Not just a chastity belt: the functional significance of mating plugs in garter snakes, revisited CHRISTOPHER R. FRIESEN 1 *, RICHARD SHINE 2 , RANDOLPH W. KROHMER 3 and ROBERT T. MASON 1 1 Department of Zoology, Oregon State University, Cordley Hall 3029, Corvallis, OR 97330, USA 2 Department of Biological Sciences, University of Sydney, Heydon-Laurence Building A08, Sydney, NSW 2006, Australia 3 Department of Biological Sciences, Saint Xavier University, 3700 W. 103rd ST., Chicago, IL 60655, USA Received 26 December 2012; revised 14 February 2013; accepted for publication 14 February 2013 During the spring emergence of red-sided garter snakes (Thamnophis sirtalis parietalis) in Manitoba, Canada, the operational sex ratio is strongly skewed towards males, who scramble to locate and court newly emerged females. A high frequency of multiple paternity litters suggests that the females are promiscuous; the gelatinous copulatory plugs (CPs) deposited by males may confer fitness benefits via passive mate guarding. Because precopulatory female choice is limited in large mating aggregations, sexual conflict may place a premium on preventing females from ejecting male sperm. In snakes, sperm are produced in the testes and delivered through the ductus deferens, and the CP is thought to be produced by the renal sexual segment and conveyed through the ureter. We manipulated the delivery of the two fluids separately by surgically ligating the ducts. Ureter-ligated males did not produce a CP, causing their sperm to leak out of the female’s cloaca immediately after copulation. Contrary to previous suggestions, histology revealed sperm distributed throughout the CP. Thus, the CP may function as a spermatophore: the protein matrix contains the sperm, which are liberated gradually as the plug dissolves. The likelihood of a male depositing a CP fell significantly after his second mating, perhaps limiting his reproductive success. These results challenge the hypothesis that passive mate guarding is the primary function of the CP in T. sirtalis parietalis. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 109, 893–907. ADDITIONAL KEYWORDS: copulatory plug – cryptic female choice – mating plug – mating system – reptile – sexual selection – sperm competition. INTRODUCTION Female sexual promiscuity is phylogenetically wide- spread (Smith, 1984; Birkhead & Møller, 1998). When a female mates with multiple males, the sperm of these males may co-mingle within her reproductive tract and compete for the fertilization of her ova: a phenomenon known as sperm competition (Parker, 1970). Males can gain an advantage in sperm compe- tition by increasing the number of sperm they insemi- nate relative to their rivals (Parker, 1990); however, an alternative strategy is to reduce the risk of sperm competition altogether by preventing a mate from remating with another male. Males can limit remat- ing opportunities by guarding their mates and/or by prolonging copulations, and thus reducing the risk of sperm competition (Birkhead & Møller, 1998; Simmons, 2001). A male may also passively guard a female by depositing substances that occlude the opening to her reproductive tract (known as copula- tory, mating, or vaginal plugs; Birkhead & Møller, 1998; Simmons, 2001). Just as female sexual promis- cuity is taxonomically widespread, so too are copula- tory plugs (CPs; Voss, 1979; Devine, 1984; Birkhead *Corresponding author. E-mail: friesenc@science.oregonstate.edu Biological Journal of the Linnean Society, 2013, 109, 893–907. With 4 figures © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 109, 893–907 893