Reproductive Co-Existence Among Five Sympatric Single-Stemmed Aloes in the Gamtoos River Valley, Eastern Cape

Reproductive co-existence among five sympatric single-stemmed aloes in the Gamtoos River Valley, Eastern Cape

Christo Botes 200303686

Thesis submitted towards the requirement for the degree of Masters in Science in the Department of Botany at the Nelson Mandela Metropolitan University, Port Elizabeth

2007

Supervised by Prof. Richard M. Cowling (NMMU) Co-Supervised by Prof. Steven D. Johnson (UKZN) i

Foreword

What started off as a study of the phenological shifts of plants has metamorphosed into an understanding of the co-existence of species within one of our most charismatic and economically important group of plants, the aloes. The collaborative input of my two supervisors contributed to the success of this project. The expert guidance of Richard, sourcing years of experience from his field work in the region, and the input of Steve on the pollination techniques, pushed me sometimes to the near ends of my abilities to do that what was needed regardless of the pain factor involved when working in spinescent vegetation, because hey “it will be worth it”, and it was. Thanks to the Kouga Municipality and the farming community of the Gamtoos River Valley for allowing unrestricted access to their land and for taking me to their secret spots at the back of their kloofs where the aloes are abundant. I am informed that there is still enquiry about the “aloe guy” from the many farmers who took a keen interest in the project, and thanks especially to the Youngs for their observational inputs and all the fruit. To my hosts for the flowering seasons, Grant and Tandi Meredith: thanks for the “foods from around the world” evenings and the long exchanges of travels over a good but not quality (it is Hankey after all) bottle of red. The 12km driveway through seven drifts each day en route to Craggy Burn after a hard day in the field was exceptional and will be sorely missed. I thank the Hankey Speurdiens for checking up on me in the remote localities and gladly shared my telescope with them for their efforts to root out crime in the township. My time in the valley has afforded me the privilege of being considered a local, and the last three years have seen me grow through some truly amazing experiences and I still can remember each one, from the enlightened conversation with Miriam, Mabel and Mavis (three local ladies funding their part-time university education as seasonal workers at the Patensie Co-op) with the white supremacists sneering from the bar, through to the vicious attack by one pissed-off swarm of Honeybees at the end, and it was all good. I found a place where time passes with a leisurely pace set by the eternal summer climate, where nature is still refreshingly wild and the culture, well is rural hence not their fault but sufficiently diverse, and will definitely be back for good someday…

Prof. Sue Nicolson, the external examiner, is thanked for her comments that improved the thesis. The NRF is greatly acknowledged for the grant holder linked bursary that was proved. ______Christo Botes Botanist, Naturalist, Sailor 2007 ii

Abstract

In this study I documented the convergence of five congeneric bird-pollinated plants (Aloe pluridens, A. lineata var. muirii, A. speciosa, A. africana, and A. ferox) into three functional groups based on size, shape, and the arrangements of flowers on the inflorescence, but also nectar rewards, pollen deposition sites on the bird-pollinators, and the degree to which bees play a role in their pollination. Individuals of similar functional groups were divergent in their peak flowering times and limited their degree of flowering overlap further by spatial aggregation and niche separation, within the Thicket of the Gamtoos River Valley. The nectar properties were especially useful in structuring the bird pollinator community, which resulted in greater ethological isolation and hence, greater reproductive assurance in the mixed co-flowering plant communities. Choice array experiments revealed that it was the fine scale aggregation of flowering individuals that ensured that bird-pollinators feed selectively, since when equal choice was available, interspecific visitation increased significantly compared to natural scenarios. Bird behaviour and the ecological intermediateness of one to the species explained its prominence in hybrid combinations. The spatial occurrence of hybrid individuals can be traced back to the energetics of foraging and its influence on bird floral constancy. The pollination ecology of similar South African Aloe species were extrapolated from these and recent findings by various authors, but emphasises the need for a robust natural phylogeny of the Aloaceae in order to draw comprehensive conclusions on the evolutionary radiation of this highly charismatic group.

Keywords: Aloe, Aloaceae, pollination, convergence, bird, bee, ethological isolation, co- flowering, hybridization iii

Table of content

Foreword ...... i Abstract...... ii Table of content ...... iii List of Figures...... iv List of Tables...... v List of Plates ...... vi GENERAL INTRODUCTION TO THE ALOES...... 1 CHAPTER 1 – REPRODUCTIVE CO-EXISTENCE AMONG FIVE SYMPATRIC ALOE SPECIES IN THE GAMTOOS RIVER VALLEY, EASTERN CAPE...... 4 ABSTRACT...... 4 INTRODUCTION...... 4 METHODS ...... 6 Breeding systems...... 6 Flowering phenology ...... 7 Floral ontogeny...... 7 Nectar ...... 7 Bird pollinators, feeding positions and pollen deposition sites ...... 8 Bees as potential pollinators...... 8 Statistical analyses...... 8 RESULTS...... 9 Breeding systems...... 9 Flowering phenology ...... 9 Floral ontogeny...... 10 Nectar ...... 13 Bird pollinators, feeding positions and pollen deposition sites ...... 14 Bees as potential pollinators...... 17 DISCUSSION...... 20 CHAPTER 2 – EXTRAPOLATING POLLINATION SYNDROMES FOR SOUTH AFRICAN ALOE SPECIES...... 24 ABSTRACT...... 24 INTRODUCTION...... 24 METHODS ...... 25 RESULTS...... 25 DISCUSSION...... 29 CHAPTER 3 – FACTORS INFLUENCING HYBRIDIZATION IN ALOE ...... 30 ABSTRACT...... 30 INTRODUCTION...... 30 METHODS ...... 32 Hybrid crosses ...... 32 Hybrid identity ...... 32 Experimental Array...... 36 Statistical Analyses ...... 36 RESULTS...... 37 Hybrid crosses ...... 37 Hybrid identity ...... 37 Experimental Array...... 39 DISCUSSION...... 41 GENERAL DISCUSSION...... 44 REFERENCES ...... 37 PERSONAL COMMUNICATIONS ...... 43 iv

List of Figures

Figure 1.1: Mean valley-wide flowering phenology from 15 sites over a two year period for the five most common Aloes in the Gamtoos River Valley. Thin lines indicate flowering time, thick lines indicate peak flowering period, and vertical lines indicate maximum flowering point……………………………………………………………………………...... 10 Figure 1.2: Ordination plot of selected floral character measurements in the five species, identifying their convergence into three floral groups. Variables that loaded on the factors were pedicle length and perianth length…………….……………………………………...... 11 Figure 1.3: Comparative floral development for the three floral groups with representative flowers from the different stages – Male phase (anthesis) at the fifth-sixth flower from the top, female phase at the sixth-seventh flower. Group1 - (a) Aloe pluridens, (b) Aloe lineata var. muirii; Group 2 - (c) Aloe africana; Group 3 - (d) Aloe speciosa, and (e) Aloe ferox. Scale 0.5x……………………………………………………………………………………..12 Figure 1.4: Comparison of Mean nectar volume and concentration from 10 bagged flowers on 12 individuals per species. Vertical bars = Standard Error of the Mean (± SE). Similar letters denote not significant differences between Means……………………………………….…..13 Figure 1.5: Feeding positions clockwise from top left: Group 1 - female Amethyst Sunbird on Aloe pluridens (Photo SD Johnson), male Greater Double-collared Sunbird on A. lineata var. muirii; Group 2 - male Greater Double-collared Sunbird on A. africana (Photo SD Johnson); and Group 3 - Yellow Weaver on A. ferox, and a Cape Weaver on A. speciosa……………..15 Figure 1.6: Pollen deposition sites (redrawn from photos) on the two classes of pollinators. Top: Sunbird feeding at Aloe pluridens, A. lineata var. muirii, and A. africana. Bottom: non- specialised weaver feeding at Aloe speciosa, A. ferox, and occasionally at A. africana. (A) denotes the main pollen deposition site on occasional nectarivores, with (B) indicating the additional site, mainly for mousebirds, when feeding at these two aloes………………….....17 Figure 1.7: Bee foraging behaviour (redrawn from photos) indicating feeding positions when selecting for pollen (P) or nectar (N), with N* indicating the other smaller bees. 1) Aloe pluridens, 2) A. lineata var. muirii, 3) A. africana, 4) A. speciosa, and 5) A. ferox……….....19 Figure 1.8: Contribution of bees and birds toward the pollination and seed-set. Sample sizes reflect individual plants, n.s. = not significant (P > 0.05)………………………………….. ...20 Figure 2.1: Distribution of flowering according to syndromes. Flowering dates taken from Reynolds (1969)………………………………………………………………………………28 Figure 3.1: Mean number of seeds per fruit attained through the hybrid and pure crosses among the three most common Aloe species. Vertical lines = Standard Error of the Mean (± v

SE). Same letter denotes no significant difference between the Means (one-way ANOVA)..…………………………………………………………………………………….37 Figure 3.2: MDS plot of 37 vegetative and floral characters, for pure species (excluding Aloe lineata var. muirii) and one individual from each of the eight hybrid variations found. Pure species are blocked. Subjective field classifications for the hybrids were: H1 = A. africana x ferox, H2 = A. ferox x africana, H3 = A. ferox x speciosa, H4 = A. speciosa x ferox, H5 = africana x speciosa, H6 = A. speciosa x africana, H7 = A. pluridens x speciosa, H8 = A. pluridens x africana…………………………………………………………………………...39 Figure 3.3: Percentage of inter plant movements during foraging bouts within the two experimental arrays where (A) is sunbirds and (B) is non-specialised birds……..………...... 40

List of Tables

Table1.1: Comparison of median fruit-set for crossed, selfed, and control treatments on bagged inflorescences from a median of 85 flowers (range 69-144) per treatment per species. N reflects the amount of individuals…………………………………………………………...9 Table 1.2: List of recorded independent observations of bird visitation to the five aloes differentiated into their different visitation actions, where P = Legitimate Pollinator, CP = Coincidental Pollinator, NR = Nectar Robber, DF, Destructive Forager…………………….16 Table 2.1: Floral descriptions forming the basis of the three pollination syndromes found in the South African Aloe species...…………………………………………………………...... 25 Table 2.2: Extrapolated pollination syndromes for the South African Aloe species, where I = insect-pollination, S = sunbird-pollination, and NS = non-specialised bird- pollination…………………………………………………………………………………….26 Table 2.3: Summary of the distribution of pollination syndromes within the Aloe Sections. I = insect-pollination, S = sunbird-pollination, NS = non-specialised bird-pollination…….28 Table 3.1: Vegetative, floral, and chemical characters identifying the five pure species…….33 Table 3.2: Median percentage fruit-set for pure and hybrid crosses among the three most common Aloe species. N = number of individuals tested, with a Median of 60 flowers (range 55-81) used in each treatment………………………………………………………………...37 Table 3.3: Frequencies of flowering natural hybrid crosses located during one season. Species rows correspond to dominant characters resembling one of the parents….…………38 Table 3.4: Bird foraging choices during visitations to the experimental array, where S = sunbird. NS = non-specialised bird. Observed values compared with expected equal choice using Goodness of fit test……………………………………………………………………..40

List of Plates

Plate 1: Map of the study area. Plate 2: Aloe pluridens Plate 3: Aloe lineata var. muirii Plate 4: Aloe africana Plate 5: Aloe speciosa Plate 6: Aloe ferox Plate 7: H1 – Aloe africana x ferox Plate 8: H2 – Aloe ferox x africana Plate 9: H3 – Aloe ferox x speciosa Plate 10: H4 – Aloe speciosa x ferox Plate 11: H5 – Aloe africana x speciosa Plate 12: H6 – Aloe speciosa x africana Plate 13: H7 – Aloe pluridens x speciosa Plate 14: H8 – Aloe pluridens x africana

General introduction to the aloes

Our current conceptual understanding of the evolutionary biogeography of the genus Aloe Linnaeus (see Holland 1978) is that the ancestral aloes appeared in south-eastern Africa during the diversification of the angiosperms in the Late Cretaceous from where they spread along the eastern parts of the continent, reaching its current Arabian distribution by the late Tertiary (Miocene-Pliocene). Consequent speciation is assumed to have occurred in eleven centres of current high species diversity and was linked to the changing pace of the orogenic processes of the African highlands. Most endemic species occur in areas that are characterised by high topographic variability and severe climatic fluctuations during the glacial-interglacial periods of the Pleistocene. Many of these highly localised endemic species are now facing declines in populations owing to various natural and anthropogenic factors (see Midgley et al. 1997, Sachedina and Bodeker 1999, Smith et al. 2000, Pfab and Scholes 2004). The environmental instability of the Pleistocene stimulated speciation within the genus probably via rampant hybridization (Riley and Majumdar 1979, Barker et al. 1996, Van Der Bank and Van Wyk 1996, Viljoen 1999); the contemporary genus today includes over 500 species1.

Aloes have evolved to fill a wide variety of niches and with the exposure to the prominent herbivory on the African continent have developed peripheral chemical defences (Gutterman and Chauser-Volfson 2000b, a, Chauser-Volfson et al. 2002), which forms the basis for a multi-billion dollar pharmaceutical industry centred predominantly around Aloe vera Linnaeus from Socotra and Aloe ferox Miller from South Africa (see Sachedina and Bodeker 1999). There are 125 Aloe species that occur naturally within South Africa (Van Wyk and Smith 2003), and the commercial importance of Aloe ferox is evident from the biased use of this species for local ecological studies into the genus. Holland et al. (1977) described a model for its habitat occupation. Holland and Fuggle (1982) reported the impacts of various management strategies on the population dynamics of this species near Swellendam in the Western Cape. Bond (1983) investigated the adaptive significance in Aloe ferox of the persistent dead leaves found on the stems of this and many other species of aloes, and confirmed its insulating function towards fire survival. Dean et al. (1992) reported the nurse

1 Support for the idea of an enlarged genus Aloe Linnaeus (consisting of the current Aloe, Astroloba, Chortolirion, Gasteria, and Haworthia) comes from the lack of variation in the structure of the ovules (Steyn and Smith 1998), and stable bi-modal karyotype (Brandham and Doherty 1998) within members of the current Alooideae (Asphodelaceae). This is strengthened by molecular work by Chase et al. (2000) identifying the Alooideae as a monophyletic group (see also Treutlein et al. 2003), and the polyphyletic nature of the five genera within the Alooideae (Treutlein et al. 2003). In light of this I join several taxonomic authors and will refer to the family as Aloaceae. 2 plant association between Aloe asperifolia Berger and Euphorbia damarana Leach in Namibia. Stokes and Yeaton (1995) studied the population ecology of Aloe candelabrum Berger, and Midgley et al. (1997) did the same for Aloe dichotoma Masson and A. pilansii Guthrie.

Oatley and Skead (1972) list several bird species (both sunbirds and non-specialised birds) as drinking nectar from aloes. The few reports on the pollination of aloes that have been published (Hoffman 1988 on Aloe ferox, Ratsirarson 1995 on the Madagascan A. divaricata Berger, and Stokes and Yeaton 1995 on A. candelabrum – considered synonymous with A. ferox) confirm this observation that the large showy aloes are visited by many bird species and that these aloes are fully dependant on the birds for reproduction. However, Ratsirarson (1995) and Johnson et al. (2006) showed that not all aloes are simultaneously pollinated by both sunbirds and non-specialised birds. Hoffman (1988) stated in accordance to earlier ideas (notably Skead 1967) that bees, being the most abundant floral visitors, might play a role in aloe pollination. However, the exclusion experiments of Stokes and Yeaton (1995) and Ratsirarson (1995), disputed this role of insects and indicated their sole interaction as resource robbers (both pollen and nectar). The nectar of Aloe Section Anguialoe (sensu Reynolds 1969), that is characteristically reddish brown and is said to have a bitter taste (attributable to phenolics), have been studied by Nicolson and Nepi (2005, Aloe castanea Schonland) and Johnson et al. (2006, A. vryheidensis Groenewald). Bees avoided the bitter nectar in both studies and only collected pollen from flowers in fresh anthesis, and although no bird visitation data are available for A. castanea, Johnson et al. did find that sunbirds (Nectarinidae) found the bitter nectar distasteful and only non-specialised birds such as the Dark-capped Bulbuls (Pycnonotus tricolour) visit the flowers regularly and act as its pollinators. This leaves us with a full spread of observations from the cosmopolitan Aloe ferox (attracting a wide array of sunbirds, non-specialised birds, and nectar and pollen collecting bees) through to A. vryheidensis and A. castanea (only non-specialised birds and pollen collecting bees). Hence, there might be more to the pollination of aloes than what was believed to be a straight forward case of pollination by sunbirds.

From this brief introduction to ecological research in aloes, it is evident that this large and highly charismatic group is not as well represented in the literature as would be expected. The subsequent chapters explore the methods of reproductive co-existence among co- occurring species and link this to the factors contributing toward the hybridization within the group. This study was undertaken in the Sub-Tropical Thicket vegetation of the Gamtoos River Valley (33°50’S, 024°55’E) in the Eastern Cape. The valley is nested in between the Indian 3

Ocean to the South and the Baviaanskloof World Heritage Site, ~50km inland to the Northwest, and is characterised by a high degree of topographic heterogeneity. The valley receives less than 500mm of rain per annum and experiences mild winters and warm to hot summers. Flat laying areas within the valley support an extensive produce farming industry, but the slopes and hills away from the main channel are still natural and boast extensive stands of the succulent rich Gamtoos Valley Thicket (sensu Vlok et al. 2003). Field experimentation was conducted over a two-year period during 2004 to 2005 and included two flowering seasons. Fifteen sites were selected for observation from near the mouth of the Gamtoos River to ~30km inland and covered both pure and mixed species stands and the full range of slopes (Plate 1). Cowling (1984) can be consulted for a detailed description of the region’s environment.

There are five single-stemmed aloes (sensu Van Wyk and Smith 2003) belonging to four different Sections (sensu Reynolds 1969) of the Aloaceae that form a conspicuous element of the Gamtoos Valley Thicket vegetation and that flower en masse during the winter-spring flowering season. The topographical heterogeneity of the valley results in many mixed populations of Aloe pluridens Haworth, A. speciosa Baker, A. africana Miller, A. ferox Miller, and A. lineata (Ait.)Haworth var. muirii (Marloth)Reynolds, with some of these populations reaching extremely large proportions (>500 individuals per species, several species per site). The seminal works by Reynolds (1969) or van Wyk and Smith (2003) can be consulted for detailed taxonomic descriptions of these five aloes. Although present within the valley, Aloe striata Haworth and several other Gasteria and Haworthia species were ignored in favour of these five more abundant and larger aloes. Even though they also flower at the same time, searching for these small scattered individuals within the Gamtoos Valley Thicket would have proven too time constraining and would in the end have diluted the quality of the outcomes from this study.

Chapter 1 – Reproductive co-existence among five sympatric Aloe species in the Gamtoos River Valley, Eastern Cape

Abstract

Aloe pluridens, A. lineata var. muirii, A. africana, A. speciosa, and A. ferox co-exist through structuring the bird pollinator community by converging into functional groups (pollinated either by sunbirds or non-specialised birds) based on nectar properties, floral architecture, and pollen deposition sites. Functionally similar species have divergent peak flowering times to further reinforce reproductive isolation. Bees played an insignificant role in the pollination of these aloes except in Aloe pluridens and A. lineata var. muirii where it was found that nectar- collecting bees did contribute towards pollination albeit to a much lesser extent than the birds.

Introduction

Aloe (Aloaceae) is considered a predominantly bird-pollinated genus by Skead (1967). The widespread trend within the genus of reddish, scentless, tubular flowers with partial exserted filaments and styles, and a copious supply of dilute nectar produced at the base of the perianth tube, do indeed predict Sunbird-pollination (Faegri and van der Pijl 1979). Specialised nectarivory is prevalent in three specialist bird lineages: the Nectarinidae (sunbirds, flowerpeckers, spiderhunters and sugarbirds) in Africa, Madagascar, Middle East, Asia and Australia; the Meliphagidae (honeyeaters) of Australia and New Zealand; and the Trochilidae (hummingbirds) of North and South America. Hummingbird radiation (> 800 species) far exceeds the diversity of that of the sunbirds (~176 species – Cheke et al. 2001), and this is also seen in the diversity of the floral architecture in some of the classical hummingbird- pollinated flowers compared to sunbird-pollinated flowers. Ollerton (1996) doubted the widespread notion of specialised pollination systems and argued that generalization in pollination is more widespread with plants conforming towards a set pollinator syndrome being visited instead by a range of potential pollinators. However, Johnson and Steiner (2000) showed that specialisation in pollination systems is in fact widespread in the southern hemisphere. Oatley and Skead (1972) list anecdotal evidence for several South African Aloe species as being frequented by an array of both sunbirds and non-specialised bird species, 5 which was confirmed to be true for Aloe ferox (Hoffman 1988, Stokes and Yeaton 1995) at least. Floral visitation by non-specialised birds (typically fruit and insect eaters that resort to nectarivory during times of low abundance of main food sources) is not unique to the African Aloe scene, and is in fact widespread globally except in Europe (Oatley and Skead 1972, Ford 1985, Bruneau 1997). Aloes attract an even wider array of insects (Skead 1967, Reynolds 1969, Hoffman 1988). Honeybees (Apis mellifera races) are frequent visitors to aloe flowers where they gather pollen and nectar. Skead (1967) mentions bees climbing into the perianth tube to reach the nectar at the base of the flower, but Hoffman (1988) did not find this behaviour in his study but suspected that pollen-collecting bees act as pollinators. However, both Ratsirarson (1995) and Stokes and Yeaton (1995) showed that bees did not pollinate aloes to the level which birds did. Elsewhere it has been found that bees visiting bird- syndrome flowers are ineffective as pollinators (Paton 1993, Castellanos et al. 2003) and reduce the reproductive success in these plants (Vaughton 1996, Castellanos et al. 2004) by stripping the pollen supply and reducing the nectar rewards.

Instead of specialising onto a single pollinator, Fenster et al. (2004) placed the emphasis on functional groups (whether taxonomically related or not). As the drivers of floral specialisation these groups exert different selective pressures on floral evolution/specialisation because of the varying degrees of successful pollination they can enact. It is doubtful whether bees, occasional nectarivores and nectarivores exert similar selective pressure on flowers, as Castellanos et al. (2004) showed with Penstemon (hummingbird and bee-pollinated). Aigner (2001) indicated that through the marginal benefits from pollination by more than one functional group, floral evolution towards more specialized forms does not necessarily rely on the most effective pollinator as the selective pressure. Flowers may simply be the product of specialisation towards the less effective pollinator whilst not loosing the services of its main pollinators in the process. The lack of diverse floral architecture in aloes might simply be a result of simultaneously specialising onto an array of functional pollinator groups (sunbirds, non-specialised birds, and bees) in order to maximise reproductive success given that pollinator communities vary over time (e.g. Herrera 1988). However, this lack in the diversity of floral architecture in the aloes make it an interesting group with respect to co-existence, considering that many aloes have mixed ranges and co-flower in an abundant floral spectacle that brightens-up the landscape.

Judging by the recent account of phenolic screening of potential pollinators in Aloe Section Anguialoe (Johnson et al. 2006), and the mixed results of the few accounts of pollination in the group (see Hoffman 1988, Ratsirarson 1995, Stokes and Yeaton 1995), there is merit in delving deeper into the pollination and subsequently reproductive co-existence within the 6 large Aloe genus. Methods of co-existence among plants pollinated by either birds or bees have been reported as divergence in flowering times (Stiles 1977), character displacement resulting in discrete pollen placement (Brown and Kodric-Brown 1979, Armbruster et al. 1994), ethological filtering through caloric nectar selection (Stiles 1975, Bruneau 1997) and the timing of pollen release (Percival 1955, Stone et al. 1998). However, not all cases of co- existence result in reproductive interference and divergence of floral characters (Murray et al. 1987). Facilitation among co-flowering species has been recorded (Gross et al. 2000) and it has been suggested that this interaction might be as important as competition in structuring flowering within communities (Bruno et al. 2003).

Here I attempted to provide an account of the pollination in five sympatric Aloe species in the Gamtoos River Valley, Eastern Cape. I studied their breeding systems, pollinators, and floral traits to determine how these species co-exist reproductively when co-flowering and potentially using the same pollinators. I also attempted to clarify the role of birds versus bees in the pollination of these aloes.

Methods

Breeding systems

The breeding system of Aloe pluridens, A. lineata var. muirii, A. africana, and A. speciosa was determined through the manipulation of flowers on inflorescences that were bagged with fine mesh pollination bags that excluded all pollinators. The breeding system for Aloe ferox was excluded here as it was previously studied by Hoffman (1988). Pollination bags were supported away from the exserted anthers and stigma with a light-weight wire frame. Randomly marked flowers on each of the inflorescences received one of three treatments that was repeated over a two day period: pollen from an individual of the same species from greater than 20m away (crossing), pollen from the same inflorescence (selfing), and no pollen (control). To avoid possible spontaneous selfing, the anthers of flowers in the crossed and selfed treatments were removed prior to anthesis (preliminary investigations showed that this has no effect on the outcome). Chi-square tests were used to test the hypothesis that the resultant fruit- and seed-set that were recorded do not differ among treatments.

Flowering phenology

Because of the impenetrable nature of the vegetation, and the steep slope of many sites, a medium strength spotting scope (20-40x magnification) was used to record the flowering phenology. The state of all the inflorescences on 60 randomly spotted individuals, per species present at each of the 15 sites, was done all on a single day, every 15 days for the duration of the flowering season between April and November 2004 and 2005. Individual inflorescences were scored according to the following six states of flowering: pre-anthesis (state 0), flowering commencing (state 1), flowering bottom half of inflorescence (state 2), flowering top half of inflorescence (state 3), flowering ending and mostly fruiting (state 4), and flowering complete (state 5). These flowering states were used to assess the degree of synchronicity within and between sites for individual species. The peak flowering period for an individual species was defined as the time period when more than 50% of the individuals that were to flower during the season, flowered. Flowering was defined as the point of anthesis where the first anthers protrude from the perianth tube.

Floral ontogeny

The floral development was investigated in 10 individuals of each species with respect to the final presentation of the individual flowers to the pollinator at anthesis, and the positional nature of the anthers and stigma. The length of the pedicel, perianth, filaments, and style were measured in 10 two-day old flowers per individual and analysed through a Principal Component Analysis.

Nectar

Nectar volume was measured in situ from 10 flowers in fresh anthesis on 12 plants for each species using a calibrated 100uL micropipette. The sugar concentration was quantified with a temperature compensated handheld nectar refractometer (Bellingham & Stanley, Inc.). Inflorescences were bagged with fine mesh pollination bags to exclude floral visitors. Results were analysed for significant differences among species with an one-way ANOVA and Tukey Pairwise Multiple Comparison Test.

Bird pollinators, feeding positions and pollen deposition sites

Bird visitors to each species were recorded opportunistically along the road whilst driving, and while working within the various sites. Formal observations were made using a medium strength spotting scope (20-40x magnification), within both mixed and pure stands, for 15min every hour on one day at a stationary position. I also walked along game paths for a distance between 300-400m at two of the larger mixed populations (Sites 8 and 12). Each transect was walked for 10min every hour between sunrise and sunset on one day during peak flowering of each species. I recorded in all cases the identity and feeding habit (with special notice on whether anthers and stigma were touched) of the bird visitors. The sites of pollen deposition on the bird visitors, which was made easy by the copious species-specific coloured pollen, were also noted.

Bees as potential pollinators

The role of bees in the pollination of these five aloes was assessed through bird exclusion treatments. I used commercially available rigid bird netting that is commonly used in fruit crop protection. The mesh size of 15mm gave the bees unhindered access to the flowers, whilst excluding the small Sunbirds (manufacturer’s claim and personal observation). The rigid net was placed around the whole inflorescence of 10 individuals, prior to anthesis, with 100-150mm spacing between the inflorescence and the net. An equal number of control inflorescences were located within 5m of each treatment plant, and were marked prior to anthesis. The resultant fruit- and seed-set were recorded for both the treatment and the control inflorescences, and tested for significant differences with a t-test. Bee observations were conducted on one day during the peak flowering of each species, from 09:00 to 16:00. The amount of bees that were active on the inflorescences of five plants was counted every five minutes. The foraging behaviour, either nectar or pollen-collecting, and their collecting methods for both, were recorded. Notice was taken whether bees contacted the stigma.

Statistical analyses

With the exception of the Principal Components Analysis of the floral measurements (conducted in STATISTICA version 7, Copyright StatSoft Inc. 1984-2004), statistical 9 analyses were conducted in SigmaStat for Windows version 2.0 (Copyright Jandel Corporation 1992-1995).

Results

Breeding systems

All four species tested were self-incompatible with fruit- and seed-set only occurring in the crossed treatments (Table 1.1).

Table1.1: Comparison of median fruit-set for crossed, selfed, and control treatments on bagged inflorescences from a median of 85 flowers (range 69-144) per treatment per species. N reflects the amount of individuals. Median fruit set (%) Species N Crossed Selfed Control X2 P A. pluridens 13 100 0 0 36.8 <0.001 A. lineata var. muirii 12 100 0 0 33.7 <0.001 A. africana 10 100 0 0 26.0 <0.001 A. speciosa 12 53.3 0 0 22.7 <0.001

Flowering phenology

The flowering of the study species was aggregated within the winter-spring flowering season from May to November, over the two consecutive years (Figure 1.1). The flowering season commenced each year at the same time, but the 2005 season was terminated a month earlier compared to the previous year, owing to a prolonged dry spell. Results indicated that the flowering peaks of these species were staggered over the flowering seasons, with varying degrees of overlap between species’ flowering phenophases. There were no obvious differences between the flowering of individuals from the pure stands and those from the mixed stands. Aloe pluridens flowered earliest in May and ceased flowering around mid- August, with peak flowering recorded around mid-June each year. Aloe speciosa peaked in its flowering around mid-July every season, flowering from around mid-June to late August. Aloe lineata var. muirii and A. ferox both peaked around September, with flowering occurring from around late June until November. Scattered individuals of Aloe africana flowered from May, but the species reached its peak flowering around mid-July, ceasing flowering in 10

October. Overall, proportionately more individuals of Aloe pluridens flowered each season than any of the other species. Median values were: for Aloe pluridens – 65%; A. africana – 35%; A. speciosa – 35%; A. ferox – 30%; and A. lineata var. muirii – 26%. Individuals from the different sites reached their peak flowering within days of each other, even though the coastal sites normally started flowering slightly sooner. Synchronicity among individuals from the same site was matched almost perfectly, and the difference between sites was only evident between the most distant of sites (coastal versus furthest inland).

Aloe pluridens

Aloe speciosa

Aloe africana

Aloe ferox

Aloe lineata var. muirii

Apr May Jun Jul Aug Sep Oct Nov Dec

Figure 1.1: Mean valley-wide flowering phenology from 15 sites over a two year period for the five most common aloes in the Gamtoos River Valley. Thin lines indicate flowering time, thick lines indicate peak flowering period, and vertical lines indicate maximum flowering point.

Floral ontogeny

Flowers of these five Aloe species were all bisexual, protandrous, and matured actopetally on the inflorescence raceme. The five species converged into three floral groups based on their floral morphology, development, and final presentation to the pollinators (Figures 1.2 and 1.3). In terms of the PCA, clustering was due to the lengths of the pedicle and perianth. Three distinct groups formed by having long pedicles and long perianths, short pedicles and long perianths, and short pedicles and short perianths. Group 1 consisted out of Aloe pluridens and A. lineata var. muirii, whose upright flower buds dropped downward prior to anthesis and lifted against the inflorescence axis after successful pollination, only to drop down again at fruit maturation. The flowers were slightly zygomorphic with the partially extruded anthers 11 in both species pressed towards the abaxial side of the perianth, which opened a small gap to the inside of the tube that was narrower in Aloe lineata var. muirii. The anthers all faced inward. Group 2 consisted of Aloe africana with its fused perianth that was curved at almost a right angle at anthesis. Its exserted anthers were presented slightly upward from the hanging position of the flower, with the anthers all facing up. The exserted filaments fully blocked the entrance to the perianth tube, which was constricted toward the tip. Flowers hung downward from the early stages of their development, which gave the raceme a sharp conical shape. No floral movement occurred after pollination. Group 3 consisted out of Aloe speciosa and A. ferox, whose short actinomorphic flowers were always near-horizontal at anthesis, and the exserted filaments fully blocked the tapering perianth opening. The anthers faced each other toward the floral axis. Flowers from Groups 2 and 3 had short pedicles, and the inflorescences were dense with typically over 300 flowers per raceme. Inflorescences of Group 1 typically had around 100 flowers per raceme, and the long pedicles elongated throughout the development of the flowers, resulting in the marked floral tropism in these two species.

3.0

2.5

2.0 Aloe ferox

1.5 Aloe speciosa 1.0

0.5 Aloe africana 0.0

-0.5

Factor46.96% 2: -1.0 Aloe lineata var. muirii -1.5 Aloe pluridens -2.0

-2.5

-3.0

-3.5 -4 -3 -2 -1 0 1 2 3 Factor 1: 52.24% Figure 1.2: Ordination plot of selected floral character measurements in the five species, identifying their convergence into three floral groups. Variables that loaded on the factors were pedicle length and perianth length.

Figure 1.3: Comparative floral development for the three floral groups with representative flowers from the different stages – Male phase (anthesis) at the fifth-sixth flower from the top, female phase at the sixth-seventh flower. Group1 - (a) Aloe pluridens, (b) Aloe lineata var. muirii; Group 2 - (c) Aloe africana; Group 3 - (d) Aloe speciosa, and (e) Aloe ferox. Scale 0.5x.

Nectar

There were significant differences in nectar volume (F = 15.34, P < 0.001) and concentration (F = 77.87, P < 0.001) among the five species (Figure 1.4). The nectar properties reflected the groups established on the basis of floral characters. Group 1, Aloe pluridens and A. lineata var. muirii, both had low volumes of more concentrated nectar than Group 3, comprising A. speciosa and A. ferox. These two species produced large quantities of nectar that was comparatively dilute. Group 2, Aloe africana, lay between these two groups. In Aloe pluridens, A. lineata var. muirii, and A. africana, the nectar was located at the base of the perianth. Nectar in Aloe speciosa and A. ferox was produced in such large quantities that it filled the perianth tube and got pushed in amongst the tight exserted filaments where it was kept by hydrostatic forces from flowing out.

A 20 A B 15 C 10 C

Concentration (%) Concentration 5

0 250 cd d

200 bc

150 ab 100 Volume (uL) a 50

0 A. lineata A. pluridens A. africana A. ferox A. speciosa var. muirii Figure 1.4: Comparison of Mean nectar volume and concentration from 10 bagged flowers on 12 individuals per species. Vertical bars = Standard Error of the Mean (± SE). Similar letters denote not significant differences between Means.

Bird pollinators, feeding positions and pollen deposition sites

In terms of bird-pollination, the five species could be readily categorized into the same three groups previously mentioned. Aloe pluridens and A. lineata var. muirii (Group 1), and A. africana (Group 2) were principally visited by sunbirds (three species) that acted as the chief pollinators, but A. africana was also frequently visited by weavers (non-specialised birds) (Table 1.2 and Figures 1.5). Although weavers were destructive in their flower probing at Aloe africana, whereby they tore open flowers (short beak probing long fused tube), they did make legitimate contact with the anthers and stigma in the same fashion as the sunbirds did. After landing on the top of the sturdy inflorescence of Aloe africana, both groups of birds climbed down to the advancing front of acropetally maturing flowers and fed upside down. The orientation of the extruded filaments (at an almost right angle to the perianth base) meant that pollen was deposited on the crown of the probing bird’s head, as it pulled the flower upward when gliding its beak along the curved perianth. Because of the curved nature of the flower, birds were only able to probe successfully, and hence reach the nectar at the base of the perianth, from this upside-down position, which resulted in the accurate placement of pollen only on the crown of the head in this species (Figure 1.6). Sunbirds feeding on Group 1 species perched on the inflorescence peduncle below the raceme from where they foraged at all the basal open flowers by extending their heads upward. Pollen of both species was deposited on the underside of the mandible and chin as the bird glided its beak over the inward facing anthers in the entrance of the perianth tube. Because the pollinator had to perch underneath the raceme whilst feeding at the downward hanging flowers, and because the anthers were all adpressed toward the abaxial side of the perianth, pollen placement on these sunbirds was species specific (Figure 1.6).

Aloe speciosa and A. ferox (Group3) were visited by a range of non-specialised birds (eight species), with weavers being the most frequent of the lot (Table 1.2, Figure 1.5). Birds probed in two ways: (1) after landing on the top of the sturdy inflorescence they climbed down to the advancing front of acropetally maturing flowers and probed upside down; and (2) after landing on the leaf rosette, they probed open flowers near the bottom and sometimes climbed onto the inflorescence below the advancing front of maturing flowers. In both cases, birds probed right through the exserted anthers that faced each other toward the floral axis, and deposited pollen in a mask-like fashion on the bird’s face (Figure 1.6). Mousebirds (the only non-passerine bird to visit the aloes), also got pollen deposited on their breasts and underbellies as they climbed awkwardly over the inflorescence. Several other bird species were visitors to the five study species, but did not seemingly play an important role in their 15 pollination. This was because their feeding behaviour was not conducive to consistent stigma contact (e.g. Collared Sunbird – nectar robbers in Groups 1 and 2); because they were not targeting the aloes as such, but the bees that were active on the flowers (e.g. Forked-tailed Drongo and Dusky Flycatcher – coincidental pollinators in Group 3); or because they were feeding destructively on the flowers (e.g. Streaky-headed Canary – destructive forager especially in Group 3).

Figure 1.5: Feeding positions clockwise from top left: Group 1 - female Amethyst Sunbird on Aloe pluridens (Photo SD Johnson), male Greater Double-collared Sunbird on A. lineata var. muirii; Group 2 - male Greater Double-collared Sunbird on A. africana (Photo SD Johnson); and Group 3 - Yellow Weaver on A. ferox, and a Cape Weaver on A. speciosa.

Table 1.2: List of recorded independent observations of bird visitation to the five aloes differentiated into their different visitation actions, where P = Legitimate Pollinator, CP = Coincidental Pollinator, NR = Nectar Robber, DF, Destructive Forager. A. pluridens A. lineata var. muirii A. africana A. speciosa A. ferox Number of Number of Number of Number of Number of Species Observations Action Observations Action Observations Action Observations Action Observations Action Malachite Sunbird (Nectarinia farmosa) 22 P 4 P 6 P 9 NR 105 NR Greater Double-collared Sunbird (Cinnyris afra) 76 P 74 P 75 P 26 NR 54 NR Amethyst Sunbird (Chalcomitra amethystina) 113 P 1 P 37 P 11 NR 5 NR Collared Sunbird (Hedydipna collaris) 4 NR 1 NR 4 NR/CP Weaver (Ploceus spp.) 10 NR/CP 34 NR/CP 121 P 344 P Speckled Mousebird (Colius striatus) 32 P 101 P Red-winged Starling (Onychognathus morio) 14 P 15 P Cape White-eye (Zosterops pallidus) 12 P 5 P Sombre Bulbul (Andropadus importunus) 2 DF 2 P Forked-tailed Drongo (Dicrurus adsimilis) 8 CP 3 CP Cape Rock Thrush (Monticola rupestris) 2 CP Dusky Flycatcher (Muscicapa adusta) 1 CP Streaky-headed Canary (Serinus gularis) 2 DF 15 DF 3 DF 17

Figure 1.6: Pollen deposition sites (redrawn from photos) on the two classes of pollinators. Top: sunbird feeding at Aloe pluridens, A. lineata var. muirii, and A. africana. Bottom: non-specialised weaver feeding at Aloe speciosa, A. ferox, and occasionally at A. africana. (A) denotes the main pollen deposition site non-specialised birds, with (B) indicating the additional site, mainly for mousebirds, when feeding at these two aloes.

Bees as potential pollinators

The Cape Honeybee (Apis mellifera capensis) was the most common bee species to visit the flowers of all the aloe species. Aloe ferox and A. lineata var. muirii were also visited by smaller bees (probably Allodapula sp. and Lasioglossum sp.) that did not visit the other three 18 species. All bee species employed the same foraging behaviours when selecting for either pollen or nectar (Figure 1.7). When selecting for pollen, bees alighted on the exserted anthers and used their front legs to gather pollen. In many cases they opened anthers prior to natural anthesis. Bees groomed themselves of loose pollen and stored it on their hind legs prior to flying to the next flower. No pollen-collecting bees were observed to make contact with the stigma, as they preferentially targeted flowers in fresh anthesis (male phase) where the stigma was still immature and shorter than the anthers. The large quantities of pollen available from the flowering aloes ensured that bees foraged with high fidelity towards a single species throughout their foraging bouts.

Nectar-collecting bees at Aloe africana, A. speciosa, and A. ferox alighted on the exserted filaments and avoided the anthers. They pushed their proboscises between the filaments at the mouth of the perianth from where they drank any available nectar. In Aloe africana, where the nectar was mostly located at the base of the perianth, bees also sometimes succeeded in making a small hole with their mouth parts at the base of the flower through the fused perianth. Nectar-collecting bees at these species crawled all over the inflorescence and only flew off to the next inflorescence after they had visited a couple of flowers. Nectar- collecting bees feeding at Aloe pluridens and A. lineata var. muirii used their front legs to grasp the partially exserted anthers and pulled themselves over them in order to crawl into the perianth tube through the small gap left by the adpressed filaments. This gap was too small in Aloe lineata var. muirii for the Cape Honeybee to crawl through; only the smaller bee species could enter. The Cape Honeybees drank nectar at the base of flower tubes in this species by pushing their proboscis in between the loose petals. Bee species were observed to crawl up the perianth as far as the ovary from where they drank nectar. Visits lasted anything from a few seconds to nearly a minute. At the end of the bout, bees reverse crawled out of the perianth and again over the anthers, with pollen deposited on the abdomen and thorax. Nectar-collecting bees mostly did not attempt to groom themselves between visiting flowers. Flowers whose anthers were depleted of pollen and where the gap to the perianth tube was enlarged by the wilting filaments were preferred over flowers in fresh anthesis, which were frequented by pollen-collecting bees. One nectar-collecting Cape Honeybee was seen to make legitimate contact with its pollen loaded abdomen on the ripe stigma of Aloe pluridens whilst exiting the perianth.

Bird exclusion experiments identified that bees negligibly contributed toward the pollination of Aloe africana, A. speciosa, and A. ferox, where they were at most accidental pollinators (Figure 1.8). The nectar-collecting bees did play a pollination role in A. pluridens and A. lineata var. muirii. These results also indicated that the pollen-collecting Cape Honeybees 19 are mainly robbers to these five aloes, and therefore could potentially suppress the reproductive success by stripping the pollen supply. Evidence for this could be seen in the fruit-set of the control inflorescences of Aloe lineata var. muirii, where the inability of the Cape Honeybees to successfully harvest the nectar resulted in prolonged pollen-collection. This left low quantities of pollen to be distributed by the sunbird-pollinators in this species, and hence resulted in a much lower fruit-set than the other four species (Figure 1.8). The seed count data also indicated that pollen transfer by bees was likely less than by birds in the study species.

Figure 1.7: Bee foraging behaviour (redrawn from photos) indicating feeding positions when selecting for pollen (P) or nectar (N), with N* indicating the other smaller bees. 1) Aloe pluridens, 2) A. lineata var. muirii, 3) A. africana, 4) A. speciosa, and 5) A. ferox.

t = 14.705 50 t = 3.042 n.s. t = 9.684 P = <0.001 t = 10.563 P = 0.014 P = <0.001 P = <0.001 40 N = 12 N = 10 30 N = 14 N = 10 20

Mean fruit set (%) set fruit Mean 10 N = 10 N = 10 N = 14 N = 10 N = 10 N = 12 0 50 t = 3.044 n.s. n.s. t = 3.483 t = 2.398 40 P = 0.008 P = 0.003 P = 0.035 N = 14 30 N = 10 N = 12 N = 10 N = 4 20

N = 10 N = 8 10 N = 3 N = 10 N = 4

Mean number seeds per fruit seeds number Mean 0 Open Bee only Open Bee only Open Bee only Open Bee only Open Bee only A. pluridens A. lineata A. africana A. speciosa A. ferox var muirii Figure 1.8: Contribution of bees and birds toward the pollination and seed-set. Sample sizes reflect individual plants, n.s. = not significant (P > 0.05).

Discussion

When the data are combined with the breeding results for Aloe ferox obtained by Hoffman (1988) it confirms that all of the study species have an obligatory mutualism with their animal pollinators for successful out-crossing. Flowering times across the study area for all the study species suggested that no selection, or very weak, occurred for altered flowering times within individual species when co-occurring with congeners. The slightly earlier start of flowering along the coast, and the delay furthest inland, is normal for plants that occur along a moisture gradient. The sites closer to the sea experience added moisture input from sea mist that periodically moves into the lower portions of the river valley. The sequence of flowering within the study area was the same for the duration of the study period; this indicated that the observed flowering sequences were the norm.

Greater reproductive success through the facilitation of regional pollinator attraction could be the driving force behind aggregated flowering in aloes in general, as this large group is otherwise not to phylogenetically constrained in their flowering (Reynolds 1969, aloes flower year-round in South Africa). However, aloes tend to flower en masse regionally within a season, co-existing with their limited variation in floral architecture through a system of staggered flowering. Feinsinger (1978) found that the staggered flowering within a successional bird-pollinated community maintained a continuous supply of pollinators to the 21 greater benefit of all the species, and promoted commitment from the bird-pollinators to the nectar sources in an area.

Birds can minimise the energetic cost of foraging through minimising floral searching times. This is done through displaying strong floral preference and consistency at times when food plants are abundant. Birds will however, tend to feed at any suitable food source during times of low food plant flowering densities (Levin and Anderson 1970). This was seen in the few incidences when weavers fed at Aloe pluridens and A. africana, and in the nectar robbing behaviour of sunbirds at A. speciosa and A. ferox. Both cases happened during times of reduced food plant flowering as happens between the flowering peaks of ecologically similar species. However, the pollinator results of this study are consistent with the prediction of optimal foraging theory (Possingham 1992, see also Rodriguez-Girones 2006), and neatly predicted the co-existence of the sunbirds and non-specialised bird-pollinators in this system, where species with two different floral types co-flower. The small bodied sunbirds have a higher mass-specific energy intake than the comparatively larger sized non-specialised bird species. Based on this, there would be the opportunity for co-occurring plants to exploit the different caloric needs of potential pollinators in order to maintain the pollinator floral constancy that is needed for reproductive success in mixed assemblages. Selection on this level has been documented in the specialised/non-specialised bird systems of the Americas for Erythrina (Feinsinger et al. 1979, Baker and Baker 1990, Bruneau 1997), a predominately bird-pollinated pantropical genus; Jacot Guillarmod et al. (1979) have found similar evidence for the species occurring in South Africa. The striking sequence in the flowering of these aloes, whereby a sunbird-pollinated species was followed by an non-specialised bird- pollinated one, with the cycle repeating itself, suggested that selection had occurred for minimal ecological overlap in flowering between those species that show convergent evolution towards the same pollinator guild, and where reproductive isolation through differential pollen placement on the pollinator was limited.

Baker and Baker (1983) have shown that the nectar sugar composition of a wide array of species show remarkable adaptation to pollinator type. van Wyk et al. (1993) however, determined the nectar sugar composition in aloes to be uniform, although it is known that purely insect-pollinated species exists (e.g. Aloe bowiea Roem. & Schult., personal observation). Insect-pollinated species have less volume, but more concentrated nectar than the sunbird-pollinated species (Unpublished data). This suggested that the pollinator differentiation in these aloes occurred through nectar concentration rather than composition, and that this was reinforced through floral architecture. Similar structuring through caloric selection and floral reinforcement happened in Penstemon (Castellanos et al. 2003, 2004, 22

Wilson et al. 2004), where species are pollinated by either bees or birds. Nectar data on most aloes are currently lacking, however, floral form was found in this study to be strongly correlated with pollination syndromes. It would be possible from the data obtained here to make reasonable predictions concerning the pollinators of floristically similar aloes, for the majority of which we lack pollinator data. The extrapolation of pollination syndromes in other South African aloes was attempted in Chapter 2.

The role of bees in the pollination of aloes is now becoming clearer, with the identification of floral types adapted toward bird-pollination where bees are resource robbers and others where they contribute to the pollination when nectar-collecting. Navarro (2000) found that nectar robbers could act as accidental pollinators, if they make occasional contact with the reproductive surfaces. Wilson and Thomson (1991) also showed that pollen-collecting bees deposited less pollen on receptive stigmas than nectar-collecting bees. Bees also tended to affect a greater amount of selfing than birds (Castellanos et al. 2003). When visiting bird syndrome flowers, bees were found both ineffective as pollinators (Paton 1993, Castellanos et al. 2003) and detrimental to the reproductive success in these plants (Vaughton 1996, Castellanos et al. 2004). The exserted anthers of the bird syndrome flowers of aloes make them highly susceptible to resource robbing by bees. In some respect, the relationship of the pollen-collecting bees, especially, could then be described as parasitic towards these aloes. These bees potentially negatively affect the reproductive success of aloes by reducing the amount of pollen available for distribution by the bird-pollinators whilst not contributing to the reproductive success of aloes themselves. However, judging by the feeding behaviour of the bird-pollinators, whereby they tended to feed at most of the flowers of the raceme, and at all the racemes of the individual plant, the stripping of the pollen supply by the bees on an individual would reduce its chances of selfing. Birds would more likely accumulate a heterosporous load of pollen over the course of their foraging bouts when feeding at bee- visited flowers, as apposed to the homosporous pollen load when feeding at hypothetically bee excluded flowers. The latter case would waste plant resources through ovule abortion, whilst the former would maximise reproductive success. Whether this is an incidental occurrence or whether bird-pollinated aloes have specifically evolved to tolerate the bees - which have been a historically constant element in the African environment where they would always have been pollen-collecting - aloes might simply have adapted to sacrifice pollen without loosing out on the services of their true bird-pollinators. The marginal benefits (see Aigner 2001) of nectar-collecting bees as pollinators in some of these aloes might be incentive not to specialise completely away from bees (given no phylogenetic restrictions of course). Competition for pollinators among individuals of the extensive populations must be 23 tough, also pollinator populations vary naturally over time (e.g. Herrera 1988). These factors would certainly have played a selective role in the floral evolution of aloes.

Chapter 2 – Extrapolating pollination syndromes for South African Aloe species

Abstract

The categorization of South African Aloe species into pollination syndromes, based on floral characteristics, was done using data from Chapter 1, other published accounts, and personal unpublished data. Of the 125 species (excluding the varieties) listed in the “Guide to the Aloes of South Africa”, 12 conformed to the insect-pollination syndrome, 91 to the sunbird- pollination syndrome, and 22 to the non-specialised bird-pollination syndrome. This confirmed Skead’s assertion that aloes are generally sunbird-pollinated. Syndromes were significantly over-represented in various Aloe Sections. Insect-pollination seemed to be basal to sunbird-pollination. Non-specialised bird-pollination was aggregated among the most derived aloes. Data from Chapter 1 would suggest that the majority of sunbird-pollinated species could be co-pollinated by nectar-collecting bees, if they are able to crawl into the perianth tube.

Introduction

While South African Aloe species are diverse in their vegetative growth forms, their floral architecture show limited variation (Reynolds 1969, Van Wyk and Smith 2003). It is the floral-trait combinations that characterize pollination syndromes (Stebbins 1970, Faegri and van der Pijl 1979). The common practise of predicting floral syndromes have been questioned recently on the basis of several pollinator types visiting a set pollination syndrome (Ollerton 1996, Waser et al. 1996). However, biologists like Wilson et al. (2004) continue, convincingly, to highlight floral radiation onto different pollinators. From available data for the aloes (Reynolds 1969, Hoffman 1988, Ratsirarson 1995, Stokes and Yeaton 1995, Johnson et al. 2006, this study), floral form and pollinator type can be linked, and the trend extrapolated onto the rest of the species for which pollinator data is lacking, but where the floral type is known. Pollinators for the majority of the aloes have not been established, but Skead (1967) claimed that most of aloes are sunbird-pollinated. Here I attempted to provide a quantitative preliminary account of the likely pollination syndromes for all the aloes found in South Africa. 25

Methods

There are three possible syndromes within the aloes based on the studies mentioned above, and personal investigations into species with a floral form for which there were no data. Table 2.1 outlines the different syndromes and how they are identified. These identifying characteristics were used to predict pollination in the 125 species (excluding varieties) listed in van Wyk and Smith (2003), with the aid of the life-sized floral photos in Reynolds (1969). Some species have one or more varieties, but these were ignored here since varieties have similar flower types and hence pollination syndromes. It was also not possible to assess which sunbird-pollinated species are co-pollinated by nectar-collecting bees as no data on the degree of perianth opening is available.

Table 2.1: Floral descriptions forming the basis of the three pollination syndromes found in the South African Aloe species. Syndrome Floral description Sunbird Long tubular flowers, anthers not exserted far, but adpressed in the perianth opening, flowers pendant, only a few open at a time, and generally rise against the inflorescence axis once pollinated. e.g. A. pluridens. Or, long tubular flowers, anthers exserted at an angle to the perianth, inflorescence dense and flowers presented downward. e.g. A. africana Non- Short flowers with prominent exserted anthers that are pressed tight at the specialised perianth opening blocking the entrance, flowers on a very dense raceme bird where they are presented close to horizontal, many open at a time. e.g. A. ferox. Or, short cup flowers with exserted anthers not blocking the opening, on a dense raceme with lots of open flowers at a time. e.g. A. castanea Insect Small very short tubular flowers, presented horizontally, with the anthers partly exserted and together with the lower petals forming a landing perch, flowers are limited in number and inflorescences are very small. Flower colour is green, light cream, or white. e.g. Aloe bowiea

Results

Table 2.2 list alphabetically, within the Sections proposed by Reynolds (1969), the predicted pollination syndromes for the South African aloes. Sections are arranged in increasing vegetative complexity, with the grass aloes considered basal and the tree aloes derived. 26

Table 2.2: Extrapolated pollination syndromes for the South African Aloe branddraaiensis S species, where I = insect-pollination, S = sunbird-pollination, and NS = non- brevifolia S specialised bird-pollination. broomii NS buhrii S Section Species Syndrome burgersfortensis S Graminialoe albida I chabaudii S bowiea I chlorantha I fouriei S ciliaris S inconspicua I claviflora NS minima I commixta S modesta I comosa S myriacantha I comptonii S parviflora I cryptopoda S saundersiae I dabenorisana S S Leptoaloe boylei S dewetii S chortolirioides S distans S cooperi S dyeri S dominella I falcata S ecklonis S fosteri S hlangapies S framesii S integra S gariepensis S kniphofioides S glauca S kraussii I gracilis S linearifolia S grandidentata S micracantha S greatheadii S nubigena S greenii S soutpansbergensis S haemanthifolia S thompsoniae I hardyi S verecunda S heroensis humilis S Eualoe affinis S immaculata S arborescens S khamiesensis S arenicola S krapohliana S aristata S lettyae S 27 lineata S umfoloziensis S longbracteata S vanbalenii S longistyla S variegata S lutescens S verdoorniae S maculata S vogtsii S melanacantha S vossii S meyeri S zebrina S microstigma S Anguialoe alooides NS monotropa S castanea NS mudenensis S spicata NS mutabilis S vryheidensis NS parvibracteata S Pachydendron aculeata NS pearsonii S africana S peglerae NS angelica NS perfoliata S excelsa NS petrophila S ferox NS pictifolia S gerstneri NS pluridens S globuligemma NS polyphylla S littoralis S pratensis S marlothii NS pretoriensis S petricola NS prinslooi S reitzii S pruinosa S rupestris NS reynoldsii S thraskii NS simii S Dracoaloe dichotoma NS speciosa NS pillansii NS striata S ramosissima NS striatula S Aloidendron barberae NS succotrina S Kumara plicatilis S suffulta S suprafoliata S swynnertonii S tenuior S thorncroftii S 28

A total of twelve species conformed to the insect-pollination syndrome, 91 to the sunbird- pollination syndrome, and 22 to the non-specialised bird-pollination syndrome. Syndromes were significantly over represented (G-test, Table 2.3) in the Sections Graminialoe (insect), Leptoaloe (sunbird), Eualoe (sunbird), and the combined Sections Anguialoe, Pachydendron, Dracoaloe, Aloidendron and Kumara (non-specialised bird), compared to the overall frequency for the three syndromes.

Table 2.3: Summary of the distribution of pollination syndromes within the Aloe Sections. I = insect-pollination, S = sunbird-pollination, NS = non-specialised bird-pollination. Pollination Syndrome Section I S NS G - value P - value Graminialoe 8 1 0 33.30 < 0.001 Leptoaloe 3 12 0 6.65 0.036 Eualoe 1 74 4 47.36 < 0.001 Anguialoe - Kumara 0 4 18 58.83 < 0.001 Total species 12 91 22

Aloes of the insect-pollination syndrome flower during the summer months (Figure 2.1). Those of the non-specialised bird-pollination syndrome flower mainly in the winter, with a cessation during the hottest months. Aloes of the sunbird-pollination syndrome flower throughout the year, but a peak during winter and a marked lull during autumn is observed.

25 Insect Sunbird Non-specialised bird 20

Number offlower species in 5

0 123456789101112 Month Figure 2.1: Distribution of flowering according to syndromes. Flowering dates taken from Reynolds (1969). 29

Discussion

Pauw’s (1998) unexpected description of Sunbird-pollination in Microloma sagittatum (Asclepiadaceae), where the pollinia are attached to the tongue of the birds, emphasises the need for caution when predicting pollination syndromes based on gross floral features. However, data from Chapter 1 and other published cases conclusively showed that sunbird- and non-specialised bird-pollinated aloes have distinct floral types. Similarly, insect- pollinated aloes have unique flowers that are unlike those of the bird-pollinated species (unpublished data for Aloe bowiea). This justifies the current use of predicting syndromes for aloes from floral-traits.

Skead (1967) was correct to propose that the South African aloes are mostly sunbird- pollinated. The probability that bees co-pollinate the majority of these sunbird-pollinated aloes is likely, if they are able to crawl into the perianth tube when nectar-collecting. This could not be assessed from the data in Reynolds (1969), and would only be possible to assess from field observations. Accepting that the current conceptual phylogeny is still to be conclusively proven, it was evident that the insect-pollination syndrome was restricted to the ancient aloes, with the non-specialised bird-pollination syndrome to the most derived.

Sunbirds have the same general beak shape, only varying in length slightly (Cheke et al. 2001). Although this probably would seem to place a limit on aloe radiation, also considering that there would appear to be only two places where pollen is deposited on sunbirds (crown of head and underside of mandible and chin), sunbirds pollinate the overwhelming majority of aloes, and aloes have radiated extensively. Only in the case of two species (Aloe africana and A. longistyla Baker) is pollen deposited on the crown of the sunbird’s head, with both species forcing the bird to probe downward from a perch. All other sunbird-pollinated aloes have the same pendant flowers where pollen would be deposited under the mandible and chin as the bird probe upwards from its perch underneath the raceme. Pollen would be deposited in a mask-like fashion in all the non-specialised bird-pollinated aloes. It would be interesting to have a look at the national database on species occurrence, and together with flowering times, see how many aloes of a set syndrome co-exist in a region. It was evident from the flowering curves that the majority of species, within the respective syndromes, flower within the same time frame. Given the limited degree of pollen separation on the bird-pollinators, a restricted number of species from the same syndrome would be able to co-flower concurrently in an area, as hybridization might become an issue when sharing pollinators. Factors influencing hybridization was investigated in Chapter 3. 30

Chapter 3 – Factors influencing hybridization in Aloe

Abstract

Natural hybridization between co-flowering Aloe species results from the breakdown in floral constancy by bird-pollinators in incidences of equal choice, where co-flowering species are not sufficiently isolated through differential pollen placement. Within a three-way choice array consisting of Aloe pluridens, A. africana, and A. speciosa, sunbirds showed strong selection towards the typical sunbird-pollinated species (A. pluridens and A. africana), whereas the non-specialised birds were indiscriminate in their choices, visiting all three species representing the three pollination types identified in chapter one. Aloe africana was more frequented during interspecies visitations by both sunbirds and non-specialised birds than the other two species, which started to explain A. africana’s high incidence in hybrid combinations. Natural hybrids between Aloe africana, A. ferox, and A. speciosa were more abundant than combinations with the two sunbird-pollinated species, implicating the non- specialised Weavers as the main interspecific pollen vector for hybridization events among these species. Aloe species were highly interfertile, although a reduced number of seeds developed through hybrid crosses compared to pure out-crossing. The spatial aggregation of species within mixed populations appear to be the main factor limiting natural hybridization, presumably because of the effect of this on the energetics of foraging. This study explains why hybrid individuals are associated mainly with ecoclinal zones where parent species are more intermixed in an equal choice manner.

Introduction

There exists a seemingly robust system of co-existence between the five co-flowering Aloe species in the Gamtoos River Valley, with species co-existing on a system of ethological attraction to a structured pollinator community, reinforced by floral characters, and staggered peak flowering (Chapter 1). If these and similar assemblages of Aloe species manage to co- exist through these mechanisms, why is it then that the incidences of hybridization is seemingly so common in this group? Reynolds (1969) lists no less than 25 commonly hybridizing species’ combinations for the 30 Aloe species occurring within the Eastern Cape Province alone, and this seems to be a widespread occurrence wherever species come into close contact (Barker et al. 1996, Van Der Bank and Van Wyk 1996). The ability to hybridize 31 readily is a trait that has been fully exploited by the horticulture industry (de Wet 2004). Members of the Aloaceae have a remarkable similarity of several reproductive characters including gross floral architecture (Reynolds 1969), the external and internal ovule characteristics (Steyn and Smith 1998), and probably most relevant here, a stable structure and similar basic number of chromosomes (Brandham and Doherty 1998). Data from Chapter 2 showed that the majority of aloes are pollinated by sunbirds and that species from the same syndrome generally flower within the same season. This would make hybridization very likely in the absence of other reproductive isolating mechanisms. Historically, hybridization might have played a pivotal role in the radiation of the group (Holland 1978, Riley and Majumdar 1979, Viljoen 1999).

Natural hybridization has been implicated in both the rise of species through the development of adaptive novel traits (Rieselberg et al. 2003), and in species’ demise through demographic swamping and genetic assimilation (Levin et al. 1996). Although the process of natural hybridization may not be uniformly frequent across all taxa (Ellstrand et al. 1996), it is seemingly a lot more common than once believed (Stebbins 1959, Abbot 1992, Arnold 1992, Rieseberg 1995). In fact, Masterson (1994) estimated that the majority of the extant angiosperms evolved from the products of ancient hybridization events. Hybridization certainly does produce far more variations at the genetic level within one generation than mutations can, leading to faster speciation (Arnold 1997). It seems then that natural hybridization, in the absence of any genetic barriers, is a pivotal process whereby evolution operates on a frequent scale. Plants can, however, isolate themselves from hybridization through spatial, ecological, and reproductive isolation means (both pre-pollination and post- pollination). It was evident that the five species in the Gamtoos River Valley managed to occur in sympatry, without the breakdown of species integrity and the formation of fertile hybrid swarms, mainly via pre-pollination reproductive isolation (Chapter 1).

The aim of this study was to understand the factors involved in promoting hybridization among the five most common sympatric Aloe species within the Gamtoos River Valley, Eastern Cape. This was done by determining the degree of interfertility among the three most abundant aloes, recording the frequencies of species pairs in natural hybrid combinations, noting hybrid spatial occurrence, and investigating the level of successful ethological isolation among species.

Methods

Hybrid crosses

Reciprocal hybridization crosses between Aloe pluridens, A. speciosa, and A. africana (the three most common of the aloes within the study site, and representing the spectrum of floral architecture and hence both sunbird and non-specialised bird-pollination syndromes), were conducted on five plants per species from monospecific populations. Inflorescences were bagged with fine mesh pollination bags prior to the onset of anthesis and treatments were randomly allocated to marked flowers with the anthers removed. Fresh pollen was collected from pure individuals each morning of applying the treatments, and treatments were repeated for the same flowers over two days ensuring good pollen coverage on the stigma. The resultant fruit- and seed-set were recorded and compared to that found for pure crosses (Chapter 1).

Hybrid identity

Since hybrid delimitation is by nature a very subjective exercise in the absence of molecular evidence, having to relay on morphological intermediaries and sometimes gestalt, I start off by defining the morphological characteristics of what I considered pure species of Aloe pluridens, A. speciosa, A. africana, A. ferox, and A. lineata var. muirii. To do this, I drew on the information in Reynolds (1969), Glen and Hardy (2000), and van Wyk and Smith (2003) stressing specific characters identified by personal field investigations into isolated monospecific populations as useful to the field ecologist. Table 3.1 lists these pure species characters, and Plates 2-6 illustrate these. Using the character states identified here, field recognisance surveys were conducted throughout the study area searching for possible hybrids among the populations. The parental lineage of the hybrids was estimated in the field based on obvious intermediate characters and a systematic gestalt based on the characters listed in Table 3.1. This field classification was verified with a Non-metric Multidimensional Scaling (MDS) exercise using 37 vegetative, floral, and leaf chemical characters, which was identified as most useful in conclusively identifying hybrids from pure species. The MDS exercise was conducted with ten random pure individuals per species, and all the flowering hybrid variations found during the 2005 flowering season.

Table 3.1: Vegetative, floral, and chemical characters identifying the five pure species. Inflorescence and Species Growth Habit Sheaths Rosette and Leaves Leaf exudate Flower Raceme Aloe pluridens Single stemmed, Papery, Upright; Choc-nut smell, Up to 3 Salmon-pink, perianth free frequently 2-3 partially Gracefully recurved clear both when simultaneously, each to base, partial exserted branched persistent, bright green, fleshy on feel; fresh and dried, up to 4 branched; filaments yellow, pedicle 1 1 /3- /2 Soft white spines, thick consistency Conical racemes ~28mm long coverage deltoid incurved, almost gel-like ~ 2mm high, abaxial spines absent Aloe lineata Single stemmed, Medium, Upright; Sweet pumpkin Always simple, up to Salmon to deep rose pink, var. muirii invariably branched persistent, Densely erect spreading, smell, clear when 4 simultaneous or perianth free to base, partial 1 2 /2- /3 yellowish-green with marked fresh, dries very consecutively; exserted filaments pale coverage striations, fleshy on feel; light yellow, Conical racemes lemon, pedicle ~30mm long Hard dark reddish-brown thick consistency spines, deltoid, ~2.5mm high, almost gel-like abaxial spines absent Aloe africana Single stemmed, Hard, Upright; Rhubarb smell, Up to 2 Dull red in bud, yellow- unbranched persistent, Spreading recurved, dull green, dark buttery consecutively, simple orange when mature with 1 2 /2-full leathery on feel; when fresh, dries sometimes 2-3 distinct curvature of the /3- 3 coverage Pungent clay brick-red marginal golden, medium branched; /4 fused perianth, exserted spines, conical shaped, consistency Cylindric-sharply filaments light orange, 34

~ 4mm high, conical racemes pedicle ~5mm short abaxial spines on apical median line Aloe speciosa Single stemmed, Medium, Tilted and untidy; Chemical smell, Always simple, up to Red in bud, greenish-white more frequently 2-5 persistent, Erect spreading, bluish green, bright golden 4 simultaneously; when mature, perianth free 1 3 branched /2- /4 pink edged, fleshy on feel; when fresh, dries Broadly cylindric to base, exserted filaments coverage Minute pink spines, shark fin reddish olive- racemes brownish-red, pedicle ~8mm shaped, ~1mm high, abaxial green, short spines absent medium-watery consistency Aloe ferox Single stemmed, Hard, Upright; Strong rhubarb- Always one, 5-8 Reddish-orange, perianth 1/3 unbranched persistent, Erect spreading sometimes urine smell, branched; fused, exserted filaments full coverage recurved, golden syrup Cylindric/narrowly deep orange, pedicle ~3mm dull green, leathery on feel; when fresh, dries conical racemes short Pungent dark brown spines, butter yellow, conical shaped, ~5mm high, thick consistency abaxial spines variably sets quickly scattered but pronounced on the apical median line

Characters used for the MDS analysis:

1. Growth habit (single stemmed branched, single stemmed unbranched) 2. Sheath consistency (soft, medium, hard) 3. Sheath stem coverage (fractions 0.25, 0.33, 0.5, 0.66, 0.75, 1) 4. Number of rosettes per plant 5. Rosette appearance (upright, tilted) 6. Number of leaves per rosette 7. Leaf feel (fleshy, leathery) 8. Leaf colour in shades of green (bright, dull, yellowish, bluish) 9. Mean leaf length (n=5) 10. Mean leaf width, measured 50mm from base (n=5) 11. Mean leaf margin spine spacing (base, mid leaf, tip; n=5 each) 12. Mean leaf margin spine height (base, mid leaf, tip; n=5 each) 13. Leaf spine colour (clear, reddish, brown) 14. Leaf spine consistency (soft, hard) 15. Leaf thorn shape (deltoid incurved, conical) 16. Mean number of abaxial leaf thorns (n=5 leaves) 17. Leaf Aloein exudate consistency (watery, medium, thick) 18. Leaf Aloein exudates smell (choc-nut, sweet pumpkin, rhubarb, chemical, urine) 19. Leaf Aloein exudates colour fresh (clear, light yellow, bright yellow, buttery, golden) 20. Leaf Aloein exudates colour air dried on white paper (clear, light yellow, butter, faded gold, ruby red, reddish olive) 21. Number of inflorescences per rosette 22. Number of racemes per inflorescence 23. Shape of raceme (conical, cylindric) 24. Height of inflorescence, measured from tip of highest raceme to point of inflorescence emergence from rosette 25. Flower colour, bud and mature (salmon orange, yellow, orange, red, white) 26. Flower presentation (pendent, sub-horizontal) 27. Mean pedicle length (n=5) 28. Mean perianth length (n=5) 29. Fused perianth length (fractions of 0, 0.25, 0.33, 0.5, 0.66, 0.75) 30. Mean filament length (n=5) 31. Exserted filament colour (yellow, orange, reddish brown) 32. Perianth mouth (open, closed) 33. Mean gynoecium length (n=5) 36

34. Ovary colour (yellow, dark green, lime green) 35. Pollen colour (salmon pink, mustard, yellow, orange) 36. Mean nectar volume (n=10) 37. Mean nectar concentration (n=10)

Experimental Array

In order to examine patterns of pollinator behaviour during foraging bouts, and whether co- existence is truly based on a robust system of ethological attraction, a three-way experimental array consisting of Aloe pluridens, A. africana, and A. speciosa was set-up in a clearing within an area where all three species occur naturally. Two sessions were conducted, each lasting three days, and each setup was placed in a different area during the time of greatest flowering overlap among the three species. Twelve similarly sized inflorescences of each species were collected at sunset the day before each session of the experiment was to start. Inflorescences were attached to 2m poles placed randomly in a 6 x 6 grid with a spacing of 1m between adjacent poles. Inflorescence placement within the array stayed the same for the duration of the session. Continuous observation between sunrise and sunset was maintained from a shelter in a nearby bush. The identity of the bird was recorded when it entered the array, and its consequent inflorescence selections were noted. The degree of successful ethological isolation between the three species was established through the fidelity of the bird’s choices in the array, and compared to field observations where species occurred in a non-random distribution.

Statistical Analyses

Hood, G. M. (2005) PopTools version 2.6.9. Available on the internet. URL http://www.cse.csiro.au/poptools, was used for conducting the statistical analyses except for the MDS exercise which was done using PRIMER for Windows version 5.2.4, Copyright 2001 PRIMER-E Ltd.

Results

Hybrid crosses

Reciprocal hybridization crosses between the three most common aloes, Aloe pluridens, A. africana, and A. speciosa, identified that these species were highly cross fertile with fruit-set from hybrid crosses comparable with pure species out-crossing (Table 3.2). An exception was Aloe speciosa, for which the hybrid crosses were more successful than pure species crosses. The amount of seeds per fruit, however, was lower in the all the hybrid crosses than for pure out-crosses (Figure 3.1).

Table 3.2: Median percentage fruit-set for pure and hybrid crosses among the three most common Aloe species. N = number of individuals tested, with a Median of 60 flowers (range 55- 81) used in each treatment. Median fruit set (%) Species crossed A. pluridens A. africana A. speciosa A. pluridens 100 (N=13) 93.3 (N=5) 88.9 (N=5) A. africana 100 (N=5) 100 (N=12) 100 (N=5) A. speciosa 100 (N=5) 94.1 (N=5) 53.3 (N=12)

70 a F = 17.282 n.s. n.s. 60 P < 0.001 50

40 bb

10 Mean number of seeds per offruit number per seeds Mean pluridens africana speciosa africana pluridens speciosa speciosa africana pluridens A. pluridens x A. africana x A. speciosa x Figure 3.1: Mean number of seeds per fruit attained through the hybrid and pure crosses among the three most common Aloe species. Vertical lines = Standard Error of the Mean (± SE). Same letter denotes no significant difference between the Means (oneway ANOVA).

Hybrid identity

A total of 573 flowering hybrid individuals were found during the field surveys. Hybrids were found to posses stark differences in their vegetative (growth habit, rosette and leaf 38 structure), and floral (inflorescence, raceme shape, and flowers) character states compared to those of pure species. A mosaic of character states was present among the hybrids of the same species crosses, but it was possible to make a prediction regarding its mixed identity in the field based on its various character resemblances to that of the pure species. No hybrid was exactly intermediate to the two parents, and individuals always resembled one parent more than the other, so that for example, Aloe africana x ferox looked different to A. ferox x africana, even though they are the same species cross. A total of eight hybrid combinations were recognised in this manner (Plates 7-14). Frequencies of these hybrid species’ combinations are given in Table 3.3 and were based on flowering individuals only. No hybrid with Aloe lineata var. muirii as one of the parents was found. Aloe africana was the most frequent species in hybrid combinations.

Table 3.3: Frequencies of flowering natural hybrid crosses located during one season. Species rows correspond to dominant characters resembling one of the parents. x lineata Species crossed x pluridens x africana x speciosa x ferox var. muirii A. pluridens - 0 13 1 0 A. lineata var. muirii 0 - 0 0 0 A. africana 0 0 - 7 181 A. speciosa 0 0 127 - 34 A. ferox 0 0 204 6 -

The MDS analysis confirmed the subjective classification which was used to classify hybrids in the field (Figure 3.2). The only exception was the hybrid cross Aloe pluridens x speciosa, which was closer to the hybrid combinations between A. speciosa and A. africana than pure A. speciosa.

Hybrid individuals were found in varying numbers across the study area. However, most hybrids were located in disturbed areas along the road verges, ephemeral drainage lines, or next to the old game paths. No hybrids were located in undisturbed bush where pure species mostly occurred. Also, no hybrids were found within the core of populations, but rather along the zone of contact between pure species’ patches within the landscape (ecoclinal zones). Hybrid individuals flowered sooner than their pure parental species, and especially in May hybrids could be easily spotted. Only a handful of bird visitations to hybrid individuals were observed, and in these cases the birds only probed one or two flowers before moving to a pure individual. Some fruit-set was noted in a few cases, and there were seeds present in some, but 39 unfortunately this was only anecdotally quantified and serves here only to point out that hybrid individuals were clearly fertile to some degree.

Stress: 0.07 Aloe africana H8 P9 A2 A3 P10P1P2P6P7P8 P5 A1A 87A 94 A6AA 510 P4P3

H1 Aloe pluridens

H2 F1F10F9F5F6 F7F8 H3 F2F3F4 H7 H5 Aloe ferox

S3S10S2 S1 Aloe speciosa S6 S7S9S5S8S4

Figure 3.2: MDS plot of 37 vegetative and floral characters, for pure species (excluding Aloe lineata var. muirii) and one individual from each of the eight hybrid variations found. Pure species are blocked. Subjective field classifications for the hybrids were: H1 = A. africana x ferox, H2 = A. ferox x africana, H3 = A. ferox x speciosa, H4 = A. speciosa x ferox, H5 = africana x speciosa, H6 = A. speciosa x africana, H7 = A. pluridens x speciosa, H8 = A. pluridens x africana.

Experimental Array

A combined total of 221 individual birds (121 nectarivores, 100 occasional nectarivores), comprising eight species, entered the experimental array during the two sessions. They performed a total of 485 foraging choices, with individual bird species (among and between sunbirds and non-specialised birds) differing in their foraging bouts (Table 3.4). Sunbirds showed a clear preference for Aloe africana and A. pluridens, with the observed ratio of visitations significantly departing from the expected equal preference among the three species (Goodness of fit, G = 79.39, P < 0.001). Non-specialised birds (mostly Weavers) were frequent visitors to all three species, although A. africana was frequented more than the other two (Goodness of fit, G = 24.48, P < 0.001). This is in contrast to what was observed outside the array within natural assemblages. Sunbirds frequented Aloe pluridens significantly more than A. africana (G-test, G = 10.16, P = 0.006), the reverse being true for the experimental array, and non-specialised birds were significantly more frequent visitors to A. speciosa 40

within natural assemblages (Table 1.2) than to the other two species (G-test, G = 62.22, P < 0.001). Birds showed no clear floral constancy in their foraging bouts within the array, although Aloe africana was more likely to be the next choice for both sunbirds and non- specialised birds, and hence experienced the most fidelity for intra-species movements of the three aloes. The frequencies of inter-plant movements were significantly different for sunbirds and non-specialised birds (G-test, G = 13.07, P = 0.001), with sunbirds mainly moving between Aloe pluridens and A. africana (two typical sunbirds-pollinated plants), versus non-specialised Weavers, which moved between all three species more frequently (Figure 3.3).

Table 3.4: Bird foraging choices during visitations to the experimental array, where S = sunbird. NS = non-specialised bird. Observed values compared with expected equal choice using Goodness of fit test. Number of visits Species A. pluridens A. africana A. speciosa G-value P-value Black Sunbird 70 111 17 77.27 < 0.001 Chalcomitra amethystina (S) Greater Double-collared Sunbird 30 36 14 10.54 0.005 Cinnyris afra (S) Malachite Sunbird 5 0 2 - - Nectarinia farmosa (S) Cape White-eye 0 0 7 - - Zosterops pallidus (NS) Weavers (4 species) 55 100 38 30.86 < 0.001 Ploceus spp. (NS)

Moving from: 35 A B 30 A. pluridens A. africana 25 A. speciosa 20 15 10 5

Percentage of inter plant visits of inter Percentage 0 A. pluridens A. africana A. speciosa A. pluridens A. africana A. speciosa Moving to: Moving to: Figure 3.3: Percentage of inter plant movements during foraging bouts within the two experimental arrays, where (A) is sunbirds and (B) is non-specialised birds. 41

Discussion

Hybrids between most species were frequently encountered in the study area. This affirmed Reynolds’ (1969) claim of widespread hybridization wherever species are growing in close proximity to each other. Co-flowering species were found to be highly interfertile. The reduced seed-set observed in the hybrid crosses compared to pure species crosses was probably due to ovule abortion, since pollen limitation was ruled out by the method. Some degree of genetic incompatibility must thus exist, albeit not very strong. Although the peak flowering periods of the study species were staggered throughout the flowering season, a great degree of overlap was observed in the study area between individual species’ total flowering times (Chapter 1), even though these were at different phenological phases. This and the interfertility of species would make hybridization a real possibility. The localities where mixed species assemblages were located were where the hybrid individuals were also found.

The MDS analysis confirmed the subjective field classification for the hybrids, but not conclusively for the suspected hybrid cross Aloe pluridens x speciosa (H7). This hybrid had most vegetative characters resembling that of Aloe pluridens and A. speciosa. However, the stiff, reddish leaf margin spines that were longer than recorded for both putative parents, and the leathery feel of the leaves, stand out as some of the characters that neither the putative parents could have contributed. The placing of this hybrid in the MDS plot, suggested that it might be a second generation hybrid between Aloe pluridens and one of the hybrids between A. africana and A. speciosa – probably A. speciosa x africana, given the sub-dominance of the A. speciosa characters in this hybrid. Some hybrids did indicate signs of some level of fertility. Barker et al. (1996) found second generation aloe hybrids in the absence of pure species. These results would suggest that aloe hybrids might not all be as sterile and enduring deleterious reproductive trait combinations as was generally claimed for plant hybrids (Arnold 1997 and references therein).

The level of interspecific pollen flow between species is dependant on the fit between the flower’s reproductive surfaces and the floral visitor (Wolf et al. 2001), also something Castellanos et al. (2004) determined when they investigated bird and bee flowers, and the adaptations toward/away from each pollinator type. Hybridization, and backcrossing, can thus be limited by an ineffective transfer of pollen as would be found in divergent flower types. Species of the same floral type was shown to be divergent in their flowering, and generally occupied discreet habitats within the landscape (Chapter 1). Aloe africana was 42 found to be prevalent in most hybrid combinations, but more frequently with species of the non-specialised bird-pollination syndrome. Aloe africana shared a common region of pollen deposition on non-specialised birds with A. speciosa and A. ferox (the crown of the head), explaining the high incidence of hybridization between these three species. Aloe pluridens and A. africana have stark floral divergences that resulted in very selective pollen placement on the same pollinator (underside of mandible and chin versus crown of head), due to the fact that pollinators are manipulated to probe in a set fashion by the flower architecture and orientation (mostly). This was probably the reason why hybrids between these two species were rarely found, even though they commonly grow together. Incidentally, the few hybrids that were found between these two species were located in a disturbed area where A. africana was penetrating the pure population of A. pluridens along a major water transfer pipeline. In fact, Aloe africana thrived in most disturbed areas, and showed altered flowering compared to the individuals within the core of the population. Lamont et al. (2003) showed that the impact of humans on the landscape promoted the hybridization between species by altering flowering phenologies, which caused greater overlap and thus probable frequency of interspecies pollen transfers. Aloe africana is well known in the region to flower in low abundance (few scattered individuals) throughout the year whenever a spell of good rains was had (RM Cowling, personal communication). The added soil moisture available to species due to run- off next to roads, within bush-cleared areas, or along ephemeral drainage lines, probably stimulates the prolonged flowering of species that occur here. These were also the areas where hybrids were most prevalent. Although hybrids were found to be localised within the clearly definable areas mentioned above, probably because these areas favour hybrid establishment, of interest further here is why they are not prevalent within the core of populations but within the narrow ecoclinal zones between species pairs.

Genera that are prone to hybridization, yet manage to maintain species integrity whilst producing fertile hybrid offspring (e.g Quercus – Whittemore and Schaal 1991, Nason, 1992; Penstemon – Wolfe and Elisens 1994, Wolfe et al 1998; Rhododendron – Milne et al 1999), seem to rely on ecological factors in order to maintain species integrity. My array results are consistent with Campbell et al. (2002,) who also found that birds tended to stray from floral constancy in equal choice situations. Within the natural situations, where aloes were aggregated spatially within the mixed assemblages, birds showed high degrees of fidelity. The energetics of foraging has been claimed to structure pollinator communities and ensure high levels of fidelity towards single floral types (Campbell et al. 1997). Within the narrow contact zones between species patches within a landscape, bird-pollinators are exposed to an altered foraging situation whereby the mixed species assemblages no longer warrant floral constancy in order to maximize foraging efficiency. Within equal choice situations, searching 43 for/targeting one type of flower will result in greater energy expenditure. Feeding at all possible flowers in these situations, even if one floral type contributes far less energy from the nectar reward as the other, gives the bird pollinator the optimum energy intake (Feinsinger 1978). The incidence of hybrid occurrence along disturbed (both anthropogenic and natural) and mixed species zones, likely reflects the altered energetic conditions for foraging by birds, with an equal choice situation created on fine scales among the mixed flowering species within these well definable zones. From the experimental array and field observations, it was evident that Weavers were more likely to forage at all available species and acquire interspecific pollen loads, than Sunbirds. Sunbirds should not, however, be ruled out as no mist-netting took place to verify their pollen loads. However, based on the observations, sunbirds would contribute far smaller interspecific pollen loads to co-occurring species than weavers, because they managed to avoid the tightly grouped anthers whilst nectar robbing at occasional nectarivore-pollinated aloes. Bird behaviour, and foraging choices (driven by energetics of foraging) within the hybrid zones were thus the influential factors in hybrid frequencies, given the high interfertility among species. 44

General discussion

In this study I set out to achieve identifying the mechanisms of reproductive co-existence between co-occurring Aloe species, trying to identify the factors that are involved in the widespread hybridization among aloes. This was done by describing the individual species’ pollination ecology (when they flowered, the type of flowers, recording bird and bee pollinators, and measuring the nectar rewards). The clear grouping of the aloes into functional groups that targeted specific pollinators (Chapter 1), allowed for the additional extrapolation of pollination in other aloes, based on floral traits (Chapter 2). The dominance of sunbird-pollination among the South African aloes highlighted one of the constraints in hybridization avoidance, namely the lack of diverse pollinators, and discreet pollen deposition sites. Hybrids were found to be abundant in very well defined zones within the study area, and it was the behaviour of the bird-pollinators and the lack of pollen separation on them that governed the ratios of hybrid combinations (Chapter 3). Although most of the aims for this study were met, the time frame did not allow me to expand my investigation into some of the issues that were consequently identified, and is discussed below for future research opportunities.

Not much was known about the migratory movements of the bird-pollinators in this system. Knowing this would begin to give us an idea of how far pollen could theoretically be transported within the study area, and to assess the degree of isolation between populations. There seemed to be a definite increase in numbers of the birds (both sunbird and non- specialised birds) during the aloe flowering season. This was based on personal observation throughout the year where there was an obvious lack of bird sights outside the aloe flowering season. The fact that there were species turnover recorded for sunbird and non-specialised birds (data not shown here) for the bird count transects that were walked over the course of the flowering season, would already suggest that these birds did track the flowering within adjacent populations. A possibility would be that many of the bird-pollinators in this study were migrants that, on local scales (<50 km), track the phenological cycles between the Thicket vegetation within the river valley and that of the adjacent Fynbos vegetation. This needs to be better assessed with more frequent bird counts at set locations throughout the study area, also using multiple groups of observers in order to get simultaneous data. Ideally, mass ringing bird-pollinators within the area, and running a consequent extensive mist netting programme, should be followed in order to hopefully track the movements of individuals. Mobile Acoustic Radar can easily track multiple marked birds within a valley with one unit and across greater distances with two or more units, but this method requires a specialist 45 operator and serious funding. However, training days for the defence force cadet operators could possibly be justified, but some strings would have to be pulled. Harmonic Radar has been successfully used to track insects too small for acoustic devises to be attached to them, this and other methods of tracking pollinators have been reviewed by Chapman et al. (2004). I have attempted to assess the phenological patterns for the known bird-pollinated plants within the Thicket and Fynbos vegetation for the study area from published data, but I failed to extract a clear trend in sequential flowering between the two vegetation Biomes due to large gaps in the data. But, within the Fynbos, bird-pollinated bulbs flower in spring and autumn, with a lull in winter when aloes are mostly flowering within the Thicket vegetation (SD Johnson, RM Cowling, and SM Pierce, personal communication). Within the Fynbos vegetation of the Western Cape it was shown that sunbirds migrated between the low- and highland Fynbos, tracking the flowering of Erica and Protea species (Rebelo et al. 1984) - two important nectar resources for sunbirds. The Gamtoos River Valley, and similar river valleys in the Eastern Cape, would make ideal corridors for migrations between the coastal- and mountain vegetation, with migrating individuals relying on the abundance of aloe nectar in these river valleys as a critical intermediate resource. Further research on this matter is needed as nectar corridors have been identified as essential to the continual conservation of plant-pollinator interactions in increasingly transformed landscapes that are traversed by migratory pollinators (van Devender et al. 2004). The interactions between migrant and resident birds through exploitative foraging interactions and its effect on breeding would also be interesting to know.

Classical pollinator observations are sorely needed to verify my pollinator predictions in Chapter 2, and investigating the insect-pollinated aloes should be of prime interest. Bees would probably turn out to be the main guild that pollinates this small group of aloes, but the scented flowers of Aloe modesta Reynolds, highly uncommon among aloes, might turn out to be butterfly pollinated.

Aloe africana, as a species, would be an interesting case to explore further. Its ecology might be in a state of flux, meaning that the speciation process is not completely stabilized (GF Smith, personal communication). It certainly behaved differently to the other aloes by flourishing in the disturbed areas; yet still occur in abundance within dense bush. It also had intermediate nectar properties and was frequented by both classes of bird-pollinators. It showed the highest hybridization frequencies with the other Aloe species, and the least degree of ovule abortion. Its preference in the arrays might have had to do with its inflorescence design, whereby it provides a prominent perch for territorial birds to be seen above the vegetation. 46

Also, what should be focused on now by future investigators are the fate of hybrids, and their ecological and evolutionary roles. Historically, mega herbivores were abundant within the Gamtoos River Valley (Skead 1987), but over two centuries of human occupation within the region has seen the complete disappearance of these species. Interestingly enough, no mention was made by the first travellers through the region of the abundant aloe displays found today along the old trek routes that converge within the study area (Skead 2007). The effect of mega herbivores on aloes can easily be observed within the Greater Addo Elephant National Park, East of Port Elizabeth. Within the park, where one of the largest elephant populations in the country is, aloes are scarce compared to the same vegetation outside the park, where elephants are absent. It would be possible that the current extensive stands of aloes within the study area are relicts of mega herbivore relaxation (RM Cowling, personal communication). If this was the case, and aloe populations were originally isolated on the steep slopes, the incidence of hybridization would be less than currently. Humans have further expanded the niche for hybridization through the road infrastructure development and agricultural practices in the study area. So, perhaps the greatest factor that governs hybridization is the human one.

Stabilizing the reproductive ecology of hybrids would be an essential part for speciation via hybridization (Grant 1981). Most of the hybrids did not co-flower with their parental taxa. Those that flowered early in the season had their inflorescences scourged by the heat, and they suffered under low bird visitation. This was probably due to a lack of regional advertising prior to the start of the main aloe flowering season, and market swamping by the mass flowering of pure species when the season start. Hybrids were spatially confined within set vegetation zones, where they probably reproduced introgressively with pure taxa on a limited scale. So, hybrid individuals currently seem utterly disadvantaged, but are they perhaps waiting for the right time? The right time, when large hybrid swarms become established and through repetitive backcrossing stabilize their reproductive ecology, might come during climatic oscillations similar as was seen during the Pleistocene. During this time, Aloe radiation took off (Holland 1978) seemingly through a process of wide scale rampant hybridization (Riley and Majumdar 1979, Viljoen 1999). Minor shifts in the flowering due to a changing climate, would result in more frequent introgression among hybrids and sympatric parental taxa. A new ecological niche that opened, for which the hybrids are more suited because of their trait combinations, would allow hybrid individuals with stabilized ecologies to spread out of their ecoclinal holding cells with a vigour that only hybrids know. If these manage to co-flower in large enough displays to warrant floral selectivity by bird-pollinators through caloric selection and the energetics of foraging, 47 speciation will be complete once more in what must have clearly been a mosaic of speciation via hybridization for this genus.

Whilst looking at Aloe ferox in the Gamtoos River Valley, a gut feel will tell you that this already happened. Although treated as the same species here, Aloe ferox undergoes a subtle change from the outskirts of the valley, where it has the classical A. ferox architecture, to the inner flood plain, where an A. ferox form that looks a lot like the hybrids between A. ferox and A. africana occur, only, with stabilized features and forming large populations. This form may very well be the product of the scenario laid out above, probably aided by the niche creation as a direct result of human activities in the valley. Only its molecules will be able to truly determine whether this is the case or whether this is just another form in what is known to be a highly variable species, but it certainly is a lot more vigorous.

Stemming from this is the need, more than ever, to get a robust natural phylogeny for the aloes. We know so much already about the distribution of the species (Van Wyk and Smith 2003), the floral and vegetative morphology (Reynolds 1969), the chemicals (Viljoen 1999, Dagne et al. 2000, Viljoen and Van Wyk 2000, Viljoen et al. 2001), and we are making solid breakthroughs in its pollination (Hoffman 1988, Ratsirarson 1995, Stokes and Yeaton 1995, Johnson et al. 2006, this and two more independent studies in prep.). Several attempts have been made in assessing the classification Reynolds (1969) proposed according to morphology (scrambling aloes are more basal than the tree aloes). So far this have been done with secondary metabolites (Viljoen 1999, found lots of shared chemicals), nectar chemistry (Van Wyk et al. 1993, the same), chromosomes (Brandham and Doherty 1998, the same), ovule structure (Steyn and Smith 1998, the same) and higher order genetics (Adams et al. 2000, Chase et al. 2000, Treutlein et al. 2003, identifying that the Aloaceae is monophyletic, groupings within it is polyphyletic, and scrambler aloes seem to be basal but the rest is a mixed story). The time is right for a whole genus natural phylogeny so that we can start to map the biogeography, chemistry, pollination, and ecology onto it to complete our understanding of the evolutionary radiation within this economically, and ecologically important group, and get a grip of really how important hybridization as a process is in the overall scheme of things.

What is needed is a coordinated Aloe Systematic Atlas Project (ASAP). (Whittemore and Schaal 1991, Nason et al. 1992, Possingham 1992, Wolfe and Elisens 1994, Wolfe et al. 1998, Milne et al. 1999, Rodriguez-Girones 2006) 37

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Personal Communications

• Prof. RM Cowling, NMMU, completed PhD (vegetation studies) within the study area, and resident within the region for a very long time. It is basically his backyard. • Prof. SD Johnson, UKZN, through his pollination studies for his PhD he became knowledgeable on the phenologies of several Fynbos plants. • Dr. SM Pierce, Consultant, author of “A synthesis of plant phenology in the Fynbos Biome” CSIR report 88, 1984. Also conducted phenological studies in the Fynbos and Thicket Biomes within the study area for her PhD. 44

• Prof. GF Smith, SANBI, taxonomic leader in the Aloaceae. I would call him Mr. Aloe myself.