J. Plant Res. 110: 393-403, 1997 Journal of Plant Research {C) by The Botanical Society of Japan 1997

Nectar, Nectaries, Flower Visitors, and Breeding System in Five Terrestrial Orchidaceae from Central Argentina

Ceonardo Galetto, Gabriel Bernardello and Guillermo L. Rivera . -

lnstituto Mwltidisciplinario de Bloiogia Vegelal. Casilla de Correo 495. 5000 Cbrdaba, Argentina

Floral nectar sugar cornpsition, nectary anatomy, and visitors are studied in five Argentine Orchidaceae, from 18 populations. Hand-pollinations were performed to evaluate their breeding system. We found two different types of perigonal nectaries located either in the spur (Habemaria gwlieana, H, hiemnymi, Habenariinae), or in the basal lateral parts of the labellurn (Beadlea dutraei, Pelexia bonariensis, SFenorrhynchos orchioides, Spir- anthinae). The spur of Hahenaria is a nonvaseularised and nanstructural nectary. The inner epidermis bears one -celled long papillae. In bud stage, the papillae are filled with starch grains, but when the flower opens and nectar secretion starts, they show no starch grains. This fact may indicate that starch is a source for some of the secreted nectar. In the remainder genera, the lateral basal parts of the labellurn are secretory. The two glands are located in the adaxial basal lateral faces of the labelturn. These nectaries are structural and nonvas- cularised. Stenorrhynchs produces abundant, coneen- trated nectar (40-5O0h). Habemria gourlieana accumu- lates copious nectar in a lower concentration (< 2Q0/4), whereas the other species produce small quantities of concentrated nectar (ca. 50%). Three of the studied species have sucrose predominant nectar (Beadlea dutraei, Habenaria gourlieana, and Pelexia bonariensis) while H, hiemnymi, Stenorrhynchos orchioides have - hexme predominant ones. Nectar removal and/or polli- nation induce flower senescence. H. gwrlieana is visit- ed by sphingids, S. orehiofdes by hummingbirds, and B. dutraei by bees. For the two other species we did not record flower visitors. Pelexia bonariensb, 5. dvtraei, and S. o r c h i d s are self-compatible species but a pol- linator is needed.

Key wards: Flower visitors - Nectar removal Meets' . - Nectar sugar composition - Nectary structure - Or-

chidaceae - Self-compatibility

Orchidaceae is one of the largest families of fFowering plants. It includes around 17,503 species and almost 800 genera of perennial herbs (Mabberley 1990). Because a f

its particular pollen presentation in pollinia, orchid flowers normally require the assistance of animals to carry out pollination and, consequently, fertilization, whereas

autogamy is rare in the family (van der Pijl and Dodson 1966, Rasrnussen 7985, Oressler 1990). Flowers have visual and olfactory attractants, as well as rewards such as nectar, oils, and scents (van der Pijl and Dodson 1966, Dressler 1990). In mamy cases, pollinators are deceived by mimic flowers which do not offer any reward (cf. Dafni 1984, DressFer 1990, Nilsson 1992)

A great variety of pollinators have been reported for the family: hymenopterans, diurnal and nocturnal lepidopter- ans, dipterans, coleopterans, and birds (e.g. van.der PijT and Dodson 1966, Burns-Balogh et 81.7987, Dressler 1990, Nilsson 1992, Trernblay 1992, Johnson 1995, Proctor ef a/. 1996). Most orchids are self-compatible, but the pollina- tion mechanisms favour outcrossing (van der Pill and Ddson 1966, Dressler 1990)

The interesting perspective of the Orchidaceae from the floral biology point of view contrasts with the compara- tively small number of investigations carried out in such a big family. Although nectar is considered one of the most important rewards, sugar composition has only been identified in some 110 specks (Daumann 1941, Ftey - Wyssling and Hauserman 1960, Percival 1961, Payne 1965, Easkin and Bliss 1969, Jeffrey ef a!. 1970) and, as tar as we know, sugar proportions have been investigated in four species (Pais and Chaves das Neves 1980, Gottsberger ef al. 1984, Pais et a / . 1986, Freeman et at. 1991). On the other hand, it is assumed the existence of floral nectaries, but little information is available on the structure and location of them (cf. van der Pijl and Dodson 1966, Daumann 1970, Pais 1982, 1987, Dressler 1990, Figueiredo and Pais 1992). These facts indicate how much it remains unexplored in this family. specially considering its great diversification and its remarkable adaptations to all sorts of environments.

The objective of this paper is to analyze different aspects of the reproductive biology of five Argentinean orchids: floral nectar sugar composition, floral nectary localization and anatomy, flower visitors, and breeding system. As these species display different flower types, the comparison of the studied aspects allow to assess their adaptaiive implications. In addition, these terrestrial orchids are the most frequent in the "Chaco Semno" forests from the central province of Cdrdoba (Cocucci 19541, where no epiphflic orchids live.

Around 200 Orchidaceae species grow spontaneously

394 L. Galetto et a/.

in Argentina (Dressier 1990), and almost 70 genera (Correa 1984). Three of the examined genera (Beadlea, Pelexia, Stenorrhynchos) are exclusively American and k l o n g to Spiranthoideae-Cranichideae-Spiranthina (Garay 1982). The remaining genus (Habenaria) is cosmopolitan and belongs to Orchidoideae-Orchideae -Habenariinae (Dress- ler 1990).

Materials and Methods

f he species examined and collection used are listed in TabPel. Voucher specimens are deposited at CORD (hrluseo Botanico de Cordoba).

Nectar was exmcted with capillarl, glass tubes without removing the flowers from the plant and avoiding damage to the nectaries. Two variables were immediately taken: volume (in L) using graduated micropipettes and sugar concentration (%, wt/total wt) with a pocket refractometer.

Nectar drops were placed on Whatman 81 chromatogra- phy paper and quickly dried. Sugar separation was accomplished by gas chromatography. Nectar was lyo- philized and silylated according to Sweeley et a/. (1963). The derivatives were then injected into a Konik KNK 3000-HRGS gas chromatograph equipped with a Spectra- Physics SP 4,290 data integrator, a flame ionization detector, and an OV 101 3% column (2 m long) on Chromosorb G/AW-DMCS mesh 100-120. Nitrogen was the carrier gas (30 rnl/min) and the following temperature program was used: 208 C/1 min, I C/rnin until 215 C, 10 C / rnin until 280 C for 5 min. Carbohydrate standards (Sigma Chem.) were prepared using the same method. Sugar ratio (r) was calculated as sucrose/fructose+ glucose (Baker & Raker 198%) and the hexose ratio (hr) as glucose/fructose.

The effect of nectar removal on flower lifetime was evaluated in 12 flowers of three individuals of Stenorhyn- chos orchioides. The inflorescences were covered -with bridal veil to exclude visitors. Ten flowers of each plant were used as control of the lifespan. Nectar was extract- ed every day until flower fade with capillary glass tubes without removing the flowers from the plant. Two vari- ables were immediately taken: volume (in pl) using

Tablel. Swrce Bf the Orchrdaaceae studied - Beadlea dumei (Schlh.) Gray : 1. i 3 d o k Villa Rivem Indarte ; 2.

Coadoba, V~lla Wmlde.

Habxwia goutlieam Gillies ex Lidley : 1. Cadoba El Dumno ; 2. Cordoba. Tart ; 3 Cb-dokq Cew Blanco.

Habenarta hfemyrnr Kranzl~n : I. Carlm Paz : 2. C&- doba, Yacanto.

Pelexia bwreoensis {Lindl.) Schltr. : 1. Md- Sierra Chica ; 2. Cdrdoba, Asmhinga ; 3. C6rdo"oq Tant~.

Sremhynchos mhtordss (Slhr.) LC. Richard: 1. Cbrdom La Calera; 2. Cbrdoba, El Diquecito; 3. W o b q Vila G~ardino; 4 Cbrdoba, Carlos Paz ; 5. C b W Casa Grande ; 6. Cdr-

graduated micropipettes and sugar concentration (%, w/ w) with a TIC pocket refractometer. The amount of sugar produced was expressed in mg and was Calculated after Bolten et a/. (1979).

Flower Fifespan was registered and compared between pollinated and non-pollinated flowers of P. bonariensis, S. orchioides and Beadlea dutraei without nectar manipula- tions.

Flowers to be sectioned were fixed in FAA. Dehydra- tion was carried out through an ethyl alcohol/xylol series and the flowers were embedded in paraffin. Sections were cut at 8 12pm, mounted serially, and stained with safranin-astral blue (MaScs and Vagas 1961).

To detect the presence of stomata or starch grains, nectaty tissue was cFeared with NaOH (1O0/0 aqueous solution), then washed with acetic acid: water (I : 3), and finally stained with I?-IK aqueous solution (Johansen 1940).

Field observations (diurnal and nocturnal when possible) - were done to register flower visitors. They were captured -

or photographed for their identification. A total of 6,4,8, 12, and 4 observation hours were spent on H, gourlieana, H. hieronymi, B. dutraei, S. orchioides, and P. bonariensis populations, respectively.

Cross- and self-pollinations were pertormed in plants of S. orchioides, B. dutraei, and P. bonariensis following the methodology suggested by Richards (1986). Total fruit set of S. orchioides was compared by a Chi-square test.

Results and Discussion

Flower Orchids display many unsurpassed floral specializa-

tions in their interaction with animal pollinators [Nilsson 1992). In the studied area, the orchids show different flower types according to the genus they belong to.

Habenaria species have yellowish-white flowers. They are fragrant at dawn and are grouped in racemes (Figs. 1 and 3C-D). The labellurn is 3-lobed and has a long tubular spur (Fig. 18-CE. The flowers of Pelexia and -7

Stenorrhynchos are grouped in spikes (Fig. 2B). They exhibit a conspicuous gullet situated at the bottom of the column (Fig. 2A). which is formed by the lateral growth of the column, the base of the lateral sepals and the label- lum. Pelexia bonariensis flowers are greenish-white, while Stenorrhynchos are dark pink or yellow. Beadlea dutraei has small greenish-white flowers grouped in spikes (Fig. 3A-B); they bear a brief gullet which is formed as in Pelexia and Stenorrhynchos. Flower morphology of the studied taxa are, in general, coincident with previous observations on these species (Cocucci 1954, Correa 1955, Garay 1982).

Orchid flowers may not have a fixed lifespan, but rather be subject to pollinator-induced senescence, triggered by deposition or removal of pollen (cf. Proctor and Harder

doba La Serran~ta ; 7. Cbrdoba, La Acequrec~ta ; 8, Tucundn, 1995). Pollinator-induced senescence may allow real- Tapias-Ram location uf resources to developing seeds or decrease All fm Argentina transpirational water loss and energy costs of maintaining

Reproductive Biology in Orchidaceae 395

Flq f Hnhnarra gour/~lr?ni?. A. I~flor~sconce. 6. Apical vlew ot n Ilovef sl?ow~ng the entranwl to the hollow spur. C. Lateral view af a recently open flower lefl and of a bud -right- shm~ng the large spur. D: A hovering sphingrd visiting an inflorescence. B m = 2 cm.

a flower whose usefulness is at an end. The flower life- from the beginning of the anthesis Fable 2). Flower time was estimated for P. bonariensis. 8. dutraei, and S. longevity is then considerably shortened: the flowers orchiaides. The flowers last up to two (in 5. dutraei) or undergo complete senescence 2-4 days after pollination. 'three weeks (P. bonariensis, S, orchioides) if they remain In S, orchioides, flower senescence can also be induced untouched (Table 2). Pollination determines the initiation by nectar removal Fable 2), being the firht report in the of the flower wilting, independently of the days lapsed family. Our results establish a link between nectar,

'7

Fig. 2. Stenmhynchos orchioldes, A: Laterat view of a flower. B: Partial vlew of an inflorescence. C: Partial cross-section of a flower. D: detail of the nectaripherous tissue. Abbreviations: I=labellurn, n= nectary, o= way s = sepal. Bars=l cm for A-6 and 1 mm for C - D.

pollinators, and flower senescence, independently of the plant female and/or male success.

Floral nectary In spite of the different flower morphologies, all the

studied species have nectaries, although not all the members of the family bear them (Daumann 1970). We found lwo different types that coincide witfl the system- atic position of the genera. They are located either in the spur, where nectar accumulates once secreted (Habena- ria, Habenariinae), or in the basal part of the labellurn and nectar accumulates in the gullet (remaining species, all Spiranthinae). Both general localizations .-inferred by nectar accumulation- are frequent in the family (van der

Pijl and Dodson 1966, Daumann 1970, Dressler 1990). According to its topographical position, the nectaries observed are defined by Daurnann (1970) and later by Fahn (1979) as perigonal. After the classification proposed by Srnets (1986,1988), they correspond to "nectaria caduca", because they are located in the petals. No septa! or gynopleural nectaries were detected in Orchidacme as the ones found in the basal stock of Liliatae (van der Pijl and Dodson 1966, Smets 1988).

The nectariferous spur of Habenaria has been reported in some of its 500 species. However, in the literature there are no specifications on the exact place and nature of the secretory tissue (cf. Thien 1969, Oaurnann 1970, Smith and Snow 1976). Anatomically, the hollow spur of

Reproductive Biology in Orchidaceae 397

Fig 3 A B Br;!dsr:a m e t . A. Sever,ll ~nilwescences B. dgapo,~ir~j~on sp. (H~1bct1dae) cartynlg a pollrnia (anow). C D. Habenarta htcfonymn~. C Partla1 ap~cal VIW of an ~nflorescew. 10 Inflaes- cence w~th two flowers me others were remwedj s w i n g its spur. Bars=50 rnm for A and 10 mm for E-D.

the two studied taxa has neither a defined nectariferous starch grains. In addition, the inner epidermis bears one- tissue nor typical secretory cells with particular vascular celled long papillae that are more abundant in its basal bundles (Fig. 4A-8). It is composed of two epidermal 213 of the hollow spur (Fig. 48-0). In bud stage. these layers and a number of parenchymatous isodiametric cells papillae are filled with starch grains (Fig. 4C). Mer , (5-6 in H gwrlieana and 4-5 in H. hieronymi). Some when the flower opens and nectar secretion starts, the scattered cells have raphides (Fig. 4B). The inner epider- papillae show no starch grains at all (Fig. 4D). This mis and some rnesophyll cells closer to it have abundant decrease in the amount of starch during the stage of

L. Galetto ef al.

TaMe 2. E M of pollination and nw3.a removal on flower senescence

Days after treatment Species Treatment

1 Ht 2nd 3rd 4th 5th

Slemj+?ymhos orchiaides HHP (rt= 12Q 120 112 56 4 0

( h m l (n=62): all flm last& 19-22 days

Pelexia h r i e n s i s HP (n=45) 45 41 26 2 0 Control (n=43): all f l m last& 18-23 days

mdlea duhei HP (n=21) 21 17 6 0 0 Control (n=20): all flm lasted 11-13days

One- to f i w x k y ~ f d flowers were treated. Data are number of flowers in anthesis. Abbreviations : HP=hand pollination. NR=raectar removal. Control : untouched flowers.

secretion may be taken as an indicator that the starch accumulated in the secretory cells serves as a source for some of the nectar, as happens in many flowering plants (Fahn 11979). Nectar seems to be exuded from the mor- phologically unchanged papillae, from the cytoplasm by vesicles which fuse with the plasmalemma, because the cuticle is thick and does not break during the process. Stomata were not detected on the inner surface of the hollow spur of these species.

According to these findings and after the classification of Zimmermann (7932)- Habenaria nectaries are nonvas- cularized and nonstructural. Only Frey-Wyssling and HBuserrnann (1960) reported this kind of nectary in the labellum of three species of Orchidaceae, belonging to Cattleya and Cymbidium, which do not have spur. On the other hand, Daumann (1970) reported the presence of structural nonvascularized nectaries in Platanthera bifolia and G ymnadenia conopea, two close relatives of Habena- ria.

The lateral basal parts of the labellum are secretory in the remaining three studied species, belonging to Stenor- rh ynchos, Beadlea, and Pekxia (Spimnthinae). The two glands are located in the adaxial face of the base of the labellum (Figs. 2C and 5). They are composed of the adaxial secretory epidermis and, below it, a number of secretory layers that reach some of the lateral bundles of the labellum (Figs. 20 and 5C-E). These strands have both xylem and phloem. The outer epidermis bears long one-celled papillae and, as the 2-3 layers underneath, is not secretory.

The labellurn of the species of Stenorrhynchos, Bea& lea, and Pelexia has a constriction in its base forming a nectariar chamber, together with the ovary apex and the base of the column (Fig. 5A, F). The base of the two adjacent lateral sepals protect the chamber (Fig. 2C). Epidermal one-celled papillae surround the chamber entrance (Fig. 5F) and are turned to the labellum base. They may contribute to retain the nectar by capillarity. The presence of raphides and starch are common fea- tures in most flower parts, but starch grains were not detected in the neighbouring tissue of the nectaries.

The differences among the species are related to the size of the nectaries and are in accordance with flower size (larger in Stenorrynchos, median in Pelexia. and srnafl in Beadlea); this correlation extends also to the number of nectariferous layers (6-9 in S, orchioides, 4-5 in P. bonari- ensis and B. dutraei) and to the number of bundles (5-8 in S. orchioides, 4 in P. bonariensis, 3 in 5. dutraei). The quantity of nectar secreted is proportional to the size of the nectary and the gullet, being B. dutraei the species which secretes less nectar.

These nectaries are structural and nonvascuTarized after the classification of Zimmermann (1932), as reported for other orchids (Daumann 1970, Pais 1982, Figueiredo and Pais 1992) with a somewhat different structure.

Nectar sugar composition The studied orchids offer nectar as reward. Among

nectar constituents, sugars are the most important to which much attention has been given because they are the basis of the energy reward to flower visitors (Baker and Baker 1983b). A comparison of the sugar composi- tion of the studied orchids is interesting considering both the different flower types and different nectaries found. In addition, according to Baker and Baker (1983b), nectar sugar composition data may give significant clues for recognizing the principal pollinator groups.

Three out of five species analysed (Table3) have sucrose predominant nectar (Beadlea dufraei, Habenaria gourlieana, Pelexia bonariensis) while the remainder two are hexose predominant (H. hferonymi and most samples of Stenorrhynchos orchioides). The overal F mean was calculated for each of the nectar carbohydrates whenever . more than one population was studied. Results indicate that although there are some differences among samples of the same species, sugar proportions are consistent (Table3). The exception is the wide distributed S.or- chioides, where nectar sugar proportions varied among the many studied samples. When analysing the monosaccharides, all the taxa present more glucose than fructose (cf. rh of Table 3): the proportions vary from about the same amount to around double glucose than fructose.

Reproductive Biology in Orchidaceae 399

Frg 4 HaLenaria gourlieana. A: Cross sectlon of the spur. B Part~al 1-1-sect~on oi the spur shornr~w the inner eprdermis with om-celled papillae. C: Superficial view of a bud spur pap~llae showing rts starch content (in black). D: Superficial VIM of an open flower papillae s w i n g no starch Bars=500 ~m for A and 10pm for €3-D.

Stenorrhynchos orchioides produces abundant (> 15 quantities (< 5 ,uL) of a concentrated nectar (ca. 5040, wt/ pL) and concentrated nectar (ca. 40-509'0, wt/wt). wt). Habenaria gourlieana also accumulates copious nectar The qualitative (Frey-Wyssling and Hauserman T960, (a. 20 PL) but in a comparatively low concentration (< Percival 1961, Baskin and Bliss 1969, Jeffrey et a!. 1970) 2G%, wtlwt). The other species studfed produce small and quantitative (Pais and Chaves das Neves 1980, Pais

L Galetto et al.

Fig. 5. Nectaty stmctwe in S t e m h y m h mY&Mes (pop. 2). A: Rower !mi-section. B-E: Swial bansections after planes indicated in A; black& areas=vascular tissue, grayed areas= nectari@- ous tissue FG: frontal and lateral view af the labellurn, respectively: arrow indicates one of the two glands. Bars=? mm, a far 4 F and G, b for the remaining figures.

et a!. 1986, Gottsberger el a/. 1984, Freeman el a/. 1991, detected for some of them. The three Habenaria species this work) nectar studies performed so far showed that studied so far in their quantitative sugar proportions Orchidaceae species have the three most common nectar present a sucrose predominant nectar and they are all sugars present in flowering plants. Raffinose was also pollinated by Sphingidae (Gottsberger et a!. 1984, this

Reproductive Biology in Orchidaceae 40 1

Table 3. Nectar sugar composition In O r c h i h e s p e ~ i e ~

Species Percwrtage of sww (msan-tlSD)

N I Vol. Cwrc. I hr S m Fm3cae Glucose

1-2 59.3k5.3

1.5 61.5k3.5

wmll mean

220 14.7t1.7

Z2Q 13222.4

215 15.521.4

werall mean

<I 53223.5

1-2 48.6f 5.3 werall mean 4 37.3t3.1

2-3 41 .at 2.4

overall mean

10-15 41.5T2-1

41 39.2136

33 51.0*4.1

25 47.521.2

54 41.523.7

6 33.323.6

22 45.924.8

10 51.31k5.9

csl~erall mean

N=$ample number, I=# of -pled plants (if 07 sampled flowen), Vol.=nectar volume per f l w (uU, Cwc=n&r oonzmtmtion (%, wt/wt), r=sugar ratio, hr=kxose ratlo.

work). Table 4 Nectar removal until flower fading in Stenwmynclros Our data show that there is not a correlation between wchhndes

nectar sugar composition and nectary structure: each Flower nectary type has different sugar proportions. lifetime bU Concentration mg of sugm

Nectar removal in Stenorrhynchos orchioides Nectar removal produces flower fading, reducing the

.- flower life span (cf. supra), and simultaneously decreases nectar production (Table 4). One-day-old flowers with- out removal present a mean of 27 p L of nectar with a concentration of 25%. There is a constant decrease in the amount of nectar secreted after each removal. As the first removal initiates the flower senescence (cf. supra), the flowers last until they are 4-days old (Table 2)

. -. - -

1 st day 27.W 14.0 24.8z 6.1 6.7922.6

2nd day 7.0k 8.2 19.22 10.8 O.%* 1 .I 3Kl day 6.8k 7.4 1 4.33+ 6.9 0.882 1.2

4th day 2.W 5.0 8.31: 1.5 0.21 ?L 0.4

and, consequently, nectar secretion decreases dramati- cally reaching a volume of 2.6 p L and 8% of concentra- tion by the end 07 the flower lifetime.

Table 5. N m k r of visitas in Argentinean Orchfdawae

Nmtuml Diurnal

Species Observation N" of visits m e w a t i i N' of vlsfts

~ l o d 4hd Sphingids mod Ihr) Humrningb~rds Bees