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Mori, T. S. Nunes, T. D. Pennington, J. R. Pirani, G. T. Prance, L. P. de Queiroz, A. Rapini, R. Riina, C. A. V. Rincon, N. Roque, G. Shimizu, M. Sobral, J. R. Stehmann, W. D. Stevens, C. M. Taylor, M. Trovo, C. van den Berg, H. van der Werff, P. L. Viana, C. E. Zartman, R. C. Forzza, Amazon plant diversity revealed

See 'Read me.rtf' (included in 'Data S1.rar') for a description of the files included in this dataset. Abstract of study: Early natural historians – Comte de Buffon, von Humboldt and De Candolle – established environment and geography as two principal axes determining the distribution of groups of organisms, laying the foundations for biogeography over the subsequent 200 years, yet the relative importance of these two axes remains unresolved. Leveraging phylogenomic and global species distribution data for Mimosoid legumes, a pantropical plant clade of c. 3,500 species, we show that the water availability gradient from deserts to rainforests dictates turnover of lineages within continents across the tropics. We demonstrate that 95% of speciation occurs within a precipitation niche, showing profound phylogenetic niche conservatism, and that lineage turnover boundaries coincide with isohyets of precipitation. We reveal similar patterns on different continents, implying that evolution and dispersal follow universal processes.

The mustard family (Brassicaceae) is a scientifically and economically important family, containing the model plant Arabidopsis thaliana and numerous crop species that feed billions worldwide. Despite its relevance, most published family phylogenies are incompletely sampled, generally contain massive polytomies, and/or show incongruent topologies between datasets. Here, we present the most complete Brassicaceae genus-level family phylogenies to date (Brassicaceae Tree of Life, or BrassiToL) based on nuclear (>1,000 genes, almost all 349 genera and 53 tribes) and plastome (60 genes, 79% of the genera, all tribes) data. We found cytonuclear discordance between nuclear and plastome-derived phylogenies, which is likely a result of rampant hybridisation among closely and more distantly related species, and highlight rogue taxa. To evaluate the impact of this rampant hybridisation on the nuclear phylogeny reconstruction, we performed four different sampling routines that increasingly removed variable data and likely paralogs. Our resulting cleaned subset of 297 nuclear genes revealed high support for the tribes, while support for the main lineages remained relatively low. Calibration based on the 20 most clock-like nuclear genes suggests a late Eocene to late Oligocene ‘icehouse origin’ of the family. Finally, we propose five new or re-established tribes, including the recognition of Arabidopsideae, a monotypic tribe to accommodate Arabidopsis. With a worldwide community of thousands of researchers working on this family, our new, densely sampled family phylogeny will be an indispensable tool to further highlight Brassicaceae as an excellent model family for studies on biodiversity and plant biology.

Early natural historians – Compte de Buffon, von Humboldt and De Candolle – established ecology and geography as two principal axes determining the distribution of groups of organisms, laying the foundations for biogeography over the subsequent 200 years, yet the relative importance of these two axes remains unresolved. Leveraging phylogenomic and global species distribution data for Mimosoid legumes, an pantropical plant clade of 3,400 species, we show that the water availability gradient from deserts to rainforests dictates turnover of lineages within continents across the tropics. We demonstrate that 95% of speciation occurs within a precipitation niche, showing profound phylogenetic niche conservatism, and that lineage turnover boundaries coincide with isohyets of precipitation. We reveal similar patterns on different continents, implying that evolution and dispersal follow universal processes. Fil: Ringelberg, Jens J. University of Zurich. Department of Systematic and Evolutionary Botany; Suiza Fil: Koenen, Erik J.M. University of Zurich. Department of Systematic and Evolutionary Botany; Suiza. Université Libre de Bruxelles. Faculté des Sciences. Evolutionary Biology & Ecology; Bélgica Fil: Sauter, Benjamín. University of Zurich. Department of Systematic and Evolutionary Botany; Suiza Fil: Aebli, Anahita. University of Zurich. Department of Systematic and Evolutionary Botany; Suiza. Abteling Umweltschutz und Energie. Departement Bau und Umwelt; Suiza Fil: Rando, Juliana G. Universidade Federal do Oeste da Bahia. Centro das Ciências Biológicas e da Saúde. Programa de Pós Graduação em Ciências Ambientais; Brasil Fil: Iganci, João R. Universidade Federal de Pelotas. Campus Universitário Capão do Leão. Instituto de Biologia; Brasil. Universidade Federal do Rio Grande do Sul. Programa de Pós-Graduação em Botânica; Brasil Fil: de Queiroz, Luciano P. Universidade Estadual de Feira de Santana. Departamento Ciências Biológicas; Brasil Fil: Murphy, Daniel J. Royal Botanic Gardens Victoria: Australia Fil: Gaudeul, Myriam. Institut de Systématique, Evolution, Biodiversité (ISYEB), MNHN-CNRS-SU-EPHE-UA: Francia Fil: Bruneau, Anne. Université de Montréal. Institut de Recherche en Biologie Végétale and Département de Sciences Biologiques; Canadá Fil: Luckow, Melissa. Cornell University. School of Integrative Plant Science. Plant Biology Section; Estados Unidos Fil: Morales, Matias. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Recursos Biológicos; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Morón. Facultad de Agronomía y Ciencias Agroalimentarias; Argentina

Strategies to breed sterile leucaena for Western Australia include plant breeding and biotechnology tools to generate sterile lines at both the tetraploid and triploid ploidy levels. For tetraploids, the main target species is the commercial Leucaena leucocephala, that is well known for its potential as a high-quality, productive and persistent forage. Gene editing technologies (CRISPR) will be utilized to edit out flowering genes and develop a non-flowering L. leucocephala and/or create male/female genic sterile lines of L. leucocephala. For triploids, the strategy is to cross tetraploid species (L. leucocephala and/or L. diversifolia) with diploid species to generate sterile triploid hybrids. The diploid parents will include species that have good forage attributes such as L. collinsii, L. macrophylla, L. shannonii and L. pulverulenta. Several of these triploid crosses have already been created by the Department of Primary Industries and Regional Development (Perth, Western Australia) and will be evaluated in the Kimberley and Pilbara regions of Western Australia for their agronomic performance and sterility. Vegetative propagation will be required for the tetraploid gene-edited non-flowering L. leucocephala. Triploids can either be vegetatively propagated, once generated, or generated via a seed production nursery. Resumen Las estrategias para producir leucaena estéril para el estado de Western Australia incluyen herramientas de fitomejoramiento y biotecnología para generar líneas estériles tanto en el nivel tetraploide como triploide. Para los tetraploides, la principal especie objetivo es la leucaena comercial, Leucaena leucocephala, que es bien conocida por su potencial como forraje de alta calidad, productividad y persistencia. Para la producción de líneas estériles, se utilizarán tecnologías de edición genética (CRISPR) para eliminar los genes de floración y desarrollar una L. leucocephala que no florece o crear líneas de L. leucocephala que tengan esterilidad genética masculina o femenina. Para los triploides, la estrategia es cruzar especies tetraploides (L. leucocephala y/o L. diversifolia) con especies diploides para generar híbridos triploides estériles. Los padres diploides incluirán especies que tengan buenos atributos forrajeros, como L. collinsii, L. macrophylla, L. shannonii y L. pulverulenta. Varios de estos cruces triploides ya han sido creados por el Department of Primary Industries and Regional Development (Perth, Western Australia) y serán evaluados en las regiones de Kimberley y Pilbara en Western Australia respecto a su desempeño agronómico y esterilidad. Para la tetraploide L. leucocephala producida por técnicas de CRISPR y que no florece, se requerirán métodos de propagación vegetativa. En cuanto a triploides, una vez generados podrán ser propagados tanto vegetativamente como con base en semilla producida en un vivero de producción de semillas.

• The Brassicaceae family comprises ca. 4000 species including economically important crops and the model plant Arabidopsis thaliana. Despite their importance, the relationships among major lineages in the family remain unresolved hampering comparative research.• Here, we inferred a Brassicaceae phylogeny using newly generated targeted enrichment sequence data of 1827 exons (>940,000 bases) representing 63 species as well as sequenced genome data of 16 species, together representing 50 of the 52 currently recognized Brassicaceae tribes. A third of the samples were derived from herbarium material, facilitating broad taxonomic coverage of the family.• Six major clades formed successive sister groups to the rest of Brassicaceae. We also recovered strong support for novel relationships among tribes, and resolved the position of 16 taxa previously not assigned to a tribe. The broad utility of these phylogenetic results is illustrated through a comparative investigation of genome-wide expression signatures that distinguish simple from complex leaves in Brassicaceae.• Our study provides an easily extended dataset for further advances in Brassicaceae systematics and a timely higher-level phylogenetic framework for a wide range of comparative studies of multiple traits in an intensively investigated group of plants. This article is protected by copyright. All rights reserved.

PREMISE: Researchers adopting target-enrichment approaches often struggle with the decision of whether to use universal or lineage-specific probe sets. To circumvent this quandary, we investigate the efficacy of a simultaneous enrichment by combining universal probes and lineage-specific probes in a single hybridization reaction, to benefit from the qualities of both probe sets with little added cost or effort.METHODS AND RESULTS: Using 26 Brassicaceae libraries and standard enrichment protocols, we compare results from three independent data sets. A large average fraction of reads mapping to the Angiosperms353 (24–31%) and Brassicaceae (35–59%) targets resulted in a sizable reconstruction of loci for each target set (x̄ ≥ 70%).CONCLUSIONS: High levels of enrichment and locus reconstruction for the two target sets demonstrate that the sampling of genomic regions can be easily extended through the combination of probe sets in single enrichment reactions. We hope that these findings will facilitate the production of expanded data sets that answer individual research questions and simultaneously allow wider applications by the research community as a whole.

Reconstructions of vascular plant mitochondrial genomes (mt-genomes) are notoriously complicated by rampant recombination that has resulted in comparatively few plant mt-genomes being available. The dearth of plant mitochondrial resources has limited our understanding of mt-genome structural diversity, complex patterns of RNA editing, and the origins of novel mt-genome elements. Here, we use an efficient long read (PacBio) iterative assembly pipeline to generate mt-genome assemblies for Leucaena trichandra (Leguminosae: Caesalpinioideae: mimosoid clade), providing the first assessment of non-papilionoid legume mt-genome content and structure to date. The efficiency of the assembly approach facilitated the exploration of alternative structures that are common place among plant mitochondrial genomes. A compact version (729 kbp) of the recovered assemblies was used to investigate sources of mt-genome size variation among legumes and mt-genome sequence similarity to the legume associated root holoparasite Lophophytum. The genome and an associated suite of transcriptome data from select species of Leucaena permitted an in-depth exploration of RNA editing in a diverse clade of closely related species that includes hybrid lineages. RNA editing in the allotetraploid, Leucaena leucocephala, is consistent with co-option of nearly equal maternal and paternal C-to-U edit components, generating novel combinations of RNA edited sites. A preliminary investigation of L. leucocephala C-to-U edit frequencies identified the potential for a hybrid to generate unique pools of alleles from parental variation through edit frequencies shared with one parental lineage, those intermediate between parents, and transgressive patterns.

The classification of the legume family proposed here addresses the long-known non-monophyly of the traditionally recognised subfamily Caesalpinioideae, by recognising six robustly supported monophyletic subfamilies. This new classification uses as its framework the most comprehensive phylogenetic analyses of legumes to date, based on plastid matK gene sequences, and including near-complete sampling of genera (698 of the currently recognised 765 genera) and ca. 20% (3696) of known species. The matK gene region has been the most widely sequenced across the legumes, and in most legume lineages, this gene region is sufficiently variable to yield well-supported clades. This analysis resolves the same major clades as in other phylogenies of whole plastid and nuclear gene sets (with much sparser taxon sampling). Our analysis improves upon previous studies that have used large phylogenies of the Leguminosae for addressing evolutionary questions, because it maximises generic sampling and provides a phylogenetic tree that is based on a fully curated set of sequences that are vouchered and taxonomically validated. The phylogenetic trees obtained and the underlying data are available to browse and download, facilitating subsequent analyses that require evolutionary trees. Here we propose a new community-endorsed classification of the family that reflects the phylogenetic structure that is consistently resolved and recognises six subfamilies in Leguminosae: a recircumscribed Caesalpinioideae DC., Cercidoideae Legume Phylogeny Working Group (stat. nov.), Detarioideae Burmeist., Dialioideae Legume Phylogeny Working Group (stat. nov.), Duparquetioideae Legume Phylogeny Working Group (stat. nov.), and Papilionoideae DC. The traditionally recognised subfamily Mimosoideae is a distinct clade nested within the recircum-scribed Caesalpinioideae and is referred to informally as the mimosoid clade pending a forthcoming formal tribal and/or clade-based classification of the new Caesalpinioideae. We provide a key for subfamily identification, descriptions with diagnostic charactertistics for the subfamilies, figures illustrating their floral and fruit diversity, and lists of genera by subfamily. This new classification of Leguminosae represents a consensus view of the international legume systematics community; it invokes both compromise and practicality of use.

Milkweeds (Asclepias) are used in wide-ranging studies including floral development, pollination biology, plant-insect interactions and co-evolution, secondary metabolite chemistry, and rapid diversification. We present a transcriptome and draft nuclear genome assembly of the common milkweed, Asclepias syriaca. This reconstruction of the nuclear genome is augmented by linkage group information, adding to existing chloroplast and mitochondrial genomic resources for this member of the Apocynaceae subfamily Asclepiadoideae. The genome was sequenced to 80.4× depth and the draft assembly contains 54,266 scaffolds ≥1 kbp, with N50 = 3415 bp, representing 37% (156.6 Mbp) of the estimated 420 Mbp genome. A total of 14,474 protein-coding genes were identified based on transcript evidence, closely related proteins, and ab initio models, and 95% of genes were annotated. A large proportion of gene space is represented in the assembly, with 96.7% of Asclepias transcripts, 88.4% of transcripts from the related genus Calotropis, and 90.6% of proteins from Coffea mapping to the assembly. Scaffolds covering 75 Mbp of the Asclepias assembly formed eleven linkage groups. Comparisons of these groups with pseudochromosomes in Coffea found that six chromosomes show consistent stability in gene content, while one may have a long history of fragmentation and rearrangement. The progesterone 5β-reductase gene family, a key component of cardenolide production, is likely reduced in Asclepias relative to other Apocynaceae. The genome and transcriptome of common milkweed provide a rich resource for future studies of the ecology and evolution of a charismatic plant family.

Milkweeds (Asclepias) are used in wide-ranging studies including floral development, pollination biology, plant-insect interactions and co-evolution, secondary metabolite chemistry, and rapid diversification. We present the first nuclear genome and transcriptome assemblies of the common milkweed, Asclepias syriaca. This is the first species in Apocynaceae subfamily Asclepiadoideae with reconstructions of the nuclear, chloroplast, and mitochondrial genomes, and the first in the Apocynaceae to have linkage group information incorporated into the nuclear assembly. The genome was sequenced to 80.4× depth and the draft assembly contains 54,266 scaffolds ≥1 kbp, with N50 = 3415 bp, representing 37% (156.6 Mbp) of the estimated 420 Mbp genome. A total of 14,474 protein-coding genes were identified based on transcript evidence, closely related proteins, and ab initio models, and 95% of genes were annotated. A large proportion of gene space is represented in the assembly, with 96.7% of Asclepias transcripts, 88.4% of transcripts from the related genus Calotropis, and 90.6% of proteins from Coffea mapping to the assembly. The progesterone 5β-reductase gene family, a key component of cardenolide production, is likely reduced in Asclepias relative to other Apocynaceae. Scaffolds covering 75 Mbp of the Asclepias assembly formed eleven linkage groups. Comparisons of these groups with pseudochromosomes in Coffea found that six chromosomes show consistent stability in gene content, while one may have a long history of fragmentation and rearrangement. The genome and transcriptome of common milkweed provide a rich resource for future studies of the ecology and evolution of a charismatic plant family.

To date, publicly available plastid genomes of legumes have for the most part been limited to the subfamily Papilionoideae. Here we report 13 new plastid genomes of legumes spanning all three subfamilies. The genomes representing Caesalpinioideae and Mimosoideae are highly conserved in gene content and gene order, similar to the ancestral angiosperm genome organization. Genomes within the Papilionoideae, however, have reduced sizes due to deletions in nine intergenic spacers primarily in the large single copy region. Our study also indicates that rps16 has been independently lost at least five times in legumes, with additional gene and intron losses scattered among the papilionoids. Additionally, genera from two distinct lineages within the papilionoids, Lupinus and Robinia, have a parallel inversion of 36 and 39 kb, respectively. This parallel inversion is novel as it appears to be caused by a 29 bp repeat within two trnS genes. This repeat is present in all available legume plastid genomes indicating that there is the potential for this inversion to be present in more species. This case of a homoplasious inversion is also evidence that some inversion events may not be reliable phylogenetic markers.

Hybrid speciation is relatively common in plants compared to other well-studied groups. Polyploidy and apomixis are strongly associated with hybrid speciation, presumably due to the opportunities they provide for both reestablishing reproductive function in hybrids with incomplete chromosomal homology and creating rapid reproductive isolation in sympatry. Boechera, a species-rich genus closely related to Arabidopsis, is a particularly fertile ground for the study of hybrid speciation. Thirty-eight apomictic triploid hybrid species are currently recognized in Boechera. Recent research has shown that apomictic diploid hybrids, although very rare in angiosperms, are common in Boechera. Given this complexity, focused studies of individual species complexes are critical to understanding speciation and diagnosing biodiversity in Boechera. Here we analyze DNA sequences from seven nuclear loci and multilocus genotypes from 15 microsatellite markers in a group of closely related taxa formerly included in ...

The insect fat body plays a central role in insect metabolism and nutrient storage, mirroring functions of the liver and fat tissue in vertebrates. Insect fat body tissue is usually distributed throughout the insect body. However, it is often concentrated in the abdomen and attached to the abdominal body wall. The mosquito fat body is the sole source of yolk proteins, which are critical for egg production. Therefore, the in vitro culture of mosquito fat body tissues represents an important system for the study of mosquito physiology, metabolism, and, ultimately, egg production. The fat body culture process begins with the preparation of solutions and reagents, including amino acid stock solutions, Aedes physiological saline salt stock solution (APS), calcium stock solution, and fat body culture medium. The process continues with fat body dissection, followed by an experimental treatment. After treatment, a variety of different analyses can be performed, including RNA sequencing (RNA-Seq), qPCR, Western blots, proteomics, and metabolomics. In our example experiment, we demonstrate the protocol through the excision and culture of fat bodies from the yellow fever mosquito, Aedes aegypti, a principal vector of arboviruses including dengue, chikungunya, and Zika. RNA from fat bodies cultured under a physiological condition known to upregulate yolk proteins versus the control were subject to RNA-Seq analysis to demonstrate the potential utility of this procedure for investigations of gene expression.

The present study aimed to develop a robust, extensively sampled phylogenetic hypothesis across all species in Leavenworthia and Selenia using chloroplast (psbJ—petA) and nuclear encoded (nrDNA ITS and LUMINIDEPENDENS) DNA sequence data. Parsimony and maximum likelihood methodologies were employed. Strong maximum likelihood bootstrap and parsimony jackknife support were recovered for the monophyly of the sister pair Leavenworthia plus Selenia and for the respective monophyly of each genus. Leavenworthia and Selenia are hypothesized to have diverged in the Ozark Mountains and results from an analysis of branch lengths leading to Leavenworthia and Selenia clades are consistent with Leavenworthia having diversified more recently than Selenia. Polyploidy and extensive aneuploidy complicate the interpretation of chromosomal evolution within and between these genera. Comparative results from phylogenetic analyses indicate that L. aurea and L. texana are hybrids with a shared set of putative parents. La...

Boechera (Brassicaceae) is a diverse genus of ±70 sexual diploid species and numerous apomictic hybrids concentrated in western North America. It is the largest genus in tribe Boechereae, which also includes seven other small genera. Boechera is closely related to Arabidopsis and is becoming a model genus for diverse studies, focusing particularly on apomixis and hybrid speciation. As part of an ongoing effort to clarify the taxonomy of the group, we present phylogenetic analyses of Boechereae, including all genera of the tribe and most of the sexual diploid species of Boechera. Ten loci are used, including two plastid loci, nrDNA ITS, and seven low-copy nuclear loci. These analyses indicate that Boechera, as currently circumscribed, is polyphyletic, comprising three main clades. Eastern North American species previously assigned to Boechera form a distinct clade with the Asian taxon Borodinia macrophylla and are herein transferred to the genus Borodinia, resulting in seven new combinations (Boro...

Here we investigate mechanisms underlying the diversification of biological forms using crucifer leaf shape as an example. We show that evolution of an enhancer element in the homeobox gene REDUCED COMPLEXITY (RCO) altered leaf shape by changing gene expression from the distal leaf blade to its base. A single amino acid substitution evolved together with this regulatory change, which reduced RCO protein stability, preventing pleiotropic effects caused by its altered gene expression. We detected hallmarks of positive selection in these evolved regulatory and coding sequence variants and showed that modulating RCO activity can improve plant physiological performance. Therefore, interplay between enhancer and coding sequence evolution created a potentially adaptive path for morphological evolution.

Gametophytic apomixis is a common form of asexual reproduction in plants. Virtually all gametophytic apomicts are polyploids, and some view polyploidy as a prerequisite for the transition to apomixis. However, any causal link between apomixis and polyploidy is complicated by the fact that most apomictic polyploids are allopolyploids, leading some to speculate that hybridization, rather than polyploidy, enables apomixis. Diploid apomixis presents a rare opportunity to isolate the role of hybridization, and a number of diploid apomicts have been documented in the genus Boechera (Brassicaceae). Here, we present the results of a microsatellite study of 1393 morphologically and geographically diverse diploid individuals, evaluating the hypothesis that diploid Boechera apomicts are hybrids. This genus-wide dataset was made possible by the applicability of a core set of microsatellite loci in 69 of the 70 diploid Boechera species and by our ability to successfully genotype herbarium specimens of widely varying ages. With few exceptions, diploid apomicts exhibited markedly high levels of heterozygosity resulting from the combination of disparate genomes. This strongly suggests that most apomictic diploid Boechera lineages are of hybrid origin, and that the genomic consequences of hybridization allow for the transition to gametophytic apomixis in this genus.

 Premise of the study: The evolutionary history of Leucaena has been impacted by polyploidy, hybridization, and divergent allopatric species diversifi cation, suggesting that this is an ideal group to investigate the evolutionary tempo of polyploidy and the complexities of reticulation and divergence in plant diversifi cation.  Methods: Parsimony- and ML-based phylogenetic approaches were applied to 105 accessions sequenced for six sequence characterized amplifi ed region-based nuclear encoded loci, nrDNA ITS, and four cpDNA regions. Hypotheses for the origin of tetraploid species were inferred using results derived from a novel species tree and established gene tree methods and from data on genome sizes and geographic distributions.  Results: The combination of comprehensively sampled multilocus DNA sequence data sets and a novel methodology provide strong resolution and support for the origins of all fi ve tetraploid species. A minimum of four allopolyploidization events are required to explain the origins of these species. The origin(s) of one tetraploid pair ( L. involucrata/L. pallida ) can be equally explained by two unique allopolyploidizations or a single event followed by divergent speciation.  Conclusions: Alongside other recent fi ndings, a comprehensive picture of the complex evolutionary dynamics of polyploidy in Leucaena is emerging that includes paleotetraploidization, diploidization of the last common ancestor to Leucaena , allopatric divergence among diploids, and recent allopolyploid origins for tetraploid species likely associated with human translocation of seed. These results provide insights into the role of divergence and reticulation in a well-characterized angiosperm lineage and into traits of diploid parents and derived tetraploids (particularly self-compatibility and year-round fl owering) favoring the formation and establishment of novel tetraploids combinations.

The relationships and taxonomy of the genus Thysanocarpus (Brassicaceae) are reassessed based on molecular phylogenetic analyses of nuclear ribosomal (ITS) and chloroplast (trnh-F) sequences and a critical re-examination of morphology and nomenclatural types. Based on these results, Thysanocarpus is well-supported as a member of tribe Thelypodieae, but no illuminating phylogenetic structure is found within the tribe. The independent origin of similar fruit morphology in Thysanocarpus and Athysanus is confirmed. Within Thysanocarpus,seven species are recognized: T. conchuliferus, T. curvipes, T. desertorum, T. erectus, T. laciniatus, T. radians, and T. rigidus comb. nov. Thysanocarpus laciniatus is found to have originated through hybridization. However, T. desertorum and T. rigidus, which have previously been included within T. laciniatus (as T. laciniatus var. hitchcockii and T. laciniatus var. rigidus,respectively), do not share that species' hybrid origin and are distinct both phylogenetically and morphologically. Within T. curvipes,five subspecies are recognized: T. curvipes subsp. amplectens comb. nov., T. curvipes subsp. curvipes, T. curvipes subsp. elegans comb. nov., T. curvipes subsp. longistylus comb. nov., and T. curvipes subsp. eradiatus comb. nov. Thysanocarpus curvipes subsp. elegans and T. curvipes subsp. longistylus form clades in ITS and/or trnL-F cladograms as well as showing morphological distinction. The remaining three subspecies are recognized based on a combination of morphology and geography.

Backyard gardens, dump heaps, and kitchen middens are thought to have provided important venues for early crop domestication via generation of hybrids between otherwise isolated plant species. However, this process has rarely been demonstrated empirically. For the majority of polyploid crops, it remains uncertain to what extent hybridization and polyploidization preceded domestication or were precipitated by human activities. Using archaeological, ethnobotanical, geographical, and genetic data, we investigate the extent and significance of predomestication cultivation, backyard sympatry, and spontaneous hybridization for the Mimosoid legume tree Leucaena , which is used as a food crop throughout south-central Mexico. We show that predomestication cultivation was widespread, involved numerous independent transitions from the wild to cultivation, and resulted in extensive artificial sympatry of 2–6 species locally and 13 species in total. Using chloroplast and rapidly evolving nuclear-encoded DNA sequences, we demonstrate that hybridization in Leucaena has been extensive and complex, spawning a diverse set of novel hybrids as a result of juxtaposition of species in cultivation. The scale and complexity of hybridization in Leucaena is significantly greater than that documented for any other Mexican plant domesticates so far. However, there are striking parallels between Leucaena and the other major Mexican perennial domesticates Agave and Opuntia , which show very similar domestication via backyard hybridization pathways. Our results suggest that backyard hybridization has played a central role in Mesoamerican crop domestication and demonstrate that the simple step of bringing species together in cultivation can provide a potent trigger for domestication.

In 2002, a monophyletic group previously unrecognized within Brassicaceae was identified through phylogenetic analysis of morphological characters in combination with data from three DNA sequences (Bailey et al.: Syst. Bot. 27: 318-332). As understood at that time, the halimolobine alliance (herein tribe Halimolobeae) comprised ca. 44 species from Halimolobos, Mancoa, Pennellia, and Sphaerocardamum. Within this group, a number of nomenclatural problems have remained unaddressed, including the polyphyly of both Halimolobos and Mancoa. With a developing understanding of Brassicaceae phylogeny, the Halimolobeae is robust in the context of higher level analyses and therefore in need of formal nomenclatural revision. The present study uses new and existing data in phylogenetic analyses based on trnL-F, ITS, and pistillata intron 1 data along with SEM studies of Synthlipsis elata and S. greggii to provide the bases for recircumscription of taxa assigned to the Halimolobeae. As recognized herein, the tribe includes five genera (Exhalimolobos, Halimolobos (including Synthlipsis elata), Mancoa, Pennellia, and Sphaerocardamum) and 39 species with two centers of distribution, one in north-central Mexico and the other in the Andes. A synopsis and keys to all taxa of the tribe are presented. Fifteen new combinations are proposed (E. arabioides, E. berlandieri, E. burkartii, E. hispidulus, E. palmeri, E. parryi, E. pazense, E. polyspermus, E. weddellii, H. elatus, H. henricksonii, H. pubens, H. stylosus, P. lechleri, and P. parvifolia).

The time needed for hand grinding and the cost of commercially available extraction kits remain to be the major limitations in plant DNA extraction for many researchers. We present inexpensive techniques for (i) simultaneously machine grinding large numbers of plant samples for DNA extraction using a commercially available reciprocating saw; and (ii) DNA recovery using silica column-based extractions similar to that used in some commercially available kits. Used together, these allow for the rapid recovery of plant DNA at relatively low cost. Furthermore, these methods appear to be widely applicable within plants with good yields recovered in test extractions across major plant groups (ferns, gymnosperms, monocots and eudicots).

The Brassicaceae is a large plant family (338 genera and 3,700 species) of major scientific and economic importance. The taxonomy of this group has been plagued by convergent evolution in nearly every morphological feature used to define tribes and genera. Phylogenetic analysis of 746 nrDNA internal transcribed spacer (ITS) sequences, representing 24 of the 25 currently recognized tribes, 146 genera, and 461 species of Brassicaceae, produced the most comprehensive, single-locus-based phylogenetic analysis of the family published to date. Novel approaches to nrDNA ITS analysis and extensive taxonomic sampling offered a test of monophyly for a large complement of the currently recognized tribes and genera of Brassicaceae. In the most comprehensive analysis, tribes Alysseae, Anchonieae plus Hesperideae, Boechereae, Cardamineae, Eutremeae, Halimolobeae, Iberideae, Noccaeeae, Physarieae, Schizopetaleae, Smelowskieae, and Thlaspideae were all monophyletic. Several broadly defined genera (e.g., Draba and Smelowskia) were supported as monophyletic, whereas others (e.g., Sisymbrium and Alyssum) were clearly polyphyletic. Analyses of ITS data identified several problematic sequences attributable to errors in sample identification or database submission. Results from parsimony ratchet and Bayesian analyses recovered little support for the backbone of the phylogeny, suggesting that many lineages of Brassicaceae have undergone rapid radiations that may ultimately be difficult to resolve with any single locus. However, the development of a preliminary supermatrix including the combination of 10 loci for 65 species provides an initial estimate of intertribal relations and suggests that broad application of such a method will provide greater understanding of relationships in the family.

Conditioned reconstruction (CR)1 represents a new phylogenetic method that has been presented as a means of utilizing vast amounts of gene absence/presence data to reconstruct phylogenetic relationships and to directly study the inXuence of genome fusion on evolution (Lake and Rivera, 2004; Rivera and Lake, 2004; Simonson et al., 2005). In the Wrst direct application of CR, the results were stated to unambiguously support the role of an archaeal and eubacterial genome fusion event in the ancestry of the eukaryotic genome (McInerney and Wilkinson, 2005; Rivera and Lake, 2004; Simonson et al., 2005). In the present manuscript, we outline the basic components of CR, discuss the logic behind their application, and subsequently identify concerns with aspects of data interpretation and the current use of conditioning genomes and aligned networks in the study of genome fusion. Conditioned reconstruction begins with the selection of a conditioning genome (CG) that will represent the full set of orthologous genes coded during matrix development (Lake and Rivera, 2004). Strong emphasis has been placed on the strict use of relatively small genomes as conditioning genomes (Lake and Rivera, 2004). This recommendation is based on the observation that the use of relatively large conditioning genomes can induce an artifact referred to as “big genome attraction,” which can mislead phylogenetic inference. All other genomes that will be used in the analysis are scored for the absence or presence of a putative ortholog to each gene in the CG. Orthologs present in other genomes included in the

Resolving phylogenies among closely related species remains a perplexing problem in plant systematics. All too often cpDNA and nrDNA ITS are insufficiently variable to provide desired resolution or support. In the present study, we have adopted a RAPD-based approach to develop sequence-characterized regions, from the nuclear genome, with levels of DNA sequence variation appropriate for resolving relationships within subclades of Leucaena (Fabaceae). RAPDs were used to amplify a set of seven Leucaena accessions. Equal length fragments amplified in two or more species were sequenced and compared. Specific primers were designed for aligned product sets displaying similar or greater levels of variation than have been found in previous ITS studies. Two regions whose DNA sequences provided greater resolution and bootstrap support than ITS or cpDNA RFLP restriction site data sets for the same sample of Leucaena species are discussed in detail.

In this work, we investigate morphological differences between Arabidopsis thaliana, which has simple leaves, and its relative Cardamine hirsuta, which has dissected leaves comprising distinct leaflets. With the use of genetics, interspecific gene transfers, and time-lapse imaging, we show that leaflet development requires the REDUCED COMPLEXITY (RCO) homeodomain protein. RCO functions specifically in leaves, where it sculpts developing leaflets by repressing growth at their flanks. RCO evolved in the Brassicaceae family through gene duplication and was lost in A. thaliana, contributing to leaf simplification in this species. Species-specific RCO action with respect to its paralog results from its distinct gene expression pattern in the leaf base. Thus, regulatory evolution coupled with gene duplication and loss generated leaf shape diversity by modifying local growth patterns during organogenesis.

In this paper, we propose a new method (uninode coding) for coding duplicate (paralogous) genes to infer species trees. Uninode coding incorporates data from duplicated and unduplicated gene copies in phylogenetic analyses of taxa. Uninode coding utilizes global parsimony through the inclusion of both duplicated and unduplicated gene copies, allows one to code all data sources from a taxon into a single terminal, and overcomes problems of character dependence among duplicated and unduplicated gene copies. We present an example of uninode coding using the phytochrome A and phytochrome C data from a study by Donoghue and Mathews.

We report the potential phylogenetic utility of DNA sequence data from the last 700 bp of a ca. 1-kb intron of the MADS-box gene pistillata from a sampling of Sphaerocardamum species and other Brassicaceae. These results are compared with nrDNA ITS and the chloroplast trnL intron for the same taxa to demonstrate the potential phylogenetic utility of this pistillata intron and to identify potential historically independent sequences for an ongoing study of relationships within Sphaerocardamum. Analyses of the DNA sequence data for Brassicaceae indicated that pairwise divergences and potentially informative characters were higher in the pistillata intron (0.6–30.8%, 284 characters) and ITS (0–24%, 94 characters) than in the chloroplast trnL intron (0–4.2%, 17 characters). A comparison of Sphaerocardamum sequences identified low divergences and numbers of informative characters for trnL intron (0–2.4%, 1 character) and nrDNA ITS (0–2.5%, 2 characters) and substantially more variation among the pistillata sequences (0.15–3.7%, 19 characters). Phylogenetic analyses of these pistillata sequences fully resolve ingroup relationships without character conflict. Results of pistillata PCR amplifications from a broader dicot sample showed that some primers may be useful in amplifying orthologous pistillata sequences. Ultimately this pistillata intron may be a valuable source of phylogenetic characters at lower taxonomic levels.

Gametophytic apomixis is a common form of asexual reproduction in plants. Virtually all gametophytic apomicts are polyploids, and some view polyploidy as a prerequisite for the transition to apomixis. However, any causal link between apomixis and polyploidy is complicated by the fact that most apomictic polyploids are allopolyploids, leading some to speculate that hybridization, rather than polyploidy, enables apomixis. Diploid apomixis presents a rare opportunity to isolate the role of hybridization, and a number of diploid apomicts have been documented in the genus Boechera (Brassicaceae). Here, we present the results of a microsatellite study of 1393 morphologically and geographically diverse diploid individuals, evaluating the hypothesis that diploid Boechera apomicts are hybrids. This genus-wide dataset was made possible by the applicability of a core set of microsatellite loci in 69 of the 70 diploid Boechera species and by our ability to successfully genotype herbarium specimens of widely varying ages. With few exceptions, diploid apomicts exhibited markedly high levels of heterozygosity resulting from the combination of disparate genomes. This strongly suggests that most apomictic diploid Boechera lineages are of hybrid origin, and that the genomic consequences of hybridization allow for the transition to gametophytic apomixis in this genus.

 Premise of the study: Leucaena comprises 17 diploid species, fi ve tetraploid species, and a complex series of hybrids whose evolutionary histories have been infl uenced by human seed translocation, cultivation, and subsequent spontaneous hybridization. Here we investigated patterns of evolutionary divergence among diploid Leucaena through comprehensively sampled multilocus phylogenetic and population genetic approaches to address species delimitation, interspecifi c relationships, hybridization, and the predominant mode of speciation among diploids.  Methods: Parsimony- and maximum-likelihood-based phylogenetic approaches were applied to 59 accessions sequenced for six SCAR-based nuclear loci, nrDNA ITS, and four cpDNA regions. Population genetic comparisons included 1215 AFLP loci representing 42 populations and 424 individuals.  Results: Phylogenetic results provided a well-resolved hypothesis of divergent species relationships, recovering previously recognized clades of diploids as well as newly resolved relationships. Phylogenetic and population genetic assessments identifi ed two cryptic species that are consistent with geography and morphology.  Conclusions: Findings from this study highlight the importance and utility of multilocus data in the recovery of complex evolutionary histories. The results are consistent with allopatric divergence representing the predominant mode of speciation among diploid Leucaena . These fi ndings contrast with the potential hybrid origin of several tetraploid species and highlight the importance of human translocation of seed to the origin of these tetraploids. The recognition of one previously unrecognized species ( L. cruziana ) and the elevation of another taxon ( L. collinsii subsp. zacapana ) to specifi c status ( L. zacapana ) is consistent with a growing number of newly diagnosed species from neotropical seasonally dry forests, suggesting these communities harbor greater species diversity than previously recognized.

 Premise of the study: As a crop and medicinal plant, the octoploid Andean endemic Lepidium meyenii suffers from taxonomic uncertainty. Few molecular markers are available to genotype individuals or track gene fl ow in wild and cultivated material.  Methods and Results: Using available sequence data, eight cpSSR primer pairs were developed for L. meyenii. Levels of polymorphism checked in 56 individual L. meyenii , including cultivated and wild material, revealed that the number of alleles per locus ranged from three to fi and intrapopulation allele frequencies ranged from 0.071 to 1.0. Polymerase-chain-reaction screens using our cpSSR primers in 27 other Lepidium species and three Coronopus species suggested a high degree of interspecifi c amplifi cation.  Conclusions: These polymorphic cpSSR markers should prove useful in characterizing genetic variation among cultivated and wild L. meyenii. Additionally, interspecifi c amplifi cations suggest that these markers will be useful for the study of related taxa.

In this study, 96 nuclear ‘conserved intron-scanning primers’ were screened across subfamilies the Arecaceae (palms) for potential use in research focused on palm evolutionary biology. Primers were evaluated based on their ability to amplify single polymerase chain reaction products in Arecaceae, the clarity of sequencing reads, and the interspecific variability observed. Ultimately, the results suggest that: (i) seven of the loci are likely to be suitable when comparing non-Arecaceae outgroups and Arecaceae ingroups; (ii) seven loci may be of use when comparing subfamilies of Arecaceae; and (iii) four of the loci may be of use when comparing closely related genera.

SummaryMorphological diversity is often caused by altered gene expression of key developmental regulators. However, the precise developmental trajectories through which morphologies evolved remain poorly understood. It is also unclear to what degree genetic changes contributing to morphological divergence were fixed by natural selection. Here we investigate these problems in the context of evolutionary developmental transitions that produced the simple unlobed leaf of the model species Arabidopsis thaliana. We demonstrate that A. thaliana leaf shape likely derived from a more complex lobed ancestral state that persists in extant Arabidopsis species. We also show that evolution of the unlobed leaf form in A. thaliana involved loss of expression of the knotted1-like homeobox gene SHOOTMERISTEMLESS (STM) in leaves and that cis-regulatory divergence contributed to this process. Further, we provide evidence for a selective sweep at the A. thaliana STM locus, indicating that loss of STM expression in A. thaliana leaves may have been fixed by positive selection. In summary, our data provide key information as to when and how the characteristic leaf form of A. thaliana evolved.

• Background and Aims The establishment of justified recommendations in conservation biology requires robust taxonomic treatments for the group(s) being considered. Controversial or poorly developed taxonomies can have a negative impact on conservation assessments. One example of a taxonomically difficult and controversial species complex that is important in conservation involves two species of Mexican palms, Chamaedorea tepejilote and C. alternans. The goal of this study was to investigate whether C. alternans and C. tepejilote are genetically distinct within the Los Tuxtlas region of Veracruz. • Methods Individuals corresponding to the morphology of C. alternans and C. tepejilote were collected from sympatric and allopatric regions within the Los Tuxtlas Biological Station. Eighty-eight samples were genotyped using amplified fragment length polymorphism (AFLP) markers. Cluster and ordination analyses were used to investigate patterns of differentiation. • Key Results UPGMA and PCO analyses of AFLP profiles recovered two divergent clusters corresponding to morphologically defined C. tepejilote and C. alternans. No intermediate genotypes were observed and five of the 45 loci were fixed for either the presence or absence between the species. The patterns of divergence observed do not identify a role for sympatric speciation. • Conclusions The observed patterns of differentiation support the recognition of C. alternans as distinct from C. tepejilote. A suite of vegetative and reproductive morphological features can be used to help distinguish these taxa in the field, but they can be difficult to differentiate from preserved material. Previous treatments of the variation found within the complex (C. alternans and C. tepejilote) as intraspecific variance is unjustified. Given that the Los Tuxtlas region has suffered from historical and ongoing deforestation and that Chamaedorea includes numerous endangered taxa, retaining conservation status for C. alternans serves to help safeguard individuals of the species as well as a region and larger taxonomic group all under considerable threat from human activities.

Many early reports of ITS region (ITS 1, 5.8S, and ITS 2) variation in flowering plants indicated that nrDNA arrays within individuals are homogeneous. However, both older and more recent studies have found intra-individual nrDNA polymorphism across a range of plant taxa including presumed non-hybrid diploids. In addition, polymorphic individuals often contain potentially non-functional nrDNA copies (pseudogenes). These findings suggest that complete concerted evolution should not be assumed when embarking on phylogenetic studies using nrDNA sequences. Here we (1). discuss paralogy in relation to species tree reconstruction and conclude that a priori determinations of orthology and paralogy of nrDNA sequences should not be made based on the functionality or lack of functionality of those sequences; (2). discuss why systematists might be particularly interested in identifying and including pseudogene sequences as a test of gene tree sampling; (3). examine the various definitions and characterizations of nrDNA pseudogenes as well as the relative merits and limitations of a subset of pseudogene detection methods and conclude that nucleotide substitution patterns are particularly appropriate for the identification of putative nrDNA pseudogenes; and (4). present and discuss the advantages of a tree-based approach to identifying pseudogenes based on comparisons of sequence substitution patterns from putatively conserved (e.g., 5.8S) and less constrained (e.g., ITS 1 and ITS 2) regions. Application of this approach, through a method employing bootstrap hypothesis testing, and the issues discussed in the paper are illustrated through reanalysis of two previously published matrices. Given the apparent robustness of the test developed and the ease of carrying out percentile bootstrap hypothesis tests, we urge researchers to employ this statistical tool. While our discussion and examples concern the literature on plant systematics, the issues addressed are relevant to studies of nrDNA and other multicopy genes in other taxa.

A previous extensive survey showed that most but not all members of the papilionoid tribe Desmodieae lack the intron for the chloroplast gene rpl2 and partially or completely lack the open reading frame 0RF184. We extended the survey of Desmodieae to include all 26 genera recognized in the most recent comprehensive taxonomic treatment of the tribe, and use slot blot hybridization with gene- or intron-specific probes for all taxa, polymerase chain reaction amplification across the rpl2 intron region for a subset of taxa, and DNA sequencing across the rpl2 intron/exon boundary in representatives of selected genera (Desmodium, Lepesdeza, and Phylacium). Our results indicate that rpl2 intron is absent from both genera of the largest subtribe, Desmodiinae, and from core Lespedezinae (Campylotropis, Kummerowia, and Lespedeza). It is indi- cated as present in both genera of Bryinae and in two Lespedezinae genera (Neocollettia and Phylacium). In contrast to the rpl2 intron, the pattern of 0RF184 loss in Desmodiinae and Lespedezinae was extremely complex and difficult to interpret from slot blots, but consistent either with partial deletion or extensive sequence divergence. Bryinae showed no such evidence of loss or partial deletion. In agreement with the distribution of the rpl2 intron, Bryinae have been considered to have affinities outside Desmodieae, while both Phylacium and Neocollettia are anomalous within Lespedezinae. Thus the rpl2 intron loss may represent a synapomorphy for a "core" Desmodieae (Desmodiinae and Lespedezinae).