<p> <b>Chapter 1.</b> We propose a method for predicting the ecological function of host-associated microbes using neural networks trained on a feature space of labeled ecological interactions from the literature. The feature space is constructed over the Laplacian spectral density distributions of the networks formed by linking the phylogenies of the host and microbial clades through their ecological associations. A classifier trained on 51 interactions with known ecology and 100 simulated controls was used predict the ecological function in the microbimes of 14 species of wild-caught cichlid fish from the Lake Tanganyika adaptive radiation observed using 16S rRNA sequencing. </p><p> <b>Chapter 2.</b> Genomic patterns of divergence are examined using whole-genome resequencing of three sympatric cichlid species pairs with similar functional and ecological differentiation but different ages, revealing a signature of genomic divergence. Regions of elevated relative differentiation exhibit increased absolute differentiation. We detect a signature of convergent evolution across all three species pairs. Our results suggest that functional phenotypic differentiation is associated with a signature of genomic divergence. </p><p> <b>Chapter 3.</b> We show that evolutionary innovations can result in competitive inferiority and extinction. The modified pharyngeal jaws of cichlid fishes and several marine fish, a classic example of evolutionary innovation, are not universally beneficial. Analysis of dietary evolution across marine fish lineages reveals that the innovation compromises access to predator niches. This competitive inferiority shaped the adaptive radiation of cichlids in Lake Tanganyika and played a pivotal, previously unrecognized role in the mass extinction of cichlid fishes in Lake Victoria after Nile perch invasion. </p><p> <b>Chapter 4.</b> We map protein sequences from the Global Ocean Survey to protein families and use non-negative matrix factorization (NMF) to approximate linear combinations of ecological components with characteristic functional and site profiles. We identify functional signatures, estimate functional distance between sites, and find that an NMF-filtered measure is more strongly correlated with environmental distance than a comparable PCA-filtered measure. We find that functional distance is more strongly correlated with environmental distance than geographic distance in agreement with prior studies. </p><p> <b>Chapter 5.</b> We describe the unique technical, logistical, organizational, and ethical issues from the 2013 Indigo V Indian Ocean Expedition research cruise from Cape Town, South Africa, to Phuket, Thailand aboard the S/Y <i>Indigo V.</i> An inventory the surface water population of bacterioplankton was collected and basic measurements of ocean physics and chemistry were tabulated. </p><p> <b>Chapter 6.</b> We report on the microbial diversity across the Indian Ocean and a lagoon in the Chagos Archipelago. The community within the lagoon differed from adjacent community despite constant water exchange, driven by photosynthetic cyanobacterium Synechococcus. Enrichment of photosynthesis-related transcripts and nutrient cycling indicate influence of primary production on community structure. A five-fold diurnal increase in viral transcripts within the lagoon suggests concomitant bacteriophage influence. </p><p> <b>Chapter 7.</b> We present a novel ChIP-seq workflow for archaea using <i>Halobacterium salinarum</i> sp. NRC-1 and map binding sites of natively expressed transcription factors. Relative to ChIP-Chip and qPCR, it improves spatial resolution and reduces cost. </p><p> <b>Chapter 8.</b> Pique is a user-friendly, freely licensed ChIP-Seq peak finding application for bacterial and archaeal ChIP-Seq experiments. Output is provided in standardized file formats for manual curation and data exploration. </p><p> <b>Chapter 9.</b> With appropriate handling, 3D printers produce sterile components from non-sterile thermoplastic feedstock without post-fabrication treatment. </p><p> <b>Chapter 10.</b> We present a method for fabricating single-use microtiter plates with volumes calibrated for each sample, allowing the use of multichannel pipettes for general liquid handling operations. Many custom plates can be 3D printed simultaneously, resulting in substantial savings in cost and time. </p><p> <b>Chapter 11.</b> The growth kinetics of 48 strains of building-associated bacteria were measured aboard the International Space Station. One strain, <i> Bacillus safensis</i> JPL-MERTA-8-2, grew 60% better in microgravity. </p><p>

<p> Plants and the insects that feed on them dominate diversity in terrestrial ecosystems: half of all named species are contained within these two groups. Herbivorous insects (herbivores) are abundant and diverse, yet paradoxically, two thirds of insect orders contain no major lineages of herbivores, implying barriers to the evolution of this trophic interaction. How herbivory evolves and why herbivores are so diverse are questions that are key to understanding the processes that have shaped global biodiversity. Yet, most lineages of herbivores are ancient with sister groups either absent or too divergent for a comparative genomic analysis to yield a mechanistic understanding of both their origin and diversification. Many of the exceptions to this pattern are among the Diptera, where lineages such as the leaf-mining drosophilids in the genus <i>Scaptomyza</i> have emerged within the last 10 million years. <i>Scaptomyza</i> is particularly well-suited for identifying the adaptations associated with the evolution of herbivory because it is embedded within the paraphyletic genus <i>Drosophila</i>, which contains species with 25 sequenced genomes, and is closely related to <i>D. melanogaster </i>, the genetic model, and a taxon with one of the most well-studied nervous systems. </p><p> Behavior is thought to be one of the earliest adaptations during the evolution of herbivory and niche shifts in general. Insects undergoing a niche shift likely lose their preferences for their ancestral diet, and also evolve an attraction to novel cues indicative of their new oviposition substrate. Once females lay eggs in a new environment, herbivores must consume the new diet, despite the fact that it may contain aversive chemicals and a different balance of macronutrients compared to the ancestral diet. Using the herbivorous <i> Scaptomyza flava</i> as a model system, the primary aim of my dissertation was to use methods in comparative genomics, chemical ecology, ethology, and neural imaging to characterize the mechanistic basis of behavioral changes associated with the evolution of herbivory in insects. </p><p> Using a comparative genomics approach, I found that targeted gain- and loss-of-function mutations were associated with the evolution of herbivory in the genus <i>Scaptomyza</i>. First, four Odorant (Olfactory) Receptor (OR) genes were lost in herbivorous species of <i>Scaptomyza </i>, which are deeply conserved among microbe-feeding drosophilids. The OR genes lost code for receptors that detect yeast-volatiles and are known to stimulate oviposition, feeding and attraction behaviors in <i>Drosophila </i> species. Consistent with these losses was also a loss of detection sensitivity to ligands of these ORs, specifically short-chain aliphatic esters such as ethyl and propyl acetate, major yeast-produced odorants. <i> S. flava</i> female flies were also unresponsive to volatiles produced by active yeast cultures, in contrast to <i>D. melanogaster</i> flies. </p><p> In contrast to some other specialized lineages of <i>Drosophila </i>, I found no evidence of increased or mass chemosensory gene loss, with one interesting and novel exception. The majority of the genes encoding the Plus-C subfamily of Odorant Binding-like proteins (OBPs) are deleted or pseudogenized in <i>Scaptomyza</i>. Additional conserved cysteine residues that form disulfide bonds that stabilize the tertiary structure characterize this subfamily. Interestingly the extra disulfide bonds in Plus-C OBPs are known to be vulnerable to attack by toxic breakdown products of glucosinolates, isothiocyanates, chemicals that are characteristic of <i>S. flava</i>'s host plants in the mustard family. Other than the loss of OBPs, I found <i> S. flava</i> to have multiple duplications of genes encoding ORs, OBPs, gustatory receptors (GRs) and ionotropic receptors (IRs), some of which showed evidence for positive selection (<i>Or67b, Obp49a, Gr33a, Ir67a</i> and <i>Ir76a</i>). Among receptors expressed in the gustatory system, losses, duplications and genes with selection regime changes were more often orthologs of genes expressed in bitter gustatory neurons in <i>D. melanogaster </i>, especially gustatory sensory neurons with a broad expression of gustatory receptor genes. Changes, such as deletions, duplications and increased amino acid substitution rates, were also found among genes encoding receptors implicated in reproductive behavior including the loss of an anti-aphrodisiac receptor, <i>Gr68a</i>, which could be associated with a switch from males chemically guarding mated females with anti-aphrodisiacs to physical guarding behavior where males remain on the backs of females post-mating. (Abstract shortened by ProQuest.)</p><p>

<p> Nutrient cycling plays a critical role in maintaining biodiversity and ecosystem services in agricultural, urban, and natural lands. However, across landscapes there is substantial unexplained heterogeneity in nutrient cycling. Classic thinking holds that abiotic factors are the source of this spatial heterogeneity with a secondary role of plant biomass. However, recent work suggests that higher trophic levels or variation in traits at the level of plant genotype may also play an important role in structuring nutrient environments. For instance, herbivores may indirectly create heterogeneity in cycling through the induction of chemical and structural changes in plants traits. Phenotypic plasticity due to anti-herbivore defense may then alter nutrient cycling rates by changing the microbial breakdown of plant litter inputs. Alternatively, variation among plant genotypes in the expression of these same traits may overwhelm the influence of phenotypic plasticity on soil processes. Both genetic and environmentally based changes in plant traits have separately been demonstrated to alter soil processes, but their interaction and the relative importance of these sources of variation across local landscapes is unknown.</p><p> I address this question by developing a plant trait-mediated, conceptual framework of nutrient cycling. I then evaluate this framework within an old-field ecosystem by focusing on the dominant plant species, <i>Solidago altissima </i>, and its dominant grasshopper herbivore, <i>Melanoplus femurrubrum </i>, using a combination of lab assays, a greenhouse pot experiment, a field mesocosm experiment, and field surveys. First, I demonstrate that goldenrod individuals exhibit both genotypic variation and phenotypic plasticity in plant defensive trait responses across a nutrient and herbivory gradient in the greenhouse. At low nutrient supply, genotypes tolerate herbivory (inducing plant physiological changes that decrease the negative impact on fitness) while at high nutrient supply, the same genotypes induce a resistance response detectable through lower herbivore growth rates. These environmentally mediated changes in plant trait expression then altered the ability of a common microbial community to decompose senesced litter harvested from the same plants. Induced resistance in the population of genotypes grown at high nutrient levels led to decreased litter decomposition of herbivore legacy litter. In contrast, at low nutrient supply, herbivore legacy litter decomposed more efficiently compared to control litter. This suggests that the interaction between herbivory and nutrient supply could cause context-dependent acceleration or deceleration of nutrient cycling. As a result, trait plasticity may mediate effects of multiple environmental conditions on ecosystem processes in this system.</p><p> I tested this hypothesis using a three-year, raised bed, field experiment examining the effect of plasticity and locally relevant genetic variation on ecosystem processes in a naturalistic setting. Genotype clone clusters were planted in homogenized soil in enclosed cages with varying nutrient supply and grasshopper herbivory. Again, I documented strong genetically and environmentally-based trait variation in plant allocation, growth, and leaf traits. I next explicitly linked these genetic and plastic functional trait changes to concurrent changes in a variety of soil processes (microbially available carbon, plant available nitrogen, nitrogen mineralization potential, and microbial biomass) and litter decomposition rates. Importantly, partitioning functional trait variation into genetic and environmental components improved explanatory power. I also documented potential differences in herbivore effects on "slow" vs. "fast" cycling in soil microbially available C pools. Within both experiments the magnitude of trait variation measured was similar to the variation expressed by individuals across a focal field.</p><p> Taken together, this dissertation demonstrates that plant genotype, herbivores, and nutrients can all modify litter decomposition and other soil processes within ecosystems through differential expression of plant functional traits. Due to the spatially clumped, clonal, and dominant nature of goldenrod, the genetic and herbivory-driven changes documented here could lead to a predictable mosaic of soil process rates across a single old field landscape. This work also highlights the complex interplay between genetically and environmentally-based trait variation in determining population and ecosystem processes within landscapes and improves our understanding of the often-overlooked indirect effects of plant/herbivore interactions on nutrient cycling It suggests that herbivores may shape not only the evolution of plant populations, but also the soil nutrient environment and microbial community in which plants live. This sets up the potential for eco-evolutionary feedbacks between plant defense expression and soil nutrient availability. More broadly, it suggests that biotic factors, in addition to abiotic ones, play a key role in determining local-scale soil nutrient availability patterns and should potentially be accounted for within ecosystem models. These results are particularly salient in a world where anthropogenic nitrogen inputs continue to rise and climate change is predicted to increase herbivory and thus plant defensive trait induction on landscapes. </p>

<p>The aim of this project is to examine the relationships between two approaches to the measurement of life history strategies. The traditional method, termed here the biodemographic approach, measures developmental characteristics like birthweight, gestation length, inter-birth intervals, pubertal timing, and sexual debut. The alternative method under exploration, termed here the psychological approach, measures a suite of cognitive and behavioral traits such as altruism, sociosexual orientation, personality, mutualism, familial relationships, and religiosity. Although both approaches are supported by a large body of literature, they remain relatively segregated. This study draws inspiration from both views, integrating measures that assess developmental milestones, including birthweight, prematurity, pubertal timing, and onset of sexual behavior, as well as psychological life history measures such as the Mini-K and a personality inventory. Drawing on previous theoretical work on the fundamental dimensions of environmental risk, these measures are tested in conjunction with several scales assessing the stability of early environmental conditions, including both &ldquo;event-based&rdquo; measures that are defined with an external referent, and measures of internal schemata, or the predicted psychological sequelae of early events. The data are tested in a three-part sequence, beginning with the measurement models under investigation, proceeding to an exploratory analysis of the causal network, and finishing with a cross-validation of the structural model on a new sample. The findings point to exciting new directions for future researchers who seek to integrate the two perspectives. </p>

A remarkable property of developing organisms is the consistency and robustness within the formation of the body plan. In many animals, morphological pattern formation is orchestrated by conserved signaling pathways, through a process of strict spatio-temporal regulation of cell fate specification. Although morphological patterns have been the focus of both classical and recent studies, little is known about how this robust process is modified throughout evolution to accomodate different morphological adaptations.

The development of cancer from a population of precancerous cells is a rapid evolutionary process. During progression, cells evolve several new traits for survive and proliferation via a few key `driver' mutations. However, these few driver alterations reside in a cancer genome alongside tens of thousands of additional `passenger' mutations. Passengers are widely believed to have no role in cancer, yet many passengers fall within functional genomic elements that may have potentially deleterious effects on the cancer cells. Here we investigate the potential of moderately deleterious passengers to accumulate and alter neoplastic progression. Evolutionary simulations suggest that moderately-deleterious passengers accumulate during progression and largely evade natural selection. Accumulation is possible because of cancer's unique evolutionary constraints: an initially small population size, an elevated mutation rate, and a need to acquire several driver mutations within a short evolutionary timeframe. Cancer dynamics can be theoretically understood as a tug-of-war between rare, strongly-beneficial drives and frequent mildly-deleterious passengers. In this formalism, passengers present a barrier to cancer progression describable by a critical population size, below which most lesions fail to progress, and a critical mutation rate, above which cancers collapse. In essence, cancer progression can be subverted by its own unique evolutionary constraints. The collective burden of passengers explain several oncological phenomena that are difficult to explain otherwise. Genomics data confirms that many passengers are likely damaging and have largely evaded negative selection, while age-incidence curves and the distribution of mutation totals suggests that drivers and passengers exhibit competing effects. These data also provide estimates of the strength of drivers and passengers. Finally, we use our model to explore cancer treatments. We identify two broad regimes of adaptive evolutionary dynamics and use these regimes to understand outcomes from various treatment strategies. Our theory explains previously paradoxical treatment outcomes and suggest that passengers could serve as a biomarker of response to mutagenic therapies. Deleterious passengers are targetable by either (i) increasing the mutation rate or (ii) exacerbating their deleterious effects. Our results suggest a unique framework for understanding cancer progression as a balance between driver and passenger mutations.

<p>In an ever-changing world, evolution is an essential process that may allow organisms to adapt to their environment through natural selection. All evolutionary processes act through a single fundamental medium: genetic variation. Polyploidy, or whole genome duplication, is a major mechanism for evolutionary change because it is both widespread across taxa and results in a proliferation of genetic material that evolution can act upon. The key questions addressed here are: (1) How does chromosome pairing during meiosis in allopolyploids affect the magnitude of genetic variation?, (2) How does the genome of polyploids evolve following formation, and what genetic mechanisms govern this evolution?, and (3) How does genetic and genomic evolution in polyploids affect phenotypic evolution? I use the shy monkeyflower, <italic>Mimulus sookensis</italic>, a tetraploid of hybrid origin between <italic>Mimulus guttatus</italic> and <italic>Mimulus nasutus</italic>, to address these focal questions. In order to develop a foundation to aid in interpretation of my findings, I first investigate the evolutionary history of <italic>M. sookensis</italic>. Chromosome counts establish that <italic>M. sookensis</italic> is indeed an allotetraploid, and a review of taxonomic literature reveals that this species is heretofore undescribed. By analysing the patterns of genetic variation at chloroplast and nuclear loci in <italic>M. guttatus</italic>, <italic>M. nasutus</italic>, and <italic>M. sookensis</italic>, I show that <italic>M. sookensis</italic> has recurrently formed from <italic>M. guttatus</italic> and <italic>M. nasutus</italic>. Crossing experiments within <italic>M. sookensis</italic> indicate that recurrent origins can contribute to genetic diversity without contributing to reproductive isolation among independently arisen polyploid lineages.</p><p>To address my focal questions, I take advantage of an intriguing and striking difference in flower size among <italic>M. sookensis</italic>, <italic>M. guttatus</italic>, and <italic>M. nasutus</italic>. The flowers of <italic>M. sookensis</italic> and <italic>M. nasutus</italic> are small and remarkably similar to one another, while the flowers of <italic>M. guttatus</italic> and diploid and tetraploid F1 hybrids between <italic>M. guttatus</italic> and <italic>M. nasutus</italic> are large and showy. This phenotypic divergence in flower size between <italic>M. sookensis</italic> and <italic>M. guttatus</italic>-like hybrids indicates that small flower size has evolved in <italic>M. sookensis</italic>. Using genetic marker data and phenotypic measurements from synthetic neoallotetraploid <italic>Mimulus</italic>, I demonstrate that there are low levels of fragment loss and phenotypic variation in neoallotetraploids; this suggests that homeologous pairing and recombination following polyploidization is not a major source of genetic variation or phenotypic evolution in <italic>M. sookensis</italic>. Analysis of the whole genome sequence of two <italic>M. sookensis</italic> lines reveals that <italic>M. sookensis</italic> is a fixed heterozygote throughout its entire genome, in that it has retained both a <italic>M. guttatus</italic>-like and <italic>M. nasutus</italic>-like subgenome, neither of which have been removed through homeologous recombination. These subgenomes have been homogenized by widespread gene conversion, and do not appear to have been differentially affected by deletions or deleterious mutations. Finally, to directly characterize the genetic architecture of flower size in <italic>M. sookensis</italic>, I cross a large-flowered synthetic neoallotetraploid <italic>Mimulus</italic> to small-flowered <italic>M. sookensis</italic>. I then employ a novel genotyping-by-sequencing approach to identify quantitative trait loci (QTL) associated with flower size. I find that there is one locus that accounts for a large proportion of phenotypic variation, and four other loci also contribute to flower size variation between the parental lines. Some of these loci co-localize with previously identified loci for flower size in diploid <italic>Mimulus</italic>, while others do not. Altogether, genetic marker data, phenotypic analysis of neoallotetraploids, whole genome sequence data, and QTL mapping data suggest that the genetic variation necessary for flower size evolution was likely caused by both gene conversion and new mutations, but not homeologous recombination. These results suggest that trait evolution in polyploids may be affected by the unique attributes of polyploids, but that new mutations are always an important source of genetic variation, regardless of ploidy level.</p> / Dissertation

<p>Recombination is the process in which genetic material is exchanged between one's homologous chromosome pairs during egg or sperm development (meiosis). Recombination is necessary for proper segregation of chromosomes during meiosis, and also plays a role in purging deleterious mutations, accelerating adaptation, and influencing the distribution of genomic features over evolutionary time. While recombination is clearly an important process, recombination rate is known to vary within and between individuals, populations, and species. Furthermore, what causes this variation remains relatively unknown. Using empirical and sequenced based estimates of recombination rate for the closely related species <italic>Drosophila pseudoobscura</italic> and <italic>Drosophila miranda</italic>, I seek to understand where recombination happens across the genome, to what extent recombination changes between species, and what genomic features are responsible for these changes. These data will deepen our understanding of mechanisms determining the recombination landscape, and shed light on generalized patterns and exceptions of recombination rate variation across the tree of life.</p> / Dissertation

Recent advances in high-throughput, genomic sequencing allow unprecedented insight into the evolution of biodiversity. Chapter 1 of this thesis is a phylogenetic study of 1,145 sequenced loci, isolated using a novel high-throughput sequence capture methodology to address the phylogenetic position of turtles within tetrapods. The results reported here unambiguously place turtles as sister to archosaurs and resolve this long-standing question. Chapter 2 investigates the genetic basis of colorful pigmentation in the Green anole (Anolis carolinensis) by sequencing complete transcriptomes from the green dorsal, white ventral and pink dewlap skin. Anoles comprise an adaptive radiation of more than 400 species and color plays a central role in their ecology and evolution, but little is known about the genetic basis of colorful pigmentation in any vertebrate. This study identified 1,719 differentially expressed genes among the three differently colored tissues. Twenty-three of these genes are involved in melanin, pteridine, and carotenoid pigmentation pathways that contribute to the coloration of anole skin. Identifying candidate genes for colorful pigmentation is a significant advance that opens the field for comparative analysis in other taxa. To determine if the genes identified in Chapter 2 are involved in population divergence and speciation, Chapter 3 investigates the complete genomes of twenty individuals from two closely related subspecies of Anolis marmoratus. While the two subspecies differ markedly in pigmentation, this study found few genetic differences between populations except in five regions of the genome, which together contained 447 genes. Of these genes, only two, melanophilin (mlph) and 'cluster of differentiation 36' (cd36), are associated with pigmentation. The intersection of the genes identified in Chapter 2 and Chapter 3 includes both cd36 and mlph, suggesting that both are involved in divergence of coloration. Cd36 is of particular interest because it regulates the uptake of carotenoid pigments and is an important candidate gene contributing to carotenoid pigmentation. Together, this research demonstrates the power of genomic approaches to address fundamental questions in systematics, micro-evolution, and speciation. The findings bolster the emerging field of phylogenomics and broadly impact future research into the genetic basis of coloration in vertebrates.

Studies have shown that facial sexual dimorphism, ranging on a continuum from very feminine to very masculine, affects observers’ ratings of dominance and threat. These studies, however, have used forced-choice paradigms, in which a pair of faces that have been manipulated to appear more masculine (masculinized) and feminine (feminized) are presented side by side. They are susceptible to demand characteristics, situational aspects of the experiment which produce the desired outcome, because participants may be able to draw the conclusion that faces which appear more masculine should be rated as more dominant. In this dissertation, I evaluated whether facial sexual dimorphism affects observers' threat perceptions in a way that minimizes the confounds caused by demand characteristics. In Experiment 1 and Experiment 2, I presented observers with masculinized and feminized faces individually, rather than in pairs, and for an extremely brief duration (Experiment 1). I predicted that observers would assign higher dominance (Experiment 1) and threat ratings (Experiment 2) to masculinized faces. Observers assigned higher dominance (Experiment 1) and threat (Experiment 2) ratings to masculinized faces, even when they were presented individually, and for as little as 100 milliseconds (ms) (Experiment 1). This would suggest that they can appraise differences in facial sexual dimorphism following very brief exposure. I proceeded to evaluate the effects of facial sexual dimorphism on men’s selective attention, while reducing the effects of demand characteristics, by testing for an attentional bias towards task-irrelevant masculinized men’s faces. In Experiment 3, forty-five men completed a Posner Cueing Paradigm in which they classified shapes, presented either right or left of center screen after a masculinized or feminized man’s face was presented in either the same or opposite location. Participants were faster to classify the shape following the presentation of a masculine face; however, they were not faster when a masculine face cued target position. In Experiment 4, forty-four men completed a Flanker Task in which they judged letter orientation (i.e., upright or upside-down), while ignoring flanking faces. Participants’ RT was not affected by Morph Type (i.e., whether the face was masculinized, feminized or unmodified). In Experiment 5, forty-one men completed a Dot Probe Task. They were presented with two facial photographs of different Morph Types to the left and right of center screen. This was followed by the presentation of a target shape, in the location of one of the faces. Participants’ objective was to classify shape orientation. Facial sexual dimorphism did not affect participants' classification speed. In Experiment 6, I primed participants with images meant to induce fear or arousal before each trial of a Dot Probe Task. Following the presentation of a fear inducing picture, participants RT to classify shapes when a masculinized face cued target position did not differ from when a feminized face cued target position. The two different presentation times did not create different patterns of results, indicating that masculinized faces did not induce either a cueing or inhibitory affect. Overall, my results do not support my hypothesis that men selectively attend to masculinized faces when they are presented as irrelevant information. I conclude by discussing future directions for evaluating the effects that facial sexual dimorphism has on observers’ interpersonal perceptions of threat and dominance, while controlling for the effects of demand characteristics.