Martin J. Ingrouille

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.

Leaf surface flavonoids in iranian species of Nepeta (Lamiaceae) and some related genera

Abstract A HPLC survey of the leaf surface flavonoids of 38 species of Nepeta (Lamiaceae) and four species of the related genera Agastache, Dracocephalum and Lallemantia revealed 14 different flavones, one of which is new (8-hydroxycirsiliol or 5,8,3′,4′-tetrahydroxy-6,7-dimethoxyflavone). In addition, two flavonols (methyl ethers of kaempferol) were found in Dracocephalum kotschyii. The most frequently encountered flavones in Nepeta were cirsimaritin (5,4′-dihydroxy-6,7-dimethoxyflavone); 8-hydroxycirsimaritin (5,8,4′-trihydroxy-6,7-dimethoxyflavone, also called isothymusin) and genkwanin (5,4′-dihydroxy-7-methoxyflavone). Apigenin and the 4′-methyl ethers of cirsimaritin and 8-hydroxycirsimaritin (salvigenin and 8-hydroxysalvigenin, respectively) were also relatively common. The distribution of surface flavones in the four genera provided some valuable data for the phylogenetic relationships at generic level. The presence of surface flavones with a 5-hydroxy-6,7-dimethoxy A-ring (as found in cirsimaritin and salvigenin) and the unusual 5,8-dihydroxy-6,7-dimethoxy A-ring substitution pattern (as found in 8-hydroxycirsimaritin, 8-hydroxysalvigenin and 8-hydroxycirsiliol), can be considered as a characteristic chemotaxonomic feature typical of the genus Nepeta. Cirsimaritin and 8-hydroxycirsimaritin were also detected in the one species examined for Agastache, but the absence of genkwanin and the presence of acacetin in A. barberi and in ten Agastache species studied previously, distinguished Agastache from Nepeta. The presence of methoxylated flavonols and absence of 8-hydroxycirsimaritin and related compounds distinguished species of Dracocephalum and Nepeta, whereas lack of flavonoids with 6- and 8-oxygenation of the A-ring characterised species of the genus Lallemantia.

Genetic diversity in Cypripedium calceolus (Orchidaceae) with a focus on north-western Europe, as revealed by plastid DNA length polymorphisms

BACKGROUND AND AIMS Cypripedium calceolus, although widespread in Eurasia, is rare in many countries in which it occurs. Population genetics studies with nuclear DNA markers on this species have been hampered by its large nuclear genome size. Plastid DNA markers are used here to gain an understanding of variation within and between populations and of biogeographical patterns. METHODS Thirteen length-variable regions (microsatellites and insertions/deletions) were identified in non-coding plastid DNA. These and a previously identified complex microsatellite in the trnL-trnF intergenic spacer were used to identify plastid DNA haplotypes for European samples, with sampling focused on England, Denmark and Sweden. KEY RESULTS The 13 additional length-variable regions identified were two homopolymer (polyA) repeats in the rps16 intron and a homopolymer (polyA) repeat and ten indels in the accD-psa1 intergenic spacer. In accD-psa1, most of these were in an extremely AT-rich region, and it was not possible to design primers in the flanking regions; therefore, the whole intergenic spacer was sequenced. Together, these new regions and the trnL-trnF complex microsatellite allowed 23 haplotypes to be characterized. Many were found in only one or a few samples (probably due to low sampling density), but some commoner haplotypes were widespread. Most of the genetic variation was found within rather than between populations (83 vs. 18%, respectively). Two haplotypes occurred from the Spanish Pyrenees to Sweden. CONCLUSIONS Plastid DNA data can be used to gain an understanding of patterns of genetic variation and seed-mediated gene flow in orchids. Although these data are less information-rich than those for nuclear DNA, they present a useful option for studying species with large genomes. Here they support the hypothesis of long-distance seed dispersal often proposed for orchids.

Phylogenetic relationships in Nepeta L. (Lamiaceae) and related genera based on ITS sequence data

The internal transcribed spacers of nuclear ribosomal DNA (nrlTS) from 43 species of Nepeta and representatives of closely related genera (Lallemantia, Dracocephalum and Agastache) and outgroups (Lavandula, Stachys, Newcustelia and Prostanthera) were sequenced. Parsimony analysis indicated that Nepeta is monophyletic and composed of five major monophyletic groups, most of which comprise species belonging to more than one section in previous classifications. These five Glades are composed of: (1) sect. Spartonepeta; (2) sects. Macronepeta + Spicatae; (3) sects. Nepeta + Micranthae p-p. + Oxynepeta + Schizocalyx + Macrostegiae; (4) sects. Capituliferae + Denudatae + Micranthae p.p. + Micronepeta p.p.; and (5) sect. Psilonepeta. The phylogenetic relationships among species of these groups are congruent with the distribution of some floral characters, including corolla shape, bract texture, color and pollen exine ornamentation.

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.

Is aristolochic acid nephropathy a widespread problem in developing countries? A case study of Aristolochia indica L. in Bangladesh using an ethnobotanical-phytochemical approach.

ETHNOPHARMACOLOGICAL RELEVANCE Species of Aristolochia are associated with aristolochic acid nephropathy (AAN), a renal interstitial fibrosis and upper urinary tract cancer (UUC). Aristolochic acid nephropathy has been reported in ten countries but its true incidence is unknown and most likely underestimated. By combining an ethnobotanical and phytochemical approach we provide evidence for the risk of AAN occurring in Bangladesh. More specifically, we assess the intra-specific variation of aristolochic acid analogues in medicinally used Aristolochia indica samples from Bangladesh. MATERIALS AND METHODS Ethnobotanical information was collected from 16 kavirajes (traditional healers) in different study locations in Bangladesh. Plant samples were obtained from native habitats, botanical gardens, herbal markets and pharmaceutical companies. The samples were extracted using 70% methanol and were analysed using LC-DAD-MS and (1)H-NMR. RESULTS Roots as well as leaves are commonly used for symptoms such as snake bites and sexual problems. Among the informants knowledge about toxicity or side effects is very limited and Aristolochia indica is often administered in very high doses. Replacement of Aristolochia indica with other medicinal plants such as Rauvolfia serpentina (L.) Benth. ex Kurz was common. Aristolochia indica samples contained a variety of aristolochic acid analogues such as aristolochic acid I, aristolochic acid II, cepharadione A and related compounds. CONCLUSIONS AAN cases are likely to occur in Bangladesh and more awareness needs to be raised about the health risks associated with the use of Aristolochia indica and other species of Aristolochia as herbal medicines.

The taxonomic treatment of agamosperms in the genusLimonium mill. (Plumbaginaceae)

Agamospermous species account for a large proportion of the species ofLimonium. Agamospermy is indicated by uneven polyploidy or aneuploidy, low pollen stainability and by the presence of monomorphic self-incompatible populations. The taxonomic treatment of agamospermous taxa varies from recognition of all of them at the same specific rank or by utilising a range of ranks in the taxonomic hierarchy. The influence of evolutionary hypotheses on taxonomic systems is considered. Molecular data provide a means of measuring the genetical relationships of taxa and establishing groups in a taxonomic hierarchy.

Gagea calcicola (Liliaceae), a new species from southwestern Iran

SummaryA new species, Gagea calcicola (Liliaceae) is described and illustrated. It differs from G. reticulata (Pall.) Schult. & Schult. f. through having a compound umbelliform inflorescence and bulbils in the axil of the lower cauline leaves. The new species is endemic to southwestern Iran. Its ecology, distribution and conservation status are discussed.

Geographical variation in the pollen of Acacia (Mimosaceae) in Sudan

Abstract The pollen grains of 14 Acacia species from different parts of Sudan are described. The number of grains in each polyad is 16 except in A. albida which has 32 grains in each polyad. The pollen diameter, and pore number and diameter, follow a geographical pattern of variation both between species and topoclines within species. Pollen grains are smaller with fewer and smaller pores in arid northern Sudan than humid southern Sudan. Patterns of morphological variation in sculpturing of the exine, which are constant for species, are reported.

Becoming fruitful and diversifying: DNA sequence phylogenetics and reproductive physiology of land plants

Publisher Summary This chapter focuses on DNA sequence phylogenetics and reproductive physiology of land plants. In the light of the new DNA sequence derived phylogenetic trees, physiological traits have been shown to have patterns of distribution substantially the same as other types of characters. They potentially have as much value in classification as other characters. Among plants, self-incompatibility (SI) is almost uniquely an angiosperm phenomenon. The amazing physiological diversity of self-incompatibility between and within groups emphasizes the importance of self-incompatibility (SI) in the diversification of the angiosperms. It also reflects on the fundamental importance of the closure of the carpel and evolution of the style and stigma within angiosperms because it was these events that provided the locus from which many kinds of self-incompatibility could arise. The distribution of different kinds of SI across groups at different ranks in the taxonomic hierarchy appears complicated because of a hitherto unsuspected flexibility in SI response. The assessment of homology is complicated because a shared common origin of an SI mechanism (true homology) may be obscured by multiple loss of SI and the presence of other kinds of SI in the families. Gene duplication and transpacific evolution, so that lineages of different SI genes in an individual taxon pre-date the divergence of the taxon itself, further complicate evolutionary patterns.