Genomic decoding of Theobroma grandiflorum (cupuassu) at chromosomal scale: evolutionary insights for horticultural innovation.

BACKGROUND: Theobroma grandiflorum (Malvaceae), known as cupuassu, is a tree indigenous to the Amazon basin, valued for its large fruits and seed pulp, contributing notably to the Amazonian bioeconomy. The seed pulp is utilized in desserts and beverages, and its seed butter is used in cosmetics. Here, we present the sequenced telomere-to-telomere genome of cupuassu, disclosing its genomic structure, evolutionary features, and phylogenetic relationships within the Malvaceae family. FINDINGS: The cupuassu genome spans 423 Mb, encodes 31,381 genes distributed in 10 chromosomes, and exhibits approximately 65% gene synteny with the Theobroma cacao genome, reflecting a conserved evolutionary history, albeit punctuated with unique genomic variations. The main changes are pronounced by bursts of long-terminal repeat retrotransposons at postspecies divergence, retrocopied and singleton genes, and gene families displaying distinctive patterns of expansion and contraction. Furthermore, positively selected genes are evident, particularly among retained and dispersed tandem and proximal duplicated genes associated with general fruit and seed traits and defense mechanisms, supporting the hypothesis of potential episodes of subfunctionalization and neofunctionalization following duplication, as well as impact from distinct domestication process. These genomic variations may underpin the differences observed in fruit and seed morphology, ripening, and disease resistance between cupuassu and the other Malvaceae species. CONCLUSIONS: The cupuassu genome offers a foundational resource for both breeding improvement and conservation biology, yielding insights into the evolution and diversity within the genus Theobroma.

Genomic Features and Phylogenetic Analysis of Antimicrobial-Resistant Salmonella Mbandaka ST413 Strains.

In recent years, Salmonella enterica subsp. enterica serovar Mbandaka (S. Mbandaka) has been increasingly isolated from laying hens and shell eggs around the world. Moreover, this serovar has been identified as the causative agent of several salmonellosis outbreaks in humans. Surprisingly, little is known about the characteristics of this emerging serovar, and therefore, we investigated antimicrobial resistance, virulence, and prophage genes of six selected Brazilian strains of Salmonella Mbandaka using Whole Genome Sequencing (WGS). Multi-locus sequence typing revealed that the tested strains belong to Sequence Type 413 (ST413), which has been linked to recent multi-country salmonellosis outbreaks in Europe. A total of nine resistance genes were detected, and the most frequent ones were aac(6')-Iaa, sul1, qacE, blaOXA-129, tet(B), and aadA1. A point mutation in ParC at the 57th position (threonine → serine) associated with quinolone resistance was present in all investigated genomes. A 112,960 bp IncHI2A plasmid was mapped in 4/6 strains. This plasmid harboured tetracycline (tetACDR) and mercury (mer) resistance genes, genes contributing to conjugative transfer, and genes involved in plasmid maintenance. Most strains (four/six) carried Salmonella genomic island 1 (SGI1). All S. Mbandaka genomes carried seven pathogenicity islands (SPIs) involved in intracellular survival and virulence: SPIs 1-5, 9, and C63PI. The virulence genes csgC, fimY, tcfA, sscA, (two/six), and ssaS (one/six) were absent in some of the genomes; conversely, fimA, prgH, and mgtC were present in all of them. Five Salmonella bacteriophage sequences (with homology to Escherichia phage phiV10, Enterobacteria phage Fels-2, Enterobacteria phage HK542, Enterobacteria phage ST64T, Salmonella phage SW9) were identified, with protein counts between 31 and 54, genome lengths of 24.7 bp and 47.7 bp, and average GC content of 51.25%. In the phylogenetic analysis, the genomes of strains isolated from poultry in Brazil clustered into well-supported clades with a heterogeneous distribution, primarily associated with strains isolated from humans and food. The phylogenetic relationship of Brazilian S. Mbandaka suggests the presence of strains with high epidemiological significance and the potential to be linked to foodborne outbreaks. Overall, our results show that isolated strains of S. Mbandaka are multidrug-resistant and encode a rather conserved virulence machinery, which is an epidemiological hallmark of Salmonella strains that have successfully disseminated both regionally and globally.

The phylogenomics and evolutionary dynamics of the organellar genomes in carnivorous Utricularia and Genlisea species (Lentibulariaceae).

Utricularia and Genlisea are highly specialized carnivorous plants whose phylogenetic history has been poorly explored using phylogenomic methods. Additional sampling and genomic data are needed to advance our phylogenetic and taxonomic knowledge of this group of plants. Within a comparative framework, we present a characterization of plastome (PT) and mitochondrial (MT) genes of 26 Utricularia and six Genlisea species, with representatives of all subgenera and growth habits. All PT genomes maintain similar gene content, showing minor variation across the genes located between the PT junctions. One exception is a major variation related to different patterns in the presence and absence of ndh genes in the small single copy region, which appears to follow the phylogenetic history of the species rather than their lifestyle. All MT genomes exhibit similar gene content, with most differences related to a lineage-specific pseudogenes. We find evidence for episodic positive diversifying selection in PT and for most of the Utricularia MT genes that may be related to the current hypothesis that bladderworts' nuclear DNA is under constant ROS oxidative DNA damage and unusual DNA repair mechanisms, or even low fidelity polymerase that bypass lesions which could also be affecting the organellar genomes. Finally, both PT and MT phylogenetic trees were well resolved and highly supported, providing a congruent phylogenomic hypothesis for Utricularia and Genlisea clade given the study sampling.

Comparative analyses of Theobroma cacao and T. grandiflorum mitogenomes reveal conserved gene content embedded within complex and plastic structures.

Unlike the chloroplast genomes (ptDNA), the plant mitochondrial genomes (mtDNA) are much more plastic in structure and size but maintain a conserved and essential gene set related to oxidative phosphorylation. Moreover, the plant mitochondrial genes and mtDNA are good markers for phylogenetic, evolutive, and comparative analyses. The two most known species in Theobroma L. (Malvaceae s.l.) genus are T. cacao, and T. grandiflorum. Besides the economic value, both species also show considerable biotechnology potential due to their other derived products, thus, aggregating additional economic value for the agroindustry. Here, we assembled and compared the mtDNA of Theobroma cacao and T. grandiflorum to generate a new genomics resource and unravel evolutionary trends. Graph-based analyses revealed that both mtDNA exhibit multiple alternative arrangements, confirming the dynamism commonly observed in plant mtDNA. The disentangled assembly graph revealed potential predominant circular molecules. The master circle molecules span 543,794 bp for T. cacao and 501,598 bp for T. grandiflorum, showing 98.9% of average sequence identity. Both mtDNA contains the same set of 39 plant mitochondrial genes, commonly found in other rosid mitogenomes. The main features are a duplicated copy of atp4, the absence of rpl6, rps2, rps8, and rps11, and the presence of two chimeric open-reading frames. Moreover, we detected few ptDNA integrations mainly represented by tRNAs, and no viral sequences were detected. Phylogenomics analyses indicate Theobroma spp. are nested in Malvaceae family. The main mtDNA differences are related to distinct structural rearrangements and exclusive regions associated with relics of Transposable Elements, supporting the hypothesis of dynamic mitochondrial genome maintenance and divergent evolutionary paths and pressures after species differentiation.

The complete organellar genomes of the entheogenic plant Psychotria viridis (Rubiaceae), a main component of the ayahuasca brew.

Psychotria viridis (Rubioideae: Rubiaceae), popularly known as chacrona, is commonly found as a shrub in the Amazon region and is well-known to produce psychoactive compounds, such as the N,N-dimethyltryptamine (DMT). Together with the liana Banisteropsis caapi, P. viridis is one of the main components of the Amerindian traditional, entheogenic beverage known as ayahuasca. In this work, we assembled and annotated the organellar genomes (ptDNA and mtDNA), presenting the first genomics resources for this species. The P. viridis ptDNA exhibits 154,106 bp, encoding all known ptDNA gene repertoire found in angiosperms. The Psychotria genus is a complex paraphyletic group, and according to phylogenomic analyses, P. viridis is nested in the Psychotrieae clade. Comparative ptDNA analyses indicate that most Rubiaceae plastomes present conserved ptDNA structures, often showing slight differences at the junction sites of the major four regions (LSC-IR-SSC). For the mitochondrion, assembly graph-based analysis supports a complex mtDNA organization, presenting at least two alternative and circular mitogenomes structures exhibiting two main repeats spanning 24 kb and 749 bp that may symmetrically isomerize the mitogenome into variable arrangements and isoforms. The circular mtDNA sequences (615,370 and 570,344 bp) encode almost all plant mitochondrial genes (except for the ccmC, rps7, rps10, rps14, rps19, rpl2 and rpl16 that appears as pseudogenes, and the absent genes sdh3, rps2, rsp4, rsp8, rps11, rpl6, and rpl10), showing slight variations related to exclusive regions, ptDNA integration, and relics of previous events of LTR-RT integration. The detection of two mitogenomes haplotypes is evidence of heteroplasmy as observed by the complex organization of the mitochondrial genome using graph-based analysis. Taken together, these results elicit the primary insights into the genome biology and evolutionary history of Psychotria viridis and may be used to aid strategies for conservation of this sacred, entheogenic species.

A Historical Perspective of Bladderworts (Utricularia): Traps, Carnivory and Body Architecture.

The genus Utricularia includes around 250 species of carnivorous plants, commonly known as bladderworts. The generic name Utricularia was coined by Carolus Linnaeus in reference to the carnivorous organs (Utriculus in Latin) present in all species of the genus. Since the formal proposition by Linnaeus, many species of Utricularia were described, but only scarce information about the biology for most species is known. All Utricularia species are herbs with vegetative organs that do not follow traditional models of morphological classification. Since the formal description of Utricularia in the 18th century, the trap function has intrigued naturalists. Historically, the traps were regarded as floating organs, a common hypothesis that was maintained by different botanists. However, Charles Darwin was most likely the first naturalist to refute this idea, since even with the removal of all traps, the plants continued to float. More recently, due mainly to methodological advances, detailed studies on the trap function and mechanisms could be investigated. This review shows a historical perspective on Utricularia studies which focuses on the traps and body organization.

Structural Features of Carnivorous Plant (Genlisea, Utricularia) Tubers as Abiotic Stress Resistance Organs.

Carnivorous plants from the Lentibulariaceae form a variety of standard and novel vegetative organs and survive unfavorable environmental conditions. Within Genlisea, only G. tuberosa, from the Brazilian Cerrado, formed tubers, while Utricularia menziesii is the only member of the genus to form seasonally dormant tubers. We aimed to examine and compare the tuber structure of two taxonomically and phylogenetically divergent terrestrial carnivorous plants: Genlisea tuberosa and Utricularia menziesii. Additionally, we analyzed tubers of U. mannii. We constructed phylogenetic trees using chloroplast genes matK/trnK and rbcL and used studied characters for ancestral state reconstruction. All examined species contained mainly starch as histologically observable reserves. The ancestral state reconstruction showed that specialized organs such as turions evolved once and tubers at least 12 times from stolons in Lentibulariaceae. Different from other clades, tubers probably evolved from thick stolons for sect. Orchidioides and both structures are primarily water storage structures. In contrast to species from section Orchidioides, G. tuberosa, U. menziesii and U. mannii form starchy tubers. In G. tuberosa and U. menziesii, underground tubers provide a perennating bud bank that protects the species in their fire-prone and seasonally desiccating environments.

A Genomic and Transcriptomic Overview of MATE, ABC, and MFS Transporters in Citrus sinensis Interaction with Xanthomonas citri subsp. citri.

The multi-antimicrobial extrusion (MATE), ATP-binding cassette (ABC), and major facilitator superfamily (MFS) are the main plant transporters families, playing an essential role in the membrane-trafficking network and plant-defense mechanism. The citrus canker type A (CC), is a devastating disease caused by Xanthomonas citri subsp. citri (Xac), affecting all citrus species. In this work, we performed an in silico analysis of genes and transcripts from MATE, ABC, and MFS families to infer the role of membrane transporters in Citrus-Xac interaction. Using as reference, the available Citrus sinensis genome and the citrus reference transcriptome from CitrusKB database, 67 MATE, 91 MFS, and 143 ABC genes and 82 MATE, 139 MFS, and 226 ABC transcripts were identified and classified into subfamilies. Duplications, alternative-splicing, and potentially non-transcribed transporters' genes were revealed. Interestingly, MATE I and ABC G subfamilies appear differently regulated during Xac infection. Furthermore, Citrus spp. showing distinct levels of CC susceptibility exhibited different sets of transporters transcripts, supporting dissimilar molecular patterns of membrane transporters in Citrus-Xac interaction. According to our findings, 4 MATE, 10 ABC, and 3 MFS are potentially related to plant-defense mechanisms. Overall, this work provides an extensive analysis of MATE, ABC, and MFS transporters' in Citrus-Xac interaction, bringing new insights on membrane transporters in plant-pathogen interactions.

The nuclear and mitochondrial genomes of Frieseomelitta varia - a highly eusocial stingless bee (Meliponini) with a permanently sterile worker caste.

BACKGROUND: Most of our understanding on the social behavior and genomics of bees and other social insects is centered on the Western honey bee, Apis mellifera. The genus Apis, however, is a highly derived branch comprising less than a dozen species, four of which genomically characterized. In contrast, for the equally highly eusocial, yet taxonomically and biologically more diverse Meliponini, a full genome sequence was so far available for a single Melipona species only. We present here the genome sequence of Frieseomelitta varia, a stingless bee that has, as a peculiarity, a completely sterile worker caste. RESULTS: The assembly of 243,974,526 high quality Illumina reads resulted in a predicted assembled genome size of 275 Mb composed of 2173 scaffolds. A BUSCO analysis for the 10,526 predicted genes showed that these represent 96.6% of the expected hymenopteran orthologs. We also predicted 169,371 repetitive genomic components, 2083 putative transposable elements, and 1946 genes for non-coding RNAs, largely long non-coding RNAs. The mitochondrial genome comprises 15,144 bp, encoding 13 proteins, 22 tRNAs and 2 rRNAs. We observed considerable rearrangement in the mitochondrial gene order compared to other bees. For an in-depth analysis of genes related to social biology, we manually checked the annotations for 533 automatically predicted gene models, including 127 genes related to reproductive processes, 104 to development, and 174 immunity-related genes. We also performed specific searches for genes containing transcription factor domains and genes related to neurogenesis and chemosensory communication. CONCLUSIONS: The total genome size for F. varia is similar to the sequenced genomes of other bees. Using specific prediction methods, we identified a large number of repetitive genome components and long non-coding RNAs, which could provide the molecular basis for gene regulatory plasticity, including worker reproduction. The remarkable reshuffling in gene order in the mitochondrial genome suggests that stingless bees may be a hotspot for mtDNA evolution. Hence, while being just the second stingless bee genome sequenced, we expect that subsequent targeting of a selected set of species from this diverse clade of highly eusocial bees will reveal relevant evolutionary signals and trends related to eusociality in these important pollinators.

DNA barcoding for molecular identification of Gynerium sagittatum(Poales: Poaceae): genetic diversity in savannah genotypes from Córdoba, Colombia / Código de barras de ADN para la identificación molecular de Gynerium sagittatum (Poales: Poaceae): diversidad genética de genotipos de las sabanas de Córdoba, Colombia

Introduction: The fiber of the Gynerium sagittatum Aubl. P. Beauv is raw material for the elaboration of several handcrafts, which are symbols of Colombian cultural identity. In the manufacture process, different genotypes are used according to the fiber quality and the type of craftsmanship, but it is believed that Gynerium is a complex species, and to date, there is no agreement on whether these genotypes belong to the same species or to different species. Objective: The aim of this study was to quickly and accurately identify wild cane plants using the nuclear ribosomal internal transcribed spacer (ITS1+ITS2), three chloroplast regions (matK, rbcL, ycf1), and their combinations. Methods: Different tests were used for discrimination: (1) inter and intraspecific distances, (2) Best Match (BM), Best Close Match (BCM), and tree-based method (3) Neighbor Joining (NJ) and (4) maximum likelihood and bayesian inference in molecular data. Results: The results showed that BM and BCM approaches revealed the low rate of correct species identification for ITS+matK (33.3 %) and ITS (28.6 %) loci, showing similarity among sequences. These results were further supported by tree-based analyses, where all individual regions and the different gene combinations had a zero discrimination rate. Conclusions: all genotypes belong to the same species of wild cane, therefore existing morphological differences can be related to phenotypic plasticity.

Introducción: La fibra de Gynerium sagittatum Aubl. P. Beauv, es materia prima esencial para la elaboración de varias artesanías, que son símbolos de la identidad cultural colombiana. En el proceso de fabricación, se utilizan diferentes genotipos de acuerdo con la calidad de la fibra y el tipo de artesanía, pero se cree que Gynerium es una especie compleja y hasta la fecha, no hay un consenso sobre si estos genotipos pertenecen a la misma especie o especies diferentes. Objetivo: Identificar de forma rápida y precisa plantas de caña silvestre utilizando el espaciador transcrito interno ribosomal nuclear (ITS1+ITS2), tres regiones de cloroplasto (matK, rbcL, ycf1) y sus combinaciones. Métodos: Se utilizaron diferentes pruebas para la discriminación: (1) distancias inter e intraespecíficas, (2) Prueba Best Match (BM), Best Close Match (BCM) y método basado en árboles (3) Neighbor Joining (NJ) y (4) Probabilidad de inferencia bayesiana mediante datos moleculares. Resultados: Los resultadosmostraron que los enfoques BM y BCM revelaron una baja tasa de identificación correcta de especies para los loci ITS+matK (33.3 %) e ITS (28.6 %), mostrando similitud entre las secuencias. Estos resultados fueron respaldados por análisis basados en árboles, donde todas las regiones individuales y las diferentes combinaciones de genes tuvieron una tasa de discriminación de cero (0 %). Conclusiones: los genotipos evaluados pertenecen a la misma especie de caña flecha y las diferencias morfológicas existentes pueden estar relacionadas con plasticidad fenotípica.

The Terrestrial Carnivorous Plant Utricularia reniformis Sheds Light on Environmental and Life-Form Genome Plasticity.

Utricularia belongs to Lentibulariaceae, a widespread family of carnivorous plants that possess ultra-small and highly dynamic nuclear genomes. It has been shown that the Lentibulariaceae genomes have been shaped by transposable elements expansion and loss, and multiple rounds of whole-genome duplications (WGD), making the family a platform for evolutionary and comparative genomics studies. To explore the evolution of Utricularia, we estimated the chromosome number and genome size, as well as sequenced the terrestrial bladderwort Utricularia reniformis (2n = 40, 1C = 317.1-Mpb). Here, we report a high quality 304 Mb draft genome, with a scaffold NG50 of 466-Kb, a BUSCO completeness of 87.8%, and 42,582 predicted genes. Compared to the smaller and aquatic U. gibba genome (101 Mb) that has a 32% repetitive sequence, the U. reniformis genome is highly repetitive (56%). The structural differences between the two genomes are the result of distinct fractionation and rearrangements after WGD, and massive proliferation of LTR-retrotransposons. Moreover, GO enrichment analyses suggest an ongoing gene birth-death-innovation process occurring among the tandem duplicated genes, shaping the evolution of carnivory-associated functions. We also identified unique patterns of developmentally related genes that support the terrestrial life-form and body plan of U. reniformis. Collectively, our results provided additional insights into the evolution of the plastic and specialized Lentibulariaceae genomes.

Intraspecific Variation within the Utricularia amethystina Species Morphotypes Based on Chloroplast Genomes.

Utricularia amethystina Salzm. ex A.St.-Hil. & Girard (Lentibulariaceae) is a highly polymorphic carnivorous plant taxonomically rearranged many times throughout history. Herein, the complete chloroplast genomes (cpDNA) of three U. amethystina morphotypes: purple-, white-, and yellow-flowered, were sequenced, compared, and putative markers for systematic, populations, and evolutionary studies were uncovered. In addition, RNA-Seq and RNA-editing analysis were employed for functional cpDNA evaluation. The cpDNA of three U. amethystina morphotypes exhibits typical quadripartite structure. Fine-grained sequence comparison revealed a high degree of intraspecific genetic variability in all morphotypes, including an exclusive inversion in the psbM and petN genes in U. amethystina yellow. Phylogenetic analyses indicate that U. amethystina morphotypes are monophyletic. Furthermore, in contrast to the terrestrial Utricularia reniformis cpDNA, the U. amethystina morphotypes retain all the plastid NAD(P)H-dehydrogenase (ndh) complex genes. This observation supports the hypothesis that the ndhs in terrestrial Utricularia were independently lost and regained, also suggesting that different habitats (aquatic and terrestrial) are not related to the absence of Utricularia ndhs gene repertoire as previously assumed. Moreover, RNA-Seq analyses recovered similar patterns, including nonsynonymous RNA-editing sites (e.g., rps14 and petB). Collectively, our results bring new insights into the chloroplast genome architecture and evolution of the photosynthesis machinery in the Lentibulariaceae.

Floral micromorphology and nectar composition of the early evolutionary lineage Utricularia (subgenus Polypompholyx, Lentibulariaceae).

Utricularia (Lentibulariaceae) is a genus comprising around 240 species of herbaceous, carnivorous plants. Utricularia is usually viewed as an insect-pollinated genus, with the exception of a few bird-pollinated species. The bladderworts Utricularia multifida and U. tenella are interesting species because they represent an early evolutionary Utricularia branch and have some unusual morphological characters in their traps and calyx. Thus, our aims were to (i) determine whether the nectar sugar concentrations and composition in U. multifida and U. tenella are similar to those of other Utricularia species from the subgenera Polypompholyx and Utricularia, (ii) compare the nectary structure of U. multifida and U. tenella with those of other Utricularia species, and (iii) determine whether U. multifida and U. tenella use some of their floral trichomes as an alternative food reward for pollinators. We used light microscopy, histochemistry, and scanning and transmission electron microscopy to address those aims. The concentration and composition of nectar sugars were analysed using high-performance liquid chromatography. In all of the examined species, the floral nectary consisted of a spur bearing glandular trichomes. The spur produced and stored the nectar. We detected hexose-dominated (fructose + glucose) nectar in U. multifida and U. tenella as well as in U. violacea. In both U. multifida and U. tenella, there were trichomes that blocked the entrance into the throat and spur. Because these trichomes were rich in chromoplasts and contained lipid droplets, they may form an additional visual attractant. Bearing in mind the phylogenetic hypothesis for the genus, we suggest that an early ancestor of Utricularia had a nectariferous spur flower with a lower lip that formed a wide landing platform for bee pollinators.

Comparative genomic analysis of Genlisea (corkscrew plants-Lentibulariaceae) chloroplast genomes reveals an increasing loss of the ndh genes.

In the carnivorous plant family Lentibulariaceae, all three genome compartments (nuclear, chloroplast, and mitochondria) have some of the highest rates of nucleotide substitutions across angiosperms. While the genera Genlisea and Utricularia have the smallest known flowering plant nuclear genomes, the chloroplast genomes (cpDNA) are mostly structurally conserved except for deletion and/or pseudogenization of the NAD(P)H-dehydrogenase complex (ndh) genes known to be involved in stress conditions of low light or CO2 concentrations. In order to determine how the cpDNA are changing, and to better understand the evolutionary history within the Genlisea genus, we sequenced, assembled and analyzed complete cpDNA from six species (G. aurea, G. filiformis, G. pygmaea, G. repens, G. tuberosa and G. violacea) together with the publicly available G. margaretae cpDNA. In general, the cpDNA structure among the analyzed Genlisea species is highly similar. However, we found that the plastidial ndh genes underwent a progressive process of degradation similar to the other terrestrial Lentibulariaceae cpDNA analyzed to date, but in contrast to the aquatic species. Contrary to current thinking that the terrestrial environment is a more stressful environment and thus requiring the ndh genes, we provide evidence that in the Lentibulariaceae the terrestrial forms have progressive loss while the aquatic forms have the eleven plastidial ndh genes intact. Therefore, the Lentibulariaceae system provides an important opportunity to understand the evolutionary forces that govern the transition to an aquatic environment and may provide insight into how plants manage water stress at a genome scale.

Molecular phylogeny of bladderworts: A wide approach of Utricularia (Lentibulariaceae) species relationships based on six plastidial and nuclear DNA sequences.

The carnivorous plant genus Utricularia L. (bladderwort) comprises about 240 species distributed worldwide and is traditionally classified into two subgenera (Polypompholyx and Utricularia) and 35 sections, based mainly on general and trap morphology. It is one out of the largest carnivorous genera, representing ca. 30% of all carnivorous plant species, and is also the most widely distributed. According to previous phylogenetic studies, most infrageneric sections are monophyletic, but there are several incongruences considering their relationships and also the dissenting position of some species as a result of a too few (mostly one or two) molecular markers analyzed. Thus, here we present a multilocus phylogeny for Utricularia species with a wide taxonomic sampling (78 species and 115 accessions) based on six plastid (rbcL, matK, rpl20-rps12, rps16, trnL-F) and nuclear DNA (ITS region) sequences. The aim is to reconstruct a well-resolved tree to propose evolutionary and biogeographic hypotheses for the radiation of lineages with inferences about the divergence times of clades using a molecular clock approach.

Intermittent gait disturbance in idiopathic normal pressure hydrocephalus.

OBJECTIVES: We identified intermittent gait disturbance (IGD) observed in the mild stage of idiopathic normal pressure hydrocephalus (iNPH). The first purpose of this study was to clarify the temporal gait profile of IGD during long-distance gait. The second purpose was to confirm the difference in treatment effect after cerebrospinal fluid (CSF) shunting in patients with and without IGD. MATERIALS AND METHODS: Fourteen consecutive iNPH patients with mild gait disturbance with a timed up-and-go (TUG) of <20 seconds were prospectively enrolled in the study. All patients were asked "Do you experience gait difficulty after over five minutes of walking?" Seven "yes" patients formed the IGD group, and seven "no" patients formed the persistent gait disturbance (PGD) group. One day before and 7 days after CSF shunting, gait function was evaluated by the 6-minute walk test (6MWT) and TUG. RESULTS: Preoperatively, all patients in the IGD group demonstrated features of IGD during the 6MWT, characterized by a progressive pattern of decreased gait speed and step length with increased cadence and absence of leg pain. Post-operatively, these features of IGD improved in all patients. In the PGD group, preoperative walking did not significantly worsen during the 6MWT and did not significantly change 7 days after treatment. Improvement of gait symptoms 1 week after CSF shunting could be detected with 6MWT instead of TUG. CONCLUSIONS: Intermittent gait disturbance is not a rare symptom in mild stage of iNPH and may serve as an important clinical diagnostic marker for identifying mild iNPH patients.

Phylogeny of the 'orchid-like' bladderworts (gen. Utricularia sect. Orchidioides and Iperua: Lentibulariaceae) with remarks on the stolon-tuber system.

Background and Aims: The 'orchid-like' bladderworts ( Utricularia ) comprise 15 species separated into two sections: Orchidioides and Iperua . These robust and mostly epiphytic species were originally grouped within the section Orchidioides by the first taxonomical systems. These species were later split into two sections when sect. Iperua was proposed. Due to the lack of strong evidence based on a robust phylogenetic perspective, this study presents a phylogenetic proposal based on four different DNA sequences (plastid and nuclear) and morphology to test the monophyly of the two sections. Methods: In comparison with all previous phylogenetic studies, the largest number of species across the sections was covered: 11 species from sections Orchidioides and Iperua with 14 species as an external group. Maximum likelihood and Bayesian inferences were applied to DNA sequences of rps16 , trnL-F , matK , the internal transcribed spacer (ITS) and three morphological characters: (1) the crest of the corolla; (2) the primary organs in the embryo; and (3) tubers. Additionally, a histochemical analysis of the stolons and tubers is presented from an evolutionary perspective. Key Results: The analyses showed the paraphyly of sect. Iperua , since Utricularia humboldtii is more related to the clade of sect. Orchidioides . Utricularia cornigera is grouped in the sect. Iperua clade based on chloroplast DNA sequences, but it is nested to sect. Orchidioides according to ITS dataset. Morphological characters do not support the breaking up of the 'orchid-like' species into two sections, either. Moreover, the stolon-tuber systems of both sections serve exclusively for water storage, according to histological analyses. Conclusions: This study provides strong evidence, based on DNA sequences from two genomic compartments (plastid and nucleus) and morphology to group the Utricularia sect. Orchidioides into the sect. Iperua . The tubers are important adaptations for water storage and have been derived from stolons at least twice in the phylogenetic history of 'orchid-like' bladderworts.

The mitochondrial genome of the terrestrial carnivorous plant Utricularia reniformis (Lentibulariaceae): Structure, comparative analysis and evolutionary landmarks.

The carnivorous plants of the family Lentibulariaceae have attained recent attention not only because of their interesting lifestyle, but also because of their dynamic nuclear genome size. Lentibulariaceae genomes span an order of magnitude and include species with the smallest genomes in angiosperms, making them a powerful system to study the mechanisms of genome expansion and contraction. However, little is known about mitochondrial DNA (mtDNA) sequences of this family, and the evolutionary forces that shape this organellar genome. Here we report the sequencing and assembly of the complete mtDNA from the endemic terrestrial Brazilian species Utricularia reniformis. The 857,234bp master circle mitochondrial genome encodes 70 transcriptionaly active genes (42 protein-coding, 25 tRNAs and 3 rRNAs), covering up to 7% of the mtDNA. A ltrA-like protein related to splicing and mobility and a LAGLIDADG homing endonuclease have been identified in intronic regions, suggesting particular mechanisms of genome maintenance. RNA-seq analysis identified properties with putative diverse and important roles in genome regulation and evolution: 1) 672kbp (78%) of the mtDNA is covered by full-length reads; 2) most of the 243kbp intergenic regions exhibit transcripts; and 3) at least 69 novel RNA editing sites in the protein-coding genes. Additional genomic features are hypothetical ORFs (48%), chloroplast insertions, including truncated plastid genes that have been lost from the chloroplast DNA (5%), repeats (5%), relics of transposable elements mostly related to LTR retrotransposons (5%), and truncated mitovirus sequences (0.4%). Phylogenetic analysis based on 32 different Lamiales mitochondrial genomes corroborate that Lentibulariaceae is a monophyletic group. In summary, the U. reniformis mtDNA represents the eighth largest plant mtDNA described to date, shedding light on the genomic trends and evolutionary characteristics and phylogenetic history of the family Lentibulariaceae.

The Chloroplast Genome of Utricularia reniformis Sheds Light on the Evolution of the ndh Gene Complex of Terrestrial Carnivorous Plants from the Lentibulariaceae Family.

Lentibulariaceae is the richest family of carnivorous plants spanning three genera including Pinguicula, Genlisea, and Utricularia. Utricularia is globally distributed, and, unlike Pinguicula and Genlisea, has both aquatic and terrestrial forms. In this study we present the analysis of the chloroplast (cp) genome of the terrestrial Utricularia reniformis. U. reniformis has a standard cp genome of 139,725bp, encoding a gene repertoire similar to essentially all photosynthetic organisms. However, an exclusive combination of losses and pseudogenization of the plastid NAD(P)H-dehydrogenase (ndh) gene complex were observed. Comparisons among aquatic and terrestrial forms of Pinguicula, Genlisea, and Utricularia indicate that, whereas the aquatic forms retained functional copies of the eleven ndh genes, these have been lost or truncated in terrestrial forms, suggesting that the ndh function may be dispensable in terrestrial Lentibulariaceae. Phylogenetic scenarios of the ndh gene loss and recovery among Pinguicula, Genlisea, and Utricularia to the ancestral Lentibulariaceae cladeare proposed. Interestingly, RNAseq analysis evidenced that U. reniformis cp genes are transcribed, including the truncated ndh genes, suggesting that these are not completely inactivated. In addition, potential novel RNA-editing sites were identified in at least six U. reniformis cp genes, while none were identified in the truncated ndh genes. Moreover, phylogenomic analyses support that Lentibulariaceae is monophyletic, belonging to the higher core Lamiales clade, corroborating the hypothesis that the first Utricularia lineage emerged in terrestrial habitats and then evolved to epiphytic and aquatic forms. Furthermore, several truncated cp genes were found interspersed with U. reniformis mitochondrial and nuclear genome scaffolds, indicating that as observed in other smaller plant genomes, such as Arabidopsis thaliana, and the related and carnivorous Genlisea nigrocaulis and G. hispidula, the endosymbiotic gene transfer may also shape the U. reniformis genome in a similar fashion. Overall the comparative analysis of the U. reniformis cp genome provides new insight into the ndh genes and cp genome evolution of carnivorous plants from Lentibulariaceae family.