Detecting Symbioses in Complex Communities: the Fungal Symbionts of Bark and Ambrosia Beetles Within Asian Pines

Separating symbioses from incidental associations is a major obstacle in symbiosis research. In this survey of fungi associated with Asian bark and ambrosia beetles, we used quantitative culture and DNA barcode identification to characterize fungal communities associated with co-infesting beetle species in pines (Pinus) of China and Vietnam. To quantitatively discern likely symbioses from coincidental associations, we used multivariate analysis and multilevel pattern analysis (a type of indicator species analysis). Nearly half of the variation in fungal community composition in beetle galleries and on beetle bodies was explained by beetle species. We inferred a spectrum of ecological strategies among beetle-associated fungi: from generalist multispecies associates to highly specialized single-host symbionts that were consistently dominant within the mycangia of their hosts. Statistically significant fungal associates of ambrosia beetles were typically only found with one beetle species. In contrast, bark beetle-associated fungi were often associated with multiple beetle species. Ambrosia beetles and their galleries were frequently colonized by low-prevalence ambrosia fungi, suggesting that facultative ambrosial associations are commonplace, and ecological mechanisms such as specialization and competition may be important in these dynamic associations. The approach used here could effectively delimit symbiotic interactions in any system where symbioses are obscured by frequent incidental associations. It has multiple advantages including (1) powerful statistical tests for non-random associations among potential symbionts, (2) simultaneous evaluation of multiple co-occurring host and symbiont associations, and (3) identifying symbionts that are significantly associated with multiple host species.