Table 1. Summary of pheromone experiments in Pseudostaurosira trainorii.

Experiment	Treatment/conditions	Results	Indications
1: ♂×♀	(1) 24h light; (2) 24h dark	successful fertilization	Pseudostaurosira trainorii is day-neutral
2: ♂×♀	each clone placed in a separate hole in an agar plate (Fig. S15A)	both clones sexualized. No amoeboid movement of ♂gametes.	compatible clones can interact through solid agar, most likely by means of chemical signals, which however do not include the one that triggers amoeboid movement of male gametes
3: ♂×F[♀]	in liquid medium	♂ sexualized (Fig. S14A)	vegetative ♀ secrete sex pheromone (ph-1)
4: ♀×F[♂]	in liquid medium	♀ not sexualized	vegetative ♂ secrete no sex pheromone
5: ♀×F[♂S]	in liquid medium	♀ sexualized (Fig. S14B).	sexualized ♂ secretes sex pheromone (ph-2)
6: ♂S×♀	in liquid medium	no movement of ♂ gametes towards vegetative ♀ (Fig. S14C).	ph-1 does not trigger the amoeboid movement of ♂ gametes. A putative sex pheromone (ph-3), which is the attractant for the ♂ gametes, can only be secreted from sexualized cells
7: ♂×F[♀] and ♂×F[♀S]	in gel medium (Fig. S15B)	♂ sexualized in both gels, but no amoeboid movement of ♂ gametes (Fig. S14D)	active ph-1, presumably secreted from both ♀ and ♀S, can be enclosed in gel, while ph3, if any , cannot be retained in gel.
8: ♂×♀	♂ and ♀ separated physically in different compartments connected by a bridge of culture medium (Fig. S15C)	both clones sexualized, but no amoeboid movement of ♂ gametes	the effective range of ph-3 is smaller than for ph-1 and ph-2
9: ♂S×dead ♀S*	in liquid medium	no amoeboid movement of ♂ gametes (Fig. S14E)	physical contact between threads and egg cells is unlikely to be required to trigger amoeboid movement of ♂ gametes

All experiments were performed in triplicate and the results were consistent. Unless stated otherwise, ♂ and ♀ are vegetative cells of male and female clones, respectively; S  =  sexualized clone where released gametes and gametangia were predominant with a small amount of vegetative cells; F  =  filtrate, so that F[♀}, F[♂S], etc. are filtrates of females, male sexualized clones, respectively.

*♀ gametes were killed by heating to 98°C for 1 h, or leaving Eppendorf tubes under 321 nm UV light (with TFX-20M transilluminator: Life Technologies, Inc, Paisley, UK) for 1 h.