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Comparison Of Four Carnivore Metabarcoding Library-Build Protocols
Get PDFDNA metabarcoding is a relatively new molecular tool for dietary analysis of wildlife populations for monitoring and conservation. Metabarcoding involves use of universal primers to amplify a
region of, for example, mitochondrial DNA (mtDNA) that is highly conserved within species and
variable among species (a “barcode”) extracted from an environmental sample. By utilizing
massively parallel sequencing of DNA extracted from predator feces, different prey mtDNA
barcodes can be sequenced simultaneously to reveal dietary patterns. Because predator DNA also
is abundant in their scats, sequences known as “blockers” are sometimes employed to block the
amplification of host mtDNA while allowing prey mtDNA to be amplified preferentially. Before
sequencing, a library must be constructed for each sample. Library construction involves
amplifying the mtDNA barcode region using universal primers and attaching unique identifiers or
indexes to those amplification products. In this study, we designed and tested four library-build
protocols on red fox (Vulpes vulpes) fecal samples. Two protocols used universal primers attached
to sample-specific inline tags, one treated with a blocker and the other without a blocker. The
other two protocols used the universal primer without inline tags, relying on a second step for
index attachment; one was treated with a blocker and the other without a blocker. We tested our
protocols with 3 mock fecal samples (mixtures of prey DNA of known content) and 38 red fox
fecal samples. We evaluated the performance of the four protocols based on total read numbers,
prey read numbers, and the number of species identified. All four protocols successfully identified
some of the prey species from the fox scats. Amplification without inline tag and with blocker
yielded the highest average number of prey reads and species identified. Thus, we conclude that
this protocol was the most efficient of the four
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Before-After-Control-Impact (BACI) Study of Large-scale Wildfire Effects on Deer Diet Using DNA Metabarcoding
Get PDFThe rising presence of wildfires in western US landscapes necessitates a better understanding of how wildlife respond to these disturbances both immediately and as scorched habitats recover. Managing for species, such as deer, requires an understanding of which forage items sustain them over different post-fire phases. We used molecular genetic analysis of feces (metabarcoding) to document changes in deer diet during springs of 2017–2021, spanning before and after a large wildfire (Ranch Fire of 2018) in Northern California. We used 707 pellets collected both on and off the burn area during pre-fire (2 yrs), post-fire (1 yr), and recovery (2 yrs) periods. We predicted that dietary diversity would decrease post-fire in the burn area as herbaceous and shrub layers were reduced, and increase during the recovery period as pioneer species began to recover. We extracted DNA from fecal pellets and sequenced a trnL gene region of the chloroplast genome on a high-throughput platform. We identified 90 plant taxa from an average of ~7,000 sequencing reads per fecal sample. Diet diversity was high in pre-fire and recovery periods in all sites but significantly reduced post-fire in burn sites only (P < 0.001), with diet dominated by a single forage category, oak (Quercus spp). Diet diversity was dominated by shrub taxa pre-fire but composed proportionally more of herbaceous taxa during recovery. Together, these findings point to the central role of oaks in buffering north-coastal California deer from immediate impacts of wildfire, underscoring further the urgency to combat the growing number of threats facing oaks
Divergence, Selection, and Demographic History of Wolves in Eurasia using Genomic Data
Get PDFSpecies are often composed of evolutionarily distinct populations and lineages. Deciphering the evolutionary history of recently diverged species with multiple lineages remains challenging because genealogical discordance is common across the genome. Emerging genomic and statistical tools are allowing unprecedented insight into the evolutionary history of complicated species that have been notoriously difficult to resolve. The gray wolf exemplifies this challenge as it has multiple recognized lineages and has only recently diverged from other canids. In this dissertation, I use genome-wide data to study the evolutionary history of gray wolves in Eurasia, with a focus on investigating the phylogenomics, demographic history, and role of adaptive introgression. For my first chapter, I investigate the genomic distinctiveness of wolves corresponding to two deeply divergent mitochondrial clades restricted to the Indian subcontinent and the Tibetan plateau, respectively. Using the first whole genome sequences of four Indian wolves along with those of two newly sequenced Tibetan wolves and 31 additional canids, I demonstrate Indian and Tibetan wolves are the two most deeply divergent wolf lineages and highly distinct from broadly distributed wolf populations corresponding to the mitochondrial Holarctic clade. Low-recombination regions of the genome provided evidence that the Indian wolf is the most basal wolf lineage, in contrast to the mitochondrial DNA, showing the Tibetan wolf as the most basal lineage. Our findings imply that southern regions of Asia have been important centers for gray wolf evolution and that the Indian wolf represents one of the world’s most endangered and evolutionarily distinct wolf lineages.
My second chapter focuses on distinguishing secondary contact zones and investigating adaptive introgression among wolf lineages in Asia. I used 5 newly and 7 recently sequenced wolf (Canis lupus) genomes from the lowland plains and high-altitude mountains of Pakistan, India, and Kyrgyzstan, along with 79 additional canid genomes, to explore the possibility that adaptive introgression from specialized basal wolf lineages into Holarctic wolves facilitated their range expansion. I detected three narrow secondary contact zones among the widespread Holarctic lineage and the divergent Indian and Tibetan lineages. Within West-Asian Holarctic wolves, I detected several gene regions that were highly differentiated compared to other Holarctic wolves and signals of higher-than expected levels of introgression from Indian or African wolves. Additionally, in the high-altitude Central Asian wolf, I found similar evidence for adaptive introgression from the Tibetan wolf including gene related to hypoxia adaptation in other mammals. Lastly, demographic analyses revealed Indian and Tibetan wolf lineages were likely isolated within separate glacial refugia in Asia while the Holarctic lineage underwent massive expansion events during the late Pleistocene.
In Chapter 3, I use genome-wide data from 5 newly sequenced gray wolves from Pakistan to more robustly investigate the locations of secondary contact zones and admixture among the three wolf lineages at these contact zones. Using a total of 8 wolves from Pakistan and an additional 48 canids, clustering and admixture analyses indicated high proportions of Indian ancestry was present in the lowlands of the Indus plains, Potwar plateau, and Western mountain ranges of Pakistan. Except for small amounts of Tibetan ancestry detected in two wolves from the Karakoram Mountains of northern Pakistan, the Tibetan lineage appeared to end to the east in the Ladakh region of India. These findings clarify the boundaries of the three divergent wolf lineages and highlight the conservation significance of Pakistan’s wolf populations, especially the wolves in Sindh and Punjab that represent the Indian lineage. Overall, this dissertation provides insight into the evolutionary and historical processes that shape genomic divergence and local adaptation in a wild and highly mobile species
Genomic Analysis of Divergence and Secondary Contact in Red Foxes (Vulpes vulpes) and Gray Foxes (Urocyon cinereoargenteus)
Get PDFSpeciation results from the accumulation of genetic differences between lineages over time, which initially decreases and eventually eliminates the probability of gene flow between them (i.e., biological species concept). Additionally, the genomes of natural populations are not only shaped by drift and selection, but also by introgression from closely related taxa following secondary contact. Secondary contact between distinct, yet interfertile, lineages may lead to outcomes ranging from complete unification to formation of stable, narrow hybrid zones permitting low levels of genetic exchange. These stable hybrid zones can be maintained either by pre-zygotic (e.g., behavioral) or post-zygotic (e.g., reduced hybrid fitness) reproductive barriers. Additionally, gene flow following long-term isolation provides opportunities for selective introgression between lineages. The process of speciation is therefore a continuum, and there is regular debate as to the classification status of related lineages that have not yet reached complete reproductive isolation and are instead in the “gray zone” of speciation. Secondary contact between lineages can result from either natural or anthropogenic forces. For example, stable hybrid zones typically arise from the natural expansions and contractions of lineages throughout geologic time due to major climatic fluctuations. Over the past century, however, human translocations have become prevalent, sometimes leading to secondary contact between introduced and native populations of the same species. Here I investigate the dynamics of secondary contact and hybridization between distinct canid lineages, focusing on two different systems, the red fox (Vulpes vulpes) and the gray fox (Urocyon cinereoargenteus). These vary both in the origins of secondary contact (anthropogenic vs. natural range expansion) and in the level of divergence between lineages (late-Pleistocene vs. mid-Pleistocene), making them valuable systems to explore the mechanisms maintaining lineage boundaries and the role of selective introgression in their evolutionIn Chapter 1, I investigated patterns of human facilitated gene flow between two lineages that are >20,000 years divergent. The native Sacramento Valley red fox (SVRF, V. v. patwin) is endemic to the semi-arid region of California’s northern Central Valley. In direct contact with the SVRF range is a population of nonnative red foxes, found primarily in the San Joaquin valley to the south of the native population and in the coastal lowland region to the west. This nonnative population was derived from multiple human translocations of fur-farmed foxes in the early 1900s. Most farmed foxes were originally sourced from eastern Canadian and Alaskan lineages in the late 1800s which were phylogenetically divergent (~20–70 kya) from the SVRF. They were bred in fur farms for several decades prior to their release or escape in California in the mid 1900s. I hypothesized that gene flow was restricted, potentially due to post-zygotic genetic mechanisms, and that some genes originating in nonnative foxes would confer higher fitness in the currently human-dominated landscape and would therefore be selectively introgressed into the native fox population. I sequenced 107 red foxes from the native (n = 59) and nonnative (n = 48) ranges at a mitochondrial fragment and >19,000 loci of the nuclear genome. Observed geographic cline widths were 6.9× (mtDNA) and 14.3× (nuDNA) narrower than expected based on simulations assuming unrestricted gene flow, consistent with the presence of reproductive barriers. Using a Bayesian genomic cline analysis, I identified 10 loci with significantly reduced levels of introgression, several of which were previously associated with reproductive fitness. Consistent with selective introgression, nine loci were identified with significantly elevated levels of gene flow, most of which originated from the nonnative population. Several genes near these outlier regions were potentially associated with adaptation to human dominated landscapes. It should be noted that pre-zygotic factors, such as assortative mating or natal habitat-biased dispersal, also could have contributed to the maintenance of the hybrid zone. Nevertheless, these findings indicate the presence of some form of reproductive barrier between the native and nonnative red fox populations, which enabled the identification of several exceptional genes that were shared at much higher rates than expected by chance. These genes flowed primarily from the nonnative population, for which ancestors had undergone strong selection for a captive environment, to the native population, which only recently (150 years) experienced the conversion of its historical range to a human-dominated landscape. In Chapters 2 and 3 I investigated patterns of divergence and gene flow between two lineages that are ~1 million years divergent, where secondary contact was presumed to be a result of natural range expansion. North American gray foxes are composed of two highly divergent, reciprocally monophyletic lineages in the western and eastern portions of their range. They currently hybridize in a relatively a narrow zone of contact in the southern Great Plains. The narrowness of their hybrid zone indicates either that secondary contact was very recent or, if ancient, that reproductive isolating mechanisms prevent their wholesale unification. Given their vagile nature and the lack of clear physical barriers separating them, we hypothesized that one or both lineages occupied smaller ranges removed from the current zone of contact throughout most of the Pleistocene and achieved contact only recently through a massive Holocene expansion. To investigate this hypothesis, we explored their demographic histories and population structure using a combination of whole-genome and reduced-representation sequencing. Additionally, we characterized the timing and extent of gene flow pulses between western and eastern gray foxes using a local ancestry inference-based approach. In Chapter 2, we used both whole-genome (n = 26) and reduced representation (n = 197) sequencing to contrast the demographic histories of western and eastern gray foxes. Pairwise sequential Markovian coalescent (PSMC) modeling, stairway plots, and summary statistics of eastern and western gray foxes on either side of the contact zone showed contrasting demographic trajectories, with the trajectory of the eastern population declining and the trajectory of the western population increasing for most of their post-divergence history; during the latter portion of the last (Wisconsinan) glacial cycle and most of the Holocene, the eastern trajectory increased and the western trajectory declined. Correspondingly, the eastern lineage exhibited much lower genetic diversity than the western lineage, a cline in diversity consistent with a westward expansion front, and minimal genetic structuring. In contrast, the western lineage exhibited population structure and locally varying demographic histories, reflecting long-term occurrence over a broad region of the continent. The recurrent declines in the eastern population may have kept them both geographically and demographically restricted to the southeast for much of their evolutionary history, resulting in the deep divergence and limited gene flow currently observed between lineages. Additionally, population structure and variable demographic histories within the western lineages may reflect separation in distinct glacial refugia and subsequent gene flow across the western gray fox range. In Chapter 3, I utilized whole genomes of gray foxes (n = 42) from both the western and eastern lineages as well as from the hybrid zone to investigate (1) the timing of secondary contact and genetic exchange, (2) the width of the hybrid zone in the context of this timing, and (3) signatures of selective introgression between lineages. I inferred the timing of admixture pulses using a local ancestry inference-based approach, which was optimized for low-coverage sequencing data. I tested whether observed patterns of admixture were consistent with expectations based on a model assuming no reproductive barriers. I then investigated specific genomic regions that were introgressed across the contact zone at unusually high frequencies, consistent with selective introgression. I identified two distinct pulses of late Holocene and historical admixture. The older pulse of admixture (3,500 YBP) reflected unidirectional gene flow from east to west, likely driven by a major demographic expansion of the eastern gray fox. In contrast, the more recent bi-directional pulse of admixture began approximately 200 YBP, coinciding with major anthropogenic landscape changes. Given the recency of genetic interchange, the narrow widths of the geographic clines provided little insight on the question of reproductive isolation but afforded an opportunity to explore selective introgression. Several genomic regions were identified as candidates for selective introgression and may have been associated with behavioral divergence, mate choice, and olfaction
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Population Structure of California’s Elk: Mapping Genetic Diversity and Hybrid Zones
Get PDFABSTRACT California is the only state or province that contains 3 unique subspecies of North American elk (Cervus canadensis): Roosevelt (C. c. roosevelti), Rocky Mountain (C. c. nelsoni), and tule (C. c. nannodes) elk. Where populations overlap, subspecies can interbreed, creating zones of hybridization. Effective management of these subspecies requires a baseline understanding of each subspecies’ range, identification of hybridization zones, and the ability to monitor and track changes in the genetic diversity and integrity of the subspecies over time. It is also important to monitor the magnitude of actual gene flow resulting from translocations and range expansion. One of the primary goals stated in California’s 2018 Elk Conservation and Management Plan was ensuring long-term persistence of all 3 subspecies (California Department of Fish and Wildlife [CDFW] 2018). To aid in these goals we conducted the most comprehensive genetic assessment of California’s elk to date. During this collaborative study, we created updated range maps for all 3 subspecies, mapped hybridization, characterized the heterozygosity and effective population sizes of subspecies and individual Elk Management Units (EMUs), and analyzed spatial population structure. Tissue, hair, and fecal samples were used for DNA extraction and genotyping of 19 polymorphic microsatellite loci. We obtained genotypes for 1,271 individual elk from California, Nevada, and Oregon. We detected hybridization between Roosevelt and Rocky Mountain elk at a natural contact zone in northern California and identified 2 introduced Rocky Mountain elk populations with little or no introgression from Roosevelt elk. An admixture analysis also revealed the existence of a genetically distinct population of Roosevelt elk in Humboldt County potentially reflecting a remnant population that survived a large decline from overhunting during the 1800s. We detected hybridization between Roosevelt and tule elk in Mendocino County and between tule and either Rocky Mountain or Roosevelt elk in San Luis Obispo County. Tule elk exhibited lower levels of heterozygosity and allelic richness than Rocky Mountain and Roosevelt elk. The Tupman and East Park Reservoir tule elk populations exhibited the lowest levels of heterozygosity and allelic richness. This study provides information that is essential for understanding the genetic landscape of elk in California, and will inform decisions about harvest management, translocations, and landscape-level conservation planning to maintain and enhance connectivity of elk populations where desired. Our study should be followed up with more intensive genomic analyses of inbreeding and admixture, but nevertheless provides a strong foundation for future genetic monitoring and informed management practices
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Get PDFWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
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