Inés Álvarez

Ribosomal ITS sequences and plant phylogenetic inference.

One of the most popular sequences for phylogenetic inference at the generic and infrageneric levels in plants is the internal transcribed spacer (ITS) region of the 18S-5.8S-26S nuclear ribosomal cistron. The prominence of this source of nuclear DNA sequence data is underscored by a survey of phylogenetic publications involving comparisons at the genus level or below, which reveals that of 244 papers published over the last five years, 66% included ITS sequence data. Perhaps even more striking is the fact that 34% of all published phylogenetic hypothesis have been based exclusively on ITS sequences. Notwithstanding the many important contributions of ITS sequence data to phylogenetic understanding and knowledge of genome relationships, a number of molecular genetic processes impact ITS sequences in ways that may mislead phylogenetic inference. These molecular genetic processes are reviewed here, drawing attention to both underlying mechanism and phylogenetic implications. Among the most prevalent complications for phylogenetic inference is the existence in many plant genomes of extensive sequence variation, arising from ancient or recent array duplication events, genomic harboring of pseudogenes in various states of decay, and/or incomplete intra- or inter-array homogenization. These phenomena separately and collectively create a network of paralogous sequence relationships potentially confounding accurate phylogenetic reconstruction. Homoplasy is shown to be higher in ITS than in other DNA sequence data sets, most likely because of orthology/paralogy conflation, compensatory base changes, problems in alignment due to indel accumulation, sequencing errors, or some combination of these phenomena. Despite the near-universal usage of ITS sequence data in plant phylogenetic studies, its complex and unpredictable evolutionary behavior reduce its utility for phylogenetic analysis. It is suggested that more robust insights are likely to emerge from the use of single-copy or low-copy nuclear genes.

Evolution and Natural History of the Cotton Genus

We present an overview of the evolution and diversity in Gossypium (the cotton genus). This framework facilitates insight into fundamental aspects of plant biology, provides the necessary underpinnings for effective utilization of cotton genetic resources, and guides exploration of the genomic basis of morphological diversity in the genus. More than 50 species of Gossypium are distributed in arid to semi-arid regions of the tropics and subtropics. Included are four species that independently have been domesticated for their fiber, two each in Africa-Asia and the Americas. Gossypium species exhibit extraordinary morphological variation, ranging from trailing herbaceous perennials to ∼15 m trees with a diverse array of reproductive and vegetative characteristics. A parallel level of cytogenetic and genomic diversity has arisen during the global radiation of the genus, leading to the evolution of eight groups of diploid (n = 13) species (genome groups A through G, and K). Data implicate an origin for Gossypium about 5–10 million years ago and a rapid early diversification of the major genome groups. Allopolyploid cottons appear to have arisen within the last 1–2 million years, as a consequence of trans-oceanic dispersal of an A-genome taxon to the New World followed by hybridization with an indigenous D-genome diploid. Subsequent to formation, allopolyploids radiated into three modern lineages, two of which contain the commercially important species G. hirsutum and G. barbadense.

Phylogeny of the New World Diploid Cottons (Gossypium L., Malvaceae) Based on Sequences of Three Low-Copy Nuclear Genes

American diploid cottons (Gossypium L., subgenus Houzingenia Fryxell) form a monophyletic group of 13 species distributed mainly in western Mexico, extending into Arizona, Baja California, and with one disjunct species each in the Galapagos Islands and Peru. Prior phylogenetic analyses based on an alcohol dehydrogenase gene (AdhA) and nuclear ribosomal DNA indicated the need for additional data from other molecular markers to resolve phylogenetic relationships within this subgenus. Toward this end, we sequenced three nuclear genes, the anonymous locus A1341, an alcohol dehydrogenase gene (AdhC), and a cellulose synthase gene (CesA1b). Independent and combined analyses resolved clades that are congruent with current taxonomy and previous phylogenies. Our analyses diagnose at least two long distance dispersal events from the Mexican mainland to Baja California, following a rapid radiation of the primary lineages early in the diversification of the subgenus. Molecular data support the proposed recognition of a new species closely related to Gossypium laxum that was recently collected in Mexico.

Phylogenetic, morphological, and chemotaxonomic incongruence in the North American endemic genus Echinacea

The study of recently formed species is important because it can help us to better understand organismal divergence and the speciation process. However, these species often present difficult challenges in the field of molecular phylogenetics because the processes that drive molecular divergence can lag behind phenotypic divergence. In the current study we show that species of the recently diverged North American endemic genus of purple coneflower, Echinacea, have low levels of molecular divergence. Data from three nuclear loci and two plastid loci provide neither resolved topologies nor congruent hypotheses about species-level relationships. This lack of phylogenetic resolution is likely due to the combined effects of incomplete lineage sorting, hybridization, and backcrossing following secondary contact. The poor resolution provided by molecular markers contrasts previous studies that found well-resolved and taxonomically supported relationships from metabolic and morphological data. These results suggest that phenotypic canalization, resulting in identifiable morphological species, has occurred rapidly within Echinacea. Conversely, molecular signals have been distorted by gene flow and incomplete lineage sorting. Here we explore the impact of natural history on the genetic organization and phylogenetic relationships of Echinacea.

CRYPTIC INTERSPECIFIC INTROGRESSION AND GENETIC DIFFERENTIATION WITHIN GOSSYPIUM ARIDUM (MALVACEAE) AND ITS RELATIVES

Abstract Interspecific gene flow is increasingly recognized as an important evolutionary phenomenon in plants. A surprising observation is that historical introgression is often inferred between species that presently have geographic and reproductive barriers that would appear to prohibit the inferred sexual exchange. A striking example concerns Gossypium aridum (subsection Erioxylum); previous analyses have shown that populations from Colima (southwestern Mexico) have a chloroplast genome (cpDNA) similar to that of a different taxonomic subsection (Integrifolia) that presently is confined to Baja California and the Galapagos Islands, whereas other G. aridum populations share a cpDNA lineage with each other and with other species in subsection Erioxylum. To evaluate further the possibility that this cpDNA evidence reflects introgression as opposed to some other evolutionary process, as well as to explore patterns of genetic diversity and similarity in both subsections, we conducted amplified fragment‐length polymorphism (AFLP) analysis using 50 populations representing all seven species in the two subsections. Genetic diversity is high in G. aridum, and is strongly correlated with geography, as are similarities among the five species in subsection Erioxylum. This subsection is genetically distant from the two species in subsection Integrifolia, whose populations are highly similar inter se. Populations of G. aridum from Colima are genetically distinct from the remainder of the species, and exhibit a comparatively high frequency of AFLP fragments that otherwise are diagnostic of the Integrifolia lineage. These data implicate intersubsectional introgression between presently allopatric and genetically isolated clades, giving rise to a morphologically cryptic, introgressant entity. Biogeographic considerations suggest that this history was initiated following migration of one or more seeds from Baja California to the Colima coast, perhaps during the Pleistocene. We suggest that cryptic and seemingly improbable interspecific introgression and molecular differentiation may be more common than appreciated in angiosperm evolution.

Selecting Single-Copy Nuclear Genes for Plant Phylogenetics: A Preliminary Analysis for the Senecioneae (Asteraceae)

Compared to organelle genomes, the nuclear genome comprises a vast reservoir of genes that potentially harbor phylogenetic signal. Despite the valuable data that sequencing projects of model systems offer, relatively few single-copy nuclear genes are being used in systematics. In part this is due to the challenges inherent in generating orthologous sequences, a problem that is ameliorated when the gene family in question has been characterized in related organisms. Here we illustrate the utility of diverse sequence databases within the Asteraceae as a framework for developing single-copy nuclear genes useful for inferring phylogenies in the tribe Senecioneae. We highlight the process of searching for informative genes by using data from Helianthus annuus, Lactuca sativa, Stevia rebaudiana, Zinnia elegans, and Gerbera cultivar. Emerging from this process were several candidate genes; two of these were used for a phylogenetic assessment of the Senecioneae and were compared to other genes previously used in Asteraceae phylogenies. Based on the preliminary sampling used, one of the genes selected during the searching process was more useful than the two previously used in Asteraceae. The search strategy described is valid for any group of plants but its efficiency is dependent on the phylogenetic proximity of the study group to the species represented in sequence databases.

Is the extremely rare Iberian endemic plant species Castrilanthemum debeauxii (Compositae, Anthemideae) a 'living fossil'? Evidence from a multi-locus species tree reconstruction

The present study provides results of multi-species coalescent species tree analyses of DNA sequences sampled from multiple nuclear and plastid regions to infer the phylogenetic relationships among the members of the subtribe Leucanthemopsidinae (Compositae, Anthemideae), to which besides the annual Castrilanthemum debeauxii (Degen, Hervier & É.Rev.) Vogt & Oberp., one of the rarest flowering plant species of the Iberian Peninsula, two other unispecific genera (Hymenostemma, Prolongoa), and the polyploidy complex of the genus Leucanthemopsis belong. Based on sequence information from two single- to low-copy nuclear regions (C16, D35, characterised by Chapman et al. (2007)), the multi-copy region of the nrDNA internal transcribed spacer regions ITS1 and ITS2, and two intergenic spacer regions of the cpDNA gene trees were reconstructed using Bayesian inference methods. For the reconstruction of a multi-locus species tree we applied three different methods: (a) analysis of concatenated sequences using Bayesian inference (MrBayes), (b) a tree reconciliation approach by minimizing the number of deep coalescences (PhyloNet), and (c) a coalescent-based species-tree method in a Bayesian framework ((∗)BEAST). All three species tree reconstruction methods unequivocally support the close relationship of the subtribe with the hitherto unclassified genus Phalacrocarpum, the sister-group relationship of Castrilanthemum with the three remaining genera of the subtribe, and the further sister-group relationship of the clade of Hymenostemma+Prolongoa with a monophyletic genus Leucanthemopsis. Dating of the (∗)BEAST phylogeny supports the long-lasting (Early Miocene, 15-22Ma) taxonomical independence and the switch from the plesiomorphic perennial to the apomorphic annual life-form assumed for the Castrilanthemum lineage that may have occurred not earlier than in the Pliocene (3Ma) when the establishment of a Mediterranean climate with summer droughts triggered evolution towards annuality.

Development of novel low-copy nuclear markers for Hieraciinae (Asteraceae) and their perspective for other tribes

PREMISE OF THE STUDY The development of three low-copy nuclear markers for low taxonomic level phylogenies in Asteraceae with emphasis on the subtribe Hieraciinae is reported. METHODS AND RESULTS Marker candidates were selected by comparing a Lactuca complementary DNA (cDNA) library with public DNA sequence databases. Interspecific variation and phylogenetic signal of the selected genes were investigated for diploid taxa from the subtribe Hieraciinae and compared to a reference phylogeny. Their ability to cross-amplify was assessed for other Asteraceae tribes. All three markers had higher variation (2.1-4.5 times) than the internal transcribed spacer (ITS) in Hieraciinae. Cross-amplification was successful in at least seven other tribes of the Asteraceae. Only three cases indicating the presence of paralogs or pseudogenes were detected. CONCLUSIONS The results demonstrate the potential of these markers for phylogeny reconstruction in the Hieraciinae as well as in other Asteraceae tribes, especially for very closely related species.

Evolution and Expression Patterns of CYC/TB1 Genes in Anacyclus: Phylogenetic Insights for Floral Symmetry Genes in Asteraceae

Homologs of the CYC/TB1 gene family have been independently recruited many times across the eudicots to control aspects of floral symmetry The family Asteraceae exhibits the largest known diversification in this gene paralog family accompanied by a parallel morphological floral richness in its specialized head-like inflorescence. In Asteraceae, whether or not CYC/TB1 gene floral symmetry function is preserved along organismic and gene lineages is unknown. In this study, we used phylogenetic, structural and expression analyses focused on the highly derived genus Anacyclus (tribe Anthemidae) to address this question. Phylogenetic reconstruction recovered eight main gene lineages present in Asteraceae: two from CYC1, four from CYC2 and two from CYC3-like genes. The species phylogeny was recovered in most of the gene lineages, allowing the delimitation of orthologous sets of CYC/TB1 genes in Asteraceae. Quantitative real-time PCR analysis indicated that in Anacyclus three of the four isolated CYC2 genes are more highly expressed in ray flowers. The expression of the four AcCYC2 genes overlaps in several organs including the ligule of ray flowers, as well as in anthers and ovules throughout development.

Molecular Confirmation of the Position of Gossypium trifurcatum Vollesen

Taxonomic understanding is a necessary prerequisite for intelligent germplasm maintenance and evaluation. Here, we use molecular evidence to address the generic position of the poorly known and morphologically unusual taxon Gossypium trifurcatum Vollesen. This species possesses dentate leaves, a feature not otherwise found in Gossypium L. but one that is common in Cienfuegosia Cav., a related genus in the small Malvaceous tribe Gossypieae. G. trifurcatum is a rare plant, restricted to deserts of Eastern Somalia and known from only two collections, the last in 1980. Using DNA extracted from an herbarium specimen, we amplified and sequenced the chloroplast gene ndhF. Phylogenetic analysis reveals G. trifurcatum to be cladistically nested within Gossypium. These data diagnose dentate leaves as an autapomorphy within a genetically diverse assemblage of African–Arabian species, which remain the least well-represented cottons in germplasm collections.