Molecular phylogeny and evolution of inflorescence types in Eperua.

PREMISE: The Amazonian hyperdominant genus Eperua (Fabaceae) currently holds 20 described species and has two strongly different inflorescence and flower types, with corresponding different pollination syndrome. The evolution of these vastly different inflorescence types within this genus was unknown and the main topic in this study. METHODS: We constructed a molecular phylogeny, based on the full nuclear ribosomal DNA and partial plastome, using Bayesian inference and maximum likelihood methods, to test whether the genus is monophyletic, whether all species are monophyletic and if the shift from bat to bee pollination (or vice versa) occurred once in this genus. RESULTS: All but two species are well supported by the nuclear ribosomal phylogeny. The plastome phylogeny, however, shows a strong geographic signal suggesting strong local hybridization or chloroplast capture, rendering chloroplast barcodes meaningless in this genus. CONCLUSIONS: With our data, we cannot fully resolve the backbone of the tree to clarify sister genera relationships and confirm monophyly of the genus Eperua. Within the genus, the shift from bat to bee and bee to bat pollination has occurred several times but, with the bee to bat not always leading to a pendant inflorescence.

Systematic review on raphide morphotype calcium oxalate crystals in angiosperms.

Calcium oxalate (CaOx) crystals are biominerals present in a wide variety of plants. Formation of these crystals is a biomineralization process occurring in vacuoles within specialized cells called crystal idioblasts. This process is dependent on two key components: deprotonated oxalic acid, and calcium ions (Ca2+), and can result in multiple crystal morphologies. Raphides are needle-like CaOx crystals found in various plant organs and tissues. Though their function is highly debated, they can potentially store calcium, sequester heavy metals, protect against herbivory and possibly programmed cell death. The last review of the taxonomic and anatomical distribution of raphides across the plant kingdom dates back to 1980, in a review by Franceschi and Horner, prompting an updated systematic review of raphides in plants. We conduct a broad literature search to record plant taxa and tissue locations containing raphides. We provide an overview of raphide-forming plant taxa, discussing phylogenetic distribution of raphides at the order level, and report on the specific locations of raphides within plants. Our review reveals raphide occurrence has been studied in 33 orders, 76 families and 1305 species, with raphides presence confirmed in 24 orders, 46 families and 797 species. These taxa represented less than 1 % of known species per family. Leaves are the most prominent raphide-containing primary location in all three major angiosperm clades investigated: Eudicots, Magnoliids, and Monocots. Roots are least reported to contain raphides. The collation of such information lays the groundwork to unveil the genetic origin and evolution of raphides in plants, and highlights targets for future studies of the presence and role of plant raphides.

The effects of standing in tutorial group meetings on learning: A randomized controlled trial.

BACKGROUND: Standing desks have been brought into the education environment to reduce sedentary behavior among students. The current study explored the effects of standing in tutorial group meetings on learning among undergraduate students. METHODS: Ninety-six participants were randomly allocated to a Sit or Stand group, with 2 h tutorial group meetings scheduled, once or twice per week, for nine weeks. Learning was analyzed using exam grades, concept maps, and tutorial interactions. RESULTS: Overall, the Sit and Stand groups did not differ from each other in terms of learning, measured through their exam, concept map, and the use of learning-oriented interactions. CONCLUSION: Standing in tutorial group meetings neither enhanced nor compromised learning. Considering the health risks associated with prolonged sedentary behavior, offering standing tutorial group meetings to undergraduate students is a recommended solution to break up prolonged sedentary behavior and encourage more physical activity, while maintaining the learning performance of students.

Chromosome-level reference genome of the soursop (Annona muricata): A new resource for Magnoliid research and tropical pomology.

The flowering plant family Annonaceae includes important commercially grown tropical crops, but development of promising species is hindered by a lack of genomic resources to build breeding programs. Annonaceae are part of the magnoliids, an ancient lineage of angiosperms for which evolutionary relationships with other major clades remain unclear. To provide resources to breeders and evolutionary researchers, we report a chromosome-level genome assembly of the soursop (Annona muricata). We assembled the genome using 444.32 Gb of DNA sequences (676× sequencing depth) from PacBio and Illumina short-reads, in combination with 10× Genomics and Bionano data (v1). A total of 949 scaffolds were assembled to a final size of 656.77 Mb, with a scaffold N50 of 3.43 Mb (v1), and then further improved to seven pseudo-chromosomes using Hi-C sequencing data (v2; scaffold N50: 93.2 Mb, total size in chromosomes: 639.6 Mb). Heterozygosity was very low (0.06%), while repeat sequences accounted for 54.87% of the genome, and 23,375 protein-coding genes with an average of 4.79 exons per gene were annotated using de novo, RNA-seq and homology-based approaches. Reconstruction of the historical population size showed a slow continuous contraction, probably related to Cenozoic climate changes. The soursop is the first genome assembled in Annonaceae, supporting further studies of floral evolution in magnoliids, providing an essential resource for delineating relationships of ancient angiosperm lineages. Both genome-assisted improvement and conservation efforts will be strengthened by the availability of the soursop genome. As a community resource, this assembly will further strengthen the role of Annonaceae as model species for research on the ecology, evolution and domestication potential of tropical species in pomology and agroforestry.

The effects of standing tutorial meetings on physical activity behavior in undergraduates: A randomized controlled trial.

This study explored the effects of using standing desks in tutorial meetings on the physical activity behavior (PAB) of undergraduate students. Standing desks have been introduced to minimize the detrimental health effects of prolonged sedentary behavior (SB). The effectiveness of using standing desks has not been explored among undergraduate students - a population showing high SB. Ninety-six undergraduate students were randomly assigned to a sitting or standing tutorial group that ran for nine weeks, and their PAB was monitored using the activPAL3™ triaxial activity monitor. To check for potential compensatory or other covarying behaviors, the students' PAB was monitored on tutorial and non-tutorial days. PAB monitoring was conducted in week 4-5, and a follow-up measurement was conducted in week 9 to examine longer-term effects. In week 4-5, the stand group (n = 41) showed less SB (ß = -0.092, SE = 0.044, 95% CI: -0.179, -0.006) and more moderate-to-vigorous physical activity (ß = 0.320, SE = 0.160, 95% CI: 0.004, 0.635) compared to the sit group (n = 36). On tutorial days, the stand group showed more light physical activity (LPA) than the sit group (p < .001, d = 1.04). In week 9, there was an exam on the last day of that week. Nonetheless, the stand group (n = 37) showed less SB (p < .001, d = 0.378) and more LPA (p = .008, d = 0.725), while breaking up prolonged SB more frequently (p = .007, d = 0.696) on the tutorial day compared to the sit group (n = 32). Overall, undergraduates attending standing tutorial meetings showed less SB and more LPA than those attending conventional, seated tutorial meetings. Standing tutorial meetings can contribute to a more active lifestyle for undergraduates.

Twelve new and exciting Annonaceae from the Neotropics.

As a result of concerted efforts of the community of Annonaceae taxonomists, increasingly detailed knowledge of the diversity of the Neotropical genera has been documented. With the exception of just two large genera, Annona and Xylopia, all Neotropical Annonaceae have been revised within the last 25 years. Subsequent to these publications, many new specimens have been collected and sent to us in Leiden for identification. These included a number that, despite the advanced state of taxonomic knowledge, proved to represent rarely collected, undescribed species. Here we describe 12 new species of Annona, Guatteria, Klarobelia, Tetrameranthus, and Xylopia. These species serve to illustrate the still underestimated diversity of the Neotropical flora, even in well studied plant groups like Annonaceae.

A novel approach to study the morphology and chemistry of pollen in a phylogenetic context, applied to the halophytic taxon Nitraria L.(Nitrariaceae).

Nitraria is a halophytic taxon (i.e., adapted to saline environments) that belongs to the plant family Nitrariaceae and is distributed from the Mediterranean, across Asia into the south-eastern tip of Australia. This taxon is thought to have originated in Asia during the Paleogene (66-23 Ma), alongside the proto-Paratethys epicontinental sea. The evolutionary history of Nitraria might hold important clues on the links between climatic and biotic evolution but limited taxonomic documentation of this taxon has thus far hindered this line of research. Here we investigate if the pollen morphology and the chemical composition of the pollen wall are informative of the evolutionary history of Nitraria and could explain if origination along the proto-Paratethys and dispersal to the Tibetan Plateau was simultaneous or a secondary process. To answer these questions, we applied a novel approach consisting of a combination of Fourier Transform Infrared spectroscopy (FTIR), to determine the chemical composition of the pollen wall, and pollen morphological analyses using Light Microscopy (LM) and Scanning Electron Microscopy (SEM). We analysed our data using ordinations (principal components analysis and non-metric multidimensional scaling), and directly mapped it on the Nitrariaceae phylogeny to produce a phylomorphospace and a phylochemospace. Our LM, SEM and FTIR analyses show clear morphological and chemical differences between the sister groups Peganum and Nitraria. Differences in the morphological and chemical characteristics of highland species (Nitraria schoberi, N. sphaerocarpa, N. sibirica and N. tangutorum) and lowland species (Nitraria billardierei and N. retusa) are very subtle, with phylogenetic history appearing to be a more important control on Nitraria pollen than local environmental conditions. Our approach shows a compelling consistency between the chemical and morphological characteristics of the eight studied Nitrariaceae species, and these traits are in agreement with the phylogenetic tree. Taken together, this demonstrates how novel methods for studying fossil pollen can facilitate the evolutionary investigation of living and extinct taxa, and the environments they represent.

Phylogenomics of the Major Tropical Plant Family Annonaceae Using Targeted Enrichment of Nuclear Genes.

Targeted enrichment and sequencing of hundreds of nuclear loci for phylogenetic reconstruction is becoming an important tool for plant systematics and evolution. Annonaceae is a major pantropical plant family with 110 genera and ca. 2,450 species, occurring across all major and minor tropical forests of the world. Baits were designed by sequencing the transcriptomes of five species from two of the largest Annonaceae subfamilies. Orthologous loci were identified. The resulting baiting kit was used to reconstruct phylogenetic relationships at two different levels using concatenated and gene tree approaches: a family wide Annonaceae analysis sampling 65 genera and a species level analysis of tribe Piptostigmateae sampling 29 species with multiple individuals per species. DNA extraction was undertaken mainly on silicagel dried leaves, with two samples from herbarium dried leaves. Our kit targets 469 exons (364,653 bp of sequence data), successfully capturing sequences from across Annonaceae. Silicagel dried and herbarium DNA worked equally well. We present for the first time a nuclear gene-based phylogenetic tree at the generic level based on 317 supercontigs. Results mainly confirm previous chloroplast based studies. However, several new relationships are found and discussed. We show significant differences in branch lengths between the two large subfamilies Annonoideae and Malmeoideae. A new tribe, Annickieae, is erected containing a single African genus Annickia. We also reconstructed a well-resolved species-level phylogenetic tree of the Piptostigmteae tribe. Our baiting kit is useful for reconstructing well-supported phylogenetic relationships within Annonaceae at different taxonomic levels. The nuclear genome is mainly concordant with plastome information with a few exceptions. Moreover, we find that substitution rate heterogeneity between the two subfamilies is also found within the nuclear compartment, and not just plastomes and ribosomal DNA as previously shown. Our results have implications for understanding the biogeography, molecular dating and evolution of Annonaceae.

Revisions of Ruizodendron and Pseudephedranthus (Annonaceae) including a new species and an overview of most up-to-date revisions of Neotropical Annonaceae genera.

We present revisions of the Neotropical genera Ruizodendron and Pseudephedranthus (Annonaceae). Ruizodendron includes a single species R. ovale. Pseudephedranthus now comprises two species, with the description of the new species P. enigmaticussp. nov. extending the range of the genus beyond the Upper Rio Negro region of Brazil (Amazonas) and adjacent Venezuela (P. fragrans), to include Guyana, Suriname, and the Brazilian state of Pará. An overview is provided of current revisions of Neotropical Annonaceae genera that will aid in accessing proper species information for this frequently encountered tropical rain forest family.

Correlated evolutionary rates across genomic compartments in Annonaceae.

The molecular clock hypothesis is an important concept in biology. Deviations from a constant rate of nucleotide substitution have been found widely among lineages, genomes, genes and individual sites. Phylogenetic research can accommodate for these differences in applying specific models of evolution. Lineage-specific rate heterogeneity however can generate bi- or multimodal distributions of substitution rates across the branches of a tree and this may mislead phylogenetic inferences with currently available models. The plant family Annonaceae is an excellent case to study lineage-specific rate heterogeneity. The two major sister subfamilies, Annonoideae and Malmeoideae, have shown great discrepancies in branch lengths. We used high-throughput sequencing data of 72 genes, 99 spacers and 16 introns from 24 chloroplast genomes and nuclear ribosomal DNA of 23 species to study the molecular rate of evolution in Annonaceae. In all analyses, longer branch lengths and/or higher substitution rates were found for the Annonoideae compared to the Malmeoideae. The Annonaceae had wide variability in chloroplast length, ranging from minimal 175,684bp to 201,723 for Annonoideae and minimal 152,357 to 170,985bp in Malmeoideae, mostly reflecting variation in inverted-repeat length. The Annonoideae showed a higher GC-content in the conserved parts of the chloroplast genome and higher omega (dN/dS)-ratios than the Malmeoideae, which could indicate less stringent purifying selection, a pattern that has been found in groups with small population sizes. This study generates new insights into the processes causing lineage-specific rate heterogeneity, which could lead to improved phylogenetic methods.

Genomic treasure troves: complete genome sequencing of herbarium and insect museum specimens.

Unlocking the vast genomic diversity stored in natural history collections would create unprecedented opportunities for genome-scale evolutionary, phylogenetic, domestication and population genomic studies. Many researchers have been discouraged from using historical specimens in molecular studies because of both generally limited success of DNA extraction and the challenges associated with PCR-amplifying highly degraded DNA. In today's next-generation sequencing (NGS) world, opportunities and prospects for historical DNA have changed dramatically, as most NGS methods are actually designed for taking short fragmented DNA molecules as templates. Here we show that using a standard multiplex and paired-end Illumina sequencing approach, genome-scale sequence data can be generated reliably from dry-preserved plant, fungal and insect specimens collected up to 115 years ago, and with minimal destructive sampling. Using a reference-based assembly approach, we were able to produce the entire nuclear genome of a 43-year-old Arabidopsis thaliana (Brassicaceae) herbarium specimen with high and uniform sequence coverage. Nuclear genome sequences of three fungal specimens of 22-82 years of age (Agaricus bisporus, Laccaria bicolor, Pleurotus ostreatus) were generated with 81.4-97.9% exome coverage. Complete organellar genome sequences were assembled for all specimens. Using de novo assembly we retrieved between 16.2-71.0% of coding sequence regions, and hence remain somewhat cautious about prospects for de novo genome assembly from historical specimens. Non-target sequence contaminations were observed in 2 of our insect museum specimens. We anticipate that future museum genomics projects will perhaps not generate entire genome sequences in all cases (our specimens contained relatively small and low-complexity genomes), but at least generating vital comparative genomic data for testing (phylo)genetic, demographic and genetic hypotheses, that become increasingly more horizontal. Furthermore, NGS of historical DNA enables recovering crucial genetic information from old type specimens that to date have remained mostly unutilized and, thus, opens up a new frontier for taxonomic research as well.

Inter-and intraspecific variation in fern mating systems after long-distance colonization: the importance of selfing.

BACKGROUND: Previous studies on the reproductive biology of ferns showed that mating strategies vary among species, and that polyploid species often show higher capacity for self-fertilization than diploid species. However, the amount of intraspecific variation in mating strategy and selfing capacity has only been assessed for a few species. Yet, such variation may have important consequences during colonization, as the establishment of any selfing genotypes may be favoured after long-distance dispersal (an idea known as Baker's law). RESULTS: We examined intra-and interspecific variation in potential for self-fertilization among four rare fern species, of which two were diploids and two were tetraploids: Asplenium scolopendrium (2n), Asplenium trichomanes subsp. quadrivalens (4n), Polystichum setiferum (2n) and Polystichum aculeatum (4n). Sporophyte production was tested at different levels of inbreeding, by culturing gametophytes in isolation, as well as in paired cultures with a genetically different gametophyte. We tested gametophytes derived from various genetically different sporophytes from populations in a recently planted forest colonized through long-distance dispersal (Kuinderbos, the Netherlands), as well as from older, less disjunct populations.Sporophyte production in isolation was high for Kuinderbos genotypes of all four species. Selfing capacity did not differ significantly between diploids and polyploids, nor between species in general. Rather selfing capacity differed between genotypes within species. Intraspecific variation in mating system was found in all four species. In two species one genotype from the Kuinderbos showed enhanced sporophyte production in paired cultures. For the other species, including a renowned out crosser, selfing capacity was consistently high. CONCLUSIONS: Our results for four different species suggest that intraspecific variation in mating system may be common, at least among temperate calcicole ferns, and that genotypes with high selfing capacity may be present among polyploid as well as diploid ferns. The surprisingly high selfing capacity of all genotypes obtained from the Kuinderbos populations might be due to the isolated position of these populations. These populations may have established through single-spore colonization, which is only possible for genotypes capable of self-fertilization. Our results therewith support the idea that selection for selfing genotypes may occur during long-distance colonization, even in normally outcrossing, diploid ferns.

Use of rbcL and trnL-F as a two-locus DNA barcode for identification of NW-European ferns: an ecological perspective.

Although consensus has now been reached on a general two-locus DNA barcode for land plants, the selected combination of markers (rbcL + matK) is not applicable for ferns at the moment. Yet especially for ferns, DNA barcoding is potentially of great value since fern gametophytes--while playing an essential role in fern colonization and reproduction--generally lack the morphological complexity for morphology-based identification and have therefore been underappreciated in ecological studies. We evaluated the potential of a combination of rbcL with a noncoding plastid marker, trnL-F, to obtain DNA-identifications for fern species. A regional approach was adopted, by creating a reference database of trusted rbcL and trnL-F sequences for the wild-occurring homosporous ferns of NW-Europe. A combination of parsimony analyses and distance-based analyses was performed to evaluate the discriminatory power of the two-region barcode. DNA was successfully extracted from 86 tiny fern gametophytes and was used as a test case for the performance of DNA-based identification. Primer universality proved high for both markers. Based on the combined rbcL + trnL-F dataset, all genera as well as all species with non-equal chloroplast genomes formed their own well supported monophyletic clade, indicating a high discriminatory power. Interspecific distances were larger than intraspecific distances for all tested taxa. Identification tests on gametophytes showed a comparable result. All test samples could be identified to genus level, species identification was well possible unless they belonged to a pair of Dryopteris species with completely identical chloroplast genomes. Our results suggest a high potential of the combined use of rbcL and trnL-F as a two-locus cpDNA barcode for identification of fern species. A regional approach may be preferred for ecological tests. We here offer such a ready-to-use barcoding approach for ferns, which opens the way for answering a whole range of questions previously unaddressed in fern gametophyte ecology.

A rapid diversification of rainforest trees (Guatteria; Annonaceae) following dispersal from Central into South America.

Several recent studies have suggested that a substantial portion of today's plant diversity in the Neotropics has resulted from the dispersal of taxa into that region rather than vicariance, but more data are needed to substantiate this claim. Guatteria (Annonaceae) is, with 265 species, the third largest genus of Neotropical trees after Inga (Fabaceae) and Ocotea (Lauraceae), and its widespread distribution and frequent occurrence makes the genus an excellent model taxon to study diversification patterns. This study reconstructed the phylogeny of Guatteria and inferred three major biogeographical events in the history of the genus: (1) a trans-oceanic Miocene migration from Central into South America before the closing of the Isthmus of Panama; (2) a major diversification of the lineage within South America; and (3) several migrations of South American lineages back into Central America via the closed Panamanian land bridge. Therefore, Guatteria is not an Amazonian centred-genus sensu Gentry but a major Miocene diversification that followed its dispersal into South America. This study provides further evidence that migration into the Neotropics was an important factor in the historical assembly of its biodiversity. Furthermore, it is shown that phylogenetic patterns are comparable to those found in Ocotea and Inga and that a closer comparison of these genera is desirable.

Prediction of energy expenditure in a whole body indirect calorimeter at both low and high levels of physical activity.

OBJECTIVES: In studies that involve the use of a room calorimeter, 24 h energy intake is often larger than 24 h energy expenditure (24 h EE) because of a decrease in activity energy expenditure due to the confined space. This positive energy balance can have large consequences for the interpretation of substrate balances. The objective of this study was to develop a method for predicting an individual's 24 h EE in a room calorimeter at both low (1.4xRMR) and high (1.8xRMR) levels of physical activity. METHODS: Two methods are presented that predict an individual's 24 h EE in a metabolic chamber. The first method was based on three components: (1) a 30 min measurement of resting metabolic rate (RMR) using a ventilated hood system; (2) measurement of exercise energy expenditure during 10 min of treadmill walking; and (3) estimation of free-living energy expenditure using a tri-axial motion sensor. Using these measurements we calculated the amount of treadmill time needed for each individual in order to obtain a total 24 h EE at either a low (1.4xRMR) or a high (1.8xRMR) level of physical activity. We also developed a method to predict total 24 h EE during the chamber stay by using the energy expenditure values for the different levels of activity as measured during the hours already spent in the chamber. This would provide us with a tool to adjust the exercise time and/or energy intake during the chamber stay. RESULTS: Method 1: there was no significant difference in expected and measured 24 h EE under either low (9.35+/-0.56 vs 9.51+/-0.47 MJ/day; measured vs predicted) or high activity conditions (13.41+/-0.74 vs 13.97+/-0.78 MJ/day; measured vs predicted). Method 2: the developed algorithm predicted 24 h EE for 97.6+/-4.0% of the final value at 3 h into the test day, and for 98.6+/-3.7% at 7 h into the test day. CONCLUSION: Both methods provide accurate prediction of energy expenditure in a room calorimeter at both high and low levels of physical activity. It equally shows that it is possible to accurately predict total 24 h EE from energy expenditure values obtained at 3 and 7 h into the study.

Substrate oxidation and energy expenditure in athletes and nonathletes consuming isoenergetic high- and low-fat diets.

Changes in substrate oxidation with isoenergetic high-carbohydrate (HC) and high-fat (HF) diets in male nonathletic subjects, aerobically trained athletes, and weight-trained athletes were examined in a crossover study. A whole-room respiration chamber was used to measure 24-h energy expenditure (EE) and substrate oxidation with control, HC, or HF diets for 7 d. The nonathletic group had higher 24-h EE (P < 0.05), exercise EE (P < 0.03), and resting metabolic rate (P < 0.04) than did the aerobically trained athletes when these measurements were corrected for lean body mass. Fat oxidation was significantly correlated with lean body mass and diet. However, athletic status had no effect on substrate oxidation. Carbohydrate oxidation across groups increased acutely by 23% after 24 h of the HC diet (P < 0.0001). Carbohydrate balance increased significantly over time with the HC diet (P < 0.002) and decreased acutely after return to the control diet (P < 0.0001). With the HF diet, carbohydrate balance increased and was significantly different from balance with the control diet by day 7 (P < 0.03). Fat balance decreased significantly with both the HF (P < 0.04) and HC (P = 0.0075) diets by day 7. Carbohydrate oxidation correlated with carbohydrate intake with both the control (r = 0.61, P < 0.01) and HC diets (r = 0.59, P < 0.02), but not the HF diet. Fat oxidation was not correlated with fat intake. In conclusion, substrate oxidation in a respiration chamber is significantly affected by diet, but not by prior athletic training.