Pollination niches of conopsea and G. densiflora in pure and mixed populations: evidence for character displacement?

Caroliné Olofsson

Degree project in biology, Master of science (2 years), 2021 Examensarbete i biologi 45 hp till masterexamen, 2021 Biology Education Centre and Department of Eclology and Evolution, Uppsala University Supervisors: Nina Joffard and Nina Sletvold External opponent: Lisette van Kolfschoten Table of content

Abstract ...... 2 Introduction ...... 3

Material and methods ...... 6

Study species ...... 6 Study populations ...... 7 Video recording ...... 8 Pollinator catching ...... 8 Statistical analysis ...... 9 Results ...... 10 Comparison between G. conopsea and G. densiflora ...... 10 Comparison between pure and mixed sites ...... 13 Comparison between diurnal and nocturnal pollinators ...... 13 Pollinator community composition ...... 14 Discussion...... 16 Visitation pattern in relation to scent data ...... 16 The pollinators ...... 17 Niche differentiation ...... 18 Conclusions ...... 19 Acknowledgments ...... 19 References...... 19

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Abstract

Reproductive isolation can be achieved through multiple types of barriers and is essential for speciation. In flowering , pre-pollination barriers (e.g. differentiation in pollination niches) are believed to be the most efficient at preventing gene flow across species boundaries. In closely related species that come into secondary contact, such barriers can evolve to prevent competition for pollinator service and/or interspecific pollen transfer, which can have fitness costs. Hence pollination niche differentiation should be stronger in sympatric populations than in allopatric populations (i.e. character displacement).

To investigate the differences in pollination niches and to see if it is consistent with a hypothesis of character displacement, I used the two closely related and phenotypically similar orchid species, Gymnadenia conopsea and G. densiflora. I sampled mixed and pure populations of G. conopsea and G. densiflora on Öland during the summer of 2020. In these populations, I used video cameras and pollinator catches to record pollinator activity and characterize the composition of pollinator communities. Estimation of pollinator efficiency was also assessed by analyzing the number of pollinia carried by each pollinator.

Contrary to my expectations, I found that both orchids had their visitation peak during the night and that the most frequent and efficient pollinators were Autographa gamma or for both of them. Furthermore, no increased differentiation between the two species was found in mixed compared to pure populations. My results suggest that plant-pollinator interactions do not act as efficient pre-pollination barriers in these two orchid species, and that competition for pollinator service and through interspecific pollen transfer seem to be too weak to drive pollination niche partitioning.

Keywords: Character displacement, Gymnadenia conopsea, Gymnadenia densiflora, Reproductive isolation.

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Introduction

One of the most fundamental aims in evolutionary biology is to gain knowledge about the process of speciation, i.e. how new species arise and how they become differentiated (Hopkins, 2013). Species can be described in several ways and one way commonly used is through the biological species concept (De Queiroz, 2007; Mallet, 1995; Noor, 2002). This concept describes species as interbreeding populations that are reproductively isolated from other populations (Mayr, 1996; Coyne and Orr 2004).

To understand the process of speciation, it is important to identify the evolutionary forces driving the divergence of populations: here, reproductive isolation plays a key role. Reproductive isolation is achieved through barriers that hinder gene flow between species and help the fixation of trait differences (Baack et al., 2015). Generally, reproductive isolation mechanisms are divided into two categories, pre-zygotic and post-zygotic barriers, depending on when they occur during the reproduction process (Baack et al., 2015). Pre-zygotic barriers are those that act before the fusion of the gametes, either before (i.e. mechanisms that limit mating opportunities) or after (i.e. incompatibility mechanisms) mating, while post-zygotic barriers are those that affect hybrid fitness (Baack et al., 2015; Hopkins, 2013). When studying plants we can, instead of talking about pre- and post-mating barriers, distinguish between pre- and post-pollination barriers (Baack et al., 2015). Examples of pre-pollination barriers include isolation caused by pollinators or flowering time (Baack et al., 2015; Lowry et al., 2008), while post-pollination barriers can act before or after the fusion of the gametes, i.e they can be either pre- or post-zygotic (Coyne and Orr 2004), and may respectively affect pollen grain germination and pollen tube growth or hybrid viability and fertility of hybrids. The individual types of reproductive isolation barriers often work together to hinder gene flow between related species (Scopece et al., 2013). However, out of pre-zygotic and post-zygotic, pre-pollination barriers have been documented to have the strongest effect in preventing gene flow between species (Lowry et al., 2008; Widmer et al., 2009).

Closely related flowering plant species can occupy different pollinator niches, which limits the opportunity for interspecific pollen transfer between them. This niche is defined by both the identity of the pollinators and the frequency at which the plant succeeds at attracting them, and by the morphological fit between the flower and the pollinator’s body, which may affect both pollen transfer efficiency and pollen placement (Barrios et al., 2016). There are several ways for plants to distinguish themselves when it comes to pollination niches. Plants may attract different pollinators using distinguishable floral characteristics, e.g. colour or scent (Grant, 1994) (i.e. ethological isolation). Alternatively, they can be visited by the same floral visitors but due to the morphology of the flowers, their pollen grains can be deposited on different parts of the pollinator’s body (i.e. mechanical isolation). Importantly, closely related plant species that share some of their floral visitors may in fact be specialised on different pollinators if these visitors differ in their respective efficiency. This efficiency depends on both the visitation rate of the pollinator and the extent to which this pollinator successfully manages to remove, carry and deposit pollen (Barrios et al., 2016). One study that investigated the interaction between three closely related Silene species and their pollinators found that although each plant species was visited by various pollinator species, the latter differed in their visitation rate and ability to remove and deposit pollen, indicating that the three plant species were in fact specialized towards distinct functional groups of pollinators (Reynolds et al., 2009).

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Even sister species can exhibit strong reproductive isolation (Scopece et al., 2013) and for some of them, pollinator-mediated reproductive isolation seems to have evolved during the early stages of speciation and to have promoted their differentiation, e.g. in Gymnadenia conopsea and G. odoratissima (Sun et al., 2015). Pollinator shifts are indeed believed to drive differentiation in floral traits (Lafon-Placette et al., 2016). This was first observed in specialized pollination systems (Van der Niet et al., 2014), but has also been observed in generalized pollination systems (Zhao and Huang, 2013). In one study, flowers of the generalist plant species Trollius ranunculoides were modified to investigate the relationship between plant-pollinator interactions and floral trait differentiation. In this study, two populations of Trollius ranunculoides were sampled, one high-altitude population and one low-altitude population. The two populations showed a differentiation in their main pollinators and floral traits. Where the high-altitude population was primarily visited by flies and produced smaller flowers with more petals whereas the low-altitude population was primarily visited by bees and produced larger flowers with less petals. By artificially removing petals in high altitude plants, the authors showed that the visitation rate of flies decreased significantly while that of bees was not affected, indicating that flies had a strong preference for flowers with more petals compared to bees (Zhao and Huang, 2013). This shows that different pollinators can differ in their ability to perceive attractive cues and in their preferences for those cues. Plants visited by distinct pollinators may experience different selective pressures and become differentiated (Chapurlat et al., 2015; Gervasi & Schiestl, 2016); however, by adapting to the locally most efficient pollinators, plants may become less attractive to (or morphologically compatible with) other pollinators. For this reason, floral divergence among conspecific populations may lead to reproductive isolation and speciation (Van der Niet et al., 2014).

For other species, pollination niche divergence seems to have evolved secondarily (Hopkins, 2013). In particular, populations that have been geographically isolated for a long time may have become incompatible (i.e. they may correspond to different species according to the biological species concept) but still rely on the same floral visitors for pollination. If these species come into secondary contact, they may suffer from stigma clogging, pollen loss or mating events resulting in low fitness hybrids. By altering traits such as flower morphology, colour or scent, selection may decrease the likelihood of pollinator sharing and interspecific pollen transfer and alleviate their costs, a process termed reinforcement (Hopkins, 2013). Selection for reproductive isolation only occurs in species that have the possibility to exchange pollen, i.e. species with overlapping distributions. In particular, it is believed that selection for reproductive isolation will be stronger in sympatric populations than in allopatric populations, which should be mirrored by a stronger differentiation between closely related species in sympatry than in allopatry (i.e. character displacement; Fig. 1; Hopkins and Rausher, 2012). The reproductive character displacement hypothesis states that selection acts on pre-mating (i.e. pre-pollination) barriers to limit the fitness costs of mating events between heterospecific individuals. Divergent selection on flowering time and/or specialization towards different pollinators may indeed reduce mating opportunities between heterospecific individuals (Hopkins, 2013). The hypothesis of ecological character displacement, on the other hand, states that rather than avoidance of gamete waste into low fitness hybrids, selection for reproductive isolation is a result of competition for resources. In sympatry, interspecific competition for pollinator visits may drive pollination niche partitioning and floral trait differentiation, since these species are likely to suffer from pollen limitation when relying on the same floral visitors for reproduction (Hopkins, 2013).

In this project, we will focus on two closely related orchid species, Gymnadenia conopsea and G. densiflora, which occur in both pure and mixed populations on the island of Öland, Sweden. These two

4 orchids are not sister species but are phenotypically very similar, although G. densiflora usually blooms a few weeks after G. conopsea, is taller and produces more flowers. Both species are fragrant, but they emit different scents, with variation between day and night emissions (i.e. G. conopsea emits more at night, while G. densiflora emits more during the day; Anderson, 2016).

The main objective of this project is to compare the extent to which plant-pollinator interactions differ between G. conopsea and G. densiflora, and between pure and mixed populations of these two species. More specifically, we will address the following questions: -Is G. conopsea mostly visited at night and G. densiflora visited both during the day and at night, as suggested by floral scent data? -Which are the most efficient pollinator species of G. conopsea and G. densiflora? Do they differ both during the day and at night, or do we observe pollinator sharing between these two species (during the day, at night, or both)? -Do we observe variation in pollination niches among conspecific populations, and is such variation consistent with a hypothesis of character displacement, i.e. decreased niche overlap between G. conopsea and G. densiflora in mixed populations compared to pure populations?

We will address these questions using data obtained from video recordings and pollinator catches.

Figure 1. Pollinator-mediated reproductive isolation between closely related species is thought to be stronger in sympatric sites where plants may be competing for pollinator visits and through interspecific pollen transfer than in allopatric populations (Joffard & Sletvold, unpublished)

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Material and methods

Study species

One of the most species-rich families of flowering plants is the orchids (). The genus Gymnadenia consists of rewarding species that have a generalized pollination system, i.e. they are visited by multiple pollinator species (Fernandez et al., 2019). Gymnadenia species are diverse both phenotypically, with variation in terms of phenology, floral sizes, shapes and scents, and genetically, with substantial genetic variation both between and within species (Travnicek et al., 2012; Valuiskikh and Teteryuk, 2014). In Sweden, there are five Gymnadenia species, namely G. conopsea, G. densiflora, G. odoratissima, G. nigra and G. runei, all with different morphologies and habitat preferences (Mossberg & Stenberg., 2010). Gymnadenia conopsea and G. densiflora were long considered to be the same species where G. densiflora was considered to be a late-flowering variant of G. conopsea (Gustafsson and Lonn, 2003). Nowadays, they are however treated as bona fide species, despite their striking morphological similarity, due to their phylogenetic distinctness (Chapurlat et al., 2020; Stark et al., 2011).

Gymnadenia conopsea (L.) is a terrestrial orchid that occurs in both temperate and boreal biomes in Europe and Asia (Meekers et al., 2012). The species usually occurs in cultivated landscapes, grazed meadows, open grasslands and areas adjacent to wetlands (Gustafsson, 2000). The highest abundance of G. conopsea can be found in alkaline, calcareous soils (Meekers et al., 2012).

Gymnadenia conopsea usually measures 10-40 cm and produces a single inflorescence containing 10- 100 fragrant flowers. Populations flower between early to late June (Sletvold et al., 2012). Flowers open successively from the bottom to the top and remain open for about a week (Chapurlat et al., 2015). Flowers can vary in color, most common is the pale and bright pink versions but white individuals can also be found (Chapurlat et al., 2015). Flowers emit a scent that can be described as spicy and vanilla (Jersakova et al., 2010) or -like (Gustafsson, 2000). Emission intensity changes between day and night, where the scents are mostly emitted at night (Meekers et al., 2012).

G. conopsea is self-compatible but to successfully generate fruits, it relies on pollinators (Chapurlat et al., 2015). The pollen grains of G. conopsea are bound together in a structure called a pollinium (Meekers et al., 2012) and each flower contains two pollinia. Pollinia are carried away by pollinators as a whole when they become attached on the pollinator’s body (Sletvold et al., 2012). The reward that the pollinators get from visiting the plants is the large amount of nectar contained within the floral spur (Gustafsson and Lonn, 2003).

Gymnadenia densiflora (Wahlenb.) has many similarities with its relative G. conopsea, is distributed across most of Europe and is especially abundant in the south-east parts. This species usually occurs in moist habitats, such as moist meadows and fens. In many places, the two species can be found growing together (Meekers et al., 2012: Mossberg & Stenberg., 2010).

Compared to G. conopsea, G. densiflora is taller and measures 30-70 cm (Mossberg & Stenberg., 2010). The plant produces a single inflorescence containing 10-100 flowers. Floral color varies in the same

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way as in G. conopsea (Gustafsson and Lonn, 2003). Gymnadenia densiflora flowers a few weeks after G. conopsea, i.e. in July, but the two species usually co-flower for one to two weeks. The scent of G. densiflora is described as being more pleasant than that of G. conopsea, with spicy and carnation-like notes (Jersakova et al., 2010). Moreover, G. densiflora emits more scents during the day than during the night (Joffard et al., in prep).

Gymnadenia conopsea and G. densiflora are visited by both diurnal and nocturnal pollinators. The diurnal pollinators mainly consist of diurnal Lepidopterans while the nocturnal pollinators consist of , hawkmoths and other nocturnal Lepidopterans. Preliminary data indicate that these two species of orchids differ in terms of pollination niches, yet some of their pollinators seems to be shared, e.g. Autographa gamma, Deilephila porcellus and Hyles gallii (Chapurlat et al., 2020).

Study populations

This study was conducted on the Swedish island of Öland. Öland is a 137 km long island located in the Baltic sea, near the east coast of Sweden. The Island has a calcareous soil due to a limestone bedrock. The island has a growing season that extends from mid-May to the end of October (Tveito et al. 2001).

In this study, a total of ten populations located at eight sites were used (Table 1). In three of the locations, pure populations of G. conopsea were sampled, in two of them pure G. densiflora populations were sampled and in the three remaining locations G. conopsea and G. densiflora grew together and were both sampled, except in one location (Hörninge) where G. densiflora could not be sampled as the population was found to be too small (<15 individuals).

Table 1. Populations sampled on the island of Öland during the summer of 2020. Video recordings were performed in all of them and pollinator catches were executed in eight of them (populations marked with an asterisk). Number Number Number of of diurnal of nocturnal Site type Species sampled plants pollinato pollinators Location Coordinates recorded rs caught caught Gråborg* 56.668797, 16.599276 Mixed G. conopsea 17 3 30

Hörninge 56.857492, 16.777293 Mixed G. conopsea 17 - -

Ismantorp* 56.746284, 16.642937 Mixed G. conopsea 50 2 13

Alböke* 56.951898, 16.776083 Pure G. conopsea 9 1 36

Sandbyborg 56.556459, 16.637859 Pure G. conopsea 16 - -

Segerstad* 56.371354, 16.561622 Pure G. conopsea 12 0 51

Ismantorp* 56.746284, 16.642937 Mixed G. densiflora 35 9 12

Gråborg* 56.668797, 16.599276 Mixed G. densiflora 56 21 42

Vanserum* 56.698447, 16.629280 Pure G. densiflora 29 22 11

Torpmossen* 56.514330, 16.564339 Pure G. densiflora 12 4 29

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Video recording

To characterize the pollinator communities in the populations, pollinator activity was recorded using four video cameras (Handycam ® FDR-AX33 & Handycam ® HDR-CX450, SONY) each equipped with a set of two SD cards and two batteries and each mounted to a tripod.

The video recordings were performed during a seven-week period during the summer of 2020, between June 8 and July 23. Populations of G. conopsea were recorded between June 8 and June 29 and populations of G. densiflora from July 3 to July 23. We had four video cameras at our disposal, two out of these had night vision (for recording night active pollinators) and an approximal recording time capacity of 6 hours, and two cameras had no night vision and an approximate recording time capacity of 12 hours. Generally, two cameras were used per population (so that pollinator activity could be recorded in several populations simultaneously), one with and one without night vision.

In eight of the populations, pollinator activity was recorded for four to five days and four to five nights distributed over one week during the flowering peak. For the remaining populations (the two populations located at Ismantorp), pollinator activity was recorded for four days and four nights distributed over two weeks during the flowering period (two days and two nights per week) to detect possible changes in pollinator activity during the flowering season. We started recording around 10-11 am during the day and around 20-21 pm during the night.

In each population, groups of 2-17 plants (or, in some cases, single plants) were marked and given an ID, the number of open flowers was recorded for each of these plants and a video camera was pointed at the group of plants to record pollinator visits. In total, 253 plants were recorded (min= 9, max=56), with an average of 25.3 plants per population. During the recording period, plant groups were switched at least one time and in some cases more if too many flowers had wilted on the plants.

After the flowering season, all videos were analysed and for each visit, the pollinator species involved, the duration of the visit and the number of flowers visited were recorded. In total 1245.25 hours of video recordings were analysed.

Pollinator catching

In eight of the populations, pollinator catches were performed to evaluate pollinator efficiency. Our initial goal was to catch ~20 individual insects per main pollinator species in each population, however the pollinator visitation rate was too low in Öland this year to achieve this and we ended up catching less (see table 1).

In the selected populations, pollinator catches were performed for three days and three nights, during the flowering peak, and catches started when we started recording (10-11 am for day catches and 20- 21 pm for night catches). The catches then went on for approximately 5 hours or till enough pollinators had been caught.

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To catch pollinators, two people wandered the study location looking for insects feeding or trying to feed from the flowers of the focal species. When a visit was observed, the insect was caught using a hand net and then killed by freezing.

When field work was over, the insects were brought back to the lab for further analyses. There, each pollinator was identified to the species level with help from the Station Linné staff and the book “Sveriges fjärilar” by Elmquist and Liljeberg (2011). For each pollinator, proboscis length was measured with a digital calliper. The insect was then put under a microscope and the number of Gymnadenia pollinia that were stuck to the pollinator was counted and the placement of these pollinia was recorded.

Statistical analysis

All statistical analyses were conducted in R version 4.0.3 using the packages lme4, dplyr, ggplot2 and car. Generalized linear mixed models (GLMM) were used to investigate variation in the day/night pollinator visitation patterns between G. conopsea and G. densiflora. Species (G. conopsea or G. densiflora), recording period (day or night) and their interaction were included as fixed effects, and population, plant ID and date as random effects; the response variable was the number of visits per hour that a single, marked plant received during a single recording event (normally distributed variable). The dataset was then divided in two so that the effect of population type (pure or mixed) could be investigated separately in each species with GLMMs including population type, recording period and their interaction as fixed effects and population, plant ID and date as random effects.

A linear model (LM) was then used to investigate how the proportion of day and night visits differed between G. conopsea and G. densiflora. Species, population type (pure or mixed) and their interaction were included as fixed effects, and the response variable was the proportion of night visits made on a single, marked plant over the course of the four days and nights of observation (normally distributed variable).

After having analysed the number and proportion of visits received by the two orchid species during the day versus during the night, we investigated the respective efficiency of these visits. To do so, we first analysed the time spent by a pollinator on an inflorescence during a single visit using a GLMM including type of pollinator (diurnal or nocturnal), orchid species and their interaction as fixed effects and pollinator species and plant ID as random effects; the response variable in this case was the duration of the visits (normally distributed variable). We further analysed the number of flowers probed during single visits (Poisson distributed variable) using a similar GLMM.

We then investigated variation in the number of pollinia carried by pollinators as estimated following pollinator catches (Poisson distributed variable). We first focused on variation between the two orchid species using data from nocturnal pollinators only as very few diurnal pollinators were caught on G. conopsea. A GLMM was applied including species on which the pollinator was caught as a fixed effect and pollinator species as a random effect. We then focused on variation between diurnal and nocturnal pollinators using data for G. densiflora only since the number of diurnal pollinators caught on G. conopsea was too low. A GLMM including type of pollinator (diurnal or nocturnal) as a fixed effect and pollinator species as a random factor was applied here.

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To investigate variation in the number of pollinia carried by the different pollinator species, a GLMM including pollinator species as a fixed effect and population as a random effect was used.

Finally, to compare the composition of the pollinator community among populations, a nMDS (non- metric multidimensional scaling) was performed. For this analysis, the number of visits made by each pollinator species in each population was used. The principle of a nMDS is that instead of using absolute values the data are ranked and put on a nMDS plot where points that are closer to each other correspond to sites that are more similar in terms of species composition. In this case, three dimensions were used to analyse the dataset, since this gave low a stress value which indicates a fairly good fit (Borcard et al., 2011).

Results

In total, 253 plants from ten populations were recorded (121 G. conopsea and 132 G. densiflora plants). From the video recordings, 845 visits, made by 482 individual pollinators, could be identified. The total number of visits observed in each population was 84.5 on average with the highest value observed for the G. densiflora population located in Gråborg (144 visits) and the lowest value observed for the G. conopsea population located in Alböke (26 visits). Plants received 0.22 visits per hour on average (max = 4.2, min = 0). The visitation rate was found to be higher during the night, with a mean of 0.42 visits/plant/h (max = 4.2, min = 0), compared to a mean of 0.04 visits/plant/h (max =0.58 , min =0) during the day.

During the pollinator catching, 286 insects (136 on G. conopsea and 150 on G. densiflora) belonging to 25 species (13 for G. conopsea and 16 for G. densiflora, with 6 species in common) were caught and in total (i.e. for both the video recordings and pollinator catching) 29 pollinator species were observed (16 for G. conopsea and 19 for G. densiflora, with 7 species in common). Only 39 % of the diurnal pollinators were found to carry pollinia, while 74 % of the nocturnal pollinators did.

Comparison between G. conopsea and G. densiflora

There was no difference in the plant visitation rate (i.e. number of visits received by one plant during a recording session divided by the number of hours of recording) between the two orchid species (P= 0.087). However, there was a significant difference between sampling periods, where the visitation rate during the night was higher than the visitation rate during the day (P= 2.2e-16) for both G. conopsea and G. densiflora (Figure 2). Gymnadenia conopsea tended to be more visited than G. densiflora during the night, with a mean of 0.5 visits/h for the former and 0.32 visits/h for the latter, whereas G. densiflora (mean=0.04 visits/h) had a higher visitation rate than G. conopsea (mean=0.03 visits/h) during the day.

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a) G. conopsea b) G. densiflora

Figure 2. Plant visitation rate (mean ± SD) for day and night recordings for a) G. conopsea (N = 121) and b) G. densiflora (N = 132).

When the number of visits received by plants during the day versus during the night was expressed in proportions, G. conopsea and G. densiflora were significantly differentiated (P=0.019). On average, G. conopsea plants received 86.9 % of their visits during the night, compared to 76.1% for G. densiflora plants (Fig. 3).

a) G. conopsea b) G. densiflora

Night visits Day visits Night visits Day visits

13% 24%

76% 87%

Figure 3. Mean proportion of day and night visits made on a) G. conopsea (N = 121) and b) G. densiflora (N = 253).

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We also observed a differentiation between G. conopsea and G. densiflora when it comes to the time spent on an inflorescence during a single visit (P=2.904e-03). Gymnadenia densiflora visits were longer with a mean duration of 24.48 seconds while G. conopsea visits lasted 11.52 seconds on average (Figure 4). Differences in duration between day and night was significant (P=2.2e-16) and visits tended to be longer during the day (mean = 28.89 seconds) than during the night (mean = 14.66 seconds). Moreover, there was a significant difference (P=1.862e-03) in the number of flowers probed during a single visit between G. conopsea and G. densiflora where visitors of G. conopsea fed from 3.91 and G. densiflora visitors from 4.12 flowers per visit on average, in line with the difference we observed in terms of time spent on the inflorescence. We also found that nocturnal pollinators captured on G. densiflora differed significantly from those captured on G. conopsea (P=1.363e-09) in terms of numbers of pollinia carried. As shown in figure 5, pollinators caught on G. densiflora carried 6.30 pollinia on average while those caught on G. conopsea carried 4.32 pollinia on average.

Figure 4. Time (measured in seconds; mean ± SD) spent on an inflorescence during a single visit in G. conopsea (N = 121) and G. densiflora (N = 253).

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Figure 5. Number of pollinia (mean ± SD) carried by nocturnal pollinators that were captured on G. conopsea (N = 136) and G. densiflora (N = 150).

Comparison between pure and mixed sites

To examine the difference between pure and mixed sites, populations of G. conopsea and G. densiflora were analyzed separately. When doing so, it was found that pure G. conopsea sites tended to have a higher visitation rate than mixed sites, with a mean of 2 visits/h for the pure sites compared to 0.75 visits/h for the mixed ones. This difference was however not significant (P=0.497). As for G. conopsea, pure and mixed G. densiflora sites did not differ significantly in terms of plant visitation rate (P=0.850). Pure and mixed populations did not differ in terms of proportion of night visits either, in both G. conopsea and G. densiflora (P=0.377).

Comparison between diurnal and nocturnal pollinators

Diurnal and nocturnal pollinators did not differ in the number of flowers probed per visit (P=0.409); they probed on average 4 flowers per visit (diurnal mean = 3.60, nocturnal mean = 4.05). In contrast, a significant difference was found in the number of pollinia carried by diurnal versus nocturnal pollinators (P=0.004), where the nocturnal pollinators carried more pollinia (6.30 on average) than the diurnal ones (1.03 on average).

There was also a significant difference in the number of pollinia carried by the different pollinator species (P=2.2e-16). Out of the nocturnal pollinators, the pollinator that carried the highest number of pollinia was Hyles gallii (14 pollinia) followed by Curculio (7.4 pollinia), Deilephila porcellus (5.6 pollinia), Aplocera plagiata (4 pollinia), and Autographa gamma (3 pollinia). Hada plebeja, Lacanobia w-latinum and Patania ruralis did not carry any pollinia.

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Pollinator community composition

Table 2 shows a compilation of all visits documented during the video recordings and based on these data and data from the pollinator catches, the pollinator species richness of every population was calculated. The result of this calculation showed that in general, populations of G. densiflora had a higher species richness than populations of G. conopsea. The populations with the highest species richness were G. densiflora populations growing in mixed sites (Ismantorp and Gråborg), whereas the population with the lowest species richness was a G. conopsea population growing in a pure site (Alböke). When focusing on Gråborg and Ismantorp, the two mixed sites where both G. conopsea and G. densiflora were sampled, G. conopsea was found to be visited by a lower number of pollinator species than G. densiflora.

It is worth mentioning that the population with the highest species richness (the G. densiflora population located in Gråborg) was also the population where the highest number of pollinators where caught, which could have introduced a bias. However, no correlation was observed between species richness and number of pollinators caught once this population was discarded.

Two of the identified pollinator species, Autographa gamma and Deilephila porcellus, were found in all ten populations. On average 83.2% and 78.2% of the visits were performed by these two pollinators for G. conopsea and G. densiflora, respectively. For the other pollinator species, seven species were observed visiting both G. conopsea and G. densiflora, that is, 43.7% of all G. conopsea pollinators and 36.8% of all G. densiflora pollinators.

Table 2. All visits observed during the video recordings. Pop Gråborg Alböke Hörninge Sandbyborg Segerstad Ismantorp Ismantorp Gråborg Vanserum Torpmossen G. G. G. G. conopsea conopsea densiflora densiflora Argynnis 0 0 0 0 0 0 7 3 0 0 paphia Autographa 12 9 17 53 39 27 41 34 49 30 gamma Bee 1 0 4 0 0 0 1 8 1 0 Bumblebee 0 0 0 0 0 0 0 1 3 0 Deilephila 31 14 46 70 49 15 35 55 10 41 porcellus Gonepteryx 1 0 1 0 0 0 1 0 4 2 rhamni Hemaris 7 0 0 0 0 0 0 0 0 0 tityus Hyles gallii 0 1 0 17 3 26 2 13 0 0 Noctua 0 2 0 3 0 0 0 15 0 0 pronuba Ochlodes 0 0 0 0 0 4 12 4 3 1 sylvanus pieris rapae 0 0 0 0 0 0 0 10 1 0 Syrphidae 0 0 0 0 1 0 1 0 0 0 zygaena 0 0 0 0 0 0 0 1 0 0 minos Unknown 1 0 0 5 0 0 0 4 0 2

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Figure 6 shows how similar the composition of the pollinator community is in the different populations which were sampled. The three pure G. conopsea populations, as well as the two mixed G. densiflora populations, seem to cluster together, indicating that these populations are quite similar in terms of species composition. One pure G. densiflora population – the one located in Torpmossen – was found to be more similar to the three mixed G. conopsea populations than to the other pure G. densiflora population (Vanserum). This is likely due to the fact that very few diurnal visits (i.e. 3 visits made by two species, Gonepteryx rhamni and Ochlodes sylvanus) were observed in Torpmossen, as typically observed for G. conopsea populations.

One of the three mixed G. conopsea populations - namely Ismantorp - was found to be quite distant from the two other populations of the same type. This is probably due to the fact that it lacks two pollinators (Bee and Gonepteryx rhamni) that were present in both Gråborg and Hörninge while two others (Hyles gallii and Ochlodes sylvanus) were not observed in these two sites but were found in Ismantorp.

Figure 6. nMDS plot showing how similar the ten sampled sites were in terms of pollinator community composition.

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Discussion

The present study set out to investigate how plant-pollinator interactions differ between two closely related orchid species of the genus Gymnadenia. In addition, we wanted to know if G. conopsea and G. densiflora has a greater differentiation in sympatric populations compared to allopatric populations based on the hypothesis of ecological and/or reproductive character displacement.

Both species received a comparable number of visits overall. Looking at the ratio of visits made by nocturnal versus diurnal pollinators, G. conopsea had a larger proportion of night active pollinators compared to G. densiflora. In turn, G. densiflora had a larger proportion of day active pollinators compared to G. conopsea. The majority of visits, however, occurred during the night for both orchid species. Autographa gamma and Deilephila porcellus were found to be the main pollinators species in all ten populations.

Visitation pattern in relation to scent data

Anderson (2016) found that G. conopsea and G. densiflora do not differ in terms of total scent emission rate when both day and night floral emissions are considered, but that G. densiflora has a higher scent emission rate at daytime while G. conopsea emits more scents during the night. As floral scents play a central role in the attraction of pollinators (Raguso, 2008: Proffit et al., 2020; Wang et al., 2020), this should have an impact on the pollinator visitation pattern. The scent emission patterns could be a result of pollinator-mediated selection, i.e. plants may have adapted so that their scent emission rate match the activity of their pollinators. Pollinator-mediated selection has been detected for various traits in plants (Benitez-Vieyra et al., 2006; Sletvold and Ågren, 2010), e.g floral color and scents (Gross et al., 2016), and has been shown to result in a close match between plant traits on the one hand and pollinator behavior on the other hand. In this study, we therefore expected no differentiation in total visitation rate between the two orchid species, but we predicted that the majority of visits would occur daytime for G. densiflora and nighttime for G. conopsea. What we found however was that the peak of pollinator activity occurred during the night for both species. The scent emission and pollinator visitation patterns were somewhat consistent in that G. densiflora plants received a higher proportion of visits daytime compared to G. conopsea plants, but the mismatch between absolute amounts of scents emitted and number of visits received during daytime and nighttime in G. densiflora is difficult to explain. It should be pointed out that the composition of pollination niches can vary a lot between consecutive years, even when abiotic conditions are relatively stable (Dupont et al., 2009). This could partly explain the results obtained in this study. Preliminary data suggested that G. densiflora was more visited daytime than nighttime in previous years, in line with the scent emission pattern. This contradicts our findings and indicates that to get an accurate picture of pollination niches, the same sites should be sampled repeatedly over several years. This also indicate that G. densiflora is not as specialized as G. conopsea, as it is able to attract both day and night active pollinators depending on their relative abundance. Whether this gives a competitive advantage to G. densiflora when pollinator activity is higher and diurnal pollinators more abundant than observed in 2020 remains to be investigated.

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The pollinators

Generally, G. densiflora populations were visited by a higher number of pollinator species than G. conopsea populations. This was particularly true for the diurnal pollinators, with G. densiflora being visited by 13 diurnal pollinator species compared to the seven that were observed visiting G. conopsea. Although the two orchid species differed in terms of pollinator species richness and were visited by different insect species, pollinator sharing occurred between them (i.e. they had different, but partially overlapping pollination niches). In total, 29 species of pollinating insects were observed and out of these, seven species were shared by G. conopsea and G. densiflora. In particular, the nocturnal pollinators Autographa gamma and Deilephila porcellus were found to be present, and to be the most frequent visitors, in all ten populations regardless of the orchid species considered.

The two orchid species were found to be mostly visited at night, but does it necessarily imply that nocturnal pollinators are the most efficient pollinators in both of them? If we look at the number of pollinia carried by diurnal versus nocturnal pollinators, there is a significant difference between the two. Diurnal pollinators carried 1.03 pollinia on average compared to the 6.3 pollinia for nocturnal pollinators. This result could be explained by the lower visitation rate of diurnal visitors, but also by a lower efficiency at removing pollinia during single visits. Daytime, the main visitors of G. densiflora were found to be diverse species of lepidopterans and according to Barrios et al., (2016), diurnal lepidopterans often act as thieves when visiting nectar rewarding plants as they often consume nectar without removing much pollen nor deposit large pollen loads on the stigmas. Our data regarding pollen loads therefore do not contradict our data regarding visitation rate and confirm that both orchid species primarily rely on nocturnal visitors for pollination. Note that we initially planned to perform single visit experiments (where pollinia removal and deposition are estimated after single visits monitored on otherwise caged plants) to get a more accurate estimation of the efficiency of the different pollinator species. However, the pollinator activity was lower than expected during summer 2020 and we could not perform this experiment. We recommend that future studies include such an experiment to assess whether nocturnal visitors do not only visit more plants (and/or more flowers per plants), but also remove and deposit more pollen when visiting them.

Interestingly, the number of pollinia carried by nocturnal pollinators was significantly different between the two orchid species. As mentioned earlier, there was no difference in the plant visitation rate between G. conopsea and G. densiflora, but we could detect a difference in the duration of single visits and in the number of flowers probed during these visits on video recordings. Pollinators observed feeding on G. densiflora spent more time on the inflorescence and probed a higher number of flowers per visit compared to those observed feeding on G. conopsea. This may explain why the pollinators caught on G. densiflora carried more pollinia that those captured on G. conopsea. Studies have shown that pollinators tend to be more attracted to plants with higher floral densities (Kunin, 1997). On average, G. densiflora produces a higher number of flowers than G. conopsea yet it did not seem to be more attractive than G. conopsea (i.e. no difference in the plant visitation rate). However, maybe because it offers more resources to pollinating insects, the latter tended to probe more flowers per visit than when feeding on G. conopsea. This might affect the rate at which geitonogamy (i.e. pollination among neighboring flowers on the same plant) occurs so that geitonogamy might be more common in G. densiflora than in G. conopsea. Since G. densiflora is self-compatible (Chapurlat et al., 2020) this

17 might not affect seed set but it may have longer term consequences as geitonogamy can cause inbreeding depression (Johnson and Nilsson, 1999).

Niche differentiation

The fact that the differentiation in terms of pollination niches between G. conopsea and G. densiflora was small is further supported by the study made by Jersakova et al. (2010), who found that pre- pollination barriers would be too weak to prevent gene flow between the two orchid species. However, this does not mean that G. conopsea and G. densiflora are free to hybridize. As mentioned in the introduction, according to Scopece et al. (2013), pre- and post-pollination barriers often work together to prevent gene flow between species, and even though G. conopsea and G. densiflora share some of their pollinator species (including the main ones), they do not flower exactly at the same time and Chapurlat et al. (2020) found divergent selection on flowering start when comparing both species. This, together with the small differences in pollination niches may work synergistically to prevent interspecific pollen transfer. Furthermore, even though pre-zygotic barriers are suggested to be the most efficient at hindering gene flow between flowering plant species (Lowry et al., 2008; Widmer et al., 2009), this may not be the case in this particular system. Widmer et al. (2009) found that in another group of closely related orchid species, post-zygotic barriers were the most efficient at hindering gene flow across species boundaries. Hence it would be interesting to explore the post-zygotic barriers putatively limiting hybridization between G. conopsea and G. densiflora in future studies. Note that we performed some reciprocal crosses during summer 2020 and that interspecific crosses produced as many fruits as intraspecific ones; however, seed viability and germination ability remain to be assessed and compared between “pure” and hybrid seeds.

Even in the case where two species are isolated by post-zygotic barriers, we expect selection against gamete waste into unfit hybrids to promote temporal and/or floral isolation in sympatry (i.e. reinforcement). One example of an increased divergence in sympatric populations when it comes to temporal isolation is that of two species of mountain roses Metrosideros nervulosa and Metrosideros sclerocarpa, which are sister species and occur in both allopatric and sympatric populations. When examining flowering time among these species the results showed that the median flowering time for M. sclerocarpa was 30 days later in sites where the two species grew together compared to sites where they grew isolated from each other (Osborne et al., 2020). Results from a previous study on our two orchid species indicate that there was no difference in flowering time between sympatric and allopatric populations of G. conopsea and G. densiflora (i.e. no earlier flowering for G. conopsea and/or later flowering for G. densiflora in mixed compared to pure populations; Joffard et al., in prep.). We therefore predicted that there would be a smaller pollination niche overlap between G. conopsea and G. densiflora in mixed sites, in order to avoid competition for pollinator service and limit interspecific pollen transfer. However, we found little evidence for this as when comparing sympatric and allopatric populations of the same species there was no clear difference in terms of visitation rate, reliance on diurnal or nocturnal pollinators nor in terms of pollinator community composition. This might indicate that the overlap in flowering time between the two species is too small for competition for pollinator services to be strong, and that if interspecific pollen transfer occurs during this time window, it does not have severe effects on plant reproductive success in mixed sites.

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Conclusions

Plant-pollinator interactions are an important component of species’ ecological niches. Pollinators can be seen as a resource for which plants can compete, and they can mediate interspecific pollen transfer with potential fitness costs. In this context, pollination niche partitioning and floral trait differentiation can promote coexistence between closely related species. In this study, we compared pollination niches of two orchid species, G. conopsea and G. densiflora, which occur in both pure and mixed sites in Öland. Despite the major difference in floral scents – especially in the relative importance of day versus night emissions – previously evidenced between the two species, we found that pollination niche differentiation was small, as both species relied on the same main pollinators, the night-active Autographa gamma and Deilephila porcellus, although G. densiflora was also visited by various day- active pollinators. We further found that the overlap in pollination niches between the two species was not smaller in mixed than in pure populations, thereby challenging the hypothesis of character displacement in this system. Further studies are needed to assess whether other reproductive barriers prevent gene flow between G. conopsea and G. densiflora in sympatry and to quantify competition between these two species and assess whether it may drive competitive exclusion or character displacement in the future.

Acknowledgments

This work would not have been possible without my supervisors Nina Sletvold and Nina Joffard, who have guided me and helped me through the whole process of this study. I would also like to thank Maria Uscka-Perzanowska, whom I worked with during the field part of the study and the staff at Station Linné. Lastly, I would like to thank Marina Åberg, who will not let me quit things just because they are hard.

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