SAJB-00783; No of Pages 4

Available online at www.sciencedirect.com

South African Journal of Botany 81 (2012) 15–18 www.elsevier.com/locate/sajb

Short Communication First record of pollination in the Afro-Eurasian Dipcadi Medik. (Hyacinthaceae): pollination of D. brevifoliumi by the owlet Syngrapha circumflexa () ⁎ J.C. Manning a,b, , P. Goldblatt c, E. Parker d, R. Kaiser a

a Compton Herbarium, South African National Biodiversity Institute, Private Bag X7, Claremont 7735, Cape Town b Research Centre for Growth and Development, School of Life Sciences, University of KwaZulu-Natal Pietermaritzburg, Private Bag X01, Scottsville 3209, South Africa c B.A. Krukoff Curator of African Botany, Missouri Botanical Garden, P.O. Box 299, St. Louis, Missouri 63166, USA d Elandsberg Nature Reserve, Bo-Hermon, South Africa

Received 4 January 2012; received in revised form 19 March 2012; accepted 20 March 2012

Abstract

Flowers of Dipcadi brevifolium (Hyacinthaceae) exhibit the characteristics associated with phalaenophily, or pollination by settling , notably a dull-coloured, shortly tubular perianth with included anthers, and nocturnal scent. Flowers are self-incompatible and produce an unusual, sour/acrid floral scent dominated by isobutyric acid, 2-methylbutyric acid and jasmine. The moth Syngrapha circumflexa (Noctuidae) was recorded as a pollinator, representing the first pollination record for the and for the . © 2012 SAAB. Published by Elsevier B.V. All rights reserved.

Keywords: Dipcadi; Hyacinthaceae; Moth pollination; Pollination biology; Scent chemistry; Syngrapha

1. Introduction southern Africa but extending into central Asia, Madagascar and India (Speta, 1998). All have characteristic, dull-coloured, tubular Hyacinthaceae are a large and florally diverse family of±900 flowers with included anthers, and several of the southern African spp. that has speciated primarily in southern Africa with a species at least become fragrant at night, leading Vogel (1954) secondary radiation in the Mediterranean (Speta, 1998). Pollina- and Manning et al. (2002) to propose that they are adapted for tion systems in the family have been remarkably little studied moth pollination. Here we provide the first documented evidence since initial speculations on the subject (Vogel, 1954), who for moth pollination in the species D. brevifolium (Thunb.) inferred pollination by bees, birds, butterflies and moths on the Fourc., including a chemical analysis of the unusual, acrid floral basis of floral morphology. It is only recently that rodent and scent and observations on self-incompatibility. other non-flying mammal pollination has been documented in the southern African genus Massonia Thunb. ex Houtt. (Johnson et 2. Materials and methods al., 2001; Wester et al., 2009; Wester, 2010, 2011). Other systems have yet to be studied, among them the pollination of the small 2.1. Study species and study site genus Dipcadi Medik., which comprises±30 spp., primarily in Dipcadi brevifolium is a small, deciduous geophyte wide- ⁎ Corresponding author at: South African National Biodiversity Institute Compton Herbarium Private Bag X7 Claremont, Cape Town 7735 South spread through the winter-rainfall zone of southern Africa Africa. Tel.: +021-799 8660; fax: +021-761 4151. (Obermeyer, 1963). Flowering occurs from spring through late E-mail address: [email protected] (J.C. Manning). summer in different localities depending on the rainfall. At this

0254-6299/$ -see front matter © 2012 SAAB. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.sajb.2012.03.012 16 J.C. Manning et al. / South African Journal of Botany 81 (2012) 15–18 time a secund of nodding, greenish to brownish, tubular 2.3. Floral visitors flowers 12–20 mm long is produced (Fig. 1). The are united in the lower half and the filaments are appressed to the were examined at various periods throughout the day inner surface of the tepals, the anthers thus apparently sessile, and early evening, from±08:00 until 21:00, on 20 and 25 Oct and included within the floral tube, along with the short style 2010 and again on 15 Oct 2011. After nightfall, examination and stigma. was carried out by the light of torches masked with red plastic We studied the species in the Elandsberg Nature Reserve foil, during which time 10–15 plants were monitored. All east of Bo-Hermon in Western Cape in October 2010 and 2011. visitors to the flowers were captured and examined for pollen Plants are widespread through the reserve in Swartland Alluvial under a dissecting microscope. Specimens are deposited at the Fynbos and Renosterveld shrublands (Mucina and Rutherford, South African Museum, Cape Town. 2006). Voucher specimens are deposited in the Compton Herbarium, South African National Biodiversity Institute. 2.4. Nectar measurement and sugar concentration

Five flowers from five randomly selected plants were 2.2. Floral longevity, self-pollination and pollination success sampled for nectar in the late afternoon, ±17:00, thus allowing a full day for nectar accumulation. Nectar was extracted by Self-compatibility was assessed by enclosing ten budding inserting a 5 μl capillary tube into the mouth of the floral tube stems in fine-mesh tubes to exclude floral visitors, and examining to the base. The percentage of sucrose equivalents in fresh them periodically over the following two weeks for fruit set. nectar was measured using a Bellingham and Stanley hand- Natural pollination success was estimated as the percentage of held refractometer (0-50%). capsules developed on twenty open (uncovered) stems from the same population. Floral longevity was initially determined on 2.5. Scent analysis five flowering stems that were picked and held in a vase indoors under natural light conditions. One mature bud from the base of Flowering stems were sniffed throughout the day and early the raceme on each of the five stems was tagged and monitored evening for any noticeable scent. Freshly picked flowers were each day until it withered. Flowers above the tagged buds sampled for floral scent in the early evening±1 h after scent continued to open successively for more than a week afterwards, emission began by placing five stems in water and enclosing suggesting that picking had no influence on flower development, the in a glass container to concentrate the scent. The and this was confirmed by checking undisturbed inflorescences head space was sampled for 4 h onto glass capillary tubes in the pollinator exclusion experiment. packed with Poropack by drawing air through the tubes with a vacuum pump. Floral scent chemistry was analysed by gas chromatography using a DB-Wax Capillary column (Kaiser, 1993).

3. Results

3.1. Floral phenology

Buds opened gradually over 2 days, with maximum perianth expansion achieved in the evening of day 3, and open flowers withered by day 10. Individual flowers were thus open for 7 nights. At anthesis on day 1 the style was short, with the stigma positioned below the anthers, but elongated slightly during the life of the flower so that by the time that the flower withered the stigma was positioned near the tips of the anthers. Flowers were entirely unscented during the day but became fragrant at sunset, ±18:30. They were again unscented by morning, 07:00.

3.2. Scent

3.2.1. Chemical analysis Analysis of floral scent identified the following constitu- ents, arranged in order of decreasing percentage composition: jasmine 57.0%, 2-methylbutyraldehyde oxime ((E)+(Z)) 20.2%, isobutyric acid 11.3%, 2-methylbutyric acid 2.4%, 2-methyl-1-nitrobutane 2.3%, 2-methyl-1-nitropropane 1.8%, Fig. 1. Dipcadi brevifolium flowers showing short tube and included anthers. butyric acid 1.3%, p-vinylphenol 0.5%, octan-3-one 0.4%, J.C. Manning et al. / South African Journal of Botany 81 (2012) 15–18 17

2-methylburyronitrile 0.1%, isobutyraldehyde oxime ((E)+(Z)) the anthers and stigmas. The probosces of the moths measured 0.6%, α-pinene 0.05%, (E)-4,8-dimethylnona-1,3,7-triene 0.05%, 12–15 mm in length, and every individual that we examined β-pinene 0.03%, 6-methylhept-5-en-2-one 0.03%, octanal 0.03%, carried few to many (b20–N200) adherent pollen grains 3-methylbutyraldehyde oxime ((E)+(Z)) 0.03%, oct-1-en-3-ol scattered along the entire length of the proboscis but usually 0.05%. concentrated in the distal half. Pollen grains were monosulcate and thus evidently monocotyledonous. Dipcadi brevifolium 3.3. Nectar was the only monocot species in flower at the locality and it is thus possible to infer that the grains were from that species. Individual flowers contain trace amounts of nectar, 0.25- 0.35 μl, visible as solitary droplets at the mouth of each of the 4. Discussion secretary pores at the base of the ovary septa. The nectar is moderately concentrated, 22–31% sucrose equivalents (Mean= We confirm that the floral syndrome of moth-pollination in 26%, n=5). Dipcadi brevifolium is indeed associated with pollination by noctuid or owlet moths as previously inferred. At our study site 3.4. Fruit set we only observed visits by Syngrapha circumflexa but it remains to be demonstrated that other moths do not visit the No fruits were set by the bagged plants. Initial swelling of species elsewhere across its range. Syngrapha circumflexa itself the ovaries occurs, suggesting that self-pollination has been is widely distributed through Africa, Europe and Asia, and in effected, but they soon abscise spontaneously, indicating that southern Africa has been documented as pollinating species of they are self-incompatible. Fruit set in open, unbagged plants is Babiana Ker Gawl. and Gladiolus L. (Iridaceae) (Goldblatt and moderate to high, with 20=83% of flowers developing capsules Manning, 2002, 2007), Satyrium Sw. (Orchidaceae) (Johnson, (Mean=45%, n=20). 1997) and Struthiola L. (Thymelaeaceae) (Makholela and Manning, 2006). Pollination by noctuids in other species that 3.5. Floral visitors we have investigated is not species-specific (Goldblatt and Manning, 2002) and it may be significant that we did not No visits were observed during the day but flowers were observe any visits to Dipcadi by the noctuid Cucullia sp., regularly visited after sunset, ±18:30, by the owlet moth which was common at the study site and active on various other Syngrapha circumflexa (L.) (= Cornutiplusia circumflexa (L.)) flowering shrubs there, including sweetly-scented Struthiola (Noctuidae) (Fig. 2). Visitation frequencies during the period cilata (L.) Lam. (Thymelaeaceae), for which both of these moth of active visitation for which we continued observation species have been recorded as pollinators (Makholela and (±18:30–21:00) were relatively low to moderate (5 visits to Manning, 2006). The probosces of all moths captured visiting 10 plants on 20 Oct. 2010; 2 visits to eight plants on 25 Oct. flowers of D. brevifolium carried adherent pollen grains in a 2010; 6 visits to 10 plants 15 Oct. 2011). position that favoured their transfer to stigmas. Individual moths visited most or all open flowers in an The flowers of Dipcadi brevifolium emit an unmistakable acrid- inflorescence before moving off, alighting on a flower and sour, butyric acid-related scent that is only slightly softened by grasping it with the legs while probing for nectar. The some floral and herbal aspects. Isobutyric acid (11.3%) and 2- entered the flowers only with the proboscis, which contacted methylbutyric acid (2.4%) dominate the olfactory profile of the species, accompanied by minor amounts of related acids such as butyric acid (1.3%). The floral and herbal aspects are contributed primarily by jasmone (57%), which has a rather broad distribution among floral scents (Knudsen et al., 2006). Isobutyraldehyde oxime (0.6%) and 2-methylbutyraldehyde oxime (20.2%), which are catabolites of the amino acids valine and isoleucine respectively, are further degraded to their corresponding acids, butyric and methylbutyric acid respectively. The occurrence of these oximes and their acids as major constituents in flower scents is highly unusual although they have been identified as minor constituents, together with 3-methylbutyraldehyde oxime and 2-phenylacetaldehyde oxime, in over 120 species (Kaiser, 2006), and minor or trace amounts of isobutyric acid and 2- methylbutyric acid are frequently encountered in flowers, e.g. in those of Beta vulgaris L. (0.4-1.3%) and Larrea tridentata J.M. Coult. (0.1-0.4%) (Knudsen et al., 2006). Many of the flowers in which these oximes and their acids have been identified exhibit the typical syndrome of moth pollination (Kaiser, 2006). Among them is Struthiola ciliata (Thymelaeaceae), also pollinated by Fig. 2. Syngrapha circumflexa. Syngrapha circumflexa (Makholela and Manning, 2006). 2- 18 J.C. Manning et al. / South African Journal of Botany 81 (2012) 15–18

Methylbutyraldoxime and the other oximes mentioned above Goldblatt, P., Manning, J.C., 2007. Floral biology of Babiana (Iridaceae: have been identified in several other moth pollinated species, in Crocoideae): adaptive floral radiation and pollination. Annals of the Missouri Botanical Garden 94, 709–733. some cases as the biologically active compounds (Dobson, 2006; Johnson, S.D., 1997. Insect pollination and floral mechanisms in South African Raguso et al., 2003; Raguso, 2006). Dipcadi brevifolium is thus species of Satyrium (Orchidaceae). Plant Systematics and Evolution 204, distinctive among moth-pollinated species essentially in the 195–206. unusually high proportion of these oximes and acids in the scent Johnson, S., Pauw, A., Midgley, J., 2001. Of mice and massonias. Veld & Flora – profile. The possibility that the distinctive floral scent in Dipcadi 87, 166 167. Kaiser, R., 1993. The Scent of Orchids: Olfactory and Chemical Investigations. brevifolium is linked to pollinator species-specificity should be Elsevier, Amsterdam. investigated further in the species and also among others in the Kaiser, R., 2006. Scent of the Vanishing Flora. Wiley, Zürich. genus. Knudsen, J.T., Eriksson, R., Gershenzon, J., 2006. Diversity and distribution of Pollination success at the study suite is moderate to high, floral scent. The Botanical Review 72, 1–120. reflecting active visitation and successful pollination of the long- Makholela, T., Manning, J.C., 2006. First report of moth pollination in Struthiola ciliata (Thymelaeaceae) in southern Africa. South African lived flowers over the flowering period. The minute amounts of Journal of Botany 72, 595–603. nectar, b0.5 μl, produced by the flowers presumably encourages Manning, J.C., Goldblatt, P., Snijman, D., 2002. The colour encyclopedia of movement between flowers and, in combination with self- Cape . Timber Press, Oregon. incompatibility, would thus favour cross-pollination. The Mucina, L., Rutherford, M.C., 2006. The Vegetation of South Africa, Lesotho Elandsberg populations of Dipcadi brevifolium are self- and Swaziland. Strelitzia 19. South African National Biodiversity Institute, Pretoria. incompatible, and herbarium collections of the species that we Obermeyer, A.A., 1963. The South African species of Dipcadi. Bothalia 8, have examined which include specimens with developing fruits 117–137. never have 100% fruit development. We thus assume that self- Raguso, R.A., 2006. Behavioral responses to floral scent: Experimental incompatibility is characteristic of D. brevifolium.Thiscontrasts manipulations and the interplay of sensory modalities. In: Dudareva, N., with the Arabian D. biflorum Ghaz. (Ghazanfar, 1996), which has Pichersky, E. (Eds.), Biology of Floral Scent. Taylor and Francis, Boca Raton, p. 297. unscented, short-lived, self-pollinated flowers lasting a day. The Raguso, R.A., Levin, R.A., Foose, S.E., Holmberg, M.W., McDade, L.A., general floral similarity among species of Dipcadi suggests that 2003. Fragrance chemistry, nocturnal rhythms and pollination “syndromes” moth-pollination is the primary pollination system in the genus, in Nicotiana. Phytochemistry 63, 265. although the possibility of self-pollination must be considered. Speta, F., 1998. Hyacinthaceae. In: Kubitzki, K. (Ed.), The Families and Genera of Vascular Plants III. Flowering Plants, , Lilianae (except Orchidaceae). Springer, Berlin, Heidelberg, New York, pp. 261–285. Acknowledgements Vogel, S., 1954. Blütenbiologische Typen als Elemente der Sippungliederung dargestellt anhand der Flora Südafrikas. Botanische Studien (Jena) 1, Anthony Magee kindly prepared the electronic figures. 1–338. Wester, P., 2010. Sticky snack for Sengis: The Cape rock elephant-shrew, References Elephantulus edwardii (Macroscelidea), as a pollinator of the Pagoda lily, Whiteheadia bifolia (Hyacinthaceae). Naturwissenschaften 97, 1107–1112. Wester, P., 2011. Sticky snack for sengis. Veld & Flora 97, 112–113. Dobson, H.E.M., 2006. Relationship between floral fragrance composition and Wester, P., Stanway, R., Pauw, A., 2009. Mice pollinate the Pagoda Lily, type of pollinator. In: Dudareva, N., Pichersky, E. (Eds.), Biology of Floral Whiteheadia bifolia (Hyacinthaceae) - First field observations and – Scent. Taylor and Francis, Boca Raton, pp. 147 198. photographic documentation of rodent pollination in South Africa. South Ghazanfar, S.A., 1996. The genus Dipcadi (Hyacinthaceae) in the Arabian African Journal of Botany 75, 713–719. peninsula. Kew Bulletin 51, 803–807. Goldblatt, P., Manning, J.C., 2002. Evidence for moth and butterfly pollination in Gladiolus (Iridaceae-Crocoideae). Annals of the Missouri Botanical Garden 89, 110–124.

Edited by AR Magee