Determining the optimal targets of genomic subsampling for phylogenomics, phylogeography, and population genomics remains a challenge for evolutionary biologists. Of the available methods for subsampling the genome, hybrid enrichment (sequence capture) has become one of the primary means of data collection for systematics, due to the flexibility and cost efficiency of this approach. Despite the utility of this method, information is lacking as to what genomic targets are most appropriate for addressing questions at different evolutionary scales. In this study, first, we compare the benefits of target loci developed for deep- and shallow scales by comparing these loci at each of three taxonomic levels: within a genus (phylogenetics), within a species (phylogeography), and within a hybrid zone (population genomics). Specifically, we target evolutionarily conserved loci that are appropriate for deeper phylogenetic scales and more rapidly evolving loci that are informative for phylogeographic and population genomic scales. Second, we assess the efficacy of targeting multiple-locus sets for different taxonomic levels in the same hybrid enrichment reaction, an approach we term hierarchical hybrid enrichment. Third, we apply this approach to the North American chorus frog genus Pseudacris to answer key evolutionary questions across taxonomic and temporal scales. We demonstrate that in this system the type of genomic target that produces the most resolved gene trees differs depending on the taxonomic level, although the potential for error is substantially lower for the deep-scale loci at all levels. We successfully recover data for the two different locus sets with high efficiency. Using hierarchical data targeting deep and shallow levels: we 1) resolve the phylogeny of the genus Pseudacris and introduce a novel visual and hypothesis testing method that uses nodal heat maps to examine the robustness of branch support values to the removal of sites and loci; 2) estimate the phylogeographic history of Pseudacris feriarum, which reveals up to five independent invasions leading to sympatry with congener Pseudacris nigrita to form replicated reinforcement contact zones with ongoing gene flow into sympatry; and 3) quantify with high confidence the frequency of hybridization in one of these zones between P. feriarum and P. nigrita, which is lower than microsatellite-based estimates. We find that the hierarchical hybrid enrichment approach offers an efficient, multitiered data collection method for simultaneously addressing questions spanning multiple evolutionary scales. [Anchored hybrid enrichment; heat map; hybridization; phylogenetics; phylogeography; population genomics; reinforcement; reproductive character displacement.]

Abstract. Long-term monitoring of frog populations is needed to understand the effects of global change. To better understand the relationships between climate variation and calling activity, we monitored an anuran assemblage in a Puerto Rican wetland by sampling the acoustic environment for one minute every 10 minutes, for 41 months. By automating data collection using passive acoustic monitoring hardware, we collected more than 110,000 recordings. These recordings were analyzed using speciesspecific identification algorithms of four Eleutherodactylus species. The peak calling activity of E. coqui (.0.3 detection frequency) and E. cochranae (.0.2) occurred between April and September, and there was a clear decline in activity during the dry months of January-March. There was no clear annual pattern in E. brittoni or E. juanariveroi, but E. juanariveroi did show a significant decline in calling activity over the 41-month study (;0.5 to ;0.35). Calling activity of E. coqui and E. cochranae was positively correlated with temperature, while E. brittoni and E. juanariveroi responded negatively to temperature and precipitation. This difference in response to temperature and precipitation could be related to differences in body size and the location of calling sites among the four species. For example, E. brittoni and E. juanariveroi are small species that call from the tips of the vegetation. High temperatures and intense precipitation may cause them to retreat into the vegetation, and reduce calling activity. In contrast, E. coqui and E. cochranae call lower in the vegetation and from leaf axils where they are more protected. Based on these findings, future scenarios of climate change could pose a threat for the survival of the populations of these four species. New climate regimes could negatively affect the calling activity, and thus diminish reproductive events.

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This study demonstrates that stage of development and origin of samples affected the structure and function of the CBB's bacterial communities. This is the first attempt to predict functional significance of the CBB microbiota in nutrition, reproduction and defence.