TY - JOUR
AU - Langkilde,, Tracy
AB - Abstract Little is known about the operation of male mate choice in systems with perceived high costs to male choosiness. Scramble mating systems are one type of system in which male choice is often considered too costly to be selected. However, in many scramble mating systems, there are also potentially high rewards of male choosiness, as females vary dramatically in reproductive output and males typically mate once per season and/or per lifetime. Using scramble mating wood frogs (Rana sylvatica), we tested whether males gain fitness benefits by mating with preferred females. We conducted choice trials (1 male presented simultaneously with 2 females) and permitted males to mate with their preferred or nonpreferred female. Offspring of preferred and nonpreferred females were reared in the laboratory and field, and we quantified various fitness-relevant parameters, including survivorship and growth rates. Across multiple parameters measured, matings with preferred females produced fewer and lower-quality offspring than did those with nonpreferred females. Our results are inconsistent with the idea that mate choice confers benefits on the choosing sex. We instead propose that, in scramble systems, males will be more likely to amplex females that are easier to capture, which may correlate with lower quality but increases male likelihood of successfully mating. Such male choice may not favor increased fitness when the operational sex ratio is less biased toward males in scramble mating systems but is, instead, a bet-hedging tactic benefitting males when available females are limited. INTRODUCTION Expanding on the traditional paradigm of female choice and male competition (Andersson 1994), male mate choice is increasingly being recognized as an important component of evolution by sexual selection. With a new appreciation of high male investment in mating, namely the costs associated with courtship and intrasexual competition, there is growing evidence that these costs lead to selection for male mate choice in a variety of mating systems (Bonduriansky 2001; Edward and Chapman 2011, and citations therein). Male mate choice may be based upon female quality, and in many instances on female body size, as larger females frequently produce more or higher-quality offspring (e.g., Ceballos and Kiorboe 2010; Liu et al. 2017). Conversely, male mate choice may rely on dynamic signals from females, particularly behavior (e.g., Swierk et al. 2013) or color (e.g., Weiss 2002) indicating sexual receptivity, which can benefit males by allowing them to mate at a low cost of courtship. Many studies have documented that males gain reproductive benefits when mating with their preferred partners (e.g., Byrne and Rice 2006; Chen et al. 2012). In fact, due to high potential female variability and its direct effect on offspring, benefits of male choice may be even greater than those of female choice (Johnstone et al. 1996). Despite its large potential benefits, substantial requirements must be met before selection can favor male mate choice (Simmons and Parker 1996; Wedell et al. 2002; Härdling et al. 2008). In some systems, for example, the costs of mate sampling (e.g., risk of physical injury or predation during search, Rowe 1994; search time, Johnstone et al. 1996; energy spent in search or competition, Watson et al. 1998) for males may be too high to warrant rejecting any current female in favor of a potential future female, as males may fail to mate altogether. Consequently, random mating for males is commonly predicted in systems in which the costs of male mate sampling are expected to be high (Dechaume-Moncharmont et al. 2016). These barriers to male choice, in conjunction with co-occurring female mate choice and intrasexual competition, has led to the prediction that male choice generally does not exert selection on female traits (Fitzpatrick and Servedio 2017), though this is far from unequivocal (Bonduriansky 2001; Clutton-Brock 2009; Yun et al. 2017). Because male choice and associated selection on female phenotype are predicted to be strongly dependent upon the relative costs and benefits of male choice to the male, it is imperative to identify these costs and benefits to have a full understanding of the operation of sexual selection in a given system. Notably, however, empirical data on the benefits of male choice are lacking for systems in which its costs are assumed to be too high to preclude its selection. One such type of system is that defined by scramble mating competition, in which courtship and combat are typically considered absent and mating success is determined by rapid mate searching (Wells 1977; Alcock 1980). Male choice in systems defined by scramble competition is assumed to be rare; due to the high opportunity cost of rejecting a current mating opportunity, it is predicted that choice thresholds must be very low and/or individuals should not sample more than 2 potential mates prior to a decision (Dechaume-Moncharmont et al. 2016). However, in many anuran scramble mating systems, the benefits of mate choice could potentially be very high, as larger females produce substantially more and higher-quality eggs, all of which are fertilized simultaneously by a single male. Furthermore, after locating a female, male anurans in scramble mating systems typically spend most of the remaining mating season in amplexus with that female; this consequently reduces the potential for any subsequent matings, which increases the likelihood of selection for male mate choice (Bonduriansky 2001). The wood frog (Rana sylvatica) is an abundant anuran found throughout Canada and the eastern United States (Conant and Collins 1998) that employs a scramble mating system. Wood frogs have a short breeding season (about one week at our study site) in early spring (Berven 1981; Howard and Kluge 1985). At the start of the season, male wood frogs enter breeding ponds and participate in a chorus that attracts conspecifics (Banta 1914; Bee 2007; Tennessen et al. 2014). Males rapidly amplex females soon after females enter breeding ponds, and amplexus with a single female can continue for the duration of the mating season (up to about 4 days) (Wells 1977; Howard 1980). This time constraint, combined with a reduced ability of males to fertilize eggs with successive matings (Swierk et al. 2015), suggests that males are limited in their ability to mate with multiple females even in the absence of competition. Females lay single-paternity clutches of up to ~1000 eggs; adult female body size strongly correlates with the number and quality of offspring produced (a 14 mm increase in female body length is associated with a 4-fold increase in egg number, and the growth in female body size during a single year can increase individual egg diameter by up to 50%; Berven 1988). Adult female wood frog body size is highly variable within a population (Howard 1980). There are mixed reports of male mate choice occurring in wood frogs, with some (Berven 1981) but not others (Howard and Kluge 1985) finding that males preferred larger females. Such contrasting findings on male mate choice are common to scramble mating anuran species (e.g., Bufo bufo, Arntzen 1999; Ceratophrys stolzmanni, Székely et al. 2018) and suggest that, if male choice on female phenotype exists, it may depend on social or environmental conditions (e.g., fluctuating operational sex ratio [OSR]; Weir et al. 2011; Janicke and Morrow 2018). Males may also vary in their mate-choice responses to females’ receptivity to mating (Hollis and Kawecki 2014) and, particularly in scramble systems, males preference may even vary with their own quality (Härdling and Kokko 2005; Franceschi et al. 2010; Wada et al. 2011). Alternatively, nonrandom male mating patterns in scramble mating systems may not necessarily reflect male choice, but instead be an emergent property of scramble mating; for example, females “preferred” by males could simply be those that are easier to catch or maintain in amplexus. In such a scenario, more easily caught females may be those more willing to mate, pointing to female choice as the driver of nonrandom mating, or they may be less-robust females with a reduced ability to dislodge males and flee. This final possibility implies that males, if given the opportunity, may inadvertently amplex with low-performance or quality females due to ease of capture. To better understand the evolution of male mate choice in scramble mating systems, we test the potential reproductive benefits of male mate choice using wood frogs as a representative scramble mating species. Because of highly variable female quality and low opportunity that males will re-mate, we predict that males will prefer larger and heavier females and that these preferred females should lay more eggs. We also expect other related reproductive benefits of mating with a preferred female, including greater offspring viability and growth, as found by other studies investigating the benefits of mate choice (e.g., Byrne and Rice 2006; Chen et al. 2012). METHODS Choice test design Adult wood frogs were collected from 3 vernal ponds within Pennsylvania State Game Lands #176 (Scotia Barrens) in March 2010 and April 2013. The 3 ponds (FP: 40.77829°N, −78.00823°W; HP: 40.77927°N, −78.00696°W; SP: 40.78135°N, −78.00292°W) were located no more than 0.6 km from each other. We captured frogs by hand or using drift fences and pitfall traps at the start of their (~1 week) breeding season. Upon capture, frogs were placed into arenas for male mate-choice tests. Each 18-L plastic arena (42.4 × 28 × 27.4 cm; L × W × H) was filled with pond water and placed at the edge of a natural pond where wood frogs were actively breeding (as per Swierk et al. 2015). One male was introduced into the mate-choice arena with 2 females. Prior to the trial, we loosely tied a thin black or white cotton sewing thread around each female’s waist for individual identification purposes (Howard and Kluge 1985); these were removed at the conclusion of the mate-choice trials. Frogs were free to move around the arena, and pilot trials confirmed that males engaged in normal mate-searching behavior within arenas and participated in the chorus. A trial commenced once the 2 females were placed into the mate-choice arena with the male, at which time a loose-fitting lid was placed over the arena. We checked the arenas every 10 min and recorded which female, if either, the male had amplexed. At each check, we gently disengaged all amplexed pairs by sliding the male’s forelimbs over the female’s head and placed the pair back into the arena with the remaining female. A proxy for male preference was defined as a male engaging in amplexus with a given female during at least 5 of the 8 checks. All trials were concluded at 80 min, at which time we measured male and female snout-urostyle length (SUL, a measure of body size; Supplementary Table 1) with a ruler and mass using a spring scale. A total of 60 mate-choice trials were conducted in the 2 years of study (50 trials in 2010, and 26 in 2013). In 4 trials, males failed to demonstrate a preference and amplexed both females 3 times; these trials were removed from the final dataset. Mating and egg mass collection At the conclusion of the mate-choice trials, we randomly allocated males to 1 of 2 treatments: preferred (in which males were permitted to mate with their preferred female) or nonpreferred (in which males were permitted to mate with their nonpreferred female). We removed the extra female and retained each assigned pair in their original choice arena, which we then furnished with leaves, a small floating log for use as a perch, and a thin branching twig for use as an oviposition site. The remaining female from each trial was released at her pond of capture. We left the mating pair undisturbed, but checked the arena every 12 h for oviposition. When oviposition had occurred, we reweighed each female to obtain a postoviposition mass measurement, and then males and females were released at their ponds of capture. On the day an egg mass was produced, it was transported to the laboratory at the Pennsylvania State University. Each egg mass was placed on a mesh sling in its own 18-L tank filled with aged, dechlorinated water. Eggs were checked daily for hatching. When 48 h had elapsed after the first larva in a clutch had hatched, we counted the number of larvae and unhatched eggs in that egg mass (Swierk 2015). We retained a total of 100 larvae per clutch (divided as described below) and immediately released the remaining larvae (up to 1000 per clutch) into their ponds of origin. Laboratory rearing We reared 50 larvae per clutch in single-clutch 18-L tanks on a 12:12 h light:dark cycle in the laboratory. Partial water changes were performed approximately once weekly and larvae were monitored daily for survival. We fed larvae a 3:1 ground mixture of alfalfa pellets (Kaytee Products, Inc., Chilton, WI) and TetraFin Goldfish Flakes (Spectrum Brands, Inc., Blacksburg, VA) ad libitum 3 times weekly. Larvae were checked daily for metamorphosis, which we defined as the emergence of forelimbs (Gosner stage 42; Gosner 1960). Upon metamorphosis, each froglet was removed from the tank, gently dried with a bleach-free paper towel, and weighed to the nearest 0.01 g. To obtain SUL measurements, we photographed each froglet next to a ruler for scale, using a tripod-mounted Cyber-shot DSC-H7B digital camera (Sony Corporation, Tokyo, Japan). We then used ImageJ software version 1.49 (Schneider et al. 2012) to measure the length between the snout and urostyle of each froglet in photographs (Lambert et al. 2017). Larger larval size and size at metamorphosis in wood frogs are directly related to postmetamorphosis survival of wood frogs in terrestrial environments (Berven 1990). Field rearing Another set of 50 larvae per clutch was reared in clutch-specific field enclosures in their ponds of origin. Each cylindrical enclosure (length, 80 cm; diameter, 30 cm) was constructed of firm 1 cm plastic mesh and covered in 1 mm fiberglass screening. A foam block was placed inside each enclosure so that enclosures floated horizontally, and dried leaf litter was added to each enclosure. Each floating enclosure was attached with a length of string to a wooden stake driven into the pond floor, which allowed the enclosure to remain at the pond’s surface despite fluctuations in water level. Enclosures were fastened at each end of the mesh tube with cable ties and floats after the 48-h-old larvae were introduced. Larvae remained in the field enclosures until they were humanely euthanized with MS-222 to obtain tissue samples for a separate study at 64 days (2010) or 62 days (2013) posthatching, prior to any metamorphosis as confirmed by periodic inspection of enclosures. In 2010, we also determined the Gosner stage (Gosner 1960) and SUL of the euthanized field-reared larvae using a dissecting microscope. Ethical note All procedures adhered to national and international standards on animal welfare and were compliant with the legal requirements of the United States, the ABS/ASAB guidelines for ethical treatment of animals, and the institutional guidelines of Penn State University (IACUC # 42015 and 33346). Animal collection was permitted by the Pennsylvania Game Commission (NC-028-2012) and the Pennsylvania Fish and Boat Commission (Scientific Collector’s Permit 483 Type 1). Statistical methods We log-transformed all continuous variables to meet assumptions of parametric analyses. In each of the following models, pond of origin and year were included as fixed factors. To test whether female SUL and condition (residuals of the regression of female SUL and mass) affected preference status (preferred vs. nonpreferred), we performed a generalized linear model (family = binomial) with preference status as the binary response variable and female SUL and condition as predictors. Using a linear model, we tested how preference status related to the total number of eggs laid, with female SUL as a covariate. To examine whether female investment per egg related to their preference status, we performed a linear regression in which the mass of the egg mass was the response variable, and preference status, total number of eggs in a clutch, and female SUL were predictors. We used the “survival” package (Therneau 2015) in R to test whether preference status affected time to oviposition and time to hatching (measured in 12-h and 24-h increments, respectively) using a Cox proportional hazards model. Hatching success per clutch was tested using a generalized linear model (family = binomial) in which the response was a 2-column matrix of the numbers of hatched and unhatched eggs per clutch (which permits the analysis of proportion data using a weighted regression with the individual sample sizes as weights; Crawley 2012) and the predictor was preference status (preferred vs. nonpreferred). For laboratory-reared larvae, we tested metamorphosis success using a generalized linear model (family = binomial), with the numbers of lab-reared larvae per clutch that did and did not survive to metamorphosis in a 2-column matrix as the response, and preference status as the predictor. We tested the effect of preference status on 1) froglet SUL at metamorphosis, with froglet mass as a covariate, and 2) larval duration, using linear mixed models in which preference was a fixed effect and clutch was the random effect. In all analyses of laboratory-reared larvae, 5 of the 56 clutches were excluded due to mortality associated with an unrelated water quality issue. For field-reared larvae, we compared whether preferred and nonpreferred females produced offspring that differed in survivorship using a generalized linear model (family = binomial) in which the numbers of surviving and deceased field-reared larvae per clutch were response variables in a 2-column matrix, with preference status as the predictor. To test whether preference status affected larval SUL at a given Gosner stage, we used a linear mixed model in which larval SUL was the response variable, preference status and Gosner stage were fixed effects, and clutch was a random effect. Year was not included as a fixed effect in this model because all data were collected in 2010. We calculated effect size indices for all analyses, with the exception of the Cox proportional hazard models (for which hazard ratios are instead reported). Cohen’s d and corresponding 95% confidence intervals (CI) were estimated for the effect of preference status on the response variable in each model according to the equations presented in Nakagawa and Cuthill (2007). For our 3 mixed models, Cohen’s d values were estimated using a GLM framework, which provides the best point estimate of Cohen’s d; however, confidence intervals for Cohen’s d of mixed models are unreliable, and those listed here are constructed from a GLM framework and do not take into account nonindependence of data (Nakagawa and Cuthill 2007). We used the “lme4” package (Bates et al. 2015) in R for mixed models. In all mixed models, P-values were obtained by performing likelihood ratio tests of each full model against its respective null model, with alpha set at 0.05. Diagnostic plots of the models revealed no deviations from homoscedasticity or normality. All analyses were performed in R (v. 3.2.1, R Foundation for Statistical Computing, Vienna, Austria). RESULTS Overall, male wood frogs tended to prefer larger females, with this effect approaching statistical significance (χ21 = 3.309, P = 0.069; Cohen’s d: 0.353, confidence interval [CI]: 0.229–0.478; Figure 1a), but not females with greater condition (χ21 = 0.102, P = 0.749, Cohen’s d: 0.062, CI: −0.061–0.185; Figure 1b) (year, χ21 = 0.634, P = 0.426; pond, χ21 = 0.125, P = 0.939). Figure 1 View largeDownload slide Male wood frog (Rana sylvatica) selection of females with respect to (a) snout-urostyle length (mm) and (b) condition (i.e., the residuals of a regression of length and mass). Filled circle indicates near significance (P = 0.05–0.07). Error bars represent ± 1 SE. Figure 1 View largeDownload slide Male wood frog (Rana sylvatica) selection of females with respect to (a) snout-urostyle length (mm) and (b) condition (i.e., the residuals of a regression of length and mass). Filled circle indicates near significance (P = 0.05–0.07). Error bars represent ± 1 SE. When males were paired with nonpreferred females, marginally more eggs were laid than those from matings with preferred females (F1,50 = 3.595, P = 0.064, Cohen’s d: 0.537, CI: −0.007–1.080; Figure 2a) (female SUL, F1,50 = 43.877, P < 0.001; year, F1,50 = 38.663, P < 0.001; pond, F1,50 = 0.569, P = 0.570). Preference status (F1,49 = 0.061, P = 0.807, Cohen’s d: 0.070, CI: −0.463–0.604) and total egg number (F1,49 = 1.134, P = 0.292) did not relate to the mass of the egg mass (female SUL, F1,49 = 38.242, P < 0.001; year, F1,49 = 0.101, P = 0.752; pond, F1,49 = 9.752, P < 0.001). No difference in time to oviposition was found for males paired with preferred versus nonpreferred females (hazard ratio = 1.016; z = 0.057; P = 0.955), but there was an effect of preference status on the time it took for eggs to hatch, such that eggs from nonpreferred pairings hatched somewhat sooner than those from preferred pairings, approaching statistical significance (hazard ratio = 0.571; z = −1.924; P = 0.054). Figure 2 View largeDownload slide (a) Average numbers of eggs per clutch and (b) the average proportions of eggs that hatched per clutch resulting from matings with preferred and nonpreferred female wood frogs (Rana sylvatica). Asterisk denotes a significant difference (P < 0.05), and filled circle indicates near significance (P = 0.05–0.07). Error bars represent ± 1 SE. Figure 2 View largeDownload slide (a) Average numbers of eggs per clutch and (b) the average proportions of eggs that hatched per clutch resulting from matings with preferred and nonpreferred female wood frogs (Rana sylvatica). Asterisk denotes a significant difference (P < 0.05), and filled circle indicates near significance (P = 0.05–0.07). Error bars represent ± 1 SE. Preference status influenced hatching success: clutches produced by matings with nonpreferred females had higher rates of hatching than did clutches produced by matings with preferred females (χ21 = 73.103, P < 0.001, Cohen’s d: 2.447, CI: 1.706–3.188; Figure 2b) (year, χ21 = 58.725, P < 0.001; pond, χ21 = 38.484, P < 0.001). Similarly, higher proportions of laboratory-reared larvae from matings with nonpreferred females survived to metamorphosis than did those from matings with preferred females (χ21 = 15.864, P < 0.001, Cohen’s d: 1.370, CI: 0.654–2.086; Figure 3a) (year, χ21 = 15.904, P < 0.001; pond, χ21 = 7.050, P = 0.029). At metamorphosis, laboratory-reared larvae from matings with nonpreferred females were larger (SUL) than those from matings with preferred females (χ21 = 4.440, P = 0.035, Cohen’s d: 0.144, CI: 0.021–0.267; Figure 3b) (year, χ21 = 60.652, P < 0.001; pond χ21 = 0.057, P = 0.972; mass, χ21 = 945.540, P < 0.001). Preference status did not affect larval duration of laboratory-reared larvae (i.e., how long it took to reach metamorphosis) (χ21 = 0.614, P = 0.433, Cohen’s d: 0.051, CI: −0.073–0.174) (year, χ21 = 64.716, P < 0.001; pond, χ21 = 3.555, P = 0.169). Figure 3 View largeDownload slide (a) Average numbers of metamorphosed wood frog (Rana sylvatica) larvae and (b) their snout-urostyle length (cm) at metamorphosis of the subset of laboratory-reared offspring resulting from matings with preferred and nonpreferred females. Asterisks denote significant differences (P < 0.05). Error bars represent ± 1 SE. Figure 3 View largeDownload slide (a) Average numbers of metamorphosed wood frog (Rana sylvatica) larvae and (b) their snout-urostyle length (cm) at metamorphosis of the subset of laboratory-reared offspring resulting from matings with preferred and nonpreferred females. Asterisks denote significant differences (P < 0.05). Error bars represent ± 1 SE. Survival of larvae in the field was not affected by preference status (χ21 = 0.120, P = 0.729, Cohen’s d: 0.106, CI: −0.428–0.640; Figure 4a) (year, χ21 = 22.015, P < 0.001; pond, χ21 = 2.090, P = 0.148), but field-reared larvae resulting from matings with nonpreferred females were larger (SUL) than those from matings with nonpreferred females (χ21 = 7.290, P = 0.007, Cohen’s d: 0.234, CI: 0.077–0.390; Figure 4b) (Gosner stage, χ21 = 974.490, P < 0.001; pond, χ21 = 0.011, P = 0.915). Figure 4 View largeDownload slide (a) Average numbers of surviving wood frog (Rana sylvatica) larvae and (b) larval snout-urostyle length (cm) of the subset of field-reared offspring from matings with preferred and nonpreferred females. Surviving larvae were euthanized at 64 days (2010) or 62 days (2013) posthatching. Asterisk denotes a significant difference (P < 0.05). Error bars represent ± 1 SE. Figure 4 View largeDownload slide (a) Average numbers of surviving wood frog (Rana sylvatica) larvae and (b) larval snout-urostyle length (cm) of the subset of field-reared offspring from matings with preferred and nonpreferred females. Surviving larvae were euthanized at 64 days (2010) or 62 days (2013) posthatching. Asterisk denotes a significant difference (P < 0.05). Error bars represent ± 1 SE. DISCUSSION Benefits of mate choice, for both males and females, have been so frequently found across taxa that it is widely accepted that individuals gain greater reproductive benefits when they are allowed to choose mates versus when they are constrained in their mate choice (e.g., Alatalo et al. 1998; Gowaty et al. 2003; Byers and Waits 2006; Rundle et al. 2007; Lancaster et al. 2009; Ihle et al. 2015; Courtiol et al. 2016). In this study, we tested for evidence of reproductive benefits of male mate choice in a scramble mating species. Contrary to expectations, male wood frogs appear to accrue reproductive costs when mating with their preferred female versus mating with a nonpreferred female. Males in this study exhibited strong preferences, with the majority of males choosing to amplex their preferred female near-exclusively during trials. Within a trial, females could differ in SUL up to 14 mm and in mass up to 15.5 g; nevertheless, males did not strongly prefer the larger female in a pair (57% of trials) over the smaller female (43%), nor did males prefer the higher-quality female (53%) more often than the lower-quality female (47%). When permitted to mate with their preferred female, males produced fewer offspring with lower viability and growth than they did with nonpreferred females. Multiple measures of offspring quantity and quality in both the field and laboratory point to the same conclusion: matings with preferred female wood frogs produced fewer and lower-quality offspring than did matings with nonpreferred females. Although mate choice is widely presumed to confer fitness advantages, fitness costs are commonly documented and may be associated with sexual conflict (Watson et al. 1998), sexual imprinting (Krüger et al. 2001), or mate copying (Dubois et al. 2012). In some systems, low-quality males may even prefer low-quality females to reduce risks associated with mating competition (Härdling and Kokko 2005). It has also been demonstrated that mating with preferred mates can shorten lifespans and accelerate aging (Friberg and Arnqvist 2003). Fitness advantages may combat the costs; for example, increased offspring resistance to disease complemented slow offspring growth of preferred mates in sticklebacks (Barber et al. 2001). A whole-picture perspective of costs and benefits is therefore required to assess the role of mate choice in a system. Despite our consistent findings in the field and laboratory that mating with preferred females correlates with reduced reproductive output and offspring fitness, we give consideration to how experimental procedures could influence our findings. First, repeated human handling of some females (preferred) more than others (nonpreferred) during trials is a potential concern. However, a separate study found no evidence that human handling and manipulation of mothers influences reproductive output or offspring characteristics in wood frogs, nor was there any evidence of an effect of repeated handling and separation of amplectant pairs on any of the reproductive or offspring parameters that we measured (Swierk and Langkilde 2018a). Second, this study involved the relocation of fertilized egg masses to a laboratory setting (at least initially, in the case of field-reared larvae), which may have shielded developing eggs and (lab-reared) larvae from natural threats such as pathogens and predators that they would normally experience in the field. It is possible that, in field conditions, a benefit to mating with preferred females may be more evident. This study’s field component did not support this idea, and numerous other studies of the benefits of preferred mates have been conducted in relatively sterile laboratory conditions and preferences have still been found (e.g., Drickamer et al. 2003; Gowaty et al. 2003). Alternatively, it is possible that our study may document only one side of a tradeoff, such that offspring of preferred females may invest in another untested (e.g., immune) benefit at the expense of one of the traits we measured (e.g., growth) (Barber et al. 2001); we further discuss this possibility below. That said, the parameters we measured are known to be highly fitness relevant in anuran larvae and should disproportionately influence offspring survival and reproduction (e.g., see Berven 1990; Chelgren et al. 2006). Lastly, it was not possible to examine the role of female preference in this study, though it could also influence the interpretation of these findings. For example, it is possible that females with low catchability in a trial were actually discriminating against a male (rather than males preferring females with high catchability). Female mate choice in anurans with explosive and scramble mating systems is particularly poorly understood. This leaves the door open for intriguing studies on the role of female choice in scramble systems, or the potential existence of mutual mate preferences based on genetic compatibility in anurans (see below). Our unanticipated findings may well reflect actual mating patterns in the field, as male wood frog mate preference may have acted primarily on a highly fitness-relevant female trait: catchability. To minimize costs associated with mate searching and pursuit, male wood frogs may have preferred females that were easier to amplex and maintain in amplexus. It is likely that in a field setting, in the presence of strong male-male competition, female catchability may be the strongest factor in male choice; discriminating among available females based on catchability helps to minimize the male risk of being left without a mate entirely. Females in our trials were not tethered or otherwise prevented from escaping a pursuing male, though females had somewhat limited escape within the mate-choice arena. Our study therefore likely provides a conservative picture of potential male mate choice based on female catchability. Although this study was not designed to test whether or not males actively chose females based on their catchability, we name it as a likely potential explanation. Female wood frogs may gain reproductive benefits by being more difficult to catch (i.e., having low catchability). By having low catchability, females may exert indirect mate choice for males, as only higher-quality mates are able to successfully capture and maintain amplexus with hard-to-get females (Cordero and Eberhard 2003). In addition, females with low catchability also gain the direct benefit of being able to escape excessive male sexual harassment (Shine et al. 2005a) or entrapment in potentially fatal mating balls of males (Howard 1980; Verrell and Mccabe 1986; Trauth et al. 2000). As virtually no wood frog female is left without a mate in a breeding aggregation (Howard 1980), it should benefit all females to have low catchability, but some females will nevertheless be more easily caught than others. As a result, it may be anticipated that females that are more easily caught (i.e., have high catchability) are in some way inferior to those with low catchability. Whether catchability is a condition-dependent trait (in which low-condition females may negatively affect offspring fitness via maternal effects) or has a stronger genetic component, females with high catchability may produce offspring with lower fitness than females with low catchability. While it is likely that male mate choice for females with high catchability guards against a major fitness risk (i.e., having no mate at all), our results suggest that this preference does carry other, perhaps less serious, costs for males if females with high catchability produce lower-fitness offspring when compared to low-catchability (i.e., potentially nonpreferred) females. Thus, male wood frog preference for female catchability may be advantageous when the breeding aggregation OSR is skewed toward males, but not so at times when the OSR is less biased. Wood frog breeding aggregations exhibit rapid changes in sex ratio despite their short duration (OSR, defined as the proportion of available males to all available breeding adults, was documented in our populations to fluctuate at most from 0.55 to 1; Supplementary Table 2), suggesting that selection for male choice on female catchability overall may outweigh its occasional costs. Our data suggest that an untested parameter of male wood frog mate choice may also play a role in determining its relative costs and benefits. In recent decades, mate choice based on genetic compatibility has been demonstrated to be an important and widely observed component of mate choice (see Brown 1997; Tregenza and Wedell 2000; Mays and Hill 2004). In particular, mate choice based on dissimilarity of major histocompatibility complex (MHC) genes has been observed in humans (Wedekind et al. 1995) and other species (e.g., Gowaty et al. 2003; Agbali et al. 2010; Rymešová et al. 2017; for review see Kamiya et al. 2014); benefits of choosing a mate with dissimilar MHC genes include provisioning offspring with a greater variety of cell-surface proteins that enable the immune system to identify and fight pathogens and parasites (Bernatchez and Landry 2003; Piertney and Oliver 2006; Ruff et al. 2012). If male mate choice in wood frogs also has an MHC-compatibility component (or similar), then it could be predicted that, when faced with challenges to the immune system, offspring of preferred females would have a fitness advantage over the offspring of nonpreferred females. When taken as a whole, our data support this idea. In the lab, in the absence of immune challenges, offspring of preferred females had lower survivorship than the offspring of nonpreferred females (Figure 3a), possibly due to reasons associated with female catchability, as proposed above. However, in the presence of numerous immune challenges in the field, this difference in offspring survivorship disappears (Figure 4a). A MHC-related fitness cost to mating with nonpreferred mates could be responsible for closing the gap between preferred and nonpreferred offspring field survivorship. While admittedly far from conclusive, these results imply that multiple types of mate choice may exist in this scramble system and could warrant future study. Unfortunately, almost no studies of MHC-related mate choice have been conducted on amphibians (but see Bos et al. 2009) and none to our knowledge have been conducted on anuran amphibians. This is despite high MHC diversity in some anurans (Lillie et al. 2015), the ability of anuran larvae to distinguish siblings based on MHC genes (Villinger and Waldman 2008), and the role of MHC genes in defense against widespread amphibian diseases including Ranavirus (Gantress et al. 2003; Teacher et al. 2009) and chytridiomycosis (Batrachochytrium dendrobatidis) (Savage and Zamudio 2011). This study highlights the importance of understanding how ease of capture factors into the mate-choice decision-making process for males (Shine et al. 2005b), particularly in systems where females frequently flee males or use indirect methods to secure higher-quality males as mates. In such systems, restraining females in mate-choice experiments unquestionably creates an artificial choice scenario for males by removing the catchability parameter (e.g., Liao and Lu 2009) which is likely a major consideration in male mating decisions as it can greatly alter the cost/benefit ratio of male choice. Conversely, disentangling male choice of catchability versus other phenotypic traits is difficult with unrestrained females in choice trials, and studies reporting choice may be confounding these variables. Future studies of male choice, including in scramble mating systems, should actively work to separate ease of capture from preference in order to better understand the targets of male choice. FUNDING This work was supported by the Animal Behavior Society (Student Research Award to L.S.), the Society for the Study of Amphibians and Reptiles (Grant-in-Herpetology to L.S.), and the National Science Foundation (grant number DGE-1255832 to L.S.; any opinions, findings, and conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the National Science Foundation). Acknowledgments We thank N. Freidenfelds for advice and field assistance, V. Braithwaite and D. Hughes for formative suggestions, C. Norjen, T. Jacobs, J. Newman, J. Williams, J. Langshaw, and E. Schlaich for their help with fieldwork and animal care, T. Adams, K. MacLeod, and D. Ensminger for comments on an early draft of this manuscript, and the Hildebrand family for their support and enthusiasm. Data accessibility: Analyses reported in this article can be reproduced using the data provided by Swierk and Langkilde (2018b). REFERENCES Agbali M , Reichard M , Bryjová A , Bryja J , Smith C . 2010 . Mate choice for nonadditive genetic benefits correlate with MHC dissimilarity in the rose bitterling (Rhodeus ocellatus) . Evolution . 64 : 1683 – 1696 . Google Scholar Crossref Search ADS PubMed Alatalo RV , Kotiaho J , Mappes J , Parri S . 1998 . Mate choice for offspring performance: major benefits or minor costs? Proc R Soc B Biol Sci . 265 : 2297 – 2301 . Google Scholar Crossref Search ADS Alcock J . 1980 . Natural selection and the mating systems of solitary bees . Am Sci . 68 : 146 – 153 . Andersson M . 1994 . Sexual selection . Princeton (NJ) : Princeton University Press . Arntzen JW . 1999 . Sexual selection and male mate choice in the common toad, Bufo bufo . Ethol Ecol Evol . 11 : 407 – 414 . Google Scholar Crossref Search ADS Banta AM . 1914 . Sex recognition and the mating behavior of the wood frog, Rana sylvatica . Biol Bull . 26 : 171 – 183 . Google Scholar Crossref Search ADS Barber I , Arnott SA , Braithwaite VA , Andrew J , Huntingford FA . 2001 . Indirect fitness consequences of mate choice in sticklebacks: offspring of brighter males grow slowly but resist parasitic infections . Proc Biol Sci . 268 : 71 – 76 . Google Scholar Crossref Search ADS PubMed Bates D , Maechler M , Bolker B , Walker S . 2015 . Fitting linear mixed-effects models using {lme4} . J Stat Softw . 67 : 1 – 48 . Google Scholar Crossref Search ADS Bee MA . 2007 . Selective phonotaxis by male wood frogs (Rana sylvatica) to the sound of a chorus . Behav Ecol Sociobiol . 61 : 955 – 966 . Google Scholar Crossref Search ADS Bernatchez L , Landry C . 2003 . MHC studies in nonmodel vertebrates: what have we learned about natural selection in 15 years? J Evol Biol . 16 : 363 – 377 . Google Scholar Crossref Search ADS PubMed Berven KA . 1981 . Mate choice in the wood frog, Rana sylvatica . Evolution . 35 : 707 – 722 . Google Scholar Crossref Search ADS PubMed Berven KA . 1988 . Factors affecting variation in reproductive traits within a population of wood frogs (Rana sylvatica) . Copeia . 1988 : 605 – 615 . Google Scholar Crossref Search ADS Berven KA . 1990 . Factors affecting population fluctuations in larval and adult stages of the wood frog (Rana sylvatica) . Ecology . 71 : 1599 – 1608 . Google Scholar Crossref Search ADS Bonduriansky R . 2001 . The evolution of male mate choice in insects: a synthesis of ideas and evidence . Biol Rev Camb Philos Soc . 76 : 305 – 339 . Google Scholar Crossref Search ADS PubMed Bos DH , Williams RN , Gopurenko D , Bulut Z , DeWoody JA . 2009 . Condition-dependent mate choice and a reproductive disadvantage for MHC-divergent male tiger salamanders . Mol Ecol . 18 : 3307 – 3315 . Google Scholar Crossref Search ADS PubMed Brown JL . 1997 . A theory of mate choice based on heterozygosity . Behav Ecol . 8 : 60 – 65 . Google Scholar Crossref Search ADS Byers JA , Waits L . 2006 . Good genes sexual selection in nature . Proc Natl Acad Sci USA . 103 : 16343 – 16345 . Google Scholar Crossref Search ADS PubMed Byrne PG , Rice WR . 2006 . Evidence for adaptive male mate choice in the fruit fly Drosophila melanogaster . Proc Biol Sci . 273 : 917 – 922 . Google Scholar Crossref Search ADS PubMed Ceballos S , Kiørboe T . 2010 . First evidences of sexual selection by mate choice in marine zooplankton . Oecologia . 164 : 627 – 635 . Google Scholar Crossref Search ADS PubMed Chelgren ND , Rosenberg DK , Heppell SS , Gitelman AI . 2006 . Carryover aquatic effects on survival of metamorphic frogs during pond emigration . Ecol Appl . 16 : 250 – 261 . Google Scholar Crossref Search ADS PubMed Chen H , Salcedo C , Sun J . 2012 . Male mate choice by chemical cues leads to higher reproductive success in a bark beetle . Anim Behav . 83 : 421 – 427 . Google Scholar Crossref Search ADS Clutton-Brock T . 2009 . Sexual selection in females . Anim Behav . 77 : 3 – 11 . Google Scholar Crossref Search ADS Conant R , Collins JT . 1998 . A field guide to reptiles and amphibians of eastern and central North America . Boston : Houghton Mifflin Company . Cordero C , Eberhard WG . 2003 . Female choice of sexually antagonistic male adaptations: a critical review of some current research . J Evol Biol . 16 : 1 – 6 . Google Scholar Crossref Search ADS PubMed Courtiol A , Etienne L , Feron R , Godelle B , Rousset F . 2016 . The evolution of mutual mate choice under direct benefits . Am Nat . 188 : 521 – 538 . Google Scholar Crossref Search ADS PubMed Crawley MJ . 2012 . The R book . 2 nd ed. Chichester (UK) : John Wiley & Sons, Inc . Dechaume-Moncharmont FX , Brom T , Cézilly F . 2016 . Opportunity costs resulting from scramble competition within the choosy sex severely impair mate choosiness . Anim Behav . 114 : 249 – 260 . Google Scholar Crossref Search ADS Drickamer LC , Gowaty PA , Wagner DM . 2003 . Free mutual mate preferences in house mice affect reproductive success and offspring performance . Anim Behav . 65 : 105 – 114 . Google Scholar Crossref Search ADS Dubois F , Drullion D , Witte K . 2012 . Social information use may lead to maladaptive decisions: a game theoretic model . Behav Ecol . 23 : 225 – 231 . Google Scholar Crossref Search ADS Edward DA , Chapman T . 2011 . The evolution and significance of male mate choice . Trends Ecol Evol . 26 : 647 – 654 . Google Scholar Crossref Search ADS PubMed Fitzpatrick CL , Servedio MR . 2017 . Male mate choice, male quality, and the potential for sexual selection on female traits under polygyny . Evolution . 71 : 174 – 183 . Google Scholar Crossref Search ADS PubMed Franceschi N , Lemaître JF , Cézilly F , Bollache L . 2010 . Size-assortative pairing in Gammarus pulex (Crustacea: Amphipoda): a test of the prudent choice hypothesis . Anim Behav . 79 : 911 – 916 . Google Scholar Crossref Search ADS Friberg U , Arnqvist G . 2003 . Fitness effects of female mate choice: preferred males are detrimental for Drosophila melanogaster females . J Evol Biol . 16 : 797 – 811 . Google Scholar Crossref Search ADS PubMed Gantress J , Maniero GD , Cohen N , Robert J . 2003 . Development and characterization of a model system to study amphibian immune responses to iridoviruses . Virology . 311 : 254 – 262 . Google Scholar Crossref Search ADS PubMed Gosner KL . 1960 . A simplified table for staging anuran embryos and larvae with notes on identification . Herpetologica . 16 : 183 – 190 . Gowaty PA , Drickamer LC , Schmid-Holmes S . 2003 . Male house mice produce fewer offspring with lower viability and poorer performance when mated with females they do not prefer . Anim Behav . 65 : 95 – 103 . Google Scholar Crossref Search ADS Härdling R , Gosden T , Aguilée R . 2008 . Male mating constraints affect mutual mate choice: prudent male courting and sperm-limited females . Am Nat . 172 : 259 – 271 . Google Scholar Crossref Search ADS PubMed Härdling R , Kokko H . 2005 . The evolution of prudent choice . Evol Ecol Res . 7 : 687 – 715 . Hollis B , Kawecki TJ . 2014 . Male cognitive performance declines in the absence of sexual selection . Proc Biol Sci . 281 : 20132873 . Google Scholar Crossref Search ADS PubMed Howard RD . 1980 . Mating behaviour and mating success in woodfrogs, Rana sylvatica . Anim Behav . 28 : 705 – 716 . Google Scholar Crossref Search ADS Howard RD , Kluge AG . 1985 . Proximate mechanisms of sexual selection in wood frogs . Evolution . 39 : 260 – 277 . Google Scholar Crossref Search ADS PubMed Ihle M , Kempenaers B , Forstmeier W . 2015 . Fitness benefits of mate choice for compatibility in a socially monogamous species . PLoS Biol . 13 : e1002248 . Google Scholar Crossref Search ADS PubMed Janicke T , Morrow EH . 2018 . Operational sex ratio predicts the opportunity and direction of sexual selection across animals . Ecol Lett . 21 : 384 – 391 . Google Scholar Crossref Search ADS PubMed Johnstone RA , Reynolds JD , Deutsch JC . 1996 . Mutual mate choice and sex differences in choosiness . Evolution . 50 : 1382 – 1391 . Google Scholar Crossref Search ADS PubMed Kamiya T , O’Dwyer K , Westerdahl H , Senior A , Nakagawa S . 2014 . A quantitative review of MHC-based mating preference: the role of diversity and dissimilarity . Mol Ecol . 23 : 5151 – 5163 . Google Scholar Crossref Search ADS PubMed Krüger O , Lindström J , Amos W . 2001 . Maladaptive mate choice maintained by heterozygote advantage . Evolution . 55 : 1207 – 1214 . Google Scholar Crossref Search ADS PubMed Lambert MR , Carlson BE , Smylie MS , Swierk L . 2017 . Ontogeny of sexual dichromatism in the explosively breeding wood frog . Herpetol Conserv Biol . 12 : 447 – 456 . Lancaster LT , Hipsley CA , Sinervo B . 2009 . Female choice for optimal combinations of multiple male display traits increases offspring survival . Behav Ecol . 20 : 993 – 999 . Google Scholar Crossref Search ADS Liao WB , Lu X . 2009 . Male mate choice in the Andrew’s toad Bufo andrewsi: a preference for larger females . J Ethol . 27 : 413 – 417 . Google Scholar Crossref Search ADS Lillie M , Grueber CE , Sutton JT , Howitt R , Bishop PJ , Gleeson D , Belov K . 2015 . Selection on MHC class II supertypes in the New Zealand endemic Hochstetter’s frog . BMC Evol Biol . 15 : 63 . Google Scholar Crossref Search ADS PubMed Liu Z , Xu B , Guo Y , Raffa KF , Sun J . 2017 . Gallery and acoustic traits related to female body size mediate male mate choice in a bark beetle . Anim Behav . 125 : 41 – 50 . Google Scholar Crossref Search ADS Mays HL Jr , Hill GE . 2004 . Choosing mates: good genes versus genes that are a good fit . Trends Ecol Evol . 19 : 554 – 559 . Google Scholar Crossref Search ADS PubMed Nakagawa S , Cuthill IC . 2007 . Effect size, confidence interval and statistical significance: a practical guide for biologists . Biol Rev Camb Philos Soc . 82 : 591 – 605 . Google Scholar Crossref Search ADS PubMed Piertney SB , Oliver MK . 2006 . The evolutionary ecology of the major histocompatibility complex . Heredity (Edinb) . 96 : 7 – 21 . Google Scholar Crossref Search ADS PubMed Rowe L . 1994 . The costs of mating and mate choice in water striders . Anim Behav . 48 : 1049 – 1056 . Google Scholar Crossref Search ADS Ruff JS , Nelson AC , Kubniak JL , Potts WK . 2012 . MHC signaling during social communication . In: López-Larrera C , editor. Self and nonself . New York : Springer Science+Business Media, LLC . p. 290 – 313 . Rundle HD , Odeen A , Mooers AØ . 2007 . An experimental test for indirect benefits in Drosophila melanogaster . BMC Evol Biol . 7 : 36 . Google Scholar Crossref Search ADS PubMed Rymešová D , Králová T , Promerová M , Bryja J , Tomášek O , Svobodová J , Šmilauer P , Šálek M , Albrecht T . 2017 . Mate choice for major histocompatibility complex complementarity in a strictly monogamous bird, the grey partridge (Perdix perdix) . Front Zool . 14 : 9 . Google Scholar Crossref Search ADS PubMed Savage AE , Zamudio KR . 2011 . MHC genotypes associate with resistance to a frog-killing fungus . Proc Natl Acad Sci USA . 108 : 16705 – 16710 . Google Scholar Crossref Search ADS PubMed Schneider CA , Rasband WS , Eliceiri KW . 2012 . NIH Image to ImageJ: 25 years of image analysis . Nat Methods . 9 : 671 – 675 . Google Scholar Crossref Search ADS PubMed Shine R , Wall M , Langkilde T , Mason RT . 2005a . Do female garter snakes evade males to avoid harassment or to enhance mate quality? Am Nat . 165 : 660 – 668 . Google Scholar Crossref Search ADS Shine R , Wall M , Langkilde T , Mason RT . 2005b . Battle of the sexes: forcibly inseminating male garter snakes target courtship to more vulnerable females . Anim Behav . 70 : 1133 – 1140 . Google Scholar Crossref Search ADS Simmons LW , Parker GA . 1996 . Parental investment and the control of sexual selection: predicting the direction of sexual competition? Proc R Soc B Biol Sci . 263 : 515 – 519 . Google Scholar Crossref Search ADS Swierk L . 2015 . Clutch size measurement . Herpetol Rev . 46 : 544 . Swierk L , Langkilde T . 2018a . Does repeated human handling of study animals during the mating season affect their offspring? J Exp Zool A Ecol Integr Physiol . 329 : 80 – 86 . Swierk L , Langkilde T . 2018b . Data from: fitness costs of mating with preferred females in a scramble mating system . Dryad Digital Repository . http://dx.doi.org10.5061/dryad.5bg107j Swierk L , Myers A , Langkilde T . 2013 . Male mate preference is influenced by both female behaviour and morphology . Anim Behav . 85 : 1451 – 1457 . Google Scholar Crossref Search ADS Swierk L , Tennessen JB , Langkilde T . 2015 . Sperm depletion may not limit male reproduction in a capital breeder . Biol J Linn Soc . 116 : 684 – 690 . Google Scholar Crossref Search ADS Székely D , Székely P , Denoël M , Cogălniceanu D . 2018 . Random size-assortative mating despite size-dependent fecundity in a neotropical amphibian with explosive reproduction . Ethology . 124 : 218 – 226 . Teacher AG , Garner TW , Nichols RA . 2009 . Population genetic patterns suggest a behavioural change in wild common frogs (Rana temporaria) following disease outbreaks (Ranavirus) . Mol Ecol . 18 : 3163 – 3172 . Google Scholar Crossref Search ADS PubMed Tennessen JB , Parks SE , Langkilde T . 2014 . Traffic noise causes physiological stress and impairs breeding migration behaviour in frogs . Conserv Physiol . 2 : cou032 . Google Scholar Crossref Search ADS PubMed Therneau T . 2015 . A package for survival analysis in S. version 2. https://CRAN.R-project.org/package=survival. (1 November 2018, date last accessed). Trauth SE , McCallum ML , Cartwright ME . 2000 . Breeding mortality in the wood frog, Rana sylvatica (Anura: Ranidae), from Northcentral Arkansas . J Ark Acad Sci 54 : 154 – 156 . Tregenza T , Wedell N . 2000 . Genetic compatibility, mate choice and patterns of parentage: invited review . Mol Ecol . 9 : 1013 – 1027 . Google Scholar Crossref Search ADS PubMed Verrell PA , Mccabe NR . 1986 . Mating balls in the common toad, Bufo bufo . Herpetol Bull . 16 : 28 – 29 . Villinger J , Waldman B . 2008 . Self-referent MHC type matching in frog tadpoles . Proc Biol Sci . 275 : 1225 – 1230 . Google Scholar Crossref Search ADS PubMed Wada S , Arashiro Y , Takeshita F , Shibata Y . 2011 . Male mate choice in hermit crabs: prudence by inferior males and simple preference by superior males . Behav Ecol . 22 : 114 – 119 . Google Scholar Crossref Search ADS Watson PJ , Arnqvist G , Stallmann RR . 1998 . Sexual conflict and the energetic costs of mating and mate choice in water striders . Am Nat . 151 : 46 – 58 . Google Scholar Crossref Search ADS PubMed Wedekind C , Seebeck T , Bettens F , Paepke AJ . 1995 . MHC-dependent mate preferences in humans . Proc Biol Sci . 260 : 245 – 249 . Google Scholar Crossref Search ADS PubMed Wedell N , Gage MJG , Parker GA . 2002 . Sperm competition, male prudence and sperm-limited females . Trends Ecol Evol . 17 : 313 – 320 . Google Scholar Crossref Search ADS Weir LK , Grant JW , Hutchings JA . 2011 . The influence of operational sex ratio on the intensity of competition for mates . Am Nat . 177 : 167 – 176 . Google Scholar Crossref Search ADS PubMed Weiss SL . 2002 . Reproductive signals of female lizards: pattern of trait expression and male response . Ethology . 108 : 793 – 813 . Wells KD . 1977 . The social behavior of anuran amphibians . Anim Behav . 25 : 666 – 693 . Google Scholar Crossref Search ADS Yun L , Chen PJ , Singh A , Agrawal AF , Rundle HD . 2017 . The physical environment mediates male harm and its effect on selection in females . Proc R Soc B Biol Sci . 284 : 20170424 . Google Scholar Crossref Search ADS © The Author(s) 2019. Published by Oxford University Press on behalf of the International Society for Behavioral Ecology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)
TI - Fitness costs of mating with preferred females in a scramble mating system
JF - Behavioral Ecology
DO - 10.1093/beheco/arz001
DA - 2019-06-13
UR - https://www.deepdyve.com/lp/oxford-university-press/fitness-costs-of-mating-with-preferred-females-in-a-scramble-mating-n8Ly0xDmCs
SP - 658
VL - 30
IS - 3
DP - DeepDyve
ER -