Visitation by putative pollinators in relation to gentian traits

Gentiana asclepiadea, like many other Gentianaceae, have colorful flowers that are attractive to bumblebees, with a size and shape compatible with bumblebee bodies (Burton, 1878;

Kozuharova, 1994; Nowotny, 2010). In our study, bumblebees were indeed the most frequent visitors of G. asclepiadea (Fig. 5.6). As they collected both pollen and nectar, this strongly

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suggests that bumblebees are important for pollination. Among the other relatively frequent visitors, only syrphid flies and other flies showed behavior that could result in pollination (i.e.

collecting nectar and/or pollen). Therefore, bumblebees, syrphid flies and other flies are the most likely pollinators that act as selective agents operating on flower traits.

Bumblebees, syrphid flies and other flies differed in their preferences for flowers and gentian traits. Bumblebees preferentially visited flowers with intermediate corolla width (Fig. 5.5). This may be due to the body size of bumblebees acting as a constraint for flower visitation. Bumblebees may have more difficulty entering narrower flowers and reaching the nectaries at the pistil base, and thus developed a preference for larger flowers. Alternatively, larger flowers may be simply easier to detect by bumblebees (Spaethe et al., 2001). Flowers with wider corollas, however, also attracted more syrphid flies, which are smaller than bumblebees, possibly because wider flowers constitute larger landing platforms. Additionally, flowers with shorter corolla lengths attracted more syrphid flies and other flies. This could be related to the comparatively shorter mouthparts of flies compared toe.g. bumblebees (Krenn et al., 2005), so that collecting nectar from flowers with shorter corollas is easier. Gentian height was important for both syrphid and other fly visitation, but in a different way. Short gentians attracted more syrphid flies, but fewer other flies than taller gentians. We do not have an explanation for this. Nevertheless, the relation between visitation frequencies and (mainly floral) gentian traits provides a first indication that pollinators may exert directional selection on gentian flower morphology.

5.5.2 Oviposition on Gentiana asclepiadea

Previous studies on theGentiana-Phengaris-Myrmicainteraction tested the effects of gentian traits and ants on Phengaris oviposition separately. In our study, we showed that gentian traits, ant-host abundance and identity, and the surrounding vegetation influenced P. alcon oviposition. Plants with more branches in areas with more Poaceae relative to Juncaceae and in areas with shorter surrounding vegetation received relatively more eggs. Oviposition also increased with decreasing Gentiana density. These results suggest thatP. alcon prefers laying eggs on plant-hosts that are exposed, and therefore can be easily found in the field.

Additionally, gentians in higher densities received relative fewer eggs, probably because Phengaris preferred to spread their eggs more equally among plant-hosts, instead of on only few. Overall, our results are in accord with the ones found by Nowicki et al. (2005a), Fürst and Nash (2010) and Wynhoff et al. (2015), but there are also differences with other studies.

For example, we did not find that more flowers per plant increased oviposition as reported for P. rebeli on G. cruciata (Kéry et al., 2001) and forP. alcon on G. pneumonanthe (Küer and Fartmann, 2004; Wynhoff et al., 2015). Nowicki et al. (2005b) also did not find that gentian density explained P. alcon oviposition in a polish population of G. pneumonanthe. Therefore,

although Phengaris oviposition is in general determined by plant-hosts traits, it may have particular differences between sites and species combinations in the Gentiana-Phengaris interaction range.

As expected, P. alcon oviposition was positively explained by ant number, particularly in regard toM. scabrinodis. Although all of the threeMyrmica species found in our study (M.

rubra,M. ruginodis andM. scabrinodis) are possible ant-hosts forP. alcon(Pech et al., 2007), Phengaris is usually specialized in one single Myrmica species as its main host in a specific area, whereas other Myrmicaspecies may be only rarely exploited (Thomas et al., 1989). We argue that M. scabrinodis is the P. alcon ant-host in the study area, similar to other reports for southern Germany (Küer and Fartmann, 2004), Spain, France, Southern Netherlands (Elmes et al., 1994), Czech Republic (Pech and Sedláček, 2016), Poland (Sielezniew and Stankiewicz, 2004) and Ukraine (Witek et al., 2008), but not for Northern Europe (Elmes et al., 1994; Als et al., 2002) or Portugal (Wynhoff et al., 2015). This supports the idea of local specificity in the Gentiana-Phengaris-Myrmica system. Other studies also suggested that ant-host presence around plant-hosts positively explains Phengaris oviposition (van Dyck et al., 2000; Nowicki et al., 2005a, but see Fürst and Nash, 2010). The link between Phengaris oviposition and ant presence, however, may have different explanations. For example, Phengaris may oviposit close to Myrmica because they may directly recognize Myrmica nests or workers around gentians. Alternatively,Phengaris may prefer to oviposit on the same plants thatMyrmica use for resources or choose for building their nests. Therefore, Phengaris oviposition may be the result of different direct and indirect ecological effects.

One factor that might influence Phengaris oviposition is the spatial environment.

Previous studies reported similar mean P. alcon egg number per plant (e.g. 9.6 + 9.2 in Thomas et al., 1991; 4.3 + 3.3in Willers, 2016). However, our relatively variable number of Phengaris eggs per plants (ranging from0to117, with a mean of10.2 + 20.6) was associated with plants closer to the forest edge and in more elevated drier areas (pers. observ.), which are potentially preferred by Myrmica for building their nests. Hence, we have indications that P. alcon oviposition is also spatially correlated. Another explanation for Phengaris oviposition might be related to thePhengaris flight behavior. Phengaris butterflies fly short distances (Nowicki et al., 2005b), therefore, butterflies in our study area may have laid relative larger egg clusters on fewer plants because they simply were born in those patches where the focal plants were scarce, and consequently did not have any other alternative for oviposition. A further explanation that could be related to the short flight behavior, and thus oviposition, ofPhengaris is that butterflies may actively prefer to oviposit where they emerge (active philopatry; Resetarits, 1996). Therefore, future studies should account for the spatial environment effect on Phengaris oviposition to bring new insights into the understanding of theGentiana-Phengaris-Myrmica system. Moreover, studies that investigate Phengaris chemical and visual preferences for example could contribute on disentangling the importance

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of Myrmica, Gentiana and environment on oviposition choices.

5.5.3 Antagonistic selection by herbivores and pollinators on