Study site
Research was conducted at the La Malinche National Park (LMNP), Tlaxcala, Mexico (19°15.205′ N, 098°02.080′ W; elevation 3,400–3,900 m; Fig. 4A). The LMNP is located in a temperate montane forest with an old-growth fir forest (Abies religiosa) frequently mixed with Pinus hartwegii above 3,500 m, and hosts a diversity of plants that rely on hummingbirds for successful reproduction74.
Study species
Penstemon gentianoides (Plantaginaceae) is an herbaceous perennial plant distributed in the major volcanic peaks of the Trans-Mexican Volcanic Belt (TMVB), and southward into Chiapas to Guatemala69,75. The species is found at middle and high elevations (3,000–4,200 m), from pine forests typically in open or disturbed sites to slightly above timberline associated with the alpine grasslands69,75. At the LMNP, P. gentianoides is found in open areas of pine and fir forests, ranging from 3,000 to 3,900 m47. Individuals (0.5–1.5 m high) bear 15–25 paniculate inflorescences, each with 2–4 pendant flowers from terminal branching stems opening per day, and ∼90 floral buds may eventually reach the flower stage during the blooming season (4 months), which extends from July to November47,74. Penstemon gentianoides flowers (Fig. 4B–D) are protandrous and long-lived47, with 8-d male phase (staminate) flowers, followed by the ∼1–7 d female phase (pistillate phase). Flowers are blue, violet or purple and vestibular flowers abruptly expanding into a broadly inflated throat and a prominent lower lip with anthers and stigmas nearly included35,47. However, in contrast to these traits that are often linked to bee pollination, nectar is abundant and dilute at morning hours, which are traits linked to hummingbird pollination47. Penstemon roseus is an herbaceous perennial plant distributed along the TMVB (Fig. 4D)69,75. This species is found at lower elevations (1,800–3,500 m) associated with oak and pine-oak forests and typically in open or disturbed sites69,75. Individuals (0.4–1.2 m high) bear 10–20 paniculate inflorescences, each with 2–4 pendant flowers from terminal branching stems opening per day, and ∼80 floral buds may eventually reach the flower stage during the blooming season (4 months), which extends from July to December46,48. Penstemon roseus flowers are protandrous and long-lived46, with 2-d male phase (staminate) flowers, followed by the ∼2 d female phase (pistillate phase). Flowers are pink to red and tubular, intermediate between hummingbird- and bee-adapted Penstemon flowers. The corolla tube with a globular shape, enlarged ventral lobes and a white throat, contains abundant and dilute nectar46,48.
The community assemblage of pollinators varies locally. At 2,900 m, large hummingbirds (Colibri thalassinus, Eugenes fulgens, Lampornis clemenciae) defend intensively the floral patches of P. gentianoides74, but bumblebees (Bombus ephippiatus, B. huntii and B. weisi) and four smaller hummingbird species (Hylocharis leucotis, Archilochus colubris, Selasphorus platycercus, Atthis heloisa) are its main floral visitors slightly above timberline47. The long-distance seasonal migrants S. platycercus and A. colubris are the most frequent (30.3% and 11.3% of all visits, respectively), followed by resident H. leucotis (2.9%) and A. heloisa (1.2%), and bumblebees and bees (Apis mellifera) account for 51.8% and 2.4% of all visits, respectively; hummingbirds probe more flowers per foraging bout than bumblebees47. While flowers experimentally pollinated by B. ephippiatus produce more fruits than those pollinated by S. platycercus (79% and 46%, respectively), single-visit efficiency measures (number of seeds per fruit) derived from bumblebee or hummingbird pollination were similar in P. gentianoides (for more details on the experimental set-up see Salas-Arcos et al.47). Eight hummingbird species visit P. roseus flowers at the LMNP: Colibri thalassinus, Hylocharis leucotis, Lampornis clemenciae, Eugenes fulgens, Archilochus colubris, Selasphorus rufus, S. platycercus, and S. sasin46. The most frequent hummingbird is the resident H. leucotis (46–65% of all visits), and bumblebees and butterflies are uncommon floral visitors at the LMNP48.
The populations of P. gentianoides and P. roseus, circumscribed to different clades69,70, are self-compatible (∼25–28% and 50%, respectively) but outcrossed flowers produce approximately two times more seeds than self-pollinated flowers46,47. The distributions of these species overlap forming a hybrid zone when growing in sympatry at the lower elevational range of P. gentianoides (Fig. 4A). At the hybrid zone, numerous individuals are similar in floral morphology to P. gentianoides, but the color of their corollas is different and likely the product of hybridization with P. roseus; no other Penstemon species is found at the LMNP69. This naturally occurring hybrid zone barely overlaps with a population of P. roseus at lower elevation. Individuals present intraspecific floral color variation (Fig. 4C), ranging from blue to fuchsia (hereafter referred as blue and fuchsia morphs) in which reflectance is higher in fuchsia flowers (F1, 37 = 26.16, P < 0.001, see details of color measurements below). Fuchsia individuals open more flowers (mean ± SD = 62.9 ± 23.7 cm tall, N = 30) than those of the blue morph (71.7 ± 19.1 cm tall, N = 43), forming patches between 3,731–3,922 m and those of the blue morph growing isolated between 3,703–3,738 m. Within the study area (Fig. 1A), the fuchsia:blue morph ratio is 3:1 (213 fuchsia individuals and 68 blue individuals in two 50 × 50 m plots spaced at least 150 m apart). The hybrid flower color morphs flower from July to November, with a flowering peak in August. Seasonal reproductive isolation is unimportant because flowering time of the two hybrid flower color morphs overlap almost completely. Within plants, flowers open asynchronously (30% per inflorescence) and are protandrous.
Floral biology
During the blooming season (July–November 2016), we selected 67 plants of each color morph, measured 2–3 fully developed flowers from each of the plants (N = 268 flowers), noting whether sexual organs were exserted, and measured the flower color (see below). Corolla tube length (distance from the base of the corolla to the corners of the corolla mouth), corolla-entrance width, corolla-entrance height, and filament and style lengths were measured with a digital calliper (error: 0.01 mm).
In September 2016, we daily inspected 40-tagged buds growing on 20 additional plants of each hybrid flower color morph until wilting to evaluate floral longevity and length of each sexual phase. We assessed stigma receptivity of 40 flowers per color morph over 12 days after flower opening by submerging stigmas into a 32% hydrogen peroxide solution and using the presence of bubbling on the stigma to infer receptivity76. We also counted the total number of ovules per ovary of 42 floral buds collected from each hybrid flower color morph, preserved in alcohol (70%), with the aid of a stereo microscope (VELAB VE-SI, USA). We used generalized linear mixed models (GLMM) using a Gaussian error distribution and logarithmic link function to assess morph differences (fixed effect) in flower size and number of ovules per flower, individual plant as a random effect, and measures as continuous response variables. All statistical analyses (here and below) were performed in R Studio version 3.2.277.
Nectar standing crops
Nectar standing crops and accumulated nectar throughout flower lifespans were quantified to determine reward availability for pollinators. Because pollinators probably respond to nectar standing crop, we extracted the nectar available in flowers that had been exposed to floral visitors and measured its volume and concentration. Data were collected in September and October 2016 from 20 individual plants (N = 153 flowers/hybrid flower color morph) at 3-h intervals, 10:00 (N = 51 flowers, 1–2 flowers sampled per plant/time interval), 13:00 (N = 51 flowers) and 16:00 (N = 51 flowers), to evaluate variation in the availability of nectar during the period of floral visitors activity. Nectar volume per flower was removed and measured by using calibrated micropipettes (5 µL) and a digital calliper (error: 0.1 mm). Sugar concentration (percentage sucrose) was measured by a hand-held pocket refractometer (range concentration 0–32° BRIX units; Atago, Tokyo, Japan), and the amount of sugar produced was expressed as milligrams of sugar76. A GLMM (Family = Gaussian, Link = logit) was also used to compare the effects of color morph and time of day (fixed effect) and plant identity (random effect) on nectar standing crops with nectar volume and amount of sugar as continuous response variables.
In a different group of 20 plants, buds about to open were randomly selected and bagged as explained above (N = 62 flowers/hybrid flower color morph) and excluded from floral visitors to let nectar accumulate. The accumulated nectar throughout the lifespan of individual flowers (6–13 d) was extracted one day before senescence. Nectar volume and sugar concentration were measured as explained above. A GLMM (Family = Gaussian, Link = logit) was used to compare the effects of color morph (fixed effect) and plant identity (random effect) on the amount of accumulated nectar (nectar volume and amount of sugar) as continuous response variables.
Floral visitors
Upon flowering (from July to November 2017), we recorded the identity and foraging patterns of the floral visitors of each hybrid flower color morph. For these observations, we haphazardly selected 17 patches that contained 72 individual flowering plants (36 of each color morph), resulting in 192 hours of observation (20-min/plant, 1–5 h/day) at different days and times of day, from 10:00 until 16:00 h. We used binoculars (10 × 42, Celestron) and recorded the beginning of the observation as time zero and subsequent foraging events of each bumblebee and hummingbird visit as minutes from start time. We estimated three metrics of pollinator visitation per focal plant and hybrid flower color morph: elapsed time between the start of the observation period and when the first visit occurs, time spent per plant of each foraging bout, and the number of flowers visited per foraging bout. A floral visitation event was defined as the arrival of any visitor at one or more of the flowers of the target patch in which the visitor contacted the sexual organs of flowers. Total visits recorded from Bombus bumblebees were grouped and coded as visits from bumblebees, while visits from different hummingbird species were grouped under the hummingbird category. We used survival analysis78 to analyze the probability of each hybrid flower color morph being visited by bumblebees and hummingbirds. The non-parametric survival analysis tool was used because the actual time of all possible events to occur in a given focal plant is not always known during the focal observation. The Kaplan–Meier method of survival analysis was used to generate and adjust survival curves using the elapsed time variable that differed between pollinator types. The censored observations are plants that a pollinator had not yet visited 20-min after the start of the observation. The log-rank (Mantel–Cox) test was used for differences between pollinator survival curves. Visitation frequency and number of flowers visited per foraging bout (continuous response variables) by pollinator type (fixed effects) were analyzed using a GLMM (Family = Poisson, Link = logit). Plant identity was included as a random effect in the model. Differences between pollinator groups per time period were evaluated with Tukey multiple comparisons. Lastly, the foraging behavior variables were analyzed as a function of hybrid flower color morph and amount and availability of nectar through a linear regression model.
Flower color measurement
The spectral reflectance of flowers of each putative parental species and hybrid flower color morph (20 flowers per species and color morph, N = 80 flowers) was measured to assess the consequences of color reflectance differences for pollinator attraction and plant reproduction. Flower reflectance of the apical, center and basal parts of the corolla were measured using a JAZ-EL 200 spectrometer with a UV-VIS deuterium tungsten halogen source (Ocean Optics Inc., Dunedin, FL, USA) connected to a computer running CLR version 1.1 (JAVA program). The light spectrum analyzed ranged from 300 to 700 nm, divided into 0.22-nm intervals, and the spectrometer sensor was fixed at an angle of 45° from the measuring areas between 215 and 1700 nm. We took all reflectance measurements at the same direction relative to the flower structure, using barium sulphate as the white standard, and analyzed spectral reflectance in four distinct wavebands (UV: 301–400 nm; blue: 400–510 nm; green: 510–605 nm; and red: 605–700 nm) for each part of the flowers. Petals were mounted on an adhesive tape to obtain a flat surface, thus minimizing reflectance variability (measured as percentage) due to uneven distances between the petals and the sensor.
We compared the flower colors from the tri- and tetrachromatic color vision systems of bumblebees and hummingbirds using the logarithm version of the receptor noise-limited model as in Bergamo et al.21. We used the standard photoreceptor sensitivities of bumblebees and hummingbirds adopting the oil droplet parameters from Hart & Vorobyev79, with noise values of 0.74, 0.67 and 0.61 as inputs in the bumblebee model for UV, blue and green photoreceptor types, respectively, while a violet sensitive (VS) vision system and noise values of 0.1, 0.07, 0.07 and 0.05 for the SWS1, SWS2, MWS and LWS photoreceptor type, respectively, for hummingbirds66,80. We also calculated the color loci of the flower colors in the respective color space models: the color hexagon for bees81 and the color tetrahedron for hummingbirds65, and the spectral purity in the color tetrahedron for hummingbirds only, since these are the two main color properties determining pollinator foraging decisions21.
Using PAVO82, we first generated a three-dimensional plot indicating the location of each point in the color tetrahedron, and then calculated the Cartesian coordinates (X, Y, Z) for the points in the tetrahedral color space, the angles theta and phi (h.theta, h.phi) in radians, which determine the hue of the color, the r vector (r.vec), which measures saturation or the distance from the achromatic center, the maximum r vector (r.max) achievable for the color’s hue, and the r.achieved, which measures the relative r distance from the achromatic center in relation to the maximum distance achievable (r.vec/r.max). In the lme4 package, we compared the pollinator’s chromatic discrimination in the average of the three floral parts between putative parental species and hybrid flower color morphs (fixed effect) performing a GLM (Family = Poisson, Link = logit). Additionally, we compared the pollinator’s chromatic discrimination between color morphs performing a one-sample t-test against the minimum discrimination threshold, and to evaluate differences in the chromatic detectability for each visual system.
Pollinator effectiveness
We conducted two pollination experiments to test for differences in female reproductive success across the two hybrid flower color morphs and across pollinator types. For the open-pollination experiment, unbagged flowers of each color morph (N = 40 plants) were exposed to natural floral visitation until fruit initiation and quantified number of fruits produced per plant. Mature fruits from each flower color morph (N = 37 fruits per morph) were marked and collected two months later, and measured (length and width; digital vernier TRUPER) and weighed with an analytic balance (to the nearest 0.01 mg; VELAB VE-1000, linearity ± 0.02), and their seeds counted.
For the second pollination experiment, we compared the effectiveness of bumblebees and hummingbirds as pollinators of each hybrid flower color morph. Buds ready to open from 30 plants were chosen (N = 62 floral buds from each color morph) and once they opened, flowers were emasculated and bagged with bridal netting until stigmatic receptivity. One or the other pollinator type pollinated half of the plants of each color morph. Individuals (N = 4) of the most common visitor bumblebee species (Bombus ephippiatus) were captured, enclosed in test tubes and then used as pollen vectors. Pollination was accomplished by placing the test-tube entrance in front of the corolla of a donor flower with dehisced anthers and allowed the bumblebee to enter and leave the flower in a single event, and then repeated this procedure into the corolla of a receptive recipient flower. We used flowers as pollen donors only from one plant to minimize possible genetic factors and to simplify our experimental design. For hummingbirds, individuals of the most frequent floral visitor (Selasphorus platycercus; N = 3) were caught and used as pollen vector by allowing to insert its bill once into the corolla of a donor flower with dehisced anthers and then into the corolla of a receptive recipient flower, as in Salas-Arcos et al.47. After pollinations were performed the bumblebees and hummingbirds were released, and flowers remained bagged until fruit production, then fruits were weighed and the seeds per fruit produced were counted and measured by pollination treatment as explained above. True effectiveness of a pollinator is determined by the proportion of pollen grains picked up from one flower and deposited in the next83, but having flowers until hummingbird visited, and marking the visited flower, was not feasible. Pollen loads carried by the hummingbirds in hand are likely different from those carried by free hummingbirds because pollen from a specific flower (donor) may persist on hummingbirds for many flowers, mixing with pollen grains. Instead, hummingbirds only performed one pollination visit with pollen from one known flower to control for pollen donor and the amount of pollen carried and to simplify our experimental design. Only one plant was used as pollen donor to minimize possible confounding factors due to individual plant. As much as possible, we removed the residual pollen with a paintbrush prior to performing the next pollination. Nonetheless, decreased seed production from hand-pollinations may not be a rare event in experimental pollination experiments84,85. Pollen grains deposited on stigmas by hand pollination using hummingbirds as tools were not counted, but pollen loads were enough to fertilize most ovules47,48. Although pollen deposition is likely different between the open-pollination experiment and the hand pollination using hummingbirds as a tool, the number of seeds produced by pollinations with hummingbirds in hand in both morphs was greater than that obtained in flowers with open pollination (see Results). With this, the reliability of our manual pollinations is strengthened.
Permission to conduct the described field study was granted by the Mexican government (Secretaría del Medio Ambiente y Recursos Naturales, Dirección General de Vida Silvestre, SGPA/DGVS/02439/16). This collecting permit specifically allowed for the capture of birds. Manipulation of birds in the field was minimal. Birds were captured with mist nets and used as pollen vectors before they were released. All procedures with birds were carried out in accordance with the Guidelines for the Use of Wild Birds in Research proposed by the North American Ornithological Council and the ethics of experimental procedures were revised and authorized by the Animal Care and Use Committee under the Graduate Studies Committee (Posgrado en Ciencias Biológicas) of the Universidad Autónoma de Tlaxcala (UAT). While the field study involves a non-threatened or protected species, no specific permits are required for bird monitoring or observational studies as the one described here.
A GLMM model (Family = Poisson, Link = logit) was used to compare fruit production (discrete response variable) between hybrid flower color morphs (fixed effect) under natural conditions. The same GLMM model was used to assess differences between morphs in fruit length, fruit width, fruit weight and number of seeds per fruit, with color morph treated as a fixed effect and fruit traits and number of seeds produced per fruit treated as continuous response variables. Plant identity was included as a random effect in the model. To evaluate differences between bumblebees and hummingbirds in their effectiveness as pollinators, two GLMM models (with Gaussian error distribution) were performed with color morph, pollinator type and their interactions treated as fixed effects, and fruit length, fruit width, fruit weight, number of fruits or number of seeds produced per fruit treated as continuous response variables. Again, plant identity was included as a random effect in the model.
Reproductive isolation
A third hand-pollination experiment was conducted on P. gentianoides, P. roseus, and the hybrid flower color morphs to determine the existence of pre-zygotic post pollination barriers (post-pollination reproductive isolation). Crosses between P. gentianoides and P. roseus, and backcrosses in both directions from naturally occurring hybrid flower color morphs were achieved by rubbing one of the dehiscent anthers on the stigmas. The pollination treatments included: (1) intraspecific crosses (e.g., pollen of P. roseus on P. roseus stigmas), (2) interspecific crosses (e.g., pollen of P. roseus on P. gentianoides stigmas), (3) intermorph or intramorph crosses (between and within hybrid flower color morphs), and (4) backcrosses (between species and hybrid flower color morphs). We performed all possible crosses (16 pollination treatments, N = 28 flowers per treatment, one flower per plant; Fig. 5) in flowers emasculated prior to pollination and previously bagged to exclude pollinators, and re-bagged after each treatment. Due to its altitudinal range and abundance, interspecific pollinations that involved P. roseus as pollen receptor were conducted at lower elevation (2,900 m) and the rest near to the hybrid zone (3,800 m). For intraspecific pollinations, we used pollen from plants in different patches (at least 200 m apart) to avoid self-pollen or pollen from clonal individuals. Fruits produced from these crosses were quantified and collected and measured two months later. A GLM model (Family = binomial, Link = logit) was used to evaluate the probability of fruit formation (%) in manual crosses between hybrid flower color morphs and putative parental species. The full GLM model included donor and recipient plant, and their interactions treated as fixed effects and fruit production as a binary response variable. In addition, we compare color morphs and parental species46,47,48 in morphology, nectar, pollinator, and fruit production data. A GLM model (Family = Gaussian, Link = logit) was used to assess differences in fruit length, fruit width, fruit weight, and seed production between color morphs. A Tukey’s post hoc test was used for multiple comparisons among pairs of means of pollination treatments.
Experimental design of a hand-pollination experiment. The pollination treatments included intraspecific crosses, interspecific crosses, intermorph or intramorph crosses, and backcrosses among Penstemon gentianoides, P. roseus, and the ‘blue’ and ‘fuchsia’ hybrid flower color morphs. J. F. Ornelas drew the flower image in figure. The figure was drawn and edited in Adobe Illustrator CS6 v16.0.0 (Adobe Systems, Inc.).
We quantified reproductive isolation (RI) to assess the contributions of post-zygotic barriers between parental species and hybrid flower color morphs (see Sobel and Chen86 for detailed explanations of RI calculations). We then compared RI values between hybrid flower color morphs, P. gentianoides and P. roseus following Sobel and Chen86: R14A = 1–2 * (H/H + C), in which H and C are fruit set of heterospecific (i.e., interspecific or intermoph) and conspecific (intraspecific or intramorph) crosses. In addition, we assessed the significance of pre-zygotic ecological isolating factors (pollinator assemblage) between morphs and species as in Sobel and Chen86: RI4C = 1 – (S/S + U), where S refers to the extent of shared pollinators (i.e., lists of shared and unshared species) and U refers to the extent of unshared pollinators. The absolute and relative cumulative strength of each barrier was also quantified (calculations also provided in Sobel and Chen86) to determine which barrier is currently contributing the most to maintaining these hybrid flower color morphs and species in sympatry.
Source: Ecology - nature.com
