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Eileen Hebets Publications Papers in the Biological Sciences

2-2016 Benefits of size dimorphism and copulatory silk wrapping in the sexually cannibalistic nursery web , mira Alissa G. Anderson University of Nebraska–Lincoln, [email protected]

Eileen Hebets University of Nebraska - Lincoln, [email protected]

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Anderson, Alissa G. and Hebets, Eileen, "Benefits of size dimorphism and copulatory silk wrapping in the sexually cannibalistic , " (2016). Eileen Hebets Publications. 54. http://digitalcommons.unl.edu/bioscihebets/54

This Article is brought to you for free and open access by the Papers in the Biological Sciences at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Eileen Hebets Publications by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. Published in Biology Letters 12: 20150957; doi: 10.1098/rsbl.2015.0957 Copyright © 2016 Alissa G. Anderson and Eileen A. Hebets. Published by the Royal Society. Used by permission. Submitted November 13, 2015; accepted January 29, 2016.

digitalcommons.unl.edu Supplementary material: Methods, Tables S1 & S2, and Figure S1 follow the References; data set attached to the repository record for this document; video link: https://youtu.be/0CpVcjT4URw

Benefits of size dimorphism and copulatory silk wrapping in the sexually cannibalistic nursery web spider, Pisaurina mira

Alissa G. Anderson and Eileen A. Hebets

School of Biological Sciences, University of Nebraska–Lincoln, Lincoln, NE 68588, USA Corresponding author — Alissa G. Anderson, email [email protected] ORCID: 0000-0003-1744-3070 (AGA)

Abstract In sexually cannibalistic , male fitness is influenced not only by successful mate acquisition and egg fertilization, but also by avoiding being eaten. In the cannibalistic nursery web spider, Pisaurina mira, the legs of mature males are longer in relation to their body size than those of females, and males use these legs to aid in wrapping a female’s legs with silk prior to and during copulation. We hypothesized that elongated male legs and silk wrapping provide benefits to males, in part through a reduced likelihood of . To test this, we paired females of random size with males from one of two treatment groups—those capable of silk wrapping versus those incapable of silk wrapping. We found that males with relatively longer legs and larger body size were more likely to mate and were less likely to be can- nibalized prior to copulation. Regardless of relative size, males capable of silk wrapping were less likely to be cannibal- ized during or following copulation and had more opportunities for sperm transfer (i.e. pedipalpal insertions). Our results suggest that male size and copulatory silk wrapping are sexually selected traits benefiting male reproductive success. Keywords: behavior, evolution, antagonistic coevolution, cannibalism, mating system evolution, sexual conflict, sexual selection

1. Introduction be beneficial to a male [5,6]. Nonetheless, even post-copulatory sexual cannibalism carries a potentially high cost (e.g. reduced For most males, reproductive success is dependent on the ability sperm transfer and loss of future reproductive opportunities) to acquire mates and successfully fertilize a female’s eggs [1,2]. and can strongly influence the evolution of male mating strate- Research across diverse animal taxa has documented elaborate gies and associated morphologies [7,8]. courtship displays and seemingly coercive strategies that exist Given the costs of sexual cannibalism, we expect positive se- to secure matings (reviewed in [1–3]). In systems with the po- lection for male traits/behaviors that reduce the likelihood of be- tential for high levels of sperm competition, males can increase ing eaten. Indeed, across cannibalistic spider taxa (Class Arach- fertilization success by preventing females from successfully re- nida, Order Araneae), sexual cannibalism is commonly associated mating through mate guarding or the production of sperm plugs with extreme morphologies and striking mating systems (re- and seminal toxins, among others (reviewed in [4]). For a small viewed in [9,10]). For example, sexual size dimorphism (SSD) number of taxa in which females are sexually cannibalistic, how- is often associated with sexual cannibalism [11,12]. It has been ever, males have an additional challenge—surviving a reproduc- suggested that longer male legs have been selected to avoid tive encounter. While cannibalism prior to sperm transfer results pre-copulatory cannibalism in a number of orb-weaving spi- in zero male fitness, cannibalism after the transfer of sperm can ders [13]. Additionally, larger male fishing (Dolomedes

1 2

laboratory under controlled conditions. To assess the effect of size dimorphism and copulatory silk wrapping, we ran mating trials in which males were assigned to either a (i) wrap (n = 15) or (ii) no wrap (n = 16) treatment and paired with females of random size. We phe- notypically manipulated males in each treatment by applying den- ® tal silicone (Take 1 Advanced™ Kerr, Orange, CA, USA) to either (i) the dorsal side of a male’s abdomen (wrap treatment) or (ii) a male’s (no wrap treatment). During mating trials, we live-scored copulation success, pre- and post-copulatory cannibalism, and the number of insertions that a male obtained. We obtained body size

measurements (i.e. carapace width (CW) and leg length) from pre-

served specimens following mating trials. For additional method-

ological details, see the supplementary material. If size dimorphism in P. mira benefits males in terms of reproduc- tive success, we predicted seeing males with higher male : female leg length or larger body size: (1a) achieve higher mating success and (1b) experience fewer pre-copulatory cannibalism events. We had no a priori predictions for the relationship between size dimor- phism and post-copulation cannibalism or insertion number, as we expected these to be influenced more by the silk wrapping. Specif- ically, we predicted that silk wrapping males would (2a) experience fewer cannibalism events during or after sperm transfer and (2b) ex- perience two versus only one pedipalpal insertions. We predicted Pisaurina mira Figure 1. Male wrapping female with silk prior to no relationship between silk wrapping and mating success or pre- copulation copulatory cannibalism, as these both occur prior to silk wrapping. We used separate binomial linear regressions to test the effects of fimbriatus) are more successful in avoiding cannibalistic attempts size and Fisher’s exact tests to examine the effects of silk wrapping by females [14]. Many species with SSD, however, are not canni- on reproductive behavior. For details on statistical analyses, see the balistic and the cause/effect relationship between SSD and sex- supplementary material. ual cannibalism is not well understood. Unusual behaviors such as death feigning [15], opportunistically mating with feeding fe- males [16,17] and rendering females unconscious with sedative 3. Results pheromones [18] are also presumably associated with avoid- ing sexual cannibalism. This study focuses on another particu- (a) Size dimorphism and proxies of fitness larly unusual mating behavior— the wrapping of females in silk Male P. mira have relatively longer legs and a larger leg length to by males in the sexually dimorphic nursery web spider Pisau- body ratio than females, but females have a larger CW (i.e. larger rina mira (Walchenaer). body size) than males (supplementary material, table S1). As pre- Pisaurina mira spiders exhibit a sexually dimorphic body dicted, male : female leg length predicted copulation success shape with males presenting seemingly elongate front legs (fore- (χ2 = 12.96, p < 0.0001; figure 2a) and pre-copulatory canni- legs), and an unusual mating strategy wherein a male will wrap 1,29 balism (χ2 = 7.76, p = 0.005; figure 2b); results are identical for a female’s legs with silk prior to and during sperm transfer [19] 1,29 male : female CW (see the supplementary material). Males were (figure 1). This unusual behavior has been postulated to reduce more likely to copulate and less likely to be cannibalized when female mobility and the likelihood of sexual cannibalism [19], but their legs were longer than those of their mating partner and this hypothesis has not been directly tested. A function in de- male : female CW was larger. For all males (i.e. those in the wrap creasing pre-copulatory cannibalism seems unlikely as receptive and those in the no wrap treatment), we found no effect of male females appear to passively allow males to initially wrap them : female leg length or body size (see the supplementary mate- with silk. Following the first act of sperm transfer (i.e. the first rial) on a male’s likelihood of being cannibalized post-copulation insertion of the male’s ), however, females begin to en- (wrap: χ2 = 0.246, p = 0.620; no wrap: χ2 = 0.412, p = 0.521) gage in movements that look like an attempt to free themselves 1,10 1,8 or on the number of insertions obtained (wrap: χ2 = 0.300, p from the silk wrapping (supplementary material, video S1). 1,10 = 0.584; no wrap: χ2 = 0.826, p = 0.363). For simplicity, we per- We hypothesized that size dimorphism influences the ability 1,8 formed both tests with the two male treatments combined and of P. mira males to initially engage a female in copulation while present these combined results in our figure (post-copulatory silk wrapping enables them to acquire additional sperm trans- cannibalism: χ2 = 0.0002, p = 0.988, figure c2 ; insertion num- fer events (hereafter termed insertions) without being cannibal- 1,19 ber: χ2 = 0.275, p = 0.600, figure d2 ). ized. We test these hypotheses by randomly pairing phenotypi- 1,17 cally manipulated males (silk wrapping versus no silk wrapping) with females of variable size and assessing proxies of fitness. (b) Silk wrapping and proxies of fitness Copulation success and rates of pre-copulatory cannibalism did not differ between males that could and could not wrap fe- 2. Material and methods males (Fisher’s exact test, p = 0.458; figure e2 and p = 0.394; We collected immature male and female Pisaurina mira (Lancaster figure 2f, respectively). In line with our predictions, copulatory County, NE, USA) in Spring 2014. Spiders were maintained in the silk wrapping affected rates of post-copulatory cannibalism and 3

Figure 2. The probability of males (a) copulating, (b) being cannibalized pre-copulation, (c) being cannibalized post-copulation and (d ) obtaining two insertions as a function of male : female leg length (n = 31). The proportion of males that (e) copulated, (f ) were cannibalized pre-copulation, (g) were cannibalized post-copulation and (h) achieved two insertions during copulation when allowed to wrap (n = 15) and not wrap (n = 16) females. The two males that obtained 1.5 insertions were excluded from the analyses presented in d and h. The asterisk signifies significance based on pa -value < 0.05.

the number of insertions a male obtained. Males that could not 4. Discussion wrap females were more likely to be cannibalized after insem- ination compared with males that could wrap females (Fisher’s Size dimorphism and male silk wrapping in the nursery web spi- exact test, p = 0.017; figure g2 ). Males in our wrap treatment der P. mira are important for male mating success and survival. were also more likely to achieve two insertions (versus one) com- Mature males possess longer forelegs, relative to body size, than pared with males in our no wrap treatment (Fisher’s exact test, their female counterparts and this dimorphism is important for p = 0.049; figure h2 ). male reproductive success as males with relatively longer legs 4 were: (i) more likely to achieve a successful mating and (ii) less copulatory silk wrapping have been sexually selected through likely to be cannibalized prior to copulation. Using phenotypic fitness benefits to males, but the putative fitness cost(s) of these manipulations to remove a male’s ability to wrap his mate, we traits for females remain unknown. also demonstrate that silk wrapping: (i) reduces the likelihood of cannibalism during or after sperm transfer and (ii) increases the number of insertions a male can achieve during mating. Though males with relatively longer legs obtained mating Ethics. The work conducted complies with the ethical regulations in advantages, male leg length and CW are strongly correlated the USA. (see the supplementary material and figure S1), making it dif- Competing interests. We have no competing interests. ficult to confirm a direct benefit of longer legs per se. Our re- sults do suggest, however, a reproductive benefit of males being Data accessibility. Datasets supporting this article have been uploaded larger (or closer in size) relative to their mating partner, a pattern as part of the supplementary material. found across a number of distinct taxa, including red-sided gar- Authors’ contributions. A.G.A. discovered the system, carried out all ter snakes [20], Drosophila [21,22] and the tobacco moth [23]. experiments and statistical analyses and wrote and revised the manu- In addition to increased mating success, longer male legs (and script. E.A.H. participated in the experimental design, the interpretation larger males) also reduced the risk of precopulatory sexual can- of data, and the writing and revising of the manuscript. All authors agree nibalism, a pattern consistent with other cannibalistic species to be held accountable for the content provided within the manuscript and give final approval for its publication. ([11,12,24] but see [25]). With precopulatory cannibalism result- ing in a complete loss of fitness for males, it is not surprising to Funding. Research funds from Graduate Assistance in Areas of National observe larger body size and/or grasping traits that reduce the Need (GAANN) provided support for this research. P. mira, risk of cannibalism. In males’ relatively longer legs ap- Acknowledgements. We thank Lincoln Parks and Recreation for park pear to aid in their ability to manipulate, wrap and restrain fe- access and Malcolm Rosenthal, Jay Stafstrom and Austin Brooks for males. As has been previously suggested across multiple can- help with spider collections. We also thank Marie-Claire Chelini, Mal- nibalistic orb-weaving spiders [13], we propose that longer leg colm Rosenthal and members of the Basolo, Hebets, Shizuka and Wag- length is a sexually selected trait in male P. mira. ner labs for providing feedback on this project. Finally, we thank Dr Ki- Sexual cannibalism can lead to obvious male fitness costs ourtsis for supplying the dental silicone used in this experiment. (e.g. lost potential to secure future matings or reduction of sperm transferred) [7,8], yet sexual cannibalism can also be an adaptive male strategy [5,6]. In the dark fishing spider (Dolom- edes tenebrosus), male consumption by females significantly in- References creases offspring number, weight and survival [6]. It is currently unknown if P. mira males receive any fitness benefits from be- 1. Andersson MB. 1994 Sexual selection. Princeton, NJ: Princeton Uni- ing cannibalized. Given that silk wrapping reduces the likelihood versity Press. of post-copulatory sexual cannibalism, however, we hypothesize 2. Parker GA. 1984 Sperm competition and the evolution of animal mat- that male silk wrapping evolved to enable males to obtain in- ing strategies. In Sperm competition and the evolution of animal mat- creased benefits from multiply mating. ing systems (ed. RL Smith), pp. 1–60. Orlando, FL: Academic Press. In addition to reducing rates of sexual cannibalism, we found 3. Arnqvist G, Rowe L. 2013 Sexual conflict. Princeton, NJ: Princeton Uni- that silk wrapping increases the number of insertions that a male versity Press. can obtain. We suspect that a male’s ability to achieve more in- 4. Simmons LW. 2001 Sperm competition and its evolutionary conse- sertions corresponds to increased sperm transfer, which in turn quences in the insects. Princeton, NJ: Princeton University Press. corresponds (to some degree) to fertilization success. In the can- 5. Andrade M. 1996 Sexual selection for male sacrifice in redback spi- Am. Assoc. Adv. Sci. nibalistic orb-weaving spider, Argiope aurantia, males experi- ders. 271, 70–72. enced 25% higher fertilization success when achieving two ver- 6. Schwartz SK. 2013 Spontaneous male death and monogyny in the dark Dolomedes tenebrosus sus one insertion [26]. Additionally, the transfer of more sperm fishing spider Hentz, 1843 (Araneae, Pisau- ridae). PhD dissertation, University of Nebraska, Lincoln, NE, USA. might out-dilute or displace rival sperm, thereby increasing the 7. Elgar MA, Crespi BJ. 1992 Ecology and evolution of cannibalism. In proportion of the female’s eggs fertilized by a given male [2,4]. Cannibalism: Ecology and evolution among diverse taxa P. mira (eds. MA El- In preliminary trials, we find evidence that female mate gar, BJ Crespi), pp. 1–12. Oxford, UK: Oxford University Press. multiply (primarily through forced copulations), suggesting a po- 8. Elgar MA, Schneider JM. 2004 Evolutionary significance of sex- tential benefit of increased sperm transfer. ual cannibalism. Adv. Study Behav. 34, 135–163. doi:10.1016/ Though females may also benefit from mating with larger S0065-3454(04)34004-0 males, the striking behavior of copulatory silk wrapping may re- 9. Schneider JM, Yael L. 1998 Intersexual conflict in spiders. OIKOS 83, flect an evolutionary history of conflicting reproductive strate- 496–506. doi: 10.2307/3546677 gies between males and females. Within mating trials, females 10. Elgar MA, Crespi BJ (eds.). 1992 Cannibalism: Ecology and evolution attempt to be freed from the silk wrapping, which suggests they among diverse taxa. Oxford: Oxford University Press. may incur a cost by being wrapped (e.g. missed post-mating 11. Elgar MA, Nash DR. 1988 Sexual cannibalism in the garden spider cannibalism). Yet, virgin females often passively allow the wrap- Araneus diadematus. Anim. Behav. 36, 1511–1517. doi: 10.1016/ ping to take place. Costs versus benefits of wrapping for vir- S0003-3472(88) 80221-5 gin versus mated females and males may differ substantially as 12. Wilder SM, Rypstra AL. 2008 Sexual size dimorphism predicts the fre- sperm competition could play an important role in this system. quency of sexual cannibalism within and among species of spiders. This study provides strong evidence that size dimorphism and Am. Nat. 172, 431–440. doi: 10.1086/589518 5

13. Elgar MA, Ghaffar N, Read AF. 1990 Sexual dimorphism in leg length 2000 Body size enhances mating success in male garter snakes. among orb-weaving spiders: a possible role for sexual cannibal- Anim. Behav. 59, F4–F11. doi: 10.1006/anbe.1999.1338 ism. J. Zool. 222, 455–470. doi: 10.1111/j.1469-7998.1990.tb04044.x 21. Sisodia S, Singh BN. 2004 Size dependent sexual selection in Dro- 14. Arnqvist G, Henriksson S. 1997 Sexual cannibalism in the fishing spi- sophila ananassae. Genetica 121, 207–217. doi: 10.1023/B:GENE.00 der and a model for the evolution of sexual cannibalism based on 00040390.90003.7f genetic constraints. Evol. Ecol. 11, 255–273. doi: 10.1023/A:10184123 22. Markow TA. 1988 Reproductive behavior of Drosophila melanogaster 02621 and D. nigrospiracula in the field and in the laboratory.J. Comp. Psy- 15. Bilde T, Tuni C, Elsayed R, Pekár S, Toft S. 2006 Death feigning in chol. 102, 169–173. doi: 10.1037/0735-7036.102.2.169 the face of sexual cannibalism. Biol. Lett. 2, 23–25. doi: 10.1098/ 23. Phelan PL, Baker TC. 1986 Male-size-related courtship success and rsbl.2005.0392 intersexual selection in the tobacco moth, Ephestia elutella. Experi- 16. Fromhage L, Schneider JM. 2005 Safer sex with feeding females: Sex- entia 42, 1291–1293. doi: 10.1007/BF01946428 ual conflict in a cannibalistic spider. Behav. Ecol. 16, 377–382. doi: 24. Persons MH, Uetz GW. 2005 Sexual cannibalism and mate choice de- 10.1093/beheco/ari011 cisions in wolf spiders: Influence of male size and secondary sexual 17. Prenter J, Elwood RW, Montgomery WI. 1994 Male exploitation of characters. Anim. Behav. 69, 83–94. doi: 10.1016/j.anbehav.2003.12. female predatory behaviour reduces sexual cannibalism in male 030 autumn spiders, Metellina segmentata. Anim. Behav. 47, 235–236. 25. Elgar MA, Fahey BF. 1996 Sexual cannibalism, competition, and doi:10.1006/anbe.1994.1031 size dimorphism in the orb-weaving spider Nephila plumipes La- 18. Becker E, Riechert S, Singer F. 2005 Male induction of female quies- treille (Araneae: Araneoidea). Behav. Ecol. 7, 195–198. doi: 10. 1093/ cence/catalepsis during courtship in the spider, Agelenopsis aperta. beheco/7.2.195 Behaviour 142, 57–70. doi: 10.1163/1568539053627767 26. Foellmer MW, Fairbairn DJ. 2004 Males under attack: Sexual canni- 19. Bruce JA, Carico JE. 1988 Silk use during mating in Pisaurina mira balism and its consequences for male morphology and behaviour (Walckenaer) (Araneae, Pisauridae). J. Arachnol. 16, 1–4. in an orb-weaving spider. Evol. Ecol. Res. 6, 163–181. 20. Shine R, Olsson MM, Moore IT, Lemaster MP, Greene M, Mason RT. Supplementary materials

Benefits of sexual size dimorphism and copulatory silk wrapping in the sexually cannibalistic nursery web spider, Pisaurina mira

Alissa G. Anderson & Eileen A. Hebets

School of Biological Sciences, University of Nebraska – Lincoln, Lincoln, NE 68588, USA [email protected]

Methods

Spider collection and maintenance

We collected immature male and female Pisaurina mira at night from Wilderness Park,

Lancaster County, Lincoln, NE from 2 May – 12 May 2014. Spiders were individually housed at the University of Nebraska-Lincoln in 87.3 mm X 87.3 mm X 112.7 mm clear plastic containers

(763C, AMAC Plastics, Petaluma, CA). We covered the outside of each container with opaque tape to visually isolate individuals. Spiders were maintained under a 12 h light/12 h dark cycle, fed three 7 mm juvenile crickets per week (Gryllus sigilatus, Ghann’s cricket farm, GA, USA) and provided water ad libitum.

I. Size Dimorphism and Proxies of Fitness

If male-biased size dimorphism in P. mira benefits males in terms of reproductive success, we predicted to see males with higher male:female leg length: (1a) achieve higher mating success and (1b) experience fewer pre-copulatory cannibalism events. We predicted no relationship between size dimorphism and post-copulatory cannibalism or insertion number.

Size measurements were taken from preserved specimens following mating trials (see II.

Silk Wrapping and Proxies of Fitness). All spiders were preserved in 70% EtOH post-death. To

1 measure leg length, we removed the first right leg from preserved individuals, and allowed them to air dry. Legs were placed lateral side up on a glass slide that sat atop a scale graduated in mm and were photographed using a Leica DM 4000 B microscope with a Di-agnostic Instruments,

Inc. Spot Flex digital camera. Our body size measure reflected the widest point of the carapace

(often referred to as cephalothorax; anterior-most body part), which is fixed in size at maturity.

To measure carapace width, we removed all appendages from each preserved spider and photographed the carapace using the same set up described previously. Measurements of leg length and carapace width were taken from the digital photographs using the program PixelStick ver. 2.8.

Additional photographs of males were taken prior to mating trials as a precaution in case males were cannibalized during the trial. Live males were placed on size scale graduated slides

(mm) and then covered by a petri dish. Covering the male with the petri dish caused them to completely stretch out their legs. We used a Nikon COOLPIX S6100 digital camera held perpendicular to the table surface to take photographs. For a subset of ten males, we used paired t-tests to confirm that leg length and body size measurements did not differ between our two measurement protocols (t9 = 0.545, p = 0.599 and t9 = -0.987, p = 0.349, respectively). In the event that males were cannibalized during mating trials, pre-mating photographs were used for morphological measurements.

Statistical Analyses

To assess differences between male and female carapace width, leg length, and leg to body ratios we used t-tests. From our body measurements we found that carapace width and leg length are strongly correlated for both males and females (males: R2 = 0.869, df = 29, p < .0001; females:

R2 = 0.829, df = 29, p < 0.0001; Figure S1). To account for male and female size differences we

2 used the ratio of male to female leg length and the ratio of male to female carapace width in the following analyses. We used separate binomial logistic regressions to test our predictions that male:female leg length and male:female carapace width (CW) affects copulation success and pre-copulatory cannibalism. We also used separate binomial logistic regressions to determine if male:female leg length and male:female CW affects post-copulatory cannibalism and the number of insertions a male obtained. Because there is an effect of our silk ablation treatment on post- copulatory cannibalism and insertion number (see results) we also performed separate binomial logistic regressions for both treatments. Within our analysis, looking at the effect of male:female leg length and CW on number of insertions, we called a male successful if he obtained two insertions (versus one). For this analysis we excluded the two trials where the females cannibalized the male in the middle of the male’s second insertion.

II. Silk Wrapping and Proxies of Fitness

If silk wrapping increases male reproductive success, especially through decreased risk of sexual cannibalism, we predicted that silk wrapping males would (2a) experience fewer cannibalism events during or after sperm transfer and (2b) experience more pedipalpal insertions.

We predicted no relationship between silk wrapping and copulation success or pre-copulatory cannibalism. To test these predictions, we randomly assigned males to a (i) wrap (n = 15) or (ii) no wrap treatment (n = 16). We ablated a male’s silk wrapping ability by covering the male’s spinnerets with dental silicone (Take 1® AdvancedTM, Kerr, Orange, CA). To control for any potential effects of our treatment, we placed dental silicone on the dorsal side of a male’s abdomen in the wrap treatment. To apply the dental silicone treatment, we first placed males into a freezer for approximately three minutes to slow their movement. We carefully restrained males onto a Styrofoam block by pinning down their legs and then applied dental silicone to the pre-

3 assigned body region (i.e. wrap – dorsal abdomen; no wrap – spinnerets). Males were never restrained for more than one minute. Once released, we gave males 30 minutes to recover prior to the start of the mating trial. Prior to trials we also confirmed by visual inspection that the ‘no wrap’ treatment males were unable to lay down any silk.

Mating trials took place from 21 May – 2 June 2014 between 1200 and 1800h and consisted of a randomly paired virgin male and female 15 days ± 1-day post-maturation. Trials were run within 200 mm dia. X 190 mm ht. covered, plastic arenas (250C, Pioneer Plastics,

North Dixon, KY) with filter paper (185 mm dia.) placed on the arena floor. To allow spiders to climb, we placed a plastic cylinder (42 mm dia. X 156 mm ht.) covered with fiberglass screen wire at the center of the arena. Two strips (40 mm wid.) of screen wire were placed on opposing walls that extended the height of the arena. The central cylinder and screen wire strips were connected near the top of the arena using two wooden craft sticks (9 mm wid.). Between mating trials we wiped down the arenas with 70% EtOH and replaced the filter paper. We placed females in mating arenas approximately 24 hours prior to the male’s introduction.

A trial began once we placed the male in the arena at the farthest point from the female’s current location. During mating trials, we live-scored copulation success, pre and post-copulatory cannibalism, and the number of insertions that a male obtained. We categorized cannibalism events as pre-copulatory cannibalism if it occurred prior to a male’s insertion or as post- copulatory cannibalism if it occurred after or during a male’s insertion. Quantification of the above variables was not preformed blindly because the dental silicone made male treatment obvious to the observer. In mating trials, the dental silicone worked as expected - all of the males in the no wrap treatment were unable to wrap females, while all males in the wrap treatment successfully wrapped the females with silk.

4

Statistical Analyses

To determine if copulation success, pre-copulatory cannibalism and post-copulatory cannibalism differed between males that could and could not wrap females, we used Fisher’s

Exact Tests. To determine if the number of insertions a male obtained differed between our two treatments we also used a Fisher’s Exact test. Because males obtained one or two insertions, we compared the proportion of males that achieved two versus one insertion. For this analysis we excluded two trials where the females cannibalized the male in the middle of the male’s second insertion. All statistical analyses were conducted using R software (ver. 3.0.3).

Results

Size Dimorphism and Proxies of Fitness

Female P. mira have a larger carapace width (i.e. larger body size) than males, but males have relatively longer legs and a larger leg length to body ratio (Table S1). As presented in the main text, males were more likely to copulate and less likely to be cannibalized when their legs were longer than those of their mating partner. Similarly, males were more likely to copulate (2 = 1,29

15.87, p < 0.0001) and less likely to be cannibalized prior to copulation ( 2 = 7.84, p = 0.005) 1,29 when male:female CW was larger.

Again, similar to our results for male:female leg length, for all males (i.e. those in the wrap and those in the no wrap treatment) we found no effect of male:female CW on a male’s likelihood of being cannibalized post-copulation (wrap:  2 = 1.136, p = 0.286; no wrap:  2 = 1,10 1,8

0.746, p = 0.388) or on the number of insertions obtained (wrap:  2 = 0.826, p = 0.363; no 1 ,10 wrap:  2 = 0.966, p = 0.326). We also present results with the two male treatments combined 1 ,8

5

(post-copulatory cannibalism:  2 = 1.811, p = 0.178; insertion number:  2 = 0.253, p = 1 ,19 1 ,17

0.615).

In order to further explore the role of size on male reproductive behavior, we used the residuals of a correlation between male leg length and male carapace width as the predictor variable for our behavioral outcomes (i.e. likelihood of copulation, pre-copulatory cannibalism, post-copulatory cannibalism and obtaining two versus one insertion). Here we found that the relationship between male body size and leg length has no effect on any of our behavioral outcomes (Tables S2).

Tables and Figures

Table S1. Average (± SE) carapace width, leg length, and leg to body ratio for male and female Pisuarina mira. Results of independent t-tests represent differences between males and females and all three variables measured.

Female mean Male mean Variable t-value df p-value (mm) ± SE (mm) ± SE

Carapace width 4.462 ± 0.086 4.000 ± 0.066 4.26 55.91 <0.001

Leg length 20.97 ± 0.382 29.53 ± 0.425 14.98 59.34 <0.001

Leg:body 0.135 ± 0.002 0.213 ± 0.001 39.66 41.63 <0.001

6

female male

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5

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5

m

.

(

4

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t

d

i

w

e

c

a

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a

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20 25 30 35

leg length (mm)

Figure S1. Relationship between carapace width (mm) and leg length (mm) for mature male and female Pisaurina mira. Dashed lines represent best-fit lines.

7

Table S2. Results from binomial linear regressions using the residuals of a correlation between male leg length and male carapace width as the predictor variable on the likelihood of copulation, pre- copulatory cannibalism, post-copulatory cannibalism and obtaining two versus one insertion (i.e. behavioral outcome).

Behavioral outcome  2 n df p-value Copulation 0.669 31 1, 29 0.416 Pre-copulatory cannibalism 2.640 31 1, 29 0.115 Post-copulatory cannibalism 2.294 21 1, 19 0.190 Insertions 0.782 19 1, 17 0.377

8