Unraveling the plant diversity of the Amazonian canga through DNA barcoding.

The canga of the Serra dos Carajás, in Eastern Amazon, is home to a unique open plant community, harboring several endemic and rare species. Although a complete flora survey has been recently published, scarce to no genetic information is available for most plant species of the ironstone outcrops of the Serra dos Carajás. In this scenario, DNA barcoding appears as a fast and effective approach to assess the genetic diversity of the Serra dos Carajás flora, considering the growing need for robust biodiversity conservation planning in such an area with industrial mining activities. Thus, after testing eight different DNA barcode markers (matK, rbcL, rpoB, rpoC1, atpF-atpH, psbK-psbI, trnH-psbA, and ITS2), we chose rbcL and ITS2 as the most suitable markers for a broad application in the regional flora. Here we describe DNA barcodes for 1,130 specimens of 538 species, 323 genera, and 115 families of vascular plants from a highly diverse flora in the Amazon basin, with a total of 344 species being barcoded for the first time. In addition, we assessed the potential of using DNA metabarcoding of bulk samples for surveying plant diversity in the canga. Upon achieving the first comprehensive DNA barcoding effort directed to a complete flora in the Brazilian Amazon, we discuss the relevance of our results to guide future conservation measures in the Serra dos Carajás.

A global-scale expert assessment of drivers and risks associated with pollinator decline.

Pollinator decline has attracted global attention and substantial efforts are underway to respond through national pollinator strategies and action plans. These policy responses require clarity on what is driving pollinator decline and what risks it generates for society in different parts of the world. Using a formal expert elicitation process, we evaluated the relative regional and global importance of eight drivers of pollinator decline and ten consequent risks to human well-being. Our results indicate that global policy responses should focus on reducing pressure from changes in land cover and configuration, land management and pesticides, as these were considered very important drivers in most regions. We quantify how the importance of drivers and risks from pollinator decline, differ among regions. For example, losing access to managed pollinators was considered a serious risk only for people in North America, whereas yield instability in pollinator-dependent crops was classed as a serious or high risk in four regions but only a moderate risk in Europe and North America. Overall, perceived risks were substantially higher in the Global South. Despite extensive research on pollinator decline, our analysis reveals considerable scientific uncertainty about what this means for human society.

Combining genotype, phenotype, and environmental data to delineate site-adjusted provenance strategies for ecological restoration.

Despite the importance of climate-adjusted provenancing to mitigate the effects of environmental change, climatic considerations alone are insufficient when restoring highly degraded sites. Here we propose a comprehensive landscape genomic approach to assist the restoration of moderately disturbed and highly degraded sites. To illustrate it we employ genomic data sets comprising thousands of single nucleotide polymorphisms from two plant species suitable for the restoration of iron-rich Amazonian Savannas. We first use a subset of neutral loci to assess genetic structure and determine the genetic neighbourhood size. We then identify genotype-phenotype-environment associations, map adaptive genetic variation, and predict adaptive genotypes for restoration sites. Whereas local provenances were found optimal to restore a moderately disturbed site, a mixture of genotypes seemed the most promising strategy to recover a highly degraded mining site. We discuss how our results can help define site-adjusted provenancing strategies, and argue that our methods can be more broadly applied to assist other restoration initiatives.

Forest Matrix Fosters High Similarity in Bee Composition Occurring on Isolated Outcrops Within Amazon Biome.

Most studies analyze fragmentation due to habitat loss caused by anthropogenic activities and few of them analyzed fragmentation on naturally fragmented areas. In the Eastern Amazon, it is possible to find areas naturally open and surrounded by pristine forest. Understanding how species respond to isolation in these areas is an important challenge for decision-making processes aiming conservation and restoration. Using standardized methods of bee collection (entomological nets, bait trap, pan trap, and nest trap), the objective of this study was to analyze the composition and diversity of bees occurring on six isolated outcrops located in two protected areas within Amazon biome. More specifically, we tested 1) if the dissimilarity in bee species composition is explained by the isolation of outcrops and 2) if bee richness, abundance, and Shannon diversity can be explained by the outcrop size. We found 118 species, with the Meliponini and Euglossini (Hymenoptera: Apidae) tribes representing the highest number of species. The similarity in species composition across all outcrops is high and is not explained by the isolation. In addition, the richness, abundance, and Shannon diversity are not explained by outcrop size. Forest does not seem to be a barrier to bee movement, and although most species probably nest in the forests, they use the highly diverse plants of the outcrops as a complementary food source.

Size and isolation of naturally isolated habitats do not affect plant-bee interactions: A case study of ferruginous outcrops within the eastern Amazon forest.

Pollination may be severely affected by the decreasing size and increasing isolation of habitat patches. However, most studies that have considered the effects of these two variables on plant-pollinator interactions have been carried out in areas that have undergone anthropogenic fragmentation, and little is known about their effects in natural habitats. The Carajás National Forest and Campos Ferruginosos National Park are two protected areas in the eastern Amazon where one can find isolated ferruginous outcrops characterized by iron-rich soil and herbaceous-shrub vegetation surrounded by Amazon forest. These patches of canga provide an opportunity to analyze plant-pollinator interactions in naturally fragmented areas. Our objective was to test whether the size and isolation of naturally isolated outcrops located in Carajás affect plant-pollinator interactions by using pollination syndromes and interaction networks. We determined the pollination syndromes of 771 plant species that occurred in eleven canga patches and performed field work to analyze plant-pollinator networks in nine canga patches. The structure of the plant-pollinator networks was not affected by the size or isolation of the canga patches. Generalist species were present in all canga areas, indicating that they are important in maintaining the plant communities in isolated canga patches. The lack of significance related to the distance between canga patches suggests that the forest does not prevent pollinator movement between canga patches.

Habitat Loss Does Not Always Entail Negative Genetic Consequences.

Although habitat loss has large, consistently negative effects on biodiversity, its genetic consequences are not yet fully understood. This is because measuring the genetic consequences of habitat loss requires accounting for major methodological limitations like the confounding effect of habitat fragmentation, historical processes underpinning genetic differentiation, time-lags between the onset of disturbances and genetic outcomes, and the need for large numbers of samples, genetic markers, and replicated landscapes to ensure sufficient statistical power. In this paper we overcame all these challenges to assess the genetic consequences of extreme habitat loss driven by mining in two herbs endemic to Amazonian savannas. Relying on genotyping-by-sequencing of hundreds of individuals collected across two mining landscapes, we identified thousands of neutral and independent single-nucleotide polymorphisms (SNPs) in each species and used these to evaluate population structure, genetic diversity, and gene flow. Since open-pit mining in our study region rarely involves habitat fragmentation, we were able to assess the independent effect of habitat loss. We also accounted for the underlying population structure when assessing landscape effects on genetic diversity and gene flow, examined the sensitivity of our analyses to the resolution of spatial data, and used annual species and cross-year analyses to minimize and quantify possible time-lag effects. We found that both species are remarkably resilient, as genetic diversity and gene flow patterns were unaffected by habitat loss. Whereas historical habitat amount was found to influence inbreeding; heterozygosity and inbreeding were not affected by habitat loss in either species, and gene flow was mainly influenced by geographic distance, pre-mining land cover, and local climate. Our study demonstrates that it is not possible to generalize about the genetic consequences of habitat loss, and implies that future conservation efforts need to consider species-specific genetic information.

Queen Execution, Diploid Males, and Selection For and Against Polyandry in the Brazilian Stingless Bee Scaptotrigona depilis.

Female mating frequency varies. Determining the causes of this variation is an active research area. We tested the hypothesis that in stingless bees, Meliponini, single mating is due to the execution of queens that make a matched mating at the complementary sex determination locus and have diploid male offspring. We studied the Brazilian species Scaptotrigona depilis. We made up 70 test colonies so that 50% (single matched mating), 25% (double mating), 12.5% (quadruple mating), or 0% (single nonmatched mating) of the emerging brood were diploid males. Queen execution following diploid male emergence was equal and high in colonies producing 50% (77% executed) and 25% (75%) diploid males versus equal and low in colonies producing 12.5% (7%) and 0% (0%) diploid males. These results show that queens that mate with two males with similar paternity suffer an increased chance of being executed, which selects against double mating. However, double mating with unequal paternity (e.g., 25∶75), which occasionally occurs in S. depilis, is selectively neutral. Single mating and double mating with unequal paternity form one adaptive peak. The results show a second adaptive peak at quadruple mating. However, this is inaccessible via gradual evolutionary change in a selective landscape with reduced fitness at double mating.

Landscape genomics to the rescue of a tropical bee threatened by habitat loss and climate change.

Habitat degradation and climate change are currently threatening wild pollinators, compromising their ability to provide pollination services to wild and cultivated plants. Landscape genomics offers powerful tools to assess the influence of landscape modifications on genetic diversity and functional connectivity, and to identify adaptations to local environmental conditions that could facilitate future bee survival. Here, we assessed range-wide patterns of genetic structure, genetic diversity, gene flow, and local adaptation in the stingless bee Melipona subnitida, a tropical pollinator of key biological and economic importance inhabiting one of the driest and hottest regions of South America. Our results reveal four genetic clusters across the species' full distribution range. All populations were found to be under a mutation-drift equilibrium, and genetic diversity was not influenced by the amount of reminiscent natural habitats. However, genetic relatedness was spatially autocorrelated and isolation by landscape resistance explained range-wide relatedness patterns better than isolation by geographic distance, contradicting earlier findings for stingless bees. Specifically, gene flow was enhanced by increased thermal stability, higher forest cover, lower elevations, and less corrugated terrains. Finally, we detected genomic signatures of adaptation to temperature, precipitation, and forest cover, spatially distributed in latitudinal and altitudinal patterns. Taken together, our findings shed important light on the life history of M. subnitida and highlight the role of regions with large thermal fluctuations, deforested areas, and mountain ranges as dispersal barriers. Conservation actions such as restricting long-distance colony transportation, preserving local adaptations, and improving the connectivity between highlands and lowlands are likely to assure future pollination services.

Climate change impact on ecosystem functions provided by birds in southeastern Amazonia.

Although the impacts of climate change on biodiversity are increasing worldwide, few studies have attempted to forecast these impacts on Amazon Tropical Forest. In this study, we estimated the impact of climate change on Amazonian avian assemblages considering range shifts, species loss, vulnerability of ecosystem functioning, future effectiveness of current protected areas and potential climatically stable areas for conservation actions. Species distribution modelling based on two algorithms and three different scenarios of climate change was used to forecast 501 avian species, organized on main ecosystem functions (frugivores, insectivores and nectarivores) for years 2050 and 2070. Considering the entire study area, we estimated that between 4 and 19% of the species will find no suitable habitat. Inside the currently established protected areas, species loss could be over 70%. Our results suggest that frugivores are the most sensitive guild, which could bring consequences on seed dispersal functions and on natural regeneration. Moreover, we identified the western and northern parts of the study area as climatically stable. Climate change will potentially affect avian assemblages in southeastern Amazonia with detrimental consequences to their ecosystem functions. Information provided here is essential to conservation practitioners and decision makers to help on planning their actions.

Mapping and quantification of ferruginous outcrop savannas in the Brazilian Amazon: A challenge for biodiversity conservation.

The eastern Brazilian Amazon contains many isolated ferruginous savanna ecosystem patches (locally known as 'canga vegetation') located on ironstone rocky outcrops on the top of plateaus and ridges, surrounded by tropical rainforests. In the Carajás Mineral Province (CMP), these outcrops contain large iron ore reserves that have been exploited by opencast mining since the 1980s. The canga vegetation is particularly impacted by mining, since the iron ores that occur are associated with this type of vegetation and currently, little is known regarding the extent of canga vegetation patches before mining activities began. This information is important for quantifying the impact of mining, in addition to helping plan conservation programmes. Here, land cover changes of the Canga area in the CMP are evaluated by estimating the pre-mining area of canga patches and comparing it to the actual extent of canga patches. We mapped canga vegetation using geographic object-based image analysis (GEOBIA) from 1973 Landsat-1 MSS, 1984 and 2001 Landsat-5 TM, and 2016 Landsat-8 OLI images, and found that canga vegetation originally occupied an area of 144.2 km2 before mining exploitation. By 2016, 19.6% of the canga area was lost in the CMP due to conversion to other land-use types (mining areas, pasturelands). In the Carajás National Forest (CNF), located within the CMP, the original canga vegetation covered 105.2 km2 (2.55% of the CNF total area), and in 2016, canga vegetation occupied an area of 77.2 km2 (1.87%). Therefore, after more than three decades of mineral exploitation, less than 20% of the total canga area was lost. Currently, 21% of the canga area in the CMP is protected by the Campos Ferruginosos National Park. By documenting the initial extent of canga vegetation in the eastern Amazon and the extent to which it has been lost due to mining operations, the results of this work are the first step towards conserving this ecosystem.

Genetic Variability of Melipona subnitida (Hymenoptera: Apidae) in Introduced and Native Populations.

Melipona subnitida (Hymenoptera: Apidae) is a stingless bee native to Caatinga biome in Brazil, well adapted to hot and dry climate of that region and has been traditionally explored for honey production. Here, we evaluate the genetic structure of 173 colonies of M. subnitida in northeast Brazil by partially sequenced mitochondrial genes cytochrome oxidase I (COI) to compare an introduced population isolated for 30 yr into the Island of Fernando de Noronha (IFN) with the continental populations. We identified high haplotype diversity (0.8220) with 14 haplotypes on the continental populations, being three new ones, compared with the database GenBank. The haplotype H4 was present at the center of network, occurring in four localities on mainland and fixed as a single haplotype on IFN. We propose that the island populations originally introduced carried one haplotype (H4), even though IFN population is suffering pressure by island effect through changes on morphology. Studies on island populations could be a model to understand the dynamics of isolated populations and sustainable management of this biome to preserve M. subnitida.

Landscape Genomic Conservation Assessment of a Narrow-Endemic and a Widespread Morning Glory From Amazonian Savannas.

Although genetic diversity ultimately determines the ability of organisms to adapt to environmental changes, conservation assessments like the widely used International Union for Conservation of Nature (IUCN) Red List Criteria do not explicitly consider genetic information. Including a genetic dimension into the IUCN Red List Criteria would greatly enhance conservation efforts, because the demographic parameters traditionally considered are poor predictors of the evolutionary resilience of natural populations to global change. Here we perform the first genomic assessment of genetic diversity, gene flow, and patterns of local adaptation in tropical plant species belonging to different IUCN Red List Categories. Employing RAD-sequencing we identified tens of thousands of single-nucleotide polymorphisms in an endangered narrow-endemic and a least concern widespread morning glory (Convolvulaceae) from Amazonian savannas, a highly threatened and under-protected tropical ecosystem. Our results reveal greater genetic diversity and less spatial genetic structure in the endangered species. Whereas terrain roughness affected gene flow in both species, forested and mining areas were found to hinder gene flow in the endangered plant. Finally we implemented environmental association tests and genome scans for selection, and identified a higher proportion of candidate adaptive loci in the widespread species. These mainly contained genes related to pathogen resistance and physiological adaptations to life in nutrient-limited environments. Our study emphasizes that IUCN Red List Criteria do not always prioritize species with low genetic diversity or whose genetic variation is being affected by habitat loss and fragmentation, and calls for the inclusion of genetic information into conservation assessments. More generally, our study exemplifies how landscape genomic tools can be employed to assess the status, threats and adaptive responses of imperiled biodiversity.

Diploid Male Production Results in Queen Death in the Stingless Bee Scaptotrigona depilis.

As in most Hymenoptera, the eusocial stingless bees (Meliponini) have a complementary sex determination (CSD) system. When a queen makes a "matched mating" with a male that shares a CSD allele with her, half of their diploid offspring are diploid males rather than females. Matched mating imposes a cost, since diploid male production reduces the colony workforce. Hence, adaptations preventing the occurrence or attenuating its effects are likely to arise. Here we provide clear evidence that in the stingless bee Scaptotrigona depilis, the emergence of diploid males induces queen death, and this usually occurs within 10-20 days of the emergence of diploid male offspring from their pupae. Queens that have not made a matched mating die when introduced into a colony in which diploid males are emerging. This shows that the adult diploid males, and not the queen that has made a matched mating herself, are the proximate cause of queen death. Analysis of the cuticular hydrocarbon profiles of adult haploid and diploid males shows six compounds with significant differences. Moreover, the diploid and haploid males only acquire distinct cuticular hydrocarbon profiles 10 days after emergence. Our data shows that the timing of queen death occurs when the cuticular hydrocarbons of haploid and diploid males differ significantly, suggesting that these chemical differences could be used as cues or signals to trigger queen death.

Reconciling Mining with the Conservation of Cave Biodiversity: A Quantitative Baseline to Help Establish Conservation Priorities.

Caves pose significant challenges for mining projects, since they harbor many endemic and threatened species, and must therefore be protected. Recent discussions between academia, environmental protection agencies, and industry partners, have highlighted problems with the current Brazilian legislation for the protection of caves. While the licensing process is long, complex and cumbersome, the criteria used to assign caves into conservation relevance categories are often subjective, with relevance being mainly determined by the presence of obligate cave dwellers (troglobites) and their presumed rarity. However, the rarity of these troglobitic species is questionable, as most remain unidentified to the species level and their habitats and distribution ranges are poorly known. Using data from 844 iron caves retrieved from different speleology reports for the Carajás region (South-Eastern Amazon, Brazil), one of the world's largest deposits of high-grade iron ore, we assess the influence of different cave characteristics on four biodiversity proxies (species richness, presence of troglobites, presence of rare troglobites, and presence of resident bat populations). We then examine how the current relevance classification scheme ranks caves with different biodiversity indicators. Large caves were found to be important reservoirs of biodiversity, so they should be prioritized in conservation programs. Our results also reveal spatial autocorrelation in all the biodiversity proxies assessed, indicating that iron caves should be treated as components of a cave network immersed in the karst landscape. Finally, we show that by prioritizing the conservation of rare troglobites, the current relevance classification scheme is undermining overall cave biodiversity and leaving ecologically important caves unprotected. We argue that conservation efforts should target subterranean habitats as a whole and propose an alternative relevance ranking scheme, which could help simplify the assessment process and channel more resources to the effective protection of overall cave biodiversity.

Beekeeping practices and geographic distance, not land use, drive gene flow across tropical bees.

Across the globe, wild bees are threatened by ongoing natural habitat loss, risking the maintenance of plant biodiversity and agricultural production. Despite the ecological and economic importance of wild bees and the fact that several species are now managed for pollination services worldwide, little is known about how land use and beekeeping practices jointly influence gene flow. Using stingless bees as a model system, containing wild and managed species that are presumed to be particularly susceptible to habitat degradation, here we examine the main drivers of tropical bee gene flow. We employ a novel landscape genetic approach to analyse data from 135 populations of 17 stingless bee species distributed across diverse tropical biomes within the Americas. Our work has important methodological implications, as we illustrate how a maximum-likelihood approach can be applied in a meta-analysis framework to account for multiple factors, and weight estimates by sample size. In contrast to previously held beliefs, gene flow was not related to body size or deforestation, and isolation by geographic distance (IBD) was significantly affected by management, with managed species exhibiting a weaker IBD than wild ones. Our study thus reveals the critical importance of beekeeping practices in shaping the patterns of genetic differentiation across bee species. Additionally, our results show that many stingless bee species maintain high gene flow across heterogeneous landscapes. We suggest that future efforts to preserve wild tropical bees should focus on regulating beekeeping practices to maintain natural gene flow and enhancing pollinator-friendly habitats, prioritizing species showing a limited dispersal ability.

Worldwide Alien Invasion: A Methodological Approach to Forecast the Potential Spread of a Highly Invasive Pollinator.

The ecological impacts of alien species invasion are a major threat to global biodiversity. The increasing number of invasion events by alien species and the high cost and difficulty of eradicating invasive species once established require the development of new methods and tools for predicting the most susceptible areas to invasion. Invasive pollinators pose serious threats to biodiversity and human activity due to their close relationship with many plants (including crop species) and high potential competitiveness for resources with native pollinators. Although at an early stage of expansion, the bumblebee species Bombus terrestris is becoming a representative case of pollinator invasion at a global scale, particularly given its high velocity of invasive spread and the increasing number of reports of its impacts on native bees and crops in many countries. We present here a methodological framework of habitat suitability modeling that integrates new approaches for detecting habitats that are susceptible to Bombus terrestris invasion at a global scale. Our approach did not include reported invaded locations in the modeling procedure; instead, those locations were used exclusively to evaluate the accuracy of the models in predicting suitability over regions already invaded. Moreover, a new and more intuitive approach was developed to select the models and evaluate different algorithms based on their performance and predictive convergence. Finally, we present a comprehensive global map of susceptibility to Bombus terrestris invasion that highlights priority areas for monitoring.

Safeguarding Ecosystem Services: A Methodological Framework to Buffer the Joint Effect of Habitat Configuration and Climate Change.

Ecosystem services provided by mobile agents are increasingly threatened by the loss and modification of natural habitats and by climate change, risking the maintenance of biodiversity, ecosystem functions, and human welfare. Research oriented towards a better understanding of the joint effects of land use and climate change over the provision of specific ecosystem services is therefore essential to safeguard such services. Here we propose a methodological framework, which integrates species distribution forecasts and graph theory to identify key conservation areas, which if protected or restored could improve habitat connectivity and safeguard ecosystem services. We applied the proposed framework to the provision of pollination services by a tropical stingless bee (Melipona quadrifasciata), a key pollinator of native flora from the Brazilian Atlantic Forest and important agricultural crops. Based on the current distribution of this bee and that of the plant species used to feed and nest, we projected the joint distribution of bees and plants in the future, considering a moderate climate change scenario (following IPPC). We then used this information, the bee's flight range, and the current mapping of Atlantic Forest remnants to infer habitat suitability and quantify local and regional habitat connectivity for 2030, 2050 and 2080. Our results revealed north to south and coastal to inland shifts in the pollinator distribution during the next 70 years. Current and future connectivity maps unraveled the most important corridors, which if protected or restored, could facilitate the dispersal and establishment of bees during distribution shifts. Our results also suggest that coffee plantations from eastern São Paulo and southern Minas Gerais States could suffer a pollinator deficit in the future, whereas pollination services seem to be secured in southern Brazil. Landowners and governmental agencies could use this information to implement new land use schemes. Overall, our proposed methodological framework could help design novel conservational and agricultural practices that can be crucial to conserve ecosystem services by buffering the joint effect of habitat configuration and climate change.

Monogamy in large bee societies: a stingless paradox.

High genetic diversity is important for the functioning of large insect societies. Across the social Hymenoptera (ants, bees, and wasps), species with the largest colonies tend to have a high colony-level genetic diversity resulting from multiple queens (polygyny) or queens that mate with multiple males (polyandry). Here we studied the genetic structure of Trigona spinipes, a stingless bee species with colonies an order of magnitude larger than those of polyandrous honeybees. Genotypes of adult workers and pupae from 43 nests distributed across three Brazilian biomes showed that T. spinipes colonies are usually headed by one singly mated queen. Apart from revealing a notable exception from the general incidence of high genetic diversity in large insect societies, our results reinforce previous findings suggesting the absence of polyandry in stingless bees and provide evidence against the sperm limitation hypothesis for the evolution of polyandry. Stingless bee species with large colonies, such as T. spinipes, thus seem promising study models to unravel alternative mechanisms to increase genetic diversity within colonies or understand the adaptive value of low genetic diversity in large insect societies.

A morphologically specialized soldier caste improves colony defense in a neotropical eusocial bee.

Division of labor among workers is common in insect societies and is thought to be important in their ecological success. In most species, division of labor is based on age (temporal castes), but workers in some ants and termites show morphological specialization for particular tasks (physical castes). Large-headed soldier ants and termites are well-known examples of this specialization. However, until now there has been no equivalent example of physical worker subcastes in social bees or wasps. Here we provide evidence for a physical soldier subcaste in a bee. In the neotropical stingless bee Tetragonisca angustula, nest defense is performed by two groups of guards, one hovering near the nest entrance and the other standing on the wax entrance tube. We show that both types of guards are 30% heavier than foragers and of different shape; foragers have relatively larger heads, whereas guards have larger legs. Low variation within each subcaste results in negligible size overlap between guards and foragers, further indicating that they are distinct physical castes. In addition, workers that remove garbage from the nest are of intermediate size, suggesting that they might represent another unrecognized caste. Guards or soldiers are reared in low but sufficient numbers (1-2% of emerging workers), considering that <1% usually perform this task. When challenged by the obligate robber bee Lestrimelitta limao, an important natural enemy, larger workers were able to fight for longer before being defeated by the much larger robber. This discovery opens up opportunities for the comparative study of physical castes in social insects, including the question of why soldiers appear to be so much rarer in bees than in ants or termites.