Salvia apiana — A carpenter bee flower?

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Salvia apiana — A carpenter bee flower? Daniela Ott ∗ , Philipp Hühn, Regine Claßen-Bockhoff ∗ Institut für Spezielle Botanik, Johannes Gutenberg-Universität, Anselm-Franz-von-Bentzel-Weg 2, 55099 Mainz, Germany

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Article history: Received 30 March 2015 Received in revised form 14 December 2015 Accepted 25 December 2015 Edited by Shahin Zarre Available online xxx Keywords: Bee pollination Breeding system Mask flower Lever mechanism Lamiaceae Video documentation

a b s t r a c t Salvia apiana has unique mask flowers restricting access to nectar by a bulged lower lip. Stamens and style protrude the flower tube. Most interesting, the staminal lever mechanism usually characterizing bee flowers in Salvia L. is lacking. In the present study, we aim to understand the peculiar pollination mechanism and to identify the pollinators and breeding system of the species. Field experiments were conducted at three natural localities in Southern California and the Rancho Santa Ana Botanical Garden, Claremont. Pollinator behavior was documented on video and interpreted considering frequency, handling time, percentage of successfully touching pollen and stigma, pollen deposition site on the pollinator’s body and duration of stay per inflorescence. Hand-pollination and bagging experiments were conducted to identify breeding system and seed set. Salvia apiana is self-compatible. The pollen/ovule ratio of 11,000:1 points to xenogamy, but our experiments indicate that the plant suffers from pollinator limitation at the study sites. The most frequent pollinators were honeybees (Apis mellifera), which, however, are too small and light to handle the flower correctly. They only occasionally transfer pollen increasing geitonogamy. We identified Bombus vosnesenskii, Xylocopa tabaniformis and X. varipuncta as rare pollinators. These large bees were able to press the lower lip down thereby lowering the stamens and positioning the pollen sacs close to the bee’s body. The largest bee, X. varipuncta, was found to fit best to the flower. Due to its foraging behavior, it particularly contributes to outcrossing. Though honeybees are the most frequent pollinators, we conclude that S. apiana is a carpenter bee flower. The species most likely suffers under the low number of Xylocopa bees which might result from the present dominance of honeybees, introduced to California three hundred years ago. © 2016 Elsevier GmbH. All rights reserved.

1. Introduction Salvia apiana Jeps. (Lamiaceae) has extraordinary flowers. They differ so much from typical Salvia L. flowers that one immediately wonders about the function of the peculiar floral traits. Unlike other Salvia species, stamens are exserted and laterally arranged, the style has a lateral position and the flower entrance is completely closed by the cushion-shaped lower lip (Fig. 1F–G). Most striking, lever-like stamens are lacking. While Salvia flowers in general are characterized by movable anthers whose lower lever arms have to be pushed back by the pollinator (reviewed by Claßen-Bockhoff et al., 2003), the two stamens in Salvia apiana have highly reduced lower lever arms resulting in almost straight, monothecate stamens each with two pollen sacs.

∗ Corresponding authors. E-mail addresses: [email protected] (D. Ott), [email protected] (P. Hühn), [email protected] (R. Claßen-Bockhoff).

Salvia apiana belongs to a group of 19 species classified as Salvia subgenus Audibertia J.B. Walker, B.T. Drew & Sytsma. All species are native to the California Floristic Province (CFP; Raven and Axelrod, 1978) and adjacent deserts. The group is well-known and its monophyly supported by morphological and molecular data (Neisess, 1983; Walker et al., 2004; Walker and Sytsma, 2007; Walker et al., 2015). Species of the Audibertia group are highly diverse in their floral traits. Salvia apiana is the only one with mask flowers, whereas the remaining species have bilabiate or tubular flowers. Most likely, all species lack the lever mechanism (R. Claßen-Bockhoff, unpubl. data), except the most common S. columbariae Benth. Based on a genus-wide phylogeny, it is evident that the New World subgenus Audibertia evolved independently from the Old World Salvia species (Will, 2013). Molecular data clearly support that staminal levers evolved several times in parallel (Walker and Sytsma, 2007; Will and Claßen-Bockhoff, 2014) pointing to their high adaptive value. It is assumed that staminal levers in bilabiate, bee-pollinated flowers contribute to pollen saving and pollinator sharing by species-specifically pollinating the insect at its back

http://dx.doi.org/10.1016/j.flora.2015.12.008 0367-2530/© 2016 Elsevier GmbH. All rights reserved.

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Fig. 1. Salvia apiana. Localities, inflorescence architecture and flowers. A: Dawson Reserve, grassland and ruderal vegetation. B: Motte Rimrock Reserve, rocky slope with Coastal sage scrub community. C: bagged plant in Motte Rimrock Reserve on open, sandy space. D: inflorescence with open flowers. E: side branch, with open terminal flower on 2nd cyme (1st cyme not in picture), *: bracts without axillar products, ll: lower lip, ul: upper lip; F: flower, side view, a: anther, j: stiff joint between filament and connective, ll: lower lip, ul: upper lip, s: stigma; G: flower, front view, ll: lower lip.

(Claßen-Bockhoff et al., 2004; Westerkamp and Claßen-Bockhoff, 2007). This hypothesis of co-evolution between (social) bees and Salvia flowers with staminal levers is indirectly supported by birdpollinated Salvia species. They show repeated and morphologically diverse reductions of the lever mechanism (Wester and ClaßenBockhoff, 2007, 2011). As far as presently known, Salvia species included in the traditional genus concept are predominantly pollinated by bees (and bee-like flies, Celep et al., 2014) or birds. Almost all bee pollinated flowers are equipped with a staminal lever mechanism. Thus, the lack of staminal levers in the Audibertia group raises the question how far the flowers with stiff stamens are adapted to bees or rather also allow flies, butterflies and birds access to nectar. Only little is known about pollination in species of Salvia subgenus Audibertia. Obviously, the large, red, tubular flowers of S. spathacea Greene are hummingbird-pollinated, while the remaining species are assumed to be insect-pollinated (Wester and Claßen-Bockhoff, 2011). As to S. apiana, Grant and Grant (1964) mention carpenter bees and bumblebees as effective pollinators. They also observed hummingbirds on the flowers, but classified them as ineffective.

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As part of a comprehensive study on the pollination and breeding biology in Californian Salvia subgenus Audibertia spp., we here present first data on S. apiana or the ’white sage‘. It is one of the common sage species in southern California. It is used for smudging, food and medical purposes by Native Americans (J. Orozco, pers. communication). We investigated the species at several natural localities to elucidate the functional morphology of the flower, to identify pollinators and to reconstruct the reproductive success of the plants. 2. Material and methods 2.1. Salvia apiana The ’white sage’ has white flowers and tomentose leaves. It is a perennial subshrub, distributed from Baja California in the south to Santa Barbara County in the north and to the western edge of the Colorado Desert in the east. Salvia apiana occurs from coastal areas to montane regions up to 1500 m elevation and prefers dry and sandy soils (Montalvo, 2004). It is a common member in the Salvia

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coastal sage scrub, chaparral and yellow pine forest communities. Flowering time is April to May (Grant and Grant, 1964). Herbarium vouchers and flower samples fixed in 70% ethanol are deposited at the Herbarium of the University at Mainz (MJG).

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Experiments were conducted in southern California from 01.04. to 01.06.2014. The species was investigated at three natural populations and in the Rancho Santa Ana Botanical Garden (RSA), Claremont (Supplementary data, S1). Distances between the sites range from 80 to 200 km. At all sites, we evaluated the surrounding vegetation with use of Jepson eFlora, Calflora.org, and the help of Jessica Orozco, RSA. We recorded the number of individuals and distances between them. To check whether S. apiana produces stolons or not, two small individuals close to bigger plants were carefully dug out.

By means of video analyses (frame by frame), we evaluated the success of each pollinator species. The number of visited flowers per minute, the handling time per flower visit, the percentage of visits with contact of pollen and/or stigma and the number of flowers visited and the time spent per inflorescence were calculated. The main pollen deposition place at the pollinator’s body was also evaluated from the videos. In addition, we caught individuals of Apis mellifera, Bombus vosnesenskii, Xylocopa tabaniformis and Xylocopa varipuncta at RSA, cooled them down to immobility and photographed the area of pollen deposition on scaled paper. These pictures were also used to measure the pollinator body sizes. To determine pollinator frequency, we counted the number of visits per insect species and plant 14 times, in each case for 30–90 min (690 min in total). We thereby compared the pollinator frequency in two wild populations, Motte Rimrock Reserve (MRR) and Dawson Los Monos Canyon Reserve (DR), with the situation in the RSA Botanical Garden.

2.3. Morphometric measurements

2.7. Reproductive system

Morphometric data were taken from 102 flowers (of 10 plants) representing different developmental stages. A slide caliper (“TCM”, Tchibo, Hamburg, Germany), accuracy ± 0.02 mm, and range of 0–100 mm was used for the following measurements (Fig. 2): 1) distance between anthers, 2, 3) lengths of left and right stamen from the point of liberation from the corolla to the anther tip, 4) shortest distance between stigma and anther, 5) distance between stigma and edge of lower lip, 6) length of the style part protruding the corolla, 7, 8) length and width of landing place on lower lip, 9) length of lower lip, 10) width of flower tube entrance, 11) length of corolla tube, 12) nectar level. Left- or right-hand style position was documented on three plants.

To evaluate the reproductive system of S. apiana, we conducted five different tests at two different localities, each with three to eight individuals. Inflorescences were marked, stabilized with plant wire and, if necessary, bagged with cotton sacs (Fig. 1C). Open flowers were taken off before the experiments started (except for control). For at least four days, the plants were checked three times the day. Newly opened flowers were treated depending on the experiment. Control: to determine seed set under natural conditions, no further treatment was done. Control plus foreign pollen: to identify maximal seed set, flowers from unbagged inflorescences were additionally hand-pollinated with pollen from a genetically different plant. Geitonogamy: to test for self-pollination among flowers, bagged flowers were hand-pollinated with pollen from the same individual by dabbing open anthers on the spread stigmatic arms. Autogamy: to test for self-pollination within a single flower, buds were bagged and not treated any more; only flowers touching neighboring flowers were removed to avoid geitonogamy. Apomixis: styles and stamens were removed with fine scissors prior to anthesis to test for seed set without pollination. After four weeks when the plants were fruiting, inflorescences were carefully cut and seeds counted. Based on maximal four seeds per flower, the percentage of seed set per flower was calculated.

2.2. Study sites

2.4. Anthesis To identify the duration of flowering and the stages of anthesis in a single flower, inflorescences from different localities were cut and kept in vases with tap water. Sixteen flowers were observed every 30–90 min from early bud opening to wilting. The stages were confirmed at 40 flowers in the field. 2.5. Flowering sequence To determine the flowering sequence, on each locality a side view of one representative inflorescence was drawn. At two inflorescences from different localities, the terminal and two lateral parts in proximal and distal position were marked with acryl color as representatives of the whole inflorescence. The development of the flowers at each of the first and third nodes was recorded over five to six days at 4 to 5 pm. 2.6. Pollinator observations During the first week, flower visitors were observed three times the day at 8 am, 1 pm and 5 pm, each time for 30 min. At all localities, it was soon evident that pollinator activity did not change during the day, but with weather conditions. Therefore, the following observations, in total about 170 h at four different sites, were made whenever weather was suitable. Insect species were determined with help of James Hung, University of California San Diego. They were defined as pollinators when they got regularly into contact with pollen and stigma. To document insect visitors and behavior, 161 videos with a total length of more than 250 min were produced using the video camera HC-V72 Full HD 20.4 Mega Pixels, Panasonic, Kadoma (Japan), and a Power Shot G9 digital camera, Canon, Tokio (Japan). Please cite this article in press as: Ott, http://dx.doi.org/10.1016/j.flora.2015.12.008

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2.8. Pollen-ovule-ratio (P/O) To determine the P/O-ratio, 18 closed anthers from nine flowers and three individuals in MRR were separately stored in Eppendorf tubes and fixed in 70% ethanol. At Mainz, the anthers were put in a new Eppendorf tube with 100 ␮l staining solution (20 ␮l Glycerol 37%, 80 ␮l H2 O, a drop of methylene blue). The anthers were squeezed until all pollen was released. The solution was homogenized with a vortex mixer and 10 ␮l were then transferred to a counting chamber (Fuchs-Rosenthal-Zählkammer). Pollen grains were counted under the microscope SM-Lux, Leitz, Wetzlar (magnification 100×). The number of grains in a chamber was used to compute the number of pollen grains per anther. Based on two anthers and four ovules per flower, the P/O ratio was calculated. 2.9. Nectar concentration To evaluate sugar concentration, nectar was taken off the flowers using a microcapillary (volume 1 mm) and put on a refractometer (28–62%), Atago, Tokyo (Japan). As the nectar volume was too small for the microcapillary, we opened corolla tubes by hand Salvia

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Fig. 2. Morphometric measurements of the flowers in Salvia apiana. A: front view of flower morph with right-sided style. B: side view of flower (one stamen removed). C: side view (style removed). D: top view. 1: Distance between anthers. 2: length of left stamen. 3: length of right stamen (from corolla insertion point). 4: distance stigma to closest anther. 5: distance stigma to lower lip. 6: length of the style part protruding the corolla. 7: length of landing place on lower lip. 8: width of landing place on lower lip. 9: length lower lip to corolla tube entrance. 10: width of corolla tube entrance.11: length of corolla tube. 12: nectar level.

and put the nectar directly on the refractometer. We conducted three measurements with ten, twenty and forty flowers in MRR, one with sixty flowers in DR and one with forty flowers in RSA (all flowers from one individual). 2.10. Statistics Means and standard deviations were calculated in “Excel 2010”, Microsoft, Redmont (USA). Mann–Whitney-test was used to compare the results of reproductive system experiments and pollinator data. Distribution of enantiostyly was evaluated with a binominal test. All statistical analyses were calculated in SPSS Statistics 22, IBM, Armonk (USA), with significance level ˛ = 0.05.

visits. However, when the inflorescences were in full bloom, they were visited by lots of bees. Corral Canyon Road (CCRd): In the Santa Monica Mountains, S. apiana was growing on a slope with coastal sage scrub vegetation. Close to the road, we investigated about ten plants on 400 m2 associated with co-flowering individuals of Salvia mellifera and S. leucophylla Greene, Hirschfeldia incana, Artemisia californica Less. and Encelia farinosa A. Gray ex Torr. Rancho Santa Ana Botanical Garden: We included 85 individuals on an area of 400 m2 in our study. The plants were surrounded by a multitude of flowering plants grown in the garden. Correspondingly, the number of pollinators (species and individuals) was much higher than at the natural localities. 3.2. Inflorescence architecture and flowering sequence

3. Results 3.1. Population size and environment The populations investigated differed in size and density. At the three natural sites, 10–25 individuals were scattered over areas of 100–2000 m2 . The plants stood solitarily or in small groups (Fig. 1A–C). They have no stolon, therefore, each plant is considered as a genetically different individual. Dawson Reserve: We found 15 plants on 250 m2 on open grassland accompanied by oaks and willows. The distance between the individuals ranged from two to ten meters. Species of Sinapis L. and Eriogonum Michx. (Fig. 1A) were co-flowering and regularly visited by bees, mainly honeybees. Motte Rimrock Reserve: We worked on three sections of a large population each with 10 (on 100 m2 ), 20 (on 2000 m2 ) and 25 (on 1600 m2 ) individuals. On rocky areas with coastal sage scrub, plants grew in high density (<1 m distance to each other) (Fig. 1B). On open areas with sandy soil (Fig. 1C), they were more loosely scattered and separated by several 10–100 m from each other. Flowering plants of Eriogonum, Hirschfeldia incana (L.) Lagr.-Foss., Phacelia Juss., Salvia mellifera Greene and S. columbariae attracted many honeybees. When S. apiana started to flower, it first received few insect Please cite this article in press as: Ott, http://dx.doi.org/10.1016/j.flora.2015.12.008

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Salvia apiana has huge, many-flowered inflorescences (Fig. 1C–D). Flowers are arranged in an open thyrse with sitting cymes composed of a terminal and more than ten lateral partial inflorescences (PIs). The terminal unit (Fig. 3A: T) bears two dichasial cymes per node. The distal branches repeat this pattern presenting PIs of 1st order (Fig. 3A: 1–7), while the more proximal ones additionally bear PIs of 2nd order (Fig. 3A: 10). Lateral thyrses terminate the basal-most branches each starting with a leafy zone followed by an inhibition zone with buds (Fig. 3A: 15–17). Interestingly, the cymes of the partial inflorescences are not dichasial throughout (Fig. 3C). In some PIs, monochasial (Fig. 3B) and dichasial cymes alternate with position, i.e. the median adaxial cymes are then lacking while the median abaxial and transversal cymes are always present (Fig. 1E). Flowering sequence within a plant starts weakly acropetalous, but looks quickly a bit chaotic (Fig. 1D). First, the most proximal flowers open, i.e. the primary flowers of the basal-most cymes (Fig. 3A: 7–1a and b). Flowering sequence proceeds in three different directions, along the main axis (Fig. 3A: 4–1a and b), along the lateral branches (Fig. 3A: 7–3a and b) and within the cymes (Fig. 3B and C). Given that an average inflorescence has about 12 lateral PIs each with more than 10 cymes branched up to the 5th order Salvia

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Fig. 3. Inflorescence architecture of Salvia apiana, schematically. A: side view of a flowering plant; T: terminal part of the thyrse. 4, 7, 10: partial inflorecences of the terminal thyrse consecutively numbered top down. 15: flowering branch with buds (black dots). 17: lateral branch with leaves and buds. 1a, b, 3a, b: position of cymes observed in detail. B: monochasial cyme (light grey in A). C: dichasial cyme (dark grey in A).

and considering that the terminal unit flowers a bit earlier than the distal PIs, is evident that the underlying acropetalous order gets completely masked. As a result, S. apiana presents mass-flowering inflorescences (Fig. 1D) with young and old flowers at the same time.

3.3. Flower morphology and anthesis Salvia apiana has zygomorphic mask flowers with minute upper lips and huge lower lips restricting access to nectar (Fig. 1F and G). The two stamens adhere laterally to the corolla wall. They protrude the flower tube as the style does which is arranged at the left or right side of the flower. During anthesis, length and position of floral structures slightly change influencing the chance of pollination (Table 1). Morphometric data were similar at all sites. Stage 1: In late bud stage, the pubescent calyx opens and the young, still closed, creme-colored corolla becomes visible. Stage 2: The lower lip starts the process of flower opening. It unfolds and the minute upper lip and narrow corolla tube entrance become visible. Filaments and connectives of the two stamens stretch and release the style, jammed by the stamens before. The stamens have long filaments which are rectangular bent upwards (Fig. 1E and F). The connectives are highly asymmetrical. While Please cite this article in press as: Ott, http://dx.doi.org/10.1016/j.flora.2015.12.008

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one side of the anther is reduced to a minute tooth-like structure (Fig. 1F: j), the fertile pollen sacs of the second side are presented far above the corolla (Fig. 1G). The lower lip is the most conspicuous structure. Close to the flower entrance it is bulged. It blocks the access to the nectar offered at the flower ground (Fig. 1F). The distal part of the lower lip is almost flat and forms the landing platform for visitors (Fig. 1G). This part bears glossy hairs and usually shows small dots in yellow, blue, rose or violet. Its size remains almost constant during flowering time, but may suffer from wilting at the end of anthesis (Table 1). Stage 3: In the male stage, the pollen sacs open. They present whitish pollen, which is sticky in the beginning but soon becomes powdery. To reach the nectar, visitors have to press down the lower lip. This stimulates the stamens to swing downwards. The pollen sacs get close to the lower lip and may touch a pollinator. Stage 4: When the weakly violet stigmatic lobes spread, the pollen sacs still present pollen. In this hermaphrodite stage, almost all flower structures have reached their final length (Table 1). Most remarkable is the elongation of the style increasing the distance between stigma and closest anther and between stigma and lower lip. The style stands usually at the right or left side of the corolla. Only rarely, it has an erect position and presents the stigma between the anthers. All stigma positions occur on the same plant.

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Table 1 Morphometric data of Salvia apiana. Means and standard deviations are given for four anthesis stages: 2, flower opening, 3, male stage, 4, hermaphrodite stage, 5, female stage. Stage

Floral structures

1 2 3 4 5 6 7 8 9 10 11 12

Distance anther – anther Length left stamen Length right stamen Distance stigma – anther Distance stigma – lower lip Length protruding style Length landing place Width landing place Length lower lip Width tube entrance Length corolla tube Average nectar level

2 (n = 8) 8.5 11.2 10.7 9.6 7.7 11.3 6.0 6.5 9.1 2.9 4.8 2.2

± ± ± ± ± ± ± ± ± ± ± ±

Counting the number of left- and right-sided styles in three different individuals revealed no statistically significant differences (114 left vs. 116 right styles, n = 230 flowers). However, as a positional peculiarity, simultaneously open flowers of two neighboring cymes showed diverging style positions. Stage 5: After the dehiscence of pollen sacs, the stigmatic lobes are still open and fresh (female phase). Each of the male, hermaphrodite and female stages usually lasts one day. The mean flower duration is thus three days (77 ± 26 h, n = 19). Observations after hand pollination indicate that successful fertilization may shorten the length of anthesis.

3.4. Flower visitors Inflorescences of S. apiana were visited at all sites by honeybees and a variety of small bees and beetles. Except at Corral Canyon Road (CCRd), we also observed bumblebees, carpenter bees and hummingbirds. Among them, Apis mellifera L., Bombus vosnesenskii Radoszk., Xylocopa tabaniformis Smith and X. varipuncta Patton were identified as pollinators. Pollen deposition onto the bee’s body and removal from it to the stigma is not very precise. Though the lateral abdomen was always the site with the highest contact to pollen and stigma (32–60% of all contacts, Table 2), pollen was also deposited at the lateral thorax, wings, legs and ventral side. During the observation time of 690 min, we recorded 502 individual pollinators visiting S. apiana plants (∼44 per h). The highest pollinator frequency (∼63 per h) and species diversity (less than 70% honeybees) was recorded in the Rancho Santa Ana BG. In contrast, the wild populations at the Motte Rimrock (MRR) and Dawson Reserves (DR) only received 35–38 pollinator visits per hour, more than 95% of them being honeybees. Apis mellifera Honeybees are the most common bees and by far the most frequent pollinators of S. apiana (Table 2: 85%). They attack other flower visitors, even larger bees (e.g. bumblebees), and thus dominate their food plant. Honeybees are the smallest bees among the pollinators (length ∼13 mm, thorax and abdomen width ∼4–5 mm, n = 3). They do not regularly touch the reproductive organs (Fig. 4A–B), but instead steal nectar to a high percentage (Table 2). When landing or leaving the flower, they occasionally touch pollen and stigma. They often pull the style and/or filaments close to their body to hold fast. Thereby, they get also contact with the reproductive surfaces. Being too light to release the flower mechanism by simply landing on the lower lip, the honeybees actively exert force to get access to nectar. In consequence, their average handling time per flower is significantly longer than the one of all other, larger bees (Tables 2 and 3). The mean time they spend on one inflorescence Please cite this article in press as: Ott, http://dx.doi.org/10.1016/j.flora.2015.12.008

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3 (n = 22)

4.3 1.0 1.6 4.7 2.4 1.0 0.7 0.8 1.0 0.4 0.8 1.0

al.,

12.5 13.3 13.3 13.7 10.9 12.6 6.5 7.4 10.4 3.5 4.8 2.5

± ± ± ± ± ± ± ± ± ± ± ±

4 (n = 53)

2.7 1.4 1.3 5.8 3.1 2.4 0.9 0.8 1.2 0.3 0.7 0.5

12.9 14.1 14.1 17.2 12.5 15.7 7.1 7.9 11.9 3.6 5.1 2.9

± ± ± ± ± ± ± ± ± ± ± ±

5 (n = 19)

2.5 1.6 1.6 5.2 2.8 2.5 1.0 0.9 1.4 0.5 0.8 0.5

12.1 13.1 12.6 15.3 12.4 15.6 6.5 6.7 10.6 3.6 5.2 3.1

± ± ± ± ± ± ± ± ± ± ± ±

3.7 1.8 1.8 6.4 2.5 2.1 1.6 1.5 1.6 0.5 0.8 1.0

is also the longest one among all pollinators, while the number of visited flowers per minute is the lowest (Table 2). Within the inflorescence, Apis mellifera prefers to crawl from flower to flower in upwards direction, using styles and stamens as steps (Fig. 4C). Bombus vosnesenskii Bumblebees count for only 10% of plant visits (Table 2). Compared to Apis mellifera, they are a bit larger (body length ∼17 mm, thorax and abdomen width ∼8 mm, n = 2), thus fitting much better to the flower proportions. They have a shorter handling time (2.7 vs. 3.8 s, Table 2) and more success in getting nectar (Tables 2 and 3) and touching pollen sacs (78% vs. 35% of all cases). Only the rate of stigma contact does not differ significantly (33% vs. 23%, Tables 2 and 3). Although bumblebees easily release the flower mechanism by landing on the lower lip, they rarely touch the reproductive surfaces during the drinking process (Fig. 4D). Contact rather takes place when the bees enter or leave the flower. When leaving, the lower lip flips back to its unreleased position and sometimes explosively disperses pollen. Like honeybees, bumblebees also occasionally pull stamens and styles under their body during the feeding process (Fig. 4E) enhancing pollen transfer. Bombus vosnesenskii usually flies to the flowers. Only in dense inflorescences, bumblebees also crawl from flower to flower whereby pollen can be exchanged among neighboring flowers (Fig. 4F). Xylocopa tabaniformis and X. varipuncta The two carpenter bee species are very rare pollinators, together only contributing 5% to plant visitation (Table 2). However, based on visits per minute, handling time per flower, success in getting nectar, touching pollen sacs and stigmas they are clearly the best pollinators (Table 2). Like Bombus vosnesenskii, they easily release the flower mechanism by landing on the lower lip and can get powdered with pollen when the lower lip springs upwards. Carpenter bees were never observed to crawl to another flower, but fly even for short distances. Nevertheless, in dense inflorescences pollen can also be exchanged among neighbor flowers (Fig. 4H). The two Xylocopa species differ in size, handling time and visits of flowers per inflorescence. • Xylocopa tabaniformis (length ∼17 mm, thorax and abdomen width ∼8–9 mm, n = 4) resembles Bombus vosnesenskii in body proportions and in almost always being successful in getting nectar and touching pollen. But like the bumblebees, it usually does not touch reproductive organs while feeding (Fig. 4G). Sometimes, the carpenter bees pull the style to the lateral abdomen when landing, exactly to the site where pollen is deposited. In more than 50% of all contacts, the lateral abdomen is touched by pollen sacs and stigma. It differs from all pollinators in having the shortest handling time per flower and the highest number of visits per minute at a single plant (Table 2).

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Table 2 Behavior of pollinators of Salvia apiana (based on video analyses).

Visits per min. Handling time [s] Nectar intake [%] Contact with pollen [%] Contact with stigma [%] Contact with stigma and pollen [%] Pollen deposition at lateral abdomen [%] Stigma contact to lateral abdomen [%] Number of flowers visited per inflorescence Duration of stay at an inflorescence [s] ** Frequency [%]

Apis mellifera

n

Bombus vosnesenskii

n

Xylocopa tabaniformis

n

Xylocopa varipuncta

n

9.33 ± 3.28 3.81 ± 2.91 86.36 35.38 22.81 12.50 48.78 38.10 4.67 ± 2.60 28.00 84

91 90 88 65 57 56 41 21 * 9 * 9

15.18 ± 3.7 2.65 ± 1.64 96.43 77.42 33.33 34.62 32.26 52.17 4.83 ± 3.82 14.5 10

65 55 56 31 27 26 31 23 * 6 * 6

22.44 ± 3.84 1.77 ± 1.01 97.10 78.43 50.00 48.28 53.33 54.55 8.63 ± 10.95 18.63 5

89 68 69 51 38 29 45 22 * 8 * 8

19.82 ± 11.17 2.37 ± 2.17 100 100 81.82 81.82 48.15 60.00 3.50 ± 2.45 9.11

31 31 29 21 11 11 27 10 * 8 * 8

n, number of flower visits in which the particular aspect could be observed. * n refers to number of inflorescences observed. ** Frequency is based on 509 bees in 690 min.

Fig. 4. Pollinators of Salvia apiana. A–C: Apis mellifera. A: regular visit, no contact to reproductive organs. B: front view showing the relative proportions of insect and flower. C: bee crawling from one flower to the next, contact to reproductive structures. D–F: Bombus vosnesenskii. A: regular visit, no contact with reproductive organs when drinking; E: bumblebee pulling the style under its abdomen, left stamen touching lateral abdomen. F: bumblebee touching reproductive surfaces when moving on dense inflorescence. G and H: Xylocopa tabaniformis. G: regular visit, no contact with pollen sacs and stigma when drinking; H: on dense inflorescence accidentally touching reproductive organs. I: Xylocopa.varipuncta drinking position, contact to reproductive surfaces.

• Xylocopa varipuncta is the largest pollinator species (length ∼25 mm, thorax and abdomen width ∼13 mm, n = 3) and fits the best to the flower (Fig. 4I). It is always successful in getting nectar (100%) and gets significantly more contact to pollen than all other pollinators (Tables 2 and 3). This carpenter bee species has also the highest contact to the stigma and the most precise pollen deposition at the lateral abdomen (Table 2: 60%), both resulting

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from body proportions. Xylocopa varipuncta is the only pollinator touching the reproductive surfaces not only when landing and leaving, but also during the feeding process. In contrast to X. tabaniformis, the large carpenter bee species visits only few flowers and leaves the plant quickly to fly to a second individual. In fact, among all pollinators, X. varipuncta visits the Salvia

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Table 3 Pair-wise comparison of pollinators of Salvia apiana. The honeybee (Apis mellifera) is significantly slower and less successful than all other pollinators. Xylocopa varipuncta is the most successful pollinator with respect to pollen contact. p-Values Mann–Whitney-test

Handling time

Nectar intake

Pollen contact

Stigma contact

A. mellifera – B. vosnesenskii A. mellifera – Xylocopa tabaniformis A. mellifera – X. varipuncta B. vosnesenskii – X. tabaniformis B. vosnesenskii – X. varipuncta X. tabaniformis – X. varipuncta

0.004* <0.001** < 0.001** < 0.001** 0.068 0.536

0.048* 0.020* 0.037* 0.832 0.306 0.357

<0.001** <0.001** <0.001** 0.915 0.020* 0.022*

0.308 0.006* < 0.001** 0.185 0.007 (exact Sig. 0.019)* 0.063

* **

Statistically significant. Highly significant.

lowest number of flowers per inflorescence and spends the shortest time (<10 s) on a single plant (Table 2). Further visitors Small bees like Halictus Latr. are not able to release the flower mechanism. They instead collect pollen from the open anthers and press their head through the small flower entrance to get access to nectar. Pollen transfer is very unlikely but may occasionally occur. Ants were found on one individual in the Dawson Reserve. They crawl across the flowers, but do not transfer pollen. Beetles collect pollen from the open anthers, but do not get in touch with the stigmas. Hummingbirds rarely visit the flowers. They do not release the flower mechanism, but pierce the lower lip to reach the nectar. Thereby, they hover in front of the flower and get not into contact with pollen sacs and stigmas. 3.5. Reproductive system The plant is self-fertile. Seed set in the test for autogamy was low (16.8%; Table 4). Geitonogamy resulted in a similar high seed set as found under natural conditions (control). Maximal seed set (85.5%) was achieved after additional hand pollination with foreign pollen. Apomixis can be excluded. Except the tests for geitonogamy and control, all results were significantly different (all p-values <0.001). Between the sites, results did not differ except for the control plants (MRR average seed set 2.7 ± 1.6, n = 76; DR 2.0 ± 1.5, n = 81, p < 0.001). We counted 22,222 ± 7355 pollen grains per anther. The pollen/ovule-ratio is thus 11,111 (±2649). Nectar volume per flower was generally too low to be measured. Only after nectar accumulation from more than 40 flowers, sugar concentration could be determined as 40.7% (±2.7, n = 3). As water could evaporate during accumulation, nectar values should be regarded with suspicion. They are probably lower than values measured. 4. Discussion Salvia apiana is a bee flower with a unique pollination mechanism. It fits best to large carpenter bees, but is most frequently visited by honeybees. 4.1. Flower construction and pollen transfer without lever mechanism Salvia apiana is clearly adapted to bees as pollinators. Flowers are zygomorphic, bilabiate, provide a landing place and offer nectar at the ground of the corolla tube (Westerkamp and ClaßenBockhoff, 2007). Flower marks and hairs increase attractiveness to bee pollinators. Though the measured nectar concentration might be too high (for technical reasons), it is in the typical range of bee flowers (Baker and Baker, 1983; Faegri and van der Pijl, 1979). In contrast to almost all other bee flowers in Salvia, a staminal lever mechanism is lacking. Pollen sacs are not hidden, but freely accessible. However, they are so far away from the landing Please cite this article in press as: Ott, http://dx.doi.org/10.1016/j.flora.2015.12.008

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place, that bees cannot collect pollen in large quantities. The specific pollen transfer mechanism guarantees nevertheless contact to pollinators. It results from the construction of the mask flower. When the lower lip is pressed down, both stamens passively lower down and present their pollen sacs to the left and right sides of the flower (Fig. 4D and I). Due to the stiff joint, filaments and extended connective arms move together as a single unit. Based on the asymmetry of the anther, each stamen is monthecate, i.e. it presents only one theca (Fig. 1G), while the other one is reduced. The fertile theca opens towards the flower realizing lateral (plagiotribic) pollination. Pollen is deposited on the lateral abdomen of the pollinator, a body site also touched by the stigma. The single style stands left or right of the flower. Both positions appear on the same individual, representing an example for monomorphic enantiostyly (Jesson and Barrett, 2003). It is evident that the bulged lower lip, exposed stamens, monothecate anthers, stiff joints and enantiostyly functionally cooperate allowing for precise pollen transfer and pollen saving at the same time. The mask flower with exposed stamens thus evolved as an alternative to the open bilabiate flower with staminal lever mechanism in Salvia. 4.2. Pollination The pollination mechanism works successfully with large bees, strong enough to press the lower lip down and broad enough to touch the reproductive surfaces. Among the observed pollinators, carpenter bees fit the best. The thorax and abdomen width of the largest pollinator, Xylocopa varipuncta, exactly matches the distance between the two anthers (both ∼13 mm). Consequently, the insect touches the pollen sacs at each flower visit (100%). Coupled with the highest percentage of stigma contact (∼82%) and the shortest stay at a single inflorescence, X. varipuncta not only proves to be a successful pollinator but also to contribute to outcrossing. Xylocopa tabaniformis handles the flowers faster than X. varipuncta, indicating that the insect easily manages the flower mechanism. However, based on its smaller body proportions, it is less successful in touching pollen sacs and stigmatic surfaces. Even more remarkable, it stays at least twice as long on a single inflorescence as each of the other pollinators. This foraging behavior may lead to more pollen dispersal and geitonogamy in an individual plant. Bombus vosnesenskii and Apis mellifera are less successful pollinators in particular due to their low contact to stigmas. They touch the reproductive surfaces when arriving and leaving the flower and when crawling from flower to flower or pulling stamens and styles under their bodies. Thereby, the bumblebee has a higher contact to pollen sacs than the honeybee. Both bee species contribute particularly to pollen dispersal within one individual. Altogether, Xylocopa varipuncta fits the best to Salvia apiana followed by X. tabaniformis, Bombus vosnesenskii and Apis mellifera. Though the honeybee is little successful and cannot easily handle the flower mechanism, it most likely is the main pollinator due to its high frequency. Grant and Grant (1964) assumed honeybees to be ineffective, but our study reveals that they are able to transfer Salvia

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Table 4 Results from pollination experiments in Salvia apiana. Data refer to all experimental plants and sites. Treatment

Control

Control plus foreign pollen

Geitonogamy

Autogamy

Apomixis

403 (179) 2.3 ± 1.6 76.0 56.3

325 (95) 3.4 ± 1 97.9 85.5

310 (139) 2.2 ± 1.5 77.7 55.8

66 (98) 0.7 ± 1.3 25.5 16.8

0 (45) 0.0 0.0 0.0

Seed and fruit set Number of seeds (flowers) Mean seeds per flower (max = 4) Fruit set [%] Seed set per fruit [%]

pollen even though to only a low degree. The sheer mass of flower visits makes them successful pollinators.

4.3. Breeding system As far as known, Salvia species are self-compatible (e.g. Haque and Goshal, 1981; Miyajima, 2001; Navarro, 1997; Ohashi, 2002; Zhang et al., 2011a). This is also confirmed for S. apiana. Though seed set is low under autogamy conditions, simulated geitonogamy results in a percentage of seeds similar to the control. The low number of seeds in the autogamy treatment can be explained by herkogamy, i.e. the spatial separation of pollen sacs and stigma, and by sexual phase separation (protandry). The latter, however, is weak as male and female stages overlap for a day. Thus, autogamy can happen when pollen falls down from a wilting anther to a receptive stigma. Geitonogamy requires the same costs as crosspollination without providing the plant with the genetic benefits of outcrossing (Lloyd and Schoen, 1992). Selection against selfpollination within an individual plant is therefore expected (Jong et al., 1993). However, in S. apiana, geitonogamy may be high due to the foraging behavior of honeybees, crawling from flower to flower, and the high number of sequential flower visits at a single inflorescence as documented for Xylocopa tabaniformis (see Jong and Klinkhammer, 2005). Furthermore, inflorescence architecture and flower construction promote geitonogamy. Due to the flowering sequence, flowers in different sexual phases are close together and their anthers and stigmas may touch each other. When inflorescences are moved by wind, even neighboring inflorescences may get in contact enhancing self-pollination. Enantiostyly is no barrier promoting outcrossing (see Fenster, 1995). Instead, the one-sided arrangement of the style may have evolved in the functional context of plagiotribic pollination. Under natural conditions, seed set is little more than 56%. Compared to maximal seed set (85%), this indicates pollinator limitation. Obviously, at the three natural localities and also in the botanical garden, pollinators promoting outcrossing are rare. The honeybee, always present at all places, contributes more to geitonogamy than to xenogamy, while ‘the best pollinator’ Xylocopa varipuncta is too rare to considerably increase seed set. Honeybees are not native to California, but were introduced in the 17th century (Goulson, 2003). Invasion by honeybees tends to have a negative impact on local bee fauna (Paini, 2004). Honeybees do not only occupy their food plants, but also chase away other flower visitors (Gross and Mackay, 1998; Roubik, 1980; Sakagami, 1959), a behavior also observed on S. apiana. They also kill larger bees in the vicinity of their hives (Thoenes, 1993), thus decreasing the abundance of native bees and pollinators. As long as the honeybees provide pollination services comparable to native bees, the plants will not suffer from a lack of fitness. Our hand pollination experiments, however, show a significant difference between maximal and natural seed set indicating that Salvia apiana suffers from the rareness of Xylocopa bees (see Bierzychudek, 1981). This might result in a loss of fitness and to a long-term decrease in population size and genetic variety (e.g. Gross and Mackay, 1998; Wilcock and Neiland, 2002).

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5. Conclusion Salvia apiana proves to be an interesting example for the replacement of native pollinators by honeybees (e.g. Zhang et al., 2011b). We assume that the flowers originated in co-evolution with Xylocopa bees which are able to handle the complex flower construction, fit perfectly to the flower proportions and promote outcrossing. This view is supported by the high pollen-ovule ratio indicating xenogamy (Cruden, 1977). Specialization to Xylocopa bees going along with a wide exclusion of other pollinator species increases reproductive success in a bee-rich environment. However, a too close specialization also bears the risk of losing flexibility. Our study shows that S. apiana in fact suffers from the rareness of its ’best pollinator‘ species, but is able to compensate by using honeybees as pollinators. The individual honeybee is much less successful in pollen transfer than a single carpenter bee, but the immense number of individuals counterbalances this incapacity. Based on the foraging behavior of the honeybees and the low seed set under natural condition, it is likely that a high percentage of seeds results from geitonogamy. Salvia apiana, thus, turns out to have a mixed mating pattern most likely tolerating the low degree of genetic diversification by the production of hundreds of flowers and perennial life form. The example of S. apiana shows that specialization to a certain pollinator should be not too narrow. Instead, allowing a range of pollinators access to nectar will help the plant to adapt to a changing environment. We assume that exactly this is demonstrated by S. apiana, a carpenter bee flower mainly pollinated by honeybees.

Acknowledgements For identification of insects, amicable cooperation and technical support we thank James Hung, PhD Candidate, University of San Diego. For plant identification we thank Jessica Orozco, Graduate Student, Rancho Santa Ana Botanic Garden. We also thank Prof. Lucinda McDade (Executive Director, Rancho Santa Ana Botanic Garden), Prof. Ken Halama (UC Riverside), Prof. Isabel Kay (UC San Diego) and Larry Cozzens (UC San Diego) for the permits to work in Rancho Santa Ana Botanic Garden resp. Motte Rimrock and Dawson Reserve, and the technical and personal support. Thanks are given to Prof. J. Mark Porter (Rancho Santa Ana Botanic Garden) and Prof. Josh Kohn (UC San Diego) for technical support. Acknowledgements also go to Maria Will (Oldenburg) and Natalie Schmalz (Mainz) for suggestions and advice. We thank Doris Franke and Maria Geyer (both Mainz) for graphic design. Finally, we want to thank the PROMOS program (University of Mainz) for financial support (given to P. Hühn).

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.flora.2015.12. 008.

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Further reading http://www.calflora.org/, 20.06.2014. http://www.cnrfc.noaa.gov/, 20.06.2014. Jepson Flora Project (eds.) 2014 Jepson eFlora, http://ucjeps. berkeley.edu/IJM.html, 20.06.2014. http://motte.ucr.edu.html, 20.06.2014. http://nrs.ucop.edu/reserves/dawson/dawson los monos.htm, 20.06.2014. http://www.rsabg.org, 20.06.2014. http://stuntranch.ucnrs.org, 20.06.2014. http://www.wieweit.net/gps koordinaten-hohe.php, 20.06.2014.

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