Honeybees (Apis mellifera) decrease the fitness of plants they pollinate.

Most flowering plants require animal pollination and are visited by multiple pollinator species. Historically, the effects of pollinators on plant fitness have been compared using the number of pollen grains they deposit, and the number of seeds or fruits produced following a visit to a virgin flower. While useful, these methods fail to consider differences in pollen quality and the fitness of zygotes resulting from pollination by different floral visitors. Here we show that, for three common native self-compatible plants in Southern California, super-abundant, non-native honeybees (Apis mellifera L.) visit more flowers on an individual before moving to the next plant compared with the suite of native insect visitors. This probably increases the transfer of self-pollen. Offspring produced after honeybee pollination have similar fitness to those resulting from hand self-pollination and both are far less fit than those produced after pollination by native insects or by cross-pollination. Because honeybees often forage methodically, visiting many flowers on each plant, low offspring fitness may commonly result from honeybee pollination of self-compatible plants. To our knowledge, this is the first study to directly compare the fitness of offspring resulting from honeybee pollination to that of other floral visitors.

Non-native honey bees disproportionately dominate the most abundant floral resources in a biodiversity hotspot.

Most plant-pollinator mutualisms are generalized. As such, they are susceptible to perturbation by abundant, generalist, non-native pollinators such as the western honey bee ( Apis mellifera), which can reach high abundances and visit flowers of many plant species in their expansive introduced range. Despite the prevalence of non-native honey bees, their effects on pollination mutualisms in natural ecosystems remain incompletely understood. Here, we contrast community-level patterns of floral visitation by honey bees with that of the diverse native pollinator fauna of southern California, USA. We show that the number of honey bees visiting plant species increases much more rapidly with flower abundance than does that of non-honey bee insects, such that the percentage of all visitors represented by honey bees increases with flower abundance. Thus, honey bees could disproportionately impact the most abundantly blooming plant species and the large numbers of both specialized and generalized pollinator species that they sustain. Honey bees may preferentially exploit high-abundance floral resources because of their ability to recruit nest-mates; these foraging patterns may cause native insect species to forage on lower-abundance resources to avoid competition. Our results illustrate the importance of understanding foraging patterns of introduced pollinators in order to reveal their ecological impacts.

The worldwide importance of honey bees as pollinators in natural habitats.

The western honey bee (Apis mellifera) is the most frequent floral visitor of crops worldwide, but quantitative knowledge of its role as a pollinator outside of managed habitats is largely lacking. Here we use a global dataset of 80 published plant-pollinator interaction networks as well as pollinator effectiveness measures from 34 plant species to assess the importance of A. mellifera in natural habitats. Apis mellifera is the most frequent floral visitor in natural habitats worldwide, averaging 13% of floral visits across all networks (range 0-85%), with 5% of plant species recorded as being exclusively visited by A. mellifera For 33% of the networks and 49% of plant species, however, A. mellifera visitation was never observed, illustrating that many flowering plant taxa and assemblages remain dependent on non-A. mellifera visitors for pollination. Apis mellifera visitation was higher in warmer, less variable climates and on mainland rather than island sites, but did not differ between its native and introduced ranges. With respect to single-visit pollination effectiveness, A. mellifera did not differ from the average non-A. mellifera floral visitor, though it was generally less effective than the most effective non-A. mellifera visitor. Our results argue for a deeper understanding of how A. mellifera, and potential future changes in its range and abundance, shape the ecology, evolution, and conservation of plants, pollinators, and their interactions in natural habitats.

The effect of removing numerically dominant, non-native honey bees on seed set of a native plant.

Pollination services are compromised by habitat destruction, land-use intensification, pesticides, and introduced species. How pollination services respond to such stressors depends on the capacity of pollinator assemblages to function in the face of environmental disruption. Here, we quantify how pollination services provided to a native plant change upon removal of the non-native, western honey bee (Apis mellifera)-a numerically dominant floral visitor in the native bee-rich ecosystems of southern California. We focus on services provided to clustered tarweed (Deinandra fasciculata), a native, annual forb that benefits from outcross pollination. Across five different study sites in coastal San Diego County, tarweed flowers attracted 70 insect taxa, approximately half of which were native bees, but non-native honey bees were always the most abundant floral visitor at each site. To test the ability of the native insect fauna to provide pollination services, we performed Apis removals within experimental 0.25 m2 plots containing approximately 20 tarweed plants and compared visitation and seed set between plants in removal and paired control plots (n = 16 pairs). Even though 92% of observed floral visits to control plots were from honey bees, Apis removal reduced seed production by only 14% relative to plants in control plots. These results indicate that native insect assemblages can contribute important pollination services even in ecosystems numerically dominated by introduced pollinators.

Global arthropod beta-diversity is spatially and temporally structured by latitude.

Global biodiversity gradients are generally expected to reflect greater species replacement closer to the equator. However, empirical validation of global biodiversity gradients largely relies on vertebrates, plants, and other less diverse taxa. Here we assess the temporal and spatial dynamics of global arthropod biodiversity dynamics using a beta-diversity framework. Sampling includes 129 sampling sites whereby malaise traps are deployed to monitor temporal changes in arthropod communities. Overall, we encountered more than 150,000 unique barcode index numbers (BINs) (i.e. species proxies). We assess between site differences in community diversity using beta-diversity and the partitioned components of species replacement and richness difference. Global total beta-diversity (dissimilarity) increases with decreasing latitude, greater spatial distance and greater temporal distance. Species replacement and richness difference patterns vary across biogeographic regions. Our findings support long-standing, general expectations of global biodiversity patterns. However, we also show that the underlying processes driving patterns may be regionally linked.

Admixture in Africanized honey bees (Apis mellifera) from Panamá to San Diego, California (U.S.A.).

The Africanized honey bee (AHB) is a New World amalgamation of several subspecies of the western honey bee (Apis mellifera), a diverse taxon historically grouped into four major biogeographic lineages: A (African), M (Western European), C (Eastern European), and O (Middle Eastern). In 1956, accidental release of experimentally bred "Africanized" hybrids from a research apiary in Sao Paulo, Brazil initiated a hybrid species expansion that now extends from northern Argentina to northern California (U.S.A.). Here, we assess nuclear admixture and mitochondrial ancestry in 60 bees from four countries (Panamá; Costa Rica, Mexico; U.S.A) across this expansive range to assess ancestry of AHB several decades following initial introduction and test the prediction that African ancestry decreases with increasing latitude. We find that AHB nuclear genomes from Central America and Mexico have predominately African genomes (76%-89%) with smaller contributions from Western and Eastern European lineages. Similarly, nearly all honey bees from Central America and Mexico possess mitochondrial ancestry from the African lineage with few individuals having European mitochondria. In contrast, AHB from San Diego (CA) shows markedly lower African ancestry (38%) with substantial genomic contributions from all four major honey bee lineages and mitochondrial ancestry from all four clades as well. Genetic diversity measures from all New World populations equal or exceed those of ancestral populations. Interestingly, the feral honey bee population of San Diego emerges as a reservoir of diverse admixture and high genetic diversity, making it a potentially rich source of genetic material for honey bee breeding.

Differential strengths of selection on S-RNases from Physalis and Solanum (Solanaceae).

BACKGROUND: The S-RNases of the Solanaceae are highly polymorphic self-incompatibility (S-) alleles subject to strong balancing selection. Relatively recent diversification of S-alleles has occurred in the genus Physalis following a historical restriction of S-allele diversity. In contrast, the genus Solanum did not undergo a restriction of S-locus diversity and its S-alleles are generally much older. Because recovery from reduced S-locus diversity should involve increased selection, we employ a statistical framework to ask whether S-locus selection intensities are higher in Physalis than Solanum. Because different S-RNase lineages diversify in Physalis and Solanum, we also ask whether different sites are under selection in different lineages. RESULTS: Maximum-likelihood and Bayesian coalescent methods found higher intensities of selection and more sites under significant positive selection in the 48 Physalis S-RNase alleles than the 49 from Solanum. Highest posterior densities of dN/dS (ω) estimates show that the strength of selection is greater for Physalis at 36 codons. A nested maximum likelihood method was more conservative, but still found 16 sites with greater selection in Physalis. Neither method found any codons under significantly greater selection in Solanum. A random effects likelihood method that examines data from both taxa jointly confirmed higher selection intensities in Physalis, but did not find different proportions of sites under selection in the two datasets. The greatest differences in strengths of selection were found in the most variable regions of the S-RNases, as expected if these regions encode self-recognition specificities. Clade-specific likelihood models indicated some codons were under greater selection in background Solanum lineages than in specific lineages of Physalis implying that selection on sites may differ among lineages. CONCLUSIONS: Likelihood and Bayesian methods provide a statistical approach to testing differential selection across populations or species. These tests appear robust to the levels of polymorphism found in diverse S-allele collections subject to strong balancing selection. As predicted, the intensity of selection at the S-locus was higher in the taxon with more recent S-locus diversification. This is the first confirmation by statistical test of differing selection intensities among self-incompatibility alleles from different populations or species.

A 15-Myr-old genetic bottleneck.

Balancing selection preserves variation at the self-incompatibility locus (S-locus) of flowering plants for tens of millions of years, making it possible to detect demographic events that occurred prior to the origin of extant species. In contrast to other Solanaceae examined, SI species in the sister genera Physalis and Witheringia share restricted variation at the S-locus. This restriction is indicative of an ancient bottleneck that occurred in a common ancestor. We sequenced 14 S-alleles from the subtribe Iochrominae, a group that is sister to the clade containing Physalis and Witheringia. At least 6 ancient S-allele lineages are represented among these alleles, demonstrating that the Iochrominae taxa do not share the restriction in S-locus diversity. Therefore, the bottleneck occurred after the divergence of the Iochrominae from the lineage leading to the most recent common ancestor of Physalis and Witheringia. Using cpDNA sequences, 3 fossil dates, and a Bayesian-relaxed molecular clock approach, the crown group of Solanaceae was estimated to be 51 Myr old and the restriction of variation at the S-locus occurred 14.0-18.4 Myr before present. These results confirm the great age of polymorphism at the S-locus and the utility of loci under balancing selection for deep historical inference.

The distribution of plant mating systems: study bias against obligately outcrossing species.

Early models of plant mating-system evolution argued that predominant outcrossing and selfing are alternative stable states. At least for animal-pollinated species, recent summaries of empirical studies have suggested the opposite-that outcrossing rates do not show the expected bimodal distribution. However, it is generally accepted that several potential biases can affect conclusions from surveys of published outcrossing rates. Here, we examine one potential bias and find that published studies of outcrossing rates contain far fewer obligate outcrossers than expected. We approximate the magnitude of this study bias and present the distribution of outcrossing rates after compensating for it. Because this study examines only one potential bias, and finds it to be large, conclusions regarding either the frequency of mixed mating or the shape of the distribution of outcrossing rates in nature are premature.

Metabarcoding of Fecal Samples to Determine Herbivore Diets: A Case Study of the Endangered Pacific Pocket Mouse.

Understanding the diet of an endangered species illuminates the animal's ecology, habitat requirements, and conservation needs. However, direct observation of diet can be difficult, particularly for small, nocturnal animals such as the Pacific pocket mouse (Heteromyidae: Perognathus longimembris pacificus). Very little is known of the dietary habits of this federally endangered rodent, hindering management and restoration efforts. We used a metabarcoding approach to identify source plants in fecal samples (N = 52) from the three remaining populations known. The internal transcribed spacers (ITS) of the nuclear ribosomal loci were sequenced following the Illumina MiSeq amplicon strategy and processed reads were mapped to reference databases. We evaluated a range of threshold mapping criteria and found the best-performing setting generally recovered two distinct mock communities in proportions similar to expectation. We tested our method on captive animals fed a known diet and recovered almost all plant sources, but found substantial heterogeneity among fecal pellets collected from the same individual at the same time. Observed richness did not increase with pooling of pellets from the same individual. In field-collected samples, we identified 4-14 plant genera in individual samples and 74 genera overall, but over 50 percent of reads mapped to just six species in five genera. We simulated the effects of sequencing error, variable read length, and chimera formation to infer taxon-specific rates of misassignment for the local flora, which were generally low with some exceptions. Richness at the species and genus levels did not reach a clear asymptote, suggesting that diet breadth remained underestimated in the current pool of samples. Large numbers of scat samples are therefore needed to make inferences about diet and resource selection in future studies of the Pacific pocket mouse. We conclude that our minimally invasive method is promising for determining herbivore diets given a library of sequences from local plants.

Contrasting patterns of floral and molecular variation across a cline in Mimulus aurantiacus.

Steep clines in ecologically important traits may be caused by divergent natural selection. However, processes that do not necessarily invoke ongoing selection, such as secondary contact or restricted gene flow, can also cause patterns of phenotypic differentiation over short spatial scales. Distinguishing among all possible scenarios is difficult, but an attainable goal is to establish whether scenarios that imply selection need to be invoked. We compared the extent of morphological and genetic differentiation between geographically structured red and yellow floral races of Mimulus aurantiacus (bush monkeyflower; Phrymaceae). Flower color was assessed in a common garden as well as in the field to determine whether variation was genetic and to quantify the extent of geographical differentiation. Population genetic differentiation at marker loci was measured for both chloroplast and nuclear genomes, and the degree of population structure within and among the floral races was evaluated. Flower color shows both a strong genetic basis and a sharp geographic transition, with pure red-flowered populations in western San Diego County and pure yellow-flowered populations to the east. In the zone of contact, both pure and intermediate phenotypes occur. Patterns of genetic differentiation at marker loci are far less pronounced, as little of the variation is partitioned according to the differences in flower color. Phenotypic differentiation (Q(ST)) between populations with different flower colors is much greater than neutral genetic differentiation (F(ST)). When comparisons are made between populations of the same flower color, the opposite trend is evident. Limited neutral genetic structure between the floral races, combined with sharp differentiation in flower color, is consistent with the hypothesis that current or recent natural selection maintains the cline in flower color.

Association of ploidy and sexual system in Lycium californicum (Solanaceae).

In North American Lycium (Solanaceae), the evolution of gender dimorphism has been proposed as a means of restoring outcrossing after polyploidization causes the loss of self-incompatibility. Previous studies of this process in Lycium focused on comparisons between species that differ in ploidy. We examined intraspecific variation in floral morphology and DNA content in populations of L. californicum to determine correlations between sexual system and cytotype. We also used nuclear ITS and GBSSI sequence data to determine whether diploid and polyploid forms represent the same phylogenetic species, and the phylogeographic relationships among populations and ploidy levels. Within populations, no variation in ploidy was found, although among populations there was a perfect correspondence between sexual system and cytotype. Diploid populations were all hermaphroditic, whereas tetraploid populations were all gender dimorphic. There was no clear geographic pattern to the occurrence of diploid and tetraploid forms. Phylogenetic analysis confirms that L. californicum, regardless of ploidy, forms a monophyletic group within the genus Lycium. Sequences from diploid and polyploid individuals did not form reciprocally monophyletic clades, indicating either multiple gains of polyploidy, ongoing gene flow between cytotypes, or lack of lineage sorting since the evolution of polyploidy. The correspondence between ploidy and sex expression is consistent with the hypothesis that polyploidization triggers the evolution of gender dimorphism in this and other Lycium species.

Range and Frequency of Africanized Honey Bees in California (USA).

Africanized honey bees entered California in 1994 but few accounts of their northward expansion or their frequency relative to European honey bees have been published. We used mitochondrial markers and morphometric analyses to determine the prevalence of Africanized honeybees in San Diego County and their current northward progress in California west of the Sierra Nevada crest. The northernmost African mitotypes detected were approximately 40 km south of Sacramento in California's central valley. In San Diego County, 65% of foraging honey bee workers carry African mitochondria and the estimated percentage of Africanized workers using morphological measurements is similar (61%). There was no correlation between mitotype and morphology in San Diego County suggesting Africanized bees result from bidirectional hybridization. Seventy percent of feral hives, but only 13% of managed hives, sampled in San Diego County carried the African mitotype indicating that a large fraction of foraging workers in both urban and rural San Diego County are feral. We also found a single nucleotide polymorphism at the DNA barcode locus COI that distinguishes European and African mitotypes. The utility of this marker was confirmed using 401 georeferenced honey bee sequences from the worldwide Barcode of Life Database. Future censuses can determine whether the current range of the Africanized form is stable, patterns of introgression at nuclear loci, and the environmental factors that may limit the northern range of the Africanized honey bee.

Hummingbird foraging position is altered by the touch-sensitive stigma of bush monkeyflower.

The two stigma lobes of bush monkeyflower (Mimulus aurantiacus) close together rapidly in response to touch by a hummingbird pollinator and usually remain closed for the life of the flower, preventing further pollen receipt. Previous work showed that hummingbirds visiting bush monkeyflowers with closed stigmas export more than twice as much pollen to recipient flowers as birds visiting flowers with open stigmas. To investigate how stigma closure increases pollen export, we used videotape to examine the interaction between bird and flower. Hummingbirds altered their method of foraging in response to whether the stigma was open or closed. When stigmas were closed, birds most frequently entered flowers from above, relative to the long axis of the corolla tube, bringing the bill close to the ceiling of the corolla where anthers and stigma are positioned. However, birds were much more likely to enter corollas from below when stigmas were open, keeping the bill farther from the sex organs, and resulting in failure to contact open stigmas 33% of the time. Distance of the bill from the corolla-tube ceiling was associated with frequency of contact with anthers and, when contact was made, explained pollen export in a non-linear fashion. Morphometric analyses showed that smaller corolla openings promoted stigma contact, but corolla openings that were wide relative to their height produced increased contact with anthers. Changes in hummingbird foraging position and reduced pollen export when visiting flowers with open stigmas helps to explain the observation of permanent stigma closure, even when less than full seed set has been achieved.

A new species of Triepeolus (Hymenoptera: Apidae), with comments on T. utahensis (Cockerell) and T. melanarius Rightmyer.

Triepeolus matildae Rightmyer, sp. nov., from Mexico (Baja California) and USA (California) is described and both genders are differentiated from the closely related species T. utahensis (Cockerell) using morphological characters. The synonymy of T. utahensis and T. heterurus was established in Rightmyer (2008); however, the younger name was used in that treatment, an error that is corrected herein. Males of both T. matildae and T. utahensis are additionally differentiated from T. melanarius Rightmyer, which is morphologically similar in that gender only. DNA barcoding evidence supporting the recognition of the new species is additionally presented.

Evolutionary genetics of an S-like polymorphism in Papaveraceae with putative function in self-incompatibility.

BACKGROUND: Papaver rhoeas possesses a gametophytic self-incompatibility (SI) system not homologous to any other SI mechanism characterized at the molecular level. Four previously published full length stigmatic S-alleles from the genus Papaver exhibited remarkable sequence divergence, but these studies failed to amplify additional S-alleles despite crossing evidence for more than 60 S-alleles in Papaver rhoeas alone. METHODOLOGY/PRINCIPAL FINDINGS: Using RT-PCR we identified 87 unique putative stigmatic S-allele sequences from the Papaveraceae Argemone munita, Papaver mcconnellii, P. nudicuale, Platystemon californicus and Romneya coulteri. Hand pollinations among two full-sib families of both A. munita and P. californicus indicate a strong correlation between the putative S-genotype and observed incompatibility phenotype. However, we also found more than two S-like sequences in some individuals of A. munita and P. californicus, with two products co-segregating in both full-sib families of P. californicus. Pairwise sequence divergence estimates within and among taxa show Papaver stigmatic S-alleles to be the most variable with lower divergence among putative S-alleles from other Papaveraceae. Genealogical analysis indicates little shared ancestral polymorphism among S-like sequences from different genera. Lack of shared ancestral polymorphism could be due to long divergence times among genera studied, reduced levels of balancing selection if some or all S-like sequences do not function in incompatibility, population bottlenecks, or different levels of recombination among taxa. Preliminary estimates of positive selection find many sites under selective constraint with a few undergoing positive selection, suggesting that self-recognition may depend on amino acid substitutions at only a few sites. CONCLUSIONS/SIGNIFICANCE: Because of the strong correlation between genotype and SI phenotype, sequences reported here represent either functional stylar S-alleles, tightly linked paralogs of the S-locus or a combination of both. The considerable complexity revealed in this study shows we have much to learn about the evolutionary dynamics of self-incompatibility systems.

Species selection maintains self-incompatibility.

Identifying traits that affect rates of speciation and extinction and, hence, explain differences in species diversity among clades is a major goal of evolutionary biology. Detecting such traits is especially difficult when they undergo frequent transitions between states. Self-incompatibility, the ability of hermaphrodites to enforce outcrossing, is frequently lost in flowering plants, enabling self-fertilization. We show, however, that in the nightshade plant family (Solanaceae), species with functional self-incompatibility diversify at a significantly higher rate than those without it. The apparent short-term advantages of potentially self-fertilizing individuals are therefore offset by strong species selection, which favors obligate outcrossing.

RNase-based self-incompatibility: puzzled by pollen S.

Many plants have a genetically determined self-incompatibility system in which the rejection of self pollen grains is controlled by alleles of an S locus. A common feature of these S loci is that separate pollen- and style-expressed genes (pollen S and style S, respectively) determine S allele identity. The long-held view has been that pollen S and style S must be a coevolving gene pair in order for allelic recognition to be maintained as new S alleles arise. In at least three plant families, the Solanaceae, Rosaceae, and Plantaginaceae, the style S gene has long been known to encode an extracellular ribonuclease called the S-RNase. Pollen S in these families has more recently been identified and encodes an F-box protein known as either SLF or SFB. In this perspective, we describe the puzzling evolutionary relationship that exists between the SLF/SFB and S-RNase genes and show that in most cases cognate pairs of genes are not coevolving in the expected manner. Because some pollen S genes appear to have arisen much more recently than their style S cognates, we conclude that either some pollen S genes have been falsely identified or that there is a major problem with our understanding of how the S locus evolves.

Population structure at the S-locus of Sorbus aucuparia L. (Rosaceae: Maloideae).

Low sequence divergence within functional alleles is predicted for the self-incompatibility locus because of strong negative frequency-dependent selection. Nevertheless, sequence variation within functional alleles is essential for current models of the evolution of new mating types. We genotyped the stylar self-incompatibility RNase of 20 Sorbus aucuparia from a population in the Pyrenees mountains of France in order to compare alleles found there to those previously sampled in a Belgian population. Both populations returned 20 different alleles from samples of 20 individuals, providing maximum-likelihood estimates of 24.4 (95% CI 20-34) alleles in each. Ten alleles occurred in both samples. The maximum likelihood (ML) estimate of the overlap in the alleles present in both populations was 16, meaning that an estimated eight alleles are private to each population, and a total of 32 alleles occur across the two populations examined. We used Fisher's (1961) missing plot method to estimate that 40 alleles occur in the species. In accord with population genetics theory, we observed at most one synonymous sequence difference between copies of alleles sampled from the different populations and no variation within populations. Phylogenetic analysis shows that nearly every allele in S. aucuparia arose prior to divergence of this species from members of three different genera of the Rosaceae subfamily, Maloideae. Lack of observable sequence variation within alleles, coupled with the slow pace of allelic relative to taxonomic diversification, implies that finding intermediate stages in the process of new allele creation will be difficult in this group.

Ancient polymorphism reveals unidirectional breeding system shifts.

Loss of complex characters is thought to be irreversible (Dollo's law). However, hypotheses of irreversible evolution are remarkably difficult to test, especially when character transitions are frequent. In such cases, inference of ancestral states, in the absence of fossil evidence, is uncertain and represents the single greatest constraint for reconstructing the evolutionary history of characters. Breeding system character transitions are of particular interest because they affect the amount and distribution of genetic variation within species. Transitions from obligate outcrossing to partial or predominant self-fertilization are thought to represent one of the most common trends in flowering plants. We use the unique molecular genetic properties (manifested as deep persistent polymorphisms) of the locus that enforces outcrossing to demonstrate that its loss is irreversible in the plant family Solanaceae. We argue that current phylogenetic methods of reconstruction are potentially inadequate in cases where ancestral state information is inferred by using only the phylogeny and the distribution of character states in extant taxa. This study shows in a statistical framework that a particular character transition is irreversible, consistent with Dollo's law.