Figures
Abstract
There is strong evidence that polyandrous taxa have evolved relatively larger testes than monogamous relatives. Sperm size may either increase or decrease across species with the risk or intensity of sperm competition. Scorpions represent an ancient direct mode with spermatophore-mediated sperm transfer and are particularly well suited for studies in sperm competition. This work aims to analyze for the first time the variables affecting testes mass, ejaculate volume and sperm length, according with their levels of polyandry, in species belonging to the Neotropical family Bothriuridae. Variables influencing testes mass and sperm length were obtained by model selection analysis using corrected Akaike Information Criterion. Testes mass varied greatly among the seven species analyzed, ranging from 1.6±1.1 mg in Timogenes dorbignyi to 16.3±4.5 mg in Brachistosternus pentheri with an average of 8.4±5.0 mg in all the species. The relationship between testes mass and body mass was not significant. Body allocation in testes mass, taken as Gonadosomatic Index, was high in Bothriurus cordubensis and Brachistosternus ferrugineus and low in Timogenes species. The best-fitting model for testes mass considered only polyandry as predictor with a positive influence. Model selection showed that body mass influenced sperm length negatively but after correcting for body mass, none of the variables analyzed explained sperm length. Both body mass and testes mass influenced spermatophore volume positively. There was a strong phylogenetic effect on the model containing testes mass. As predicted by the sperm competition theory and according to what happens in other arthropods, testes mass increased in species with higher levels of sperm competition, and influenced positively spermatophore volume, but data was not conclusive for sperm length.
Citation: Vrech DE, Olivero PA, Mattoni CI, Peretti AV (2014) Testes Mass, but Not Sperm Length, Increases with Higher Levels of Polyandry in an Ancient Sex Model. PLoS ONE 9(4): e94135. https://doi.org/10.1371/journal.pone.0094135
Editor: Wei Yan, University of Nevada School of Medicine, United States of America
Received: October 18, 2013; Accepted: March 14, 2014; Published: April 15, 2014
Copyright: © 2014 Vrech et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: Funding was provided by FONCYT PICT 2010-096, Argentina, CONICET PIP 2009-2012. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Sperm competition is a widespread phenomenon that influences several sexual characters [1]–[4]. It is defined as the competition of ejaculates of two or more males to fertilize a given set of ova [5]–[7]. In a polyandrous mating system, and when the competition is numeric [6], [7], there is a selective pressure on males to increase investment in sperm production, thus showing relatively larger testes compared to monogamous relatives [7]–[11]. Larger testes may produce more sperm and give the male an advantage in numerical sperm competition [12]. Nevertheless, sperm is costly [13], [14] and males should optimize their investment in ejaculates allocating sperm strategically according to sperm competition risk [6], [7], [15], [16]. Testes size is considered a reliable index of sperm competition [17], but there are other tactics that can affect testes size as well (e.g. [18]–[20]).
Sperm size is another trait that may vary according to sperm competition risk. Sperm changes considerably in size (mainly length) among species and sperm size may either increase ([21] (butterflies); [22] (moths); [23], [24] (fishes); [25] (frogs); [26] (snakes); [27]–[29] (birds); [30], [31] (eutherian mammals); [32] (marsupial mammals)), or decrease (e.g. [33] (fishes)) across species with the risk or intensity of sperm competition.
Among arthropods, sperm competition has been widely studied in insects [5], [7], but spiders have also been a good model for sperm competition [34]–[36]. Classic reviews provided by Austad [37] and Thomas and Zeh [38] focused on the influence of sperm competition in shaping mating strategies in spiders and other arachnids. Unfortunately, studies of the influence of sperm competition over testes mass are still lacking in arachnids.
Scorpions are particularly well suited models for studies on sperm competition. Traditionally, they have been considered to be among the most basal arachnids [39], representing an ancient sex model. Sperm is transferred indirectly to the female genital opening, by means of a sclerotized spermatophore deposited in the substrate by the male [40]. Scorpion spermatozoa are long and commonly transferred as sperm packages [41], [42]. All scorpion species are viviparous [40] and in general females are polyandrous, capable of storing sperm in paired seminal receptacles [43]. Males of some species deposit a genital plug that occludes the female’s genital opening after sperm transfer [44]–[46]. Although some courtship characteristics and spermatozoa have been studied in this order (e.g. [41], [42], [47]–[50]), details of sperm competition mechanisms remain unexplored.
In this context, the present study aims to analyze variables affecting testes mass and sperm length in scorpion species of the Neotropical family Bothriuridae. This is a novel approach in these arachnids. In accordance with sperm competition theory, and assuming that greater testes produce more spermatozoa, we predict a positive association between the risk of sperm competition (measured as level of polyandry) and testes mass due to direct selection arising from sperm competition. The ejaculate, in the form of the spermatophore volume, should increase accordingly. Besides, and according to analyses in other groups, we initially expect sperm length to increase with sperm competition risk to confer individual spermatozoa a competitive advantage in sperm competition.
Materials and Methods
Analyzed Species
Adult males of eight scorpion species belonging to the Bothriuridae family were analyzed, as listed below together with capture sites, capture technique and number of males: Bothriurus bonariensis (C. L. Koch, 1842), Mendiolaza, Córdoba, Argentina, Ultraviolet light (UV light) (N = 11); Bothriurus cordubensis Acosta, 1995, Villa Berna, Córdoba, Argentina, turning rocks during the day (N = 6); Bothriurus rochensis San Martín, 1965, Piedras de Afilar, Montevideo, Uruguay, UV light (N = 10); Brachistosternus ferrugineus (Thorell, 1876), Chancaní Reserve, Córdoba, Argentina, UV light (N = 18); Brachistosternus pentheri Mello-Leitão, 1931, Chancaní Reserve, Córdoba, Argentina, UV light (N = 6); Timogenes elegans (Mello-Leitão, 1931) Chancaní Reserve, Córdoba, Argentina, UV light (N = 19); Timogenes dorbignyi (Guérin Méneville, 1843) Chancaní Reserve, Córdoba, Argentina, UV light (N = 9) Urophonius brachycentrus (Thorell, 1876), Tanti, Córdoba, Argentina, turning rocks during the day (N = 9). In all species, captures were between 2009 and 2012. Detailed data of voucher specimes and collection deposition are included as Table S1. The authors confirm that no specific permissions were required for capturing those sample sizes (N<20 individuals per species) in their respective localities and that the study did not involve endangered or protected species. In the laboratory, individuals were kept inside individual plastic boxes (10×13×8 cm) with moistened cotton as water supply and fed with larvae of Tenebrio molitor Linné (Insecta, Coleoptera). Data on testes mass was only obtained from recently dissected males. Specimens were euthanized using ethyl ether, and dissections were performed in ethanol 80%.
Comparative Design
Body mass and testes mass (bm, tm).
Specimens were cleaned and dried with tissue paper in order to remove ethanol excess. When dried, they were weighted in a microbalance (Ohaus Pioneer PA114) to the nearest 0.0001 g to obtain the male’s body mass. Males were then dissected under stereoscopic dissection microscope (Nikon SMZ1500). Paired paraxial organs, that produce the hemispermatophores, were removed and testes were cleaned and cut out from them (Figs. 1 A, B). Testes from both paraxial organs were dried from liquid excess using a tissue paper and were weighted together to the nearest 0.0001 g to get the male’s testes mass. In all species, measurements were taken 30 seconds after the value of the microbalance was stabilized. For comparison with other works, we also computed relative testes mass, in the form of a Gonadosomatic Index (GSI: gonad weight/body weight×100) [51]. The growth of testes mass and body mass was described by the ‘allometric relation’ [52], also termed ‘allometric equation’ [53] y = a*xb. This allometric relationship will also be described in the following two variables, sperm length and spermatophore’s volume.
A: Right paraxial organ. B: Right testis. C: Sperm package. D: Spermatozoa forming the sperm package. E: Spermatophore deposited in the substrate. F: Mating pair immediately after sperm transfer (the used spermatophore appears in yellow). Abbreviations: Cm: Cementing material which sticks the spermatophore in the substrate; Fe: female; H: Sperm package’s head; where all the spermatozoa heads concur; Ma: male; Po: Paraxial organ; S: Spermatophore; Sd: sperm drop; which is transferred to the female; Smtz: spermatozoa; St: Spermatophore’s trunk; Sv: seminal vesicle; Te: testes.
Sperm length (sl).
Paraxial organs also contain seminal vesicles [54] (Fig. 1A). Spermatozoa are stored inside seminal vesicles as sperm packages [42], [49] (Fig. 1 C, D). Sperm packages were photographed in a phase contrast microscope (Nikon Eclipse 50i) with an attached digital camera (Nikon DS-fi1). Sperm packages were then measured from the digital images with ImageJ 64 bit software [55]. Sperm packages are elongated and spermatozoa are easily identified (Fig. 1 C, D). Sperm length was measured indirectly as the length of sperm packages per species. Sperm package’s length is considered a reliable measure of sperm length and both measures, spermatozoa and sperm length, correlate [42], [48]. Mean value of sperm package’s length was recorded per male (N = 10 sperm packages per individual) and then averaged per species.
Spermatophore’s volume (sv).
Besides testes mass, sperm volume from pre-insemination spermatophores was observed. For this purpose, area and total length of spermatophore’s trunk were taken from one to three males depending on material availability. Volume was estimated using the trunk´s area and its width. We were able to calculate a volume in mm3 multiplying the area in mm2 by the trunk’s width in mm. The total volume of the spermatophore’s trunk can be taken as an estimative of the sperm volume that is transferred to the female during mating (Fig. 1 E).
Risk of sperm competition: polyandry levels (pol).
Polyandry levels were considered as the average number of males a female accepted during the reproductive season, based in controlled laboratory assays [44], [47], [48], [56]–[58] (Fig. 1 F). Urophonius brachycentrus is a species whose males deposit a very effective mating plug that occludes the female genital opening [44], [48]. The females only mate once [48], (Costa-Schmidt, Romero-Lebrón, unpublished data). Thus, this species is the only species considered monandrous in this study. In all other species females accepted at least two matings with different males [44], [47], [48], [56]–[58], (Peretti, Vrech, unpublished data) (average number of males accepted per reproductive season: Timogenes elegans, 1,5; T. dorbignyi, 2; Bothriurus rochensis, 2,5; B. cordubensis, 2,5; Brachistosternus ferrugineus, 2,5; Br. pentheri, 3; B. bonariensis, 4).
Statistical Analysis
Measurements were Log10 transformed to normalize their distributions (Shapiro-Wilks normality test on Log10 transformed data: lbm w = 0.968, p = 0.880; ltm w = 0.889, p = 0.228; lsv w = 0.928, p = 0.502; lsl w = 089, p = 0.263).
We used model selection analysis [59] for choosing among biological meaningful models. All these models were chosen because they could be explained by a hypothesis or an observation in nature. We tested 18 models divided into three categories aiming to explain one response variable in each category. First we tested the effect on testes mass (ltm = Log10 Testes mass). The first model was the null model (ltm∼1). This model will have the greatest chance of being chosen when the others models perform poorly in explaining the response variable. This is why the null model was tested with the three response variables. The second model for testes mass was ltm∼lbm for testing the allometric effect of body mass on testes mass. Third, we tested the effect of sperm competition on testes mass by using polyandry levels as a predictor (ltm∼pol). Finally, we used a model assuming an additive effect between these last two variables (ltm∼lbm+pol). If body mass was affecting the response variable, the effect of body mass on this particular variable was controlled for by including body mass in a multiple regression [60]. This multiple regression analysis was performed using a sequential (Type I) sum of squares, in which the predictor variables were added to the model only in the following order: body mass, polyandry to control for the possible effects of body mass. The second set of models was aimed to explain the performance of sperm length. First we tested the null model. Second, as for testes mass, we tested single models with body mass and polyandry as single variables. Here, we added the effect of testes mass on sperm length, as bigger testes could explain the production of larger spermatozoa in some organisms (e.g. [61]). Finally, we added two additive models to test polyandry and testes mass influence with the control of the possible effects of body mass (lsl∼lbm+pol; lsl∼lbm+ltm). The last set of variables tried to explain spermatophore volume. Besides the null model we tested four single models (lsv∼lbm; lsv∼pol; lsv∼ltm, lsv∼lsl). The influence of testes mass is important as a positive influence could be supported by the sperm competition model [17]. The last model, tried to corroborate if bigger spermatozoa influences ejaculate volume. Finally, we tested the influence of the last three variables controlled for body mass as in the other set of models.
We compared these models for each response variable, and we used Akaike’s Information Criterion corrected for small sample size (AICc) to infer the maximum likelihood of the current models, as suggested by Burnham & Anderson [59]. The model with the smallest value of AICc and models whose change in AICc was smaller than 2 (ΔAICc<2) were selected, because this difference suggests substantial evidence for the model (see [63]). It is noteworthy that models with four parameters (y∼a*b) were excluded as their changes in AICc were bigger than 10 (ΔAICc>10). We used Akaike weights to assess the relative strength of the model compared to the other models tested [59]. This value can give the idea of how many times one model is better than the other.
Species data may not be free of phylogenetic association. They may share character values because of a common ancestry rather than independent evolution [52], [62]. Because of this lack of independence, regressions were performed using a generalized least-squares approach within a phylogenetic framework (pGLS) [63]. This method estimates a phylogenetic scaling parameter lambda (λ), which represents the transformation that makes the data fit a Brownian motion evolutionary model. If λ values are close to 0, the variables are likely to have evolved independently of phylogeny, whereas λ values close to 1 indicate strong phylogenetic association of the variables. As an advantage, GLS allows a variable degree of phylogenetic control according to each tested model, accounting for differences in the level of phylogenetic association between different traits. The estimation of λ values and GLS analyses were performed using a code written by R. Freckleton for the statistical package R v.2.15.1 (R Foundation for Statistical Computing 2012) and the maximum likelihood value of λ was compared against models with λ = 1 and λ = 0. Using the current phylogenetic hypotheses available [64], (Mattoni, unpublished data), a cladogram was built for the Bothriuridae studied species (Fig. 2). Branch lengths were assumed equal, thereby assuming a punctuated model of evolution. Data was analyzed with R v. 2.15.1 64 bit open source statistical package [65].
Phylogeny of species used in the analysis and their respective Gonadosomatic index values.
Results
Testes Mass, Ejaculate Volume and Sperm Length: Description and Allometric Values
Testes mass.
Male reproductive allocation differed markedly among the analyzed scorpion species (Table 1). There was an order of magnitude of difference between the species with the lowest testes mass and the species with the highest testes mass values. Testes mass ranged from 1.6±1.1 mg in Timogenes dorbignyi to 16.3±4.5 mg in Brachistosternus pentheri. The average absolute testes mass among analyzed species was 8.4±5.0 mg. Two species showed low absolute testes mass values (both belonging to Timogenes) and two showed high values (Br. pentheri and B. bonariensis), the rest had intermediate values. Urophonius brachycentrus, the species with mating plug, had 3.2±1.4 mg, twice the mass found in T. dorbignyi. Timogenes elegans, the biggest species in the dataset, had really small testes mass compared to species with big body sizes like Bothriurus bonariensis or Br. pentheri. In proportion to male’s body size (Gonadosomatic Index, GSI), B. cordubensis and Br. ferrugineus allocated more in testes mass compared to other analyzed species, in opposition to what happened in both Timogenes species which allocated very little in testes mass (see Table 1, Fig. 1).
The linear logarithmic equation tested was Log10 (Testes mass) = −2.0901+0.2521*Log10 (Body mass) (Fig. 3 A). This relationship between testes mass and body mass lacked statistical significance (ltm∼lbm, Table 2).
A: Effect of body mass on testes mass. B: Effect of body mass on sperm length. C: Effect of body mass on spermatophore’s volume. D: Effect of polyandry on testes mass. E: Effect of polyandry on sperm length. F: Effect of polyandry on spermatophore’s volume. Abbreviations: Td: Timogenes dorbignyi, Te: Timogenes elegans, Br: Bothriurus rochensis, Bb: Bothriurus bonariensis, Bc: Bothriurus cordubensis, Brf: Brachistosternus ferrugineus, Brp: Brachistosternus pentheri, Ub: Urophonius brachycentrus.
Sperm length.
Sperm length varied from 208±15 µm to 302±3 µm. Urophonius brachycentrus together with B. cordubensis had the longest spermatozoa in the dataset (Table 1). On the opposite side, Br. pentheri had the shortest sperm, and the rest of the species had similar values oscillating from 233 to 269 µm. There was a significant effect of body mass on sperm length (lsl∼lbm, Table 2). The pendant was negative (b<0) (Fig. 3 B). The allometric equation found was Sperm length = 212.96*body mass−0.14.
Spermatophore’s volume (Ejaculate volume).
Spermatophore’s trunk volume varied but not as much as testes mass. The greatest difference among analyzed species is of about five times, for example between T. dorbignyi and Br. pentheri (see Table 1). The linear logarithmic equation tested was Log10 (spermatophore volume) = 0.72+0.43*Log10 (Body mass) (Fig. 3 C). With those parameters, the allometric equation was as follows: Testes mass = 5.22*body mass0.43. However, the relationship between testes mass and body mass was marginally not significant (lsv∼lbm, Table 2).
Statistical Phylogenetic Analysis on Testes Mass and Sperm Package Length in Relation to Levels of Polyandry
Models explaining testes mass.
Model selection analysis for testes mass, sperm package length and ejaculate volume resulted in one best model for each dependent variable (Table 2, highest wt values). The best-fitting model for testes mass considered only polyandry as predictor with almost 70% of chance of being the best model over 22% of the null model, over 3 times better at explaining ltm (Table 2). The phylogenetically controlled GLS regression analysis showed a positive influence of polyandry on testes mass (Fig. 3D, Table 2). The lambda value for this model was significantly closed to 1 (Table 2).Body mass performed poorly in tested models explaining response variables, as seen the previous allometric analysis (see Fig. 3A).
Models explaining sperm length.
Model selection analysis on sperm length showed that there is also only one model that best fit this data. In this case, according to the allometric analysis, the model incorporated only body mass and had a 46% chance of being the best model (Table 2). This value was greater than the Akaike’s weight for the null model (1.5 times bigger). There was a negative influence of body mass in sperm package length (Fig. 3 B). Polyandry levels (Fig. 3 E) and Testes mass (both predictors associated with sperm competition) alone were not good predictors of sperm length. Nevertheless, the additive models of these variables with body mass were significant or marginally significant as shown in Table 2. Nevertheless these two models were not selected by model selection analysis.
Models explaining spermatophore volume.
The best model for explaining ejaculate volume had body mass and testes mass as predictors. This model beared an astonishing chance of being the best model. The Akaike’s weight value was almost 100%. The relationship between spermatophore volume, both body mass and testes mass was highly significant with a positive relationship. Lambda value was not significantly close to 0 or 1. Both relationships with spermatophore volume were positive (See Fig. 3 C and F). Although there were other models with significant values (p<0.05), model selection analysis did not choose them.
Discussion
In this study we have analyzed the relative importance of body mass and polyandry over testes mass, sperm length and ejaculate volume, as well as some biological important relationships among these last three variables. For accomplishing this task, we used scorpions that represent an ancient sex model. To date, this is the first analysis of this kind performed on arthropods with indirect sperm transfer. The addition of a phylogenetically comparative analysis gives extra support to the findings made. The results are supported by classic sperm competition theories, and some of the patterns are found in other organisms closely related to arachnids as well as distant related groups.
We found that testes mass varied widely among analyzed species, both in relative (GSI) and absolute values. In some cases, the difference was of an order of magnitude across species. This pattern of variation in testes mass has been widely described in similar analyses in other invertebrates (e.g. [21] (butterflies); [61] (fruit flies); [66] (fireflies); [67] (bushcrickets); [9], [68] (Beetles)). In Table S2, we have reviewed some studies that used GSI values for assessing relative testes mass in insects, which represent a well-studied group in this subject. We compared these GSI values with the ones obtained for scorpions. In general, values for scorpions are low, with Timogenes species showing the lowest values of the reviewed dataset (less than 1%). Bothriurus cordubensis ranks higher in the table but far from the colossal values shown by some bushcrickets (eg. Sepiana sepium or Platycleis affinis), which are almost 3 times bigger than B. cordubensis’ GSI value.
Our results showed that body mass did not influence testes mass. In some insects, a clear positive relationship between body and testes was observed (eg. [21], [61]). Nevertheless, Wedell and Hosken [69] suggest that in fact, there is usually no relationship between both variables (see also [70], [71]), as we found for scorpions in the present study. As predicted, we found a clear positive influence of polyandry (a reliable estimator of sperm competition risk) over testes mass. The positive effect of sperm competition on testes mass have been widely demonstrated in various other taxa (e.g. [21] (butterflies); [25] (anurans); [66] (fireflies); [67] (bushcrickets); [72] (primates); [73] (bats); [74], [75] (birds); [76] (ants); [77] (seed beetle)). The results suggest that testes mass in scorpions would be a reliable predictor of sperm competition risk as suggested by many studies (e.g. [19], [78]). In this scenario, sperm would compete numerically following the fair raffle principle [6], [79], [80]. Increased gonadal investment would be traduced directly to sperm numbers because testes would only produce sperm (following what was suggested for insects, see [81]). The strong positive association between testes mass and spermatophore volume suggests, indeed, that bigger testes produce a bigger volume of ejaculate for transferring to the female. At the moment, we do not know if a greater volume of ejaculate is traduced directly to an increase in sperm number in scorpions. Preliminary analyses in seminal vesicle volume for these species, suggested that sperm volume was positively related to sperm count, (Vrech, unpublished data).
All analyzed scorpions were polyandrous except for U. brachycentrus. In this species, females mate only once because males deposit a very efficient mating plug [44], [48]. The presence of this mating plug seems to make females unreceptive and/or unattractive to new males (Romero-Lebrón, Vrech & Peretti, unpublished data). The mating plug in this species should have appeared in the past as a strategy to overcome the risk of sperm competition [45]. Sperm competition should have been huge before the appearance of this strategy. The investment in sperm plug should have favored the reduction of testes mass over evolutionary time. However, from a wide evolutionary perspective, the appearance of an efficient genital plug would be recent in this species [48], (Mattoni and Peretti unpublished data) without a clear optimization in gonadal investment yet.
Similarly, both Timogenes species did not show the expected testes mass inferred by their polyandry levels. Unfortunately, we do not know why these species show such small testes mass, but we can speculate there is a phylogenetic component, as both species belong to the same genus.
Some other possible explanations could be given for this unexpected result in Timogenes species: a.- Testes could be nonfunctional during some periods of time or they could stop producing sperm after the last molt, as happens in some other arachnids [82], [83]. b.- They could be nearly monandrous in nature, however they behave as polyandrous in controlled laboratory essays. c.- Sex ratio could be biased towards males, although sex ratio in these species are not yet fully evaluated (M. Nime, unplublished data). The study of physiological testes function as well as resource allocation influence over sex ratio (e.g. [84], [85]) in these species could give in the future interesting insights that could delimit better the interpretation of the data of this analysis. For example, the use of different evolutionary strategies on resource allocation for male function either through the increase of testes mass or other traits. However, for this purpose new information from field studies on sex ratio is needed.
Sperm Transfer, Ejaculate Volume and Sperm Length
The positive effect of body mass on ejaculate volume could be related to the size of the spermatophore itself. Bigger species produce bigger spermatophores with bigger storing capacity. The results suggest that this character scales negatively with body mass, as the pendant b is positive but smaller than one [86]. Although, studies show that positive allometry is generally shown in characters under directional sexual selection, one clear exception for this pattern is insect genitalia where negative allometry is usually observed (see [86], reviewed in [87]).
It is important to point out that sperm transfer in scorpions has distinct characteristics. In contrast to what happens in all other arthropods usually analyzed under the sperm competition theory, scorpions transfer their ejaculate using a sclerotized spermatophore deposited on soil [1], [48], [88]. The features implied in this ancient sex model drive to interesting questions at a macroevolutionary level. For example, how sperm competition pressures shaped the use of an indirect mode of sperm transfer. This question is particularly important in scorpions, considering its ancient phylogenetic position. Indeed, it is important to point out that natural selection promoted the appearance of spermatophores as a solution to the desiccation problem of some terrestrial organisms, including scorpions [38], [89], [90]. Nevertheless, sexual selection via sperm competition is directly involved in the evolution of direct sperm transfer, passing first through indirect spermatophore transfer (e.g. scorpions, amblypigids, uropygids) [5], [42], [91], [92]. In resume, spermatophores were the first attempt to put the sperm close to the ova. Spermatophores usually appear in basal systematic position groups, such as scorpions [89], [93], [94]. They are solitary predators that in general show relative low density and require more intimate contact compared to other groups with spermatophores [89], [92]. Spermatophores in scorpions have a relatively fixed volume [52], and this volume varies up to a certain maximum value (Vrech, unpublished data). Therefore, males will face a clear limitation in the ejaculate volume, a fact that would be relevant in a context of sperm competition [1], [48]. Interestingly, this could imply that, even though spermatogenesis is continuous in adulthood [95], [96], the great majority of the sperm production would not be transferred to females during the male’s life. Nevertheless, males could produce several spermatophores during their life [48], [57]. Some preliminary results suggest that scorpion males can adjust the volume of sperm deposited in the spermatophore (D. Vrech, C. I. Mattoni & A. V. Peretti unpublished data), but it is yet unknown if there is a real mechanism of sperm allocation in scorpions.
The volume of the sperm drop was smaller than the volume of the whole spermatophore trunk, as preliminary tested in both Timogenes species (Vrech, unpublished data). Volumes of ejaculated sperm and spermatophore’s trunk covary, both showing a positive relationship with testes mass (Vrech unpublished data). This pattern may suggest that bigger testes would be associated with bigger ejaculates. This idea has the support of the sperm competition theory [5], [12] where polyandry promotes bigger testes in males generating more spermatozoa to prevail in a numeric type of competition [6].
The influence of sperm competition risk has been widely tested on spermatozoa length. There is strong support of a positive influence of sperm competition over sperm length (eg. [21] (butterflies); [22] (moths); [23], [33] (fishes); [27], [29], [97] (birds); [32], [98], [99] (mammals); [72] (bats), [100] (Beetle), [101] (fruit flies); [102] (Coenorabditis elegans)). However, our results showed no influence of sperm competition risk (measured as the level of female polyandry and testes mass) on total sperm length. This result would agree with those published mostly in vertebrates (e.g. [27], [72], [99], [100]). According to Parker [12], sperm size should not necessary increase with an increase in the risk of sperm competition. Indeed, there must be some selective advantage in increased sperm size. With no advantage, sperm would tend to remain at a minimum size (due to energetic reasons) independent of sperm competition influence [33].
Unlike with testes mass, body mass did influenced sperm length. The negative association found suggests that species with greater body masses had smaller spermatozoa. Similar association is hard to find among invertebrates, but in vertebrates, many studies suggest a positive relationship or no relationship at all (see [21], [22], [30], [101], [102]). Cummins and Woodall [103] found a similar pattern in mammals, but their findings were not supported by posterior analyses [30], [99]. In snakes, Tourmente et al. [26] found a similar negative association between body mass and medial piece length, but found no relationship between total sperm length and body mass. This negative pattern between body mass and sperm size in scorpions could be partially explained by the dilution effect that species with great body volumes usually experiment [104]–[106]. Bearing in mind the hypothetical occurrence of a trade-off between sperm length and number, sperm in scorpions could be small and numerous in bigger species relative to small ones. Females from bigger species show a relative increase in the volume of the genital tract where the ejaculate is distributed [107]. In this situation, males from bigger species should produce a greater amount of smaller spermatozoa to fill the increased volume of the female genital tract [108].
In conclusion, the present study shows a variation in testes mass that was influenced by polyandry but not by body mass. Body mass positively related to ejaculate volume, and negatively to sperm length. Ejaculate volume and testes mass were strongly associated and both increased linearly in the relationship. Some models were under phylogenetic effects but the great majority lacked phylogenetic influence. Timogenes species showed disproportionally small testes relative to the expected for their body mass. Urophonius brachycentrus produces a very effective mating plug, but testes mass is not as small as would be expected in this situation, suggesting a very strong sperm competition over evolutionary time. Sexual selection mechanisms such as sperm competition are poorly known in scorpions. In the future, studies with a bigger sample of species are strongly needed. Unfortunately, this is no easy task as species should have a well-known mating system, and they should be easy to collect, as fresh material is needed. This last condition is essential as fixed material is difficult and inexactly to use, contrary to what happens for example in vertebrates. Besides, sperm concentration analyses of scorpions are strongly needed to elucidate the real sperm competition tactics affecting these arachnids. Furthermore, a detailed analysis of how sperm competition shapes different components of spermatozoa is also needed. Polyandry levels should be enhanced with more field observations, as well as additional knowledge of population parameters such as operational sex ratio.
Supporting Information
Table S1.
List of voucher specimens. All specimens were deposited in the Scientific Collection of the Institute of Animal Diversity and Ecology (IDEA), CONICET- Universidad Nacional de Córdoba, Argentina. In the following list each voucher specimen is described (species name, catalog number, collecting date and site, collectors’ names).
https://doi.org/10.1371/journal.pone.0094135.s001
(DOC)
Table S2.
Comparative table reviewing GSI values from different species of invertebrates. Species are ordered from the smallest to the highest GSI value.
https://doi.org/10.1371/journal.pone.0094135.s002
(DOC)
Acknowledgments
We would like to thank M. Tourmente and L. E. Costa-Schmidt for their kindness in revising the manuscript, for their useful suggestions, and for helping in the phylogenetic comparative analysis and in the use of R statistical software. We are also immensely grateful to A. Aisenberg, K. Vahed, W. Yan, Alejandro Cordoba-Aguilar, and one anonymous referee for their kindness in revising previous versions of the manuscript. We would like to thank E. Romero-Lebrón, M. Nime, F. Bollatti, M. González, and S. Burela for helping in field collecting material for this work. Special thanks to R. Freckleton for writing and supplying the R code to conduct the analysis. We thank L. C. Lee for helping with the English text. Finally, we would like to thank open source software and specially the R Core Team for providing the statistical software.
Author Contributions
Conceived and designed the experiments: AVP CIM DEV. Performed the experiments: DEV PAO. Analyzed the data: DEV PAO CIM AVP. Contributed reagents/materials/analysis tools: DEV CIM AVP. Wrote the paper: AVP CIM DEV.
References
- 1.
Smith R (1984) Sperm competition and the evolution of animal mating systems. Orlando: Academic Press. 687p.
- 2. Ginsberg J, Huck U (1989) Sperm competition in mammals. Trends Ecol Evol 4: 74–79.
- 3. Simmons LW, Craig M, Llorens T, Schinzig M, Hosken D (1993) Bushcricket Spermatophores Vary in Accord with Sperm Competition and Parental Investment Theory. Proc R Soc Lond B Biol Sci 251(1332): 183–186.
- 4.
Birkhead TR, Møller AP (1998) Sperm Competition and Sexual Selection. London: Academic Press. 826p.
- 5. Parker GA (1970) Sperm competition and its evolutionary consequences in the insects. Biol Rev 45 (4): 525–567.
- 6. Parker GA, Pizzari T (2010) Sperm competition and ejaculate economics. Biol Rev 85: 897–934.
- 7.
Simmons LW (2001) Sperm Competition and Its Evolutionary Consequences in the Insects. Princeton University Press. 448p.
- 8. Gage MJG, Parker GA, Nylin S, Wiklund C (2002) Sexual selection and speciation in mammals, butterflies and spiders. Proc R Soc Lond B Biol Sci 269: 2309–2316.
- 9. Simmons LW, García-González F (2008) Evolutionary reduction in testes size and competitive fertilization success in response to the experimental removal of sexual selection in dung beetles. Evolution 62: 2580–2591.
- 10. Hosken DJ, Ward PI (2001) Experimental evidence for testis size evolution via sperm competition. Ecol Lett 4: 10–13.
- 11. Soulsbury CD (2010) Genetic patterns of paternity and testes size in mammals. PLoS One 5(3): e9581.
- 12.
Parker GA (1998) Sperm competition and the evolution of ejaculates: Towards a theory base. In: Birkhead TR, Møller AP editors. Sperm Competition and Sexual Selection New York: Academic. 3–54.
- 13. Dewsbury DA (1982) Ejaculate cost and male choice. Am Nat 119: 601–610.
- 14. Nakatsuru K, Kramer DL (1982) Is sperm cheap? Limited male fertility and female choice in the lemon tetra (Pisces, Characidae). Science 216: 753–755.
- 15. Wedell N, Gage MJG, Parker GA (2002) Sperm competition, male prudence and sperm-limited females. Trends Ecol Evol 17: 313–320.
- 16. Parker GA (1982) Why are there so many tiny sperm? Sperm competition and the maintenance of two sexes. J Theor Biol 96: 281–294.
- 17. Parker G, Ball M (2005) Sperm competition, mating rate and the evolution of testis and ejaculate sizes: a population model. Biol Lett 1(2): 235–238.
- 18.
Short RV (1977) Sexual selection and the descent of man. In: Calaby JH, Tyndale-Biscoe CH, editors. Reproduction and evolution: proceedings of the fourth symposium on comparative biology of reproduction. Canberra: Australian Academy of Science. 3–19.
- 19. Short RV (1979) Sex determination and differentiation. Br Med Bull 35: 121–127.
- 20. Vahed K, Parker DJ (2012) The Evolution of Large Testes: Sperm Competition or Male Mating Rate? Ethology 118: 107–117.
- 21. Gage MJG (1994) Associations between body size, mating pattern, testis size and sperm lengths across butterflies. Proc R Soc Lond B Biol Sci 258 (1353): 247–254.
- 22. Morrow E, Gage MJG (2000) The evolution of sperm length in moths. Proc R Soc Lond B Biol Sci 267: 307–313.
- 23. Balshine S, Leach BJ, Neat F, Werner NY, Montgomerie R (2001) Sperm size of African cichlids in relation to sperm competition. Behav Ecol 12(6): 726–731.
- 24. Fitzpatrick JL, Montgomerie R, Desjardins JK, Stiver KA, Kolm N, et al. (2009) Female promiscuity promotes the evolution of faster sperm in cichlid fishes. Proc Natl Acad Sci USA 106(4): 1128–1132.
- 25. Byrne PG, Roberts JD, Simmons LW (2002) Sperm competition selects for increased testes mass in Australian frogs. J Evol Biol 15: 347–355.
- 26. Tourmente M, Gomendio M, Roldan ERS, Giojalas LC, Chiaraviglio M (2009) Sperm competition and reproductive mode influence sperm dimensions and structure among snakes. Evolution 63: 2513–2524.
- 27. Briskie JV, Montgomerie R (1992) Sperm Size and Sperm Competition in Birds. Proc R Soc Lond B Biol Sci 247: 89–95.
- 28. Briskie JV, Montgomerie R, Birkhead TR (1997) The Evolution of Sperm Size in Birds. Evolution 51(3): 937–945.
- 29. Johnson D, Briskie J (1999) Sperm competition and sperm length in shorebirds. Condor 101 (4): 848–854.
- 30. Gomendio M, Roldan ERS (1991) Sperm competition influences sperm size in mammals. Proc R Soc Lond B Biol Sci 243: 181–185.
- 31.
Tourmente M, Gomendio M, Roldan ERS (2011a) Sperm competition and the evolution of sperm design in mammals. BMC Evol Biol 11–12.
- 32. Tourmente M, Gomendio M, Roldan ERS (2011b) Mass-specific metabolic rate and sperm competition determine sperm size in marsupial mammals. PloS one 6(6): e21244.
- 33. Stockley P, Gage MJG, Parker GA, Møller AP (1997) Sperm Competition in Fishes: The Evolution of Testis Size and Ejaculate Characteristics. Am Nat 149(5): 933–954.
- 34.
Elgar M (1998) Sperm competition and sexual selection in Spiders and other arachnids. In: Birkhead TR, Møller AP, editors. Sperm Competition and Sexual Selection. London: Academic Press, 307–332.
- 35.
Schneider JM, Andrade MCB (2011) Mating behaviour and sexual selection. In: Herberstein ME, editor. Spider Behaviour; Flexibility and Versatility. Cambridge: Cambridge University Press. 215–274.
- 36. Herberstein M, Schneider J, Uhl G, Michalik P (2011) Sperm dynamics in spiders. Behav Ecol 22(4): 692–695.
- 37. Austad SN (1984) A classification of alternative reproductive behaviors, and methods for field testing ESS models. Am Zool 24: 309–320.
- 38.
Thomas RH, Zeh DW (1984) Sperm transfer and utilization strategies in arachnids: ecological and morphological constraints. In Smith RL, editor. Sperm competition and the evolution of animal mating systems. New York: Academic Press. 179–221.
- 39. Weygoldt P, Paulus HF (1979) Untersuchungen zur Morphologie, Taxonomie und Phylogenie der Chelicerata1 I. Morphologische Untersuchungen. J Zool Syst Evol Res 17(2): 85–116.
- 40.
Polis GA, Sissom WD (1990) Life history. In Polis GA, editor. The biology of scorpions. California: Stanford University Press. 161–223.
- 41. Michalik P, Mercati D (2010) First investigation of the spermatozoa of a species of the superfamily Scorpionoidea (Opistophthalmus penrithorum, Scorpionidae) with a revision of the evolutionary and phylogenetic implications of sperm structures in scorpions (Chelicerata, Scorpiones). J Zool Syst Evol Res 48: 89–101.
- 42. Vrech DE, Peretti AV, Mattoni CI (2011) Sperm package morphology in scorpions and its relation to phylogeny. Zool J Linn Soc 161: 463–483.
- 43.
Hjelle JT (1990) Anatomy and morphology. In: Polis GA, editor. The biology of scorpions. Stanford, California: Stanford University Press. 9–63.
- 44. Mattoni CI, Peretti AV (2004) The giant and complex genital plug of the asper group of Bothriurus (Scorpiones, Bothriuridae): morphology and comparison with other genital plugs in scorpions. Zool Anz 243(1–2): 75–84.
- 45. Contreras-Garduño J, Peretti AV, Córdoba-Aguilar A (2006) Evidence that Mating Plug is Related to Null Female Mating Activity in the Scorpion Vaejovis punctatus. Ethology 112: 152–163.
- 46. Althaus S, Jacob A, Graber W, Hofer D, Nentwig W, et al. (2010) A double role of sperm in scorpions: The mating plug of Euscorpius italicus (Scorpiones: Euscorpiidae) consists of sperm. J Morphol 271: 383–393.
- 47. Peretti AV (2003) Functional morphology of spermatophores and female genitalia in bothriurid scorpions: genital courtship, coercion and other possible mechanisms. J Zool 261(2): 135–153.
- 48.
Peretti AV (2010) An ancient indirect sex model: single and mixed patterns in the evolution of scorpion genitalia. In: Leonard J, Córdoba-Aguilar A, editors. The evolution of primary sexual characters in animals. Oxford: Oxford University Press. 218–248.
- 49. Peretti AV, Battán-Horenstein M (2003) Comparative analysis of the male reproductive system in Bothriuridae scorpions: structures associated with the paraxial organs and the presence of sperm packages (Chelicerata, Scorpiones). Zool Anz 242: 21–31.
- 50. Vignoli V, Klann AE, Michalik P (2008) Spermatozoa and sperm packages of the European troglophylous scorpion Belisarius xambeui Simon, 1879 (Troglotayosicidae, Scorpiones). Tissue Cell 40: 411–416.
- 51.
Olsson M, Madsen T (1998) Sexual Selection and sperm Competition in Reptiles. In: Birkhead TR, Møller AP, editors. Sperm Competition and Sexual Selection. Academic Press. 503–577.
- 52.
Harvey PH, Pagel MD (1991) The comparative method in evolutionary biology. Oxford: Oxford University Press. 248 p.
- 53.
Reiss MJ (1991). The allometry of growth and reproduction. Cambridge University Press. 197 p.
- 54. Lautié N, Soranzo L, Lajarille MC, Stockmann R (2008) Paraxial organ of a scorpion: structural and ultrastructural studies of Euscorpius tergestinus paraxial organ (Scorpiones, Euscorpiidae). Invertebr Reprod Dev 51: 77–90.
- 55. Schneider CA, Rasband WS, Eliceiri KW (2012) NIH Image to ImageJ: 25 years of image analysis. Nat Meth 9: 671–675.
- 56. Peretti AV (2001) Patrones de resistencia femenina y respuesta del macho durante el apareamiento en escorpiones Bothriuridae y Buthidae:¿ qué hipótesis puede explicarlos mejor? Rev Etol (Brasil) 3: 25–45.
- 57. Peretti AV (1996) Comportamiento de transferencia espermática de Bothriurus flavidus (Scorpiones: Bothriuridae).Rev Soc Entomol Argentina. 55: 7–20.
- 58. Toscano-Gadea CA (2010) Sexual behavior of Bothriurus buecherli (Scorpiones: Bothriuridae) and comparison with the B. prospicuus group.J Arachnol 38. (2): 360–363.
- 59.
Burnham KP, Anderson DR (2002) Model Selection and Multi- model Inference: A Practical Information-theoretic Approach. New York: Springer. 515 p.
- 60. Tomkins JL, Simmons LW (2002) Measuring Relative Investment: a case study of testes investment in species with alternative male reproductive tactics. Anim Behav 63(5): 1009–1016.
- 61. Pitnick S (1996) Investment in testes and the cost of making long sperm in Drosophila. Am Nat 148(1): 57–80.
- 62. Felsenstein J (1985) Phylogenies and the comparative method. Am Nat 125: 1–15.
- 63. Freckleton R, Harvey PH, Pagel MD (2002) Phylogenetic analysis and comparative data: a test and review of evidence. Am Nat 160: 712–726.
- 64.
Mattoni CI, Prendini L (2007) Phylogeny and biogeography of the family Bothriuridae (Scorpiones). Darwiniana 45 (suppl): 96–98.
- 65.
R Core Team (2012) R: a language and environment for statistical computing. Vienna, Austria. Available: http://www.R-project.org/. Accessed 2012 Oct 2.
- 66. Demary KC, Lewis SM (2007) Male reproductive allocation in fireflies (Photinus spp.). Invertebr Biol 126: 74–80.
- 67. Vahed K, Parker DJ, Gilbert JDJ (2011) Larger testes are associated with a higher level of polyandry, but a smaller ejaculate volume, across bushcricket species (Tettigoniidae). Biol Lett 7: 261–264.
- 68. Simmons LW, Emlen DJ, Tomkins JL (2007) Sperm competition games between sneaks and guards: a comparative analysis using dimorphic male beetles. Evolution 61(11): 2684–2692.
- 69.
Wedell N, Hosken DJ (2010) The evolution of male and female internal reproductive organs in insects. In Leonard JL, Córdoba-Aguilar A, editors. The evolution of primary sexual characters in animals. New York: Oxford University Press. 307–331.
- 70. Presgraves DC, Baker RH, Wilkinson GS (1999) Coevolution of sperm and female reproductive tract morphology in stalk-eyed flies. Proc R Soc Lond B Biol Sci 266(1423): 1041–1047.
- 71. Minder AM, Hosken DJ, Ward PI (2005) Co-evolution of male and female reproductive characters across the Scathophagidae (Diptera). J Evol Biol 18(1): 60–69.
- 72. Harcourt AH, Purvis A, Liles L (1995) Sperm competition, mating system, not breeding season, affects testes size of primates. Funct Ecol 9: 468–476.
- 73. Hosken DJ (1998) Sperm competition in bats. Proc R Soc Lond B Biol Sci 264: 385–392.
- 74. Pitcher T, Dunn P, Whittingham L (2005) Sperm competition and the evolution of testes size in birds. J Evol Biol 18(3): 557–567.
- 75. Pyron M (2000) Testes mass and reproductive mode of minnows. Behav Ecol Sociobiol 48: 132–136.
- 76. Baer B, Boomsma JJ (2004) Male reproductive investment and queen mating-frequency in fungus-growing ants. Behav Ecol 15: 426–432.
- 77. Katvala M, Rönn JL, Arnqvist G (2008) Correlated evolution between male ejaculate allocation and female remating behaviour in seed beetles (Bruchidae). J Evol Biol 21: 471–479.
- 78. Iossa G, Soulsbury C, Baker P, Harris S (2008) Sperm competition and the evolution of testes size in terrestrial mammalian carnivores. Funct Ecol 22(4): 655–662.
- 79. Parker GA (1982) Why are there so many tiny sperm? Sperm competition and the maintenance of two sexes. J Theor Biol 96: 281–294.
- 80. Parker GA (1993) Sperm competition games: sperm size and sperm number under adult control. Proc R Soc Lond B Biol Sci 253: 245–254.
- 81.
Chapman RF (1998) The Insects: Structure and Function. Cambridge: Harvard University press. 961 p.
- 82. Michalik P, Rittschof CC (2011) A Comparative Analysis of the Morphology and Evolution of Permanent Sperm Depletion in Spiders. PloS One 6(1): e16014.
- 83. de Armas LF (2004) Arácnidos de República Dominicana: Palpigradi, Schizomida, Solifugae y Thelyphonida (Chelicerata: Arachnida). Rev Iber Aracnol, volumen especial monográfico (2): 3–63.
- 84. Wang RW, Wang YQ, He JZ, Li YT (2013) Resource elasticity of offspring survival and the optimal evolution of sex ratios. Plos One 8(1): e53904.
- 85. Zhang DY, Wang G (1994) Evolutionarily stable reproductive strategies in sexual organisms: an integrated approach to life-history evolution and sex allocation. Am Nat 144: 65–75.
- 86.
Eberhard WG, Huber BA, Rodriguez RLS, Briceño RD, Salas I, et al. (1998) One size fits all? Relationships between the size and degree of variation in genitalia and other body parts in twenty species of insects and spiders. Evolution 415–431.
- 87. Hosken DJ, Stockley P (2004) Sexual selection and genital evolution. Trends Ecol Evol 19: 87–93.
- 88. Francke 0F (1979) Spermatophores of some North American scorpions (Arachnida, Scorpiones). J Arachnol 7: 19–32.
- 89. Schaller F (1971) Indirect sperm transfer by soil arthropods. Ann Rev Entomol 16(1): 407–446.
- 90. Davey KG (1960) The evolution of spermatophores in insects. Proc R Entomol Soc Lond A Gen Entomol 35(7–9): 107–113.
- 91. Parker GA (1978) Evolution of competitive mate searching. Ann Rev Entomol 23: 173–96.
- 92. Proctor HC (1998) Indirect sperm transfer in arthropods: behavioral and evolutionary trends. Ann Rev Entomol 43(1): 153–174.
- 93.
Coddington JA, Giribet G, Harvey MS, Prendini L, Walter DE (2004) Arachnida. In: Cracraft J, Donoghue MJ, editors. Assembling the Tree of Life. 296–318.
- 94. Prendini L, Wheeler WC (2005) Scorpion higher phylogeny and classification, taxonomic anarchy, and standards for peer review in online publishing. Cladistics 21(5): 446–494.
- 95. Jespersen Å, Hartwick R (1973) Fine structure of spermiogenesis in scorpions from the family Vejovidae. J Ultrastruct Res 45: 366–383.
- 96. Alberti G (1983) Fine structure of scorpion spermatozoa (Buthus occitanus; Buthidae, Scorpiones). J Morphol 177: 205–212.
- 97. Kleven O, Laskemoen T, Fossøy F, Robertson RJ, Lifjeld JT (2008) Intraspecific variation in sperm length is negatively related to sperm competition in passerine birds. Evolution 62: 494–499.
- 98. Harcourt AH (1991) Sperm competition and the evolution of nonfertilizing sperm in mammals. Evolution 45: 314–328.
- 99. Breed WG, Taylor J (2000) Body mass, testes mass, and sperm size in murine rodents. J Mamm 8(3): 758–768.
- 100. Gay L, Hosken D, Vasudev R, Tregenza T, Eady P (2009) Sperm competition and maternal effects differentially influence testis and sperm size in Callosobruchus maculatus. J Evol Biol 22: 1143–1150.
- 101. Pitnick S, Markow TA, Spicer GS (1995) Delayed male maturity is a cost of producing large sperm in Drosophila. Proc Nat Acad Sc 92(23): 10614–10618.
- 102. Schulte-Hostedde AI, Millar JS (2004) Intraspecific variation of testis size and sperm length in the yellow-pine chipmunk (Tamias amoenus): implications for sperm competition and reproductive success. Behav Ecol Sociobiol 55(3): 272–277.
- 103. Cummins JM, Woodall PF (1985) On mammalian sperm dimensions. J Reprod Fertil 75: 153–175.
- 104.
Brody S (1945).Bioenergetics and growth. Reinhold publishing corporation.
- 105.
Peters RH (1983) The ecological implications of body size. Cambridge: Cambridge University Press. 297 p.
- 106.
Calder WA (1996). Size, function, and life history. Courier Dover Publications. 384 p.
- 107.
Short RV (1981) Sexual selection in man and the great apes. In Short RV, editor. Reproductive biology of the great apes. New York: Academic Press. 319–341.
- 108. Gage MJG (1998) Influences of sex, size, and symmetry on ejaculate expenditure in a moth. Behav Ecol 9(6): 592–597.