Proposal Draft Jackie Lungmus Title Comparative Histology of Lagomorph and Sciuridae Long Bones Across an Elevation Gradient.

Abstract Further research is needed on the influence of oxygen level on the development and histological characteristics of mammalian bones. This research will work to identify and analyze the influence of elevation on the characteristics of long bones from the order Lagomorpha and the family Sciuridae. Data will be collected on vasculature, bone thickness, primary osteon diameter, and growth rate. The methods are classic histological bone preparation, then analysis through a photographic microscope. Data will be collected using a variety of computer software, from which it will be analyzed using statistical methodology. Limitations fall mainly within the degree to which one can make assumptions about growth patterns across the class Mammalia. The implications of this research are possible inference about the universal effects of elevation (or as a proxy, oxygen level) on mammalian bones and thus possibly human bone, increasing our broad level understanding on how an organisms habitat influences bone growth. Research will be conducted on a timeline of 13 months. Keywords: osteology, histology, lagomorpha, elevation Introduction There have long been a variety of gaps in the literature on the physical influences of the environment on the mammalian body. For example, it is only weakly understood how oxygen level and hypoxia may influence the osteology of mammalian bones. My research will work to identify and analyze the influence of elevation on the microstructure of long bones of the order Lagomorpha and the family Sciuridae. This will hopefully allow for possible assumptions across a variety of species, and provide a better grasp of environmental effects on bones, thus increasing the scientific knowledge on how the environment can influence the characteristics of an organism. This project will be conducted using classic bone histology as the primary methodology. Histology is the study of the microscopic anatomy of animals and plant tissue. This study will concentrate on bone, and generally speaking focuses on the microscopic details within a section of bone around a hundred microns thick. Bone histology offers a unique lens through which the analysis of bones can be conducted, and allows for an understanding of what is taking place within the microstructure of bones by analyzing the bone on a cellular level. This allows for a much more complete understanding of how an animals osteology is changing than can be accomplished by looking at macrostructure alone. Oxygen plays a fundamental role in every aspect of mammalian biology, however it is often overlooked as a possible agent of evolution. The influences and pressures that can be placed upon an organism by oxygen in the environment has only recently began to be explored. Elevation is the most obvious proxy for oxygen level, and animals are often !"

exposed to changes in oxygen level associated with changes in altitude. Environmental atmospheric hypoxia, as defined by Ramirez et al., is a state of deficiency in oxygen availability to body tissues. By conducting a comparative bone histology project evaluating animals from different elevations, and thus animals that were subject to different levels of environmental atmospheric hypoxia, the influences of oxygen level on the osteology of non-captive small mammals can be explored. Research Objectives The specific research question we aim to answer is how does elevation affect the characteristics of the femora of Lagomorpha and Sciuridae? The over-arching goal of this research project is to better understand the effects of hypoxia on growth and various discreet characteristics of the femora of Lagomorpha and Sciuridae. By focusing on this order and family we will attempt to allow for possible assumptions across a variety of species, and provide a better grasp of environmental effects on bones. Specifically this will enhance the scientific understanding of the environments influence on bone growth and development, and will thus strengthen the types of inferences that can be made about an organisms life history patterns, based on its osteology. We hypothesize that as elevation increases the femora will increase in robusticity, as well as increase in overall vascularity, probably through both an increase in percent vascularity and an increase in primary osteon size. Literature Review The effect of metabolism on bones has a robust literature, and it is understood that higher metabolic rates can result in increased rate of bone formation but a decrease in overall bone robusticity (Kohler et al., 2012; Plochoki, 2008; Arnett et al., 2003). Similarly, it is established that larger mass is associated with lower metabolic rates (Ramirez, 2007). These studies have examined metabolic rate in terms of total bone growth and the development of lines of arrested growth. More work has been done looking at blood cells in bones under different circumstances, and it has been observed that diving animals, who are subject to extreme environmental hypoxia, have increased 02 storage across their bodies, and red blood cells mass can be positively correlated with depth (Ramirez, 2007). Studies done so far on the effects of hypoxia focus mostly on the mass and rate of flow of blood vessel and red blood cell uptake throughout the organism (Russell & Dombkowski, 2008; Holland & Forster, 1965), but little work has been done on hypoxia due to elevation increase and any research on this that has been conducted has focused primarily on the dimensions of red blood cells (Yamaguchi et al., 1986) as opposed to other histological characteristics that have to do with growth. Research that has been conducted on hypoxia in relation to bones up to this point has looked at the relationship of bone remodeling and oxygen uptake, focusing on blood flow within the bone (Sim et al., 1970), along with some work on the role of hypoxia as a stimulator to osteoclast formation and the resorption of bone (Arnett et al., 2003). However, there appears to be no comparative work done within and across species focusing on the changes of discreet characteristics of mammalian limb bones due to atmospheric environmental hypoxia.

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Overall, science has historically been very interested in the effects and influence on the environment on the phenotype of an organism, especially in relation to bone, and there is a good comparative literature on the nature of mammalian histology (Enlow & Brown, 1956; Currey, 2003; Polk et al., 2000). Force, such as speed, gait (Rubin & Lanyon, 1982), structural adaptation (Ruff & Runestad, 1992), and tapering in mammalian limb bones (Lieberman et al., 2003) have been looked at in a comparative nature, but not much has been explored beyond those few forces. In other areas of the field there has been a new emphasis on the role of oxygen as a major driving force in evolution (Berner et al. 2007). Hypoxia is increasingly being understood to have an influence on multiple levels of an organisms biology. Researchers have been looking at how extant animals have successfully adapted to hypoxia in order to infer how they may have been able to do so in the past as well (Ramirez, 2006), specifically in relation to the Permian-Triassic mass extinction (Huey & Ward, 2005). More comparative work on mammalian limb bones needs to be conducted to look at discreet changes in osteology and bone histology, in order to better understand how hypoxia and other environmental effects may influence bone development and growth. Methods For the purposed of this study we chose to focus on two groups of small mammals. Lagomorpha is the order that includes rabbits and pika, and Sciuridae is the family name for squirrels, chipmunks, and prairie dogs. Lagomorpha is a relatively small order, and the animals that are classified as Lagomorpha do not vary extensively in their anatomy or life-history strategies. However, the order Rodentia that includes the family Sciuridae was considered too broad for this specific study, and so the decision was made to narrow the study group down just to the family Sciuridae. It is also worth noting that these were two groups of animals that had a substantial collection housed at the Burke Museum of Natural History, making destructive research on the specimens much more justifiable. In addition, small mammals were chosen as the focus of the project because it would allow for several important variables to be controlled. The process of bone remodeling can be a large inconvenience when attempting to study the microstructure of animal bone and usually needs to be taken heavily into consideration. However, the bones of small mammals do no undergo a significant amount of remodeling throughout their lifespan, and thus the issues associated with remodeling can be avoided. In addition, Lagomorpha and Sciuridae all have small home-ranges, and thus are subject to the same level of environmental atmospheric hypoxia for their entire lives. The main independent variable for in this study is elevation, and changes in histology will be analyzed and compared as the elevation at which the animals lived changes. To understand the changes taking place we will be look specifically at bone robusticity, vasculature, and general bone size as well as primary osteon diameter. Sample size will be a minimum of 10 Lagomorpha femora and 10 Sciuridae femora collected from the United States and in Central Asia, and obtained from the collections of the Burke Museum of Natural History. Animals were noncaptive, and all data collection will be done on skeletally mature femora. For purposed of the study elevation will be analyzed both as discreet points and in categorizations. Femora where chosen as the focus

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bone due to their large size and certain characteristics of femora, such as the long and straight single shaft, makes them ideal for histological study. Bone robusticity will be analyzed looking at K area, which is measured as a ratio of the inner cavity of the bone to the total bone area. This results in a value that represents the amount of area taken up by the bone wall, thus the robusticity. By comparing K-area across specimens we will be able to see if bone thickness changes across elevations. Data on vasculature is collected by thresholding images of the histological specimens and taking percentage measurements of ten areas within the bone wall. This results in ten data points that are representative of how much of the measured bone area is devoted to transporting blood through the bone. By taking an average across 10 values that come from a variety of spots on the entire bone we can get an average for vascularity within the specimens femora. Primary osteon diameter is measured by eye using a simple computer program. 10 primary osteons from across the entire bone wall, measured across their smaller diameter to ensure conservative results are then averaged to provide an estimate of the primary osteon size within the femur. Data will be collected using a microscope and computer programming to measure and record data. After it is collected it will be compiled and analyzed using various statistical methods. Through these methods we will look at how different character states change or do not change as we move across elevation gradients and between discreet elevation points. To allow for as much generalization as possible we will be comparing the same feature within orders at different heights, and will be looking to see changes in two separate types of small mammal. Significance The significance of my project relates primarily to the discipline of bone histology and the larger fields of evolutionary biology and ecology. If significant results are found, there are large-scale implications for the way we understand how the environment can influence the development of an organism, in this case specifically how elevation is affecting the femora of small mammals. This would influence our larger understanding of how animals develop, and how individual families or orders can change across extremely different environments and under different environmental pressures, in this case the amount of oxygen in the enviroment. In addition, even if there are no significant results that come out of the project, the data collected can be useful for future histological studies on small mammals that are being conducted by other scientists. Similarly, the histological slides will be housed under the Washington State Burke Museum of Natural History, and will thus be available to other researchers for further study. Limitations Possible limitations include the ability to make larger generalizations to higher orders from Lagomorpha and Sciuridae. There is also a chance that an N of 10 may be small for making analysis, and this could be exacerbated by the sole use of femora. Finally, while I am confident in the elevation data recorded for the specimens, there is the possibility of a certain degree of human error present with the recording of elevation data that could have taken place when the specimens were collected. The timeline for this %"

project is 12 months. In July the preparation and mounting of specimens will be completed, in August through October time will be spent collecting the data. November and December will be primarily analyzing the data, and finally January and February of 2014 will be for the writing of the paper. The plan is for completion by March of 2014. References
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