Attractiveness of Ornamental Flowers for Pollinating Insects in an Urban Area

Attractiveness of ornamental flowers for pollinating insects in an urban area

Master thesis

Written by

Lea Annina Kretschmer Matr.-no.: 529248

University of Hohenheim

2017

First auditor: Priv. Doz. Dr. rer. nat. Rosenkranz, Peter Second auditor: Prof. Dr. Dieterich, Martin Excecutive summary 2

Excecutive summary

This master thesis is focusing on the potential of ornamental flowers as food source for pollinating insects in urban areas. Many insects are functioning as pollinators and thereby collecting pollen and nectar as food source from the flowers they visit. Objectives were to identify attractive ornamental flowers as well as their visiting insects. Methods for the flower evaluation should be tested and moreover the current literature should be reviewed. More than one hundred articles were reviewed to get an overview of the current knowledge on pollination. The focus was laid on botanical as well as entomological aspects. Studies on the botanical point of view were often focusing on different floral traits as for example : scent, floral morphology, pollen and nectar. Concerning the pollinators many studies have been conducted on Apis mellifera and their pollination performance. Highly controversial terms like pollination syndrome and-crisis were explained. Relating to pollinators in urban areas it was shown that this topic is gaining more importance. Nevertheless only few articles were found studying the topic of insect friendly ornamental flowers. The field trial of this study was conducted at the research area of the Lehr- und Versuch- sanstalt für Gartenbau (LVG) in Heidelberg from end of June till beginning of September in 2016. 20 varieties of Calibrachoa - a popular bed and balcony cultivar were compared in regards to the number of insect visits. Two replicates were studied at two different locations. Observations per variety were recorded for one minute with a total of 18 observation days. The visiting insects were classified into Apis mellifera, Bombus sp., wild bees, Syr- phidae sp. and others. Calibrachoa visiting rate was relatively low compared to other ornamental flowers. Nevertheless, additional insect sampling identified ten different species visiting the Calibrachoas. Moreover pollen of the sampled insects was analyzed and resulted in relative high accordance with the pollen of Calibrachoa. This indicates that Cali- brachoa can be an additional food source of several species from the Apiodea superfamily. Apart from this more than 120 different ornamental flower varieties have been observed with regards to their insect visiting rate. Main plant species were Bidens ferulifolia, Dahlia, Euphorbia hypericifolia, Salvia and Scaevola as well as other popular bed and balcony cultivars. Insect visitation rate differed greatly between species, as well as within the species. Number of visitors ranged from zero to 27 insects per minute per species. Moreover the composition of visiting insects differed highly between the species. Euphorbia hypericifolia had the highest insect diversity with 15 identified species whereas Scaevola was mainly visited by Apis mellifera. To evaluate the observation method and its validity a ‘whole day every hour observation’ was conducted at ten days in August. Therefore every hour an observation of one minute and an additional observation of two minutes were performed. The overall three minute Excecutive summary 3 observation was then compared with the one minute observation. Concluding that a one minute observation turn is long enough for flowers with high visitation rates whereas flowers with a lower visitation rate might need a longer observation turn. In total 106 Insects were sampled which resulted in 35 species belonging to 19 genera. The sampling was conducted subjective to catch those species that could not be identified on the field. Taking into account the few selective sampling dates, a relatively high number of different species was found. Out of the sampled insects, 75 individuals were taken to analyse their pollen thus providing information on their foraging source. Pollen was compared with the plant source on which the insects were sampled. A high number of samples corresponded to the flower source on which the insects were sampled. As a summary, the field trial indicates that ornamental flowers can function as an additional food source for insects in urban areas. Due to high variations of ornamental flowers within the species, recommendations can only be provided on the variety level. Neverthe- less more studies are needed to build up a broader base of information concerning ornamental flowers and their benefits for insects.

Abstract 4

Abstract

This study conducted a field trial to investigate the attractiveness of ornamental flowers as food source for pollination insects in urban areas. The decline in insects’ diversity can be mainly attributed to ongoing land use change, which is also influenced by ongoing urbanization. Less is known about food demand and supply of pollination insects in urban areas, whereas the topic of insect friendly parks and gardens is gaining more interest. Ornamental flowers are strongly represented in urban areas; nevertheless, knowledge of their benefits for insects is limited. This field trial evaluated more than 140 popular bed and balcony varieties and counted visiting insects. The main species of the bed and balcony varieties were Bidens ferulifolia, Calibrachoa, Dahlia, Euphorbia hypericifolia, Salvia and Scaevola. Real time snap shot observations of one minute were used to grade the flowers in terms of their insect visitation rates. Visiting insects were classified into Apis mellifera, Bombus sp., wild bees, Syrphidae sp. and others. The number of visiting insects as well as their composition differed greatly between species. Moreover, high variation within species occurred from varieties having zero visitors per minute compared to those having more than 25 visitors per minute. Selective insect sampling was conducted to identify present wild bees and other species. Overall 106 caught insects represented 35 species. Out of the 106 insects, 75 were used for further pollen analysis. The pollen analysis compared pollen carried by the insect with the flower source on which insects were sampled. A large number of the pollen samples corresponded to the plant source. The observation protocols and additional pollen analysis show that insects can use ornamental flowers as an additional food source. Never- theless, the strong variations in between varieties can only justify recommendations on the variety level. Moreover, further studies are needed to investigate the characteristics of ornamental flowers in different urban environments.

Table of contents 5

Table of contents

Excecutive summary ...... 2

Abstract ...... 4

Table of contents ...... 5

List of figures ...... 7

List of tables ...... 9

List of appendix ...... 9

1 Introduction ...... 10 1.1 Objectives ...... 11

2 Literature review ...... 12 2.1 Pollination ...... 12 2.1.1 Pollination – a botanical point of view ...... 12 2.1.2 Pollination – from the pollinators point of view ...... 16 2.1.3 Pollination crisis – decline of pollinators ...... 18 2.2 Pollination insects in urban areas ...... 20

3 Material and Methods ...... 26 3.1 Study area ...... 26 3.2 Ornamental flowers ...... 26 3.2.1 Calibrachoa ...... 29 3.3 Ornamental plant monitoring ...... 29 3.3.1 One minute observation ...... 29 3.3.2 Whole day every hour observation ...... 30 3.4 Insect sampling ...... 30 3.4.1 Sampling ...... 30 3.4.2 Preparation and identification ...... 31 3.5 Pollen sampling ...... 31 3.5.1 Ornamental flowers – stamen ...... 31 3.5.2 Pollination insects – pollen load ...... 31 3.5.3 Pollen analysis ...... 32

4 Results ...... 33 4.1 Ornamental plant monitoring ...... 33 4.1.1 Calibrachoa variety testing ...... 33 Table of contents 6

4.1.2 One minute observation ...... 35 4.1.3 Whole day every hour observation ...... 46 4.2 Insect sampling ...... 50 4.3 Pollen sampling ...... 51

5 Discussion ...... 53

6 Conclusion and outlook ...... 58

Appendix ...... 59

List of references ...... 63

Acknowledgement ...... 71

Declaration ...... 72 List of figures 7

List of figures Figure 1: Ternary diagram by Abrahamczyk et al. (2016) showing the different pollinators and their preferences of the Nectar sugar composition in a sucrose-, fructose- and glucose triangle ...... 15 Figure 2: The five species for the whole day observation. From left to right: 1. Euphorbia hypericifolia, 2. Scaevola, 3. Salvia farinacea, 4. Bidens ferulifolia, 5. Dahlia ...... 30 Figure 3: Bombus sp. collecting nectar from Calibrachoa. Left hand: Bombus pascuorum collecting nectar in an open flower. Middle: Bombus pascuorum collecting nectar in a filled flower. Right side: Bombus terrestris nectar robbing from outside of the flower...... 34 Figure 4: Observation protocols with the belonging observation dates of the Bidens species with the two varieties ‘golden star’ which is having yellow coloured flowers and the variety ‘Beedance Painted Red’ with red flowers. Each bar per observation day is representing one minute of observation...... 35 Figure 5: ‚Pirate’s Princess‘and ‘Pirates Pearl White’ with their observed visiting pollinators. Each bar per observation day is representing one minute of observation...... 36 Figure 6: Two varieties of the species Brachyscome iberidifolia and their counted pollinators with the belonging observation dates. Each bar per observation day is representing one minute of observation...... 36 Figure 7: Two open flower shaped Dahlia species and their visiting pollinators with the belonging observation dates. Each bar per observation day is representing one minute of observation...... 37 Figure 8 two species of Dahlia, ‘Dalaya Yogi’ with half filled flowers and less anthers and ‘Bicolor White/Red’ with almost filled flowers and their visiting pollinators. Each bar per observation day is representing one minute of observation...... 38 Figure 9: Observation protocols of the variety ‘Igloo’ and ‘Glamour’ with the belonging observation dates of the Euphorbia hypericifolia varieties...... 39 Figure 10: Observation protocols of the variety ‘Diamont Frost’ and ‘Starpleasure’ ..... 39 Figure 11: Lantana varieties ‘Sun Fun Yellow‘and ‘Sun Fun Red’ with their observed pollinator protocols. Each bar per observation day is representing one minute of observation...... 40 Figure 12: Mercadonia varieties ‚Golddust‘and ’Garden Frechles’ with their present pollinators with the belonging observation dates. Each bar per observation day is representing one minute of observation...... 40 Figure 13: Observation protocols of the two Salvia farinacae varieties ‘Sallyfun Snowwhite’ and ‘Sallyfun Bicolor Blue’ with the belonging observation dates. Each bar per observation day is representing one minute of observation...... 41 Figure 14: Variety ‘Love and Wishes‘and ‘Black and Bloom’ and their counted pollinators. Each bar per observation day is representing one minute of observation...... 42 Figure 15: ‚GOGO Coral‘and ‘GOGOScarlet‘with their observed pollinators. Each bar per observation day is representing one minute of observation...... 42 List of figures 8

Figure 16: Observed pollinators of ‘Grandstand Salmon’ and ‘Grandstand Blue Bicolor’. Each bar per observation day is representing one minute of observation...... 43 Figure 17: Observation protocol of the two Scaevola varieties ‘Fancy Blush’ and ‘Fancy white’ with the belonging observation dates. Each bar per observation day is representing one minute of observation...... 43 Figure 18: Observation protocol of the two Scaevola varieties ‘Fancy Exp Pink’ and ‘Exp. Bicolor Purple-White’. Each bar per observation day is representing one minute of observation...... 44 Figure 19: Days of pollinator observation for the varieties ‘Snowbird’ and ‘Chers Yellow’. Each bar per observation day is representing one minute of observation...... 45 Figure 20: Variety ‘Copper Apricot’ with its visited pollinators. Each bar per observation day is representing one minute of observation...... 45 Figure 21: Whole day observation of the species Euphorbia hypericifolia; represented by the variety ‘Glamour’. Each bar is representing the observed pollinator composition of either one or three minutes of observation...... 46 Figure 22: Whole day observation of the species Salvia farinacea; represented by the variety ‘Midnight Candle’. Each bar is representing the observed pollinator composition of either one or three minutes of observation...... 47 Figure 23: Whole day observation of the species Scaevola aemula; represented by the variety ‘Exp. Bicolor Purple White’. Each bar is representing the observed pollinator composition of either one or three minutes of observation...... 48 Figure 24: Whole day observation of the species Bidens ferulifolia; represented by the variety ‘Pirate's Pearl White’. Each bar is representing the observed pollinator composition of either one or three minutes of observation...... 49 Figure 25: Whole day observation of the genus Dahlia; represented by the variety ‘Dahlegria White’. Each bar is representing the observed pollinator composition of either one or three minutes of observation...... 50

List of tables 9

List of tables Table 1: Six main observed ornamental flower species with their origin and a short description based on Altmann (2008) and SanMarcosGrower (2016)...... 28 Table 2: Number of flower visiting insects of the 18observation days in the observed Calibrachoa varieties...... 33 Table 3 the ten days of the whole day observations with the belonging dates ...... 46 Table 4: Genus and species of the sampled insects. Insects were sampled between 25.07.2016 and 30.08.2016 and caught individually from ornamental flowers. . 51 Table 5: Pollen analysis from insects sampled while visiting the Calibrachoa flower. The results are given in a qualitative form. Samples were high amount of Calibracoa pollen was visable lacking other pollen was expressed with (99,9%)52

List of appendix Appendix 1: Caught and identified insects with their genus, species, sex and the date and place of collection...... 59 Appendix 2: The location flower on which the insects were caught compared to the botanical pollen groups which were found on the microscope slide...... 61

1 Introduction 10

1 Introduction

Urbanization and its ecological consequences is not a new topic anymore nevertheless the dimension of subjects which are discussed became much more complex and multifaceted. Pollination is one of the ecosystem services influenced by land use changes which is highly controversially debated. Steffan-Dewenter et al. (2008) synthesised 23 studies focusing on the topic of pollination. They came to the conclusion that land use change, especially the increase of distance between pollination insects and their natural habitat had significant effects on the decline of the pollinators in the study areas. Pollination services can even decline in areas with rich biodiversity if distances which have to be flown by the pollinators are to huge as Carvalheiro et al. (2010) could show in his field research about different aspects of Mangifera indica L. pollination. Both, Carvalheiro et al. (2010) and Steffan- Dewenter et al. (2008) proposed the preservation and support of those natural habitats. Nevertheless most of those pollination studies were performed either in rural areas or in agricultural managed fields. Little is known about the consequences for pollinators in urban areas. Therefore more often the question arises which barriers insects are facing in urban areas and whether an urban area can be managed in a pollinator’s friendly way. Bates et al. (2011) showed that the numbers of pollinators in cities like Birmingham UK decreased on a gradient from rural to urban area; most bees and hoverflies were negative affected by a higher urbanization and only few could benefit from it. Urban areas are mainly man made and due to this many alien ornamental species can be found in public areas as well as in private gardens or balconies. Only few studies were focusing on the question if insects can benefit from those ornamental flowers. Nevertheless, public as well as the ornamental flower market are sensitized on the idea of pollinator friendly flowers. Thru whole Europe NGO’s and governmental organisations are focusing on the question of how pollinators can be supported in the future. The SUPER-B Action is a European cooperation in science and technology which tries to develop conservation strategies for pollinators based on scientific evidence combined with public participation. In total 30 countries are participating in the SUPER-B Action including Germany as a member (Biesmeijer 2016). However, wide disparity between the different memberships and their national activities are found. Countries like Ireland or the Unit- ed Kingdom can present national pollination plants whereas Germany is lacking this kind of national action. Besides the German governmental actions many nongovernmental activities are taking place like the Netzwerk Blühende Landschaften or smaller regional banded projects like Bienenretter and Bunte Wiese (Mellifera e.V. 2016; FINE e.V. 2016; Bunte-Wiese 2016). 1 Introduction 11

Nevertheless the disparity to other countries as well as the knowledge gap concerning pollinators in urban areas cannot be ignored. A more detailed focus should be set to the urban flower pollinator network, especially to the ornamental flowers which are common in urban and suburban areas. This Master thesis focuses on common ornamental flowers which can be found on the German bed and balcony market. Flowers are evaluated on their attractiveness for pollinators to value their potential concerning pollination benefits in urban areas.

1.1 Objectives

This study aims to value the attractiveness of ornamental flowers for pollinators which further on should help to understand the role of ornamental flowers in urban areas. In total five main objectives can be listed. Review the current literature regarding the topic of insect friendly ornamental flowers. Pointing out suitable methods in the assessment of insect potential of ornamental flowers. Identifying attractive ornamental plants for insect pollinators and showing the regional pollinator spectrum for the identified varieties. Comparing different varieties within the species Calibrachoa to identify attractive flower traits for pollinators Catching pollinators and analysing their collected pollen grains to gather information about the visited flowers. 2 Literature review 12

2 Literature review

This literature review should serve as an introduction to the complex field of pollination and further on gives an overview of the current knowledge regarding urban pollinators and their preferences on ornamental flowers.

2.1 Pollination

Pollination is the sexual reproduction of plants in which the pollen grains, the male parts of the plants, are transferred to the stigma which is the female part of the flower. Depending on the plant different styles of pollination exist. In general two main types occur namely the self- and the cross pollination (Dharam 2012). Defined by Faegri & van Der Pijl (1979) the dispersal of the pollen can either be abiotic by which physical transport agents such as water or wind play a role or biotic transport by which animals are transferring the pollen grains. Biotic pollination, also called zoophily, can be split in pollination by vertebrates and invertebrates; whereby this study is focusing on the pollination of invertebrates. Inver- tebrate pollinators are beetles, flies, wasps, bees, ants, butterflies and moths (Dharam 2012). Ollerton et al. (2009) published their research “A global test of the pollination syndrome hypothesis” to question the pollination syndrome which was developed in the 1870s and is almost consensually accepted by scientists. The pollination syndrome describes the adaptation between angiosperms and their pollinators. Therefore mainly phenotypic flower traits are assigned to attract specific pollinators. This hypothesis and its conclusion are currently controversial debated in many studies and reviews (Ollerton et al. 2009; Aguilar et al. 2015; Ollerton et al. 2015; Harder 2000; Rosas-Guerrero et al. 2014; Fenster et al. 2011). Nevertheless phenotypic flower traits can be good indicators to draw conclusions on the main pollinators. Good examples are studies focusing on different floral tube lengths and shapes to predict the matching pollinator based on the pollination syndrome (Joly et al. 2016; Venail et al. 2010; Wilson et al. 2004). Typical floral traits which are distinguished can be the flower morphology including the size, the rewards system which implies pollen and nectar, scent, colour as well as phenotype and many more expressions depending on the species.

2.1.1 Pollination – a botanical point of view Based on the pollination syndrome the floral traits play a major role in the interaction of flower and insect. The composition of the single trades can be highly divers within the an- giosperm and lead to different pollination systems. 2 Literature review 13

The morphology of inflorescences and single flowers can have strong influences on the pollinator range. Wilson et al. (2004) set up a research on the pollination syndromes of penstemon; mainly focusing on the morphology of the flower and the presence of pollinators. In total the floral characteristics of 49 species were evaluated. Five flowers per species were sampled. Nine floral characteristics were measured to specify the different species for further conclusions on their pollinators. Wilson et al. (2004) could show that different flower characteristics can be used to separate hummingbird-pollinated flowers from such flowers which are pollinated by hymenopterans. They concluded that the main factor for this separation was the classification between blue and yellow flowers for the hymenopterans in comparison to reds and oranges flowers for the hummingbirds. 37 out of the 49 species were identified as “hymenopteran-flowers” and could further on be split into two main groups depending on the flower shape and the size of bee and their tongue length. Nevertheless Wilson et al. (2004) pointed out that there is still a huge knowledge gap as well as there are no characteristics which can explain pollination syndromes to100%. Also Gómez et al. (2014) studied the floral morphology and questioned whether pollinators influenced the corolla shape of an pollination generalist plant. He focused on 40 Erysimum species from Northwest Africa, Western and Central Europe. By conducting phylogenetic comparative methods the corolla-shape integration was low but significant different. Low- est significances were found in those species with a high diverse pollination system. Due to this Gómez et al. (2014) concluded that plants with more diverse pollination systems are under lower selection pressure and therefore result in less specific corolla trades. Looking to the genus Petunia which is a common ornamental flower on the European market many studies are focusing on quantitative trait loci (QTL) to compare species which are naturally isolated from hybridisation due to their different pollinators (Galliot et al. 2006; Klahre et al. 2011; Venail et al. 2010; Stuurman et al. 2004). Klahre et al. (2011) performed interspecific crosses between the two species Petunia axillaris and Petunia exserta. Petunia axillaris was having white flowers and known to be pollinated by hawk moths whereas Petunia exserta was having red flowers and pollinated by hummingbirds. The F2 generation out of the interspecific cross was used to identify QTL which differ between the two species. Two QTL could be identified which are playing a role in the floral scent production and differ between Petunia axillaris and Petunia exsert. Further on Klahre et al. (2011) could show, that Moths which had the chance to choose flowers on their own were preferring those which were producing scent no matter their shape or colour. This study indicates how important the floral characteristic scent can be when it comes to pollination. Further on it shows which serious effects cross breeding can have for pollinators Also Parachnowitsch et al. (2012) proved that scent can play an important role in the phenotypic flower selection of pollinators. He tested floral characteristics like inflorescence height, corolla colour scent and many more of Penstemon digitalis. By testing the effects 2 Literature review 14 of sampled characteristics under field conditions it turned out that the scent of the flower has a higher effect on the selection than other commonly measured characteristics of flowers. Flower shape but also the colour is often used to simplify the classification of flowers into their belonging pollinator groups. Miller et al. (2011) are summarizing the different coloured plant parts beside the green ones which are responsible for the photosynthesis. Not only the corolla must be coloured, also other parts like pollen and nectar can be coloured to attract pollinators(Miller et al. 2011). Some colours are assigned to be preferred by typical groups of pollinators. Purple is known to be more attractive for hymenopterans compared to red which is assigned to bird pollination(Revert et al. 2016; Shrestha et al. 2013). Polli- nators recognize flowers different than human beings; Bees can see in ultraviolet colour range which can lead to a total different colour and flower characteristic than expected (Chittka & Raine 2006; Miller et al. 2011; Rohde et al. 2013). But also in the case of colours and pollination syndrome there doesn’t exist an always applicable rule. Revert et al. (2016) conducted their study in four Mediterranean communities at the province of Barce- lona and confirmed that different pollinators have specific colour preferences. The results also pointed out the preference of purple flowers for bees. Pink coloured flowers were attractive for lepidopterans, white and yellow for coleopterans and UV-yellow flowers for wasps and dipterans. Nevertheless by looking at the pollinator composition from the flower point of view it turned out that flowers having similar colours do not directly attract similar pollinators. Revert et al.( 2016) explain this result with pointing to the principal that many flowers are functioning as pollination generalist and therefore do not only attract one group of pollinators. The mean reason why pollinators are visiting plants is the reward in form of pollen and nectar for their pollination service which makes them depending on each other. As shown in the previous mentioned studies due to evolution flowers and pollinators have adapted to each other. Also nectar and its composition differs depending on the primary pollinator of a flower (Abrahamczyk et al. 2016; Perret et al. 2001). According to Baker & Baker (1983) nectar mainly consist of three different sugars; the glucose, fructose and sucrose. They are differentiating between four different types of nectar which are the sucrose-rich, sucrose dominant, hexose rich and hexose dominant nectar type. Abrahamczyk et al. (2016) studied the nectar sugar composition of more than 2100 Astrids taking into account their common pollinator groups. Plotting the results in a ternary diagram, which is shown in Figure 1, they could show the separation of different pollination groups along the sucrose axis. Plants which are pollinated by specialists have a higher nectar sucrose proportion (NSP) than those which are pollinated by generalists. Beside this overall ranking Abrahamczyk et al. (2016) pointed out that within the single pollinator groups high variations occur. This variations cannot be completely explained and need following research. Further on they tested the hypothesis that the different NSP have developed due to the adaptation of the 2 Literature review 15 different pollination groups. This hypothesis could not be rejected and it is evincing that the pollinator groups play an important role in the evolution of the NSP, nevertheless other factors need to take into account to fully explain this effects.

Figure 1: Ternary diagram by Abrahamczyk et al. (2016) showing the different pollinators and their preferences of the Nectar sugar composition in a sucrose-, fructose- and glucose triangle

Beside nectar, pollen is the other part of the flower reward system which is important for those pollinators which are feeding their offspring with the protein and amino acid rich pollen (Moerman et al. 2015; Vaudo et al. 2016). From the angiosperms point of view the pollen transport service from the anthers with its successful transfer to the stigma is indispensable for their reproduction. Also pollen varies in the nutrient composition and different pollinators have different preferences. Vaudo et al. (2016) could proof that bumblebees can select the nutrient composition in pollen which is fitting their diet best. This leads to the conclusion that also in the case of pollen pollinators have their preferred host plant species to reach a good composition in their diet. Not all pollinators are good pollinators; in this regard the adaptation between flowers and pollinators play a major role. Focusing on the architecture of solanum, Falcão et al. (2016) tried to find out how important the anthers morphology is for a pollination strategy. He 2 Literature review 16 focused on the Solanum luridifuscescens to identify whether the species belongs to the so called pollen flowers or perfume flowers. Solanum pollen flowers are such flowers which are lacking nectar and are only releasing pollen after the pollinators vibrated the anthers which is also called buzz pollination(Corbet & Huang 2014). Solanum perfume flowers are those flowers visited by (perfume collecting) euglossine male bees which release their pollen due to pneumatic anthers. By observing the pollination mechanism of Solanum luridifuscescens Falcão et al. (2016) concluded that morphological traits of flowers are not enough to draw their pollination system. A high number of present pollinators which were carrying big amounts of pollen loads did not directly ensure a good pollination (Delmas et al. 2016). The facultative autogamous plant Rhododendron ferrugineum was also investigated on the efficiency of insect pollination. Therefore Delmas et al. (2016) focused on the pollinators and their pollen load as well as on the stigmas and the deposited pollen. By comparing intact and emasculated flowers they found out that the attending pollinators, in this case mostly bumblebees, contributed only half of the conspecific pollen on the stigma. In the case of Rhododendron ferrugineum the self pollination buffered the insufficient pollination by the pollinators. Concluding these examples of pollination from the botanical point of view it can be said that many floral characteristics as well as their interaction with each other play an important role for the subsequent pollinators.

2.1.2 Pollination – from the pollinators point of view This part of the review should only give a short overview on the super family of Apiodea and their state as pollinator. An overview of the German fauna, their main characteristics and their importance will be summarized. However the super family of Syrphidae and other occurring insects like Lepidoptera and Vespinae should not be underestimated in their importance as pollinators. In the case of pollination the honeybee Apis mellifera is probably the most studied insect. Apis mellifera belongs to the family of Apoidea and is living in big colonies with up to 40 000 individuals per colony. Such eusocial organisms as the honeybees are not common under the German Apoidea; most bees are solitary bees often called wild bees. The family of Apiodea in general collects nectar as source for their energy demand and pollen as protein source mainly for their offspring (Amiet & Krebs 2012). Studies focusing on the pollination success of Apis mellifera in agricultural products like almonds, apples, pumpkins or others are often found (Brittain et al. 2013; Park et al. 2016; Petersen et al. 2013). Nevertheless those studies are also questioning on the quality of pollination performed by honeybees compared to other pollinators. It could be shown, that for example in the case of pumpkins, the overall yield cannot be increased by the managed use of Apis mellifera (Petersen et al. 2013; Petersen et al. 2014). In huge monocultures like 2 Literature review 17 almonds in California the pollination is managed with Apis mellifera. In this case the pollination by Apis mellifera is indispensable, but nevertheless honeybees alone do not lead to the best pollination result. Brittain et al. (2013) could show that Prunus dulcis orchards with a more diverse pollinator’s spectrum compared to those which are only pollinated by Apis mellifera lead to a more efficient pollination and with this to a greater fruit set of the trees. Not only from a global point of view but also with a focus on the German structure of bee- keeping it can be said that from 2007 to 2015 the number of bee hives constantly increased (BLE 2016; Moritz & Erler 2016). Also German beekeepers are working in collaboration with farmers and placing their hives into orchards to ensure a good pollination. Honeybees are favored for their relative easy handling and their huge number of individuals per colony. Another positive aspect is that due to the hibernation of the whole colony already in the early spring a big number of pollinators is available. Further on Apis mellifera can fly long distances which can ensure pollination in a wide radius from where they were placed (Danner et al. 2016). Beside the managed honeybees also bumblebees are commonly used for commercial pollination (Velthuis & Doorn 2006). Bumblebees, the genus Bombus is as well as the genus Apis living in colonies but not eusocial and only reaches a colony size of 30 to 600 individuals depending on the genus (Amiet & Krebs 2012). Compared to the honeybees only the queens are hibernating Nevertheless as cited by Velthuis & Doorn (2006) bumblebees are often the more efficient pollinators as for example they can pollinate Solanaceae with the so called buzz-pollination(De Luca & Vallejo-Marín 2013). Further on they can be used in green houses where they are mainly found for tomato pollination. Another advantage is their cold temperature tolerance; bumblebees fly until 10°C whereas honeybees only fly until 16°C air temperature (Velthuis & Doorn 2006). Both Apis mellifera and the genus Bombus are living in colonies as well as they are known as generalists. Generalists are those bees acting polylectic. On the other hand there are those bees only collecting pollen from one plant family. Bees which depend on one source of pollen are called oligolectic. Beside the generalists Apis and Bombus, 38 more genera are found in Germany. In total 561 species of wild bees are known for Germany. Wild bees can differ in various habits as for example life cycle, nesting sides and materials as well as their foraging behavior (Amiet & Krebs 2012). It was shown by many studies that the pollination effectiveness of wild bees is often superior to that of Honeybees. Moreover it was shown having higher diversity in pollinators can enhance the pollination of Apis mellifera (Garibaldi et al. 2014; Lowenstein et al. 2015; Mallinger & Gratton 2015). The study of Mallinger & Gratton in 2015 showed that honeybees are having different foraging preferences compared to wild bees. In this case wild bees were also visiting apple trees having a low density of flowers whereas honey bees had preferences for apple trees with high flower densities. 2 Literature review 18

2.1.3 Pollination crisis – decline of pollinators The European red list of bees and the red list of butterflies are shown the decline of pollinators. Nearly every tenth of them is threatened (Swaay et al. 2010; Nieto et al. 2015). The European red lists are following the International Union for Conservation of Nature (IUCN) guidelines which are worldwide recognized. Nieto et al. (2015) were pointing out that the major threat for the bees in Europe is the change of landscape and with this the loss of habitats. The intensification of agriculture and their change in practice is the main reason for the decline in bee population (Nieto et al. 2015). Also butterflies are facing this problem; the intensification of grassland or even the conversion to cropland leads to less flowering areas and with this to scarcity of essential food sources (Swaay et al. 2010). This decline in number of species turned out even more drastic when valuing them under a long time frame like Habel et al. (2016) did for the butterfly and burnet moths communities in southeast Germany. By the long term observation over nearly two century (1840–2013) they could show a significant loss of the entire set of species for the researched region. Moreover they found out that the composition of species switched from a community with many niche specialists to a community with more generalized species (Habel et al. 2016). Also the German red list of bees shows this drastic loss of species; 53% (293 species) are more or less endangered. Two main factors which are on the basis of human intervention were specified; the damage and loss of suitable nesting sites as well as the decline of food sources (Westrich et al. 2011). The term “pollination crisis” describes this decline of pollinators and its consequences in a decrease of the important ecosystem service of pollination. The fact that 35% of the worldwide crops are depending on pollination (Klein et al. 2007) and with this directly influencing the human welfare(Cunningham 2016) makes the term highly controversial discussed (Deguines et al. 2014; González-Varo et al. 2013; Goulson et al. 2015; Lever et al. 2014; Potts et al. 2010). There are several drivers for the pollinator decline. Those drivers are often seen as single pillars of the whole problem but they are inescapable connected to each other(Potts et al. 2010). The main drivers are the land-use change leading in de- struction of habitats (Deguines et al. 2014; Patrício-Roberto & Campos 2014; Weiner et al. 2014; Xie et al. 2013; Westrich 1996), the decrease of food sources (Potts et al. 2003; Biesmeijer et al. 2006; Pauw & Hawkins 2011); environmental pollution and the use of pesticides (Stanley et al. 2015; Feltham et al. 2014), the global spread of pathogens and introduction of non-native species (Evison et al. 2012) as well as the climate change and its consequences (Giannini et al. 2012; Kerr et al. 2015). As already stated by the European red lists land-use change is the major threat for pollinators. This threat is becoming even bigger when taking into account that many pollinators only fly few hundred meters to reach their food source (Zurbuchen et al. 2010). Therefore pollination systems can be in risk if the distance between pollinator nesting side and food source is too far (Kremen & Cunningham 2011). Studies focused on different landscapes 2 Literature review 19 from rural to urban areas could show that pollinator diversity is negatively affected by areas with high urbanization rate (Bates et al. 2011; Bengtsson et al. 2009; Guilbaud et al. 2014; McFrederick & Lebuhn 2005). The decline in pollinators is resulting in less pollination. The decline leads to a lower reproduction of those flowers which are depending on pollination. This causal connection was tested by using available entomological and botanical data for the Netherlands and UK. Biesmeijer et al. (2006) couldn’t proof this theory; nevertheless transnational results as the decline of bee-dependent plants and the increase of abiotic pollinated plants are referring to this theory. This theory is strongly connected with the change from communities having more specialized pollinators to groups with more generalized pollinators in relation. A national wide research across France focusing on the flower visitor communities showed the decline in pollinator diversity with having more generalist closer to the urban area (Deguines et al. 2016). 1606 sites of France along different rural-urban gradients were evaluated on flowers and their visitors and showed a significant negative effect on urbanization for pollinators. The use of pesticide an it’s consequences for pollinators is controversial discussed, many studies were focusing only on honey bees and less is known about the pollinators in total (Blacquière et al. 2012; Krupke et al. 2012). However Brittain et al. (2010) pointed out that the species richness might be negatively affected by the use of pesticides and that mainly wild bees are vulnerable. Nevertheless more knowledge about pesticide effects has to be compiled. Beside this also other environmental pollutions like air pollution or soil pollution can harm pollinators. Even low air pollution can influence the scent of flowers by deg- radation of the floral volatiles. This change and decrease of the original flower scent leads to a more difficult recognition for pollinator’s (Fuentes et al. 2016). Soil pollution based on metals is known to damage many groups of organisms; Meindl & Ashman (2013) were suggesting that plants growing on contaminated soils can negatively affect visiting pollinators. Flowers were reacting different on soil contamination. The study of Mulder et al. (2005) showed that flowers mainly visited by butterflies where much more sensitive so contaminated soils than flowers of moths or other pollinators. Another aspect which influences the pollinator decline is the spread of pests and pathogens as well as the introduction of alien species. Also in this case many studies were focusing on Apis mellifera which is the main managed pollinator. Varroa destructor is the major ectoparasite of Apis mellifera and known to cause many colony losses in Europe and USA (Nazzi & Le Conte 2016; Rosenkranz et al. 2010). The mite is acting as a vector for many other diseases as for example the deformed wing virus (DWV); fortunately Varroa destructor is only known to be found on the Apis genus, however it cannot be excluded that Virus infections transmitted by Varroa mites jump over to other bee species living sympat- ric with honey bees (Fürst et al. 2014). Nevertheless other pests like the microsporidian Nosema ceranae have been spreading from the genus Apis to other pollinators. It is now 2 Literature review 20 known that Bombus and even other wild bees are infected with Nosema (Graystock et al. 2013; Ravoet et al. 2014). Nosema in Bumblebees was shown to have a higher virulence than in Apis and is seen as a big threat for Bombus (Graystock et al. 2013). A screening of solitary bees showed that many parasites were corresponding from those of the honeybee. It was presumed that either the hives of the honeybees can be seen as a source of pathogens for the solitary bees or the other way round (Ravoet et al. 2014). Beside pests and pathogens also alien introduced species can lead to an imbalance in the pollination network. Traveset & Richardson (2006) were splitting this disruptions into for main disruptive ele- ments; alien pollinators, alien herbivores, alien predators and alien plants. Alien plants for example can lead to a competition between the native and introduced species, leading in decline of pollinator visits which leads to a lower pollination and seed set as it was shown for the introduced Lythrum salicaria and the native L. alatum (Brown et al. 2002). As all the other organisms also pollinators are affected by the climate change. Models were developed to understand the complex possible changes attributed to climate change (Giannini et al. 2012; Polce et al. 2014); as well as existing long period data sets were evaluated to understand previous changes(Kerr et al. 2015; Pyke et al. 2016; Schweiger et al. 2010). Not only pollinators also their native flower sources will be affected by the climate change. Schweiger et al. (2010) is expecting advantages for some introduced alien species which might suppress the native ones. The shift in flowers could in a second step lead to a shift towards the more generalized species. Those might be the more prepared once for the global change (Schweiger et al. 2010). Nevertheless models and expected hypothesis can never face the real world difficulty. Pyke et al. (2016) compared bumblebee and flower data from 1974 with new data from 2007 and expected a shift to higher alti- tudes for both components due to the given temperature increase. Although they could con- firm their hypothesis for some species they had to reject it for most of the species. This demonstrates how difficult such environmental changes are predictable. As mentioned with explaining the pollination crisis, all those single drivers are interacting with each other wherefore an integrated consideration of the single parts will never lead to a significant prediction.

2.2 Pollination insects in urban areas

This part of the review gives an overview of the current scientific knowledge regarding the topic of urban pollination. It will show current study questions and further on introduce to the topic of ornamental flowers and their benefits for pollinators. The term “urban” cannot be described by a single definition. Referring to the United Na- tions (UN) the definition is depending on the country in particular, different distinguishing features are used to define rural and urban areas(UN 2016). In 2007 the global population exceeded the 50 percent bounder of people living in urban areas; currently is approximate- 2 Literature review 21 ly 54 percent of the world population living in urban areas. Urban areas can vary in size and characteristics; more than half of the urban population is living in cities with around 500 000 inhabitants. Most of the cities will face ecological problems and therefore have to seek sustainable solutions (UN 2014). Ecosystem services as Pollination in urban areas are rated high in the term of a sustainable urban area management. As already mentioned in the previous chapter, pollinators are sensitive to environmental changes and have certain requirements regarding their environment. The last research dec- ades mainly were focusing on pollination science in rural areas whereas information’s on urban areas were missing (Hernandez et al. 2009). The review of Hernandez et al. (2009) compared 59 publications focusing on urban bee ecology and concluded there are existing huge knowledge gaps. Comparing different studies turned out to be complicate due to lack of detailed background information of the study areas. But rising interests on this topic lead to an increase in the field of urban pollination science within the last ten years. Differ- ent fields were investigated; landscape effects (Bates et al. 2011; Cusser et al. 2015; Ferreira et al. 2013; Matteson et al. 2013; Wray & Elle 2014) abundance and composition of pollinators in an urban area (Guilbaud et al. 2014; Gunnarsson & Federsel 2014; Matteson & Langellotto 2010); urban gardens (Kaluza et al. 2016; Pardee & Philpott 2014; Salisbury et al. 2015); plant-pollinator interactions (Hennig & Ghazoul 2011; Ushimaru et al. 2014) as well as pollinator friendly ornamental flowers (Monique et al. 2015; Garbuzov et al. 2015; Garbuzov & Ratnieks 2014; Garbuzov & Ratnieks 2015). In general most of the time the composition of pollinators in urban areas from those in rural areas differ; nevertheless an overall statement cannot be made because of the high variation within the urban areas. Depending on the city, the research question as well as the timeframe of the research the results might have huge variations. Comparing the pollinator abundance and assemblage of pollinators along an urbanization gradient can indicate which landscape factors are making a difference. Bates et al. (2011) carried out an insect sampling in and around Birmingham, UK at 24 sample sides which differed in their degree of urbanization. The insects were sampled with pan traps and additional sweep netting on five sessions. To test the effects of different variables on the bee and hoverfly abundance and richness they generated a generalized linear model (GLM). Variables of the model were ‘flowering tree abundance’, ‘flower abundance’, ‘altitude’, ‘exposure’, ‘percentage built place’ and ‘percentage gardens’. To compare the total abundance and richness of pollinators a one-way analysis of variance (ANOVA) was used. Flower abundance and the percentage of built space turned out to have the highest significance. In this case they played the most important role in pollinator abundance and richness under the given model. In addition significant differences were shown in the abundance and richness between rural, suburban and urban sides. The total species abundance and richness was highest for the rural areas(Bates et al. 2011). Another study focussed on the same topic comparing rural to urban areas in the UK resulted in different outcomes. Baldock et al. (2015) com- 2 Literature review 22 pared three different landscapes; urban, farmland and nature reserves. In total they researched 36 sites at 12 cities focusing on flower-visitation networks. Insect sampling was done along a 2 m x 1km transect; all insects where collected while walking along the transect. Sampling was performed once per site. Although most pollinators were sampled in nature reserve followed by farmland and concluded in urban areas in total numbers; no significant differences were found. Also in the case of insect richness between the three different landscapes no significant difference was found. Testing exclusive bee species richness even resulted in significant higher bee richness in urban areas compared to farmland. Considering the results Baldock et al. (2015) concluded that also urban areas can be good habitats for pollinators. Nevertheless both studies recommended further research on urban areas to identify important landscape factors for a pollinator friendly environment in urban areas (Bates et al. 2011; Baldock et al. 2015). A two years study was conducted; sampling insects at 24 sites around Lyon, France to evaluate the wild bee community structure along an urbanization gradient. The sites were differentiated by the degree of impervious surface at a radius of 2km. Sampling was performed with three different coloured pan traps and an additional net survey on a monthly basis. Overall Guilbaud et al. (2014) sampled 79% of the predicted bee species from the regions. The diversity of species was higher for areas with lower percentage of impervious in this case equating with a lower urbanisation level. Nevertheless Lyon and the surrounding areas were hosting a diverse bee community. Therefore Guilbaud et al. (2014) recommended wild bees as flagship genera to raise awareness on the biodiversity conservation in urban areas. Beside this also other studies were focused on urban pollinator communities. By sampling hoverflies and bees along an urban-rural gradient in Flanders, Belgium, Verboven et al. (2014) could show that hoverflies and bees were reacting different concerning landscape changes. Hoverfly abundance as well as diversity significantly decreased in urban areas whereas bee abundance and diversity significantly decreased under rural- agricultural sides (Verboven et al. 2014). Concerning on the current studies the main effect for a lower abundance of pollinators is the strong surface sealing which directly leads to the problem of missing nesting areas as well as needed food resources. Nevertheless in all of the studies it was mentioned that urban areas can provide good quality habitat for pollinators if enough food sources and nesting sides are available(Baldock et al. 2015; Bates et al. 2011; Guilbaud et al. 2014; Verboven et al. 2014). In some cases urban areas might even have advantages compared to rural-agricultural sides. In cases of urban areas with a high proportion of late and persistent (ornamental) flowers pollinators can profit from the late season food source (Matteson et al. 2013; Wray & Elle 2014). Urban areas can be an attractive habitat, also indicated by the number of pollinator species found in the different urban areas around the world. 2 Literature review 23

In total 291 bee species were sampled in the Lyon region over a time period of two years (Guilbaud et al. 2014). In Flanders, Belgium Verboven et al. (2014) counted 42 bee species and, 43 hoverfly species within one year. Gunnarsson & Federsel (2014) which were only focusing on the genus bombus in Gothenburg, Sweden sampled 12 species of bumblebees. Sampling neighborhood blocks and green spaces in New York City resulted 47 bee species and 29 other flower-visiting insect in 2008 and 2009 (Matteson et al. 2013). 34 hymenopterans, 24 dipterans, 8 lepitopteran, 3 coleoptera and one heteroptera were found in Zü- rich, Switzerland in 2008 (Hennig & Ghazoul 2011). In total 18 community gardens in the high population dense quarters of Harlem and Bronx, New York were sampled resulting in 24 butterfly species and 54 bee species(Matteson & Langellotto 2010).Investigating Bir- mingham and its surrounding area showed a occurrence of 24 bee species and 50 species of hoverflies (Bates et al. 2011). Having this huge differences in counted species between the cities and studies can first of all be explained by the different characteristics of study area and secondly by the different study design mainly the sampling method. Lyon for example was surveyed for two years and the sampling was done with pan traps and additional sweep netting five times a year (Guilbaud et al. 2014); whereas other studies often sampled only by netting and less often. Nevertheless even studies showing lower species richness declare the importance of urban areas as habitat for pollinators. Urban areas are known for a different flower range compared to the surrounding rural environment; especially many alien species can be found in the public green areas or at private gardens. Whether such typical urban gardens can base as habitat and good food source has been researched by surveying those gardens on the visiting pollinators. In comparing eight backyard gardens with native plant structure to eight gardens without native flower composition in Ohio, USA Pardee & Philpott (2014) wanted to value the role of plant composition in urban gardens. Native with non-native gardens were paired in nearby backyards and flower assemblage as well as bee occurrence were measured. Further on the surrounding landscape was specified to understand whether local garden characteristics or the surrounding environment plays an important role in the pollinator presence. Bees have been sampled with direct observation, hand-netting and pan trapping between May and August in 2010. The overall abundance of bees in native gardens was significant higher as well as for native bees. Comparing ground nesting bees and cavity nesting bees it showed that only the cavity nesting bees had a significant higher appearance; whereas the ground nesting bees did not significant differ between native and non-native gardens. Pardee & Philpott (2014) were concluding that mainly the local conditions, especially the floral composition are playing an important role in attracting bees. They went even further calling native plants the “bee’s knees” and recommended to plant more native in urban areas (Pardee & Philpott 2014). Ten gardens in Ottawa, Canada which were observed on the pollinator diversity by Hennig and Ghazoul in 2012 also showed the preference of pollinators for native gardens (Science 2 Literature review 24

2016). Flower species in the surveyed gardens were determined and pollinators were observed. Nine observation dates between July and September 2013 with a two times ten minute observation period were carried out. Beside the high variation in pollinator composition along the observation timescale gradient; a significant interaction between pollinators and the richness in native flower species could be proved(Science 2016). Focusing on the question how effective this native species can be in an urban environment; thirty wildflower plots in Stirlingshire, UK were observed by Blackmore & Goulson (2014). Compar- ing this wildflower pots with control plots resulted in average 50 times more bumblebees and 13 times more hoverflies visiting the plots with wildflowers than those without. In South East Queensland, Australia Kaluza et al. (2016) tried to evaluate different landscape effects from the pollinators point of view by focusing on a wild stingless bee Tetragonula carbonaria common in Australia. Colonies were monitored over three years under different landscapes; their natural habitat forest and two landscapes affected by man, agricultural macadamia plantations and suburban gardens. Tetragonula carbonaria colonies were monitored over two years mainly focusing on their foraging behavior and resource intake. Un- expectedly it turned out that not the natural habitat but rather the suburban gardens lead to be the best for the food supply of the colonies. Kaluza et al. (2016) discussed that the mixture of native and exotic plants in the suburban gardens lead to stable food availability and a continuous supply of pollen and nectar thru out the whole season. Whereas native plants are usually accepted as good food source and have been proved on their attractive regarding pollinators few is known about the value of exotic plants. There- fore more often it is discussed which ornamental flowers can be recommended for urban gardens to attract pollinators. Also Salisbury et al. (2015) were asking “should we plant native or exotic species?”. To answer this question a four years field study was performed in which native, near-native and exotic ornamental flowers were tested regarding the visit of pollinators under common horticultural practice. Insects were observed by Salisbury throughout the whole flowering season and clustered in different pollinator groups as well as flowering units from 1-100 were listed. High flower units resulted in an increase of pollinator visits; which indicates that gardens having a higher amount of flowers can supply more pollinators, no matter the flowers are exotic, near-native or native. Nevertheless exotic plants were less often visited by pollinators. Compared to the native and near-native flowers 40% less pollinators were counted. This resembles what other studies stated. How- ever flowering units of exotic plants often continuously increased while season and flow- ered longer than native once. In three of the four study years most pollinators in September were counted on exotic flowers. This means exotic plants could extend the flowering period and with this supported still active pollinators in late summer season. Concluding Salisbury et al. (2015) were recommending planting a wide range of native and near-native flowers which can then be supported by carefully selected exotic species. The advice of a careful exotic plant selection concerning the attractiveness of insects was confirmed by 2 Literature review 25 other studies focusing only on certain ornamental species(Garbuzov & Ratnieks 2015; Garbuzov et al. 2015). Out of 228 different varieties of Aster only three could be rated as highly attractive for insects, 74 have been moderately attractive but 151 turned out to be relative unattractive(Garbuzov & Ratnieks 2015). Comparing the ornamental flower Dahl- ia, mainly species with open flowers were attractive for insect. In this case two species out of eleven had significant higher numbers of pollinator visits (Garbuzov et al. 2015). Focus- ing on 32 different ornamental flowers in UK horticulture with specific view to 13 Laven- dula species also showed the huge differences between varieties within one species. There- fore different Lavendula varieties showed different results in pollination attractiveness (Garbuzov & Ratnieks 2014b). Garbuzov and his colleges who were mainly focusing on ornamental flowers and their attractiveness for pollinators were concluding that open parks as well as private gardens can be easily designed pollinator friendly. This can be done without increasing workload or time and without losing attractiveness of the flowerbeds. Nevertheless they pointed out that still more research is needed to get an understanding of which ornamental flowers can be used to support pollination insects(Garbuzov & Ratnieks 2015; Garbuzov et al. 2015; Garbuzov & Ratnieks 2014b). Beside scientific research many public available list can be found; informing about pollinator friendly gardens and advisable flowers. Garbuzov & Ratnieks (2014a) evaluated 15 of those lists to get an idea of the quality of information. The plant genera recommended in this lists ranged from 29 to 257 in the single lists and represented a total spectrum of 455 genera. One of the main weaknesses was that the overlapping among the lists was rather low. Some lists where lacking good pollinator plants whereas others sometimes recommended poor quality flowers. Also references of the published information were missing excepting one list. Nevertheless Garbuzov & Ratnieks (2014a) conclude that such lists can rise public interests and might be a good step to a more pollinator friendly horticulture. Also many suggestions on often listed genera were from good quality. But further lists should be based on hard data wherefore more research on ornamental flowers and their attractiveness on insects have to be conducted (Garbuzov & Ratnieks 2014a). Concluding this literature review it can be said that the topic of urban pollinators is multifaceted studied. Nevertheless studies are often not comparable due to different research methods. Concerning ornamental flowers and their benefits for pollinators more basic research is needed to give reliable statements. 3 Material and Methods 26

3 Material and Methods

The field research was performed in the year 2016 in collaboration between the Lehr- und Versuchsanstalt für Gartenbau (LVG) in Heidelberg and the Landesanstalt für Bienenkun- de (LAB) in Hohenheim.

3.1 Study area

The study area was placed at the research area of the LVG in Heidelberg. Heidelberg is located in the south west of Germany and is a city of approximately 150 000 inhabitants (Heidelberg 2016). Based on the climate maps of Köppen and Geiger the region belongs to the warm temperate, fully humid and warm summer (Cfb) climate zones (Kottek et al. 2006). The LVG is based in the district Pfaffengrund which is found south west to the river Neckar. The total research area of the LVG covers 5,5 ha and combines open land fields as well as glass houses. Five areas at the LVG are used to present the current product range of ornamental flowers in Germany. Three of those areas were presenting the flowers in common plastic flower- pots. Each pot had a volume of 30 litres and was planted with three individuals per variety; the potting soil was a common peat based soil. Each pot was connected with an irrigation system which was watered two to three times per day. Fertilisation was done in a liquid form thru the irrigation system and was done manually depending on the flowers and their demand. The fourth area was representing perennial flowers wherefore each individual was planted in a single 9 litre pot. The perennial flowers have been planted in soils with a long term fertilization buffer wherefore additional fertilization was not needed. Irrigation of the perennial flowers was done with an irrigation trolley two to three times per day. The fifth field was representing a certain number of the varieties which have already been presented in the 30 litres pot but which were furthermore represented under field conditions; therefore the individuals have been directly planted into the soil in a one square me- ter area for each variety. The fifth field was irrigated with trip tubes and no additional fertilization was done.

3.2 Ornamental flowers

The flowers grown at the LVG Heidelberg are representing ornamental flowers which can be found on the German bedding and balcony market; furthermore new breeds are introduced and tested there. The most represented species are the Pelargonium, Petunia, Cali- 3 Material and Methods 27 brachoa, Salvium and Bidens; beside those other common species are also represented with different varieties. Due to the high number of different varieties it was not possible to evaluate the whole range of flowers. A meeting in the early spring of 2016 was set up to discuss plants from interest which should be evaluated on their attractiveness for pollinating insects. Participants were members of the LVG Heidelberg, Ms. Dr. Andrea Dohm, the head of breeding and development of SelectaOne and the breeder Ms. Dipl. Katharina Zerr from Floricultz. The Request from the breeders’ side was to identify different trades which are responding for pollinator attractiveness depending on one species but several varieties. For the identification of those trades which led to a higher rate of insect visits the species Calibrachoa was chosen. Beside this members of the LVG were interested to get more information about pollinator friendly flowers in general, therefore a larger amount of the available ornamental flowers at the LVG area should be evaluated on pollinator visits. Already existing public access literature from the Netzwerk Blühende Landschaft as well as from the Bienenweidekatalog was used as a starting point. The first observations were done on more than 250 varieties. Due to the huge sample size of flowers a reduction of the sample size was done after three observations for each of the varieties. All those varieties which had less or equal one insect per minute visited were removed from the list of observation as well as the perennial species. The perennial species have been removed to concentrate on the more typical annual balcony species. Furthermore those typical balcony flowers are known to have a longer flowering period than the perennial once. In Table 1 the six main groups are explained to get an overview

3 Material and Methods 28

MAIN FLOWER GROUPS

Table 1: Six main observed ornamental flower species with their origin and a short description based on Altmann (2008) and SanMarcosGrower (2016).

Calibrachoa origin: Brasil short description: closely connected to the species Petunia but more weather resistant. Having huge number of smaller flowers – compared to Petunia. High variation within variety morphologies and flower colours. Filled and non-filled varieties are available on the market. Can flower throughout the whole summer until autumn. Bidens ferulifolia origin: Mexico and south Arizona short description: high variance between varieties having yellow as main colour with new varieties in white white-purple. High number of small open flowers. Flowering time throughout summer until autumn.

Dahlia origin: Mexico and other mountain areas in central America short description: facultative day-neutral flower and developing tubes under short days. Nowadays mainly smaller phenotypes are propagated. Flower shapes can vary from huge and open flowers to small and filled flowers. Moreover having huge variations in colours. Flowering period lasts until autumn. Euphorbia hypericifolia origin: Mexico and North America short description: warm climate evergreen subshrub. Having huge amount of small white flowers. Flowering time can last until first freeze. No huge differences between flower shapes and colour on the market until now.

Salvia farinacea, Salvia interspecific origin: Texas and Mexico short description: Salvia farinacea mainly white to blue coloured. Plant reaches height to 70 cm flowering period from June until September. Sal- via interspecific has huge variations in flower shape and colour due to different species. Scaevola aemula origin: west- Australia and Tasmania short description: expected to be a facultative long-day plant. Having a long flowering period until the first freeze. Main flower colours are found between purple, blue and white. Variation between morphology is very low.

3 Material and Methods 29

3.2.1 Calibrachoa Twenty different Calibrachoa varieties should be observed and compared to evaluate different plant trades which might influence insects foraging behaviour. Two different shapes per colour were chosen. Two fully double flowers per colour and three none fully flowers per colour were used. Except for the yellow colour where only one filled variety was available and for the white colour which had four open varieties. Three individuals per variety were potted in a 30 litres pot; all pots were connected to an irrigation system. The twenty varieties were randomly placed in a row; due to plant losses only two replicates in total were possible. Two different locations at the LVG area were chosen; one between two building complexes and the other one close to an open area. The Calibrachoa cuttings were produced by the SelectaOne Company.

3.3 Ornamental plant monitoring

To evaluate the attractiveness of ornamental flowers for pollination insect the foraging behaviour of food either pollen or nectar should be used as a measurement. In this case an observation gives the opportunity to make sure that insects really were foraging at the observed plant. Observations with fixed timeframes can be used to compare different plant- insect interactions of flowers with each other. The measuring unit is quantitative and counts insects per minute or insects per counting period.

3.3.1 One minute observation The one minute sampling can be seen as a snapshot observation which gives only a short impression of the whole situation. Therefore it is important to sample at best possible standardised environmental factors. Warm, sunny, non cloudy and wind free days were chosen to observe the flower insect interaction. Each flower was observed for exactly one minute and several parameters were noted. Before the observation started the number of flowers per pot was counted and the percentage of cloudiness was noted. Beginning the observation the first step was counting those insects which already were present on the plant; insects on the plant were only counted once. After counting the present insects the new arriving once where counted. The insects were split into five groups; Apis mellifera, Bombus spp, Syrphidae spp, wild bees and others. Wild bees have not been divided into the genus due to the complicate field identification. The observations started in June 2016 with the outset sample of more than 250 varieties; due to the huge sample size it was not possible to observe the whole selection within one day wherefore the environmental factors might differ between the observations. To mini- mize this error factor the sampling order always changed. Bound to the good weather con- 3 Material and Methods 30 ditions the observation days were not stringed together. Observation took place between 10.00 o’clock and 18.00 o’clock each observation day.

3.3.2 Whole day every hour observation Since the one minute observation sampling gives only snapshot information a whole day observation was implemented. The whole day sampling started at 8.30 o’clock in the morning and was carried out in an one hour cycle until 17.30 o’clock. In total ten observations per day were documented. Five different balcony plants shown in Figure 2 were evaluated. To evaluate whether the one minute time frame is long enough to get an idea of the flower attractiveness for insects, the single observations were done with the one minute time frame and an additional two minutes timeframe. The total three minute observation should afterwards be compared with the one minute observation in terms of efficiency and the informative value.

Figure 2: The five species for the whole day observation. From left to right: 1. Euphorbia hypericifolia, 2. Scaevola, 3. Salvia farinacea, 4. Bidens ferulifolia, 5. Dahlia

3.4 Insect sampling

Insect sampling was performed to identify wild bees’ genus and species as well as to identify their carried pollen load.

3.4.1 Sampling Observed flowers should be attractive and healthy throughout the whole observation period wherefore a careful insect sampling had to be carried out. Every insect was caught individually and stored separately in a 100 ml plastic container. The insects have been sampled selectively with an exhauster and then transferred into the plastic container or were directly 3 Material and Methods 31 caught into the plastic container. Caught insects were immediately frozen at -40°C and stored until the preparation. The sampling took place at warm and non-cloudy days. Only unknown insects were caught to identify them and get more details about the pollinator spectrum of a flower. Each plastic container was labelled with the date and the flower on which the insect was caught.

3.4.2 Preparation and identification Collected insects were defrosted and fixed with insect needles. Binoculars were used to fix the smaller insects; they were transfixed thru the Mesonotum to consider those parts which are important for the identification. Genus and species were identified; genus was identified with the key of ‘Bienen Mitteleuropas’ (Amiet & Krebs 2012). Depending on the genus the species were identified with the specific identification keys. Daniela Warzecha was afterwards verifying the identified species.

3.5 Pollen sampling

To identify pollinators’ preference a pollen analysis can produce a good data basis.

3.5.1 Ornamental flowers – stamen Many of the ornamental flowers are neither endemic nor are they used by Apis mellifera and therefore their pollen shape and structure is often not well documented. Identification of the insects collected pollen is only possible by comparing it to species pure flower pollen. Pollen samples directly from the flower stamen were taken to get pure and comparative samples. Depending on the flower and the pollen occurrence several anthers were taken. Pollen grains from the anthers were then transferred into Eppendorf tubes filled with some drops of ethanol.

3.5.2 Pollination insects – pollen load Those insects which had a pollen load or which showed pollen on their surveys where used for the pollen analysis. For the pollen analysis pollen should be removed from the insect and transferred to a microscope slide. Two binoculars with the external ‘KL 1500 compact’ cold light sources from SCHOTT were placed and used next to each other for a faster pollen transfer. The first binocular was used to recognize the pollen on the insects with a 40-45x magnification. The second binocular was focused on the microscope slide with a 45x magnification. Depending on the quantity of pollen loads the insects were handled different; pollen of insects with lower pollen quantities was removed manually with an insect pins. The insect pin with the removed pollen grains were then transferred to the microscope slide and stripped of. 3 Material and Methods 32

Bigger pollen loads on the legs have been removed with a tweeze and transferred into a one millilitre Eppendorf tube to liquidate them with some drops of 95,5 % ethanol. Each Eppendorf tube was shaken for 15 up to 30 minutes with the MULTI VORTEX-GENIE from Scientific Industries. After the pollen load dissolved in the ethanol, the ethanol pollen mixture was pipette to the microscope slide and dried on the heating plate at 40°C. The microscope slides with the pollen samples were fixed with a cover glass dabbed with Kaiser's glycerol gelatine and then dried for 24 hours at room temperature. Same principal was conducted for the collected flower pollen.

3.5.3 Pollen analysis A qualitative pollen analysis gives information about the insects food source and about the question on how many flowers they have been collected. The microscope slides with the insect pollen samples have been evaluated manually by Dr. Dr. Horn with the light microscope to identify the plant genus; depending on the amount of pollen a tendency of the quantitative composition was given. 4 Results 33

4 Results

4.1 Ornamental plant monitoring

The ornamental plant observation started in June and concluded until first of September 2016. All the observed flower visiting insects were counted by one person to limit the error of counting.

4.1.1 Calibrachoa variety testing The Calibrachoa observation was performed from the 23th of June until the 24th of Au- gust; in total 18 observation days were carried out. 18 observation days result in a total observation time of 720 minutes for the 40 observed plants. Only warm, cloudless and windless days have been used for the observation. Relative low abundance of flower visiting insects was noticed. Overall 319 insects were counted during the whole observations. The precise number of flower visiting insects per variety and location can be found in Ta- ble 2.

Table 2: Number of flower visiting insects of the 18 observation days in the observed Calibrachoa varieties.

varieties: Blue Blue_violet Dark_Blue_WG double_blue Neo_Royal_Blue Deep_Yellow_WG double_yellow Neo_true_yellow Yellow compact_double_white Neo_white_12 white white_yellow_ring white_72 White_643 Double_Dark_Red Double_Purple_red Neo_cherry_red Neo_Sangria Red number of pollinators; location no. 1: 181152161171592741219203554 number of pollinators; location no. 2: 7010315205112305158150541042 sum of pollinators : 25125531311226322121920342571596 sum of pollinators ‐ for the flower colours: 87 101 89 42

Based on the low visitation rate of flower visiting insects per minute no statistical comparison was performed. The yellow varieties had the highest number of insect visits. Present flower visiting insects have been mainly bumblebees almost only Bombus pascuorum and little wild bees. Little wild bees were often Lasioglossum or Halictus species. Only few Apis mellifera have been seen. Filled flower varieties (blue violet, double blue, double yellow, compact double white, white 643, double dark red and double purple red) had fewer visitors than open flowers; nevertheless filled flowers were visited by nectar robbing bumblebees (Figure 3). Some filled flowers were even visited by bumblebees without robbing. 4 Results 34

Robbing flower visiting insects were counted equal as flower entering insects. Little wild bees were observed collecting big amounts of pollen for their pollen sacs as well as Bum- blebees. Therefore pollinators also have been caught to analyze whether pollen was only collected at Calibrachoa. Location preference was not measurable as well as the effect of flowers numbers. Number of flowers ranged between varieties and within the season.

Figure 3: Bombus sp. collecting nectar from Calibrachoa. Left hand: Bombus pascuorum collecting nectar in an open flower. Middle: Bombus pascuorum collecting nectar in a filled flower. Right side: Bombus terrestris nectar robbing from outside of the flower.

Beginning with a range of 50 to 200 flowers per plant in June and increasing in number of up to 600 flowers per plant in August. Highest visitation rate was noted for the variety yellow at location two which was 1,28 insects per minute. Lowest visitation range was zero flower visiting insects per minute and found for the varieties ‘blue violet’, ‘compact double white’, ‘white 643’ and ‘double dark red’.

4 Results 35

4.1.2 One minute observation Based on the huge sample size of more than 120 varieties observed thru the flowering period not all the observation protocols will be shown. The results will ratther show exempla- ry those varieties having a common flower visiting insect set resembliing the other varieties. The same species were always observed at the same date to compare varieties under same environmental bias; whereas the time at which the observation was conducted can differ.

4.1.2.1 Bidens ferulifolia Bidens ferulifolia was observed on fourteen days from end of June ttill first September, shown in Figure 4. Twelve different varieties were observede ; out of those nine were coloured yellow some with light red appeearance and three were white coloured. Main Bidens ferulifolia varieties on the LVG research area were yellow coloured which reflects the common ornamental flower market. Number of flowers increased while the observation periood ranging from 50-400 flowers per plant at the end of June and increasing up to 600 floweers at the first of September. The variety ‘golden star’ represented a common yellow coloured species with 400 to 500 flowers throughout the sampling period. As shown in Figure 4 mainly Apis melliffera was visiting the flowers of ‘golden star’. Highest visitation rate was eight flower visiting insects per minute. ‘Beedance Painted Red’ was a slightly red coloured variety and also had 400 to 500 flower while observation time. In total 70 floweer visiting insects were counted on ‘Beedance Painted Red’ which were 21 more than for the ‘golden star’. Compared to the white varieties less flower visiting insects were observed on the common yellow types.

Bidens ferulifolia dates of observation: 23.06.16 11.07.16 22.07.16 27.07.16 05.08.16 08.08.16 15.08.16 16.08.16 17.08.16 18.08.16 23.08.16 25.08.16 26.08.16 01.09.16 day: 1234567891011 12 13 14 Figure 4: Observation protocols with the bellonging observation dates of the Bidens species with the two varieties ‘golden star’ which is having yellow coloured flowers and the variety ‘Beeddance Painted Red’ with red flowers. Each bar per observation day is representing one minute of observation. 4 Results 36

‘Pirate’s Princess’ (Figure 5) had white primed flowers with a pink collour shift. The plant had 60 to 300 flowers. In total 130 pollinators were counted dominated by 125 Apis mellifera. ‘Pirates Pearl White’ (Figure 5) had the highest visitation rate withh almost reaching an average of 19 pollinators per minute. Apis mellifere a represented more than 90% of the pollinators counted on ‘Pirates Pearl Whhite’. Other pollinators have been hoverflies as for example Eristalis tenax and wild bees as the Halictus scabiosae which were mainly represented by male individuals.

Figure 5: ‚Pirate’s Princess‘and ‘Pirates Pearl White’ with their observed visiting pollinators. Each bar per observation day is representing one minute off observation.

4.1.2.2 Brachyscome iberidifolia The species were observed on twelve days from end of June until first of September (Figure 6). Both observed varieties had decreasing flower numbers from 300 flowers at the beginning to 120/150 at the end of observation time. Flowers had lightt purple colour. Ob- served pollinators were more diverse, not having a primary pollinator shown. Butterflies have been present under “other” pollinators. The new variety ‘Obb 0800268’ had more Syr- phidae sp. present as pollinators than ‘Brasco Violet’.

Brachyscome iberidifolia dates of observation: 29.06.16 12.07.16 22.07.16 27.07.16 08.08.16 11.08.16 15.08.16 16.08.16 17.08.16 18.08.16 25.08.16 01.09.16 day: 1 2345678 9 10 11 12 Figure 6: Two varieties of the species Brachyscome iberidifolia and their counted pollinators with the belonging observation dates. Each bar per observation day is representing one minute of observation. 4 Results 37

4.1.2.3 Dahlia Overall a set of thirteen different Dahlia varieties was tested. The main differences between those varieties were the flower shapes and number of flowers preesent. Flower shapes varied between open flowers, half filled flowers with fewer anthers and fully filled flowers. Colours differed within the different colour shades of white, yellow, red and purple. De- pending on the flower shapes anthers were available for pollinators from opening of the floweers or at a later stage when corolla even sometimes wilted. The varieties were observed from end of June until end of August as shown in Figure 7. ‘Dahlegria White’ and ‘Red Pink Bicolor’ were open shaped flower varieties (Figure 7). ‘Dahlegria White’ started with 15 flowers and increased in flower number until day eight and then remained stable with around 25 flowers. Whereas ‘Red Pink Bicolor’ increased from 16 flowers to 35 floweers at day eleven and then decreased again to 16 flowers at the end. Dominant pollinator was Apis mellifera for both varietiies whereby ‘Dahlegria White’ haad more Bombus sp. mainly represented by Bombus terresstris present. ‘Dahlegria White’ haad 105 visitors and ‘Red Pink Bicolor’ 110 visitors in total. Also other open flower shaped Dahlia varieties had higher numbers in insect visits compared to filled flower shapes. The open flower variety ‘Purple off Home Run’ for example reached 80 pollinators in total..

Dahlia dates of observation: 24.06.16 11.07.16 22.07.16 26.08.16 27.07.16 08.08.16 11.08.16 15.08.16 16.08.16 17.08.16 18.08.16 23.08.16 25.08.16 26.08.16 day: 1234567891011 12 13 14 Figure 7: Two open flower shaped Dahlia species and their visiting pollinators with the belonging observation dates. Each bar per observation day is representing one minute of observation.

Majority of the varieties at the LVG research area had filled flowers.. Some flowers had more or less half filled flower at the beginning of the observation periood and then changed to filled flowers. Those filled varieties had lower visitation rates of pollinators than the varieties with open flowers. The varieties ‘Dalaya Yogi’ and ‘Bicolor White/Red’ were representing those half filled flower varieties (Figure 8). Flower number of ‘Bicolor White/Red’ varied between 40 and 50 while observation time. ‘Dalaya Yogi’ varied be- 4 Results 38 tween 30 and 40 flowers from day two to ten but then declined to around 10 flowers. Same as for the open flowers they were visiited mainly by Apis mellifera and ‘Dalaya Yogi’ also was vvisited by some Bombus sp.; in thhis case mainly Bombus terrestris. The total number of pollinators foor ’Bicolor White/Red’ was 19 and 35 for ‘Dalaya Yogi’. Other half filled floweers had similar visitor rates whereas filled flowers had lower rates up to zero.

Figure 8 two species of Dahlia, ‘Dalaya Yogi’ with half filled flowers and less anthers and ‘Bicolor White/Red’ with almost filled flowers and their visiting pollinators. Each bar per observation day is representing one minute of observation.

4.1.2.4 Euphorbia hypericifolia Euphorbia hypericifolia was observed on twelve days from end of June till end of August, shown in Figure 9. Plants had a high number of flowers ranging from 1000 to 2000 small white flowers per plant. Visitation rate per variety was highly diverse;; ranging from zero pollinators in the variety ‘Diamond Cloud’ or three pollinators at ‘Stardust Super Flash’ to 125 in ‘Igloo’ or even 184 pollinators in ‘Glamour’ during the entire observation time. Flowers with none visitors showed different floral structure with retarded anthers. Varieties which were visited by pollinators reseembled a highly diverse pollinatoor set. Most pollinators were Apis mellifera and wild bees followed by Syphidea sp. and other pollinators. Other pollinators were dominantly represented by Vespidea sp.. Wild bees were represented by smaller species as for example Lasioglossum morio, Lasioglossum latiaps or species from the genus Hylaeus. There was only one Boombus pascuorum sampled on the variety ‘Snow Mountain’ within the entire observation time. The varieties ‘Iglloo’ and ‘Glamour’ turned out to be those with the higheest number of pollinator visits as shown in Figure 9. Both varieties showed an increase in pollinator visitation rate from miid of August. From the 15.08.16 until the 26.08.16 the vissitation rate remained almost stablle. ‘Igloo’ had more Syphidea sp. visited had a visitation rate around 15 pollinators per minute and ‘Glamour’ reached a rate around 20 pollinators per minute.

4 Results 39

Euphorbia hypericifolia dates of observation 29.06.16 12.07.16 22.07.16 27.07.16 08.08.16 11.08.16 15.08.16 16.08.16 17.08.16 18.08.16 24.08.16 26.08.16 day: 123 45678 9 10 11 12 Figure 9: Observation protocols of the variety ‘Igloo’ and ‘Glamour’ with the belongiing observation dates of the Euphorbia hypericifolia varieties.

The two varieties ‘Diamond Frost’ and ‘Starpleasure’ shown in Figure 10, had a lower visitation rate but also showed an increase from the 15.08.16 with a continuous stable pollinator per minute rate of ±10; whereas ‘Diamond Frost’ was superior ‘Starpleasure’.

Figure 10: Observation protocols of the variety ‘Diamont Frost’ and ‘Starpleasure’ In total ten varieties of Euphorbia hypericifolia were observed on their visiting pollinators. Three varieties ‘Diamond Cloud’, ’Diamond Star’ and ‘Stardust Super Flash’ had either no visitors or only very view. The varieeties ‘Snow Mountain’,’ Loreen compact white’ and ‘Diamond Ice’ had similar visitor rates to ‘Diamond Frost’ and ‘Starpleasure’.

4.1.2.5 Lantana Eight varieties of Lantana were observed. Observation dates were same as for Bra- chyscome shown in Figure 6. The varieties varied in their colour from lemon yellow, orange, red to violet. Plants varied between 100 and 300 inflorescence with more than 20 little flowers per inflorescence. Overall main visitors were Bombus spp. Followed by Apis mellifera. Both pollinators were not able to reach the nectaries with theiir proboscis. There- fore nectar collection was performed by nectar robbing. Under the group of other pollina- 4 Results 40 tors Butterflies were observed. Butterflies entered their proboscis flower in the regular way. ‘Sun Fun Yellow’ had 15 pollinators and ‘Sun Fun Red’ had 37 pollinators in total (Figure 11). Other observed varieties were within this range or even lower.

Figure 11: Lantana varieties ‘Sun Fun Yellow‘and ‘Sun Fun Red’ with their observeed pollinator protocols. Each bbar per observation day is representing one minute of observation.

4.1.2.6 Mecardonia Two Mecardonia species were observed for fourteen days from end of June until first of September as shown in Figure 12. The two varieties had a high disparity between their numbers of flowers. ‘Golddust’ varied between 400 and 800 flowers and ‘Garden Frechles’ between 80 and 350 flowers. ‘Golddust’ had 60 visitors and ‘Garden Frechles’ 29 visitors in total shown in Figure 12.

Mecardonia dates of observation: 23.06.16 11.07.16 22.07.16 27.07.16 08.08.16 11.08.16 15.08.16 16.08.16 17.08.16 18.08.16 23.08.16 25.08.16 26.08.16 01.09.16 day: 1234567891011 12 13 14 Figure 12: Mercadonia varieties ‚Golddust‘and ’Garden Frechles’ with their preseent pollinators with the belonging observation dates. Each bar per obsservation day is representing one minute oof observation.

4.1.2.7 Salvia A highly diverse set of 18 different plants from the genus Salvia was represented at the research area of the LVG. The species Salvia Coccinea, Salvia coerula, Salvia nemorosa, Salvia splendens, and some interspecific hybrids were available. All the varieties were ob- 4 Results 41 served at thirteen days from beginning of July until beginning of September, shown in Fig- ure 13. Five of the observed varieties belonged to the Salvia farinacea species. Flowers were mainly blue coloured; one was white and one bicoloured. Overrall the species was mainly visited by Bombus sp. and wild bees. Bombus sp. was represented by Bombus terrestris/locorum complex the wild bees which were identified ranged from small once as Lasioglossum morio, to bigger once as Anthidium manicatum, and Xylocopa sp.. Due to the territorial sexual behaviour of Anthidium manicatum other bees were offtten driven away.

Salvium dates of observation: 08.07.16 20.07.16 25.07.16 01.08.16 08.08.16 11.08.16 16.08.16 17.08.16 18.08.16 23.08.16 25.08.16 26.08.16 01.09.16 day: 123 4 5 678910 11 12 13 Figure 13: Observation protocols of the two Salvia farinacae varieties ‘Sallyfun Snowwhite’ and ‘Sallyfun Bicolor Blue’ with the belonging observation dates. Each bar per observation day is rrepresenting one minute of observation.

The white variety ‘Sallyfun Snowwhite’ had 50 inflorescences at the beginning and increased to 90 inflorescences while observation time. Inflorescences number of the bicolour type ‘Sallyfun Bicolor Blue’ stayed stable at 150 for the whole observattion time. Pollinator protocols are shown in Figure 13.‘Sallyfun Snowwhite’ was visited by 76 and ‘Sallyfun Bicolor Blue’ by 89 pollinators in total. The “other” pollinators of ‘Salllyfun Bicolor Blue” were butterflies. For some varieties the species were unknown as for Salvia ‘Love and Whishes’. ’Love and Wishes’had purple and ‘Black and Bloom’ blue coloured flowers. The varieties ’Love and Wishes’ and ‘Black and Bloom’ had long narrow shaped flowers in which most of the pollinators couldn’t enter. Nectar collection by Apis mellifera and Bombus sp. was done by nectar robbing. Only little wild bees entered the flowers and were also observed collecting pollen. Due to the nectar robbing floweres were easily damaged and often falled down befor afternoon. Therefore observation was usually done before twelve o’cklock.’Love and Wishes’ had around 500 flowers and ‘Black and Bloom’ around 300. ‘Love and Wishes’ 4 Results 42 reached the total number of 39 pollinators (Figure 14). ‘Black and Bloom’ reached 31 pollinators in total (Figure 14).

Figure 14: Variety ‘Love and Wishes‘and ‘Black and Bloom’ and their counted pollinators. Each bar per observation day is representing one minute off observation. The interspecific group ‘GOGO’ was standing out because of their bright coloured sepals. ‘GOGO Coral’ was cream orange coloured having 30 increasing to 60 inflorescence while observation. ‘GOGO Scarlet’ was red coloured having 30 inflorescence at the beginning and 90 inflorescence at the end of observation. They also lost flowers due to nectar robbing wherefore observation was completed before twelve in the afternoon. Main visitors were Apis mellifera. Some Bombus sp. annd small wild bees which were enntering the flowers were observed. Total number of pollinatos for ‘GOGO Coral’ was 61 and 91 for ‘GOGO Scarlet’

Figure 15: ‚GOGO Coral‘and ‘GOGOScarlet‘with their observed pollinators. Each baarr per observation day is representing one minute of observation.

Also the species Salvia Splendens haad striking sepal coloures varyinng from cream, red, lavender and blue. Nectar robbing as well as corolla lost was also common. Concerning the species splendens, ‘Grandstand Salmon’ had the lowest number in infloorescence increasing from 30 to 60. Pollinators have also been lowest for the variety with a total number of 30. ‘Grandstand Blue Bicolor’ had 80 pollinators visited with a inflorescences number increasing frfrom 50 until 70. 4 Results 43

Figure 16: Observed pollinators of ‘Grandstand Salmon’ and ‘Grandstand Blue Bicolor’. Each bar per observation day is representing one minute of observation.

4.1.2.8 Scaevola aemula 17 different varieties of the species Scaevola where present at the LVG research area. The observation time was from beginning of July until beginning of Septteember as shown in Figure 17. The varieties differed in flower colour from white, yellow, pink or purple whereas the flower shapes did not noticeable differ. The main visiting pollinator was Apis mellifera for all the observed varieties. Varieties differed in flower numbers and number of visited insects. Two species with a lower visitation rate of pollinators were ‘Fancy Blush’ and ‘Fancy white’ (Figure 17). ‘Fancy Blush’ was ranging between 600 and 800 flowers while the observation time. ‘Fancy white’ had 300 flowers at the beginning and increased up to 700 flowers until the end of observation time. ‘Fancy Blush’ had a average pollination rrate of 3,3 pollinators per minutee which results from a total observed pollinator number of 46 pollinators. ‘Fancy white’ had a total number of 55 pollinators which lead to an average rate of 3,9 pollinators per minnuute.

Scaevola dates of observation: 04.07.16 20.07.16 25.07.16 01.08.16 08.08.16 11.08.16 15.08.16 16.08.16 17.08.16 18.08.16 24.08.16 25.08.16 26.08.16 01.09.16 day: 1234567891011 12 13 14 Figure 17: Observation protocol of the two Scaevola varieties ‘Fancy Blush’ and ‘‘Fancy white’ with the belonging observation dates. Each bar per obsservation day is representing one minute oof observation. 4 Results 44

Two varieties with a higher visitation rate were Scaevola ‘Fancy Exp. Pink’ and ‘Exp. Bi- color Purple White’ (Figure 18). The flower number of ‘Fancy Exp. Piink’ increased from 600 to 900 and decreased again to 800 while observation. ‘Fancy Exp. Pink’ had 92 visited pollinaters which was a rate of 6,6 pollinators per minute. ‘Exp. Bicolor Purple White’ floweer number increased from 600 until 1000 while observation. The average pollinators rate of ‘Exp. Bicolor Purple White’ was 7,8 pollinators per minute witth a total number of 109 observed pollinators.

Figure 18: Observation protocol of the two Scaevola varieties ‘Fancy Exp Pink’ and ‘Exp. Bicolor Purple- White’. Each bar per observation day is representing one minute of observation.

4.1.2.9 Others Other single varieties which species are common as ornamental flowerrs in urban gardens like Gaura lindheimi, Calendula and antirrhinum majus were also observed. Gaura lindheimi is a perennial flower found in puplic parcs. The observed variety was ‘Snowbird’ (Figure 19). The flower number of ‘Snowbird’ ranged between 150 and 200 flowers. Main visitor was Apis mellifera which was followed by Syrphidae sp. and wiild bees. In total 67 pollinators visited ‘Snowbird’ within the 13 observation minutes whiich was an average rate of 5,2 pollinators per minute. The Calendula variety observed was ‘Chers Yellow’ having smaller yellow flowers compared to common Calendula varieties. The flower number decreased from 300 flowers at the beginning to 200 flowers at the end of observation. Beside one observation day ‘Chers Yeellow’ had low numbers of visitors as it is shown in Figure 19. Main visitors were the Syrphidae sp.. The total number of polllinators was 27. 4 Results 45

Figure 19: Days of pollinator observation foor the varieties ‘Snowbird’ and ‘Chers Yellow’. Each bar per observation day is representing one minute off observation. The AAntirrhinum majus is normally known for its two lips which are closing the corolla tube. These lips are only opened by heavy pollinators as for example Bombus sp.. The observed variety ‘Copper Apricot’ did not had this typical shape. The fflowers of ‘Copper Apriccot’ were open flowers with easy accessible anthers and nectaries. IIf ‘Copper Apricot’ was visited; the species was mainly visited by small wild bees as it is shown in Figure 20. The total number of visited pollinators was 39 from which 36 were wild bees.

Figure 20: Variety ‘Copper Apricot’ with its visited pollinators. Each bar per observaattion day is representing one minute of observation.

4 Results 46

4.1.3 Whole day every hour obseervation Five different species were taken for a whole day observation. In total the flowers were observed for ten days. Observation started mid of August and ended fiirrst of September as shown in Table 3. The weather was quite stable throughout the observation days. The first observation started 8.30 o’clock and was then continued in a one hour rhythm until 17.30 o’clock. The created figures (Figure 21- Figure 25) are showing the mean values of the ten observation days. Due to the small sample size and high variation of pollinator sometimes huge standard errors occurred. Figures are made without standard errors to avoid misun- derstanding of the diagram.

Table 3 the ten days of the whole day observaattions with the belonging dates

Whole day observations dates of observation: 15.08.16 16.08.16 17.08.16 18.08.16 23.08.16 24.08.16 25.08.16 26.08.16 30.08.16 01.09.16 day: 1 2 345 678 910 The vvariety ‘Glamour’ was chosen as a representative for the species Euphorbia hypericifolia. The flower number was stable around 2000 flowers for the wholle observation time. Due to huge amount of pollinators and the opaque architecture of Euphorbia hypericifolia it was complicate to count the pollinators. Distinguishing new arriving pollinators from the present once also became difficult in the two additional minutes due to high throughput. As shown in Figure 21 the one minute and three minute observation do nott noticeable differ in relation of composition. The number of pollinators within the three miinute observation is not three times higher as for the one minute observation. The curve of tthhe Figure 21 shows an increase of pollinators until afternoon followed by a decrease from 17.30 o’clock.

Figure 21: Whole day observation of the species Euphorbia hypericifolia; represented by the variety ‘Glam- our’. Each bar is representing the observed pollinator composition of either one or thrree minutes of observation. 4 Results 47

Within the genus Salvia the species Salvia farinacea was taken for the wwhole day observation. The represented variety was ‘Miidnight Candle’. The flower number increased from 100 inflorescences to 160 inflorescences while the observation time. Due to the clear flower arcchitecture the observation of pollinators was simple to conduct. Loower number of pollinators made it easy to exactly differentiate between present and new arrived pollinators. Wild bees were mainly represented by Anthidium manicatum, Xylocopa sp. and by small bees from the genus Lasioglossum or Halictus. The curve in Figure 22 shows a more or less stable number of visitors between 11.35 and 17.35 o’clock. Samme as for Euphorbia hypeericifolia the three minute observation did not result in a three times higher number of pollinators.

Figure 22: Whole day observation of the speccies Salvia farinacea; represented by the variety ‘Midnight Can- dle’. Each bar is representing the observed pollinator composition of either one or thrree minutes of observation.

4 Results 48

The species Scaevola aemula was reppresented by the variety ‘Exp. Biccolor Purple White’. The number of flowers ranged between 900 and 1000 flowers throughout the time of observation. The huge overhanging plant shape made it difficult to survey all plant parts at once. Nevertheless due to the relative low number of pollinators all could be easily detect- ed. Also new arriving pollinators were easy to observe. Between 11.40 and 16.40 o’clock the number of pollinators was relative stable.

Figure 23: Whole day observation of the species Scaevola aemula; represented by the variety ‘Exp. Bicolor Purple White’. Each bar is representing the observed pollinator composition of either one or three minutes of observation.

4 Results 49

The Bidens ferulifolia species was represented by the highly frequentted white flowering variety ‘Pirate's Pearl White’. The number of flowers ranged between 400 and 500. The parabolic shape in Figure 24 shows a saturation of pollinator number between 11.45 and 13.45 o’clock. Compared to the other species number of pollinators decreases earlier in the observed variety ‘Pirate's Pearl White’.

Figure 24: Whole day observation of the species Bidens ferulifolia; represented by thhe variety ‘Pirate's Pearl White’. Each bar is representing the observed pollinator composition of either one or three minutes of observation.

4 Results 50

The observed variety of the genus Dahlia was ‘Dahlegria White’. Exceppt for one day were the number of flowers was 16 all the other observation dates ranged at a number of 25. The wild bees were mainly represented by Halictus scabiosae. Counted poollinators remained relative stable between 10.50 and 16.550 o’clock.

Figure 25: Whole day observation of the genus Dahlia; represented by the variety ‘Dahlegria White’. Each bar is representing the observed pollinator composition of either one or three minutes of observation.

4.2 Insect sampling

In total 106 individuals were caught while the observation time. 34 different species were identified in the laboratory, shown in Table 4. Those 34 species belonged to 19 genera. The genus Xylocopa was not caught because of simple identification in the field. Therefore an overall number of 35 species were iddentified on the observed flowers. As explained in 3.4.1 the sampling was rather conducted subjective to catch those individuals which could not be identified in the field. The sampling was conducted either before or after the observation. Most insects were sampled while walking between the different flower varieties and looking for unknown species. Standing at single varieties for longer time often resulted in viewer catches. The flower on whicch each individual insect was caught was also noted for the further pollen analysis. The total table of all sampled insects and their flowers from which they were collected can be looked up in the Appendix 1. Depending on the species more female or male individuals were noticeed in the field. The species Halictus scabiosae was mainly represented by male individuals. Halictus scabiosae has often been recognized on Dahlia. Due to the late season samppling also Queens of the genus Bombus were present. As for example two queens of the species Bombus sylvarus were sampled. 4 Results 51

Table 4: Genus and species of the sampled insects. Insects were sampled between 25.07.2016 and 30.08.2016 and caught individually from ornamental flowers. no. Genus Species no. Genus Species 1 Andrena flavipes 19 Heriades crenulatus 2 Anthidium manicatum 20 Hylaeus difformis 3 Anthophora quadrimaculata 21 Hylaeus hyalinatus 4 Apis mellifera 22 Hylaeus communis 5 Bombus terrestris complex 23 Hylaeus signatus 6 Bombus sylvarus 24 Isodontia mexicana 7 Bombus pascuorum 25 Lasioglossum politum 8 Cerceris rybyensis 26 Lasioglossum malachurum 9 Cerceris arenaria 27 Lasioglossum laticeps 10 Episyrphus balteathus 28 Lasioglossum morio 11 Eristalinea helophilus 29 Lasioglossum calcaetum 12 eristalinus sepulchralis 30 Lasioglossum pauxillum 13 Eristalis arbustorum 31 Megachile willughbiella 14 Eristalis tenax 32 platycheirus pipiens 15 Halictus subauratus 33 Sphaerophoria scripta 16 Halictus tumulorum 34 Syrphidae vitripennis 17 Halictus scabiosae 35 Xylocopa valga/Violacea 18 Halictus tumulorum

A special discovery was the sampled species Hylaeus signatus which is described as a oligolectic species on Reseda (Amiet & Krebs 2012). One male individual was sampled on Euphorbia hypericifolia. In total 15 different pollinator species were identified on the Eu- phorbia hypericifolia. Which were mainly smaller wild bees from the Halictus, Lasioglos- sum and Hylaeus genera and some Crabroidae from the genus Cerceris. Ten different species of pollinators were caught on the Calibrachoa including Apis mellifera. Bigger insects from the Bombus genus were sampled as well as smaller bees like La- sioglossum morio.

4.3 Pollen sampling

The collected 106 insects from Appendix 1 were evaluated on their pollen load. All insects were analyzed under the binocular to detect present pollen grains. 75 out of the 106 insects had a visible amount of pollen grains on their surveys. Removing single pollen grains with a preparation needle turned out being very time consuming. Whereas removing pollen from pollen sacs or other parts of the body with huge amounts of pollen went fast. Due to sometimes very low number of pollen, four prepared microscope slides of the pollen samples couldn’t be evaluated. The Kaiser’s glycerol-gelatine which sometimes flooded the pollen grains beside the cover glass might be the reason why some pollen was not visible anymore. The results were highly diverse from pollinators carrying only one type of pollen to pollinators carrying nine different pollen types; showing in Appendix 2. The pollen was 4 Results 52 only evaluated qualitative. Evaluation of the quantitative composition was not possible because of to low quantity of pollen. Nevertheless for most of the sampled insects it was possible to identify the pollen they carried on their surveys. It was also possible to identify whether pollinators were only on the plant on which they were sampled or whether they have been somewhere else before. For the genus Galibrachoa it was from interest to find out whether pollinators are only using the nectar or whether wild bees collect pollen for the rearing of their larva. As shown in Table 5 most of the sampled pollinators from Calibrachoa had either dominant occurrence or 99,9% of its transported pollen from Calibrachoa.

Table 5: Pollen analysis from insects sampled while visiting the Calibrachoa flower. The results are given in a qualitative form. Samples were high amount of Calibracoa pollen was visable lacking other pollen was expressed with (99,9%)

no. flower sampled identified pollen genus species sex 18 Calibrachoa Calibrachoa (99,9%) Apis mellifera ♀ 5 Calibrachoa Calibrachoa (99,9%) Bombus terrestris complex ♀ 6 Calibrachoa Malvacea (Hibiscus) Bombus terrestris complex ♀ Calibrachoa (dominant), Mentha (some), 7 Calibrachoa Circium cardus (one pollen grain) Bombus sylvarus ♀ 8 Calibrachoa Calibrachoa (99,9%) Bombus sylvarus ♀ 2 Calibrachoa Calibrachoa (dominant), Bidens Bombus pascuorum ♀ 3 Calibrachoa Calibrachoa (99,9%) Bombus pascuorum 55 Calibrachoa Trifolium repens, Trifolium pratense Bombus pascuorum ♀ 56 Calibrachoa Calibrachoa (99,9%) Bombus pascuorum ♀ 57 Calibrachoa Calibrachoa, Malvaceae Bombus pascuorum ♀ 38 Calibrachoa calibrachoa (99,9%) Halictus tumulorum ♀ 30 Calibrachoa Calibrachoa (dominant), Allium Lasioglossum laticeps ♀ 31 Calibrachoa Calibrachoa (dominant), Circium cardus Lasioglossum politum ♀ 33 Calibrachoa Calibrachoa, Bidens, Achelleae Lasioglossum malachurum ♀ 35 Calibrachoa Calibrachoa (99,9%) Lasioglossum morio ♀ Calibrachoa (dominant), Asteraceae, 36 Calibrachoa Callesthecia Lasioglossum malachurum ♀ 39 Calibrachoa calibrachoa (99,9%) Lasioglossum malachurum ♀ 40 Calibrachoa calibrachoa (99,9%) Lasioglossum pauxillum ♀ Asteraceae, Calibrachoa,Taraxacum (one 37 Calibrachoa pollen grain), plantago (one pollen grain) Lasioglossum calcaetum ♀ 11 Calibrachoa Campanula (dominant), Achillea Megachile willughbiella ♂

5 Discussion 53

5 Discussion

This discussion first of all will evaluate the overall field trial regarding the practicability of the used methods. The single results of the insect attractiveness regarding the main observed flowers will then be clarified as a second step. Focusing on the used observation method in this thesis it can be said that observing the pollination behaviour is up to now the best possibility to ensure that pollinators indeed collected nectar and pollen. Even more it is possible to specify the pollinator by catching and identifying. This gives the opportunity to identify the composition of the present pollinators for a plant. The additional pollen sampling than further on proofed the plant source of pollen which the insect was carrying. Catching pollinators with pan traps or other traps can achieve higher sample numbers as shown for other studies (Guilbaud et al. 2014; Bates et al. 2011). Nevertheless these sampled insects cannot be assigned to the flower they pollinated. Further on it is known that some of the coloured pen traps attract insects. Whereas pan traps also are poor in catching some species which might distort the results (Guilbaud et al. 2014; Roulston et al. 2007). The observation method of this field trial resulted in re- peatable and informative results wherefore it can be seen as a convincing method. To get significant results out of it at least three samples or even five should be used for further field trials. As 3.3.2 shows, the different observation times of one and three minutes resulted in relative same composition rates of pollinators. The number of pollinators for the three minute observation was not three times as high as for the one minute. This can be assigned to the point that already present pollinators have been counted when starting observation at the plant. The one minute observation turned out to be long enough in case of observing plants with a higher range of present insects. Therefore having several short observation points within one day can be recommended. Also other study designs used short term or so called snap shot observations for the evaluation of flower attractiveness (Salisbury et al. 2015; Garbuzov & Ratnieks 2014b). Beside this for flowers with a huge number of pollinators if often became difficult after a long time of observation to separate between new and already present pollinators. Based on this the snap shot observation turned out as a good method. For flowers with a lower number of pollinators a longer observation time might be necessary. Longer observation times can increase the chance of identifying pollinators of semi-attractive plants. But longer observation times also decrease the number of possible observations per day. The observation time has to be weighing out with the number of observations to be conducted. Another important aspect is the time at which the observation takes place. The whole day every hour observation showed that for most of the flowers a timeframe of four to five hours at the afternoon achieved stable results. Therefore the observations should always be conducted within this timeframe.

5 Discussion 54

Although the conducted sampling of pollinators was done selectively and without any fixed sampling protocol it reached relative high number in species. Compared to pan trap sampling this direct sampling has three advantages. First of all the method is pollinator preserving by sampling only those pollinators needed. Second the method can ensure that the insect were visiting the sampled plant variety. At last there are no biases concerning any pollinator species which often occur when using traps. The main disadvantage is the huge time amount needed for the observation. But for further insect preparation steps the direct sampling is more efficient. Sampled insects from pan traps need additional time for sorting, cleaning and trying before fixing is possible. Another important point is the purity of the single caught insect. The pollen of the individually caught insects isn’t contaminated with pollen of other insects or plants. Therefore the sampled insects Practical feasibility for the insect pollen samples were depending the quantity of pollen the insects carried. Insects carrying only small amounts of pollen grains were very time consuming. Every pollen grain had to be removed individually. In addition only few pollen grains could be used for the following analysis which decreases the significance of the results. Making pollen preparations from insects carrying bigger quantities of pollen was from higher informative value. Moreover the time consumption was less and the imple- mentation more practical. Those pollen preparations had a high number of pollen countable which made a specification of the distribution possible. Not all insects could be used for a pollen analysis which is mirroring the point that some pollinators were carrying none or only few amounts of pollen. Many pollen samples fitted to the flowers on which the insects have been collected. For Calibrachoa, Salvia and Dalia the majority of pollen samples corresponded. Sixteen of the samples from Calibrachoa had either dominant or 99,9% of the pollen corresponding. This shows that ornamental flowers even having a low visitation rate can provide pollinators with pollen as protein source. Nevertheless to evaluate the nutritional aspects of the different ornamental flowers a nutritional analysis has to be conducted. This pollen analysis can be recommended for further research. To reach higher significance only those insects carrying bigger amounts of pollen should be sampled. As the relative high corresponding rate is indicating this pollen analysis can be seen as a good field method to analyse caught insects on the flower they visited. Moreover it is possible to identify pollen sources of pollinators without knowing the plant source they visited. Never- theless to make the insect catching and pollen analysis statistical valuable a more detailed and random method design is needed. Due to the selective sampling some plants were sampled more often as for example the different Calibrachoa varieties. This was mainly done to have more samples for the following pollen sampling. Therefore there are only qualitative conclusions possible.

5 Discussion 55

Overall it can be said that ornamental flowers can be an attractive food source. However as already stated by literature attractiveness can highly differ between varieties even within one species (Garbuzov & Ratnieks 2015; Garbuzov & Ratnieks 2014b). In the following section the observed ornamental flowers will be discussed regarding their insect visiting rates. The observation of Calibrachoa resulted in very little number of visiting pollinators for all the tested varieties compared to other observed species. Due to this little number of pollinators no conclusions concerning pollinators preferences was made. As it was shown in other researches; pollinators have strong preferences concerning their choice of food source (Vaudo et al. 2016; Klahre et al. 2011). In the case of this conducted outdoor experiment it can be expected that pollinators preferred other flowers towards Calibrachoa. To test different Calibrachoa varieties on their decisive flower traits concerning pollinator preferences another study design has to be chosen. This improved study design should ensure Calibrachoa as the only source of food for the pollinators. One option could be a semi-outdoor experiment in which Calibrachoa and pollinators are located in a tent or green house. Even smaller indoor set ups, like Klahre et al. (2011) used when testing different Petunia species in wind tunnels, might be a good option. These controlled environments could give more precise information on favourable Calibrachoa traits. For statistical analysis a randomised block design with at least five replicates could be a good option. Even without having statistical verification; colour preferences of the pollinators can be expected. Yellow had the highest number of visited pollinators followed by blue and white. The red coloured flowers were visited lowest. Similar principals were found in literature where red coloured flowers were assigned to bird pollinators (Revert et al. 2016; Shrestha et al. 2013). Nevertheless having a little number of visited pollinators at least ten different species were identified visiting Calibrachoa. In this case Calibrachoa was used as nectar or pollen source. The pollen analysis proofed that some species had pure Calibrachoa- pollen-sacs. Concerning the relative short observation time and little number of caught insects it can be assumed that even more species can be fed by Calibrachoa. This fact can be from interest concerning future plans of breeding a pollinator friendly Calibrachoa variety. But as already stated by Salisbury et al. (2015) exotic ornamental flowers as Cali- brachoa are less preferred by pollinator compared to native plants. Therefore from the current position it cannot be expected to result in a pollinator friendly variety reaching or even overtaking native flower species concerning their benefits for urban pollinators. Variation between the different ornamental flower species and even within a species concerning the visitation rate of pollinators was quite high. Also the composition of pollinators differed highly between the species. Other studies already proofed that ornamental flower can have huge ranges in their pollinator attractiveness (Garbuzov & Ratnieks 2014b). Nev- ertheless only little is documented about the attractiveness for pollinators on ornamental flowers. If recommendation lists were available the information were often lacking quality 5 Discussion 56 and good background knowledge (Garbuzov & Ratnieks 2014a). This makes it even more important to continue the evaluation of ornamental flower varieties. However it is not yet decided what the term ‘pollinator friendly’ means and how it has to be examined. This thesis tried to determine pollinator friendly flowers by real time observation. Therefore flowers with high number of visiting pollinators will be defined as relative pollinator friendly within this discussion. But it should be mentioned that this kind of definition excludes many other important factors. In this case the nutritional quality of nectar and pollen is not from interest. This quality aspect should play a role in further research to consider the quality aspect of pollination in terms of pollinator food intake. The preferences of insects concerning the species Dahlia differed mainly in-between the flower shapes. Gathered results can be compared with the results of Garbuzov et al. (2015). Open shaped flowers can be assigned as relative pollinator friendly flowers. Whereas filled flowers had only few pollinators visited and can therefore not be recommended as pollinator friendly flowers. The main reason for those preferences might be the accessibility of the anthers and nectaries which are retarded for the filled species. Dahlia seems to be interest- ing for bigger pollinators as Bombus sp. and Apis mellifera; whereas smaller species do not profit from Dahlia. Compared to Dahlia the species Euphorbia hypericifolia resulted in the most divers’ pollinator composition within this field study. Visiting pollinators where mainly smaller species. The numbers of visitors increased while the overall observation time. This might indicate the end of native flower season. One advantage of exotic ornamental flowers compared to native species is their lasting flower period (Matteson et al. 2013; Wray & Elle 2014). In this case attractive species of Euphorbia hypericifolia can be recommended as a late season food source for pollinators. The high preference variation in-between the species might also be based on the flower architecture. Some varieties with low or even no pollinators seemed to be sterile types. Therefore same as for Dahlia is valid; not all species are pollinator friendly and a precise choice of varieties is important. Euphorbia hypericifolia had a huge range of pollinators and therefore many species can profit from it. As already mentioned there is no definition concerning the term ‘pollinator friendly’ and it would be too simple to value a flower more important because of a more diverse attracted pollinator range. Bidens ferulifolia and Scaevola for example had Apis mellifera as their main pollinator. Having a low diversity in pollinators but a high number of those pollinators can also be seen as ‘pollinator friendly’. This shows once more that a more detailed definition and a list of criteria is needed to define this term. How diverse the attractiveness for pollinators can be within one genus was shown by the different salvia species and their individual varieties. They differed in composition and abundance of pollinators. Indicating that recommendations on the basis of genera are impossible and also on the species level cannot be advised. Crucial is a deep valuation of ornamental flowers based on the variety level. 5 Discussion 57

This variety level is that important because of high morphological variation due to ornamental flower breeding. One example is the species Antirrhinum majus known for its typical flower shape. On the ornamental flower market some varieties exist having an open shaped flower without the regular ‘lips’ that are closing the corolla tube. Therefore little pollinator were observed to enter this flower which was not expected from the original flower shape. This is pointing out that different varieties from the same species can benefit several different groups of pollinators. Pollinators can also profit from flowers which they normally cannot access due to morphological characteristics. Lantana for example is a species having long and narrowed corolla tube only accessible by pollinators with long tongues. Nevertheless Lantana was visited by species having relative short tongues. This was possible due to nectar robbing wherefore the Bombus sp. was biting holes into the corolla tube of Lantana. This example shows how flexible pollinators can deal with exotic species. And points out once more that exotic ornamental flower can be seen as an additional support of food source beside the native plant species. 6 Conclusion and outlook 58

6 Conclusion and outlook

This master thesis had the objective to gain information on insects’ preferences concerning ornamental flowers and moreover evaluating methods to estimate these preferences. As shown by the literature review the topic of insect friendly flowers in urban areas is from growing interest although only little has been researched. The used observation methods in this thesis generated a good basic knowledge about ornamental flower varieties and their flower visiting insects. It can be said that there exists a high variation among and even within species in regard to their visitation rate of insects. Furthermore the composition of visiting insects between different flower species can have high variations More insect friendly varieties are wanted for the bed and balcony market. The generated results of this field trial can be seen as a starting point for further research. A European Innovation Partnership (EIP) Project starting in March 2017 is addressing the idea of breeding insect friendly ornamental flower varieties. Based on insect preferences focusing on the Calibrachoa - a popular bet and balcony cultivar - an innovative breeding program will be designed. To determine important floral traits for the further breeding steps semi- outdoor experiments will be conducted. This kind of controlled environment will force insects feeding from Calibrachoa. With this the abundance of visitors per plant can be increase compared to the low abundance in the outdoor experiments. Planting ornamental flowers in urban areas do not directly ensure insect biodiversity within a city. To better understand the foraging behavior of insects in urban areas, studies have to be conducted in different urban environments. Urban research fields can than clarify whether insects rely on the provided ornamental flowers or not. Further on recommendation lists on insect friendly ornamental flowers have to be improved with scientific based data. Overall a broader scientific knowledge platform on insect friendly ornamental flowers has to be established. Appendix 59

Appendix

Appendix 1: Caught and identified insects with their genus, species, sex and the date and place of collection.

Genus Species sex collection place/plant date Andrena flavipes ♀ Sanvitalia 08.08.16 Anthidium manicatum ♂ Salvia interdisciplinary 01.08.16 Anthidium manicatum ♀ Salvia farinacea 01.08.16 Anthophora quadrimaculata ♀ Lantana 15.08.16 Apis mellifera ♀ Calibrachoa 30.08.16 Bombus terrestris complex ♀ ‐ ‐ Bombus terrestris complex ♀ Calibrachoa 30.08.16 Bombus terrestris complex ♀ ‐ ‐ Bombus terrestris complex ♀ Calibrachoa 30.08.16 Bombus terrestris complex ♀ Calibrachoa 30.08.16 Bombus terrestris complex ♂ Salvia farinacea 01.08.16 Bombus sylvarus ♀ Calibrachoa 30.08.16 Bombus sylvarus ♀ Calibrachoa 26.07.16 Bombus sylvarus ♀ Calibrachoa 30.08.16 Bombus pascuorum ♀ Salvia farinacea 01.08.16 Bombus pascuorum ♀ Euphorbia hypericifolia 23.08.16 Bombus pascuorum ♀ Calibrachoa 30.08.16 Bombus pascuorum ♀ Calibrachoa 30.08.16 Bombus pascuorum ♀ ‐ ‐ Bombus pascuorum ♀ Calibrachoa 26.07.16 Bombus pascuorum ♀ Antirrhinum majus 16.08.16 Bombus pascuorum ♀ Calibrachoa 30.08.16 cerceris rybyensis ♀ Euphorbia hypericifolia 23.08.16 cerceris rybyensis ♂ Euphorbia hypericifolia 16.08.16 cerceris rybyensis ♂ Euphorbia hypericifolia 23.08.16 cerceris rybyensis ♂ Euphorbia hypericifolia 23.08.16 cerceris rybyensis ♂ Euphorbia hypericifolia 08.08.16 cerceris arenaria ♂ Euphorbia hypericifolia ‐ Episyrphus balteathus ♀ ‐ ‐ Episyrphus balteathus ♀ ‐ ‐ Eristalinea helophilus ♀ ‐‐ Eristalinea helophilus ♀ ‐ ‐ eristalinus sepulchralis ♀ ‐‐ Eristalis arbustorum ♀ Euphorbia hypericifolia 01.08.16 Eristalis arbustorum ♀ Euphorbia hypericifolia 23.08.16 Eristalis arbustorum ♀ Euphorbia hypericifolia 23.08.16 Eristalis tenax ♀ Xerochrysum bracteatum 01.08.16 Eristalis tenax ♀ Euphorbia hypericifolia 01.08.16 Eristalis tenax ♀ Bidens ferulifolius 30.08.16 Eristalis tenax ♀ Bidens ferulifolius 30.08.16 Eristalis tenax ♀ Bidens ferulifolius 23.08.16 Halictus subauratus ♀ Bidens ferulifolius 08.08.16 Halictus subauratus ♀ Xerochrysum bracteatum 01.08.16 Halictus subauratus ♀ Bidens ferulifolius 08.08.16 Halictus subauratus ♀ Xerochrysum bracteatum 01.08.16 Halictus subauratus ♀ Xerochrysum bracteatum 16.08.16 Halictus subauratus ♀ Xerochrysum bracteatum 01.08.16 Halictus subauratus ♂ Euphorbia hypericifolia 16.08.16 Halictus tumulorum ♀ Antirrhinum majus 16.08.16 Halictus tumulorum ♂ Xerochrysum bracteatum 01.08.16 Appendix 60

Halictus scabiosae ♂ Dahlia 08.08.16 Halictus scabiosae ♂ Bidens ferulifolius 24.08.16 Halictus scabiosae ♂ Dahlia 08.08.16 Halictus scabiosae ♂ Dahlia 23.08.16 Halictus scabiosae ♂ Bidens ferulifolius 23.08.16 Halictus scabiosae ♂ Dahlia 23.08.16 Halictus scabiosae ♂ Bidens ferulifolius 08.08.16 Halictus scabiosae ♂ Dahlia 08.08.16 Halictus scabiosae ♂ Xerochrysum bracteatum 16.08.16 Halictus scabiosae ♂ Bidens ferulifolius 08.08.16 Halictus scabiosae ♂ Dahlia 23.08.16 Halictus scabiosae ♀ ‐‐ Halictus scabiosae ♀ Xerochrysum bracteatum 01.08.16 Halictus scabiosae ♀ Dahlia 23.08.16 Halictus scabiosae ♀ Bidens ferulifolius 24.08.16 Halictus scabiosae ♀ Dahlia 26.07.16 Halictus tumulorum ♀ ‐ ‐ Heriades crenulatus ♀ Xerochrysum bracteatum 01.08.16 Isodontia mexicana ♀ Euphorbia hypericifolia 23.08.16 Isodontia mexicana ♀ Euphorbia hypericifolia 23.08.16 Lasioglossum politum ♀ Calibrachoa 26.07.16 Lasioglossum malachurum ♀ Osteospermum 26.07.16 Lasioglossum malachurum ♀ Calibrachoa 26.07.16 Lasioglossum malachurum ♀ Calibrachoa 26.07.16 Lasioglossum malachurum ♀ Calibrachoa 26.07.16 Lasioglossum laticeps ♀ ‐ ‐ Lasioglossum laticeps ♀ calibrachoa 26.07.16 Lasioglossum laticeps ♀ ‐ ‐ Lasioglossum laticeps ♂ Euphorbia hypericifolia 23.08.16 Lasioglossum laticeps ♂ Euphorbia hypericifolia ‐ Lasioglossum morio ♀ Euphorbia hypericifolia 01.08.16 Lasioglossum morio ♀ Calibrachoa 30.08.16 Lasioglossum morio ♀ Salvia farinacea 01.08.16 Lasioglossum morio ♀ hand 25.07.16 Lasioglossum morio ♀ Sanvitalia 08.08.16 Lasioglossum morio ♀ Antirrhinum majus 16.08.16 Lasioglossum morio ♀ Scaevola 08.08.16 Lasioglossum morio ♀ Euphorbia hypericifolia 08.08.16 Lasioglossum morio ♀ Scaevola 08.08.16 Lasioglossum morio ♀ Salvia farinacea 01.08.16 Lasioglossum morio ♀ Scaevola 08.08.16 Lasioglossum morio ♀ ‐‐ Lasioglossum morio ♂ Euphorbia hypericifolia 23.08.16 Lasioglossum morio ♂ ‐‐ Lasioglossum morio ♂ Euphorbia hypericifolia 08.08.16 Lasioglossum calcaetum ♀ ‐‐ Lasioglossum pauxillum ♀ Calibrachoa 26.07.16 Lasioglossum pauxillum ♂ Euphorbia hypericifolia 23.08.16 Megachile willughbiella ♂ Calibrachoa 26.07.16 Megachile willughbiella ♀ Salvia farinacea 01.08.16 platycheirus pipiens ♀ ‐‐ sphaerophoria scripta ♂ Euphorbia hypericifolia 23.08.16 sphaerophoria scripta ♂ ‐‐ sphaerophoria scripta ♂ ‐‐ sphaerophoria scripta ♂ ‐ ‐ syrphidae vitripennis ♀ Bidens ferulifolius 08.08.16

Appendix 61

Appendix 2: The location flower on which the insects were caught compared to the botanical pollen groups which were found on the microscope slide.

No. location flower botanical groups date genus species sex

54 Antirrhinum majus Salvia, Xerochrysum bracteantum, Calibrachoa 16.08.16 Bombus pascuorum ♀

59 Argyranthemum no Pollen present ‐ Sphaerophoria scripta ♂

43 Bidens ferulifolia Bidens, Helianthus annuus, Xerochrysum bracteantum 08.08.16 Halictus subauratus ♀

44 Bidens ferulifolia Taraxacum, Helianthus annuus, Senecio 24.08.16 Halictus scabiosae ♀

49 Bidens ferulifolia Dalia, Xerochrysum bracteantum 24.08.16 Halictus scabiosae ♂ Dalia, Xerochrysum bracteantum, Helianthus annuus, Urtica 52 Bidens ferulifolia (one pollen grain) 23.08.16 Halictus scabiosae ♂

60 Bidens ferulifolia no Pollen present 08.08.16 Syrphidae vitripennis ♀ Asteraceae, Apiaceae, Ambrosia, (2 pollen grains), Yuk‐ 67 Bidens ferulifolia landaceae (2 pollen grains) 30.08.16 Eristalis tenax ♀

69 Bidens ferulifolia Apiaceae, Helianthus annuus (1 pollen grain) 23.08.16 Eristalis tenax ♀

70 Bidens ferulifolia Helianthus annuus, Ambrosia 30.08.16 Eristalis tenax ♀

4 Calibrachoa Anthriscus, Senecio, Mentha, Centaurea cyanus, Salvia ‐ ‐ ‐ ‐

2 Calibrachoa Calibrachoa (dominant), Bidens 30.08.16 Bombus pascuorum ♀

3 Calibrachoa Calibrachoa (99,9%) 26.07.16 Bombus pascuorum

5 Calibrachoa Calibrachoa (99,9%) 30.08.16 Bombus terrestris complex ♀

6 Calibrachoa Malvacea (Hibiscus) 30.08.16 Bombus terrestris complex ♀ Calibrachoa (dominant), Mentha (some), Circium cardus (one 7 Calibrachoa pollen grain) 26.07.16 Bombus sylvarus ♀

8 Calibrachoa Calibrachoa (99,9%) 30.08.16 Bombus sylvarus ♀

11 Calibrachoa Campanula (dominant), Achillea 26.07.16 Megachile willughbiella ♂

18 Calibrachoa Calibrachoa (99,9%) 30.08.16 Apis mellifera ♀

30 Calibrachoa Calibrachoa (dominant), Allium 26.07.16 Lasioglossum laticeps ♀

31 Calibrachoa Calibrachoa (dominant), Circium cardus 26.07.16 Lasioglossum politum ♀

33 Calibrachoa Calibrachoa, Bidens, Achelleae 26.07.16 Lasioglossum malachurum ♀

35 Calibrachoa Calibrachoa (99,9%) 30.08.16 Lasioglossum morio ♀

36 Calibrachoa Calibrachoa (dominant), Asteraceae, Callesthecia 26.07.16 Lasioglossum malachurum ♀

38 Calibrachoa calibrachoa (99,9%) ‐ Halictus tumulorum ♀

39 Calibrachoa calibrachoa (99,9%) 26.07.16 Lasioglossum malachurum ♀

40 Calibrachoa calibrachoa (99,9%) 26.07.16 Lasioglossum pauxillum ♀

55 Calibrachoa Trifolium repens, Trifolium pratense ‐ ‐ ‐ ‐

56 Calibrachoa Calibrachoa (99,9%) 30.08.16 Bombus pascuorum ♀

57 Calibrachoa Calibrachoa, Malvaceae 30.08.16 Bombus pascuorum ♀ Asteraceae, Calibrachoa,Taraxacum (one pollen grain), 37 Calibrachoa plantago (one pollen grain) ‐ Lasioglossum calcaetum ♀

12 Dahlia Taraxacum, Helianthus annuus, Dahlia 26.07.16 Halictus scabiosae ♀

13 Dahlia Dahlia, Helianthus annuus 23.08.16 Halictus scabiosae ♀

47 Dahlia Dahlia (dominant), Helianthus annuus 08.08.16 Halictus scabiosae ♂

48 Dahlia Dahlia (dominant), Apiaceae ‐ Halictus scabiosae ♂

50 Dahlia Dalia, Xerochrysum bracteantum, Helianthus annuus 23.08.16 Halictus scabiosae ♂

51 Dahlia Dalia, Xerochrysum bracteantum 08.08.16 Halictus scabiosae ♂

53 Dahlia Asteraceae , Xerochrysum bracteantum 08.08.16 Halictus scabiosae ♂

34 Euphorbia hypericifolia Euphorbia (99,9%) 01.08.16 Lasioglossum morio ♀

58 Euphorbia hypericifolia Linum, Pinus 23.08.16 Bombus pascuorum

63 Euphorbia hypericifolia Mentha, Apiaceae 01.08.16 Eristalis arbustorum ♀ Appendix 62

65 Euphorbia hypericifolia Salvia, Asteraceae, Apiaceae,Circium cardus 23.08.16 Eristalis arbustorum ♀

66 Euphorbia hypericifolia no Pollen present 23.08.16 Eristalis arbustorum ♀

73 Euphorbia hypericifolia Senecio, Euphorbiaceae, Apiaceae ‐ ‐ ‐ ‐

74 Euphorbia hypericifolia Asteraceae, Poaceae 23.08.16 Isodontia mexicana ♀ Pinus, Apiaceae, Robus, Bidens, Asteraceae, Borago, Euphor‐ 75 Euphorbia hypericifolia biaceae, Mentha, Salvia,Circium cardus ‐ Isodontia mexicana ♀

29 Osteospermum Euphorbiaceae 26.07.16 Lasioglossum malachurum ♀

1 Salvia farinacea Mentha, Helianthus annuus 01.08.16 Bombus pascuorum ♀

16 Salvia farinacea Salvia (dominant), Mentha 01.08.16 Anthidium manicatum ♀

17 Salvia farinacea Salvia (dominant), Malvacea ( one pollen grain) 01.08.16 Anthidium manicatum ♂

19 Salvia farinacea Salvia, Mentha, 01.08.16 Megachile willughbiella ♀

25 Salvia farinacea Salvia (dominant), Mentha 01.08.16 Lasioglossum morio ♀

32 Salvia farinacea Salvia (99,9%) 01.08.16 Lasioglossum morio ♀

10 Sanvitalia Anthriscus, Achillea, Helianthus annuus 08.08.16 Andrena flavipes ♀ Pinus (only one pollen grain), Helianthus annuus (only one 24 Sanvitalia pollen grain) 08.08.16 Lasioglossum morio ♀

22 Scaevola Scaevola 08.08.16 Lasioglossum morio ♀

28 Scaevola Xerochrysum bracteantum, Mentha 08.08.16 Lasioglossum morio ♀

14 Xerochrysum bracteatum Helianthus annuus 16.08.16 Halictus subauratus ♀

15 Xerochrysum bracteatum Helianthus annuus 01.08.16 Heriades crenulatus ♀

41 Xerochrysum bracteatum Xerochrysum bracteantum (99,9%) 01.08.16 Halictus subauratus ♀

42 Xerochrysum bracteatum Xerochrysum bracteantum (50%), Trifolium repens (50%) 01.08.16 Halictus subauratus ♀ Xerochrysum bracteantum (dominant), Apiaceae, Helianthus 46 Xerochrysum bracteatum annuus 01.08.16 Halictus scabiosae ♀

45 Xerochrysum bracteatum Xerochrysum bracteantum (dominant), Plantago, Buddleija 16.08.16 Halictus scabiosae ♂ Xerochrysum bracteatum/ Xerochrysum bracteantum (dominant), Euphorbiaceae, 64 Salvia Armeria, Taraxacum 01.08.16 Eristalis tenax ♀

27 ‐ ‐ ‐ Lasioglossum morio ♀

62 ‐ Asteraceae, Helianthus annuus ‐ Platycheirus pipiens ♀

68 ‐ Sinapis, Asteraceae, Taraxacum, Circium cardus ‐ Eristalinea helophilus ♀

Apiaceae, Achillea, Taraxacum, Circium cardus, phacelia 71 ‐ tannacetifolia, Asteraceae, Taraxacum, Helianthus annuus ‐ Eristalinea helophilus ♀

61 ‐ Helianthus annuus, Asteraceae,chenopodiacea ‐ ‐ ‐

72 ‐ Asteraceae, Centaurea jacea, Trifolium pratense ‐ Eristalinus sepulchralis ♀ Helianthus annus (dominant),Taraxacum, Centaurea cyanus, 9 ‐ Senecio ‐ Halictus scabiosae ♀

20 ‐ Dahlia, Malvaceae ‐ Episyrphus balteathus ♀

26 ‐ Lamiaceae (few) 25.07.16 Lasioglossum morio ♀ Taraxacum, Salvia, Helianthus annuus, Asteracea, Mentha, 23 ‐ Anthriscus ‐ Lasioglossum laticeps ♀

21 ‐ Euphorbiaceae, Asteracea, Taraxacum, Salvia ‐ Lasioglossum laticeps ♀

List of references 63

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Acknowledgement 71

Acknowledgement

Dankeschön, thank you, merci and mille grazie to all those who have supported me for the last months while counting bees, typing data and writing this thesis.

First of all I wish to thank Ute Ruttensperger; she gave me a call and asked me if I would like to cooperate with her on the topic of insect friendly ornamental flowers. At the time I told her that she was talking to the wrong person – but as you can see it turned out that Ute was right!

Then I would like to thank PD Dr. Peter Rosenkranz for his continuing guidance throughout the whole process of this master thesis. Especially, for his patience and for his kindness in placing his trust in me.

Prof. Dr. Martin Dieterich, I really appreciated having you as my second supervisor. I will always remember the day that we spend together in the field – it feels like yesterday.

Thanks, to Dr. Helmut Horn for helping me with the pollen analysis and for always being open for questions. Thanks, to Daniela Warzecha who spent days with me identifying all those fascinating bees – I really appreciate your work and enthusiasms

Of course, I want to thank all my colleagues from the Institute – I actually don’t know where to start and stop. I want to thank Birgit for always being such great company! Thanks to Franzi for being the best office mate ever. Also I want to thank Eva who always gave me positive reinforce- ment when I needed it. Thanks to the boys, Klaus, Raphael, Stefan and Daniel who could always calm me down. Thanks to Mervat for supporting me in the laboratory and making me the best Syri- an dishes. Thanks to, Bo, Manu and Dana for helping me in the laboratory.

What would my thesis look like without all the beautiful flowers I observed? Therefore I want to thank all those ‘green thumbs’ at the LVG-Heidelberg. Thanks to all the gardeners who did the cultivating and took care of the flowers; without their efforts it wouldn’t have been possible. Thanks to Susanne Bonk who helped me in the field. Also a great thank you to Rainer Koch who always had a motivating sentence for me. Thanks as well to Bernd Hoffstedde, who introduced me to the world of the 360° camera and all the other technical innovations.

Those which have always been there if I needed them are my friends. No words are enough to thank them; nevertheless they have to be written here in black letters: FELIX, MAXI, JANINA, INGA, LEA, FRANZI, SIMON, JANINA, ANJA, LISA, SARAH, JANE, MANU, HANNA, AN- NA, FALK, SARA, SARA, IVAN, LISA, LAURA, CLAIRE, CHRISTIANE, DOMINIK – but you are not black and white, you too colourful and bright in this world. I’m glad to have you!

I hope nobody got lost – if it happened I am sorry for that!

Thanks once more to all of you for trusting in me. Declaration 72

Declaration

I Name, First name Kretschmer, Lea Annina

Born on 07.06.1990

Matriculation number 529248

hereby declare on my honour that the attached declaration, to my Homework/Presentation Bachelor Thesis x Master Thesis has been independently prepared, solely with the support of the listed literature references, and that no information has been presented that has not been officially acknowledged.

Supervisor PD Dr. Rosenkranz, Peter

Thesis topic Attractiveness of ornamental flowers for pollinating insects in an urban area

Semester 5th

I declare, here within, that I have transferred the final digital text document (in the format doc, docx, odt, pdf, or rtf) to my mentoring supervisor and that the content and wording is entirely my own work. I am aware that the digital version of my document can and/or will be checked for pla- giarism with the help of an analyses software program. Also, I agree that the thesis is put into the department’s library and can be consulted by third par- ties. In addition, I agree that an abstract of my thesis may be put on the department’s website.

City, Date Signature