Large Bees Research Articles

High gene flow through pollen partially compensates spatial limited gene flow by seeds for a Neotropical tree in forest conservation and restoration areas

Gene flow may occur both through pollen movement and seed dispersal, although their relative contribution to overall species gene flow is not understood for most tropical trees, which compromises management recommendations. We investigated the implications of seed dispersal limitation, pollen dispersal capacity, and mating system to the spatial genetic structure of populations of the Neotropical legume tree, Centrolobium tomentosum, in Brazil’s Atlantic Forest. We estimated seed dispersal distribution by counting individuals around adult trees; pollen dispersal distribution and outcrossing rates from adult and offspring genotypes, using seven microsatellite loci. Spatial genetic structure was inferred through the correlation among kinship coefficients and geographical distances between pairs of individuals, using nuclear and chloroplast microsatellite markers. We observed restricted seed dispersal, with most seeds (78%) falling up to 10 m from the adult tree trunk. The best-fitted pollen dispersal distribution was the exponential power distribution, with a heavy tail and average pollen dispersal distance of 3191 m. The mating system was preferably allogamous, with an average of eight pollen donors. There was significant spatial genetic structure in all populations, with stronger structure obtained from chloroplast DNA, which suggests that the restricted gene flow by seed dispersal may be compensated by high outcrossing rates and long-distance pollen flow promoted by the large bees that pollinate the species. Our results emphasize the importance of pollination services in fragmented tropical landscapes, where most tree species are pollinated by animals and a large number of species experience seed dispersal limitation.

Rediscovering the eusocial sweat bee Lasioglossum marginatum (Hymenoptera: Halictidae) in Sicily through DNA barcoding

Sicily (Italy) is an important hotspot diversity for bees and new species records are constantly reported for the island. Here, we rediscovered after 45 years without records, the occurrence in Eastern Sicily of Lasioglossum marginatum (Brullé 1832) confirmed by DNA barcoding. This is the only known species of eusocial sweat bees (Halictidae) with perennial colonies. Since a single female was collected in our large 2-years bee sampling, combined with its absence in previous collections, this species is suggested to be rare in Sicily.

Andromonoecy in Solanum lycocarpum A. St. ‐Hil. (Solanaceae): Floral attributes, visitors and variation in sexual expression over time

AbstractSexual expression in andromonoecious species—those in which a single individual can bear both staminate and hermaphroditic flowers—may vary among reproductive events in the same plant, among individuals and across populations. This variation influences, in turn, the individual contribution of hermaphroditic plants via male and female fitness functions (i.e., Lloydʼs phenotypic gender). However, temporal variation in sexual expression in andromonoecious species and its relationship with seasonal changes in climatic conditions remain poorly understood. Here we analyze floral attributes, visitors and variation in sexual expression in three populations of Solanum lycocarpum A. St. ‐Hil. Seasonality in the production of floral types, the mating system and floral visitors were also investigated. Hermaphroditic flowers produced more pollen grains, but the pollen of staminate flowers had higher viability. Only hermaphroditic flowers produced fruits, and ovules in staminate flowers were sterile. Solanum lycocarpum is mainly pollinated by large bees with the ability to vibrate flowers. Phenotypic gender varied throughout the year, and the seasonal production of staminate flowers is associated with the local climate. We suggest that the higher and seasonally variable relative abundance of staminate flowers compared to the low and uniform production of hermaphroditic flowers may be explained by (a) the very high energetic costs incurred in producing large fruits, which in turn make hermaphroditic flower production very costly, and (b) the potentially lower energy expenditure of the smaller staminate flowers with reduced pistils and non‐viable ovules that allow them to rapidly respond to climate variability.

Pollination in thecampo rupestre: a test of hypothesis for an ancient tropical mountain vegetation

AbstractThe campo rupestre is a Neotropical OCBIL (old, climatically buffered infertile landscape), a grassy-shrub vegetation with high species richness and endemism, characterized by rocky outcrops surrounded by grasslands distributed in South American ancient mountaintops. We tested one OCBIL prediction: the prevalence of long-distance pollinators ensuring cross-pollination across the archipelago-like landscapes of the campo rupestre. We described the pollination systems and tested whether their frequency differed across vegetation types and elevation, focusing on long-distance systems. We performed non-systematic and systematic surveys of plants and plant-pollinator interactions across the elevation gradient and vegetation types. We also reviewed the literature on campo rupestre pollination and applied an accuracy criterion to infer 11 pollination systems. The bee system was split into large bee (long-distance) and small bee (shorter distances) to test the prevalence of long-distance pollination systems. We surveyed 413 pollinator species, mostly bees (220) and flies (69). Among the 636 plant species studied, the bee pollination system was dominant (56%), followed by wind and hummingbird. Wind, small-bee and fly pollination systems increased with elevation, and small-bee and wind pollination systems prevailed in grasslands. Large-bee and hummingbird long-distance pollination systems remained unchanged with elevation and were more frequent in the highly isolated rocky outcrops corroborating the OCBIL theory.

Population‐level plant pollination mode is influenced by Quaternary climate and pollinators

AbstractPatterns in ecology are the products of current factors interacting with history. Nevertheless, few studies have attempted to disentangle the contribution of historical and current factors, such as climate change and pollinator identity and behavior, on plant reproduction. Here, we attempted to separate the relative importance of current and historical processes on geographical patterns of the mating system of the tree speciesCuratella americana(Dilleniaceae). Specifically, we asked the following: (a) How do Quaternary and current climate affect plant mating system? (b) How does current pollinator abundance and diversity relate to plant mating system? (c) How does mating system relate to fruit/seed quantity and quality inC. americana? We recorded pollinators (richness, frequency, and body size) and performed pollination tests in ten populations ofC. americanaspread over 3,000 km in the Brazilian savannah. The frequency of self‐pollination in the absence of pollinators was strongly influenced by historical climatic instability and not by present‐day pollinators. In contrast, seed set from hand‐cross and natural pollination were affected by pollinators (especially large bees) and temperature, indicating the importance of current factors on out‐cross pollination. Two populations at the Southern edge of the species’ distribution showed high level of hand‐cross‐pollination and high flower visitation by large bees, but also a high level of autogamy resulting from recent colonization. Our results indicate that historical instability in climate has favored autogamy, most likely as a reproductive insurance strategy facilitating colonization and population maintenance over time, while pollinators are currently modulating the level of cross‐pollination.

Ectoparasite Habrobracon Hebetor Say Is an Efficient Biological Control Agent of Lepidopteran Pests

Ectoparasite Habrobracon hebetor Say is one of the most widely used biological controllers in biological plant protection against a number of harmful lepidopterans, including especially dangerous pests of corn, soy, fruit and vegetable crops. As a result of research conducted in 2017, food specialization and parasitic activity of three different populations of H.hebetor were studied. Two races have been identified for mass rearing and application: pyralid and leaf roller (against corn moth, bean pod borer, apple and plum moths), and pyralid owl-moth (against cotton moth, corn borer, bean pod borer and boxwood moth). As a result of studies of biological features and trophic needs, it has been determined that caterpillars of mill moth (Ephestia cuhniellia Zella) should be used as a host insect for laboratory cultivation of the stock population of the Habrobracon pyralid and leaf roller race (race No. 1). For the introduced from South Kazakhstan the H.hebetor pyralid and noctuid race the most productive rearing is on the caterpillars of large bee moth (Galleria mellonela L.). Optimal temperature for rearing of both races is 26-28 ° C, relative air humidity is 70% and photoperiod is not less than 16 hours. It has been noticed that before laying eggs on the host’s caterpillars, the Habrobracon female preliminarily paralyzes the victim, piercing the sheath with ovipositor. As a result, the caterpillar stops eating and is immobilized. In 3-4 days larvae hatch out of the laid on the caterpillar eggs. The larvae feed on the contents of the caterpillars for 4-5 days, then pupate and after 6-8 days an adult insect leaves the cocoon. The development of one generation lasts 13-16 days, one cocoon includes one parasite. 1,000 large bee moth caterpillars used for infection provide on average 5.8-6.0 thousand cocoons, of which an average of 4.5-4.7 thousand parasites fly out.

Relationships between wild bee abundance and fruit set of Camellia oleifera Abel

AbstractCamellia oleifera Abel. is an economically important plant for edible oils. To investigate the importance of wild bee abundance at different canopy and spatial scale for fruit set of Camellia oleifera, we evaluated the relationship of bee abundance with fruit set using yellow pan trap, with additional focus on distance and wild bee abundance counts at a farm in China for 2 years. We found that yellow traps collected 95% more individual bees than the second‐best (pink) trap colour. Looking across canopy positions, the number of wild bees (including Colletes gigas, Andrena camellia, Andrena striata, Andrena hunanensis and Andrena chekiangensis) varied significantly in each year. The fruit set also varied significantly based on canopy position. In 2018, there was with an average fruit set of 34.13% in the lower canopy, 54.30% in the middle canopy and 47.41% in the upper canopy. In 2019, fruit set was 29.67% in the lower canopy, 54.15% in the middle canopy and 47.36% in the upper canopy. Comparing inter‐annual changes, only the fruit set and number of bees in the low canopy showed significant differences between 2018 and 2019. Fruit set and wild bee abundance were positively correlated in all canopy layers in each year, and there was an interactive effect between canopy and bee abundance on fruit set. Across 205 trees, growth indicators, crown size and tree height, increased significantly from 2018 to 2019, but the fruit set did not increase with the significant increase in the number of flowers. From the spatial perspective, wild bee abundance and fruit set were positively correlated, but fruit set was negatively correlated with distance from a large bee nesting aggregation nearby. Among the variables investigated, bee abundance seems to have the strongest influence on fruit set in C. oleifera.

Tools to Tie: Flower Characteristics, VOC Emission Profile, and Glandular Trichomes of Two Mexican Salvia Species to Attract Bees.

A plant can combine physical and chemical tools to interact with other organisms. Some are designed for pollinator attraction (i.e., colors and volatile organic compounds-VOCs); others can act to discourage herbivores (i.e., non-glandular trichomes). Few studies fully address available tools in a single species; notwithstanding, this information can be pivotal in understanding new interactions out of the home range. We characterized flower traits, emission profiles of constitutive compounds from flowers and leaves, micro-morphology of the glandular trichomes, and listed flower visitors of two Mexican bird-pollinated Salvia species (S. blepharophylla and S. greggii), growing in an Italian botanical garden. Flowers were highly variable in their morphometric characteristics. In both species, four trichome morphotypes with similar histochemistry and distribution were documented for leaves and flowers except the calyx abaxial side. The vegetative emission profiles were qualitatively more complex than the floral ones; however, common compounds occurring in high relative percentages were β-caryophyllene and germacrene D. Floral bouquets were dominated by limonene and β-pinene in S. greggii and by 1,8-cineole in S. blepharophylla. Two potential (non-bird) pollinators were especially abundant: small bees belonging to the genus Lasioglossum and large bees belonging to the species Xylocopa violacea. Our study highlights the plasticity of these plants, as well as tools that can be conveniently used to establish novel interactions.

First record of the gregarious parasitoid Leucospis leucotelus Walker (Leucospidae) attacking nests of Xylocopa (Neoxylocopa) frontalis (Olivier) (Apidae)

Notes on three nests of the large carpenter bee Xylocopa (Neoxylocopa) frontalis (Olivier) (Apidae) attacked by the parasitoid Leucospis leucotelus Walker (Leucospidae) are presented. We observed that host brood cells contained from four to 12 parasitoids (adults or immature) which allowed us to identify this Leucospis species as a gregarious parasitoid. This was the first record of a gregarious Leucospis species attacking X. frontalis nests.

Thermoregulation in the large carpenter bee Xylocopa frontalis in the face of climate change in the Neotropics

There is evidence that climate change may worsen the ecological conditions needed by many pollinator species for their survival; however, little is known about how the rise in ambient temperature might affect the survival of large bee species in tropical regions of the planet. This study investigated the thermoregulation mechanisms of the large carpenter bee Xylocopa frontalis when nesting spontaneously in nests designed to allow direct measurement of the bees’ temperature while carrying out different activities. It was seen that the species divides its activities throughout the day according to the amount of body heat generated and the ambient temperature, performing activities that generate more body heat at cooler times of the day. The bees also use strategies of thermoregulation, such as heat loss by air convection, evaporative cooling by nectar dehydration, and not foraging during the hottest times of the day to avoid overheating. The study shows that X. frontalis is well adjusted to the ecological conditions of the areas in which it lives but suggests that future rises in the ambient temperature could pose a serious threat to the survival and pollination services provided by this bee and other similar tropical species.

Using ITS2 metabarcoding and microscopy to analyse shifts in pollen diets of honey bees and bumble bees along a mass‐flowering crop gradient

Worldwide pollinator declines lead to pollination deficits in crops and wild plants, and managed bees are frequently used to meet the increasing demand for pollination. However, their foraging can be affected by flower availability and colony size. We investigated how mass-flowering oilseed rape (OSR) can influence the pollen resource use of small and large honey bee (Apis mellifera L.) and bumble bee (Bombus terrestris L.) colonies. Colonies were placed adjacent to strawberry fields along a gradient of OSR availability in the landscapes. We used ITS2 metabarcoding to identify the pollen richness based on ITS2 amplicon sequencing and microscopy for quantification of target pollen. Bumble bees collected pollen from more different plant genera than honey bees. In both species, strawberry pollen collection decreased with high OSR availability but was facilitated by increasing strawberry flower cover. Colony size had no effect. The relationship between next-generation sequencing-generated ITS2 amplicon reads and microscopic pollen counts was positive but pollen type-specific. Bumble bees and, to a lesser degree, honey bees collected pollen from a wide variety of plants. Therefore, in order to support pollinators and associated pollination services, future conservation schemes should sustain and promote pollen plant richness in agricultural landscapes. Both bee species responded to the availability of flower resources in the landscape. Although honey bees collected slightly more strawberry pollen than bumble bees, both can be considered as crop pollinators. Metabarcoding could provide similar quantitative information to microscopy, taking into account the pollen types, but there remains high potential to improve the methodological weaknesses.

Visuo-motor biases in buff-tailed bumblebees (Bombus terrestris)

ABSTRACT Bees provide a good model to investigate the evolution of lateralization. So far, most studies focused on olfactory learning and memories in tethered bees. This study investigated possible behavioural biases in free-flying buff-tailed bumblebees (Bombus terrestris) by analysing their turning decisions in a T-maze. Bees of various size were trained to associate a syrup reward with a blue target placed at the centre of the T-maze. The bees were then tested over 16 trials by presenting them with blue targets at the end of the maze’s arms. The maze was rotated 180° after the first 8 trials to control for environmental factors. The number of turnings to the left and right arms were analysed. The bees sampled exhibited a population-level rightward turning bias. As bumblebees vary significantly in size with large bees being better learners than smaller ones, we measured the thorax width to identify a possible relationship between size and bias. No significant correlation was identified. This study shows that bees present lateralization in a visuo-motor task that mimics their foraging behaviour, indicating a possible specialization of the right side of the nervous system in routine tasks.

First Records of Xylocopa virginica (Linnaeus) (Hymenoptera: Apidae) in Montana and Idaho

A species of large carpenter bee, Xylocopa virginica virginica (Linnaeus, 1771), is reported from Montana and Idaho, USA for the first time. This new Montana distribution record adds to a growing list of bee species found in the state and contributes to an ongoing inventory of the bees of Montana. The Montana collection likely does not represent an established population but a transient accidental introduction resulting from human transportation since Montana is considerably geographically separated from the published distribution of this species and no additional adults have been found. The Idaho collections may represent an established local population as there are several specimens and online records in or near Boise on multiple dates and years. An online record of a specimen of X. californica arizonensis Cresson, 1879 from Montana proved to be erroneous. The distribution of X. virginica is reviewed and the economic, social, and ecological implications of the occurrence or establishment of this species in Montana and Idaho are discussed.

Genetic structure across urban and agricultural landscapes reveals evidence of resource specialization and philopatry in the Eastern carpenter bee, Xylocopa virginica L.

Human activity continues to impact global ecosystems, often by altering the habitat suitability, persistence, and movement of native species. It is thus critical to examine the population genetic structure of key ecosystemservice providers across human‐altered landscapes to provide insight into the forces that limit wildlife persistence and movement across multiple spatial scales. While some studies have documented declines of bee pollinators as a result of human‐mediated habitat alteration, others suggest that some bee species may benefit from altered land use due to increased food or nesting resource availability; however, detailed population and dispersal studies have been lacking. We investigated the population genetic structure of the Eastern carpenter bee, Xylocopa virginica, across 14 sites spanning more than 450 km, including dense urban areas and intensive agricultural habitat. X. virginica is a large bee which constructs nests in natural and human‐associated wooden substrates, and is hypothesized to disperse broadly across urbanizing areas. Using 10 microsatellite loci, we detected significant genetic isolation by geographic distance and significant isolation by land use, where urban and cultivated landscapes were most conducive to gene flow. This is one of the first population genetic analyses to provide evidence of enhanced insect dispersal in human‐altered areas as compared to semi‐natural landscapes. We found moderate levels of regional‐scale population structure across the study system (GʹST = 0.146) and substantial co‐ancestry between the sampling regions, where co‐ancestry patterns align with major human transportation corridors, suggesting that human‐mediated movement may be influencing regional dispersal processes. Additionally, we found a signature of strong site‐level philopatry where our analyses revealed significant levels of high genetic relatedness at very fine scales (<1 km), surprising given X. virginica's large body size. These results provide unique genetic evidence that insects can simultaneously exhibit substantial regional dispersal as well as high local nesting fidelity in landscapes dominated by human activity.

Within-individual phenotypic plasticity in flowers fosters pollination niche shift

Phenotypic plasticity, the ability of a genotype of producing different phenotypes when exposed to different environments, may impact ecological interactions. We study here how within-individual plasticity in Moricandia arvensis flowers modifies its pollination niche. During spring, this plant produces large, cross-shaped, UV-reflecting lilac flowers attracting mostly long-tongued large bees. However, unlike most co-occurring species, M. arvensis keeps flowering during the hot, dry summer due to its plasticity in key vegetative traits. Changes in temperature and photoperiod in summer trigger changes in gene expression and the production of small, rounded, UV-absorbing white flowers that attract a different assemblage of generalist pollinators. This shift in pollination niche potentially allows successful reproduction in harsh conditions, facilitating M. arvensis to face anthropogenic perturbations and climate change.

Buzz pollination

Pritchard and Vallejo-Marín introduce the process and evolution of buzz pollination.

Optimizing yield and flower resources for pollinators in intensively managed multi-species grasslands

Intensively managed grasslands, typically containing a few high-yielding species, play a key role in providing biomass feedstock for animal feed and bioenergy production. However, these grasslands often contain few resources for flower-visiting insects, which provide pollination services for crops. In this study, we investigate if both high yield and floral resources for pollinators can be achieved in intensively managed grasslands by varying species diversity and composition, and cutting frequency. Four perennial grassland mixtures containing 3, 5, 11 and 13 species of grasses and/or forbs were established at three sites varying in surrounding landscape, and plots were managed with three cutting strategies (four cuts per year, two cuts per year and no cut). Biomass production was measured as annual herbage yield. Every 2–4 weeks throughout the flowering season, flower abundance, richness, temporal stability and flower-visiting insects were monitored. The 11- and 13-species mixtures, designed to enhance pollinators, produced similar or higher herbage yield than the 3-species mixture under a two-cut strategy or the 5-species mixture under a four-cut strategy. The 3- and 5- species mixtures had a high accumulated flower abundance due to excessive flowering of lucerne under the two-cut strategy and white clover under the four-cut strategy. Interestingly, flower abundance was not significantly reduced under the two-cut strategy compared to no cut. As expected, the 11- and 13- species mixtures presented a higher diversity of flowers during the flowering season. Flower temporal stability was generally higher in 11- and 13-species mixtures under the two-cut strategy, while it was highest in the 5-species mixture under the four-cut strategy. Pollinator profiles (visits by different functional groups of insects) were plant-species specific, and varied very little among sites. Legume species mainly attracted large bees (honey bees and bumblebees), while the most abundant forbs attracted other insect groups, in particular syrphids and other flies. Our results suggest that multi-species grasslands can be designed to support a high diversity of pollinators without compromising herbage yield. In particular, adding forbs to the grass-legume seed mixtures and managing grasslands with a two-cut strategy may increase flower resources available for a larger range of wild pollinators.

Timing of male territorial flight and foraging of the large carpenter bee Xylocopa appendiculata related to serotonin in the brain.

To determine how males of the large carpenter bee, Xylocopa appendiculata, maximize access to females while minimizing energy cost and acquiring energy for territorial flights, we investigated the times of territorial flights by males and foraging by males and females. Males were present continuously in territories from 8:00 to 12:00. They approached, chased, and excluded conspecific males from their territories. In the laboratory, males showed higher locomotor and flight activities in the morning and lower activities in the afternoon. Both males and females visited flowers from 8:00 to 16:00, but the most frequent visits were earlier in females (10:00-12:00) than in males (12:00-13:00). Relative body weights in territorial males often increased. These results indicate that the males time their territorial flights to maximize contact with females and obtain nectar as fuel between and after the territorial flights. The time-related territorial flight in males might be based on a time-keeping system in the brain. Brain levels of serotonin and its precursor tryptophan were significantly higher in males collected at 16:00 than at 11:00, suggesting a relation between time-related territorial flight and serotonin synthesis in the brain.

The long spur of Impatiens macrovexilla may reflect adaptation to diurnal hawkmoth pollinators despite diversity of floral visitors

Many tropical and subtropical Impatiens species possess conspicuously long and narrow spurs and have been assigned to the butterfly pollination syndrome in investigations of floral evolution of this genus. However, this contradicts some previous studies documenting abundant hawkmoth visitors rather than butterflies. In this study, we explored the pollination ecology of Impatiens macrovexilla, a long-spurred species in Asia. We investigated a series of floral traits, tested the breeding system and contributions of different floral visitors to reproductive success, observed floral visitors and compared the spur length with the proboscis length of the nectar-collecting floral visitors. I. macrovexilla shows floral traits that cannot clearly be associated with a certain group of long-proboscid insects. Results showed that the species was visited by large nectar-collecting bees, diurnal hawkmoths, small pollen-collecting insects and rarely butterflies at different sites and in different years. Pollination treatments indicated that I. macrovexilla is self-compatible (Self-Incompatibility Index = 0.02) but completely relies on floral visitors for pollination. A treatment which excluded hawkmoths but permitted pollen-collecting insects to visit flowers revealed that diurnal hawkmoths contributed considerably to reproductive success of this species but that pollen-collecting insects acted as pollen thieves. Large bees firmly contacted anthers and stigmas when visiting flowers so probably also act as pollinators. The proboscises of large bees were shorter than half of the spur of I. macrovexilla in length, suggesting that they could only access the nectar accumulated near the mouth of the spur. However, hawkmoths can access most or all of the nectar because their proboscides match the length of the floral spur. Thus, the long nectar spur presumably reflects adaptation to diurnal hawkmoths. Using evidence from multiple lines of investigation, our study is the first to confirm the role of diurnal hawkmoths as pollinators in Impatiens.

Xylocopa sonorina Smith, 1874 from Vancouver, British Columbia, Canada (Hymenoptera: Apidae, Xylocopinae) with comments on its taxonomy.

BackgroundOnly one species of large carpenter bee, Xylocopa virginica (Linnaeus, 1771), has been recorded from Canada, albeit restricted to southern Ontario and Quebec. However, a single female specimen identified by Hurd in 1954 as X. varipuncta Patton, 1879 from British Columbia is in the C.A. Triplehorn Insect Collection at The Ohio State University (OSUC), suggesting that this species was accidentally introduced into coastal western Canada. As wood-nesters, many large carpenter bees are likely capable of expanding their range great distances by natural and unnatural transport methods while nesting inside suitable substrates, the presumed mode of transport into western Canada, and likely elsewhere. The ease at which the nests are transported has likely contributed to the nomenclatural and distributional ambiguity surrounding this species due to morphological similarities of specimens from North America, Hawaii, and several South Pacific islands.New informationBy comparing DNA barcodes of specimens from the western United States to specimens from Hawaii, we confirm the early opinion of P.H. Timberlake (Timberlake 1922) that specimens long established on the Hawaiian Islands are the same X. varipuncta from continental North America. Furthermore, these DNA barcode sequences also match those of specimens identified as X. sonorina Smith, 1874 from the French Polynesian and Samoan Islands, thus fully supporting the opinion of Groom et al. (2017) that all are likely conspecific. As X. sonorina, a species described from and likely introduced to Hawaii is the oldest name available, X. varipuncta is here placed into synonymy. Additional research will be needed to trace the timing and pathway of introduction and establishment of X. sonorina; it is presumed that the species is native to the southwestern United States but has been established in Hawaii since the mid-1800s. It is also established in French Polynesia, the Samoan Islands, and likely other south Pacific islands, with additional records of occurrence from Java, New Zealand, and now Canada.