Differences in the biochemical content and radical scavenging activity of propolis from different parts of a Meliponula ferruginea hive.

Research on stingless bee products has increased in recent years, and of particular interest is propolis because of its biological activities such as antioxidant and antimicrobial. However, there is paucity of information regarding intra-hive variations in the biochemical composition and biofunctional properties of this propolis. In this study, we investigated the phytochemicals and radical scavenging activity (RSA) of Meliponula ferruginea propolis from 10 wooden hives (n = 49). The samples were collected from five different locations comprising the entrance, involucrum, pillars, pots and sealant. Principal component analysis showed that there is an intra-hive variation in phytochemical content and RSA. Phenolic content constituted the highest phytochemical content in all the locations. The sealant and entrance had the highest amounts of phytochemicals compared to the involucrum, pillars and pots. Further analysis of propolis extracts by gas chromatography-mass spectrometry revealed occurrence of different compounds such as monoterpenoids, hydrocarbons, triterpenoids and alkaloids. Hydrocarbons were common in all parts while monoterpenes and triterpenes were present in the entrance. The findings of our study indicates that there is an intra-hive variation in propolis of M. ferruginea and hence this information will provide further insight into better understanding of stingless bee propolis.

The wild solitary bees Andrena vaga, Anthophora plumipes, Colletes cunicularius, and Osmia cornuta microbiota are host specific and dominated by endosymbionts and environmental microorganisms.

We characterized the microbial communities of the crop, midgut, hindgut, and ovaries of the wild solitary bees Andrena vaga, Anthophora plumipes, Colletes cunicularius, and Osmia cornuta through 16S rRNA gene and ITS2 amplicon sequencing and a large-scale isolation campaign. The bacterial communities of these bees were dominated by endosymbionts of the genera Wolbachia and Spiroplasma. Bacterial and yeast genera representing the remaining predominant taxa were linked to an environmental origin. While only a single sampling site was examined for Andrena vaga, Anthophora plumipes, and Colletes cunicularius, and two sampling sites for Osmia cornuta, the microbiota appeared to be host specific: bacterial, but not fungal, communities generally differed between the analyzed bee species, gut compartments and ovaries. This may suggest a selective process determined by floral and host traits. Many of the gut symbionts identified in the present study are characterized by metabolic versatility. Whether they exert similar functionalities within the bee gut and thus functional redundancy remains to be elucidated.

Traditional ecological knowledge and non-food uses of stingless bee honey in Kenya's last pocket of tropical rainforest.

BACKGROUND: Stingless bee honey (SBH) is a natural remedy and therapeutic agent traditionally used by local communities across the (sub-)tropics. Forest SBH represents a prime non-timber forest product (NTFP) with a potential to revitalize indigenous foodways and to generate income in rural areas, yet it is also used in a variety of non-food contexts that are poorly documented in sub-Saharan Africa and that collectively represent a significant part of the local traditional ecological knowledge (TEK) passed on across generations. Documenting TEK of local communities in African tropical forests facing global change is a pressing issue to recognize the value of their insights, to evaluate their sustainability, to determine how they contribute to enhancing conservation efforts, and how TEK generally contributes to the well-being of both the natural environment and the communities that rely on it. This is particularly important to achieve in Kenya's only tropical rainforest at Kakamega where SBH production and non-food uses have evolved and diversified to a remarkable extent. METHODS: We used ethnographic techniques and methods, including semi-structured questionnaires and recorded interviews. We used snowball sampling, a non-probability sampling method where new interviewees were recruited by other respondents, to collectively form a sample consisting of 36 interviewees (including only one woman). RESULTS: Our results indicate that local communities in Kakamega were able to discriminate between six different and scientifically recognized stingless bee species, and they provided detailed accounts on the species-specific non-food uses of these SBH. Collectively, we recorded an array of 26 different non-food uses that are all passed on orally across generations in the Kakamega community. CONCLUSION: Our results uncover the vast and hitherto unexpected diversity of TEK associated with SBH and pave the way for a systematic survey of SBH and their non-food uses across a network of communities in different environments and with different cultural backgrounds in the Afrotropics. This, along with parallel and more in-depth investigations into honey chemistry, will help develop a comprehensive understanding of SBH, offering insights into holistic ecosystem management, resilience and adaptation while in the mid- to long-term promoting cross-cultural exchanges and pathways for the revitalization of cultural practices and traditions.

Microbiota and pathogens in an invasive bee: Megachile sculpturalis from native and invaded regions.

The present study aimed to characterise the bacterial, fungal and parasite gut community of the invasive bee Megachile sculpturalis sampled from native (Japan) and invaded (USA and France) regions via 16S rRNA and ITS2 amplicon sequencing and PCR detection of bee microparasites. The bacterial and fungal gut microbiota communities in bees from invaded regions were highly similar and differed strongly from those obtained in Japan. Core amplicon sequence variants (ASVs) within each population represented environmental micro-organisms commonly present in bee-associated niches that likely provide beneficial functions to their host. Although the overall bacterial and fungal communities of the invasive M. sculpturalis in France and the co-foraging native bees Anthidium florentinum and Halictus scabiosae, were significantly different, five out of eight core ASVs were shared suggesting common environmental sources and potential transmission. None of the 46 M. sculpturalis bees analysed harboured known bee pathogens, while microparasite infections were common in A. florentinum, and rare in H. scabiosae. A common shift in the gut microbiota of M. sculpturalis in invaded regions as a response to changed environmental conditions, or a founder effect coupled to population re-establishment in the invaded regions may explain the observed microbial community profiles and the absence of parasites. While the role of pathogen pressure in shaping biological invasions is still debated, the absence of natural enemies may contribute to the invasion success of M. sculpturalis.

Convivina is a specialised core gut symbiont of the invasive hornet Vespa velutina.

We provide a culturomics analysis of the cultivable bacterial communities of the crop, midgut and hindgut compartments, as well as the ovaries, of the invasive insect Vespa velutina, along with a cultivation-independent analysis of samples of the same nest through 16S rRNA amplicon sequencing. The Vespa velutina bacterial symbiont community was dominated by the genera Convivina, Fructobacillus, Lactiplantibacillus, Lactococcus, Sphingomonas and Spiroplasma. Lactococcus lactis and Lactiplantibacillus plantarum represented generalist core lactic acid bacteria (LAB) symbionts, while Convivina species and Fructobacillus fructosus represented highly specialised core LAB symbionts with strongly reduced genome sizes. Sphingomonas and Spiroplasma were the only non-LAB core symbionts but were not isolated. Convivina bacteria were particularly enriched in the hornet crop and included Convivina intestini, a species adapted towards amino acid metabolism, and Convivina praedatoris sp. nov. which was adapted towards carbohydrate metabolism.

A phylogenomic and comparative genomic analysis of Commensalibacter, a versatile insect symbiont.

BACKGROUND: To understand mechanisms of adaptation and plasticity of pollinators and other insects a better understanding of diversity and function of their key symbionts is required. Commensalibacter is a genus of acetic acid bacterial symbionts in the gut of honey bees and other insect species, yet little information is available on the diversity and function of Commensalibacter bacteria. In the present study, whole-genome sequences of 12 Commensalibacter isolates from bumble bees, butterflies, Asian hornets and rowan berries were determined, and publicly available genome assemblies of 14 Commensalibacter strains were used in a phylogenomic and comparative genomic analysis. RESULTS: The phylogenomic analysis revealed that the 26 Commensalibacter isolates represented four species, i.e. Commensalibacter intestini and three novel species for which we propose the names Commensalibacter melissae sp. nov., Commensalibacter communis sp. nov. and Commensalibacter papalotli sp. nov. Comparative genomic analysis revealed that the four Commensalibacter species had similar genetic pathways for central metabolism characterized by a complete tricarboxylic acid cycle and pentose phosphate pathway, but their genomes differed in size, G + C content, amino acid metabolism and carbohydrate-utilizing enzymes. The reduced genome size, the large number of species-specific gene clusters, and the small number of gene clusters shared between C. melissae and other Commensalibacter species suggested a unique evolutionary process in C. melissae, the Western honey bee symbiont. CONCLUSION: The genus Commensalibacter is a widely distributed insect symbiont that consists of multiple species, each contributing in a species specific manner to the physiology of the holobiont host.

The Megachilidae (Hymenoptera, Apoidea, Apiformes) of the Democratic Republic of Congo curated at the Royal Museum for Central Africa (RMCA, Belgium).

Natural history collections are a cornerstone of entomology, and the conservation of specimens is the essential prerequisite for the development of research into systematics, biogeography, ecology, evolution and other disciplines. Yet, specimens collected during decades of entomological research conducted in less developed countries across Sub-Saharan Africa on pests, beneficial insects and insect biodiversity in general have largely been exported to be permanently preserved in developed countries, mainly in Europe and the United States of America. This is particularly true for the Democratic Republic of the Congos (DRC) diverse wild bee fauna, which has been investigated throughout the colonial period by visiting or resident entomologists and missionaries who have then transferred their collected material primarily to Belgium as part of a wider legacy of scientific exploration and colonialism. Digitizing NHC is one way to mitigate this current bias, by making samples accessible to researchers from the target post-colonial countries as well as to the wider international scientific community. In this study, we compiled and digitized 6,490 specimens records relevant to 195 wild bee species grouped in 18 genera within the biodiverse family Megachilidae, essentially from the colonial era (i.e., mostly between 19051960, with additional records up to 1978), and curated at the Royal Museum for Central Africa in Belgium. We provide a detailed catalogue of all records with updated locality and province names, including 29 species only available as type specimens. We also explore the historical patterns of diversity and distribution across DRC, and we provide a list of the research entomologists involved. This study is an important first step that uses digital technologies to democratize and repatriate important aspects of DRCs natural heritage of insect biodiversity, to stimulate more contemporary field surveys and modern taxonomic revisions, as well as to identify and characterize research gaps and biodiversity shortfalls in some of the less-explored regions of Sub-Saharan Africa.

Honey compositional convergence and the parallel domestication of social bees.

Honey collection evolved from simple honey hunting to the parallel and independent domestication of different species of bees in various parts of the world. In this study, we investigate the extent to which the composition of Apis and stingless bee honeys has been a driver in the selection of different bee species for domestication in Mesoamerica (Mexico) and Asia (Thailand) using a sampling design that combines peak honey profiling by H1 NMR spectroscopy with the collection of honeys from domesticated and undomesticated bee species. Our results show that, independently of the region of the world considered, domesticated stingless bees produce honey whose compositional profiles differ from those of the non-domesticated species and exhibit more similarities towards honeys produced by the domesticated Apis species. Our results provide evidence for the first time that the search for natural sweeteners in the environment by our ancestors led to the parallel and independent domestication of social bees producing honeys with similar compositional profiles.

Signals of adaptation to agricultural stress in the genomes of two European bumblebees.

Human-induced environmental impacts on wildlife are widespread, causing major biodiversity losses. One major threat is agricultural intensification, typically characterised by large areas of monoculture, mechanical tillage, and the use of agrochemicals. Intensification leads to the fragmentation and loss of natural habitats, native vegetation, and nesting and breeding sites. Understanding the adaptability of insects to these changing environmental conditions is critical to predicting their survival. Bumblebees, key pollinators of wild and cultivated plants, are used as model species to assess insect adaptation to anthropogenic stressors. We investigated the effects of agricultural pressures on two common European bumblebees, Bombus pascuorum and B. lapidarius. Restriction-site Associated DNA Sequencing was used to identify loci under selective pressure across agricultural-natural gradients over 97 locations in Europe. 191 unique loci in B. pascuorum and 260 in B. lapidarius were identified as under selective pressure, and associated with agricultural stressors. Further investigation suggested several candidate proteins including several neurodevelopment, muscle, and detoxification proteins, but these have yet to be validated. These results provide insights into agriculture as a stressor for bumblebees, and signal for conservation action in light of ongoing anthropogenic changes.

Phylogenomic Analyses of Snodgrassella Isolates from Honeybees and Bumblebees Reveal Taxonomic and Functional Diversity.

Snodgrassella is a genus of Betaproteobacteria that lives in the gut of honeybees (Apis spp.) and bumblebees (Bombus spp). It is part of a conserved microbiome that is composed of a few core phylotypes and is essential for bee health and metabolism. Phylogenomic analyses using whole-genome sequences of 75 Snodgrassella strains from 4 species of honeybees and 14 species of bumblebees showed that these strains formed a monophyletic lineage within the Neisseriaceae family, that Snodgrassella isolates from Asian honeybees diverged early from the other species in their evolution, that isolates from honeybees and bumblebees were well separated, and that this genus consists of at least seven species. We propose to formally name two new Snodgrassella species that were isolated from bumblebees: i.e., Snodgrassella gandavensis sp. nov. and Snodgrassella communis sp. nov. Possible evolutionary scenarios for 107 species- or group-specific genes revealed very limited evidence for horizontal gene transfer. Functional analyses revealed the importance of small proteins, defense mechanisms, amino acid transport and metabolism, inorganic ion transport and metabolism and carbohydrate transport and metabolism among these 107 specific genes. IMPORTANCE The microbiome of honeybees (Apis spp.) and bumblebees (Bombus spp.) is highly conserved and represented by few phylotypes. This simplicity in taxon composition makes the bee's microbiome an emergent model organism for the study of gut microbial communities. Since the description of the Snodgrassella genus, which was isolated from the gut of honeybees and bumblebees in 2013, a single species (i.e., Snodgrassella alvi), has been named. Here, we demonstrate that this genus is actually composed of at least seven species, two of which (Snodgrassella gandavensis sp. nov. and Snodgrassella communis sp. nov.) are formally described and named in the present publication. We also report the presence of 107 genes specific to Snodgrassella species, showing notably the importance of small proteins and defense mechanisms in this genus.

On the road: Anthropogenic factors drive the invasion risk of a wild solitary bee species.

Complex biotic networks of invaders and their new environments pose immense challenges for researchers aiming to predict current and future occupancy of introduced species. This might be especially true for invasive bees, as they enter novel trophic interactions. Little attention has been paid to solitary, invasive wild bees, despite their increasing recognition as a potential global threat to biodiversity. Here, we present the first comprehensive species distribution modelling approach targeting the invasive bee Megachile sculpturalis, which is currently undergoing parallel range expansion in North America and Europe. While the species has largely colonised the most highly suitable areas of North America over the past decades, its invasion of Europe seems to be in its early stages. We showed that its current distribution is largely explained by anthropogenic factors, suggesting that its spread is facilitated by road and maritime traffic, largely beyond its intrinsic dispersal ability. Our results suggest that M. sculpturalis is likely to be negatively affected by future climate change in North America, while in Europe the potential suitable areas at-risk of invasion remain equally large. Based on our study, we emphasise the role of expert knowledge for evaluation of ecologically meaningful variables implemented and interpreted for species distribution modelling. We strongly recommend that the monitoring of this and other invasive pollinator species should be prioritised in areas identified as at-risk, alongside development of effective management strategies.

Characterization of honeys produced by sympatric species of Afrotropical stingless bees (Hymenoptera, Meliponini).

We investigated the effect of bee species identity and harvesting methods on the chemical composition and antiradical activity of 53 honey samples, produced by six stingless bee species in western Kenya (Kakamega forest). Our results illustrate that none of the assayed parameters significantly varied between the honey samples harvested by "punching holes" (n = 25) and "squeezing" (n = 28) methods. By contrast, species identity drove significant differences in the assayed parameters. Positive correlations between the antiradical activity and the phytochemicals (phenols and flavonoids) were observed, and honeys from Liotrigona sp. exhibited the highest amounts of phenols (214 mg GAE/100 g), flavonoids (73.0 mg QE/100 g) and antiradical activity (76.2%). The physicochemical analyses confirm the need to establish separate stingless bee honey standards for moisture, free acidity, invertase, electrical conductivity, and HMF, as these parameters significantly diverged from the set limits for Apis mellifera honey.

Bee flowers drive macroevolutionary diversification in long-horned bees.

The role of plant-pollinator interactions in the rapid radiation of the angiosperms have long fascinated evolutionary biologists. Studies have brought evidence for pollinator-driven diversification of various plant lineages, particularly plants with specialized flowers and concealed rewards. By contrast, little is known about how this crucial interaction has shaped macroevolutionary patterns of floral visitors. In particular, there is currently no empirical evidence that floral host association has increased diversification in bees, the most prominent group of floral visitors that essentially rely on angiosperm pollen. In this study, we examine how floral host preference influenced diversification in eucerine bees (Apidae, Eucerini), which exhibit large variations in their floral associations. We combine quantitative pollen analyses with a recently proposed phylogenetic hypothesis, and use a state speciation and extinction probabilistic approach. Using this framework, we provide the first evidence that multiple evolutionary transitions from host plants with accessible pollen to restricted pollen from 'bee-flowers' have significantly increased the diversification of a bee clade. We suggest that exploiting host plants with restricted pollen has allowed the exploitation of a new ecological niche for eucerine bees and contributed both to their colonization of vast regions of the world and their rapid diversification.

Opportunities to reduce pollination deficits and address production shortfalls in an important insect-pollinated crop.

Pollinators face multiple pressures and there is evidence of populations in decline. As demand for insect-pollinated crops increases, crop production is threatened by shortfalls in pollination services. Understanding the extent of current yield deficits due to pollination and identifying opportunities to protect or improve crop yield and quality through pollination management is therefore of international importance. To explore the extent of "pollination deficits," where maximum yield is not being achieved due to insufficient pollination, we used an extensive dataset on a globally important crop, apples. We quantified how these deficits vary between orchards and countries and we compared "pollinator dependence" across different apple varieties. We found evidence of pollination deficits and, in some cases, risks of overpollination were even apparent for which fruit quality could be reduced by too much pollination. In almost all regions studied we found some orchards performing significantly better than others in terms of avoiding a pollination deficit and crop yield shortfalls due to suboptimal pollination. This represents an opportunity to improve production through better pollinator and crop management. Our findings also demonstrated that pollinator dependence varies considerably between apple varieties in terms of fruit number and fruit quality. We propose that assessments of pollination service and deficits in crops can be used to quantify supply and demand for pollinators and help to target local management to address deficits although crop variety has a strong influence on the role of pollinators.

Impact of intraspecific variation on measurements of thermal tolerance in bumble bees.

Climate change is an important driver of bee decline despite the fact that many species might respond to climate change differently. One method to predict how a species will respond to climate change is to identify its thermal tolerance limits. However, differences in thermal tolerance might also occur among distant populations of the same species based on their local environment or even among castes of social insects. Here, we investigated intraspecific differences in thermal tolerance among subspecies of the large earth bumble bee, Bombus terrestris (Apidae). We determined the critical thermal minima and maxima (CTmin and CTmax, respectively) of workers and queens from three lab-reared B. terrestris subspecies (B. t. terrestris, B. t. audax, and B. t. canariensis) which originated from different thermal environments. Our results showed that caste has an influence on critical thermal minima, with queens being most cold-tolerant, but the values of critical thermal maxima were not correlated to caste or size. The thermal tolerance of workers did not differ among the subspecies. Although heat tolerance was similar in queens, B. t. canariensis queens (originating from the warmest environments) were the least cold tolerant. Overall, we showed that B. terrestris may be generally robust against climate warming, but that particular subspecies and/or populations may be more vulnerable to extreme temperature variability. Future research should focus on responses of B. terrestris populations to short, extreme thermal events.

Bumblebee resilience to climate change, through plastic and adaptive responses.

Bumblebees are ubiquitous, cold-adapted eusocial bees found worldwide from subarctic to tropical regions of the world. They are key pollinators in most temperate and boreal ecosystems, and both wild and managed populations are significant contributors to agricultural pollination services. Despite their broad ecological niche at the genus level, bumblebee species are threatened by climate change, particularly by rising average temperatures, intensifying seasonality and the increasing frequency of extreme weather events. While some temperature extremes may be offset at the individual or colony level through temperature regulation, most bumblebees are expected to exhibit specific plastic responses, selection in various key traits, and/or range contractions under even the mildest climate change. In this review, we provide an in-depth and up-to-date review on the various ways by which bumblebees overcome the threats associated with current and future global change. We use examples relevant to the fields of bumblebee physiology, morphology, behaviour, phenology, and dispersal to illustrate and discuss the contours of this new theoretical framework. Furthermore, we speculate on the extent to which adaptive responses to climate change may be influenced by bumblebees' capacity to disperse and track suitable climate conditions. Closing the knowledge gap and improving our understanding of bumblebees' adaptability or avoidance behaviour to different climatic circumstances will be necessary to improve current species climate response models. These models are essential to make correct predictions of species vulnerability in the face of future climate change and human-induced environmental changes to unfold appropriate future conservation strategies.

Long-term effects of global change on occupancy and flight period of wild bees in Belgium.

Global change affects species by modifying their abundance, spatial distribution, and activity period. The challenge is now to identify the respective drivers of those responses and to understand how those responses combine to affect species assemblages and ecosystem functioning. Here we correlate changes in occupancy and mean flight date of 205 wild bee species in Belgium with temporal changes in temperature trend and interannual variation, agricultural intensification, and urbanization. Over the last 70 years, bee occupancy decreased on average by 33%, most likely because of agricultural intensification, and flight period of bees advanced on average by 4 days, most likely because of interannual temperature changes. Those responses resulted in a synergistic effect because species which increased in occupancy tend to be those that have shifted their phenologies earlier in the season. This leads to an overall advancement and shortening of the pollination season by 9 and 15 days respectively, with lower species richness and abundance compared to historical pollinator assemblages, except at the early start of the season. Our results thus suggest a strong decline in pollination function and services.

A primer of host-plant specialization in bees.

The bee-flower biological association is one of the most famous examples of insect-plant interactions, and it is axiomatic that these are of critical importance for sustaining thriving terrestrial ecosystems. Yet, the most familiar associations are often artificially managed agricultural ecosystems, reflecting an exceptionally narrow range of bee species (often only one) and a concomitantly restricted range of associated behaviors, morphologies, and mechanisms tied to pollination. Here we provide a brief account of the range of bee-floral associations encompassing floral specialization in terms of diet, behavior, and morphology. These natural associations not only promote healthy ecosystems, but also can be integrated in sustainable ways for more efficient pollination of crops by targeting bee species whose diets, behaviors, and pollen-gathering structures evolved precisely to visit such floral species rather than less efficient, and often non-native, generalists that are otherwise exploited for such purposes.

Flower Colour Polymorphism, Pollination Modes, Breeding System and Gene Flow in Anemone coronaria.

The flower colour of Anemone coronaria (Ranunculaceae) is a genetically inherited trait. Such intra-specific flower colour polymorphism might be driven by pollinators, other non-pollinating agents, or by abiotic factors. We investigated the genetic relations among red, white and purple-blue flower colour morphs growing in 10 populations of A. coronaria in Israel, in relation to their breeding system, pollination modes, differential perception by bees and visitors' behaviour. Flowers of these three morphs differed in their reflectance that could be perceived by bees. Honeybees, solitary bees and flies demonstrated only partial preferences for the different colour morphs. No spontaneous self-pollination was found; however, fruit set under nets, excluding insects but allowing wind pollination, was not significantly lower than that of natural free pollinated flowers, indicating a potential role of wind pollination. Anemone coronaria flowers were visited by various insects, honeybees and Andrena sp. preferred the white and purple-blue morphs, while the syrphid flies preferred the white flowers. Thus, visitor behaviour can only partially explain the evolution or maintenance of the colour polymorphism. No significant genetic differences were found among the populations or colour morphs. Wind pollination, causing random gene flow, may explain why no significant genetic divergence was found among all studied populations and their colour morphs. The existence of monomorphic red populations, along other polymorphic populations, might be explained by linked resistance to aridity and/or grazing.

Shift in size of bumblebee queens over the last century.

Species can respond differently when facing environmental changes, such as by shifting their geographical ranges or through plastic or adaptive modifications to new environmental conditions. Phenotypic modifications related to environmental factors have been mainly explored along latitudinal gradients, but they are relatively understudied through time despite their importance for key ecological interactions. Here we hypothesize that the average bumblebee queen body size has changed in Belgium during the last century. Based on historical and contemporary databases, we first tested if queen body sizes changed during the last century at the intraspecific level among four common bumblebee species and if it could be linked to global warming and/or habitat fragmentation as well as by the replacement by individuals from new populations. Then, we assessed body size changes at the community level, among 22 species, taking into account species population trends (i.e. increasing, stable or decreasing relative abundance). Our results show that the average queen body size of all four bumblebee species increased over the last century. This size increase was significantly correlated to global warming and habitat fragmentation, but not explained by changes in the population genetic structure (i.e. colonization). At the community level, species with stable or increasing relative abundance tend to be larger than declining species. Contrary to theoretical expectations from Bergmann's rule (i.e. increasing body size in colder climates), temperature does not seem to be the main driver of bumblebee body size during the last century as we observed the opposite body size trend. However, agricultural intensification and habitat fragmentation could be alternative mechanisms that shape body size clines. This study stresses the importance of considering alternative global change factors when assessing body size change.