Plant Biology ISSN 1435-8603 RESEARCH PAPER Reproductive biology and pollination mechanisms of Epidendrum secundum (Orchidaceae). Floral variation: a consequence of natural hybridization? E. R. Pansarin & M. C. E. Amaral Departamento de Botaˆ nica, Instituto de Biologia, Universidade Estadual de Campinas, Sa˜ o Paulo, Brazil Keywords ABSTRACT Epidendroideae; Epidendrum; Laeliinae; Orchidaceae; pollination; reproductive biology. The phenology, flower morphology, pollination mechanism and reproductive biology of Epidendrum secundum were studied in a semi-deciduous forest at Correspondence the Serra do Japi (SJ), and in the Atlantic rain forest of Picinguaba, both E. R. Pansarin, Departamento de Biologia natural reserves in the State of Sa˜o Paulo, southeastern Brazil. E. secundum Aplicada, Universidade Estadual Paulista, flowers all year round, with a flowering peak between September and FCAV, 14884-900, Jaboticabal, SP, Brazil. January. This species is either a lithophytic or terrestrial herb in the SJ, E-mail: [email protected] whereas, in Picinguaba, it grows mainly in disturbed areas along roadsides. E. secundum is pollinated by several species of diurnal Lepidoptera at both Editor study sites. In Picinguaba, where E. secundum is sympatric with E. fulgens M. Ayasse and both share the same pollinators, pollen transference between these two species was recorded. E. secundum is self-compatible but pollinator-depen- Received: 25 March 2007; Accepted: 22 May dent. It is inter-compatible with E. fulgens, producing fertile seeds. In con- 2007 trast to the population of the SJ, in the Picinguaba region, floral morphology is quite variable among plants and some individuals present doi:10.1111/j.1438-8677.2007.00025.x flowers with characteristics in-between both sympatric species, suggesting that natural hybridization occasionally occurs. The anthropogenic perturba- tion is probably the cause of the occurrence of E. secundum in the Picingu- aba region, enabling its contact with E. fulgens. hummingbirds of the genus Amazilia as further pollina- INTRODUCTION tors of this orchid species. According to Van der Pijl & Epidendrum L. (tribe Epidendreae, subtribe Laeliinae) is a Dodson (1966), an unidentified skipper and Papilio large genus belonging to the orchid family. It has almost polyxenes Fabricius (Papilionidae) are pollinators of 1000 native species distributed throughout tropical E. secundum in Costa Rica. Some Epidendrum species America, from Florida to northern Argentina (Dressler closely related to the ‘E. secundum complex’, such as 1993; Morrison 1997). As in most of the South American E. ibaguense and E. radicans Pavo´ n ex Lindl., do not species that are often misnamed Epidendrum ibaguense produce nectar and mimicry species of Lantana and Lindl., Epidendrum secundum Jacq. is a member of the Asclepias are thus pollinated by deceit (Boyden 1980). ‘Epidendrum secundum complex’ (Dressler 1989). The Food-deception is a common pollination system in many numerous forms belonging to the ‘E. secundum complex’ Orchidaceae (e.g. Ackerman 1986; Montalvo & Ackerman are common along roadsides at high elevations in Central 1987; Ackerman & Montalvo 1990; Calvo 1990; and South America and in the West Indies (Dressler Christensen 1992; Johnson 2000), which are estimated to 1989), and occur in disturbed areas of Venezuela (Duns- represent at least one-third of the orchid family terville 1979). (Ackerman 1986). Orchid species pollinated by deceit According to Dodson & Frymire (1961), E. secundum have low-reproductive success under natural conditions, is pollinated by Urbanus proteus L. (Hesperiidae). mainly because of the low frequency of effective Dodson (1962a) reported skippers as co-pollinators pollinators on their flowers (e.g. Montalvo & Ackerman of E. secundum, and mentioned having observed 1987; Ackerman 1989; Zimmerman & Aide 1989). Plant Biology 10 (2008) 211–219 ª 2008 German Botanical Society and The Royal Botanical Society of the Netherlands 211 Reproductive biology of Epidendrum secundum Pansarin & Amaral Pre-pollination barriers are the main factor to prevent (Leita˜o-Filho 1992). In Picinguaba, the annual rainfall is hybridization in Orchidaceae (Dodson 1962a,b; Van der about 2624 mm, and the average temperature circa 21 °C Pijl & Dodson 1966; Dressler 1981). When these barriers (Nimer 1977). This region is characterized by evergreen are broken or do not exist, however, hybrids occasionally Atlantic rain forests on steep slopes and ‘restinga’ vegeta- occur (e.g. Dodson 1962a,b; Van der Pijl & Dodson 1966; tion covering the coastal plain on sandy saline soils. Romero & Carnevalli 1990, 1991a,b, 1992; Borba & Semir 1998; Levin 2000). In southern Ecuador, two colour Phenology and floral features forms of E. secundum grow side-by-side on rock faces and seldom is an intermediate form found. It appears that The phenological data on E. secundum were gathered by each form is pollinated by a different species of hum- visiting both study areas each month, from March 1998 mingbird, each of them attracted by a different colour. In to February 2000. During these visits, the production of northern Ecuador a bewildering hybrid swarm occurs, flowers, their duration and fruit dehiscence were with the two colour forms of E. secundum as parents. The recorded. Fresh flowers of E. secundum collected at both whole complex seems to be pollinated and sustained by a study sites, as well as flowers of E. fulgens and putative third hummingbird species, but does not occur in south- hybrids collected in the Picinguaba region, were studied ern Ecuador (see Van der Pijl & Dodson 1966). Hybrid- under a binocular stereomicroscope and photographed. ization between different forms of E. secundum and other The measurements were made directly from floral struc- species belonging to the ‘E. secundum complex’ was also tures using a caliper rule. To understand the variation in documented by Dressler (1989). One of these hybrids is floral characters, as many flowers taken from different Epidendrum xobrienianum, an artificial cross between plants as possible were examined. Plant vouchers were E. radicans and E. evectum Hook. According to Dressler deposited in the herbarium UEC at the Universidade (1989), this hybrid possibly ‘escapes’ from gardens, being Estadual de Campinas. Vouchers numbers and location sub-spontaneous in areas as far apart as Mexico and are presented in Table 1. Africa. The introduction of these specimens in herbaria For anatomical study, fresh flowers of E. secundum col- causes taxonomic difficulties to taxonomists studying lected at the SJ were fixed in 50% FAA. The labella were them (Van der Pijl & Dodson 1966; Dressler 1989). dehydrated in an ethanol series and embedded in glycol The present study reports the pollination mechanism, methacrylate. Cross sections were obtained using a micro- reproductive biology and fruit set of E. secundum under tome. Sections 9–12-lm thick were stained with toluidin natural conditions. Although information about pollina- blue (Sakai 1973). tor species (Van der Pijl & Dodson 1966) and hybridiza- tion among species belonging to the ‘E. secundum Pollinators and pollination mechanisms complex’ (Dressler 1989) are present in the literature, they are fragmentary. Furthermore, this work provides Field visits were undertaken at both sites to observe and the first report on the breeding system and reproductive record the pollination process and visitation frequencies, success of a species belonging to the ‘E. secundum com- and to capture pollinators of Epidendrum secundum for plex’. Based on flower variation and on evidence of pollen transference between plants of E. secundum and another Table 1. Epidendrum species and putative hybrids, with their respec- related species (E. fulgens Brongn.), which occur sympat- tive location and voucher numbers. rically in the Picinguaba region, the possible occurrence of natural hybridization is discussed. species voucher location Putative hybrid 1 E.R. Pansarin 528 Ubatuba Putative hybrid 2 E.R. Pansarin & Ubatuba MATERIALS AND METHODS L. Mickeliunas1203 Epidendrum E.R. Pansarin 530 Ubatuba Study sites secundum Jacq. The pollination mechanisms and reproductive biology of E. secundum Jacq. E.R. Pansarin 190 Serra do Japi E. secundum were studied in a mesophytic semi-decidu- Putative hybrid 3 E.R. Pansarin 529 Ubatuba ous forest in the Serra do Japi (SJ), district of Jundiaı´ Putative hybrid 4 E.R. Pansarin 713 Ubatuba (approx. 23°11¢ S, 46°52¢ W; 700–1300 m.a.s.l.), and in Putative hybrid 5 E.R. Pansarin 531 Ubatuba the Atlantic rain forest of Picinguaba (approx. 23°33¢ S, Putative hybrid 6 E.R. Pansarin 210 Ubatuba 45°04¢ W; 0–50 m.a.s.l.), district of Ubatuba. Both areas Putative hybrid 7 E.R. Pansarin Ubatuba are natural reserves of the State of Sa˜o Paulo, Brazil. The L. Mickeliunas 1207 SJ is located inland and Picinguaba is on the coast. The Putative hybrid 8 E.R. Pansarin & Ubatuba SJ is about 350 km from Picinguaba. In the SJ, annual L. Mickeliunas 1205 rainfall is about 1500 mm, and annual average tempera- Putative hybrid 9 E.R. Pansarin & Ubatuba L. Mickeliunas 1206 ture circa 17.5 °C (Pinto 1992). This region is mainly Epidendrum E.R. Pansarin 712 Ubatuba characterized by semi-deciduous mesophytic forests fulgens Brongn of medium altitude with occasional rocky outcrops 212 Plant Biology 10 (2008) 211–219 ª 2008 German Botanical Society and The Royal Botanical Society of the Netherlands Pansarin & Amaral Reproductive biology of Epidendrum secundum later identification. In the SJ, the visits occurred from 20 In both study areas E. secundum flowers all year round, March to 17 April 1998, 29 July to 23 November 1998, but presents a main flowering peak between September and from 2 to 4 December 1999, a total of 43 h of obser- and January. Inflorescences of E. secundum can be vation. In the Picinguaba region, visits took place from observed simultaneously with buds, open flowers and 21 to 26 July 1998, 23 to 25 April 1999, and 9 to 13 fruits. Flower opening is diurnal and each flower lasts November 1999, a total of 37 h. In the Picinguaba region, about 7 days.