Journal articles on the topic 'Spectral diversity hypothesi'

Plant diversity (PD) plays an important role in maintaining the healthy function of an ecosystem through affecting the productivity, stability, and nutrient utilization of a terrestrial ecosystem. Remote sensing is a vital way to monitor the status and changes of PD. Most of the existing methods rely on a field botany survey to construct a statistical relationship between PD and remote sensing observations. However, a field botany survey is too costly to be applied widely. In this study, we constructed a new remote sensing index of PD (RSPD), combining the spectral variation hypothesis and productivity hypothesis. Concretely, the RSPD integrated the multi-band spectral reflectance and several spectral greenness, moisture, and red-edge vegetation indices with the principles of Shannon information entropy and Euclidean distance. The RSPD was evaluated by comparing the classical coefficient of variation (CV) method and the Shannon and Simpson diversity indices based on vegetation classification results. Two cases were selected, where Case I was in Beijing and Case II was located in part of Huai’an, China. Sentinel-2 data in three years of 2016, 2018, and 2020 and higher-resolution Pléiades-1 data in 2018 were also utilized. The results demonstrate that: (1) the RSPD is basically consistent with the CV in spatiotemporal variation; (2) the RSPD outperforms the CV as compared with Shannon and Simpson diversity indices that are based on vegetation classification results with Sentinel-2 and Pléiades-1 data; (3) the RSPD outperforms the CV as compared with visual interpretations with Google Earth image. The suggested index can reflect the richness and evenness of plant species, which is inherent in its calculation formula. Moreover, it has a great potential for large-scale regional and long-term series monitoring.

Grassland species diversity monitoring is essential to grassland resource protection and utilization. “Spectral variation hypothesis” (SVH) provides a remote sensing method for monitoring grassland species diversity at pixel scale by calculating spectral heterogeneity. However, the pixel spectrum is easily affected by soil and other background factors in natural grassland. Unmanned aerial vehicle (UAV)-based imaging spectroscopy provides the possibility of soil information removal by virtue of its high spatial and spectral resolution. In this study, UAV-imaging spectroscopy data with a spatial resolution of 0.2 m obtained in two sites of typical alpine steppe within the Sanjiangyuan National Nature Reserve were used to analyze the relationships between four spectral diversity metrics (coefficient of variation based on NDVI (CVNDVI), coefficient of variation based on multiple bands (CVMulti), minimum convex hull volume (CHV) and minimum convex hull area (CHA)) and two species diversity indices (species richness and the Shannon–Wiener index). Meanwhile, two soil removal methods (based on NDVI threshold and the linear spectral unmixing model) were used to investigate the impact of soil on species diversity estimation. The results showed that the Shannon–Wiener index had a better response to spectral diversity than species richness, and CVMulti showed the best correlation with the Shannon–Wiener index between the four spectral diversity metrics after removing soil information using the linear spectral unmixing model. It indicated that the estimation ability of spectral diversity to species diversity was significantly improved after removing the soil information. Our findings demonstrated the applicability of the spectral variation hypothesis in natural grassland, and illustrated the impact of soil on species diversity estimation.

Combining field collected and remotely sensed (RS) data represents one of the most promising approaches for an extensive and up-to-date ecosystem assessment. We investigated the potential of the so called spectral variability hypothesis (SVH) in linking field-collected and remote-sensed data in Mediterranean coastal dunes and explored if spectral diversity provides reliable information to monitor floristic diversity, as well as the consistency of such information in altered ecosystems due to plant invasions. We analyzed alpha diversity and beta diversity, integrating floristic field and Remote-Sensing PlanetScope data in the Tyrrhenian coast (Central Italy). We explored the relationship among alpha field diversity (species richness, Shannon index, inverse Simpson index) and spectral variability (distance from the spectral centroid index) through linear regressions. For beta diversity, we implemented a distance decay model (DDM) relating field pairwise (Jaccard similarities index, Bray–Curtis similarities index) and spectral pairwise (Euclidean distance) measures. We observed a positive relationship between alpha diversity and spectral heterogeneity with richness reporting the higher R score. As for DDM, we found a significant relationship between Bray–Curtis floristic similarity and Euclidean spectral distance. We provided a first assessment of the relationship between floristic and spectral RS diversity in Mediterranean coastal dune habitats (i.e., natural or invaded). SVH provided evidence about the potential of RS for estimating diversity in complex and dynamic landscapes.

Abstract. Tackling the accelerated human-induced biodiversity loss requires tools able to map biodiversity and its changes globally. Remote sensing (RS) offers unique capabilities of characterizing Earth surfaces; therefore, it could map plant biodiversity continuously and globally. This approach is supported by the Spectral Variation Hypothesis (SVH), which states that spectra and species (taxonomic and trait) diversities are linked through environmental heterogeneity. In this work, we evaluate the capability of the DESIS hyperspectral imager to capture plant diversity patterns as measured in dedicated plots of the network FunDivEUROPE. We computed functional and taxonomical diversity metrics from field taxonomic, structural, and foliar measurements in vegetation plots sampled in Spain and Romania. In addition, we also computed functional diversity metrics both from the DESIS reflectance factors and from vegetation parameters estimated via inversion of a radiative transfer model. Results showed that only metrics computed from spectral reflectance were able to capture taxonomic variability in the area. However, the lack of sensitivity was related to the insufficient plot size and the lack of spatial match between remote sensing and field data, but also the differences between the information contained in the field traits and remote sensing data, and the potential uncertainties in the remote estimates of vegetation parameters. Thus, while DESIS showed some sensitivity to plant diversity, further efforts are needed to deploy suitable biodiversity evaluation and validation plots and networks that support the development of biodiversity remote sensing products.

As there is an urgent need to protect rapidly declining global diversity, it is important to identify methods to quickly estimate the diversity and heterogeneity of a region and effectively implement monitoring and conservation plans. The combination of remotely sensed and field-collected data, under the paradigm of the Spectral Variation Hypothesis (SVH), represents one of the most promising approaches to boost large-scale and reliable biodiversity monitoring practices. Here, the potential of SVH to capture information on plant diversity at a fine scale in an ecological network (EN) embedded in a complex landscape has been tested using two new and promising methodological approaches: the first estimates α and β spectral diversity and the latter ecosystem spectral heterogeneity expressed as Rao’s Quadratic heterogeneity measure (Rao’s Q). Both approaches are available thanks to two brand-new R packages: “biodivMapR” and “rasterdiv”. Our aims were to investigate if spectral diversity and heterogeneity provide reliable information to assess and monitor over time floristic diversity maintained in an EN selected as an example and located in northeast Italy. We analyzed and compared spectral and taxonomic α and β diversities and spectral and landscape heterogeneity, based on field-based plant data collection and remotely sensed data from Sentinel-2A, using different statistical approaches. We observed a positive relationship between taxonomic and spectral diversity and also between spectral heterogeneity, landscape heterogeneity, and the amount of alien species in relation to the native ones, reaching a value of R2 = 0.36 and R2 = 0.43, respectively. Our results confirmed the effectiveness of estimating and mapping α and β spectral diversity and ecosystem spectral heterogeneity using remotely sensed images. Moreover, we highlighted that spectral diversity values become more effective to identify biodiversity-rich areas, representing the most important diversity hotspots to be preserved. Finally, the spectral heterogeneity index in anthropogenic landscapes could be a powerful method to identify those areas most at risk of biological invasion.

The emergence of the spectral variation hypothesis (SVH) has gained widespread attention in the remote sensing community as a method for deriving biodiversity information from remotely sensed data. SVH states that spectral heterogeneity on remotely sensed imagery reflects environmental heterogeneity, which in turn is associated with high species diversity and, therefore, could be useful for characterizing landscape biodiversity. However, the effect of phenology has received relatively less attention despite being an important variable influencing plant species spectral responses. The study investigated (i) the effect of phenology on the relationship between spectral heterogeneity and plant species diversity and (ii) explored spectral angle mapper (SAM), the coefficient of variation (CV) and their interaction effect in estimating species diversity. Stratified random sampling was adopted to survey all tree species with a diameter at breast height of > 10 cm in 90 × 90 m plots distributed throughout the study site. Tree species diversity was quantified by the Shannon diversity index (H′), Simpson index of diversity (D2) and species richness (S). SAM and CV were employed on Landsat-8 data to compute spectral heterogeneity. The study applied linear regression models to investigate the relationship between spectral heterogeneity metrics and species diversity indices across four phenological stages. The results showed that the end of the growing season was the most ideal phenological stage for estimating species diversity, following the SVH concept. During this period, SAM and species diversity indices (S, H′, D2) had an r2 of 0.14, 0.24, and 0.20, respectively, while CV had an r2 of 0.22, 0.22, and 0.25, respectively. The interaction of SAM and CV improved the relationship between the spectral data and H′ and D2 (from r2 of 0.24 and 0.25 to r2 of 0.32 and 0.28, respectively) at the end of the growing season. The two spectral heterogeneity metrics showed differential sensitivity to components of plant diversity. SAM had a high relationship with H′ followed by D2 and then a lower relationship with S throughout the different phenological stages. Meanwhile, CV had a higher relationship with D2 than other plant diversity indices and its relationship with S and H′ remained similar. Although the coefficient of determination was comparatively low, the relationship between spectral heterogeneity metrics and species diversity indices was statistically significant (p < 0.05) and this supports the assertion that SVH could be implemented to characterize plant species diversity. Importantly, the application of SVH should consider (i) the choice of spectral heterogeneity metric in line with the purpose of the SVH application since these metrics relate to components of species diversity differently and (ii) vegetation phenology, which affects the relationship that spectral heterogeneity has with plant species diversity.

Landscape heterogeneity, as measured by the spectral diversity of satellite imagery, has the potential to provide information on the resources available within the movement capacity range of arthropod vectors, and to help predict vector abundance. The Spectral Variation Hypothesis states that higher spectral diversity is positively related to a higher number of ecological niches present in the landscape, allowing more species to coexist regardless of the taxonomic group considered. Investigating the landscape heterogeneity as a proxy of the resources available to vectors may be relevant for complex and continuous agro-forest mosaics of small farmlands and degraded forests, where land cover classification is often imprecise. In this study, we hypothesized that larger spectral diversity would be associated with higher tick abundance due to the potentially higher number of hosts in heterogeneous landscapes. Specifically, we tested whether spectral diversity indices could represent heterogeneous landscapes, and if so, whether they explain Amblyomma and Hyalomma tick abundance in Benin and inform on their habitat preferences. Benin is a West-African country characterized by a mosaic landscape of farmland and degraded forests. Our results showed that both NDVI-derived and spectral predictors are highly collinear, with NDVI-derived predictors related to vegetated land cover classes and spectral predictors correlated to mosaic landscapes. Amblyomma abundance was not related to the predictors considered. Hyalomma abundance showed positive relationships to spectral diversity indices and negative relationships to NDVI-derived-ones. Though taxa dependent, our approach showed moderate performance in terms of goodness of fit (ca. 13–20% R2), which is a promising result considering the sampling and scale limitations. Spectral diversity indices coupled with classical SRS vegetation indices could be a complementary approach for providing further ecological aspects in the field of disease biogeography.

Over the last 20 years, there has been a surge of interest in the use of reflectance data collected using satellites and aerial vehicles to monitor vegetation diversity. One methodological option to monitor these systems involves developing empirical relationships between spectral heterogeneity in space (spectral variation) and plant or habitat diversity. This approach is commonly termed the ‘Spectral Variation Hypothesis’. Although increasingly used, it is controversial and can be unreliable in some contexts. Here, we review the literature and apply three-level meta-analytical models to assess the test results of the hypothesis across studies using several moderating variables relating to the botanical and spectral sampling strategies and the types of sites evaluated. We focus on the literature relating to grasslands, which are less well studied compared to forests and are likely to require separate treatments due to their dynamic phenology and the taxonomic complexity of their canopies on a small scale. Across studies, the results suggest an overall positive relationship between spectral variation and species diversity (mean correlation coefficient = 0.36). However, high levels of both within-study and between-study heterogeneity were found. Whether data was collected at the leaf or canopy level had the most impact on the mean effect size, with leaf-level studies displaying a stronger relationship compared to canopy-level studies. We highlight the challenges facing the synthesis of these kinds of experiments, the lack of studies carried out in arid or tropical systems and the need for scalable, multitemporal assessments to resolve the controversy in this field.

What are the ecological causes and consequences of variation in phytochemical diversity within and between plant taxa? Despite decades of natural products discovery by organic chemists and research by chemical ecologists, our understanding of phytochemically mediated ecological processes in natural communities has been restricted to studies of either broad classes of compounds or a small number of well-characterized molecules. Until now, no studies have assessed the ecological causes or consequences of rigorously quantified phytochemical diversity across taxa in natural systems. Consequently, hypotheses that attempt to explain variation in phytochemical diversity among plants remain largely untested. We use spectral data from crude plant extracts to characterize phytochemical diversity in a suite of co-occurring plants in the tropical genus Piper (Piperaceae). In combination with 20 years of data focused on Piper-associated insects, we find that phytochemical diversity has a direct and positive effect on the diversity of herbivores but also reduces overall herbivore damage. Elevated chemical diversity is associated with more specialized assemblages of herbivores, and the cascading positive effect of phytochemistry on herbivore enemies is stronger as herbivore diet breadth narrows. These results are consistent with traditional hypotheses that predict positive associations between plant chemical diversity, insect herbivore diversity, and trophic specialization. It is clear from these results that high phytochemical diversity not only enhances the diversity of plant-associated insects but also contributes to the ecological predominance of specialized insect herbivores.

This paper analyzes, through computational simulations, which spectral filters increase the number of discernible colors (NODC) of subjects with normal color vision, as well as red–green anomalous trichromats and dichromats. The filters are selected from a set of filters in which we have modeled spectral transmittances. With the selected filters we have carried out simulations performed using the spectral reflectances captured either by a hyperspectral camera or by a spectrometer. We have also studied the effects of these filters on color coordinates. Finally, we have simulated the results of two widely used color blindness tests: Ishihara and Farnsworth–Munsell 100 Hue (FM100). In these analyses the selected filters are compared with the commercial filters from EnChroma and VINO companies. The results show that the increase in NODC with the selected filters is not relevant. The simulation results show that none of these chosen filters help color vision deficiency (CVD) subjects to pass the set of color blindness tests studied. These results obtained using standard colorimetry support the hypothesis that the use of color filters does not cause CVDs to have a perception similar to that of a normal observer.

The central aim in ecometabolomics and chemical ecology is to pinpoint chemical features that explain molecular functioning. The greatest challenge is the identification of compounds due to the lack of constitutive reference spectra, the large number of completely unknown compounds, and bioinformatic methods to analyze the big data. In this study we present an interdisciplinary methodological framework that extends ultra-performance liquid chromatography coupled to electrospray ionization quadrupole time-of-flight mass spectrometry (UPLC/ESI-QTOF-MS) with data-dependent acquisition (DDA-MS) and the automated in silico classification of fragment peaks into compound classes. We synthesize findings from a prior study that explored the influence of seasonal variations on the chemodiversity of secondary metabolites in nine bryophyte species. Here we reuse and extend the representative dataset with DDA-MS data. Hierarchical clustering, heatmaps, dbRDA, and ANOVA with post-hoc Tukey HSD were used to determine relationships of the study factors species, seasons, and ecological characteristics. The tested bryophytes showed species-specific metabolic responses to seasonal variations (50% vs. 5% of explained variation). Marchantia polymorpha, Plagiomnium undulatum, and Polytrichum strictum were biochemically most diverse and unique. Flavonoids and sesquiterpenoids were upregulated in all bryophytes in the growing seasons. We identified ecological functioning of compound classes indicating light protection (flavonoids), biotic and pathogen interactions (sesquiterpenoids, flavonoids), low temperature and desiccation tolerance (glycosides, sesquiterpenoids, anthocyanins, lactones), and moss growth supporting anatomic structures (few methoxyphenols and cinnamic acids as part of proto-lignin constituents). The reusable bioinformatic framework of this study can differentiate species based on automated compound classification. Our study allows detailed insights into the ecological roles of biochemical constituents of bryophytes with regard to seasonal variations. We demonstrate that compound classification can be improved with adding constitutive reference spectra to existing spectral libraries. We also show that generalization on compound classes improves our understanding of molecular ecological functioning and can be used to generate new research hypotheses.

This paper describes recent studies of the FS CMa-type objects, a group of stars showing the B[e] phenomenon defined in 2007. The objects exhibit strong emission-line spectra with both permitted and forbidden lines suggesting the presence of a B-type star as well as strong IR excesses due to radiation of circumstellar dust. These properties are hard to explain in the framework of the evolution of single stars with luminosities between ~300 and ~30,000 L⊙ typical of most B-type stars. We explore the hypothesis that the gaseous-and-dusty envelopes of FS CMa objects are due to either earlier or ongoing mass transfer between the binary system components. It is hard to detect the secondary components in these systems because of veiling and distortions by the circumstellar matter because of the relative faintness of the companions. Nevertheless, we detected regular radial velocity variations of the spectral lines in MWC 728, 3 Pup, and AS 386 and we found absorption lines typical of cool stars in the spectra of MWC 645, AS 174, and several other objects. The diversity of the secondary components in FS CMa objects is discussed in the context of non-conservative binary evolution.

Classic linguistic theory ascribes language change and diversity to population migrations, conquests, and geographical isolation, with the assumption that human populations have equivalent language processing abilities. We hypothesize that spectral and temporal characteristics make some consonant manners vulnerable to differences in temporal precision associated with specific population allele frequencies. To test this hypothesis, we modelled association between RU1-1 alleles of DCDC2 and manner of articulation in 51 populations spanning five continents, and adjusting for geographical proximity, and genetic and linguistic relatedness. RU1-1 alleles, acting through increased expression of DCDC2 , appear to increase auditory processing precision that enhances stop-consonant discrimination, favouring retention in some populations and loss by others. These findings enhance classical linguistic theories by adding a genetic dimension, which until recently, has not been considered to be a significant catalyst for language change.

Groundwater-Dependent Ecosystems (GDEs) are under threat from groundwater over-abstraction, which significantly impacts their conservation and sustainable management. Although the socio-economic significance of GDEs is understood, their ecosystem services and ecological significance (e.g., biodiversity hotspots) in arid environments remains understudied. Therefore, under the United Nations Sustainable Development Goal (SDG) 15, characterizing or identifying biodiversity hotspots in GDEs improves their management and conservation. In this study, we present the first attempt towards the spatial characterization of vegetation diversity in GDEs within the Khakea-Bray Transboundary Aquifer. Following the Spectral Variation Hypothesis (SVH), we used multispectral remotely sensed data (i.e., Sentinel-2 MSI) to characterize the vegetation diversity. This involved the use of the Rao’s Q to measure spectral diversity from several measures of spectral variation and validating the Rao’s Q using field-measured data on vegetation diversity (i.e., effective number of species). We observed that the Rao’s Q has the potential of spatially characterizing vegetation diversity of GDEs in the Khakea-Bray Transboundary Aquifer. Specifically, we discovered that the Rao’s Q was related to field-measured vegetation diversity (R2 = 0.61 and p = 0.00), and the coefficient of variation (CV) was the best measure to derive the Rao’s Q. Vegetation diversity was also used as a proxy for identifying priority conservation areas and biodiversity hotspots. Vegetation diversity was more concentrated around natural pans and along roads, fence lines, and rivers. In addition, vegetation diversity was observed to decrease with an increasing distance (>35 m) from natural pans and simulated an inverse piosphere (i.e., minimal utilization around the natural water pans). We provide baseline information necessary for identifying priority conservation areas within the Khakea-Bray Transboundary Aquifer. Furthermore, this work provides a pathway for resource managers to achieve SDG 15 as well as national and regional Aichi biodiversity targets.

Biodiversity monitoring in the Niger delta has become pertinent in view of the incessant spillages from oil production activities and the socio-economic impact of these spillages on the inhabitants who depend on the resources for their livelihood. Conventional methods of post-impact assessments are expensive, time consuming, and cause damage to the environment, as they often require the removal of affected samples/specimens for laboratory analysis. Remote sensing offers the opportunity to track biodiversity changes from space while using the spectral variability hypothesis (SVH). The SVH proposes that the species diversity of a sampled area is linearly correlated with the variability of spectral reflectance of the area. Several authors have tested the SVH on various land cover types and spatial scales; however, the present study evaluated the validity of the SVH against the backdrop of oil pollution impact on biodiversity while using vascular plant species as surrogates. Species richness and diversity indices were computed from vegetation data collected from polluted and non-polluted transects. Spectral metrics that were derived from Sentinel 2 bands and broadband vegetation indices (BVIs) using various algorithms, including averages, spread, dimension reduction, and so on, were assessed for their ability to estimate vascular plants species richness and diversity. The results showed significant differences in vegetation characteristics of polluted and control transects (H = 76.05, p-value = <0.05 for abundance and H = 170.03, p-value < 0.05 for richness). Spectral diversity metrics correlated negatively with species data on polluted transects and positively on control transects. The metrics computed using Sentinel 2A bands and vegetation indices proved to be sensitive to changes in vegetation characteristics following oil pollution. The most robust relationship was observed between the metrics and indices on control transects, whereas the weakest relationships were observed on polluted transects. Index-wise, the Simpson’s diversity index regressed better with spectral metrics (R2 > 0.5), whereas the Chao-1 richness index regressed the least (R2 < 0.5). The strength of the relationship resulted in successfully estimating species richness and diversity values of investigated transects, thereby enhancing biodiversity monitoring over time and space.

The spatial monitoring of plant diversity in the endangered species-rich grasslands of European mountain pastoral systems is an important step for fairer and more efficient Agri-Environmental policy schemes supporting conservation. This study assessed the underlying support for a spatially explicit monitoring of plant species richness at parcel level (policy making scale) in Southern European mountain grasslands, with statistical models informed by Sentinel-2 satellite and environmental factors. Twenty-four grassland parcels were surveyed for species richness in the Peneda-Gerês National Park, northern Portugal. Using a multi-model inference approach, three competing hypotheses guided by the species-scaling theoretical framework were established: species–area (P1), species–energy (P2) and species–spectral heterogeneity (P3), each representing a candidate spatial pathway to predict species richness. To evaluate the statistical support of each spatial pathway, generalized linear models were fitted and model selection based on Akaike information criterion (AIC) was conducted. Later, the performance of the most supported spatial pathway(s) was assessed using a leave-one-out cross validation. A model guided by the species–energy hypothesis (P2) was the most parsimonious spatial pathway to monitor plant species richness in mountain grassland parcels (P2, AICc = 137.6, ∆AIC = 0.0, wi = 0.97). Species–area and species–spectral heterogeneity pathways (P1 and P3) were less statistically supported (ΔAICc values in the range 5.7–10.0). The underlying support of the species–energy spatial pathway was based on Sentinel-2 satellite data, namely on the near-infrared (NIR) green ratio in the spring season (NIR/Greenspring) and on its ratio of change between spring and summer (NIR/Greenchange). Both predictor variables related negatively to species richness. Grassland parcels with lower values of near-infrared (NIR) green ratio and lower seasonal amplitude presented higher species richness records. The leave-one-out cross validation indicated a moderate performance of the species–energy spatial pathway in predicting species richness in the grassland parcels covered by the dataset (R2 = 0.44, RMSE = 4.3 species, MAE = 3.5 species). Overall, a species–energy framework based on Sentinel 2 data resulted in a promising spatial pathway for the monitoring of species richness in mountain grassland parcels and for informing decision making on Agri-Environmental policy schemes. The near-infrared (NIR) green ratio and its change in time seems a relevant variable to deliver predictions for plant species richness and further research should be conducted on that.

AbstractStomatopod crustaceans possess apposition compound eyes that contain more photoreceptor types than any other animal described. While the anatomy and physiology of this complexity have been studied for more than two decades, few studies have investigated the molecular aspects underlying the stomatopod visual complexity. Based on previous studies of the structure and function of the different types of photoreceptors, stomatopod retinas are hypothesized to contain up to 16 different visual pigments, with 6 of these having sensitivity to middle or long wavelengths of light. We investigated stomatopod middle- and long-wavelength-sensitive opsin genes from five species with the hypothesis that each species investigated would express up to six different opsin genes. In order to understand the evolution of this class of stomatopod opsins, we examined the complement of expressed transcripts in the retinas of species representing a broad taxonomic range (four families and three superfamilies). A total of 54 unique retinal opsins were isolated, resulting in 6–15 different expressed transcripts in each species. Phylogenetically, these transcripts form six distinct clades, grouping with other crustacean opsins and sister to insect long-wavelength visual pigments. Within these stomatopod opsin groups, intra- and interspecific clusters of highly similar transcripts suggest that there has been rampant recent gene duplication. Some of the observed molecular diversity is also due to ancient gene duplication events within the stem crustacean lineage. Using evolutionary trace analysis, 10 amino acid sites were identified as functionally divergent among the six stomatopod opsin clades. These sites form tight clusters in two regions of the opsin protein known to be functionally important: six in the chromophore-binding pocket and four at the cytoplasmic surface in loops II and III. These two clusters of sites indicate that stomatopod opsins have diverged with respect to both spectral tuning and signal transduction.

Incident solar radiation (insolation) passing through the forest canopy to the ground surface is either absorbed or scattered. This phenomenon, known as radiation attenuation, is measured using the extinction coefficient (K). The amount of radiation reaching the ground surface of a given site is effectively controlled by the canopy’s surface and structure, determining its suitability for plant species. Menhinick’s and Simpson’s biodiversity indexes were selected as spatially explicit response variables for the regression equation using canopy structure metrics as predictors. Independent variables include modeled area solar radiation, LiDAR-derived canopy height, effective leaf area index data derived from multi-spectral imagery and canopy strata metrics derived from LiDAR point-cloud data. The results support the hypothesis that (1) canopy surface and strata variability may be associated with understory species diversity due to radiation attenuation and the resultant habitat partitioning and that, (2) such a model can predict both this relationship and biodiversity clustering. The study data yielded significant correlations between predictor and response variables and were used to produce a multiple–linear model comprising canopy relief, the texture of heights, and vegetation density to predict understory plant diversity. When analyzed for spatial autocorrelation, the predicted biodiversity data exhibited non-random spatial continuity.

Species body size spectra have been constructed for macrofauna assemblages from four sites with contrasting sediment granulometry and heterogeneity in and around Plymouth Sound. The number of species and species turnover (β diversity) were higher on coarse sediment. While the fauna were distinct between sites, the median geometric size-class was conservative (class 14; 0.153–0.305 mg dry blotted weight). Only one site had significantly lower heterogeneity within the species size spectrum, yet this was the most heterogeneous sediment. As such, we were unable to reject the null hypothesis that species body size distribution patterns are conservative despite differences in sediment granulometry and heterogeneity.

Multimodal signals facilitate communication with conspecifics during courtship, but they can also alert eavesdropper predators. Hence, signallers face two pressures: enticing partners to mate and avoiding detection by enemies. Undefended organisms with limited escape abilities are expected to minimize predator recognition over mate attraction by limiting or modifying their signalling. Alternatively, organisms with anti-predator mechanisms such as aposematism (i.e. unprofitability signalled by warning cues) might elaborate mating signals as a consequence of reduced predation. We hypothesize that calls diversified in association with aposematism. To test this, we assembled a large acoustic signal database for a diurnal lineage of aposematic and cryptic/non-defended taxa, the poison frogs. First, we showed that aposematic and non-aposematic species share similar extinction rates, and aposematic lineages diversify more and rarely revert to the non-aposematic phenotype. We then characterized mating calls based on morphological (spectral), behavioural/physiological (temporal) and environmental traits. Of these, only spectral and temporal features were associated with aposematism. We propose that with the evolution of anti-predator defences, reduced predation facilitated the diversification of vocal signals, which then became elaborated or showy via sexual selection.

Colour vision is highly variable in New World monkeys (NWMs). Evidence for the adaptive basis of colour vision in this group has largely centred on environmental features such as foraging benefits for differently coloured foods or predator detection, whereas selection on colour vision for sociosexual communication is an alternative hypothesis that has received little attention. The colour vision of uakaris ( Cacajao ) is of particular interest because these monkeys have the most dramatic red facial skin of any primate, as well as a unique fission/fusion social system and a specialist diet of seeds. Here, we investigate colour vision in a wild population of the bald uakari, C. calvus , by genotyping the X-linked opsin locus. We document the presence of a polymorphic colour vision system with an unprecedented number of functional alleles (six), including a novel allele with a predicted maximum spectral sensitivity of 555 nm. This supports the presence of strong balancing selection on different alleles at this locus. We consider different hypotheses to explain this selection. One possibility is that trichromacy functions in sexual selection, enabling females to choose high-quality males on the basis of red facial coloration. In support of this, there is some evidence that health affects facial coloration in uakaris, as well as a high prevalence of blood-borne parasitism in wild uakari populations. Alternatively, the low proportion of heterozygous female trichromats in the population may indicate selection on different dichromatic phenotypes, which might be related to cryptic food coloration. We have uncovered unexpected diversity in the last major lineage of NWMs to be assayed for colour vision, which will provide an interesting system to dissect adaptation of polymorphic trichromacy.

Representatives of the family <em>Cactaceae</em> are characterized by a wide range of phyllotaxis. To assess the origin of this diversity, early stages of phyllotactic pattern formation were examined in seedlings. The analysis of the sequence of areole initiation revealed intertribal differences. In seedlings from the <em>Trichocereeae</em> (<em>Gymnocalycium</em>, <em>Rebutia</em>) and <em>Notocacteae</em> (<em>Parodia</em>) tribes, two opposite cotyledonal areoles developed as the first elements of a pattern. Usually, next pair of areoles was initiated perpendicularly to cotyledonal areoles, starting the decussate pattern. This pattern was subsequently transformed into bijugate or into simple spiral phyllotaxis. In seedlings from the <em>Cacteae</em> tribe (<em>Mammillaria</em> and <em>Thelocactus</em>), cotyledonal areoles were never observed and the first areoles always appeared in the space between cotyledons. It was either areole pair (mainly in <em>Mammillaria</em>), starting a decussate pattern, or a single areole (mainly in <em>Thelocactus</em>) quickly followed by areoles spirally arranged, usually in accordance with the main Fibonacci phyllotaxis. Differences in the initial stages of pattern formation do not fully explain the phyllotaxis diversity in mature cacti. Only two, the most common phyllotactic patterns occurred in the early development of studied seedlings, i.e. the main Fibonacci and the decussate pattern. Discrepancy in the range of phyllotactic spectra in seedlings and in mature plants suggests that phyllotaxis diversity emerges during further plant growth. Initial phyllotactic transformations, occurring already in the very early stages, indicate great plasticity of cactus growth and seem to support the hypothesis of the ontogenetic increase of phyllotaxis diversity due to transformations.

Measurement of water content in forest habitats is considered essential in ecological research on forests, climate change, or forest management. In the traditional forest habitat classification, two systems of habitat conditions analysis are found: single factor and multifactor methods. Both are laborious and therefore costly. Remote sensing methods provide a low-cost alternative. The aim of the presented study was to find the relationship between the spectral indices obtained from satellite images and the forest habitats moisture indices used traditionally in the Polish forest habitats classification. The scientific hypothesis of the research is as follows: it is possible to assess the variation in the humidity of forest habitats on the basis of spectral indices. Using advanced geographic information system (GIS) technology, 923 research plots were tested, where habitat studies performed with the traditional methods were compared with the analysis of 191 spectral indices calculated for Sentinel-2 image data. The normalized difference vegetation index (NDVI) has proved to be the most useful to the assessing of moisture of forest habitats. The ranking of the most correlated indices was calculated as Eintg—the product of the absolute value of the slope and the mean square error complement, and for the top five indices was as follows: NDVI = 0.248619, EXG = 0.242112, OSAVI = 0.239412, DSWI-4 = 0.238784, and RDVI = 0.236995. The results also highlight the impact of water reservoirs on the humidity and trophicity of forest habitats, showing a decrease in the fertility of habitats with an increase in distance from the water reservoir. The results of the study can be used to preparing maps of the diversity of forest types, especially in hard-to-reach places, as well as to assess changes in the moisture status of habitats, which may be useful, for example, in the assessment of the fire risk of forest habitats. We have proved that NDVI can be used in applications for which it was not originally designed.

Abstract Color is among the most striking features of organisms, varying not only in spectral properties like hue and brightness, but also in where and how it is produced on the body. Different combinations of colors on a bird’s body are important in both environmental and social contexts. Previous comparative studies have treated plumage patches individually or derived plumage complexity scores from color measurements across a bird’s body. However, these approaches do not consider the multivariate nature of plumages (allowing for plumage to evolve as a whole) or account for interpatch distances. Here, we leverage a rich toolkit used in historical biogeography to assess color pattern evolution in a cosmopolitan radiation of birds, kingfishers (Aves: Alcedinidae). We demonstrate the utility of this approach and test hypotheses about the tempo and mode of color evolution in kingfishers. Our results highlight the importance of considering interpatch distances in understanding macroevolutionary trends in color diversity and demonstrate how historical biogeography models are a useful way to model plumage color pattern evolution. Furthermore, they show that distinct color mechanisms (pigments or structural colors) spread across the body in different ways and at different rates. Specifically, net rates are higher for structural colors than pigment-based colors. Together, our study suggests a role for both development and selection in driving extraordinary color pattern diversity in kingfishers. We anticipate this approach will be useful for modeling other complex phenotypes besides color, such as parasite evolution across the body.

Abstract. Propagule traits vary among biomes along geographical gradients such as longitude, but the mechanisms that underlie these variations remain unclear. This study aims to explore seed mass variation patterns of different biome types along a longitudinal gradient and their underlying variation mechanisms by involving an in-depth analysis on the variation of seed mass, fruit type spectra, growth forms and dispersal mode spectra in Inner Mongolia and northeastern China. Plant community characterization and seed collection were conducted in 26 sites spreading over five vegetation types and covering 622 species belonging to 66 families and 298 genera. We found there are significantly declining trends for mean seed mass, vertebrate-dispersed species richness and fleshy-fruited species richness along a longitudinal gradient from forests to desert grasslands. However, we also found the lowest average seed mass and the smallest proportion of species dispersed by vertebrates occurring at typical grasslands in the five biomes. The variations of average seed mass display high congruence with transition of growth form spectra. The selection for these propagule attributes is driven mainly by climatic factors such as precipitation, temperature, soil moisture and evaporation, as well as by internal biotic factors such as growth forms, canopy coverage and leaf area (Ackerly and Donoghue, 1998). A hypothesis was provided that environmental factors and botanical traits that favor greater water availability lead to emergence (or speciation) of species with large seeds or fleshy fruits with high water content. Due to greater water availability and increasing leaf area, much more photosynthate (photosynthesis production) and allometric growth then ultimately increase the biome average seed mass from west to east. Phylogenetic signal or diversity are not found to be significantly involved in the effect on the patterns. A novel mechanistic framework and mathematical model are provided to expound seed variation among species or biomes.

Characterization of metabolites and microbiota composition from human stool provides powerful insight into the molecular phenotypic difference between subjects with normal weight and those with overweight/obesity. The aim of this study was to identify potential metabolic and bacterial signatures from stool that distinguish the overweight/obesity state in children/adolescents. Using 1H NMR spectral analysis and 16S rRNA gene profiling, the fecal metabolic profile and bacterial composition from 52 children aged 7 to 16 was evaluated. The children were classified into three groups (16 with normal-weight, 17 with overweight, 19 with obesity). The metabolomic analysis identified four metabolites that were significantly different (p < 0.05) among the study groups based on one-way ANOVA testing: arabinose, butyrate, galactose, and trimethylamine. Significantly different (p < 0.01) genus-level taxa based on edgeR differential abundance tests were genus Escherichia and Tyzzerella subgroup 3. No significant difference in alpha-diversity was detected among the three study groups, and no significant correlations were found between the significant taxa and metabolites. The findings support the hypothesis of increased energy harvest in obesity by human gut bacteria through the growing observation of increased fecal butyrate in children with overweight/obesity, as well as an increase of certain monosaccharides in the stool. Also supported is the increase of trimethylamine as an indicator of an unhealthy state.

The properties of soil constitute one of the most important features of the environment that determine the potential for food production in a given region. Knowledge of the soil texture and agroclimate allows for the proper selection of species and agrotechnics in plant production. However, in contrast to the agroclimate, the soil may show a large spatial variation of physical and chemical characteristics within the plot. In regions where the soil diversity is so high that the available soil maps are not sufficient, the only method that allows for precise mapping of the soil mosaic is remote sensing. This paper presents the concepts of using Sentinel-2 multispectral satellite images to detail the available soil-agriculture map at a scale of 1:25,000. In the presented work, the following research hypothesis has been formulated: spatial and temporal analysis of high-resolution satellite images can be used to improve the quality of a large-scale archival soil-agriculture map. It is possible due to the spatial differentiation of the spectral reflection from the field (canopy), which is influenced by soil conditions—especially the differentiation of physical properties (granulometric composition) in soil profiles which determine the possibility of water retention during drought conditions. The research carried out as a case study of maize remote sensing confirmed the hypothesis. It was based on the selection of the most appropriate term (maize development period: BBCH 79, 6-decade drought index: CBW = −206 mm) and the vegetation index (NDVI). This made it possible to make the scale of the map 10 times more detailed. The obtained results are the first step in developing a general model (based on remote sensing) for detailing the soil-agriculture map for Poland, which will significantly improve the accuracy of the drought monitoring system developed by the Institute of Soil Science and Plant Cultivation (Poland).

Replication of RNA viruses is characterized by exploration of sequence space which facilitates their adaptation to changing environments. It is generally accepted that such exploration takes place mainly in response to positive selection, and that further diversification is boosted by modifications of virus population size, particularly bottleneck events. Our recent results with hepatitis C virus (HCV) have shown that the expansion in sequence space of a viral clone continues despite prolonged replication in a stable cell culture environment. Diagnosis of the expansion was based on the quantification of diversity indices, the occurrence of intra-population mutational waves (variations in mutant frequencies), and greater individual residue variations in mutant spectra than those anticipated from sequence alignments in data banks. In the present report, we review our previous results, and show additionally that mutational waves in amplicons from the NS5A-NS5B-coding region are equally prominent during HCV passage in the absence or presence of the mutagenic nucleotide analogues favipiravir or ribavirin. In addition, by extending our previous analysis to amplicons of the NS3- and NS5A-coding region, we provide further evidence of the incongruence between amino acid conservation scores in mutant spectra from infected patients and in the Los Alamos National Laboratory HCV data banks. We hypothesize that these observations have as a common origin a permanent state of HCV population disequilibrium even upon extensive viral replication in the absence of external selective constraints or changes in population size. Such a persistent disequilibrium—revealed by the changing composition of the mutant spectrum—may facilitate finding alternative mutational pathways for HCV antiviral resistance. The possible significance of our model for other genetically variable viruses is discussed.

The limits of the visible spectrum are set by the light available for vision, and by the visual pigment absorbance. The hundreds of visual pigments studied to the present day have absorbance maxima spread within the range from 350 to 620 nm. Yet this diversity is used for vision quite nonuniformly: rod and cone visual pigments are tightly clustered around a few preferred positions in the spectrum, eg near 500 nm in the rods of land animals. The so-called ‘sensitivity hypothesis’ assumes that the clustering is to maximise the number of absorbed photons available in the animals' light environment. In most cases, however, visual pigments are substantially more short-wave (blue-shifted) than is necessary for maximum quantal absorption. Examples of the ‘blue shift’ are the Purkinje shift during cone - rod transition in dark adaptation, the hypsochromic shift of rod visual pigments in deep-water fish, and a similar shift in the cone pigments of geckos and some snakes as a result of evolutionary adaptation to nocturnal habits. It is argued that an important limiting factor in vision is the dark noise produced by thermal isomerisation of the chromophore. Measurements of the dark noise in rods with different visual pigments show that the noise increases steeply when the absorbance maximum is shifted to longer wavelengths, thus precluding the use of long-wave pigments for vision at low intensities. The optimum spectral position of a pigment may be that which ensures a maximum light-to-noise ratio in a particular photic environment.

Abstract Aims Bee-pollinated flowers are rarely red, presumably because bees (which lack red receptors) have difficulty detecting red targets. Although the response of bees to red colour has been investigated in lab experiments, most stimuli have been pure red, while the subtle diversity of red as perceived by humans (human-red) has received very limited attention. Here we test the hypothesis that ultraviolet (UV) reflected from human-red flowers enhances their attractiveness to bees, through increased chromatic contrast. Methods Using Onosma confertum (Boraginaceae), a plant with UV-reflecting red flowers that are pollinated by bumblebees, we investigated the effects of UV reflection on pollinator responses by conducting phenotypic manipulation experiments in the field. Colour preferences of flower-naïve bumblebees were also examined. Colour perception by bumblebees was estimated in terms of chromatic and achromatic contrast, based on two different colour perception models. Important Findings We found that both natural and flower-naïve bumblebees strongly preferred visiting UV-reflecting targets compared with UV-absorbing ones. Colour models show that the UV-reflecting flowers exhibit higher spectral purity and higher chromatic contrast against the foliage background, whereas they have similar achromatic contrast in terms of green receptor contrast. These results indicate that the component of UV reflection increases chromatic contrast in O. confertum, enhancing the visual attractiveness of these red flowers to bumblebees. We further infer that the secondary reflectance might be a necessary component in human-red flowers that are primarily pollinated by animals without red receptors, such as bees.

In plant ecology, biochemical analyses of bryophytes and vascular plants are often conducted on dried herbarium specimen as species typically grow in distant and inaccessible locations. Here, we present an automated in silico compound classification framework to annotate metabolites using an untargeted data independent acquisition (DIA)–LC/MS–QToF-sequential windowed acquisition of all theoretical fragment ion mass spectra (SWATH) ecometabolomics analytical method. We perform a comparative investigation of the chemical diversity at the global level and the composition of metabolite families in ten different species of bryophytes using fresh samples collected on-site and dried specimen stored in a herbarium for half a year. Shannon and Pielou’s diversity indices, hierarchical clustering analysis (HCA), sparse partial least squares discriminant analysis (sPLS-DA), distance-based redundancy analysis (dbRDA), ANOVA with post-hoc Tukey honestly significant difference (HSD) test, and the Fisher’s exact test were used to determine differences in the richness and composition of metabolite families, with regard to herbarium conditions, ecological characteristics, and species. We functionally annotated metabolite families to biochemical processes related to the structural integrity of membranes and cell walls (proto-lignin, glycerophospholipids, carbohydrates), chemical defense (polyphenols, steroids), reactive oxygen species (ROS) protection (alkaloids, amino acids, flavonoids), nutrition (nitrogen- and phosphate-containing glycerophospholipids), and photosynthesis. Changes in the composition of metabolite families also explained variance related to ecological functioning like physiological adaptations of bryophytes to dry environments (proteins, peptides, flavonoids, terpenes), light availability (flavonoids, terpenes, carbohydrates), temperature (flavonoids), and biotic interactions (steroids, terpenes). The results from this study allow to construct chemical traits that can be attributed to biogeochemistry, habitat conditions, environmental changes and biotic interactions. Our classification framework accelerates the complex annotation process in metabolomics and can be used to simplify biochemical patterns. We show that compound classification is a powerful tool that allows to explore relationships in both molecular biology by “zooming in” and in ecology by “zooming out”. The insights revealed by our framework allow to construct new research hypotheses and to enable detailed follow-up studies.

A developed economy provides a large selection of goods and services that can be consumed. To meet their needs for goods and services, consumers do not always have to pay in cash because many companies offer financing services. Financing services are specialized in accordance with the development of product diversity and customer needs. Finance service companies must be able to improve the quality of their services to embrace new customers and retain existing customers. This study was made with the aim of knowing and analyzing how the Quality of Service provided by the Spectra Business Unit at FIFGROUP Tanjung Pos Buntok Branch can influence the Decision of Making Consumer Credit RO (Repeat Order). This type of research is a descriptive quantitative type with the data collection process using questionnaires, observations, interviews, library research, and documentation. The selected sample of 124 people determined by the Random Sampling method and then processed by the Multiple Linear Regression method. The results showed that the calculation of multiple linear regression is Y = 4.718 + 1.270X1 + 0.702X2 +0.645X3 + 1.097X4 + 0.656X5. The results of the hypothesis test show that simultaneously service quality has a positive and significant effect on consumer credit decisions at RO FIFGROUP Tanjung Pos Buntok Branch with a calculated F value of 58.402 > F table 2.29. While partially. obtained t count Tangibles (X1) 4.187, Reliability (X2) 2.585, Responsiveness (X3) 2.592, Assurance (X4) 3.739 and Empathy (X5) 2.146, which means that these values ​​are values ​​that greatly affect Credit Decisions by consumers repeat order FIFGROUP Tanjung Pos Buntok Branch because tcount > ttable

Bats represent one of the largest and most striking nocturnal mammalian radiations, exhibiting many visual system specializations for performance in light-limited environments. Despite representing the greatest ecological diversity and species richness in Chiroptera, Neotropical lineages have been undersampled in molecular studies, limiting the potential for identifying signatures of selection on visual genes associated with differences in bat ecology. Here, we investigated how diverse ecological pressures mediate long-term shifts in selection upon long-wavelength ( Lws ) and short-wavelength ( Sws1 ) opsins, photosensitive cone pigments that form the basis of colour vision in most mammals, including bats. We used codon-based likelihood clade models to test whether ecological variables associated with reliance on visual information (e.g. echolocation ability and diet) or exposure to varying light environments (e.g. roosting behaviour and foraging habitat) mediated shifts in evolutionary rates in bat cone opsin genes. Using additional cone opsin sequences from newly sequenced eye transcriptomes of six Neotropical bat species, we found significant evidence for different ecological pressures influencing the evolution of the cone opsins. While Lws is evolving under significantly lower constraint in highly specialized high-duty cycle echolocating lineages, which have enhanced sonar ability to detect and track targets, variation in Sws1 constraint was significantly associated with foraging habitat, exhibiting elevated rates of evolution in species that forage among vegetation. This suggests that increased reliance on echolocation as well as the spectral environment experienced by foraging bats may differentially influence the evolution of different cone opsins. Our study demonstrates that different ecological variables may underlie contrasting evolutionary patterns in bat visual opsins, and highlights the suitability of clade models for testing ecological hypotheses of visual evolution.

Radial age gradients hold the cumulative record for the multitude of physical processes driving the build-up of stellar populations and the ensuing star formation (SF) quenching process in galaxy bulges and, therefore, potentially sensitive discriminators between competing theoretical concepts on bulge formation and evolution. Based on spectral modeling of integral field spectroscopy (IFS) data from the CALIFA survey, we derived mass- and light-weighted stellar age gradients (∇(t⋆, B)ℒ, ℳ) within the photometrically determined bulge radius (RB) of a representative sample of local face-on late-type galaxies that span 2.6 dex in stellar mass (8.9 ≤ log ℳ⋆, T ≤ 11.5). Our analysis documents a trend of decreasing ∇(t⋆, B)ℒ, ℳ with increasing ℳ⋆, T, with high-mass bulges predominantly showing negative age gradients and vice versa. The inversion from positive to negative ∇(t⋆, B)ℒ, ℳ occurs at log ℳ⋆, T ≃ 10, which roughly coincides with the transition from lower-mass bulges whose gas excitation is powered by SF to bulges classified as composite, LINER, or Seyfert. We discuss two simple limiting cases for the origin of radial age gradients in massive late-type galaxy bulges. The first one assumes that the stellar age in the bulge is initially spatially uniform (∇(t⋆, B)ℒ, ℳ ≈ 0), thus the observed age gradients (∼ − 3 Gyr/RB) arise from an inside-out SF quenching (ioSFQ) front that is radially expanding with a mean velocity vq. In this case, the age gradients for massive bulges translate into a slow (vq ∼1–2 km s−1) ioSFQ that lasts until z ∼ 2, suggesting mild negative feedback by SF or an active galactic nucleus (AGN). If, on the other hand, negative age gradients in massive bulges are not due to ioSFQ but primarily due to their inside-out formation process, then the standard hypothesis of quasi-monolithic bulge formation has to be discarded in favor of another scenario. This would involve a gradual buildup of stellar mass over 2–3 Gyr through, for instance, inside-out SF and inward migration of SF clumps from the disk. In this case, rapid (≪1 Gyr) AGN-driven ioSFQ cannot be ruled out. While the ℳ⋆, T versus ∇(t⋆, B)ℒ, ℳ relation suggests that the assembly history of bulges is primarily regulated by galaxy mass, its large scatter (∼1.7 Gyr/RB) reflects a considerable diversity. This calls for an in-depth examination of the role of various processes (e.g., negative and positive AGN feedback, bar-driven gas inflows) with higher-quality IFS data in conjunction with advanced spectral modeling codes.

Almost a year after the COVID-19 pandemic had begun, new lineages (B.1.1.7, B.1.351, P.1, and B.1.617.2) associated with enhanced transmissibility, immunity evasion, and mortality were identified in the United Kingdom, South Africa, and Brazil. The previous most prevalent lineages in the state of Rio Grande do Sul (RS, Southern Brazil), B.1.1.28 and B.1.1.33, were rapidly replaced by P.1 and P.2, two B.1.1.28-derived lineages harboring the E484K mutation. To perform a genomic characterization from the metropolitan region of Porto Alegre, we sequenced viral samples to: (i) identify the prevalence of SARS-CoV-2 lineages in the region, the state, and bordering countries/regions; (ii) characterize the mutation spectra; (iii) hypothesize viral dispersal routes by using phylogenetic and phylogeographic approaches. We found that 96.4% of the samples belonged to the P.1 lineage and approximately 20% of them were assigned as the novel P.1.2, a P.1-derived sublineage harboring signature substitutions recently described in other Brazilian states and foreign countries. Moreover, sequences from this study were allocated in distinct branches of the P.1 phylogeny, suggesting multiple introductions in RS and placing this state as a potential diffusion core of P.1-derived clades and the emergence of P.1.2. It is uncertain whether the emergence of P.1.2 and other P.1 clades is related to clinical or epidemiological consequences. However, the clear signs of molecular diversity from the recently introduced P.1 warrant further genomic surveillance.

Abstract. Two dimensional gas chromatography (GC × GC) with detection by time-of-flight mass spectrometry (TOFMS) was applied in the rapid analysis of air samples containing highly complex mixtures of volatilizable biogenic organic compounds (VBOCs). VBOC analytical methodologies are briefly reviewed, and optimal conditions are discussed for sampling with both adsorption/thermal desorption (ATD) cartridges and solid-phase microextraction (SPME) fibers. Air samples containing VBOC emissions from leaves of two tree species (Cedrus atlantica and Calycolpus moritzianus) were obtained by both ATD and SPME. The optimized gas chromatographic conditions utilized a 45 m, 0.25 mm I.D. low-polarity primary column (DB-VRX, 1.4 μm film) and a 1.5 m, 0.25 mm I.D. polar secondary column (Stabilwax® 0.25 μm film). Excellent separation was achieved in a 36 min temperature programmed GC × GC chromatogram. Thousands of VBOC peaks were present in the sample chromatograms; hundreds of tentative identifications by NIST mass spectral matching are provided. Very few of the tentatively identified compounds are currently available as authentic standards. Method detection limit values for a 5 l ATD sample were 3.5 pptv (10 ng m−3) for isoprene, methyl vinyl ketone, and methacrolein, and ~1.5 pptv (~10 ng m−3) for monoterpenes and sesquiterpenes. Kovats-type chromatographic retention index values on the primary column and relative retention time values on the secondary column are provided for 21 standard compounds and for 417 tentatively identified VBOCs. 19 of the 21 authentic standard compounds were found in one of the Cedrus atlantica SPME samples. In addition, easily quantifiable levels of at least 13 sesquiterpenes were found in an ATD sample obtained from a branch enclosure of Calycolpus moritzianus. Overall, the results obtained via GC × GC-TOFMS highlight an extreme, and largely uncharacterized diversity of VBOCs, consistent with the hypothesis that sesquiterpenes and other compounds beyond the current list of typically determined VBOC analytes may well be important contributors to global atmospheric levels of organic particulate matter.

Abstract. Two dimensional gas chromatography (GC × GC) with detection by time-of-flight mass spectrometry (TOFMS) was applied in the rapid analysis of air samples containing highly complex mixtures of volatilizable biogenic organic compounds (VBOCs). VBOC analytical methodologies are briefly reviewed, and optimal conditions are discussed for sampling with both adsorption/thermal desorption (ATD) cartridges and solid-phase microextraction (SPME) fibers. Air samples containing VBOC emissions from leaves of two tree species (Cedrus atlantica and Calycolpus moritzianus) were obtained by both ATD and SPME. The optimized gas chromatographic conditions utilized a 45 m, 0.25 mm I.D. low-polarity primary column (DB-VRX, 1.4 μm film) and a 1.5 m, 0.25 mm I.D. polar secondary column (StabilwaxTM, 0.25 μm film). Excellent separation was achieved in a 36 min temperature programmed GC × GC chromatogram. Thousands of VBOC peaks were present in the sample chromatograms; hundreds of tentative identifications by NIST mass spectral matching are provided. Very few of the tentatively identified compounds are currently available as authentic standards. Minimum detection limit values for a 5 l ATD sample were 3.5 pptv (10 ng m−3) for isoprene, methyl vinyl ketone, and methacrolein, and ~1.5 pptv (~10 ng m−3) for monoterpenes and sesquiterpenes. Kovats-type chromatographic retention index values on the primary column and relative retention time values on the secondary column are provided for 21 standard compounds and for 417 tentatively identified VBOCs. 19 of the 21 authentic standard compounds were found in one of the Cedrus atlantica SPME samples. In addition, easily quantifiable levels of at least 13 sesquiterpenes were found in an ATD sample obtained from a branch enclosure of Calycolpus moritzianus. Overall, the results obtained via GC × GC-TOFMS highlight an extreme, and largely uncharacterized diversity of VBOCs, consistent with the hypothesis that sesquiterpenes and other compounds beyond the current list of typically determined VBOC analytes may well be important contributors to global atmospheric levels of organic particulate matter.

Linking supernovae to their progenitors is a powerful method for furthering our understanding of the physical origin of their observed differences while at the same time testing stellar evolution theory. In this second study of a series of three papers where we characterise type II supernovae (SNe II) to understand their diversity, we derive progenitor properties (initial and ejecta masses and radius), explosion energy, and 56Ni mass and its degree of mixing within the ejecta for a large sample of SNe II. This dataset was obtained by the Carnegie Supernova Project-I and is characterised by a high cadence of SNe II optical and near-infrared light curves and optical spectra that were homogeneously observed and processed. A large grid of hydrodynamical models and a fitting procedure based on Markov chain Monte Carlo methods were used to fit the bolometric light curve and the evolution of the photospheric velocity of 53 SNe II. We infer ejecta masses of between 7.9 and 14.8 M⊙, explosion energies between 0.15 and 1.40 foe, and 56Ni masses between 0.006 and 0.069 M⊙. We define a subset of 24 SNe (the ‘gold sample’) with well-sampled bolometric light curves and expansion velocities for which we consider the results more robust. Most SNe II in the gold sample (∼88%) are found with ejecta masses in the range of ∼8−10 M⊙, coming from low zero-age main-sequence masses (9−12 M⊙). The modelling of the initial-mass distribution of the gold sample gives an upper mass limit of 21.3$ ^{+3.8}_{-0.4} $ M⊙ and a much steeper distribution than that for a Salpeter massive-star initial mass function (IMF). This IMF incompatibility is due to the large number of low-mass progenitors found – when assuming standard stellar evolution. This may imply that high-mass progenitors lose more mass during their lives than predicted. However, a deeper analysis of all stellar evolution assumptions is required to test this hypothesis.

Abstract. In this study we introduce a hybrid ensemble consisting of air quality models operating at both the global and regional scale. The work is motivated by the fact that these different types of models treat specific portions of the atmospheric spectrum with different levels of detail, and it is hypothesized that their combination can generate an ensemble that performs better than mono-scale ensembles. A detailed analysis of the hybrid ensemble is carried out in the attempt to investigate this hypothesis and determine the real benefit it produces compared to ensembles constructed from only global-scale or only regional-scale models. The study utilizes 13 regional and 7 global models participating in the Hemispheric Transport of Air Pollutants phase 2 (HTAP2)–Air Quality Model Evaluation International Initiative phase 3 (AQMEII3) activity and focuses on surface ozone concentrations over Europe for the year 2010. Observations from 405 monitoring rural stations are used for the evaluation of the ensemble performance. The analysis first compares the modelled and measured power spectra of all models and then assesses the properties of the mono-scale ensembles, particularly their level of redundancy, in order to inform the process of constructing the hybrid ensemble. This study has been conducted in the attempt to identify that the improvements obtained by the hybrid ensemble relative to the mono-scale ensembles can be attributed to its hybrid nature. The improvements are visible in a slight increase of the diversity (4 % for the hourly time series, 10 % for the daily maximum time series) and a smaller improvement of the accuracy compared to diversity. Root mean square error (RMSE) improved by 13–16 % compared to G and by 2–3 % compared to R. Probability of detection (POD) and false-alarm rate (FAR) show a remarkable improvement, with a steep increase in the largest POD values and smallest values of FAR across the concentration ranges. The results show that the optimal set is constructed from an equal number of global and regional models at only 15 % of the stations. This implies that for the majority of the cases the regional-scale set of models governs the ensemble. However given the high degree of redundancy that characterizes the regional-scale models, no further improvement could be expected in the ensemble performance by adding yet more regional models to it. Therefore the improvement obtained with the hybrid set can confidently be attributed to the different nature of the global models. The study strongly reaffirms the importance of an in-depth inspection of any ensemble of opportunity in order to extract the maximum amount of information and to have full control over the data used in the construction of the ensemble.

Three drafts of Bos indicus cross steers (initially 178–216 kg) grazed Leucaena–grass pasture [Leucaena leucocephala subspecies glabrata cv. Cunningham with green panic (Panicum maximum cv. trichoglume)] from late winter through to autumn during three consecutive years in the Burnett region of south-east Queensland. Measured daily weight gain (DWGActual) of the steers was generally 0.7–1.1 kg/day during the summer months. Estimated intakes of metabolisable energy and dry matter (DM) were calculated from feeding standards as the intakes required by the steers to grow at the DWGActual. Diet attributes were predicted from near infrared reflectance spectroscopy spectra of faeces (F.NIRS) using established calibration equations appropriate for northern Australian forages. Inclusion of some additional reference samples from cattle consuming Leucaena diets into F.NIRS calibrations based on grass and herbaceous legume–grass pastures improved prediction of the proportion of Leucaena in the diet. Mahalanobis distance values supported the hypothesis that the F.NIRS predictions of diet crude protein concentration and DM digestibility (DMD) were acceptable. F.NIRS indicated that the percentage of Leucaena in the diet varied widely (10–99%). Diet crude protein concentration and DMD were usually high, averaging 12.4 and 62%, respectively, and were related asymptotically to the percentage of Leucaena in the diet (R2 = 0.48 and 0.33, respectively). F.NIRS calibrations for DWG were not satisfactory to predict this variable from an individual faecal sample since the s.e. of prediction were 0.33–0.40 kg/day. Cumulative steer liveweight (LW) predicted from F.NIRS DWG calibrations, which had been previously developed with tropical grass and grass–herbaceous legume pastures, greatly overestimated the measured steer LW; therefore, these calibrations were not useful. Cumulative steer LW predicted from a modified F.NIRS DWG calibration, which included data from the present study, was strongly correlated (R2 = 0.95) with steer LW but overestimated LW by 19–31 kg after 8 months. Additional reference data are needed to develop robust F.NIRS calibrations to encompass the diversity of Leucaena pastures of northern Australia. In conclusion, the experiment demonstrated that F.NIRS could improve understanding of diet quality and nutrient intake of cattle grazing Leucaena–grass pasture, and the relationships between nutrient supply and cattle growth.

Aim. Our work was aimed at investigating the specificities in the formation of the genetic structure of populations depending on the productivity direction of cattle, bred in Ukraine, using single locus DNA fragments, and studying the impact of the parental form on genetic polymorphism of modern intensive specialized breeds as a factor. Methods. The following methods were used in the work: veterinary methods (peripheral blood sampling); molecular-genetic meth- ods (the isolation and genotyping of DNA samples of dairy (83 animals), meat (192 animals) and aboriginal (43 ani- mals) cattle, bred in Ukraine, were performed by 10 microsatellite loci from the list, recommended by the International Society for Animal Genetics (ISAG); the complex of statistics methods was used for mathematic-statistical analysis, using modern software. Results. The analysis by 10 microsatellite loci demonstrated the specificities of genetic dif- ferentiation and the similarities between the investigated cattle populations, bred in Ukraine. Our results provide new information about the impact of artificial selection factors on single locus DNA fragments under the specialization of cattle breeds. The impact of the factor of ancestral form on the genetic structure was determined and confirmed by the same polymorphism spectra of the investigated DNA fragments in the maternal and derivative breeds. Another con- firmation was found in the differences, observed in animals of different productivity directions, which are a probable result of the breed-forming process, demonstrated by the results of the mathematic calculations of the data obtained. It was shown that microsatellite DNA loci are highly informative markers of genetic processes, occurring in domestic cattle populations. Conclusions. The specificities in the formation of the genetic structure of populations depending on the productivity direction of animals were determined. The impact of the parental form on genetic polymorphism of modern intensive specialized breeds was noted. It was found that among 10 microsatellite loci, used by us, there were loci in each group of animals, regarding which the hypothesis about their neutrality was reliably rejected accord- ing to the results of Ewens-Watterson test: for dairy cattle (INRA023, ETH3, ETH225, BM1824, BM2113, ETH10 and SPS115), for meat cattle (TGLA122 and ETH225), and for aboriginal cattle (TGLA126, INRA023 and TGLA227). We determined a high level of genetic diversity, remarkable for each investigated cattle population, bred in Ukraine, and general tendencies of differentiation in the selected populations depending on the targeted breeding work, on the level of polymorphism of microsatellite DNA loci (Friedman’s test: P < 0.01), and a similar genetic picture for a number of loci of investigated DNA fragments, which may be related to the history of creating these breeds.

Introduction. Intensification of various aspects of modern agricultural production, based on the use of a large number of mineral fertilizers and chemical means of plant and animal protection, actualizes the development and mastering of natural-like methods for restoring the quality of soils and bottom deposits of inland water bodies. The goal is their successful use in cultivating cultivated plants and conducting remediation measures. One such approach is vermiculturing – breeding of earthworms of the family Lumbricidae, for biotransformation of depleted soils and organic wastes in order to obtain biohumus. Relevance. Earthworms differ significantly in biological characteristics from animals, traditionally bred in agriculture. When working with them, a selection problem arises to assess their phenotypes – it is difficult to identify individuals and assess their performance. In this regard, the main task of effective selection becomes more complicated. Today in scientific sources on the study of the genetic structure of earthworms natural populations, their karyotypes and morphological features are not few data [3]. But the development of vermiculture, based on breeding and industrial use for biotransformation of earthworms, requires a clear species identification of cultural lines of invertebrate animals of the family Lumbricidae. Research objective. The aim of the study was to investigate the morphological and cytogenetic features of the Eisenia worms population that is being forms. Materials and methods. While forming of the new population, the worms that were purchased by the Department of Biotechnology of the UGHTU in the association "Bioconversion" and previously described as E. foetida were used. Of the array of these animals, 6 worms were selected that became the founders of the new population. After increasing the number of up to 300 animals, we formed groups of 20 individuals, each group irradiated with a laser of the LGN-208b type With a power of 1 mW, a wavelength of 633 nm, a beam diameter of 14 mm, various exposures in time from 5 to 30 min. The control was not irradiated. Irradiated animals were bred in separate groups and studied morphological, biochemical and cytogenetic features. Morphological study of the species affiliation was carried out that was determined and compared with the descriptions of these species given in the works of foreign taxonomy [5]. The intensity of the pigmentation of the integument of the body was determined in animals. All further morpho-metric studies were carried out on worms fixed in 75% ethanol. By the method of microscopy, parameters such as length and diameter of the body, total number of segments, location of the segments of the girdle, pubertal ridges and the first dorsal pore, type of setae were analyzed. Karyological analysis was performed with worms selected at the time of highest sexual activity. Preparations were prepared from the tissue of the seminal sacks by the method previously successfully used to study karyotypes of lumbricids [6]. The worms were injected with 0.1% solution of colchicine into the pre-lobe zone for 19 hours. 20 min to the autopsy. The animals were immobilized in 75% ethanol solution and digested along the median spinal line. Removed spermatic bags hypotone 50 min. in the distillate and fixed in three steps in a mixture of acetic acid and ethanol in a ratio of 1:3. Chromosome preparations were made by imprinting. Genetic markings were performed by electrophoresis in a 7.5% polyacrylamide gel Tris-EDTA • Na2-borate system with pH = 8.5 [7] 1 hour 20 minutes at a voltage of 200 V and a current strength of 140–mA. Extract of enzymes and proteins was obtained by grinding the final segments of the body with a size of 5–10 mm in the distillate in the ratio 1:1. After switching of the electric current, the gel was treated with a solution containing a special substrate that specifically reacts with the enzyme under study, forming spots corresponding to the spectra of the enzymes on the gels. The genotype of the individual according to the locus encoding the enzyme being studied is determined by the nature of the distribution of the spots on the gel [8]. Results of the research. The carried out researches have shown that the groups of animals under study belong to the species E. foetida, and by other attributes to the species E. venetta, which caused the problem of the species affiliation of the earthworm array when working to form a new population. Thus, the morphological indices studied have revealed that animals for all morphological features refer to the species Eisenia foetida. During the study of cytogenetic, it was found that the karyotype of the animals was 36 chromosomes but it should be 22. Just kind of Eisenia foetida is composed of 22 chromosomes and karyotypes of other species of the genus Eisenia have 36 chromosomes. Therefore, biochemical gene marking was carried out on enzyme systems, in particular nonspecific esterases. Nonspecific esterases of different species of the genus Eisenia differ in molecular weight. Esterаs of E. foetida have a lower mass than esterases of E. veneta. Our studies showed that in the place of the locus of spectra of nonspecific esterases, the individuals under study belong to the species E. veneta. Nonspecific esterases of different species of the genus Eisenia differ in molecular weight. Esterases E. foetida have a lower mass than E. veneta esterase. Our studies have shown that individuals at the locus of spectra of non-specific esterases belong to the species E. veneta. Conclusions The research may be a theoretical hypothesis for certain types of animal identification in vermiculture and creating biological diversity in its population. Despite the fact that it originates from 6 individuals obtained from a single array of animals, polymorphism by esterases showed a fairly high level of genetic variability in the forming line that indicates the reserve of its genetic variability and allows it to hope for its successful development in the future.

Isolation, characterization of natural products dimeric amide alkaloids from roots of the Piper chaba Hunter. The synthesis of these products using intermolecular [4+2] cycloaddition reaction has been described. Obtained products were characterized using IR, 1HNMR, 13CNMR and Mass Spectroscopy. Introduction The awesome structural diversity and complexity of natural products inspire many chemists to consider how nature creates these molecules. Nature’s biosynthetic enzymes offer a powerful and practical route to many organic compounds, and synthetic chemists sometimes seek to imitate the efficiency and elegance of the biosynthetic machinery by designing biomimetic reactions that approximate natural reaction pathways. Probably the most astonishing biomimetic reactions1 are tandem processes that combine several transformations in sequence and produce complicated structures from comparably simple starting materials in a single laboratory operation. Biosynthesis is described as “the reaction or reaction sequence occurred in organism or its immediate environment will be viewed as biosynthesis” where as biomimetic synthesis describes as “A specific reaction or a sequence of reactions that mimic a proposed biological pathway is defined as bimimetic synthesis. An early example is Sir Robert Robinson’s landmark synthesis of tropinone in 1917.2 Forty-two years later, Gilbert Stork and Albert Eschenmoser independently proposed that the steroid ring system could be formed by tandem cation-π cyclizations of a polyene in an ordered transition state.3 A non-enzymatic version of this reaction type was demonstrated in W. S. Johnson’s classic synthesis of progesterone in 1971.4 Chapman’s synthesis of carpanone is a striking example of the power of biomimetic strategies.5 In 1980, Black proposed that the endiandric acids could arise biosynthetically from linear polyenes.6 In 1982, K. C. Nicolaou gave chemical support to Black’s hypothesis by chemically synthesizing endiandric acids A-G.7 Biomimetic Synthesis of Natural Products which involves, The biomimetic polyene carbocyclizations reaction, The biomimetic cycloaddition reaction, The biomimetic electrocyclization reaction, The polyether biomimetic synthesis, The biomimetic oxidative coupling of phenol, Some other interesting biomimetic synthesis, The present biomimetic synthesis of chabamides or dimeric amide alkaloids involves cycloaddition reactions. The Diels Alder reaction In the Diels-Alder reaction a six membered ring is formed through fusion of a 4 π component, usually a diene and a 2 π component which is commonly referred to as the Figure 1. dienophile. The Diels Alder reaction has proven to be great synthetic value, forming a key-step in the construction of compounds containing six-membered rings. Cyclohexene ring generated all the way through the formation of two new σ-bonds and one π bond with four adjacent stereocenters. The reaction is named after Otto Diels and Kurt Alder, two German chemists who studied the synthetic and theoretical aspects of this reaction in great detail.8 Their efforts have been rewarded with the 1950 Noble prize. Figure 2 Schematic representation of the Diels-Alder reaction. Cis principle In Diels-Alder reactions, the stereoselectivity is generally high due to the “cis principle”, which states that Diels-Alder reactions require a cisoid conformation for the diene and suprafacial-suprafacial mode of reaction, meaning that both ends of the diene attack from the same face of the dienophile in a syn fashion. Frontier Molecular Orbital (FMO) Approach Diels-Alder rections can be devided into, normal electron demand and inverse electron demand additions. This difference is based on the way the rate of the reaction responds to the introduction of electron withdrawing and electron donating substituents. Normal electron demand Diels-Alder reactions are promoted by electron donating substituents on the diene and electron withdrawing substituents on the dienophile. In contrast, inverse electron demand reactions are accelerated by electron withdrawing substituents on the diene and electron donating ones on the dienophile. There also exists an intermediate class, the neutral Diels-alder reaction, which is accelerated by both electron withdrawing and donating substitutents. The way the substituents affect the rate of the reaction can be rationalized with aid of Frontier Molecular Orbital (FMO) theory. This theory was developed during a study of the role of orbital asymmetry in pericyclic reactions by Woodward and Hoffmann9 and, independently, by fukui10 Later, Houk contributed significantly to the understanding of the reactivity and selectivity of these processes.11 The FMO theory states that a reaction between two compounds is controlled by the efficiency with which the molecular orbitals of the individual reaction partners interact. The interaction is most efficient for the reactivity is completely determined by interactions of the electrons that are highest in energy of the of the reaction partners (those in the Highest Occupied Molecular Orbital, the HOMO) with the Lowest Unoccupied Molecular Orbital (LUMO) of the other partner, applied to the Diels-alder reactions, two modes of interaction are possible. The reaction can be controlled by the interaction of the HOMO of the diene and the LUMO of the Dienophile (normal electron demand), or by the interaction between the LUMO of the diene and the HOMO of the dienophile (inverse electron demand), as illustrated in Fig-B. In the former case, a reduction of the diene-HOMO and dienophile-LUMO energy gap can be realized by either raising the energy of the HOMO of the diene by introducing electron donating substituents or lowering the energy of the dienophile LUMO by the introduction of electron donating substituents or lowering the energy of the dienophile LUMO by the introduction of electron withdrawing substituents. A glance at Fig-A confirms that in the formation of two new bonds, orbital symmetry is conserved so that, according to Woodward and Hoffmann, the reaction is concerted. In other words, no intermediate is involved in the pericyclic process such as the Diels-Alder reaction.12 This conclusion is consistent with a number of experimental observations. The cis or trans conformation of the dienophile is fully conserved in the configuration of the cycloadduct, which proves that there is no intermediate involved with a lifetime long enough to allow rotation around C-C bond. Selectivity can arise when substituted dienes and dienophiles are employed in the Diels-Alder reaction. Two different cycloadducts denoted as endo and exo are possible. Under the usual conditions their ratio is kinetically controlled. Alder and Stein already discerned that there usually exists a preference for formation of the endo isomer i.e formulated as tendency of maximum accumulation of unsaturation, (the Alder-Stein rule)13 Indeed, there are only very few examples of Diels-Alder reactions where the exo isomer is major product.14 The interactions underlying this behavior have been subject of intensive research. Since the reactions leading to endo and exo product share the same initial state, the difference between the respective transition-state energies fully account for the observed selectivity. These differences are typically in the range of 10-15 kJ per mole.15 Woodward and Katz16 suggested that secondary orbital interactions are of primary importance. These interactions are illustrated in fig-B for the normal electron demand (HOMO-diene, LUMO-dienophile controlled). The symmetry allowed overlap between π-orbital of the carbonyl group of the dienophile and the diene-HOMO is only possible in the endo activiated complex. Hence, only the endo transition state is stabilized so that the reaction forming the endo adduct is faster than that yielding exo product. This interpretation has been criticized by Mellor, who attributed the endo selectivity to steric interactions. Steric effects are frequently suggested as important in determining the selectivity of Diels-Alder reactions, particularly of α-subsituted dienophiles, and may ultimately lead to exo-selectivity. 17 For other systems, steric effects in the exo activated complex can enhance endo selectivity. 18 In summary, it seems for most Diels-Alder reactions secondary orbital interactions afford a satisfactory rationalization of the endo-exo selectivity. However, since the endo-exo ratio is determined by small differences in transition state energies, the influence of other interactions, most often steric in origin and different for each particular reaction is likely to be felt. The compact character of the Diels-Alder activated complex (the activation volume of the retro Diels-Alder reaction is negative) will attenuate these effects.19 Results and Discussions Chabamides F & G as dimeric amide alkaloids were isolated from this plant Piper chaba Hunter. These two dimers were formed by Diels-Alder reaction employing monomer trichostachine. This hypothesis was further confirmed by the mass spectrum, which showed a significant peak at m/z 294.113 [M++Na], assigned to the trichostachine ion arising by the Retro-Diels–Alder cleavage of molecular ion into two halves. Finally, to confirm the existence of the compounds F and G, we extracted the roots of P.chaba with MeOH at room temperature followed HPLC/electron spray ionization (ESI) MS experiments. In HPLC/ESIMS of the MeOH extract showed the presence of peaks at m/z 563 [M++Na] and 543 [M++1] at about 8.8 min and 10.6 min of LC retention time, respectively. To prove this biosynthetic hypothesis we have carried out the intermolecular [4+2] cycloaddition reaction with the trichostachine under solvent free conditions (Scheme 1). Reaction mixture was analysed by the LC-MS, which clearly indicted the presence of the compounds 1 and 2 (retention time and mass). In HPLC analysis, retention times of the synthetic 1 and 2 were identical to those of chabamide F and G, confirming the structure and stereochemistry are same as that of isolated alkaloids. Based on above result during Diels-Alder reaction of trichostachine, we developed two kinds of methodologies for this biomimetic synthesis of dimeric amide alkaloids based on catalytic. On the basis of a biosynthetic hypothesis (described in Chapter I) by the intermolecular Diels-Alder reaction, we chosen piperine (1a), pellitorine (1c) and trans-fagaramide (1c) as substrates to perform the biomimetic synthesis of the dimeric chabamides (Compound H-K) and this study also identified plausible products between piperine (1a) and pellitorine (1c). This study not only explains formation of cyclo adducts but also explains the different mechanistic aspects in Diels-Alder reaction (endo and exo products) of copper salts in aqueous medium. Under normal conditions only combinations of dienes and dienophiles that have FMO’s of similar energy can be transformed into a Diels-Alder adduct. When the gap between the FMO’s large, forcing conditions are required, and undesired side reactions and retro Diels-Alder reactions can easily take over. These cases challenge the creativity of the organic chemist and have led to the invention of a number of methods for promoting reluctant Diels-Alder reactions under mild conditions.20 Plausible mechanism for Diels-Alder reaction: Sijbren Otto. et. al studied extensively on copper (II) catalyzed Diels-Alder reactions on various moieties. 25, 26 Based on these reports we proposed plausible mechanism for this copper catalyzed Diels-Alder reaction. The first step in the cycle comprises rapid coordination of the lewis acid to the dienophile leading to a complex in which the dienophile is activated for reaction with the diene. The cycloadduct has dissociated from the lewis acid in order to make the catalyst available for another cycle. However we didn’t carry any kinetic studies to prove this mechanism. Plausible mechanism of Diels-Alder reaction catalyzed by copper (II) salts Use of lewis acids in Diels-Alder reaction is to lower LUMO dienophile energy to result in the decrease of the LUMO dienophile-HOMO diene gap (normal electron demand) or reduce LUMO diene energy to result in the decrease of the LUMO diene-HOMO dienophile gap (inverse electron demand). The presence of Lewis acids, the Diels-Alder dimerization of piperine, pellitorine, piperine with fagaramide, peperine with pellitorine, gave much lower combined yields in neat conditions. Wie et al. previously reported 21, 22 Diels-Alder reaction of piperine and in both thermal and by lewis acid of Co(II) Cl2.6H2O/P(Ph)3/Zn (1:10:10 mol %) in 3-octanol at 170oC with isomerised product (24 %) and 77 % over all yield. To find the optimum conditions towards the catalyst, piperine (1a) was taken to perform the Diels-Alder reaction in presence of variety of lewis acids and metal salts (Table 1). The highest catalytic activity was attained for the reaction using 10 mol % of Cu (II) salts. The role of copper salts in this reaction can be attributed to its Lewis acid ability, which enhances both the electron donating capacity of diene and electron withdrawing capacity of the dienophile (required for normal electron demand for Diels-Alder reaction). The The catalytic activitiy of Lewis acids like Cu+2 mainly relies on their coordinating character to assemble both dienophile and diene to such a way that promote the reaction to wards the reaction barrier. To find the optimum conditions towards the solvent several reactions were carried out under the solvents like benzene, toluene, xylene, water and results were tabulated (Table 2). Among organic solvents xylene is better to get considerable yield with copper salts. Later water was found to be the best for both yield and selectivity of this cycloaddition. Cycloaddition reactions of piperine (1a): Lewis acids catalyzed cycloaddition reactions (Scheme 2) of piperine (1a) under organic and aqueous solvent conditions to give resultant cycloadducts 2a, 3a, 4a, 5a and 6a, among them 2a is major ortho-exo cyclohexene type dimeric amide alkaloid and also known as chabamide, which is previously isolated23 from this plant, isomer 3a is previously isolated from Piper nigrum21 Remaining isomers (4a-6a) were synthesized from piperine by Diels-Alder reaction by Kun Wei. et al. its physical and spectroscopic data were identical with reported data22 (1H-NMR, 13C-NMR & Mass spectra). In the cycloaddition of piperine (1a), solvents toluene, xylene and water were used in presence of cuper (II) salts. Reaction showed good overall yield and more exo selectivity in organic solvent like xylene. Water catalyzed reactions were ended with good overall yield and minute decrease in exo selectivity, infinitesimal increase in endo selectivity (Table 2). This reaction showed completely regioselectivity (yield of 2a+3a>4a+5a+6a) due to maximum involvement of α-double bond rather than γ-double bond of 1a during Diels-Alder reaction. Cycloaddition reactions of pellitorine (1b): Same catalytic and solvent conditions were employed for pellitorine (1b) as used in piperine (1a) for the biomimetic synthesis (Scheme 3) of chabamide J & K (Chapter-II). These dimers were plausibly generated by monomer pellitorine by cycloaddtion reactions in biosynthesis. During cycloaddition of pellitorine (1b), solvents like toluene xylene and water were used in presence of cupper (II) salts. In former catalyzed reaction showed good overall yield and more endo selectivity in both organic (xylene) and water. Increase in endo selectivity is more in aqueous medium rather than organic solvent like xylene (Table 2). Cycloaddition of pellitorine under above said catalytic conditions gave corresponding cycloadducts 2b, 3b, 4b and 5b. Physical and spectral data of adducts 2b & 3b are identical with compound J & K (chabamide J & K mentioned in Chapter-II) and all physical and spectral data of adduct 4b is identical with nigramide O which is isolated previously from piper nigrum.21 The structure of 5b a new cycloadduct formed during this biomoimetic synthesis employ pellitorine as monomer, its structure was elucidated by 1D and 2D spectral data. This reaction showed completely regioselectivity (yield of 2b+5b≈3b+4b) due to maximum involvement of α-double bond rather than γ-double bond of 1b during Diels-Alder reaction. Structure elucidation of compound 5b: Compound 5b was obtained as a pale yellow oil, had the molecular formula of C28H50N2O2, as deduced from the HRESIMS (Fig-9) m/z, 447.3958 [M++H]. IR spectrum (Fig-1) implied the presence of carbonyl (1648 cm-1) and NH (3304 cm-1). The 1H NMR spectrum of 5b revealed the presence of a trans double bond at δ 5.28 (dd, J = 15.0, 10.0 Hz, H-4"), 5.63 (m, H-5"), two isobutylamide groups at δ 3.15 (m), 3.17 (m), 3.17 (m, H2-1'), 1.74 (m, H-2'), 0.91 (d, J = 6.7 Hz, H-3'), 0.90 (d, J = 6.7, H- 3'), 5.53 (br t, J = 5.7 Hz, NH) and δ 2.96 (m, H1-1'''), 2.97 (m, H2-1'''), 1.73 (m, H-2'''), 0.87 (d, J = 6.7 Hz, H-3'''), 0.86 (d, J = 6.7 Hz, H-3'''), 3.15 (br t, J = 6.0 Hz, NH), n-amyl group and 1-heptene unit at δ 1.96 (m, H-6), 1.40 (m, H-7), 1.20 (m, H-8), 1.27 (m, H-9), 0.86 ( t, J = 6.5 Hz, H-10) and δ 5.28 (dd, J = 15.0, 10.0 Hz, H-4"), 5.63 (m, H-5"), 1.89 (m, H-6"), 1.30 (m, H-7"), 1.28 (m, H-8"), 1.27 (m, H-9"), 0.88 (t, J = 6.5 Hz, H-10"), respectively (Table 3). The 13C NMR spectrum (Fig-3) displayed the presence of 28 carbon atoms and were further classified by DEPT experiments (Fig-4) into categories of 6 methyls, 10 methylenes, 10 methines and 2 quaternary carbons including two carbonyls (δ 173.80 and 173.04). ' The analyses of the 1H and 13C NMR spectral data of 5b showed a high degree of similarity to dimeric alkaloid, compound J naturally isolated from this plant (Chapter-II) compound is meta-endo while 5b is meta-exo product. Furthermore, the detailed elucidation of the 2D NMR data (COSY, HSQC and HMBC) had determined the planar structure of 5b. The 1H homodecoupling NMR (Fig-7) experiments of 5b revealed the connectivities H-2 (δ 2.45, m) to H-3 (δ 5.56, ddd, J = 10.0, 4.3, 2.6 Hz) to H-4 (δ 5.98, dt, J = 10.0, 1.8 Hz) to H-5 (δ 2.41, m) to H-2"( δ 2.68, dd, J = 11.3, 10.0 Hz) to H-3" (δ 2.82, ddd, J = 10.1, 10.0, 5.0 Hz ) via cyclohexene ring protons. The meta-orientation of the carbonyl and isobutylamide groups were established by HMBC (Fig-6) correlations for δ 2.45 (m, H-2), 5.56 (ddd, J = 10.0, 4.3, 2.6 Hz, H-3), 2.82 (ddd, J = 10.3, 10.0, 5.0 Hz, H-3")/δ 173.80 (C-1) and δ 2.68 (dd, J = 10.3, 10.0 Hz, H-2"), 2.41 (m, H-5), 2.82 (ddd, J = 10.3, 10.0, 5.0 Hz, H-3")/δ 173.04 (C-1"). Furthermore, the 1H-1H COSY (Fig-7) cross-peaks between δ 2.82 (ddd, J = 10.3, 10.0, 5.0 Hz, H-3") and δ 5.28 (dd, J = 15.0, 10.0 Hz, H-4"), and δ 5.63 (m, H-5") and δ 2.41 (m, H-5), 1.96 (m, H-6), 1.40 (m, H-7), coupled with the HMBC correlation for δ 5.63 (m, H-5'') to δ 28.35 (C-7"), δ 1.40 (m, H-7) to δ 37.04 (C-5) established the attachment of the 1-heptene and n-amyl groups at C-3" and C-5, respectively. The analysis of the 1H-1H coupling constants and NOESY (Fig- 8) data allowed us to determine the relative stereochemistry of compound 5b. The coupling constants of H-2"/H-5 and H-2"/H-3" (10.3 Hz) indicated anti relations of H-2"/H-5 and H-2"/H-3". In the NOESY spectrum correlations were observed at δ 2.45 (H-2) δ 2.82 (H-3") and δ 2.41 (H-5) and correlations were not observed at δ 2.68 (H-2") with δ 2.82 (H-3") and δ 2.68 (H-2") with δ 2.41 (H-5). These data were in agreement with the β-orientation for H-2" and α-orientation for H-3" and H-5. Thus, based on these spectral data the stereostructure of 5b was confirmed and trivially named as chabamide L. Cycloaddition reaction between piperine (1a) and pellitorine (1b): Our aim of this cycloaddition reaction is to explain to study different cycloadducts and selectivity of diene among piperine and pellitorine (Scheme 4). This biomimetic synthesis will explain the probability of diene, which participated in Diels-Alder reaction between piperine (1a) and pellitorine (1b) both were isolated from same plant (P. chaba). Nigramide N, which is formed biosynthetically via cycloaddition reaction between piperine and pellitorine, this adduct previously isolated from roots of P. nigrum 21 by Wei. et. al. Lewis acid catalyzed cycloaddition reactions of piperine (1a) and pellitorine (1b) under organic and aqueous solvent conditions to give resultant cycloadducts 2c, 3c, 4c, 2a and 3b. Cycloadduct 2c and 3c is new cycloadducts and their structures were illustrated by 1D and 2D spectral data. Structure elucidation of compound 2c: Compound 2c was obtained as pale yellow liquid. The molecular formula of 2c was established as C31H44N2O4 by HRESIMS (Fig-18), which provided a molecular ion peak at m/z 509.3381 [M++H], in conjunction with its 13C NMR spectrum (Fig-12). The IR spectrum displayed absorption bands diagnostic of carbonyl (1640 cm-1) (Fig-10). The 300 MHz 1H NMR spectrum (in CDCl3) indicated the presence of two signals at δ 5.86 (dd, J = 15.6, 10.1 Hz) and 6.27 (d, J = 15.6 Hz), which were assigned to trans-olefinic protons by the coupling constant of 15.6 Hz. It also displayed aromatic protons due to two 1, 3, 4-trisubstituted aromatic rings at δ 6.82 (1H, br s), 6.76 (1H, dd, J = 7.8, 1.4 Hz), 6.75 (1H, d, J = 7.8 Hz) (Fig-11), (Table 4). In addition to the above-mentioned moieties, combined inspection of 1H NMR and 1H–1H COSY revealed the presence of cyclohexene ring, one isobutylamide and one pyrrolidine ring. The 13C NMR spectrum displayed the presence of 31 carbon atoms and were further confirmed by DEPT experiments into categories of 11 methylenes, 12 methines and 5 quaternary carbons including two carbonyls (δ 173.01 and 172.50). On the basis of these characteristic features, database and literature search led the skeleton of compound 2c as a dimeric alkaloidal framework. A comprehensive analysis of the 2D NMR data of compound 2c facilitated the proton and carbon assignments. 1H–1H COSY spectrum suggested the sequential correlations of δ 3.51 (dq, J = 5.0, 2.6 Hz)/5.62 (dt, J = 9.8, 2.6 Hz)/6.10 (ddd, J = 9.8, 1.5 Hz)/2.20 (m)/2.72 (ddd, J = 11.1, 10.1, 5.2 Hz)/3.35 (dd, J = 11.1, 9.8 Hz) assignable to H-2-H-3-H-4-H-5-H-3"-H-2" of the cyclohexene ring. Concerning the connections of the n-amyl and 3, 4-methylenedioxy styryl groups, HMBC spectrum (Fig-15) showed correlations of H-4, H-6, H-7/C-5; H-5", H-4"/C-3", which implies that these units were bonded to the cyclohexene ring at C-5 and C-3". Further, HMBC correlations of two methylene protons at δ 5.95 with 147.91 (C-8"), 146.87 (C-9"), confirmed the location of methylenedioxy group at C-8", and C-9". Remaining units, isobutylamine and pyrrolidine (rings) were connected through carbonyl groups at C-2 and C-2", which was confirmed by HMBC correlations of H-2 and H-1' to C-1 (δ 173.01) and H-2" and H-1''' to C-1" (δ 172.50). The assignment of the relative configuration of compound 2c, and confirmation of overall structure were achieved by the interpretation of the NOESY spectral data and by analysis of 1H NMR coupling constants. The large vicinal coupling constants of H-2"/H-2 (11.1 Hz) and H-2"/H-3" (11.1 Hz) indicated anti-relations of H-2"/H-2 and H-2"/H-3" and the axial orientations for these protons. In the NOESY spectrum (Fig-17), the occurrence of the correlations between H-2/H-3" and the absence of NOE effects between H-2/H-2" and H-2"/H-3" supported the above result. This data indicated β-orientation for H-2" and α-orientation for H-2 and H-3". The α-orientation of H-5 was suggested by the coupling constant of H-5/H-3" (5.2 Hz) and the absence of the NOESY correlations between H-3" and H-2". On the basis of these spectral data, the structure of compound 2c was unambiguously established and trivially named as chabamide M. Structure elucidation of compound 3c: Compound 3c was obtained as pale yellow liquid. The molecular formula of 3c was established as C31H44N2O4 by HRESIMS (Fig-27), which provided a molecular ion peak at m/z 509.3391 [M++H], in conjunction with its 13C NMR spectrum (Fig-21). The IR spectrum displayed absorption bands diagnostic of carbonyl (1624 cm-1) moiety (Fig-19). The 300 MHz 1H NMR spectrum (in CDCl3) indicated the presence of two signals at δ 4.63 (dd, J = 15.6, 10.0 Hz) and 5.46 (dt, J = 15.6, 6.8 Hz), which were assigned to trans-olefinic protons by the coupling constant of 15.6 Hz. It also displayed aromatic protons due to two 1, 3, 4-trisubstituted aromatic ring at δ 6.75 (1H, br s), 6.73 (1H, d, J = 7.8, 1.4 Hz), 6.71 (1H, d, J = 7.8 Hz) (Fig-20). In addition to the above-mentioned moieties, combined inspection of 1H NMR and 1H–1H COSY revealed the presence of cyclohexene ring, one isobutylamide and one pyrrolidine ring. The 13C NMR spectrum displayed the presence of 31 carbon atoms (Table 5), and were further classified by DEPT experiments (Fig-22) into categories of 11 methylenes, 12 methines and 5 quaternary carbons including two carbonyls (δ 173.34 and 173.88). On the basis of these characteristic features, database and literature searches led the skeleton of compound 3c as a dimeric alkaloidal framework. A comprehensive analysis of the 2D NMR data of compound 3c facilitated the proton and carbon assignments. 1H–1H COSY spectrum (Fig-25) suggested the sequential correlations of δ 2.82 (m)/5.63 (dt, J = 9.7, 1.9 Hz)/5.82 (ddd, J = 9.7, 4.8, 1.9 Hz)/3.94 (dq, J =.10.0, 1.9 Hz)/2.76 (ddd, J = 11.7, 10.0 Hz)/3.36 (dt, J = 11.7, 4.8 Hz) assignable to H-2-H-3-H-4-H-5-H-3"-H-2" of the cyclohexene ring. Concerning the connections of the 3, 4-methylenedioxyphenyl and 1-heptene groups, HMBC spectrum (Fig-24) showed correlations of H-7, H-11, H-3"/C-5; H-5", H-4", H-5/C-3", which implies that these units were bonded to the cyclohexene ring at C-5 and C-3". Further, HMBC correlations of two methylene protons at δ 5.92 with 147.42 (C-8"), 146.49 (C-9"), confirmed the location of methylenedioxy group at C-8", and C-9". Remaining units, pyrrolidine and isobutylamine were connected through carbonyl groups at C-2 and C-2", which was confirmed by HMBC correlations of H-2 and H-1' to C-1 (δ 171.34) and H-2" and H-1''' to C-1" (δ 173.88). The assignment of the relative configuration of compound 3c, and confirmation of overall structure were achieved by the interpretation of the NOESY spectral data and by analysis of 1H NMR coupling constants. The large vicinal coupling constants of H-3"/H-2" (11.7 Hz) and H-5/H-3" (10.0 Hz), indicated anti-relations of H-3"/H-5 and H-3"/H-2" and the axial orientations for these protons. In the NOESY spectrum (Fig-26), the occurrence of the correlations between H-2"/H-5 and the absence of NOE effects between H-3"/H-2" and H-3"/H-5 supported the above result. These data indicated β-orientation for H-2" and α-orientation for H-2 and H-3". The α-orientation of H-2 was suggested by the coupling constant of H-2/H-2" (4.8 Hz) and the occurrence of the NOESY correlations between H-2" and H-2. On the basis of this spectral data, the structure of compound 3c was unambiguously established and trivially named as Chabamide N. Cycloaddition reaction between piperine (1a) and E-fagaramide (1c) Lewis acid catalyzed cycloaddition reactions (Scheme 5) of piperine (1a) and trans-fagaramide (1c) under aqueous solvent conditions to give resultant cycloadducts 2d, 3d and 2a. To carry this biomimetic synthesis to explain compound H and I (mentioned in chapter-II), we taken piperine (1a) which is isolated from same plant and trans fagaramide was synthesized by reported method.24 Cycloaddition reaction between 1a and 1c end up with overall yield 70% in xylene and 75% in water. In both solvents ortho products were formed dominantly compared with meta products. Spectral data 1D and 2D of cycloadducts 2d & 3d were identical with compound H & I (see chapter I, compound H & K). Cycloadduct 2a is identical with chabamide. This cycloaddition reaction practically proved as biomimetic synthesis for compound H and I. Acknowledgements: The authors are thankful to Director IICT for his constant encouragement and CSIR New Delhi for providing the fellowship References and Notes Braun, M. Synth. Highlights 1991, 232 Robinson, R. Chem. Soc. 1917, 762. Stork, G.; Burgstahler, A. W. Am. Chem. Soc. 1955, 38, 1890. Johnson, W. S.; Gravestock, M. B.; McCarry, B. E. Am. Chem. Soc. 1971, 93, 4332. Chapman, O. L.; Engel, M. R.; Springer, J. P.; Clardy, J. C. Am. Chem. Soc. 1971, 93, 6696. Bandaranayake, W. M.; Banfield, J. E.; Black, D. St. C. Chem. Soc., Chem Commun. 1980, 902. Nicolaou, K. C.; Zipkin, R. E.; Petasis, N. A. Am. Chem. Soc. 1982, 104, 5558. O.; Alder, K. Ann. 1928, 460, 98. Woodward, R. B.; Hoffmann, R. Chem. 1969, 81, 797. Fakui, K. Chem. Res. 1971, 4, 57. Houk, K. N. Chem.. Res. 1975, 8, 361. Houk, K. N.; Li, Y.; Evanseck, D. Angew Chem., Ed. Engl. 1992, 31, 682. Alder, K.; Stein, G. Chem. 1937, 50, 510. Fotiadu, F.; Michel, F.; Buono, G. Tetraheron Lett. 1990, 34, 4863. Gleiter, R.; Bohm, M. C. Pure Appl. Chem. 1983, 55, 237. Woodward, R. B.; Katz, T. J. Terahedron 1958, 5, 70. Kakushima, M. J. Chem. 1979, 57, 2564. Houk, K. N. Tetrahedron Lett. 1970, 30, 2621. Houk, K. N.; Luskus, L. J. Am. Chem. Soc. 1971, 93, 4606. Otto, S.; Bertoncin, F.; Engberts, J. F. N. Am. Chem. Soc., 1996, 118, 7702–7707. Wei, K.; Li, W.; Koike, K.; Chen, Y-J.; Nikaido, T. Org. Chem. 2005, 70, 1164. Wei, K.; Li, W.; Koike, K.; Chen, Y-J.; Nikaido, T. Lett. 2005, 7, 2833–2835. Rukachaisirikul, T.; Prabpai, S.; Champung, P.; Suksamrarn, A. Planta Med. 2002, 68, 850-853. Nagao, Y.; Seno, K.; Kawabata, K.; Miyasaka, T.; Takao, S.; Fujita, Tetrahedron Lett. 1980, 21, 841. Otto, S.; Boccaletti, G.; Engberts, J. B. F. N. Am. Chem. Soc. 1998, 120, 4238–4239. Otto, S.; Bertoncin, F.; Engberts, J. B. F. N. Am. Chem. Soc. 1996, 118, 7702–7707. O.; Alder, K. Ann. 1931, 490, 243. Woodward, R. B.; Baer, H. Am. Chem. Soc. 1948, 70, 1161. Breslow, R.; Rideout, D. C. Am. Chem. Soc. 1980, 102, 7816. Breslow, R.; Guo, T. Am. Chem. Soc. 1988, 110, 5613. Grieco, P.A.; Nunes, J. J.; Gaul, M. D. Am. Chem. Soc. 1990, 112, 4595.

Introduction: Classical Hodgkin lymphoma (cHL) is characterized by a robust and complex immune cell infiltrate and the rare presence of malignant Hodgkin-Reed-Sternberg (HRS) cells. At the genetic level, HRS cells recurrently acquire alterations that lead to defective antigen presentation (β2 microglobulin mutations) and mediate T cell dysfunction (PD-L1 copy gains/amplifications) in order to subvert host immune surveillance. The clinical relevance of PD-L1 protein over-expression in cHL is clear, as response rates to PD-1 blockade therapy are extremely high among patients with relapsed/refractory (r/r) disease. Despite its remarkable efficacy, the cells that mediate response to anti-PD-1 therapy in cHL remain undefined. Recent analyses have highlighted a possible role for CD4+ T cells in mediating the clinical activity of anti-PD-1 therapy in cHL. CD4+ T cells significantly outnumber CD8+ T cells in cHL lesions and are more frequently juxtaposed to HRS cells in situ. Furthermore, HLA class II expression on HRS cells predicted higher complete response rates to PD-1 blockade therapy in r/r cHL patients. However, a candidate T cell population capable of specific reactivity to antigens expressed by HRS cells has yet to be identified. This information is critical as such T cells might be functionally reinvigorated to mediate HRS cell elimination following PD-1 blockade therapy. In order to address this key knowledge gap, we analyzed data at single cell (sc) resolution using paired RNA and T cell receptor (TCR) sequencing in 9 diagnostic cHL and 5 reactive lymph node (RLN) specimens. Methods: Sequencing was performed using the 10x Genomics Chromium Single Cell 5' Gene Expression and V(D)J workflows. B-cell depletion of each sample was achieved using CD19 microbeads and negative selection to enrich T cell populations. Reads were analyzed and aligned with CellRanger (v3.1.0) and Seurat (v3.2.0) was used to conduct clustering by a shared nearest neighbor (SNN) graph on scRNA data. TCR sequencing data was integrated using scRepertoire (v1.0.0). Results: A detailed map of the immune cell states in cHL was created using scRNA-seq (10X) data on 79,085 cells from 9 cHL (52,602 cells) and 5 RLN samples (26,484 cells) expressing a total of 21,421 genes (mean 5649 cells/sample; mean 2849 mRNA reads/cell). Dimensionality reduction and unsupervised graph-based clustering revealed 21 distinct cell type and activation state clusters, including T cells, NK cells, macrophages, and dendritic cells (Fig 1A-B). A cluster identifying HRS cells was not observed, consistent with a recently published report. Ten T cell clusters were identified (47,573 cells), including naive- and memory-like T cells, effector/cytotoxic CD8+ T cells, regulatory T cells, and T follicular helper cells. Unexpectedly, a putative exhausted T cell cluster was not clearly observed. The relative contributions of cHL and RLNs cases to these clusters are shown in Fig 1C. Paired TCR sequencing was available for 23,943 cells. Overall TCR diversity was lower among cHL samples compared to RLN specimens (Fig 1D). In cHL samples, modest clonal expansion within regulatory T cell and memory CD4+ T cell clusters was observed, but the most striking clonal expansion occurred among cells assigned to effector/cytotoxic CD8+ T cell clusters - a finding not observed in most RLN specimens (Fig 1E). Clonally-expanded effector/cytotoxic CD8+ T cells displayed high expression of granzymes (GZMA, GZMH, GZMK), cytokines (TNF, IFNG) and chemokines (CCL4/CCL5), and modest expression of exhaustion markers (PDCD1, ENTPD1, HAVCR2, ITGAE), contrasting with data from single-cell analyses of solid tumors. Clonal expansion of effector/cytotoxic CD8+ T cells was particularly robust in EBV-positive cHLs, likely due to recognition of viral-derived epitopes displayed on HRS cells (Fig 1F). Phenotypic and functional validation of key immune cell clusters in cHL specimens using spectral cytometry is underway and will be reported at the meeting. Conclusions: For the first time, our data have unveiled the nature of the T cell repertoire in cHL at single cell resolution. Our results reveal a recurrent pattern of clonal expansion within effector CD8+ cells, which may be the HRS antigen-specific T cells that mediate response to PD-1 blockade. This hypothesis requires confirmation through similar analyses of pre- and on-treatment biopsies of cHL patients receiving anti-PD-1 therapy. Disclosures Godfrey: Gilead: Research Funding; Merck: Research Funding; Verastem: Research Funding. Venkataraman:EUSA Pharma: Speakers Bureau. Smith:Janssen: Consultancy; BMS: Consultancy; TG Therapeutics: Consultancy, Research Funding; Genentech/Roche: Consultancy, Other: Support of parent study and funding of editorial support, Research Funding; Karyopharm: Consultancy, Research Funding; FortySeven: Research Funding; Pharmacyclics: Research Funding; Acerta: Research Funding; Celgene: Consultancy, Research Funding. Kline:Kite/Gilead: Speakers Bureau; Seattle Genetics: Membership on an entity's Board of Directors or advisory committees; Merck: Research Funding; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Verastem: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Rhizosphere bacteria can have wide-ranging effects on their host plants, influencing plant biochemical and structural characteristics, and overall productivity. The implications of plant-microbe interactions provides an opportunity to interfere agriculture ecosystem with exogenous regulation of soil microbial community. Therefore, how to efficiently predict soil bacterial community at low cost is becoming a practical demand. Here, we hypothesize that foliar spectral traits can predict the diversity of bacterial community in orchard ecosystem. We tested this hypothesis by studying the ecological linkages between foliar spectral traits and soil bacterial community in a peach orchard in Yanqing, Beijing in 2020. Foliar spectral indexes were strongly correlated with alpha bacterial diversity and abundant genera that can promote soil nutrient conversion and utilization, such as Blastococcus, Solirubrobacter, and Sphingomonas at fruit mature stage. Certain unidentified or relative abundance &lt;1% genera were also associated with foliar spectral traits. We selected specific indicators (photochemical reflectance index, normalized difference vegetable index, greenness index, and optimized soil-adjusted vegetation index) of foliar spectral indexes, alpha and beta diversities of bacterial community, and quantified the relations between foliar spectral traits and belowground bacterial community via SEM. The results of this study indicated that foliar spectral traits could powerfully predict belowground bacterial diversity. Characterizing plant attributes with easy-accessed foliar spectral indexes provides a new thinking in untangling the complex plant-microbe relationship, which could better cope with the decreased functional attributes (physiological, ecological, and productive traits) in orchard ecosystem.

By combining amplicon-based diversity survey with recently developed nonlinear analytical tools, we successfully determined the interaction networks of more than 150 natural soil microbial genera under less or more temperature stress and explored the applicability of the stress gradient hypothesis to soil microbiota, shedding new light on the well-known hypothesis.