Urbanization modifies ecosystem conditions and evolutionary processes. This includes air pollution, mostly tropospheric ozone (O3), which leads to urban and peri-urban forest decline. Such is the case of fir (Abies religiosa) forests in the peripheral mountains west of Mexico City, which have been severely affected by O3 pollution since the 1970s. Interestingly, some young individuals with low O3—related damage have been observed within a zone of great O3 incidence, which might suggest rapid tolerance/adaptation to this pollutant. We compared asymptomatic and damaged individuals of the same age (≤15 years old; n = 10) using histological, metabolomic and transcriptomic methods. Plants were sampled during days of high (170 ppb) and moderate (87 ppb) O3 concentration periods. After verifying that all individuals clustered within the same 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 in needles from damaged than from asymptomatic individuals; such differences increased with needle age. Furthermore, damaged individuals had lower concentrations of various terpenoids (ß-Pinene, ß-Caryophylene Oxide, α-Caryophylene, ß-α-Cubebene and α-Muurolene) than asymptomatic trees, as evidenced through GC-MS. Finally, transcriptomic analyses revealed differential expression for thirteen genes related to carbohydrate metabolism, plant defense, and gene regulation. Our results indicate a rapid and contrasting phenotypic response between plants, likely modeled by standing genetic variation and/or plastic mechanisms. They open the door to future evolutionary studies for understanding how O3 tolerance develops in urban environments, and for identifying tolerant germplasm for forest restoration.

Secondary contact of species that have evolved partial reproductive isolation in allopatry may result in several outcomes, which range from rampant hybridization to barrier reinforcement. Reinforcement arises from reduced hybrid fitness, which promotes assortative mating and hence speciation. In plants, self-fertilization and disjunction in reproductive phenology are often cited as evidence of reinforcement. However, local adaptation and pleiotropic effects during colonization can also lead to reproductive isolation without reinforcement. We explored these competing possibilities in a fir species complex (Abies flinckii - A. religiosa) distributed in ‘sky-islands’ along the Trans-Mexican Volcanic Belt (TMVB), in central Mexico. Despite co-occurring in two independent sympatric regions (west and center), these two taxa seem to rarely interbreed because of disjunct reproductive phenologies. We genotyped 1,147 SNPs, generated by GBS across 23 populations, and compared multiple demographic scenarios, built based on the geological history of the TMVB. The best-fitting model revealed one of the most rapid and complete speciation cases for a conifer species-pair, dating back to ~1.2 Ma, which included an early asymmetric gene flow (mostly from A. flinckii into A. religiosa) limited to the most ancient sympatric region. Coupled with the lack of support for colonization models, our demographic inferences point to an early development of reinforcement, as a putative mechanism for avoiding hybridization. Reinforcement should be considered as a key player for originating and maintaining species diversity in the tropics and subtropics and incorporated in studies with other model systems.

In land plants, heteroblasty broadly refers to a drastic change in morphology during growth through ontogeny. Juniperus flaccida and Pinus cembroides are conifers of independent lineages known to exhibit leaf heteroblasty between the juvenile and adult life stage of development. Juvenile leaves of P. cembroides develop spirally on the main stem and appear decurrent, flattened and needle-like; whereas adult photosynthetic leaves are triangular or semi-circular needle-like, and grow in whorls on secondary or tertiary compact dwarf shoots. By comparison, J. flaccida juvenile leaves are decurrent and needle-like, and adult leaves are compact, short, and scale-like. Comparative analyses were performed to evaluate differences in anatomy and gene expression patterns between developmental phases in both species. RNA from twelve samples was sequenced and analyzed with available software. They were assembled de novo from the RNA-Seq reads. Following assembly, 63,741 high quality transcripts were functionally annotated in P. cembroides and 69,448 in J. flaccida. Evaluation of the orthologous groups yielded 4,140 shared gene families among the four references (adult and juvenile from each species). Activities related to cell division and development were more abundant in juveniles than adults in P. cembroides, and more abundant in adults than juveniles in J. flaccida. Overall, there were 509 up-regulated and 81 down-regulated genes in the juvenile condition of P. cembroides and 14 up-regulated and 22 down-regulated in J. flaccida. Gene interaction network analysis showed evidence of co-expression and co-localization of up-regulated genes involved in cell wall and cuticle formation, development, and phenylpropanoid pathway, in juvenile P. cembroides leaves. Whereas in J. flaccida, differential expression and gene interaction patterns were detected in genes involved in photosynthesis and chloroplast biogenesis. Although J. flaccida and P. cembroides both exhibit leaf heteroblastic development, little overlap was detected, and unique genes and pathways were highlighted in this study.Keywords : Heteroblasty, RNA-seq analysis, cell wall and cutin biosynthesis, conifer leaf development, Juniperus flaccida, Pinus cembroides. 

Secondary contact of species that have evolved partial reproductive isolation in allopatry may result in several outcomes, which range from rampant hybridization to barrier reinforcement. Reinforcement arises from reduced hybrid fitness, which promotes assortative mating and hence speciation. In plants, self-fertilization and disjunctions in reproductive-phenology are often invoked as evidence of reinforcement. However, local adaptation and pleiotropic effects during colonization can also lead to reproductive isolation without reinforcement. We explored these possibilities in a fir species complex (Abies flinckii - A. religiosa) distributed in ‘sky-islands’ along the Trans-Mexican Volcanic Belt (TMVB), in central Mexico. Despite co-occurring in two independent sympatric regions (west and center), these two taxa seem to rarely interbreed because of disjunct reproductive phenologies. We genotyped 1,147 SNPs, generated by GBS across 23 populations, and compared multiple demographic scenarios, built based on the geological history of the TMVB. The best-fitting model suggested a recent species split (for a conifer), dating back to ~1.2 Ma, together with early asymmetric gene flow (mostly from A. flinckii into A. religiosa), limited to the central sympatric region. Coupled with the lack of support for colonization models, the summary statistics (f, Hobs, FST, θπ, etc.) and historical demographic inferences made herein point to a rapid speciation with an early development of reinforcement, as a putative mechanism for avoiding hybridization. The role of reinforcement should be thus further explored in the (sub)tropics, as likely explanation for how species diversity is generated and maintained.