Genetic structure and differentiation of Plantago major reveals a pair of sympatric sister species

Seeds of the widespread weed Plantago major were collected from 10 European countries, as well as Trinidad and North America. The seed collections were from populations of two taxa which are ecologically rather than geographically separated and formally recognized as the subspecies Plantago major ssp. major and P.m. ssp. intermedia (also called P.m. ssp. pleiosperma). Eight polymorphic allozyme loci and 73 random-primed DNA fragments were scored, as well as 11 morphological characters. Complete concordance between morphological traits and genetic data provides evidence that these two taxa, although very similar, are distinct species. They are both widespread, they are broadly sympatric and capable of interbreeding. However, slight morphological and ecological differences coincide with genetic clustering of populations from widely separated locations. In addition, P. major and P. intermedia differ in their population structure: P. intermedia has greater genetic diversity among populations and less genetic variance within populations than P. major. We suggest that differences between the two species in their levels of selfing may explain the distinctive genetic structure of each species. We hypothesize a link between selfing rate and lifespan of the two taxa. P. major is characterized by lower genetic variation among populations, a higher rate of outcrossing, longer lifespan and production of fewer seeds per seed capsule. P. intermedia is more highly structured with much differentiation among populations, a higher rate of inbreeding and it often grows as an annual.     

Evolution of the Portulaca oleracea L. aggregate in Egypt on molecular and phenotypic levels revealed by morphology,inter-simple sequence repeat (ISSR) and 18S rDNA gene sequence markers

Molecular markers including inter simple sequence repeats (ISSR) and 18S rDNA gene sequence markers were combined with a detailed morphological analysis to seek characters that discriminate four taxa of Portulaca oleracea s.l. These taxa were identified as the microspecies Portulaca nitida, Portulaca oleracea, Portulaca rausii and Portulaca granulatostellulata. Morphological characters did not provide a clear distinction among the four taxa of P. oleracea s.l. It was found that mixed populations of the taxa occur in several locations and morphological similarities between the microspecies were detected. ISSR analysis indicates that gene flow among populations of different taxa was high and most of the genetic variation (61.9%) occurs within population. These results are inconsistent with the general characteristics of P. oleracea as a self- or 5% cross-pollinated species. A close relationship between P. oleracea, P. rausii and P. granulatostellulata was supported by ISSR and 18S rDNA gene sequence. The ISSR and 18s data support the specific status of P. nitida as a recently evolved taxon. We conclude that P. oleracea exists as a polymorphic species and is not divisible into microspecies based on seed surface as the primary morphological trait, and inter simple sequence repeats (ISSR) and 18S rDNA gene sequence markers. We suggest that the taxa do not merit specific rank as morphological characters and absence of a breeding barrier fail to separate the different populations when they become sympatric.     

Lipid Composition of the Roots of Plantago Species: Response to Alteration of the Level of Mineral Nutrition and Ecological Significance

In a study of the adaptation of Plantago species to their specific environment the lipid composition of the roots of several species: Plantago major L. ssp. major, Plantago major L. ssp. pleiosperma Pilger, Plantago lanceolata L., Plantago media L., Plantago maritima L., Plantago coronopus L. was studied as well as the effect of the nutritional regime. Upon exposure to low-salt conditions Plantago major L. ssp. major L. and Plantago maritima L. maintained the level of free sterols in the roots, despite a depressed level of total sterols, and the root lipids were more saturated than under high-salt conditions. Both factors may reduce nutrient leakage from the roots to the low-salt condition. Upon exposure to low-salt conditions, all Plantago species showed a decreased level of galactolipid (exception: Plantago coronopus) and a decreased level of sitosterol (exception: Plantago maritima); the latter being compensated by an elevated level of cholesterol + tocopherol in Plantago major ssp. pleiosperma, Plantago lanceolata and Plantago media. Plantago coronopus was the only species which under low-salt conditions showed an increased level of free sterols, among which cholesterol was the most important; thus indicating a high degree of regulation of membrane permeability under alternating nutritional conditions. The level of sulfolipid was kept constant in all Plantago species, with the highest level observed in Plantago maritima. The role which various lipids may play in maintenance of membrane integrity under alternating nutritional conditions is discussed.     

A genetic investigation of the esters of caffeic acid with rhamnose or glucose in leaves of Plantago major

The genetic relationship between two sugar esters of caffeic acid in the leaves of Plantago major has been investigated. The difference is shown to be due to a single gene. Glucose accounts for the sugar part in the dominant phenotype while in the recessive type the sugar is rhamnose. This difference is only partly correlated with the division of the species into subspecies. The rhamnose type is found only in P. major ssp. pleiosperma Pilger, although only in less than half of the plants investigated.     

Genetic differentiation in Plantago major L. in growth and P uptake under conditions of P limitation

To study possible adaptive mechanisms inbred lines from three populations of Plantago major from sites that were found to differ in P availability were compared. In a pot experiment the growth and P uptake either in the presence or absence of Glomus fasciculatum was determined. Under these P-limited conditions it was shown by partitioning the relative growth rate (RGR, in mg g^-1 day^-1) in the components root weight ratio (RWR, in g_roots g_plant ^-1), specific P uptake rate (SPUR, in mol P g_roots ^-1 day^-1), and P-efficiency (PEFF, in mg mol P^-1), that the increase in RGR of mycorrhizal infected plants was related to an increase in SPUR, and a decrease in RWR and PEFF. P. major ssp. major had a lower RGR (related to a lower PEFF and SPUR) and a higher RWR than P. major ssp. pleiosperma. In a second experiment three inbred lines were compared upon P depletion in a nutrient solution. The P. major ssp. major line had a lower RGR and higher RWR, and a higher accumulation of P in the roots than the P. major ssp. pleiosperma lines under optimal growing conditions. There were no differences among the inbred lines in the relative contribution of inorganic P to the total P concentration in the shoot. The results are discussed in relation to the characteristics of the habitats of the investigated P. major populations.     

Genetic differentiation and postglacial migration of the Dactylorhiza majalis ssp. traunsteineri/lapponica complex into Fennoscandia

Eight variable regions (microsatellites, insertion/deletion and duplication regions) from the plastid DNA genome were analyzed for 91 populations belonging to Dactylorhiza majalis ssp. traunsteineri and closely related taxa. A total of 36 composite plastid haplotypes were found. The two dominating haplotypes had a clear geographic distribution suggesting at least two separate immigration routes into Scandinavia after the last glaciation: one southwestern route and one or two southeastern routes. D. majalis ssp. traunsteineri could not be clearly separated from any of the other taxa included in the study except for D. majalis ssp. sphagnicola. The morphologically similar taxa D. majalis ssp. traunsteineri, D. majalis ssp. lapponica and D. majalis ssp. russowii showed no genetic differentiation, and therefore we suggest an amalgamation of the three taxa into one broadly circumscribed subspecies; D. majalis ssp. lapponica. The plastid data also revealed incidents of hybridization and possible introgression between D. majalis ssp. lapponica and other members of the genus, e.g., D. incarnata.     

Genetic variability in Plantago species in relation to their ecology

Summary A survey of enzyme variability in several populations of Plantago major in the Netherlands has been made. Nine of 36 loci were found to be polymorphic. The most extensively studies population showed 7 polymorphic loci (19%). The average heterozygosity was 0.005, a low value since P. major is predominantly inbreeding; a first estimate of the outcrossing rate is only 10%. All nine variable loci show simple Mendelian inheritance, seven of them could be placed into four different linkage groups. Marked differences in allele frequencies were found between two subspecies: ssp. major and ssp. pleiosperma. Two enzyme loci possess subspecies-specific alleles, Pgm-1 and Got-1. The most likely explanation of this phenomenon is the existence of fitness differences, caused either by the enzyme loci themselves or by linked loci.Grassland Species Research Group Publication no. 14     

Meloidogyne javanica Parasitic on Peanut

Peanut fields in four governorates of Egypt were surveyed to identify species of Meloidogyne present. Fourteen populations obtained from peanut roots were all identified as M. javanica based on perineal patterns, stylet and body lengths of second-stage juveniles, esterase phenotypes, and restriction fragment length polymorphisms of mtDNA. Three of 14 populations, all from contiguous fields in the Behara governorate, had individuals with a unique two-isozyme esterase phenotype. All populations of M. javanica tested on peanut had levels of reproduction on the M. arenaria-susceptible peanut cultivar Florunner that were not different from M. arenaria (P = 0.05), and had lower levels of reproduction on the M. arenaria-resistant genotype TxAG-7 than on Florunner (P = 0.05). Reproduction of the five Egyptian populations of M. javanica tested was lower on root-knot nematode resistant tomato cultivars Better Boy and Celebrity than on the root-knot nematode susceptible cultivar Rutgers (P = 0.05). These data are evidence that some populations of M. javanica are parasitic on peanut and that the peanut and tomato genotypes resistant to M. arenaria are also resistant to these populations of M. javanica.     

Growth Responses of Plantago major L. ssp. pleiosperma (Pilger) to Changes in Mineral Supply : Evidence for Regulation by Cytokinins

Plants of an inbred line of Plantago major ssp. pleiosperma were subjected to an alteration in mineral supply. Observed responses of growth rate and shoot to root ratio are thought to be induced by changes in endogenous cytokinin concentration and not by mineral concentration in plant tissue.     

A search for Y-chromosomal species-specific markers and their use for hybridization analysis in ground squirrels (Spermophilus: Rodentia, sciuridae)

In four ground squirrel species from the Volga region—yellow (Spermophilus fulvus), russet (S. major), little (S. pygmaeus), and speckled (S. suslicus)—four hybridization variants (major/fulvus, major/pygmaeus, major/suslicus, and pygmaeus/suslicus) have been reliably described. Earlier we have shown that populations of S. major from the Volga region were characterized by wide introgression of mtDNA from S. fulvus and S. pygmaeus, which probably, resulted from ancient hybridization [5]. In this study, the same populations were used to analyze the introgression of the Y chromosome, which (unlike mtDNA) is paternally inherited. Three genes, ZfY, SRY, and SmcY were tested as Y-chromosomal candidate markers. It was demonstrated that Y chromosome of ground squirrels lacked the ZfY gene, while its homologous structure, ZfY(X), was presumably linked to the X chromosome. The SRY region examined was rather conservative. In particular, the sequences determined in S. major and S. fulvus were identical, while three out of four substitutions found in S. pygmaeus were located in the coding region. The SmcY gene was found to be the most suitable marker, providing distinguishing of all of the four ground squirrel species by nine nucleotide substitutions. Introgression at the Y chromosome was observed only in two cases: in one S. major individual (out of 51 phenotypically pure animals) caught in the major/fulvus sympatry zone, and in four (one litter) out of fourteen S. fulvus individuals caught in close vicinity of the sympatry zone of these two species. Among 28 S. pymaeus and 9 S. suslicus individuals, no foreign SmcY genes were detected. Two colonies of the “hybrid swarm” type were examined with eight major/suslicus hybrids analyzed in the first and seventeen major/fulvus hybrids in the second colony. The prevalence of the S. major paternal lineages was observed in both colonies (87.5 and 82.4%, respectively). The data obtained suggest that compared to wide mtDNA introgression, introgression of Y chromosome in the Volga region ground squirrels is statistically significantly less frequent event.     

Systematics and phylogeography of the Dactylorhiza maculata complex (Orchidaceae) in Scandinavia: insights from cytological, morphological and molecular data

Flow cytometry, morphometry and molecular markers [plastid DNA and internal transcribed spacers (ITS) of nuclear ribosomal DNA] were used to determine taxonomic and phylogeographic patterns in Dactylorhiza maculata s.l. from Scandinavia. A total of 238 individuals from 27 populations from throughout all of Scandinavia, including the adjacent Kola Peninsula of Russia, were analyzed. Diploid D. maculata ssp. fuchsii and autotetraploid D. maculata ssp. maculata are morphologically differentiated. Fragment size variants from 10 plastid DNA loci (seven microsatellite loci and three loci with indel variation) were combined to give 43 haplotypes. Three major groups of haplotypes were found. Group I haplotypes were prevalent in the north and the northeast, whereas Group II haplotypes were prevalent in the south and the southwest. Group III was represented by only a single haplotype and appeared to be the result of introgression from D. incarnata s.l. Group I and Group II haplotypes did not correspond with cytologically and morphologically defined D. maculata ssp. fuchsii or D. maculata ssp. maculata. Past introgressive gene flow rather than recent hybridization is envisaged. Intermediate Group I haplotypes between Group II and the rest of Group I were detected in a zone of contact in central Sweden, which may suggest plastid DNA recombination. The six ITS alleles scored showed strong positive correlation with taxonomy. All data sets obtained for ssp. maculata were significantly correlated with geography. Three different autotetraploid lineages are hypothesized. One lineage may represent postglacial immigration from the south and the other two lineages may represent eastern immigration routes. Morphology and ITS data suggested that subarctic populations of ssp. maculata should be recognized as var. kolaënsis.     

High genetic differentiation among wild populations of alien Medicago sativa in Lithuania

Alfalfa (Medicago sativa; =M. sativa ssp. sativa) in Lithuania is sown as albuminous forage for cattle due to favourable climatic condition. Over many generations, alfalfa plants have escaped from cultivation fields into natural ecosystems and established wild populations. We collected and analyzed individuals from seventeen wild populations of M. sativa. Using random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) analyses, 117 RAPD and 64 ISSR reproducible and highly polymorphic (90.8% for RAPD and 86.3% for ISSR) loci were established. AMOVA showed a high genetic differentiation of M. sativa populations for both types of DNA markers utilized. According to RAPD markers, the genetic variability among populations was 63.1% and 57.0% when ISSR markers were used. Taken together, these results demonstrate that wild populations of M. sativa possess a high potential of genetic variability, that could potentially result in colonization of natural ecosystems. The UPGMA cluster analysis also showed that the DNA markers discovered in this study can distinguish between M. sativa and M. falcata (=M. sativa ssp. falcata) populations and therefore may be used to study the genetic impact of M. sativa on the native populations of M. falcata.     

Chemical characterisation of wild populations of Thymus from different climatic regions in southeast Spain

Thymus is taxonomically a very complex genus with a high frequency of hybridisation and introgression among sympatric species. The variation in accumulation of leaf-surface flavonoids was investigated in 71 wild populations of Thymus from different putative hybrid swarm areas in Andalucia, Spain. Twenty-two flavones, five flavanones, two dihydroflavonols, a flavonol and two unknowns were detected by HPLC–DAD combined with LC–APCI-MS analysis. The majority of compounds were flavones with a luteolin-type substitution of the B-ring, in contrast to previous reports on Macedonian taxa, which predominantly accumulate flavones with apigenin-type substitution of the B-ring. Anatomical and morphometric studies, supported by cluster analysis, identified pure Thymus hyemalis and Thymus baeticus populations, and a large number of putative hybrids. Flavonoid variation was closely related to morphological variation in all populations and is suspected to be a result of genetic polymorphism. Principal component analysis identified the presence of species-specific and geographically linked chemotypes and putative hybrids with mixed morphological and chemical characteristics. Qualitative and quantitative flavonoid accumulation appears to be genetically regulated, while external factors play a secondary role. Flavonoid profiles can thus provide diagnostic markers for the taxonomy of Thymus and are also useful in detecting hybridising taxa.     

Comparison of the extent of genetic variation of Vallisneria natans and its sympatric congener V. spinulosa in lakes of the middle-lower reaches of the Yangtze River

Vallisneria natans and Vallisneria spinulosa are two morphologically very similar and sympatrically dominant submerged macrophytes in lakes of the middle-lower reaches of the Yangtze River. Genetic variation was compared based on a total of 196 individuals from six V. natans populations and 201 individuals from seven V. spinulosa populations. Using eight ISSR primers, a total of 139 and 129 DNA fragments were generated with 121 being polymorphic in V. natans and 99 in V. spinulosa. The two species maintained higher genetic variation both at the species and population levels in comparison with other aquatic macrophytes. A higher level of genetic diversity among populations was found in V. natans than in V. spinulosa: the percentage of polymorphic loci (PPL) in V. natans was 52-62% vs. 38-47% in V. spinulosa; gene diversity (H) was 0.21 in V. natans vs. 0.17 in V. spinulosa.Both an analysis of molecular variance (AMOVA) and F-estimation (F_ST) showed that most of the total genetic variation resided within populations of both species (AMOVA: 85% and 80%; F_ST: 0.132 and 0.202), indicating low genetic differentiation between populations. Principal coordinates analysis (PCA) indicated evident gene flow between populations of both species. The outcrossing reproductive mode and pervasive gene flow might have played important roles in maintaining high genetic diversity and in shaping low population differentiation of the two Vallisneria species, while the extent of clonal growth might account for the different levels of population divergence between them.     

Genetic variability in Plantago species in relation to their ecology

Summary Twelve Plantago major plants, good representatives of their populations, appeared to be genetically different for several characters which are important for adaptation to the respective habitat conditions. These characters are: juvenile growth, leaf morphology, production of secondary rosettes, flowering time, seed production, seed size and adult leaf production. The adaptive value of some of these characters was investigated by transplantation experiments in the field and by intraspecific competition experiments. The roadside type of ssp. major was adapted to trampling by being erect and elastic. The lawn type of ssp. major was adapted to a short, frequently cut, vegetation by being prostrate and by producing leaves with short petioles throughout the growth season. In the natural situations in which ssp. pleiosperma occurs, growth rate and first-year seed production of this subspecies were considerably higher than that of ssp. major. In a number of experiments, F₁s and F₂s were included, derived from crosses between the original plants. The F₁s were generally rather well adapted to both parental habitats, whereas the F₂s appeared to be less fit. The various alternatives in spending resources relevant for fitness optimization in different habitats are discussed.Grassland Species Research Group Publication No. 93     

Pure species in a continuum of genetic and morphological variation: sympatric oaks at the edge of their range

Background and Aims Studies on oaks (Quercus spp.) have often been hampered by taxonomic confusion, a situation further compounded by the occurrence of extensive interspecific hybridization. In the present study, a combination of genetic and morphological analyses was used to examine sympatric populations of Q. petraea and Q. robur at the north-western edge of their ranges in Northern Ireland, since it had previously been suggested that hybridization could facilitate the apparent rapid, long-distance dispersal of oaks following the glaciations.Methods Samples were collected from 24 sites across Northern Ireland that had been previously designated as ancient or semi-natural woodland. Genotypes were obtained from a total of 950 trees using 12 nuclear microsatellite loci, and admixture coefficients were calculated based on a Bayesian clustering approach. Individuals were also classified as Q. petraea, Q. robur or hybrids based on two objective morphometric characters shown previously to delineate pure individuals effectively. Genetically ‘pure’ individuals of both species, as defined by the Bayesian clustering, were also genotyped for five chloroplast microsatellites.Key Results Genetic and morphological analyses both indicated the presence of pure individuals of both species, as well as a continuum of intermediates. There was a good agreement between the molecular and morphological classification, with a generally clear separation between pure individuals.Conclusions Despite millennia of hybridization and introgression, genetically and morphologically pure individuals of both Q. petraea and Q. robur can be found at the edge of their range, where both species occur sympatrically. The high proportion of individuals exhibiting introgression compared with previous studies may reflect the historical role of hybridization in facilitating dispersal following the glaciations. This is further supported by the significantly higher chloroplast diversity in Q. robur compared with Q. petraea.     

The genus Micatagla Argaman, 1994 in Egypt,with three new species and a new record (Hymenoptera,Bradynobaenidae, Apterogyninae)

The genus Micatagla Argaman (Bradynobaenidae: Apterogyninae) is reviewed from Egypt, based on specimens collected from Wadi Allaqi (Aswan, Southern Egypt) and Kom Osheim (Fayoum) and those deposited in Egyptian insect collections as well as recorded data from the literature. A single species, Micatagla klugi (André), was previously recorded from Egypt. Micatagla allaqiensis sp. n., Micatagla ezzati sp. n. and Micatagla pseudorainerii sp. n. are described here. Micatagla antropovi Pagliano is also newly recorded from the Egyptian fauna. An illustrated key and a faunistic list comprising all Micatagla species recorded from Egypt are given.     

Efflux of Potassium from Wheat Roots Induced by Changes in the Water Potential of the Root Medium

Growth of Plantago major L., ssp. major L. and ssp. pleiosperma Pilger and P. maritime L. was followed at two levels of mineral nutrition (low-salt and high-salt). In addition the response of transfer of plants from low-salt conditions to high-salt conditions and vice versa was studied. Growth of the studied Plantago species was strongly stimulated by high-salt conditions. The Ca²⁺- and Mg²⁺-stimulated ATPase activity of microsomal preparations of the roots was also studied. In P. major ssp. major and P. maritime a major pH optimum was observed at pH 6.5, and in addition a second pH optimum was found at pH 8.0. High-salt plants of these two species were characterized by biphasic stimulation curves for Ca²⁺ and Mg²⁺, whereas P. major ssp. pleiosperma showed a monophasic pattern. The ATPase activity per g dry weight of P. major and P. maritima was highest in high-salt plants. The species investigated here are adapted to relatively nutrient-rich conditions, and the properties of ATPases (K_m, K_max, protein content) and the growth responses are discussed in relation to this ecological property.     

Highly differentiated populations of the narrow endemic and endangered species Primula cicutariifolia in China,revealed by ISSR and SSR

The perennial herb Primula cicutariifolia Pax is an endangered and endemic species with narrow distribution in eastern Anhui and Zhejiang provinces of China. In this study, the levels of genetic variation and the pattern of genetic structure in five natural populations of P. cicutariifolia were assessed by inter-simple sequence repeats (ISSR) and simple sequence repeat (SSR) markers. Both markers revealed that there was remarkably low genetic variation within populations (e.g., H_e = 0.19 and 0.18, for ISSR and SSR respectively) and high differentiation among populations (G_ST = 0.714 and 0.611; Φ_ST = 0.698 and 0.599, for ISSR and SSR respectively). The level of population genetic diversity was correlated to population size only detected by ISSR markers. The genetic structure of P. cicutariifolia may be explained by limited gene flow that was caused by habitat fragmentation and limited seeds and pollen dispersal ability, self breeding system and biennial life form. To protect and avoid disappearance of P. cicutariifolia, much more attentions should be paid to protect all the remnant populations and their habitats, and three management units, i.e. Tianmushan, Damodao, and Panan units, were proposed.     

Inter-simple sequence repeat (ISSR) analysis of genetic variation of Chondrus crispus populations from North Atlantic

ISSR analysis was used to investigate genetic variations of 184 haploid and diploid samples from nine North Atlantic Chondrus crispus Stackhouse populations and one outgroup Yellow Sea Chondrus ocellatus Holmes population. Twenty-two of 50 primers were selected and 163 loci were scored for genetic diversity analysis. Genetic diversity varied among populations, percentage of polymorphic bands (PPB) ranged from 27.0 to 55.8%, H (Nei's genetic diversity) ranged from 0.11 to 0.20 and I (Shannon's information index) ranged from 0.16 to 0.30. Estimators PPB, H and I had similar values in intra-population genetic diversity, regardless of calculation methods. Analysis of molecular variance (AMOVA) apportioned inter-population and intra-population variations for C. crispus, showing more genetic variance (56.5%) occurred in intra-population, and 43.5% variation among nine populations. The Mantel test suggested that genetic differentiation between nine C. crispus populations was closely related with geographic distances (R = 0.78, P = 0.002). Results suggest that, on larger distance scale (ca. >1000 km), ISSR analysis is useful for determining genetic differentiations of C. crispus populations including morphologically inseparable haploid and diploid individuals.