Genomic Investigations of Diversity within the Milkweed Genus Asclepias, at Multiple Scales

At a time when the biodiversity on Earth is being rapidly lost, new technologies and methods in genomic analysis are fortunately allowing scientists to catalog and explore the diversity that remains more efficiently and precisely. The studies in this dissertation investigate genomic diversity within the milkweed genus, Asclepias, at multiple scales, including diversity found within a single individual, diversity within and among populations in a species, and diversity across the entire genus. These investigations contribute to our understanding of the genomic content and architecture within Asclepias and the Gentianales, patterns of population diversification in the western United States, and the evolutionary history of select loci within Asclepias, with implications across flowering plants. Chapter 2 investigates patterns of polymorphisms among paralogous copies of nuclear ribosomal DNA (nrDNA) within individual genomes, and presents a bioinformatic pipeline for characterizing polymorphisms among copies of a high-copy locus. Results are presented for intragenomic nrDNA polymorphisms across Asclepias. The 18S-26S portion of the nrDNA cistron of Asclepias syriaca served as a reference for assembly of the region from 124 samples representing 90 species of Asclepias. Reads were mapped back to each individual’s consensus and at each position reads differing from the consensus were tallied using a custom Perl script. Low frequency polymorphisms existed in all individuals (mean = 5.8%). Most nrDNA positions (91%) were polymorphic in at least one individual, with polymorphic sites being less frequent in subunit regions and loops. Highly polymorphic sites existed in each individual, with highest abundance in the “noncoding” ITS regions. Phylogenetic signal was present in the distribution of intragenomic polymorphisms across the genus. Intragenomic polymorphisms in nrDNA are common in Asclepias, being found at higher frequency than any other study to date. The high and variable frequency of polymorphisms across species highlights concerns that phylogenetic applications of nrDNA may be error-prone. The new analytical approach provided in this chapter is applicable to other taxa and other high-copy regions characterized by low coverage genome sequencing (genome skimming). Chapter 3 presents Hyb-Seq, a new method combining target enrichment and genome skimming to allow simultaneous data collection for low-copy nuclear genes and high-copy genomic targets for plant systematics and evolution studies. A program is presented that takes genome and transcriptome assemblies and locates loci likely to be low copy and phylogenetically informative, to be used for probe development and enrichment in sequence libraries. A workflow is presented for processing data, from raw sequence reads to assembled exons and reconstructed trees. Genome and transcriptome assemblies for Asclepias syriaca were used to design enrichment probes for 3385 exons from 768 genes (>1.6 Mbp) followed by Illumina sequencing of enriched libraries. Hyb-Seq of 12 individuals (10 Asclepias species and two related genera) resulted in at least partial assembly of 92.6% of exons and 99.7% of genes and an average assembly length >2 Mbp. Importantly, complete plastomes and nrDNA cistrons were assembled using off-target reads. Phylogenomic analyses demonstrated signal conflict between genomes. The Hyb-Seq approach enables targeted sequencing of thousands of low-copy nuclear exons and flanking regions, as well as genome skimming of high-copy repeats and organellar genomes, to efficiently produce genome-scale data sets for phylogenomics. Chapter 4 presents an assembly of the genome of the common milkweed, Asclepias syriaca. It uses principles from Chapter 3 to target SNPs and reconstruct linkage groups, enabling an analysis of chromosomal evolution within Gentianales, the order containing Asclepias. Asclepias syriaca is the first species in Apocynaceae with reconstructions of the nuclear, chloroplast, and mitochondrial genomes, and the first to have linkage group information incorporated into the nuclear assembly. The final assembly of Asclepias syriaca contains 54,266 scaffolds ≥1 kbp, with N50 = 3415 bp, representing 37% (156.6 Mbp) of the estimated 420 Mbp genome. Scaffolds ≥200 bp sum to 229.7 Mbp, with N50 = 1904 bp. A total of 14,474 protein coding genes were identified based on transcript evidence, closely related proteins, and ab initio models, and 95% of genes were annotated based on genes from Coffea canephora and Catharanthus rosea. A large proportion of gene space is represented in the assembly, with 96.7% of Asclepias transcripts, 88.4% of transcripts from the related genus Calotropis, and 90.6% of proteins from Coffea mapping to the assembly. Analyses were performed for three gene families, involved in rubber production, light sensing, and cardenolide production, with the finding that the cardenolide related progesterone 5β-reductase gene family is likely reduced in Asclepias relative to other Apocynaceae. Scaffolds covering 75 Mbp of the Asclepias assembly were grouped into eleven linkage groups. Comparisons of these groups with pseudochromosomes in Coffea found that six chromosome show consistent stability in gene content, while one may have a long history of fragmentation and rearrangement. Finally, in Chapter 5, diversity within a species across its entire range is investigated with a phylogeographic study of the jewel milkweed, Asclepias cryptoceras. This study applies the SNP targets developed from Chapter 4 to populations of A. cryptoceras, asking whether two recognized subspecies are genetically distinct, and searching for the origin of populations that are morphologically intermediate between the two. A total of 54,673 SNPs were found on 7372 contigs, across 96 individuals from ten populations. Principal component analysis and measures of allelic differentiation indicate a clear disjunction between subspecies cryptoceras and davisii (F[subscript ST] = 0.092 between geographic regions). For intermediate populations, estimates of hybrid index below 0.25 and measures of allelic diversity and private alleles, argue against a hybrid origin due to secondary contact, and instead support their origin as stepping stone populations during expansion along a southern corridor from east to west

A dopaminergic switch for fear to safety transitions

Overcoming aversive emotional memories requires neural systems that detect when fear responses are no longer appropriate so that they can be extinguished. The midbrain ventral tegmental area (VTA) dopamine system has been implicated in reward and more broadly in signaling when a better-than-expected outcome has occurred. This suggests that it may be important in guiding fear to safety transitions. We report that when an expected aversive outcome does not occur, activity in midbrain dopamine neurons is necessary to extinguish behavioral fear responses and engage molecular signaling events in extinction learning circuits. Furthermore, a specific dopamine projection to the nucleus accumbens medial shell is partially responsible for this effect. In contrast, a separate dopamine projection to the medial prefrontal cortex opposes extinction learning. This demonstrates a novel function for the canonical VTA-dopamine reward system and reveals opposing behavioral roles for different dopamine neuron projections in fear extinction learning

The Edinger-Westphal-lateral septum urocortin pathway and its relationship to alcohol consumption

Identifying and characterizing brain regions regulating alcohol consumption is beneficial for understanding the mechanisms of alcoholism. To this aim, we first identified brain regions changing in expression of the inducible transcription factor c-Fos in the alcohol-preferring C57BL/6J (B6) and alcohol-avoiding DBA/2J (D2) mice after ethanol consumption. Drinking a 5% ethanol/10% sucrose solution in a 30 min limited access procedure led to induction of c-Fos immunoreactivity in urocortin (Ucn)-positive cells of the Edinger-Westphal nucleus (EW), suppression of c-Fos immunoreactivity in the dorsal portion of the lateral septum (LS) of both strains of mice, and strain-specific suppression in the intermediate portion of the LS and the CA3 hippocampal region. Because the EW sends Ucn projections to the LS, and B6 and D2 mice differ dramatically in EW Ucn expression, we further analyzed the Ucn EW–LS pathway using several genetic approaches. We find that D2 mice have higher numbers of Ucn-immunoreactive processes than B6 mice in the LS and that consumption of ethanol/sucrose in the F2 offspring of a B6D2 intercross positively correlates with Ucn immunoreactivity in the EW and negatively correlates with Ucn immunoreactivity in the LS. In agreement with these findings, we find that alcohol-avoiding male B6.D2Alcp1 line 2.2 congenic mice have lower Ucn immunoreactivity in the EW than male B6.B6 mice. Finally, we also find that HAP mice, selectively bred for high alcohol preference, have higher Ucn immunoreactivity in EW, than LAP mice, selectively bred for low alcohol preference. Taken together, these studies provide substantial evidence for involvement of the EW–LS Ucn pathway in alcohol consumption

The use of chloroplast genome sequences to solve phylogenetic incongruences in Polystachya Hook (Orchidaceae Juss)

Background: Current evidence suggests that for more robust estimates of species tree and divergence times, several unlinked genes are required. However, most phylogenetic trees for non-model organisms are based on single sequences or just a few regions, using traditional sequencing methods. Techniques for massive parallel sequencing or next generation sequencing (NGS) are an alternative to traditional methods that allow access to hundreds of DNA regions. Here we use this approach to resolve the phylogenetic incongruence found in Polystachya Hook. (Orchidaceae), a genus that stands out due to several interesting aspects, including cytological (polyploid and diploid species), evolutionary (reticulate evolution) and biogeographical (species widely distributed in the tropics and high endemism in Brazil). The genus has a notoriously complicated taxonomy, with several sections that are widely used but probably not monophyletic. Methods: We generated the complete plastid genome of 40 individuals from one clade within the genus. The method consisted in construction of genomic libraries, hybridization to RNA probes designed from available sequences of a related species, and subsequent sequencing of the product. We also tested how well a smaller sample of the plastid genome would perform in phylogenetic inference in two ways: by duplicating a fast region and analyzing multiple copies of this dataset, and by sampling without replacement from all non-coding regions in our alignment. We further examined the phylogenetic implications of non-coding sequences that appear to have undergone hairpin inversions (reverse complemented sequences associated with small loops). Results: We retrieved 131,214 bp, including coding and non-coding regions of the plastid genome. The phylogeny was able to fully resolve the relationships among all species in the targeted clade with high support values. The first divergent species are represented by African accessions and the most recent ones are among Neotropical species. Discussion: Our results indicate that using the entire plastid genome is a better option than screening highly variable markers, especially when the expected tree is likely to contain many short branches. The phylogeny inferred is consistent with the proposed origin of the genus, showing a probable origin in Africa, with later dispersal into the Neotropics, as evidenced by a clade containing all Neotropical individuals. The multiple positions of Polystachya concreta (Jacq.) Garay & Sweet in the phylogeny are explained by allotetraploidy. Polystachya estrellensis Rchb.f. can be considered a genetically distinct species from P. concreta and P. foliosa (Lindl.) Rchb.f., but the delimitation of P. concreta remains uncertain. Our study shows that NGS provides a powerful tool for inferring relationships at low taxonomic levels, even in taxonomically challenging groups with short branches and intricate morphology.Swedish Research Council [B0569601]; European Research Council under the European Union's Seventh Framework Programme (ERC) [331024]; Swedish Foundation for Strategic Research; Knut and Alice Wallenberg Foundation; Biodiversity and Ecosystems in a Changing Climate programme; Wenner-Gren Foundations; David Rockefeller Center for Latin American Studies at Harvard University; Faculty of Science at the University of Gothenbur

Intragenomic polymorphisms among high-copy loci: a genus-wide study of nuclear ribosomal DNA in Asclepias (Apocynaceae)

Despite knowledge that concerted evolution of high-copy loci is often imperfect, studies that investigate the extent of intragenomic polymorphisms and comparisons across a large number of species are rarely made. We present a bioinformatic pipeline for characterizing polymorphisms within an individual among copies of a high-copy locus. Results are presented for nuclear ribosomal DNA (nrDNA) across the milkweed genus, Asclepias. The 18S-26S portion of the nrDNA cistron of Asclepias syriaca served as a reference for assembly of the region from 124 samples representing 90 species of Asclepias. Reads were mapped back to each individual’s consensus and at each position reads differing from the consensus were tallied using a custom perl script. Low frequency polymorphisms existed in all individuals (mean = 5.8%). Most nrDNA positions (91%) were polymorphic in at least one individual, with polymorphic sites being less frequent in subunit regions and loops. Highly polymorphic sites existed in each individual, with highest abundance in the “noncoding” ITS regions. Phylogenetic signal was present in the distribution of intragenomic polymorphisms across the genus. Intragenomic polymorphisms in nrDNA are common in Asclepias, being found at higher frequency than any other study to date. The high and variable frequency of polymorphisms across species highlights concerns that phylogenetic applications of nrDNA may be error-prone. The new analytical approach provided here is applicable to other taxa and other high-copy regions characterized by low coverage genome sequencing (genome skimming).This is the publisher’s final pdf. The published article is copyrighted by the author(s) and published by PeerJ. The published article can be found at: https://peerj.com/.Keywords: Evolutionary studies, Bioinformatics, Intragenomic polymorphism, Plant science, Asclepias, Genomics, High-copy, Partial SNP (pSNP), ITS, Genetics, Concerted evolution, Nuclear ribosomal DNA (nrDNA), Intra-individual site polymorphism, Genome skimming, 2IS

Hyb-Seq: Combining target enrichment and genome skimming for plant phylogenomics

PREMISE OF THE STUDY: Hyb-Seq, the combination of target enrichment and genome skimming allows simultaneous data collection for low-copy nuclear genes and high-copy genomic targets for plant systematics and evolution studies. METHODS AND RESULTS: Genome and transcriptome assemblies for milkweed (Asclepias syriaca) were utilized to design enrichment probes for 3385 exons from 768 genes (>1.6 Mbp) followed by Illumina sequencing of enriched libraries. Hyb-Seq of twelve individuals (ten Asclepias species and two related genera) resulted in at least partial assembly of 92.6% of exons and 99.7% of genes and an average assembly length >2 Mbp. Importantly, complete plastomes and nrDNA cistrons were assembled using off-target reads. Phylogenomic analyses demonstrated signal conflict between genomes. CONCLUSIONS: The Hyb-Seq approach enables targeted sequencing of thousands of low-copy nuclear exons and flanking regions, as well as genome skimming of high-copy repeats and organellar genomes, to efficiently produce genome-scale datasets for phylogenomics.This is an author's peer-reviewed final manuscript, as accepted by the publisher. The article is in press and will be published in Applications in Plant Sciences, Vol. 2, no. 9, September 2014.Keywords: Genome skimming, Hyb-Seq, Target enrichment, Phylogenomics, Species tree, Nuclear lociKeywords: Genome skimming, Hyb-Seq, Target enrichment, Phylogenomics, Species tree, Nuclear loc

Two Functionally Distinct Serotonergic Projections into Hippocampus

Hippocampus receives dense serotonergic input specifically from raphe nuclei. However, what information is carried by this input and its impact on behavior has not been fully elucidated. Here we used in vivo two-photon imaging of activity of hippocampal median raphe projection fibers in behaving male and female mice and identified two distinct populations: one linked to reward delivery and the other to locomotion. Local optogenetic manipulation of these fibers confirmed a functional role for these projections in the modulation of reward-induced behavior. The diverse function of serotonergic inputs suggests a key role in integrating locomotion and reward information into the hippocampal CA1

eDNA as a tool for identifying freshwater species in sustainable forestry: A critical review and potential future applications

Environmental DNA (eDNA) is an emerging biological monitoring tool that can aid in assessing the effects of forestry and forest manufacturing activities on biota. Monitoring taxa across broad spatial and temporal scales is necessary to ensure forest management and forest manufacturing activities meet their environmental goals of maintaining biodiversity. Our objectives are to describe potential applications of eDNA across the wood products supply chain extending from regenerating forests, harvesting, and wood transport, to manufacturing facilities, and to review the current state of the science in this context. To meet our second objective, we summarize the taxa examined with targeted (PCR, qPCR or ddPCR) or metagenomic eDNA methods (eDNA metabarcoding), evaluate how estimated species richness compares between traditional field sampling and eDNA metabarcoding approaches, and compare the geographical representation of prior eDNA studies in freshwater ecosystems to global wood baskets. Potential applications of eDNA include evaluating the effects of forestry and forest manufacturing activities on aquatic biota, delineating fish-bearing versus non fish-bearing reaches, evaluating effectiveness of constructed road crossings for freshwater organism passage, and determining the presence of at-risk species. Studies using targeted eDNA approaches focused on fish, amphibians, and invertebrates, while metagenomic studies focused on fish, invertebrates, and microorganisms. Rare, threatened, or endangered species received the least attention in targeted eDNA research, but are arguably of greatest interest to sustainable forestry and forest manufacturing that seek to preserve freshwater biodiversity. Ultimately, using eDNA methods will enable forestry and forest manufacturing managers to have data-driven prioritization for conservation actions for all freshwater species

Capturing variation in Lens (Fabaceae): Development and utility of an exome capture array for lentil

Premise of the Study: Lentil is an important legume crop with reduced genetic diversity caused by domestication bottlenecks. Due to its large and complex genome, tools for reduced representation sequencing are needed. We developed an exome capture array for use in various genetic diversity studies. Methods: Based on the CDC Redberry draft genome, we developed an exome capture array using multiple sources of transcript resources. The probes were designed to target not only the cultivated lentil, but also wild species. We assessed the utility of the developed method by applying the generated data set to population structure and phylogenetic analyses. Results: The data set includes 16 wild lentils and 22 cultivar accessions of lentil. Alignment rates were over 90%, and the genic regions were well represented in the capture array. After stringent filtering, 6.5 million high-quality variants were called, and the data set was used to assess the interspecific relationships within the genus Lens. Discussion: The developed exome capture array provides large amounts of genomic data to be used in many downstream analyses. The method will have useful applications in marker-assisted breeding programs aiming to improve the quality of cultivated lentil

Phylogenetic marker development for target enrichment from transcriptome and genome skim data: the pipeline and its application in southern African Oxalis (Oxalidaceae)

Phylogenetics benefits from using a large number of putatively independent nuclear loci and their combination with other sources of information, such as the plastid and mitochondrial genomes. To facilitate the selection of orthologous low‐copy nuclear (LCN) loci for phylogenetics in nonmodel organisms, we created an automated and interactive script to select hundreds of LCN loci by a comparison between transcriptome and genome skim data. We used our script to obtain LCN genes for southern African Oxalis (Oxalidaceae), a speciose plant lineage in the Greater Cape Floristic Region. This resulted in 1164 LCN genes greater than 600 bp. Using target enrichment combined with genome skimming (Hyb‐Seq), we obtained on average 1141 LCN loci, nearly the whole plastid genome and the nrDNA cistron from 23 southern African Oxalis species. Despite a wide range of gene trees, the phylogeny based on the LCN genes was very robust, as retrieved through various gene and species tree reconstruction methods as well as concatenation. Cytonuclear discordance was strong. This indicates that organellar phylogenies alone are unlikely to represent the species tree and stresses the utility of Hyb‐Seq in phylogenetics