247 Key words: orchids, insects, arthropods, protection, plant ecology. Ključne besede: orhideje, žuželke, členonožci, zavarovanje, rastlinska ekologija. Corresponding author: A. M. Gnatiuk E-mail: gnatiukalla@gmail.com Received: 30. 10. 2022 Accepted: 23. 2. 2023 Diversity of Epipactis palustris (L.) Crantz (Orchidaceae) pollinators and visitors in conditions of Kyiv city (Ukraine) Abstract Epipactis palustris (L.) Crantz is a protected species in Ukraine. In order to organize optimal protection measures comprehensive studies of various aspects of its biology are necessary. The aim of this study was to determine the pollinators and visitors of E. palustris. The study was conducted in Kyiv city (Ukraine) between 2020 and 2022. The research focused on arthropod visitors in two human-made populations. Specimens of 31 families and 48 genera from 9 arthropod orders were identified and established. The greatest number of species represented Hymenoptera, Coleoptera and Diptera. Evaluation of visitor activity show that Hymenoptera were the most frequent visitors. E. palustris in the conditions of Kyiv is mainly characterized by entomophily. Halictidae, Apidae and Coccinellidae played an important role in E. palustris pollination. It was found that populations of E. palustris in the conditions of the Kyiv city form a unique self-regulating biocenosis that provides shelter and food for 53 identified species of arthropods. Iz vleček Epipactis palustris (L.) Crantz je v Ukrajini zavarovana vrsta in da bi zagotovili njeno optimalno varovanje, so potrebne raziskave različnih vidikov njene biologije. S to raziskavo smo hoteli ugotoviti, katere vrste jo oprašujejo in obiskujejo. Raziskavo smo izvedli v mestu Kijev (Ukrajina) med letoma 2020 in 2022. Osredotočili smo se na obiskovalce iz skupine členonožcev v dveh umetnih populacijah. Osebki so pripadali 31 družinam in 48 rodovom iz 9 redov členonožcev. Največ vrst je bilo iz skupin Hymenoptera, Coleoptera in Diptera. Spremljanje obiskovalcev je pokazalo, da so bili najbolj pogosti obiskovalci iz skupine Hymenoptera. Vrsta E. palustris je v razmerah v Kijevu večinoma entomofilna. Halictidae, Apidae in Coccinellidae so pomembne za opraševanje vrste E. palustris. Ugotovili smo, da je v populacijah E. palustris v mestu Kijev nastala samo regulirajoča biocenoza, ki nudi zavetje in hrano 53 vrstam členonožcev. Alla M. Gnatiuk1  , Mykola B. Gaponenko1  , Hanna Y . Honchar2  & Andrii M. Gaponenko3  DOI: 10.2478/hacq-2022-0018 22/2 • 2023, 247–262 1 M. M. Gryshko National Botanical Garden of National Academy of Science of Ukraine, Kyiv, Ukraine. 2 Institute for Evolutionary Ecology, National Academy of Sciences of Ukraine, Kyiv, Ukraine. 3 National University of Life and Environmental Sciences of Ukraine, Kyiv, Ukraine. 22/2 • 2023, 247–262 248 Gnatiuk et al. Diversity of Epipactis palustris pollinators and visitors in Kyiv Introduction Epipactis palustris (L.) Crantz (Orchidaceae) is a Palearc- tic orchid species that is included in Appendix II of CITES and the Red Book of Ukraine (Didukh, 2009). It occasionally occurs in Ukrainian Polissia, Roztochcha, Opillia, Forest-steppe, Steppe, the Carpathians and the Mountainous Crimea. It rarely occurs in northern part of Polissia, and in the south of the Forest-steppe. In the Steppe, it is known in the basin of the Dnipro and Siver- skyi Donets, in the Kherson region on the Tendrivska (T endra) Spit and on Dzharylgach. It occurs frequently in the Carpathians, found in the Chornogora, in the Gor- gans, in the Eastern Beskids. In Transcarpathia, it grows from the lowlands to the foothills. Plants very rarely occur on the Southern coast of Crimea. E. palustris often settle in peat bogs, marshy meadows and are phytocoenotically associated with sedge-sphagnum plant communities, as well as with grass-herbaceous marsh meadows (Gaponen- ko, 2013). Natural populations of the species were found in the vicinity of Kyiv (the village of Bobrytsia, Kyiv-Svy- atoshinsky district and Svyatoshin Lakes) (Sobko, 1989; Parnikosa & Shevchenko, 2006). The study of antecological features of E. palustris in the conditions of Kyiv is extremely important, as they dem- onstrate the prospects of preserving this species outside its natural habitats and its role in the formation of ur- ban ecosystems. Anthropogenic habitat transformation regarded as a principal cause of pollinator decline on a global scale, as well as an important factor directly and indirectly changing the species structure of pollinating in- sects (Rewicz et al., 2017). The presence of a forage base in urban conditions is decisive in preserving the biodiver- sity of useful insects (Honchar & Gnatiuk, 2020). Epipactis palustris is a nectar-bearing orchid that is vis- ited by a large number of insects. Marsh Helleborine’s nectar is rich in aromatic compounds. The main attract- ants found in nectar are: nonanal (pelargonaldehyde), decanal, eicosanol and its derivatives (Jakubska-Busse & Kadej, 2011). Nectar of Epipactis is a rich source of amino acids and carbohydrates, which are essential for the de- velopment of animals (Jakubska et al., 2005). Studies of pollinators and visitors of this orchid in different regions have shown that the species that dominate in pollination differ depending on the geography of the region of the research conducted (Jacquemyn et al., 2014). In a semi-wild population in Haren, the Netherlands, E. palustris was visited by honeybees, sawflies, parasitic hymenoptera, ants, hoverflies and other flies. Bumblebees rejected the flowers. Of the hoverflies, only Lejogaster met- allina (Fabricius, 1777) and males of Syritta pipiens (Lin- naeus, 1758) were pollinators. S. pipiens was common enough (20% of the visits) to substantially effect polli- nation. Small flies of several families were effective pol- linators but in low abundance (1%). Ants were the most frequent visitors (50%) and effected self- and cross-polli- nation. Honeybees (25%) pollinated and collected pol- linia for larval food. Other hymenopterans were very rare on the flowers (1%). Vespula vulgaris (Linnaeus,  1758) neglected the flowers. E. palustris was not autogamous here. The epichile bent down under heavy insects, and worked as a ‘springboard’ for light hymenopterans. On the epichile a taste nectar guide was found, effective for flies, ants, experienced honeybees and other hymenopter- ans (Brantjes, 1981). Epipactis palustris is well visited by insects, primarily by honey bees, solitary bees and hoverflies (Claessens & Kleynen, 2016). Van der Pijl and Dodson (1966) state that Marsh Helleborine is pollinated mostly by nectar feeding solitary wasps (Hymenoptera: Eumenidae) as well as by ants, bumblebees and hoverflies. Brantjes (1981) notes that Syrphidae (Diptera), Myrmi- ca sp. (Hymenoptera), Sarcophaga carnaria (Diptera) and Coelopa frigida (Fabricius, 1805) (Diptera) also belong to E. palustris pollinators. Observations on the pollination of E. palustris from the island of Oland (southern Sweden) reported a broad spectrum of insects feeding on the nectar too. Ants, bum- blebees and hover-flies were noted as the most frequent visitors, but generally played a minor role as pollinators. Solitary wasps (Hymenoptera, Eumenidae) were the most important pollen vectors, although present only at low frequencies. Ants were important pollinators in one lo- cality. Entomogamy prevails and the frequency of visits is much lower if the plant is growing in association with entomophilous competitors. On Oland in Sweden, Hy- menoptera (49.7%) and Diptera (40.8%) were the most frequent visitors, whereas Coleoptera (5.8%), Lepidoptera (2.8%) and Araneida (0.9%) were only occasionally ob- served visiting flowers of E. palustris (Nilsson, 1978). Kryvosheev (2012) notes that in the southern Urals, E. palustris is highly specialized to Vespidae. Research car- ried out in natural populations of Crimea also showed pollination by Vespidae: Polistes dominulus (Christ, 1791), Vespula vulgaris (Ivanov et al., 2009). Vast research on representatives of genus Epipac- tis and their pollination peculiarities were provided by A. Jakubska-Busse et al. in Poland, Czech Republic and Lithuania (Jakubska et al., 2005a, Jakubska-Busse & Kadej, 2011; Jakubska-Busse, 2011). It has been proven that Hymenoptera (Apis, Bombus) and representatives of Diptera (Empis, Episyrphus) are the main pollina- tors of E. palustris (Jakubska et al., 2005b; Jakubska- Busse & Kadej, 2011). Jakubska-Busse (2011) notes that 22/2 • 2023, 247–262 249 Gnatiuk et al. Diversity of Epipactis palustris pollinators and visitors in Kyiv H y m e n o p ter a : Apis mellifera (Linnaeus, 1758), Bombus lapidarius (Linnaeus, 1758), Bombus lucorum (Linnaeus, 1758), Diptera: Empis livida (Linnaeus, 1758), Episyrphus balteatus (De Geer 1776), Cetema cereris (Fallen 1820), Chloromyia formosa (Scopoli, 1763) belong to the true pollinators in Central Europe. On the other hand Dip - tera: Calyptrata sp., Coleoptera: Anomala dubia (Scopoli, 1763), Cantharis pellucida (Fabricius, 1792), Rhagonycha fulva (Scopoli, 1763), Gaurotes virginea (Linnaeus 1758), Oedemera femorata (Scopoli, 1763) and Lepidoptera: Ochlodes sylvanus (Esper, 1777), Aphantopus hyperantus (Linnaeus, 1759) belong to the flower visitors, that can carry out the pollination. In order to organize optimal protection measures for E. palustris in Ukraine, comprehensive studies of various aspects of its biology and ecology are necessary. The inter- action of plants and animals is one of the links to adap- tation to environmental conditions and coexistence with other living organisms. The aim of this study was to determine the visitors (pollinators and concomitant arthropods) of E. palustris plants in Kyiv (Ukraine). Material and methods Kyiv is located on the border of the Forest and Forest- steppe physiographic zones. The climate is moderately continental, with mild winters and warm summers. Aver- age monthly temperatures in January are −3.5 °C, in July +20.5 °C. The average annual amount of precipitation is 649 mm, with the maximum precipitation falling in July (88 mm), the minimum in October (35 mm). In winter, Kyiv has a snow cover with an average height of 20 cm of snow in February and a maximum of 44 cm (Osadchiy et al., 2010). The study of E. palustris was carried out on two artificial populations: 1) in M.M. Gryshko National Bo- tanical Garden of NAS of Ukraine (hereinafter¸– NBG) and 2) in the private garden that is inside the natural range of E. palustris. The area belongs to the Right-Bank Forest-steppe of Ukraine, a temperate climate zone. NBG is located in the Pechersk District of Kyiv in South-Eastern part of city. The population is located at the collection plot “Rare species of Ukrainian Flora” of the Natural Flora Department where E. palustris was planted since 1978 (plants originate from Vorokhta, which is an urban-type settlement located in the Carpathian Moun- tains on the Prut River) (Gaponenko, 2013). The popu- lation is local with vegetative reproduction. Plants grow along the pathway near the artificial pond, which pro- vides soil moisture in a semi-shaded condition. The soil in the study area is dark gray, sandy, with a humus content of about 2.6%, pH 6.5. The number of the population is not stable and depends on the lighting and moisture conditions during the growing season. The population had 55–75 generative shoots on a 0.9 m 2 in 2020–2022. Private garden is located in Solomianskyi District of Kyiv in the western part of the city, in the basin of Ly- bid river near the Sovki Lakes. The soil in the study area is gray, podzolic, sandy, with a low humus content, pH 6.5–7. The artificial population is local with vegetative re- production. Plants originate from NBG, planted in 1995. Artificial irrigation is provided during the growing season for soil moisture. There are 60–83 generative stems in population on a 1.4 m 2 area in a semi-shaded condition. The research was carried out in the growing seasons of 2020–2022. Observations of entomofauna related to Marsh Helleborine were carried out in field conditions during the flowering period. They were made over a span of 2–6 h, covering daylight hours (08.00–18.00 h). The sites were visited during sunny days, when pollina- tors have been shown to be most active. The arthropods were observed and photographed in natural conditions (Appendix 1–4). For the documentation purposes and identification some of observed insects were caught using entomological net. In the case when the species affiliation could be determined visually, the capture was not carried out, and the fact of the visit was only recorded in the field diary. Uncaught insects, the species of which cannot be determined from the photo, are assigned to the genus. The species composition of insects was studied based on collection materials and photos. Species were identi- fied using special literature (Medvedev, 1978; Gaetan du Chatenet, 1990; Oosterbroek, 2006; Pauly, 2015) as well as through specialized communities (iNaturalist, 2022) and a database (GBIF.org, 2022). Species are listed in an alphabetical order. Arthropod representatives observed during the study on E. helleborine have been divided into 5 groups (sta- tus) using a modified classification system introduced by Jakubska et al. (2005). Arthropods: 1) N – potential pollinators: nectar feeding insects visiting the flowers, but not demonstrated to carry pollen; 2) P – effective true pollinators or pollen vectors: flowers visitors touching the androecium and demonstrated to carry Epipactis pollen or pollinia on the body; 3) H – aphid hunters; 4) P – pests of Epipactis plants; 5) V– other visitors on Epipactis plant. The activity of visitors was evaluated on a 5-point scale: 1) very rare visitors – occur once or twice per observation season; 2) rare visitors – three or more occurrences; 3) un- common visitors – do not occur often or there are few of them; 4) common visitors – present on the plants almost every day, but their number is not large; 5) active visitors – occur every day in fairly large quantities. 22/2 • 2023, 247–262 250 Gnatiuk et al. Diversity of Epipactis palustris pollinators and visitors in Kyiv The surface of the flower’s lip and the structure of pol- linia were studied by the Stemmi 2000 light microscope using the “Axio Vision” program (Figure 2). Results and Discussion. Epipactis palustris (L.) Crantz is the most showy and conspicuous orchid of the genus. Together with E. vera - trifolia Boiss. & Hohen., it belongs to the section Ar - throchilium Irmisch, characterised by a non cup-shaped hypochile with lateral lobes and a hinged lip (Claessens & Kleynen, 2011). E. palustris is a perennial herbaceous plant. The plant consists of separate stems growing from a common rhizome. In studied populations the stems of plants reach a height of 20–60 cm and produce 15–30 flowers. The inflorescence is panicle-like, 8–19 cm long, drooping before flowering, then straight, sparse. The flowers are hanging, without a noticeable smell; the main color is white (due to the color of the lip). The flower perianth is bell-shaped and has a bipartite lip with the epichilium (part that serves as a landing place for insects) and a basal, cup shaped part where nectar is secreted (hy - pochile) (Figure 2). Perianth leaves are brownish-green or greenish-gray, greenish-white or reddish from the in - side, 9–12 mm long. The lip is almost as long as the outer petals of the perianth; hypochile is 4.5–5.5 mm long, grooved, with two blunt lobes in front, pinkish-white below, white above, with light purple veins, and yellow relief dots at the base. The epichile is 5.5–6.5 (8) mm wider than the hypochile, with which it is connected by a narrow bridge. The epichile is almost flat, broadly ovoid or round, blunt, wavy-toothed, white, with 2 longitudi - nal, front yellow combs at the base, its surface is smooth. Pollinaria about 1.8 mm posterior, yellow 2-lobed, with - out a stalk, connected by a small sticky viscidium. The flowers produce small amounts of nectar that is present on the labellu.The viscidium glues pollinia to the polli- nator and prevents pollinia from sliding out of the anther and contacting the stigmatic surface. The conspicuous flowers produce nectar in the hypochile mostly. In order to enable autogamy the pollinia must be able to contact the stigma. Figure 1: Locations of the Epipactis palustris populations in Kyiv: 1) M. M. Gryshko National Botanical Garden of NAS of Ukraine (hereinafter – NBG); 2) private garden. Slika 1: Lokacije populacij vrste Epipactis palustris v Kijevu: 1) Nacionalni botanični vrt M. M. NAS Ukrajine (v nadaljevanju – NBG); 2) zasebni vrt. 22/2 • 2023, 247–262 251 Gnatiuk et al. Diversity of Epipactis palustris pollinators and visitors in Kyiv Epipactis palustris has a flowering period from the mid-June until the end of 1st decade of July. In the years 2020–2022, flowering in populations in Kyiv starts be - tween June 13 and 20, peak flowering took place be- tween June 25 and 30. The earliest record for the end of flowering was 6 July 2020 and the latest was 10 July 2021. Plants of E. palustris are very attractive for insects in Kyiv. We noted visitors, pollinators, and other arthro - pods of E. palustris (Table 1). Figure 2: Parts of Epipactis palustris flower: a – labellum; b, d – pollinia; c, e – hypochile; f – epichile. Slika 2: Deli cveta Epipactis palustris: a – medena ustna; b, d – poliniji; c, e – hipohil; f – epihil. 22/2 • 2023, 247–262 252 Gnatiuk et al. Diversity of Epipactis palustris pollinators and visitors in Kyiv Order, Family, Species Status/ Activity 2020 2021 2022 Order Araneida Phalangiidae: Phalangium opilio (Linnaeus, 1758) V/3 V/3 V/3 Thomisidae: Misumena vatia (Clerck, 1757) V/4 V/4 V4 Thomisus sp. V/3 V/3 V/3 Order Coleoptera Cantharidae: Cantharis livida (Linnaeus, 1758) V/3 - N/1 Cerambycidae: Pseudovadonia livida (Fabricius, 1776) N/1 - N,V/1 Coccinellidae: Coccinella septempunctata (Linnaeus, 1758) - H/4 H,P/5 Harmonia axyridis (Pallas, 1773) H, P/5 H/5 P , H/5 Propylea quatuordecimpunctata (Linnaeus, 1758) H/4 - H,P/4 Scymnus frontalis (Fabricius, 1787) - H,P/1 - Tytthaspis sedecimpunctata (Linnaeus, 1758) - H,P/3 H/2 Curculionidae: Gymnetron rostellum (Herbst, 1795) - - V/1 Otiorhynchus ligustici (Linnaeus, 1758) - V/1 - Oedemeridae: Oedemera virescens (Linnaeus, 1767) N/1 - N/1 Order Diptera Anthomiidae: Leucophora sp. N/3 N/3 N/3 Hydrophoria lancifer (Harris, 1780) V/1 - - Bombyliidae: Hemipenthes morio (Linnaeus, 1758) N/3 N/3 N/3 Polleniidae: Pollenia sp. N/3 N/2 N/3 Sarcophagidae: Sarcophaga carnaria (Linnaeus, 1758) N/3 N/3 N/3 Stratiomyidae: Chloromyia formosa (Scopoli, 1763) V/2 V/2 V/1 Syrphidae: Eupeodes corollae (Fabricius, 1794) N,H/1 N,H/2 N,H/2 Sphaerophoria scripta (Linnaeus, 1758) H,N/3 H,N/4 N/4 Syritta pipiens (Linnaeus, 1758) - N/4 N/4 Order Hemiptera Aphidae: Aphis epipactis (Theobald, 1927) A/5 A/5 A/5 Flatidae: Metcalfa pruinosa (Say, 1830) A/3 A/4 A/3 Miridae: Deraeocoris ruber (Linnaeus, 1758) H/3 H/5 H/5 Lygus rugulipennis (Poppius, 1911) - - N/1 Stenotus binotatus (Fabricius, 1794) - - N/1 Pentatomidae: Palomena prasina (Linnaeus, 1761) V/1 V/1 V/1 Rhopalidae: Corizus hyoscyami (Linnaeus, 1758) - - V/1 Order Hymenoptera Apidae: Apis mellifera (Linnaeus, 1758) P/5 P/5 P/5 Bombus hortorum (Linnaeus, 1761) V/1 N/2 N/2 Bombus terrestris (Linnaeus, 1758) N/1 - N/1 Bombus pascuorum (Scopoli, 1763) V/1 - V/1 Halictidae: Lasioglossum malachurum (Kirby, 1802) P/4 P/4 P/4 Lasioglossum morio (Fabricius, 1793) P/4 P/4 P/5 Lasioglossum politum (Schenck, 1853) P/5 P/5 P/5 Lasioglossum zonulum (Smith, 1848) P/4 P/4 P/4 Colletidae: Hylaeus confusus (Nylander, 1852) - - N/1 Table 1: List of visitors of Epipactis palustris and their activity on population in Kyiv (Ukraine) during 2020–2022. Tabela 1: Seznam obiskovalcev vrste Epipactis palustris in njihova aktivnost v populaciji v Kijevu (Ukrajina) med 2020–2022. 22/2 • 2023, 247–262 253 Gnatiuk et al. Diversity of Epipactis palustris pollinators and visitors in Kyiv Epipactis palustris was visited by honeybees, bumble- bees, parasitic hymenopterans, ants, wasps, hover flies, soldier flies, flesh flies, dung flies and other flies, bugs, grass bugs, aphids, butterflies, dragonflies, spiders and grasshoopers (Apendix 1–4). 53 species from 28 families and 46 genera were identified and established: Phalangii- dae (Phalangium Linnaeus, 1758), Thomisidae (Mi- sumena, Latreille, 1804, Thomisus Walckenaer, 1805), Cantharididae Imhoff, 1856 (Cantharis Linnaeus, 1758), Cerambycidae Latreille, 1802 (Pseudovadonia Lobanov, Danilevsky & Murzin, 1981), Coccinellidae Latreille, 1807 (Harmonia Mulsant, 1850; Coccinella Linnaeus, 1758; Propylea Mulsant, 1846; Scymnus Kugelann, 1794; Tytthaspis Crotch, 1874), Curculionidae Latreille, 1802 (Otiorhynchus Germar, 1822, Gymnetron Schönherr, 1825), Oedemeridae Latreille, 1810 (Oedemera Olivier, 1763), Stratiomyidae Latreille, 1802 (Chloromyia Dun- can, 1837); Sarcophagidae Macquart, 1834 (Sarcophaga Meigen, 1826); Syrphidae Latreille, 1802 (Sphaerophoria Le Peletier & Serville, 1828; Syritta Le Peletier & Ser- ville, 1828; Eupeodes Osten Sacken, 1877), Bombyliidae Latreille, 1802 (Hemipenthes Loew., 1861), Anthomiidae Robineau-Desvoidy, 1830 (Leucophora Robineau-Des- voidy, 1830; Hydrophoria Robineau-Desvoidy, 1830), Polleniidae Brauer & Bergenstamm, 1889 (Pollenia Rob - ineau-Desvoidy, 1830), Aphididae Latreille, 1802 (Aphis Linnaeus, 1758), Miridae Hahn, 1831 (Lygus Hahn, 1833; Deraeocoris Kirschbaum, 1856), Rhopalidae Amyot & Serville, 1843 (Corizus Fallén, 1814), Pentatomidae Leach, 1815 (Palomena Mulsant & Rey, 1866); Flatidae Spinola, 1839 (Metcalfa Caldwell & Martorell, 1951), Apidae (Apis Linnaeus, 1758; Bombus Latreille, 1802), Halictidae Thomson, 1869 (Lasioglossum Curtis, 1833), Colletidae Lepetier,1841 (Hylaeus Fabricius, 1793), Cra- bronidae (Pemphredon Latreille, 1796, Cerceris Latreille, 1802), Eumenidae Leach, 1815 (Stenodynerus Saussure, 1863, Euodynerus Dalla Torre, 1904, Eumenes Latreille, 1802), Chrysididae Latreille, 1802 (Elampus Spinola, 1806), Formicidae Latreille, 1802 (Lasius Fabricius, 1804; Formica Linnaeus, 1758; T etramorium (Mayr, 1855), Pieridae Duponchel, 1835 (Pieris Schrank, 1801), Chrys- opidae Schneider, 1851 (Chrysoperla Steinmann, 1964), Platycnemididae Yakobson & Bianchi, 1905 (Platycnemis Burmeister, 1839; Enallagma Charpentier, 1840), Acridi- dae MacLeay, 1819 (Chorthippus Fieber, 1852, T ettigonii- dae Krauss, 1902, Phaneroptera Serville, 1831). Taxonomic diversity of visitors in Kyiv on E. palustris show that Hymenoptera (34%), Coleoptera (19%) and Diptera (17%) are represented by the greatest diversity, whereas, Neuroptera (2%) – by the smallest number of Order, Family, Species Status/ Activity 2020 2021 2022 Chrysididae: Elampus panzeri (Fabricius, 1804) - N/1 N/1 Crabronidae: Cerceris arenaria (Linnaeus, 1758) - - N/2 Pemphredon sp. H/3 H,P/4 P ,H/3 Eumenidae: Eumenes pedunculatus (Panzer, 1799) - - N/1 Euodynerus quadrifasciatus (Fabricius, 1793) - N\1 - Stenodynerus chevrieranus (Saussure, 1855) - - N/2 Formicidae: Formica cunicularia (Latreille, 1798) N,P/5 N/5 N/5 T etramorium caespitum (Linnaeus, 1758) - N/1 - Lasius niger (Linnaeus, 1758) N/5 N/5 N/5 Order Lepidoptera Pieridae: Pieris napi (Linnaeus, 1758) N/2 N/2 N/2 Order Neuroptera Chrysopidae: Chrysoperla carnea (Stephens, 1836) H/1 - H/1 Order Odonata Coenagrionidae: Enallagma cyathigerum (Charpentier, 1840) V/3 V/3 V/- Platycnemididae: Platycnemis pennipes (Pallas, 1771) V/5 V/5 V/5 Order Orthoptera Acrididae: Chorthippus sp. - - V/1 Tettigoniidae: Phaneroptera sp. N/1 V/1 N/1 Notes: Status: 1) N – potential pollinators; 2) P – true pollinators; 3) H – aphid hunters; 4) P – pests; 5) V – other visitors. Activity: 1 – very rare visitors; 2 – rare visitors ; 3 – uncommon visitors; 4 – common visitors; 5 – active visitors. 22/2 • 2023, 247–262 254 Gnatiuk et al. Diversity of Epipactis palustris pollinators and visitors in Kyiv species (Figure 2.). Evaluation of visitor activity show that Hymenoptera (36%) were the most frequent visitors, whereas, Lepidoptera (2%) and Neuroptera (0%) were only occasionally visiting of E. palustris (Figure 3, 4). It makes our data somewhat different from those reported in the literature. The species diversity of wild bees in the parks of Kyiv is about 115 species (Radchenko & Gonchar, 2019). Gaponenko et al., 2017). The Apis mellifera and species of Lassioglossum are in the lists of E. palustris visitors in the south-western Poland (Jakubska-Busse, 2005; Jakubska- Busse & Kadej, 2011), British Isles ( Jacquemyn et. al., 2014), and Crimea (Fateryga & Ivanov, 2012). Hymenoptera Apis and Lasioglossum were active polli- nators that were purposefully interested in the flowers of E. palustris and often carried pollinia. Bees from the genus Lasioglossum (body length ≈ 0.5 cm) carried pollinia on their backs, crawling from flower to flower, as they often could not fly away under their weight. The presence and frequent visitation of flowers by these bees is not surpris- ing, since these are the most common polylectic species, which occur in different biotopes. Despite their non-food specialization, these bees play an important role in the pollination of many plants, and this species of orchid is probably no exception. Bumblebees visited flowers most- ly by accident and did not linger on them for a long time. Wasps (Crabronidae, Eumeninae) and flies visited flow- ers, fed on nectar and could transfer pollen, but we did not manage to see these insects with pollinia (except for Pemphredon). Flowers with nectar attracted beetles (Oede- meridae and Coccinellidae) and butterflies. From 10 to 25 insects were observed per hour on the studied population of E. palustris flowers, and by 1:00 p.m., all open flowers had no pollinia. Nilsson (1978) noted, that «deposition and frequency of pollinaria, morphology and action of the hinged label- lum, and the time of flowering all suggest that E. palustris is adapted to pollination by solitary wasps, perhaps pri- marily to males of the genus Eumenes Latr.». However, our observations have not confirmed the main role of Vespidae in pollination. Solitary wasps were the rare visi- tors on E. palustris flowers. We would like to note the symbiosis of ants and the aphids, which plays a significant role in the life of these orchids. Among the recorded species of arthropods 13 species (25%) prey on aphids or feed on their secretions. Every year there are aphids on the E. palustris shoots. They populate the inflorescence zone even at the stage of budding. In the population, 15–40% of the shoots have aphids every year. Ants were regular visitors to E. palus- tris in all years of observation: they cared for aphids and drank nectar from flowers. We observed an ant with a pollinia fragment only once. The presence of aphids in the inflorescences attracted insects that prey on them even before the flowers open. During the flowering of plants, Syrphidae and Coccinellidae, both adults and their larvae, were active consumers of the Aphis. Coccinellidae adults often had pollinia or pollen attached to their backs and visited flowers. The Deraeocoris ruber, which simultane- ously numbered in 3–7 adults at the peak of flowering of Figure 3: Taxonomic diversity of visitors on E. palustris in 2020–2021. Slika 3: Taksonomska raznolikost obiskovalcev vrste E. palustris med 2020–2021. Figure 4: Activity of visitors on E. palustris in 2020–2021. Slika 4: Aktivnost obiskovalcev vrste E. palustris med 2020–2021. 147 species from 6 families and 35 genera were found in NBG (Gonchar & Gnatiuk, 2018). However, bees from only three genuses (Apis, Halictus and Lasioglossum) were observed on E. palustris. Only Apis and Lasioglossum were active pollinators. Surveys of pollination of Orchidaceae in the NBG in previous years also indicate Halictidae: La- sioglossum: L. zonulum, L. politum, L. morio, and A. mellifera as main pollinators of E. palustris (Gnatiuk et al., 2017; 22/2 • 2023, 247–262 255 Gnatiuk et al. Diversity of Epipactis palustris pollinators and visitors in Kyiv Epipactis population, had a significant effect on the num- ber of aphids. Mirid Bugs visited flowers rarely. Wasps of genus Pemphredon also prey on aphids quite actively, they sometimes sat on the flowers. In all the years of observa- tion, even before the end of flowering, the aphids were completely eradicated by predator insects. The presence of aphids had almost no effect on the formation of fruits and seeds. Other researchers also confirm the presence of aphids, particularly Aphis ilicis (Kaltenbach, 1843), on E. palus- tris (Jakubska et al. 2005, Jakubska-Busse & Kadej, 2008) and connect it with the presence of Chrysopa carnea (Ste- phens, 1836) and Syrphidae. Some authors separate the aphids that settle and feed on E. palustris into a species Aphis epipactis (Jacquemyn et al., 2014), which is close to other species of “long-haired black aphids” of Aphis fabae group: A. ilicis, A. viburni (Scopoli, 1763), and A. mor- dwilkoi (Börner, 1926) in terms of morphological and biological characteristics (Jorg et al., 1995). A vital role of ladybirds in pollination biology of Epi - pactis species were noted by A. Jakubska and others in Poland. They consider that: «the presence of Coccinelli- dae should not be connected only with the plant louses which occur often on the vegetative sprouts of the or- chids. Ladybirds were present numerously on the plants without plant louses» (Jakubska et al., 2005). However, we observed the presence of ladybirds mostly in correla- tion with aphids. The largest number of Coccinellidae and their larvae (like other aphid-hunters) was observed most- ly during the period of maxim growth of aphid colonies on E. palustris stems. At the same time several species of aphidophagous hoverflies (the females of which sometime lay eggs on the flowers) and wasps actively visit flowers. The fact that hoverfly’s females lay eggs on the flowers of the orchid may point to specific flower attraction. During the flowering time we observed imago and larvae of Coc- cinellidae actively visit open flowers. Based on this fact we suggested that the E. palustris might attract aphidophago- us by generalized mimicry of aphid pheromone in floral odour profile as it is described for Epipactis veratrifolia Boiss. & Hohen (Stökl J. et al., 2011). Colonies and young individuals of Metcalfa pruinosa (Say, 1830) were found on the shoots of E. palustris in Kyiv. This polyphage is an invasive pest that has recently spread in Ukraine. We have not established the role of other Hemiptera as pests of E. palustris. E. palustris plants proved to be a convenient shelter for dragonflies and spiders, which actively prey on flies and bees. Some Phaneropterinae nymphs feed on flower’s nec- tar and Phaneropterinae imago sit on the plants. Among Lepidoptera, flowers with nectar attracted only Pieris napi butterflies in all years of our observations. Anna Jakubska-Busse noted Ochlodes sylvanus (Esper, 1777) butterflies as flower visitors (Jakubska-Busse, 2011). In “Biological Flora of the British Isles” Autographa gamma (Linnaeus, 1758) noted as a pollinator ( Jacquemyn et al., 2014). In the conditions of Kyiv, these butterflies are quite numerous, but they ignored the flowers of E. palus- tris in the studied populations. We were interested in the research of Jennifer Brod- mann with colleagues (2008). They investigated that E. helleborine (L.) Crantz flowers emit four common “green leaf volatiles” (GLVs) that can also be found in cabbage infested by Pieris caterpillars. GLVs are emitted by plants while herbivorous insects, for example caterpil- lars, feed on them. E. helleborine flowers produce GLVs in order to attract preyhunting social wasps for pollination (Brodmann et al., 2008). The fact that only Pieris napi butterflies were observed on E. palustris flowers in Kyiv led us to believe that its flowers might produce similar GLVs that could attract these butterflies. Epipactis helleborine is distant from E. palustris, both chemically and genetically (Jakubska-Busse & Kadej, 2011). However, plants of the same genus may have simi- lar adaptations, in particular chemical mimicry for the attraction of pollinating insects and predatory insects for biological protection against pests. But, the presence of such mimicry in E. palustris should be checked by de- tailed chemical analyses. All insects that visited E. palustris flowers accidentally or purposefully can be pollinators or pollen vectors. E. palus- tris has a rather sticky viscidium and after 1–2 visits by insects the pollinia are removed from the flower. On larger insects (especially A. mellifera), it sticks to the head, in smaller insects to the back (Lassioglossum spp., Pemphredon sp.) or to the elytra (Coccinellidae). Often pollinia simply lie removed from the flower on the leaves and stems of E. palustris or nearby plants. In our study, only 23.4% of insect species were recorded with pollinia or their frag- ments on the body. We observed geitonogamy induced by bees, bugs and ants on E. palustris. Lassioglossum and Coccinellidae mainly ensured self-compatibility within the same inflorescence by crawling from flower to flower. Conclusions It was found that populations of E. palustris in the condi- tions of the Kyiv city form a unique self-regulating bio- cenosis that provides shelter and food for 53 identified species of arthropods. Taxonomic diversity of visitors on E. palustris show that Hymenoptera (34%), Coleoptera (19%) and Diptera (17%) are represented by the great- est diversity, whereas, Neuroptera and Lepidoptera by the smallest number of species. Evaluation of visitor activity 22/2 • 2023, 247–262 256 Gnatiuk et al. Diversity of Epipactis palustris pollinators and visitors in Kyiv shows that Hymenoptera (36%) were the most frequent visitors, whereas, Lepidoptera, Orthoptera and Neuroptera were only occasionally observed on E. palustris. E. palustris in the conditions of Kyiv is mainly charac- terized by entomophilia (melitophilia and cantharophil- ia). Potential pollen vectors or pollinators belong to Co- leoptera, Diptera, part of Hemiptera, most of Hymenoptera. 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Diversity of Epipactis palustris pollinators and visitors in Kyiv Appendix I 1 – Sarcophaga carnaria; 2 – Hemipenthes morio; 3 – Pollenia sp.; 4 – Eupeodes corollae; 5 – Hydrophoria lancifer; 6 – Chloromyia Formosa; 7 – Syritta pipiens; 8 – Sphaerophoria scripta ♀; 9 – Leucophora sp. 22/2 • 2023, 247–262 259 Gnatiuk et al. Diversity of Epipactis palustris pollinators and visitors in Kyiv Appendix II 10 – Corizus hyoscyami; 11 – Aphis epipactis; 12 – Metcalfa pruinosa; 13 – Deraeocoris ruber; 14 – Palomena prasina; 15 – Stenotus binotatus; 16 – Phalangium opilio; 17 – Misumena vatia; 18 – Thomisus sp. 22/2 • 2023, 247–262 260 Gnatiuk et al. Diversity of Epipactis palustris pollinators and visitors in Kyiv Appendix III 19 – Harmonia axyridis; 20 – Harmonia axyridis larvae; 21 – Coccinellidae pupae; 22 – Cantharis livida; 23 – Coccinella septem- punctata; 24 – Pseudovadonia livida; 25 – Propylea quatuordecimpunctata; 26 – Otiorhynchus ligustici, 27 – Oedemera virescens. 22/2 • 2023, 247–262 261 Gnatiuk et al. Diversity of Epipactis palustris pollinators and visitors in Kyiv Appendix IV 28 – Lasioglossum zonulum; 29 – Lassioglossum politum; 30 – Apis mellifera; 31 – Lasioglossum morio; 32 – Bombus terestris; 33 – Formica fusca; 34 – Eumenes pedunculatus; 35, 36 – Pemphedron sp. 22/2 • 2023, 247–262 262 Gnatiuk et al. Diversity of Epipactis palustris pollinators and visitors in Kyiv Appendix V 37 – Lasioglossum malachurum; 38 – Hylaeus confusus; 39 – T etramorium caespitum; 40 – Pieris napi; 41 – Platycnemis pennipes; 42 – Phaneroptera sp. nymph; 43, 44 – Chorthippus sp. ; 45 – Chrysoperla carnea.