Pollination of Echinodorus grandiflorus (Alismataceae

ELSEVIER Aquatic Botany 58 (1997) 89 98

Aquatic botany

Pollination of Echinodorus grandiflorus (Alismataceae)

Milene Faria Vieira ~, Nat~.lia A. de Souza Lima Departamento de Biologia Vegetal, Universidade Federal de Vi¢osa, 36571-000, Vi~osa, Minas Gerais, Brasif

Accepted 8 April 1997

Abstract

Studies on the pollination and breeding system of Echinodorus grand(florus (Chamisso and Schlechtendal) Micheli were carried out on two natural populations in Vi~osa, Minas Gerais State. southeastern Brazil. One population was formed by individuals of ssp. grandiflorus and the other by individuals of ssp. aureus (Fassett) Haynes and Holm-Nielsen. The flowering periods were within the rainy season. Flower anthesis occurred during the morning hours and flowers lasted ca. 8 h. E. grandiflorus ssp. grandiflorus is self-incompatible: few fruits, but many adventitious plantlets are produced at the inflorescences. E. grando%rus ssp. aureus is self-compatible; many fruits and few adventitious plantlets are formed. Bees are the main flower visitors. The bee Protodiscelis echinodori (Colletidae) presents oligolectic foraging behavior, i.e., collects pollen from a single plant species or a group of related plant species. © 1997 Elsevier Science B.V.

Keywords." Bees; Breeding system; Echinodorus; Floral b o ogy; Oligolecty; Protodisceli.~

1. Introduct ion

The family Alismataceae is cosmopolitan and consists of I I genera and about 75 aquatic or semi-aquatic species, the majority occurring in the New World. The principal neotropic genera are Echinodorus with 26 species and Sagittaria. with about 25 species:

in Brazil, this family is represented by 18 species of Echinodorus and six Sa~ittaria species (Haynes and Holm-Nielsen, 1994).

With the exception of preliminary studies of Lima and Vieira (1994) on Echinodorus

grand(17orus, no information is available on the relationship between neotropic species

Corresponding author.

0304-3770/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved. I'll S0304-3770(97)00019-3

90 M.F. Vieira, N.A. de Souza Lima~Aquatic Botany 58 (1997) 89-98

of the Alismataceae and their pollinators, Taking into consideration only floral features, Cook (1988) proposed that 10 out of 11 genera are insect-pollinated.

The aim of the present study was to provide data on the pollination biology of E. grandiflorus in natural populations in southeastern Brazil.

2. Materials and methods

2.1. Study species

Echinodorus grandiflorus (Chamisso and Schlechtendal) Micheli has a wide geo- graphic distribution from Mexico to southern South America, and is found in many Brazilian states (Fassett, 1955a,b,c; Rataj, 1978; Haynes and Holm-Nielsen, 1994). It is a perennial up to 0.9 m tall marsh herb growing from a creeping rhizome. Its leaves are basal, sheathing, with cordate blades and long petioles. Inflorescences are erect, 1.5-1.9 m tall, terminate on a leafless scape, and contain many flowers and vegetative buds. At the sites (S 1 and $2) where this study was carried out, E. grandiflorus forms colonies.

Collections of E. grandiflorus from sites S 1 and $2 were sent to Haynes (Univ. of Alabama) for identification. According to him (pers. comm.), the two subspecies of E. grandiflorus in Minas Gerais overlap in distribution, which makes determination to the subspecies level difficult. The individuals at S1 are probably ssp. grandiflorus and $2 individuals are ssp. aureus (Fassett) Haynes and Holm-Nielsen. The two subspecies are distinguished by the presence or absence of lines in the pellucid markings in the leaf (Haynes and Holm-Nielsen, 1994).

E. grandiflorus ssp. grandiflorus occurs in Central Brazil south to Paraguay, northern Argentina, and Uruguay; flowering from October to May. E. grandifiorus ssp. aureus occurs in Cuba, southern Mexico to Central America, Colombia, Venezuela, and Brazil; flowering year round, according to Haynes and Holm-Nielsen (1994).

2.2. Study sites

The study was carried out in the municipality of Vi~osa (20o46 ' S, 42o54 ' W), Minas Gerais State, where altitude ranges from 600 to 800 m; its average annual temperature is 19°C and annual rainfall ranges from 1300 to 1400 ram, with most of it falling from October through March.

Two study sites (SI and $2), 12 km apart, were chosen. S1 has an area of approximately 160 m 2. The period of field work at S1 was from November 1991 through January 1992, and October 1992 through January 1993, for a total of 82 daylight h concentrated in the morning. Site $2 has an area of approximately 260 m e. The period of field work at $2 was from October 1992 through January 1993, for a total of 28 daylight h.

2.3. Flowering phenology and floral biology

Part of the SI population in an area of about 35 m 2 was used for studying flowering phenology. The number of individuals with open flowers were counted throughout the flowering period. In addition, inflorescences were chosen at random and tagged when

M.F. Vieira, N.A. de Souza Lima/Aquatic Bolanv 58 (1997) 89-98 91

they were in the first day of flowering. From this day on, daily counts of open flowers were done and opening sequences were determined for each inflorescence.

At S1 and $2, data were obtained about anthesis period, flower longevity, availability of pollen, nectar period, and odor production. In order to test pollen viability, flowers from S I and $2 were collected during the morning, before insect visits. Slides were prepared with macerated anthers over a drop of aceto-carmine glycerol jelly (Radford et al.. 1974). For odor verification flowers at the anthesis stage were placed in glass containers.

2.4. Breeding system

Breeding experiments were conducted during the morning hours as follows: (a) cross-pollination (between 7:45 and 10:00 am): buds were bagged one day belk)re anthesis and emasculated before flower opening, in order to receive pollen from S I or $2 flowers (always utilizing pollen from a different site): (b) open pollination: buds were tagged but not bagged; (c) control: buds were left untouched in bags.

2.5. Visitors

Flower visitors from SI and $2 were collected, examined lor pollen, and identified. The number of flower visits by the principal species (frequency of visits) was recorded during the flowering period. Records were made during the peak of flower visitation (between 8:30 and 10:30 am), at selected S1 locations over five non-consecutive days, between the end of October and the beginning of December. The choice of location was related to flower number (around 55). Seven consecutive 1 rain counts were made each day (except on the third, when five counts were made) at 10 rain intervals.

3. Results

3. I. Inflorescence and.floral morphology

The inflorescences and flowers of the two populations had different morphologies. lnflorescences from S I had a main axis and three verticillate branches. Along the main axis nine lo 10 whorls (av. = 9.5; N = 13) of three bracts were found, in which flowers and vegetative buds (producing adventitious plantlets) were born. Each whorl produced 1 to 15 flowers and up to three propagules, one in each bract. Along each branch there were three to six whorls (av. = 4.0; N = 39) with three bracts in which the flowers (from I to 13) and vegetative buds (up to three) were born. Branches originated from one of the bracts at the first whorl of the main axis. 90% of 240 observed inflorescences developed propagules.

The inflorescences from $2 developed up to 12 branches, commonly five or six, localized at the first (three branches) and second (two or three branches) whorls of the main axis; they rarely developed propagules (only 7.6% of the 104 inflorescences observed).

92 M.F. Vieira, N.A. de Souza Lima/Aquatic Botany 58 (1997) 89-98

Flowers were actinomorphic, perfect; receptacle convex; sepals 3, green, 6 - 7 mm long; petals 3, white, separate, about 18-20 m m long; 22 -29 stamens (av. = 26.9; N = 29) with yellow (S1) or greenish yellow ($2) versatile anthers; head of yellow (S1) or greenish yellow ($2) pistils (av. = 124.6; N = 10 for S1 and av. = 181.4; N = 47 for $2) which developed into nutlets. Stamens and pistils constitute the flower center, which had a diameter of about 8 mm.

3.2. Flowering phenology and floral biology

The plants at S 1 were in flower from the beginning of October to mid-January, with a peak in November (Fig. 1A). On the first day of flowering, the inflorescence developed one or two open flowers at the second whorl of the main axis, and on the second day, flowers at the first a n d / o r second a n d / o r third whorl could open. Afterwards, flower opening was acropetal. The number of flowers formed on the main axis ranged from 91 to 120 (av. = 106.5; N = 10). Flower opening on lateral branches started two to four days after that on the main axis; flowering was acropetal. The number of flowers on each lateral branch ranged from 22 to 48 (av. = 36.6; N = 27). Daily number of open

2 5 0 -

e~ o i r -

=. 200 t

~ q = 100 ~ $ ) I

Z 0 - - ; : , ~ ,

10/20 10/29 11112 11/19 12/8 12123 01/8

Flowering pedod (Month/day)

B

10/29 11/17 1t/26 12/3 12J8

Observations (Mon~day) <~ x - Bees

Fig. 1. Flowering period (A) and frequency of visits by different bee species (B) to flowers of Echinodorus grandiflorus. AM = Apis rnellifera; HA = Halictidae; OB = Other bees; PE = Protodiscelis echinodori; TS = Trigona spinipes

M.F. Vieira, N.A. de Sot<a Lima/Aquatic Botany 58 (1997) 89-98 93

flowers per whorl varied from zero to three, with flower opening in each whorl

occurring commonly at intervals of one to four days. The flowering period of an inflorescence ranged from 26 to 39 days (av. = 33.4; N = 10).

Flower anthesis was characterized by petal expansion and the exposure of the androecium and gynoecium. The process of flower opening lasted about one hour. It

began at about 7:00 am. At about 7:30 am, the flowers presented the petals in a position parallel to the floral axis. At this moment, the sexual organs were visible and the first insect visits could be observed. When the process was completed, the flowers exhibited

the petals arranged at an angle of approximately 45 ° in relation to the floral axis. The flowers were odorless. During the morning hours pollen and nectar were available to

visitors. Anther dehiscence occurred at anthesis. On average, pollen viability was 73.7% (N = 25) at SI and 52.4% (N = 25) at $2. Nectar accumulated in the form of droplets

between the petals and the stamens, and between the stamens and the pistils. The results of manual cross-pollination (Table 1) showed that the stigmas were receptive between 7:45 and 10:00 am. At about 11:00 am, the anthers were practically empty and chestnut-colored, while the stigmas were covered with pollen.

Flowers lasted for about 8 h. At approximately 2:30 pro, the process of petal closure

commenced: The petals became united at the apex and wilt over stamens and pistils, they acquired a cream color, and remained on the flower until deterioration. The calyx

persisted while the receptacle enlarged and accommodated the ripening nutlets, which

turned green or reddish green soon after fertilization.

3.3. Breed ing sys t em

Table 1 summarizes the results of the breeding experiments. Individuals from each site presented different reproductive behavior. Plants at S I appeared to be self-incompatible, since bagged flowers (control) did not set fruits. This incompatibility also occurred

among individuals, as shown by the results of open pollination. $2 plants were sell-compatible; but a significant difference was found between open pollination and the control ( k e = 52.40, I df, P < 0.01): open pollination permitted a higher rate of fruit

set in relation to the control.

Table 1 Results of controlled pollination treatments in Echinodorus grandifloru~

Treatments Study sites Plants (No.) F lowers Flowers Fruits setting fruit (No.) g,

Control J S 1 14 14 0 f) 0.0 $2 10 10 10 434 28.7

Open pollination S1 h 50 718 120 171 0.2 $2 . . . . "m "~o _~,','~ 1607 :;9.1

Cross-pollination S 1 8 8 8 722 72.9 $2 8 8 8 559 45.2

~' Buds were left untouched in bags. h Due to the large number of flowers utilized in this test, 125 pistils per flower were considered l~)r the calculation of fruit set percentage.


3.4. Floral visitors

Among the floral visitors (Table 2), bees (Insecta, Hymenoptera) were the most diverse and important group. All bees, except Protodiscelis echinodori (Colletidae), presented a similar visiting behavior. They either landed on the petals and moved to the flower center or landed directly on the flower center, using the stamens as main support. For pollen collection, bees used the mouth parts and the anterior and middle legs, making circular movements and exploring all anthers. During collection, they contacted the stigmas with their legs and their ventral body parts, where loads of pollen were observed. Bees also took nectar, introducing their heads either between the petals and stamens or between the stamens and pistils.

Halictidae species (e.g., Augochloropsis spp. ) and Andrenidae species (e.g., Acamp- topoeum prinii) sometimes grasped some stamens and started vibratory movements ('buzz'). Males of A. prinii were seen flying near the flowers, possibly to meet females, and pairs were often seen mating on the flowers. Flying in copulation posture, pairs visited further flowers where the female continued to collect pollen. A pair

Table 2 List of insect visitors of Echinodorus grandiflorus

Group Species Insect length (mm)

Coleoptera

Cantharidae Not identified 6.5 Coccinelidae Coleomegilla maculata (De Geer, 1775) 5.0 Crysomelidae Diabrotica sp. 8.0

Not identified 2.5 Curculionidae Not identified 1 3.0

Not identified 2 4.5

Hymenoptera

Andrenidae

Anthophoridae

Apidae

Colletidae

Halictidae

Acamptopoeum prinii (Holmberg, 1884) Psaenythia bergi Holmberg, 1884 Exomalopsis fulvopilosa Spinola, 1853 E. auropilosa Spinola, 1854 Apis mellifera Linnaeus, 1758 Schwarziana quadripunctata (Lepeletier, 1836) Trigona spinipes (Fabricius, 1793) Colletes punctatissimus Schrottky, 1902 Protodiscelis echinodori (de Melo, 1996) a Augochlora sp. Augochlorella sp. Augochloropsis ( Paraugochloropsis) sp. A. ( P.) cleopatra (Schrottky, 1902) A. ( P.) electra (Smith, 1853) Dialictus (Chloralictus) sp. Pereirapsis sp. Pseudagapostemon brasiliensis Cure, 1989 Tectochlora alaris (Vachal, 1904)

9.5 12.0 10.0 8.0

15.0 8.0 8.0

10.0 7.5 8.5 7.0 9.5

10.0 11.0 4.0 5.0 7.5 7.0

a New species, described from individuals collected during this study.

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remaining in copula for 7 min was observed on a flower. Males of this species were not observed visiting flowers of E. grandiflorus.

Bees of the species Protodiscelis echinodori (Colletidae) landed on the flowers like other bees, but their pollen and nectar collecting behavior was very peculiar. They leaned onto the stamens and stayed on or between them and the corolla, with the body perpendicular to the stamens and the back turned to the petals in the latter case. Then. with circulatory movements, they collected pollen using mouth pans, the anterior and middle legs, and the abdomen simultaneously. The abdomen was used to beat the anthers, appearing to brush them. When taking nectar, the bees kept beating the anthers and probably collected pollen at the same time. Males of Protodiscelis were seen visiting the flowers for nectar collection, but they were not seen in copulation.

Fig. 1B shows that bee visitation frequency changed during the flowering period, f . spinipes was observed very frequently until the flowering peak in mid-November (Fig. lA), when the bee P. echinodori started to appear. This species showed a rapid increase in frequency, with three times more visits than T. spinipes, and apparently displacing all the other bee species.

Small beetles (Insecta, Coleoptera, Table 2) were observed foraging on pollen and flower parts including corolla and androecium. Movements of these beetles between flowers were rarely observed. One of the Curculionidae species was often observed in copulation on the flowers.

4. Discussion

E. grandiflorus individuals from the two studied sites presented morphological differences (e.g., number of verticillate branches in the inflorescences and flower color) as well as reproductive differences (e.g., number of propagules per inflorescence and compatibility systems). These characteristics help to distinguish the two taxa and confirm Haynes' identification of the subspecies.

Flowers of E. grandiflorus are actinomorphic, flat, with numerous exposed stamens. pollen-flower features which can be characterized as Papa~,er-type according to the classification of Vogel (1978). In this type, pollen is predominantly utilized by bees: they gather the pollen by brushing, vibrating or scrambling around the flower, a behavior that was observed among bees visiting E. grandiflorus flowers, thus affecting "mess and soil' pollination, a melittophilous specialization (Vogel, 1978).

The marcescent corolla of E. grandiflorus may provide protection against pistil damage caused by insects (e.g., curculionid beetles which copulate on the flowers), at least during the first days after pollination. Gottsberger (1977) has observed that in flowers of Dat,illa elliptica (Dilleniaceae), a shrub in the Brazilian cerrado vegetation pollinated by bees and beetles and whose morphological type is similar to that of E. ,~randiflorus, the ovary is completely enclosed by the two inner sepals alter flowering. Some of the curculionid beetles use the ovary as a breeding place and the folding of the sepals seems to be an adaptation to save some of the ovaries from destruction (Gottsberger, 1977).

Although E. grandiflorus is visited mainly by bees, these insects seem to have no importance for pollination of E. grand~florus ssp. grand(llorus, which produced only a


few or no fruits with the pollen of their own population. This demonstrates self-incompatibility within and between individuals. The E. grandiflorus ssp. grandiflorus population probably is formed by clones that originated from a few individuals. The high production of propagules may be related to the low fruit production (cf. Grant, 1981). Self-compatibility among E. grandiflorus ssp. aureus individuals seems to be a derived condition, considering that E. grandiflorus flowers are insect-pollinated. Similar results indicating different compatibility systems between subspecies were obtained by Vuille (1988) for Baldellia, an Old World genus of the Alismataceae. Using herbarium material and plants of the genus Echinodorus cultivated in a greenhouse, Vuille made observations which suggested that the section Echinodorus, to which E. grandiflorus belongs, is strongly self-compatible and that the section Helianthemum (probably Helianthium, cf. Rataj, 1978) may be self-incompatible. The results of our study show that more field experiments are necessary for a better understanding of the reproductive systems in this genus.

The shift from obligate crossing to self-fertilization occurs chiefly in species that occupy pioneer habitats, as are the marshy habitats where individuals of E. grandiflorus are found. Self-fertilization may be the only way in which new colonizers can produce offspring (Stebbins, 1970). Production of large numbers of fruits, as observed in individuals of E. grandiflorus ssp. aureus, seems to be an advantageous condition for rapid population growth. For E. grandiflorus ssp. grandiflorus, high propagule production does not mean a rapid increase of individuals within the population. Propagule establishment only occurs when the inflorescence falls and reaches the soil. This occurred only in plants at the population's margin. Most of the other inflorescences did not touch the soil, due to the proximity of the plants, causing propagule withering.

The bees' foraging behavior and frequency of visits suggest that these insects may be important pollinators for E. grandiflorus ssp. aureus, as shown by the significant difference in fruit set between plants with (open pollination) and without (control) their presence (see $2, Table 1). For E. grandiflorus ssp. grandiflorus pollination, however, insects are apparently not required (see S 1, Table 1). On the other hand, their flowers are important as a food source and/or as a meeting site for mating for the bees. Flowers are a common place for bee mating (Eickwort and Ginsberg, 1980).

The relevance of E. grandiflorus flowers as a source of pollen and nectar is more conspicuous for Protodiscelis echinodori (Colletidae). The visiting behavior of this species (simultaneous extraction of nectar and pollen and constancy of pollen collection) and the appearance of these bees during the blooming period of their host plant indicate oligolectic foraging behavior (Linsley, 1958). The genus Protodiscelis Br&hes was treated as a subgenus of the genus Leioproctus (Michener, 1989). It includes at least six species which are all restricted to South America (de Melo, pers. comm.). Ducke (1908) recorded the bees Panurginus alismatis and P. palpalis (now Protodiscelis alismatis and P. palpalis; de Melo, pers. comm.; de Melo, 1996) in flowers of Alisma sp. in northeastern Brazil. The genus Alisma, however, does not occur in Brazil, and probably the author was referring to the genus Echinodorus, whose species are widely distributed throughout the country (Rataj, 1978). Bees of the genus Leioproctus, in Australia, tend to be oligolectic on certain plant species (Michener, 1965, 1970; Houston, 1989, 1991). Some of these plants belong to the Myrtaceae (Michener, 1965) and present floral

M.F. Vieira, N.A. de Souza Lima~Aquatic Botany 58 (1997) 89-98 97

morphologies similar to those of E. grandiflorus (e.g., actinomorphic, flat, and with numerous exposed stamens). These characteristics permit short-tongued bees, as P. echinodori, to obtain nectar and pollen easily. Further studies on the relationship between neotropic species of Echinodorus and their floral visitors may clarify whether that relationship between plants and oligolectic Protodiscelis bees is common.

Acknowledgements

We thank E.F. Morato, G.A.R. de Melo, and J.R. Cure for insect identification, and R.R. Haynes for identification of Echinodorus grandifforus. We are also indebted to M.F. Soares for help with the fieldwork. This work was supported by CNPq/UFV.

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Pollination of Echinodorus grandiflorus (Alismataceae

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