DIVERSIFICATION OF SPIDER SILK PROPERTIES IN AN ADAPTIVE RADIATON OF HAWAIIAN ORB-WEAVING SPIDERS. A Thesis. Presented to

Transcription

1 DIVERSIFICATION OF SPIDER SILK PROPERTIES IN AN ADAPTIVE RADIATON OF HAWAIIAN ORB-WEAVING SPIDERS A Thesis Presented to The Graduate Faculty of The University of Akron In Partial Fulfillment of the Requirements for the Degree Master of Science Angela M. Alicea-Serrano May, 2017

2 DIVERSIFICATION OF SPIDER SILK PROPERTIES IN AN ADAPTIVE RADIATON OF HAWAIIAN ORB-WEAVING SPIDERS Angela M. Alicea-Serrano Thesis Approved: Accepted: Advisor Department Chair Dr. Todd A. Blackledge Dr. Stephen Weeks Committee Member Dean of the College Dr. Randall Mitchell Dr. John Green Committee Member Dean of the Graduate School Dr. Brian Baggatto Dr. Chand Midha Committee Member Date Dr. Ali Dhinojwala ii

3 ABSTRACT The design of biological structures and the materials composing them are intimately connected to their functions in biological systems. A lineage of Hawaiian orb weavers present an excellent system in which the study of the role of material properties in the evolution of performance is simplified. Web architecture evolved rapidly among Hawaiian Tetragnatha, with divergence of web forms in the same island but repeated convergence of web forms on different islands. However, whether silk properties also evolve during adaptive radiations remains unknown. In this study we tested for diversification in silk properties during rapid evolutionary events. We predicted a relationship between silk properties and the performance of webs, driven by prey selection. As silk density decrease in the webs of some species, we expected that toughness for major ampullate silk will decrease, allowing sparse webs to stop small and slow prey compared to webs constructed with larger amounts of silk. A correlation between stickiness of the capture spiral and toughness of radial silk is expected because stopping and retention should be functionally related to trap prey effectively. To test diversification of material properties during adaptive radiations, orb webs from endemic Hawaiian Tetragnatha were collected in two islands of the archipelago. Radial and capture spiral silk where obtained directly from orb webs in the field and tensile and adhesion tests were performed using a Nano Bionix test system. Since low molecular weight compounds present in the glue determine in part work of adhesion, solution state NMR was done to determine if these compounds diversified. Results showed differences in toughness of radial silk among four population of Hawaiian Tetragnatha, correlated with changes in tensile strength, while extensibility remained the same. Stickiness changed in the same predictable way among the four population of orb weavers, with spiders that produced tougher radial silk in their web iii

4 also producing stickier capture spirals. No conspicuous qualitative differences in low molecular weight compound composition of aggregate glue was found. While our small sampling of species prevents robust testing of our hypotheses, here we show that silk material properties are changing over relatively short timescales in an adaptive radiation of Hawaiian orb-weaving spiders. iv

5 DEDICATION be here today. To my parents and loving family that with their infinite support made possible for me to v

6 ACKWNOLEDGEMENTS Thanks to my committee members for providing guidance and important insight to make this project possible. Special thanks to my advisor Dr. Blackledge for all of his support through this process. Thanks to Pat Bily from The Nature Conservancy of Hawaii and to Joey S. Mello, Raymond McGuire and Cynthia B. King from the Department of Forestry and Wildlife of Hawaii for all their help in logistic and field work. Thanks to Susan Kennedy for all her input and web pictures, and Bricey Kepner who did all silk measurements. This work was partially funded by The American Arachnological Society Research Fund. vi

7 TABLE OF CONTENTS Page LIST OF TABLES.... ix LIST OF FIGURES.. x CHAPTER I. INTRODUCTION.. 1 II. METHODOLOGY.. 10 Spiders and silk collection Biomaterial properties.. 11 Low molecular weight compounds composition Coevolution of silk Data analysis. 15 III. RESULTS.. 16 Biomaterial properties.. 16 Radial silk. 16 Viscid silk.. 20 Low molecular weight compounds composition Coevolution of silk vii

8 IV. DISCUSSION 28 LITERATURE CITED APPENDIX viii

9 LIST OF TABLES Table Page 1 List of species collected S1 Spider s size measurements S2 Tensile properties for radial silk.. 43 S3 Glue droplets morphology measurements and adhesive properties S4 Web architecture measurements ix

10 LIST OF FIGURES Figure Page 1 Orb webs are made of two types of fibrous silk that vary in their properties Differences in the material properties of radial threads for T.hawaiensis_M, T.hawaiensis_H, T.stelarobusta and T.trituberculata Covariation of material properties of radial threads for T.hawaiensis_M (open red), T.hawaiensis_H (closed red), T.stelarobusta (open green) and T.trituberculata (open blue) Differences in the adhesive properties of viscid silk for T.hawaiensis_M, T.hawaiensis_H, T.stelarobusta and T.trituberculata Viscid silk images representing the four populations Stickiness of Hawaiian Tetragnatha compared to two local Ohio, USA species: T.laboriosa and T.elongata H solution-state NMR results for soluble molecules of aggregate glue in viscid silk Covariation of material properties between radial threads and sticky capture spirals for T.hawaiensis_M (open red), T.hawaiensis_H (closed red), T.stelarobusta (open green) and T.trituberculata (open blue) Correlation of architectural aspects of webs and its silks material quality to determine potential prey type and/or the amount of insects that could potentially be capture x

11 S1 Differences in the adhesive properties of viscid silk for species at three humid conditions: 50% (blue), 70% (dark blue) and 85% (grey) xi

12 CHAPTER I INTRODUCTION Adaptive radiations play an important role in the rise of biological diversity and are ideal systems to study how that diversity was generated. Closely related species often diversify in their use of resources, by diverging in traits (character displacement) (Darwin 1859). Character displacement has been observed, for example, in Anolis lizards from the Caribbean Islands (Losos 1990), cichlid fishes in Africa (Schluter 1993), and Darwin s finches in the Galapagos Islands (Lack 1945). These studies show examples of character displacement, and document extensive coevolution between behavioral and morphological traits (e.g. leg shape and locomotion in Anolis, and jaw or beak shape and feeding in cichlids and finches). However, the degree to which the underlying materials from which those structures are built also coevolve with function is unknown. Do biomaterials remain largely unchanged during character displacement or can they undergo the same rapid evolutionary changes seen in other types of traits? The design of biological structures and the materials composing them are intimately connected to their functions in biological systems (André Meyers et al. 2008), and hence organismal performance and fitness. The study of the morphology or integrity of structures, for example correlating shapes of finches beaks for species eating larger or smaller seeds (Soons et al. 2015), is often easier than characterizing the materials from which they are made (the bone, keratinous ramphotheca, or the jaw closure muscles). Study of these materials often requires methods that are too invasive using in vivo techniques that present both technical and ethical challenges (Vuskovic et al. 2001). Spider webs present an ideal model in which the study of biological materials is simplified, as they are considered to be an extended phenotype of spiders 1

13 (Craig 2003) reflecting their behavior and physiology (Heiling & Herberstein 2000). There are many advantages that webs provide. Webs are readily accessible, making the material easy to obtain, to measure and to compare between individuals. Moreover, spider silk is one of the toughest known biomaterials and, the focus of many scientific studies because of its easy access and incredible properties (Andersen 1970; Gosline 1987; Gillespie 1991; Harmer et al. 2011; Boutry & Blackledge 2013; Sensenig et al. 2013). Hawaiian Tetragnatha offers an excellent model system to test diversification of biomaterials in adaptive radiations for the following reasons. First, the Hawaiian Islands present a natural laboratory for the study of evolution given that isolation among them allowed repeated and explosive taxonomic diversification generating endemism (Simon 1987). Second, the evolutionary history, ecology and behavior of Hawaiian Tetragnatha is well-studied (Luczak & Dabrowsk.E 1966; Levi 1981; Gillespie 1991, 1992, 1994, 1999, 2002, 2003, 2005; Gillespie et al. 1994; Gillespie & Croom 1995; Blackledge & Gillespie 2004). Tetragnatha are the only endemic nocturnal spiders in the archipelago and character displacement has been observed to occur among orb-weaving spiders from this genus showing divergence of web architecture within sympatric species, while showing convergence of web architecture across the Hawaiian Islands Oahu, Maui and Hawaii (Blackledge & Gillespie 2004). Orb webs are stereotypical structures built using two types of materials: the major ampullate (MA) silk builds the frame and radii, while the glue-coated flagelliform (Flag) viscid silk forms the adhesive capture spirals (Figure 1A). To be successful, orb webs need to (1) intercept a flying insect, (2) stop it by withstanding the kinetic energy with which flying insects strikes it, and (3) hold onto the captured prey once its intercepted long enough for the spider to reach it (Eberhard 1986). How effective a web is at catching prey will have an impact on a spider s diet, survival and fitness. Each one of these steps can be influenced by the spiders web spinning behaviors that are highly diverse, as well as the silk material properties (Heiling & Herberstein 2000; Blackledge 2012). Spinning behaviors of spiders are framed in the architectures of webs. 2

14 And, by looking at parameters like the size of the web, the area of the capture spiral, the spacing between the rows of capture spiral (mesh width) and number of radial threads, these behaviors are easily quantifiable (Figure 1A). Structural properties (e.g. size of fibers and the morphology of glue droplets in viscid silk) can also reflect the behavior of spiders, because spiders can manipulate these properties in response to stimuli (Blackledge 2012). Both the web architectures and the structural components are of great importance for orb webs when intercepting prey. For example, having a bigger capture area with a smaller mesh width and higher number of spiral turns could facilitate interception of prey by increasing the likelihood that an insect will contact silk. On the other hand, having fibers with smaller diameters and a wider mesh width of capture spirals will make a web invisible to an insect s eyes. Differences in web shapes and size can be used as a predictor of how spiders use microhabitats and the types of insects those webs might target (Blackledge et al. 2003). In the Hawaiian archipelago resource differentiation has been described for sympatric species of Tetragnatha (Blackledge et al. 2003), with different webs architectures. Different species utilize discrete microhabitats and their wide range of web architectures correlate with the capture of different types of insects. In general among orb-weavers, two extremes in web performance have been described based on their ability to dissipate the kinetic energy inflicted by a striking prey: high energy absorbing webs and low energy absorbing webs (Craig 1987). High energy absorbing webs are typically larger, tight webs that have many radii per spiral turn and absorb kinetic energy by relying more on tension and tensile deformation rather than aerodynamic damping. High energy webs are hypothesized to intercept and stop bigger flying prey (Craig 1987) and as size increases so will the quality of the silk materials necessary for this task. In contrast, low energy absorbing webs are smaller structures with fewer radii per spiral turn, are under less tension and absorb energy primarily by displacement, architectural design (Craig 1987). This type of web, since smaller in size, is targeting smaller low energy prey and is spun from silk of slightly lower quality (e.g. less tough) (Sensenig et al. 2010). 3

15 Figure 1. Orb webs are made of two types of fibrous silk that vary in their properties. (A) Measurements of architectural components of orb webs include the length of capture spiral and the spacing between them (referred to as mesh width), and the length and number of radii. (B-C) Structural components of webs include silk diameters, as well as glue droplets morphology in the viscid silk. Molecular composition give silks its unique properties. Silks unique properties are dictated by their amino acid sequences and how they are assembled (Blackledge 2012). Silk proteins are largely composed of repetitive motifs of amino acids in a block copolymer-like arrangement. The amino acids comprising a motif change depending on the type of silk, thereby altering its physical properties and functions (Tokareva et al. 2014). For example the toughness of major ampullate silk is accredited to the formation of beta-sheet crystals made of poly-alanine and poly-glycinealanine repeats, while the extensibility of flagelliform silk is attributed to the glycine-rich beta-spirals (Hayashi & Lewis 2001). (D) Stress-strain behavioral curve of two types of silk: major ampullate and flagelliform silk. Differences in the properties of silk make webs compared to other man-made materials. 4

16 Silk material properties enhance web structures and are important for stopping and retaining the prey. These properties can be measured by looking at the stress-strain behavior of the threads, which are normalized to the dimensions of the sample being tested to facilitate comparison across different lengths or thicknesses of materials. Due to its viscoelastic nature, silk properties change as it stretches. The nonlinear behavior of silk after yielding, softening followed by stiffening of the silk (major ampullate curve in Figure 1D), has been suggested to play an important part in the performance of webs (Cranford et al. 2012). Therefore is important to look at different parts of the stress-strain curve as the material stretches because it gives us different information. Parameters like tensile strength, extensibility, stiffness and toughness are typically used to define silks performance. Ultimate or breaking strength refers to how much force a strand can resist before failure, while extensibility is a measurement of how much it deforms until this point. Stiffness is indicated by the initial resistance of the silk before permanent deformation (designated by the yield point). Toughness is defined as how much work per volume of material can the fiber sustain before it breaks. As silks are deforming, they get rid of the energy by releasing it as heat, a behavior known as hysteresis or energy damping. These properties differ significantly between the types of silks making an orb web. Major ampullate silk is most important for stopping prey due to its toughness (Swanson et al. 2006) and high energy damping (Kelly et al. 2011), while flagelliform silk is less strong than major ampullate but highly extensible, deforming over 500% of its original length (Blackledge & Hayashi 2006). These properties make webs very efficient in stopping prey by dissipating the energy of impact of flying insects hitting them and then dissipating this energy (Kelly et al. 2011; Blackledge 2012), so that webs won t act as trampolines but rather stop the insects with minimal oscillations. To understand the ecology of spider webs, performance of silks is determined by more just its material properties and is an outcome of mechanics combined with the structures of the fibers. Thus, by using these materials in different structural arrangements it is possible for spiders to change the mechanics of whole webs. 5

17 Flying prey can struggle free within seconds after hitting an orb web, less than a second for an insect like a fly (Blackledge & Zevenbergen 2006). The capture spiral is spun from viscid silk and plays a key role in retaining prey (Opell 1999). Aggregate glue coating the flagelliform silk is composed of glycoproteins and hygroscopic low molecular weight compounds (LMWCs) that are important in maintaining silk stickiness. (Vollrath et al. 1990; Sahni et al. 2011; Amarpuri et al. 2015a). LMWCs in the droplets solvates the glycoproteins to stabilize them and also aid in the absorption of water from the environment. By sequestering water from the environment glue droplets can change their viscosity, altering how the glues spread on surfaces and deform during detachment. Differences in salt profiles of glue between different species of spiders results in differences in viscosity and hence adhesion (Amarpuri et al. 2015b). Stickiness can be further enhanced by structural properties of the capture spiral. The suspension bridge mechanism describes how viscous threads operates when pulling off after coming in contact with a surface (Opell & Hendricks 2007). Because glue droplets are evenly spaced in a beads on string arrangement, multiple contact points on the axial fibers work effectively to distribute the force when pulling off is occurring (Figure 1B). Therefore adhesive recruitment can be affected by glue droplets size and the spacing between them, which could also result in differences in adhesion. Changes in silk properties through evolutionary time across araneoids show biomechanical and behavioral traits coevolving in the evolution of the stopping potential of orb webs (Sensenig et al. 2010). However, this investigation was at a very deep phylogenetic level and the biomechanical properties of silks from a range of web structures within a closely related group of spiders still needs to be examined to understand how biomaterials, may enhance function of behaviors and diversification. Evolution of the behavioral component of web building (architectures), as well as the adaptation to microhabitats to target different prey types in Hawaiian Tetragnatha has been already studied (Blackledge et al. 2003; Blackledge and Gillespie 2004), but whether or not silk biomaterials also change with these behaviors remains unknown. In this study we explore the question: are material properties of spider silk diversifying during adaptive radiations? To do this we look at the quality of silk for different species of Hawaiian orb 6

18 weavers. Through this study we refer to quality as how tough and sticky silk is. Toughness is used as a measurement of quality because this property best describes the amount of energy per volume that silks can sustain and thus how well they are performing when an insect hits a web. Therefore the toughest silk can potentially stop bigger and fastest insect, while highest stickiness can retain insects longer. If we find that there is diversification of material properties, the following hypothesis of why properties might be changing were developed. I. Major ampullate radial silk A. Prey-specialization hypothesis For any given amount of silk, spiders may spin orb webs that could maximize interception of prey, maximize stopping of prey, or webs that may maximize retention of prey. While one or more of these have a positive effect on prey capture, one or more may be negatively affecting it. For example, the most efficient architecture of an orb web that maximizes the number of prey contacting silk is constructed by spacing threads just larger than the average insect s wingspan (Eberhard 1986), however this type of design may negatively affect stopping and retention of insects (Blackledge & Eliason 2007; Blackledge 2012). Because radial silk is more important for stopping prey, changes in its toughness selects for the type of insect webs are trapping. If Hawaiian Tetragnatha are building webs to specialize to catch prey of different energy it is expected that sparse architectures will have a lower toughness of radial silk to capture small low energy prey. Shift towards this type of behavior may be as consequence of selective advantages. Firstly, by building webs that rely on the silk properties in localized areas of prey impact rather than on the web structure as a whole can help to localize damage, keeping the rest of the web functional and allow it to be reusable more times (Harmer et al. 2011). Also by decoupling structure from function in webs, spiders may modify their webs to almost any building environment and eventually facilitate the exploration of new foraging niches because this webs don t rely on structures for prey retention (Craig 1987; Griswold et al. 1998; Harmer et al. 2011). 7

19 B. Compensation hypothesis As an alternative hypothesis, if Hawaiian Tetragnatha are not selecting for different type of prey a compensatory behavior is expected. If spiders are building sparser structures by using better quality materials then it is expected that webs with sparse architectures and lower silk density (loose, open sticky spirals and few radii) will have tougher major ampullate silk than webs with compact architectures and higher silk density (longer, tightly packed spirals of sticky silk and many radii). If this is happening potential to stopping same energy prey should be similar. II. Viscid silk A. Prey-specialization hypothesis Changing the architectures of webs is an easy way orb-weaving spiders can control overall stickiness of webs. For example, decreasing mesh width can increase retention time of insects in webs (Blackledge and Zevenbergen 2006). If spiders are specializing for a type of prey then it is expected that webs with sparser architectures will have a low single thread stickiness to target smaller prey, when compared to webs with compact architectures selected for larger prey. B. Compensation hypothesis If spiders are not specializing for different types of prey, it is hypothesized that webs with sparse architectures will have higher single thread adhesiveness than dense compact architectures. We expect this because single threads may have to do more work retaining prey in webs with sparser architecture, while in compact structures with a smaller mesh width multiple threads may collaborate to do the same amount of work. C. Chemistry-based hypothesis If a difference in stickiness of viscid silk is found between species, then is hypothesized that these differences are due to differences in glue chemistry. Water uptake from the environment by viscid silk and adhesion of glue are facilitated by the hygroscopic low molecular weight compounds present in the droplets (Sahni et al. 2014) with maximum adhesion being 8

20 achieved at the relative humidity of the environment where spiders lives (Amarpuri et al. 2015). By modifying low molecular weight compounds, spiders can tune the viscosity of their glue to one that is ideal for its environment. III. Coevolution of silk Properties of both radial and viscid silk act together to improve functions of whole webs. Webs with high stopping potential also improve stickiness (Sensenig et al. 2010), likely to allow better retention of difficult to capture large insects. Thus we expect stopping potential of webs to positively correlate with stickiness. To test these hypotheses here we study the material and chemical properties of capture spiral and major ampullate silk of three species of Tetragnatha in an adaptive radiation in the Hawaiian archipelago. 9

21 CHAPTER II METHODOLOGY Spiders and silk collection For this study orb-weaving spiders (Tetragnatha) were collected during the month of July in 2015, at two sites in the Hawaiian archipelago: Waikamoi Nature Conservancy Preserve, Maui Island and Upper Waiakea Forest Reserve, Hawaii Island (Table 1). Three species (T.stelarobusta Gillespie 1992, T.trituberculata Gillespie 1992 and T.hawaiensis Simon 1900) where commonly found at Waikamoi Preserve. Four other species (T.eurychasma Gillespie 1992, T.filiciphilia Gillespie 1992, T.paludicola Gillespie 1992 and T.acuta Gillespie 1992) were found cohabiting in the same area, but were not as abundant and thus were not included because of low sample size. At Upper Waiakea Forest Reserve only T.hawaiensis was found. With the assumption that no gene flow is occurring between Tetragnatha hawaiensis collected at Waikamoi Nature Conservancy Forest Preserve, Maui and T.hawaiensis collected at Upper Waiakea Forest Reserve, Hawaii Island, these will be treated as distinct populations, respectively called T.hawaiensis_M and T.hawaiensis_H from this point forward. Table 1. List of species collected. *For some webs it was not possible to get good thread samples. For this reason sample size was not the same. Species Island N* N* N* Total webs Radial silk Viscid silk Tetragnatha hawaiensis Maui Tetragnatha hawaiensis Hawaii Tetragnatha stelarobusta Maui Tetragnatha trituberculata Maui

22 Hawaiian Tetragnatha are nocturnal foragers and orb-weavers that build their webs during the night and take them down during the day. Webs for each species were located in the field (Table 1 for sample size) during the hours of 20:00 till 02:00. Whole webs were collected using circle frames (15 cm in diameter) attaching them using double-sided tape and cutting the radial threads from its attachment using heat. Using a butane lighter, radial silk threads were cut to retain the natural tension of the web. In cases where webs were larger than the circle frame only the bottom half of the web was collected. Webs were carried back to the field station using a water-proof box. Spiders where also collected, preserved in 95% EtOH and brought back to the University of Akron where prosoma, opisthosoma and the femur of the first left leg were measured. A total of up to six samples of radii (major ampullate silk) and 20 samples of capture spiral (viscid silk) were obtained from each web using cardboard holders across mm gaps (Sensenig et al. 2010). Major ampullate threads were secured with cyanoacrylate glue (Superglue ). Capture spiral threads were secured with water soluble glue (Elmers ) to avoid desiccation of glue droplets. Threads were kept in a dark box to prevent damage from UV light (Lai & Goh 2015) until being tested and shipped to the University of Akron. The remains of the web after threads collection was gathered using a clean glass pipette for chemical composition analysis of the glue, and pooled within species. All insects or debris stick to the web were removed prior collection of capture spirals. For some webs capture threads were stuck together and it was not possible to get good thread samples so they were only collected for chemical composition purposes. For this reason sample size for the different tests was not the same (Table 1). Biomaterial properties To test how silk properties may have evolved in response to the second step of catching insects, stopping the prey, the potential ability of silks to dissipate energy was studied by generating force-extension curves for major ampullate radial silk using a Nano Bionix test system (Agilent Technologies, Oak Ridge, TN, USA) following methods previously described (Blackledge et al. 2005; Sensenig et al. 2010). Radial threads were tested three months after collection. 11

23 Cardboard mounted fibers were extended at a strain rate of s -1. A minimum of three threads per web for each type of silk were tested at room humidity (46%RH). Each thread is composed of two axial fibers generated by a pair of posterior spinnerets. Diameter of threads was measured using polarized light microscopy (100X). Photographs of the suspended radial threads were taken using a 5 Megapixel Olympus Q-Color5 TM imaging system (Olympus America, Inc., Melville, NY) attached to the microscope. Using Image-Pro Plus 6.2 software (Media Cybernetics, Inc., Bethesda, MD) measurements at three points were taken and averaged for one axial fiber per thread. Material properties (tensile strength, extensibility, toughness and stiffness) for each thread were obtained as previously done (Vollrath et al. 2001; Sensenig et al. 2010). Tensile strength or true breaking stress was calculated as the amount of force required to break a fiber relative to the instantaneous cross-sectional area. Extensibility or true breaking strain was calculated as the natural log of one plus the ratio of breaking length to the original length and refers to how much the material has stretched normalized to the instantaneously length. Toughness or the energy absorbed by a fiber prior to rupture was calculated as the area under the stress-strain curve. Stiffness or Young s modulus was obtained from the initial slope of the force-extension curve in which the relationship of stress and strain was almost linear. To test how silk properties may have evolved in response to the third step of catching insects, retaining the prey, adhesiveness of capture spiral silk was measured by looking at the following mechanical properties: work to release also referred to as work of adhesion, peak load or force of adhesion and extension. Samples were tested days after collection from nature. Cardboard-mounted viscid silk threads were pressed to a clean 5 mm wide glass surface and pulled off at an extension of 0.1 mm s -1 using the Nano Bionix test system (Agilent Technologies, TN, USA) to generate force-extension curves (Agnarsson and Blackledge 2009). The thread was lowered into the surface until contact and then further pressed until a reading force of 50 mn to ensure adhesion. These conditions were kept for 6 seconds before pulling off. The area under the load-displacement curve represents the energy required to separate the thread from the glass 12

24 substrate (referred to as work to release). Peak load was the maximum force of adhesion achieved by the system and extensibility was the distance the capture spiral displaced until reaching the maximum force of adhesion. This includes both stretching of the flagelliform fiber and stretching of the glue droplets themselves. To control for humidity, adhesion tests were performed inside a humidity chamber. Nine capture spiral threads were tested per web, three threads being tested at a control humid conditions of 85% RH, to mimic natural conditions of the spider s habitat. Threads were also tested at 70% RH and 50 % RH, to see the effect of low humidity on the glue (Figure S1). Samples were placed inside the humidity chamber and acclimates for 2-3 minutes prior to testing, enough time for droplets to swell or contract to equilibrium. To control for differences in glue quantities across species, the glue volume in 5 mm of thread to come in contact with the substrate and the space between glue droplets were measured by looking at the suspended capture silk under a polarized light microscope (100X). Photographs of the suspended viscid silk were taken at room humidity (~46%RH) using a 5 Megapixel Olympus Q-Color5 TM imaging system (Olympus America, Inc., Melville, NY) attached to the microscope. Glue morphology measurements were done using Image-Pro Plus 6.2 software (Media Cybernetics, Inc., Bethesda, MD). Total glue volume of glue and the distance between the droplets were calculated as in (Liao et al. 2015). Stickiness parameters were normalized by glue volume in 5 mm of thread. Because glue droplets swell when exposed to higher humidities (Opell et al. 2013) and measurements were taken at room humidity, normalized stickiness parameters for relative humidities over 50% might by overestimated. Low molecular weight compounds composition Glue droplets are composed of glycoproteins, water, and hygroscopic salts. Salts capture atmospheric water (Amarpuri et al. 2015b; Jain et al. 2015) and directly facilitate adhesion by solvating the glycoproteins (Sahni et al. 2014; Jain et al. 2016). The difference in types of salts in the glue droplets of different species may explain differences in humidity dependence and can help to further understand adhesiveness in humid environments. To 13

25 establish the chemical nature of salts present in the capture glue, 1 H Solution-State Nuclear Magnetic Resonance (NMR) experiments were performed (Sahni et al. 2014; Jain et al. 2015). Firstly capture spiral material collected in glass pipettes (T.hawaiensis_H ~ 22, T.hawaiensis_M ~13, T.trituberculata ~ 9 and T.stelarobusta ~ 14) were washed with deionized water (~10 ml) to remove the soluble salt components. Next, the soluble residue was lyophilized for about 10 hours in order to remove the water and obtain the dried water soluble salt components. Finally, the dried extracts were dissolved in about 1 ml of 99.96% deuterated water (Sigma Aldrich) and filled in 5 mm NMR glass tubes (Norell). All experiments were performed on a Varian 300 MHz spectrometer at 298 K. The spectra were recorded with scan size ~ 512, acquisition time ~3 s and 90 0 pulse width of µs. Once the spectras were processed, different peaks were identified for the presence of salts on the basis of findings reported in the literature (Anderson & Tillinghast 1980; Tillinghast & Christenson 1984; Tillinghast et al. 1987; Vollrath et al. 1990; Townley et al. 1991, 2006, 2012; Townley & Tillinghast 2013; Sahni et al. 2014; Jain et al. 2015) and unpublished database provided by M.A. Townley. Coevolution of silk To understand whole web performance, estimated stopping potential of webs and stickiness per area were calculated. Estimated stopping potential is the total breaking energy of silk per unit area of the web, and is calculated as the product of the volume of radial silk in the web times the average toughness of the radial silk per species divided by the capture area of the web (Sensenig et al. 2010). Using parameters of web architectures for Hawaiian Tetragnatha (Figure 1A; Table S4; Blackledge & Gillespie 2004), volume of radial silk (Vrad) is obtained using Formula 1, Formula1. Vrad = (ru + dhorizontal + rl)/4(rn)(a) where ru is the length of radial threads in the upper half of the web, rl is the length of radial threads in the lower part of the web, dhorizontal is the horizontal diameter of the capture area, Rn is the number of radial threads in the web and A refers to the total cross-sectional area of the radial silk threads. Previous attempts to estimate stopping potential of orb webs, using this approach, 14

26 found that values were consistently overestimated by approximately seven-fold compared to actual maximum work performed by webs stopping a variety of projectiles, but that it provided a very precise measure of the relative variation in performance among webs, allowing for comparison between species (Sensenig et al. 2010). Stickiness per area (Sta) was calculated using Formula 2, Formula 2. Sta = (WR)(CTL)/Cg/Ac where WR is the average work to release for each species, CTL refers to the average total length of capture spiral placed in the web, Cg is the average length of contact with the glass substrate (5 mm), and Ac is the average capture area of the web. Stickiness per unit area of webs is used because this best represents the maximum adhesiveness that could be applied to an insect of a given size (Sensenig et al. 2010). Web architecture information was only available for only Big Island T.hawaiensis (Blackledge & Gillespie 2004). For this study it was assumed that webbuilding behaviors are conserved among individuals of the same species, thus identical architectural parameter values were used for both populations of T.hawaiensis included in this study. Data analysis All data was analyzed using the software Statistica 13 Academic TM (StatSoft Inc. Tulsa, OK, USA). One-way Analysis of Variance (ANOVA) was used to compare each of the silk material properties between the four populations of Tetragnatha: T.stelarobusta, T.trituberculata, T.hawaiensis_M and T.hawaiensis_H. Biomaterial properties (strength, extensibility, toughness and stiffness) of major ampullate threads and adhesiveness of capture spiral threads (work of adhesion, normalized work of adhesion, force of adhesion, normalized force of adhesion, and extension) at three humidity (50, 70, 85% RH) between the species were compared. Values were averaged across all threads tested for an individual spider. Pairwise comparisons were done by using Tukey s range test for unequal sample size. Regression analysis were performed to look at 15

27 the covariation of silks properties among populations as well as the covariation of whole web performance between the four populations. 16

28 CHAPTER III RESULTS Using carapace width as a proxy of spider size, size differed among species included in this study (F(3,17)=16.706, p= ). Pairwise comparison showed Tetragnatha stelarobusta (1.9±0.1 mm) and T.trituberculata (1.6±0.04 mm) were significantly larger in size than T.hawaiensis_M and T.hawaiensis_H (1.3±0.1 and 1.2±0.1 mm respectively) (Table S1). Biomaterial properties Radial silk Radial thread diameter did not differ between the four populations of Tetragnatha spiders (F(3,25)=0.74, p=0.538) (Table S2). However, the material properties of radial threads differed among the species (Figure 2 and 3; Table S2). One-way analysis of variance revealed a significant difference among silks from different populations for toughness (F(3,25)=3.9513, p=0.0196, Figure 2A), stiffness (F(3,25)=4.0012, p=0.0187, Figure 2B), and ultimate strength (F(3,25)=5.1523, p= , Figure 2D). Extensibility of radial silk did not differ between the four populations (F(3,25)=1.8893, p=0.1572, Figure 2C). Tetragnatha trituberculata had the highest strength (1.5 ± 0.2 GPa), followed by T.stelarobusta (1.2 ± 0.4 GPa), while both populations of T.hawaiensis had the least strong radial threads (Maui: 0.66 ± 0.16 GPa and Hawaii: 0.64 ± 0.10 GPa) (Table S2). Pairwise comparison revealed significant difference in means only between T.trituberculata and T.hawaiensis_H (p=0.0174). It is possible that no differences in strength for T.stelarobusta and T.hawaiensis_M were observed because of our low sample size which gave us low statistical power. Not surprisingly a similar pattern is observed for radial toughness, where 17

29 T.trituberculata had tougher silk (0.16 ± 0.04 GPa) and T.hawaiensis_H had the least tough silk (0.05 ± 0.01 GPa) (p= ). T.hawaiensis_M (0.06 ± 0.02 GPa) and T.stelarobusta (0.08 ± 0.04 GPa) had in between toughness with T.stelarobusta being more similar to T.trituberculata and T.hawaiensis_M being more similar to T.hawaiensis_H (Figure 2). This is expected because no difference in extensibility was found. There is a tight correlation in radial silk between strength and toughness within each species, with toughness increasing up to 60% faster as strength increases (T.hawaiensis_M: y= x, p=0.0399, r 2 =0.8019; T.hawaiensis_H: y= x, p= , r 2 = ; T.stelarobusta: y= x, p=0.0026, r 2 = ; T.trituberculata: y = x, p = , r 2 = ) (Figure 3D). On the other hand toughness and extensibility was found to relate significantly for only T.trituberculata (y = x, p=0.0302; r 2 =0.6425) and T.stelarobusta (y= x, p=0.0115, r 2 =0.9115) but not for either population of T.hawaiensis (Maui: y= x, p=0.1899, r 2 =0.4872; Hawaii: y= x, p=0.7331, r 2 =0.0121) (Figure 3A). Even though ANOVA results for stiffness revealed a difference in means between the groups, Tukey s for unequal N HSD test showed no significant pairwise differences. Nevertheless figure 3B and 3C depicts differences in stiffness with the largest difference being T.trituberculata and T.stelarobusta distinctly grouping at higher values than T.hawaiensis_M and T.hawaiensis_H. Analysis was rerun without a value stiffness for T.trituberculata which was 5X lower than the 2 nd lowest value. A pairwise difference was then found between T.trituberculata and T.hawaiensis_H (p=0.0459). Stiffness is the initial slope of the stress-strain behavior curve of a material, so it should scale with strength. Our results showed a significant relationship between toughness and stiffness of radial silk for T.hawaiensis_H (y= x, p=0.0019, r 2 =0.6368) and T.stelarobusta (y = x, p=0.1057, r 2 =0.6366), while no significance for T.hawaiensis_M (y= x, p=0.7465, r 2 =0.0402) and T.trituberculata (y = x, p=0.7973, r 2 =0.0145) (Figure 3B). On the other hand no significant relationship was found between stiffness and extensibility of radial threads (T.hawaiensis_M: y= x, p=0.4879, r 2 =0.1717; T.hawaiensis_H: y= x, p=0.1214, r 2 =0.2226; T.stelarobusta: y= x, 18

30 Figure 2. Differences in the material properties of radial threads for T.hawaiensis_M, T.hawaiensis_H, T.stelarobusta and T.trituberculata. Letters represent significance difference (p<0.05). Bars represent mean values with standard error. (E) Representative stress-strain curves for each of the groups. Tests were performed at room humidity (46%RH). 19

31 p=0.2995, r 2 =0.3429; T.trituberculata: y= x, p=0.2795, r 2 =0.2272) (Figure 3C). Although not significant negative regression coefficients were obtained for T.hawaiensis_M, T.hawaiensis_H and T.trituberculata, but no for T.stelarobusta. It is possible that some variation seen in the biomaterial properties comes from measurement type error. Natural variation in silk may be present as spiders can change the diameter of their silks depending on environmental and physiological circumstances (Boutry & Blackledge 2008; Ortlepp & Gosline 2008). Silk spinning conditions might also result in some variation (Vollrath et al. 2001) even within different locations of a single orb web (Blackledge et al. Figure 3. Covariation of material properties of radial threads for T.hawaiensis_M (open red), T.hawaiensis_H (closed red), T.stelarobusta (open green) and T.trituberculata (open blue). (A-C) Dash-lines are used to delimit the major differences separating the groups. (D) Line of best fit correlating toughness and strength of radial major ampullate silk. 20

32 2005). Random error may also be present in our study. Since silk fibers approach longer wavelengths of light (Blackledge et al. 2005), using light microscope for measurement of diameters is a bit challenging. This particular tasks gets even harder when the silk being tested is collected from native structures, like is the case of our samples, that in addition of having irregular diameters also have two intercoiled axial fibers. We try to overcome this by measuring diameter for a single axial fiber in three random points across the 12.7 mm gage length thread length and averaging these for each web. In addition all diameter measurements were made by the same person to avoid additional error. Therefore it is likely that some error is observed in this properties that are highly sensitive on the cross-sectional area of the material. Viscid silk Adhesive properties of viscid silk differed between the different populations of spiders (Figure 4, Table S3). Tetragnatha trituberculata had a higher work of adhesion (7.9X10-7 ± 1.16X10-7 J) and higher force of adhesion (170.9 ± 23.6 un), followed by T.stelarobusta (6.77X10-7 ± 2.09X10-7 J and ± 38.4 un respectively), while T.hawaiensis_M and T.hawaiensis_H showed the lowest stickiness with a work of adhesion of 3.8X10-7 ± 0.9X10-7 J and 2.8X10-7 ± 0.6X10-7 J, and a force of adhesion of 91.3 ± 18.5 un and ± un. One-way ANOVA showed significant differences in work and force of adhesion (F(3,30)=5.469, p=0.004 Figure 4E; F(3,30)=4.814, p=0.008 Figure 4D respectively). Extension was not different between the four groups (F(3,30)=0.276, p=0.842) (Figure 4C). Pairwise comparisons revealed significant differences of work (p=0.0148) and force of adhesion (p=0.319) only between T.trituberculata and T.hawaiensis_H. Differences in stickiness among the four groups in this study may be due to structural properties of the glue (e.g. volume, shape) (Figure 5 and 6). Adhesive recruitment can be affected by the size of the glue droplets and the spacing between them, as well as the size of the substrate that sticks too (Opell & Hendricks 2007). 21

33 Figure 4. Differences in the adhesive properties of viscid silk for T.hawaiensis_M, T.hawaiensis_H, T.stelarobusta and T.trituberculata. Letters represent significance difference (p<0.05). Bars represent mean values with standard error. (A-B) Show stickiness parameters normalized to glue volume. (C-E) Show absolute values of stickiness. (F) Representative force-extension curves for each of the groups. Tests were performed at a relative humidity of 85 ± 5 %. 22

34 Figure 5. Viscid silk images representing the four populations. Pictures were taken at room humidity (46 %RH). Substrate used for this experiment were 5 mm in diameter and adhesion for T.stelarobusta at fewer larger points of contact might be making transfer of forces to axial fibers not as effective. This is observed in our study with structural properties of glue varying among the four groups [glue volume: F(3,30)=7.219, p=0.001; spacing: F(3,30)=4.578, p=0.009] (Figure 5 and 6). Tetragnatha stelarobusta had on average the highest glue volume (572X10 3 ± 149X10 3 um 3, and the largest spacing between glue droplets (105 ± 29 um), which means that glue is getting packed in larger droplets that are relatively farther apart from each other (Figure 5 and 6, table S3). Tetragnatha trituberculata had the second largest volume of glue in 5 mm of thread (476X10 3 ± 88X10 3 um 3 ), followed by T.hawaiensis_M (265X10 3 ± 59X10 3 um 3 ). However T.hawaiensis_M had larger spacing between glue droplets than T.trituberculata (91 ±12 um and 79 ± 10 um respectively), which means that in average T.trituberculata have a larger amount of droplets that are bigger while T.hawaiensis_M have less amount of droplets that are smaller in size. Tetragnatha hawaiensis_h had in contrast the lowest glue volume (143X10 3 ± 35X10 3 um 3 ), and the least space in between them (43±8 um) (Figure 5 and 6, table S3). Since variation in these structural properties of glue in viscid silk is present among our groups, force and work of adhesion were normalized to glue volume. By doing this we see that T.hawaiensis_H had normalized peeling forces 1.5X higher than T.trituberculata and 23

35 Figure 6. (A) Stickiness of Hawaiian Tetragnatha compared to two local Ohio, USA species: T.laboriosa and T.elongata. (B-C) Covariation between adhesive properties of viscid and structural properties of glue for T.hawaiensis_M (open red), T.hawaiensis_H (closed red), T.stelarobusta (open green) and T.trituberculata (open blue). Dash-lines are used to delimit the major differences separating the groups. (D-E) ANOVA results of structural properties of viscid silk between the four populations included in this group. Measurements of glue were done at room humidity (46 ± 1 %RH). 24

36 T.hawaiensis_M, and up to 2X higher than T.stelarobusta (F(3,30)=4.3644, p=0.0115) (Figure4A). Tetragnatha stelarobusta had relatively less adhesion forces (3.15X10-4 un/um 3 ) significantly lower than T.hawaiensis_H (p=0.0481). Other work done in our lab studying stickiness properties of two other species of Tetragnatha from the Northeast Ohio area show T.laboriosa Hentz 1850 work of adhesion to be two times less to that of T.hawaiensis_H (1.5 X10-7 J, Figure 6A) and T.elongata Walckenaer 1841 about the same as T.trituberculata (8.3X10-7 J, Figure 6). Low molecular weight compounds composition Figure 7 shows the 1H NMR spectra illustrating different small molecules present in the glues of Tetragnatha spiders. The major small molecules present include betaine, choline, glycine and alanine. Little variation seen in the LMWCs across the Hawaiian species. The quantitative comparison of LMWCs among species was challenging due to small sample leading to poor signal to noise ratio. However, we compared the LMWCs profiles of Hawaiian Tetragnatha spiders to that of mainland (Ohio, USA) Tetragnatha laboriosa and found no major differences except for the lack of N-acetyl taurine in Hawaiian spiders. While the absolute lack of N-acetyl taurine in Hawaiian species is not clear because the starred peaks around 2 and 3 ppm in T.trituberculata and T.hawaiensis (Hawaii and Maui) may represent low concentrations of the compound, these unidentified peaks are clearly absent in the better resolved spectrum of the glue of T.stelarobusta. LMWCs found in the glues of other local US species such as T.versicolor Walckenaer 1841 and T.elongata also show the presence of N-acetyl taurine and in some cases proline (Townley unpublished data), in addition to presently reported LMWCs in Tetragnatha from the Hawaiian archipelago. Coevolution of silk Spider orb web performance depends on the intrinsic properties of radial supporting threads and capture spiral viscid silk, in addition to the web s architecture. We took a look to see how 25

37 Figure 7. 1 H solution-state NMR results for soluble molecules of aggregate glue in viscid silk. *Indicates ambiguity of presence of molecules. 26

38 radial silk and adhesion relates to one another. When looking at the covariation between viscid silk work of adhesion and radial toughness we see that spiders with the least sticky viscid silk have the least tough radial silk. A significant relationship is observed for T.hawaiensis_H (y= x, p=0.0299, r 2 =0.4243) and T.stelarobusta (y= x, p=0.0291, r 2 =0.8382), with stickiness of capture spiral threads scaling only about 45% of toughness of radial threads increase (Figure 8A). No relation was found for the other two species (T.hawaiensis_M: y= x, p=0.3801, r 2 =0.260; T.trituberculata: y= x, p=0.7040, r 2 =0.0314), however is worth noting that although not significant, an inverse relationship was found for stickiness of capture spirals and toughness of radial threads for T.trituberculata. Finally toughness and stickiness were used to calculate respectively stopping potential of webs and stickiness per area to look at whole webs performance of Tetragnatha. A significant relationship was found between these two variables with stickiness per area scaling with the stopping potential (y= e5x, p=0.0208, r 2 =0.9588) (Figure 8B). Both T.stelarobusta and T.trituberculata had over 1.5X higher stopping potential and over 3.9X stickiness per area than T.hawaiensis (Maui and Hawaii). 27

39 Figure 8 Covariation of material properties between radial threads and sticky capture spirals for T.hawaiensis_M (open red), T.hawaiensis_H (closed red), T.stelarobusta (open green) and T.trituberculata (open blue). (A) Dash-lines are used to delimit the major differences separating the groups. (B) Line of best fit correlating stopping potential of webs and stickiness per area. (C) Line of best fit correlating stopping potential and normalized stickiness per area. 28

40 CHAPTER IV DISCUSSION In this study we examined silk material properties of closely related species in an adaptive radiation of Hawaiian Tetragnatha to test if biomaterial properties diversify as behaviors are diversifying. We found evolutionary shifts in the material properties of spider silks over the short timescale of radiation in Hawaiian Tetragnatha. By quantifying the intrinsic material properties of silk we were able to characterize mechanical performance and chemical properties of two types of silks for four populations of orb weaving spiders in Hawaii, and showed that silk properties diversified in this adaptive radiation. Previous research showed a coevolution of biomaterials and orb web architectures over 60 million years of diversification (Sensenig et al. 2010; Garrison et al. 2016). Our results on the other hand revealed that like behaviors, spiders are changing the properties of their silk even on a relatively short time scale (~1.2 MYA). We evaluate three hypotheses for why the properties of major ampullate radial silk are changing by exploring correlation of web-building behaviors and material properties of silks. First we look at the prey-specialization hypothesis that spider webs are evolving under selection to specialize upon prey of different flight energy and thus dense webs will have better major ampullate silk. The opposite prediction was expected for our second hypothesis, the compensation hypothesis, were spiders are targeting insects of similar flight kinetic energy by adding less material of better quality (sparser webs) in comparison to spiders that are building denser architectures. The third hypothesis we looked for major ampullate silk was the microhabitat hypothesis where some species of spiders are expected to have properties of silks that will allow their webs to withstand rough windy conditions in open microhabitats compared to species that live in more secure ones. While our small sampling of species prevents robust 29

41 testing of these hypotheses, we could not reject any of these hypothesis. Toughness and strength of radial silk scale one fold among these four Hawaiian Tetragnatha (Figure 2A, 2E; Table S2). Tetragnatha trituberculata have half the number of radii in its web (Table S1) compared to the other two species: T.stelarobusta and T.hawaiensis, and we found that T. trituberculata has tougher and stronger radial silk. High performing radial silk improves with sparser architectures among distantly related species like Verrucosa, Nuctenea, Caerostris, Nephila and Mangora (Sensenig et al. 2010) and yet we still see this trend showing up among closely related species. Stiffness of silk permits webs to sustain constant wind stress and maintain a structural rigidity (Cranford et al. 2012). Radial silk stiffness varied significantly, although not at the level of pairwise comparisons (Figure 2B, Table S2). Even though results showed no significance between groups, stiffness of T.stelarobusta and T.trituberculata radial silk was much higher than T.hawaiensis. It can be argued therefore that T.stelarobusta and T.trituberculata have webs better equipped to handle more windy conditions. A study looking at resource use in Tetragnatha at Maui Island show no difference in openness of website between T.trituberculata and T.stelarobusta, but also no difference in openness was found among the others species included in the study (T.eurychasma and T.filiciphilia) (Blackledge et al. 2003). Important to mention is that although visually T.trituberculata was found to share same openness of website as T.stelarobusta only three webs for T.trituberculata were described and therefore no statistical determination was possible. On the other hand, other studies have located webs of T.stelarobusta in drier open areas of the forest, while T.trituberculata was found to often build webs close to wet tree bark (Gillespie et al. 1997) but a quantitative approach was not used, like in the case of (Blackledge et al. 2003). An idea of how sympatric species are sharing the habitat is available, however more detail is needed about both habitat utilization and the material properties of silk to start building more complete synthesis of the performance of webs. We also proposed two hypotheses to explain different evolutionary patterns in viscid silk performance. Stemming from the same rationale as for the radial silk, a compensation hypothesis and a prey specialization hypothesis were evaluated. If a spider web is aiming to stop high energy insects it needs a viscid silk that will retain it. Our results showed that adhesion force 30

42 values for stickiness of viscid silk varied one fold, while a 3X variation in work of adhesion was observed (Figure 3D, 3E). The same trend as radial silk was observed for stickiness, with T.trituberculata yielding higher adhesive forces and Big Island T.hawaiensis the lowest. A positive correlation of stickiness of viscid silk and toughness of radial silk was found (Figure 8A), which means that this two properties are acting together to aid in the webs function as a trapping snare of flying insects. This trend was not only seen for the performance of single threads but also for performance of whole webs (Figure 8B). It has been previously shown that webs with high stopping potential also improve stickiness to retain insects that are intercepted by webs (Agnarsson & Blackledge 2009; Sensenig et al. 2010), consistent with our results. It is interesting that the different architectures of T.stelarobusta and T.trituberculata have similar whole webs performance, while T.hawaiensis from both islands even though having similar architectures as T.stelarobusta have a different and lower performance. This might indicate that rather than structures, the silks intrinsic properties are a more important factor determining the type of prey a web can catch, or that material and structures can evolve independently of one another. A third hypothesis for viscid silk chemistry was proposed. Low molecular weight compounds in glue aids in the absorption of atmospheric water and in the solvation of glycoproteins in aggregate glue droplets (Edmonds & Vollrath 1992; Opell et al. 2011; Sahni et al. 2014), tuning the viscosity of glue to the environment in which species forage (Amarpuri et al. 2015b). To see if there were differences from mainland species we looked at small molecule composition of the glue. Overall our results showed a similar profile pattern of salts present in the aggregate glue of webs, and no novel compounds where found in Hawaiian Tetragnatha when compared to a mainland species, T.laboriosa. Unexpectedly, T.stelarobusta lacks a salt present in the glue of mainland T.laboriosa and possibly in other groups of Hawaiian Tetragnatha (Figure 7). Tetragnatha stelarobusta diet is composed of 70 to 90% moths, while other species of Hawaiian orb weaving Tetragnatha have a diet consisting of small Drosophila and Tipulidae (Gillespie et al. 1997; Blackledge et al. 2003). Moths are not a common food for orb weavers as they are difficult prey to catch by webs because of their scales, which rub off easily allowing the moths to pull free from webs (Stowe 1986). This unique low molecular weight salt composition 31

43 might be a mechanism of T.stelarobusta to select for different food type as we also found evidence in our study that they are sticking differently. Results of stickiness for T.stelarobusta show a difference in adhesiveness per volume of glue (Normalized values) than its relatives, being significantly lower than Big Island T.hawaiensis (Figure 4, Table S3). Moth catching is also associated with dense webs that evolved elongated vertical architectures known as ladder orb webs (Eberhard 1975), that allow moths to be entangled multiple times as is struggling to get free and this might be a reason for why T.stelarobusta have denser architectures. Tetragnatha stelarobusta thus have potentially diverged behaviors to specialize to a different prey type that is not easily recognize by just looking at the web architecture but at the whole hierarchical picture of form and material properties of their webs. Other than the chemistry we should also consider the morphology of glue droplets in the viscid silk, as stickiness can be influenced by adhesive recruitment determine by glue volume and the spacing between them (Opell & Hendricks 2007). Of all species in this study T.stelarobusta had the largest glue droplets with the largest distance between them (Figure 6, Table S3). Droplets need to transfer forces effectively to the axial threads in order to get the more effective stickiness and the number of droplets that come in contact with the surface can have an impact on the amount of work performed by viscous silk. By increasing the width of substrate Opell and Hendricks (2007) found an increase in stickiness. Also adhesive recruitment depends not only on the properties of the glue but also on the extensibility of flagelliform axial threads in about a ratio (Sahni et al. 2011). But this may vary with humidity, temperature and rate of pealing, and although assume to be a general trend we don t know if viscid silk of Tetragnatha behaves in the same way. In the future it would be ideal to have information on the mechanical properties of the flagelliform silk to determine the amount of work done by glue droplets and how much work is done by the flagelliform silk to get a better idea of how viscid silk is behaving among the different taxa, especially at very humid environments that this species are exposed to. We need to consider that all tests done in this study were performed using a standard 5 mm hydrophilic glass surface, which have the advantage of providing a control and comparable measurements, but have the challenge that doesn t give a proper representation of natural conditions, because 32

44 insects cuticle are hydrophobic. Even though we have information on stickiness we have little knowledge of the mechanism behind Tetragnatha viscid silk. Water content in glue droplets have an impact on adhesiveness (Opell & Hendricks 2009). Droplet volume measurements for this study were done at room humidity so we don t know if and how much water is gained and lost with changes in humidity, but it might be a predictor for stickiness in the species in this study. Among the traditional orb webs we can simplify architectures by describing size of webs (big or small) and the amount of silk used to build it (dense or low and sparse or high). Hypothetically, quality of the material can correlate with size and volume of silk to catch prey as shown in Figure 9. For example bigger webs have a higher chance of intercepting a higher number of insect than smaller webs, and having a higher volume of material can increase that effect. But having material of better quality can potentially increase the chances of trapping prey of higher energy; faster and bigger insects (Craig 1987). Ideally, all orb-weavers should build bigger webs because it significantly increase the chance of catching any type of prey. However silk volume is limited and spiders may get the best energy return from building smaller webs with that silk, to catch big prey (Venner & Casas 2005; Blackledge 2011). Among the radiation of Hawaiian Tetragnatha you can find webs all over this range, from relatively small and sparse to big and dense (Blackledge & Gillespie 2004). However, our study on silk properties includes a limited sample of this web-building behaviors with little variation of size and two types of material volume (big-sparse and big-dense). Results of our study suggest that there can be three possibilities among the webs tested: (1) good-quality material, big and sparse web, (2) good-quality material, big and dense web, and (3) low-quality material, big and dense web (Blue rectangles Figure 9). Because we found T.trituberculata to have the toughest radial silk and stickier viscid silk, compared to the other groups, we discarded low-quality material, big and sparse web as a possible explanation. What is more likely the case for Hawaiian orb weavers is that they have adapted to the type of flying prey present in the archipelago. Indigenous insect fauna in the islands is dominated by Lepidoptera, Diptera, Hymenoptera and Coleoptera (Nishida 1994; Wigfull 1997) and Diptera and 33

45 Lepidoptera are the most common prey found in webs of Tetragnatha (Nishida 1994; Gillespie et al. 1997; Wigfull 1997; Blackledge et al. 2003). Because webs of spiders were collected directly in the field we have no idea or control of the diet and conditions of spiders prior to collection. Spiders may adjust web architectures, glue droplet morphologies and/or glue properties based on dietary input (Blamires et al. 2016). Results suggest that webs from populations of Tetragnatha stelarobusta and T.trituberculata may be targeting similar energy prey, but of higher energy than prey capture by webs of T.hawaiensis. We also saw that T.stelarobusta use a different mechanism to specialize, using chemistry of glue to target insect from the Lepidoptera family. Apart from the material intrinsic properties other diverse selective forces may promote patterns of coevolution. First, there s a tradeoff on the thickness of silk diameters making an orb web, fibers too thick may be visible by prey and predators (Craig 1986), but too thin may compromise the properties of the silks. Second, the number of radii in the web correlates with the mesh width of orb webs, many spirals per turn help to avoid the risk of capture silk sticking together by adding many radii (Sensenig et al 2010). Third, in addition of prey selection other habitat characteristics, like wind, may be co-acting and influencing web-building behaviors. When looking close at the data, T.hawaiensis_M is similar to T.hawaiensis_H, while T.stelarobusta is most similar to T.trituberculata (See stress-strain curves force-extension curves in Figure 2E and 4D). This make sense when looking at the evolutionary history of Hawaiian web-building clade of Tetragnatha. T.stelarobusta and T.trituberculata are sister taxa and part of a radiation that s about 1.2 MYA (Gillespie et al 1999). T.hawaiensis on the other hand is part of a later natural colonization to the islands, most closely related to species in the continental area (Gillespie et al 1994, 1997). Furthermore, when comparing radial silk toughness to a species of North American Tetragnatha, the likely origin of Hawaiian Tetragnatha, similar work was performed by radial threads of T.versicolor (0.16 ± 0.05 GPa; Sensenig et al. 2010) and T.trituberculata (0.16 ± 0.04 GPa), having a higher toughness than the other species T.stelarobusta and T.hawaiensis. Additionally viscid silk stickiness was compared to two other species from the Ohio area, T.laboriosa (1.5 X 10-7 J) and T.elongata (8.3 X 10-7 J) (Figure 6A; 34

46 Figure 9. Correlation of architectural aspects of webs and its silks material quality to determine potential prey type and/or the amount of insects that could potentially be capture. Prey type refers to high (+) Energy insects that are faster and bigger, and low (-) Energy insects that are smaller and slower. Following from (Craig 1987) two extremes of webs (II) big, dense high-energy absorbing webs with many radial per capture spiral turn ratio and (III) small, sparse low-energy absorbing webs with low volume of silk (yellow square). Bigger webs have the potential to intercept a larger amount (+) of Insects, while smaller webs have a less (-) chance. Unpublished data from our lab), and stickiness values for the Hawaiian species range in between these. Tetragnatha in the archipelago of Hawaii have evolved a diverse range of morphologies and web spinning behaviors making them distinct from their congeners around the world who are relatively homogeneous in their ecology and morphologies (Gillespie & Croom 1995; Blackledge & Gillespie 2004). This brings the question of phylogenetic inertia (Hansen & Orzack 2005). Is phylogenetic inertia constraining the evolution of silks or have not enough ecological time been pass to see a change in the biomaterial properties? Most likely no since we already see a change in silk properties but silk properties seem to be constrained by other factors, like body size, and the genes determining mechanical functions (Craig 2003). For example Sensing et al. 35

47 (2010) found a link between spider size and performance of webs and species in this study range very little in size (Carapace width: mm; Table S1) so we might not be seeing this size effect. Also at a deep phylogenetic level mechanical properties of silk scale up to two folds for toughness of major ampullate silk among orb weavers (Agnarsson et al. 2010; Sensenig et al. 2010), showing this constraint. Many factors are determining success of webs and it will be really interesting to look at the silk gene differences in this radiation to see were effects on function starts. Minimal attention has been given to the role of material properties in the evolution of performance. Spider webs present an ideal system to the study this, and we show that silk material properties are changing in an adaptive radiation of Hawaiian orb-weaving spiders. A variety of web behaviors have evolved among this closely related species in the archipelago but we only look at a limited range so widening our sampling will also widen the scope of our understanding of form and function of orb-webs. By doing this we could also explore other interesting questions about the behaviors of this orb-weavers such as the loss of the behavior and the effects on material properties. This study shows potential to answer big picture questions about the form and functions of structures in nature, and how those functions may be enhanced by the properties that make them at a short time scale. 36

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53 APPENDIX Table S1. Spider s size measurements. Values represent mean + standard error. Species Location / N Carapace Width Carapace Length Opisthosoma Width Opisthosoma Length Femur Width Femur Length (mm) (mm) (mm) (mm) (mm) (mm) T.hawaiensis_M Maui / ± ± ± ± ± ± 0.3 T.hawaiensis_H Hawai'I / ± ± ± ± ± ± 0.5 T.stelarobusta Maui / ± ± ± ± ± ± 0.3 T.trituberculata Maui / ± ± ± ± ± ±

54 Species Table S2. Tensile properties for radial silk. Values represent mean ± standard error. *Diameter of one axial thread. Location / N Diameter* Toughness Strength Extensibility Stiffness (um) (GPa) (=True Stress GPa) (=True Strain ln mm/mm) (=Young s Modulus GPa) T.hawaiensis_M Maui / ± ± ± ± ± 3.05 T.hawaiensis_H Hawai'i / ± ± ± ± ± 1.95 T.stelarobusta Maui / ± ± ± ± ± T.trituberculata Maui / ± ± ± ± ±

55 Table S3. Glue morphology measurements and adhesive tensile properties. Values represent mean ± standard error. Glue measurements were done at room humidity (46 ± 1 %RH). Species Location / N T.hawaiensis_M Maui / 6 T.hawaiensis_H Maui / 14 T.stelarobusta Maui / 6 T.trituberculata Maui / 8 RH Glue Vol. Space between Droplets Peak Load Normalized Peak Load Work to Release Normalized Work to Release Extension (%) (um 3 X10 3 ) (um) (un) (un X10-4 /um 3 ) (J X10-7 ) (J X10-12 /um 3 ) (mm) ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

56 Table S4. Web architecture measurements. Values represent mean ± standard error. Species Location / N # Radii T.hawaiensis_H Hawai I / ± 0.45 T.stelarobusta Maui / ± 0.32 T.trituberculata Maui / ± 0.33 Spiral Total Length Mesh Width Total area of web (mm) (mm) (cm 2 ) ± ± ± Type of Architecture 3.04 ± ± Strong 2.99 ± ± Strong 8.23 ± ± Weak 45

57 Figure S1. Differences in the adhesive properties of viscid silk for species at three humid conditions: 50% (red), 70% (grey) and 85% (blue). Letters represent significance difference (p<0.05). (A-B) Show absolute values of stickiness. (D-E) Show stickiness parameters normalized to glue volume. (F) Percent of change in absolute and normalized stickiness parameters for between humidities (85-70, 70-50, 85-50) of each species. Red values represent significant differences. 46