Cross‐species amplification of microsatellite loci in Aquilegia and Semiaquilegia (Ranunculaceae)

We developed 16 microsatellite loci from an F₂ hybrid between Aquilegia formosa and Aquilegia pubescens. In samples of 28 individuals, we found an average of 14 alleles per locus from each parental species. We tested these loci for cross‐amplification in 10 additional species of Aquilegia and found that all 16 loci amplified in other North American species and 12 consistently amplified in European or Asian species. Nine loci amplified in the sister species to Aquilegia, Semiaquilegia adoxoides. The success of cross‐species amplification suggests that these microsatellites should prove useful for studies in a broad range of Aquilegia species.     

Local adaptation to distinct elevational cores contributes to current elevational divergence of two Aquilegia vulgaris subspecies

Aims Phenotypic plasticity and local adaptation of populations at their distributional limits are crucial to understand species colonization and persistence in novel or marginal environments, as well as species divergence and niche width evolution. We assess the contribution of these processes to shape current elevational limits and determine elevational phenotypic divergence between two subspecies of Aquilegia vulgaris (subsp. vulgaris and nevadensis).Methods We conducted sowing and transplant experiments considering four elevations throughout the current elevational range of A. vulgaris in southern Iberian Peninsula. Experiments were designed to explore, on the one hand, local adaptation through three components of performance (germination, survival and growth) and, on the other hand, the phenotypic differentiation and/or plasticity associated to local adaptation. Four populations per subspecies (three from the elevational core and one from the elevational boundary) were used as seed sources. Patterns of local adaptation and phenotypic differentiation are examined in the context of the 'centre-periphery' hypothesis.Important findings Central populations of both subspecies performed better at their local elevations while marginal populations were maladapted, confirming the hypothesis and contributing to explain the current elevational segregation of these subspecies. Density of glandular pubescence and germination timing seem to be related to local adaptation, through phenotypic differentiation between subspecies or elevations. The widespread subsp. vulgaris showed signals of adaptive plasticity in the timing of germination while it was not the case in the endemic subsp. nevadensis .     

Different photosynthetic responses of wild and cultivated plants to high irradiance

In addition to other factors, high altitude (HA) environment is characterized by high photosynthetic photon flux density (PPFD). Photosynthetic characteristics of wild and cultivated plants were studied at different irradiances at Losar, India (altitude 4 200 m). Wild plants were tolerant to high PPFDs. Slopes of curve between net photosynthetic rate (P _N) and intercellular CO₂ concentration (C _i) or stomatal conductance (g _s) increased with increase in irradiance suggesting insensitivity or tolerance of these plants to higher PPFD. Cultivated plants, however, were sensitive to higher PPFD, their slopes of curves between P _N and C _i or g _s decreased with increased PPFD. Tolerance or insensitivity to higher PPFD was an important parameter affecting plant performance at HA.     

Impact of insect pollinator group and floral display size on outcrossing rate

Despite the strong influence of pollination ecology on the evolution of selfing, we have little information on how distinct groups of insect pollinators influence outcrossing rate. However, differences in behavior between pollinator groups could easily influence how each group affects outcrossing rate. We examined the influence of distinct insect pollinator groups on outcrossing rate in the rocky mountain columbine, Aquilegia coerulea. The impact of population size, plant density, size of floral display, and herkogamy (spatial separation between anthers and stigmas) on outcrossing rate was also considered as these variables were previously found to affect outcrossing rate in some plant species. We quantified correlations between all independent variables and used simple and two-factor regressions to determine direct and indirect impact of each independent variable on outcrossing rate. Outcrossing rate increased significantly with hawkmoth abundance but not with the abundance of any of the other groups of floral visitors, which included bumblebees, solitary bees, syrphid flies, and muscidae. Outcrossing rate was also significantly affected by floral display size and together, hawkmoth abundance and floral display size explained 87% of the variation in outcrossing rate. None of the other independent variables directly affected the outcrossing rate. This is the first report of a significant impact of pollinator type on outcrossing rate. Hawkmoths did not visit fewer flowers per plant relative to other pollinator groups but preferred visiting female-phase flowers first on a plant. Both the behavior of pollinators and floral display size affected outcrossing rate via their impact on the level of geitonogamous (among flower) selfing. Given that geitonogamous selfing is never advantageous, the variation in outcrossing rate and maintenance of mixed mating systems in populations of A. coerulea may not require an adaptive explanation.     

Comparative floral and vegetative differentiation between two European Aquilegia taxa along a narrow contact zone

As a first step in determining the identity and relative importance of the evolutionary forces promoting the speciation process in two closely related European taxa of Aquilegia, we investigated the levels of morphological variation in floral and vegetative characters over the narrow region where their ranges enter into contact, and evaluate the relative importance of both types of traits in their differentiation. A total of 12 floral and ten vegetative characters were measured on 375 plants belonging to seven A. vulgaris populations and six A. pyrenaica subsp. cazorlensis populations located in southeastern Spain. Floral and vegetative morphological differentiation occur between taxa and among populations within taxa, but only vegetative characters (particularly plant height and leaf petiolule length) contribute significantly to the discrimination between taxa. Differentiation among populations within taxa is mostly explained by variation in floral traits. Consequently, morphological divergence between the two taxa cannot be interpreted as an extension of among-population differences occurring within taxa. Multivariate vegetative, but not floral, similarity between populations could be predicted from geographical distance. Moreover, the key role of certain vegetative traits in the differentiation of A. vulgaris and A. p. cazorlensis could possibly be attributable to the contrasting habitat requirements and stress tolerance strategies of the two taxa. These preliminary findings seem to disagree with the currently established view of the radiation process in the genus Aquilegia in North America, where the differentiation of floral traits seems to have played a more important role.     

Conserved and non-conserved triggers of 24-nucleotide reproductive phasiRNAs in eudicots

Small RNAs play important roles in plant growth and development by modulating expression of genes and transposons. In many flowering plant species, male reproductive organs, the anthers, produce abundant phased small interfering RNAs (phasiRNAs). Two classes of reproductive phasiRNAs are generally known, mostly from monocots: (i) pre-meiotic 21-nucleotide (nt) phasiRNAs triggered by miR2118 and (ii) meiotic 24-nt phasiRNAs triggered by miR2275. Here, we describe conserved and non-conserved triggers of 24-nt phasiRNAs in several eudicots. We found that the abundant 24-nt phasiRNAs in the basal eudicot columbine (Aquilegia coerulea) are produced by the canonical trigger miR2275, as well as by other non-canonical triggers, miR482/2118 and miR14051. These triggering microRNAs (miRNAs) are localized in microspore mother cells and tapetal cells of meiotic and post-meiotic stage anthers. Furthermore, we identified a lineage-specific trigger (miR11308) of 24-nt phasiRNAs and an expanded number of 24-PHAS loci in wild strawberry (Fragaria vesca). We validated the presence of the miR2275-derived 24-nt phasiRNA pathway in rose (Rosa chinensis). Finally, we evaluated all eudicots that have been validated for the presence of 24-nt phasiRNAs as possible model systems in which to study the biogenesis and function of 24-nt phasiRNAs. We conclude that columbine (Aquilegia coerulea) would be a strong model because of its extensive number of 24-PHAS loci and its diversity of trigger miRNAs.     

Characterization of Aquilegia Polycomb Repressive Complex 2 homologs reveals absence of imprinting

Epigenetic regulation is important for maintaining gene expression patterns in multicellular organisms. The Polycomb Group (PcG) proteins form several complexes with important and deeply conserved epigenetic functions in both the plant and animal kingdoms. The plant Polycomb Repressive Complex 2 (PRC2) contains four core proteins, Enhancer of Zeste (E(z)), Suppressor of Zeste 12 (Su(z)12), Extra Sex Combs (ESC), and Multicopy Suppressor of IRA 1 (MSI1), and functions in many developmental transitions. In some plant species, including rice and Arabidopsis, duplications in the core PRC2 proteins allow the formation of PRC2s with distinct developmental functions. In addition, members of the plant specific VEL PHD family have been shown to associate with the PRC2 complex in Arabidopsis and may play a role in targeting the PRC2 to specific loci. Here we examine the evolution and expression of the PRC2 and VEL PHD families in Aquilegia, a member of the lower eudicot order Ranunculales and an emerging model for the investigation of plant ecology, evolution and developmental genetics. We find that Aquilegia has a relatively simple PRC2 with only one homolog of Su(z)12, ESC and MSI1 and two ancient copies of E(z), AqSWN and AqCLF. Aquilegia has four members of the VEL PHD family, three of which appear to be closely related to Arabidopsis proteins known to associate with the PRC2. The PRC2 and VEL PHD family proteins are expressed at a relatively constant level throughout Aquilegia vulgaris development, with the VEL PHD family and MSI1 expressed at higher levels during and after vernalization and in the inflorescence. Both AqSWN and AqCLF are expressed in Aquilegia endosperm but neither copy is imprinted.     

Temporal floral sex allocation in protogynous Aquilegia yabeana contrasts with protandrous species: support for the mating environment hypothesis

We tested one of the predictions of Brunet and Charlesworth (1995) that relative floral sex allocation will vary temporally with the mating environment and that the form of dichogamy (protandry vs. protogyny) will select for the pattern of variation in male versus female resource allocation. In many hermaphroditic plant species, allocation to female function (ovule number) decreases from early to late flowers within inflorescences as a result of resource limitation or ontogenetic changes. This pattern may obscure the effects of the mating environment and dichogamy on selection for allocation patterns in protandrous species (male allocation increases regardless). By examining a protogynous species the alternative pattern of temporal variation in resource allocation is predicted, namely that allocation to male function should decrease (or female allocation increase) throughout the flowering sequence. This pattern was observed in protogynous Aquilegia yabeana (Ranunculaceae), in which ovule number per flower remained constant whereas pollen number decreased in sequentially blooming flowers. These observations support the temporal sex allocation hypothesis of Brunet and Charlesworth (1995).