Caught in the Act: Incipient Speciation at the Southern limit of Viburnum in the Central Andes.

A fundamental objective of evolutionary biology is to understand the origin of independently evolving species. Phylogenetic studies of species radiations rarely are able to document ongoing speciation; instead, modes of speciation, entailing geographic separation and/or ecological differentiation, are posited retrospectively. The Oreinotinus clade of Viburnum has radiated recently from north to south through the cloud forests of Mexico and Central America to the Central Andes. Our analyses support a hypothesis of incipient speciation in Oreinotinus at the southern edge of its geographic range, from central Peru to northern Argentina. Although several species and infraspecific taxa of have been recognized in this area, multiple lines of evidence and analytical approaches (including analyses of phylogenetic relationships, genetic structure, leaf morphology, and climatic envelopes) favor the recognition of just a single species, V. seemenii. We show that what has previously been recognized as V. seemenii f. minor has recently occupied the drier Tucuman-Bolivian forest region from Samaipata in Bolivia to Salta in northern Argentina. Plants in these populations form a well-supported clade with a distinctive genetic signature and they have evolved smaller, narrower leaves. We interpret this as the beginning of a within-species divergence process that has elsewhere in the neotropics resulted repeatedly in Viburnum species with a particular set of leaf ecomorphs. Specifically, the southern populations are in the process of evolving the small, glabrous, and entire leaf ecomorph that has evolved in four other montane areas of endemism. As predicted based on our studies of leaf ecomorphs in Chiapas, Mexico, these southern populations experience generally drier conditions, with large diurnal temperature fluctuations. In a central portion of the range of V. seemenii, characterized by wetter climatic conditions, we also document what may be the initial differentiation of the leaf ecomorph with larger, pubescent, and toothy leaves. The emergence of these ecomorphs thus appears to be driven by adaptation to subtly different climatic conditions in separate geographic regions, as opposed to parapatric differentiation along elevational gradients as suggested by Viburnum species distributions in other parts of the neotropics.

Radiocarbon and wood anatomy as complementary tools for generating tree-ring records in Bolivia.

The science of tropical dendrochronology is now emerging in regions where tree-ring dating had previously not been considered possible. Here, we combine wood anatomical microsectioning techniques and radiocarbon analysis to produce the first tree-ring chronology with verified annual periodicity for a new dendrochronological species, Neltuma alba (commonly known as "algarrobo blanco") in the tropical Andes of Bolivia. First, we generated a preliminary chronology composed of six trees using traditional dendrochronological methods (i.e., cross-dating). We then measured the 14C content on nine selected tree rings from two samples and compared them with the Southern Hemisphere (SH) atmospheric 14C curves, covering the period of the bomb 14C peak. We find consistent offsets of 5 and 12 years, respectively, in the calendar dates initially assigned, indicating that several tree rings were missing in the sequence. In order to identify the tree-ring boundaries of the unidentified rings we investigated further by analyzing stem wood microsections to examine anatomical characteristics. These anatomical microsections revealed the presence of very narrow terminal parenchyma defining several tree-ring boundaries within the sapwood, which was not visible in sanded samples under a stereomicroscope. Such newly identified tree rings were consistent with the offsets shown by the radiocarbon analysis and allowed us to correct the calendar dates of the initial chronology. Additional radiocarbon measurements over a new batch of rings of the corrected dated samples resulted in a perfect match between the dendrochronological calendar years and the 14C dating, which is based on good agreement between the tree-ring 14C content and the SH 14C curves. Correlations with prior season precipitation and temperature reveal a strong legacy effect of climate conditions prior to the current Neltuma alba growing season. Overall, our study highlights much potential to complement traditional dendrochronology in tree species with challenging tree-ring boundaries with wood anatomical methods and 14C analyses. Taken together, these approaches confirm that Neltuma alba can be accurately dated and thereby used in climatic and ecological studies in tropical and subtropical South America.

FunAndes - A functional trait database of Andean plants.

We introduce the FunAndes database, a compilation of functional trait data for the Andean flora spanning six countries. FunAndes contains data on 24 traits across 2,694 taxa, for a total of 105,466 entries. The database features plant-morphological attributes including growth form, and leaf, stem, and wood traits measured at the species or individual level, together with geographic metadata (i.e., coordinates and elevation). FunAndes follows the field names, trait descriptions and units of measurement of the TRY database. It is currently available in open access in the FIGSHARE data repository, and will be part of TRY's next release. Open access trait data from Andean plants will contribute to ecological research in the region, the most species rich terrestrial biodiversity hotspot.

Travel Tales of a Worldwide Weed: Genomic Signatures of Plantago major L. Reveal Distinct Genotypic Groups With Links to Colonial Trade Routes.

Retracing pathways of historical species introductions is fundamental to understanding the factors involved in the successful colonization and spread, centuries after a species' establishment in an introduced range. Numerous plants have been introduced to regions outside their native ranges both intentionally and accidentally by European voyagers and early colonists making transoceanic journeys; however, records are scarce to document this. We use genotyping-by-sequencing and genotype-likelihood methods on the selfing, global weed, Plantago major, collected from 50 populations worldwide to investigate how patterns of genomic diversity are distributed among populations of this global weed. Although genomic differentiation among populations is found to be low, we identify six unique genotype groups showing very little sign of admixture and low degree of outcrossing among them. We show that genotype groups are latitudinally restricted, and that more than one successful genotype colonized and spread into the introduced ranges. With the exception of New Zealand, only one genotype group is present in the Southern Hemisphere. Three of the most prevalent genotypes present in the native Eurasian range gave rise to introduced populations in the Americas, Africa, Australia, and New Zealand, which could lend support to the hypothesis that P. major was unknowlingly dispersed by early European colonists. Dispersal of multiple successful genotypes is a likely reason for success. Genomic signatures and phylogeographic methods can provide new perspectives on the drivers behind the historic introductions and the successful colonization of introduced species, contributing to our understanding of the role of genomic variation for successful establishment of introduced taxa.

A highly contiguous, scaffold-level nuclear genome assembly for the fever tree (Cinchona pubescens Vahl) as a novel resource for Rubiaceae research.

The Andean fever tree (Cinchona L.; Rubiaceae) is a source of bioactive quinine alkaloids used to treat malaria. C. pubescens Vahl is a valuable cash crop within its native range in northwestern South America, however, genomic resources are lacking. Here we provide the first highly contiguous and annotated nuclear and plastid genome assemblies using Oxford Nanopore PromethION-derived long-read and Illumina short-read data. Our nuclear genome assembly comprises 603 scaffolds with a total length of 904 Mbp (∼82% of the full genome based on a genome size of 1.1 Gbp/1C). Using a combination of de novo and reference-based transcriptome assemblies we annotated 72,305 coding sequences comprising 83% of the BUSCO gene set and 4.6% fragmented sequences. Using additional plastid and nuclear datasets we place C. pubescens in the Gentianales order. This first genomic resource for C. pubescens opens new research avenues, including the analysis of alkaloid biosynthesis in the fever tree.

Historical chemical annotations of Cinchona bark collections are comparable to results from current day high-pressure liquid chromatography technologies.

ETHNOPHARMACOLOGICAL RELEVANCE: Species of the genus Cinchona (Rubiaceae) have been used in traditional medicine, and as a source for quinine since its discovery as an effective medicine against malaria in the 17th century. Despite being the sole cure of malaria for almost 350 years, little is known about the chemical diversity between and within species of the antimalarial alkaloids found in the bark. Extensive historical Cinchona bark collections housed at the Royal Botanic Gardens, Kew, UK, and in other museums may shed new light on the alkaloid chemistry of the Cinchona genus and the history of the quest for the most effective Cinchona barks. AIM OF THE STUDY: We used High-Pressure Liquid Chromatography (HPLC) coupled with fluorescence detection (FLD) to reanalyze a set of Cinchona barks originally annotated for the four major quinine alkaloids by John Eliot Howard and others more than 150 years ago. MATERIALS AND METHODS: We performed an archival search on the Cinchona bark collections in the Economic Botany Collection housed in Kew, focusing on those with historical alkaloid content information. Then, we performed HPLC analysis of the bark samples to separate and quantify the four major quinine alkaloids and the total alkaloid content using fluorescence detection. Correlations between historic and current annotations were calculated using Spearman's rank correlation coefficient, before paired comparisons were performed using Wilcox rank sum tests. The effects of source were explored using generalized linear modelling (GLM), before the significance of each parameter in predicting alkaloid concentrations were assessed using chi-square tests as likelihood ratio testing (LRT) models. RESULTS: The total alkaloid content estimation obtained by our HPLC analysis was comparatively similar to the historical chemical annotations made by Howard. Additionally, the quantity of two of the major alkaloids, quinine and cinchonine, and the total content of the four alkaloids obtained were significantly similar between the historical and current day analysis using linear regression. CONCLUSIONS: This study demonstrates that the historical chemical analysis by Howard and current day HPLC alkaloid content estimations are comparable. Current day HPLC analysis thus provide a realistic estimate of the alkaloid contents in the historical bark samples at the time of sampling more than 150 years ago. Museum collections provide a powerful but underused source of material for understanding early use and collecting history as well as for comparative analyses with current day samples.

Anestesia para cirugía fetal: Consideraciones anestésicas y alcances terapéuticos / Implications and anesthetic considerations for fetal surgery

Fetal surgery is a field that has experienced great progress in recent decades. Advances in prenatal imaging techniques have allowed treatment of in-utero fetal pathologies during the prenatal period, so that it is currently possible to intervene in the natural history of certain alterations in the development of the fetus, avoiding sequelae in the newborn and in its subsequent development in extrauterine life. The perioperative management of fetal surgery requires a multidisciplinary team, constituting a challenge for the anesthesiologist to maintain the homeostasis of the mother and the fetus. The understanding of the maternal-fetal physiology together with an adequate management of the anesthetic techniques constitute the cornerstone for the success of the surgery.

La cirugía fetal es un campo que ha experimentado gran progreso en las últimas décadas. El avance en las técnicas de diagnóstico prenatal por imágenes ha permitido tratamiento de patologías fetales in utero durante el período prenatal, de modo que actualmente es posible modificar la historia natural de determinadas alteraciones en el desarrollo del feto evitando secuelas en el recién nacido y en su posterior desarrollo en la vida extrauterina. El manejo perioperatorio de la cirugía fetal precisa de un equipo multidisciplinario, constituyendo un desafío para el anestesiólogo mantener la homeostasis de la madre y el feto. El entendimiento de la fisiología materno-fetal junto con un adecuado manejo de las técnicas anestésicas constituyen un factor fundamental para el éxito de la cirugía.

An Optimised Method for Routine Separation and Quantification of Major Alkaloids in Cortex Cinchona by HPLC Coupled with UV and Fluorescence Detection.

INTRODUCTION: Authentication of herbal products to ensure efficacy and safety require efficient separation and quantification of constituents. Standard assays for Cinchona bark used for the treatment of malaria and production of quinine, either use only spectrophotometry to detect two pairs of diastereoisomers of quinine and cinchonine type alkaloids (European Pharmacopoeia, Ph.Eur.) or liquid chromatography primarily optimised for detection of the four major alkaloids. However, numerous minor alkaloids occur in Cinchona and related species and efficient separation including gradient elution is necessary in order to obtain the full pattern of constituents in bark samples. OBJECTIVE: To develop an optimised HPLC method for separation and quantitative analysis of the four major alkaloids in Cinchona bark using UV detection. METHODOLOGY: Dimethyl sulphoxide (DMSO) extracts of 50 mg of pulverised barks were prepared using ultrasonication. The chromatographic separation was performed on an XB-C18 column packed with 2.6 µm particles. Gradient elution using an ammonium formate buffer and methanol as organic modifier over 26 min was based on non-chiral separation of the diastereoisomers and the high solvent selectivity of methanol. Post column UV detection was performed at 250 nm and 330 nm. Fluorescence detection was performed using 330 nm for excitation and 420 nm for emission. RESULTS: The optimised HPLC method facilitates efficient separation and quantification of the four major alkaloids in 26 min with a limit of quantification of 5 µg/g from 50 mg bark sample. CONCLUSION: The optimised HPLC method offers a simple and efficient quantification of the four major alkaloids. Copyright © 2017 John Wiley & Sons, Ltd.

Phylogeny Predicts the Quantity of Antimalarial Alkaloids within the Iconic Yellow Cinchona Bark (Rubiaceae: Cinchona calisaya).

Considerable inter- and intraspecific variation with respect to the quantity and composition of plant natural products exists. The processes that drive this variation remain largely unknown. Understanding which factors determine chemical diversity has the potential to shed light on plant defenses against herbivores and diseases and accelerate drug discovery. For centuries, Cinchona alkaloids were the primary treatment of malaria. Using Cinchona calisaya as a model, we generated genetic profiles of leaf samples from four plastid (trnL-F, matK, rps16, and ndhF) and one nuclear (ITS) DNA regions from twenty-two C. calisaya stands sampled in the Yungas region of Bolivia. Climatic and soil parameters were characterized and bark samples were analyzed for content of the four major alkaloids using HPLC-UV to explore the utility of evolutionary history (phylogeny) in determining variation within species of these compounds under natural conditions. A significant phylogenetic signal was found for the content of two out of four major Cinchona alkaloids (quinine and cinchonidine) and their total content. Climatic parameters, primarily driven by changing altitude, predicted 20.2% of the overall alkaloid variation, and geographical separation accounted for a further 9.7%. A clade of high alkaloid producing trees was identified that spanned a narrow range of altitudes, from 1,100 to 1,350 m. However, climate expressed by altitude was not a significant driver when accounting for phylogeny, suggesting that the chemical diversity is primarily driven by phylogeny. Comparisons of the relative effects of both environmental and genetic variability in determining plant chemical diversity have scarcely been performed at the genotypic level. In this study we demonstrate there is an essential need to do so if the extensive genotypic variation in plant biochemistry is to be fully understood.

Unexpectedly High Beta-Diversity of Root-Associated Fungal Communities in the Bolivian Andes.

Bolivia is one of the most biologically diverse countries on the planet. Between the Andes and the Amazon drainage basin spans the Yungas, a vast forested region shown to be extremely species rich in macro-organisms. However, it remains unclear whether this high diversity is also reflected in microbial diversity. Here we assess the genetic, taxonomic and functional diversity of root-associated fungi surrounding Cinchona calisaya trees, a typical element of the intermediate altitudes of the Bolivian Yungas. We determine the relative effects of edaphic properties, climate, and geography in regulating fungal community assembly. We show that α-diversity for these fungal communities was similar to temperate and arid ecosystems, averaging 90.1 operational taxonomic units (OTUs) per sample, with reads predominantly assigned to the Ascomycota phylum and with a saprotrophic lifestyle. ß-diversity was calculated as the distance-decay rate, and in contrast to α-diversity, was exceptionally high with a rate of -0.407. Soil properties (pH and P) principally regulated fungal community assembly in an analogous manner to temperate environments, with pH and phosphorus explaining 7.8 and 7.2% of community variation respectively. Surprisingly, altitude does not influence community formation, and there is limited evidence that climate (precipitation and temperature) play a role. Our results suggest that sampling should be performed over a wide geographical and environmental range in order to capture the full root-associated fungal diversity in subtropical regions. This study sheds further light on the diversity and distribution of the world's "hidden biodiversity."

Estimating species diversity and distribution in the era of Big Data: to what extent can we trust public databases?

AIM: Massive digitalization of natural history collections is now leading to a steep accumulation of publicly available species distribution data. However, taxonomic errors and geographical uncertainty of species occurrence records are now acknowledged by the scientific community - putting into question to what extent such data can be used to unveil correct patterns of biodiversity and distribution. We explore this question through quantitative and qualitative analyses of uncleaned versus manually verified datasets of species distribution records across different spatial scales. LOCATION: The American tropics. METHODS: As test case we used the plant tribe Cinchoneae (Rubiaceae). We compiled four datasets of species occurrences: one created manually and verified through classical taxonomic work, and the rest derived from GBIF under different cleaning and filling schemes. We used new bioinformatic tools to code species into grids, ecoregions, and biomes following WWF's classification. We analysed species richness and altitudinal ranges of the species. RESULTS: Altitudinal ranges for species and genera were correctly inferred even without manual data cleaning and filling. However, erroneous records affected spatial patterns of species richness. They led to an overestimation of species richness in certain areas outside the centres of diversity in the clade. The location of many of these areas comprised the geographical midpoint of countries and political subdivisions, assigned long after the specimens had been collected. MAIN CONCLUSION: Open databases and integrative bioinformatic tools allow a rapid approximation of large-scale patterns of biodiversity across space and altitudinal ranges. We found that geographic inaccuracy affects diversity patterns more than taxonomic uncertainties, often leading to false positives, i.e. overestimating species richness in relatively species poor regions. Public databases for species distribution are valuable and should be more explored, but under scrutiny and validation by taxonomic experts. We suggest that database managers implement easy ways of community feedback on data quality.

An Efficient, Robust, and Inexpensive Grinding Device for Herbal Samples like Cinchona Bark.

An effective, robust, and inexpensive grinding device for the grinding of herb samples like bark and roots was developed by rebuilding a commercially available coffee grinder. The grinder was constructed to be able to provide various particle sizes, to be easy to clean, and to have a minimum of dead volume. The recovery of the sample when grinding as little as 50 mg of crude Cinchona bark was about 60%. Grinding is performed in seconds with no rise in temperature, and the grinder is easily disassembled to be cleaned. The influence of the particle size of the obtained powders on the recovery of analytes in extracts of Cinchona bark was investigated using HPLC.