Collection of flowers

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 flower buds were collected at different sites with abundant flowering 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 flowering 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 home-made traps of 1.6 l of transparent plastic flasks, 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 30–40 min above a near shore seagrass meadow. This meadow had none or very few flowers to avoid collecting fauna with pollen attached as much as possible.

Aquarium experiment 1

T. testudinum flowers 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 filled with additional seawater until 3 l. The density of organisms in the aquaria was ≈500 individuals per liter; the majority being small crustacean larvae (Supplementary Table 1). The flowers were presented in pairs: the first flower always was a recently opened male flower with abundant pollen embedded in mucilage, and the second flower a recently opened female flower. The flowers were placed in small trays (5 × 6 cm), divided in two sections with a 3 cm high separation, to avoid pollen transport between the flowers during manipulation when placing the flowers (especially the sticky mucilage of the male flowers is difficult to handle). The trays were introduced into the aquaria with fauna (Supplementary Fig. 1). We conducted six trials with different flowers and fauna. Both flowers received equal illumination to allow filming during 15 min. But only the first minute of each film was analysed for behaviour, because some organisms were trapped in the sticky mucilage mass of the male flowers in the absence of water movement. We determined the number of visits per flower, and they were added for all female or male trials, and a χ2 analysis was carried out to test whether the type of visits was independent of the flower type (female versus male).

The number of pollen on the female flowers 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 flower and one foliar shoot of T. testudinum, and two small powerheads to induce water movement (Supplementary Fig. 1C). Either the female flower or the foliar shoot was placed in the centre and filmed during 1 min; with and without current (powerheads on or off). This was repeated four times with different flowers, 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 filled with seawater from a closed water circuit treated with UV light, to eliminate the possible influx of external viable pollen. The cores with flowers were transported within 1–1.5 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 flowers in the same mesocosm. The treatments (distance, with fauna or control), were assigned randomly to the mesocosms. The distances between the male and female flowers were 15, 30, 45, 60, 90 and 150 cm on different nights. The female flowers were always placed at the end of the separation of the Y-maze and the position of the male flowers was changed depending on the treatment. The female flowers in the same mesocosm were treated as independent replicates, which was reasonable because the flowers 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 flowers. The density of the fauna in the mesocosms varied between ≈30 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 flowers were left overnight (from ∼22:00 until 05:30–06:00 next morning) in the dark. The following morning, the concrete blocks with the shoots bearing the female flowers 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 fixed in FAA (formalin–acetic–alcohol). The fauna was collected in sieves when water was removed from the tanks, and ∼5% 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 floral 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 fixed flowers, were detected under a fluorescent 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 flowers, as hydrophilous pollination is unlikely due to the absence of water movement.

When pollen tubes are abundant it is difficult to determine their exact number in the stigmas and style of the female flower, therefore we established the following categories: 0: without pollen tubes, 1: less than 10 pollen tubes, 2: regular amount of pollen tubes (>10 but<100), 3: many pollen tubes (>100). χ2 Analysis was carried out to test if the abundance of pollen tubes in the female flowers was independent of treatment: pooled data for all mesocosms with or without fauna (Control). Another χ2 analysis was carried out to determine whether the abundance of pollen tubes in the female flowers was independent of the distance between the male and female flowers for the mesocosms with fauna.

Invertebrate fauna with pollen

The fauna from the mesocosms was sampled after conclusion of the experiments, and fixed in alcohol. The principal groups were identified (Supplementary Table 1), and specimens with attached spheres resembling pollen were separated. Fauna from the aquaria was fixed in alcohol within 10–15 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 fluorescent dye that only stains exine and lights up under fluorescent 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 fluorescent microscope (Olympus BX41).

Data availability

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