G Model FLORA-50724; No. of Pages 9 ARTICLE IN PRESS Flora xxx (2013) xxx–xxx Contents lists available at ScienceDirect Flora j ournal homepage: www.elsevier.com/locate/flora Low legitimate pollen flow in distylic Turnera hermannioides (Passifloraceae) and its consequences on fruit and seed set a,∗ b c Paulo Milet-Pinheiro , Diana Corrêa de Andrade Penante , Clemens Schlindwein a Institute of Experimental Ecology, University of Ulm, Albert-Einstein-Allee 11, 89069 Ulm, Germany b Centro de Ciências Biológicas, Universidade Federal de Pernambuco, Av. Prof. Moraes Rego, 1235, 50670-901, Brazil c Departamento de Botânica, Universidade Federal de Minas Gerais, Av. Antônio Carlos, 6627, 31270-901 Belo Horizonte, MG, Brazil a r t i c l e i n f o a b s t r a c t Article history: Turnera hermannioides is a ruderal distylic subshrub, native to NE-Brazil. In the Catimbau National Received 16 May 2013 Park, situated within the semi-arid Caatinga region, we studied the pollination ecology of this species, Accepted 23 September 2013 emphasizing (1) effective pollinators; (2) characteristics of short- and long-styled flowers; (3) intra- Available online xxx and intermorph pollen flow; and (4) fruit and seed set. Short and long-styled morphs differ in pollen size and ornamentation, stigmatic surface, style and stamen length and nectar production. The flowers Keywords: are obligate intermorphic outcrossers and depend on animals for pollination. The flowers of T. herman- Heterostyly nioides attracted insect visitors of 25 species, among them butterflies, beetles, but mainly bees. Polylectic Oligolectic bees bees, such as Apis mellifera, stingless bees, and solitary Callonychium brasiliense were the most frequent Polylectic bees visitors and the principal pollinators. The frequency of visits, however, was very low (on average 1.9 Pollen flow Pollination visits/flower/day), resulting in a low and unbalanced legitimate pollen flow, i.e. a much lower number Turneraceae-clade of pollen grains from short-styled flowers reaching stigmas of long-styles than vice versa, which in turn compromised fruit and seed set. Surprisingly, Protomeliturga catimbaui and P. turnerae, both oligolectic on flowers of the Turneraceae-clade of the Passifloraceae s.l., were very rare flower visitors and did not contribute significantly to the pollination of Turnera hermannioides. We discuss how the low visitation rate and the inefficiency of pollinators (i.e. inefficiency in promoting legitimate pollination) compromise pollen flow and, consequently, fruit and seed set in this species. © 2013 Elsevier GmbH. All rights reserved. Introduction fruit set (Ganders, 1979). In some cases, associations involve oligolectic bees, which restrict pollen collection to a few plant Heterostyly is a heteromorphic self-incompatibility system in species of a given genus or family to rear their offspring (Cane which individuals of the same species present a floral polymor- and Sipes, 2006; Robertson, 1925). Recent studies documented phism that is expressed in reciprocal differences in anther and very tight relationships of oligolectic bees to their heterosty- stigma length, and often by additional features, such as number, lous host plants, showing mutual reproductive dependence. In size and ornamentation of pollen grains, and length of stigmatic these cases, the pollinating bees are provided with morphologi- lobes and papillae (Barrett, 1990; Darwin, 1877; Ganders, 1979). In cal and/or behavioral adaptations to optimize resource gathering the two (short-and long-styled; distyly) or three (short-, mid- and in those plants (Alves-dos-Santos and Wittmann, 2000; Carvalho long-styled; tristyly) flower morphs of heterostylic plants, fecun- and Schlindwein, 2011; Milet-Pinheiro and Schlindwein, 2010; dity is only possible (or often seed set is maximized) if legitimate Schlindwein and Wittmann, 1995, 1997a, 1997b). The absence of pollination occurs, i.e. if intermorphic pollen grains of the appropri- the specialized pollinators generally results in diminished fruit set ate height level are deposited on the stigmas. This heteromorphic (Carvalho and Schlindwein, 2011; Milet-Pinheiro and Schlindwein, self-incompatibility system has evolved independently in several 2010) and in di- or tristylic species even in breakdown of het- angiosperm lineages and has been recorded in at least 28 families erostyly (Alves-dos-Santos, 2002; Barrett, 1988). In other cases, (Vuilleumier, 1967). oligolectic bees depend on their specific host plants as exclu- Heterostylous plants are often obligate outcrossers and, accord- sive pollen sources, but the plants, in general, do not depend on ing to the pollination system, pollinators are required to guarantee the bees as exclusive pollinators (Minckley and Roulston, 2006; Schlindwein, 2004). Turnera Smith (Passifloraceae) is the largest genus of the Turn- ∗ eraceae clade of Passifloraceae s.l. (formerly Turneraceae; APG III, Corresponding author. E-mail address: [email protected] (P. Milet-Pinheiro). 2009; Thulin et al., 2012), distributed mainly in the neotropics, and 0367-2530/$ – see front matter © 2013 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.flora.2013.09.005 Please cite this article in press as: Milet-Pinheiro, P., et al., Low legitimate pollen flow in distylic Turnera hermannioides (Passifloraceae) and its consequences on fruit and seed set. Flora (2013), http://dx.doi.org/10.1016/j.flora.2013.09.005 G Model FLORA-50724; No. of Pages 9 ARTICLE IN PRESS 2 P. Milet-Pinheiro et al. / Flora xxx (2013) xxx–xxx most of the about 130 species are distylous (Shore et al., 2006). flower opening and wilting, anther dehiscence, stigma receptivity Flowers in this genus are pollinated mainly by bees, including both (with H2O2 at 20%, Kearns and Inouye, 1993), and nectar availabil- polylectic and oligolectic species (Barrett, 1978; Schlindwein and ity. Nectar amount was measured at 12:00 h in bagged flowers of Medeiros, 2006). Protomeliturgini is a monogeneric bee tribe rep- 20 plants for each morph (n = 1 flower per individual) with micro- ® resented by two species, Protomeliturga turnerae (Ducke, 1912) and capillaries (0.5 ␮l, Duran ). P. catimbaui Schlindwein and Moure, 2005. Protomeliturga turnerae The proportion of short and long-styled individuals in the pop- is an oligolectic species that collects pollen exclusively in flow- ulation was determined for 65 plants. ers of Turnera. In addition to the use as a food source, this bee uses flowers of T. subulata as mating site; the males establish ter- Pollen ritories in flowers for several days, where they patrol in search of females (Medeiros and Schlindwein, 2003). In spite of this food and To describe exine ornamentation and measure the size of the reproductive dependence by P. turnerae, its absence does not com- pollen grains, we prepared microscope slides from short- and long- promise the fruit set of T. subulata, since polylectic flower visitors styled flowers (n = 10 for each morph) of different individuals. are also effective pollinators (Schlindwein and Medeiros, 2006). Grains were removed from anthers with a small piece of glyc- Protomeliturga catimbaui is a rare species endemic to the Brazilian erin gelatine, mounted with a cover glass and sealed with paraffin Caatinga (a tropical dry forest, characterized by succulent plants wax (Louveaux et al., 1978). Under a Zeiss Axiolab microscope (at × and thorn-shrubs) and so far it was recorded only in the Catimbau 1000 ), we measured the pollen grains in equatorial view (50 per National Park (Northeastern Brazil) in flowers of Piriqueta sidifolia flower, n = 10 flowers per morph) and analyzed the ornamentation (Cambess) Urban/Passifloraceae, Turneraceae clade (Schlindwein of the exine. Unviable empty pollen grains were not considered for and Moure, 2005). the measurements of size. We studied the pollination ecology of Turnera hermannioides, a Pollen viability was determined using the methods of Radford distylic subshrub (Barrett and Shore, 1985), which is common to et al. (1974). Grains from short- and long-styled flowers (n = 10 the Catimbau National Park, and asked: (1) which features diverge for each morph) were removed from anthers fixed in FAA, and between short- and long-styled flowers? (2) What is the spectrum transferred to microscope slides with acetocarmine solution. Three and frequency of flower visitors? (3) How does pollen flow between hundred pollen grains per flower were counted under the micro- and within morphs? (4) What is the fruit and seed set of short- and scope to determine the proportion of stained viable vs. non-stained long-styled morphs? unviable grains. The number of pollen grains per flower was determined in Materials and methods flower buds of 15 plants for each morph (n = 1 flower per indi- vidual). The grains were removed from all anthers available in a Study site flower, placed in a Petri dish, and counted under a stereomicro- scope (Zeiss, Stemi SV8). We placed a grid paper below the Petri dish The study was performed between August 2007 and November to avoid counting grains twice or more. The ovaries of short- and ◦ ◦ 2008 in the Catimbau National Park (S 08 32 54,2 ; W 37 14 49,6 ), long-styled flowers (n = 15 for each morph) were dissected and the situated in the municipalities of Buíque, Tupanatinga, and Ibimirim, ovules counted under a Zeiss Stemi SV8 stereomicroscope (Dafni State of Pernambuco, Northeastern Brazil. The Park covers about et al., 2005). 62,000 hectares with altitudes varying from 700 to 1100 m (IBAMA, 2008). The climate is tropical semi-arid and the average annual Breeding system ◦ temperature and precipitation is 21.5 C and 1100 mm, respectively (ITEP, 2008). The vegetation of the Park is composed by ever- To determine the breeding system of T. hermannioides, the fol- green shrubs and small trees among which typical species of the lowing pollination experiments were performed for both morphs: surrounding Caatinga intermingle, the common vegetation of semi- (a) spontaneous self-pollination – flowers were bagged and left arid NE-Brazil (Andrade et al., 2004; Gomes et al., 2006; Rodal et al., unmanipulated; (b) hand self-pollination – bagged flowers were 1998).