32RNA-sequencing is a popular next-generation sequencing technique for assaying 33 genome-wide gene expression profiles. Nonetheless, it is susceptible to biases that are 34 introduced by sample handling prior gene expression measurements. Two of the most 35 common methods for preserving samples in both field-based and laboratory conditions 36 are submersion in RNAlater and flash freezing in liquid nitrogen. Flash freezing in liquid 37 nitrogen can be impractical, particularly for field collections. RNAlater is a solution for 38 stabilizing tissue for longer-term storage as it rapidly permeates tissue to protect cellular 39RNA. In this study, we assessed genome-wide expression patterns in 30 day old fry 40 collected from the same brood at the same time point that were flash-frozen in liquid 41nitrogen and stored at -80°C or submerged and stored in RNAlater at room 42 temperature, simulating conditions of fieldwork. We show that sample storage is a 43 significant factor influencing observed differential gene expression. In particular, genes 44 with elevated GC content exhibit higher observed expression levels in liquid nitrogen 45 flash-freezing relative to RNAlater-storage. Further, genes with higher expression in 46RNAlater relative to liquid nitrogen experience disproportionate enrichment for 47 functional categories, many of which are involved in RNA processing. This suggests 48that RNAlater may elicit a physiological response that has the potential to bias biological 49interpretations of expression studies. The biases introduced to observed gene 50 expression arising from mimicking many field-based studies are substantial and should 51 not be ignored. 52 53

Compared to selection on new mutations and standing genetic variation, the role of gene flow in generating adaptive genetic variation has been subject to much debate. Theory predicts that gene flow constrains adaptive evolution via natural selection by homogenizing allele frequencies among populations and introducing migrant alleles that may be locally maladaptive. However, recent work has revealed that populations can diverge even when high levels of gene flow are present and that gene flow may play an underappreciated role in facilitating local adaptation by increasing the amount of genetic variation present for selection to act upon. Here, we investigate how genetic variation introduced by gene flow contributes to adaptive evolution of complex traits using an emerging eco-evolutionary model system, the Mexican tetra (Astyanax mexicanus). The ancestral surface form of the Mexican tetra has repeatedly invaded and adapted to cave environments. The Chica cave is unique in that it contains several pool microenvironments inhabited by putative hybrids between surface and cave populations, providing an opportunity to investigate the dynamics of complex trait evolution and gene flow on a local scale. Here we conduct high-resolution genomic mapping and analysis of eye morphology and pigmentation in fish from multiple pools within Chica cave. We demonstrate that hybridization between cave and surface populations contributes to highly localized variation in behavioral and morphological traits. Analysis of sleep and locomotor behaviors between individual pools within this cave revealed reduced sleep associated with an increase in ancestry derived from cave populations, suggesting pool-specific ecological differences may drive the highly-localized evolution of sleep and locomotor behaviors. Lastly, our analyses uncovered a compelling example of convergent evolution in a core circadian clock gene in multiple independent cavefish lineages and burrowing mammals, indicating a shared genetic mechanism underlying circadian disruption in subterranean vertebrates. Together, our results provide insight into the evolutionary mechanisms that promote adaptive genetic variation and the genetic basis of complex behavioral phenotypes involved in local adaptation.

Sensory systems display remarkable plasticity and are under strong evolutionary selection. The Mexican cavefish, Astyanax mexicanus, consists of eyed river-dwelling surface populations, and multiple independent cave populations which have converged on eye loss, providing the opportunity to examine the evolution of sensory circuits in response to environmental perturbation. Functional analysis across multiple transgenic populations expressing GCaMP6s showed the total activity and predicted functional connectivity of the optic tectum largely did not differ between populations, revealing the maintenance of neural connectivity in the absence of retinal inputs. The correlation between activity events in the tectum revealed the selective loss of negatively correlated activity in cavefish that are suggestive of differences in selective pressure on excitatory and inhibitory connections within the tectum. Further, analysis of surface-cave hybrid fish reveals that changes in the tectum are genetically distinct from those encoding eye-loss. Together, these findings uncover the independent evolution of multiple components of the visual system and establish the use of functional imaging in A. mexicanus to study neural circuit evolution.