Refuge Update – March/April 2006, Volume 3, Number 2

Table of Contents: Energy Company Fine Benefits Ottawa Refuge, page 3 Hide and Seek…but Mostly Hide page 4 Focus on …Pollinators, page 10 Great Storytellers, page 18

An Investigation of Wild Bee Diversity and Abundance in Plots Managed by The Nature Conservancy in South-Central Nebraska and of Beneficial Arthropods Associated with Native Nebraska Flora

Insect pollination is an essential ecosystem service, and bees are the principal pollinators of wild and cultivated plants. Habitat management and enhancement are a proven way to encourage wild bee populations, providing them with food and nesting resources. I examined bee diversity and abundance in plots managed by The Nature Conservancy near Wood River, NE. The plots were seeded with 2 seed mixes at 2 seeding rates: high diversity mix at the recommended rate, high diversity mix double the recommended rate, Natural Resources Conservation Service (NRCS) conservation planting (CP) 25 mix at one-half the recommended rate, and NRCS CP25 mix at the recommended rate. I measured wild bee abundance and diversity, and established a database of wild bees associated with the plots. I also compared genus richness and abundance among the plots using and aerial net and blue vane traps to collect bees. Significant differences were not observed in genus richness and diversity among the plots; however, plot size and the ability of blue vane traps to draw bees from a long distance may have influenced my results. In 2008, 15 genera and 95 individual bees were collected using an aerial net and in 2009, 32 genera and 6,103 individual bees were collected using blue vane traps. I also studied the beneficial insects associated with native Nebraska flora. Seventeen species of native, perennial flora were established in 3 separate plots located in eastern Nebraska. I transplanted four plants of each species in randomized 0.61 m x 0.61 m squares of a 3.05 m x 9.14 m plot. Arthropods were sampled using a modified leaf blower/vacuum. Insects and other arthropods were identified to family and organized into groups of predators, parasites, pollinators, herbivores, and miscellaneous. Associations between plant species and families of beneficial arthropods (predators, parasites, and pollinators) were made. Pycnanthemum flexuosum Walter attracted significantly more beneficial arthropod families than 7 other species of plants tested. Dalea purpurea Vent and Liatris punctata Hook also attracted significantly fewer beneficial arthropod families than 4 other species of plants tested. In total, 31 predator, 11 parasitic, 4 pollinator, 31 herbivore, and 10 miscellaneous families of arthropods were recorded.

Techniques for the In Vitro Production of Queens in Stingless Bees (Apidae, Meliponini)

Considering the ecological importance of stingless bees as caretakers and pollinators of a variety of native plants makes it necessary to improve techniques which increase of colonies' number in order to preserve these species and the biodiversity associated with them. Thus, our aim was to develop a methodology of in vitro production of stingless bee queens by offering a large quantity of food to the larvae. Our methodology consisted of determining the amount of larval food needed for the development of the queens, collecting and storing the larval food, and feeding the food to the larvae in acrylic plates. We found that the total average amount of larval food in a worker bee cell of E varia is approximately 26.70 +/- 3.55 mu L. We observed that after the consumption of extra amounts of food (25, 30, 35 and 40 mu L) the larvae differentiate into queens (n = 98). Therefore, the average total volume of food needed for the differentiation of a young larva of F. varia queen is approximately 61.70 +/- 5.00 mu L. In other words; the larvae destined to become queens eat 2.31 times more food than the ones destined to become workers. We used the species Frieseomelitta varia as a model, however the methodology can be reproduced for all species of stingless bees whose mechanism of caste differentiation depends on the amount of food ingested by the larvae. Our results demonstrate the effectiveness of the in vitro technique developed herein, pointing to the possibility of its use as a tool to assist the production of queens on a large scale. This would allow for the artificial splitting of colonies and contribute to conservation efforts in native bees.

The importance of plant diversity in maintaining the pollinator bee, Eulaema nigrita (Hymenoptera: Apidae) in sweet passion fruit fields

The euglossine bee Eulaema nigrita plays an important role for the pollination of native and economically important plants, such as the sweet passion-fruit Passiflora alata. E. nigrita uniquely collects the nectar from the flowers of P alata, nevertheless, it needs to visit other plants to collect pollen, nectar and other resources for its survival. There are two methods to identify the species of plants used by bees in their diet: by direct observation of the bees in the flowers, and through identification of pollen grains present in brood cells, feces, or in the bees' body. In order to identify the other plants that E. nigrita visits, we analyzed samples of pollen grains removed from the bee's body in the course of the flowering period of P alata. Among our results, the flora visited by E. nigrita comprised 40 species from 32 genera and 19 families, some of them used as a pollen source or just nectar. In spite of being a polyletic species, E. nigrita exhibited preference for some plant species with poricidal anthers. P alata which has high sugar concentration nectar was the main source of nectar for this bee in the studied area. Nonetheless, the pollinic analysis indicated that others nectariferous plant species are necessary to keep the populations of E. nigrita. Studies such as this one are important since they indicate supplementary pollen-nectar sources which must be used for the conservation of the populations of E. nigrita in crops neighbouring areas. In the absence of pollinators, growers are forced to pay for hand pollination, which increases production costs; keeping pollinators in cultivated areas is still more feasible to ensure sweet passion fruit production. Rev. Biol. Trop. 60 (4): 1553-1565. Epub 2012 December 01.

Biotic versus abiotic pollination in Oeceoclades maculata (Lindl.) Lindl. (Orchidaceae)

Geographical variation in the reproductive biology of widespread species often occurs at their distributional boundaries. We sought to determine whether such variation has occurred in an invasive orchid, Oeceoclades maculata, across its naturalized range. We compared its reproductive biology in a Brazilian population with that published for a population on the Caribbean island of Puerto Rico. In the state of Sao Paulo, O. maculata flowers between December and February, at the height of the rainy season. Similar fruit sets were observed in manual self (76%) and cross (70.4%) pollination treatments. The fruit set of plants protected from both pollinators and rainfall was 6.1%, whereas plants exposed only to rainfall had a fruit set of 41.4%, slightly less than the controls (48.3%). Like the Puerto Rico population, reproduction is primarily through rain-assisted autogamy, but unlike observations made on the island, outcrossing can eventually occur. We observed two butterfly species (Heliconius ethilla narcaea and Heliconius erato phyllis) pollinating O. maculata. Secretory epidermal cells and trichomes of the spur lumen produced 0.7 mu L of 25% (sucrose equivalents) nectar per flower each morning, which was stored in a dilated basal portion of the spur and reabsorbed by the afternoon. Thus, geographical variation in reproductive biology exists across the broad invasive range of O. maculata.

The Ecological Basis for Biogeographic Classification: an Example in Orchid Bees (Apidae: Euglossini)

Biogeography has been difficult to apply as a methodological approach because organismic biology is incomplete at levels where the process of formulating comparisons and analogies is complex. The study of insect biogeography became necessary because insects possess numerous evolutionary traits and play an important role as pollinators. Among insects, the euglossine bees, or orchid bees, attract interest because the study of their biology allows us to explain important steps in the evolution of social behavior and many other adaptive tradeoffs. We analyzed the distribution of morphological characteristics in Colombian orchid bees from an ecological perspective. The aim of this study was to observe the distribution of these attributes on a regional basis. Data corresponding to Colombian euglossine species were ordered with a correspondence analysis and with subsequent hierarchical clustering. Later, and based on community proprieties, we compared the resulting hierarchical model with the collection localities to seek to identify a biogeographic classification pattern. From this analysis, we derived a model that classifies the territory of Colombia into 11 biogeographic units or natural clusters. Ecological assumptions in concordance with the derived classification levels suggest that species characteristics associated with flight performance, nectar uptake, and social behavior are the factors that served to produce the current geographical structure.

Hox Gene Expression Leads to Differential Hind Leg Development between Honeybee Castes

Beyond the physiological and behavioural, differences in appendage morphology between the workers and queens of Apis mellifera are pre-eminent. The hind legs of workers, which are highly specialized pollinators, deserve special attention. The hind tibia of worker has an expanded bristle-free region used for carrying pollen and propolis, the corbicula. In queens this structure is absent. Although the morphological differences are well characterized, the genetic inputs driving the development of this alternative morphology remain unknown. Leg phenotype determination takes place between the fourth and fifth larval instar and herein we show that the morphogenesis is completed at brown-eyed pupa. Using results from the hybridization of whole genome-based oligonucleotide arrays with RNA samples from hind leg imaginal discs of pre-pupal honeybees of both castes we present a list of 200 differentially expressed genes. Notably, there are castes preferentially expressed cuticular protein genes and members of the P450 family. We also provide results of qPCR analyses determining the developmental transcription profiles of eight selected genes, including abdominal-A, distal-less and ultrabithorax (Ubx), whose roles in leg development have been previously demonstrated in other insect models. Ubx expression in workers hind leg is approximately 25 times higher than in queens. Finally, immunohistochemistry assays show that Ubx localization during hind leg development resembles the bristles localization in the tibia/basitarsus of the adult legs in both castes. Our data strongly indicate that the development of the hind legs diphenism characteristic of this corbiculate species is driven by a set of caste-preferentially expressed genes, such as those encoding cuticular protein genes, P450 and Hox proteins, in response to the naturally different diets offered to honeybees during the larval period.

An Extreme Case of Plant-Insect Codiversification: Figs and Fig-Pollinating Wasps

It is thought that speciation in phytophagous insects is often due to colonization of novel host plants, because radiations of plant and insect lineages are typically asynchronous. Recent phylogenetic comparisons have supported this model of diversification for both insect herbivores and specialized pollinators. An exceptional case where contemporaneous plant-insect diversification might be expected is the obligate mutualism between fig trees (Ficus species, Moraceae) and their pollinating wasps (Agaonidae, Hymenoptera). The ubiquity and ecological significance of this mutualism in tropical and subtropical ecosystems has long intrigued biologists, but the systematic challenge posed by >750 interacting species pairs has hindered progress toward understanding its evolutionary history. In particular, taxon sampling and analytical tools have been insufficient for large-scale cophylogenetic analyses. Here, we sampled nearly 200 interacting pairs of fig and wasp species from across the globe. Two supermatrices were assembled: on an average, wasps had sequences from 77% of 6 genes (5.6 kb), figs had sequences from 60% of 5 genes (5.5 kb), and overall 850 new DNA sequences were generated for this study. We also developed a new analytical tool, Jane 2, for event-based phylogenetic reconciliation analysis of very large data sets. Separate Bayesian phylogenetic analyses for figs and fig wasps under relaxed molecular clock assumptions indicate Cretaceous diversification of crown groups and contemporaneous divergence for nearly half of all fig and pollinator lineages. Event-based cophylogenetic analyses further support the codiversification hypothesis. Biogeographic analyses indicate that the present-day distribution of fig and pollinator lineages is consistent with a Eurasian origin and subsequent dispersal, rather than with Gondwanan vicariance. Overall, our findings indicate that the fig-pollinator mutualism represents an extreme case among plant-insect interactions of coordinated dispersal and long-term codiversification.

Geopropolis from Melipona scutellaris decreases the mechanical inflammatory hypernociception by inhibiting the production of IL-1 beta and TNF-alpha

Ethnopharmacological relevance: The pharmacological activity of geopropolis collected by stingless bees (important and threatened pollinators), a product widely used in folk medicine by several communities in Brazil, especially in the Northeast Region, needs to be studied. Objective: The aim of this study was to evaluate the antinociceptive activity of Melipona scutellaris geopropolis (stingless bee) using different models of nociception. Material and methods: The antinociceptive activity of the ethanolic extract of geopropolis (EEGP) and fractions was evaluated using writhing induced by acetic acid, formalin test, carrageenan-induced hypernociception, and quantification of IL-1 beta and TNF-alpha. The chemical composition was assessed by quantification of total flavonoids and phenolic compounds. Results: EEGP and its hexane and aqueous fractions showed antinociceptive activity. Both EEGP and its aqueous fraction presented activity in the mechanical inflammatory hypernociception induced by the carrageenan model, an effect mediated by the inhibition of IL-1 beta and TNF-alpha. The chemical composition of EEGP and its hexane and aqueous fractions showed a significant presence of phenolic compounds and absence of flavonoids. Conclusion: Our data indicate that geopropolis is a natural source of bioactive substances with promising antinociceptive activity. (C) 2012 Elsevier Ireland Ltd. All rights reserved.

Pollination services at risk: Bee habitats will decrease owing to climate change in Brazil

Native bees are important providers of pollination services, but there are cumulative evidences of their decline. Global changes such as habitat losses, invasions of exotic species and climate change have been suggested as the main causes of the decline of pollinators. In this study, the influence of climate change on the distribution of 10 species of Brazilian bees was estimated with species distribution modelling. We used Maxent algorithm (maximum entropy) and two different scenarios, an optimistic and a pessimistic, to the years 2050 and 2080. We also evaluated the percentage reduction of species habitat based on the future scenarios of climate change through Geographic Information System (GIS). Results showed that the total area of suitable habitats decreased for all species but one under the different future scenarios. The greatest reductions in habitat area were found for Melipona bicolor bicolor and Melipona scutellaris, which occur predominantly in areas related originally to Atlantic Moist Forest. The species analysed have been reported to be pollinators of some regional crops and the consequence of their decrease for these crops needs further clarification. (C) 2012 Elsevier B.V. All rights reserved.

Consequences of plant population size for pollinator visitation and plant reproductive success

Habitat loss and fragmentation have a prominent role in determining the size of plant populations, and can affect plant-pollinator interactions. It is hypothesized that in small plant populations the ability to set seeds can be reduced due to limited pollination services, since individuals in small populations can receive less quantity or quality of visits. In this study, I investigated the effect of population size on plant reproductive success and insect visitation in 8 populations of two common species in the island of Lesvos, Greece (Mediterranean Sea), Echium plantagineum and Ballota acetabulosa, and of a rare perennial shrub endemic to north-central Italy, Ononis masquillierii. All the three species depended on insect pollinators for sexual reproduction. For each species, pollen limitation was present in all or nearly all populations, but the relationship between pollen limitation and population size was only present in Ononis masquillierii. However, in Echium plantagineum, significant relationships between both open-pollinated and handcrossed-pollinated seed sets and population size were found, being small populations comparatively less productive than large ones. Additionally, for this species, livestock grazing intensity was greater for small populations and for sparse patches, and had a negative influence on productivity of the remnant plants. Both Echium plantagineum and Ballota acetabulosa attracted a great number of insects, representing a wide spectrum of pollinators, thereby can be considered as generalist species. For Ballota acetabulosa, the most important pollinators were megachilid female bees, and insect diversity didn’t decrease with decreasing plant population size. By contrast, Ononis masquillierii plants generally received few visits, with flowers specialized on small bees (Lasioglossum spp.), representing the most important insect guild. In Echium plantagineum and Ballota acetabulosa, plants in small and large populations received the same amount of visits per flower, and no differences in the number of intraplant visited flowers were detected. On the contrary, large Ononis populations supported higher amounts of pollinators than small ones. At patch level, high Echium flower density was associated with more and higher quality pollinators. My results indicate that small populations were not subject to reduced pollination services than large ones in Echium plantagineum and Ballota acetabulosa, and suggest that grazing and resource limitation could have a major impact on population fitness in Echium plantagineum. The absence of any size effects in these two species can be explained in the light of their high local abundance, wide habitat specificity, and ability to compete with other co-flowering species for pollinators. By contrast, size represents a key characteristic for both pollination and reproduction in Ononis masquillierii populations, as an increase in size could mitigate the negative effects coming from the disadvantageous reproductive traits of the species. Finally, the widespread occurrence of pollen limitation in the three species may be the result of 1) an ongoing weakening or disruption of plantpollinator interactions derived from ecological perturbations, 2) an adaptive equilibrium in response to stochastic processes, and 3) the presence of unfavourable reproductive traits (for Ononis masquillierii).

Ornithophily in the genus Salvia L. (Lamiaceae)

This dissertation presents for the first time a survey of bird pollinated (ornithophilous) Salvia species. Within the approximately 1000 species of the worldwide distributed genus roughly 20% (186 spp.) are bird pollinated. Excepting four species in the Old World (South Africa and Madagascar), ornithophilous species are restricted to the New World where they represent about one third of the species. They occur mainly in higher altitudes (1500-3000m) and usually grow as shrubs or perennial herbs (97%). The bilabiate to tubular flowers are often red (at least 49%), averaging 35mm (7-130mm) in length and produce a large to medium volume of nectar with rather low sugar concentration. Pollination by sunbirds and white-eyes is documented in a South African species, and that by hummingbirds in 16 species of the New World (USA, Mexico, Guatemala and Bolivia). Beside pollinator observations, the functionality of the staminal levers, the process of pollen transfer and the fitting between flowers and birds are tested by inserting museum skins and metal rods into fresh flowers. The most surprising result is the finding of two different main pollen transfer mechanisms. In at least 54% of the species an active staminal lever mechanism enables pollen deposition on the birds body. This is illustrated in detail in the South African S. lanceolata at which birds were observed to release the lever mechanism and became dusted with pollen. In contrast, the lever mechanism in about 35% of the New World species is reduced in different ways. Pollen transfer by inactive ‘levers’ is demonstrated in detail in S. haenkei in Bolivia, at which four pollinating hummingbird species could be observed. The tubular corolla forced the birds in a specific position, thereby causing pollen transfer from the exserted pollen-sacs to the birds body. With respect to the floral diversity and systematic affiliation of the species, parallel evolution of ornithophily and lever reduction is likely. Considering that bird pollinated species might have derived from bee pollinated species and that the staminal levers have become secondarily inactive, it is concluded that the shift in pollinators induced phenotypic changes even disabling such a sophisticated structure as the staminal lever mechanism.

Taxonomy, phylogeny and reproductive ecology of Gentiana lutea L.

This research focuses on taxonomy, phylogeny and reproductive ecology of Gentiana lutea. L.. Taxonomic analysis is a critical step in botanical studies, as it is necessary to recognize taxonomical unit. Herbarium specimens were observed to assess the reliability of several subspecies-diagnostic characters. The analysis of G. lutea genetic variability and the comparison with that of the other species of sect. Gentiana were performed to elucidate phylogenetic relationships among G. lutea subspecies and to propose a phylogenetic hypothesis for the evolution and the colonization dynamics of the section. Appropriate scientific information is critical for the assessment of species conservation status and for effective management plans. I carried out field work on five natural populations and performed laboratory analyses on specific critical aspects, with special regard to G. lutea breeding system and type and efficiency of plant-pollinator system. Bracts length is a reliable character to identify subsp. vardjanii, however it is not exclusive, hence to clearly identify subsp. vardjanii, other traits have to be considered. The phylogenetic hypotheses obtained from nuclear and chloroplast data are not congruent. Nuclear markers show a monophyly of sect. Gentiana, a strongly species identity of G. lutea and clear genetic identity of subsp. vardjanii. The little information emerging from plastid markers indicate a weak signal of hybridization and incomplete sorting of ancestral lineages. G. lutea shows a striking variation in intra-floral dichogamy probably evolved to reduce pollen-stigma interference. Although the species is partially self-compatible, pollen vectors are necessary for a successful reproduction, and moreover it shows a strong inbreeding depression. G. lutea is a generalist species: within its spectrum of visitors is possible to recognize "nectar thieves" and pollinators with sedentary or dynamic behaviour. Pollen limitation is frequent and it could be mainly explained by poor pollen quality.

Evolutionary tendencies in African Marantaceae

Die Marantaceae (550 Arten) sind eine weltweit verbreitete Familie von Stauden und Lianen aus dem Unterwuchs tropischer Tieflandregenwälder. Der morphologisch-ökologische Vergleich des basal abzweigenden Sarcophrynium-Astes mit dem in abgeleiteter Position stehenden Marantochloa-Ast, soll beispielhaft evolutionäre Muster in der Familie beleuchten. So wird in der Doktorarbeit zum ersten Mal ein Überblick über die Blütenbiologie und Phylogenie von rund 30 der 40 afrikanischen Marantaceae Arten präsentiert. Die Analysen basieren auf Daten von drei mehrmonatigen Feldaufenthalten in Gabun jeweils zwischen September und Januar. Vier Blütentypen werden beschrieben, die jeweils mit einer spezifischen Bestäubergilde verbunden sind (kleine, mittlere, große Bienen bzw. Vögel). Bestäubungsexperimente belegen, dass 18 Arten selbstkompatibel, aber nur zwei Arten autogam sind, also keine Bestäubungsvermittler benötigen. Der Fruchtansatz ist generell gering (10 -30 %). Die komplexe Synorganisation der Blüte ermöglicht in den Marantaceae einen explosiven Bestäubungsmechanismus. Um dessen ökologische Funktionalität zu verstehen, werden die Blüten von 66 Arten, alle wichtigen Äste der Marantaceae abdeckend, unter einem morphologisch-funktionalen Gesichtspunkt untersucht. Es gibt große Übereinstimmungen zwischen allen untersuchten Arten im Zusammenspiel (Synorganisation) der wichtigsten Bauelemente (Griffel, Kapuzenblatt, Schwielenblatt), die eine präzise Pollenübertragung ermöglichen. Basierend auf Daten von nrDNA (ITS, 5S) und cpDNA (trnL-F) wird für ein nahezu komplettes Artenspektrum die Phylogenie der zwei afrikanischen Äste erstellt. Hierauf werden morphologische und ökologische Merkmale sowie geographischer Verbreitungsmuster nach dem Parsimonieprinzip rekonstruiert, um so deren evolutionäre Bedeutung für die Marantaceae abschätzen zu können. Die Ergebnisse weisen auf die Beteiligung einer Vielzahl verschiedener Artbildungsfaktoren hin.

Pollen-placement and pollen-portioning in diverse Salvia-species

This dissertation addresses the staminal lever mechanism of the genus Salvia. Various hypotheses referring to its purpose and function are tested and elucidated. The first hypothesis maintains that the lever is a mechanical selection mechanism which excludes weak pollinators from the flower. This hypothesis is refuted and the respective results of force measurements and morphological investigations are presented, statistically evaluated and discussed. The force measurements and morphological investigations were conducted on the staminal levers and flowers of 8 bee pollinated (melittophilous) and 6 bird pollinated (ornithophilous) species. For comparison a ninth melittophilous species that lacks the staminal lever was investigated. In this species the force measurements were conducted on floral structures that were suspected to hinder a flower visitor. The hypotheses, which state that the staminal lever is a tool for pollen portioning and reduces the risk of pollen loss as well as hybridisation due to its ability to perform a repeatable, accurate and species-specific pollen placement on a wide range of diverse pollinators, are confirmed. Investigations with respect to pollen portioning were carried out on 13 sages. The lever mechanism can be released several times in a row, while the pollen sacs leave a dosed pollen portion on a well defined spot on the pollinator‘s body. Pollen placement was investigated for 12 sages. In sympatric sages, lever length and the area of pollen placement are of particular interest. A shared pollinator bears species-specific areas of pollen placement for different sages. The accurate pollen placement ensures an efficient pollination. However, the question of the functionality of the lever mechanism can not be answered with absolute certainty. The lever‘s backswing is not caused by the adaxial lever arm; the adaxial lever arm is too light and too short to be an adequate counterweight to the abaxial lever arm. Therefore, the adaxial lever arm can not pull the abaxial lever arm to return it to its neutral position. But there are indications of a cellular mainspring in the filament. According to the current state of knowledge, this is the most plausible explanation for the lever's backswing, but further histological investigations on the joint of the lever mechanism are necessary to confirm this assumption.