Persistence of DNA adducts, hypermutation and acquisition of cellular resistance to alkylating agents in glioblastoma.

Glioblastoma is a lethal form of brain tumour usually treated by surgical resection followed by radiotherapy and an alkylating chemotherapeutic agent. Key to the success of this multimodal approach is maintaining apoptotic sensitivity of tumour cells to the alkylating agent. This initial treatment likely establishes conditions contributing to development of drug resistance as alkylating agents form the O6-methylguanine adduct. This activates the mismatch repair (MMR) process inducing apoptosis and mutagenesis. This review describes key juxtaposed drivers in the balance between alkylation induced mutagenesis and apoptosis. Mutations in MMR genes are the probable drivers for alkylation based drug resistance. Critical to this interaction are the dose-response and temporal interactions between adduct formation and MMR mutations. The precision in dose interval, dose-responses and temporal relationships dictate a role for alkylating agents in either promoting experimental tumour formation or inducing tumour cell death with chemotherapy. Importantly, this resultant loss of chemotherapeutic selective pressure provides opportunity to explore novel therapeutics and appropriate combinations to minimise alkylation based drug resistance and tumour relapse.

Pitfalls of using estimations of glomerular filtration rate in an intensive care population.

BACKGROUND: Accurate knowledge of the glomerular filtration rate (GFR) is imperative in the intensive care unit (ICU) as renal status is important for medical decisions, including drug dosing. AIMS: Recently, an estimation of GFR (eGFR) was suggested as a method of estimating GFR. How well this formula predicts GFR in unwell patients with normal initial serum creatinine concentrations has not been examined. METHODS: The accuracy of the eGFR (before and after adjustment for actual body surface area (BSA)) was compared with measured and with estimated creatinine clearance using the Cockcroft Gault (CG) formula adjusted for total and lean body weight. RESULTS: A total of 237 observations was recorded in 47 subjects. These were initially analysed independently, and then using the first observation only. Overall the mean difference between measured creatinine clearance and eGFR was -12 mL/min (95% confidence interval (CI) -20 to -3), between measured creatinine clearance and CG +17 mL/min (95% CI 9-24), between measured creatinine clearance and CG adjusted for ideal body weight +12 mL/min (95% CI 4-21) and between measured creatinine clearance and eGFR 'unadjusted' for BSA 5 mL/min (95% CI -2-13). CONCLUSIONS: Using either eGFR or CG formulae to estimate renal function in ICU subjects with normal serum creatinine concentrations is inaccurate. Although correcting for BSA improves the eGFR, this requirement to measure height and weight removes a major attraction for its use. We suggest that eGFR should not be automatically calculated in the ICU setting.

Molecular systematics of Gagea and Lloydia (Liliaceae; Liliales): implications of analyses of nuclear ribosomal and plastid DNA sequences for infrageneric classification.

BACKGROUND AND AIMS: Gagea is a Eurasian genus of petaloid monocots, with a few species in North Africa, comprising between 70 and approximately 275 species depending on the author. Lloydia (thought to be the closest relative of Gagea) consists of 12-20 species that have a mostly eastern Asian distribution. Delimitation of these genera and their subdivisions are unresolved questions in Liliaceae taxonomy. The objective of this study is to evaluate generic and infrageneric circumscription of Gagea and Lloydia using DNA sequence data. METHODS: A phylogenetic study of Gagea and Lloydia (Liliaceae) was conducted using sequences of nuclear ribosomal internal transcribed spacer (ITS) and plastid (rpl16 intron, trnL intron, trnL-F spacer, matK and the psbA-trnH spacer) DNA regions. This included 149 accessions (seven as outgroups), with multiple accessions of some taxa; 552 sequences were included, of which 393 were generated as part of this research. KEY RESULTS: A close relationship of Gagea and Lloydia was confirmed in analyses using different datasets, but neither Gagea nor Lloydia forms a monophyletic group as currently circumscribed; however, the ITS and plastid analyses did not produce congruent results for the placement of Lloydia relative to the major groups within Gagea. Gagea accessions formed five moderately to strongly supported clades in all trees, with most Lloydia taxa positioned at the basal nodes; in the strict consensus trees from the combined data a basal polytomy occurs. There is limited congruence between the classical, morphology-derived infrageneric taxonomy in Gagea (including Lloydia) and clades in the present phylogenetic analyses. CONCLUSIONS: The analyses support monophyly of Gagea/Lloydia collectively, and they clearly comprise a single lineage, as some previous authors have hypothesized. The results provide the basis for a new classification of Gagea that has support from some morphological features. Incongruence between plastid and nuclear ITS results is interpreted as potentially due to ancient hybridization and/or paralogy of ITS rDNA.

Genome size diversity in orchids: consequences and evolution.

BACKGROUND: The amount of DNA comprising the genome of an organism (its genome size) varies a remarkable 40 000-fold across eukaryotes, yet most groups are characterized by much narrower ranges (e.g. 14-fold in gymnosperms, 3- to 4-fold in mammals). Angiosperms stand out as one of the most variable groups with genome sizes varying nearly 2000-fold. Nevertheless within angiosperms the majority of families are characterized by genomes which are small and vary little. Species with large genomes are mostly restricted to a few monocots families including Orchidaceae. SCOPE: A survey of the literature revealed that genome size data for Orchidaceae are comparatively rare representing just 327 species. Nevertheless they reveal that Orchidaceae are currently the most variable angiosperm family with genome sizes ranging 168-fold (1C = 0.33-55.4 pg). Analysing the data provided insights into the distribution, evolution and possible consequences to the plant of this genome size diversity. CONCLUSIONS: Superimposing the data onto the increasingly robust phylogenetic tree of Orchidaceae revealed how different subfamilies were characterized by distinct genome size profiles. Epidendroideae possessed the greatest range of genome sizes, although the majority of species had small genomes. In contrast, the largest genomes were found in subfamilies Cypripedioideae and Vanilloideae. Genome size evolution within this subfamily was analysed as this is the only one with reasonable representation of data. This approach highlighted striking differences in genome size and karyotype evolution between the closely related Cypripedium, Paphiopedilum and Phragmipedium. As to the consequences of genome size diversity, various studies revealed that this has both practical (e.g. application of genetic fingerprinting techniques) and biological consequences (e.g. affecting where and when an orchid may grow) and emphasizes the importance of obtaining further genome size data given the considerable phylogenetic gaps which have been highlighted by the current study.

Genetic diversity and differentiation processes in the ploidy series of Olea europaea L.: a multiscale approach from subspecies to insular populations.

Geographical isolation and polyploidization are central concepts in plant evolution. The hierarchical organization of archipelagos in this study provides a framework for testing the evolutionary consequences for polyploid taxa and populations occurring in isolation. Using amplified fragment length polymorphism and simple sequence repeat markers, we determined the genetic diversity and differentiation patterns at three levels of geographical isolation in Olea europaea: mainland-archipelagos, islands within an archipelago, and populations within an island. At the subspecies scale, the hexaploid ssp. maroccana (southwest Morocco) exhibited higher genetic diversity than the insular counterparts. In contrast, the tetraploid ssp. cerasiformis (Madeira) displayed values similar to those obtained for the diploid ssp. guanchica (Canary Islands). Geographical isolation was associated with a high genetic differentiation at this scale. In the Canarian archipelago, the stepping-stone model of differentiation suggested in a previous study was partially supported. Within the western lineage, an east-to-west differentiation pattern was confirmed. Conversely, the easternmost populations were more related to the mainland ssp. europaea than to the western guanchica lineage. Genetic diversity across the Canarian archipelago was significantly correlated with the date of the last volcanic activity in the area/island where each population occurs. At the island scale, this pattern was not confirmed in older islands (Tenerife and Madeira), where populations were genetically homogeneous. In contrast, founder effects resulted in low genetic diversity and marked genetic differentiation among populations of the youngest island, La Palma.

Within-population spatial genetic structure in four naturally fragmented species of a neotropical inselberg radiation, Alcantarea imperialis, A. geniculata, A. glaziouana and A. regina (Bromeliaceae).

Studies of organisms on 'terrestrial islands' can improve our understanding of two unresolved issues in evolutionary genetics: the likely long-term effects of habitat fragmentation and the genetic underpinnings of continental species radiations in island-like terrestrial habitats. We have addressed both issues for four closely related plant species of the adaptive radiation Bromeliaceae, Alcantarea imperialis, A. geniculata, A. regina and A. glaziouana. All four are adapted to ancient, isolated inselberg rock outcrops in the Brazilian Atlantic rainforest and are thus long-term fragmented by nature. We used eight nuclear microsatellites to study within-population spatial genetic structure (SGS) and historical gene dispersal in nine populations of these species. Within-population SGS reflected known between-species differences in mating systems. The strongest SGS observed in A. glaziouana (Sp=0.947) was stronger than literature estimates available for plants. Analysis of short- and long-distance components of SGS identified biparental inbreeding, selfing and restricted seed dispersal as main determinants of SGS, with restricted pollen dispersal by bats contributing in some localities. The ability of Alcantarea spp. to colonize isolated inselbergs probably stems from their flexible mating systems and an ability to tolerate inbreeding. Short-ranging gene dispersal (average sigma=7-27 m) is consistent with a loss of dispersal power in terrestrial island habitats. Population subdivision associated with sympatric colour morphs in A. imperialis is accompanied by between-morph differences in pollen and seed dispersal. Our results indicate a high potential for divergence with gene flow in inselberg bromeliads and they provide base-line data about the long-term effects of fragmentation in plants.

Punctuated genome size evolution in Liliaceae.

Most angiosperms possess small genomes (mode 1C = 0.6 pg, median 1C = 2.9 pg). Those with truly enormous genomes (i.e. > or = 35 pg) are phylogenetically restricted to a few families and include Liliaceae - with species possessing some of the largest genomes so far reported for any plant as well as including species with much smaller genomes. To gain insights into when and where genome size expansion took place during the evolution of Liliaceae and the mode and tempo of this change, data for 78 species were superimposed onto a phylogenetic tree and analysed. Results suggest that genome size in Liliaceae followed a punctuated rather than gradual mode of evolution and that most of the diversification evolved recently rather than early in the evolution of the family. We consider that the large genome sizes of Liliaceae may have emerged passively rather than being driven primarily by selection.

A preliminary study of genetic variation in populations of Monstera adansonii var. klotzschiana (Araceae) from North-East Brazil, estimated with AFLP molecular markers.

BACKGROUND AND AIMS: This study sought genetic evidence of long-term isolation in populations of Monstera adansonii var. klotzschiana (Araceae), a herbaceous, probably outbreeding, humid forest hemi-epiphyte, in the brejo forests of Ceará (north-east Brazil), and clarification of their relationships with populations in Amazonia and the Atlantic forest of Brazil. METHODS: Within-population genetic diversity and between-population dissimilarity were estimated using AFLP molecular markers in 75 individuals from eight populations located in Ceará, the Brazilian Atlantic Forest and Amazonia. KEY RESULTS: The populations showed a clinal pattern of weak genetic differentiation over a large geographical region (F(ST) = 0.1896). A strong correlation between genetic and geographical distance (Mantel test: r = 0.6903, P = 0.002) suggests a historical pattern of isolation by distance. Genetic structure analysis revealed at least two distinct gene pools in the data. The two isolated Ceará populations are significantly different from each other (pairwise Phi(PT) = 0.137, P = 0.003) and as diverse (Nei's gene diversity, average H(e) = 0.1832, 0.1706) as those in the Atlantic and Amazon forest regions. The population in southern Brazil is less diverse (Nei's gene diversity, average H(e) = 0.127) than the rest. The Ceará populations are related to those of the Atlantic forest rather than those from Amazonia (AMOVA, among-groups variation = 11.95 %, P = 0.037). CONCLUSIONS: The gene pools detected within an overall pattern of clinal variation suggest distinct episodes of gene flow, possibly correlated with past humid forest expansions. The Ceará populations show no evidence of erosion of genetic diversity, although this was expected because of their isolation. Their genetic differentiation and relatively high diversity reinforce the importance of conserving the endangered brejo forests.

Population differentiation and species cohesion in two closely related plants adapted to neotropical high-altitude 'inselbergs', Alcantarea imperialis and Alcantarea geniculata (Bromeliaceae).

Isolated granitic rock outcrops or 'inselbergs' may provide a window into the molecular ecology and genetics of continental radiations under simplified conditions, in analogy to the use of oceanic islands in studies of species radiations. Patterns of variability and gene flow in inselberg species have never been thoroughly evaluated in comparison to related taxa with more continuous distribution ranges, or to other species in the same kingdom in general. We use nuclear microsatellites to study population differentiation and gene flow in two diploid, perennial plants adapted to high-altitude neotropical inselbergs, Alcantarea imperialis and Alcantarea geniculata (Bromeliaceae). Population differentiation is pronounced in both taxa, especially in A. imperialis. Gene flow in this species is considerably lower than expected from the literature on plants in general and Bromeliaceae in particular, and too low to prevent differentiation due to drift (N(e)m < 1), unless selection coefficients/effect sizes of favourable alleles are great enough to maintain species cohesion. Low gene flow in A. imperialis indicates that the ability of pollinating bats to promote gene exchange between inselbergs is smaller than previously assumed. Population subdivision in one inselberg population of A. imperialis appears to be associated with the presence of two colour morphs that differ in the coloration of rosettes and bracts. Our results indicate a high potential for inselbergs as venues for studies of the molecular ecology and genetics of continental radiations, such as the one that gave rise to the extraordinary diversity of adaptive strategies and phenotypes seen in Bromeliaceae.

Admixture in European Populus hybrid zones makes feasible the mapping of loci that contribute to reproductive isolation and trait differences.

The use of admixed human populations to scan the genome for chromosomal segments affecting complex phenotypic traits has proved a powerful analytical tool. However, its potential in other organisms has not yet been evaluated. Here, we use DNA microsatellites to assess the feasibility of this approach in hybrid zones between two members of the 'model tree' genus Populus: Populus alba (white poplar) and Populus tremula (European aspen). We analyzed samples of both species and a Central European hybrid zone (N=544 chromosomes) for a genome-wide set of 19 polymorphic DNA microsatellites. Our results indicate that allele frequency differentials between the two species are substantial (mean delta=0.619+/-0.067). Background linkage disequilibrium (LD) in samples of the parental gene pools is moderate and should respond to sampling schemes that minimize drift and account for rare alleles. LD in hybrids decays with increasing number of backcross generations as expected from theory and approaches background levels of the parental gene pools in advanced generation backcrosses. Introgression from P. tremula into P. alba varies strongly across marker loci. For several markers, alleles from P. tremula are slightly over-represented relative to neutral expectations, whereas a single locus exhibits evidence of selection against P. tremula genotypes. We interpret our results in terms of the potential for admixture mapping in these two ecologically divergent Populus species, and we validate a modified approach of studying genotypic clines in 'mosaic' hybrid zones.

Adaptation to environmental stress: a rare or frequent driver of speciation?

Recent results of evolutionary genomics and other research programmes indicate an important role for environment-dependent selection in speciation, but the conceptual frameworks of speciation genetics and environmental stress physiology have not been fully integrated. Only a small number of model systems have been established for cross-disciplinary studies of this type in animals and plants. In these taxa (e.g. Drosophila and Arabidopsis/Arabis), studies of the mechanistic basis of various stress responses are increasingly combined with attempts to understand their evolutionary consequences. Our understanding of the role of environmental stress in speciation would benefit from studies of a larger variety of taxa. We pinpoint areas for future study and predict that in many taxa 'broad' hybrid zones maintained by ecological selection will be valuable venues for addressing the link between environmental stress, adaptation, and speciation.

Barrier to gene flow between two ecologically divergent Populus species, P. alba (white poplar) and P. tremula (European aspen): the role of ecology and life history in gene introgression.

The renewed interest in the use of hybrid zones for studying speciation calls for the identification and study of hybrid zones across a wide range of organisms, especially in long-lived taxa for which it is often difficult to generate interpopulation variation through controlled crosses. Here, we report on the extent and direction of introgression between two members of the "model tree" genus Populus: Populus alba (white poplar) and Populus tremula (European aspen), across a large zone of sympatry located in the Danube valley. We genotyped 93 hybrid morphotypes and samples from four parental reference populations from within and outside the zone of sympatry for a genome-wide set of 20 nuclear microsatellites and eight plastid DNA restriction site polymorphisms. Our results indicate that introgression occurs preferentially from P. tremula to P. alba via P. tremula pollen. This unidirectional pattern is facilitated by high levels of pollen vs. seed dispersal in P. tremula (pollen/seed flow = 23.9) and by great ecological opportunity in the lowland floodplain forest in proximity to P. alba seed parents, which maintains gene flow in the direction of P. alba despite smaller effective population sizes (N(e)) in this species (P. alba N(e)c. 500-550; P. tremula N(e)c. 550-700). Our results indicate that hybrid zones will be valuable tools for studying the genetic architecture of the barrier to gene flow between these two ecologically divergent Populus species.

Patterns of variability and gene flow in Medicago citrina, an endangered endemic of islands in the western Mediterranean, as revealed by amplified fragment length polymorphism (AFLP).

Medicago citrina is an endangered western Mediterranean endemic that grows only on small islets of the Balearic archipelago and off the eastern Spanish coast. Only 10 isolated subpopulations are currently known (four from Ibiza, three from Cabrera, two from Columbretes and one from an offshore islet in northern Alicante province), constituting a severely fragmented genetic system. Data were analysed with the unweighted pair-group method using arithmetic averages (UPGMA) and principle coordinates analysis (PCOA), revealing several distinct groups. Genetic diversity indices indicated that Ibizan subpopulations had the highest genetic variability (Nei's index: 0.1463; Shannon's index: 0.228), whereas the lowest variability was found in Alicante (Nei's index: 0.035; Shannon's index: 0.050) and Cabrera (Nei's index: 0.068; Shannon's index: 0.104). These latter populations show the highest FST values (FST = 0.548) revealing high differentiation between them. Columbretes subpopulations formed a defined single group, although it also included some Ibizan samples. The smallest FST values, obtained between Ibiza and Columbretes (FST = 0.185), are not correlated with geographical proximity, but appear to be related to the geologically recent volcanic origin of the Columbretes islands (300,000 years ago). According to the distribution of the Ibizan samples in the dendrogram and the FST values, the best hypothesis is to regard the Ibizan subpopulations as the centre of genetic diversity of the currently known subpopulations. Our results suggest migratory scenarios from Ibiza to Columbretes based mainly on zoochory probably by seabirds. Finally, recommendations are provided for management strategies to facilitate the conservation of this endangered species.

Genetics of colonization in Hypochaeris tenuifolia (Asteraceae, Lactuceae) on Volcán Lonquimay, Chile.

Understanding the genetics of colonizing populations has been, and continues to remain, an important focus in evolutionary biology. Different theoretical models predict varying levels of genetic variation in colonizing populations depending upon strength of founder effect, gene flow and rate of population growth and immigration following colonization. We analyse overall genetic variation using amplified fragment length polymorphism markers in colonizing populations of Hypochaeris tenuifolia (Asteraceae) in the southern Andes. Volcán Lonquimay newly erupted on 25 December 1988, producing a side cone, La Navidad, and sent lava and ash into surrounding areas. Many domesticated animals (estimated at 10 000) and many natural plant populations were destroyed. Into this new open habitat have come immigrant populations of several angiosperm species, most conspicuously H. tenuifolia that forms leaf rosettes with flowering scapes to 15 cm and orange-yellow heads 1-2 cm in diameter. Genetic diversity in five founder populations in the eruption zone is compared with that from five nearby survivor populations, as well as with eight isolated northern and four southern populations from throughout the entire range of the species in Chile. Results from 477 individuals representing 447 different multilocus phenotypes, yielded 170 DNA fragments of which 144 (85%) were polymorphic. Genetic diversity within founder populations is neither lower than in survivor populations nor in isolated populations throughout the range of the species, but it is lower among founder populations than among other populations immediately and distantly outside the zone of disturbance. Closest genetic similarity occurs between founders and nearby survivor populations as well as those in adjacent southern regions.

Rapid and recent origin of species richness in the Cape flora of South Africa.

The Cape flora of South Africa grows in a continental area with many diverse and endemic species. We need to understand the evolutionary origins and ages of such 'hotspots' to conserve them effectively. In volcanic islands the timing of diversification can be precisely measured with potassium-argon dating. In contrast, the history of these continental species is based upon an incomplete fossil record and relatively imprecise isotopic palaeotemperature signatures. Here we use molecular phylogenetics and precise dating of two island species within the same clade as the continental taxa to show recent speciation in a species-rich genus characteristic of the Cape flora. The results indicate that diversification began approximately 7-8 Myr ago, coincident with extensive aridification caused by changes in ocean currents. The recent origin of endemic species diversity in the Cape flora shows that large continental bursts of speciation can occur rapidly over timescales comparable to those previously associated with oceanic island radiations.

Ancient flowering plants: DNA sequences and angiosperm classification.

Phylogenetic analyses of gene sequences provide a clear pattern of which extant flowering plant genera diversified earliest. Combined with complete genomic sequences, these data will vastly improve understanding of the genetic basis of plant diversity.

Amplified fragment length polymorphisms (AFLP) reveal details of polyploid evolution in Dactylorhiza (Orchidaceae).

The utility of the PCR-based AFLP technique (polymerase chain reaction; amplified fragment length polymorphisms) was explored in elucidating details of polyploid evolution in the Eurasian orchid genus Dactylorhiza. We emphasized Swedish taxa but also included some material from the British Isles and elsewhere in Europe. Three different sets of primers, amplifying different subsets of restriction fragments, independently revealed similar patterns for relationships among the Dactylorhiza samples investigated. The AFLP data support the general picture of polyploid evolution in Dactylorhiza, i.e., that allotetraploid derivatives have arisen repeatedly as a result of hybridization beween the two parental groups D. incarnata s.l. (sensu lato; diploid marsh orchids) and the D. maculata group (spotted orchids). Within the incarnata s.l. group, morphologically defined varieties were interdigitated. The D. maculata group consisted of two distinct subgroups, one containing autotetraploid D. maculata subsp. maculata and the other containing diploid D. maculata subsp. fuchsii. Allotetraploids showed a high degree of additivity for the putative parental genomes, and relationships among them were partly correlated to morphologically based entities, but also to geographic distribution. Thus, allotetraploid taxa from the British Isles clustered together, rather than with morphologically similar plants from other areas.