Temporal, but not spatial, changes in expression patterns of petal identity genes are associated with loss of papillate conical cells and the shift to bird pollination in Macaronesian Lotus (Leguminosae).

In the generally bee-pollinated genus Lotus a group of four species have evolved bird-pollinated flowers. The floral changes in these species include altered petal orientation, shape and texture. In Lotus these characters are associated with dorsiventral petal identity, suggesting that shifts in the expression of dorsal identity genes may be involved in the evolution of bird pollination. Of particular interest is Lotus japonicus CYCLOIDEA 2 (LjCYC2), known to determine the presence of papillate conical cells on the dorsal petal in L. japonicus. Bird-pollinated species are unusual in not having papillate conical cells on the dorsal petal. Using RT-PCR at various stages of flower development, we determined the timing of expression in all petal types for the three putative petal identity genes (CYC-like genes) in different species with contrasting floral morphology and pollination syndromes. In bird-pollinated species the dorsal identity gene, LjCYC2, is not expressed at the floral stage when papillate conical cells are normally differentiating in bee-pollinated species. In contrast, in bee-pollinated species, LjCYC2 is expressed during conical cell development. Changes in the timing of expression of the above two genes are associated with modifications in petal growth and lateralisation of the dorsal and ventral petals in the bird-pollinated species. This study indicates that changes in the timing, rather than spatial distribution, of expression likely contribute to the modifications of petal micromorphology and petal size during the transition from bee to bird pollination in Macaronesian Lotus species.

Recent Y chromosome divergence despite ancient origin of dioecy in poplars (Populus).

All species of the genus Populus (poplar, aspen) are dioecious, suggesting an ancient origin of this trait. Despite some empirical counter examples, theory suggests that nonrecombining sex-linked regions should quickly spread, eventually becoming heteromorphic chromosomes. In contrast, we show using whole-genome scans that the sex-associated region in Populus trichocarpa is small and much younger than the age of the genus. This indicates that sex determination is highly labile in poplar, consistent with recent evidence of 'turnover' of sex-determination regions in animals. We performed whole-genome resequencing of 52 P. trichocarpa (black cottonwood) and 34 Populus balsamifera (balsam poplar) individuals of known sex. Genomewide association studies in these unstructured populations identified 650 SNPs significantly associated with sex. We estimate the size of the sex-linked region to be ~100 kbp. All SNPs significantly associated with sex were in strong linkage disequilibrium despite the fact that they were mapped to six different chromosomes (plus 3 unmapped scaffolds) in version 2.2 of the reference genome. We show that this is likely due to genome misassembly. The segregation pattern of sex-associated SNPs revealed this to be an XY sex-determining system. Estimated divergence times of X and Y haplotype sequences (6-7 Ma) are much more recent than the divergence of P. trichocarpa (poplar) and Populus tremuloides (aspen). Consistent with this, in P. tremuloides, we found no XY haplotype divergence within the P. trichocarpa sex-determining region. These two species therefore have a different genomic architecture of sex, suggestive of at least one turnover event in the recent past.

A 34K SNP genotyping array for Populus trichocarpa: design, application to the study of natural populations and transferability to other Populus species.

Genetic mapping of quantitative traits requires genotypic data for large numbers of markers in many individuals. For such studies, the use of large single nucleotide polymorphism (SNP) genotyping arrays still offers the most cost-effective solution. Herein we report on the design and performance of a SNP genotyping array for Populus trichocarpa (black cottonwood). This genotyping array was designed with SNPs pre-ascertained in 34 wild accessions covering most of the species latitudinal range. We adopted a candidate gene approach to the array design that resulted in the selection of 34 131 SNPs, the majority of which are located in, or within 2 kb of, 3543 candidate genes. A subset of the SNPs on the array (539) was selected based on patterns of variation among the SNP discovery accessions. We show that more than 95% of the loci produce high quality genotypes and that the genotyping error rate for these is likely below 2%. We demonstrate that even among small numbers of samples (n = 10) from local populations over 84% of loci are polymorphic. We also tested the applicability of the array to other species in the genus and found that the number of polymorphic loci decreases rapidly with genetic distance, with the largest numbers detected in other species in section Tacamahaca. Finally, we provide evidence for the utility of the array to address evolutionary questions such as intraspecific studies of genetic differentiation, species assignment and the detection of natural hybrids.

Chloroplast microsatellite primers for cacao (Theobroma cacao) and other Malvaceae.

PREMISE OF THE STUDY: Chloroplast microsatellites were developed in Theobroma cacao to examine the genetic diversity of cacao cultivars in Trinidad and Tobago. METHODS AND RESULTS: Nine polymorphic microsatellites were designed from the chloroplast genomes of two T. cacao accessions. These microsatellites were tested in 95 hybrid accessions from Trinidad and Tobago. An average of 2.9 alleles per locus was found. CONCLUSIONS: These chloroplast microsatellites, particularly the highly polymorphic pentameric repeat, were useful in assessing genetic variation in T. cacao. In addition, these markers should also prove to be useful for population genetic studies in other species of Malvaceae.

Evolution in reverse gear: the molecular basis of loss and reversal.

Three types of regressive evolution are reviewed: loss, reversal, and regain after loss. Loss refers to the loss of a physical entity, either a structure or an organ, whereas reversals apply to character states returning to plesiomorphic from apomorphic conditions. The regain of characters after their loss represents a third type of evolutionary character change. The reconstruction of multiple losses and gains of characters by mapping on phylogenies is often problematic because of a lack of information about the relative likelihood of losses and gains. A developmental genetic approach using morphological, developmental, and molecular analysis is therefore an extremely important adjunct to phylogenetic approaches in interpreting losses, reversals, and regains. The molecular developmental basis of character loss and reversal is gradually becoming better understood. Loss of organs can occur by gain-of-function mutations (suppression) and loss-of-function mutations (that often leave a vestigial structure). The regain of characters after loss may occur by regulatory capture (a gain-of-function mutation) or by loss of function in suppressor genes. Reversals may occur by cryptic innovation (the formation of a new structure that mimics the old structure by gain-of-function mutations) or by loss of gene function associated with the apomorphic state (although this may have pleitropic or neomorphic effects). The genetic landscape of reversal is illustrated by the reversal to polysymmetry from monosymmetry in flowers. The range of observed phenotypes, loss with vestige, cryptic innovation, and loss with neomorphism matches the range of changes predicted."In plants with separated sexes, the male flowers often have a rudiment of a pistil; and Kolreuter found that by crossing such male plants with an hermaphrodite species, the rudiment of the pistil in the hybrid offspring was much increased in size; and this shows that the rudiment and the perfect pistil are essentially alike in nature" (Darwin 1859).

The genome of black cottonwood, Populus trichocarpa (Torr. & Gray).

We report the draft genome of the black cottonwood tree, Populus trichocarpa. Integration of shotgun sequence assembly with genetic mapping enabled chromosome-scale reconstruction of the genome. More than 45,000 putative protein-coding genes were identified. Analysis of the assembled genome revealed a whole-genome duplication event; about 8000 pairs of duplicated genes from that event survived in the Populus genome. A second, older duplication event is indistinguishably coincident with the divergence of the Populus and Arabidopsis lineages. Nucleotide substitution, tandem gene duplication, and gross chromosomal rearrangement appear to proceed substantially more slowly in Populus than in Arabidopsis. Populus has more protein-coding genes than Arabidopsis, ranging on average from 1.4 to 1.6 putative Populus homologs for each Arabidopsis gene. However, the relative frequency of protein domains in the two genomes is similar. Overrepresented exceptions in Populus include genes associated with lignocellulosic wall biosynthesis, meristem development, disease resistance, and metabolite transport.

Plant eco-devo: the potential of poplar as a model organism.

Ecological developmental genetics is the study of how ecologically significant traits originate in the genome and how the allelic combinations responsible are maintained in populations and species. Plant development involves a continuous feedback between growth and environment and the success of individual genotype x environment interactions determines the passage of alleles to the next generation: the adaptive recursion. Outbreeding plants contain a large amount of genetic variation, mostly in the form of single nucleotide polymorphisms (SNPs). One of the challenges of eco-devo is to distinguish neutral SNPs from those with ecological consequences. The complete genome sequence of Populus trichocarpa Torr. & A. Gray will be a significant aid in this endeavour. Occurring from California to Alaska, this is the first ecologically 'keystone' species to be sequenced. It has a rich natural history and is an obligate outbreeder. The individual sequenced, Nisqually-1, appears to be heterozygous on average about every 100 bp over the c. 500 million bp of the genome. Overlaid on this within-individual variation is some ecologically based between-individual genotypic variation evident across the distribution of the species. The synthesis of information from genomics and ecology is now in prospect. This 'ecomolecular synthesis' is likely to provide a rich insight into the genomic basis of plant adaptation.

Plant evolution and development in a post-genomic context.

Large-scale gene-sequencing projects that have been undertaken in animals have involved organisms from contrasting taxonomic groups, such as worm, fly and mammal. By contrast, similar botanical projects have focused exclusively on flowering plants. This has made it difficult to carry out fundamental research on how plants have evolved from simple to complex forms - a task that has been very successful in animals. However, in the flowering plants, the many completely or partially sequenced genomes now becoming available will provide a powerful tool to investigate the details of evolution in one group of related organisms.

Evolution and alignment of the hypervariable arm 1 of Aeschynanthus (Gesneriaceae) ITS2 nuclear ribosomal DNA.

Comparative ITS2 sequencing in the plant genus Aeschynanthus(Gesneriaceae) reveals an insertion/deletion (indel) hot spot in the ITS2 sequences that is difficult to align. Examination of other Gesneriaceae sequences shows that this is a widespread phenomenon in this plant family. Minimum free-energy secondary structure analyses localize the hot spot to the terminal part of arm 1. Arm 1 is twice as long in Gesneriaceae than in other asterids. In addition, the pattern of indels is consistent with this secondary structure model. The high variability of the extended terminal part of arm 1 in Gesneriaceae and the fact that it can be deleted altogether imply that it is functionally superfluous. In contrast, the base of arm 1 is relatively conserved and may function as an exonuclease recognition site. This study illustrates how comparative secondary structure analyses can be helpful in fine-scale alignment. Alignment based on secondary structure conflicts with our initial manual alignment and, to a lesser extent, with a CLUSTAL X alignment with default parameters.

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.

Evolution of morphological novelty: a phylogenetic analysis of growth patterns in Streptocarpus (Gesneriaceae).

Streptocarpus shows great variation in vegetative architecture. In some species a normal shoot apical meristem never forms and the entire vegetative plant body may consist of a single giant cotyledon, which may measure up to 0.75 m (the unifoliate type) or with further leaves arising from this structure (the rosulate type). A molecular phylogeny of 87 taxa (77 Streptocarpus species, seven related species, and three outgroup species) using the internal transcribed spacers and 5.8S region of nuclear ribosomal DNA suggests that Streptocarpus can be divided into two major clades. One of these broadly corresponds to the caulescent group (with conventional shoot architecture) classified as subgenus Streptocarpella, whereas the other is mainly composed of acaulescent species with unusual architecture (subgenus Streptocarpus). Some caulescent species (such as S. papangae) are anomalously placed with the acaulescent clade. Available cytological data are, however, completely congruent with the two major clades: the caulescent clade is x = 15 and the acaulescent clade (including the caulescent S. papangae) is x = 16 (or polyploid multiples of 16). The genera Linnaeopsis, Saintpaulia, and Schizoboea are nested within Streptocarpus. The sequenced region has evolved, on average, 2.44 times faster in the caulescent clade than in the acaulescent clade and this is associated with the more rapid life cycle of the caulescents. Morphological variation in plant architecture within the acaulescent clade is homoplastic and does not appear to have arisen by unique abrupt changes. Instead, rosulate and unifoliate growth forms have evolved several times, reversals have occurred, and intermediate architectures are found. An underlying developmental plasticity seems to be a characteristic of the acaulescent clade and is reflected in a great lability of form.

A phylogenetic analysis of Rhamnaceae using rbcL and trnL-F plastid DNA sequences.

Previous tribal classifications of Rhamnaceae have been based on fruit characters, resulting in the delimitation of large and otherwise heterogeneous groups. We evaluated the most recent classification with DNA sequences of two regions of the plastid genome, rbcL and trnL-F, from 42 genera of Rhamnaceae and representatives of the related families Elaeagnaceae, Barbeyaceae, Dirachmaceae, Urticaceae, Ulmaceae, Moraceae, and Rosaceae. The trnL-F trees have higher consistency and retention indices than the rbcL trees, and patterns of change in rbcL and trnL-F are compared. The closest relatives of Rhamnaceae are Dirachmaceae and Barbeyaceae, followed by the urticalean families. The plastid trees support the monophyly of the family and provide the basis for a new tribal classification. Three strongly supported clades are identified, but morphological characters could not be found to underpin a formal taxonomic description of these three clades as subfamilies. We therefore only recognize groups that are also defined by morphological characters. The biogeography of Rhamnaceae is discussed with reference to the molecular trees.

High intraindividual variation in internal transcibed spacer sequences in Aeschynanthus (Gesneriaceae): implications for phylogenetics.

Aeschynanthus (Gesneriaceae) is a large genus of tropical epiphytes that is widely distributed from the Himalayas and China throughout South-East Asia to New Guinea and the Solomon Islands. Polymerase chain reaction (PCR) consensus sequences of the internal transcribed spacers (ITS) of Aeschynanthus nuclear ribosomal DNA showed sequence polymorphism that was difficult to interpret. Cloning individual sequences from the PCR product generated a phylogenetic tree of 23 Aeschynanthus species (two clones per species). The intraindividual clone pairs varied from 0 to 5.01%. We suggest that the high intraindividual sequence variation results from low molecular drive in the ITS of Aeschynanthus. However, this study shows that, despite the variation found within some individuals, it is still possible to use these data to reconstruct phylogenetic relationships of the species, suggesting that clone variation, although persistent, does not pre-date the divergence of Aeschynanthus species. The Aeschynanthus analysis revealed two major clades with different but overlapping geographic distributions and reflected classification based on morphology (particularly seed hair type).

Origin and relationships of Saintpaulia (Gesneriaceae) based on ribosomal DNA internal transcribed spacer (ITS) sequences.

Phylogenetic relationships of eight species of Saintpaulia H. Wendl., 19 species of Streptocarpus Lindl. (representing all major growth forms within the genus), and two outgroups (Haberlea rhodopensis Friv., Chirita spadiciformis W. T. Wang) were examined using comparative nucleotide sequences from the two internal transcribed spacers (ITS) of nuclear ribosomal DNA. The length of the ITS 1 region ranged from 228 to 249 base pairs (bp) and the ITS 2 region from 196 to 245 bp. Pairwise sequence divergence across both spacers for ingroup and outgroup species ranged from 0 to 29%. Streptocarpus is not monophyletic, and Saintpaulia is nested within Streptocarpus subgenus Streptocarpella. Streptocarpus subgenus Streptocarpus is monophyletic. The ITS sequence data demonstrate that the unifoliate Streptocarpus species form a clade, and are also characterized by a unique 47-bp deletion in ITS 2. The results strongly support the monophyly of (1) Saintpaulia, and (2) Saintpaulia plus the African members of the subgenus Streptocarpella of Streptocarpus. The data suggest the evolution of Saintpaulia from Streptocarpus subgenus Streptocarpella. The differences in flower and vegetative characters are probably due to ecological adaptation leading to a relatively rapid radiation of Saintpaulia.

Phylogeny and disjunct distribution: evolution of Saintpaulia (Gesneriaceae).

The molecular phylogeny of African violets (Saintpaulia H. Wendl.), based on ribosomal DNA internal transcribed spacer (ITS) sequences, follows the disjunct biogeography of the genus. Sequence analysis by parsimony of 19 accessions, representing 17 currently recognized Saintpaulia species, resulted in four trees of 182 steps. The first major division is between S. goetzeana, from the Uluguru Mts, Tanzania, and the rest of the genus. The basal position of S. goetzeana, and its putative primitive characters, may indicate an Uluguru origin for Saintpaulia and subsequent colonization of the more northerly mountains. Of the remainder, S. teitensis, from the Teita Hills of Kenya, is sister taxon to the other species (which occur mainly in the Usambara Mts of north-east Tanzania). A group of nine Usambaran species that we call the 'ionantha complex' show minimal ITS genetic differentiation and are also taxonomically critical. Species diversity in the Usambara Mts appears to be the result of rapid, recent (possibly Pleistocene) radiation. This study reveals the limitations of ITS sequences for elucidating the radiation of poorly differentiated species (the ionantha complex). However, the molecular data strongly suggest that conservation of the Uluguru and Teita populations is essential for the protection of the full range of diversity within the genus.

Cell-context signalling.

In plants, cells differentiate according to their position with relation to their cell neighbours. Monoclonal antibody (MAb) probes to polysaccharide epitopes, present at the surfaces of all plant cells, have defined a family of proteoglycan antigens which signify cellular position. These MAbs have been used to sort the single cells present in carrot somatic cell cultures on the basis of the presence or absence of specific polysaccharide epitopes. This sorting allows embryo initial cells to be cultured among different cell collectives (based on their polysaccharide epitope expression) and thus in altered contextual backgrounds. These experiments have shown that specific populations of embryo initial precursor cells induce and sustain the early development of the embryo initials, revealing that the populations of different cell collectives which are defined by different polysaccharide epitopes (cell-context) serves important regulatory function in early plant development. Somatic embryo initials deprived of the influence of the cell collective-defined by the presence of the polysaccharide epitope recognised by the MAb JIM8-establish unorganised first divisions and develop as callus. However, in the presence of the JIM8-reactive cell collective, or medium conditioned by the collective, the initials develop into somatic embryos. This demonstrates that the cells defined by the JIM8 polysaccharide epitope are necessary to sustain the meristematic activity which drives the renewed development. Transfer of a cell-wall signal from the JIM8-reactive cells to cellular situations in carrot seedlings in which they would not normally occur (out-of-context signals) stimulates lateral root production, thus demonstrating that the inductive signal operative in suspension cultures can be reinterpreted by specific cells later in development and reinitiate meristematic activity. The communication between the precursor cells defined by JIM8 and embryo initials defines an early cell-cell interaction in developing carrot plants. Labelling of flower sections suggests that the same interaction exists between embryo apical and basal cells early in normal development.

The history of the endemic flora of St Helena: late Miocene:'Trochetiopsis-like' pollen from St Helena and the origin of Trochetiopsis.

Re-examination of a fossiliferous sediment from St Helena has revealed the presence in the late Miocene of pollen grains very similar to modern Trochetiopsis pollen. Trochetiopsis is found only in St Helena and is assumed to be a palaeoendemic genus. The late Miocene material provides striking confirmation of this view. The two extant Trochetiopsis species are compared with other species in the Sterculiaceae: Dombeyeae, using multivariate methods. It is suggested that the two extant Trochetiopsis species diverged on St Helena comparatively recently, probably as a result of disruptive selection followed by ecological isolation caused by environmental change at the beginning of the Quaternary. It is also suggested that the ancestor of the extant Trochetiopsis species arrived in St Helena by long distance dispersal from African or Madagascar Dombeyeae stock at least 9 million years ago. However, since this time the pollen morphology of the Trochetiopsis lineage has changed little. The taxonomic isolation of Trochetiopsis can thus be explained partly by evolution on St Helena, but mainly by evolution and extinction in the Dombeyeae elsewhere (reliction).

THE HISTORY OF ENDEMIC FLORA OF ST HELENA: A RELICTUAL SERIES.

When the endemic plant species of St Helena are ordered according to their increasing taxonomic isolation, their corresponding 'sister groups' show a 'relictual series' of increasing geographical disjunction. This is best explained by progressive extinction and evolution in continental areas rather than by particularly high rates of evolutionary change on the island (i.e. the taxonomically isolated endemics are ancient relicts). Few of the supposed trends of oceanic island evolution are convincingly evident in the flora of St Helena. St Helena was formed in the Miocene, and it is likely that the relict genera colonized St Helena from Southern Africa before the wet forest flora in this area largely disappeared as the climate deteriorated from the late Miocene onwards.