Does reproductive isolation reflect the segregation of color forms in Spiranthes sinensis (Pers.) Ames complex (Orchidaceae) in the Chinese Himalayas?

Isolation between species, or taxa sharing a common lineage, depends primarily on the relative strengths of various reproductive barriers. Previous studies on reproductive isolation between orchids emphasized mechanical and ethological barriers in flowers of species showing food and/or sexual mimicry. In this study, we investigated and quantified a series of prepollination and postpollination barriers between pink and white forms of Spiranthes sinensis sl, a nectar‐secreting complex. We generated ML trees based on trnS‐G and matK to explore phylogenetic relationships in this species complex. Spiranthes sinensis sl segregated from some other congeners, but the white form constituted a distinct clade in relation to the pink form. The white form secreted 2‐Phenylethanol as it is a single‐scent compound and was pollinated almost exclusively by native, large‐bodied Apis cerana and Bombus species (Apidae). Apis cerana showed a high floral constancy to this form. The scentless, pink form was pollinated primarily by smaller bees in the genera Ceratina (Apidae), and members of the family Halictidae, with infrequent visits by A. cerana and Bombus species. Fruit set and the production of large embryos following interform pollination treatments were significantly lower compared to intraform pollination results for the white form. Our results suggested that pollinator isolation, based on color and scent cues, may result in greater floral constancy in white populations when both forms are sympatric as two different, guilds of pollinators forage selectively preventing or reducing prospective gene flow. Postpollination barriers appear weaker than prepollination barriers but they also play a role in interform isolation, especially in the white form. Our findings suggest that floral color forms in S. sinensis do not represent an unbalanced polymorphism. Interpretations of the evolutionary status of these forms are discussed.

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title: A LOCAL FLORA AND THE BIOLOGICAL SPECIES CONCEPT
abstract: Up to the end of the 19th century the typological species concept was universally accepted, in which degree of morphological difference was the species criterion. This concept was increasingly replaced (particularly in zoology) beginning around 1900 by the biological species concept because the criterion of morphological distinctness broke down in the case of sibling species and of highly distinct intrapopulational variants. The yardstick for the recognition of biological species is the noninterbreeding of coexisting species in a local situation. This yardstick can be applied to isolated populations only by inference. It is discussed whether the making of such inferences is too arbitrary to be acceptable. A local flora is analyzed to determine whether polyploidy, apomixis, hybridization, and other deviations from regular sexual reproduction are sufficiently frequent in plants to make the biological species concept illusory. It is found that the number of difficulties is small, validating use of the sympatric situation as yardstick for the ranking of allopatric populations.

title: The strength and genetic basis of reproductive isolating barriers in flowering plants
abstract: Speciation is characterized by the evolution of reproductive isolation between two groups of organisms. Understanding the process of speciation requires the quantification of barriers to reproductive isolation, dissection of the genetic mechanisms that contribute to those barriers and determination of the forces driving the evolution of those barriers. Through a comprehensive analysis involving 19 pairs of plant taxa, we assessed the strength and patterns of asymmetry of multiple prezygotic and postzygotic reproductive isolating barriers. We then reviewed contemporary knowledge of the genetic architecture of reproductive isolation and the relative role of chromosomal and genic factors in intrinsic postzygotic isolation. On average, we found that prezygotic isolation is approximately twice as strong as postzygotic isolation, and that postmating barriers are approximately three times more asymmetrical in their action than premating barriers. Barriers involve a variable number of loci, and chromosomal rearrangements may have a limited direct role in reproductive isolation in plants. Future research should aim to understand the relationship between particular genetic loci and the magnitude of their effect on reproductive isolation in nature, the geographical scale at which plant speciation occurs, and the role of different evolutionary forces in the speciation process.

title: Ecological Reproductive Isolation of Coast and Inland Races of Mimulus guttatus
abstract: Adaptive divergence due to habitat differences is thought to play a major role in formation of new species. However it is rarely clear the extent to which individual reproductive isolating barriers related to habitat differentiation contribute to total isolation. Furthermore, it is often difficult to determine the specific environmental variables that drive the evolution of those ecological barriers, and the geographic scale at which habitat-mediated speciation occurs. Here, we address these questions through an analysis of the population structure and reproductive isolation between coastal perennial and inland annual forms of the yellow monkeyflower, Mimulus guttatus. We found substantial morphological and molecular genetic divergence among populations derived from coast and inland habitats. Reciprocal transplant experiments revealed nearly complete reproductive isolation between coast and inland populations mediated by selection against immigrants and flowering time differences, but not postzygotic isolation. Our results suggest that selection against immigrants is a function of adaptations to seasonal drought in inland habitat and to year round soil moisture and salt spray in coastal habitat. We conclude that the coast and inland populations collectively comprise distinct ecological races. Overall, this study suggests that adaptations to widespread habitats can lead to the formation of reproductively isolated species.

title: Evolution of reproductive isolation in plants
abstract: Reproductive isolation is essential for the process of speciation and much has been learned in recent years about the ecology and underlying genetics of reproductive barriers. But plant species are typically isolated not by a single factor, but by a large number of different pre- and postzygotic barriers, and their potentially complex interactions. This phenomenon has often been ignored to date. Recent studies of the relative importance of different isolating barriers between plant species pairs concluded that prezygotic isolation is much stronger than postzygotic isolation. But studies of the patterns of reproductive isolation in plants did not find that prezygotic isolation evolves faster than postzygotic isolation, in contrast to most animals. This may be due to the multiple premating barriers that isolate most species pairs, some of which may be controlled by few genes of major effect and evolve rapidly, whereas others have a complex genetic architecture and evolve more slowly. Intrinsic postzygotic isolation in plants is correlated with genetic divergence, but some instrinsic postzygotic barriers evolve rapidly and are polymorphic within species. Extrinsic postzygotic barriers are rarely included in estimates of different components of reproductive isolation. Much remains to be learned about ecological and molecular interactions among isolating barriers. The role of reinforcement and reproductive character displacement in the evolution of premating barriers is an open topic that deserves further study. At the molecular level, chromosomal and genic isolation factors may be associated and act in concert to mediate reproductive isolation, but their interactions are only starting to be explored.

title: The origins of reproductive isolation in plants.
abstract: Reproductive isolation in plants occurs through multiple barriers that restrict gene flow between populations, but their origins remain uncertain. Work in the past decade has shown that postpollination barriers, such as the failure to form hybrid seeds or sterility of hybrid offspring, are often less strong than prepollination barriers. Evidence implicates multiple evolutionary forces in the origins of reproductive barriers, including mutation, stochastic processes and natural selection. Although adaptation to different environments is a common element of reproductive isolation, genomic conflicts also play a role, including female meiotic drive. The genetic basis of some reproductive barriers, particularly flower colour influencing pollinator behaviour, is well understood in some species, but the genetic changes underlying many other barriers, especially pollen-stylar interactions, are largely unknown. Postpollination barriers appear to accumulate at a faster rate in annuals compared with perennials, due in part to chromosomal rearrangements. Chromosomal changes can be important isolating barriers in themselves but may also reduce the recombination of genes contributing to isolation. Important questions for the next decade include identifying the evolutionary forces responsible for chromosomal rearrangements, determining how often prezygotic barriers arise due to selection against hybrids, and establishing the relative importance of genomic conflicts in speciation.

title: Strong post-pollination pre-zygotic isolation between sympatric, food-deceptive Mediterranean orchids
abstract: Mediterranean orchids that grow in admixed, co-flowering populations, and frequently show hybrid progenies are interesting to use to study the nature and the strength of post-zygotic barriers. However, examination of pre- and post-pollination pre-zygotic isolating mechanisms requires sympatric, co-flowering species pairs that do not produce hybrid swarms. In this study, we analyzed a contact zone between Orchis italica and O. papilionacea, in which hybrid forms have never been reported, although hybridization between members of their groups of appurtenance has been signaled. We investigated pre-pollination barriers observing the floral phenology of both species and identified pollinators by means of molecular analysis of pollinaria collected on the insects captured in the study site. Post-pollination barriers were tested performing manual crosses in order to evaluate pollen germination/pollen tube growth in vivo and fruit and seed formation. Floral phenologies of O. italica and O. papilionacea display nearly overlapping trends, and two common pollinators have been identified by molecular analysis of pollinaria. Thus, pre-pollination barriers are very weak or nonexistent. Bidirectional crosses have shown that the growth of heterospecific pollen tubes is fully blocked in stigmatic cell layers. Since no fruit formation was detected in bidirectional interspecific crosses, we assume that reproductive isolation between the examined species is fully guaranteed by post-pollination pre-zygotic mechanisms acting at stigmatic level. Such condition has been rarely described and may mask the potential action of post-zygotic mechanisms.

title: Strong premating reproductive isolation drives incipient speciation in Mimulus aurantiacus
abstract: Determining which forms of reproductive isolation have the biggest impact on the process of divergence is a major goal of speciation research. These barriers are often divided into those that affect the potential for hybridization (premating isolation), and those that occur after mating (postmating isolation), and much debate has surrounded the relative importance of these categories. Within the species Mimulus aurantiacus, red‐ and yellow‐flowered ecotypes occur in the southwest corner of California, and a hybrid zone occurs where their ranges overlap. We show that premating barriers are exclusively responsible for isolation in this system, with both ecogeographic and pollinator isolation contributing significantly to total isolation. Postmating forms of reproductive isolation have little or no impact on gene flow, indicating that hybrids likely contribute to introgression at neutral loci. Analysis of molecular variation across thousands of restriction‐site associated DNA sequencing (RAD‐seq) markers reveals that the genomes of these taxa are largely undifferentiated. However, structure analysis shows that these taxa are distinguishable genetically, likely due to the impact of loci underlying differentiated adaptive phenotypes. These data exhibit the power of divergent natural selection to maintain highly differentiated phenotypes in the face of gene flow during the early stages of speciation.

title: FLORAL ISOLATION IS THE MAIN REPRODUCTIVE BARRIER AMONG CLOSELY RELATED SEXUALLY DECEPTIVE ORCHIDS
abstract: Floral isolation is an important component of pollinator‐driven speciation. However, up to now, only a few studies have quantified its strength and relative contribution to total reproductive isolation. In this study, we quantified floral isolation among three closely related, sympatric orchid species of the genus Ophrys by directly tracking pollen flow. Ophrys orchids mimic their pollinators’ mating signals, and are pollinated by male insects during mating attempts. This pollination system, called sexual deception, is usually highly specific. However, whether pollinator specialization also conveys floral isolation is currently under debate. In this study, we found strong floral isolation: among 46 tracked pollen transfers in two flowering seasons, all occurred within species. Accounting for observation error rate, we estimated a floral isolation index ≥0.98 among each pair of species. Hand pollination experiments suggested that postpollination barriers were effectively absent among our study species. Genetic analysis based on AFLP markers showed a clear species clustering and very few F1 hybrids in natural populations, providing independent evidence that strong floral isolation prevents significant interspecies gene flow. Our results provide the first direct evidence that floral isolation acts as the main reproductive barrier among closely related plant species with specialized pollination.

title: Pre- and post-zygotic reproductive isolation between co-occurring Mussaenda pubescens var. alba and M. shikokiana (Rubiaceae).
abstract: Reproductive isolation is a fundamental requirement for speciation and includes several sequential stages. Few studies have determined the relative contributions of pre- and post-zygotic reproductive isolation in plants, especially between relative species with clear differentiation in flower form. To investigate the mechanisms responsible for reproductive isolation in sympatric Mussaenda pubescens var. alba and Mussaenda shikokiana (Rubiaceae) in Guangxi Province, China, we made observations of flowering phenology, patterns of insect visitation, and conducted pollination experiments, including artificial hybridization. The two species had overlapping flowering times and were pollinated by overlapping pollinators; however, their relative importance differed significantly with M. pubescens visited more commonly by bees and M. shikokiana more frequently by butterflies. Using vegetative and floral characters and molecular evidence based on nuclear ribosomal internal and external transcribed spacer regions we detected seven naturally occurring hybrids among a sample of approximately 125 individuals. Hybrids were characterized by morphologies that most closely resembled their maternal parents based on chloroplast evidence. Studies of artificially synthesized and natural hybrids demonstrated that hybrid seed had very low germination rates and naturally occurring hybrids exhibited pollen sterility. Post-zygotic reproductive isolating mechanisms play a primary role in limiting gene exchange between co-occurring species and maintaining species integrity in areas of sympatry.

title: Post-zygotic reproductive isolation between sympatric taxa in the Chamaecrista desvauxii complex (Leguminosae-Caesalpinioideae).
abstract: Differences in the mating systems and the mechanisms of reproductive isolation between Chamaecrista desvauxii var. graminea and C. desvauxii var. latistipula were examined in the Chapada Diamantina, Brazil. These taxa occur sympatrically, and their populations demonstrate marked morphological differences. The objective of the present work was to determine if reproductive isolation mechanisms exist between these two populations of C. desvauxii, and to determine the influence of these putative mechanisms on their genetic differentiation.



Field observations were made of floral biology, phenology and floral visitation, and experiments on intra- and interpopulation pollination and germination rates of the resultant seeds were performed. A genetic examination of the populations was undertaken using four allozyme loci.



The varieties examined demonstrated overlapping of flowering periods during the months of June to September. The main pollinator for both varieties was the bee Bombus brevivillus. Both varieties are self-compatible, and a large number of fruits are formed in cross-pollinations with high seed germination rates. Inter-taxa pollinations result in high levels of fruit production, but no seeds are formed. Two of the four loci examined were diagnostic for the varieties, and exclusive high-frequency alleles were encountered at the other loci, leading to a high genetic distance between the two populations (0.495).



Pre-zygotic barriers were not found between the two varieties, and these remain isolated due to post-zygotic events. The two varieties demonstrate marked differences in their morphology, floral biology, phenology and genetic make-up, all of which indicate that they should be treated as two distinct species. A complete revision involving the other varieties of the C. desvauxii complex will be necessary in order to define these two taxa formally.

title: Isolation mechanisms between two sympatric Sophronitis (Orchidaceae) species endemic to Northeastern Brazil
abstract: Sophronitis sincorana and S. pfisteri (Orchidaceae) are endemic to the campo rupestre vegetation, northeastern Brazil, occurring sympatrically and flowering synchronously. In this work we studied the reproductive biology of both species and investigated reproductive barriers between them. Both species are partially self-compatible, pollinator dependent, and are pollinated by males and worker bees of Bombus brevivillus. Pollinaria are deposited on the anterior portion of the mesonotum of these insects in S. pfisteri, and on the scutellum in S. sincorana. The flowers of both species are deceptive, as they have a dry cuniculus. Morphological differences in the labellum and the gynostemium impede inter-specific pollination of S. pfisteri with pollen from S. sincorana. Very low fruiting results from inter-specific crosses of S. sincorana with pollen from S. pfisteri. Although viable seeds can be produced from experimental inter-specific crosses, no natural hybrids were found. Species integrity appears to be maintained by additive unidirectional reproductive barriers between them.

title: Generalization versus specialization in plant pollination systems.
abstract: The long-standing notion that most angiosperm flowers are specialized for pollination by particular animal types, such as birds or bees, has been challenged recently on the basis of apparent widespread generalization in pollination systems. At the same time, biologists working mainly in the tropics and the species-rich temperate floras of the Southern hemisphere are documenting pollination systems that are remarkably specialized, often involving a single pollinator species. Current studies are aimed at understanding: (1) the ecological forces that have favoured either generalization or specialization in particular lineages and regions; (2) the implications for selection on floral traits and divergence of populations; and (3) the risk of collapse in plant-pollinator mutualisms of varying specificity.

title: Pollinator‐mediated isolation in sympatric milkweeds (Asclepias): do floral morphology and insect behavior influence species boundaries?
abstract: •We explored whether mechanical or ethological differences provide pollinator-mediated floral isolation capable of reinforcing existing species barriers among sympatric Asclepias with divergent floral morphologies: A. incarnata, A. verticiallata and A. syriaca. 
•In a common garden, we quantified pollinator visitation and flight patterns, differences in corporal attachment of pollinia to insects, and the potential outcome of putative floral barriers for interspecific pollination and fruit set. 
•We detected significant variation in the importance, constancy, and behavior of major pollinators on sympatric asclepiads, including Bombus, Xylocopa and large sphecid wasps. Pollinia attach differentially to the arolium on insect legs for A. syriaca, but to the tarsal hairs in other asclepiads. Fruit-set was lower in mixed than unispecific patches of Asclepias. 
•We detected mechanical isolation between A. syriaca and its congeners and a tendency toward wasp pollination in A. verticillata. All three species appear to show some specialization for long-tongued hymenoptera and lepidopterans. Pre–mating barriers provide a potentially effective means of reducing interspecific pollination, but more study is needed in species visited by generalists.

title: Generalization in Pollination Systems, and Why it Matters
abstract: One view of pollination systems is that they tend toward specialization. This view is implicit in many discussions of angiosperm evolution and plant-pollinator coevolution and in the long-standing concept of pollination syndromes. But actual pollination systems often are more generalized and dynamic than these traditions might suggest. To illustrate the range of specialization and generalization in pollinators' use of plants and vice versa, we draw on studies of two floras in the United States, and of members of several plant families and solitary bee genera. We also summarize a recent study of one local flora which suggests that, although the colors of flowers are aggregated in phenotype space, there is no strong association with pollinator types as pollination syndromes would predict. That moderate to substantial generalization often occurs is not surprising on theoretical

title: Variation in sexual reproduction in orchids and its evolutionary consequences: a spasmodic journey to diversification
abstract: The great taxonomic diversity of the Orchidaceae is often attributed to adaptive radiation for specific pollinators driven by selection for outcrossing. However, when one looks beyond the product to the process, the evidence for selection is less than overwhelming. We explore this problem by discussing relevant aspects of orchid biology and asking which aspects of reproduction explain the intricate pollination mechanisms and diversification of this family. We reaffirm that orchids are primarily pollination limited, the severity of which is affected by resource constraints. Fruit set is higher in temperate than in tropical species, and in species which offer pollinator rewards than those that do not. Reproductive success is skewed towards few individuals in a population and effective population sizes are often small. Population structure, reproductive success and gene flow among populations suggest that in many situations genetic drift may be as important as selection in fostering genetic and morphological variation in this family. Although there is some evidence for a gradualist model of evolutionary change, we believe that the great diversity in this family is largely a consequence of sequential and rapid interplay between drift and natural selection.  2005, 84, 1–54.

title: Orchid phylogenomics and multiple drivers of their extraordinary diversification
abstract: Orchids are the most diverse family of angiosperms, with over 25 000 species, more than mammals, birds and reptiles combined. Tests of hypotheses to account for such diversity have been stymied by the lack of a fully resolved broad-scale phylogeny. Here, we provide such a phylogeny, based on 75 chloroplast genes for 39 species representing all orchid subfamilies and 16 of 17 tribes, time-calibrated against 17 angiosperm fossils. A supermatrix analysis places an additional 144 species based on three plastid genes. Orchids appear to have arisen roughly 112 million years ago (Mya); the subfamilies Orchidoideae and Epidendroideae diverged from each other at the end of the Cretaceous; and the eight tribes and three previously unplaced subtribes of the upper epidendroids diverged rapidly from each other between 37.9 and 30.8 Mya. Orchids appear to have undergone one significant acceleration of net species diversification in the orchidoids, and two accelerations and one deceleration in the upper epidendroids. Consistent with theory, such accelerations were correlated with the evolution of pollinia, the epiphytic habit, CAM photosynthesis, tropical distribution (especially in extensive cordilleras), and pollination via Lepidoptera or euglossine bees. Deceit pollination appears to have elevated the number of orchid species by one-half but not via acceleration of the rate of net diversification. The highest rate of net species diversification within the orchids (0.382 sp sp−1 My−1) is 6.8 times that at the Asparagales crown.

title: Floral isolation, specialized pollination, and pollinator behavior in orchids.
abstract: Floral isolation is a form of prepollination reproductive isolation mediated by floral morphology (morphological isolation) and pollinator behavior (ethological isolation). Here we review mechanisms and evolutionary consequences of floral isolation in various pollination systems. Furthermore, we compare key features of floral isolation, i.e., pollinator sharing and specialization in pollination, in different orchid pollination systems. In orchid pollination, pollinator sharing is generally low, indicating strong floral isolation. The pollinators' motivation to visit flowers (specifically) can be due to both foraging or reproductive behavior. In both types of behavior, innate preferences for floral signals can be quickly overruled by learning. In pollination systems in which reproductive behavior of pollinators triggers flower visits, lower pollinator sharing was evident compared with systems with foraging behavior, probably because pollinators displaying reproductive behavior show higher fidelity in their visitation patterns. Orchids pollinated through reproductive behavior also use fewer pollinators than orchids pollinated through foraging behavior. No association between specialization and pollinator sharing was found. Thus, generalized pollination does not impede floral isolation, as orchids with many pollinators may nonetheless have low pollinator sharing. Specialization in pollination was, however, linked to orchid species richness in our analysis. Flower size, spur, and column morphology are most important for morphological isolation, and floral scent is most important for ethological isolation. These traits may be based on few genes, implying that floral isolation can be brought about by few genes of large effect.

title: Phylogeny and classification of the orchid family
abstract: 1.  Structure and other evidence of relationship 3. Orchid phylogeny 4. Orchids with two or three fertile anthers 5. Primitive orchids with a single fertile anther 6. Lady's tresses and relatives, the subfamily Spiranthoideae 7. Orchis and its allies, the subfamily Orchidoideae 8. The advanced Epidendroideae, general discussion and phylogeny 9. The cormous or cymbidioid phylad 10. The reed-stem or epidendroid phylad 11. Misfits, parallelisms, and miscellaneous problems 12. Classification and phylogenetic analysis Glossary Appendices Literature cited Index.

title: Specificity in pollination and consequences for postmating reproductive isolation in deceptive Mediterranean orchids
abstract: The type of reproductive isolation prevalent in the initial stages of species divergence can affect the nature and rate of emergence of additional reproductive barriers that subsequently strengthen isolation between species. Different groups of Mediterranean deceptive orchids are characterized by different levels of pollinator specificity. Whereas food-deceptive orchid species show weak pollinator specificity, the sexually deceptive Ophrys species display a more specialized pollination strategy. Comparative analyses reveal that orchids with high pollinator specificity mostly rely on premating reproductive barriers and have very little postmating isolation. In this group, a shift to a novel pollinator achieved by modifying the odour bouquet may represent the main isolation mechanism involved in speciation. By contrast, orchids with weak premating isolation, such as generalized food-deceptive orchids, show strong evidence for intrinsic postmating reproductive barriers, particularly for late-acting postzygotic barriers such as hybrid sterility. In such species, chromosomal differences may have played a key role in species isolation, although strong postmating–prezygotic isolation has also evolved in these orchids. Molecular analyses of hybrid zones indicate that the types and strength of reproductive barriers in deceptive orchids with contrasting premating isolation mechanisms directly affect the rate and evolutionary consequences of hybridization and the nature of species differentiation.

title: Floral biology of large-flowered Thelymitra species (Orchidaceae) and their hybrids in Western Australia
abstract: Historically, only a few large flowered species in the genus Thelymitra were identified as obligate out-breeders. We compared floral presentation, pollen-pistil interactions, pollination ecology, and interspecific hybridization in two populations of T. macrophylla where its flowering periods overlapped with T. antennifera (Tenterden) and T. crinita (Lesmurdie) respectively. Pollen-pistil interactions were studied using glasshouse collections of T. crinita and T. macrophylla at the Kings Park and Botanic Garden (Perth, WA). The number of flowers/inflorescence in T. macrophylla varied significantly between sites. Climatic conditions influenced flower opening and closing regimes differently in T. crinita vs. T. macrophylla. While all three Thelymitra spp. opened on warm, sunny mornings and closed by late afternoon, T. crinita at Lesmurdie was significantly more likely to open its perianth segments on cool days compared to the co-blooming, sympatric flowers of T. macrophylla. The floral lifespans of individual flowers of T. macrophylla and T. crinita were reduced significantly following application of Thelymitra pollen onto the stigmatic surface but were not reduced by pollinarium removal. Flowers of both species were self-compatible but neither species self-pollinated mechanically (autogamy). Fluorescence microscopy also showed that both species were inter-compatible. Natural rates of pollinarium removal by insects were low in all three species at both sites. Natural rates of pollen deposition on receptive stigmas were significantly higher in T. crinita vs. T. macrophylla but pollen deposition was less than 12% in both species. Observations and collections of pollinators were infrequent and pollinaria vectors were restricted to a few polylectic, female bees in the families Apidae, Colletidae, and Halictidae. We found a large number of hybrids between T. antennifera and T. macrophylla at Tenterden but few obvious hybrids between T. crinita and T. macrophylla at Lesmurdie. As expected, all hybrids showed characteristics intermediate between their two putative parent species, including pollen configuration in the T. crinita X T. macrophylla specimens. Due to malformations of the column, the majority of T. antennifera X T. macrophylla flowers appeared unable to attach their pollinia to their respective rostella.

title: COMPONENTS OF REPRODUCTIVE ISOLATION BETWEEN ORCHIS MASCULA AND ORCHIS PAUCIFLORA
abstract: Studies of the strength and nature of reproductive isolation (RI) between species can greatly contribute to our understanding of speciation. Although the role of RI in speciation is well recognized, there is a dearth of information on the contributions of different barriers between related plant species. Here, we estimated multiple components of RI between two Mediterranean orchid sister species (Orchis mascula and Orchis pauciflora), disentangling the strength and absolute contributions of seven different isolating mechanisms. Our survey includes one prepollination, two postpollination prezygotic (pollen–stigma incompatibility, conspecific pollen precedence), two intrinsic postzygotic (embryo mortality and hybrid sterility) and two extrinsic postzygotic (hybrid habitat differentiation and hybrid pollination) isolating mechanisms. We found strong RI between the investigated species, although none of the barriers were able to completely impede gene flow. Five isolating mechanisms contributed positively to the maintenance of species boundaries. Contrary to most surveys of isolating mechanisms, our data speak against a clear predominance of prepollination or of prezygotic barriers but confirm the emerging pattern of multiple barriers contributing to the maintenance of species integrity. These findings suggest an allopatric condition during early phases of species divergence. We discuss our data in the wider context of previous studies carried out in this orchid group by using a comparative approach.

title: PATTERNS OF REPRODUCTIVE ISOLATION IN MEDITERRANEAN DECEPTIVE ORCHIDS
abstract: The evolution of reproductive isolation is of central interest in evolutionary biology. In plants, this is typically achieved by a combination of pre- and postpollination mechanisms that prevent, or limit, the amount of interspecific gene flow. Here, we investigated and compared two ecologically defined groups of Mediterranean orchids that differ in pollination biology and pollinator specificity: sexually deceptive orchids versus food-deceptive orchids. We used experimental crosses to assess the strength of postmating prezygotic, and postzygotic reproductive isolation, and a phylogenetic framework to determine their relative rates of evolution. We found quantitative and qualitative differences between the two groups. Food-deceptive orchids have weak premating isolation but strong postmating isolation, whereas the converse situation characterizes sexually deceptive orchids. Only postzygotic reproductive isolation among food-deceptive orchids was found to evolve in a clock-like manner. Comparison of evolutionary rates, within a common interval of genetic distance, showed that the contribution of postmating barriers was more relevant in the food-deceptive species than in the sexually deceptive species. Asymmetry in prezygotic isolation was found among food-deceptive species. Our results indicate that postmating barriers are most important for reproductive isolation in food-deceptive orchids, whereas premating barriers are most important in sexually deceptive orchids. The different rate of evolution of reproductive isolation and the relative strength of pre- and postmating barriers may have implication for speciation processes in the two orchid groups.

title: Pollen transfer efficiency and its effect on inflorescence size in deceptive pollination strategies.
abstract: Pollination systems differ in pollen transfer efficiency, a variable that may influence the evolution of flower number. Here we apply a comparative approach to examine the link between pollen transfer efficiency and the evolution of inflorescence size in food and sexually deceptive orchids. We examined pollination performance in nine food-deceptive, and eight sexually deceptive orchids by recording pollen removal and deposition in the field. We calculated correlations between reproductive success and flower number (as a proxy for resources allocated during reproductive process), and directional selection differentials were estimated on flower number for four species. Results indicate that sexually deceptive species experience decreased pollen loss compared to food-deceptive species. Despite producing fewer flowers, sexually deceptive species attained levels of overall pollination success (through male and female function) similar to food-deceptive species. Furthermore, a positive correlation between flower number and pollination success was observed in food-deceptive species, but this correlation was not detected in sexually deceptive species. Directional selection differentials for flower number were significantly higher in food compared to sexually deceptive species. We suggest that pollination systems with more efficient pollen transfer and no correlation between pollination success and number of flowers produced, such as sexual deception, may allow the production of inflorescences with fewer flowers that permit the plant to allocate fewer resources to floral displays and, at the same time, limit transpiration. This strategy can be particularly important for ecological success in Mediterranean water-deprived habitats, and might explain the high frequency of sexually deceptive species in these specialised ecosystems.

title: Evolution of Postzygotic Reproductive Isolation in a Guild of Deceptive Orchids
abstract: The evolution of reproductive barriers is of central importance for speciation. Here, we investigated three components of postzygotic isolation—embryo mortality, hybrid inviability, and hybrid sterility—in a group of food‐deceptive Mediterranean orchids from the genera Anacamptis, Neotinea, and Orchis. In these orchids, pollinator‐mediated isolation is weak, which suggests that postpollination barriers exist. Based on crossing experiments and a literature survey, we found that embryo mortality caused complete reproductive isolation among 36.3% of the species pairs, and hybrid inviability affected 55.6% of the potentially hybridizing species pairs. Hybrid sterility was assessed experimentally for seven species pairs. A strong reduction of fertility in all investigated hybrids was found, together with clear differences between male and female components of hybrid sterility. Postzygotic isolation was found to evolve gradually with genetic divergence, and late postzygotic isolation (i.e., hybrid inviability and sterility) evolved faster than embryo mortality, which is an earlier postzygotic isolation stage. These results reveal that intrinsic postzygotic isolation strongly contributes to maintaining species boundaries among Mediterranean food‐deceptive orchids while establishing a prominent role for these reproductive barriers in the early stage of species isolation.

title: Orchid diversity: an evolutionary consequence of deception?
abstract: The Orchidaceae are one of the most species-rich plant families and their floral diversity and pollination biology have long intrigued evolutionary biologists. About one-third of the estimated 18,500 species are thought to be pollinated by deceit. To date, the focus has been on how such pollination evolved, how the different types of deception work, and how it is maintained, but little progress has been made in understanding its evolutionary consequences. To address this issue, we discuss here how deception affects orchid mating systems, the evolution of reproductive isolation, speciation processes and neutral genetic divergence among species. We argue that pollination by deceit is one of the keys to orchid floral and species diversity. A better understanding of its evolutionary consequences could help evolutionary biologists to unravel the reasons for the evolutionary success of orchids.

title: Processes of isolation and introgressive interplay between Platanthera bifolia (L.) Rich and P. chlorantha (Custer) Reichb. (Orchidaceae)
abstract: NILSSON, L. A., 1983. Processes of isolation and introgressive interplay between Platanthera bifolia L. Rich, and P. chlorantha (Custer; Reichb. (Orchidaceae). Platanthera bifolia and P. chlorantha are highly specialized for pollination by crepuscular and nocturnal Lepidoptera. The plants are largely sympatric, have overlapping flowering-times, and are completely interfertile. Principal differences exist in floral fragrance and column morphology. Isolation is effected by a set of integrated morphological, ethological, and seasonal mechanisms. Odour-based constancy by moths, differences in column structure, attachment of pollinaria, spurlength, nectar availability, and flowering-peaks reduce interspecific pollen transfer. The plants form a functional species pair in relation to their pollination environment and locally share the same main pollinator species. 
 
 
 
Hybrids, which infrequently occur in areas of sympatry, emit a blend of the species-specific fragrance compounds and have intermediate column structure. They are frequently visited by-moths but suffer a serious reduction in seed production due to imperfect interaction vis-a-vis the pollen vectors. Hybrid viscidia, in an intermediate position on the column, are touched by moths' scaly and hairy palps and get insufficient attachment, evidently causing hybrids to act solely as female recipient. First backcross individuals largely join the functional range of either species which promotes rapid introgression. The two gene-pools interplay, although always at a low level. Patterns of introgressive hybridization were exemplified.

title: Floral syndromes accurately predict pollination by a specialized oil-collecting bee (Rediviva peringueyi, Melittidae) in a guild of South African orchids (Coryciinae).
abstract: The long-standing notion of pollination syndromes, which postulates that plants form recognizable groups according to pollinator type, has been challenged recently on the basis of apparent widespread generalization in pollination systems. As a test of the pollination syndrome concept, I examined the pollination biology of a group of 15 orchids that share a recognizable syndrome of floral features that includes yellow-green coloration, oil secretion, pungent scent, shallow flowers, and a September peak in flowering. The orchids occur in sympatry in the Cape Floral Region of South Africa. According to the pollination syndrome concept, the similar floral features of this group indicate a shared pollinator. To test this prediction, I observed pollinators on Pterygodium alatum, P. caffrum, P. catholicum, P. volucris, Corycium orobanchoides, and Disperis bolusiana subsp. bolusiana. They shared a single species of pollinator, the oil-collecting bee, Rediviva peringueyi. Female bees collected oil from the lip appendage using modified front tarsi. The orchids reduce interspecific reproductive interference through differences in pollinarium length or the use of mutually exclusive pollinarium attachment sites on the body of the bee. The results are contrary to the expectation of generalization in pollination systems and suggest that pollinators play an important role in mediating selection on floral traits.

title: Eye Attached Hemipollinaria in the Hawkmoth and Settling Moth Pollination of Habenaria (Orchidaceae): A Study on Functional Morphology in 5 Species from Subtropical South America
abstract: : 
 
Morphological adaptations to sphingophily and pollination by moths was studied in 5 South American Habenaria species. For H. gourlieana and H. hieronymi direct evidence of hawkmoth (Agrius cingulatus and Manduca sexta) and settling moth (Rachiplusia nu) pollination, respectively, by hemipollinaria attachment on the eyes, is presented. In two other species (H. paucofilia, H. rupicola) pollination by settling moths and eye attachment of the hemipollinaria can be inferred by indirect evidence (placement of scales and massulae on the flowers) and by flower structure. For the fifth species (H. montevidensis) pollination by small moths or mosquitoes with hemipollinaria attachment on the proboscis is postulated. A comparative study in floral features revealed clear morphological divergence between sphingophilous and phalaenophilous species. In addition to deeper spurs the former have slender, exerted, and upturned petal lobes (acting as mechanical guide to the hovering visitors), a much developed median rostellar lobe (acting as deflecting surface of the hawkmoths towards the viscidia), flexible and sinuous hemipollinarium caudicles (appropriate for frontal strikes against the stigma when hemipollinaria are brought by the hawkmoths dangling against the flower). Male efficiency was compared between 4 species with overlapping flowering time in the same area. Male efficiency factors were unexpectedly low in all species. Only in one species (H. hieronymi) each pollen donation accounted for more than one pollination. H. gourlieana is part of a more or less rich sphingophilous flora interacting with the same two long-tongued hawkmoth species. Interspecific competition for pollen placement on the pollinator's body surface is probably low on account of different pollination mechanisms.

title: Quechua, a New Monotypic Genus of Andean Spiranthinae (Orchidaceae)
abstract: The systematic position of the little-known Andean species Spiranthes glabrescens, previously known only from Peru but here recorded for the first time from Ecuador, was investigated by means of parsimony and Bayesian analyses of cpDNA and nuclear DNA sequences. Our results indicate that this species is only distantly related to both Spiranthes and Cyclopogon, the genera in which it has been included by previous taxonomists. Instead it is recovered as sister to a clade formed by Eurystyles and Lankesterella, with strong support from the Bayesian analysis. The new, monotypic genus Quechua is proposed to accommodate former S. glabrescens, which differs from both Eurystyles and Lankesterella in habit as well as in vegetative and reproductive characters. The main ecological and structural features of Quechua glabrescens are discussed and compared with those of other genera of Spiranthinae.

title: Global Collaborative Research on the Pollination Biology of Rare and Threatened Orchid Species (Orchidaceae)1
abstract: Charles Darwin (1862, 1877) conducted field and lab research on orchid pollination mechanisms before any protocols or laws protected orchid species or individual populations. Information on the reproductive ecology of rare and threatened orchids remains intrinsic to their conservation as populations continue to diminish during the Anthropocene. We provide case studies of species representing five genera (Calanthe R. Br., Corunastylis Fitzg., Cypripedium L., Spiranthes Rich., and Thelymitra J. R. Forst. & G. Forst.) in three countries (the United States, Australia, and China). We compare differences in their pollination systems and seasonal variations in their fertility rates. Declining rates of fruit set and/or seed development are compared to breeding systems, pollinator life histories/foraging behaviors, and climate cycles. We conclude that, while some orchid species are pollinator limited, several interrelated factors influence rates of fecundity in terrestrial/lithophytic orchids.

title: Phylogenetics of Cranichideae with emphasis on Spiranthinae (Orchidaceae, Orchidoideae): evidence from plastid and nuclear DNA sequences.
abstract: DNA sequences from plastid rbcL and matK genes and the trnL-F region, as well as the nuclear ribosomal ITS region, were used to evaluate monophyly and subtribal delimitation of Cranichideae and generic relationships in Spiranthinae. Cranichideae are moderately supported as monophyletic, with Chloraeinae and Pterostylis-Megastylis indicated as their collective sisters. Within Cranichideae, Pachyplectroninae and Goodyerinae form a well-supported monophyletic group sister to a "core spiranthid" clade that includes, according to their branching order, Galeottiellinae, Manniellinae, and a Prescottiinae-Cranichidinae-Spiranthinae subclade. Inclusion of Galeottiella in Spiranthinae, as in previous classifications, renders the latter paraphyletic to all other spiranthid subtribes. Cranichidinae and Spiranthinae (minus Galeottiella) are monophyletic and strongly supported, but Prescottiinae form a grade that includes a strongly supported prescottioid Andean clade and a weakly supported Prescottia-Cranichidinae clade sister to Spiranthinae. Well-supported major clades in Spiranthinae identified in this study do not correspond to previous alliances or the narrowly defined subtribes in which they have been divided recently. Morphological characters, especially those that have been used for taxonomic delimitation in Cranichideae, are discussed against the framework of the molecular trees, emphasizing putative synapomorphies and problems derived from lack of information or inadequate interpretation of the characters.

title: A rapid DNA isolation procedure for small quantities of fresh leaf tissue
abstract: A stopped flow kinetic analysis has been performed with a homogeneous protein fraction of plant glutamate dehydrogenase. The enzyme exerts strong negative cooperativity with ammonium as variable substrate. The limiting initial rate constants for low substrate concentrations, as calculated from the kinetic data, indicate that the catalytic efficiency of the enzyme increases at low ammonium concentrations. From this it becomes evident that the reductive amination reaction is highly adaptive to the ammonium environment.

title: DNA barcoding of Pedicularis L. (Orobanchaceae): Evaluating four universal barcode loci in a large and hemiparasitic genus
abstract: One application of DNA barcoding is species identification based on sequences of a short and standardized DNA region. In plants, various DNA regions, alone or in combination, have been proposed and investigated, but consensus on a universal plant barcode remains elusive. In this study, we tested the utility of four candidate barcoding regions (rbcL, matK, trnH‐psbA, and internal transcribed spacer (ITS)) as DNA barcodes for discriminating species in a large and hemiparasitic genus Pedicularis (Orobanchaceae). Amplification and sequencing was successful using single primer pairs for rbcL, trnH‐psbA, and ITS, whereas two primer pairs were required for matK. Patterns of sequence divergence commonly showed a “barcoding gap”, that is, a bimodal frequency distribution of pairwise distances representing genetic diversity within and between species, respectively. Considering primer universality, ease of amplification and sequencing, and performance in discriminating species, we found the most effective single‐region barcode for Pedicularis to be ITS, and the most effective two‐region barcode to be rbcL + ITS. Both discriminated at least 78% of the 88 species and correctly identified at least 89% of the sequences in our sample, and were effective in placing unidentified samples in known species groups. Our results suggest that DNA barcoding has the potential to aid taxonomic research in Pedicularis, a species‐rich cosmopolitan clade much in need of revision, as well as ecological studies in its center of diversity, the Hengduan Mountains region of China.

title: MUSCLE: multiple sequence alignment with high accuracy and high throughput.
abstract: We describe MUSCLE, a new computer program for creating multiple alignments of protein sequences. Elements of the algorithm include fast distance estimation using kmer counting, progressive alignment using a new profile function we call the log-expectation score, and refinement using tree-dependent restricted partitioning. The speed and accuracy of MUSCLE are compared with T-Coffee, MAFFT and CLUSTALW on four test sets of reference alignments: BAliBASE, SABmark, SMART and a new benchmark, PREFAB. MUSCLE achieves the highest, or joint highest, rank in accuracy on each of these sets. Without refinement, MUSCLE achieves average accuracy statistically indistinguishable from T-Coffee and MAFFT, and is the fastest of the tested methods for large numbers of sequences, aligning 5000 sequences of average length 350 in 7 min on a current desktop computer. The MUSCLE program, source code and PREFAB test data are freely available at http://www.drive5. com/muscle.

title: SequenceMatrix: concatenation software for the fast assembly of multi‐gene datasets with character set and codon information
abstract: We present SequenceMatrix, software that is designed to facilitate the assembly and analysis of multi‐gene datasets. Genes are concatenated by dragging and dropping FASTA, NEXUS, or TNT files with aligned sequences into the program window. A multi‐gene dataset is concatenated and displayed in a spreadsheet; each sequence is represented by a cell that provides information on sequence length, number of indels, the number of ambiguous bases (“Ns”), and the availability of codon information. Alternatively, GenBank numbers for the sequences can be displayed and exported. Matrices with hundreds of genes and taxa can be concatenated within minutes and exported in TNT, NEXUS, or PHYLIP formats, preserving both character set and codon information for TNT and NEXUS files. SequenceMatrix also creates taxon sets listing taxa with a minimum number of characters or gene fragments, which helps assess preliminary datasets. Entire taxa, whole gene fragments, or individual sequences for a particular gene and species can be excluded from export. Data matrices can be  into their component genes and the gene fragments can be exported as individual gene files. SequenceMatrix also includes two tools that help to identify sequences that may have been compromised through laboratory contamination or data management error. One tool lists identical or near‐identical sequences within genes, while the other compares the pairwise distance pattern of one gene against the pattern for all remaining genes combined. SequenceMatrix is Java‐based and compatible with the Microsoft Windows, Apple MacOS X and Linux operating systems. The software is freely available from http://code.google.com/p/sequencematrix/.

title: The colour hexagon: a chromaticity diagram based on photoreceptor excitations as a generalized representation of colour opponency
abstract: SummaryA chromaticity diagram which plots the 3 photoreceptor excitations of trichromatic colour vision systems at an angle of 120° is presented. It takes into acount the nonlinear transduction process in the receptors. The resulting diagram has the outline of an equilateral hexagon. It is demonstrated by geometrical means that excitation values for any type of spectrally opponent mechanism can be read from this diagram if the weighting factors of this mechanism add up to zero. Thus, it may also be regarded as a general representation of colour opponent relations, linking graphically the Young-Helmholtz theory of trichromacy and Hering's concept of opponent colours. It is shown on a geometrical. basis that chromaticity can be coded unequivocally by any two combined spectrally opponent mechanisms, the main difference between particular mechanisms being the extension and compression of certain spectral areas. This type of graphical representation can qualitatively explain the Bezold-Brücke phenomenon. Furthermore, colour hexagon distances may be taken as standardized perceptual colour distance values for trichromatic insects, as is demonstrated by comparison with behavioural colour discrimination data of 3 hymenopteran species.

title: The spectral input systems of hymenopteran insects and their receptor-based colour vision
abstract: SummarySpectral sensitivity functions S(λ) of single photoreceptor cells in 43 different hymenopteran species were measured intracellularly with the fast spectral scan method. The distribution of maximal sensitivity values (λmax) shows 3 major peaks at 340 nm, 430 nm and 535 nm and a small peak at 600 nm. Predictions about the colour vision systems of the different hymenopteran species are derived from the spectral sensitivities by application of a receptor model of colour vision and a model of two colour opponent channels. Most of the species have a trichromatic colour vision system. Although the S(λ) functions are quite similar, the predicted colour discriminability curves differ in their relative height of best discriminability in the UV-blue or bluegreen area of the spectrum, indicating that relatively small differences in the S(λ) functions may have considerable effects on colour discriminability. Four of the hymenopteran insects tested contain an additional R-receptor with maximal sensitivity around 600 nm. The R-receptor of the solitary bee Callonychium petuniae is based on a pigment (P596) with a long λmax, whereas in the sawfly Tenthredo campestris the G-receptor appears to act as filter to a pigment (P570), shifting its λmax value to a longer wavelength and narrowing its bandwidth. Evolutionary and life history constraints (e.g. phylogenetic relatedness, social or solitary life, general or specialized feeding behaviour) appear to have no effect on the S(λ) functions. The only effect is found in UV receptors, for which λmax values at longer wavelengths are found in bees flying predominantly within the forest.

title: Foraging dynamics of bumble bees: correlates of movements within and between plant species
abstract: What rules determine whether bumble bee* continue exploiting plants of the species just visited or switch to another species? To tackle this question, we recorded handling times and flight times from bees foraging in a natural meadow containing five plant species. Interand ultra-specific plant distances were quantified. The bee-subjective colors of the five species were determined; two of these species had similar colors and structures, while three species were distinct from all others. The following rules were identified: (1) The decision to switch species was correlated with previous flower handling time, which we assume is a function of the reward amount received at the flower. After short handling times, the probability of switching to another species increased, whereas it decreased after long handling times. This difference became even greater if the bee had had a run of several short or several long handling times. (2) Constant flights (those between flowers of the same species) and transition flights (those between flowers of different species) followed stereotyped temporal patterns independent of the distances between flowers. Constant flights within five plant species consistently had median durations of about 2 seconds, whereas median transition times between species took 3-6 seconds. (3) This temporal rule broke down, however, if the flowers of two species had similar colors, in which case transition flights had equal dynamics as constant flights. (4) Bees switched more frequently from rare than from common species but even more frequently between similar species. We conclude that the bees' choices were determined by a set of rules that guided them to stay with the current plant species as long as flowers were rewarding and available within close distance but to switch to another species if flowers offered low rewards or were not encountered at close range. Kty words: bumble bees, Bombus, flower color, flower constancy, foraging, pollination ecology, switching.

title: Which food-mimic floral traits and environmental factors influence fecundity in a rare orchid, Calanthe yaoshanensis?
abstract: Orchid species that are food mimics produce fewer fruits than species offering rewards, but few studies have shown the impact of environmental factors (e.g. anthropogenic activity, frost and herbivores) on their reproductive success over several seasons. In this study, we focused on the sole population of the endangered Calanthe yaoshanensis as it secretes no nectar. We investigated its floral biology, fruit set rates and prevailing environmental factors over three seasons (2008-2010). Mechanical self-pollination did not occur in C.yaoshanensis, but hand-selfed and crossed flowers produced equal numbers of fruit. However, seed viability and embryo size were significantly higher in cross-pollinated fruits maximizing embryonic fitness. Large hoverflies (Syrphidae) and Bombus patagiatus (gynes) were the only pollinarium vectors, but they often failed to disperse pollinaria. We interpret the temporary retention of the anther cap over the pollinarium as an adaptation lowering self-pollination. Insect-mediated rates of pollinarium removal were always higher than rates of pollinia deposition on stigmas. Over 3 years, natural rates of pollinarium removal differed significantly, whereas natural rates of fruit set were not significantly different (<22%). Climate, herbivory and anthropogenic collections also inhibited some fruit set and maturation. Both biotic and abiotic factors appear to lower the fecundity of this endangered population.(c) 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 176, 421-433.

title: UNIFICATION OF METHODS FOR ESTIMATING THE STRENGTH OF REPRODUCTIVE ISOLATION
abstract: Understanding the evolution of reproductive isolation is tantamount to describing the origin of species. Therefore, a primary goal in evolutionary biology is to identify which reproductive barriers are most important to the process. To achieve this goal, the strength of multiple forms of isolation must be compared in an equivalent manner. However, a diversity of methods has been used to estimate barrier strength, falling into several mathematically distinct categories. This study provides a unified method for calculating isolation that relates the amount of gene flow experienced by taxa to random expectations in a simple linear framework. This approach has three distinct advantages over previous methods: (1) it is directly related to gene flow, (2) it is symmetrical, such that measures in both the positive and negative range are comparable, and (3) it is equivalent between broad categories of reproductive isolation, allowing for appropriate comparisons. This linear formulation can be adjusted for use in all forms of isolation, and can accommodate cases in which null expectations for con‐ and heterospecific gene flow differ. Additionally, this framework can be used to calculate total reproductive isolation and the relative contributions of individual barriers.

title: Does the Flower Constancy of Bumble Bees Reflect Foraging Economics
abstract: We examined the effects of floral reward level and spatial arrangement on the propensity of bumble bees to exhibit flower constancy. In three separate experiments, we compared the flower constancy of bees on dimorphic arrays of blue and yellow flowers that differed either in reward concentration, reward volume, or inter-flower distance. Overall, flower choice patterns varied among bees, ranging from random selection to complete constancy. When flowers contained greater reward volumes and were spaced farther apart, bees showed less flower constancy and more moves to closely neighbouring flowers. Changes in reward concentration had no effect on flower constancy; however, more dilute rewards produced shorter flight times between flowers. In addition, there was a strong positive relationship between degree of flower constancy and net rate of energy gain when flowers were spaced farther apart, indicating that constant bees were more economic foragers than inconstant bees. Together, these results support the view that the flower constancy of pollinators reflects an economic foraging decision.

title: An R Companion to Applied Regression
abstract: Preface 1. Getting Started With R 2. Reading and Manipulating Data 3. Exploring and Transforming Data 4. Fitting Linear Models 5. Fitting Generalized Linear Models 6. Diagnosing Problems in Linear and Generalized Linear Models 7. Drawing Graphs 8. Writing Programs References Author Index Subject Index Command Index Data Set Index Package Index About the Authors

title: Simultaneous inference in general parametric models.
abstract: Simultaneous inference is a common problem in many areas of application. If multiple null hypotheses are tested simultaneously, the probability of rejecting erroneously at least one of them increases beyond the pre-specified significance level. Simultaneous inference procedures have to be used which adjust for multiplicity and thus control the overall type I error rate. In this paper we describe simultaneous inference procedures in general parametric models, where the experimental questions are specified through a linear combination of elemental model parameters. The framework described here is quite general and extends the canonical theory of multiple comparison procedures in ANOVA models to linear regression problems, generalized linear models, linear mixed effects models, the Cox model, robust linear models, etc. Several examples using a variety of different statistical models illustrate the breadth of the results. For the analyses we use the R add-on package multcomp, which provides a convenient interface to the general approach adopted here.

title: Flower colour and visitation rates of Costus arabicus support the ‘bee avoidance’ hypothesis for red‐reflecting hummingbird‐pollinated flowers
abstract: Floral colour mediates plant–pollinator interactions by often signalling floral resources. In this sense, hummingbird-pollinated flowers are frequently red-coloured, and there are two tentative hypotheses to explain this pattern: 1. hummingbirds are attracted to red due its easier detection and 2. bees are sensorially excluded from red flowers. The second hypothesis is based on bees’ red colour blindness, which lead them to be less frequent and less important than hummingbirds as pollinators of red-reflecting flowers.
Here, we untangled the role of different flower traits mediating plant–pollinator interactions and empirically tested the above hypotheses. We chose Costus arabicus due to its synchronopatric white- and pink-flowered individuals and its bee and hummingbird pollination system. Although pink flowers are not totally achromatic as pure red ones, they show an achromaticity degree that could drive bee exclusion. Specifically, we tested whether differences on red reflectance work attracting hummingbirds or excluding bees and the consequent implications for the plant's reproduction.
Flower colour morphs of C. arabicus do differ neither in morphology nor in nectar sugar content. Moreover, white and pink flowers can be discriminated by the bees’ and hummingbirds’ colour vision system. Both groups are able to discriminate the red colour variation morph on the flower petals, the white flowers being more easily detected by bees and the pink flowers by hummingbirds. Bees preferentially visited the white flowers, whereas hummingbirds visited both colours at the same rate – both patterns corroborating the second hypothesis.
Pollen loads deposited on stigmas did not differ between flower colour morphs, indicating that bees and hummingbirds play a similar role in the overall pollen deposition. However, bees are more likely to self-pollinate than hummingbirds. Self-pollination limits C. arabicus reproduction, and red-reflecting flowers may be better pollinated by discouraging bee visitation. Therefore, the intraspecific colour variation is driving flowers to show colour-related different levels of generalization.
Our results support the ‘bee avoidance’ rather than the ‘hummingbird preference’ hypothesis. Sensory exclusion of bees seems to be the pressure for red-reflecting flowers evolution, driving specialization in hummingbird-pollinated flowers due to the costs of bee pollination on plant reproduction.

title: Avoidance of achromatic colours by bees provides a private niche for hummingbirds
abstract: That hummingbird-pollinated plants predominantly have red flowers has been known for decades, but well-investigated research studies are still rare. Preference tests have shown that hummingbirds do not have an innate preference for red colours. In addition, hummingbirds do not depend solely upon red flowers, because white-flowered hummingbird-pollinated plants are also common and temporarily abundant. Here we show that both white and red hummingbird-pollinated flowers differ from bee-pollinated flowers in their reflection properties for ultraviolet (UV) light. Hummingbird-pollinated red flowers are on average less UV reflective, and white hummingbird-pollinated flowers are more UV reflective than the same coloured bee-pollinated ones. In preference tests with artificial flowers, neotropical orchid bees prefer red UV-reflecting artificial flowers and white UV-nonreflecting flowers over red and white flowers with the opposite UV properties. By contrast, hummingbirds showed no preference for any colour in the same tests. Plotting floral colours and test stimuli into the honeybees' perceptual colour space suggests that the less attractive colours are achromatic for bees and therefore more difficult to detect against the background. This underlying colour preference in bees might provide hummingbirds with a private niche that is not attractive to bees.

title: The Orchids: Natural History and Classification
abstract: This lively examination of the structure, classification, evolution, and ecology of the Orchidaceae will appeal to anyone with an eye for beauty or a bent for natural history. It will provide professional biologists and amateur orchidists alike with a deeper understanding--and a thoroughly new classification--of this, the largest flowering family in the plant kingdom. The book is richly illustrated, with 95 color photographs and 99 line drawings emphasizing orchid morphology. But in the context of serious orchidology, aesthetic richness is not a superficial vanity. It is the result of evolutionary processes that demand adaptation and reward diversity, and Dressler's exploration of these mechanisms demonstrates how well the orchids have met the challenge, with over 20,000 species, a geographical range from Sweden to Tierra del Fuego, and, at times, as many as 47 different species on a single tree. But both the orchids' success and their profligate beauty are grounded, in part, in their voluptuary nature, and one of Dressler's most engaging themes is the reproductive adaptations, worthy of a Renaissance courtesan, that orchids use to attract, deceive, and manipulate pollinating insects. The orchid family is actively evolving, providing rich possibilities for comparative study. Yet no one, whether professional or amateur, could reasonably proceed without first addressing Robert Dressler's "The Orchids," Its new classification alone, including pollination biology, phyletic trends, chromosome counts, and generic relationships, will stand as a watershed contribution to the field.

title: Scent of the Vanishing Flora
abstract: 1. Introduction to the 'Scent of the Vanishing Flora' 1.1. Extinction of Species and Monitoring Endangered Species 1.2. Investigating the Scent of the Vanishing Flora 2. Illustrating the Scent of the Vanishing Flora 2.1. No More Existing in Nature 2.2. Ten Icon Species of the Vanishing Flora 2.3. The Scent of 'Living Fossils' 2.4. A Brief Look into Rainforests and Associated Biotopes 2.5. Hawaii's Vanishing Flora 2.6. The Cape Floral Kingdom, a Closer Look at One of the Hotspots of Biodiversity 2.7. Endangered Conifers 2.8.  Orchids of the African Tropic 3.3. Orchids of the Indo-Australian Tropics and Subtropics 4. Analytics 4.1. General Remarks 4.2. Equipment Used 4.3. Compositions of the Presented Scents 4.4. Synthesis of New Compounds Index Acknowledgement

title: Multicomponent floral signals elicit selective foraging in bumblebees
abstract: Flower constancy, or the tendency of individual pollinators to visit sequentially a single flower type even when other equally rewarding types are available, has important implications for animal-pollinated plants. Yet, the proximal reason for the behaviour still remains poorly understood. Here I show that bumblebees visiting equally rewarding flowers that differ in size and odour are more flower constant and less efficient (visited fewer flowers per minute) than bees visiting flowers that differ in size only and odour only. These results are consistent with the view that flower constancy in pollinators is related to their inability to perceive, process or recall multicomponent floral signals. I discuss these findings in the context of pollinator behavioural mechanisms and the evolution of floral diversity.

title: Dosage-Dependent Impacts of a Floral Volatile Compound on Pollinators, Larcenists, and the Potential for Floral Evolution in the Alpine Skypilot Polemonium viscosum
abstract: All volatile organic compounds (VOCs) vary quantitatively, yet how such variation affects their ecological roles is unknown. Because floral VOCs are cues for both pollinators and floral antagonists, variation in emission may have major consequences for costs and benefits in plant-pollinator interactions. In Polemonium viscosum, the emission rate for the floral VOC 2-phenylethanol (2PE) spans more than two orders of magnitude. We investigated the ecological and evolutionary impacts of this immense phenotypic variation. The emission rate of 2PE varies independently of nectar rewards and thus is uninformative of profitability. Emission is elevated in flowers that are morphologically vulnerable to ant larcenists, suggesting that chemical deterrence may compensate for weak physical barriers. In nature, plants emitting more 2PE than their neighbors escape ant damage. Flower-damaging ants die when exposed to 2PE in the laboratory, and they avoid high 2PE emitters in the field. High 2PE also reduces bumblebee visitation and pollination, suggesting an ecological cost of defense in pollinator service. However, at more moderate emission rates, 2PE enhances the amount of nectar left in flowers, at no pollination cost. In conclusion, repellency of 2PE is highly sensitive to dosage, giving it a key role in shaping ecological interactions between skypilot plants and their floral visitors.

title: Orchid Pollination: An Observation on Pollination-Pollinator Interaction in Cymbidium pendulum (Sw.) Roxb.
abstract: The path of pollination in Cymbidium pendulum (Sw.) Roxb. has been traced in the present studies. The honey been identified as Apis mellifera was found to act as main pollinator and this bee is the only insects, among others, who succeeded in performing pollination because probably due to its structural compatibility with the plant species. Pollination by Apis mellifera bees was suggested to occur in a number of families but rare phenomenon in orchids and it is first time that the species was observed to pollinate the Cymbidium pendulum flowers. Bee moved around the flowers for some times, entered the flower and carried on pollinia along with on the back during its journey. It revisited the different flower and deposited its pollinia on to it and the act of pollination was accomplished. SEM study showed an intricate network on the back of bee thus clearly indicates its role in firm attachment to pollinia.

title: Mycorrhizal associations and reproductive isolation in three closely related Orchis species.
abstract: The maintenance of species boundaries in sympatric populations of closely related species requires some kind of reproductive isolation that limits gene flow among species and/or prevents the production of viable progeny. Because in orchids mycorrhizal fungi are needed for seed germination and subsequent seedling establishment, orchid-mycorrhizal associations may be involved in acting as a post-mating barrier.



We investigated the strength of post-mating barriers up to the seed germination stage acting between three closely related Orchis species (Orchis anthropophora, O. militaris and O. purpurea) and studied the role of mycorrhizal fungi in hybridization by burying seed packets of pure and hybrid seeds. After retrieval and assessment of seed germination, the fungi associating with protocorms originating from hybrid and pure seeds were determined and compared with those associating with adult individuals using DNA array technology.



Whereas pre-zygotic post-mating barriers were rather weak in most crosses, post-zygotic post-mating barriers were stronger, particularly when O. purpurea was crossed with O. anthropophora. Germination trials in the field showed that seed germination percentages of hybrid seeds were in most cases lower than those originating from pure crosses. In all species pair combinations, total post-mating reproductive isolation was asymmetric. Protocorms associated with a smaller range of fungal symbionts than adult plants, but there was considerable overlap in mycorrhizal associations between protocorms and their respective parents.



Our results suggest that mycorrhizal associations contribute little to reproductive isolation. Pre-mating barriers are probably the main factors determining hybridization rates between the investigated species.

title: Evidence for reproductive isolate selection in Mediterranean orchids: karyotype differences compensate for the lack of pollinator specificity
abstract: Mediterranean orchids of the subtribe Orchidinae are highly diverse and display a range of fascinating pollination strategies. Based on observations that orchid–pollinator relationships are often highly specialized and species specific, Darwin and others have argued that selection for different pollinators has been the driving force behind the evolutionary diversification of orchids. This may be true for orchids that attract different, specialized pollinators that act as prezygotic reproductive barriers. It is, however, not clear how closely related co–flowering Mediterranean orchids that share pollinators survive the challenge of sympatry. We show that species pairs with a generalized pool of pollinators have significantly more divergent karyotypes compared with species pairs with different pollinators. These results show that karyotype differences that act as postzygotic reproductive barriers may have played an important role in the evolution of Mediterranean orchid diversity.

title: Inbreeding and outbreeding depression in Stylidium hispidum: implications for mixing seed sources for ecological restoration
abstract: The benefits of composite rather than local seed provenances for ecological restoration have recently been argued, largely on the basis of maximizing evolutionary potential. However, these arguments have downplayed the potentially negative consequences of outbreeding depression once mixed provenances interbreed. In this study, we compared intraspecific F1 hybrid performance and molecular marker differentiation among four populations of Stylidium hispidum, a species endemic to Southwestern Australia. Multivariate ordination of 134 AFLP markers analyzed genetic structure and detected two clusters of paired sites that diverged significantly for marker variation along a latitudinal boundary. To test for outbreeding depression and to determine the consequences of molecular population divergence for hybrid fitness, we conducted controlled pollinations and studied germination and survival for three cross categories (within-population crosses, short- and long-distance F1 hybrids) for paired sites distributed within and between the two genetically differentiated regions. We found evidence of outbreeding depression in long-distance hybrids (111–124 km), and inbreeding depression among progeny of within-population crosses, relative to short-distance (3–10 km) hybrids, suggesting an intermediate optimal outcrossing distance in this species. These results are discussed in light of the evolutionary consequences of mixing seed sources for biodiversity restoration.