Morphological divergence and the Quaternary speciation of Actaea purpurea (Ranunculaceae) and its relatives

To understand the process and mechanism of speciation, a detailed analysis of origin and demographic history of recently diverged species pairs is necessary. Here, we investigate the evolutionary history of Actaea purpurea (P.K. Hsiao) J. Compton and its closest relatives, A. japonica Thunb. and A. biternata (Siebold and Zuccarini) Prantl. We aim to estimate important parameters of the divergence event, and to lay the foundation for further investigation of the speciation mechanism of this system. Floral and vegetative traits were measured and analyzed. Genetic structure, divergence history, and historical gene flow were also inferred from the plastid and single nucleotide polymorphism data. Floral traits were divergent, and a strong match between pollinator and floral traits was revealed. Genetically the two species were also well diverged, and the time of divergence was dated to the Pleistocene. The demographic modelling results suggest that A. purpurea had continuous limited gene flow with A. japonica and A. biternata since divergence. More work is now needed to confirm that floral trait divergence was selected by pollinators, as well as to understand how pollinator isolation acts in conjunction with other reproductive barriers to reduce gene flow between the two species.     

Floral traits and isolation of three sympatric Aquilegia species in the Qinling Mountains, China

Floral isolation has been considered to be an important reproductive mechanism governing the species diversification in many genera. In a classic example Aquilegia, sympatric species from North America with diverse floral traits are generally associated with specialized pollinators that prohibit interspecific hybridization. It remains unclear whether species diversification in the genus from Eurasia is also maintained by floral isolation. We investigated floral phenology, floral characteristics and pollinators in three sympatric Aquilegia species (A. ecalcarata, A. incurvata and A. yabeana) in the Qinling Mountains, Shanxi Province, China from 2001 to 2005. The spurless A. ecalcarata flowers earlier than the other two species with nectar spurs but their floral phenology overlaps. Major pollinators of A. ecalcarata are syrphid flies while bumblebees are major for A. incurvata and A. yabeana. Therefore our observations confirm that mechanical isolation through differential pollinators could contribute reproductive isolation between spurless and spurred species, as demonstrated by studies from North America. Whether floral isolation plays a major role in the reproductive isolation between two spurred species (A. incurvata and A. yabeana), however, remains to be seen. Further studies are required to quantify the potential role of geographical isolation because they occupy different habitats.     

Ancestral deceit and labile evolution of nectar production in the African orchid genus Disa

An outstanding feature of the orchid family is that approximately 30–40% of the species have non-rewarding flowers and deploy various modes of deception to attract pollinators, whereas the remaining species engage in pollination mutualisms based on provision of floral rewards. Here, we explore the direction, frequency and reversibility of transitions between deceptive and rewarding pollination systems in the radiation of the large African genus Disa, and test whether these transitions had consequences for diversification. By optimizing nectar production data for 111 species on a well-resolved phylogeny, we confirmed that floral deception was the ancestral condition and that nectar production evolved at least nine times and was lost at least once. Transitions to nectar production first occurred ca 17 million years ago but did not significantly affect either speciation or extinction rates. Nectar evolved independently of a spur, which was lost and gained multiple times. These results show that nectar production can be a highly labile trait and highlight the need for further studies of the genetic architecture of nectar production and the selective factors underlying transitions between deception and mutualism.     

Floral specialization for different pollinators and divergent use of the same pollinator among co‐occurring Impatiens species (Balsaminaceae) from Southeast Asia

Floral variation among closely related species is thought to often reflect differences in pollination systems. Flowers of the large genus Impatiens are characterized by extensive variation in colour, shape and size and in anther and stigma positioning, but studies of their pollination ecology are scarce and most lack a comparative context. Consequently, the function of floral diversity in Impatiens remains enigmatic. This study documents floral variation and pollination of seven co‐occurring Impatiens spp. in the Southeast Asian diversity hotspot. To assess whether floral trait variation reflects specialization for different pollination systems, we tested whether species depend on pollinators for reproduction, identified animals that visit flowers, determined whether these visitors play a role in pollination and quantified and compared key floral traits, including floral dimensions and nectar characteristics. Experimental exclusion of insects decreased fruit and seed set significantly for all species except I. muscicola, which also received almost no visits from animals. Most species received visits from several animals, including bees, birds, butterflies and hawkmoths, only a subset of which were effective pollinators. Impatiens psittacina, I. kerriae, I. racemosa and I. daraneenae were pollinated by bees, primarily Bombus haemorrhoidalis. Impatiens chiangdaoensis and I. santisukii had bimodal pollination systems which combined bee and lepidopteran pollination. Floral traits differed significantly among species with different pollination systems. Autogamous flowers were small and spurless, and did not produce nectar; bee‐pollinated flowers had short spurs and large floral chambers with a wide entrance; and bimodally bee‐ and lepidopteran‐pollinated species had long spurs and a small floral chamber with a narrow entrance. Nectar‐producing species with different pollination systems did not differ in nectar volume and sugar concentration. Despite the high frequency of bee pollination in co‐occurring species, individuals with a morphology suggestive of hybrid origin were rare. Variation in floral architecture, including various forms of corolla asymmetry, facilitates distinct, species‐specific pollen‐placement on visiting bees. Our results show that floral morphological diversity among Impatiens spp. is associated with both differences in functional pollinator groups and divergent use of the same pollinator. Non‐homologous mechanisms of floral asymmetry are consistent with repeated independent evolution, suggesting that competitive interactions among species with the same pollination system have been an important driver of floral variation among Impatiens spp.     

Pollinators and nectar robbers cause directional selection for large spur circle in Impatiens oxyanthera (Balsaminaceae)

Nectar spurs have an important role in floral evolution and plant–pollinator coadaptation. The flowers of some species possess spurs curving into a circle. However, it is unclear whether spur circle diameter is under direct selection pressure from different sources, such as pollinators and nectar robbers. In this study, we quantified selection on some floral traits, such as spur circle diameter in Impatiens oxyanthera (Balsaminaceae) using phenotypic selection analysis and compared the relative importance of pollinators and nectar robbers as selective agents using mediation analysis. The study showed that pollinators caused significant selection on corolla length, spur curvature and spur circle diameter while nectar robbers only imposed strong selection on spur circle diameter. Pollinators favored flowers with large corolla, curly spurs and large spur circle while nectar robbers preferred flowers with small spur circle. More pollinator visits resulted in higher female reproductive success, while robbery reduced female fitness. Conflicting selection on spur traits from pollinators and nectar robbers was not found. Mediation analysis showed that selection on floral traits through nectar robbing was stronger than selection through pollination. The results suggested that pollinators and nectar robbers jointly mediated the directional selection for large spur circle, and nectar robbers caused stronger selection than pollinators on floral traits.     

Pollinators of the Rocky Mountain columbine: temporal variation, functional groups and associations with floral traits

Background and Aims

Pollinators together with other biotic and some abiotic factors can select for floral traits. However, variation in pollinator abundance over time and space can weaken such selection. In the present study, the variation in pollinator abundance over time and space was examined in populations of the Rocky Mountain columbine. The variation in three floral traits is described and correlations between pollinator type, functional pollinator groups or altitude and floral traits are examined.

Methods

Pollinator observations took place in six Aquilegia coerulea populations over 1–4 years and spur length, flower colour and sepal length were measured in 12 populations. Pollinator abundance, measured as visits per flower per hour, was compared among populations and years. Pollinators were grouped into two functional groups: pollen or nectar collectors. The following associations were examined: annual presence of hawkmoths and whiter flowers with longer spurs; the presence of Sphinx vashti and longer spurs; and higher altitudes and whiter flowers. The study looked at whether an increase in the proportion of hawkmoths in a population was associated with whiter and larger flowers with longer spurs.

Key Results

The abundance of different pollinator groups varied over time and space. Floral traits varied among populations. Higher altitude was correlated with bluer flowers. Whiter flowers were associated with the annual presence of hawkmoths. Populations visited by Sphinx vashti had longer spurs than populations visited only by Hyles lineata. Populations with greater percentage of nectar-collecting pollinators did not have whiter, larger flowers with longer spurs.

Conclusions

Despite the large variation in pollinator abundance over time and space, one species of bumble-bee or hawkmoth tended to predominate in each population each year. Future studies of Aquilegia coerulea should examine the specific influences of pollinators and the environment on flower colour and of hawkmoth species on spur length.Key words: Aquilegia coerulea, columbine, pollinator abundance, bumble-bee, hawkmoth, flower colour, spur length, functional pollinator group, altitude, floral trait     

Nectar robbers influence the trait–fitness relationship of Primula secundiflora

• The trait–fitness relationship influences the strength and direction of floral evolution. To fully understand and predict the evolutionary trajectories of floral traits, it is critical to disentangle the direct and indirect effects of floral traits on plant fitness in natural populations.
• We experimentally quantified phenotypic selection on floral traits through female fitness and estimated the casual effects of nectar robbing with different nectar robbing intensities on trait–fitness relationships in both the L‐ (long‐style and short‐anther phenotype) and S‐morph (short‐style and long‐anther phenotype) flowers among Primula secundiflora populations.
• A larger number of flowers and wider corolla tubes had both direct and indirect positive effects on female fitness in the P. secundiflora populations. The indirect effects of these two traits on female fitness were mediated by nectar robbers. The indirect effect of the number of flowers on female fitness increased with increasing nectar robbing intensity. In most populations, the direct and/or indirect effects of floral traits on female fitness were stronger in the S‐morph flowers than in the L‐morph flowers. In addition, nectar robbers had a direct positive effect on female fitness, but this effect varied between the L‐ and S‐morph flowers.
• These results show the potential role of nectar robbers in influencing the trait–fitness relationships in this primrose species.

     

四川攀西地区云南松群落物种多样性和谱系多样性对紫茎泽兰入侵的影响

外来植物入侵严重威胁着入侵地本土植物多样性和生态系统功能, 认识外来物种的入侵机制有助于提高对入侵植物的防控能力。本文以攀西地区云南松(Pinus yunnanensis)林下外来入侵植物紫茎泽兰(Ageratina adenophora)为研究对象, 基于大量野外群落调查, 从群落可入侵性入手, 分析了环境因子和群落物种多样性、谱系多样性等群落生态学特征对紫茎泽兰入侵的影响。结果表明: 海拔、坡向和火烧强度等环境因子和冠层郁闭度、灌木层盖度等生物因子对研究区紫茎泽兰入侵强度没有显著影响(P > 0.05); 但群落灌木层物种多样性和草本层组分种与紫茎泽兰的亲缘关系显著影响紫茎泽兰的入侵强度(P < 0.05), 说明灌木层对光照等环境资源的占用和草本层(同层)物种对相似资源的竞争能够在很大程度上抑制紫茎泽兰的入侵。     

大蜜蜂的生物学特性、面临威胁与保护策略

昆虫传粉在维持植物的有性繁殖、物种形成及生态系统稳定中扮演着重要角色, 而野生传粉昆虫为生态系统提供了巨大的传粉服务功能。大蜜蜂(Apis dorsata)为亚洲特有的一种野生传粉昆虫, 是热带地区多种植物和农作物的有效传粉者, 在保障热带生物多样性及作物产量中有不可或缺的作用。但受全球气候变化、人类活动和生境恶化等因素的影响, 其种群数量日益减少, 开展大蜜蜂种质资源保护势在必行。本文综述了大蜜蜂筑巢、迁飞和传粉服务功能, 分析了人为猎取蜂巢, 栖息生境遭受破坏, 杀虫剂和除草剂滥用, 昆虫、螨类和病原物侵染, 气候变化等威胁种群的因素, 以期从强化大蜜蜂基础研究和保护、推动生态农业发展、建立适合大蜜蜂迁飞生态廊道、加强检验检疫及科学合理利用大蜜蜂种质资源等方面制定相应的保护措施。     

自交与异交象牙参属植物的蜜腺结构及其生物学意义

蜜腺是有花植物与传粉昆虫构建互惠关系的关键花部结构,解析不同繁殖特性物种间蜜腺结构的差异可为理解有花植物繁殖特性的演化提供理论依据。以传粉系统高度特化、异交实现有性生殖的早花象牙参（Roscoea cautleoides）和缺乏有效传粉者、主动自交实现有性生殖的无柄象牙参（R. schneideriana）为材料,通过野外测量2个物种蜜腺特性、超景深显微镜观察和石蜡切片染色,探究早花象牙参与无柄象牙参在蜜腺分布位置、外部形态及内部结构的异同。结果表明：2个物种均具有结构蜜腺,从外部形态来看,早花象牙参蜜腺体积较大、分泌糖浓度较高的花蜜,而无柄象牙参蜜腺体积较小、无花蜜分泌;从内部结构来看,早花象牙参蜜腺结构特化、各个组成部分有明显的区分,而无柄象牙参蜜腺出现一定程度的退化。该研究结果为揭示早花象牙参与无柄象牙参在花部特征、传粉机制及繁育系统的差异以及无柄象牙参蜜腺无花蜜分泌的可能机理提供了重要的形态学依据,也为深入地探讨植物与传粉动物间的协同进化关系以及理解蜜腺多样性的起源与维持机制奠定了科学基础。     

Nectar reward and advertisement in hummingbird-pollinated Silene virginica (Caryophyllaceae)

We tested for an association between nectar and various floral traits and investigated their roles as primary and secondary pollinator attractants in hummingbird-pollinated Silene virginica. Our goal was to gain insight into the mechanisms of pollinator-mediated selection that underlies floral trait divergence within the genus. In a field population of S. virginica, we measured five floral and eight vegetative traits and quantified nectar volume, nectar sugar concentration, and total sugar reward (nectar volume × nectar sugar concentration). All three components of nectar reward were positively correlated to flower size, and nectar volume varied significantly among individuals within the population. To ascertain whether the correlation of specific floral traits with nectar reward influences the behavior of the primary pollinator of S. virginica, the ruby-throated hummingbird, Archilochus colubris, we investigated whether A. colubris preferred the expression of floral traits associated with high nectar volume and total sugar reward. We accomplished this by constructing floral arrays consisting of artificial flowers that had equal nectar quantity and total sugar reward but that differed in petal area and corolla tube diameter, which were positively correlated with nectar quantity and total sugar reward in our field study. In observations of visitation frequencies to the various floral-trait combinations, hummingbirds preferentially visited artificial floral phenotypes with larger petal displays, with the greatest preference for floral phenotypes with both larger petals and wider corolla-tube diameters. This association between primary and secondary floral attractants and hummingbird discrimination of floral features supports the concept that the floral traits of S. virginica reflect pollinator-mediated selection by the principal pollinator.     

Floral Biology and Pollination Mechanisms in Two Viola Species—From Nectar to Pollen Flowers?

The genus Viola is represented by four related species in Brazil belonging to section Leptidium, one of the most primitive sections in the genus. Floral biology and pollination by bees were studied in Viola cerasifolia and V. subdimidiata in high-altitude areas in south-eastern Brazil. Flowers are zygomorphic and spurred. The five stamens are arranged in a cuff around the ovary, and pollen is released by means of apical connective projections, which form a cone surrounding the base of the style. The connective projections of the inferior stamens are elongated and curved to form a hook-shaped structure. Nectar-secreting tissue can occur in the basal connective appendages of the inferior stamens, which project into the spur. Flowers of V. subdimidiata secreted a mean volume of 0.14 micro l nectar over a 24-h period; approx. 40 % of flowers did not secrete any nectar. The main pollinators of these Viola species are female bees belonging to the genus Anthrenoides (Andrenidae), which search mainly for pollen. These bees seem to be oligolectic and obtain large amounts of pollen from Viola by vibrating the flowers or by moving the hook repeatedly back and forth. Males of Anthrenoides patrol Viola clusters and also feed on nectar, acting as secondary pollinators. The basic floral structure in the genus Viola fits that of 'nectar flowers'. The uncommon hook-shaped projections, scanty nectar production, and behaviour of pollinators suggest that V. cerasifolia and V. subdimidiata are shifting their reward for pollinators from nectar to pollen. Based on floral morphology, this shift may be widespread in Viola sect. Leptidium.     

Are spurred cyathia a key innovation? Molecular systematics and trait evolution in the slipper spurges (Pedilanthus clade: Euphorbia, Euphorbiaceae)

The study of traits that play a key role in promoting diversification is central to evolutionary biology. Floral nectar spurs are among the few plant traits that correlate with an enhanced rate of diversification, supporting the claim that they are key innovations. Slight changes in spur morphology could confer some degree of premating isolation, explaining why clades with spurs tend to include more species than their spurless close relatives. We explored whether the cyathial nectar spur of the Pedilanthus clade (Euphorbia) may also function as a key innovation. We estimated the phylogeny of the Pedilanthus clade using one plastid (matK) and three nuclear regions (ITS and two G3pdh loci) and used our results and a Yule model of diversification to test the hypothesis that the cyathial spur correlates with an increased diversification rate. We found a lack of statistical support for the key innovation hypothesis unless specific assumptions regarding the phylogeny apply. However, the young age (hence small size) of the group may limit our ability to detect a significant increase in diversification rate. Additionally, our results confirm previous species designations, indicate higher homoplasy in cyathial than in vegetative features, and suggest a possible Central American origin of the group.     

Behavioral genomics and the study of speciation at a porous species boundary

Porous species boundaries are characterized by differential gene flow, where some regions of the genome experience divergent evolution while others experience the homogenizing effects of gene flow. If species can arise or remain distinct despite gene flow between them, speciation can only be understood on a gene by gene level. To understand the genetics of speciation, we therefore must identify the targets of selection that cause divergent evolution and identify the genetic architecture underlying such “speciation phenotypes”. This will enable characterization of genomic regions that are “free to flow” between species, and those that diverge in the face of gene flow. We discuss this problem in the genus Laupala, a morphologically cryptic, flightless group of crickets that has radiated in Hawaii. Because songs are used in courtship and always distinguish close relatives of Laupala as well as species in sympatry, we argue that songs in Laupala are speciation phenotypes. Here, we present our approaches to identify the underlying genomic regions and song genes that differentiate closely related species. We discuss what is known about the genetic basis of this species difference derived from classic quantitative genetics and quantitative trait locus mapping experiments. We also present a model of the molecular expression of cricket song to assist in our goal to identify the genes involved in song variation. As most species are sympatric and exchange genes with congeners, we discuss the importance of understanding the genetic and genomic architecture of song as a speciation phenotype that must be characterized to identify differential patterns of gene flow at porous species boundaries.     

木棉黄花个体的适应意义

木棉(Bombax ceiba)花大、红色, 多为鸟媒传粉, 但也存在频率极低的黄花个体, 有蜜蜂访花。本文对比研究了海南岛木棉红花、黄花的花部特征及传粉过程, 以解释黄花个体的适应意义。木棉两种花色表型的花蜜组分(葡萄糖和果糖)及挥发性物质(烷烃类、酯类、酚类等)没有显著差异, 但黄花花瓣的反射波长范围均在鸟类与蜜蜂视觉范围内, 可同时吸引鸟和蜜蜂。木棉黄花的雌雄异熟程度较小(花粉活性时期与柱头可受期重叠较大), 可在传粉媒介访花次数有限的情况下同时输出或接受花粉。另一方面, 黄花有着更大程度的柱头探出式雌雄异位, 降低了较小程度雌雄异熟可能导致的自交与雌雄功能干扰。木棉存在明显的花粉限制, 且黄花的坐果率(1.08 ± 0.56)%显著低于红花(3.27 ± 0.93)%。我们推测, 黄花个体可能通过增加木棉访花者类群多样性、降低雌雄异熟程度而提高了木棉的繁殖成功率, 也通过吸引蜜蜂而降低了蜜蜂对红花鸟媒传粉的干扰。     

基于线性混合模型研究海州湾六丝钝尾虾虎鱼摄食生态

随着我国近海多种传统经济渔业资源的衰退,六丝钝尾虾虎鱼已成为海州湾等海域的优势鱼种之一,并在海洋食物网中发挥着重要作用。本研究根据2011年和2013—2016年春季和秋季在海州湾及其邻近海域进行的渔业资源底拖网调查,基于线性混合模型研究了海州湾六丝钝尾虾虎鱼的摄食习性以及影响其摄食的主要因素,并对其摄食策略进行分析。结果表明: 海州湾六丝钝尾虾虎鱼以虾类、双壳类、端足类等14个饵料生物类群为食,其中端足类、虾类、双壳类、蛇尾类是其主要的饵料类群。从饵料生物种类来看,六丝钝尾虾虎鱼摄食的饵料种类有37种,优势饵料生物有钩虾、细螯虾、独眼钩虾、彩虹明樱蛤和鲜明鼓虾等。线性混合模型分析表明,海州湾六丝钝尾虾虎鱼的摄食强度受体长、水深、季节、底层盐度的显著影响,其中,摄食强度与水深和底层盐度呈显著负相关关系,而与体长呈显著正相关关系,且春季的摄食强度显著高于秋季。聚类分析表明,以体长70 mm为界,可将六丝钝尾虾虎鱼划分为食性差异较大的两个体长组,其摄食随体长的变化主要与自身摄食能力与饵料生物丰度的变化有关。摄食策略分析表明,六丝钝尾虾虎鱼的营养生态位具有较高的表型间成分,对食物的种内竞争较低。本研究将有助于揭示六丝钝尾虾虎鱼的生活史特征,并为其资源的可持续利用和科学管理提供参考依据。     

Flower colour adaptation in a mimetic orchid

Although the tremendous variability in floral colour among angiosperms is often attributed to divergent selection by pollinators, it is usually difficult to preclude the possibility that floral colour shifts were driven by non-pollinator processes. Here, we examine the adaptive significance of flower colour in Disa ferruginea, a non-rewarding orchid that is thought to attract its butterfly pollinator by mimicking the flowers of sympatric nectar-producing species. Disa ferruginea has red flowers in the western part of its range and orange flowers in the eastern part--a colour shift that we hypothesized to be the outcome of selection for resemblance to different local nectar-producing plants. Using reciprocal translocations of red and orange phenotypes as well as arrays of artificial flowers, we found that the butterfly Aeropetes tulbaghia, the only pollinator of the orchid, preferred both the red phenotype and red artificial flowers in the west where its main nectar plant also has red flowers, and both the orange phenotype and orange artificial flowers in the east, where its main nectar plant has orange flowers. This phenotype by environment interaction demonstrates that the flower colour shift in D. ferruginea is adaptive and driven by local colour preference in its pollinator.     

Unraveling independent origins of two tetraploid Achillea species by amplicon sequencing

Allopolyploidy is a significant mechanism of plant speciation, and many allopolyploid species have arisen recurrently. However, the probability that allopolyploidization between the same two parental species could lead to the origin of different taxa has received little attention. Here we used a new progenitor‐specific amplicon sequencing method to demonstrate the independent origins of two yarrow species, Achillea alpina L. and Achillea wilsoniana Heimerl ex Hand.‐Mazz., through allotetraploidy from the same diploid progenitor species pair, Achillea acuminata (Ledeb.) Sch. Bip. and Achillea asiatica Serg. Based on the sequences of 17 nuclear genes from 21 wild populations of the four Achillea species investigated, a clear view of genetic structure and demographic history was obtained with each species. Significant genetic differentiation was evident between the two tetraploid species. Two genetically distinguishable groups were detected within one of the progenitor, A. acuminata, and ancestors belonging to those two groups contributed to the two tetraploid species. Excluding fixed heterozygosity, we detected extremely low genetic diversity in many populations of both tetraploid species. Approximate Bayesian computation indicated that both tetraploid species originated before the Last Glacial Maximum, and nearly all diploid lineages went through population declines after the allopolyploidization events. Our study indicates that independent allopolyploidization events between the same Achillea parental species have generated two genetically and ecologically distinct taxa.     

Effects of fire frequency on oak litter decomposition and nitrogen dynamics

Rapid speciation within some plant families has been attributed to the evolution of floral spurs and to the effect of spur length on plant reproductive success. The flowers of Impatiens capensis (jewelweed) possess a long, curved spur in which nectar is produced and stored. Spur length and curvature varies among plants within one population. Here I document that spur shape is variable in natural populations, variation within plants is less than variation among plants, and spur shape is correlated with components of female and male reproductive success. The apparent natural selection is weakly directional in 1 of 2 years, with greatest seed production and pollen removal occurring in flowers with the greatest spur curvature. Bee pollinator visit length is longest at flowers with highly curved spurs, and they leave less nectar in these spurs than in flowers with straighter spurs. Spur angle evolution may be limited, at least in part, by opposing selection by nectar-robbers who prefer to visit flowers with greater spur curvature. Other factors that might contribute to the maintenance of spur angle variation are temporal variation in the strength of selection and potential genetic correlations of spur shape with other traits under selection.     

How generalists coexist: the role of floral phenotype and spatial factors in the pollination systems of two Ranunculus species

Aims Competition for pollinators between phenotypically similar flowers is believed to play an important role in floral trait diversification in the angiosperms. However, in many plant communities, species with apparently similar floral phenotypes and generalist pollination systems co-flower. Here, the pollination systems of Ranunculus acris L. and Ranunculus repens L. were investigated to determine the factors enabling the species to coexist within apparently overlapping pollination niches.Methods Sympatrically flowering populations of R. acris and R. repens were investigated at three study sites in West Wales. The floral phenotypes of the two species were compared using measurements of floral morphology and spectral analyses of petal reflectance, using principal component analysis and bee and fly colour-space models. Evidence of inter-specific discrimination by foraging insects was tested for in the field and using floral arrays. The relative roles of behavioural constancy and spatial patchiness in maintaining pollinator fidelity were estimated.Important findings The floral phenotypes of R. acris and R. repens differed significantly. Social bees were highly constant when foraging at flowers of the two species and patchy floral distribution explained some of the observed fidelity. Dipterans visiting mixed floral arrays appeared to discriminate between the species, visiting more R. acris than R. repens flowers, but there was no difference in the number of visits to single-species arrays. Social bees were more likely to display constancy to flowers of R. repens in the field. Patchiness in floral distribution, subtle differences in floral phenotype, pollinator preferences and behavioural constancy are all likely to contribute to the continued coexistence of R. acris and R. repens, despite apparent overlap in their pollination niches. Such differences have the potential to facilitate the maintenance of species diversity in plant communities, even where plants appear to share similar floral phenotypes.