Scarab Beetle Pollination of the Neotropical Aroid Taccarum Ulei (Araceae: Spathicarpeae)

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Biological Journal of the Linnean Society, 2013, 108, 22–34. With 5 ﬁgures

The cowl does not make the monk: scarab beetle pollination of the Neotropical aroid Taccarum ulei (Araceae: Spathicarpeae)

ARTUR CAMPOS DÁLIA MAIA1*, MARC GIBERNAU2, AIRTON TORRES CARVALHO3, EDUARDO GOMES GONÇALVES4† and CLEMENS SCHLINDWEIN4

1Departamento de Química Fundamental, Universidade Federal de Pernambuco, 50740-560 Recife, Brazil 2CNRS – Ecofog (UMR 8172), Campus Agronomique BP 316, F-97379 Kourou cedex, France 3Programa de Pós-graduação em Ciências Biológicas (Zoologia), Universidade Federal da Paraíba, 58059-900 João Pessoa, Brazil 4Departamento de Botânica, Universidade Federal de Minas Gerais, 31270-901 Belo Horizonte, Brazil

Received 19 May 2012; revised 28 June 2012; accepted for publication 28 June 2012

Taccarum ulei (Araceae, Spathicarpeae) is a seasonal geophytic aroid, native to north-eastern Brazil, that flowers during two months of the rainy season. Patterns of floral thermogenesis, pollination biology, and floral traits associated with pollination syndromes were studied and compared with those of other Araceae. Two species of cyclocephaline scarabs (Scarabaeidae, Cyclocephalini) were recognized as effective pollinators: Cyclocephala celata and Cyclocephala cearae. Larvae of an unidentified species of fruit fly (Melanoloma spp., Richardiidae, Diptera) were also frequently observed in inflorescences at various maturation stages, feeding on the connectives of male florets and fruits, and thus lowering the reproductive success of individual plants. Beetles were attracted by odoriferous inflorescences in the early evening of the first day of anthesis, during the female phase. The emission of attractive volatiles was coupled with intense thermogenic activity in the entire spadix, unlike other aroids in which only certain zones of the spadix heat up. Pollen release, which marks the beginning of the male phase on the subsequent evening, was not related to floral thermogenesis. Comparative multivariate analysis of the floral traits of T. ulei points to a beetle-pollinated aroid, although some of the observed traits of the species are not common to other taxa sharing this pollination strategy. Such incongruence might be explained by the evolutionary history of the tribe Spathicarpeae and potential pollinator shifts. © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 108, 22–34.

ADDITIONAL KEYWORDS: ﬂower predation – nocturnal pollinators – pollination syndromes – Scarabaeidae – thermogenesis.

INTRODUCTION ponderantly entomophilous, aroid inflorescences have co-evolved in their interactions with arthropod visi- The aroids (Araceae) are a rich group of cosmopolitan tors, both generalist and highly specialized, from flies monocots that presently encompass over 3300 species and beetles to bees, thrips, and perhaps even mites of herbs and vines, mostly found in tropical and (Grayum, 1990; Mayo, Bogner & Boyce, 1997). Accord- subtropical environments (Haigh et al., 2012). Pre- ing to the most recent overview of the family, however, our knowledge of effective pollinators is restricted to less than 50% of the currently described *Corresponding author. genera (Gibernau, 2011). E-mail: [email protected] th †Current address: Pro Reitoria de Pesquisa e Pós Graduação, It has been known since the end of the 19 century Universidade Católica de Brasília, 70790-160 Brasilia, Brazil. that several aspects of basic floral structure are

22 © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 108, 22–34 POLLINATION OF TACCARUM ULEI 23 directly associated with the reproductive biology of dieae (~150 spp.), and several of the Spathicarpeae angiosperms (Müller, 1873; Vogel, 1954). Such floral (~165 spp.) (Gibernau, 2011). characters have been selected through evolution by Although rather diverse and taxonomically well- the increasing effectiveness of pollen transfer to the defined, the tribe Spathicarpeae has been commonly stigmas, and as such are also particularly influenced disregarded with respect to their reproductive biology by the strategies that optimize the use of specific and phenology (Grayum, 1990; Mayo et al., 1997; Gib- pollen vectors (Fenster et al., 2004). Among aroids, ernau, 2011). The tribe is mostly restricted to extra- Chouteau, Gibernau & Barabé (2008) and Gibernau, Amazonian South America, and is presently composed Chartier & Barabé (2010) have presented associations of 13 genera: Asterostigma (10 spp.), Bognera (1 sp.), between a wide set of floral traits and specific polli- Croatiella (1 sp.), Dieffenbachia (~135 spp.), Gearum nation syndromes, to the point that a few extrapola- (1 sp.), Gorgonidium (5 spp.), Incarum (1 spp.), Loren- tions could be safely assumed when enough of these zia (1 sp.), Mangonia (2 spp.), Spathantheum (2 spp.), characters are understood. For instance, large pollen Spathicarpa (4 spp.), Synandrospadix (1 sp.), and grains sticky with stigmatic exudates, typical of Taccarum (6 spp.) (Gonçalves, 2002, 2012; Gonçalves aroids pollinated by large scarab beetles, are unlikely et al., 2007). The monophyly of the Spathicarpeae is to be carried efficiently by smaller-sized insects. On strongly supported by the most recent molecular phy- the other hand, the glabrous body surface of beetles is logenies of the Araceae family (Cabrera et al., 2008; inadequate to carry the loads of powdery pollen found Cusimano et al., 2011). in some fly-pollinated taxa (Sannier et al., 2009). The Together with the Anchomanes clade and the pollen to ovule ratio (P : O) of bee- and fly-pollinated Homalomena clade, the Spathicarpeae form the Zant- species is similar, suggesting that bees and flies have edeschia clade, a pantropical, heterogeneous group of comparable pollination efficiency (Chouteau et al., evergreen or dormant ground and climbing herbs 2008). The much higher P : O of beetle-pollinated (Mayo et al., 1997; Cusimano et al., 2011). From species lends credence to the hypothesis that beetles the standpoint of reproductive biology, the Zantede- may be less efficient pollinators, thus requiring a schia clade is intriguing. Pollination within the much higher investment in pollen production by their Anchomanes clade is mainly achieved by flies and/or plant hosts (Gibernau et al., 2010). beetles, whereas in the Homalomena clade flies are Adaptations towards specific sets of pollinators also the main pollinators of Indo-Malayan species, and result in deeper modifications of flower morphology only cyclocephaline scarab beetles have been impli- and physiology, and this is clearly evident in the cated in the reproductive success of the Neotropical Araceae (Grayum, 1990; Mayo et al., 1997). Mediter- taxa (Gibernau, 2011; Fig. 1). In this phylogenetic ranean Helicodiceros muscivorus (L. f.) Engl., polli- context, the study of pollination systems of the most nated by blowflies, bears foul-smelling inflorescences derived genera of the Spathicarpeae may provide new that closely resemble the anal orifice of a large insights into the evolution of floral characters in mammalian carcass (Angioy et al., 2004). The kettle- relation to pollinator selection within the tribe. shaped inflorescences of European Arum and Indo- The genus Taccarum Brongn. ex Schott, one of Malayan Arisaema are designed to entrap small the most derived taxa within the Spathicarpeae pollinating flies to enhance their contact with recep- (Gonçalves et al., 2007; Cusimano et al., 2011), com- tive female flowers (Vogel & Martens, 2000; Gibernau, prises six species of seasonally dormant geophytes Macquart & Przetak, 2004), whereas the funnel- found in most of the extra-Amazonian Brazilian ter- shaped inflorescences of all known species of the large ritory, as well as in Peru, Bolivia, Paraguay, and Neotropical genus Philodendron offer food and warm northern Argentina. These plants can be recognized shelter for mating scarab beetles (Gottsberger & by the usually large and solitary leaf that is strongly Amaral, 1984; Gibernau et al., 1999; Seymour, White bipinnately divided. They grow preferably in well- & Gibernau, 2009; Maia et al., 2010). drained soils and at low-to-medium altitudes (up to In the Neotropics, the diverse scarab beetles of 850 m a.s.l.), usually in areas with pronounced sea- the tribe Cyclocephalini (Scarabaeidae, Dynastinae; sonality. Populations often consist of scattered indi- ~350 spp.) comprise one of the most preponderant viduals, seldom bearing inflorescences (Gonçalves, groups of scent-driven pollinators of several Mag- 2002). noliaceae, Annonaceae, Cyclanthaceae, Arecaceae, We studied the flowering, phenology, and the pol- Nymphaeaceae, and Araceae (Gottsberger, 1986; lination biology of Taccarum ulei Engl. & K. Krause, Schatz, 1990; Bernhardt, 2000). Among the latter a geophytic aroid native to north-eastern Brazil, and group of plants, these night-active pollinators are addressed the following questions: (1) what is the directly involved with the reproductive success of floral cycle and the pattern of floral thermogenesis the Montrichardieae (two species) and Philo- for the species; (2) what are the flower visitors and dendreae (over 500 spp.), the majority of the Cala- effective pollinators; (3) how do the floral traits

Indo-Malaya / Australasia Anchomanes Aglaonema fly

Afrotropic Aglaodorum fly / beetle

Nephthytis fly / beetle clade

Afrotropic Anchomanes fly / beetle Homalomena Culcasia fly / beetle

fly / beetle Indo Malaya / Neotropic Cercestis

Homalomena fly / beetle ld Tribe Spathicarpeae clade Afrotropic Philodendron beetle Zantedeschia

Zantedeschia beetle

Lorenzia ?

Bognera ? ‘beetle’ beetle

Gearum clade

Dieffenbachia beetle Neotropic Mangonia * ?

Incarum ?

Spathantheum ? ‘fly’

Gorgonidium ?

Croatiella ?

Synandrospadix ? ‘fly’

Spathicarpa ? ‘fly’

Taccarum this study

Asterostigma ?

Figure 1. Simpliﬁed phylogeny of the Zantedeschia clade, redrawn from Cusimano et al. (2011) and Gonçalves et al. (2007). Geographical information is taken from Mayo, Bogner & Boyce (1997), and pollinator records are from Gibernau (2011). Within the Spathicarpeae, the boxes outlined by dotted lines indicate taxa with biogeographical distribution essentially restricted to lowland forests. Taxa restricted to the Andean highlands are indicated by the grey box. position T. ulei in relation to its effective polli- MATERIAL AND METHODS nators with regard to the pollination systems of SPECIES STUDIED associated genera? These questions are discussed within a broader phylogenetic context to improve our Taccarum ulei is a seasonally dormant rhizomatous knowledge about evolutionary trends in the tribe geophyte that is adapted to a wide array of habitats, Spathicarpeae. from humid forests to dry shrublands (Gonçalves,

DEF0.25 cm 0.5cm

Figure 2. Taccarum ulei: A, habit; B, Cyclocephala celata (Scarabaeidae, Cyclocephalini) over pistillate flowers on day 1 of anthesis; C, Cyclocephala cearae crowding inside a thermogenic and odoriferous inflorescence on day 1 of anthesis; D, pollen-covered Cyclocephala cearae over the male zone of the spadix on day 2 of anthesis, ready to fly away; E, Melanoloma sp. (Richardiidae) visiting an inflorescence on day 2 of anthesis; F, pupae of Melanoloma sp. attached to the inner surface of the spathe. Photos: A.C.D. Maia and C. Schlindwein.

2002). Each individual produces a single erect leaf emerge along with the leaf or a few days later (pedun- that may reach up to 150 cm high, with a bipinnatiﬁd cle 10–23 cm high). The spathe (13–22 cm long) is leaf blade of up to 90 cm in diameter (Fig. 2A). The leathery and brown in colour, with light–pink specks funnel-shaped inﬂorescences are also solitary, and (Fig. 2B, C). The spadix is slightly longer than the

© 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 108, 22–34 26 A. C. D. MAIA ET AL. spathe (14–23 cm long), and bears male and female FLORAL THERMOGENESIS perianth-less flowers that occupy distinct zones of its To determine floral temperature patterns (thermogen- vertical axis. The male flowers are synandria, found esis) during anthesis, measurements from both the in the upper portion of the spadix. They are charac- spadix and ambient air were taken at regular 5-min terized by well-developed button-shaped connectives, intervals with a portable logging thermocouple ther- which are pale pink in colour, bearing a ring of mometer (accuracy 0.1 °C, Hanna Instruments). Data between six and ten bright-yellow anthers (Fig. 2C, were recorded from inflorescences of three individuals D). The transition between male and female zones is carefully removed from the field a few days before abrupt. The female zone is 5–10% shorter than the anthesis and kept inside a glasshouse. For each inflo- male zone, and occupies the basal portion of the rescence, thermal probes (0.8 mm in diameter) were spadix. Female flowers are yellow and always sur- inserted about 3 mm into the middle of the male zone rounded by four or five milky white club-shaped of the spadix (Tmz), into the connectives of male staminodes (Fig. 2B). flowers (Tco), and into the middle of the female zone of Vouchers of T. ulei are deposited at ‘Herbário UFP the spadix (Tfz), carefully avoiding any tissue damage. – Geraldo Mariz’, Universidade Federal de Pernam- Another probe was kept about 50 cm distant from the buco (UFPE), Recife, Brazil (A.T. Carvalho 830; inflorescence, and was used to record ambient air UFP 69 808). temperatures.

STUDY SITES BREEDING SYSTEM AND FLORAL CHARACTERS We studied indigenous populations of T. ulei in May– Controlled pollination experiments were performed as June 2005, April–May 2009, and April–June 2010 at follows: (1) spontaneous self-pollination – immature two locations in the Atlantic Forest of the northern inflorescences were enclosed within fine organdie coastal region of the state of Pernambuco, north- bags throughout anthesis (n = 25); (2) hand self- eastern Brazil. A total of ~60 scattered individuals pollination – immature inflorescences were bagged were found growing in clayey soil along the border of until day 2 of anthesis, then stigmas were manually a small forest patch (~130 ha) on the grounds of the brushed with self pollen (n = 4); (3) hand cross- Usina São José SA sugarcane factory and plantation pollination – immature inflorescences were bagged (7°48′–7°49′S, 35°01′–35°02′W; ~110 m a.s.l.). Flower- until day 1 of anthesis, then stigmas were manually ing plants (~40 individuals) were also found growing brushed with pollen from other inflorescences (n = 3); in mildly urbanized areas along a ~1-km section of (4) emasculation (test for apomixis) – immature inflo- the BR101 North Road segment, connecting the rescences were bagged until day 1 of anthesis, then municipalities of Abreu e Lima and Igarassu, no more their upper spadices (containing fertile male flowers) than 10 km from the other location (7°47′S, 34°55′W; were extirpated (n = 3); and (5) open pollination ~80 m a.s.l.). (control group) – unbagged inflorescences were left The local climate is perhumid, although over 80% of accessible to flower visitors. Reproductive success was the average annual precipitation (~1800 mm) falls determined by direct observation of fruit develop- between February and August, whereas in the drier ment, interpreted as the visible enlargement of the months of September to January monthly precipita- ovaries (n = 22). tion is commonly less than 100 mm. The mean We have analysed selected floral characters of monthly temperatures vary between 26 °C in March T. ulei and used the data for comparisons with those and 23 °C in August (AGRITEMPO, 2012). from other species of Araceae, in order to determine feasible correspondences with the adopted pollination strategy (49 genera, 69 species, from Gibernau et al., PHENOLOGY AND FLORAL CYCLE 2010). We used data from the same set of floral Plant phenology was observed at both sites during the characters for comparisons within the Zantedeschia 3 years of the study. We performed weekly surveys in clade (13 genera, 26 species, from Gibernau et al., which we documented the dates when blooming indi- 2010) in order to determine possible correspondences viduals were first and last witnessed in the field. with the adopted pollination strategy (details in Four inflorescences were observed throughout the Chouteau et al., 2008): (1) stigma area per flower entire flowering cycle at 1-hour intervals to determine (mm2); (2) pollen grains volume (mm3); (3) number of the sequence of events at anthesis. Female and male connectives per inflorescence; (4) number of female phases were defined as the beginning of stigma recep- flowers per inflorescence; (5) number of pollen grains tivity (determined by the addition of droplets of a per male flower; (6) number of locules per female

3% solution of H2O2) and anther dehiscence (pollen flower; (7) number of ovules per flower; and (8) release), respectively (Kearns & Inouye, 1993). P : O ratio of inflorescences. Multiple floral-trait

© 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 108, 22–34 POLLINATION OF TACCARUM ULEI 27 comparisons were performed with the method of non- Early in the morning on day 1 of anthesis, the metric multidimensional scaling (NMDS) using the female flowers were moist with viscous, sticky exu- Gower similarity measure, which allows the use of dates, and stigma receptivity was evident. At this both categorical and continuous data (PAST 1.74; time the spadix was fully turgid and protruded, sur- Hammer, Harper & Ryan, 2001). This method maxi- passing the spathe in length, then fully opening to mizes the representation of the ranked difference completely expose the male flowers (Fig. 2C). By late between samples in a two-dimensional plot using a afternoon, around 17:00 h, the spadix became warm distance matrix (Gower index). The degree of the fit is to the touch and gave off a strong floral scent, which estimated by the stress value, ranging from 0 (best was sensed for the subsequent 3–4 h. To the human solution) to 1 (worst solution) (Rabinowitz, 1975). nose, the peculiar aroma is characterized by agree- able floral notes accompanied by a pungent, POLLINATION AND FLOWER PREDATION fermented-like odour that resembles that of old We observed the behaviour of flower-visiting insects cheese. It was easily sensed in the air up to 15 m in four inflorescences of T. ulei throughout the entire away from the warmed-up inflorescences. On the fol- duration of anthesis. Thirty-three inflorescences were lowing day, the spadix was less turgid and no longer also inspected during the second day of anthesis, protruded. The male phase was evident around between 11:00 and 17:00 h, to characterize the insects 13:00–14:30 h, when the anthers opened and bright- inside the floral chambers. During the months of yellow threads of pollen were abundantly released. April–June 2010, inflorescences at various stages of There was clear overlapping of the two phases, as the maturation (n = 52) were additionally examined at stigmas remained receptive even after pollen dis- both study locations for any damage caused by the charge. Between 2 and 5 days after anthesis, the feeding activity of flower predators. Differences in upper third of the spadix withered, and its remainder the abundance of the different scarab beetles inside was enclosed by the spathe along with the fertilized T. ulei inflorescences were assessed by a Z-test. female flowers. Reproductive success was evident The insects collected for identification are deposited 5–8 days after the end of anthesis by the distinct in the entomological collections of: Universidade enlargement of the ovaries. Non-pollinated inflores- Federal de Pernambuco (UFPE), Recife, Brazil; cences withered and rotted away 7–12 days after the ‘Coleção Entomológica Pe. Jesus Santiago Moure end of anthesis. (DZUP)’, Curitiba, Brazil; and Universidade Estadual de Campinas (UNICAMP), Instituto de Biologia, FLORAL THERMOGENESIS Departamento de Parasitologia. Throughout the experiment the temperatures inside the glasshouse ranged between 25 °C and 31 °C. RESULTS Between 16:30 and 17:00 h of day 1 of anthesis, the PHENOLOGY AND FLORAL CYCLE spadices of T. ulei inflorescences gradually heated up. The first flowering individuals of T. ulei were Temperatures rose haphazardly, until reaching a peak observed by early May in 2005 and early April in 2009 in the subsequent 60–90 min (Fig. 3A). Highest tem- and 2010, during the intense seasonal rainy period. peratures were recorded from the tissue of the axis in

The last open inflorescences were seen by late June the middle of the male zone (Tmz = 38.4 ± 1.4 °C SD; in 2005 and late May in 2009 and 2010, giving a 11.0 ± 2.7 SD over the ambient air). The connectives flowering period of little less than 2 months. of male flowers and the female zone of the spadix From the moment the inflorescence emerged above were also thermogenic, and the temperatures of the soil it took 13–20 days to reach maturity. The all measured points oscillate in a similar pattern spadices of newly emerged inflorescences were totally (Fig. 3B). Once they had peaked, the temperatures of enclosed by the spathe, and were exposed only the spadices dropped unevenly, but remained notice- 3–5 days before anthesis. Through maturation, we ably warmer than those of the surrounding air for observed a gradual vertical growth and swelling of a period of over 9 h, after which floral thermogenesis the axial tissue of the spadix, which by day 1 of was no longer detectable by Tmz measurements anthesis would have increased between two- and (Fig. 3A). Through the remainder of the flowering three-fold in length and girth, compared with when it cycle no unambiguous floral thermogenesis was was enclosed within the spathe. The connectives of recorded from the spadices. the male flowers changed colour, turning from an intense lilac coloration to a faded pink tone. Inflores- cences of T. ulei exhibited a 2-day protogynous flow- BREEDING SYSTEM AND FLORAL CHARACTERS ering cycle, undergoing female and male phases, Hand cross-pollination always led to fruit devel- consecutively. opment, whereas non-manipulated inflorescences

middle of male zone 36 connective of male flower

middle of female zone 34 ambient air

30 A temperature (°C) temperature 28

15:00 19:00 23:00 03:00 07:00 11:00 15:00 19:00 23:00 03:00 time of day

10 inflorescence 1

inflorescence 2 8

6 4 B 2

temperature differencetemperature (°C) 0

-2

15:00 19:00 23:00 03:00 07:00 11:00 15:00 19:00 23:00 03:00 time of day

Figure 3. Thermogenesis in Taccarum ulei during the 2-day flowering cycle: A, temperature curves of different zones of the spadix of one inflorescence; B, temperature differences between the middle of the male zone of the spadix and ambient air, measured from two inflorescences. The vertical grey bands represent the heating interval during the female phase (day 1). accessible to flower visitors set fruit in 60% and hand floral structures by flower predators, rather than to self-pollinated inflorescences set fruit in 25% of the the absence of effective pollinators (see below). Only observed cases (Table 1). Limitations of fruit develop- 8% of the inflorescences set fruit after spontaneous ment were attributed to the damage caused to fertile self-pollination.

Table 1. Controlled pollination experiments in Taccarum Dechambre, 1980 (total body length, 1.5–1.8 cm) and ulei (Araceae) Cyclocephala cearae Höhne, 1923 (total body length, 1.7–2.0 cm). With the strong odour emission at dusk Fruit and early evening, between 17:00 and 19:30 h, beetles Treatment N Fruit set set (%) of both species arrived at the inflorescences on day 1 of anthesis (Fig. 2B, C, D). Intense activity was observed Emasculation (apomixis) 3 0 0 upon arrival, including copulating and feeding on the Hand cross-pollination 3 3 100 staminodes inside the floral chambers, where they Hand self-pollination 4 1 25 remained hidden through the rest of the night and the Spontaneous self-pollination 25 2 8 following day. Even though less active, the beetles still Open pollinated inflorescences 22 13 59.1 fed on the nutritious staminodes all through the daytime, during which we also observed copulation in N = sample size; fruit set = number of inflorescences start- ing to develop fruits (i.e. with enlargement of the ovaries). both species. By dusk on day 2 of anthesis the scarab beetles slowly climbed up the spadix. With their bodies sticky with stigmatic exudates and pollen, they then Table 2. Floral traits of Taccarum ulei (Araceae) flew away in search of other odoriferous inflorescences (mean ± [SD]) in an interval of no more than 2 h. Occasionally, however, beetles were observed inside inflorescences of Floral traits Mean ± SD T. ulei up to 2 days after anthesis. Length male zone (N = 6) 125.1 ± 47.5 mm All inflorescences of T. ulei that were inspected on Length female zone (N = 6) 88.3 ± 37.3 mm day 2 of anthesis bore individuals of at least one Diameter, male zone (N = 6) 15.8 ± 4.1 mm species of Cyclocephala inside the floral chambers. Diameter, female zone (N = 6) 15.9 ± 3.3 mm Among individuals of the population located in the Stigmatic area per flower 2.9 ± 1.2 mm2 grounds of Usina São José, we recorded an overall Stigmatic area per inflorescence 738.3 ± 493.4 mm2 mean of 10.3 ± 5.1 (SD) beetles per inflorescence Pollen diameter (N = 25) 80.2 ± 4.4 mm (n = 8). Beetles of both C. celata and C. cearae were Pollen grains volume 0.27 mm3 observed, and there was no significant difference in Number of connectives per 270.5 ± 78.6 the abundance of either species (Z = 1.22; d.f. = 7; inflorescence (N = 6) P = 0.22). Only C. cearae were recovered from flower- Number of anthers per connective 7.6 ± 1.1 ing individuals growing along the BR101 North Road, (N = 100) giving an overall mean of 15.0 ± 23.2 (SD) beetles per Number of pollen grains per 1512 ± 521 inflorescence (n = 25). Both C. cearae and C. celata anther (N = 15) were identified as effective pollinators of T. ulei, con- Number of female flowers per 273.3 ± 104.7 sidering that when visiting female-phase inflores- inflorescence (N = 6) cences they came into contact with receptive stigmas 6 Number of pollen grains per 3.11 ± 0.904 ¥ 10 carrying large loads of cross-pollen on their bodies. inflorescence Exceptionally large crowds of beetles inside a single Number of ovules per flower 6.1 ± 0.6 inflorescence (> 30 individuals) often resulted in (N = 100) damage to both staminate and pistillate flowers, Number of ovules per inflorescence 1448 ± 638 through indiscriminate feeding after the depletion of Pollen/ovule ratio 2375 ± 899 nutritious staminodes. In bold, floral traits used for the multivariate comparison At both study locations, flies of an unidentified NMDS analysis (see the Results section). Sample sizes, species of the genus Melanoloma (Diptera, Richardii- when applicable, are indicated for each floral trait. dae) (total body length, 0.5–0.6 cm; Figure 2E) were also frequent visitors to T. ulei inflorescences, which they used as oviposition/brooding sites. At various The complete set of analysed floral characters is times throughout the day, adults landed on the spadi- presented in Table 2. Inflorescences of T. ulei pro- ces and unopened spathes of inflorescences at various duced on average 3.11 ± 0.904 ¥ 106 (SD) large pollen maturation stages, from recently emerged inflores- grains (80.2 mm in diameter) and 1448 ± 638 (SD) cences to late male-phase ones. Flower-visiting ovules, leading to a low average P : O ratio of 2375 : 1. Melanoloma flies did not venture lower towards the female flowers of mature inflorescences, and none of the individuals carried pollen loads on their bodies POLLINATION AND FLOWER PREDATION (n = 10). Female flies laid their eggs on the male zone Cyclocephaline scarabs of two species were frequent of the spadix and the spathe. After 3–10 days, larvae visitors of T. ulei at the study sites, Cyclocephala celata hatched and started to feed on the connectives of

0.2

Beetle

0.15 Bee

0.10

0.05

-0.25 -0.20 -0.15 -0.10 -0.05 0.05 0.10 0.15 0.20 0.25

-0.05

Fly bee -0.10 Generalist beetle

fly -0.15 generalist

unknown -0.20

Figure 4. Non-metric multidimensional scaling (NMDS) representation of eight selected floral traits (stress = 0.088, R2 axis 1 = 0.73, R2 axis 2 = 0.42) obtained from 69 species of the family Araceae (taken from Gibernau et al., 2010). Each different plot marker corresponds to a pollination syndrome (Chouteau et al., 2008), detailed at the bottom caption. Taccarum ulei is represented by the larger grey triangle. male flowers, ingesting pollen from damaged anthers Zantedeschia clade indicates that the species pos- as well. Infestation was observed in 67% of the inflo- sesses a global set of floral characters that are typical rescences, and was predominantly limited to 25– of scarab beetle-pollinated taxa (Fig. 4). It is the 50 larvae, which caused floral tissue damage to a few taxon of Spathicarpeae most similar to the Homalom- male flowers. Over-infested inflorescences (< 5%), ena clade, lying between species of the beetle- however, hosted > 200 feeding larvae, which severely pollinated genera Homalomena and Philodendron damaged not only the male zone of the spadix but (Fig. 5). On the other hand, Dieffenbachia species also female flowers, and even the axial tissue of the (Spathicarpeae tribe), which are also pollinated by spadix. Between 7 and 12 days after hatching, the scarab beetles, are plotted outside the Homalomena larvae developed into pupae that attached themselves clade, and are unrelated to T. ulei (Fig. 5). to the inner surface of the spathe (Fig. 2F). Adults emerged 10–15 days after pupation. Thus, the larvae DISCUSSION of Melanoloma were flower predators of T. ulei inflorescences. Taccarum ulei is pollinated exclusively by two species Other occasional insect visitors included stingless of Cyclocephala: C. celata and C. cearae. Along the bees of the genus Trigona, seen sporadically, and a north-eastern coast of Brazil where our study sites single record of an unidentified species of predator bug are located, the former species was also identified as (Heteroptera, Reduviidae). These cases were deemed the sole pollinator of Caladium bicolor (Aiton) Vent. irrelevant to the reproductive biology of T. ulei. by Maia & Schlindwein (2006), and of Philodendron acutatum Schott by Maia et al. (2010). These studies have shown that C. celata is an effective pollinator of FLORAL TRAITS at least three genera of Araceae. The multivariate comparison of the floral characters When considering the uniqueness of the inflores- of T. ulei with those of other known Araceae from the cence, compared with those of other cyclocephaline-

0.4

0.3 Homalomena Dieffenbachia

0.2 Taccarum ulei

0.1

-0.4 -0.3 -0.2 -0.10.1 0.2 0.3 0.4

-0.1

Philodendron Cercestis / Culcasia -0.2

Anchomanes clade

-0.3 Homalomena clade

Tribe Spathicarpeae -0.4

Figure 5. Non-metric multidimensional scaling (NMDS) representation of eight selected floral traits (stress = 0.088, R2 axis 1 = 0.73, R2 axis 2 = 0.42), comprising all known taxa from the Zantedeschia clade (13 genera, 26 species; taken from Gibernau et al., 2010). Each different plot marker corresponds to a particular clade or tribe, detailed in the bottom caption. Black elipses delimit the genera Dieffenbachia, Homalomena and Philodendron. pollinated aroids, the overall colour pattern observed Spathantheum orbignyanum Schott, and Spathicarpa in T. ulei stands out clearly. Vivid and bright tonali- hastifolia Hook. would best be regarded as fly- ties have not been documented previously in other pollinated taxa (Gibernau et al., 2010; Fig. 4), but this taxa visited by these beetles, among which floral assumption is yet to be validated by field observa- structures are usually whitish or faintly coloured tions. Nonetheless, fruit flies (aff. Drosophilidae, (Gottsberger & Silberbauer-Gottsberger, 1991; Bern- Tephritidae, Richardiidae) have been observed visit- hardt, 2000). To the human eye, inflorescences of ing inflorescences of Spathicarpa and Spathantheum T. ulei resemble those of unrelated fly-pollinated taxa (E. G. Gonçalves, unpubl. data). It is particularly of the tribe Areae, e.g. Dracunculus, Helicodiceros, noteworthy that all basal groups of the Spathicarpeae and Sauromatum (Mayo et al., 1997). Spathe colours are lowland geophytes pollinated by cyclocepha- and patterns are known to be important in the attrac- line beetles, such as Dieffenbachia and Gearum tion of pollinators by myiophilous aroids (Grayum, (Gibernau, 2011), and most likely Bognera (E. G. 1990). Floral structures in such taxa are frequently Gonçalves, unpubl. data). related to visual deception, and may resemble the A more detailed investigation of the overall floral colours of rotting fruits or carrion, the preferred features of T. ulei evidences clear adaptations towards oviposition sites of several fly taxa (Raguso, 2004). scarab beetle pollination. Its inflorescences are large Multivariate analyses of floral characters suggested and sturdily built, and are able to endure the vigorous that Synandrospadix vermitoxicus (Griseb.) Engl., activity of the insects. A funnel-shaped spathe

© 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 108, 22–34 32 A. C. D. MAIA ET AL. provides a sheltered chamber that can accommodate cyclocephaline scarabs, there is only slight heating even large crowds of visitors. Protogynous anthesis (or no heating at all) during the male phase of occurs approximately 1 day between thermogenesis anthesis, which culminates with the complete abscis- during the female phase and pollen release during the sence of the petals and anthers from the floral male phase: a presentation common to many species receptacle. This ensures that pollinating scarabs of Philodendron subgenus Philodendron (Gibernau, leave the flowers at the end of anthesis (Silberbauer- Barabé & Labat, 2000; Gibernau & Barabé, 2002; Gottsberger, Gottsberger & Webber, 2003). In many Maia et al., 2010), Philodendron subgenus Mecon- species of Philodendron there is a physical repulsion ostigma (Gibernau et al., 1999; Seymour, 1999), of the beetles at the end of anthesis by the retraction Caladium (Maia & Schlindwein, 2006), Xantho- of the spadix and closure of the spathe (Gottsberger & soma (García-Robledo et al., 2004), Montrichardia Amaral, 1984; Gibernau et al., 2000; Gibernau & (Gibernau et al., 2003), and Dieffenbachia (Young, Barabé, 2002; Maia et al., 2010). We did not observe 1986; Beath, 1999). The pollination syndrome of any of these strategies in T. ulei, in which case the T. ulei is also supported by the multivariate analysis departure of pollinator C. celata and C. cearae from of floral traits (Fig. 4), even if some characters male-phase inflorescences could be exclusively related such as the number of pollen grains per inflorescence to the depletion of edible staminodia and the scent- and the number of ovules per flower were considered attraction promoted by other receptive female-phase low for beetle-pollinated taxa. This could be inter- inflorescences. preted as an expression of facultative xenogamy in The atypical cyclocephaline-pollinated inflores- T. ulei (see Cruden, 1977), considering that one- cences of T. ulei, together with the recent origin of the quarter of the inflorescences set fruit after hand self- genus (interpreted as the sister group of Aster- pollination, and could thus be occasionally liable to ostigma; Gonçalves et al., 2007; Cusimano et al., sporadic selfing. Other characters, such as the large 2011), may be a clue for a possible recent adaptive pollen grain volume and wide stigmatic area are reversion to beetle pollination. Such an assumption is shared with other cantharophilous aroids. The rela- supported by the evolutionary history of the tribe, tively low number of male flowers in T. ulei inflores- which strongly suggests a correlation between biogeo- cences, although more generally associated with graphical distribution and pollination systems. The fly-pollinated taxa, also occurs among aroids visited basal groups of the Spathicarpeae, such as Dieffen- by scarab beetles (Gibernau et al., 2010). bachia and Gearum, are lowland geophytes pollinated We verified no temperature elevation or odour by cyclocephaline beetles (Gibernau, 2011; Fig. 1). emission from T. ulei inflorescences during day 2 of Posterior invasions of colder and dryer habitats in the anthesis. For Philodendron subgenus Philodendron Andean Cordillera are presumably associated with (Gibernau et al., 2000; Gibernau & Barabé, 2002; shifts to fly pollination (Fig. 1). Interestingly, T. ulei is Maia et al., 2010), Caladium (Maia & Schlindwein, not plotted next to other scarab beetle-pollinated 2006), Montrichardia (Gibernau et al., 2003), and Spathicarpeae (Dieffenbachia spp.), but instead is Homalomena (Barabé & Gibernau, 2000), there are found in the opposite direction (Fig. 5). Such results two thermogenic events, occurring on consecutive imply that different sets of floral characters might days (during the female and male phases). The impor- have evolved in Dieffenbachia spp. and T. ulei to tance of a temperature increase during the male exploit the pollination services of the same group of phase has been the object of conjecture. Pollen release specialized flower-visiting insects. This hypothesis following a second thermogenic episode has raised the should be validated by studying other genera of this hypothesis that the two events might be co-related tribe using a biogeographical approach, particularly (Gibernau et al., 2000; Gibernau & Barabé, 2002). It the closely related Asterostigma, the pollinators of was also suggested that the increase of temperature which are so far unknown. A comparative approach of during the male phase allows the endothermic polli- the pollination ecology of Spathicarpeae genera is nator beetles to save the energy required to warm up needed in order to further explore evolutionary shifts their bodies prior to flight activity (Seymour et al., between fly and beetle pollination. This must include 2009). In T. ulei, anther dehiscence and the departure detailed floral scent chemistry analyses and investi- of beetles from the floral chambers take place without gation of olfactory attraction cues, which are probably any temperature increase from the spadix, indicating a major radiative force in the group. that pollen release in this aroid is not related to temperature, and that thermogenesis might not be ACKNOWLEDGEMENTS used as an energy-saver by the pollinators, at least in this species and under the ambient temperatures We wish to thank Frederico Cavalcanti de Petribú of the lowland Atlantic Rainforest. Also, in flowers Vilaça and Roberto Siqueira as representatives of of Annona and other Annonaceae pollinated by Usina São José SA for the allowing fieldwork at its

© 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 108, 22–34 POLLINATION OF TACCARUM ULEI 33 facilities, and also for logistic support. We would like specialization. Annual Review of Ecology, Evolution and to thank Gerhard Gottsberger, Thomas Croat, and Systematics 35: 375–403. two anonymous reviewers for improving the article García-Robledo C, Kattan G, Murcia C, Quintero-Marín with valuable suggestions and comments. We thank P. 2004. Beetle pollination and fruit predation of Xantho- Paschoal Coelho Grossi and Ângelo Pires do Prado for soma daguense (Araceae) in an Andean cloud forest in the identification of the beetles and the flies. This Colombia. Journal of Tropical Ecology 20: 459–469. research was part of the project ‘Sustainability of Gibernau M. 2011. Pollinators and visitors of aroid inflores- remnants of Atlantic rainforest in Pernambuco and cences: an addendum. Aroideana 34: 70–83. Gibernau M, Barabé D. 2002. 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