T HE FLORAL BAT LURE DIMETHYL DISULPHIDE DOES NOT ATTRACT THE PALAEOTROPICAL D AWN BAT

—In the neotropics, dimethyl disulphide (DMDS) is innately attractive to flower-visiting bats, and acts as a powerful bat lure in the scent bouquets of many bat-pollinated flowers. At first, DMDS appeared to be part of a general bat pollination syndrome. However, DMDS is absent in many bat-pollinated flowers of West Africa, and it is unclear whether palaeotropical flower-visiting bats are also attracted to it. Furthermore, DMDS was previously observed in neotropical, but not palaeotropical, populations of Ceiba pentandra (Malvaceae, Bombacoideae). We tested for an attraction to DMDS in the most common flower-visiting bat in Thailand, the dawn bat Eonycteris spelaea. We gave bats choices of Ceiba pentandra flowers, where one random flower was scented with DMDS. Rather than preferring the DMDS-treated flower, 21 of 22 bats chose an untreated flower, showing no attraction to DMDS. Alongside past evidence, this result suggests that the role of DMDS in bat pollination syndromes may result from an adaptive convergence that is limited to the neotropics. This hypothesis could be tested through comparative studies of (1) attraction across bats, (2) floral DMDS presence across bat-pollinated plants in Asia, and (3) floral DMDS measures across New and Old World populations of Ceiba pentandra.


INTRODUCTION
The odourous sulphur compound, dimethyl disulphide (DMDS), is a powerful attractant of neotropical nectarivorous bats (Glossaphaga sp., von Helversen et al. 2000;Anoura sp., N. Muchhala, pers. comm.). Flowervisiting bats use DMDS as a beacon to find flower locations and to locate the nectar reward within the flower. Captiveborn long-tongued bats Glossaphaga soricina with no previous experience of the scent will hover and stick their heads into tubes of DMDS, which demonstrates that their attraction to the scent is innate (von Helversen et al. 2000;Carter et al. 2009). DMDS appears to have evolved independently in the scent bouquets of many neotropical bat-pollinated flowers (Knudsen et al. 1995;Bestmann et al. 1997;von Helversen et al. 2000), but its evolutionary origin as a bat lure is unclear. Given that DMDS is attractive to the males of some mammalian species (e.g. Singer et al. 1976), one hypothesis is that its presence in flowers represents sensory exploitation of olfactory communication in bats or other mammals (von Helversen et al. 2000). DMDS may also have originally attracted flies (Jürgens et al. 2006;Shuttleworth & Johnson 2010;Stensmyr et al. 2002), and then later became innately attractive to bats. Based on early reports of floral odours in both the palaeotropics and neotropics, von Helversen et al. Perhaps most remarkable is the case of the silk-cotton tree Ceiba pentandra (Malvaceae, Bombacoideae), which is pollinated by bats on both hemispheres. In Central America, C. pentandra flowers possess a substantial amount of DMDS (von Helversen et al. 2000), but in West Africa, they contain no detectable DMDS (Petterson et al. 2004). In India, the short-nosed fruit bat (Cynopterus sphinx) sometimes feeds on nectar but it does not prefer the scent of DMDS over other odours or scentless controls (Elangovan et al. 2006). These two pieces of evidence suggest that DMDS may only be a component of the pollination syndrome for bats in the neotropics but not the palaeotropics.
Nectarivory in bats arose independently in the neotropical Phyllostomidae (subfamily Glossophaginae) and the palaeotropical Pteropodidae, lineages that diverged about We also used a two-way binomial test to compare the E. spelaea selection rate to expected rates based on choices by the neotropical bat Glossophaga soricina. To calculate this expected rate, we used unpublished data from a pilot test that was most similar in design to this test. At the Montreal Biodome (see Carter et al. 2009) 19 naïve captive-born G. soricina chose from arrays of four tubes, one randomly marked with the same amount of DMDS as used in the tests above. Under these similar conditions, the mean DMDS choice rate across G. soricina bats weighted by number of visits per bat (N = 1 to 33) was 50.46% (chance = 25 %, 95% C.I. = 38-63 %, Carter et al. 2009, unpublished data).

RESULTS AND DISCUSSION
Eonycteris spelaea chose the flower marked with dimethyl disulphide in only one of 22 trials (selection rate = 4.5 %), fewer times than expected based on either random chance (P = 0.025, Fig. 1) or the G. soricina preference (P < 0.0001). This result suggests that E. spelaea, unlike G. soricina, is not innately attracted to DMDS. Instead, we found evidence that the bats avoided the DMDS-treated flower, presumably because the DMDS masked the flower's natural odour. This adds to a growing body of knowledge on how bat pollination syndromes differ between the neotropics and palaeotropics (Pettersson et al. 2004;Fleming et al. 2009). showed that mutations leading to the presence and absence of DMDS alone have likely led to evolutionary transitions among insect pollinators. They studied four closely related species of Eucomis (Hyacinthaceae) that are pollinated by either wasps or carrion flies (which are attracted to DMDS).
All four Eucomis species have flowers of similar colour, nectar, and morphology, but differ in scent compounds. In a field test, they experimentally manipulated floral scents and demonstrated that the presence of dimethyl disulphide and dimethyl trisulphide led to shifts from wasp to fly pollinators. Hence, evolutionary shifts between pollinators can occur based on DMDS alone, without changes in floral shape, colour or nectar. Similar co-evolutionary transitions may also have occurred for palaeotropical and neotropical flower-visiting bats.
Comparative studies would provide great insight into the coevolution of bat nectarivory and floral DMDS. First, to determine the extent of innate DMDS attraction in bats, it would be informative to test other captive-born or wild palaeotropical pteropodids as well as the neotropical Lonchophyllinae-a lineage which may have evolved their nectarivory independent of the Glossophaginae (Griffiths 1982;Datzmann et al. 2010; see also critical review by Dávalos et al. 2012). Second, to confirm a dichotomy in floral DMDS between neotropical and palaeotropical plant species, one could test for DMDS in other bat-pollinated flowers in Asia. Finally, the hypothesis of a neotropical versus palaeotropical dichotomy in floral DMDS within C. pentandra could be tested by mapping the occurrence of floral DMDS across the species range.