ORAL PRESENTATIONS *Presenter SESSION I

1) Shoaling preferences in juvenile convict affected by group composition Theresa Marlin* and Joseph M. Leese; DeSales University (Undergraduate)

Shoaling is a widespread behavior found in diverse taxa of marine and freshwater . Its ubiquity is presumably due to the number of fitness benefits that it confers, including protection from predators and increased foraging efficiency. The potential costs, however, are also substantial and likely change during ontogeny. For instance, if a fish’s shoal mates are larger, the individual may suffer reduced fitness from competition when foraging as a group and a smaller individual may be more likely to suffer predation if an attack were to occur. To explore these effects, juvenile convict shoaling patterns were studied with respect to three factors: shoal size, age of individuals in a shoal, and size of a focal individual. In each trial, a focal fish was presented with a choice of two shoals. Shoals differed in size (one-to-one or three-to-one ratio) and age composition (juveniles or adults). The data suggest that focal individuals prefer to associate with larger shoals, and spend more time shoaling when shoals are comprised of juveniles rather than adults. By studying the interaction among these three factors, a clearer understanding can be gained regarding ontogenically-dependent variations in shoaling patterns, as well as other social behaviors.

2) How the number and sex of parents present during intruder events affects the defense and behavior of offspring: a field study of the biparental in Lake Xiloá, Nicaragua Layla Al-Shaer*, Murray Itzkowitz; Lehigh University (Graduate)

The convict cichlid ( siquia) is a serially monogamous, biparental fish endemic to streams and lakes in Central America. During the day, fry emerge from their nests and move around the habitat foraging in a dense shoal while being shepherded by their parents. This leaves the fry exposed, and successful parents must be able to exclude predators from the immediate vicinity while also influencing the movement and antipredator behavior of their fry. Alarmed parents will sometimes "call" their offspring by using subtle fin flicks, causing the fry shoal to tighten, descend closer to the substrate and move towards their parents. Whether or not both parents are equally effective, or necessary, when it comes to defending against intruders and manipulating their fry's antipredator behavior is unknown. Although both parents are capable of performing all parental care duties, there are known sex differences in preferred parental roles, with females typically spending more time in direct contact with the offspring than males. To gain insight into how each parent and their offspring responds to intruders, a field study was conducted on convict cichlids in Lake Xiloá, Nicaragua. Preliminary analysis of the data suggests that varying the number and sex of parents present during intruder events influences the antipredator behavior of both adults and offspring.

1

3) The influence of sex-ratio on reproductive success in the sheepshead minnow (Cyprinodon variegatus) Timothy Paciorek*, Michael Kuchka, and Murray Itzkowitz, Lehigh University (Graduate)

Male pupfish (Cyprinodontidae) utilize several mating strategies in order to increase reproductive success. Dominant males establish territories in order to attract promiscuous females while also defending territories from neighboring males. Subordinate males lacking the ability to hold territories engage in sneaker tactics, attempting to steal mating opportunities from dominant males. Since several pupfish species live in confined habitats, the success of each strategy may depend on the amount of available space to establish territories and proportion of individuals present. This project aimed to determine whether alterations to sex-ratio affected the ability of territorial and non-territorial male sheepshead minnows (Cyprinodon variegatus) to father eggs. Artificial pools were established with a 1:2 male to female sex ratio and four territories for males to utilize. Territorial males were identified based on their aggression levels. Eggs were collected from each territory over two days and matched to potential fathers using microsatellite markers. Territorial males fathered significantly fewer eggs compared to non-territorial males throughout the entire pool. Territorial and non-territorial males showed no difference in eggs fathered within the territorial male’s territory. This study demonstrates how territorial male reproductive success may be limited compared to non-territorial males, which may influence future populations of this species.

4) Do Conspecific or Heterospecific Fish Affect Territorial Male Reproductive Success in the Leon Springs Pupfish (Cyprinodon bovinus)? Andrew Bloch*; Lehigh University (Graduate)

Using the critically endangered Leon Springs pupfish (Cyprinodon bovinus), this study examined whether male mating behavior was affected by the presence of either conspecific or heterospecific individuals in a male’s territory. This study shows that females prefer to spawn in territories that contain another female, which suggests that female mate choice copying may exist in this system.

5) A Test of Cognitive Ability as a Reproductive Isolating Barrier Michael McQuillan*, Timothy C. Roth, and Amber M. Rice; Lehigh University (Graduate)

Identifying the barriers that prevent gene flow between closely related species is a fundamental goal in evolutionary biology. A potential reproductive isolating barrier that has not yet been tested is cognitive ability. Cognitive traits, such as spatial memory and learning ability, are important for fitness and survival, especially in ‘scatter hoarding’ species. Scatter hoarding species will cache, or store, hundreds of food items throughout the habitat, and must rely on spatial memory to accurately retrieve caches. This ability is important for survival in harsh or unpredictable environments. Here, using a population of hybridizing, scatter-hoarding songbirds (black- capped and Carolina chickadees), I test the hypothesis that hybrids are cognitively deficient relative to pure species individuals. Specifically, I test the spatial memory and learning abilities of pure species and naturally occurring hybrids using a set of behavioral experiments in an outdoor aviary. My preliminary data suggest that hybrids are cognitively deficient relative to pure species individuals. While much work has focused on identifying the genetic underpinnings of hybrid dysfunction in controlled laboratory environments, almost nothing is known about the effects of hybridization on cognition, particularly in wild hybrids. Understanding these potential sources of selection against hybrids is crucial if we are to fully understand how new species are formed.

2

SESSION II

6) Food preference and feeding rates in a native and invasive species of terrestrial slug Melissa Mayol* and Erika V. Iyengar; Muhlenberg College (Undergraduate)

Terrestrial slugs may seem like just slimy pests, but they are important in nutrient cycling and maintaining a healthy habitat, especially in the temperate rainforest of the Pacific Northwest. Global climate change may alter the food sources and habitat of these slugs, exacerbating competition. Last summer we conducted experiments on the two largest and most common terrestrial slugs on San Juan Island, Washington state: the native species Ariolimax columbianus and the invasive species Arion rufus. The invader has multiple color morphs, but jet-black and chocolate brown morphs dominate. We studied the slugs’ food preferences among white button mushrooms, lichen, sword fern, and pathfinder. All slugs preferred white button mushrooms. However, in a follow-up experiment where we oven-dried the mushrooms and soaked the lichen, all the slugs now preferred hydrated lichen to dried mushrooms. Thus, the moisture content of food may be more important than taste and nutrition. Additionally, we tested the effect of temperature (4°C, 10°C, 20°C) on feeding rates, but this relationship was complicated, perhaps because moisture levels confounded weak temperature effects. This summer, we will conduct similar experiments in the field in the complex array of natural scent and habitat cues for comparison. We will also explore the effects of salinity on habitat preference. These data will be useful in predicting how the two slug species, and perhaps their interactions, will react to global climate change, as food sources, moisture levels, temperatures, and soil salinities may alter.

7) "Agar-based diets for assessing nutritional value of sediment ingestion: the picky-eater sea urchin conundrum!" Dana I. Grieco*; Michael P. Russell; Villanova University (Undergraduate)

The purple sea urchin, Strongylocentrotus purpuratus, is an abundant member of marine communities on the west coast of North America. It typically resides in “pits” in rocky intertidal substrata. Previous studies showed that urchins excavate pits and that erosion rates on different types of rocks – sandstone, mudstone, and granite – vary from 5 years to over a century. These studies also showed that urchins ingest the rock as they erode it and urchins that ingest rock grow faster. However, these studies could not separate ingestion of the sedimentary rock (which has an organic component) from the microbial “biofilm” as the source of additional nutrition. My study attempted to test whether rock ingestion provides nutritional value independent of a natural biofilm. I planned to keep the biofilm constant across all treatment groups by growing all urchins on glass plates and feeding them an agar-based feed. Over four months and many agar-based feed “recipes” later, the urchins still did not consume the feed at an adequate pace for quantifiable growth. In the time following, four different 24-hour feed studies were performed to assess the feeding rates and behaviors of the urchins, and to distinguish the specific issues with the feed. Overall, we found that agar-based diets are not a successful vehicle for assessing nutritional value of sediment ingested by sea urchins. It is important to note this pickiness by such an opportunistic eater, as it exposes the difficulties with agar-based feed that are not readily expressed in literature.nutritional value of sediment ingested by sea urchins. It is important to note this pickiness by such an opportunistic eater, as it exposes the difficulties with agar-based feed that are not readily expressed in literature.

8) Using Pollen-Tube Germination to Examine the Incompatibility System of Schlumbergera xbuckleyi var. Magenta Briana N. Ferguson* and Amy E. Faivre, Cedar Crest College (Undergraduate)

Schlumbergera xbuckleyi var. Magenta is one of many varieties of S. xbuckleyi, a hybrid known as the Christmas cactus. Some species within the Schlumbergera genus have been reported to be self-incompatible, but at least 3 one variety of S. xbuckleyi, has exhibited self-compatibility. Previous studies over three years in our lab on self- and out-crosses of S. xbuckleyi var. Magenta have given mixed results, with neither cross leading to pollen tubes reaching ovules. After pollination, flowers were harvested, stained, and observed using fluorescence microscopy to measure pollen tube germination. The first year flowers were left on the plant for one day following pollination but yielded no significant difference in the distance of pollen tube growth between selfed and outcrossed flowers (t=1.75, d.f.=60, p<0.085). In the second year flowers were collected three days after pollination, and showed no significant difference in pollen tube growth between crosses (t=0.53, d.f.=26, p<0.599). In the third year flowers were left on the plant for seven days, approximately the amount of time until flower dehiscence, and still no significance between crosses was observed, (t=0.43, d.f.=110, p<0.670). Results show variable lengths in pollen tube growth among individual flowers, but when comparisons were made of pollen tube growth among plants, there were no significant differences among plants. These data are important for horticulturalists to enhance the preservation of this variety.

9) The Chemical Ecology of a Hybridizing Chickadee System: Olfaction and Reproductive Isolation Alex Huynh*, Lehigh University (Graduate)

A major question in evolutionary biology asks why species stay separate from one another when hybridization and gene flow can act to bring them together? Pre-mating isolating mechanisms, such as sexual signaling and preference, in hybridizing species are selectively favored when hybrid offspring possess lower fitness, i.e. reinforcement. This leads to reduced gene flow, maintaining species integrity. In songbirds, visual and auditory cues have been well studied modes of communication that have been shown to undergo reinforcement in hybrid zones. However, communication via olfaction has historically been overlooked in birds and its role in the evolution of hybridizing songbirds remains unknown. The aim of this work is to investigate the chemical composition of oils secreted by the black-capped chickadee (Poecile atricapillus), Carolina chickadee (Poecile carolinensis), and their hybrid offspring, which possess reduced fitness. Through principal component analysis of ~60 oil profiles analyzed via gas chromatography mass spectrometry, I show that these two species and their hybrid offspring produce chemically distinct odors, which may act as a signal in mate choice. Future directions for this project include investigating chemically-mediated mate choice (female preference for con- versus hetero-specific male odor) as well as comparing odor and female preferences between sympatric and allopatric populations, i.e. reinforcement. In conclusion, this work begins to investigate how chemical communication mediates the evolution of two hybridizing songbird species, a taxon whose chemical ecology is nearly unknown.

10) Effect of Bisphenol A on Sea Urchin Embryo Spicule Development Michael Hovan*, Muhlenberg College (Undergraduate)

Bisphenol A (BPA) is a monomer used in the production of many industrial polycarbonate plastic products and its use in industry led it to become a prominent environmental pollutant, especially in aquatic environments. It has been demonstrated to be “weakly estrogenic” and to cause developmental malformations at low doses across many organisms including fish and marine invertebrates. It has also been established that Bisphenol A appears to affect the formation of spicules by disturbing the action of the enzyme carbonic anhydrase. This study aimed to determine the effects of environmentally relevant concentrations of BPA on the development of the larval spicules of two species of sea urchins, Lytechinus variegatus and Arbacia punctulata. The concentrations of BPA the urchins were exposed to ranged from 0.1 μg/L to 100 μg/L. As the concentration of Bisphenol A increased, increased malformations were seen in the spicules including decreased thickness, decreased length of aboral arms and inconsistencies in the organization of fenestrations on the arms. This is one of the first studies that has described specific, clear skeletal alterations at sublethal levels of Bisphenol A exposure.

4

11) Reduction of achaete-scute like expression in Nematostella vectensis leads to loss of endodermal neurons Dylan Z. Faltine-Gonzalez*, Jamie Havrilak, Michael J. Layden; Lehigh University (Graduate)

The presence of a central nervous system (CNS) is believed to be a major contributing factor to success of the radiation of bilaterians. Cnidarians, the sister clade to bilaterians, possess a diffuse ectodermal and endodermal nerve net, which is thought to represent the ancestral nervous system. Investigating the cnidarian nerve net provides insight that allows us to infer the organization and molecular mechanisms that patterned the nervous system in the cnidarian-bilaterian ancestor. Thus, aiding in our understanding of the evolution of the central nervous system. Here we aim to understand the role of the proneural transcription factor achaete-scute A (NvashA), which is known to regulate neurogenesis in bilaterians, during neurogenesis in the anthozoan Nematostella vectensis. We constructed a reporter line, NvLWamide::mCherry, which allows us to visualize the effects of NvashA manipulation in vivo. NvLWamide is expressed in a salt and pepper pattern within the aboral domain during gastrulation. Its expression expands in both the ectoderm and endoderm during planula larva stages. This expression pattern was recapitulated in the NvLWamide::mCherry transgenic reporter line. We screened NvLWamide::mcherry expression in NvashA knock down experiments and observed a loss of endodermal neurons, which indicates a role for NvashA in endodermal neurogenesis. This conserved role of NvashA in both the endoderm and ectoderm hints at the evolutionary origin of the CNS from an ancestral ectoderm or endoderm.

SESSION III

12) Quantifying host genome response to gene drive using experimental evolution. Katie Fisher* & G. Lang Lehigh University Department of Biological Sciences (Graduate)

Gene drives—selfish alleles that bias inheritance in their favor—have great potential for solving epidemiological and agricultural problems. Seeding populations with transgenic individuals containing drive elements linked to a trait of interest, such as resistance, has been proposed as strategy to control disease vectors and agricultural pests. The most predictable, and consequently useful, of these elements are targeted endonucleases that recognize and insert themselves into homologous alleles in which they are absent by cleaving a target sequence and allowing homologous recombination to result in gene conversion. Homing endonucleases and CRISPR endonucleases have been demonstrated to be effective knockout drivers with reliable activity and specificity in laboratory populations (Windbichler et al. 2011; DiCarlo et al, 2015; Gantz & Bier, 2015), and thus are possible tools for this type of population level genetic manipulation in the field. Models of gene drive suggest that the gains from distorting inheritance can overcome selection such that even strongly deleterious alleles can spread through drive (Deredec et al. 2008; Unckless et al. 2015). These studies, however, do not consider coevolutionary dynamics between drive elements and host genomes. Homing endonucleases rarely exert deleterious effects in their native hosts, suggesting that coevolution readily occurs in response to drive to lessen intragenomic antagonism. Changes in response to selection on the part of either drive elements or host genomes could potential derail the intended effects of synthetic gene drives. To date, no studies have assessed the efficacy and reproducibility of gene drive in evolving populations or to the predictability of host genome response. Here we propose to directly test the efficacy of gene drive in evolving populations of the budding yeast, Saccharomyces cerevisiae. We have successfully constructed several drive elements. Alleles have been designed so that we can vary the fitness cost of drive across experimental replicates. Drive and target alleles will be fluorescently tagged to quantify their frequencies over time. Whole genome sequencing will be used to characterize reproducibility of host genome responses. We predict that host genomes will evolve in response to

5 drive and that fates of drive elements will be predictable as a function of increasing selection, but unpredictable between replicate populations of the same selective regime due to the stochastic nature of molecular adaptation.

13) Rate of Adaptation and Spectrum of Beneficial Mutations in Haploids and Diploids Daniel Marad*, Greg Lang, Department of Biological Sciences, Lehigh University (Graduate)

Our lab studies the fundamental process of evolution by using the baker’s yeast, Saccharomyces cerevisiae. Previous work from our group suggests that haploid yeast increase in fitness primarily through recessive loss-of- function mutations. To test this hypothesis, we are directly comparing adaptation in haploid versus diploid yeast. This hypothesis makes two predictions: (1) that diploid populations adapt slower and (2) that diploid populations acquire dominant or partially-dominant beneficial mutations. We quantify adaptation using flow cytometry-based competition assays measuring relative growth rates. We perform whole genome sequencing of our evolved populations and analyze the results with a computational pipeline to identify the mutations that are driving adaptation.

We have evolved haploid MAT-a, haploid MAT-α, and diploid MAT-a/α populations in rich medium for 3,000 generations and identified the mutations driving adaptation in these populations via next-generation sequencing. The results confirm that the mutational spectra do indeed differ between haploids and diploids. We have also analyzed the dynamics of adaptation in two diploid populations, providing insight into the mechanisms by which beneficial mutations arise and sweep through diploid populations.

14) Variable Effects of eRpL22 Family Paralogue Depletion on Eye Development in Drosophila melanogaster Brett W. Gershman* and Vassie C. Ware, Department of Biological Sciences, Lehigh University (Graduate)

In D. melanogaster, ribosomal protein (Rp) eRpL22-like has a tissue-specific expression pattern in the testis and eye (Kearse et al., 2011), compared to its ubiquitously expressed paralogue eRpL22. Consistent with a ribosomal role, these paralogues are structurally similar within the rRNA binding domain (C terminus). However, there is considerable sequence divergence within the N terminal domain of each paralogue, suggesting the possibility of divergent roles as components of ribosomes or other pathways. Here we focus on the requirements for eRpL22 paralogues in three stages (3rd instar larva, midpupa, adult) of eye development using an RNAi-mediated Gal4- UAS strategy for tissue-specific paralogue knockdown, followed by immunohistochemistry to determine paralogue localization and score differences in eye development. eRpL22 is ubiquitously expressed, but eRpL22- like localization is tissue/cell-specific, depending on developmental stage. Non-overlapping localization patterns in different cell/tissues are suggestive of specific functional roles for eRpL22 paralogues. Notably, utilizing a single promoter, knockdown of either paralogue results in paralogue specific-phenotypes, suggesting non-redundant (and specialized) roles for paralogues during eye development. Tissue-specific paralogue requirements may originate from subfunctionalization or neofunctionalization of eRpL22 paralogues over evolutionary time. Future work will include attempts to rescue paralogue-specific knockdown phenotypes with overexpression of the cognate paralogue to determine the degree, if any, of functional redundancy in the eRpL22 family. Current and future data will provide further insight into functional diversification and redundancy of duplicated Rp genes.

15) Whole-genome duplication provides a fitness advantage to galactose-evolved yeast Kenneth Brill*, Biological Sciences, Lehigh University (Undergraduate)

During an experiment involving Saccharomyces cerevisiae evolving in glucose, galactose, and alternating between the two media, we noticed that the populations evolving in galactose had two unusual characteristics to their fitness improvement. The strains evolved in galactose had a much higher fitness level than the strains evolving in

6 glucose alone or alternating media, as well as developing this fitness advantage much faster than the other backgrounds. In addition, both haploids and diploids evolved the significant fitness advantage at nearly the same rate, which would not have occurred if the fitness advantage was caused by a recessive beneficial mutation as was expected. The goal was to determine the actual cause. We initially hypothesized that the evolved strains must have acquired a large-effect, dominant beneficial mutation. In order to test this, we crossed the evolved strain with its ancestor. Unexpectedly, the spore viability of these test crosses was poor, suggesting a karyotype change in the evolved strains. To determine if this was a duplication or translocation event, we crossed evolved MATα haploids, MATa/a diploids, and evolved MATa haploids, and used DNA content staining with propidium iodide to determine the ploidy of the evolved strains. The test crosses yielded high sporulation and spore viability, indicating a uniform karyotype change and possibly a whole genome duplication. The PI staining and FACS analysis confirmed the latter, but only in evolved haploids. To determine if this whole genome duplication was a solution to a GAL cluster gene dosage issue, we will construct haploid strains containing a duplication of the GAL cluster alone and a deletion of the GAL cluster and compete these against the reference strains to determine if duplication of the GAL cluster provides the fitness advantage observed in the original evolution experiment.

16) Characterizing patterns of epistasis in experimentally-evolved yeast Sean W. Buskirk*, Ryan Emily Peace, Gregory I. Lang Department of Biological Sciences, Lehigh University Bioengineering Program, Lehigh University (Post-Doc)

Understanding how genes interact is a central challenge in biology. Of particular interest for evolutionary biologists, these genetic interactions (i.e. epistasis) directly impact which evolutionary pathways are accessible. The effect of a specific mutation often depends on the genetic background in which it exists. Current methods for investigating epistatic interactions are only sufficient for studying a limited number of mutations. To this end, we developed a high-throughput method to assay fitness and epistasis then applied this method to twelve populations from a previously published yeast evolution experiment.

By crossing each evolved clone with its ancestor, we generated large pools of recombinant progeny containing random combinations of evolved mutations. For each of the twelve crosses we determined the distribution of fitness among 192 randomly selected recombinant individuals. Next, we assayed fitness for all 111 mutations across our twelve evolved populations by propagating the recombinant pools and measuring the change in allele frequency over time. We developed a theoretical framework that combines these two data sets to reveal the structure of epistasis among the mutations in each population.

Our analysis demonstrates that most interactions between evolved mutations follow the model of “global diminishing returns epistasis” in which the selective advantage of a beneficial mutation and the fitness of the underlying genetic background are negatively correlated. Additionally, this method also succeeded in identifying several new examples of idiosyncratic epistasis. This is the first step towards understanding how epistatic interactions arise in the context of evolution and how epistasis influences the dynamics of adaptation.

7