Chromosome numbers and pollen fertility in the Senecio nemorensis group (Compositae) in the Carpathians

New chromosome numbers for two species from the Senecio nemorensis group: S. dacicus (2n = 40) and S. ucranicus (2n = 40) have been ascertained. The counts for S. germanicus Wallr. subsp. germanicus (2n = 40), S. hercynicus Herborg subsp. hercynicus (2n = 40), S. ovatus (P. Gaertn. et al.) Willd. subsp. ovatus (2n = 40) occurring in the Carpathians are also reported. The study confirmed only the known tetraploid chromosome number for the taxa of this group. The pollen fertility ranged from 82.09 to 92.99% in all examined species and subspecies, including their hybrids.     

Morphological and phytochemical variation, genetic structure and phenology in an introgressive hybrid swarm of Senecio hercynicus and S. ovatus (Compositae, Senecioneae)

Introgressive hybridisation between Senecio hercynicus and Senecio ovatus (Compositae, Senecioneae) was studied in a mixed stand of the two species on the S- and SW-facing slopes of the German part of the Bohemian Forest (SE Germany). Morphological variation based on multivariate analysis of 14 diagnostic characters, along with genetic data from an amplified fragment length polymorphism (AFLP) fingerprinting analysis, indicates that the surveyed stand forms an intensively introgressed hybrid swarm. The majority of individuals were found to be intermediate between the two parental taxa, and strong statistical correlation between phenetic and genetic distances was observed. In contrast to that, flowering time of individuals (expressed as the time when 50% of the capitula of a plant were in flower) was found to follow a bimodal distribution in the hybrid swarm and lacked any correlation with the genetic and morphological relationships among plants. The same was true for the spectra of pyrrolizidine alkaloids (PA) revealed by a gas-chromatographic survey: most of the 142 individuals surveyed fell into one of two main chemotypes, only a few plants exhibited an intermediate and additive PA spectrum, and no correlation with the genetic and morphological relationships among plants was observed. Assuming that most of the AFLP markers are neutral, we conclude that the correlation of morphology with the genetic pattern may argue for the neutrality of morphological features analysed. Consequently, we interpret the lack of correlation between phenological and phytochemical data with the neutral genetic markers as evidence that both phenology and phytochemistry may be either under selection or that these features are genetically or biosynthetically constrained.     

Lost in the hybridisation vortex: high-elevation <Emphasis Type="Italic">Senecio hercynicus</Emphasis> (Compositae,Senecioneae) is genetically swamped by its congener <Emphasis Type="Italic">S. ovatus</Emphasis> in the Bavarian Forest National Park (SE Germany)

Hybridisation is an important evolutionary process. The investigation of hybridisation along elevational gradients, with their steep abiotic and biotic clines, provides insight into the adaptation and maintenance of species in adjacent habitats. The rare Senecio hercynicus and its spreading congener S. ovatus are vertically vicariant species that show hybridisation in their range overlaps. In the present study, we used AFLP fingerprinting of 689 individuals from 38 populations to analyse population structure and introgression patterns along four elevational transects (650–1350 m) in the Bavarian Forest National Park, Gemany. Subsequently, we searched for loci putatively under divergent selection that may maintain ‘pure’ species despite hybrid formation by identifying taxon-specific alleles. A maximum-likelihood based hybrid index shows that the overall genetic differentiation among all populations was very low with a vanishingly small number of pure parental individuals. Almost 75% of the investigated individuals were classified as backcrosses towards S. ovatus. The highest S. hercynicus ancestry was found in the uppermost populations of two transects. Further, we found seven taxon-specific alleles being under divergent selection that correlated with climatic variables and deviating from neutral introgression. According to our results, hybridisation of S. ovatus and S. hercynicus has reached an advanced state of genetic swamping and there seems to be no driving force that is strong enough to keep both species as different lineages. Rather, S. ovatus appears to benefit through putatively adaptive introgression.     

Introgressive hybridization of Senecio hercynicus and S. ovatus (Compositae,Senecioneae) along an altitudinal gradient in Harz National Park (Germany)

Introgressive hybridization of Senecio hercynicus and S. ovatus (Compositae, Senecioneae) was studied in a hybrid zone on the southern slopes of Mt Brocken (Harz Mountains, Germany). A total of 415 plants representing 10 stands along an altitudinal gradient were investigated using multivariate statistical analyses of morphological characters and molecular markers (random amplified polymorphic DNA[RAPD]). Both types of traits detected pure S. hercynicus stands on the summit plateau, pure S. ovatus stands at the lowest elevations, and hybrid swarms at intermediate elevations. While morphological and molecular patterns coincided, some individuals in hybrid stands combined morphological patterns typical of S. ovatus with RAPD patterns typical of S. hercynicus, and vice versa. In general, introgression was symmetrical within stands, though one stand combined S. ovatus characters with the glandular hair typical for S. hercynicus, and two stands combined a S. hercynicus typical RAPD genotype with morphological characters shifted towards S. ovatus. Because pure stands of S. hercynicus occurred only on the summit plateau of Mt Brocken, and markers typical for S. ovatus were detectable in stands up to 1040 m a.s.l., future fusion or assimilation of the rare form, S. hercynicus, by the more widespread S. ovatus appears possible at Mt Brocken.     

Morphological differentiation supports the genetic pattern of the geographic structure of Juniperus thurifera (Cupressaceae)

Juniperus thurifera is an important component of woodland communities of dry sites within the West Mediterranean region and is characterised by a strongly disjunctive geographic range. Two subspecies were recognised, subsp. thurifera in Europe and subsp. africana in Africa. The aim of the study was the comparison of phenetic diversity to the pattern of AFLP geographic differentiation of the species described in the literature. The examination of phenetic diversity was based on the biometrical analysis of 17 populations using 12 morphological characters of cone and seed. The differences among populations were analysed using Student’s t test, analysis of discrimination, UPGMA agglomeration and hierarchical analysis of variance. The majority of morphological characters differentiated at a statistically significant level between populations and between J. thurifera subsp. thurifera and subsp. africana. Three groups of populations were detected using multivariate statistical analyses. The first, well separated, is subsp. africana, while the following two concern subsp. thurifera. The morphological differentiation of populations appeared similar to that described on the AFLP. The Gibraltar Straight appeared to be the most important barrier.     

Genetic structure of Eurasian and North American Leymus (Triticeae) wildryes assessed by chloroplast DNA sequences and AFLP profiles

Leymus is a genomically defined allopolyploid of genus Triticeae with two distinct subgenomes. Chloroplast DNA sequences of Eurasian and North American species are distinct and polyphyletic. However, phylogenies derived from chloroplast and nuclear DNA sequences are confounded by polyploidy and lack of polymorphism among many taxa. The AFLP technique can resolve phylogenetic relationships between closely related species, with a curvilinear relationship expected between the proportion of shared bands and nucleotide substitution rate (D), up to about 0.100 D. The objective of this study was to compare D and phylogenetic relationships among 16 Leymus taxa, based on chloroplast DNA sequences and multi-locus AFLP genotypes. Estimates of chloroplast D between taxa were 0.002 and 0.013 within and among continental regions, respectively. Estimates of AFLP D between taxa were 0.076 and 0.093 compared within and between continental regions, respectively, versus 0.024 within taxa. Bayesian and neighbor-joining cluster analyses effectively separated all AFLP genotypes by species, but showed that North American L. ambiguus is a hybrid species with nearly equal contributions from sympatric L. cinereus and L. salinus taxa. Two hierarchical AFLP clades, containing six North American taxa and four Eurasian taxa, had more than 98% bootstrap confidence with 0.071 and 0.055 D among taxa. Three other Eurasian taxa clustered with 79% and 89% confidence, with up to 0.79 D between taxa. These estimates provide benchmarks for phylogenetic comparisons of AFLP profiles, but three taxa could not be reliably grouped, which may reflect concurrent radiation of multiple lineages or lack of homologous AFLP characters caused by a high D.     

PFGE and AFLP genotyping of Staphylococcus aureus subsp. anaerobius isolated from goats with Morel’s disease

Staphylococcus aureus subsp. anaerobius is the etiological agent of the Morel’s disease in sheep and goats. The disease presents with subcutaneous abscesses, located mainly in the superficial lymph nodes. Forty-one isolates of S. aureus subsp. anaerobius were collected from two outbreaks of the Morel’s disease in Poland in years 2006–2008. Analysis of DNA SmaI digests by PFGE showed that 35 of 41 isolates belonged to the same PFGE type, identical to the type strain of S. aureus subsp. anaerobius ATCC 35844, confirming high level of clonality of the species. The DNA patterns of the remaining identical 6 isolates, different from the reference strain only by two bands, were found closely related. Genotyping performed with AFLP technique revealed two clonal groups including 16 and 25 isolates, respectively. The study indicated that AFLP technique might be a better discriminatory tool for genetic analysis of S. aureus subsp. anaerobius isolates, when compared to PFGE.     

Human-induced hybridization among congeneric endemic plants on Tenerife, Canary Islands

Endemic genera on oceanic islands often evolved striking morphological and ecological differences among species, with weak postzygotic reproductive isolation. Human activities can lead to increased connectivity and can thereby promote secondary contact and hybridization between previously isolated species. We studied this phenomenon in three species of the genus Pericallis (Asteraceae) on Tenerife, Canary Islands. A total of 53 populations was sampled, including morphologically uniform as well as mixed populations containing morphologically diverse individuals. All plants were analyzed both genetically using AFLP markers and morphometrically. As expected, morphological analysis clearly separated the plants from the uniform populations in three clusters, with distinguishing characters corresponding to those used in species identification. The three species were also grouped into distinct genetic clusters in a structure analysis, although no private alleles were observed. Adding the mixed population data to the analyses provided evidence for extensive hybridization among species, predominantly between P. cruenta and P. echinata, and between P. cruenta and P. tussilaginis, and morphological and genetic signals were congruent. The mixed populations were geographically located in between the uniform populations and were found in road verges significantly more often than the uniform populations. The observed distribution strongly suggests that hybridization between originally isolated species is recent, promoted by secondary contact due to human disturbance and the construction of roads.     

Infraspecific differentiation in the Balkan diploidCardamine acris (Brassicaceae): Molecular and morphological evidence

Molecular and morphological variation in the Balkan diploidCardamine acris was studied by amplified fragment length polymorphism (AFLP) and multivariate morphometric analyses. Principal coordinate analysis and neighbour-joining analysis of the AFLP data showed three genetically differentiated groups of populations corresponding to their geographic origin. Following the results of molecular analysis three subspecies were recognized by a combination of morphological characters as shown by separate analyses of field-collected and cultivated plants. These subspecies are allopatric;C. acris subsp.acris occurs in most parts of the Balkan Peninsula, and newly described subsp.pindicola and subsp.vardousiae are endemic to the Northern Pindhos Mts. in Northwestern Greece, and Central Greece, respectively. The differentiation into geographically isolated subspecies is most probably caused by long-term isolation of populations in different mountain ranges of the Balkan Peninsula, possibly initiated during the cold periods of the Pleistocene.     

Evolution of the polyploid north-west Iberian Leucanthemum pluriflorum clan (Compositae,Anthemideae) based on plastid DNA sequence variation and AFLP fingerprinting

Background and Aims

The genus Leucanthemum is a species-rich polyploid complex from southern and central Europe, comprising 41 species with ploidy ranging from 2x to 22x. The present contribution aims at reconstructing the evolutionary history of a geographically isolated species group (the L. pluriflorum clan) from the north-west Iberian Peninsula comprising the diploid L. pluriflorum, the tetraploids L. ircutianum subsp. pseudosylvaticum and L. × corunnense (a putative hybrid taxon based on crossing between L. pluriflorum and L. merinoi), and the hexaploids L. sylvaticum and L. merinoi.

Methods

Chromosome number variation (determined flow cytometrically) and sequence variation were analysed for two intergenic spacer regions on the plastid genome (psbA-trnH and trnC-petN) for individuals from 54 populations in combination with amplified fragment length polymorphism (AFLP) fingerprinting of 246 representative individuals from these populations.

Key Results

Plastid sequence data revealed that all surveyed members of the L. pluriflorum clan possess plastid haplotypes that are closely related to each other and distinctly separated from other Leucanthemum species. AFLP fingerprinting resulted in allopolyploid fragment patterns for most of the polyploid populations, except for the tetraploid L. × corunnense and a further tetraploid population in northern Galicia, which cluster with the diploids rather than with the other polyploids. In silico modelling of (auto)tetraploid AFLP genotypes further corroborates the allopolyploid nature of L. ircutianum subsp. pseudosylvaticum, L. sylvaticum and L. merinoi.

Conclusions

The present study provides evidence for recognizing one diploid (L. pluriflorum), one autotetraploid (L. corunnense), one allotetraploid (L. pseudosylvaticum) and one allohexaploid (L. sylvaticum with the two geographically and ecologically differentiated subspecies subsp. sylvaticum and subsp. merinoi) in the L. pluriflorum clan. It also has implications for the understanding of biogeographical patterns in the Iberian Peninsula.     

Cytogenetic studies on natural intergeneric hybridization inAster alliances

In addition to various types of natural hybrids betweenAster ageratoides subsp.ovatus andKalimeris incisa reported earlier, a new backcross type has been discovered. This new type, characterized by the chromosomes of 2n=27L+54S, was most probably produced through fertilization of a normally-reduced gamete of the F₁ plant (2n=72=18L+54S) and an unreduced gamete of subsp.ovatus (2n=36=18L+18S).     

Cytogenetics, geographical distribution, pollen stainability and fecundity of five diploid taxa of Santolina rosmarinifolia L. aggregate (Asteraceae: Anthemideae)

The chromosome analysis of Santolina rosmarinifolia subsp. rosmarinifolia, S. oblongifolia, S. semidentata subsp. semidentata, S. semidentata subsp. melidensis, S. canescens and the hybrid complex (S. rosmarinifolia subsp. rosmarinifolia, S. oblongifolia and their putative hybrids) shows that all the taxa are diploids (2n = 2x = 18; 18 + 1 or more B chromosomes, with 2n = 19, 20 only in the hybrid complex). The results show a conserved general structure of the karyotype (14m + 2sm + 2st), but in S. semidentata subsp. melidensis it is variable, with 14m + 2sm + 2st in ten individuals, 14m + (1m ? 1sm) + (1 m ? 1st) in nine individuals and 12m + (1m ? 1sm) + (1m ? 1st) + 2st + 1B in five individuals. Tetraploid individuals occurred in the diploid populations of S. rosmarinifolia subsp. rosmarinifolia and S. canescens, and their autopolyploid origin is discussed. Multivalent configurations at diakinesis, simple and double chromosome bridges and delayed disjunction of homologous and non-homologous chromosomes at anaphase I have negative effects on pollen stainability. The mean fructification percentage is moderate. The results suggest that the complex is a mosaic of introgressive hybrids.     

Comparative phylogeography and species boundaries in Echinolittorina snails in the central Indo-West Pacific

Aim This study aimed to test monophyly and geographical boundaries in five marine intertidal snail species from the central Indo‐West Pacific. We tested the prediction that phylogenetic breaks between the Indian and Pacific Ocean basins should be more pronounced in continental than oceanic settings, and sought common geographical patterns of interspecific boundaries and intraspecific phylogenetic breaks in the region. Location The tropical seas of the Indo‐West Pacific. Methods We sequenced over 1200 bp of the mitochondrial cytochrome oxidase subunit I gene (COI) from 18–92 individuals sampled from throughout the ranges of each of five species of Echinolittorina (Littorinidae): three members of the Echinolittorina trochoides species complex; Echinolittorina reticulata; and Echinolittorina vidua, together with sister species, in order to test species boundaries. In addition, 630 bp of the nuclear 28S rRNA gene were sequenced from E. reticulata and its sister Echinolittorina millegrana. Phylogenetic structure was assessed using neighbour‐joining and parsimony analyses. Results COI data confirmed species boundaries and geographical distributions for all species except the pair E. reticulata and E. millegrana, which were nevertheless reciprocally monophyletic for 28S rRNA. The species from ecologically ‘continental’ habitats (E. trochoides A and E. vidua, but not E. trochoides B) mostly showed strong interoceanic breaks (with age estimates 0.58–4.4 Ma), while the ecologically ‘oceanic’E. trochoides D and E. reticulata did not. The sister species E. trochoides A and B occupy the shores of the continental shelves of Southeast Asia and Australasia respectively; between them lies the oceanic ‘eastern Indonesian corridor’ occupied by E. trochoides D and E. reticulata. The widespread continental species E. vidua showed a complex pattern of deep division into six haplotype clades with apparently parapatric distributions. Main conclusions Our results show that ecological differences (in this case continental vs. oceanic habitat) influence both intraspecific phylogenetic structure and interspecific boundaries in these snails of intertidal rocky shores. Two of the three species restricted to continental shelves show phylogenetic breaks between the Indian and Pacific Oceans, consistent with vicariant separation during Plio‐Pleistocene low sea levels. The two oceanic species do not show breaks, suggesting that they maintained interoceanic connections through the eastern Indonesian corridor. The geographical location of the interspecific boundary between continental E. trochoides A and oceanic E. trochoides D mirrors intraspecific breaks reported in other species. The sister relationship of E. trochoides A and B in Asia and Australasia, respectively, is an example of a ‘marine Wallace's line’ distribution, and we suggest that it is the result of separation of two continental species by a barrier of unsuitable oceanic habitat.     

Morphological variation in populations of Bletia purpurea (Orchidaceae) and description of the new species B. riparia

Bletia purpurea is the most widespread species in its genus. Morphological variation has been recognized throughout the range of its distribution. In this paper, the morphological variation from 63 populations (583 individuals) ofBletia purpurea is assessed to determine whether more than one species were present. Forty-four quantitative and qualitative characters were examined by univariate analyses and exploratory multivariate analyses. Univariate analyses indicate that quantitative characters such as lateral sepal width, petal. width, lip length, and lip width are significantly different for populations from Acazónica, Mexico. Floral parts in the populations from Acazónica are the smallest among all populations. Qualitative characters such as petals covering the lip midlobe and horizontal lip position are found exclusively in the same populations. We concluded that these populations should be described as a new species,B. riparia. Multivariate analyses indicated that morphological variation among the other populations cannot be ascribed to geographic distribution or ecological factors.     

Cytogenetic studies on natural intergeneric hybridization inAster alliances

Natural intergeneric hybrids betweenAster ageratoides subsp.ovatus (2n=36) andKalimeris incisa (2n=72) were found. All of the hybrids studied were found to have 2n=72, 18 more chromosomes than a regular F₁ hybrid. The hybrids were found to be of two types: one having 18 large chromosomes ofovatus, and the other having 9 large chromosomes of the same subspecies. In meiosis of the PMCs of the hybrid with 18 large chromosomes, a regular chromosome configuration, 36_II, was observed. In PMCs of the hybrid with 9 large chromosomes an irregularity of chromosome pairings was observed, showing varied chromosome configurations: 35_II+2_I, 34_II+4_I, 33_II+6_I, I_III+33_II+3_I, 1_IV+32_II+4_I, 32_II+8_I, 31_II+10_I, 29_II+14_I, 3_III+29_II+5_I. Most univalents were large, but a few were small. The hybrids with 18 large chromosomes were found to be partial amphidiploid and possessing double chromosome complements ofovatus. The hybrids with 9 large chromosomes were found to be the first backcrossed generation between the hybrid with 18 large chromosomes andK. incisa.     

Genetic variation in Mediterranean Helichrysum italicum (Asteraceae; Gnaphalieae): do disjunct populations of subsp. microphyllum have a common origin?

The yellow‐flowered everlasting daisy Helichrysum italicum (Asteraceae, Gnaphalieae) is widely distributed in the Mediterranean basin, where it grows in continuous and widespread populations in diverse open habitats. Helichrysum italicum subsp. microphyllum has a disjunct distribution in the Balearic Islands (Majorca and Dragonera), Corsica, Sardinia, Crete and Cyprus. Numerous morphological intermediates between subsp. italicum and subsp. microphyllum are known from Corsica, where the two subspecies co‐occur. The aims of the study were to investigate if subsp. microphyllum has a common origin, constituting an independent gene pool from subsp. italicum, or if the morphological differences between subsp. microphyllum and subsp. italicum have arisen independently in different locations from a common wider gene pool. Our analyses of AFLP, cpDNA sequences and morphological characters show that there is geographic structure to the genetic variation within H. italicum, with eastern and western Mediterranean groups, which do not correspond with the division into subsp. microphyllum and subsp. italicum as currently circumscribed. Local selection on quantitative trait loci provides sufficient explanation for the morphological divergence observed and is consistent with genetic data. Within the western Mediterranean group of the species we found considerable polymorphism in chloroplast DNA sequences among and within some populations. Comparison with chloroplast DNA sequences from other Helichrysum species showed that some chloroplast haplotypes are shared across species.     

Morphological and seed protein characterization of the cultivated and the wild taxa of Sesamum L. (Pedaliaceae)

Seed protein analysis and morphological characterization were carried out in one cultivated and one wild species of Sesamum, Sesamum indicum L. and S. occidentale Regel and Heer. Data on 13 quantitative and 33 qualitative characters of the cultivated species and seven accessions of the wild taxa were analyzed. The genetic diversity of the taxa was assessed using UPGMA dendrogram and one-way ANOVA (p?<?0.05). Principal component analysis (PCA) was executed to identify the significant characters to delimit the taxa. Seed protein analysis showed diverse bands, ranging from 16 to 88?kDa. A dendrogram based on UPGMA analysis of seed protein suggested intraspecific relationships of the wild taxa as evidenced from the morphological characterization.     

Morphologic and AFLP analysis of relationships between tulip species Tulipa biebersteiniana (Liliaceae)

In populations of four species of tulips (Tulipa biebersteiniana, T. patens, T. scytica and T. riparia) from the Volgograd, Kurgansk, Orenburg, and Chelyabinsk regions and the Republic of Bashkortostan, genetic diversity was studied by means of morphological and AFLP analysis. A morphological analysis of seven quantitative and two qualitative criteria was carried out. Three selective EcoRI/MseI primer pairs allowed one to genotype 81 individuals from 13 tulip populations with 87 loci. The low level of variability by AFLP loci were revealed in all species, including T. biebersteiniana (P = 20.41%, UH _e = 0.075), T. patens (26.97%, 0.082), T. scytica (27.53%, 0.086), and T. riparia (27.72%, 0.096). According to the AMOVA results, the variability proportion that characterizes the differences between the four Tulip species was lower (F _CT = 0.235) than between populations within species (F _ST = 0.439). Tulipa patens is well differentiated by means of Nei’s distances, coordination, and analysis in the STRUCTURE program. An analysis in the STRUCTURE revealed four genetic groups of tulips that are not completely in accordance with the analyzed species. This acknowledges the presence of complicated genetic process in the tulip population.     

Application of Amplified Fragment Length Polymorphism Fingerprinting for Taxonomy and Identification of the Soft Rot Bacteria Erwinia carotovora and Erwinia chrysanthemi

The soft rot bacteria Erwinia carotovora and Erwinia chrysanthemi are important pathogens of potato and other crops. However, the taxonomy of these pathogens, particularly at subspecies level, is unclear. An investigation using amplified fragment length polymorphism (AFLP) fingerprinting was undertaken to determine the taxonomic relationships within this group based on their genetic relatedness. Following cluster analysis on the similarity matrices derived from the AFLP gels, four clusters (clusters 1 to 4) resulted. Cluster 1 contained Erwinia carotovora subsp. carotovora (subclusters 1a and 1b) and Erwinia carotovora subsp. odorifera (subcluster 1c) strains, while cluster 2 contained Erwinia carotovora subsp. atroseptica (subcluster 2a) and Erwinia carotovora subsp. betavasculorum (subcluster 2b) strains. Clusters 3 and 4 contained Erwinia carotovora subsp. wasabiae and E. chrysanthemi strains, respectively. While E. carotovora subsp. carotovora and E. chrysanthemi showed a high level of molecular diversity (23 to 38% mean similarity), E. carotovora subsp. odorifera, E. carotovora subsp. betavasculorum, E. carotovora subsp. atroseptica, and E. carotovora subsp. wasabiae showed considerably less (56 to 76% mean similarity), which may reflect their limited geographical distributions and/or host ranges. The species- and subspecies-specific banding profiles generated from the AFLPs allowed rapid identification of unknown isolates and the potential for future development of diagnostics. AFLP fingerprinting was also found to be more differentiating than other techniques for typing the soft rot erwinias and was applicable to all strain types, including different serogroups.