Overcoming an evolutionary conflict: Removal of a reproductive organ greatly increases locomotor performance

Margarita Ramos*, Duncan J. Irschick*†, and Terry E. Christenson‡

*Department of Ecology and Evolutionary Biology, 310 Dinwiddie Hall, and ‡Department of Psychology, 2007 Stern Hall, Tulane University, New Orleans, LA 70118

Communicated by Thomas W. Schoener, University of California, Davis, CA, January 15, 2004 (received for review February 26, 2003) One potential consequence of sexual size dimorphism is conflict Here, we examine a functional conflict imposed by differential among characters. For example, a structure evolved for reproduc- scaling and extreme sexual size dimorphism in a , and tion can impair performance during other activities (e.g., locomo- describe how a novel behavior overcomes this conflict. We tion). Here we provide quantitative evidence for an over- demonstrate that male gain a substantial locomotor coming an evolutionary conflict generated by differential scaling performance advantage through the removal of a structure that and sexual size dimorphism by obligatorily removing an undam- is normally essential for reproduction (a pedipalp). Biologists aged reproductive organ, and thus dramatically enhancing its have shown that the removal of a structure occurs in a variety of locomotor performance. The spider (Araneae, taxa [lizards and tail loss (11, 12); harvestmen and leg loss (13); ) is interesting because, within several species present- damselfly larvae and loss of lamellae (14)]. However, these ing extreme sexual size dimorphism (males representing Ϸ1% of examples are cases where remove structures faculta- the total mass of the female), males voluntarily remove one of their tively in relatively specific situations. Here, we document a case two disproportionately large pedipalps (modified copulatory or- in which all males of the species remove a reproductive organ in gans; a single one represents Ϸ10% of the body mass in an adult) an obligatory manner, thus representing a case of dramatic before achieving sexual maturity. Whether the left or right pedi- evolutionary change in behavior. palp is removed appears to be random. Previous researchers have Although sexual size dimorphism is common in spiders (15– hypothesized that pedipalp removal might enhance locomotor 17), it is extreme in the cobweb spider, Tidarren (Araneae, performance, a prediction that has remained untested. We found Theridiidae) (18–20). In this genus, sexual size dimorphism that, for male Tidarren sisyphoides, maximum speed increased results from a decrease in male size (19, 21, 22) and an increase (44%) significantly and endurance increased (63%) significantly in female size (20). The male pedipalps (copulatory organs) are after pedipalp removal. Furthermore, spiders with one pedipalp considered large in relation to male body size (refs. 21, 23, and moved Ϸ300% greater distances before exhaustion and had a 24, but see ref. 25) (Fig. 1). Vollrath (21) has suggested that the higher survival after exertion than those with two pedipalps. pedipalp is large to enable mechanical coupling with the repro- Removal of the pedipalp may have evolved in male Tidarren ductive organ of the relatively large female. In other words, as because of enhanced abilities to search for females (higher endur- males decreased in body size, their pedipalps did not decrease in ance and survival after exertion) and to out-compete rival males on size at the same rate (unpublished data). In most spiders, males the female’s web (higher maximum speed). Our data also highlight use as copulatory organs a pair of modified appendages discon- how the evolution of conflicts can result in the evolution of a novel nected from the gonads. After loading or inducting them with behavior. sperm, males search for females. During copulation, males generally use both pedipalps in an alternating fashion to insem- EVOLUTION central tenet of optimality theory is that natural selection inate the paired spermathecae in the female. In Tidarren, Awill optimize structure and performance resulting in an however, one pedipalp is removed (either left or right pedipalp, overall highly fit organism (1, 2). Many studies, however, have seemingly at random, see ref. 26) before sexual maturation (20, shown that the evolution of high performance in one task can 22, 25, 26), which has also been described for another spider of lead to decreased performance during another task (e.g., refs. similar size and closely related to Tidarren (, ref. 3–5). In extreme cases of such apparent conflicts, a structure 27). Just after molting to the penultimate instar, the male secures evolved for one activity can substantially impair performance one of its pedipalps to a silk scaffold and then twists it off by during another activity. For example, Darwin (6) described how turning in circles and pushing the bulb with the third and fourth sexual selection for sex differences might lead to such functional pairs of legs (26). conflicts, particularly in males. He depicted how the relatively Previous authors have suggested that having two extremely elaborate feathers in some male birds result in enhanced repro- large pedipalps presents a functional constraint (23), and that the ductive success via female mate choice, yet also reduces or removal of one pedipalp would enhance locomotion. Because constrains flight capacities, thus potentially making the animal adult male spiders actively move about in search of females more susceptible to predators (7). A relatively unexplored area (28–31) and participate in often intense intermale competition is how organisms cope with these constraints imposed by factors (29, 32), any decrement in locomotor ability would have impor- such as sexual selection, natural selection, or allometry. One tant negative consequences for their reproductive fitness. This view of constraints is that they limit or hinder morphological or proposed explanation for the origin of the pedipalp removal behavioral change, but another possibility is that constraints can behavior has remained untested; no studies have examined the result in a novel phenotype or behavior (8, 9). For example, as potential functional consequences of locomotion with these plethodontid salamanders undergo evolutionary miniaturization large pedipalps. (become smaller), they shift from a terrestrial to an arboreal lifestyle (10). This transition occurs because of developmental constraints on limb growth, resulting in increased toe webbing, †To whom correspondence should be addressed. E-mail: [email protected]. and hence enhanced climbing ability in small salamanders. © 2004 by The National Academy of Sciences of the USA

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0400324101 PNAS ͉ April 6, 2004 ͉ vol. 101 ͉ no. 14 ͉ 4883–4887 Downloaded by guest on September 29, 2021 Materials and Methods Subjects. Male T. sisyphoides emerge from the egg sac in the second instar and attain adulthood in three subsequent molts (M.R., personal observation). Thirteen adult individuals and 10 egg sacs were collected at the F. Edward Hebert Center of Tulane University in Belle Chase, LA, and maintained in the laboratory at room temperature (all locomotion trials also took place at 22°C). The 956 resulting spiderlings were housed individually in 20-ml glass scintillation vials with several nylon strands for web support. They were fed small mealworms (Ϸ5 mm long), wingless Drosophila flies, and newborn crickets, and were spritzed with water twice a week. A total of 31 males in the fourth instar (penultimate) and 38 males in the fifth instar (adult) were used in this study.

Quantification of Pedipalp Mass. The male spiders’ mean weights were estimated by weighing 15 adult specimens simultaneously. The weight of the pedipalp was estimated by calculating the volume of the different body segments of three adult male spiders by approximating them to the closest geometric figure. The bodies were divided into cephalothorax (cylinder), abdomen (sphere), legs (cone), and pedipalp (ellipse), and then each segment was photographed from two different angles and digitized.

Maximum Speed. Sixteen penultimate instar spiders were ob- served at least once before and after pedipalp removal. Pedipalp removal usually occurs within a few hours of the molt to this instar. Individual spiders were filmed while moving on a strand of tightly stretched silk (from a virgin adult female) the observer had placed between two 15-cm-long sticks placed 5 cm apart. A small ruler (7 cm long) was glued to a second set of sticks parallel to the first. A Redlake high-speed motionscope PCI camera (60 frames per second) filmed all locomotion and a PC digitally recorded all trials. The spiders were removed from the vial by using a brush, and were placed on one of the sticks. The trial began once the animal moved onto the silk, and the trial ended Fig. 1. A male T. sisyphoides before (A) and after (B) removing a pedipalp. when it reached the second stick. Maximum speed was defined Note the pedipalps overlap in the two-pedipalp condition (A), whereas the as the maximum instantaneous velocity achieved (quantified by one pedipalp is carried in a central position after pedipalp removal (B). (The using PEAK PERFORMANCE software) by an animal during the scale bars represent 1 mm.) trial while it was chased with a microcapillary tube. We analyzed only locomotion in which the spider moved continuously to ensure that we examined steady-state locomotion. In this study, we had several objectives. We first attempted to measure the size of Tidarren pedipalps to estimate the loss of Potential Confounding Variables: Hardening of the Exoskeleton, Ex- mass after pedipalp removal. Second, we tested the hypotheses perience, and Instar Differences. After emerging from their old that removal of the pedipalp by male Tidarren sisyphoides would exoskeleton, spiders are soft and relatively immobile (30). We result in an increase in maximum speed (potentially linked to therefore filmed eight adult males to examine the effects of intermale competition) and endurance (potentially linked to hardening of the exoskeleton on maximum speed. Each spider enhanced mate searching abilities and intermale competition). was run 20, 60, 120, 180, 240, and 300 min after molting. To Third, we examined the effects of pedipalp removal on survi- control for a practice effect in a manner such that there were no interactions with the effects of a recent molt, we collected 13 vorship after the above taxing endurance trials. We hypothesized adults in the field from female webs and filmed them twice (1-h that individual male spiders that have removed their pedipalp rest between trials) following the previously outlined procedure. will be significantly less likely to die after endurance trials These males were filmed at least 5 days after molting, because compared to males that have not yet removed their pedipalp. If the process of sperm induction takes several days after the last the presence of the two palps causes high mortality in males, then molt. Finally, to demonstrate that speed did not differ according pedipalp removal may thus provide a fitness advantage, and to instar, we compared maximum speed in penultimate instar could provide some insight into why this genus has evolved the (n ϭ 16) and adult (n ϭ 8) males bearing only one pedipalp. palp-removal behavior. To test these hypotheses, we measured maximum speed in individual males before and after pedipalp Endurance. Time to exhaustion was measured in 15 penultimate removal, measured the endurance of spiders with one or two instar subjects bearing two pedipalps (different from the subjects pedipalps by chasing them until exhaustion, and measured used to measure maximum speed) and 17 adult subjects with one survivorship after the endurance trials. Finally, we accounted for pedipalp. The same spiders could not be examined under both the potential confounding variables of the molting process and one- and two-pedipalp conditions because the endurance trials practice effects by measuring maximum speed several times in were extremely taxing for the animals. experience a two groups of spiders. high rate of water loss for a period after the molt (33). All

4884 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0400324101 Ramos et al. Downloaded by guest on September 29, 2021 Fig. 3. Box plot of maximum speed in a group of spiders (n ϭ 16) before and after pedipalp removal. The bottom of the box represents the 25th percentile, the dotted line represents the mean, the solid line represents the median, the top of the box represents the 75th percentile, and the whiskers represent the 5th and 95th percentile.

time since molting (independent variable) (homogeneity of ϭ Ͼ slopes test; F7,31 0.65, P 0.50), and time since molting also ϭ Ͼ did not affect maximum speed (F1,38 0.26, P 0.50). A linear least-squares regression of the first (independent variable) and second (dependent variable) runs also did not reveal a significant ϭ ϭ ϭ Ͼ Fig. 2. Male and female T. sisyphoides in copula. The minute male (indicated relationship between them (F1,11 1.2, r 0.31, df 13, P by the arrow) on the female’s ventrum is Ϸ1% of the female’s mass. (The scale 0.25, slope ϭ 0.38). Thus, how spiders performed on their first bar represents 1 mm.) run did not predict how they would perform on their second run. In addition, we did not find a significant difference in maximum speed between penultimate instar and adult males with a single animals therefore were tested4haftermolting to minimize a pedipalp (two sample t test, t ϭ 0.26, df ϭ 22, P Ͼ 0.1). potential confounding effect of water loss. Subjects were chased with a small paintbrush on a sheet of paper until exhaustion, that Endurance. For the endurance trials, spiders with two pedipalps is, when the spider did not respond to 10 consecutive touches. were able to move for an average of 17 min 30 s Ϯ 55 s, whereas During the second minute of each test, the animal’s path was spiders with one pedipalp were able to move for an average of traced by following the spider with a pencil to estimate the 28 min 30 s Ϯ 45 s (Fig. 4; two-sample t test, t ϭ 9.3, df ϭ 30, distance moved during that minute, and to provide an estimate P Ͻ 0.001). Thus, spiders with one pedipalp had endurance of the average running speed (distance͞time) during that capacities that were Ϸ63% greater than spiders with two pedi- minute. We quantified only the second minute because handling palps. In addition, spiders with one pedipalp moved significantly and placement of the spider on the test arena precluded an faster (1.4 Ϯ 0.04 cm͞s) than those with two pedipalps (0.8 Ϯ EVOLUTION accurate trace of the path during the first minute of each test and 0.03 cm͞s) (t ϭ 11.5, df ϭ 24, P Ͻ 0.001). Assuming constant because tracing the entire path would have resulted in too speeds throughout the duration of the tests, we estimated that complex a record (375–1,930 s in range). The track was measured by laying a piece of string over the path and measuring its total length with a ruler. Mean speed per path was calculated by dividing this total distance by 60 s. SYSTAT (SPSS Institute, Chicago) was used for statistical analysis, and SIGMAPLOT was used for creating graphs (SPSS Institute). Results Quantification of Pedipalp Mass. We estimate the adult male pedipalp to account on average for 10% of body mass (Fig. 1). Tidarren males have an average body length of 1.4 mm and weight of 7.1 ϫ 10Ϫ4 g, whereas females are on average 6 mm in length and weigh 5.8 ϫ 10Ϫ2 g (Fig. 2).

Maximum Speed. Pedipalp removal significantly increased maxi- mum speed (Fig. 3) from an average of 2.7 Ϯ 0.2 (SE) cm͞sto 3.8 Ϯ 0.3 cm͞s (paired t test, t ϭ 4.41, df ϭ 15, P Ͻ 0.001). Thus, spiders ran 44% faster after they had removed one pedipalp than when they still possessed both pedipalps. We used analysis of Fig. 4. Box plot of time until exhaustion in two different groups of spiders covariance (ANCOVA) to test whether the potentially con- with two (n ϭ 15) and one (n ϭ 17) pedipalp, respectively. The bottom of the founding process of exoskeleton hardening (time after molt) box represents the 25th percentile, the dotted line represents the mean, the affected maximum speed. Individual spiders did not differ in solid line represents the median, the top of the box represents the 75th their slope between maximum speed (dependent variable) and percentile, and the whiskers represent the 5th and 95th percentile.

Ramos et al. PNAS ͉ April 6, 2004 ͉ vol. 101 ͉ no. 14 ͉ 4885 Downloaded by guest on September 29, 2021 animals with one pedipalp moved an average of 2,435 Ϯ 118 cm molting and pedipalp removal (fourth instar) or sperm induction or 17,393 body lengths (based on a mean body length of 1.4 mm (fifth instar) events. taken from ref. 23) before exhaustion, whereas those with two A more likely possibility to explain the large difference in pedipalps moved and average of 822 Ϯ 61 cm or 5,870 body locomotor performance between spiders bearing one or two lengths. In other words, spiders with one pedipalp moved on pedipalps is that the presence of both pedipalps might mechan- average 296% greater distances than those with two pedipalps. ically interfere with locomotion, such as impairing limb move- Of 11 spiders that died immediately after an endurance trial, ment or by dragging on the locomotor surface (Fig. 1). Loco- significantly more spiders bore two pedipalps (eight individuals, motion would become more energetically expensive for spiders or 53% of the total number of spiders measured bearing two moving long distances, and maximum speed would be reduced. pedipalps) than one pedipalp (two individuals, or 12% of the The structure of male spider pedipalps is complex, and dragging total number of spiders measured bearing one pedipalp) (Fish- when moving on flat surfaces could damage pedipalpal compo- ers’ exact test, P ϭ 0.021). A multiple regression using survival nents needed for coupling with female genitalia. Another non- (1 ϭ spider died, 2 ϭ spider lived) as the dependent variable and exclusive possibility is that the loss of mass caused by removal of endurance and average speed during the second minute as the the pedipalp enables Tidarren to move both faster and longer ϭ independent variables was statistically significant (F2,23 7.09, relative to their body size. Tidarren males are very small, so the P Ͻ 0.01), but endurance was the only statistically significant effects of gravity on both horizontal and vertical locomotion are (and positive) predictor of survival (P ϭ 0.028, standardized minor compared to larger animals (34, 35). Loss of mass will coefficient ϭ 0.77). Thus, spiders with high endurance capacities likely have a lesser impact on the locomotion of a smaller animal tended to more often survive after endurance trials. In addition, compared to the effect of a proportional loss in mass on a larger those individuals with two pedipalps that survived the endurance animal. Ideally, this latter hypothesis would most profitably be trial (n ϭ 4) took up to 5 days to remove the pedipalp (mean ϭ tested by adding or subtracting weights equaling the mass of a 3.0 Ϯ 0.9 days), although this process typically takes place within pedipalp, but adding such a small mass would be extremely a few hours after the molt. difficult. However, further experiments that examine the posi- tion of the pedipalps during locomotion with one and two Discussion pedipalps might differentiate among these hypotheses. In addi- One potential consequence of extreme sexual size dimorphism is tion, pedipalp removal does not seem to have significant prox- conflict among characters (e.g., refs. 6 and 7). A central issue in imal costs for the spider. For instance, the animals twist in circles evolutionary biology concerns the evolution of how organisms to remove the pedipalp, thus effectively sealing the wound and cope with such conflicts. In T. sisyphoides, male body size has apparently preventing excessive fluid loss and infection. Tidarren been reduced (19–22), whereas the reproductive organs might The endurance capacities of male are remarkably high for a spider (36–42) and are comparable to many mammal not have changed evolutionarily in an equal manner (21). The species (43, 44). This high endurance is especially intriguing in apparent cost of such conflict resulting from this differential the light of the lack of a sophisticated circulatory system in scaling is a relatively large pair of pedipalps (Fig. 1) that spiders that would enable high rates of oxygen exchange, such as substantially impair both maximum speed and endurance. Re- found in birds and mammals (45). More remarkably, Tidarren markably, rather than evolving one pedipalp to overcome this males ran for very long distances at relatively fast speeds, which functional conflict, male Tidarren have evolved the behavior of should lead to high levels of lactate in their tissues caused by pedipalp removal. In this regard, our data provide evidence for anaerobic processes (39, 40). Indeed, in cases where the spider an organism overcoming an evolutionary conflict by evolving a died after the endurance trails, the most likely cause was the novel behavior. build-up of lactate (39, 40). The fact that males with two Which proximate factors could explain why Tidarren have pedipalps died significantly more often than males with one greater maximum speed and endurance with one compared to indicates that long movements with two pedipalps are especially two pedipalps? One explanation is that the observed perfor- exhausting. mance difference is not caused by removal of the pedipalp, but The negative effect of pedipalp size on both aerobic and rather to gross anatomical, physiological, or behavioral changes anaerobic locomotion could have profound implications for the in male spiders as they molt from the fourth (penultimate) to the reproductive ecology of Tidarren. Several lines of evidence fifth (adult) instar. We consider this possibility unlikely. First, suggest that Tidarren males move long distances (relative to their male size and overall morphology change very little between the size). First, a large body of literature shows that male spiders, on fourth and fifth instars in Tidarren (C. Linn, personal commu- reaching adulthood, shift from being sedentary to actively nication), and we found no difference in maximum speed searching for females. Male spiders frequently travel long dis- between spiders bearing one pedipalp in these two instars. In tances, often vertically, to find a mate, because females can be Ϸ fact, relative pedipalp size increases dramatically ( 370%) from dispersed widely (31, 46). Second, some simple considerations the third to the fourth instar, whereas it increases only slightly show that Tidarren males may be forced to move long distances Ϸ from the fourth to the fifth instar ( 2%) (C. Linn, personal because of their very small size. For example, if a Tidarren male communication). Therefore, the two pedipalps become a hin- moves only 2 meters, that distance is equivalent to Ϸ1,400 body drance only after the molt into the fourth instar, and this might lengths, which is similar to a 3-m tetrapod (e.g., an alligator) explain why the pedipalp is not removed earlier. In terms of moving Ϸ4.3 km. Added to this is intense intermale competition physiological changes, spiders produce hormones (e.g., ecdy- that might result in males often moving from female to female sone) that play a vital role in the molting process (29). However, as is the case in other species of spiders. If Tidarren males do we are not aware of any hormones that would restructure the indeed move long distances, then the significantly lower mor- physiological system to provide the dramatic changes in perfor- tality of one-pedipalp males compared to two-pedipalp males mance that we observed after pedipalp removal. Moreover, the after endurance trials may provide an immediate fitness advan- oxygen transport system of spiders is not known to undergo tage for palp-removal. However, to test this idea rigorously, one dramatic changes between instars (29). Finally, it is unlikely that would need detailed field data on both movement distances and any differences in general natural history and behavior between speeds of Tidarren males during mate searching. individuals in the fourth and fifth instars could explain the Another context in which locomotion might be important for observed performance increase after pedipalp removal, because Tidarren is during scramble competition for a female (T.E.C., our subjects were in a period of relative quiescence, between the C. Linn, J. DuFatta, M.R., J. Feldman, and T. Bukowski,

4886 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0400324101 Ramos et al. Downloaded by guest on September 29, 2021 unpublished data). For example, once a male has located a inate only one of the female’s two spermathecae. Consequently, female, maximum speed may be especially important for out- removal of a pedipalp might result in conservation of gametes competing rival males (47–50) on the female’s web. Up to 25 because only one pedipalp is used in copulation. If both pedi- male T. sisyphoides have been observed at one time on a single, palps were filled and only one used for copulation, the sperm in unrestrained penultimate instar female’s web (T.E.C., C. Linn, the unused palp would be wasted. Males could simply leave one J. DuFatta, M.R., J. Feldman, and T. Bukowski, unpublished palp unfilled at sperm induction. The fact that they do not data). Furthermore, in staged encounters with a newly molted suggests that there are benefits to palp removal, as noted earlier. female, the first male to reach the female’s abdomen usually Finally, it is not clear why Tidarren males have not evolved a copulates. Endurance may also be important for the prolonged single-pedipalp condition, which would obviate the need for efforts expended attempting to dislodge a rival male in copula removing a seemingly functional organ. Pedipalps might play an (T.E.C., C. Linn, J. DuFatta, M.R., J. Feldman, and T. Bukowski, important role in other functions during the early instars, such unpublished data). as in prey handling. In addition, the genetic traits underlying the It is possible that ancestral male Tidarren spiders that first development of two pedipalps may be more difficult to overcome exhibited pedipalp removal were at a selective advantage relative than simply evolving a behavior of pedipalp removal. Thus, data to males that did not because of the enhanced locomotor that shed light on the developmental genetics of pedipalps in performance that a one-pedipalp condition provides. However, spiders would be especially interesting within this evolutionary one must also consider the possibility that the behavior of context. pedipalp removal has evolved for reasons more related to reproduction. For instance, as Tidarren and Echinotheridion We thank T. Sherry, Anthony Herrel, Bieke VanHooydonck, and three males die in copula (Tidarren cuneolatum, Tidarren argo, T. anonymous reviewers for helpful comments on previous versions of this sisyphoides, Echinotheridion gibberosum: refs. 25, 22, 20, and 27, manuscript. This work was supported by National Science Foundation respectively), they can insert one pedipalp only once and insem- Grant IBN 9983003 (to D.J.I.).

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