Showing papers in "Plant Systematics and Evolution in 2009"

Mutational dynamics and phylogenetic utility of noncoding chloroplast DNA

Abstract: Introns and spacers are a rich and well-appreciated information source for evolutionary studies in plants. Compared to coding sequences, the mutational dynamics of introns and spacers is very different, involving frequent microstructural changes in addition to substitutions of individual nucleotides. An understanding of the biology of sequence change is required for correct application of molecular characters in phylogenetic analyses, including homology assessment, alignment coding, and tree inference. The widely used term “indel” is very general, and different kinds of microstructural mutations, such as simple sequence repeats, short tandem repeats, homonucleotide repeats, inversions, inverted repeats, and deletions, need to be distinguished. Noncoding DNA has been indispensable for analyses at the species level because coding sequences usually do not offer sufficient variability. A variety of introns and spacers has been successfully applied for phylogeny inference at deeper levels (major lineages of angiosperms and land plants) in past years, and phylogenetic structure R in intron and spacer data sets usually outperforms that of coding-sequence data sets. In order to fully utilize their potential, the molecular evolution and applicability of the most important noncoding markers (the trnT–trnF region comprising two spacers and a group I intron; the trnS–G region comprising one spacer and a group II intron in trnG; the group II introns in petD, rpl16, rps16, and trnK; and the atpB–rbcL and psbA–trnG spacers) are reviewed. The study argues for the use of noncoding DNA in a spectrum of applications from deep-level phylogenetics to speciation studies and barcoding, and aims at outlining molecular evolutionary principles needed for effective analysis.

Phylogenetic utility of ycf1 in orchids: a plastid gene more variable than matK

Abstract: Plastid DNA sequences have been widely used by systematists for reconstructing plant phylogenies. The utility of any DNA region for phylogenetic analysis is determined by ease of amplification and sequencing, confidence of assessment in phylogenetic character alignment, and by variability across broad taxon sampling. Often, a compromise must be made between using relatively highly conserved coding regions or highly variable introns and intergenic spacers. Analyses of a combination of these types of DNA regions yield phylogenetic structure at various levels of a tree (i.e., along the spine and at the tips of the branches). Here, we demonstrate the phylogenetic utility of a heretofore unused portion of a plastid protein-coding gene, hypothetical chloroplast open reading frame 1 (ycf1), in orchids. All portions of ycf1 examined are highly variable, yet alignable across Orchidaceae, and are phylogenetically informative at the level of species. In Orchidaceae, ycf1 is more variable than matK both in total number of parsimony informative characters and in percent variability. The nrITS region is more variable than ycf1, but is more difficult to align. Although we only demonstrate the phylogenetic utility of ycf1 in orchids, it is likely to be similarly useful among other plant taxa.

Non-coding nuclear DNA markers in phylogenetic reconstruction

Abstract: Molecular DNA based data sets are the most important resource for phylogenetic reconstruction. Among the various marker systems, which were introduced and optimized within the last decade, coding sequences played an important role, especially when molecular clock approaches and multi-gene datasets were assembled. However, non-coding DNA sequences do not only play a quantitatively dominant role, as demonstrated by the two examples nuclear ITS (Internal transcribed spacer regions of nuclear ribosomal DNA) and plastidic trnL-F region, but there is also a wide range of different marker systems that can be applied in different ways. Herein, we review the application of several non-coding nuclear DNA marker systems for phylogenetic reconstructions and summarize valuable information for future research.

Contribution to ITS phylogeny of the Brassicaceae, with special reference to some Asian taxa

Abstract: Sequence data from the nuclear ribosomal internal transcribed spacer (ITS) region for 189 accessions representing 184 species in 121 genera of Brassicaceae were used to determine monophyly of tribes and genera, tribal boundaries, and component genera Parsimony analysis and Bayesian inference suggest that the tribes Camelineae and Arabideae are polyphyletic and should be subdivided into smaller tribes The study also supports the recent recognition of the new tribes Aphragmeae, Biscutelleae, Buniadeae, Calepineae, Conringieae, Dontostemoneae, Erysimeae, Malcolmieae, Megacarpaeeae, and Turritideae The data argue for the placement of Borodinia in the tribe Boechereae, Litwinowia and Pseudoclausia in the Chorisporeae, Atelanthera and Streptoloma in the Euclidieae, and Megacarpaea and Pugionium in the Megacarpaeeae, and exclusion of Asperuginoides, Didymophysa, and Ptilotrichum from the Alysseae, Macropodium, Pseudoturritis, and Stevenia from the Arabideae, and Crucihimalaya, Irenepharsus, Pachycladon, and Turritis from the Camelineae Finally, the findings support the expansion of Stevenia to include both Berteroella and Ptilotrichum, Sterigmostemum to include Oreoloma and one species of Anchonium, Crucihimalaya to include Transberingia and several species of Arabis, and Parrya to include Pseudoclausia The data also suggest that Calymmatium and Olimarabidopsis may be congeneric

The systematic value of nuclear genome size for ''all'' species of Tulipa L. (Liliaceae)

Abstract: Nuclear genome size, as measured by flow cytometry with propidium iodide, was used to investigate the relationships within the genus Tulipa L. (Liliaceae). More than 400 accessions representing 123 taxa from mainly wild-collected plants were investigated. Most species of Tulipa have the same basic chromosome number, 2n = 2x = 24. However, the somatic DNA 2C value (2C) is shown to range from 32 to 69 pg for the diploids. The largest genome contains roughly 3.4 × 1010 more base pairs than the smallest and has chromosomes that are more than twice as large. These large differences in the amount of nuclear DNA predict that the hybrids, if any arise, are usually sterile. Depending on the size of the total genome, 1 pg amounts to several thousand genes. Moreover, genome sizes are evaluated here in combination with available morphological, geographical, and molecular data. Therefore, the taxonomy proposed here is not a single-character taxonomy based on genome size alone. The genus Tulipa, as here determined, has 87 species, 29 more than accepted by van Raamsdonk et al. [Acta Hort (ISHS) 430:821–828, 1997], but including 25 species that were not available to them. Of these 87 species, 28 were not seen by Hall (The genus Tulipa, The Royal Horticultural Society, London, 1940) in a living state and placed by him in an addendum. Species of the subgenus Clusianae (Baker) Zonn. differ strongly in nuclear DNA content (DNA 2C value), 32 versus 40–68 pg for all other tulips, and are placed here in a separate subgenus. Also Orithyia, the only group with a style and with only 38–39 pg is placed in a separate subgenus. Therefore, all tulips are attributed to four subgenera, Clusianae (Baker) Zonn., Tulipa, Eriostemones Raamsd., and Orithyia (D. Don) Baker and divided further into 12 sections. Seven of the eight series of section Eichleres (A.D. Hall) Raamsd. are now placed in four sections: (1) section Lanatae (Raamsd.) Zonn., mainly confined to species from the Pamir-Alay and including series Lanatae Raamsd., (2) section Multiflorae (Raamsd.) Zonn. (including series Glabrae Raamsd.), (3) section Vinistriatae (Raamsd.) Zonn. (including series Undulatae Raamsd.), and (4) section Spiranthera Vved. ex Zonn. and Veldk. Triploids, tetraploids, and pentaploids were found in several species. DNA content confirmed the close relationships of the species within the different sections. The rather similar looking and therefore often confused T. armena Boiss. (51.8 pg), T. systola Stapf (56.3 pg), and T. julia K., Koch (61.6 pg) could be clearly distinguished. The same is true for T. biebersteiniana Schult. f. (56.9 pg), T. sylvestris ssp. australis (Link) Pamp. (62.0 pg), and T. primulina Baker (64.6 pg). T. doerfleri Gand. and T. whittalli (Dykes) Hall could be placed as polyploid forms of T. orphanidea Boiss. ex Heldr. On the basis of DNA content, a systematic association between T. julia K. Koch and the triploid T. aleppensis Boiss. and between T. systola Stapf and the triploid T. praecox Tenore was suggested. The new species T. lemmersii Zonn., Peterse, and de Groot is described, and four possible new species are indicated. Genome size as measured by using flow cytometry may conveniently be used to produce systematic data. It is applicable even in the case of dormant bulbs or sterile plants for monitoring the trade in bulbous species.

Application of non-coding DNA regions in intraspecific analyses.

Abstract: In this review we discuss the use of non-coding DNA at the intraspecific level in plants. Both nuclear and organelle non-coding regions are widely used in interspecific phylogenetic approaches. However, they are also valuable in analyses on the intraspecific level. Besides taxonomy, that is, defining subspecies or varieties, large fields for the application of non-coding DNA are population genetic and phylogeographic studies. Population genetics tries to explain the genetic patterns within species mostly by the amount of extant gene flow among populations, while phylogeography explicitly tries to reconstruct historic events. Depending on the study different molecular markers can be used, varying between very fast evolving microsatellites or some more slowly changing regions like intergenic spacers and introns. Here, we focus mainly on the use of non-coding regions in phylogeographic analyses. Mostly used in this context are regions of the genomes of the chloroplasts and mitochondria. In phylogeography, the correct estimation of allele or haplotype relationships is particularly important. As tree-based methods are mostly insufficient to depict relationships within species, network approaches are better suitable to infer gene or locus genealogies. Problematic for phylogeographic studies are alleles shared among multiple species, which could result from either hybridization or incomplete lineage sorting. Especially the latter can severely influence the interpretation of the phylogeographic patterns. Therefore, it seems necessary for us to also include close relatives of the species under study in phylogeographic analyses. Not only the sample design but also the analysis methods are currently changing, as some new methods such as statistical phylogeography were emerging recently and widely used methods like nested clade analysis might not be reliable in every case. During the last few years, a multitude of studies were published, which mainly analyzed phylogeographic patterns in European and North American plants. Phylogeographic studies in other regions of the earth are still comparably rare, although questions like the influence of the ice age on the vegetation in the tropics or southern hemisphere are still open and phylogeography provides an excellent remedy to answer them.

A framework for phylogenetic sequence alignment

Abstract: A phylogenetic alignment differs from other forms of multiple sequence alignment because it must align homologous features. Therefore, the goal of the alignment procedure should be to identify the events associated with the homologies, so that the aligned sequences accurately reflect those events. That is, an alignment is a set of hypotheses about historical events rather than about residues, and any alignment algorithm must be designed to identify and align such events. Some events (e.g., substitution) involve single residues, and our current algorithms can successfully align those events when sequence similarity is great enough. However, the other common events (such as duplication, translocation, deletion, insertion and inversion) can create complex sequence patterns that defeat such algorithms. There is therefore currently no computerized algorithm that can successfully align molecular sequences for phylogenetic analysis, except under restricted circumstances. Manual re-alignment of a preliminary alignment is thus the only feasible contemporary methodology, although it should be possible to automate such a procedure.

Trade-offs between sexual and asexual reproduction in a monoecious species Sagittaria pygmaea (Alismataceae): the effect of different nutrient levels

Abstract: Available resources could influence the trade-offs among different reproductive components in plants. Here, we created three nutrient levels to test the nutrient effects on trade-offs among sexual reproduction, clonal propagation and vegetative growth in a monoecious clonal herb Sagittaria pygmaea. The results of this study showed that the plant exhibited different trade-off patterns among different nutrient levels. When the nutrient level was low, there were weak trade-offs between sexual reproduction and vegetative growth and between clonal propagation and vegetative growth; when the nutrient level was moderate, we found a strong trade-off between sexual reproduction and clonal propagation; but when the nutrient level was high, we found no trade-offs among these three different reproductive components. These results indicated that the plant could adjust its trade-off patterns to fit the nutrient variation and suggested that trade-offs are unlikely to constrain the evolution of reproductive strategy in this species.

Intraspecific diversity and relationship between subspecies of Origanum vulgare revealed by comparative AFLP and SAMPL marker analysis

Abstract: The genus Origanum is often referred to as an under-utilized taxon because of its complex taxonomy. Origanum vulgare L., the most variable species of the genus, is a spice and medicinal herb that is characterized by high morphological diversity (six subspecies). In this study, the relative efficiencies of two PCR-based marker approaches, amplified fragment length polymorphism (AFLP) and selectively amplified microsatellite polymorphic loci (SAMPL), were used for comparable genetic diversity surveys and subspecies discrimination among 42 oregano accessions. Seven assays each of AFLP and SAMPL markers were utilized. Effective multiplex ratio (EMR), average heterozygosity (Hav-p), marker index (MI), and resolving power (RP) of the primer combinations were calculated for the two marker systems. UPGMA and Structure analysis along with PCoA plots derived from the binary data matrices of the two markers depicted the genetic distinction of accessions. Our results indicate that both marker systems are suitable but SAMPL markers are slightly more efficient in differentiating accessions and subspecies than AFLPs.

Molecular phylogeny of Ceropegia (Asclepiadoideae, Apocynaceae) from Indian Western Ghats

Abstract: Ceropegia includes more than 200 species distributed in the Old World ranging from the Canary Islands to Australia. In India, there are about 50 species described on a morphological basis as belonging to Ceropegia, and most of them are endemic to the Western Ghats. To investigate evolutionary relationships among Indian Ceropegia taxa and their allies, a phylogenetic analysis was conducted to include 31 Indian taxa of Ceropegia and Brachystelma and their congeners from other geographical regions using nuclear ribosomal internal transcribed spacer (ITS) and three noncoding chloroplast DNA (cpDNA) sequences, including intergenic spacers trnT-L and trnL-F, and trnL intron. The Western Ghats Ceropegia species were found to be most closely related to Indian Brachystelma, with the two genera being placed sister to each other in the ITS phylogeny or with the Brachystelma clade nested within one of the two subclades of Indian Ceropegia in the cpDNA phylogeny. In contrast, Ceropegia species from other regions and African Brachystelma all formed separate clades basal to the Indian Ceropegia-Brachystelma clade. Thus, it can be concluded that the classical morphology-based delineation of the two genera needs revision to reflect their phylogenetic relationships, which are more in accordance with their geographical origin than with morphology.

Deep divergences in the coffee family and the systematic position of Acranthera

Abstract: Despite extensive efforts, there are unresolved questions on evolutionary relationships in the angiosperm family Rubiaceae. Here, information from six loci and 149 Rubiaceae taxa provide new insights. Acranthera and Coptosapelta are strongly supported as sisters. Pollen grains of Acranthera possess several features common in Rubiaceae, but amongst potential similarities with the unusual grains of Coptosapelta are the nature of the apertures and the structure of the sexine. Luculia, Acranthera and Coptosapelta are excluded from the three subfamilies Ixoroideae, Cinchonoideae and Rubioideae. Sipaneeae and Condamineeae form a clade, sister to remaining Ixoroideae. Rondeletieae and Guettardeae are sisters to remaining Cinchonoideae. Colletoecema is sister to remaining Rubioideae, followed by the Urophylleae–Ophiorrhizeae clade. Nuclear ITS provided structured information at all phylogenetic levels, but the main gain from adding nrITS was the increased resolution. Average support values also increased but were generally high also without nrITS and the increase was not statistically significant.

Phylogeny of the eudicot order Malpighiales: analysis of a recalcitrant clade with sequences of the petD group II intron

Abstract: Malpighiales are one of the most diverse orders of angiosperms. Molecular phylogenetic studies based on combined sequences of coding genes allowed to identify major lineages but hitherto were unable to resolve relationships among most families. Spacers and introns of the chloroplast genome have recently been shown to provide strong signal for inferring relationships among major angiosperm lineages and within difficult clades. In this study, we employed sequence data of the petD group II intron and the petB-petD spacer for a set of 64 Malpighiales taxa, representing all major lineages. Celastrales and Oxalidales served as outgroups. Sequence alignment was straightforward due to frequent microstructural changes with easily recognizable motifs (e.g., simple sequence repeats), and well defined mutational hotspots. The secondary structure of the complete petD intron was calculated for Idesia polycarpa as an example. Domains I and IV are the most length variable parts of the intron. They contain terminal A/T-rich stem-loop elements that are suggested to elongate independently in different lineages with a slippage mechanism earlier reported from the P8 stem-loop of the trnL intron. Parsimony and Bayesian analyses of the petD dataset yielded trees largely congruent with results from earlier multigene studies but statistical support of nodes was generally higher. For the first time a deep node of the Malpighiales backbone, a clade comprising Achariaceae, Violaceae, Malesherbiaceae, Turneraceae, Passifloraceae, and a Lacistemataceae–Salicaceae lineage received significant statistical support (83% JK, 1.00 PP) from plastid DNA sequences.

Genetic variation of Pinus uncinata (Pinaceae) in the Pyrenees determined with cpSSR markers

Abstract: The genetic variation within and between 13 populations (385 individuals) of Pinus uncinata was analyzed with ten chloroplast microsatellite markers. Both the infinite allele mutation and stepwise mutation model (SMM) have been applied to the analysis of the genetic structure and the geographical distribution of haplotypic variation. High level of genetic diversity and low but significant differentiation among compared population were found. Three marginal populations, Sierra de Cebollera, Margaride Mountains and Sierra de Gudar are strongly differentiated from the rest. Mutations following SMM-like process contributed significantly to the regional differentiation. The pattern of genetic structure observed in mountain pine is common in conifers with a wide distribution range. Lack of significant genetic structuring may be a result of a recent fragmentation of a historically larger population and/or interspecific hybridization and introgression. The southernmost populations from the Sierra Cebollera and the Sierra de Gudar are the most genetically distinct. This suggests a long period of spatial isolation and/or origin from different ancestral populations.

Genealogical analysis of subspecies divergence and spikelet-shape diversification in central Eurasian wild wheat Aegilops tauschii Coss.

Abstract: The genealogical and geographic structure of variation in spikelet morphology was analyzed for central Eurasian wild wheat Aegilops tauschii Coss. using a diverse array of 203 sample accessions that represented the entire species range. In this sample set, two subspecies were identified on the basis of sensu-stricto criteria: only the accessions having markedly moniliform spikes were assigned to Ae. tauschii Coss. subspecies strangulata (Eig) Tzvel., whereas those having mildly moniliform and cylindrical spikes to Ae. tauschii Coss. subspecies tauschii. In a graph of the first two axes from a principal component analysis based on nine spikelet traits, the plots of the two subspecies formed separate clusters, indicating that subspecies strangulata sens. str. is a practically usable taxon. Chloroplast-DNA-based genealogical analyses suggested that subspecies strangulata diverged from an ancestor that carried a specific chloroplast DNA type, whereas, after divergence, this subspecies became polyphyletic, likely through hybridization. Geographically, significant longitudinal and latitudinal clines were detected for spikelet size, with spikelets tending to be small in the eastern and southern regions. These results shed some light on the patterns of subspecies divergence and spikelet-shape diversification in the course of Ae. tauschii’s long-distance dispersal from the Transcaucasus to China.

Hybridization drives speciation in Gagea (Liliaceae)

Abstract: Hybridization seems to play an important role in speciation of Gagea Salisb., a genus which is characterised by polyploid taxa lines and in which diploids (2n = 24) appear only to be common in basal sections. Hybrid detection was applied utilising direct and cloning nrDNA ITS data (ITS1, 5.8S rDNA, ITS2) combined with neighbour and ribotype networks and discussed in connection with previously published cpDNA, morphological and karyological data of the authors. We have evidence of the hybrid origin of taxa within the section Gagea (G. pomeranica, G.megapolitana) and the monophyletic clade of sections Didymobulbos and Fistulosae (G. microfistulosa, G. polidorii, G. cf. bohemica). Morphologically and karyologically differentiated Gageamegapolitana and G. pomeranica, adapted to synanthropic habitats, represent both hybrids of G. pratensis × G. lutea. Gagea microfistulosa represents a hybrid of G. villosa × G. fragifera; Gagea polidorii could represent the reverse hybrid. G. glacialis is also closely related to the latter complex.

Identifying a mysterious aquatic fern gametophyte

Abstract: Suswassertang, a popular aquatic plant that is sold worldwide in aquarium markets, has been long considered a liverwort because of its ribbon-like thallus. However, its antheridia are remarkably fern-like in morphology. To corroborate the hypothesis that Suswassertang is a fern gametophyte and to determine its closest relative, we have sequenced five chloroplast regions (rbcL, accD, rps4–trnS, trnL intron, and trnL-F intergenic spacer), applying a DNA-based identification approach. The BLAST results on all regions revealed that Suswassertang is a polypod fern (order: Polypodiales) with strong affinities to the Lomariopsidaceae. Our phylogenetic analyses further showed that Suswassertang is nested within the hemi-epiphytic fern genus Lomariopsis (Lomariopsidaceae) and aligned very close to L. lineata. Our study brings new insights on the unexpected biology of Lomariopsis gametophytes—the capacity of retaining a prolonged gametophytic stage under water. It is of great interest to discover that a fern usually known to grow on trees also has gametophytes that thrive in water.

Genetic diversity and structure among Iranian tall fescue populations based on genomic-SSR and EST-SSR marker analysis

Abstract: Tall fescue (Festuca arundinacea Schreb. subsp. arundinacea) is one of the most economically important forage grasses in cold and temperate regions of the world. In this study, we have assessed the genetic diversity and structure of wild Iranian tall fescue populations. Thirty-seven individuals from nine natural populations from northern, western, and southern Iranian mountain ranges were analyzed using six genomic-SSRs and five EST-SSRs primer pairs. Our analysis has demonstrated that transcribed EST-SSR regions showed levels of polymorphism similar to genomic-SSR regions. UPGMA, repeated bisection, and model-based Bayesian STRUCTURE clustering methods coupled with neighbor-net network were used to establish six divergent groups of individuals. FST estimates among clusters showed moderate to low genetic structure. Within-group genetic diversity estimate H and partial correlations between genetic and geographic distances among populations suggested that western Zagros population was related to the rest of the Iranian individuals. The isolation-by-distance hypothesis was not supported by SSR data and the present geographical sampling.

Distribution and diversity of Arctic-Alpine species in the Balkans

Abstract: The distributions of 77 Arctic-Alpine species in the Balkans are mapped and the centers of their richness and diversity presented. Within the Dinaric Alps these are Mts Vranica, Durmitor, and Prokletije; in the Scardo-Pindhic mountains, Sarplanina–Rudoka–Korab form a continuous chain; in the Rhodope-Rila mountain system there are Mts Vitosa, Rila, and Pirin; while in the Balkan mountain system there are the West and Central part of Stara planina. A comparison of floristic richness and distribution of Arctic-Alpine flora in relation to altitude, geographical location, and geological substrate is made. Correlations between floristic richness and geographical distance of the Balkan mountains from the two main centers of Arctic-Alpine flora in Central Europe are also provided.

Genetic similarity among cultivars of Phyllostachys pubescens

Abstract: Phyllostachys pubescens is the most important economic bamboo species in China, which grows widely in the South of China There are more than ten cultivars in this species but their genetic relationship still remains unknown We used both amplified fragment length polymorphism (AFLP) and inter-simple sequence repeat (ISSR) techniques to determine genetic similarity among ten cultivars of P pubescens and two related species Eight hundred and twenty seven bands, in which 495 are polymorphic, were detected using 15 pairs of AFLP primers whereas total 231 bands, in which 154 bands are polymorphic, were scored using 16 ISSR primers Statistic analysis showed that the genetic similarity matrices obtained from these two sets of molecular markers had a significant correlation (R = 0959, P = 0013) The dendrogram generated with AFLP and ISSR markers could clearly genetically identify ten cultivars of P pubescens that had high similarity with genetic distances ranging from 0023 to 0108, and could be divided into three groups based on their genetic variation and similarity Our results suggest that these molecular markers are useful to genetically classify cultivars or varieties of a species, particularly a bamboo species

Systematics and evolution of Carex sects. Spirostachyae and Elatae (Cyperaceae)

Abstract: Carex sect. Spirostachyae comprises 25 species displaying the centre of diversity in Eurasia, while sect. Elatae comprises 22 species displaying the centre of diversity in tropical African mountainous regions. Phylogenetic analysis of the 136 ITS and 108 5′trnK intron sequences of 23 species of sect. Spirostachyae and 20 species of sect. Elatae revealed that neither section is monophyletic. With the exclusion of C. montis-eeka (sect. Spirostachyae) and C. insularis, C. iynx and C. longibrachiata (sect. Elatae), the sects. Spirostachyae and Elatae constitute a monophyletic group of 38 species, 22 of which were previously included in sect. Spirostachyae and 16 of which were in sect. Elatae (considering C. thomasii as C. mannii ssp. thomasii). Two main groups with different edaphic preferences were identified in the core Spirostachyae, in congruence with some morphological features. One group comprises primarily acidophilus species including 11 species of sect. Spirostachyae and 16 species of sect. Elatae. The other group includes 11 basophilic species of sect. Spirostachyae. Incongruence between nuclear and plastid genomes was detected, suggesting hybridization or lineage sorting processes in the evolution of the core Spirostachyae.

Low levels of genetic differentiation between Opuntia echios varieties on Santa Cruz (Galapagos)

Abstract: Three processes play an important role in plant speciation: isolation, hybridization and polyploidization. Galapagos endemic Opuntia display putatively all of these processes. On this archipelago most islands are inhabited by a single Opuntia taxon. Santa Cruz, however, houses two morphologically distinct O. echios varieties (echios and gigantea). Morphological intermediates are found where these two geographically isolated varieties meet. Here we used ten microsatellite loci to reveal the population genetic structure of this system. In contrast to earlier studies, we found high genetic variability within localities. Genetic structuring was weak and no evidence for the existence of hybrids was found. The reasons for this weak genetic structure may include: the species’ hexaploid nature, high levels of gene flow, recent colonization, and the lack of geographic barriers. This first detailed genetic study on these threatened species will be important for further conservation planning.

Genome size variation and morphological differentiation within Ranunculus parnassifolius group (Ranunculaceae) from calcareous screes in the Northwest of Spain.

Abstract: Ranunculus parnassifolius is an orophilous plant distributed throughout Central and Southwestern Europe (Alps, Pyrenees and Cantabrian Mountains). Its evolutionary history and taxonomy are often complicated, having been little studied before now. The purpose of this article is to present flow cytometry measurements and multivariate morphometric analyses to ascertain cytotype distribution patterns and the morphological differentiation of R. parnassifolius s.l. from calcareous screes in the Northwest of Spain. DNA ploidy level and morphometric analysis were determined for plants of R. parnassifolius s.l. using flow cytometry (112 individuals) and multivariate analysis (152 individuals). Specimens were collected in eight localities in the Northwest of the Iberian Peninsula. Different sample preservation methods (fresh, frozen, and herbarium specimens) were employed as well as the use of various buffers and internal standards, in order to test the reproducibility of DNA flow cytometry. Three ploidy levels were detected in the study area (diploid, tetraploid, and pentaploid), and mixed-cytotype populations were also found. The mean nuclear DNA content of the R. parnassifolius group ranged from 7.43 ± 0.185 to 7.63 ± 0.339 pg/2C in diploids and from 15.09 ± 0.161 to 15.85 ± 0.587 pg/2C in tetraploids. The analysis of the monoploid genome sizes (1Cx) did not reveal a clear difference among cytotypes. These results suggest low intraspecific variation, at least among the populations studied. In addition, a comparison of different DNA reference standards was conducted. A new value for the chicken genome size was used as internal reference standard (2C = 3.14 ± 0.155 pg), with similar results found using both animal and plant standards (Pisum sativum and Solanum lycopersicum). Finally, herbarium vouchers and frozen tissue were proved to be suitable for DNA ploidy level measurements. This study provided a first assessment of C values in the R. parnassifolius group using flow cytometry. The weak morphological distinction of the cytotypes and the existence of mixed-cytotype populations in the Northwest of Spain are reported here for the first time. The different distribution pattern of the two cytotypes is discussed.

What phylogeny and gene genealogy analyses reveal about homoplasy in citrus microsatellite alleles

Abstract: Sixty-five microsatellite alleles amplified from ancestral citrus accessions classified in three separate genera were evaluated for sequence polymorphism to establish the basis of inter- and intra-allelic genetic variation, evaluate the extent of size homoplasy, and determine an appropriate model (stepwise or infinite allele) for analysis of citrus microsatellite alleles. Sequences for each locus were aligned and subsequently used to determine relationships between alleles of different taxa via parsimony. Interallelic size variation at each SSR locus examined was due to changes in repeat copy number with one exception. Sequencing these alleles uncovered new distinct point mutations in the microsatellite region and the region flanking the microsatellite. Several of the point mutations were found to be genus, species, or allele specific, and some mutations were informative about the inferred evolutionary relationships among alleles. Overall, homoplasy was observed in alleles from all three loci, where the core microsatellite repeat was changed causing alleles of the same size class to be identical in state but not identical by descent. Because nearly all changes in allele size (with one exception) were due to expansion or contraction of the repeat motif, this suggests that a stepwise mutation model, which assumes homoplasy may occur, would be the most appropriate for analyzing Citrus SSR data. The collected data indicate that microsatellites can be a useful tool for evaluating Citrus species and two related genera since repeat motifs were reasonably well retained. However, this work also demonstrated that the number of microsatellite alleles is clearly an underestimate of the number of sequence variants present.

Homology in coding and non-coding DNA sequences: a parsimony perspective

Abstract: Putative synapomorphy assessment (primary homology assessment) is distinct for DNA strings having a codon structure (hereafter, coding DNA) versus those lacking it (hereafter, non-coding DNA). The first requires the identification of a reading frame and of usually few in-frame insertions and deletions. In non-coding DNA, where length variation is much more common, putative synapomorphy assessment is considerably less straightforward and highly depends on the alignment method. Appreciating the existence of evolutionary constraints, alignments that consider patterns associated with specific putative evolutionary events are favored. Once the sequences have been aligned, the postulated putative evolutionary events need to be coded as an additional step. In order for the alignments and the alignment coding to be falsifiable, they should be carried out using justified and explicitly formulated criteria. Alternative coding methods for the most common patterns present in alignments of non-coding DNA are discussed here. Simpler putative synapomorphy assessment will not always correlate to more reliable phylogenetic information because simplicity does not necessarily correlate to the degree of homoplasy. The use of non-coding DNA can result in more laborious coding, but at the same time in more corroborated hypotheses, mirroring their accuracy for phylogenetic inference.

Pollen grain size, stigma depth, and style length: the relationships revisited

Abstract: I examine data and review information in the literature to test hypotheses proposed by Delpino and Darwin to explain the source of nutrients utilized by pollen tubes. In 1867, Delpino, in his discussion of distyly, suggested that the positive relationship between pollen grain size and style length was based on the pollen grains containing sufficient nutrients to sustain the growth of their pollen tubes through their respective styles. Darwin (The different forms of flowers on plants of the same species, 2nd edn. J. Murray, London, 1884) rejected Delpino’s suggestion based on his examination of distylous species whose morphs produced pollen grains whose sizes were not proportionate to the lengths of their respective styles. Darwin then proposed that pollen tubes first grow autotrophically, i.e., through the stigma, then heterotrophically in the style. This should result in a positive relationship between pollen grain size and stigma depth, if pollen tubes grow autotrophically through the stigma. I examined 15 species in Fabaceae and 20 species in Proteaceae to test the two hypotheses. Pollen grain size was correlated with stigma depth among the Fabaceae, i.e., consistent with Darwin’s hypothesis, and was not correlated with style length in either family, i.e., inconsistent with Delpino’s proposal. Comparisons of related species, in general, were consistent with Darwin’s hypothesis. In addition, information in the literature provided no evidence that pollen tubes obtain resources on or in the stigma, i.e., pollen tube growth from the stigmatic surface to the style was autotrophic. In contrast, pollen tubes obtain an array of resources from the transmission tissue, thus there is little reason for pollen grains to contain those resources. In addition, I suggest that positive correlations between pollen grain size and style/pistil length may be a result of both being correlated with stigma depth.

The evolution of cultivated plant species: classical plant breeding versus genetic engineering

Abstract: Agriculture is the most intensive form of environmental exploitation performed by mankind. It involves replacing the natural ecosystem with an artificial plant community comprising one or more crop species, and weeds can invade the cleared land. Initially, the adoption of agriculture did not necessarily imply an improvement in standard of living (there is, in fact, evidence to the contrary), but as agricultural efficiency improved, surpluses were generated on top of mere subsistence levels. It may take many years of labor in order to obtain a crop that has all of the desired traits. It is not possible to control which genes are transferred from the parents to the offspring, and the results are often uncertain. In comparison, the utilization of genetic engineering to improve crops can be a faster and more precise approach. Unlike traditional breeding, genetic engineering makes it possible to select the specific traits desired and insert the genes that code for them into the plant.

Self-incompatibility and myophily in Octomeria (Orchidaceae, Pleurothallidinae) species

Abstract: Pleurothallidinae (ca. 4,000 spp.) is the largest myophilous group in Orchidaceae. Recently three self-compatible bird-pollinated genera were included at the base of the group, while Octomeria (plus Brachionidium) is the sister group to the remaining subtribe in the former circumscription. Studies of reproductive biology of three Octomeria species were carried out to determine the occurrence of myophily and self-incompatibility in the genus, to help trace the evolution of these characteristics in the subtribe. The three species showed a strong self-incompatibility and are intercompatible. Octomeria crassifolia and O. grandiflora are pollinated by Sciaridae (Diptera) species, presenting high specificity. These results indicate that both self-incompatibility and myophily are basal characteristics in the former delimitation of the Pleurothallidinae and possibly have been kept in the derived lineages, representing biological synapomorphies of the group.

High level of genetic differentiation of Juniperus phoenicea (Cupressaceae) in the Mediterranean region: geographic implications

Abstract: Fourteen natural populations of Juniperus phoenicea L. from the quite entire species range have been compared using isoenzyme polymorphism. Among 17 loci, 5 (Got1, 6Pgd3, Pgi2, Pgm2 and Shdh2) appeared to be differentiated sufficiently to provide useful information for discrimination between the subspecies phoenicea and turbinata (Guss.) Nyman. Two distinct groups of populations were detected using the Nei’s genetic distance unweighted pair group method with arithmetic mean (UPGMA) and discrimination analyses, one including the inland populations of the eastern Iberian Peninsula and southern France (subsp. phoenicea), and the second from the Mediterranean and Atlantic shores, and from the Atlas mountains in Africa (subsp. turbinata). The high level of differences confirms a long period of isolation, probably during the whole Pleistocene. The population from the Aegean Sea shore differed from the other Mediterranean shore plus Atlas mountain population. It also suggests spatial isolation between them, at least during the last Glaciation.

Relationships among arctic and non-arctic haplotypes of the moss species Scorpidium cossonii and Scorpidium scorpioides (Calliergonaceae).

Abstract: Relationships among Scorpidium cossonii and Scorpidium scorpioides haplotypes from most of the species’ distribution areas were analyzed based on ITS and rpl16. The haplotype networks were produced by TCS and were rooted by neighbor joining (ITS, recombination present) or maximum parsimony analysis. The haplotype closest to the root of the ITS network and some poorly represented haplotypes close to this have S. cossonii morphology and are arctic to subarctic, suggesting a northern origin of the complex. Additionally, two major lineages evolved from the root haplotype; one with S. cossonii morphology samples and one with all S. scorpioides morphology ones. The basal haplotypes in these major lineages include numerous temperate zone representatives, suggesting that adaptations for relatively warmer environments are present. Among haplotype groups that evolved later, one group in each major lineage is absent from America. rpl16 provides a scenario similar to that revealed by ITS for S. cossonii (no variation in S. scorpioides). Fossil and molecular evidence suggest that ancestral populations of S. cossonii evolved at least almost 4 Myr bp.

Comparison of four molecular markers for genetic analysis in Diospyros L. (Ebenaceae).

Abstract: Four molecular markers, including inter-retrotransposon amplified polymorphism (IRAP), retrotransposon-microsatellite amplified polymorphism (REMAP), sequence-specific amplified polymorphism (SSAP), and amplified fragment length polymorphism (AFLP), were compared in terms of their informativeness and efficiency for analysis of genetic relationships among 28 genotypes in the genus Diospyros. The results were as follows: (1) the highest level of detected polymorphism were observed for IRAP; (2) AFLP was the most efficient marker system due to the simultaneous detection of abundant polymorphism markers per single reaction; (3) the marker index (MI) value was lower for SSAP than for AFLP, but SSAP had a higher level of detected polymorphism than AFLP; (4) the correlation coefficients of similarity were statistically significant for all four marker systems; (5) the four molecular markers yielded similar phylogenetic trees. To our knowledge, this was the first detailed report of a comparison of performance among three retrotransposon-based molecular markers (IRAP, REMAP, SSAP) and the AFLP technique (DNA-based molecular marker) on a set of samples of Diospyros. The results provide guidance for future efficient use of these molecular methods in the genetic analysis of Diospyros.