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Dickeya dianthicola has caused an outbreak of blackleg and soft rot of potato in the eastern half of the United States since 2015. To investigate genetic diversity of the pathogen, a comparative analysis was conducted on genomes of D. dianthicola strains. Whole genomes of 16 strains from the United States outbreak were assembled and compared with 16 previously sequenced genomes of D. dianthicola isolated from potato or carnation. Among the 32 strains, eight distinct clades were distinguished based on phylogenomic analysis. The outbreak strains were grouped into three clades, with the majority of the strains in clade I. Clade I strains were unique and homogeneous, suggesting a recent incursion of this strain into potato production from alternative hosts or environmental sources. The pangenome of the 32 strains contained 6,693 genes, 3,377 of which were core genes. By screening primary protein subunits associated with virulence from all U.S. strains, we found that many virulence-related gene clusters, such as plant cell wall degrading enzyme genes, flagellar and chemotaxis related genes, two-component regulatory genes, and type I/II/III secretion system genes, were highly conserved but that type IV and type VI secretion system genes varied. The clade I strains encoded two clusters of type IV secretion systems, whereas the clade II and III strains encoded only one cluster. Clade I and II strains encoded one more VgrG/PAAR spike protein than did clade III. Thus, we predicted that the presence of additional virulence-related genes may have enabled the unique clade I strain to become predominant in the U.S. outbreak.

Macroevolutionary studies frequently emphasize the importance of pollinator shifts for driving speciation within the angiosperms. Pollinators have been identified as a driver of diversification in the mega-diverse genus Erica in the Cape Floristic Region of South Africa, but basic information on pollinator-driven divergence at the initial stages of speciation in this genus is lacking. We focus on two populations of Erica junonia (var. junonia and var. minor) that occur on adjacent mountain ranges separated by c. 10 km and which differ in floral tube length, and ask whether these differences in tube length are associated with a shift between pollinators with different proboscis lengths. In addition, we assess whether the two forms differ in their ability to reproduce in the absence of pollinators. Our findings show that different Erica junonia varieties are visited by different nemestrinid fly species in the genus Moegistorhynchus, and that their floral tube lengths correspond to the proboscis lengths of these flies. Pollinator exclusion experiments show that the short-tubed var. minor is capable of producing seeds in the absence of pollinators, whereas the long-tubed var. junonia depends fully on pollinators. Our study provides an example of pollinator-driven divergence on a small spatial scale and contributes to an understanding of diversification of the genus Erica.

An outbreak of blackleg and soft rot of potato, caused primarily by the bacterial pathogen Dickeya dianthicola, has resulted in significant economic losses in the northeastern United States since 2015. The spread of this seedborne disease is highly associated with seed distribution; therefore, the pathogen likely spread with seed tubers. To describe the blackleg epidemic and track inoculum origins, a total of 1,183 potato samples were collected from 11 states associated with blackleg outbreak from 2015 to 2019. Of these samples, 39.8% tested positive for D. dianthicola. Seventeen isolates of D. dianthicola were recovered from these samples and the genetic diversity of these isolates was examined. Fingerprinting with BOX-A1R-based repetitive extragenic palindromic PCR and phylogenetic analysis based on sequences of the 16S rRNA and gapA genes indicated that D. dianthicola isolates were divided into three genotypes, denoted types I, II, and III. Ninety-five percent of samples from Maine were type I. Type II was found in Maine only in 2015 and 2018. Type II was present throughout the 5 years in some states at a lower percentage than type I. Type III was found in Pennsylvania, New Jersey, and Massachusetts, but not in Maine. Therefore, type I appears to be associated with Maine, but type II appeared to be distributed throughout the northeastern United States. The type II and rarer type III strains were closer to the D. dianthicola type strain isolated from the United Kingdom. This work provides evidence that the outbreak of blackleg of potato in the northeastern United States was caused by multiple strains of D. dianthicola. The geographic origins of these strains remain unknown.

Background The universal nature of the human–companion animal relationship and their shared ticks and tick-borne pathogens offers an opportunity for improving public and veterinary health surveillance. With this in mind, we describe the spatiotemporal trends for blacklegged tick (Ixodes scapularis) submissions from humans and companion animals in Ontario, along with pathogen prevalence. Methods We tested tick samples submitted through passive surveillance (2011–2017) from humans and companion animals for Borrelia burgdorferi, Borrelia miyamotoi, Anaplasma phagocytophilum and Babesia microti. We describe pathogen prevalence in ticks from humans and from companion animals and constructed univariable Poisson and negative binomial regression models to explore the spatiotemporal relationship between the rates of tick submissions by host type. Results During the study, there were 17,230 blacklegged tick samples submitted from humans and 4375 from companion animals. Tick submission rates from companion animals were higher than expected in several public health units (PHUs) lacking established tick populations, potentially indicating newly emerging populations. Pathogen prevalence in ticks was higher in PHUs where established blacklegged tick populations exist. Borrelia burgdorferi prevalence was higher in ticks collected from humans (maximum likelihood estimate, MLE = 17.5%; 95% confidence interval, CI 16.97–18.09%) than from companion animals (9.9%, 95% CI 9.15–10.78%). There was no difference in pathogen prevalence in ticks by host type for the remaining pathogens, which were found in less than 1% of tested ticks. The most common co-infection B. burgdorferi + B. miyamotoi occurred in 0.11% of blacklegged ticks from humans and animals combined. Borrelia burgdorferi prevalence was higher in unengorged (21.9%, 95% CI 21.12–22.65%) than engorged ticks (10.0%, 95% CI 9.45–10.56%). There were no consistent and significant spatiotemporal relationships detected via regression models between the annual rates of submission of each host type. Conclusions While B. burgdorferi has been present in blacklegged ticks in Ontario for several decades, other tick-borne pathogens are also present at low prevalence. Blacklegged tick and pathogen surveillance data can be used to monitor risk in human and companion animal populations, and efforts are under consideration to unite surveillance efforts for the different target populations. Graphical Abstract

Experimental studies of the use of visual and olfactory cues by flower-visiting animals can shed light on the evolution of floral signalling traits. We examined the functional significance of floral traits in Clivia miniata (Amaryllidaceae). This forest lily with large orange trumpet-shaped flowers is pollinated mainly by swallowtail butterflies and belongs to a lineage with ancestral bird pollination. We used C. miniata flowers varying in colour, orientation and scent, and arrays of artificial flowers varying in colour, pattern, orientation, size, shape, and scent to assess foraging preferences of the butterflies that pollinate C. miniata. Butterflies preferred orange over yellow colour morphs of C. miniata and preferred red and orange model flowers over yellow ones. Orange models with a central yellow target ‘nectar guide’ were favoured over plain orange models. Butterflies also favoured large over small model flowers and preferred to alight on upward-facing flowers. Addition of scent compounds emitted by C. miniata flowers increased butterfly visitation to model and natural flowers. These results identify the importance of particular combinations of visual and olfactory signals for attraction of swallowtail butterflies and shed light on the floral modifications associated with a shift from bird to butterfly pollination.

Premise of research. In hermaphroditic plants, pollen production can enhance fitness through siring success by attracting pollen-seeking pollinators. However, pollen production can also decrease fe...

Faecal mimicry by seeds is a recently discovered phenomenon in plants. Here we demonstrate that seeds of Ceratocaryum pulchrum (Restionaceae) are dispersed and buried by dung beetles, making it the second species, after its sister species C. argenteum, known to exhibit this mode of dispersal. Seeds of C. pulchrum are similar in size, shape and scent to those of C. argenteum and the dung of various mammalian herbivores. The distribution range of C. pulchrum entirely overlaps with that of C. argenteum, but they flower and release seeds six months apart. Several dung beetle species, including both of those associated with C. argenteum, were found to be attracted to C. pulchrum seeds. Seeds were buried in situ by the tunnelling species Copris victorini. Since seeds of both plant species attract several generalist dung beetles, this suggests that speciation in these two species has not been due to evolution for different or specific dispersers but may instead be related to exploitation of temporal niche opportunities.

Premise of research. Breeding systems dictate the potential for self-fertilization in plants and may therefore have a strong influence on evolutionary and ecological processes. Previous investigati...

Potato virus Y (PVY) is one of the main viruses affecting potato in Australia. However, molecular characterization of PVY isolates circulating in potato in different states of Australia has not yet been thoroughly conducted. Only nonrecombinant isolates of three biological PVY strains collected from potato were reported previously from Western Australia and one from Queensland. Here, PVY isolates collected from seed potato originating in Victoria, Australia, and printed on FTA cards, were subjected to strain typing by RT-PCR, with three isolates subjected to whole genome sequencing. All the 59 PVY isolates detected during two growing seasons were identified to be recombinants based on two RT-PCR assays. No nonrecombinant PVY isolates were identified. All the RT-PCR typed isolates belonged to the PVYNTN strain. Sequence analysis of the whole genomes of three isolates suggested a single introduction of the PVYNTN strain to Australia but provided no clues as to where this introduction originated. Given the association of the PVYNTN strain with potato tuber damage, growers in Australia should implement appropriate strategies to manage PVYNTN in potato.

Mutualisms between nectarivorous birds and the plants they pollinate are functionally diverse. Nectarivorous birds which hover while feeding (the majority of hummingbirds) tend to have straight bills, while those that perch while feeding (some hummingbirds and almost all passerine nectarivores) tend to have decurved bills. Sunbirds typically use their curved bills to feed in an arc from a perching position and we thus predicted that they would prefer, and feed most efficiently on, flowers that are curved towards a perching position. To test this, we examined the responses of sunbirds to model flowers differing in curvature (straight or curved), orientation (facing upwards or downwards), and availability of a top perch (present or absent). Birds did not show preferences among model flower types in terms of number of landings to feed or number of probes. In general they preferred to use perches above model flowers, particularly those that curved upwards, but they tended to perch below model flowers that curve downwards and in such cases also took the least time to insert their bills. These results are consistent with the idea that perching birds with curved beaks will feed most efficiently from flowers that are curved towards the perching position. We discuss the implications of these results for the evolution of floral architecture, including provision of perches.

Objectives The nested variant of urothelial carcinoma (NVUC) is a rare bladder tumor that may possess a luminal molecular phenotype. We sought to determine whether a small immunohistochemical (IHC) panel using common surrogates for molecular phenotypes would reliably classify a cohort of pure NVUC cases. Methods IHC staining with a panel composed of markers for basal subtypes (CK5/6, CK14) and luminal subtypes (FOXA1, GATA3) was performed on pure small NVUC cases (n = 23) and 5 large NVUC cases (n = 5). Scoring of IHC stains was performed semiquantitatively. Individual cases were analyzed using previously reported IHC-based surrogates for molecular subtype. Results The phenotype of NVUC was classified as luminal from 60.1% (FOXA1+/CK5/6−) to 100% (GATA3+/CK14−) of cases using composite phenotypes. No cases possessed a basal or squamous cell carcinoma–like phenotype. The majority of small NVUC cases (69.5%) showed subset CK5/6 expression distinctly localized to the basal layers of tumor cell nests. Intratumoral heterogeneity was also noted in CK5/6 (21.7% of small NVUC cases) but no other markers. Conclusions NVUC appears to express markers of both basal and luminal bladder tumors. Definitive gene expression profiling may be valuable to further characterize this unique histologic variant.

Geographical variation in pollinator assemblages can result in divergent selective pressures and consequently drive the evolution of phenotypically distinct pollination ecotypes. Intraspecific divergence in pollination systems is well known among angiosperms, but is poorly studied among gymnosperms, often considered a relictual group not undergoing recent radiations. We investigated whether geographical divergence in the chemistry of volatiles emitted by cones of the cycad Encephalartos ghellinckii can be linked with functions for attracting different pollinators. We tested the pollination effectiveness of different insects in terms of deposition of fluorescent dye on micropyles and investigated whether daily fluctuations in cone temperature are correlated with insect pollinator activity. We also performed field bioassays to test the responses of insects to key volatiles and laboratory y-maze experiments to determine whether insects discriminate among cones of the two forms. Experiments confirmed that the beetles Metacucujus goodei and Erotylidae sp. nov. are the most effective pollinators of the mountain and lowland forms, respectively. A peak in cone temperatures in the afternoon was associated with extensive movement of insects among cones of mountain plants, but only limited movement of insects among cones of lowland plants. In field bioassays, Metacucujus goodei beetles were attracted mainly to (3E)-1,3-octadiene, a dominant component of the scent of mountain plants, whereas Erotylidae sp. nov. responded to camphene, a dominant component of the scent of lowland plants. When offered a choice, beetles preferred cones from the local population over those from a different population. The results suggest that E. ghellinckii consists of two pollination ecotypes with modifications in scent chemistry being correlated with an apparent shift between different pollinators. The relationship between thermogenesis and pollinator activities varies between ecotypes, with mountain plants showing a push–pull type of system and the lowland plants a system in which insects depart from cones with declining quality. This study highlights the role of insect pollinators in driving the evolution of cycads and the importance of thermogenesis and volatile emissions for mediating some beetle pollination systems.

Long-proboscid nemestrinid flies are keystone pollinators of dozens of Southern African plants and, consequently, their taxonomic status might have important consequences for insect and plant conservation. We focus on Prosoeca peringueyi, considered to be a single, morphologically variable species, upon which a guild of ~28 plants in the winter rainfall region depends for pollination. We quantified morphological variation and established whether it was associated with genetic variation within and among sites. Phylogenetic analyses of the mitochondrial COI gene revealed two well-supported clades. One clade contains long-proboscid individuals that conform morphologically to the holotype of P. peringueyi. The sister clade contains individuals that frequently occur sympatrically with P. peringueyi and have shorter proboscides, with additional diagnostic characters that set it apart from P. peringueyi. A haplotype analysis based on nuclear ribosomal 28S DNA sequences of a subset of individuals corroborated these results. Based on our results, we propose the recognition of two species: P. peringueyi and Prosoeca torquata sp. nov., which is described here. Future research is required to quantify the interaction networks of these two fly species and the plant guilds with which they interact, to facilitate conservation in the global biodiversity hotspot where they occur.

Taxa relegated to synonymy with other superficially similar taxa based on herbarium-based taxonomy may be distinct species that are only ‘rediscovered’ on the collection of new, multi-disciplinary evidence. Here we investigate the case of Satyrium jacottetiae that has been included in the concept of S. membranaceum (Orchidaceae). The two taxa share similarities in some conspicuous traits, such as the dry and membranous floral bracts, light pink labellum and laceration of the petals, but a quantitative morphometric analysis based on 23 floral traits showed that the two taxa differentiate in ordination space. We found that the taxa can be distinguished by features of the rostellum (short and broad in S. jacottetiae and elongate and notched in S. membranaceum), which are not readily apparent from examination of herbarium specimens. A phylogenetic analysis of nuclear ribosomal DNA sequences further revealed that accessions of S. jacottetiae belong to a clade that contains two other Satyrium spp. but not S. membranaceum. The two taxa have largely non-overlapping distributions on slightly different soil types, but they also maintain their integrity in the few zones of overlap despite evidence indicating that both are primarily pollinated by nocturnal moths. Coexistence in these cases of sympatry appears to be facilitated by a sequential difference in flowering times. We conclude that S. jacottetiae and S. membranaceum are morphologically, phylogenetically, geographically and ecologically distinct and should therefore be recognized as separate species. This study highlights the importance of multi-disciplinary taxonomic effort to avoid under-assessment of biodiversity.

Background and Aims Complex modifications of angiosperm flowers often function for precise pollen placement on pollinators and to promote cross-pollination. We explore the functional significance of the unusually elaborate morphology of Gloriosa superba flowers, which are divided into one hermaphrodite meranthium and five male meranthia (functional pollination units of a single flower). Methods We used controlled pollination experiments, floral measurements, pollen load analyses and visitor observations in four populations of G. superba in South Africa to determine the breeding system, mechanism of pollination and role of flower in the promotion of cross-pollination. Key Results We established that G. superba is self-compatible, but reliant on pollinators for seed production. Butterflies, in particular the pierid Eronia cleodora, were the primary pollinators (>90 % of visitors). Butterflies brush against the anthers and stigma during nectar feeding and pollen is carried on their ventral wing surfaces. Butterfly scales were positively correlated with the number of pollen grains on stigmas. We demonstrate that the styles were orientated towards clearings in the vegetation and we confirm that the highest proportion of initial visits was to hermaphrodite meranthia pointing towards clearings. Conclusions The flower morphology of G. superba results in effective pollen transfer on the wings of butterfly visitors. The style-bearing hermaphrodite meranthium of the flowers orientates towards open spaces in the vegetation, thus increasing the probability that butterflies land first on the hermaphrodite meranthium. This novel aspect of flower orientation is interpreted as a mechanism that promotes cross-pollination.

Understanding the evolution of floral morphology requires information about the identity of pollinators as well as the specific mechanisms of pollen transfer. Based on preliminary field observations and floral structure, we hypothesized that pollination mechanisms involving the transfer of pollen on butterfly wings occur in several lineages of South African Amaryllidaceae. Here we report findings from a detailed study of butterfly-wing pollination in two subspecies of Scadoxus multiflorus and review the prevalence of this pollination mechanism among other Amaryllidaceae in southern Africa. We established that S. multiflorus subsp. katherinae is genetically self-incompatible and thus entirely reliant on pollinators for seed production. We determined that this subspecies is pollinated almost exclusively by large swallowtail butterflies, principally males of the mocker swallowtail Papilio dardanus cenea. Flowers of S. multiflorus subsp. multiflorus are pollinated by pierid and swallowtail butterflies. Pollen is deposited on the ventral surface of the wings of butterflies as they flutter over the strongly exserted stamens. We predict that butterfly-wing pollination occurs in at least nine species of South African Amaryllidaceae, which may reflect several independent origins of this mechanism. The flowers of these species are red or orange with strong herkogamy and are either bowl-brush or open-brush in shape. We provide maps of the distribution of pollen on the ventral surface of the wings of pollinators for four of these species. All four appear to be pollinated via the ventral surface of large butterfly wings, with the floral structure facilitating the process. These findings illustrate the importance of investigating pollen transfer mechanisms in order to understand patterns of floral diversification and floral convergence.

We discuss the modification of an old method of connecting two capillary columns in a confluent configuration which elegantly allows introduction of make‐up gas at a controlled flow rate when the effluent is split for electroantennographic detection experiments, and allows for easy switching of the stationary phase between successive GC runs. Being able to run samples on different phases is extremely beneficial, both for optimal resolution of active compounds and for compound identification. This method warrants greater consideration by GC‐EAD practitioners.

We reported recently that activators of AMP-activated protein kinase (AMPK) slow the rundown of current evoked by the D2 autoreceptor agonist quinpirole in rat substantia nigra compacta (SNC) dopamine neurons. The present study examined the effect of AMPK on current generated by dopamine, which unlike quinpirole, is a substrate for the dopamine transporter (DAT). Using whole-cell patch-clamp, we constructed current–voltage (I–V) plots while superfusing brain slices with dopamine (100 μM) for 25 min. Two minutes after starting superfusion, dopamine evoked a peak current with an average slope conductance of 0.97 nS and an estimated reversal potential (Erev) of −113 mV, which is near that expected for K+. But after 10 min of superfusion, dopamine-evoked currents had shifted to more depolarized values with a slope conductance of 0.64 nS and an Erev of −83 mV. This inward shift in current was completely blocked by the DAT inhibitor GBR12935. However, an AMPK blocking agent (dorsomorphin) permitted the emergence of inward current despite the continued presence of the DAT inhibitor. When D2 autoreceptors were blocked by sulpiride, I–V plots showed that dopamine evoked an inward current with an estimated slope conductance of 0.45 nS with an Erev of −57 mV. Moreover, this inward current was completely blocked by the trace amine-associated receptor 1 (TAAR1) antagonist EPPTB. These results suggest that dopamine activates a TAAR1-dependent non-selective cation current that is regulated by AMPK.

Lymphoepithelioma-like carcinoma of the bladder (LELC-B) is a rare subtype of urothelial carcinoma consisting of undifferentiated epithelial cells within a dense inflammatory cell infiltrate. We set out to molecularly characterize LELC-B through RNA expression profiling as well as immunohistochemistry (IHC) to understand its underlying biology. Sixteen cases of LELC-B were identified at Johns Hopkins University. RNA sequencing was performed on 14 cases. IHC staining for programmed cell death ligand 1 (PD-L1) and mismatch repair proteins MutL homolog 1 (MLH1), MutS homolog 2 (MSH2), MSH6, and PMS1 homolog, mismatch repair system component 2 (PMS2) was performed. Transcriptomic profiling of LELC-B showed that they are enriched in a basal-like phenotype, with 12 of 14 LELC-B cases correlating to the basal centroid of the bladder cancer analysis of subtypes by gene expression 47 (BASE47) predictive analysis of microarrays (PAM) classifier. Gene signature analysis confirmed the lymphocyte infiltration profile consistent with the histomorphology. LELC-B lacked features to explain the robust lymphocytic infiltrate, such as loss of mismatch repair protein expression or expression of Epstein-Barr virus transcripts. Nonetheless, PD-L1 IHC was positive in 93% of LELC cases. Our study demonstrates that LELC-B tumors are enriched in a basal-like molecular subtype and share a high level of immune infiltration and PD-L1 expression, similar to basal tumors. The basal-like phenotype is consistent with the known sensitivity of LELC-B to chemotherapy and suggests that immune checkpoint therapy should be explored in this rare disease.

Florivores and rainfall generally have negative impacts on plant fecundity. However, in some cases, they can mediate fruit set. Some plants face severe pollen-limited fecundity and any additional fruit set, even if from self-pollination, can be advantageous. This is the case in some tropical deceptive orchids, such as the threatened Cyrtopodium hatschbachii. Here we test the hypothesis that florivory of the anther cap would facilitate rain-assisted autogamy in this species. In the field, we followed flowers in which the anther cap was removed by the orthopteran Stenopola sp. and found cases where pollinia self-deposited after rainfall and in one case this resulted in swelling of the column typical of fruit development. This event comprised 33% of all fruit set in the population in 2019. We then experimentally varied anther cap removal and rainfall in a factorial design and found increased fruit set in the group with cap removal (simulated florivory) followed by rain. The water absorption by pollinia makes them heavier, causing the stipe to bend. The droplet of water on the stigma then shrinks and pulls the pollinia back onto the stigma, causing self-pollination. Seeds from self-pollination have considerable viability and may allow population persistence, given that bee-mediated cross-pollination is uncertain and even absent in some years. Our study provides a unique example of how two unrelated factors (i.e., florivory and rain) that are detrimental alone may together promote fruit set.

An outbreak of bacterial soft rot and blackleg of potato has occurred since 2014 with the epicenter being in the northeastern region of the United States. Multiple species of Pectobacterium and Dickeya are causal agents, resulting in losses to commercial and seed potato production over the past decade in the Northeastern and North Central United States. To clarify the pathogen present at the outset of the epidemic in 2015 and 2016, a phylogenetic study was made of 121 pectolytic soft rot bacteria isolated from symptomatic potato, also included were 27 type strains of Dickeya and Pectobacterium species, and 47 historic reference strains. Phylogenetic trees constructed based on multilocus sequence alignments of concatenated dnaJ, dnaX and gyrB fragments revealed the epidemic isolates to cluster with type strains of D. chrysanthemi, D. dianthicola, D. dadantii, P. atrosepticum, P. brasiliense, P. carotovorum, P. parmentieri, P. polaris, P. punjabense, and P. versatile. Genetic diversity within D. dianthicola strains was low, with one sequence type (ST1) identified in 17 of 19 strains. Pectobacterium parmentieri was more diverse, with ten sequence types detected among 37 of the 2015–2016 strains. This study can aid in monitoring future shifts in potato soft rot pathogens within the U.S. and inform strategies for disease management.

A recent claim that evening primrose flowers adaptively secrete nectar in response to vibrations from hovering bees lacks supporting evidence. The authors fail to demonstrate that bees can access the concealed nectar and that their visits enhance plant fitness. Reanalysis of the authors' data raises additional concerns about their conclusions.

Human granulocytic anaplasmosis (HGA), caused by the bacteria Anaplasma phagocytophilum, is transmitted to humans by blacklegged ticks (Ixodes scapularis) in eastern North America. To assess the emergence of A. phagocytophilum in Ontario, we analyzed patient serological and clinical data in combination with pathogen detection in blacklegged ticks from 2011 to 2017. Our sample population included all patients who had Anaplasma serological testing ordered by their physicians (n = 851). Eighty-three patients (10.8%) were A. phagocytophilum seropositive (IgG titers ≥ 1:64) and 686 (89.2%) were seronegative (IgG titers < 1:64). Applying published surveillance case definitions, we classified zero as confirmed, five as probable, and 78 as suspected cases. The percentage of seropositive patients remained generally stable at 13.6%. Seropositive patients were most often adult females, 40–59 years of age, and reported nonspecific signs and symptoms, such as fatigue, headache, and fever. Higher seropositivity rates (≥ 1.5 patients per 100,000 population) occurred in eastern and northwestern Ontario. The percentage of A. phagocytophilum-positive blacklegged ticks, through passive and active surveillance, was 0.4 and 1.1%, respectively, and increased over time. Serological and entomological indicators of A. phagocytophilum activity increased in areas of the province with established blacklegged tick populations. The risk of HGA is presently low in Ontario; however, further research is required to document the epidemiology of HGA in the province. To minimize the impact of HGA emergence in Ontario, increased awareness and education of the public and health-care providers is recommended, with consideration to making HGA a reportable infection in Ontario.

Seeds of many Amaryllidaceae are unorthodox (recalcitrant) and toxic, and cannot survive ingestion, yet are packaged in brightly colored fruits suggestive of zoochory. Seed dispersal and germination of the understory amaryllid, Clivia miniata, were investigated in KwaZulu‐Natal, South Africa. Motion‐activated cameras revealed that samango monkeys (Cercopithecus mitis labiatus) are the primary disperser of C. miniata seeds. They eat the mesocarp and, to a lesser extent, the exocarp, and spit the large (13 mm diameter) seeds whole and cleaned onto the forest floor. Most seeds were dispersed farther than 1 m from the parent. Experimental removal of the fruit pulp had a small positive effect on the rate of seed germination, but did not affect subsequent seedling growth rates. The main advantages of monkey dispersal of Clivia seeds appear to be short‐distance dispersal away from the dense foliage of clumped parent plants and occasional long‐distance dispersal through cheek‐pouching behavior.

Trade-offs are considered a major factor in the evolution of ecological specialization, as organisms can rarely specialize on more than one niche. Here we ask whether flowers can be specialized for more than one pollinator group. We studied an outcrossing plant, Aloe kraussii (Asphodelaceae), whose floral morphology is intermediate between that of exclusively bird- and exclusively insect-pollinated aloes. Previous field experiments showed that bees effectively pollinate A. kraussii; while sunbirds are frequent visitors their effectiveness as pollinators was unknown, especially as their long bills prevent head feathers—the normal location of pollen transfer by birds—from touching A. kraussii's relatively short anthers and stigmas. We tested whether sunbirds can pollinate A. kraussii, and whether this might be facilitated by flowers' narrow corolla opening, which could increase pollen transfer via narrow sunbird bills. Aviary experiments showed that a Malachite sunbird's bill can successfully pick up and deposit A. kraussii pollen, so long-billed sunbirds can be effective pollinators despite the apparent mismatch between bill and corolla length. Flowers with experimentally widened corolla openings experienced lower pollen removal and deposition by sunbirds than unmanipulated flowers with narrow openings. Aloe kraussii's floral morphology therefore enables dual specialization on two divergent pollinator guilds; short corollas facilitate bee pollination by enabling access to nectar, while narrow corolla openings facilitate pollen transfer via sunbird bills.

Current treatments for Mycobacterium tuberculosis infections require long and complicated regimens that can lead to patient non-compliance, increasing incidences of antibiotic-resistant strains, and lack of efficacy against latent stages of disease. Thus, new therapeutics are needed to improve tuberculosis standard of care. One strategy is to target protein homeostasis pathways by inhibiting molecular chaperones such as GroEL/ES (HSP60/10) chaperonin systems. M. tuberculosis has two GroEL homologs: GroEL1 is not essential but is important for cytokine-dependent granuloma formation, while GroEL2 is essential for survival and likely functions as the canonical housekeeping chaperonin for folding proteins. Another strategy is to target the protein tyrosine phosphatase B (PtpB) virulence factor that M. tuberculosis secretes into host cells to help evade immune responses. In the present study, we have identified a series of GroEL/ES inhibitors that inhibit M. tuberculosis growth in liquid culture and biochemical function of PtpB in vitro. With further optimization, such dual-targeting GroEL/ES and PtpB inhibitors could be effective against all stages of tuberculosis – actively replicating bacteria, bacteria evading host cell immune responses, and granuloma formation in latent disease – which would be a significant advance to augment current therapeutics that primarily target actively replicating bacteria.

Although moths and butterflies are closely related, they tend to respond to different floral cues for foraging and thus select for different suites of floral traits. Shifts between moth and butterfly pollination in the same plant clade provide an opportunity to assess the traits that are required to impart a shift between these pollination systems. The orchid genus Bonatea consists mostly of night-scented moth-pollinated taxa, but here we document a butterfly pollination system in Bonatea cassidea, a century after Mansel Weale's initial observations of butterflies carrying pollinaria of this taxon in South Africa were communicated to the Linnean Society by Charles Darwin. We recorded visits by a number of species of butterflies from the family Hesperiidae and Pieridae, but contrary to Weale, we found that the majority of pollinaria were firmly affixed between the palpi and not on the sternum. Removal of pollinaria from flowers during the day, but not at night, suggested that moths do not contribute to pollination, despite the white flower colouration. Floral traits found in B. cassidea but not its moth-pollinated congeners include diurnal anthesis, scent emission which is weak in the day and virtually absent at night, and the production of small amounts of relatively dilute sucrose-rich nectar in short spurs. These features are consistent with those expected for butterfly-pollinated plants in general. This study represents the first detailed report of butterfly-pollination in Bonatea and one of very few cases of orchid pollination by pierid butterflies.

Motion-activated cameras (“camera traps”) are useful for recording plant–animal interactions that are difficult to observe using conventional means. We used camera traps to document interactions between animal flower visitors and the orchid Satyrium neglectum subsp. woodii and used mesh exclosures which selectively exclude access of larger animals to flowers to test the importance of bird visits for pollination. Camera footage revealed that the orchid is visited by two sunbird species (amethyst and greater double-collared) which carry pollinaria on the upper mandible of their beaks. Plants which could only be accessed by bees and other small insects had few pollinaria removed and set very few fruits relative to plants which could also be accessed by larger animals such as sunbirds. The sturdy inflorescence stem, and thick fleshy perianth tissue, orange–red colour of the flowers, low scent production and large viscidia, are traits shared with other, distantly related bird-pollinated Satyrium species and can therefore be considered likely to play a functional role in bird pollination. We previously made anecdotal observations of sunbird visits to this orchid species in south-central Africa, and this study conducted in populations in South Africa confirms that sunbird pollination is consistent across the highly disjunct distribution of the taxon.

All living organisms contain a unique class of molecular chaperones called 60 kDa heat shock proteins (HSP60 – also known as GroEL in bacteria). While some organisms contain more than one HSP60 or GroEL isoform, at least one isoform has always proven to be essential. Because of this, we have been investigating targeting HSP60 and GroEL chaperonin systems as an antibiotic strategy. Our initial studies focused on applying this antibiotic strategy for treating African sleeping sickness (caused by Trypanosoma brucei parasites) and drug-resistant bacterial infections (in particular Methicillin-resistant Staphylococcus aureus – MRSA). Intriguingly, during our studies we found that three known antibiotics – suramin, closantel, and rafoxanide – were potent inhibitors of bacterial GroEL and human HSP60 chaperonin systems. These findings prompted us to explore what other approved drugs, natural products, and known bioactive molecules might also inhibit HSP60 and GroEL chaperonin systems. Initial high-throughput screening of 3680 approved drugs, natural products, and known bioactives identified 161 hit inhibitors of the Escherichia coli GroEL chaperonin system (4.3% hit rate). From a purchased subset of 60 hits, 29 compounds (48%) re-confirmed as selective GroEL inhibitors in our assays, all of which were nearly equipotent against human HSP60. These findings illuminate the notion that targeting chaperonin systems might be a more common occurrence than we previously appreciated. Future studies are needed to determine if the in vivo modes of action of these approved drugs, natural products, and known bioactive molecules are related to GroEL and HSP60 inhibition.

Volatile emissions may play a key role in structuring pollination systems of plants with morphologically unspecialised flowers. Here we test for pollination by small mammals in Eucomis regia and investigate whether its floral scent differs markedly from fly- and wasp-pollinated congeners and attracts mammals. We measured floral traits of E. regia and made comparisons with insect-pollinated congeners. We observed floral visitors and examined fur and faeces of live-trapped mammals for pollen. We determined the contributions of different floral visitors to seed set with selective exclusion and established the breeding system with controlled pollination experiments. Using bioassays, we examined whether mammals are attracted by the floral scent and are effective agents of pollen transfer. Eucomis regia differs from closely related insect-pollinated species mainly in floral scent, with morphology, colour and nectar properties being similar. We found that mice and elephant-shrews pollinate E. regia, which is self-incompatible and reliant on vertebrates for seed production. Mammals are strongly attracted to the overall floral scent, which contains unusual sulphur compounds, including methional (which imparts the distinctive potato-like scent and which was shown to be attractive to small mammals). The results highlight the important role of scent chemistry in shifts between insect and mammal pollination systems.

Geographical variation in flower color of a plant species may reflect the outcome of selection by pollinators or may reflect abiotic factors such as soil chemistry or neutral processes such as genetic drift. Here we document striking geographical structure in the color of capitula of the endemic South African grassland daisy Gerbera aurantiaca and ask which of these competing explanations best explains this pattern. The color of capitula ranges from predominantly red in the southwest to yellow in the center, with some northern populations showing within-population polymorphism. Hopliine scarab beetles were the most abundant flower visitors in all populations, apart from a yellow-flowered one where honeybees were frequent. In a mixed color population, yellow, orange and red morphs were equally attractive to hopliine beetles and did not differ significantly in terms of fruit set. Beetles were attracted to both red and yellow pan traps, but preferred the latter even at sites dominated by the red morph. We found no strong associations between morph color and abiotic factors, including soil chemistry. Plants in a common garden retained the capitulum color of the source population, even when grown from seed, suggesting that flower color variation is not a result of phenotypic plasticity. These results show that flower color in G. aurantiaca is geographically structured, but the ultimate evolutionary basis of this color variation remains elusive.

Despite increased focus on elucidating the various reproductive strategies employed by orchids, we still have only a rather limited understanding of deceptive pollination systems that are not bee- or wasp-mediated. In Europe, the orchid Neotinea ustulata has been known to consist of two phenologically divergent varieties, neither of which provide rewards to its pollinators. However, detailed studies of their reproductive biology have been lacking. Our study aimed to characterize and understand the floral traits (i.e., morphology, color, and scent chemistry) and reproductive biology of N. ustulata. We found that the two varieties differ in all their floral traits; furthermore, while Neotinea ustulata var. ustulata appears to be pollinated by both bees (e.g., Anthophora, Bombus) and flies (e.g., Dilophus, Tachina), var. aestivalis is pollinated almost entirely by flies (i.e., Nowickia, Tachina). Tachinids were also found to be much more effective than bees in removing pollinaria, and we show experimentally that they use the characteristic dark inflorescence top as a cue for approaching inflorescences. Our results thus suggest that while both N. ustulata varieties rely on tachinids for pollination, they differ in their degree of specialization. Further studies are, however, needed to fully understand the reproductive strategy of N. ustulata varieties., publishedVersion

Insects thermoregulate using both canalized and plastic mechanisms. Populations of insects utilize these mechanisms to different extents, and while it is posited that the degree of thermal fluctuation a population experiences can determine the optimal combination of mechanisms to utilize, this is still being elucidated. We used three populations of the generalist grasshopper, Melanoplus differentialis (Thomas, 1856), from sites experiencing different degrees of thermal heterogeneity to test for correlations between thermal heterogeneity and 1) behavioral thermoregulation, 2) upper temperature tolerance, 3) the ability to thermally acclimate, and 4) gene expression. We found that 1) behavioral thermoregulation did not differ among sites, 2) CTMax of males, but not females, was higher at more thermally heterogeneous sites, 3) there was acclimation in some of the tested traits, but thermally heterogeneous sites did not always have the most plastic individuals, and 4) there were differences in gene expression among sites, but these differences were not between the most and least thermally heterogeneous sites. We concluded that thermal heterogeneity may play a selective role in some, but not all, of the measured thermoregulatory traits and their plasticity.

Key Points We report a bilineal process with truncal U2AF1 mutation and lineage-specific secondary mutations in B-lymphoblast and myeloid populations.This case highlights an atypical mechanism of BLL leukemogenesis and illustrates the potential advantage of fractionated sequencing., Rare hematologic malignancies display evidence of both myeloid and lymphoid differentiation. Here, we describe such a novel bilineal event discovered in an adult woman with B-lymphoblastic leukemia (BLL). At the time of BLL diagnosis, the patient had a normal karyotype and a bulk sequencing panel identified pathogenic variants in BCOR, EZH2, RUNX1, and U2AF1, a genotype more typical of myeloid neoplasia. Additionally, the patient was noted to have 3-year history of cytopenias, and morphologic dyspoiesis was noted on post-treatment samples, raising the possibility of an antecedent hematologic disorder. To investigate the clonal architecture of her disease, we performed targeted sequencing on fractionated samples enriched for either B-lymphoblasts or circulating granulocytes. These studies revealed a truncal founder mutation in the spliceosome gene U2AF1 in both fractions, while distinct secondary mutations were present only in B-lymphoblasts (BCOR, NRAS) or myeloid cells (ASXL1, EZH2, RUNX1). These results indicate that both processes evolved from a common U2AF1-mutated precursor, which then acquired additional mutations during a process of divergent evolution and bilineal differentiation. Our findings highlight an atypical mechanism of BLL leukemogenesis and demonstrate the potential utility of fractionated sequencing in the characterization of acute leukemia.

The structure of a 10-kDa protein complex is one of the smallest to be determined using cryo-electron microscopy at high resolution. The structure reveals that C1-INH is sequestered in an inactivated state by burial of the reactive center loop in Vag8., Complement, contact activation, coagulation, and fibrinolysis are serum protein cascades that need strict regulation to maintain human health. Serum glycoprotein, a C1 inhibitor (C1-INH), is a key regulator (inhibitor) of serine proteases of all the above-mentioned pathways. Recently, an autotransporter protein, virulence-associated gene 8 (Vag8), produced by the whooping cough pathogen, Bordetella pertussis, was shown to bind to C1-INH and interfere with its function. Here, we present the structure of the Vag8–C1-INH complex determined using cryo-electron microscopy at a 3.6-Å resolution. The structure shows a unique mechanism of C1-INH inhibition not employed by other pathogens, where Vag8 sequesters the reactive center loop of C1-INH, preventing its interaction with the target proteases.

Dickeya and Pectobacterium spp. both cause blackleg and soft rot of potato, which can be a yield-reducing factor to potato production. The purpose of this study was to examine the interaction between these two bacterial genera causing potato infection, and subsequent disease development and yield responses under field conditions. Analysis of 883 potato samples collected in Northeastern USA using polymerase chain reaction determined that Dickeya dianthicola and P. parmentieri were found in 38.1% and 53.3% of all samples, respectively, and that 20.6% of samples contained both D. dianthicola and P. parmentieri. To further investigate the relationship between the two bacterial species and their interaction, field trials were established. Potato seed pieces of “Russet Burbank”, “Lamoka”, and “Atlantic” were inoculated with bacterial suspension of D. dianthicola at 107 colony-forming unite (CFU)/mL using a vacuum infiltration method, air dried, and then planted in the field. Two-year results showed that there was a high correlation (p &lt, 0.01) between yield loss and percent of inoculated seed pieces. In a secondary field trial conducted in 2018 and 2019, seed pieces of potato “Shepody”, “Lamoka” and “Atlantic” were inoculated with D. dianthicola, P. parmentieri, or mixture of both species, and then planted. In 2019, disease severity index, as measured by the most sensitive variety “Lamoka”, was 16.2 with D. dianthicola inoculation, 10.4 with P. parmentieri, 25.4 with inoculation with both bacteria. Two-year data had a similar trend. Thus, D. dianthicola was more virulent than P. parmentieri, but the co-inoculation of the two species resulted in increased disease severity compared to single-species inoculation with either pathogen.

The GroES/GroEL chaperonin from E. coli has long served as the model system for other chaperonins. This assumption seemed valid because of the high conservation between the chaperonins., As the GroES/GroEL chaperonin system is the only bacterial chaperone that is essential under all conditions, we have been interested in the development of GroES/GroEL inhibitors as potential antibiotics. Using Escherichia coli GroES/GroEL as a surrogate, we have discovered several classes of GroES/GroEL inhibitors that show potent antibacterial activity against both Gram-positive and Gram-negative bacteria. However, it remains unknown if E. coli GroES/GroEL is functionally identical to other GroES/GroEL chaperonins and hence if our inhibitors will function against other chaperonins. Herein we report our initial efforts to characterize the GroES/GroEL chaperonins from clinically significant ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species). We used complementation experiments in GroES/GroEL-deficient and -null E. coli strains to report on exogenous ESKAPE chaperone function. In GroES/GroEL-deficient (but not knocked-out) E. coli, we found that only a subset of the ESKAPE GroES/GroEL chaperone systems could complement to produce a viable organism. Surprisingly, GroES/GroEL chaperone systems from two of the ESKAPE pathogens were found to complement in E. coli, but only in the strict absence of either E. coli GroEL (P. aeruginosa) or both E. coli GroES and GroEL (E. faecium). In addition, GroES/GroEL from S. aureus was unable to complement E. coli GroES/GroEL under all conditions. The resulting viable strains, in which E. coli groESL was replaced with ESKAPE groESL, demonstrated similar growth kinetics to wild-type E. coli, but displayed an elongated phenotype (potentially indicating compromised GroEL function) at some temperatures. These results suggest functional differences between GroES/GroEL chaperonins despite high conservation of amino acid identity.

Elaborated kettle trap flowers to temporarily detain pollinators evolved independently in several angiosperm lineages. Intensive research on species of Aristolochia and Ceropegia recently illuminated how these specialized trap flowers attract particular pollinators through chemical deception. Morphologically similar trap flowers evolved in Riocreuxia, however, no data about floral rewards, pollinators, and chemical ecology were available for this plant group. Here we provide data on pollination ecology and floral chemistry of R. torulosa. Specifically, we determined flower visitors and pollinators, assessed pollen transfer efficiency, and analysed floral scent chemistry. R. torulosa flowers are myiophilous and predominantly pollinated by Nematocera. Pollinating Diptera included, in order of decreasing abundance, male and female Sciaridae, Ceratopogonidae, Scatopsidae, Chloropidae, and Phoridae. Approximately 16% of pollen removed from flowers was successfully exported to conspecific stigmas. The flowers emitted mainly ubiquitous terpenoids, most abundantly linalool, furanoid (Z)-linalool oxide, and (E)-β-ocimene—compounds typical of rewarding flowers and fruits. R. torulosa can be considered to use generalized food (and possibly also brood-site) deception to lure small nematocerous Diptera into their flowers. These results suggest that R. torulosa has a less specific pollination system than previously reported for other kettle trap flowers but is nevertheless specialized at the level of Diptera suborder Nematocera.

South Africa is a megadiverse country with three globally recognised biodiversity hotspots within its borders. Bees in particular show high diversity and endemism in the western part of the country. Not much is currently known about the floral host preferences of indigenous bees in South Africa, with data only available from observational studies. Pollen metabarcoding provides provenance information by utilising DNA analyses instead of floral visitation and traditional microscopic identification to identify pollinator food plants, which can be time consuming and imprecise. In this study, we sampled pollen from leaf-cutter bees (Megachilidae) specimens maintained in a historic insect collection (National Collection of Insects, South Africa) that were originally collected from two florally important areas in South Africa (Succulent Karoo and Savanna) and used metabarcoding to determine pollen provenance. We also sampled pollen from leafcutter bee species with wider distributions, that extend across many different biomes, to determine if these ‘generalist’ species show relaxed floral host specificity in some biomes. Metabarcoding involved sequencing of the nuclear internal transcribed spacer 2 (ITS2) region. Amplicons were compared to a sequence reference database to assign taxonomic classifications to family level. Sequence reads were also clustered to OTUs based on 97% sequence similarity to estimate numbers of plant species visited. We found no significant difference in the mean number of plant taxa visited in the Succulent Karoo and Savanna regions, but the widespread group visited significantly more floral hosts. Bees from the widespread group were also characterised by a significantly different composition in pollen assemblage. The time since specimens were collected did not have an effect on the mean number of taxa visited by any of the bee species studied. This study highlights national history collections as valuable sources of temporal and spatial biodiversity data.

The soft rot Pectobacteriaceae (SRP) infect a wide range of plants worldwide and cause economic damage to crops and ornamentals but can also colonize other plants as part of their natural life cycle. They are found in a variety of environmental niches, including water, soil and insects, where they may spread to susceptible plants and cause disease. In this chapter, we look in detail at the plants colonized and infected by these pathogens and at the diseases and symptoms they cause. We also focus on where in the environment these organisms are found and their ability to survive and thrive there. Finally, we present evidence that SRP may assist the colonization of human enteric pathogens on plants, potentially implicating them in aspects of human/animal as well as plant health.

Siring success of flowering plants depends on the fates of male gametophytes, which compete for access to stigmas, stylar resources, and ovules. Although rarely considered, pollen may often compete during dispersal, affecting the processes required for export to stigmas: pollen pickup, transport, and deposition. We quantified dispersal interference by tracking bee-mediated dispersal of stained Anacamptis morio (Orchidaceae) pollen from individual donor flowers and inferred the affected dispersal mechanisms on the basis of the fit of a process-based model. During individual trials, all recipient flowers were either emasculated, precluding interference with donor pollen, or intact, adding potentially interfering pollen to the pollinator. The presence of competing pollinaria on bees reduced pickup of additional pollinaria, doubled the overall proportion of lost donor pollen, and reduced total pollen export by 27%. Interference specifically increased loss of donor pollen between successive flower visits and variation in deposition among trials, and it likely also reduced pollen contact with stigmas and pollen deposition when contact occurred. Thus, by altering pollen removal, transport, and deposition, male-male interference during pollen dispersal can significantly—and perhaps commonly—limit plant-siring success.

Soft rot Pectobacteriaceae (SRP) are ubiquitous on earth as there are records of findings from all continents where host plants are grown. This chapter describes information on soft rot diseases on these continents. For some countries, detailed information is provided by local experts on the SRP present, their economic damage, and the management strategies applied for their control. The focus of the chapter is mainly on SRP as causative agents of potato blackleg, although in specific cases details are provided on SRP in other host plants. In Europe, the SRP cause important economic losses mainly on potato, with most species described in the literature being found. In Latin America significant losses are also reported due to potato diseases caused by various Dickeya and Pectobacterium species, while in Australia and Oceania, recent outbreaks of D. dianthicola in potato have resulted in high economic losses. In Asia, however, SRP cause economic losses mainly in vegetable crops other than potato, while in North America SRP cause diseases on a wide range of crops (including potato and ornamental plants) in both field and storage. In Africa SRP are only known to occur in 17 of the 54 African countries but where it is known, potato is the most affected crop.

1. Floral nectar harbours a diverse microbiome of yeasts and bacteria that depend predominantly on animal visitors for their dispersal. Since pollinators visit specific sets of flowers and carry their own unique microbiota, we hypothesize that plant species visited by the same set of pollinators may support non-random nectar microbial communities linked together by the type of pollinator. 2. Here we explore the importance of plant–pollinator interactions in the assembly of nectar microbiome and study the role of plant geographic location as a determinant of microbial community composition. We intensively sampled the nectar of 282 flowers of 48 plant species with beetles, birds, long-tongued and short-tongued insects as pollinators in wild populations in South Africa, one of the world's biodiversity hotspots, and using molecular techniques we identified nectar yeast and bacteria taxa. The analyses provided new insights into the richness, geographic structure and phylogenetic characterization of nectar microbiome, and compared patterns of composition of bacteria and yeast communities in relation to plant and pollinator guild. 3. Our results showed that plant–pollinator interactions played a crucial role in shaping nectar microbial communities. Plants visited by different pollinator guilds supported significantly different yeast and bacterial communities. The pollinator guild also contributed to the maintenance of beta diversity and phylogenetic microbial segregation. The results revealed different patterns for yeast and bacteria; whereas plants visited by beetles supported the highest richness and phylogenetic diversity of yeasts, bacteria communities were significantly more diverse in plants visited by other insect groups. We found no clear microbial spatial segregation at different geographical scales for bacteria, and only the phylogenetic similarity of yeast composition was correlated significantly with geography. 4. Synthesis. Interactions of animal vector, plant host traits and microbe physiology contribute to microbial community assemblages in nectar. Our results suggest that plants visited by the same pollinator guild have a characteristic nectar microbiota signature that may transcends the geographic region they are in. Contrasted patterns for yeast and bacteria stress the need for future work aimed at better understanding the causes and consequences of the importance of plants and pollinators in shaping nectar microbial communities in nature.

Floral color shifts are thought to be one of the most common evolutionary transitions in plants, and pollinators are often proposed as important selective agents driving these transitions. However, shifts in flower color can also be related to neutral genetic processes or pleiotropy linked with selection via other biotic agents or abiotic factors. Here we ask whether abiotic factors or pollinators provide the best explanation for divergence in flower color among populations of the sundew Drosera cistiflora s.l. (Droseraceae). This species complex in the Greater Cape Floristic Region contains at least five distinctive floral color forms. Abiotic factors do not appear to play a significant role in color determination, as the forms are not specific to a single soil or vegetation type, sometimes co-occur in the same habitat, and maintain their color traits in common-garden and soil switching experiments. Instead, we found strong associations between flower color and the composition of pollinator assemblages which are dominated by hopliine scarab beetles. Pollinator assemblages show geographical structuring, both within and among color forms. This makes it difficult to dissect the roles of geography versus floral traits in explaining pollinator assemblages, but strong pollinator partitioning among color forms at sites where they are sympatric indicates that pollinators may select strongly on color. These results suggest that beetle pollinators are a significant factor in the evolution of D. cistiflora s.l. flower color.

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 relative regional and global importance of eight pressures driving 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 for the first time how the importance of drivers, and risks from pollinator decline, differ among regions. For example, losing access to managed pollinators was only considered a serious risk to people in North America, whereas yield instability in pollinator-dependent crops, classed as a serious or high risk in four regions, presented only 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.