Plant Biology ISSN 1435-8603 RESEARCH PAPER Spatio-temporal patterns in pollination of deceptive Aristolochia rotunda L. (Aristolochiaceae) B. Oelschlagel€ 1, M. von Tschirnhaus2, M. Nuss3, T. Nikolic4, S. Wanke1,†,S.Dotterl€ 5,† & C. Neinhuis1,† 1 Institut fur€ Botanik, Technische Universitat€ Dresden, Dresden, Germany 2 Fakultat€ Biologie, Universitat€ Bielefeld, Bielefeld, Germany 3 Senckenberg Naturhistorische Sammlungen Dresden & Museum fur€ Tierkunde, Dresden, Germany 4 Department of Botany, Faculty of Science, University of Zagreb, Zagreb, Croatia 5 Department of Ecology & Evolution, University of Salzburg, Salzburg, Austria Keywords ABSTRACT Aristolochia rotunda; autogamy; Chloropidae; deceptive pollination; floral life span; flower biology; pollinator variability. • Pollination success of highly specialised flowers is susceptible to fluctuations of the pollinator fauna. Mediterranean Aristolochia rotunda has deceptive trap flowers Correspondence exhibiting a highly specialised pollination system. The sole pollinators are kleptopara- B. Oelschlagel,€ Institut fur€ Botanik, Technische sitic flies in search of food. This study investigates these pollinators on a spatio-tem- Universitat€ Dresden, Zellescher Weg 20b, poral scale and the impact of weather conditions on their availability. Two potential 01062 Dresden, Germany. strategies of the plants to cope with pollinator limitation, i.e. autonomous selfing and E-mail: [email protected] an increased floral life span, were tested. • A total of 6156 flowers were investigated for entrapped pollinators in 10 Croatian † Authors contributed equally to this study. populations. Availability of the main pollinator was correlated to meteorological data. Artificial pollination experiments were conducted and the floral life span was recorded Editor in two populations according to pollinator availability. A. Dafni • Trachysiphonella ruficeps (Chloropidae) was identified as dominant pollinator, along with less abundant species of Chloropidae, Ceratopogonidae and Milichiidae. Pollina- Received: 16 March 2016; Accepted: 22 tor compositions varied among populations. Weather conditions 15–30 days before August 2016 pollination had a significant effect on availability of the main pollinator. Flowers were not autonomously selfing, and the floral life span exhibited considerable plasticity doi:10.1111/plb.12503 depending on pollinator availability. • A. rotunda flowers rely on insect pollen vectors. Plants are specialised on a guild of kleptoparasitic flies, rather than on a single species. Pollinator variability may result in differing selection pressures among populations. The availability/abundance of polli- nators depends on weather conditions during their larval development. Flowers show a prolonged trapping flower stage that likely increases outcrossing success during peri- ods of pollinator limitation. short-term weather conditions (Dunkeloh€ & Jacobeit 2003; INTRODUCTION Martin-Vide & Lopez-Bustins 2006). It has been reported that Deceptive pollination has evolved several times independently weather conditions during flowering influence the frequency of in flowering plants (Proctor et al. 1996; Renner 2006). Decep- pollinator visits and therefore pollination success (Baker et al. tive flowers advertise a reward to their pollinators that they do 2000; Jacquemyn et al. 2009). Such strong variability of polli- not provide in the end, and exploit the instinctive behaviour of nator abundance has earlier been shown for Mediterranean often non-typical flower visitors (e.g. Brodmann et al. 2008; Aristolochia species (Berjano et al. 2009). Stokl€ et al. 2010). This results in highly specialised pollination The genus Aristolochia (Aristolochiaceae) comprises over that is usually mediated by only one to a few pollinator species 450 species and is distributed in the tropics and in temperate (Dafni 1984; Proctor et al. 1996). Highly specialised plants (in- regions (Wagner et al. 2014). In the Mediterranean about 60 dependent of whether deceptive or not) benefit from highly species are known, representing a diversity hotspot for the specific pollen delivery and high outcrossing rates compared to genus (Wanke 2007). Virtually all Aristolochia species exhibit less specialised rewarding species (Scopece et al. 2010). How- deceptive pollination strategies and it was assumed that flowers ever, they are counter-balanced by a high dependence on spa- mimic the brood sites of their fly pollinators (Vogel 1978; tiotemporal fluctuation of the pollinator fauna (Waser et al. Proctor et al. 1996). However, detailed knowledge on the 1996; Armbruster et al. 2000) and overall lower reproductive mechanism of pollinator attraction and deception is presently success (Scopece et al. 2010). The Mediterranean climate is only available for A. rotunda. A. rotunda flowers are klepto- characterised by strong seasonality and high variability of myiophilous, a pollination strategy recently described 928 Plant Biology 18 (2016) 928–937 © 2016 German Botanical Society and The Royal Botanical Society of the Netherlands Oelschlagel,€ Tschirnhaus, Nuss, Nikolic, Wanke, Dotterl€ & Neinhuis Spatio-temporal patterns in pollination (Oelschl€agel et al. 2015). Representatives of Chloropidae and populations and years. The impact of weather conditions on Ceratopogonidae were found pollinating this species A. rotunda’s highly specialised pollination system is discussed (Oelschl€agel et al. 2015), and members of these families are and potential mechanisms promoting reproductive success known kleptoparasites, stealing food of invertebrate predators during pollinator limitation are evaluated. With respect to the (e.g. Sivinski et al. 1999). The flowers of A. rotunda employ an highly specialised pollinator attraction we test the following olfactory mimicry of the heteropteran prey to attract and hypothesis: (i) A. rotunda flowers are pollinated by the same deceive their kleptoparasitic pollinators (Oelschl€agel et al. pollinator species in different populations; (ii) pollinator avail- 2015). Due to this particular attraction mechanism, A. rotunda ability is strongly influenced by weather conditions during is a highly specialised plant species that could make the plant’s flowering of A. rotunda, and unfavourable weather conditions, reproductive success susceptible to fluctuations in pollinator such as high precipitation or low temperatures, result in peri- fauna. ods with pollinator absence; (iii) autonomous selfing is a strat- Known strategies of plants to cope with pollinator limitation egy of A. rotunda to ensure reproductive success in periods of and increase reproductive fitness are autonomous selfing and pollinator limitation; and (iv) flowers of A. rotunda show a increased floral longevity (e.g. Ashmann & Schoen 1994, 1996; prolonged trapping flower stage (female stage and interphase) Fenster & Marten-Rodrıguez 2007; Castro et al. 2008). as compared to tropical Aristolochia species. Although flowers of Aristolochia show strong adaptations for cross-pollination, the ability for delayed autonomous selfing has been proven for several of its species (Razzak et al. 1992; MATERIAL AND METHODS Hall & Brown 1993; Sakai 2002; Berjano et al. 2006; Bliss et al. Sampling sites 2013). However, it is yet unknown if A. rotunda is outcrossing or selfing. Aristolochia flowers possess an elaborated perianth Aristolochia rotunda is endemic to the northwestern Mediter- morphology evolved for trapping, retention and release of pol- ranean region and occurs from western Turkey to eastern linators (Correns 1891; Oelschl€agel et al. 2009; Fig. 1A). The Spain. It colonises damp grassy areas in woodlands, on river- perianth consists of three main parts: the limb, the tube and banks and boulders (Nardi 1984, 1991). The study was the utricle that contains the gynostemium. The flowers show performed at ten localities along the Croatian coast (Fig. 1G): pronounced protogyny, with functional adaptations in the dif- Dragonja (45°27051.7″ N, 013°37020.3″ E, 0 m a.s.l.), Most Rasa ferent sexual stages. Erect and receptive stigmas, closed pollen (45°03046.5″ N, 014°02044.4″ E, À2 m a.s.l.) both Istria; sacks (Fig. 1B) and functional trapping devices characterise the Caska (44°32059.7″ N, 014°55016.3″ E, 2 m a.s.l.), Dinjisko female flower stage (i.e. bendable trichomes inside the flower polje (44°23.5940 N, 015°07.9120 E, 73 m a.s.l.), Mt. Sveti tube; Fig. 1C). Pollinators are attracted and trapped during this Vid (44°30045.6″ N, 014°57025.6″ E, 40 m a.s.l.), Stari stage. During the interphase stigmas wilt and bend together grad (44°25048″ N, 015°03052″), Vlasici’ (44°19021.5″ N, while pollen sacs open (Fig. 1D). The trapping devices remain 015°12031.7″ E, 20 m a.s.l.) all Isle of Pag; Omisalij lake turgescent, enabling the flower to cover the trapped flies with (45°10022.3″ N, 14°34027.3″ E 6 m a.s.l., Isle of Krk), Veli its pollen. During the male flower stage, the trapping devices Losinj (44°31017.1″ N, 014°30059.8″ E, 5 m a.s.l., Isle of Los- wilt (Fig. 1E) and enable the flies, loaded with pollen, to leave inje) and Dracevac Ninski (44°10054.9″ N, 015°18036.6″ E, the flower (Correns 1891; Daumann 1971; Gonzalez & Steven- 49 m a.s.l., Ravni Kotari, Croatia mainland coast). Climate of son 2000; Oelschl€agel et al. 2009). the study region corresponds to the typical seasonal climate of Stotz & Gianoli (2013) hypothesised an adaptation of the the Mediterranean, with dry summer and wet winter seasons. floral longevity within the genus to pollinator limitation fac- tors. Species in habitats with low net