Experimental evidence of pollination in marine flowers by invertebrate fauna

Abstract

Pollen transport by water-flow (hydrophily) is a typical, and almost exclusive, adaptation of plants to life in the marine environment. It is thought that, unlike terrestrial environments, animals are not involved in pollination in the sea. The male flowers of the tropical marine angiosperm Thalassia testudinum open-up and release pollen in mucilage at night when invertebrate fauna is active. Here we present experimental evidence that, in the absence of water-flow, these invertebrates visit the flowers, carry and transfer mucilage mass with embedded pollen from the male flowers to the stigmas of the female flowers. Pollen tubes are formed on the stigmas, indicating that pollination is successful. Thus, T. testudinum has mixed abiotic-biotic pollination. We propose a zoobenthophilous pollination syndrome (pollen transfer in the benthic zone by invertebrate animals) which shares many characteristics with hydrophily, but flowers are expected to open-up during the night.

ARTICLE

Received 28 Oct 2015 | Accepted 23 Aug 2016 | Published 29 Sep 2016

Pollen transport by water-ow (hydrophily) is a typical, and almost exclusive, adaptation of plants to life in the marine environment. It is thought that, unlike terrestrial environments, animals are not involved in pollination in the sea. The male owers of the tropical marine angiosperm Thalassia testudinum open-up and release pollen in mucilage at night when invertebrate fauna is active. Here we present experimental evidence that, in the absence of water-ow, these invertebrates visit the owers, carry and transfer mucilage mass with embedded pollen from the male owers to the stigmas of the female owers. Pollen tubes are formed on the stigmas, indicating that pollination is successful. Thus, T. testudinum has mixed abioticbiotic pollination. We propose a zoobenthophilous pollination syndrome (pollen transfer in the benthic zone by invertebrate animals) which shares many characteristics with hydrophily, but owers are expected to open-up during the night.

DOI: 10.1038/ncomms12980 OPEN

Experimental evidence of pollination in marine owers by invertebrate fauna

Brigitta I. van Tussenbroek1, Nora Villamil1,w, Judith Mrquez-Guzmn2, Ricardo Wong2,L. Vernica Monroy-Velzquez1 & Vivianne Solis-Weiss1

1 Unidad Acadmica de Sistemas Arrecifales-Puerto Morelos, Instituto de Ciencias del Mar y Limnologa, Universidad Nacional Autnoma de Mxico, Prolongacin Ninos Hroes S/N, Puerto Morelos, Quintana Roo 77580, Mexico. 2 Laboratorio del Desarrollo en Plantas, Facultad de Ciencias, Universidad Nacional Autnoma de Mxico, Circuito Exterior, Ciudad Universitaria, Del. Coyoacn, Ciudad de Mxico 04510, Mexico. w Present address: Nora Villamil, L.

4.21 Ashworth Laboratories, Institute of Evolutionary Biology, The Kings Buildings, West Mains Road, University of Edinburgh, Edinburgh EH9 3JT, UK. Correspondence and requests for materials should be addressed to B.I.v.T. (email: mailto:[email protected]

Web End [email protected] ).

NATURE COMMUNICATIONS | 7:12980 | DOI: 10.1038/ncomms12980 | http://www.nature.com/naturecommunications

Web End =www.nature.com/naturecommunications 1

ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms12980

Pollination is a key process in the life cycle of owering plants (angiosperms). It typically involves a vector responsible for the transport which can be either biotic

(animals) or abiotic: usually wind. Another abiotic vector is water-ow beneath the surface of the water, which is named true hydrophily (or hydrophilic pollination). Hydrophily only occurs in 14 out of the B14,000 angiosperm genera, and 10 out of these 14 genera occur in the marine environment1.

Flowering plants in the marine environment are commonly known as seagrasses2. All seagrass species, but one, are truly hydrophilic1; although some can also pollinate at the water surface when growing in or just below the intertidal zone3. Seagrasses form extensive meadows in shallow marine waters; and are amongst the worlds most productive ecosystems. They improve water transparency, stabilize coastlines and store carbon, and also provide food and shelter to a diverse faunal community4. Seagrasses are clonal plants, and owering has long been considered of little importance in this group of plants5. But gradually, this paradigm of the insignicance of sexual reproduction has been changing. Newly developed genetic markers have revealed that seagrass populations can be genetically highly variable. In addition, sexual reproduction is important for re-colonization after major disturbances and maintaining the gene-ow among populations6. However, knowledge of the processes involved in the reproduction and pollination of seagrasses is still limited1, which can partly be attributed to the inconspicuousness of the owers2. In addition, seagrass owers are ephemeral once they opened-up, which may be a consequence of owering in an aqueous medium; alternatively, it may be an adaptation to synchronize reproduction optimizing the chance of fertilization7 and/or avoid predation by sh8.

A suite of oral adaptations is associated with the water-mediated pollen transport, such as separation of the male and female structures in different owers on the same plant (monoecy) or different plants (dioecy), and reduced perianths. The female owers have tentacle-like stigmas, and the male owers produce copious pollen. Pollen with a reduced exine layer, is either elongated (liform) or spherical released in elongated strands of mucilage1,3,5,7. Thalassia testudinum, the species under study, is the dominant seagrass throughout the Caribbean and subtropical western Atlantic. It is dioecious with separate male and female plants with owers situated 12 cm above the sea-oor within the canopy. Male owers occur in clusters of one to ve owers (usually two or three) and produce in the order of 1.6 105 pollen per ower7. Pollen grains

(diameter B56 mm) are released in strands or masses of almost neutrally buoyant mucilage7,9. Female owers usually occur singly with an inferior ovary below sediment level that develops into a relatively large fruit (diameter 2025 mm) with 16 (usually 24) seeds. The male owers open-up at sunset and release the pollen within 12 h at night9,10. The female owers open-up during the day and are viable up to 72 h (ref. 7).

Until recently, it was thought that in the sea, unlike terrestrial environments, animals are not involved in pollination. However, van Tussenbroek et al.11 describe a highly abundant and diverse faunal community visiting the owers of T. testudinum at night. Seawater is almost 800 times as dense as air; and small crustaceans (0.68 mm long; mostly o1 mm) and polychaetes (1 20 mm long; mostly o10 mm) are swept by water motion.

But they show directional movements when approaching and visiting the male owers, probably being attracted to the mucilage-pollen mass, mainly composed of nutritious polysaccharides and proteins11. These visitors can be a potential vector of pollen transfer between owers; and for a visitor to be conrmed as a pollinator, the following conditions should be

demonstrated12: (1) both male and female organs are visited,(2) the visitor carries pollen, (3) the visitor transfers pollen between male and female sexual organs, (4) pollen deposition by the visitor results in successful fertilization, estimated as pollen germination on the stigmas, pollen tube growth or seed set.

We tested these four requirements to conrm whether the visiting invertebrates were pollinators on T. testudinum in three different experimental set-ups. The main challenge to disclose whether the fauna potentially pollinates this seagrass is excluding pollen transfer by water. We achieved this by placing owers and fauna in small aquaria or mesocosms without water-ow (Supplementary Figs 1 and 2). Before each trial, the fauna was captured with 1.6 l light traps after sunset. The rst set-up served to observe visitation behaviour of fauna, and deposition of pollen on the stigmas. Recently dehisced male and female owers were placed 23 cm apart in an aquarium, and lmed in absence or presence of abundant fauna (densityE500 individuals per liter).

The aim of a second aquarium set-up was to verify attractiveness of the female owers to fauna. Visits to female owers were registered on video, in absence or presence of water movement (generated with two small powerheads), with a foliar shoot ofT. testudinum as control substrate. A third set-up tested pollination success in a more natural setting in mesocosms (B100 l) with or without fauna (density B3090 individuals per liter). Male- and female owers were placed 15 to 150 cm apart (corresponding to distances in a meadow with relatively abundant owering) to determine if the proximity of a male ower was determinant in the success of pollination. The owers were left in the mesocosm during the night. Afterwards, the female owers were removed and left in a separate tank to permit the growth of pollen tubes, which were detected in preserved stigmas and styles under a uorescent microscope after staining13.

With this experimental evidence we demonstrate that marine invertebrates are pollinators of T. testudinum. They visit both female and male owers, carry pollen grains on their bodies, and transfer pollen between male and female owers in aquaria experiments. In the mesocosms, the transferred pollen grains germinate on the stigmas and form pollen tubes, indicating successful pollination. Thus, T. testudinum has both hydrophilous and zoophilous (transport by animals) pollination, revoking the paradigm that pollen in the sea is only transported by water.

ResultsBoth male and female organs are visited. Conrming contact with the reproductive organs of the owers is the rst step towards proving that a visitor is a pollinator. In the rst experimental set-up, comparing the behaviour of fauna on male and female owers in aquaria, we identied four types of visitation behaviour: (1) touching: the fauna touched the plant parts, the contact only lasting a fraction of a second; (2) multi-contact: the fauna touched these the parts at least two times consecutively; (3) visit: the fauna settled for 41 s on the parts;

(4) foraging: behaviour indicating feeding; either by moving along the plant parts or exhibiting abrupt movements of retreat. The rst three behaviour types were witnessed on both male and female owers; however, foraging was only observed on male owers (Fig. 1). We identied spheres in the digestive tract of the transparent zoea (Fig. 2a). The shape and size of several spheres corresponded with that of pollen of T. testudinum, which was conrmed by histochemical staining with auramine-O (Fig. 3), because exine exhibits uorescence with this staining technique14. The pollen grains in the digestive tract of the crustacean larvae indicated that they ingested the mucilage-pollen matrix.

In the second aquarium experiment, we tested the attractiveness of the female owers to fauna in comparison with

2 NATURE COMMUNICATIONS | 7:12980 | DOI: 10.1038/ncomms12980 | http://www.nature.com/naturecommunications

Web End =www.nature.com/naturecommunications

NATURE COMMUNICATIONS | DOI: 10.1038/ncomms12980 ARTICLE

12

a b c

0.5

10

(Number min1 )

0.2

8

Frequency

6

4

2

0

Male Female Flower type

d

Touching Multi-contact Visit Foraging

Figure 1 | The frequency of the visits to male or female owers of Thalassia testudinum. One male and one female ower were placed together in an aquarium and lmed in six trials at high faunal density (B500 individuals per liter). The result of the Chi-squared analysis was: w2 26.99, df 3, Po0.001; rejecting H0 (the number of visits of each type

is independent of the sex of the ower). Mean (s.e.m.), n 6.

0.5

0.1

Figure 2 | Fauna with pollen grains. (a) Majid zoea larva (stage I) with a pollen grain in digestive tract (detail was obtained with different illumination). (b) Thallasinidea zoea (stage I) with a pollen grain near rostrum and antenna. (c) Brachyuran zoea (stage I) with a pollen grain attached to abdomen. (d) Young Syllid polychaete with two pollen grains attached to segments; the almost hidden second grain is indicated by arrow. The pollen grains are indicated by circles. The bars represent mm.

vegetative plant parts. Although both owers and foliar shoots were visited, there was no difference in the number and type of visit to the female owers and foliar shoots in absence of water movement. However, at water movement, the female owers were visited more frequently than the foliar shoots (Fig. 4), indicating that under natural conditions with ow, these owers can effectively capture small fauna. The stigmas are very sticky, and in situ small sand grains, debris and fauna remain stuck until swept away by water movement (Supplementary Fig. 3).

The visitor carries pollen. Fauna caught in the light traps consisted mainly of small crustaceans and polychaetes (Supplementary Table 1). Some specimens, collected after termination of the mesocosm experiments, had one or many spheres resembling pollen attached to their bodies. Crustaceans had spheres on antennae, mouthparts (maxillipeds), pereopods or abdomen (Fig. 2b,c), and polychaetes usually had many attached to their segments or bristles (chaetae, Fig. 2d). Histochemical staining with auramine-O conrmed that these spheres were pollen grains (Fig. 3c).

The visitor transfers pollen between male and female organs. Deposition of pollen on the stigmas by fauna was demonstrated in the rst experimental aquaria set-up observing the fauna on a male and female ower. We counted the number of pollen grains on stigmas at the beginning and after 15 min. When we initiated our observations, most female owers had already some grains attached; most likely deposited during manipulation of the owers. After 15 min, on average 9 (3.9 s.e.m., N 6) grains were added, and 3 (0.8 s.e.m., N 6) grains were removed from the stigmas of the owers (Fig. 5). Only fauna could have moved the pollen because there was no water-ow in the aquaria. No pollen grains were gained or lost on the stigmas of female owers in the control treatments without fauna (Fig. 5).

The deposited pollen results in successful pollination. In the third experimental set-up with the mesocosms, the pollen transported by fauna resulted in successful pollination, as most female owers showed pollen tubes (Fig. 6; Supplementary Fig. 4). The control treatments without fauna, in contrast, had only few pollen tubes on rare occasions (Fig. 6). We expected to nd no pollen tubes at all in the control treatments, because the owers dehisced after we placed these in the mesocosms; however, they

could have been from pollen grains left over in the tanks or introduced with the newly collected male shoots, since some male shoots had a ower that had opened-up the previous night with possibly some remnants of pollen, and B1% of pollen grains is still viable after 24 h (ref. 10). But the number of pollen tubes on the control owers was always minimal, and it was signicantly different from the number on the female owers in presence of fauna (w2 38.75, df 4, Po0.001). In the tanks with fauna, distance

a b

200 50

10 10

c d

Figure 3 | Tests for verication of pollen grains of Thalassia testudinum. (a,b). Pollen grain (indicated by arrow) in the digestive tract of a decapod zoea I (Brachyura), stained with auramine-O. Images were taken with confocal microscope with excitation wavelength 405 nm and emission wavelength 422 nm (the chitin of the exoskeleton of crustaceans exhibits natural uorescence). In the detailed image (b), the microechinate ornamentation lights up as yellow points. (c) Pollen attached to the abdomen of a crustacean stained with auramine-O which lights up the exine in yellow, under uorescent microscope. (d) Photograph of pollen grain with a scanning electron microscope showing the microechinate ornamentation. The bars represent mm.

NATURE COMMUNICATIONS | 7:12980 | DOI: 10.1038/ncomms12980 | http://www.nature.com/naturecommunications

Web End =www.nature.com/naturecommunications 3

ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms12980

No water movement

Water movement

12

12

Plant part Plant part

Control

With fauna Color key

Without pollen (No. tubes = 0)

10

(number min1)

10

5

No record

8

8

Frequency

6

6

4

4

4

High pollen load (No. tubes 100)

Trial

Regular pollen load(No. tubes 10 but < 100)

2

2

3

0

0 Foliar shoot Female ower Foliar shoot Female ower

2

Few pollen grains (No. tubes <10)

Touching Multi-contact Visit

1

Figure 4 | The frequency of visits to female ower or foliar shoot of Thalassia testudinum. The female owers and foliar shoots in aquaria were lmed in absence and presence of water movement in four trials (Faunal density B100 individuals per liter). In absence of water movement, the result of the w2 analysis was w2 0.13, df 2, P 0.993; failing to reject H0

(The number of visits of each type is independent of plant part). With water movement, w2 9.26, df 2, P 0.001; rejecting H0. Mean (s.e.m.),

n 4.

15

30

45

60

90

150

15

30

45

60

90

150

Distance between male and female flower (cm)

Figure 6 | Developed pollen tubes in absence or presence of fauna. Heatmap of the abundance of pollen tubes in female owers of Thalassia testudinum in the mesocosms in absence (control) or presence (with fauna) of fauna. The male and female owers were separated by a range of distances from 15 to 150 cm.

12

8

6

2

0

4

10

Added

Removed

Number of pollen

With fauna

Control

Treatment

Figure 5 | Pollen transfer by fauna. Boxplot of the number of added and removed pollen grains on the stigmas of Thalassia testudinum in the rst aquaria experiments with (N 6) or without fauna (control, N 4), after

15 min. The horizontal bars are the median, the boxes show 25th and 75th percentiles, and the whiskers indicate spread. No pollen was added or removed in control treatments without fauna.

between male and female owers had no signicant effect on the number of pollen tubes (w2 10.465, df 15, P40.90; Fig. 6), suggesting that fauna can transport the pollen over relatively large distances.

DiscussionIn this study, we show that the invertebrate fauna visiting the seagrass T. testudinum comply with the four prerequisites to be considered pollinators. The pollination process involves the drifting fauna approaching the mucilage-pollen mass of the male owers. The fauna forages on this mass, and some pollen grains remain attached to their body parts due to the sticky nature of mucilage. The fauna removed from the ower by water movement is then captured by the tentacle-like stigmas of the female owers, and pollen grains are deposited to subsequently germinate, forming pollen tubes. The mucilage of the male owers forms a cloud when dissolving in the water, increasing viscosity of the water and decreasing ow velocity15; thereby assisting in the approach of the fauna. The mucilage-pollen mass

is a likely food reward for the pollinators, in exchange for pollen dispersal services similar to many terrestrial plants16. The tentacle-like stigmas and bracts of the female owers change the water-ow patterns facilitating the capture of pollen1 and fauna, which subsequently remain stuck to the stigmas. Pollination also occurs in the absence of water movement in the mesocosms, suggesting that the fauna can move actively towards both male and female owers in the absence of ow, possibly in search for substrate for settlement or driven by chemotaxis (movement driven by a chemical stimulus)17.

We do not discard water as the principal vector for pollen transport in this seagrass species. But similar to many wind-pollinated terrestrial plants that have shared traits with insect-pollinated plants18, T. testudinum likely has a mixed pollination syndrome: hydrophilous and zoobenthophilous. Zoobenthophily is a new type of pollination derived from benthos (community of organisms that live in, near or on the sea-bottom), because the pollination occurs near the seabed and many invertebrates belong to zoo-benthos; although zoea are free-swimming or free-oating19, and occur both in the benthic and planktonic zone. Many characteristics of the zoobenthophilous pollination syndrome coincide with that of hydrophily; such as pollen release in abundant mucilage and tentacle-like stigmas. However, similar to nocturnal owering of plants associated with pollination by bats, beetles or moths20,21, we expect nocturnal pollen release to be a trait of the zoobenthophilous pollination syndrome. Many small pollinating invertebrates hide in the sediment and in the seagrass canopy during the day, and become active at night22,23. Very little is known about the timing of pollen release in seagrasses, and the only records of other seagrass species with nocturnal opening-up of male owers (in aquaria) concern Halophila hawaiiana24.

Zoobenthophily likely enhances the reproductive success of the seagrass T. testudinum. It can be a mechanism of reproductive assurance in absence of water movement (although this is uncommon in the marine environment), and it likely extends the water-mediated pollination range which is limited25. At distances 420 cm between male and female owers, the probability of fertilization already decreases26. Marine invertebrates with semi-active locomotion27 can travel much further, potentially extending the dispersal range of pollen. This is conrmed by our results from the mesocosm experiment with fauna, where the distance between male and female owers (range 15150 cm) had no effect on the number of developed pollen tubes. In this context, it is important to mention that our light traps only

4 NATURE COMMUNICATIONS | 7:12980 | DOI: 10.1038/ncomms12980 | http://www.nature.com/naturecommunications

Web End =www.nature.com/naturecommunications

NATURE COMMUNICATIONS | DOI: 10.1038/ncomms12980 ARTICLE

captured the smaller invertebrates, as the larger ones usually escaped from the traps. We registered a higher frequency of larger specimens in the eld11, and we observed that larger specimens carried more pollen (http://www.youtube.com/watch?v=B7VLBhQ-rQo

Web End =www.youtube.com/watch?v= http://www.youtube.com/watch?v=B7VLBhQ-rQo

Web End =B7VLBhQ-rQo ); thus, we expect that the contribution of fauna to pollen transport in our experiments was underestimated. Negative consequences of the consumption of pollen grains by the invertebrate fauna are thought to be negligible, as only a very small fraction of the 1.6 105 pollen grains per ower is

consumed. The actual contribution of zoobenthophily versus hydrophily in the pollination success of this seagrass has yet to be established in the sea, and will most likely depend on environmental settings.

Pollination by water-ow is uncommon, and most freshwater aquatic plants have owers that emerge into the air28. Marine angiosperms probably evolved from freshwater ancestors1,3,29, and the Hydrocharitaceae (the family of T. testudinum) were likely insect pollinated28. Terrestrial abiotic pollination usually occurs by wind, which probably evolved from insect pollination in response to pollinator limitations and changes in the abiotic environment18. Similar to insect pollination, pollination by invertebrate marine fauna may have played a role in the transition from terrestrial to abiotic submarine pollination by water-ow. However, seagrasses are polyphyletic30 and the transition from freshwater to the marine environment may have differed among lineages. Further investigations into the reproductive ecology of this group of plants may reveal whether mixed abioticbiotic pollination syndrome occurs more frequently in the marine environment.

Methods

Collection of owers. The experiments were performed from 20 to 24 April 2014, and 13 to 14 May 2016. At midday, before the experiments, T. testudinum foliar shoots with closed male or female ower buds were collected at different sites with abundant owering in Puerto Morelos reef lagoon, Mexico. Only buds extending above the sediment level having a stretched pedicel were selected, because they were expected to open-up that same evening10. Foliar shoots with the buds for the aquarium experiments, were cut with a knife below substratum, and they were kept separately in closed seawater tanks until used. Flowers for the mesocosm experiments were sampled with a PVC corer (diameter 4.3 cm, 15 cm depth), to collect a whole owering shoot with a small sod of sediment.

Collection of fauna. Invertebrate fauna was collected immediately before each experiment. Collection occurred after sunset (after 20:30 local DLS time), in homemade traps of 1.6 l of transparent plastic asks, with inverted entrance, tied to a rod with a diving lamp. The light of the diving lamp attracted the fauna, and the traps were left 3040 min above a near shore seagrass meadow. This meadow had none or very few owers to avoid collecting fauna with pollen attached as much as possible.

Aquarium experiment 1. T. testudinum owers and fauna were observed in small aquaria placed in the dark. The seawater with fauna from a trap was very carefully poured into the aquaria (Supplementary Fig. 1) and lled with additional seawater until 3 l. The density of organisms in the aquaria was E500 individuals per liter;

the majority being small crustacean larvae (Supplementary Table 1). The owers were presented in pairs: the rst ower always was a recently opened male ower with abundant pollen embedded in mucilage, and the second ower a recently opened female ower. The owers were placed in small trays (5 6 cm), divided in

two sections with a 3 cm high separation, to avoid pollen transport between the owers during manipulation when placing the owers (especially the sticky mucilage of the male owers is difcult to handle). The trays were introduced into the aquaria with fauna (Supplementary Fig. 1). We conducted six trials with different owers and fauna. Both owers received equal illumination to allow lming during 15 min. But only the rst minute of each lm was analysed for behaviour, because some organisms were trapped in the sticky mucilage mass of the male owers in the absence of water movement. We determined the number of visits per ower, and they were added for all female or male trials, and a w2 analysis was carried out to test whether the type of visits was independent of the ower type (female versus male).

The number of pollen on the female owers was counted at the beginning of the experiment and after 15 min. We also included four control treatments without fauna.

Aquarium experiment 2. The aquaria were prepared as above, with one female ower and one foliar shoot of T. testudinum, and two small powerheads to induce water movement (Supplementary Fig. 1C). Either the female ower or the foliar shoot was placed in the centre and lmed during 1 min; with and without current (powerheads on or off). This was repeated four times with different owers, shoots and fauna. The types of visits were registered as above.

Experimental trial 3 with mesocosms. Experiments were carried out from 7 until 24 of April 2013 in Y-maze mesocosms (1.7 0.8 0.4 m depth, length partial

separation 1.0 m, Supplementary Fig. 2). The mesocosms were lled with seawater from a closed water circuit treated with UV light, to eliminate the possible inux of external viable pollen. The cores with owers were transported within 11.5 h of collection to the mesocosms and the sods with the shoots were placed in concrete blocks of 15 15 17(h) cm with a central hole of the size of the core samples.

Four shoots (two male and two female) were placed per mesocosm, and there were four mesocosms in total. Similar treatments were applied to all owers in the same mesocosm. The treatments (distance, with fauna or control), were assigned randomly to the mesocosms. The distances between the male and female owers were 15, 30, 45, 60, 90 and 150 cm on different nights. The female owers were always placed at the end of the separation of the Y-maze and the position of the male owers was changed depending on the treatment. The female owers in the same mesocosm were treated as independent replicates, which was reasonable because the owers in the same tanks received different quantities of pollen. Seawater with fauna from two 1.6 l traps, was very carefully poured into the mesocosm (of the fauna treatment) away from the male owers. The density of the fauna in the mesocosms varied between E30 and 90 individuals per liter. For each distance there was a control (no fauna) and a faunal trial. The control trials received the same volume of seawater. The owers were left overnight (from B22:00 until 05:3006:00 next morning) in the dark. The following morning, the concrete blocks with the shoots bearing the female owers were carefully removed from the mesocosms and placed in separate tanks (0.6 0.6 0.3 m depth) for at least 16 h

to allow for the growth of the pollen tubes before they were xed in FAA (formalinaceticalcohol). The fauna was collected in sieves when water was removed from the tanks, and B5% was analysed to determine the composition of the fauna until Family level, and to check whether they had pollen grains attached.

The treatments were rotated among the mesocosms randomly after thorough cleaning. The oral buds did not always open-up; therefore, the number of replicates might vary per treatment.

Observation on pollen tubes. The pollen tubes, in squash preparations of stigmas and styles of the xed owers, were detected under a uorescent microscope after staining with anile-blue13 (Supplementary Fig. 4). It is expected that if the fauna transports pollen, pollen tubes will be registered in the female owers, as hydrophilous pollination is unlikely due to the absence of water movement.

When pollen tubes are abundant it is difcult to determine their exact number in the stigmas and style of the female ower, therefore we established the following categories: 0: without pollen tubes, 1: less than 10 pollen tubes, 2: regular amount of pollen tubes (410 buto100), 3: many pollen tubes (4100). w2 Analysis was carried out to test if the abundance of pollen tubes in the female owers was independent of treatment: pooled data for all mesocosms with or without fauna (Control). Another w2 analysis was carried out to determine whether the abundance of pollen tubes in the female owers was independent of the distance between the male and female owers for the mesocosms with fauna.

Invertebrate fauna with pollen. The fauna from the mesocosms was sampled after conclusion of the experiments, and xed in alcohol. The principal groups were identied (Supplementary Table 1), and specimens with attached spheres resembling pollen were separated. Fauna from the aquaria was xed in alcohol within 1015 min after termination of the experiments, and the gut contents of the preserved almost transparent zoeas were examined. We applied the stain auranina-O, a uorescent dye that only stains exine and lights up under uorescent light, to verify whether the spheres inside or attached to the fauna were pollen grains. Spheres inside a zoea suspected to be pollen were examined under a confocal microscope (Olympus FV 1000) with excitation wavelength 405 nm and emission wavelength 422 nm. Selected fauna with spheres attached were observed under a uorescent microscope (Olympus BX41).

Data availability. The data that support the ndings of this study are included within the Article and Supplementary Information Files or available from the authors upon request.

References

1. Ackerman, J. D. in Seagrasses: Biology, Ecology and Conservation (eds Larkum,A. W. D., Orth, R. J. & Duarte, C. M.) Ch. 4 (Springer, 2006).2. Den Hartog, C. The sea-grasses of the world. Verh. Kon. Ned. Akad. Wetens. Afd. Natuurk. Ser. 59, 1275 (1970).

3. Cox, P. A. Hydrophilous pollination. Annu. Rev. Ecol. Syst. 19, 261279 (1988).

NATURE COMMUNICATIONS | 7:12980 | DOI: 10.1038/ncomms12980 | http://www.nature.com/naturecommunications

Web End =www.nature.com/naturecommunications 5

ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms12980

4. Duarte, C. M. The future of seagrass meadows. Environ. Conserv. 29, 192206 (2002).

5. Les, D. H. Breeding systems, population structure, and evolution in hydrophilous angiosperms. Ann. Missouri Bot. Gard. 3, 819835 (1988).

6. Waycott, M., Procaccini, G., Les, D. H. & Reusch, T. B. H. in Seagrasses: Biology, Ecology and Conservation (eds Larkum, A. W. D., Orth, R. J. & Duarte,C. M.) Ch. 2 (Springer, 2006).7. Van Tussenbroek, B. I., Marquz Guzmn, J. & Wong, R. in Functional Approach to Sexual Plant Reproduction (eds Gamboa-deBuen, A., Orozco-Segovia, A. & Cruz-Garcia, F.) Ch. 2 (Research Signpost, 2006).

8. Van Tussenbroek, B. I. & Muhlia Montero, M. Can oral consumption by sh shape traits of seagrass owers? Evol. Ecol. 27, 269284 (2013).

9. Cox, P. A. & Tomlinson, P. B. Pollination ecology of a seagrass, Thalassia testudinum (Hydrocharitaceae), in St Croix. Am. J. Bot. 75, 958965 (1988).

10. Van Tussenbroek, B. I., Wong, J. G. R. & Mrquez-Guzmn, J. Synchronized anthesis and predation on pollen in the marine angiosperm Thalassia testudinum (Hydrocharitaceae). Mar. Ecol. Prog. Ser. 354, 119124 (2008).11. Van Tussenbroek, B. I., Monroy-Velazquez, L. V. & Solis-Weis, V. Meso-fauna foraging on seagrass pollen may serve in marine zoophilous pollination. Mar. Ecol. Prog. Ser. 469, 16 (2012).

12. Kearns, C. A. & Inouye, D. W. Techniques for Pollination Biologists (University Press of Colorado, 1993).

13. Martin, F. W. Staining and observing pollen tubes in the style by means of uorescence. Stain Technol. 34, 125128 (1959).

14. Heslop-Harrison, Y. The pollen-stigma interaction: pollen-tube penetration in Crocus. Ann. Botany 41, 913922 (1977).

15. Margalef, R. Turbulence and marine life. Sci. Mar. 61, 109163 (1997).16. Willmer, P. Pollination and Floral Ecology (Princeton University Press,

2011).

17. Martens, E. A. et al. Size structures sensory hierarchy in ocean life. Proc. R. Soc. B 281, 20151346 (2015).

18. Culley, T. M., Weller, S. G. & Sakai, A. K. The evolution of wind pollination in angiosperms. Trends Ecol. Evol. 17, 361369 (2002).

19. Anger, K. The Biology of Decapod Crustacean Larvae Vol. 14 (Lisse: AA Balkema Publishers, 2001).

20. Gribel, R., Gibbs, P. E. & Queirz, A. L. Flowering phenology and pollination biology of Ceiba pentandra (Bombacaceae) in central Amazonia. J. Trop. Ecol. 15, 247263 (1999).

21. Miyake, T. & Yahara, T. Theoretical evaluation of pollen transfer by nocturnal and diurnal pollinators: when should a ower open? Oikos 86, 233240 (1999).

22. Robertson, A. I. & Howard, R. K. Diel trophic interactions between vertically migrating zooplankton and their sh predators in an eelgrass community. Mar. Biol. 48, 207213 (1978).

23. Bauer, T. R. Diel and seasonal variation in species composition and abundance of caridean shrimps (Crustacea, Decapoda) from seagrass meadows on the north coast of Puerto Rico. Bull. Mar. Sci. 36, 150162 (1985).

24. Herbert, D. A. Staminate owers of Halophila hawaiiana: description and notes on its owering ecology. Aquat. Bot. 25, 97102 (1986).

25. Van Tussenbroek, B. I. et al. Coping with potential bi-parental inbreeding: limited pollen and seed dispersal and large genets in the dioecious marine angiosperm Thalassia testudinum. Ecol. Evol 6, 55425556 (2016).

26. Van Tussenbroek, B. I., Montero, M. M., Wong, R., Santos, M. B. & Guzmn, J. M. Pollen limitation in a dioecious seagrass: evidence from a eld experiment. Mar. Ecol. Prog. Ser. 419, 283288 (2010).

27. Brusca, R. C. & Brusca, G. J. Invertebrates 2nd edn (Sinauer Associates Inc., 2003).

28. Hu, S., Dilcher, D. L., Jarzen, D. M. & Taylor, D. W. Early steps of angiosperm pollinator coevolution. Proc. Natl Acad. Sci. USA 105, 240245 (2008).

29. Sculthorpe, C. D. Biology of Aquatic Vascular Plants (Cambridge University Press, 1967).

30. Les, D. H., Cleland, M. A. & Waycott, M. Phylogenetic studies in Alismatidae, II: evolution of marine angiosperms (seagrasses) and hydrophily. Syst. Bot. 22, 443463 (1997).

Acknowledgements

We thank M.G. Barba Santos, L.F. Gonzlez Morales, and F. Negrete for their logistic help, K. Jimnez Durn for her support on the confocal microscope, and T. Godfrey for his comments on previous versions of the manuscript.

Author contributions

B.I.v.T., N.V., L.V.M-V. and V.S-W.: eldwork and execution of aquarium and mesocosm experiments. J.M-G. and R.W.: histochemical analysis of pollen and pollen tubes.

Additional information

Supplementary Information accompanies this paper at http://www.nature.com/naturecommunications

Web End =http://www.nature.com/ http://www.nature.com/naturecommunications

Web End =naturecommunications

Competing nancial interests: The authors declare no competing nancial interests.

Reprints and permission information is available online at http://npg.nature.com/reprintsandpermissions

Web End =http://npg.nature.com/ http://npg.nature.com/reprintsandpermissions

Web End =reprintsandpermissions .

How to cite this article: van Tussenbroek, B. I. et al. Experimental evidenceof pollination in marine owers by invertebrate fauna. Nat. Commun. 7, 12980 doi: 10.1038/ncomms12980 (2016).

This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the articles Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

Web End =http://creativecommons.org/licenses/by/4.0/

r The Author(s) 2016

6 NATURE COMMUNICATIONS | 7:12980 | DOI: 10.1038/ncomms12980 | http://www.nature.com/naturecommunications

Web End =www.nature.com/naturecommunications