Assessing the biodiversity and the impact of pollinators on carob production

current work. The potential impacts of low visitation rates of diurnal species to flowers of female trees may result in pollen limitation. Nocturnal pollinator species carry less pollen than diurnal species [29] and result in lower pollination success [26]. Our finding that carob pollen grains were more likely to be present in pollen traps closer to a male tree suggests that insects are indispensable to carob pollination, as female trees that are distant from male trees might not receive adequate pollen loads from wind alone. However, we note that the pollen transfer study in the current work lasted for 24 h, a very short time period. According to Ortiz et al. [46], the low production of pods is very common in carob trees and does not depend only on the distance from the nearest pollen source but also on blooming phenology. The open pollination treatment resulted in significantly higher production of pods than wind pollination as described by Dafni et al. [26], with the increase ranging from 4x to 16x higher production. Open-pollinated inflorescences produced pods with a significantly higher weight and length, and higher seed numbers per pod, than wind pollinated flowers. A previous work reported that open-pollinated flowers produced heavier seeds with higher germination rates than bagged flowers [45]. The differences in pod production between the study areas are probably the result of carob landrace / agro-environmental factors as discussed by [34]. Although we did not quantify the exact number of flowers for each inflorescence, our obser-

vations at the beginning of flowering suggest that the ca.14 mature flowers per inflorescence reported by Retana et al. [47] in north-east Spain are towards the high end of the numbers we observed in the current work. Retana et al. [47] relying on their own anecdotal observations and past studies report that few C. siliqua inflorescences produce fruit, and only a small pro- portion produces more than two fruits, with fruit set at around 5%. In contrast, Ortiz et al. [46] report a mean of almost 41 flowers per inflorescence on carob trees in south Spain, with an average of nine pods per inflorescence, and Arista et al. [44] report a mean of ca. 2.7 pods per inflorescence suggesting that there is high variability within the species and a potential interaction with environmental factors. Pod yield may be influenced by endogenous factors related to alternate bearing, although unfavorable environmental conditions may significantly reduce yield by fruit set reduction [20]. In the current work, each inflorescence produced less than 0.5 pods, which is lower than that reported in previous studies. The low production was similar across regions. In their detailed study Arista et al. [44] showed that even though hand pollination did not increase the number of initiated flowers per inflorescence, it did increase the number of fruits, suggesting an effect of pollen limitation. Yet, even in hand pollinated inflorescences, only a mean of three fruits were produced, suggesting that resources for fruit production are limited. In other

words, it seems that fruit production in C. siliqua is both pollen and resource limited, and in the current study it seems that pollen limitation was an important impediment to production. The impact of pollinators on production could be major, as Arista et al. [44] showed that flow- ers located towards the apex of the inflorescence have a higher probability of developing into fruits, because pollinators tend to visit the apical flowers first [30, 44]. The positive effect of pollination is not limited to carobs, for instance Nasare et al. [48] recorded heavier shea kernels under open pollination compared with bagged inflorescences. Similarly, tomatoes from non-bagged flowers were larger, heavier, and with more seeds com- pared with those produced by bagged flowers [49]. Several studies reported that open-pollina- tion can benefit fruit-set, and increase weight and quality [e.g. 11, 12, 50, 51]. Open pollination in kiwifruit resulted in higher fruit set due to insect and especially bee visitation [52]. Several studies on avocado, litchi, longan, guava trees and strawberries highlighted the importance of open pollination to fruit set [53–55]. PLOS ONE Biodiversity and the impact of pollinators on carobs PLOS ONE | https://doi.org/10.1371/journal.pone.0291431 October 20, 2023 12 / 16