Interactions between the asymmetrical flower of Cochliasanthus caracalla (Fabaceae: Papilionoideae) with its visitors

Accepted Manuscript Title: Interactions between the asymmetrical flower of Cochliasanthus caracalla (Fabaceae: Papilionoideae) with its visitors Authors: Angela Virginia Etcheverry, Stefan Vogel PII: DOI: Reference:

S0367-2530(17)33363-7 https://doi.org/10.1016/j.flora.2017.10.006 FLORA 51204

To appear in: Received date: Revised date: Accepted date:

15-5-2016 19-4-2017 25-10-2017

Please cite this article as: Etcheverry, Angela Virginia, Vogel, Stefan, Interactions between the asymmetrical flower of Cochliasanthus caracalla (Fabaceae: Papilionoideae) with its visitors.Flora https://doi.org/10.1016/j.flora.2017.10.006 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Interactions between the asymmetrical flower of Cochliasanthus caracalla (Fabaceae: Papilionoideae) with its visitors

Angela Virginia Etcheverrya, Stefan Vogel b.

a

Cátedra de Botánica, Laboratorio de Biología Reproductiva de Plantas,

Facultad de Ciencias Naturales, Universidad Nacional de Salta, Calle Buenos Aires 177, 4400 Salta, Argentina. b Former

affiliation: Department of Botany and Biodiversity Research, Division of

Structural and Functional Botany, University of Vienna, Rennweg 14, A-1030 Vienna, Austria.

Corresponding author: [email protected]; [email protected]

Highlights  

 

The floral parts present connections which allow mechanical cooperation. The pollen was deposited onto the dorsal side of pollinators. We observed morphological variation in the positioning of the stigma. The flowers produced a high volume of nectar.

Abstract 1

The South American Cochliasanthus caracalla (L.) Trew (=Vigna caracalla (L.) Verdc.) has the most complex flower among Papilionoideae. In this study we describe a) floral functional morphology, b) nectary and nectar traits, and c) floral visitors’ behaviour. The flower presents an intricate connection of flower parts which allows mechanical cooperation. Mean nectar production per flower was 24.0 ± 4.0 µl. Mean sugar concentration and mass were 41.4 ± 2.2 % and 11.7 ±1.8 mg respectively. Despite the floral complexity, Bombus morio, Xylocopa eximia and Centris bicolor triggered the pollination mechanism successfully. The pollen was deposited onto the dorsal side of pollinators. Most visitors are adapted to flower characteristics in their morphology and behavior, although some act as thieves due to some of their morphological features. We observed morphological variation in the positioning of the stigma at the level of individuals. To the authors’ knowledge, the present study is the first one that describes a variability of the stigma positioning in Papilionoideae.

Keywords: asymmetry; complex flowers; floral morphology; nectar; melittophily; pollination mechanism.

1. Introduction Papilionoideae flowers present an extremely elaborate architecture by the synorganisation of all its parts, and their evolution has been predominantly determined by pollinating bees, at least in the initial stages (Leppik 1966, Arroyo 1981; Endress, 1994, 2001). The petals of this flower consist of a vexillum or standard, two wings and a keel, the latter composed by two petals, which 2

enclose rewards and reproductive parts; the typical symmetry is zygomorphic. This type of symmetry “is a successful system because of its potential for efficient precision mechanisms in pollination biology” (Endress, 2001). The pollination mechanisms in Papilionoideae were described since Delpino (1868/9) and four types are recognised, each one related to a special floral architecture: explosive, valvular, pump and brush. Activation of each mechanism requires a certain degree of strength; consequently, only strong and specialized pollinators are physically able to manipulate the flower and access the rewards successfully (Faegri and van der Pijl, 1979; Córdoba and Cocucci, 2011), but some robbers can also access the rewards, despite their lack of the necessary strength to operate the flower mechanism (Amaral-Neto et al., 2015). In the brush type, the stigma and a pollen-loaded stylar brush emerge from the keel, touching the insect ventrally (sternotriby). In this way, pollen donation and reception are concentrated at the same spot. Apart from the general symmetrical type, there is a group of not closely related taxa possessing asymmetrical flowers, among them Phaseoleae (in part), Robinieae and Fabeae (Yeo, 1993). Within the asymmetrical Phaseoleae, especially the subtribe Phaseolinae, some taxa show highly evolved coadaptive combinations between wings and keel, and the prolongation of the keel in a narrow and pipe-like rostrum, which conducts to coiling of the keel (Einsiedel, 1976). Furthermore, these features are related to the occurrence of a stylar brush (Lavin and Delgado, 1990). Depending on the turns of the keel, the place of contact is the base of the proboscis, or the side or the back of the insect’s body (Yeo, 1993). Whilst Goebel (1924) and Troll (1951) asserted that asymmetry did not have any functional significance, other authors tried to find 3

the biological advantages of asymmetry in legume flowers. More recent studies (Brizuela et al., 1993; Westerkamp, 1993; Hoc and Amela, 1998, 1999; Etcheverry et al., 1998, 1999, 2001, 2008; de Souza et al., 2017) suggest that asymmetry could play an important role in economic pollination and interspecific genetic isolation. This is supported by the fact that the pollen is deposited at a small spot (difficult to collect by grooming) on the pollinator’s body. These spots are “safe sites” which are not contaminated by pollen from other species, with another type of symmetry (Endress, 1999). The genus Vigna and relatives (Delgado-Salinas et al. 2011) (ca. 150 sp.), show diversity in floral symmetry: some species have simple, monosymmetric flowers, e.g., V. luteola (Agulló et al. 1993), and others are highly asymmetric. The South American Cochliasanthus caracalla (L.) Trew (=Vigna caracalla (L.) Verdc.) is positioned within the last group. It has probably the most complex flower among Papilionoideae. This fact attracted attention of Lindman (1902) and Troll (1951), who observed 4-5 turns of the keel rostrum. This raises several important questions: 1) How does the pollination mechanism of a flower with so many turns of the keel work? 2) Are the stigma and the style able to move through all these turns to expose and receive pollen? It was hypothesised that this extreme complexity could lead to a total loss of functionality of the flower, delimiting reproduction to obligate autogamy without any insect interaction. Etcheverry et al. (2008) partly answered these questions, reporting that despite its complexity, insect pollination was possible, with a brush mechanism activated by Bombus morio, Centris bicolor, Eufriesea mariana and Xylocopa eximia (Apoideae, Himenoptera).

4

In the present work, we studied: (a) floral functioning morphology, (b) nectar production, and (c) the behaviour of the floral visitors. Regarding floral morphology, we present new aspects that complete the description given in Etcheverry et al. (2008)

2. Materials and methods Cochliasanthus caracalla is a tuberous, winding, perennial plant native in South America (Argentina, Brazil, Bolivia, Colombia, Ecuador, Paraguay, Peru, Uruguay) and Central America, (Guatemala, Nicaragua, Costa Rica, Mexico and Panama) (Tropicos.org Missouri Botanical Garden, 2016). The species has long been cultivated worldwide for its spectacular flowers (Delgado-Salinas et al., 2011). It is a self-compatible species but depends on pollinators to set fruits and seeds (Etcheverry et al., 2008). The field work was carried out during the period from January to March (rainy season) of 2001, 2002 and 2003 at three natural populations in the province of Salta, Argentina, one located in Vaqueros (1200 m. a. s.) and the others located in La Caldera (1400 m. a. s.) and Campo Quijano (1570 m. a. s.). The studied populations are situated in the Yungas, a seasonal rain forest of the Eastern Andes that occurs between 500 and 2500 meters above sea level. More precisely, Vaqueros is part of the Tipa and Pacará District, while La Caldera and Campo Quijano belong to the Montana Rain Forest District (Cabrera 1971).The total extent of the field observations was 67 days. Inflorescence morphology was studied in January 2001. In each population, we collected 60 inflorescences of similar phenological stage from ten plants (six 5

inflorescences per plant). From each inflorescence, total numbers of flowers in bud and in anthesis were determined. Flower functional morphology was analysed in detail in January 2001 on 30 selected individuals, ten from each population. On each plant we marked 10 flower buds of similar length which we observed until floral senescence. Ten flowers from different plants were removed in each sample. In the lab, we used a dissecting microscope with a drawing tube. In case of buds, we opened them, and made histological cuts by hand. Besides, at the Vaqueros population we collected

20

randomly

selected

opened

flowers.

For

these

flowers,

measurements were taken with a caliper of floral length (from the vexillum to the margin of the right wing) and width (a perpendicular line to the first measure), length of wings, keel and depth of the nectar chamber. Based on the observations of the interactions between the flowers and their visitors, we identified the floral structures involved in its functioning. Measurements were also taken from these structures (Figs. 1A, 1B, 1C). The terminology to describe the floral structures and the interacting parts of flower visitors follows AmaralNeto et al. (2015). For histological observations, sections from basal and central parts of opened flowers were cut free hand or with a microtome from material embedded in paraffin. The sections were stained in safranin-fast green and mounted in balsam. For the purposes of this work, the left and right sides of the flower were described with reference to a human observing the flower from the back of the blade of the standard petal. The stigma position around the circumference formed by the last turn of the keel rostrum was measured as an angle for 635 flowers collected from eight 6

individual plants from the Vaqueros population. Flowers were grouped as follows: those with the stigma situated on the left hemi-circumference were designated as left type, while the opposite were designated as right type. To determine the location of stomata on the nectary, dissected flowers that were previously fixed in FAA (formaldehydeacetic acid-alcohol) solution, were cleared with NaOH (10%) and stained with a I2-IK solution (Galetto et al., 2000) in January 2003. Nectar was collected from 60 flowers from 12 individuals at Vaqueros population, which were bagged with voile bags (five flowers per plant) before anthesis. Nectar samples were collected around 13.00 hours, just before the time in which the resorption phase in nectar secretion begins (Etcheverry, 2006). Nectar was extracted with microcapillary tubes without removing the flowers from the plant and avoiding damage to nectaries. Immediately after nectar extraction, volume and sugar concentration were estimated. Volume (µl) was estimated using graduated micropipettes, whereas sugar concentration was determined with a pocket refractometer. The amount of sugar produced was expressed in milligrams (Dafni, 1992). Floral visitor’s behavior at flowers was observed during all flowering seasons from 2001 to 2003. We collected all visitors at various dates during this period. We used photography and video-recording. The duration at the Vaqueros population of each visit was recorded. Voucher specimens are deposited in the Museo de la Facultad de Ciencias Naturales de la Universidad Nacional de Salta (MCNS). The results were analysed using SYSTAT (1992) for Windows. Floral traits were compared by one-way analysis of variance (ANOVA) and with chi-square 7

tests. For comparison of visit duration, we performed a t test. Data are presented as mean ± standard deviation (S.D.). 3. Results Cochliasanthus caracalla bloomed from January to March in the studied populations (Fig. 2A). Length and width of flowers were 48.72 ± 5.11 mm and 34.04 ± 2.81 mm, respectively. The flowers are arranged in axillary inflorescences

(“pseudoracemes”

sensu

Tucker,

1987)

with

acropetal

maturation. It is a compound raceme of several (range 4 - 21) modified simple racemes. Each simple raceme has a contracted rachis with two (rarely three) flowers; apparently the other floral buds degenerate in early stages of development and transform into an extra-floral nectary with a swollen appearance. The total number of flowers produced per inflorescence varies between 8 and 42, while the mean values do not differ significantly among populations (Vaqueros, 18.65 ± 1.03; La Caldera, 20.36 ± 1.29; Campo Quijano, 21.03 ± 0.91; F = 0.899, d.f. = 2,135, P = 0.49). The number of flowers opened per day and per inflorescence is one or rarely two (85.7 % and 14.3%, respectively), in this case from the same node or consecutive nodes. In the peak of flowering, floral density varied between 12 and 22 opened flowers per square metre. Pedicel length ranged from 8.59 to 11.39 mm (mean 9.68 ± 2.82). We observed pedicels to be flexible, therefore, flowers hung down loosely from the inflorescence rachis. As a consequence of this flexibility, the right wing petal (used as landing area) adopted a horizontal position independently of the orientation of the rachis (Figs. 2B, 2C).

8

3.1. Floral functional morphology The morphology of the flowers from all populations was generally similar, so we will treat them as uniform in the following description, except where they differ. The strong floral asymmetry of C. caracalla results of growth processes occurring late in ontogeny. Young buds exhibit zygomorphic symmetry, and, in profile, are incurved. The standard or vexillum is folded along the midvein and completely covers the other developing petals. Some days before anthesis, the keel begins to perform a curvature to the right that finally reaches a value of ca. 720°. This curvature is accompanied by the staminal tube, wings and standard. As a result, the flower bud becomes totally asymmetric, with a dextrorotatory torsion. When the flower opens by separation of the standard margins from the proximal end, one additional turn (beyond 720º) is performed by the keel. Registering the sequence of movements, we noted that once the keel is released from the standard, it is retracted with some force, performing rapidly the additional turns, resulting in a total number of up to five turns. As a consequence, the keel adopts a more or less hidden position under the left wing, which comes to lie in the upper side of the flower. The calyx is bilabiate and succulent, with a tubular form and five lobes in its distal part. It constitutes a strong structure which encloses tightly the bases of petals, stamens and carpel. The lowest lobe is slightly longer than the two lateral ones, and its sinuses interlock with the margins of the base of the standard blade and the claws of the wings (Fig. 3A, arrows). This feature allows that the standard remains fixed during insect visits. The two upper lobes are slightly dented (i.e., their sinuses are almost absent) and they are facing the standard blade. There is a bulging in the adaxial side of the calyx which is 9

indicative of nectar for visitors, as Westerkamp (1997) suggested for many Papilionoideae. All petals are separated into claw and blade presenting a sturdy consistency. Anatomically, some isolated short sclereids were visible, probably related to a supporting function. The standard is emarginated and bent backwards. The distal portion of the blade is curled, describing a turn to the right, adopting a curious figure. In the proximal portion, it presents a yellow marking which apparently functions as a visual guide to the nectar, and two prominent appendages on inner face above region of nectary entrance. The claw of the vexillum is wide, stiff, and thickened. If one tries to reach the nectar chamber by pulling up the standard by force, a rupture occurs at the base of its blade. In comparison with the other petals, the vexillum has the shortest claw (0.55 ± 0.14 cm). The unequal wings are panduriform (Fig. 3B). They serve as main attractants, exposing their coloured sides through a torsion which positions them in a transversal orientation. Only the right wing, which comes to lie at the lower side of the flower, functions as a landing area for visitors, with its morphological outer side oriented upwards. It is noteworthy that this wing petal is 2 mm longer than the right one (Table 1), and it presents a less pronounced curvature. The left wing adopts the vexillum location within the flower, and covers the keel with its concave surface .The claws of both wings are long, with a similar length (1.48 ± 0.10-1.47 ± 0.13 cm, right and left wing, respectively), slightly curved, and form an angle of ca. 130° with its blade. A fold is visible between claw and blade, due to the proximity of the blade to its claw, which permits downward movements during insect visits. Proximally, the wing claws 10

are adnated to the staminal tube (Fig. 3C). The wings are unilaterally auriculate; the auricle of the right wing covers the margin of the auricle of the left wing. Both petals have on their outer faces transversal foldings or sculpturings on the proximal half of their blades which are oriented towards the nectar entrance (Fig. 3B, arrow). The sculpturings can be described applying Stirton’s (1981) classification for wing morphology as follows: with a lamellatepattern, located in the upper basal and central area of the wing, crest cells striate, through cells flattish and no striate. There were differences between the sculpturings area of the right and left wing petals (3.99 ± 0.16 vs. 30.6 ± 0.12 cm2, respectively). The keel is the part of the corolla most strongly transformed by asymmetry, and encloses androecium and gynoecium. The distal part is coiled and forms several turns (Fig. 3E). The keel petals have long and curved claws, with similar length (1.29 ± 0.1 and 1.25 ± 0.1cm, respectively), which at their bases are adnated to the staminal tube (Fig. 3B). The basal parts of androecium and gynoecium (which form a central column) run inside, close to the abaxial side. Distally, the keel petals narrow abruptly, forming a channel 1.20-1.50 mm in diameter and 80-90 mm long (rostrum), which encloses the style and the distal part of androecium and forms revolutions, which are always dextrorotatory. The last revolution, which encloses the stylar brush and the free, distal part of the filaments, is wider (2.50-3.00 mm). In all populations full revolutions were observed (4 and 5) but also intermediates occur (see Supplemental data). So, the tip of the keel rostrum and, consequently, the stigma had different positions (see below), but the complete rostrum always occupied the upper side in the floral space, close to the nectar guide. The spiral of the rostrum has a diameter 11

of 7.69 to 11.67 mm. The keel petals are joined along their lower margins, from above the claws to the apex by genuine post-genital fusion. On their upper edges, the keel petals show rows of unicellular trichomes (Fig. 3F), which are very small in the proximal part, while in the distal part (last turn of the keel) are longer and interdigitated (“capillinection”; Sigmond, 1930). These trichomes are interpenetrating like a zipper, closing the keel except at the very tip, leaving a small opening (Fig. 3E, arrow). The bases of the wing blades and the keel petals are connected, resulting in a “wings/keel complex” (Westerkamp, 1997) (Fig. 3C, 3G). This connection is realized in two ways: First, protuberances of the external face of the keel, (“vaultings” in the sense of Westerkamp, 1997), grip into fitting depressions of the internal faces of the wings in the manner of a “press-stud”, the right of which is more pronounced than the left (see Table 1). This difference plays a supportive role, considering that the right wing acts as a landing area for visitors. Also, this “press-stud” holds the wing and keel petals together during the visits. This characteristic is very important for flower mechanics, since the most force is applied by the bee only to the right wing (with his hind legs) and keel (with its back); as a consequence all flower parts move in synchrony. Secondly, wing and keel petals are connected epidermically at the same point by means of interlocking cuticular indentations, distributed on a circular area in the right wing and on a triangular, smaller area in the left one (Fig. 3B, shaded area). The fusion is so strong that it is impossible to detach the petals from each other without tearing out keel tissue. The androecium is typically diadelphous, with free vexillary stamen (9+1pattern). Longer and shorter filaments alternate, but all anthers are similar in 12

size and shape. The widened basal part of the staminal tube forms a very spacious chamber, where the secreted nectar is collected. The filament of the free stamen divides the opening to this chamber in two holes (fenestrae), which allow pollinators’ tongues to reach the nectar. The free parts of the filaments are thread shaped with strongly curled distal parts (Fig. 3D). During the visits, only the stylar brush and the stigma are exposed, while the stamens are retracted due to the flexibility of filaments. The gynoecium of C. caracalla is borne on a short gynophore. The ovary (2 2.5 cm in length) bears glandular and no-glandular trichomes, and runs almost straight, while the style has an upward curve, describing distally the same number of revolutions as the keel. Totally expanded, it reaches a length of 8.5 9.5 cm, being 5 - 15 mm longer than the keel. This difference is significant in the exposing mechanism of the style from the keel. The distal part of it (2 - 2.5 cm) is stiff and thickened, according to its role in applying pollen to visitors. This structure is termed entasis and was cited for six tribes of Papilionoideae, including Phaseoleae (Shivana and Owens, 1989). The stigma is globose, terminal, but presents a lateral position at the same side as the stylar brush, due to a rotation of the style at its end. This position is ergonomically favorable because it allows a closer contact with the insect body during pollination. The pollen is presented to pollinators by means of the stylar brush. The brush is composed by several rows of trichomes, slightly curved towards the stigma (Fig. 3H) that can brush pollen upward.

13

In the 635 flowers from the Vaqueros population, six different positions of the stigma were recorded: 90º; 135º; 180º; 225º; 270º and 315º. The first three positions were considered as to be right type, while the remaining positions were considered as to be left type. The number of flowers of each type was almost similar, and did not differ from a 1:1 ratio (P > 0.05, see Supplemental data). As a consequence of this variability, the stylar brush of visited flowers applied pollen on different arcs of a circle on the visitors’ pronotum (Fig. 4A) and the stigma made contact with different points of this circular spot of pollen (Fig. 4B).

3.2. Nectary and nectar traits There is a discoid nectary located at the base of the gynophore, within the staminal tube. Its margin shows ten crenulations and open stomata. Nectar secretion starts in the bud and continues for about 6-8 h after flower opening. The secreted nectar is collected between the nectary and the staminal tube. Mean nectar production per flower was 24.0 ± 4.0 µl. Mean sugar concentration and mass were 41.4 ± 2.2 % and 11.7 ±1.8 mg respectively. Independently of the position adopted by the flower, the nectar always remained in the chamber, and never flew out. Nectar foraging ants in extrafloral nectaries located in the nodes of the inflorescence were observed in all populations (Fig. 4C) during flowering and fruit formation.

14

3.3. Pollination mechanism and floral visitors’ behaviour The complex pollination mechanism of C. caracalla was legitimately worked by females of Bombus morio Swederus, Xylocopa eximia Pérez and Centris bicolor Lepeletier (Apidae) in the studied populations. Bees land on the flower’s right wing, and proceeding to the center of the flower, they introduce the head and thorax under the spiral of the keel (Fig. 4A), i.e., the pollination chamber (Fig. 1C). Then, they introduce their proboscis between the base of standard and the wing-keel complex (which are in contact), following the nectar guide mark. This insertion is facilitated by an initial pressing-down of the right wing, which as a consequence, opens the tongue channel between the wing auricles. Some millimeters deeper (ca. 7 mm), pollinators introduce their tongue through the central channel of the standard, which is closed laterally by two protuberances, and adaxially by the protuberance of the free stamen. To execute this action and consequently reach the nectar chamber, pollinators have to lower the right wing (mainly with their hind legs), while the other parts of the flower are pulled upwards with its back (Fig. 4A). This effort results in the appearance of the stigma and the stylar brush. This is consequence of a relative movement: the central column formed by the androecium and gynoecium is rigid and keeps its place in floral space, while the keel and the right wing are pulled down ca. 6 mm (measured at the base of the keel), thereby exposing the style, which receives and applies pollen, respectively. The cited visitors have the size and strength to behave as primary pollinators, and their morphologies are adapted to C. caracalla flowers (see Table 2).

15

We observed small drops of lipids on the epidermis of the keel (positive reaction with Sudan IV), that could facilitate the described movements of the style within the tube formed by the keel. The stigma emerges first and contacts the pronotum of the insect and eventually any pollen, that may have been deposited. Then, the stylar brush slides over the same part during its run of a ½ or ¾ turn, applying pollen (arc of 7-13 mm). The distal part of the androecium, because of its flexibility (see above), remains enclosed within the rostrum during visits. The floral mechanism can be activated several times. At the end of each visit, all floral parts return to the original position immediately, except for the style, which needs 1-20 min to withdraws within the turns of the keel. Apis mellifera (4D) and one species of Meliponini take advantage of this delay, collecting the remaining pollen from the stylar brush. These visitors were unable to reach the nectar and to trigger the pollination mechanism of C. caracalla, and are characterized by smaller body heights and shorter mouthparts compared with the height of the pollination chamber and the deep of the alignment channel. Besides, some species of Lepidoptera acted as nectar thieves without trigger the pollination mechanism. We found significant differences in duration of visits (in seconds), comparing B. morio workers and X. eximia: 18.72 ± 1.63 s. vs. 37.44 ± 7.77 s., respectively; t = -2.35, g.l.= 87, P = 0,044. It is noteworthy that flowers visited just before were avoided by approaching insects, probably due to the odour left behind by the previous visitor. 4. Discussion

16

Cochliasanthus caracalla certainly is the most complex flower among Papiloinoideae. The highly elaborated floral morphology includes traits to attract pollinators and also represents mechanical barriers that delimit the access to nectar. The flowers are the largest among the American Phaseolinae and their unique features are their pronounced asymmetry, and the high number of revolutions of the keel. Despite their complexity, the breeding system is xenogamous (Etcheverry et al., 2008) and the brush mechanism works perfectly, released by pollinators capable of exerting the considerable strength necessary to manage such a strange construction. The observed pollinators met the expectations based on floral syndromes (Vogel, 1954), and applying Ollerton’s et al. (2007) classification, C. caracalla is a phenotypic specialist, and has a functionally and ecologically specialized pollination system. Fabaceae are a morphologically variable group that exhibit a diverse range of pollination systems, the highest expression of which is found in the highly, specialized papilionate flowers (Arroyo, 1981; Yeo, 1993). Papilionoideae appear to represent lineages that are monophyletic, which are sisters of Caesalpinioideae (LPWG, 2017). The early diverging lineages have flowers that look more like those of a wild rose, and many modifications exist, from the reduced flowers of Amorpha (tribe Amorpheae, with only one petal), to spectacular orchid-like flowers of species that share only a more distant ancestry with these models (Doyle, 2003). In contrast to Mimosoideae and Cesalpinoideae, the functions of individual petals in Papilionoideae are clearly defined. Thus, in a typical flower the enlarged, dorsal vexillum is associated with attraction, the ventral petals (keel) protect the reproductive organs, and the 17

lateral wings, together with the keel act as landing area to pollinators. Nevertheless, these functions and positions could be interchanged, as in several genera of Phaseoleae. For example, in Macroptilium the wing petals are large and prominent, the left-hand wing petal twists upward to assume the function of the standard petal, whereas the standard is reduced in size and coloration and positions itself as a support structure of the wing petal that takes its place (Delgado-Salinas et al., 2011). The brush pollination mechanism has arisen independently in six tribes, specifically in the following taxa: 1) Crotalaria (Crotalarieae), 2) Coluteinae (Galegeae), 3) Tephrosia subgenus Barbistyla (Millettieae), 4) Adenodolichos (Phaseoleae subtribe Cajaninae), 5) Clitoria (Phaseoleae subtribe Clitoriinae), 6) Phaseolinae (Phaseoleae), 7) Robinieae, and 8) Fabeae (Vicieae) (Delpino, 1868/9; Leppik, 1966; Arroyo, 1981; Polhill, 1976; Lavin and Delgado, 1990;Yeo, 1993; Westerkamp, 1997; Galloni, 2007). It is noteworthy that many species of Papilionoideae with brush mechanism have asymmetric flowers (i.e., a loss of bilateral symmetry). The cited asymmetry may be notable only in some part of the flower, for example, the style and pollen brush (e.g., Coursetia, tribe Robinieae, Lavin, 1988, and Adenodolichos, Phaseoleae, Lavin and Delgado, 1990). Various species of Vicieae (e.g., Lathyrus odoratus, Lavin and Delgado, 1990; L. latifolius, Westerkamp, 1992) have a slightly twisted keel, and also Crotalaria spp. (Le Roux and Van Wyk, 2012). Incurved, or spiralized keels appear to have evolved several times in different taxa of the subfamily, e.g., Ancistrotropis, Helicotropis, Leptospron, Oxyrhynchus, Phaseolus, Sigmoidotropis, Wajira (Phaseoleae, Thulin, 2004; Delgado-Salinas et al., 2011); Bolusia (Crotalarieae, Van Wyk et 18

al., 2010, Le Roux and Van Wyk, 2012) and in the Lebeckia pauciflora group (Le Roux and Van Wyk, 2012). When the asymmetry involves the entire flower, the wings adopt a more or less horizontal position, and one of them serves as the landing area. This characteristic was observed in Vigna vexillata (Hedström and Thulin, 1986); Macropitlium bracteatum (Brizuela et al., 1993); M. lathyroides, (Etcheverry et al., 1998); M. erythroloma (Etcheverry et al., 1999); Phaseolus vulgaris var. aborigineus (Hoc and Amela, 1999); M. panduratum (Etcheverry et al., 2001); Leptospron adenanthum (=Vigna adenantha) and Condylostylis candida (=Vigna candida) (Hoc and Ojeda, 2013), and Vigna longifolia (de Souza et al., 2017). Cochliasanthus caracalla could be positioned in this group. Specifically, the morphology of the keel petals (i.e., twisted, incurved or coiled) may be related to which body part of the bee touches the stigma and the stylar brush, thus suggesting a functional significance for asymmetry. In the twisted type, the style brushes the side of the abdomen of a visiting bee (e.g., Crotalaria spp., (Le Roux and Van Wyk, 2012; Amaral-Neto et al., 2015). In the other types, pollen deposition site can vary. Flowers possessing a long, incurved or coiled keel were related to nototribic pollination, and apparently have evolved more than once along different evolutionary lines within Vigna. An independent, apparently nototribic line in the Old World is subg. Macrorhynchus (now included in Wajira, Thulin et al., 2004), and in subg. Plectrotropis (e.g., V. vexillata, Hedström and Thulin, 1986). Nototribic or pleurotribic lines were cited in the subg. Lasiospron (nototribic; e.g. V. longifolia (de Souza et al., 2017), and in the American Phaseolineae (nototribic; e.g., Macroptilium bracteatum, Brizuela et al., 1993; Phaselus vulgaris var. aborigineus, Hoc and Amela, 1999; 19

C.

caracalla,

Etcheverry

et

al.,

2008,

Leptospron

adenanthum

and

Condylostylis candida, Hoc and Ojeda, 2013; pleurotribic; e.g., Macropitlium erythroloma, Etcheverry et al., 1999). Besides, sternotribic pollination could be present in this group, (e.g. sternotribic; e.g., V. minima, Gopinathan and Babu, 1987; Macroptilium atropurpureum and M. longipedunculatum, Torres-Colín, 2006). The observed variation in pollen placement is probably related to a mechanism for minimizing competitive interactions in synchronopatric plant species with similar pollinators (e.g. Etcheverry et al., 2008; de Souza et al., 2017), and appear to be subjected to selective pressure towards pollen placement in safe sites on the bee’s body, as Westerkamp and ClaßenBockhoff (2007) have suggested for complex keel flowers as well as for bilabiate flowers. As previously stated, pollen is presented secondarily on a stylar brush; this observation coincides with Lindman (1902) in a Brazilian population, although movements of the style during the pollination process were different. We observed that the style moves over the body and the insect was not hugged by it. Leppik (1966) and Arroyo (1981) stated that more efficient pollen transfer systems appear to be a major trend in the evolution of the Papilionoideae. Thus, some tribes can be expected to have a more efficient pollination mechanism; such as Phaseoleae, which mainly display the brush mechanism (Lavin and Delgado, 1990). In a comparative study of P/O ratios in Papilionoideae in Argentina, the species pertaining to the cited tribe Phaseoleae presented the lowest P/O ratios (Macroptilium spp., Phaseolus vulgaris var. aborigineus and C. caracalla, Etcheverry et al., 2012), confirming, at least for these species, this

20

trend. As a consequence, pollen is not a resource available to pollinators and the flowers behave like nectar flowers (Etcheverry et al, 2012). In the studied population of C. caracalla, the revolutions of the keel varied in number, but this variability was tolerated by the flowers without loosing their ability to deliver and receive pollen. Also, Einsiedel (1976) observed a variation between 1.5 to 2.5 turns in a Brazilian population of C. caracalla. For many Phaseolus species, Lackey (1983) reported variability in the number of turns (2.75-2.9

in

P.

ambylosepalus,

P.

galactioides,

P.

nelsonii

and

P.

xanthotrichos). In one Argentinean population of Vigna hookeri, Gamba (2001) reported a variation of 4-5 turns. In the present study, and, as a direct consequence of the observed variability, the stigma and the stylar brush differ in position as well. This implies different sites of stigma contact with the pollinator’s body (around a circular area on the pronotum) and could be related to a strategy of favoring cross-pollination in this self-compatible species. In relation to the last aspect, similar conclusions were obtained in enantiostylous species, as Chamaecrista (e.g. de Almeida et al., 2013) and Senna (Caesalpinioideae) (Westerkamp 2004; Laporta 2005), but in these cases, only two floral morphs are described. To the authors’ knowledge, the present study is the first one that describes a variability of the stigma and the stylar brush’s positioning in Papilionoideae. The sense of asymmetry of the flower was always right-handed, and this feature consistently marks all of the American genera of Phaseolinae (DelgadoSalinas et al., 2011), for example, Macroptilium (e.g., Barbosa-Fevereiro, 1986/7; Etcheverry et al., 1998, 1999, 2001), Phaseolus (e.g. Lackey, 1983; Hoc and Amela, 1998) and Leptospron adenanthum,(e.g., Castro and Agulló, 21

1998). Within the group, Condylostylis candida is the exception (Hoc and Ojeda, 2013). This floral morphology sets this clade apart from the rest of the primarily Old World Vigna s.s., which have floral morphologies that are either bilaterally symmetric or with left-handed asymmetries (e.g., V. vexillata, Hedström and Thulin, 1986). Endress (2001) hypothesized that there is a selective pressure to produce only one floral morph of asymmetry in plants with this characteristic. He pointed out that flowers with enclosed reproductive organs are more difficult to handle by pollinators than those without this character, but if they are always worked from the same direction, their disadvantage may be reduced due to pollination taking less time. Besides, bees are able to retain a limited amount of manipulation skills needed to obtain rewards when they forage between plants with different floral structures (e.g., Menzel, 2001; Amaral–Neto, 2015). Several features of C. caracalla could enhance the support of large and heavy pollinators, e.g., the stiffness and general shape of sepals and petals, connections between different structures, esclerids in the mesophyll of the petals and sculpturings on the wings. In a comparative study of the floral morphology of Papilionoideae in Argentina, it was ascertained that the sculpturings of the asymmetrical flowers of Macroptilium spp., were deeper in the right wing, which serves as landing area for pollinators (Alemán, 2014). In the present study, this wing petal presented a longer blade, with more and deeper sculpturings than the other wing. Besides, the interlocking zone between the right wing and keel petals was more pronounced than the opposite side, due to its supportive role. All these observations in C. caracalla, reinforce the idea that its floral architecture is related to pollinators.

22

Considering the combined occurrence of certain floral features in Papilionoideae, Lavin and Delgado (1990) concluded that many species with pollen brushes have asymmetric flowers, distally thickened styles, basal fenestrae on the staminal tube, rostrate and keel petals fused with trichomes along the upper margins except at the extreme tip. These interlocking trichomes serve to bind the two keel petals along their upper edges and ensure that the keel protects the reproductive parts and functions as a single unit during pollination (Le Roux and Van Wyk, 2012). Thus, traits involved in pollen transfer (deposition and removal) are expected to be integrated into a module due to the selective pressures exerted by pollinators (Specht and Bartlett, 2009; Gómez et al., 2014; Castellanos et al., 2004). However, it is possible that traits from the same or different whorls cooperate in a particular function, thus promoting compartmentalization and functional integration (Herrera et al., 2002; Córdoba and Cocucci, 2011). Considering flowers with forcible pollination mechanisms, the traits associated with these mechanisms would need to be intercorrelated to ensure flower functionality. Córdoba and Cocucci (2015) tested this hypothesis in keel flowers of Collaea argentina (Papilionoideae) and identified a module that involves traits from the keel and wings being directly involved in the mechanism of keel flowers. Besides, no trait from the flag was part of this module, probably because the flag is usually associated with attractiveness, and in only a few Papilionoideae genera does the flag function as a landing area for visitors (e.g., Centrosema and Clitoria, Faegri and van der Pijl ,1979; Amaral-Neto et al., 2015). With respect to the visitors of C. caracalla, Lindman (op. cit.) reported the same genera as we do (Bombus and Xylocopa), although he did not identify 23

the species. We observed a third pollinator, Centris bicolor, visiting legitimally the flowers of C. caracalla. In V. vexillata (Hedström and Thulin, op.cit.) also large bees were observed (X. gualanensis). Recently, de Souza et al. (2017) reported B. morio, C. decolorata, C. tarsata, Megachile susurrans, X. brasilianorum and X. frontalis as pollinators of V. longifolia in a Brazilian population. All these observations confirm the hypothesis that large bees pollinate asymmetric flowers (Endress, 1999). Taking into account nectar production, we observed that flowers of C. caracalla produced a higher volume of more concentrated nectar than other Phaseoleae, e.g., Phaseolus coccineus (Búrquez, 1979) and P. augusti (Hoc and Amela García, 1998). According to Cruden et al. (1983), species pollinated by Hymenoptera, as C. caracalla, produce between 6.5-8 µl of nectar per flower, with a concentration of 29-35 % and 5-6 mg of sugar. The results obtained in this study exceed the referred values. This could be related to the adaptation of this flower morphology to large bees, because these bees demand more nectar with higher sugar concentration (e.g. Heinrich, 1975), and it was also demonstrated in artificial flowers that bumblebees prefer to forage on floral types associated with more concentrated nectar (Cnanni et al., 2006). However, a study of 26 plant species in southern South America did not show a clear-cut association between nectar concentration and pollinators (Chalcoff et al., 2006). Faegri and van der Pijl (1979) suggested that visits to C. caracalla flowers could last a half minute, due to the high amount of produced nectar. Our observations agree with these authors. As well as the greater reward, long visits could be related to handling of this complex flower. Handling times can differ by 24

a factor of 10 between simple and complex flowers (Laverty, 1994; Ohashi, 2002). However, it is often advantageous for bees to forage on complex flowers, because they are exploited by fewer individuals and thus may contain more reward than simple flowers (Heinrich, 1975). It is noteworthy that flowers that had been visited recently were avoided by approaching insects, probably due to the odour left behind by the previous visitor, as Pijl (1954) and Hedstrom and Thulin (1986) have suggested for Xylocopa spp. in V. vexillata. This behaviour allows animals to avoid time consuming inspections of unprofitable food sources. Saleh et al. (2006) demonstrated that bees appear to be minimizing the energy and time they spend probing flowers by selectively rejecting marked flowers. In C. caracalla, floral nectar is a renewing resource, but the time it takes a flower to refill with nectar exceeds the duration of a foraging bout or visits within a patch; therefore, bees should avoid returning to flowers that they have recently visited (Etcheverry, 2006). Considering ant feeding on extrafloral nectaries, there is some evidence that ant presence has a positive and significant effect on the plant, reflected in a decrease in herbivory and an increase in the production of unripe fruit, e.g., Koptur et al., 1998. In the case of C. caracalla, Ojeda et al. (2008) demonstrated that fruit production was significantly higher in plants that were exposed to ants when compared to those that weren’t, and the same result was reported for V. adenantha (Ojeda and Amela, 2010). Specialized, complex flowers may have evolved to achieve two primary, complementary, functions: first, to attract and permit access to efficient

25

pollinators; and secondly, to protect the reproductive organs and rewards against unwanted visitors (including robbers, herbivores and predators) and weather conditions through mechanical barriers (e.g., Brantjes, 1981; SánchezLafuente, 2007; but see Waser et al., 1996). Apart from the Papilionoid family, complex floral morphologies exist in many independent families, e.g., Labiatae (e.g., Ohashi, 2002), Schrophulariaceae (Sánchez-Lafuente, 2007) and Polygalaceae (Westerkamp and Weber, 1999). For keel flowers and inverted keel-flowers, the main mechanical barrier is the wings/keel complex (Westerkamp, 1997; Amaral-Neto, 2015). In the case of C. caracalla, considerable force is required to lower the wings/keel complex, which is evident from the lesions that legitimate visitors leave with their claws on the right wing. But, also butterflies can reach the nectar although they are incapable of activate the flower mechanism, so strength and size is needed for the pollinator. Córdoba and Cocucci (2011) measured the strength exerted by bees and found that body size is positively correlated to strength. To consider which visitors would operate properly, keel-flowers or inverted keel-flowers, Amaral-Neto et al. (2015) suggest analyzing the following features (1) body size (as a measure of strength); (2) length of their mouthparts; and (3) the ability to position their mouthparts forward, so becoming functionally prognathous. To our knowledge, the bee flowers that are the hardest to trigger are those of Lathyrus pubescens (Papilionoideae: Fabaceae), which requires a force of 0.36 N (Córdoba and Cocucci, 2017), while L. latifolius, requires a force of 0.1 N (Westerkamp, 1993). Considering a gullet-blossom as Phlomis fruticosa (Lamiaceae), the measured force was 0.068 N (Brantjes, 1981). The forces that pollinators can exert on flowers are greater than the forces that correspond to their body mass

26

(Westerkamp, 1997). Recently, Códoba and Cocucci (2017) reported that pollinator assemblage compositions were associated with the force needed to open the keel in five Argentinean co-occurring legume species. Up until now, several works have provided detailed accounts of the floral mechanism of the typical papilionate flower, but little is known about the functioning of the asymmetrical ones, considered as “grotesque” derivations of the normal type, (Faegri and van der Pijl, 1979). Inverted keel flowers are another case that has received little attention (Amaral-Neto et al., 2015). Asymmetry is not uncommon, especially in groups with highly elaborated and otherwise

monosymmetric

flowers

(e.g.,

Lamiales,

Orchidaceae

and

Zingiberales; Endress, 1999). Moreover, in Caesalpinioideae flowers, despite lack of a keel, in species of Chamaecrista and Senna asymmetry is also present, given that the gynoecium and stamens are curved to one side. Complex leguminous flowers include several kinds of asymmetry in different organs of different floral whorls, and unrelated species have superficially similar asymmetric flowers. Detailed knowledge on the diverse floral morphology within a functional context with emphasis on morphological and anatomical features related to pollination mechanisms and breeding systems is essential for hypotheses on floral evolution in the group. Aknowledgements This study is part of the Ph. D. Thesis of AVE (2000-2005), supervised by S. Vogel, and was supported by the “Consejo de Investigación de la Universidad Nacional de Salta”. We thank three anonymous reviewers for important and constructive suggestions. We are grateful to Arturo Roig-Alsina, Museo de 27

Ciencias Naturales “Bernardino Rivadavia”, for identification of the bees, and to Trinidad Figueroa, Paula Narváez, Antonella Ducci and Mercedes Alemán for field and lab assistance. Diego López Spahr helped with the figures. Maria Schulze and Hugo Lesser improved the English style.

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Fig. 1. Schematic views showing measured parts of Cochliasanthus caracalla. A. Lateral view of the flower. B. Lower view, vexillum removed. C. Frontal view. ac: depth of alignment channel; rp: right protuberance of the wings/keel complex; lp: left protuberance of the wings/keel complex; pc: height of pollination chamber; la: length of landing area; fl: floral length; fw: floral width.

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Fig. 2. Cochliasanthus caracalla of the Vaqueros population, Salta province, Argentina. A. View of the plant, showing their climbing habit. B. Inflorescence, with one flower in anthesis, with the apex of the rachis (asterisk) upwards oriented (arrow); rw, rigtht wing. C. Same inflorescence, but in an inverted position. Note the position of the rigth wing (landing area) independent of the orientation of the rachis, as a consequence of the flexibility of the pedicel.

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Fig. 3. Floral morphology of C. caracalla. A. Lower view of the basal part of the flower, showing articulation between standard and the claws of the wings with the lower lobes of the calyx, (arrows). B. Right wing, outer face, showing transversal sculpturing on the proximal half of its blade (arrow). The zone of adnation with the corresponding keel petal (asterisk) is shaded. C. Lateral view of the wings/keel complex, calyx and standard removed; note the protuberance of free stamen (arrow); asterisk, claw of the keel petal. D. Distal part of the stamens, stigma (arrow) and style, rostrum removed. Note stigma-anthers separation, and the curled, flexible distal part of the filaments. E. Rostrum of the keel, with four revolutions, and the open tip (arrow) (2x magnification). F. Right keel petal, showing the marginal trichomes (arrow) that interlock with corresponding ones from the other keel petal (6x magnification). G. Transverse section of the flower at the level of the adnation between keel (k) and wings (w); sc, staminal column; g, gynoecium ; arrow, right protuberance (7x magnification). H. Stigma and stylar brush (asterisk), charged with pollen grains (4x magnification). Scale bars: Fig. A= 1mm, Fig. B= 3.6 mm, Fig. C= 4.5 mm, Fig. D= 2 mm.

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Fig. 4. Floral visitors of C. caracalla. A. Bombus morio worker, visiting a lefttype flower of C. caracalla (body length approx. 2 cm). Note the stylar brush applying pollen on the visitor’s pronotum (arrow). B. Spot of C. caracalla pollen on the right area of the pronotum of a B. morio worker. C. Nectar foraging ants in extrafloral nectaries located at the nodes of the inflorescence. D. Apis mellifera acting as a pollen thief (body length approx. 1 cm).

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Table 1. Measurements in centimeters (sculpturing areas in cm2), of interacting floral parts of C. caracalla in Vaqueros population, Salta province, northwestern Argentina (N = 20). Measurement

Mean ± SD

Floral length

4.80 ± 0.26

Floral width

3.40 ± 0.28

Height of pollinator chamber (pc)

0.53 ± 0.13

Depth of alignment channel (ac)

1.57 ± 0.09

Length of landing area(la)

2.38 ± 0.29

Protuberance of right keel petal(rp)

0.71 ± 0.06

Protuberance of left keel petal(lp)

0.54 ± 0.06

Length of right wing

3.20 ± 0.11

Length of left wing

3.01 ± 0.17

Sculpturing area, right wing

3.99 ± 0.16

Sculpturing area, left wing

3.05 ± 0.11

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Table 2. Measurements (mean and standard deviation), in centimeters, of interacting parts of floral visitors collected at C. caracalla flowers in Vaqueros population, Salta province, north-western Argentina. (N= 7). Bee species

Body height Mouthpart length

Body length Head width

Apis mellifera

0.37 ± 0.08 0.70 ± 0.16

1.28 ± 0.10 0.36 ± 0.10

morio, 0.68 ± 0.08 1.09 ± 0.03

1.89 ± 0.08 0.41 ± 0.02

Centris bicolor

0.74 ± 0.09 1.22 ± 0.02

1.49 ± 0.04 0.71 ± 0.14

Xylocopa eximia

0.80 ± 0.06 1.52 ± 0.03

2.45 ± 0.06 0.72 ± 0.01

Bombus workers

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