined using propidium iodide flow cytometry for 376 homogeneously cultivated individuals of Hieracium sub-genus Pilosella, including 24 species (271 individuals), five recent natural hybrids (seven individuals) and experimental F(1) hybrids from four parental combinations (98 individuals). Chromosome counts were available for more than half of the plant accessions. Base composition (proportion of AT/GC bases) was cytometrically estimated in 73 individuals. KEY RESULTS: Seven different ploidy levels (2x-8x) were detected, with intraspecific ploidy polymorphism (up to four different cytotypes) occurring in 11 wild species. Mean 2C-values varied approx. 4.3-fold from 3.53 pg in diploid H. hoppeanum to 15.30 pg in octoploid H. brachiatum. 1Cx-values ranged from 1.72 pg in H. pilosella to 2.16 pg in H. echioides (1.26-fold). The DNA content of (high) polyploids was usually proportional to the DNA values of their diploid/low polyploid counterparts, indicating lack of processes altering genome size (i.e. genome down-sizing). Most species showed constant nuclear DNA amounts, exceptions being three hybridogenous taxa, in which introgressive hybridization was suggested as a presumable trigger for genome size variation. Monoploid genome sizes of hybridogenous species were always between the corresponding values of their putative parents. In addition, there was a good congruency between actual DNA estimates and theoretical values inferred from putative parental combinations and between DNA values of experimental F(1) hybrids and corresponding established hybridogenous taxa. CONCLUSIONS: Significant differences in genome size between hawkweed species from hybridogenous lineages involving the small-genome H. pilosella document the usefulness of nuclear DNA content as a supportive marker for reliable delineation of several of the most problematic taxa in Hieracium sub-genus Pilosella (including classification of borderline morphotypes). In addition, genome size data were shown to have a good predictive value for inferring evolutionary relationships and genome constitution (i.e. putative parental combinations) in hybridogenous species.     

Genome size is negatively correlated with effective population size in ray-finned fish

A recent theory suggesting that genome size and complexity can increase as a passive consequence of small effective population size has generated much controversy. In this article, we demonstrate that freshwater fish species, which have smaller effective population sizes than marine fish species, have larger genomes. We show that genome size is negatively correlated with genetic variability, independent of phylogeny, body size and generation time. Genome duplication is also observed predominantly in freshwater fish. These results suggest that the raw materials of complexity originate under conditions of reduced selection efficiency.     

Genome size in Bulgarian Centaurea s.l. (Asteraceae)

Thirty-nine species and subspecies of the genera Centaurea, Colymbada, Psephellus and Cyanus (all included in Centaurea s.l.) including many rare and endemic taxa of preponderantly Bulgarian distribution have been investigated with Feulgen DNA image densitometry for holoploid and monoploid genome size (C- and Cx-values). Cyanus varies gradually 2.17-fold between 0.74 pg and 1.56 pg (1Cx). In the remaining taxa two major genome size groups are found, which differ about 1.8-fold in Cx-value. Low values occur in Centaurea subgenera Acrolophus, Solstitiaria, Phalolepis (0.77 pg to 0.90 pg, 1Cx) and Jacea (0.95 pg to 1.09 pg, 1Cx), high values in the genera Colymbada (1.65 pg to 1.93 pg, 1Cx) and Psephellus (1.79 pg, 1Cx, in P. marschallianus). Cx-values support a distinction of Colymbada from Centaurea. Genome size variation is discussed with regard to phylogeny, life form (annual versus perennial), polyploidy, chromosome basic numbers, altitude of occurrence and climate, endemism, and rarity.     

A new typification of Hieracium umbellatum (Asteraceae)

The Linnaean name Hieracium umbellatum has generally been understood in a very strict and clear sense. The original material comprises three specimens and a few illustrations, with a lectotype designated here in Burser's Hortus siccus (UPS). The earlier choice published by W. Lack was made from non-original material and was therefore not effective. The application of this name is confined to a variety generally known as H. umbellatum var. commune Fr. nom. illeg. (restricted here to the forms with almost entire, sublinear leaves), most proximate to H. umbellatum var. coronopifolium (Bernh. ex Hornem.) Wimm. et Grab.     

Genome size in Echinops L. and related genera (Asteraceae, Cardueae): karyological, ecological and phylogenetic implications

Genome size was assessed by flow cytometry in 33 species belonging to seven genera of the tribe Cardueae (Asteraceae), which can be grouped in three taxonomic complexes. 2C nuclear DNA content ranged from 1.49 to 16.98 pg, which is more than elevenfold variation. Genome size correlated well with some karyological traits. Nuclear DNA amount variations also have systematic and evolutionary implications and/or are linked to adaptations to ecological conditions.     

Cytotype distribution and phylogeography of Hieracium intybaceum (Asteraceae)

Using flow cytometry and amplified fragment length polymorphism (AFLP), we explored the cytogeography and phylogeography of Hieracium intybaceum, a silicicolous species distributed in the Alps and spatially isolated in the Vosges Mountains and the Schwarzwald Mountains. We detected two ploidies, diploid and tetraploid, but no triploid or mixed‐ploidy populations. Whereas diploids are sexual and distributed all across the Alps, tetraploids are apomictic and seem to be confined to the western Alps and the Vosges. We detected a low level of genetic variation. Bayesian clustering identified four clusters/genetic groups, which are partly congruent with the ploidal pattern. The first two groups consisting exclusively of diploids dominate the whole distribution range in the Alps and show east–west geographical separation with a diffuse borderline running from eastern Switzerland to the eastern part of North Tyrol. The third genetic group lacks a defined geographical range and includes diploid and tetraploid plants. The last genetic group comprises tetraploid plants in the French Alps and the Vosges. We suppose that diploids colonized the deglaciated areas from source populations most likely located mainly in the southern part of the recent distribution range and occasionally also in the western Alps. Gene flow and further differentiation likely took place. Apomictic tetraploids most likely originated in the western Alps or in the refugium at the south‐western foot of the Alps. Their rather limited geographical range (partly contrasting with the theory of geographical parthenogenesis) can be explained by their rather recent origin. © 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2015, 179 , 487–498.     

Genome size shifts: karyotype evolution in Crepis section Neglectoides (Asteraceae)

Plant genome size evolution is a very dynamic process: the ancestral genome of angiosperms was initially most likely small, which led to a tendency towards genome increase during evolution. However, findings in several angiosperm lineages demonstrate mechanisms that also led to genome size contraction. Recent molecular investigations on the Asteraceae genus Crepis suggest that several genomic reduction events have occurred during the evolution of the genus. This study focuses on the Mediterranean Crepis sect. Neglectoides, which includes three species with some of the smallest genomes within the whole genus. Crepis neglecta has the largest genome in sect. Neglectoides, approximately twice the size of the two species Crepis cretica and Crepis hellenica. Whereas C. cretica and C. hellencia are more closely related to each other than to C. neglecta the karyotypes of the latter species and C. cretica are similar, while that of C. hellenica differs considerably. Here, the karyotypic organisation of the three species is investigated with fluorescence in‐situ hybridisation and studied in a molecular phylogenetic framework based on the nuclear markers Actin, CHR12, CPN60B, GPCR1 and XTH23. Our findings further corroborate the occurrence of genome size contraction in Crepis, and suggest that the difference in genome size between C. neglecta and C. cretica is mostly due to elimination of dispersed repetitive elements, whereas chromosomal reorganisation was involved in the karyotype formation of C. hellenica.     

Lectotypification of names of south Swedish Hieracium species (Asteraceae)

Ninety-six south Swedish taxa in Hieracium L. sect. Hieracium and H. sect Vulgata (Griseb.) Willk. & Lange are lectotypified. The authorship of each name is analysed and the identity and synonomy of some dubious taxa are discussed.-Flora Nordica Note no. 24     

Pollen and chromosome studies in Hieracium sect. Alpestria (Asteraceae)

Potential pollen viability has been investigated in British apomictic Hieracium section Alpestria and related species using Alexander's Stain. New and existing chromosome counts are given. Some species produced no pollen, others varied in the amount they produced, and a few species consistently produced reasonable quantities. The three triploid section Alpestria species produced very little or no pollen, whilst the amount produced by the tetraploids varied widely. Section Alpestria species had significantly lower pollen viability than the non‐section Alpestria species. The generally low pollen production in most species of section Alpestria is consistent with them being of hybrid origin.     

A taxonomic and nomenclatural note on Hieracium caesium (Asteraceae)

The only specimen suitable for typification of Hieracium caesium was discovered in UPS and is here designated as the lectotype. This name appears to have been misapplied and superfluous when originally published at specific rank, but is legitimate according to Art. 52.3. Correct names proposed for three species of the group Caesia (H. caesium = H. basifolium, H. laeticolor and H. plumbeum) are given with infraspecific variants and some more important synonyms. The names H. caesium subsp. laeticolor, H. imitans, H. caesium var. nemorum, H. plumbeum are also lectotypified. Two new combinations H. caesium var. basifolium and H. caesium var. imitans are proposed.     

A new diploid Hieracium (Asteraceae: Lactuceae) from Bulgaria

Hieracium kittanae Vladimirov, a new diploid (2 n  = 18) species restricted to a few gorges in the Central Rhodope Mountains, south Bulgaria, is described and illustrated. It occurs in crevices of limestone rock in shady to sunny places with high atmospheric humidity. Morphologically, it to some extent resembles taxa from the H. glaucinum and H. schmidtii groups, but is otherwise very distinct, without close relatives in the Rhodope and neighbouring mountains.  © 2003 The Linnean Society of London, Botanical Journal of the Linnean Society, 2003 , 143 , 213−218.     

On Hieracium incurrens (Asteraceae) and similar species in central Sweden

Hieracium incurrens Saelan ex Norrl., originally described from Finland, has been reported from Sweden several times and independently by different authors. Still, however, its presence in Sweden has remained dubious and it has become obvious that the different reports are referable to different taxa and the name has thus been, at least partially, misapplied. In the present study, all Swedish material and putative taxa that have been suggested to belong to H. incurrens , as well as some additional closely similar taxa, is critically compared with authentic material of the latter from Finland. It is concluded that H. incurrens do indeed occur in central Sweden, namely in the eastern coastal provinces closest to Finland. However, the relevant material has mostly been referred to H. longilingua Johanss., which is here considered to be a later synonym of H. incurrens. Material previously referred to H. incurrens from other parts of Sweden does, however, represent other taxa. Five accepted species of this affinity are described and discussed in some detail and one species, viz. H. oestmanii T. Tyler is described anew. In addition, the unrelated species H. arnoldii T. Tyler is described anew based on material from the provinces of Hälsingland and Medelpad.     

Genome size distributions in bacteria and archaea are strongly linked to evolutionary history at broad phylogenetic scales

The evolutionary forces that determine genome size in bacteria and archaea have been the subject of intense debate over the last few decades. Although the preferential loss of genes observed in prokaryotes is explained through the deletional bias, factors promoting and preventing the fixation of such gene losses often remain unclear. Importantly, statistical analyses on this topic typically do not consider the potential bias introduced by the shared ancestry of many lineages, which is critical when using species as data points because of the potential dependence on residuals. In this study, we investigated the genome size distributions across a broad diversity of bacteria and archaea to evaluate if this trait is phylogenetically conserved at broad phylogenetic scales. After model fit, Pagel’s lambda indicated a strong phylogenetic signal in genome size data, suggesting that the diversification of this trait is influenced by shared evolutionary histories. We used a phylogenetic generalized least-squares analysis (PGLS) to test whether phylogeny influences the predictability of genome size from dN/dS ratios and 16S copy number, two variables that have been previously linked to genome size. These results confirm that failure to account for evolutionary history can lead to biased interpretations of genome size predictors. Overall, our results indicate that although bacteria and archaea can rapidly gain and lose genetic material through gene transfers and deletions, respectively, phylogenetic signal for genome size distributions can still be recovered at broad phylogenetic scales that should be taken into account when inferring the drivers of genome size evolution.     

Reproductive strategy and population variability in the facultative apomict Hieracium pilosella (Asteraceae)

Molecular studies of apomictic plant species often detect more genotypic variation than predicted from their assumed reproductive mode. The two most commonly invoked mechanisms to explain these high levels of variation are recombination, via facultative sexuality, and mutation. The potential for sexual reproduction in the facultative apomict Hieracium pilosella (Asteraceae) was determined at three field sites by artificially pollinating with the closely related, but morphologically distinct, H. aurantiacum. The level of genotypic variation at the three sites was recorded using inter-simple sequence repeats (ISSRs). There was a significant, positive relationship between the measured potential for sexual reproduction and population genotypic variability, indicating that sex has played a role in the structuring of these populations; however, a causal relationship cannot be stated because of the use of regression. We also applied the recently developed method of compatibility analysis. Compatibility analysis can determine, using the occurrence of "character incompatibilities," whether patterns of variation observed in populations are most parsimoniously explained by mutation or recombination. Compatibility analysis also indicated that sexual reproduction had played a role in generating genotypic diversity in these populations. Combining these different types of data may give a greater understanding of the potential for the generation of genotypic diversity in facultative apomictic populations.     

Genome size dynamics in Artemisia L. (Asteraceae): following the track of polyploidy

Polyploidy is a key factor in the evolution of higher plants and plays an important role in the variation of plant genomes, leading to speciation in some cases. During polyploidisation, different balancing processes take place at the genomic level that can promote variation in nuclear DNA content. We estimated genome size using flow cytometry in 84 populations of 67 Artemisia species and one population of Crossostephium chinense. A total of 73 sequences of nrDNA ITS and 3′‐ETS were newly generated and analysed, together with previously published sequences, to address the evolution of genome size in a phylogenetic framework. Differences in 2C values were detected among some lineages, as well as an increase of genome size heterogeneity in subgenera whose phylogenetic relationships are still unclear. We confirmed that the increase in 2C values in Artemisia polyploids was not proportional to ploidy level, but 1Cx genome size tended to decrease significantly when high ploidy levels were reached. The results lead us to hypothesise that genome size in polyploids tends to a maximum as it follows saturation behaviour, in agreement with the Michaelis–Menten model. We tested different arithmetic functions with our dataset that corroborated a non‐linear relationship of genome size increase in polyploids, allowing us to suggest a theoretical upper limit for the DNA content of this genus.     

A new species of Brickellia,subgenus Phanerostylis (Asteraceae)

Brickellia nesomii, a new species from northeastern Mexico, is described. It belongs to the subgenus Phanerostylis, which Gray originally placed in Eupatorium; King and Robinson elevated the taxon to generic rank; it is herein kept at the subgeneric level but transferred to Brickellia. Reasons for this transfer are given. Barroetea glutinosa, positioned by King and Robinson in Phanerostylis, is excluded.     

Intra-individual polymorphism in diploid and apomictic polyploid hawkweeds (Hieracium, Lactuceae, Asteraceae): disentangling phylogenetic signal, reticulation, and noise

Background  

Hieracium s.str. is a complex species-rich group of perennial herbs composed of few sexual diploids and numerous apomictic polyploids. The existence of reticulation and the near-continuity of morphological characters across taxa seriously affect species determination, making Hieracium one of the best examples of a 'botanist's nightmare'. Consequently, its species relationships have not previously been addressed by molecular methods. Concentrating on the supposed major evolutionary units, we used nuclear ribosomal (ETS) and chloroplast (trnT - trnL) sequences in order to disentangle the phylogenetic relationships and to infer the origins of the polyploids.     

Genome size variation in the Artemisia arborescens complex (Asteraceae, Anthemideae) and its cultivars.

Different wild Mediterranean populations of Artemisia arborescens from diverse locations representing its geographical distribution, as well as some of its well-known cultivars and some specimens cultivated as ornamentals in gardens, streets, roads and nurseries, were analysed for genome size. Other closely related species endemic to Macaronesia, Artemisia canariensis, Artemisia argentea, and Artemisia gorgonum, were also analysed, and their nuclear DNA amount has been related to the biogeography of this group of species. Additionally, 5 populations of the closely related Artemisia absinthium were analysed to establish comparisons. Measurements acquired by flow cytometry ranged from 8.29 to 11.61 pg for 2C values. Statistically significant differences of 2C nuclear DNA amounts with respect to factors such as insularity or domestication have been detected. However, quite a low intraspecific genome size variation has been found in these species. Furthermore, the study also addressed the possible hybrid origins and possible misidentifications of some of the supposed cultivars of A. arborescens.     

Genetic structure of the rare and endangered Hieracium wiesbaurianum group (Asteraceae) in Bavaria

The rare and endangered Hieracium wiesbaurianum species group shows a scattered relictual distribution in Bavaria. Recently, a couple of populations were discovered which clearly differ from all other populations. If these must be considered as taxonomically independent units, they would be of crucial conservation interest, because of the sole responsibility that Bavaria has for these worldwide endemics. We therefore analysed the genetic structure of H. wiesbaurianum in a comparative approach. Our analysis comprised 37 populations of 13 taxa of H. wiesbaurianum, H. bifidum and H. laevigatum, including three potentially new taxa. We applied amplified fragment length polymorphism (AFLP) analysis and observed only limited genetic variation within populations and taxa. Nevertheless, each studied individual exhibited a unique genotype. An analysis of molecular variance revealed high levels of genetic variation between taxa, but populations were genetically less different. The clear genetic differentiation between the studied taxa was supported by neighbor‐joining cluster analyses and principal coordinate analyses in which every individual was clearly assigned to its respective taxon. The three potentially new taxa were genetically as well differentiated as the other taxa included in our study. This supports the assumption that they should be treated as taxonomically independent units of high conservation interest. Therefore, the genetic analysis confirmed the morphologically based classification of the studied Hieracium taxa. © 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2015, 177 , 112–123.