Fluidigm2PURC: automated processing and haplotype inference for double-barcoded PCR amplicons

Abstract
Premise of the study: Targeted enrichment strategies for phylogenomic inference are a time- and cost-efficient way to collect DNA sequence data for large numbers of individuals at multiple, independent loci. Automated and reproducible processing of these data is a crucial step for researchers conducting phylogenetic studies. Methods and Results: We present Fluidigm2PURC, an open source Python utility for processing paired-end Illumina data from double-barcoded PCR amplicons. In combination with the program PURC (Pipeline for Untangling Reticulate Complexes), our scripts process raw FASTQ files for analysis with PURC and use its output to infer haplotypes for diploids, polyploids, and samples with unknown ploidy. We demonstrate the use of the pipeline with an example data set from the genus Thalictrum L. (Ranunculaceae). Conclusions: Fluidigm2PURC is freely available for Unix-like operating systems on GitHub [https://github.com/pblischak/fluidigm2purc] and for all operating systems through Docker [https://hub.docker.com/r/pblischak/fluidigm2purc].

Abstract
Moderate severity disturbances, which only kill a subset of canopy trees (e.g., via insects, pathogens, and windthrow), are increasingly widespread in North America, and can alter forest structure and production. Whether the net primary production (NPP) of forest stands differing in pre-disturbance site quality and composition respond similarly to moderate severity disturbance, however, is unknown, but critical to understanding the disturbance response dynamics of patchy landscapes. We experimentally disturbed three, 2-ha stands varying in pre-disturbance primary production and community composition, temporarily reducing live stand basal area by 38% to 66% through the stem girdling of all mature early successional aspen (Populus tremuloides Michx. and Populus grandidentata Michx.) and birch (Betula papyrifera Marshall). Disturbance significantly altered stand-scale physical and biological structure and prompted a similar decade-long pattern of wood NPP decline and recovery. All stands exhibited an initial reduction in wood NPP, followed by a recovery period and eventual return to pre-disturbance levels within eight years, with the most productive stand exhibiting an increase in primary production following recovery. Following wood NPP recovery, more biologically diverse forest canopies with higher leaf area indexes intercepted more light, and, consequently, had higher rates of wood NPP. We conclude that, despite substantial pre-disturbance differences in productivity and community composition, relative wood NPP recovery patterns can be similar, though long-term post-recovery primary production may trend higher in more productive and compositionally diverse stands. We suggest that improved mechanistic understanding of different forest ecosystems’ responses to disturbances remains critical to informing management decisions across diverse landscape mosaics.

Abstract
Venom toxicity assessments are often based upon non-native surrogate prey species that are not consumed in the wild by the venomous predator. This raises questions about the relevance of toxicity results on these “model” prey in addressing ecological or evolutionary questions about venom effects on native prey. We explore this issue by comparing the toxicity of venom from pygmy rattlesnakes (Sistrurus miliarius) on taxonomically-diverse sets of model (non-native) and native prey. Specifically, we compared rattlesnake venom toxicity for nine species from three broad taxonomic groups of prey (reptiles, mammals, and amphibians) to determine whether estimates of venom toxicity for the non-native model species of each group was representative of species which were native prey. In all three groups, model species (Anolis sagrei, Mus musculus, and Lithobates pipiens) had a significantly different mortality response from one or more of the native prey species (Anolis carolinensis, Peromyscus gossypinus, Lithobates sphenocephalus, Hyla cinerea, and Hyla squirella) that the models were meant to represent. Two features of our results suggest an importance of evolutionary history in understanding these differences. First, there was a phylogenetic component to prey responses to venom in that in each group, non-native models and congeneric native prey showed more similar responses than prey from other genera suggesting that venom may act on common prey targets that result from common ancestry. Second, native prey generally showed higher LD50 values than their non-native counterparts, suggesting greater resistance to venom from a predator with which they interact in nature. Our results suggest that researchers should use native prey to generate measures of venom toxicity that are ecologically and evolutionarily relevant. If this is not possible using “model” prey species that are close taxonomic relatives to natural prey may be a reasonable alternative.

Abstract
Phytophagous stink bugs are globally distributed and many harbor vertically inherited bacterial symbionts that are extracellular, yet little is known about how the symbiont’s genomes have evolved under this transmission strategy. Genome reduction is common in insect intracellular symbionts but limited genome sampling of the extracellular symbionts of distantly related stink bugs has precluded inferring patterns of extracellular symbiont genome evolution. To address this knowledge gap, we completely sequenced the genomes of the uncultivable bacterial symbionts of four neotropical stink bugs of the Edessa genus. Phylogenetic and comparative analyses indicated that the symbionts form a clade within the Pantoea genus and their genomes are highly reduced ( 0.8 Mb). Furthermore, genome synteny analysis and a jackknife approach for phylogenetic reconstruction, which corrected for long branch attraction artifacts, indicated that the Edessa symbionts were the result of a single symbiotic event that was distinct from the symbiosis event giving rise to Candidatus “Pantoea carbekii,” the extracellular symbiont of the invasive pentatomid stink bug, Halyomorpha halys. Metabolic functions inferred from the Edessa symbiont genomes suggests a shift in genomic composition characteristic of its lifestyle in that they retained many host-supportive functions while undergoing dramatic gene loss and establish- ing a stable relationship with their host insects. Given the undersampled nature of extracellular insect symbionts, this study is the first comparative analysis of these symbiont genomes from four distinct Edessa stink bug species. Finally, we propose the candidate name “Candidatus Pantoea edessiphila” for the species of these symbionts with strain designations according to their host species.

Abstract
Identifying the evolutionary and ecological mechanisms that drive lineage diversification in the species-rich tropics is of broad interest to evolutionary biologists. Here, we use phylogeographical and demographic analyses of genome-scale RADseq data to assess the impact of a large geographical feature, the Amazon River, on lineage formation in a venomous pitviper, Bothrops atrox. We compared genetic differentiation in samples from four sites near Santarem, Brazil, that spanned the Amazon and represented major habitat types. A species delimitation analysis identified each population as a distinct evolutionary lineage while a species tree analysis with populations as taxa revealed a phylogenetic tree consistent with dispersal across the Amazon from north to south. Phylogenetic analyses of mitochondrial DNA variation confirmed this pattern and suggest that all lineages originated during the mid- to late Pleistocene. Historical demographic analyses support a population model of lineage formation through isolation between lineages with low ongoing migration between large populations and reject a model of differentiation through isolation by distance alone. The results provide a rare example of a phylogeographical pattern demonstrating dispersal over evolutionary timescales across a large tropical river and suggest a role for the Amazon River as a driver of in situ divergence both by impeding (but not preventing) gene flow and through parapatric differentiation along an ecological gradient.

Abstract
While genetic exchange between non-sister species was traditionally considered to be rare in mammals, analyses of molecular data in multiple systems suggest that it may be common. Interspecific gene flow, if present, is problematic for phylogenetic inference, particularly for analyses near the species level. Here, we explore how to detect and account for gene flow during phylogeny estimation using data from a clade of North American Myotis bats where previous results have led researchers to suspect that gene flow among lineages is present. Initial estimates of phylogenetic networks and species trees indicate that subspecies described within M. lucifugus are paraphyletic. In order to explore the extent to which gene flow is likely to interfere with phylogeny estimation, we use posterior predictive simulation and a novel ABC approach based on gene tree distances. The former indicates that the species tree model is a poor fit to the data, and the latter provides evidence that a species tree with gene flow is a better fit. Taken together, we present evidence that the currently recognized M. lucifugus subspecies are paraphyletic, exchange alleles with other Myotis species in regions of secondary contact, and should be considered independent evolutionary lineages despite their morphological similarity.

Abstract
Understanding how historic habitat changes have impacted species and searching the past for clues to better understand the current plight of threatened species can help inform and improve future conservation efforts. We coupled species distribution modeling with historical imagery analysis to assess how changes in land use/land cover have influenced the distribution of eastern massasauga rattlesnake (Sistrurus catenatus), a federally threatened species, and its habitat in northeastern Ohio over the past ∼75 years. We also examined land use/land cover changes throughout southern Michigan for a broader perspective on the influence of historical processes on contemporary habitat. There was a pronounced shift in northeastern Ohio land cover from 1938 to 2011 with forest cover becoming the predominant land cover type as agricultural fields were abandoned and succession occurred. Most known eastern massasauga locations in the area were at some point used for agriculture and higher habitat suitability values were associated with agricultural fields that were eventually abandoned. We observed more stable habitat conditions across southern Michigan populations indicating agricultural abandonment was not as necessary for habitat creation in this part of their range. We present a new approach for linking historical landscapes to present day habitat suitability models; permitting inferences on how prior land use/land cover states have influenced the current distribution of species and their habitats. We demonstrate how agricultural abandonment was an important source of early successional habitat for a species that requires an open canopy, a finding applicable to a broad array of species with similar habitat requirements.