Dioecious fruit trees

Dioecious fruit trees

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Dioecious fruit trees were cross pollinated. Pollen grains stained blue with aniline blue were counted per mm^2^ of aerenchyma on non-green stems. This was done on 15 per population (three trees per species). We performed repeated measures ANOVAs with SPSS 15.0. The main and interactive effects of species and substrate were tested. Species was a factor and substrate was a repeated measure with 20 repetitions (see [Fig. 3](#pone.0118632.g003){ref-type="fig"}). Population was a random factor nested in the species factor. Type of stimulus was also a random factor and tree nested in population and type of stimulus was a factor. As a post hoc test we performed an ANOVA with treatment as a factor and species as a repeated measure. Tukey tests were used to separate species means.

![Cross pollination treatment.

The graphs illustrate means ± SE (10 repetitions) of the number of pollen grains per mm^2^ of aerenchyma on non-green stems.](pone.0118632.g003){#pone.0118632.g003}

In this experiment, plants were used as sender and pollinators as receivers. Cross pollination resulted in green offspring when a plant was the sender and a pollinator the receiver, whereas black offspring were produced when cross pollination occurred between plants of different species. Furthermore, according to Chiagas (2006), if there was any reciprocal pollen transfer between two plants of the same species, the plant that produced the larger number of ovules should have been the maternal plant.

In *M. rubra*, there was no difference in the number of seeds per fruit between groups that were crossed by unrelated and those that were crossed by relatives of the same sex (U: 5.36 ± 0.36, R: 5.64 ± 0.33). In *M. nigra*, there was no difference in the number of seeds per fruit between groups that were crossed by unrelated and those that were crossed by relatives of the same sex (U: 5.11 ± 0.55, R: 6.43 ± 0.85). However, in both species the number of seeds per fruit was higher when the cross was between unrelated individuals of different sexes (10.06 ± 0.52) than when the cross was between individuals of the same sex (5.51 ± 0.24). Therefore, individuals were classified in two groups based on their reproductive strategy: conspecifics (females and males), heterospecifics (females from *M. nigra* and males from *M. rubra*), and outcrossing (crossing of individuals of the same and different species).

The type of fruit on which pollen deposition was performed depended on the fruit colour. Brown fruits represented by the 5th and 6th clusters were marked with coloured lids and were presented to pollinators after the experiment. These plants did not show any pollination but the presence of the lid might still have had an effect on the response of the pollinators to the nectar. Hence, we calculated the number of pollen grains on each cluster. Pollen deposition was carried out during the flowering period of each fruit to ensure its colouration was already complete. Furthermore, this procedure avoided any variation in the number of seeds produced on each cluster.

The number of seeds produced in each group was different. Brown fruits produced from crosses between plants of the same species had 6.51 ± 0.61 seeds and brown fruits produced from crosses between plants of different species had 5.43 ± 0.43. Therefore, each brown cluster was regarded as a unit for analysis. We then compared the number of seeds produced between groups and the number of seeds deposited on each cluster.

In the simultaneous choice test, pollinators were presented with two brown fruits in order to ascertain whether they were familiar with one or both of them. Each fruit was attached to a blue plastic plate and was at 10 cm from the pollinator. The perching height of pollinators was noted to determine whether they were sessile, semi-flying, or flying. The height of each pollinator was also recorded using an Erect Vision^®^. Individuals that showed less than 10 cm of length for the walking stage (sessile pollinators) were not included in the choice test. Pollinator choices were recorded for 1 min, as per previous studies [[@pone.0118632.ref035],[@pone.0118632.ref036],[@pone.0118632.ref037]]. They were observed during the first 2 min and the choice for each fruit was recorded. Then, the number of pollen grains deposited on each fruit was counted and the fruit that the pollinator chose was the one where the greater number of pollen grains was deposited. The observation period was 4 min, as in other studies [[@pone.0118632.ref035],[@pone.0118632.ref038],[@pone.0118632.ref039]].Individuals that had chosen the wrong fruit or had taken less than 1 min to make a choice were excluded from the analysis.

In *M. rubra*, there were five colonies of *M. nigra*, four of which were studied. In the simultaneous choice test there was a significant effect of colony on pollinator choice, with pollinators preferentially choosing fruit from one colony ([Table 3](#pone.0118632.t