Revealing potential bridge vectors for BTV and SBV: a study on Culicoides blood feeding preferences in natural ecosystems in Spain.

Several species of Culicoides (Diptera: Ceratopogonidae) are vectors of pathogens, such as the bluetongue (BTV) and Schmallenberg (SBV) viruses, which cause important diseases in domestic and wild ruminants. As wild ruminants can contribute to overwintering and epizootics of both diseases, knowledge of the host-feeding behaviour of Culicoides in natural ecosystems is important to better understand their epidemiology. Blood-engorged Culicoides females trapped in natural areas inhabited by different wild ruminant species were genetically analysed to identify host species. The origin of bloodmeals was identified in 114 females of 14 species of Culicoides. A total of 104 (91.1%) Culicoides fed on mammals and 10 (8.9%) on birds. The most abundant host identified was red deer (66.7%), followed by humans (13%) and fallow deer (6.1%). Eleven of the 14 species of Culicoides fed exclusively on mammalian hosts. Among them, five are mammalophilic species considered to be important BTV and/or SBV vectors. The results of the present study confirm that Culicoides imicola, Culicoides obsoletus, Culicoides scoticus, Culicoides pulicaris and Culicoides punctatus fed on wild ruminants, and therefore support the hypothesis that these species can act as bridge vectors by facilitating the circulation of pathogens between wild and domestic ruminant communities.

Experimental study of the susceptibility of a European Aedes albopictus strain to dengue virus under a simulated Mediterranean temperature regime.

Dengue virus (DENV) has re-emerged in Europe driven by the geographic expansion of the mosquito species Aedes albopictus and Aedes aegypti (Diptera: Culicidae) and the introduction of the virus by viraemic travellers. In the present study, the vector competence (VC) of Ae. albopictus collected in Catalonia (northeast Spain) was evaluated for two different DENV strains, DENV-1 and DENV-2, the serotypes responsible for all outbreaks of dengue that have occurred in Europe. Mosquitoes were reared under environmental conditions mimicking the mean temperature and humidity recorded in July on the Mediterranean coast of Catalonia. Mosquitoes were fed on an artificial infectious bloodmeal and, at 14 days post-exposure, infection, disseminated infection and transmission rates (IR, DIR, TR) and transmission efficiency (TE) were determined by testing the virus in the body, legs and saliva. The tested Ae. albopictus strain was found to be susceptible to both DENV-1 and DENV-2 and to be able to transmit DENV-1. This is the first time that the VC of Ae. albopictus for DENV has been tested in Europe in this specific context (i.e. mimicking the Mediterranean temperature and humidity recorded in Catalonia in July). This study confirms the potential of Ae. albopictus to start autochthonous DENV transmission cycles in the Mediterranean basin.

Morphology and DNA barcoding reveal three species in one: description of Culicoides cryptipulicaris sp. nov. and Culicoides quasipulicaris sp. nov. in the subgenus Culicoides.

Species of the genus Culicoides (Diptera: Ceratopogonidae) are well known for their importance in the field of medical and veterinary entomology. Culicoides spp. transmit a wide variety of pathogens, primarily viruses that affect animals and humans. In Europe, the most economically important disease transmitted by Culicoides is bluetongue (BT). Culicoides spp. have been recently involved as primary vectors for Schmallenberg disease. The taxonomy within the subgenus Culicoides has been historically difficult and reorganizations have been proposed regularly. The subgenus Culicoides includes species that are considered to be potential vectors for BT. High morphological intraspecific variability has been attributed to these species. This highlights the apparent presence of previously undetected cryptic species diversity in the subgenus. In the present study, a detailed morphological and molecular study of specimens belonging to Culicoides pulicaris s.l. and specimens resembling a cross between C. pulicaris and Culicoides punctatus revealed the presence of two new species: Culicoides cryptipulicaris and Culicoides quasipulicaris. Females of C. quasipulicaris and males of both species were morphologically distinguished from C. pulicaris (Linnaeus, 1758), whereas females of C. cryptipulicaris were identified using molecular techniques exclusively.

Rift Valley fever virus and European mosquitoes: vector competence of Culex pipiens and Stegomyia albopicta (= Aedes albopictus).

Rift Valley fever (RVF) is a mosquito-borne disease caused by the Rift Valley fever virus (RVFV). Rift Valley fever affects a large number of species, including human, and has severe impact on public health and the economy, especially in African countries. The present study examined the vector competence of three different European mosquito species, Culex pipiens (Linnaeus, 1758) form molestus (Diptera: Culicidae), Culex pipiens hybrid form and Stegomyia albopicta (= Aedes albopictus) (Skuse, 1894) (Diptera: Culicidae). Mosquitoes were artificially fed with blood containing RVFV. Infection, disseminated infection and transmission efficiency were evaluated. This is the first study to assess the transmission efficiency of European mosquito species using a virulent RVFV strain. The virus disseminated in Cx. pipiens hybrid form and in S. albopicta. Moreover, infectious viral particles were isolated from saliva of both species, showing their RVFV transmission capacity. The presence of competent Cx. pipiens and S. albopicta in Spain indicates that an autochthonous outbreak of RVF may occur if the virus is introduced. These findings provide information that will help health authorities to set up efficient entomological surveillance and RVFV vector control programmes.

Culex pipiens and Stegomyia albopicta (= Aedes albopictus) populations as vectors for lineage 1 and 2 West Nile virus in Europe.

The emerging disease West Nile fever is caused by West Nile virus (WNV), one of the most widespread arboviruses. This study represents the first test of the vectorial competence of European Culex pipiens Linnaeus 1758 and Stegomyia albopicta (= Aedes albopictus) (both: Diptera: Culicidae) populations for lineage 1 and 2 WNV isolated in Europe. Culex pipiens and S. albopicta populations were susceptible to WNV infection, had disseminated infection, and were capable of transmitting both WNV lineages. This is the first WNV competence assay to maintain mosquito specimens under environmental conditions mimicking the field (day/night) conditions associated with the period of maximum expected WNV activity. The importance of environmental conditions is discussed and the issue of how previous experiments conducted in fixed high temperatures may have overestimated WNV vector competence results with respect to natural environmental conditions is analysed. The information presented should be useful to policymakers and public health authorities for establishing effective WNV surveillance and vector control programmes. This would improve preparedness to prevent future outbreaks.

Phenotypic differentiation and phylogenetic signal of wing shape in western European biting midges, Culicoides spp., of the subgenus Avaritia.

In the past decade biting midges of the subgenus Avaritia (Diptera: Ceratopogonidae) have been popular subjects of applied entomological studies in Europe owing to their implication as biological vectors in outbreaks of bluetongue and Schmallenberg viruses. This study uses a combination of cytochrome oxidase subunit I barcode sequencing and geometric morphometric analyses to investigate wing shape as a means to infer species identification within this subgenus. In addition the congruence of morphological data with different phylogenetic hypotheses is tested. Five different species of the subgenus Avaritia were considered in the study (C. obsoletus (Meigen); C. scoticus Kettle and Lawson; C. chiopterus (Meigen); C. dewulfi Goetghebuer and C. imicola (Kieffer)). The study demonstrated that over 90% of individuals could be separated correctly into species by their wing shape and that patterns of morphological differentiation derived from the geometric morphometric analyses were congruent with phylogenies generated from sequencing data. Morphological data produced are congruent with monophyly of the subgenus Avaritia and the exclusion of C. dewulfi from the group containing C. obsoletus, C. scoticus and C. chiopterus. The implications of these results and their importance in a wider context of integrating multiple data types to interpret both phylogeny and species characterization is discussed.

Molecular phylogeny and systematics of the highly polymorphic Rumex bucephalophorus complex (Polygonaceae).

Rumex bucephalophorus is a very polymorphic species that has been subjected to various taxonomic studies in which diverse infraspecific taxa have been recognised on the basis of diaspore traits. In this study we used molecular markers (ITS and AFLP) to explore this remarkable diversity, to test previous hypotheses of classification, and attempt to explain biogeographic patterns. Results show that R. bucephalophorus forms a monophyletic group in which diversification began around 4.2 Mya, at the end of Messinian Salinity Crisis. The two molecular markers clearly show a deep divergence separating subsp. bucephalophorus from all other subspecific taxa, among which subsp. canariensis also constitutes a separate and well distinguishable unit. In contrast, subspecies hispanicus and subsp. gallicus constitute a monophyletic group in which three subgroups can be recognised: subsp. hispanicus, subsp. gallicus var. gallicus and subsp. gallicus var. subaegeus. However, these three subgroups are not clearly distinguished genetically or morphologically, so that in formal classification it would be preferable to treat them at the varietal level.

Geometric morphometrics of the wing in the subgenus Culicoides (Diptera: Ceratopogonidae): from practical implications to evolutionary interpretations.

Biting midges of the genus Culicoides Latreille have been incriminated in transmission of bluetongue. Since 1998, the disease has spread across Europe provoking the largest epidemic ever recorded with important economic loses. Some species of the subgenus Avaritia and Culicoides have been described as candidate vectors involved in these epizootics. Both subgenera contain groups of cryptic species that could differ in their vectorial capacity. For this reason, the correct identification of vector species is considered an essential issue in epidemiological programs. In the current study, the usefulness of wing form in differentiating morphologically similar species of the subgenus Culicoides by means of geometric morphometric techniques is assessed in specimens previously identified through molecular analyses based on cytochrome oxidase subunit I gene sequences. Significant differences between species were detected in the two components of form, i.e., size and shape. Although wing size was affected by temperature, wing shape showed a more stable specific variation, allowing the proper classification of a high percentage of specimens. In addition, the concordance between phylogenies inferred from molecular data and phenetic clusters suggests the existence of a phylogenetic signal in wing shape. These findings enhance the use of this complex phenotypic trait not only to infer genetic relationships among species of the subgenus Culicoides but also as apotentially powerful tool to differentiate cryptic species within the genus.

Type 2 diabetes mellitus metabolic control correlates with the phenotype of human monocytes and monocyte-derived macrophages.

AIMS: Monocytes and macrophages express cell-surface markers indicative of their inflammatory and activation status. In this study, we investigated whether these markers are affected or correlated in non-obese T2D subjects, or glycemic/metabolic control variables. METHODS: Clinical data was recorded, and peripheral blood drawn from T2D patients (n = 28) and control subjects (n = 27). Isolated monocytes were evaluated by flow cytometry for the expression of CD14, CD16, and the phenotypic markers for the different states of activation spectrum, such as pro-inflammatory (M1) (HLA-DR, CD86), anti-inflammatory/pro-resolving (M2) (CD163, CD206, MERTK, PD-L1) and metabolically-activated (MMe) (CD36, ABCA-1). From a subset of individuals, monocytes-derived macrophages (MDM) were obtained and evaluated for phenotypic markers. A correlation analysis was performed between the clinical variables and the marker expression. RESULTS: The frequency of CD14++CD16- monocytes was lower in T2D patients and it correlates negatively with poor control in glycemic and metabolic variables. T2D monocytes expressed lower levels of HLA-DR, CD86, PD-L1, and CD163, which correlated negatively with poor metabolic control. In MDM from T2D patients, HLA-DR, CD86 and CD163 expression was lower and it inversely correlated with deficient glycemic or metabolic control parameters. CONCLUSION: The glycemic/metabolic control associated with T2D influences monocyte and MDM phenotypes toward an immune-suppressive phenotype.

Factors affecting Culicoides species composition and abundance in avian nests.

Mechanisms affecting patterns of vector distribution among host individuals may influence the population and evolutionary dynamics of vectors, hosts and the parasites transmitted. We studied the role of different factors affecting the species composition and abundance of Culicoides found in nests of the blue tit (Cyanistes caeruleus). We identified 1531 females and 2 males of 7 different Culicoides species in nests, with C. simulator being the most abundant species, followed by C. kibunensis, C. festivipennis, C. segnis, C. truncorum, C. pictipennis and C. circumscriptus. We conducted a medicationxfumigation experiment randomly assigning bird's nests to different treatments, thereby generating groups of medicated and control pairs breeding in fumigated and control nests. Medicated pairs were injected with the anti-malarial drug Primaquine diluted in saline solution while control pairs were injected with saline solution. The fumigation treatment was carried out using insecticide solution or water for fumigated and control nests respectively. Brood size was the main factor associated with the abundance of biting midges probably because more nestlings may produce higher quantities of vector attractants. In addition, birds medicated against haemoparasites breeding in non-fumigated nests supported a higher abundance of C. festivipennis than the rest of the groups. Also, we found that the fumigation treatment reduced the abundance of engorged Culicoides in both medicated and control nests, thus indicating a reduction of feeding success produced by the insecticide. These results represent the first evidence for the role of different factors in affecting the Culicoides infracommunity in wild avian nests.

Pollinators, flowering phenology and floral longevity in two Mediterranean Aristolochia species, with a review of flower visitor records for the genus.

The pollination of Aristolochia involves the temporary confinement of visitors inside the flower. A literature review has shown that some species are visited by one or a few dipteran families, while others are visited by a wider variety of dipterans, but only some of these are effective pollinators. We observed flowering phenology and temporal patterns of pollinator attendance in diverse populations of Aristolochia baetica and A. paucinervis, two species that grow in SW Spain, frequently in mixed populations. The two species had overlapping floral phenologies, extended flowering periods and long-lived flowers. A. baetica attracted a higher number of visitors than A. paucinervis. Drosophilids and, to a lesser extent, phorids, were the main pollinators of A. baetica, whereas in A. paucinervis, phorids were the only pollinators. Attendance to A. paucinervis flowers by phorids in mixed populations was markedly lower than in pure populations. This effect was more evident in years with lower pollinator density. Our results suggest that A. baetica and A. paucinervis may compete for pollinators in mixed populations.

The interaction between Cistaceae and a highly specific seed-harvester ant in a Mediterranean scrubland.

We studied the interaction between the ant Goniomma kugleri and Cistaceae in a Cistus ladanifer-dominated scrubland, in southwestern Spain. We monitored seed harvesting, and studied ant preferences among Cistaceae seeds and their capture efficiencies for preferred seeds. For the stand of C. ladanifer, we estimated seed losses due to the ants. Harvesting was restricted to two seasons: mid-autumn to late winter, and late spring. Ant diet relied on Cistaceae seeds: during autumn and winter 90% of seeds returned to nests were of C. ladanifer, and the remaining fraction also comprised Cistaceae seeds. At this time, the ants harvested seeds directly from the plants. In late spring, the ant diet consisted of Tuberaria guttata s.l. seeds. Goniomma kugleri selectively collected Cistaceae seeds. For preferred species, seed removal rates at the colony level and seed capture times invested by individual workers were correlated with seed size. Because of shorter capture time and higher success frequency, capture efficiency in terms of number of seeds captured per unit time was higher for small-seeded species. Although each ant colony collected large numbers (up to 10(5)) of C. ladanifer seeds over the autumn-winter season, the impact of ant removal on the annual seed output was moderate, at around 20%. It is likely that, in C. ladanifer, the staggered seed release period, and the pulsed exposure of seed clumps in capsules through progressive locule dehiscence, effectively minimise seed losses to the ants.

Phylogeography of the invasive weed Hypochaeris radicata (Asteraceae): from Moroccan origin to worldwide introduced populations.

In an attempt to delineate the area of origin and migratory expansion of the highly successful invasive weedy species Hypochaeris radicata, we analysed amplified fragment length polymorphisms from samples taken from 44 populations. Population sampling focused on the central and western Mediterranean area, but also included sites from Northern Spain, Western and Central Europe, Southeast Asia and South America. The six primer combinations applied to 213 individuals generated a total of 517 fragments of which 513 (99.2%) were polymorphic. The neighbour-joining tree presented five clusters and these divisions were supported by the results of Bayesian analyses: plants in the Moroccan, Betic Sierras (Southern Spain), and central Mediterranean clusters are all heterocarpic. The north and central Spanish, southwestern Sierra Morena, and Central European, Asian and South American cluster contain both heterocarpic (southwestern Sierra Morena) and homocarpic populations (all other populations). The Doñana cluster includes two homocarpic populations. Analyses of fragment parameters indicate that the oldest populations of H. radicata are located in Morocco and that the species expanded from this area in the Late Quaternary via at least three migratory routes, the earliest of which seems to have been to the southwestern Iberian Peninsula, with subsequent colonizations to the central Mediterranean area and the Betic Sierras. Homocarpic populations originated in the southwestern Iberian Peninsula and subsequently spread across north and central Spain, Central Europe and worldwide, where they became a highly successful weed.

Schmallenberg virus detection in Culicoides biting midges in Spain: First laboratory evidence for highly efficient infection of Culicoides of the Obsoletus complex and Culicoides imicola.

Since Schmallenberg disease was discovered in 2011, the disease rapidly spread across Europe. Culicoides biting midges have been implicated as putative Schmallenberg vectors in Europe. The detection of Schmallenberg virus (SBV) in field collected Culicoides was evaluated through retrospective (2011-2012) collections and captures performed in 2013. This study represents the first detection of SBV in field collected Culicoides in Spain. Infectious midges were detected at the foothills of Pyrenees, Aramunt, in the summer 2012. All the specimens infected with Schmallenberg were of the species Culicoides obsoletus s.s. confirming its putative vector status in Spain. Experimental infection on field collected Culicoides provided evidence of atypical high efficiency for SBV vector infection and transmission potential in local populations of Culicoides imicola and in Culicoides of the Obsoletus complex. However, captured individuals of C. imicola were more susceptible to SBV infection than C. obsoletus s.l. (p < .001), with an infection ratio of 0.94 and 0.63, respectively. In contrast, a Culicoides nubeculosus colony appeared to be refractory to SBV infection.

Polyembryony and apomixis in Eriotheca pubescens (Malvaceae - Bombacoideae).

Apomixis and adventitious polyembryony have been reported for several species of Bombacoideae, including Eriotheca pubescens, a tree species of the Neotropical savanna (Cerrado) areas in Brazil. However, the origin of polyembryonic seeds and their importance for the reproduction of the species remained to be shown. Here, we analyzed the early embryology of this species to establish the apomictic origin of extranumerary embryos. We also observed the geographic distribution of polyembryony in E. pubescens, and tested if apomixis was related to the source of pollen (self or cross) and population density. Moreover, we tested if polyembryonic apomictic embryos would develop normally into seedlings. In the observed seed primordia, after a relatively long quiescent period, the zygote developed into a sexual embryo concurrently with adventitious apomictic embryos which developed from nucellus cells. Adventitious embryos develop faster than sexual ones and are morphologically similar, so that 44 days after anthesis it was virtually impossible to distinguish and trace the fate of the sexual embryo. Polyembryony is widely distributed in populations some 400 km distant, and only one strictly monoembryonic individual was observed during the study. The number of embryos per seed varied between fruits and individuals but was significantly higher in seeds from cross-pollinations than from selfs, although fruit and seed set after crosses were much lower than after selfs. Embryo development into seedlings depended on their weight at germination, but polyembryonic seeds germinated and produced up to seven seedlings per seed in greenhouse conditions. Adventitious embryony and apomictic seedlings would explain the mostly clonal populations suggested by molecular studies.

Andromonoecy in an Old World Papilionoid legume, Erophaca baetica.

Andromonoecy (i.e. the occurrence on individual plants of hermaphroditic and male flowers) is a rare sexual system among the angiosperms, regarded by some authors as a transitional stage from hermaphroditism to monoecy. Having discovered the occurrence of andromonoecy in Erophaca baetica (a Mediterranean shrubby legume with two subspecies), a novelty for Old World papilionoid legumes, we investigated the morpho-functional correlates and the geographical distribution of this phenomenon in the species. The relative frequencies of hermaphrodite and male flowers were determined in two field and 111 herbarium populations. Biomass allocation within flowers, pollen production and viability, pollen tube growth, nectar production and the temporal pattern of male flower production were also studied in two nearby southern Spanish populations. Virtually all of the studied populations were andromonoecious. Male flowers tended to appear at apical positions within the inflorescence, and became more abundant by the end of the flowering season. Male flowers were externally similar to hermaphroditic flowers (although with less biomass and smaller parts) and released equivalent amounts of pollen and nectar; however, their pollen germinated significantly better. Erophaca is the first example of an andromonecious Papilionoid in the Old World. Since the main difference among floral morphs in this species is functional (i.e. pollen germination rate) rather than morphological, andromonoecy is not readily noticeable, and very careful inspection may be required to reveal it. The potential effect of andromonoecy in enhancing outcrossing rate in this species is discussed.

Persistently low fruiting success in the Mediterranean pipevine Aristolochia baetica (Aristolochiaceae): a multi-year study.

Low fruit set is common in many plant species and may be caused by a variety of factors, such as predation, resource limitation or deficient pollination, or it may be an evolutionary strategy. In this paper, we investigate factors that affect fruit set in Aristolochia baetica (Aristolochiaceae), a Mediterranean pipevine found in southwest Spain. Fruit production was monitored in two populations over 4 years (2002-2005), and the causes of flower or fruit loss were determined. Experimental hand-pollinations were performed, and germinated pollen grains on the stigmas of open-pollinated flowers were quantified. Fruit set was always very low (4-14%). Floral abscission initially reduced reproductive output by more than 50%; then herbivory (6-12%) and fruit abortion (8-26%) caused further reductions. Given that the number of efficiently pollinated flowers was always higher than that of ripe fruits, and that xenogamous hand-pollination did not increase fruit set in relation to open-pollination, the final fruit production of A. baetica seems not to be pollen-limited. Fruit abortion of effectively pollinated flowers supports the idea that resource availability limits fruit set. In A. baetica, fruit abortion could lead to mate selection of the best quality fruits. Moreover, the initiated fruits that finally abort could also satiate predators, contributing to increase progeny fitness.

Identification of cryptic species of Culicoides (Diptera: Ceratopogonidae) in the subgenus Culicoides and development of species-specific PCR assays based on barcode regions.

Culicoides biting midges (Diptera: Ceratopogonidae) are vectors of important diseases affecting wild and domestic animals. During the last decade they have played a major role in the epidemiology of the largest bluetongue epizootic ever recorded in Europe, the disease is transmitted between hosts almost exclusively by bites of Culicoides midges and affects both domestic and wild ruminants however severe disease usually occurs in certain breeds of sheep and some species of deer. An accurate vector identification is of major importance in arthropod borne diseases surveillance, as great differences in vectorial capacity are found even between close species. Unfortunately, specialized taxonomic knowledge of Culicoides identification is rarely available in routine surveillance, mainly based on wing morphology. Recently, some European species of Culicoides belonging to the subgenus Avaritia Fox, 1955 and Culicoides Latreille, 1809 have been described as new bluetongue virus vectors. In the present study, by using a fragment of the barcode region (COI gene) we report the presence of up to 11 species within the subgenus Culicoides in Catalonia (NE Spain), a region recently affected by a bluetongue epizootic. The molecular analysis revealed new non-described cryptic species which were grouped in three complexes of morphologically similar species, two in the Pulicaris complex resembling Culicoides pulicaris, two in the Fagineus complex resembling Culicoides fagineus and three in the Newsteadi complex resembling Culicoides newsteadi. The phylogenetic relationships among them showed that cryptic species detected in both Pulicaris and Fagineus complexes were closely related, whereas those in the Newsteadi complex were more distant. Accurate analysis of all species using morphological and molecular approaches resulted in the detection of diagnostic metric traits for cryptic species and the design of several new species-specific single and multiplex PCR assays to identify unambiguously all the species, most of them still lacking a specific molecular diagnosis.

Population structure of Hypochaeris salzmanniana DC. (Asteraceae), an endemic species to the Atlantic coast on both sides of the Strait of Gibraltar, in relation to Quaternary sea level changes.

To detect potential Pleistocene refugia and colonization routes along the Atlantic coast, we analysed amplified fragment length polymorphisms (AFLPs) in 140 individuals from 14 populations of Hypochaeris salzmanniana (Asteraceae), an annual species endemic to the southwestern European and northwestern African coastal areas. Samples covered the total distributional range of the species, with eight populations in southwestern Spain and six populations in northwestern Morocco. Using nine primer combinations, we obtained 546 fragments in H. salzmanniana and its sister species H. arachnoidea of which 487 (89.2%) were polymorphic. The neighbour-joining tree shows that the populations south of the Loukos river in Morocco are clearly differentiated, having more polymorphic, private, and rare fragments, and higher genetic diversity, than all the other populations. The southernmost populations in Morocco, south of the river Sebou, form a large panmictic population. They are probably the oldest populations that have been relatively unaffected by stochastic processes resulting from Pleistocene glaciations. Northward migration of populations during this period may have resulted in loss of genetic diversity in specific regions, perhaps due to bottlenecks caused by rise in sea level during interglacial periods, and, in some cases, by changes in the breeding system.

Apical pattern of fruit production in the racemes of Ceratonia siliqua (Leguminosae: Caesalpinioideae): role of pollinators.

Fruit production and arrangement within the raceme were studied in two dioecious populations of Ceratonia siliqua (Leguminosae: Caesalpinioideae), an arboreal species that produces caulogenous racemes (emerging only from the old branches) with numerous flowers. Fruit production per raceme was low and similar between years and populations and even between individuals. During flowering, there were considerable flower losses from predation and lack of pollination. A mean of nine flowers per raceme began the transformation into fruits, of which 77% aborted. The final fruit production per raceme increased significantly following hand pollination, but was always very much lower than the availability of flowers in the raceme. The results suggest that fruit production of each raceme is limited by both availability of resources and a deficient pollination. In racemes setting fruit arrangement follows a definite pattern that remains constant between years and populations: fruit production was significantly higher in the apical zone of the raceme and lower in the basal zone. The pollinators of C. siliqua (flies and wasps) showed a clear preference for beginning their visits at the apex of a raceme. As a result, the pollen load deposited on the stigmas decreased from apex to base of the raceme. In most of the flowers situated in the central and basal zone of the raceme, the number of pollen grains deposited on their stigmas was lower than the number of their ovules. The high number of seeds in developed fruits suggests that the plant selectively aborts flowers that receive a smaller pollen load. The results indicate that the final pattern of fruit arrangement within the raceme is a direct result of pollinator activity.