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. 2011 Feb 2;107(4):611–619. doi: 10.1093/aob/mcr008

Reproductive biology of Abolboda pulchella and A. poarchon (Xyridaceae: Poales)

Aline Oriani 1,*, Vera Lucia Scatena 1
PMCID: PMC3064540  PMID: 21292675

Abstract

Background and Aims

Abolboda (Xyridaceae) belongs to the Poales, a predominantly wind-pollinated order whose phylogeny has been widely studied in recent years. The reproductive biology of Abolboda pulchella and A. poarchon was studied to determine the main pollination system of these species, providing the first experimental data on reproduction in the Xyridaceae.

Methods

A field study was conducted, including observations on the morphology and biology of the flowers, insect visits and pollinator behaviour. Experimental pollination treatments were performed to assess agamospermy, spontaneous self-pollination and self-compatibility. Pollination success was determined by pollen tube growth, and reproductive success was assessed by fruit- and seed-set.

Key Results

Abolboda pulchella and A. poarchon were pollinated by Apidae, Megachilidae and Halictidae bees. The floral resources were pollen and nectar that was produced by stylar appendages, an uncommom nectary type for monocotyledons. The species were self-compatible, and pollen tube growth from self-pollen was similar to that of cross-pollen. However, herkogamy prevented spontaneous selfing, rendering the plants dependent on the pollinator's activity. There was no production of seeds by agamospermy.

Conclusions

Melittophily is the main pollination system of these two Abolboda species. Nectar production was first recorded here for Xyridaceae, and along with self-compatibility, herkogamy and bee pollination, is an informative characteristic that can be used in future phylogenetic analyses of the family as well as Poales.

Keywords: Abolboda pulchella, Abolboda poarchon, bee pollination, herkogamy, nectar, Poales, reproduction, self-compatibility, stylar appendages, Xyridaceae

INTRODUCTION

Xyridaceae includes five genera and about 300 herbaceous species (Kral, 1998). Abolboda is the second largest genus of the family, including a total of 23 species restricted to South America (Campbell, 2005), with its diversity centred in the northern Amazon river basin from the Guyanas to south-eastern Colombia (Smith and Downs, 1968). The monophyly of Xyridaceae is still under investigation (Michelangeli et al., 2003; Davis et al., 2004). Abolbodaceae was suggested to be a distinct family, including Abolboda, Aratitiyopea and Orectanthe, genera that share the same inflorescence type, stamen number and the presence of stylar appendages (Steyermark, 1984).

Xyridaceae is included in the Poales, which comprises 16 families and about 19 000 shrubby–herbaceous species (Soltis et al., 2005; APG III, 2009). Because of its representativeness, the order has been widely studied in recent years, especially from the phylogenetic point of view (Linder and Rudall, 2005). However, specific studies on reproductive biology, although informative to phylogeny, are restricted to Eriocaulaceae (Ramos et al., 2005; Sawyer et al., 2005; Oriani et al., 2009), Bromeliaceae (e.g. Sazima et al., 1989; Wendt et al., 2001; Canela and Sazima, 2005; Kaheler et al., 2005; Vosgueritchian and Buzato, 2006), Poaceae (e.g. Soderstrom and Calderón, 1971; Adams et al., 1981; Guilherme and Ressel, 2001; Koshy et al., 2001; Fang et al., 2004) and Cyperaceae (Leppik, 1955; Snyder and Richards, 2005).

A majority of the families in Poales have reduced and non-attractive flowers that are probably wind-pollinated (Linder and Rudall, 2005). Xyridaceae, Bromeliaceae, Rapateaceae and Mayacaceae are exceptions because they have large, colourful and attractive flowers, indicating animal pollination. Experimental studies on Bromeliaceae have demonstrated that they are pollinated by different pollinator guilds, including hummingbirds, bats and bees (Kaheler et al., 2005). For Xyridaceae, it was suggested that Orectanthe species are ornitophilous, and the remaining species are bee-pollinated (Kral, 1998). According to Kral (1998), the Xyridaceae flowers do not produce nectar, and the pollinators are attracted by the pollen. In pollination studies in Venezuelan plant communities, Ramírez (1992, 1993) also attested that Xyridaceae and Rapateaceae species are bee-pollinated.

This study aimed to investigate the reproductive biology of two species of Abolboda, providing the first experimental data on reproduction in Xyridaceae and contributing to the understanding of this trait in Poales. We investigated the floral morphology and biology, pollination and breeding systems of A. pulchella and A. poarchon, addressing the following questions: What is the main pollination system of these species? What is the behaviour of the floral visitors and what floral resources are used by them? Do the Abolboda species share pollinators? Are they self-compatible and self-fertile?

MATERIALS AND METHODS

Species and study area

Abolboda pulchella Humb. & Bonpl. and A. poarchon Seub are restricted to Brazil, occurring in wetland and swamp areas (Kral, 1992). They flower from October to November and fruit from November to December (Coan et al., 2009).

The studied area is a swamp grassland in a characteristic ‘cerrado’ (Brazilian savanna) area. It is located on private property between the cities of Itirapina and Brotas in the state of São Paulo (22 °16′5·3″S, 47 °54′32·7″W). The regional climate is mesotermic, with two well-defined seasons: dry from April to September and wet from October to March (Tannus and Assis, 2004). The experiments were carried out from October to December 2009. Voucher specimens were deposited in the Herbarium Rioclarense (HRCB) at São Paulo State University (UNESP) in Rio Claro, São Paulo (Coan and Oriani 61 and Coan and Oriani 72).

Floral morphology and biology

Flowers were fixed in FAA50 (37 % formaldehyde, glacial acetic acid, 50 % ethanol, 1 : 1 : 18, v/v) (Johansen, 1940), dehydrated in an absolute ethanol series, critical-point dried, coated with gold and examined with a scanning electron microscope (JSM 5410, JEOL, Tokyo, Japan). Fixed flowers were also dehydrated in an n-butyl alcohol series (Feder and O'Brien, 1968), embedded in (2-hydroxyethyl)-methacrylate (Leica Historesin Embedding Kit, Nussloch, Germany) and sectioned at 6–8 µm on a microtome (Model 2040, Reichert-Jung, Nussloch, Germany). The sections were stained with periodic acid–Schiff's reagent (PAS reaction) and toluidine blue (Feder and O'Brien, 1968) and mounted on permanent slides using Entellan (Merck, Darmstadt, Germany).

Floral biology was studied over 25 d. Data regarding the phases of flowering, time of anthesis, inflorescence maturation, stigmatic receptivity, anther dehiscence, pollen availability and viability, pollen composition and nectar production were recorded.

Stigmatic receptivity was tested with hydrogen peroxide (Dafni, 1992). Pollen viability was analysed in pollen grains that were removed from the anthers of three individuals and stained with acetocarmine (Kearns and Inouye, 1993). In addition, the number of pollen grains and ovules from four individuals were counted to calculate the pollen/ovule ratio. The anthers were placed in Petri dishes with glycerine and the number of pollen grains was counted under a stereomicroscope (MZ 125, Leica, Heerbrugg, Switzerland). The presence of lipidic substances (pollenkitt) and starch in the pollen grains was tested with Sudan IV and Lugol, respectively (Johansen, 1940).

To locate the nectariferous tissues, flowers were submerged in a 1 : 10 000 neutral red solution (Kearns and Inouye, 1993). Nectar production was analysed by chemical tests conducted in the field with Diabur-test 5000 test strips (Roche, Mannheim, Germany), which are sensitive to glucose.

Floral visitors

Floral visitors were collected from 0900 to 1700 h, the period during which the flowers were open. Each insect was placed in a separate vial, and the presence or absence of pollen grains adhered to their bodies was determined in the laboratory with a stereomicroscope. Species were identified by specialists. Voucher specimens were deposited in the Instituto de Biociências collection at UNESP in Rio Claro, São Paulo.

The times and frequencies of visits to A. pulchella and A. poarchon flowers were recorded over 15 consecutive days from 0900 to 1700 h, totalling 45 h of observation for A. pulchella (n = 196 flowers) and 48 h for A. poarchon (n = 242 flowers). Temperature and relative humidity were monitored with a thermohygrometer (Incoterm, Porto Alegre, Brazil). The behaviour and the type of resource used by the floral visitors were analysed by direct visual observations, complemented with photographs taken during the visits. Based on their visitation frequency and behaviour on the flowers, the insects were classified as effective pollinators, occasional pollinators or thieves. Effective pollinators represented the frequent insects (with a higher number of visits) that carried pollen grains from an anther to the stigma of another flower. Occasional pollinators represented those insects showing the same behaviour, but with low visitation frequency. Those insects that only collected pollen and/or nectar without pollinating the flowers were classified as thieves (Inouye, 1980).

Breeding systems

The breeding systems of A. pulchella and A. poarchon were evaluated using inflorescences with floral buds marked at random and isolated in polyester bags (25 × 25-μm mesh). The bags were attached to iron frames to avoid contact with the scapes (inflorescence stalks) and sewn on the bottom. We conducted the following treatments. (1) Agamospermy: the flowers were emasculated soon after anthesis and before anther dehiscence, and the inflorescences were bagged. (2) Spontaneous self-pollination: the inflorescences were bagged without any manipulation of the flowers. (3) Experimental self-pollination: inflorescences with hand self-pollinated flowers were bagged. (4) Experimental cross-pollination: pollen from different individuals was transferred to the stigma of emasculated flowers, and the inflorescences were bagged. (5) Natural pollination (free exposure): inflorescences with floral buds were marked at random and maintained under natural conditions.

Each treatment was conducted on ten inflorescences of A. pulchella and six inflorescences of A. poarchon, using four flowers from each inflorescence. After 90 d, each of the marked inflorescences was collected and placed in a separate plastic bag. Fruit-set was calculated in the laboratory. The numbers of seeds and aborted ovules per flower were also counted under a stereomicroscope to calculate seed-set, which was compared statistically among the treatments by a one-way ANOVA and Tukey's test (α < 0·05) (Zar, 1999).

The self-fertility index (SFI) and self-compatibility index (SCI) were also calculated (Zapata and Arroyo, 1978; Lloyd and Schoen, 1992). SFI is the average fruit-set after spontaneous self-pollination divided by the average fruit-set after experimental cross-pollination. SCI is the average fruit-set after experimental self-pollination divided by that of experimental cross-pollination. SCI values of ≤0·2 indicate self-incompatibility, whereas values >0·2 indicate self-compatibility (Zapata and Arroyo, 1978).

Pollen tube growth in the styles was examined by fluorescence microscopy (Martin, 1959). Two treatments were compared: experimental self-pollination and experimental cross-pollination. The hand-pollinated flowers were fixed in FAA50 at 1, 3, 6, 9, 12, 24, 36, 48, 60 and 72 h after pollination, using three flowers for each interval. Before the observation, the flowers were clarified with 8 m NaOH (at 60 °C for 20 min) and dyed in 0·1 % aniline blue solution.

RESULTS

Floral morphology and biology

The inflorescences of A. pulchella have from four to 13 hermaphrodite flowers (mean = 8; n = 30 inflorescences). The purplish petals are fused from the base up to the middle region, forming a tube approx. 0·6 cm long. The style, approx. 1·2 cm long, has three secreting appendages, with the two lateral appendages being more developed than the posterior one (Fig. 1C, arrow, and D). These appendages are inserted approx. 2·8 mm from the base of the style and are located inside the floral tube. The distance from the stigma to the beginning of the floral tube is 0·8 cm. The anthers and pollen grains are yellow and are located below the stigmas.

Fig. 1.

Fig. 1.

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Floral morphology and nectary anatomy of Abolboda pulchella and A. poarchon. (A, B) Detail of the stylar nectariferous appendages of A. poarchon. (C, D) Detail of the stylar nectariferous appendages of A. pulchella. (E, F) Longitudinal sections of the stylar nectariferous appendages of A. pulchella. (G–I) Cross-sections of the stylar nectariferous appendages of A. poarchon. (J) Pollen grains of A. poarchon with lipidic substances (pollenkitt) on their surfaces. White arrow: stylar nectariferous appendages; black arrow: phloem; arrowhead: xylem. Abbreviations: E, epidermis; P, parenchyma; S, style; A, stylar nectariferous appendages. Scale bars: (A, C) = 1·5 mm; (B, D) = 200 µm; (E, F, H, I) = 50 µm; (G) = 150 µm; (J) = 340 µm.

In A. poarchon, the inflorescences have from four to ten flowers (mean = 6; n = 15). The petals are bluish purple, forming a tube approx. 1·0 cm long. The secreting appendages (Fig. 1A, arrow, and B) are inserted 4 mm from the base of the style, which is 1·7 cm long. The distance from the stigma to the beginning of the floral tube is 1·2 cm.

Anatomically, the stylar appendages consist of an epidermis subtended by specialized parenchymatous tissue with vascular bundles (Fig. 1E–I). The epidermal cells are isodiametric (Fig. 1E–I) and have rounded external periclinal walls (Fig. 1B, D). The parenchymatous cells are longitudinally elongated with thin walls and dense granular cytoplasm, a large nucleus and starch storage (Fig. 1F). The vascular bundles are composed of phloem and xylem (Fig. 1F). Figure 1(E, G) show the ramifications of vascular bundles from the style to the appendages. The xylem is present only in the proximal portion of the appendages (Fig. 1H, arrowhead), and the last branches reaching the distal portion are phloem elements (Fig. 1I, arrow).

In A. pulchella, the flowers began to open around 0930 h (Fig. 2A, B) and were completely open at about 1030 h. Each flower lasted a single day, began to close at about 1500 h and was completely closed at about 1630 h. The A. poarchon flowers also lasted a single day, beginning to open around 0900 h and closing at about 1530 h. In both species, the flower opening and duration were influenced by temperature and humidity because the flowers opened and closed earlier on warmer, drier days.

Fig. 2.

Fig. 2.

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Flowers and pollinating bees of Abolboda pulchella and A. poarchon. (A, B) Flower of A. pulchella opening. (C) Flower of A. poarchon. (D, E) Arhysoceble cf. xanthopoda (Apidae), collecting nectar and pollen from A. poarchon (D) and A. pulchella (E) flowers. (F, G) Megachile sp. (Megachilidae), collecting nectar from A. poarchon flower. (H) Dialictus sp. (Halictidae) and (I) Augochloropsis sp. (Halictidae), collecting nectar and pollen from A. poarchon flower. Arrow: pollinated flower, with pollen grains on the stigma. Scale bars: (A–C) = 4 mm; (D–I) = 6 mm.

Inflorescence maturation was acropetal and lasted from 20 to 50 d in A. pulchella and from 15 to 45 d in A. poarchon. A single flower (or less often two) opened per day in each inflorescence.

In both species, when the flower began to open, the style was straight (Fig. 2A), but it became curved shortly after flower opening, moving the stigma away from the anthers and forming a landing platform (Fig. 2B, C). The stigma was receptive from flower opening until closing. In non-pollinated flowers, the stigma became pinkish at about 1100 h, and small droplets of stigmatic secretion could be observed on its surface.

Anther dehiscence began after the flowers opened (Fig. 2A). The pollen grains contained lipidic substances on their surfaces (Fig. 1J) and stored starch. Pollen viability was 95·2 % (s.d. = 1·9 %; n = 3 flowers) for A. pulchella and 95·9 % (s.d. = 3·4 %; n = 3 flowers) for A. poarchon. Pollen grain availability was higher in A. poarchon with a mean of 956 pollen grains per anther (s.d. = 30; n = 4 flowers), while the mean in A. pulchella was 501 (s.d. = 41; n = 4 flowers). However, the mean pollen/ovule ratio was similar in both species, 47 (s.d. = 1·2; n = 4 flowers) for A. pulchella and 51 (s.d. = 2·2; n = 4 flowers) for A. poarchon. The pollen grain size was also similar in both species, measuring 0·08–0·1 mm in diameter (n = 10 flowers).

Nectar was secreted by the stylar appendages during the entire period of flower anthesis and accumulated in the floral tube in both species. The appendages stained intensely in neutral red solution, indicating their secretory function, and the glucose test was positive.

Floral visitors

The most frequent visitors to the A. pulchella and A. poarchon flowers were bees (Hymenoptera), but butterflies (Lepidoptera) were also recorded in addition to ants (Hymenoptera), flies (Diptera), grasshoppers (Orthoptera) and beetles (Coleoptera) (Table 1). These insects visited the flowers from their opening until closing, and the floral rewards were nectar and pollen.

Table 1.

Floral visitor species and visitation frequencies to Abolboda pulchella and A. poarchon flowers

Number of visits
Species A. pulchella A. poarchon Classification*
HYMENOPTERA
Apidae
 Arhysoceble cf. xanthopoda 42 78 Effective pollinator
 Ceratina (Crewella) sp. 4 5 Occasional pollinator
Megachilidae
 Megachile sp. 28 42 Effective pollinator
Halictidae
 Dialictus sp. 4 11 Occasional/effective pollinator
 Augochloropsis sp. 2 32 Occasional/effective pollinator
Formicidae
 Pseudomyrmex sp.1 4 0 Nectar thief
 Pseudomyrmex sp.2 1 21 Nectar thief
 Camponotus sp. 1 1 Nectar thief
 Ata sp. 0 5 Herbivore
LEPIDOPTERA
Hesperiidae
 Vidius vidius 1 5 Nectar thief
 Hesperiinae sp. 0 4 Nectar thief
Lepidoptera sp.1 20 4 Nectar thief
Lepidoptera sp.2 5 0 Nectar thief
Lepidoptera sp.3 2 0 Nectar thief
Lepidoptera sp.4 1 0 Nectar thief
DIPTERA
Bombyllidae
 Poecilognathus sp. nov. 2 0 Nectar thief
Syrphidae sp. 1 1 Pollen thief
COLEOPTERA
Chrysomelidae
 Diabrotica sp. 1 0 Pollen thief
 Chrysomelidae sp.1 3 0 Pollen thief
 Chrysomelidae sp.2 1 1 Pollen thief
Coccinellidae sp. 1 0 Pollen thief
ORTHOPTERA
Acrididae
 Dichromatos sp. 0 2 Herbivore
 Acrididae sp. 1 0 Herbivore
Tettigoniidae sp.1 4 0 Herbivore
Tettigoniidae sp.2 5 0 Herbivore
Tettigoniidae sp.3 2 0 Herbivore
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* Classification based on insect behaviour and visitation frequency.

Five bee species were registered, belonging to three families: Apidae (two species), Megachilidae (one species) and Halictidae (two species), which were considered effective or occasional pollinators (Table 1). These bees collected pollen and nectar and transported the pollen from one flower to another, with activity peaking from 0900 to 1400 h. There were pollen grains from A. pulchella or A. poarchon adhered to the bodies of all individuals collected.

The Apidae and Megachilidae species were the most frequent pollinators (Table 1). Arhysoceble cf. xanthopoda (Apidae) (Fig. 2D, E) measured 0·6–0·7 cm in length and landed on the stamens with their heads turned toward the flower tube, from which they extracted the nectar with their proboscis. While they extracted the nectar, they also collected pollen from the anthers, which became attached to their legs and abdomen. Part of that pollen was left on the upper region of the stigma when these bees moved from one flower to the next (Fig. 2E, arrow). They visited almost all of the flowers in anthesis that were nearby and returned to feed on the same flowers visited previously. Visits peaked from 0900 to 1100 h in A. pulchella and from 1100 to 1200 h in A. poarchon.

Megachile sp. (Megachilidae) (Fig. 2F, G) individuals were larger, measuring 1·0–1·2 cm in length, and presented a visiting behaviour similar to that of Arhysoceble cf. xanthopoda. Due to their larger body size, as they landed on the flowers to extract nectar their abdomens were necessarily over the stigmas, thus pollinating the flowers. Their visit peak occurred from 1000 to 1200 h in A. pulchella and from 1200 to 1400 h in A. poarchon.

The Halictidae species visited A. poarchon flowers with a higher frequency as compared with the A. pulchella flowers. Because of this they were considered effective pollinators for A. poarchon and occasional pollinators for A. pulchella (Table 1). Dialictus sp. (Fig. 2H) individuals measured 0·5–0·6 cm in length and also presented a visiting behaviour similar to that of Arhysoceble cf. xanthopoda. Visits peaked from 1000 to 1100 h in A. pulchella and from 1200 to 1300 h in A. poarchon. Augochloropsis sp. (Fig. 2I) measured 0·8–1·0 cm in length, and due to their body size, as they landed on the flowers, their abdomens and hind legs necessarily touched the stigmas, achieving pollination. Visits peaked from 1000 to 1200 h in A. poarchon. For A. pulchella, only two visits were recorded between 1000 and 1100 h.

The least frequent bee species was Ceratina (Crewella) sp. (Apidae), with four visits recorded for A. pulchella between 1100 and 1200 h and five visits recorded for A. poarchon between 1400 and 1500 h (Table 1). Their representatives measured 0·9–1·1 cm in length and also collected pollen and nectar.

The Lepidoptera species behaved as nectar thieves. They landed on the petals without touching the stigma and collected nectar from the floral tube with their proboscis, remaining on each flower for only a few seconds. Their visits peaked from 1100 to 1200 h in A. pulchella and from 1400 to 1500 h in A. poarchon. Several species of Formicidae, Diptera and Coleoptera also behaved as nectar and/or pollen thieves (Table 1).

In addition to the nectar/pollen thieves, several herbivore species were observed to feed on both A. pulchella and A. poarchon flowers. These included Atta sp. (Formicidae – Hymenoptera), grasshoppers (Tettigoniidae – Orthoptera) and locusts (Acrididae – Orthoptera) (Table 1). Larvae of two species of Psychidae (Lepidoptera) were also observed to feed on the flowers.

Breeding systems

The fruit- and seed-set obtained in each pollination treatment are shown in Table 2. In both species, no fruits were formed by agamospermy or by spontaneous self-pollination (SFI = 0). Fruit-set in experimental self-pollination, experimental cross-pollination and natural pollination was equivalent and higher than 89 %. The SCI was 0·99 for A. pulchella and 1·0 for A. poarchon, indicating self-compatibility.

Table 2.

Fruit- and seed-set in different pollinations treatments in Abolboda pulchella and A. poarchon

Species Treatment Fruit-set Seed-set
Aboldoda pulchella Natural pollination 0·92 0·841 ± 0·057; n = 25 a*
Experimental cross-pollination 0·91 0·848 ± 0·067; n = 23 a
Experimental self-pollination 0·90 0·619 ± 0·035; n = 20 b
Spontaneous self-pollination 0 0; n = 24
Agamospermy 0 0; n = 23
Abolboda poarchon Natural pollination 0·95 0·631 ± 0·052; n = 20 a
Experimental cross-pollination 0·89 0·801 ± 0·069; n = 18 b
Experimental self-pollination 0·89 0·599 ± 0·058; n = 18 a
Spontaneous self-pollination 0 0; n = 20
Agamospermy 0 0; n = 19
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* Different letters represent a significant difference between treatments according to Tukey's test (P < 0·05).

In A. pulchella, seed-set in experimental self-pollination was smaller compared with the other treatments (Tukey test, P < 0·05), whereas seed-set in experimental cross-pollination did not differ statistically from natural pollination (Tukey test, P > 0·05). In A. poarchon, seed-set in experimental cross-pollination was higher (Tukey test, P < 0·05), whereas that in experimental self-pollination did not differ statistically from natural pollination (Tukey test, P > 0·05).

Fluorescence microscopy revealed that pollen tube growth from self-pollen did not differ from that of cross-pollen in either species. In A. pulchella, 6 h after pollination, the pollen tubes had already reached the ovules, while in A. poarchon, fecundation occurred 9 h after pollination.

DISCUSSION

Abolboda pulchella and A. poarchon are pollinated by bees that collect and transport the pollen, promoting crossing and selfing. Another floral resource used by the visitors was nectar, which is produced by the stylar appendages. These appendages have been described previously in taxonomic studies of Xyridaceae (Steyermark, 1984; Dahlgren et al., 1985; Stützel, 1990; Kral, 1992, 1998), but here we provide anatomical evidence other than field observations that these structures are nectaries.

Nectaries are characterized anatomically by an epidermis, a specialized parenchyma and vascular bundles (Fahn, 1979, 2000; Pacini et al., 2003), as demonstrated in the Abolboda species. The parenchyma produces and stores nectar solutes, while the vascular bundles convey water and nutrients to the parenchyma (Pacini et al., 2003). The starch storage observed in the parenchyma cells of the Abolboda nectaries probably provides part of the source of nectar carbohydrates. According to Pacini and Nepi (2007), the sugar component of nectar is derived from photosynthesis by the nectary itself, or more commonly by photosynthesis in other parts of the plant, with or without starch as an intermediary storage product.

In monocotyledons, nectar is often produced by septal nectaries (Daumann, 1970; Fahn, 1979). These structures, however, are rare in Poales, being present in only two early divergent families of the order: Bromeliaceae and Rapateaceae (Givnish et al., 1999; Smets et al., 2000; Linder and Rudall, 2005). Thus, the nectariferous appendages of the style found in Xyridaceae are an uncommon type of nectary for monocotyledons and are similar to that of Eriocaulaceae, a closely related family (Rosa and Scatena, 2007).

For Eriocaulaceae, the nectariferous function of the stylar appendages has been recently demonstrated in Paepalanthoideae (Rosa and Scatena, 2007). These nectariferous appendages are probably a modification of the simple stigmas of Eriocauloideae that occurred during the evolution of the family in response to animal pollination (Rosa and Scatena, 2007).

Recent phylogenetic analyses of Poales (Givnish et al., 2005, 2006; Linder and Rudall, 2005) show Bromeliaceae, Typhaceae and the remaining Poales as a polytomy. Bromeliaceae and Thypaceae are sister to each other and diverged earliest within the order. Rapateaceae is the next divergent family and is sister to the remaining Poales. Eriocaulaceae is the sister-group of Xyridaceae and both families are in one of the two major clades of the core Poales. Thus, the fact that the studied Abolboda species produce nectar is not only important in reproductive terms but also from a phylogenetic perspective. The septal nectary present in Bromeliaceae (Böhme, 1988; Bernardello et al., 1991; Sajo et al., 2004) and Rapateaceae (Venturelli and Bouman, 1988; Givnish et al., 2000), and the stylar nectariferous appendages present in Eriocaulaceae (Rosa and Scatena, 2007) and Xyridaceae have a homoplastic origin.

In addition to the presence of nectaries, all of these families are pollinated by animals. Ramírez (1993) observed bees pollinating the flowers of Stegolepis angustata (Rapateaceae) and Xyris setigera (Xyridaceae). Studies on the pollination biology of Syngonanthus (Eriocaulaceae) (Ramos et al., 2005; Oriani et al., 2009) demonstrated that these species are pollinated by several small insects, including beetles, flies and bees. Thus, nectar production and pollination by animals are probably labile features that appeared independently in Poales. It can be inferred also that nectar production and pollination by animals is an ancestral condition for the order, while wind pollination is a derived characteristic, in accordance with the hypothesis of Linder and Rudall (2005) on the evolution of the Poales reproductive system.

Abolboda pulchella and A. poarchon shared the same pollinators as expected, as these species are sympatric (Kral, 1992) and show a very similar floral morphology. Arhysoceble cf. xanthopoda was the species with the highest visitation frequency, but Megachile sp. was observed to have the most suitable size and behaviour for performing pollination. The genus Arhysoceble is restricted to Argentina and Brazil, with only four species recorded in Brazil, including solitary bees that gather floral oils (Silveira et al., 2002). Arhysoceble xanthopoda individuals were observed collecting floral resources from different plant families, such as Malpighiaceae, Malvaceae and Scrophulariaceae (Gonçalves and Melo, 2005). Megachile is a cosmopolite genus that includes a great number of species. They are also solitary bees, and the females of the non-parasitic species have a characteristic scopa in the ventral surface of the abdomen for removing and transporting pollen (Silveira et al., 2002).

In addition to the pollinators, several insect species were observed to act as herbivores or nectar/pollen thieves. The most frequent thieves observed on A. pulchella and A. poarchon flowers were butterflies (Lepidoptera) and ants (Hymenoptera), which mainly collected nectar. However, the activity of these thieves probably does not influence the reproductive success of the Abolboda species because their visits peaked during a later period than the peak of visits made by pollinating bees.

The controlled pollination tests showed that A. pulchella and A. poarchon are self-compatible (SCI = 1); however, spontaneous self-pollination is prevented by herkogamy, i.e. the spatial separation of the male and female reproductive organs (Faegri and Van der Pijl, 1979). Because of this, the self-fertility index of A. pulchella and A. poarchon is null (SFI = 0), indicating complete dependence on bees for their reproductive success. Because both are self-compatible, the activity of bees may result in both cross- and self-pollination. However, the floral morphology forces the pollinators to touch the stigmas before the anthers, favouring outcrossing.

For A. pulchella, the similar seed-set in both experimental cross-pollination and natural pollination demonstrates the effectiveness of bees in pollinating the species. For A. poarchon, the higher seed-set in experimental cross-pollination compared with that in natural pollination indicates that the amount of pollen deposited on the stigma by the bees was not sufficient to fertilize all of the ovules, the number of which was twice that observed in A. pulchella.

For both species, the smaller seed-set in experimental self-pollination compared with that in experimental cross-pollination is probably a result of inbreeding depression, in which deleterious or lethal recessive alleles cause the random abortion of embryos (cf. Wiens et al., 1987). Therefore, genetic and physical barriers (herkogamy) that prevent self-fertilization, in addition to bee pollination, probably maintain the genetic variability of the populations studied and define the reproductive system of A. pulchella and A. poarchon.

Nectar production, herkogamy and bee pollination in Abolboda species are demonstrated for the first time in this study and are important traits for future phylogenetic analyses in Xyridaceae and Poales. The present study also opens perspectives for future studies involving genetic variability, hybridization and population dynamics as well as for intra- and inter-familiar comparative studies.

ACKNOWLEDGEMENTS

We thank R. B. Gonçalves, E. Giannotti, O. H. H. Mielke, C. J. E. Lamas, F. J. Zara and A. Ferreira for identifications of floral visitors, and A. Bianconi for assistance with the statistical tests. We also thank the researchers who helped us with the fieldwork, especially A. I. Coan and M. T. Eichemberg. The anonymous reviewers are acknowledged for critical comments that improved the manuscript. This work was supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico – CNPq (Grants 302105/2007-7 and 471202/2008-8 to V.L.S., 141582/2007-3 to A.O.).

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