Horizontal gene transfer mediated by broad-host-range plasmids is an important mechanism of antibiotic resistance spread. While not all bacteria maintain plasmids equally well, plasmid persistence can improve over time, yet no general evolutionary mechanisms have emerged. Our goal was to identify these mechanisms, and to assess if adaptation to one plasmid affects the permissiveness to others. We experimentally evolved Pseudomonas sp. H2 containing multi-drug resistance plasmid RP4, determined plasmid persistence and cost using a joint experimental-modeling approach, resequenced evolved clones, and reconstructed key mutations. Plasmid persistence improved in fewer than 600 generations because the fitness cost turned into a benefit. Improved retention of naive plasmids indicated that the host evolved towards increased plasmid permissiveness. Key chromosomal mutations affected two accessory helicases and the RNA polymerase β-subunit. Our and other findings suggest that poor plasmid persistence can be caused by a high cost involving helicase-plasmid interactions that can be rapidly ameliorated.

The World Health Organization has declared the emergence of antibiotic resistance to be a global threat to human health. Broad-host-range plasmids have a key role in causing this health crisis because they transfer multiple resistance genes to a wide range of bacteria. To limit the spread of antibiotic resistance, we need to gain insight into the mechanisms by which the host range of plasmids evolves. Although initially unstable plasmids have been shown to improve their persistence through evolution of the plasmid, the host, or both, the means by which this occurs are poorly understood. Here, we sought to identify the underlying genetic basis of expanded plasmid host-range and increased persistence of an antibiotic resistance plasmid using a combined experimental-modeling approach that included whole-genome resequencing, molecular genetics and a plasmid population dynamics model. In nine of the ten previously evolved clones, changes in host and plasmid each slightly improved plasmid persistence, but their combination resulted in a much larger improvement, which indicated positive epistasis. The only genetic change in the plasmid was the acquisition of a transposable element from a plasmid native to the Pseudomonas host used in these studies. The analysis of genetic deletions showed that the critical genes on this transposon encode a putative toxin-antitoxin (TA) and a cointegrate resolution system. As evolved plasmids were able to persist longer in multiple naïve hosts, acquisition of this transposon also expanded the plasmid's host range, which has important implications for the spread of antibiotic resistance.

Teleost fish regenerate their retinas after damage, in contrast to mammals. In zebrafish subjected to an extensive ouabain-induced lesion that destroys all neurons and spares Müller glia, functional recovery and restoration of normal optic nerve head (ONH) diameter take place at 100 days post-injury. Subsequently, regenerated retinas overproduce cells in the retinal ganglion cell (RGC) layer, and the ONH becomes enlarged. Here we test the hypothesis that a selective injury, which spares photoreceptors and Müller glia, results in faster functional recovery and fewer long-term histological abnormalities. Following this selective retinal damage, recovery of visual function required 60 days, consistent with this hypothesis. In contrast to extensively damaged retinas, selectively damaged retinas showed fewer histological errors and did not overproduce neurons. Extensively damaged retinas had RGC axons that were delayed in pathfinding to the ONH, and showed misrouted axons within the ONH, suggesting that delayed functional recovery following an extensive lesion is related to defects in RGC axons exiting the eye and/or reaching their central targets. The atoh7, fgf8a, shha, and netrin-1 genes were differentially expressed, and the distribution of Hh protein was disrupted following extensive damage as compared with selective damage. Confirming a role for Shh signaling in supporting rapid regeneration, shhat4+/− zebrafish showed delayed functional recovery following selective damage. We suggest that surviving retinal neurons provide structural/molecular information to regenerating neurons, and that this patterning mechanism regulates factors such as Shh. These factors in turn control neuronal number, retinal lamination, and RGC axon pathfinding during retinal regeneration.

Zebrafish have the remarkable capacity to regenerate retinal neurons following a variety of damage paradigms. Following initial tissue insult and a period of cell death, a proliferative phase ensues that generates neuronal progenitors, which ultimately regenerate damaged neurons. Recent work has revealed that Müller glia are the source of regenerated neurons in zebrafish. However, the roles of another important class of glia present in the retina, microglia, during this regenerative phase remain elusive. Here, we examine retinal tissue and perform QuantSeq. 3′mRNA sequencing/transcriptome analysis to reveal localization and putative functions, respectively, of mpeg1 expressing cells (microglia/macrophages) during Müller glia-mediated regeneration, corresponding to a time of progenitor proliferation and production of new neurons. Our results indicate that in this regenerative state, mpeg1-expressing cells are located in regions containing regenerative Müller glia and are likely engaged in active vesicle trafficking. Further, mpeg1+ cells congregate at and around the optic nerve head. Our transcriptome analysis reveals several novel genes not previously described in microglia. This dataset represents the first report, to our knowledge, to use RNA sequencing to probe the microglial transcriptome in such context, and therefore provides a resource towards understanding microglia/macrophage function during successful retinal (and central nervous tissue) regeneration.

The signaling molecule retinoic acid (RA) regulates rod and cone photoreceptor fate, differentiation, and survival. Here we elucidate the role of RA in differential regulation of the tandemly-duplicated long wavelength-sensitive (LWS) cone opsin genes. Zebrafish embryos were treated with RA from 48 hours post-fertilization (hpf) to 75 hpf, and RNA was isolated from eyes for microarray analysis. ~170 genes showed significantly altered expression, including several transcription factors and components of cellular signaling pathways. Of interest, the LWS1 opsin gene was strongly upregulated by RA. LWS1 is the upstream member of the tandemly duplicated LWS opsin array and is normally not expressed embryonically. Embryos treated with RA 48 hpf to 100 hpf or beyond showed significant reductions in LWS2-expressing cones in favor of LWS1-expressing cones. The LWS reporter line, LWS-PAC(H) provided evidence that individual LWS cones switched from LWS2 to LWS1 expression in response to RA. The RA signaling reporter line, RARE:YFP indicated that increased RA signaling in cones was associated with this opsin switch, and experimental reduction of RA signaling in larvae at the normal time of onset of LWS1 expression significantly inhibited LWS1 expression. A role for endogenous RA signaling in regulating differential expression of the LWS genes in postmitotic cones was further supported by the presence of an RA signaling domain in ventral retina of juvenile zebrafish that coincided with a ventral zone of LWS1 expression. This is the first evidence that an extracellular signal may regulate differential expression of opsin genes in a tandemly duplicated array.

Understanding the process of adaptation during rapid environmental change remains one of the central focal points of evolutionary biology. The recently formed White Sands system of southern New Mexico offers an outstanding example of rapid adaptation, with a variety of species having rapidly evolved blanched forms on the dunes that contrast with their close relatives in the surrounding dark soil habitat. In this study, we focus on two of the White Sands lizard species, Sceloporus cowlesi and Aspidoscelis inornata, for which previous research has linked mutations in the melanocortin-1 receptor gene (Mc1r) to blanched coloration. We sampled populations both on and off the dunes and used a custom sequence capture assay based on probed fosmid libraries to obtain >50 kb of sequence around Mc1r and hundreds of other random genomic locations. We then used model-based statistical inference methods to describe the demographic and adaptive history characterizing the colonization of White Sands. We identified a number of similarities between the two focal species, including strong evidence of selection in the blanched populations in the Mc1r region. We also found important differences between the species, suggesting different colonization times, different genetic architecture underlying the blanched phenotype and different ages of the beneficial alleles. Finally, the beneficial allele is dominant in S. cowlesi and recessive in A. inornata, allowing for a rare empirical test of theoretically expected patterns of selective sweeps under these differing models.

Sex is determined genetically in most fishes, but the gene responsible for sex determination is not known for the vast majority of fish species, including Chinook Salmon Oncorhynchus tshawytscha. The purpose of this study was to characterize a putative sex‐determining gene (“sexually dimorphic on the Y‐chromosome” [sdY] gene) in Chinook Salmon and develop a method to test genomic DNA (gDNA) samples for genetic sex assignment. Using next‐generation sequencing and salmonid DNA sequence data from GenBank, the entire genomic organization of Chinook Salmon sdY was described. The corresponding full‐length complementary DNA (cDNA) sequence generated from total RNA was determined by using a combination of genomic‐based primers and “rapid amplification of cDNA ends” (RACE) PCR assays. A phylogenic analysis was conducted by comparing the Chinook Salmon sdY cDNA sequence with sdY sequences (GenBank) from 10 other teleost species. A multisequence alignment was performed, and a phylogenetic tree was inferred from the alignment, providing evidence for sdY identity. Based upon a TaqMan real‐time PCR assay, a genetic test for male sex using sdY was developed and tested on gDNA isolated from phenotypic female and male Chinook Salmon representing populations in Alaska, Idaho, and Washington. The results provide compelling evidence that sdY is the sex‐determining gene in Chinook Salmon. We developed a reliable assay for sdY that can be used for genetic sex assignment in this species. However, discordance between phenotype and genotype was noted in 13 of 107 phenotypic female Chinook Salmon from Alaska and Washington. Several explanations for this discordance are discussed.Received January 14, 2014; accepted November 25, 2014

Evidence from natural systems suggests that hybridization between animal species is more common than traditionally thought, but the overall contribution of introgression to standing genetic variation within species remains unclear for most animal systems. Here, we use targeted exon capture to sequence thousands of nuclear loci and complete mitochondrial genomes from closely related chipmunk species in the Tamias quadrivittatus group that are distributed across the Great Basin and the central and southern Rocky Mountains of North America. This recent radiation includes six overlapping, ecologically distinct species (Tamias canipes, Tamias cinereicollis, Tamias dorsalis, T. quadrivittatus, Tamias rufus, and Tamias umbrinus) that show evidence for widespread introgression across species boundaries. Such evidence has historically been derived from a handful of markers, typically focused on mitochondrial loci, to describe patterns of introgression; consequently, the extent of introgression of nuclear genes is less well characterized. We conducted a series of phylogenomic and species-tree analyses to resolve the phylogeny of six species in this group. In addition, we performed several population-genomic analyses to characterize nuclear genomes and infer coancestry among individuals. Furthermore, we used emerging quartets-based approaches to simultaneously infer the species tree (SVDquartets) and identify introgression (HyDe). We found that, in spite of rampant introgression of mitochondrial genomes between some species pairs (and sometimes involving up to three species), there appears to be little to no evidence for nuclear introgression. These findings mirror other genomic results where complete mitochondrial capture has occurred between chipmunk species in the absence of appreciable nuclear gene flow. The underlying causes of recurrent massive cytonuclear discordance remain unresolved in this group but mitochondrial DNA appears highly misleading of population histories as a whole. Collectively, it appears that chipmunk species boundaries are largely impermeable to nuclear gene flow and that hybridization, while pervasive with respect to mtDNA, has likely played a relatively minor role in the evolutionary history of this group. [Cytonuclear discordance; hyridization; introgression, phylogenomics; SVDquartets; Tamias.]