Plant Syst Evol (2012) 298:931–936 DOI 10.1007/s00606-012-0602-z ORIGINAL ARTICLE Floral functional structure, sexual phases, flower visitors and aspects of breeding system in Roella ciliata (Campanulaceae) in a fragmented habitat Christopher N. Cupido • Leanne J. Nelson Received: 7 October 2011 / Accepted: 24 January 2012 / Published online: 16 March 2012 Ó Springer-Verlag 2012 Abstract Continued habitat destruction in lowland areas of the Western Cape, South Africa, due to urbanization has reduced Roella ciliata to a few isolated populations and it is unknown whether this endemic shrublet will persist. Here, we examine the reproductive biology of a small population of R. ciliata occurring in a residential nature reserve 30 km east of Cape Town to provide baseline information for future studies. Data were recorded on the functional structure of the flower, duration of the morphological gender phases, floral visitor profile and the relationship between breeding system and habitat. Observations revealed that this species possesses reproductive characteristics typical of Campanulaceae such as dichogamy, protandry and secondary pollen presentation via pollen collecting hairs. Floral longevity ranges between 1 and 3 days. Anthesis occurs in synchronicity at 0700 hours and individual flowers close between 0930 and 1500 hours daily. The staminate phase lasts between 2 and 29 h and is significantly influenced by the removal of pollen by floral visitors, while the pistillate phase lasts between 21 and 69 h. R. ciliata maintains a generalized, entomophilous pollination system although floral visitors are generally scarce. Despite some intraspecific variation, the pollen/ ovule ratio indicates that R. ciliata has a facultatively autogamous breeding system, in contrast to its floral attributes which might favour xenogamy. This reproductive mechanism may ensure survival under these habitat C. N. Cupido (&) South African National Biodiversity Institute, Compton Herbarium, Private bag X7, Claremont 7735, South Africa e-mail: c.cupido@sanbi.org.za L. J. Nelson Biodiversity Program, Queensland Museum, South Brisbane, Queensland, Australia conditions and could represent a shift from outcrossing to selfing at the cost of reducing genetic variation over time. Keywords Campanulaceae
Cape Floristic Region
Habitat fragmentation
Roella ciliata
Reproductive biology Introduction The continued existence of an organism depends on its ability to regenerate itself in a stable as well as a changing natural environment. Failure of its reproductive process to adapt to change could lead to its extinction. The use of anthropogenic interventions to reduce the risk of extinction or loss requires an understanding of the organism’s reproductive biology. For flowering plants, this includes floral biology, seed dispersal, germination ability, pollination and life span. Studies on the reproductive biology of the Campanulaceae have focused almost entirely on Campanula L. (e.g. Richardson and Stephenson 1989; Evanhoe and Galloway 2002; Giblin 2005) and Wahlenbergia Schrad. ex Roth (e.g. Lloyd and Yates 1982; Anderson et al. 2000). Evidence from these studies suggests that the reproductive biology of the family is complex and detailed studies of individual taxa are required to understand the reproductive characteristics (Anderson et al. 2000). Little is known about the reproductive biology of the South African bellflower and how finely tuned it is to the diverse flower structure, pollinators and habitat, and its significance in systematics (Cupido 2009). The South African endemic genus, Roella L. is largely confined to the Cape Floristic Region (CFR) with one species occurring along the east coast from the Eastern 123 932 C. N. Cupido, L. J. Nelson Cape to KwaZulu-Natal. Species of Roella are summer flowering dwarf shrubs or perennial herbs with an erect to prostrate habit. In a checklist of Campanulaceae, Lammers (2007) followed the treatment by Goldblatt and Manning (2000) and Welman and Cupido (2003) in recognizing 20 species. More recently, Cupido (2010) described a new species of Roella from Hermanus on the east coast of the south-western Cape. Fire is critical in the growth and survival of Roella species in the CFR. Plants are most abundant and flower profusely within the first 3 years after fire. When fire is absent for extended periods, plants begin to disappear from the landscape. In the midst of ongoing habitat destruction in lowland areas of the Western Cape, South Africa, due to urbanization, R. ciliata L. has become restricted to a few small populations which occur in vacant residential plots, small nature reserves and farmland. Data on the reproductive biology of this species are limited, and it is uncertain whether these populations will persist in this fragmented landscape where fire is often absent. Through developing a detailed understanding of the reproductive biology of the species, it is hoped that R. ciliata may be conserved despite ongoing transformation of its natural habitat. To this end in order to provide baseline information for future studies on the effects of habitat fragmentation on the reproductive biology of R. ciliata, we address the following: 1. 2. 3. 4. Functional structure in bud, at anthesis and senescence. Duration of the morphological gender phases of the flower. The relationship between breeding system and habitat. Visitor profile. et al. 2006). Over 250 indigenous plant species occur in the reserve amongst a considerable number of exotic species. The population of the study species, R. ciliata, occurs amongst grass on a gentle slope near the eastern border of Bracken in granite-derived sandy to loamy soils. Adams (2010) estimated a population size in excess of 150 individuals and reported that the last recorded fire occurred in 2006. R. ciliata is an erect or sprawling dwarf shrub up to 45 cm high. Its leaves are linear, ciliate with axillary clusters of smaller leaves present. Its bell-shaped flowers are blue with a dark ring or spot on the lobes, often edged with white, and borne solitary on the tips of branches. This species is endemic to the CFR, where it ranges from the Cape Peninsula to Caledon on slopes or flats. A voucher of the species was deposited at the Compton Herbarium (NBG). Field study The population was initially visited in early January 2010 to confirm historical records of R. ciliata in the Brackenfell area. Subsequent to this visit, the population was monitored from 29 August 2010 to observe the onset of flowering. In early October, 15 plants with five or more buds were selected and tagged for the study. Over the ensuing weeks, the population was visited at different times of the day to ascertain the most suitable period for data collection. This became necessary as closing flowers were noted during morning and late afternoon visits, irrespective of the degree of sunlight, creating difficulty in distinguishing newly opening from senescing flowers. Data were collected during the period 0700–1500 hours. Phenology and floral longevity Material and methods Site and species studied Field studies were conducted at the Bracken Nature Reserve, which is located in the residential area of Brackenfell, approximately 30 km east of Cape Town. Prior to its proclamation in 1976, a portion of the land was utilized as a granite quarry and later converted to a land-fill site which was closed in 2000. The area is currently undergoing rehabilitation by the City of Cape Town and forms part of the 39-ha reserve which conserves two isolated remnants of vegetation. The smaller of the two, a granite outcrop, Perdekop, is approximately 4 ha in extent while the remaining larger area is generally referred to as Bracken. Floristically, the reserve falls within the CFR and its vegetation is described as the critically endangered, Cape Flats Sand Fynbos and Swartland Granite Renosterveld (Rebelo 123 The flowering period was defined as the period between the onset of anthesis of the first and senescence of the last flower. Anthesis was recorded as the time the petals separated to expose the style to pollinators, following the definition of Evanhoe and Galloway (2002). The scoring of senescence at two stages, wilt and complete senescence, proved difficult in practice since the turgidity of the style could not be ascertained without damaging the floral parts. Therefore, flowers were considered as senesced when the wilted petals had completely enclosed the style, rendering it inaccessible to floral visitors. Floral longevity was determined by randomly selecting three flowers per plant, and recording the date/time of anthesis and senescence. Flower functional structure The stages of flower development and function were studied in bud immediately prior to anthesis, during Floral functional structure, sexual phases, flower visitors and breeding system in Roella ciliata anthesis, and when 1, 2 and 3 days old. Flowers were collected and fixed in 70% ethanol. Pollen/ovule ratio To determine the pollen/ovule (P/O) ratio of R. ciliata, three buds on 15 plants (N = 15) were randomly collected and fixed in 70% ethanol. Pollen and ovule quantities were estimated using a modification of the technique described by Cruden (1977). The number of pollen grains per bud was calculated by suspending mature anthers in 0.5 ml of water. Anthers were ruptured and shaken to release the pollen grains which were counted with a Neubauer haemacytometer. The number of ovules per ovary was determined by counting under a dissecting microscope. The P/O ratio was calculated by dividing the total pollen count by the total ovule count. Duration of the morphological gender phases The morphological gender of one randomly selected flower on 15 plants was tracked every hour between 0700 and 1500 hours under consistent environmental conditions to determine the duration of the staminate and pistillate phases. The procedure was later replicated in 14 additional plants increasing the sample size to 29. Flowers were considered staminate from the time of anthesis to the full expansion of the stigmatic lobes and pistillate from the time of stigmatic lobe expansion to complete senescence. The removal of [50% pollen by floral visitors during the staminate phase was estimated by eye and recorded as considerable. A one-way analysis of variance was performed to test the effects of considerable pollen removal on the duration of the staminate phase. Statistical analyses were conducted using Minitab 16 software (Minitab 2010). Floral visitors All plants in our sample were observed simultaneously for floral visitors from 0700 to 1500 hours, by which time most flowers had closed. Floral visitor diversity and behaviour were recorded at 10-min intervals. Several individuals were captured for identification. Results Flower structure and function When the flower is in bud, the corolla is enclosed by five broad leathery calyx lobes interlocked by their marginal hairs and cilia. The apices of the calyx lobes taper to a sharp point and extend beyond the corolla. The base of the 933 bud is surrounded by rigid leaf-like bracts. As the corolla elongates and the calyx lobes begin to separate, its exposed surface is protected by hispid hairs. Prior to anthesis, the stamens occupy three-quarters of the space inside the closed corolla. The stamens extend beyond and completely surround a hollow style. The anthers enclose the portion of the style with pollen-collecting hairs. The lower portion of the style is covered by hispid hairs and is surrounded by the filaments. Its upper flattened portion forms the unexpanded stigmatic lobes with a gland on each side of its base. Prior to and at anthesis, the flower is in the morphological male phase (Fig. 1a). Flowers are actinomorphic and the corolla is campanulate. The corolla comprises a short tube with an inner ring of hairs near its base and five longer lobes with hairs on the midrib and at each sinus. The corolla lobes are alternate with the calyx and the stamens. The five stamens are inserted at the upper part of the hypanthium between the corolla tube and the nectariferous disc. The filament bases are arching and dilated with hairy shoulders. They form a dome over a nectariferous disc by interlocking the shoulder hairs with those on the tube base and lower portion of the style. Inside the bud, the anthers dehisce introrsely by longitudinal slits and pollen is shed and deposited onto collecting hairs. As the style extends up through the anthers, pollen is brushed up and presented to potential insect visitors as a ‘club’. Soon after shedding pollen, the anthers wither and remain at the lower part of the flower. The inferior ovary is two-locular with numerous ovules on axile placentae. The top of the ovary is covered by a plug with a nectariferous disc. The onset of the female phase occurs when the stigmatic lobes separate and the pollen-collecting hairs retract (Fig. 1b). At this stage the flower is ready for pollination. Occasionally pollen remains present below the separated lobes and may fall to the bottom of the flower as the collecting hairs retract. This phase is interrupted when the petals wilt and enclose the style while the stigmatic lobes remain expanded (Fig. 1c). Phenology and floral longevity The population of approximately 150 individuals flowered from late October to late January (mid-spring to midsummer), and was considered to be at its optimal growth period due to recent fire. Each day, floral anthesis occurred in synchronicity across the population at 0700 hours and closing took place between 0930 and 1500 hours. The process of floral senescence occurred during the late afternoon and/or overnight because all flowers had completely senesced by 0700 hours when observations began the following day. Flowers that had not wilted by 1500 hours the day before but had wilted by 0700 hours the following day were scored as 1 day. The longevity of 123 934 C. N. Cupido, L. J. Nelson Fig. 1 Stages of floral development: a morphological male phase, b morphological female phase, c complete senescence individual flowers ranged between 1 and 3 days with the majority lasting 2 days. Early senescence (\3 days) appears to be triggered by considerable pollen removal ([50%) within the first few hours after anthesis. The duration of the morphological male phase across our sample population (N = 29) varied between two and 29 h (14.8 ± 11.3 h; mean ± SD). The majority of flowers had a considerable amount of pollen removed by floral visitors within 3 h of anthesis. These same flowers had a significantly lower mean staminate duration (11.04 ± 10.6 h) than flowers which experienced little to no pollen removal (21.6 ± 9.1 h; F (1, 27) = 6.83, p = 0.014). Onset of the pistillate phase generally occurred within a few hours after pollen dissemination; however, some flowers reached female onset the following day. The pistillate duration for all flowers ranged between 21 and 69 h (34.1 ± 12.7 h) before complete senescence. Pollen/ovule ratio There was considerable intraspecific variation in pollen and ovule quantities within the population (Fig. 2). Pollen grain and ovule quantities per bud ranged from 6,250 to 86,250 (N = 15, 32,522 ± 1,654) and 116 to 462 (N = 15, 239 ± 58), respectively. Individual P/O ratios show corresponding variations, ranging from 32 to 285. Based on the mean pollen and ovule counts across the population, the P/O ratio of R. ciliata is estimated at 136 and falls within the range of facultative autogamy (Cruden 1977). However, a few buds had relatively low P/O ratios approaching those characteristic of obligate autogamy (Cruden 1977). Floral visitors Floral visitors were observed from the time the flowers opened at 0700 hours. All flowers were entomophilous, visited by insect species from multiple orders. Honey bees 123 300 Pollen-ovule ratio Duration of the morphological gender phases 350 250 200 150 100 50 0 Population (N=15) Fig. 2 Within-population variation in P/O ratio (Apis mellifera capensis) were the most abundant and active visitors; some individuals were observed visiting up to ten flowers on the same plant in 1 min. Monkey beetles (Peritrichia sp.) were common, generally remaining on the same plant for long periods ([20 min) and possibly overnighting in closed flowers. Flies (Diptera) and blister beetles (Ceroctis capensis) were relatively infrequent and were observed on fewer than five occasions. Floral visitors were observed foraging for nectar, incidentally collecting pollen on their bodies through contact with the pollen collecting hairs. Foraging activity occurred primarily in the morning hours (0800–1100 hours) prior to the flowers closing. Discussion Field observations of R. ciliata show that flowering extends over a period of 12 weeks from October to January, after the main flowering period for most bulbs and annuals in this vegetation type. Flower colour and size, and plant height in the population are relatively uniform. Individual flowers begin closing daily between 2 and 8 h after opening Floral functional structure, sexual phases, flower visitors and breeding system in Roella ciliata irrespective of weather conditions and have an average longevity of 2 days. R. ciliata has the typical functional structure known elsewhere in the Campanulaceae: flowers are dichogamous whereby the order of presentation of the sexual phases is protandrous and pollen is presented secondarily via pollen-collecting hairs. At the end of summer, plants become moribund while bearing unopened capsules. New vegetative growth appears in late winter. Our study demonstrated that the duration of the morphological male phase varies depending on the amount of pollen removed by floral visitors. We found a relationship between high levels of pollen removal and shorter staminate phases. On average, flowers which had a considerable amount of pollen removed had approximately half the staminate length of those which had little to no pollen removed. The dissemination of pollen is believed to trigger the onset of the morphological female phase, thereby making pollinator activity an important factor in the longevity of R. ciliata’s flowers. These data are consistent with several studies of protandrous species in the Campanulaceae family (Devlin and Stephenson 1984; Evanhoe and Galloway 2002; Richardson and Stephenson 1989, etc.) which show that gender phase durations are plastic in response to pollen removal. The amount of pollen removed by floral visitors may not be the only factor controlling the duration of the gender phases. Studies in Campanula afra Cav. (Nyman 1992) have shown that stimulation of pollencollecting hairs and the frequency thereof reduce the length of the male phase. The effects of pollen removal and stimulation of the pollen-collecting hairs on the length of the staminate phase in R. ciliata needs to be determined, as it is unclear whether they act independently or in combination. While our study did not examine the effects of pollen deposition on the length of the pistillate phase, it is plausible that the amount of pollen received by stigmas or pollen hair stimulation influences the longevity of the female phase and time of senescence. Dichogamy and the functioning of pollen-collecting hairs are among the mechanisms known to restrict selfpollination (Proctor and Yeo 1972; Nyman 1993) and promote out-breeding in plants. Contrary to possessing outcrossing attributes, the mean P/O ratio suggests that R. ciliata is a facultative inbreeder characteristic of disturbed or early successional habitats according to the categories of Cruden (1977). There was some intraspecific variation in the pollen and ovule numbers across our sample population (Fig. 2). This trend has been identified within individuals and among populations (Cruden 2000). Such variation could be associated with levels of selfcompatibility among individuals as we noted that some P/O ratios were approaching obligate autogamy. Cruden’s generalizations are not always consistent with P/O ratios of known breeding systems (Lord 1980; Arnold 1982; Böcher 935 and Phillips 1985; Anderson et al. 2001). Factors which may influence variation in P/O ratios such as types/efficiency of pollen vector, differences among sets of stamens, intra-inflorescence variability and flower type (unisexual or bisexual) should be taken into consideration when calculated (Preston 1986). Field observations reveal that R. ciliata maintains a generalized, entomophilous pollination system. Honey bees and monkey beetles are considered the primary pollinators due to their relative frequency while blister beetles and flies are thought to play a minor role as pollen vectors. Actinomorphy and an open floral design may facilitate generalization by enabling morphologically diverse taxa to fit and access rewards. Flower colour, particularly the dark spots on the petal lobes (beetle marks) may attract monkey beetles as demonstrated by Van Kleunen et al. (2007) for members of the Iridaceae. The capacity to exploit a range of nonspecific pollinators may benefit this species because it occurs in degraded areas where insect activity may be unreliable. In the absence of suitable pollinators, the progressive flower closure by midday could be a mechanism to prevent access by illegitimate visitors or to promote autonomous self-pollination despite being protandrous and possessing floral attributes which favour outcrossing. Direct habitat loss has reduced R. ciliata to a few isolated populations and it is unknown how the local pollinating fauna has been affected. Changes to the abundance and composition of insect pollinators can have negative impacts on plant reproductive success (Jennerston 1988; Aizen and Feinsinger 1994; Steffan-Dewenter and Tscharntke 1999; Cunningham 2000; Donaldson et al. 2002). Ecological factors which may influence plant sensitivity to habitat disturbance are degree of pollinator specialization and breeding system. Self-compatibility and a generalized pollination system may provide some resilience in a changing environment (Johnson and Steiner 2000; Bond 1994; but see Aguilar et al. 2006). It is also expected that insects visiting surrounding home gardens may be reciprocally available to the flowers of R. ciliata. However, more data on the pollination ecology of this endemic shrublet are needed, particularly in relation to ascertaining pollinator efficiency, pollination rates and reproductive output. The isolated and fragmented habitat, disturbance caused by recent fire and unreliability of floral visitors to deliver outcross pollen might meet the conditions for these plants to shift to an autogamous mating system. The advantage of inbreeding under these conditions is that it promotes reproductive assurance albeit at the cost of reducing genetic variability over time. Acknowledgments We are grateful to the following individuals for contributing to the study. Tshepo Mamabolo, Manager of Bracken Nature Reserve, for the research permit and assistance locating the 123 936 plant population, Prof. Jeremy Klaasen and Danielle Cupido, Medical Bioscience Department at the University of the Western Cape, for assistance in the pollen counting, Dr Jonathan Collville, Applied Biodiversity Research Division at the South African National Biodiversity Institute, who kindly identified the insects, and Ursula Cupido for general field assistance. References Adams R (2010) An evaluation of the distribution and status of Roella ciliata (Blue-eyed Roella) within the central district of the City of Cape Town. Report to the Cape Peninsula University of Technology Aguilar R, Ashworth L, Galetto L, Aizen MA (2006) Plant reproductive susceptibility to habitat fragmentation: review and synthesis through a meta-analysis. 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