Hierarchical Hybrid Enrichment: Multitiered Genomic Data Collection Across Evolutionary Scales, With Application to Chorus Frogs (Pseudacris).

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.].

Growth Dynamics and Survival of Liberibacter crescens BT-1, an Important Model Organism for the Citrus Huanglongbing Pathogen "Candidatus Liberibacter asiaticus".

Liberibacter crescens is the only cultured member of its genus, which includes the devastating plant pathogen "Candidatus Liberibacter asiaticus," associated with citrus greening/Huanglongbing (HLB). L. crescens has a larger genome and greater metabolic flexibility than "Ca Liberibacter asiaticus" and the other uncultured plant-pathogenic Liberibacter species, and it is currently the best model organism available for these pathogens. L. crescens grows slowly and dies rapidly under current culture protocols and this extreme fastidiousness makes it challenging to study. We have determined that a major cause of rapid death of L. crescens in batch culture is its alkalinization of the medium (to pH 8.5 by the end of logarithmic phase). The majority of this alkalinization is due to consumption of alpha-ketoglutaric acid as its primary carbon source, with a smaller proportion of the pH rise due to NH3 production. Controlling the pH rise with higher buffering capacity and lower starting pH improved recoverability of cells from 10-day cultures by >1,000-fold. We have also performed a detailed analysis of L. crescens growth with total cell numbers calibrated to the optical density and the percentage of live and recoverable bacteria determined over 10-day time courses. We modified L. crescens culture conditions to greatly enhance survival and increase maximum culture density. The similarities between L. crescens and the pathogenic liberibacters make this work relevant to efforts to culture the latter organisms. Our results also suggest that growth-dependent pH alteration that overcomes medium buffering should always be considered when growing fastidious bacteria.IMPORTANCELiberibacter crescens is a bacterium that is closely related to plant pathogens that have caused billions of dollars in crop losses in recent years. Particularly devastating are citrus losses due to citrus greening disease, also known as Huanglongbing, which is caused by "Candidatus Liberibacter asiaticus" and carried by the Asian citrus psyllid. L. crescens is the only close relative of "Ca Liberibacter asiaticus" that can currently be grown in culture, and it therefore serves as an important model organism for the growth, genetic manipulation, and biological control of the pathogenic species. Here, we show that one of the greatest limitations to L. crescens growth is the sharp increase in alkaline conditions it produces as a consequence of consumption of its preferred nutrient source. In addition to new information about L. crescens growth and metabolism, we provide new guidelines for culture conditions that improve the survival and yield of L. crescens.

Anchored hybrid enrichment generated nuclear, plastid and mitochondrial markers resolve the Lepanthes horrida (Orchidaceae: Pleurothallidinae) species complex.

Phylogenetic relationships in species complexes and lineages derived from rapid diversifications are often challenging to resolve using morphology or standard DNA barcoding markers. The hyper-diverse genus Lepanthes from Neotropical cloud forest includes over 1200 species and many recent, explosive diversifications that have resulted in poorly supported nodes and morphological convergence across clades. Here, we assess the performance of 446 nuclear-plastid-mitochondrial markers derived from an anchored hybrid enrichment approach (AHE) coupled with coalescence- and species network-based inferences to resolve phylogenetic relationships and improve species recognition in the Lepanthes horrida species group. In addition to using orchid-specific probes to increase enrichment efficiency, we improved gene tree resolution by extending standard angiosperm targets into adjacent exons. We found high topological discordance among individual gene trees, suggesting that hybridization/polyploidy may have promoted speciation in the lineage via formation of new hybrid taxa. In addition, we identified ten loci with the highest phylogenetic informativeness values from these genomes. Most previous phylogenetic sampling in the Pleurothallidinae relies on two regions (ITS and matK), therefore, the evaluation of other markers such as those shown here may be useful in future phylogenetic studies in the orchid family. Coalescent-based species tree estimation methods resolved the phylogenetic relationships of the L. horrida species group. The resolution of the phylogenetic estimations was improved with the inclusion of extended anchor targets. This approach produced longer loci with higher discriminative power. These analyses also disclosed two undescribed species, L. amicitiae and L. genetoapophantica, formally described here, which are also supported by morphology. Our study demonstrates the utility of combined genomic evidence to disentangle phylogenetic relationships at very shallow levels of the tree of life, and in clades showing convergent trait evolution. With a fully resolved phylogeny, is it possible to disentangle traits evolving in parallel or convergently across these orchid lineages such as flower color and size from diagnostic traits such as the shape and orientation of the lobes of the petals and lip.