Premise: The isolation of RNA from trees is challenging due to the interference of
polyphenols and polysaccharides with downstream processes. Furthermore, many
RNA extraction protocols are time consuming and involve hazardous chemicals. To
address these issues, we aimed to develop a safe protocol for high‐quality RNA
extraction from diverse Metrosideros taxa representing a broad range of leaf
toughness, pubescence, and secondary metabolites.
Methods and Results: We tested popular RNA isolation kits and protocols that were
effective on other recalcitrant trees, including a broad range of optimization and
purification steps. We optimized a protocol involving two silica‐membrane columnbased
kits that yielded high‐quantity RNA with an RNA integrity number >7 and
without DNA contamination. All RNA samples were used successfully in a follow‐on
RNA‐Seq experiment.
Conclusions: We present an optimized high‐throughput RNA extraction protocol
that yielded high‐quality and high‐quantity RNA from three contrasting leaf
phenotypes within a hyperdiverse woody species complex.
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Abstract Interspecies hybrids can express phenotypic traits far outside the range of parental species. The atypical traits of hybrids provide insight into differences in the factors that regulate the expression of these traits in the parental species. In some cases, the unusual phenotypic traits of hybrids can lead to phenotypic dysfunction with hybrids experiencing reduced survival or reproduction. Cuticular hydrocarbons (CHCs) in insects are important phenotypic traits that serve several functions, including desiccation resistance and pheromones for mating. We used gas chromatography mass spectrometry to investigate the differences in CHC production between two closely related sympatric Hawaiian picture-wing
Drosophila species,Drosophila heteroneura andD. silvestris , and their F1 and backcross hybrid offspring. CHC profiles differed between males of the two species, with substantial sexual dimorphism inD. silvestris but limited sexual dimorphism inD. heteroneura . Surprisingly, F1 hybrids did not produce three CHCs, and the abundances of several other CHCs occurred outside the ranges present in the two parental species. Backcross hybrids produced all CHCs with greater variation than observed in F1 or parental species. Overall, these results suggest that the production of CHCs was disrupted in F1 and backcross hybrids, which may have important consequences for their survival or reproduction. -
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Premise The drivers of isolation between sympatric populations of long‐lived and highly dispersible conspecific plants are not well understood. In the Hawaiian Islands, the landscape‐dominant tree,
Metrosideros polymorpha , displays extraordinary phenotypic differences among sympatric varieties despite high dispersibility of its pollen and seeds, thereby presenting a unique opportunity to investigate how disruptive selection alone can maintain incipient forms. StenophyllousM. polymorpha var.newellii is a recently evolved tree endemic to the waterways of eastern Hawai'i Island that shows striking neutral genetic differentiation from its ancestor, wet‐forestM. polymorpha var.glaberrima , despite sympatry of these forms. We looked for evidence for, and drivers of, differential local adaptation of these varieties across the range ofM. polymorpha var.newellii .Methods For paired populations of these varieties, we compared seedling performance under contrasting light conditions and a strong water current characteristic of the riparian zone. We also conducted a reciprocal transplant experiment and contrasted adult leaf anatomy.
Results Results suggest that the riparian zone is harsh and that selection involving the mechanical stress of rushing water, and secondarily, light, led to significant reciprocal immigrant inviability in adjacent forest and riparian environments. The strongest adaptive divergence between varieties was seen in leaves and seedlings from the site with the sharpest ecotone, coincident with the strongest genetic isolation of
M. polymorpha var.newellii observed previously.Conclusions These findings suggest that disruptive selection across a sharp ecotone contributes to the maintenance of an incipient riparian ecotype from within a continuous population of a long‐lived and highly dispersible tree species.
