Histologic, metabolomic, and transcriptomic differences in fir trees from a peri-urban forest under chronic ozone exposure.

Urbanization modifies ecosystem conditions and evolutionary processes. This includes air pollution, mostly as tropospheric ozone (O 3 ), which contributes to the decline of urban and peri-urban forests. A notable case are fir ( Abies religiosa ) forests in the peripheral mountains southwest of Mexico City, which have been severely affected by O 3 pollution since the 1970s. Interestingly, some young individuals exhibiting minimal O 3 -related damage have been observed within a zone of significant O 3 exposure. Using this setting as a natural experiment, we compared asymptomatic and symptomatic individuals of similar age (≤15 years old; n  = 10) using histologic, metabolomic, and transcriptomic approaches. Plants were sampled during days of high (170 ppb) and moderate (87 ppb) O 3 concentration. Given that there have been reforestation efforts in the region, with plants from different source populations, we first confirmed that all analyzed individuals clustered within the local genetic group when compared to a species-wide panel (Admixture analysis with ~1.5K SNPs). We observed thicker epidermis and more collapsed cells in the palisade parenchyma of needles from symptomatic individuals than from their asymptomatic counterparts, with differences increasing with needle age. Furthermore, symptomatic individuals exhibited lower concentrations of various terpenes (ß-pinene, ß-caryophylene oxide, α-caryophylene, and ß-α-cubebene) than asymptomatic trees, as evidenced through GC-MS. Finally, transcriptomic analyses revealed differential expression for 13 genes related to carbohydrate metabolism, plant defense, and gene regulation. Our results indicate a rapid and contrasting phenotypic response among trees, likely influenced by standing genetic variation and/or plastic mechanisms. They open the door to future evolutionary studies for understanding how O 3 tolerance develops in urban environments, and how this knowledge could contribute to forest restoration.