Journal of Zoology. Print ISSN 0952-8369

Sexual dimorphism and physiological correlates of horn length in a South African isopod crustacean D. S. Glazier1,2, S. Clusella-Trullas3 & J. S. Terblanche2

1 Department of Biology, Juniata College, Huntingdon, PA, USA 2 Department of Conservation Ecology and Entomology, Centre for Invasion Biology, Stellenbosch University, Stellenbosch, South Africa 3 Department of Botany and Zoology, Centre for Invasion Biology, Stellenbosch University, Stellenbosch, South Africa

Keywords Abstract allometry; body condition; Deto echinata; exaggerated traits; rates of metabolism and Although isopod crustaceans frequently show sexual dimorphism in body size, sex- water loss; sexual selection. ual differences in other non-reproductive (secondary sexual) traits have been rarely studied. Furthermore, little is known about the physiological correlates of variation Correspondence in the expression of sexually dimorphic traits in animals generally. Here, we show Douglas S. Glazier, Department of Biology, that the unusual dorsal horns of the South African horned isopod Deto echinata Brumbaugh Academic Center, 1700 Moore St, exhibit significantly different allometric scaling relationships with body size in Juniata College, Huntingdon, PA 16652, USA. males versus females. Not only are the horns of adult males substantially longer Tel: 814-641-3584 than those of adult females but also their log–log scaling slope with body length is Email: [email protected] significantly steeper than that of females and juveniles. Furthermore, in males rela- tive horn length is positively correlated with body condition, as estimated by body Editor: Robert Knell mass relative to body length, and with resting metabolic (CO2 release) rate, also adjusted to body length. However, although showing a weak positive trend, the rel- Received 6 June 2015; revised 22 December ative horn length of males is not significantly correlated with body length adjusted 2015; accepted 11 February 2016 active metabolic rate. It is also not significantly correlated with the relative rates of whole body or cuticular water loss rates or with two measures of activity level. doi:10.1111/jzo.12338 We hypothesize that the sexual dimorphism of horn length in D. echinata is the result of sexual selection and that horn length is a reliable indicator of the ability of a male to acquire and process resources, which in turn may be critical to female mate choice and (or) male–male competition for mates. However, this hypothesis requires further testing based on direct observations of female mate choice and male–male behavioral interactions, which have not yet been carried out.

Introduction related to the magnitude of a sexually dimorphic structure in any kind of animal (cf. Hill, 2011, 2014). Several studies have Isopod crustaceans frequently show sexual dimorphism in body examined how a sexually dimorphic behavior (e.g. mate calling size that has been considered to be the result of sexual selec- or displaying) is related to metabolic rate or aerobic capacity tion (Jormalainen, 1998; Bertin et al., 2002). However, sexual (reviewed by Kotiaho, 2001; Lailvaux & Irschick, 2006), but differences in other secondary sexual traits that are not directly only two studies have done this for sexually dimorphic struc- related to reproduction are little known and understood in iso- tures (Basolo & Alcaraz, 2003; Allen & Levinton, 2007). This pods (see Lefebvre, Limousin & Caubet, 2000; Emlen, 2008). knowledge is important for evaluating the degree to which the Furthermore, although the degree of expression of sexually expression of secondary sexual traits depends on the ability of selected traits has been related to nutrition and body condition an organism to acquire, utilize and conserve resources. This in many kinds of animals (Andersson, 1994; Johnstone, 1995; dependence is expected to be substantial, especially when sec- Bonduriansky, 2007b; Emlen et al., 2012; Warren et al., ondary sexual traits have exaggerated proportions that require 2013), this has never been studied in isopods. In such studies, high resource investment (Andersson, 1994; Bonduriansky, body condition is often measured as body mass adjusted to 2007b; Emlen et al., 2012; Shingleton & Frankino, 2013; body length, or as relative fat content, level of oxidative stress, Johns et al., 2014; Morehouse, 2014; Lavine et al., 2015). immuno-competence or parasitic infection (Andersson, 1994; This knowledge is also critical for testing leading hypotheses von Schantz et al., 1999; Cotton, Fowler & Pomiankowski, (e.g. the ‘handicap’ and ‘good genes’ models) that propose that 2004). the expression of sexually selected traits is a reliable indicator However, little is known about how various vital cellular of ‘health’ or ‘strength’ and ultimately evolutionary fitness, and physiological processes, such as metabolic rate, may be which is in turn used in female mate choice (Zahavi, 1975;

Journal of Zoology 300 (2016) 99–110 ª 2016 The Zoological Society of London 99 Sexually dimorphic isopod horns D. S. Glazier, S. Clusella-Trullas and J. S. Terblanche

Kodric-Brown & Brown, 1984; Johnstone, 1995; Johnstone, (Emlen, 2008, 2014; Kim, Jang & Choe, 2011; McCullough & Rands & Evans, 2009; Lavine et al., 2015) or male–male com- Emlen, 2013; To€ıgo, Gaillard & Loison, 2013), but none so petition for mates (Andersson, 1994; Emlen, 2008, 2014). In far in isopods, which rarely have horns (see Emlen, 2008). addition, studies of the physiological correlates of variation in Therefore, the aims of this study were (1) to document the secondary sexual traits could improve basic understanding of sexual dimorphism of both the size and body size scaling of how traits affected by sexual and natural selection are related, the highly distinctive dorsal horns of the horned isopod; (2) to and thus how different fitness-related components of the phe- test whether relative horn size is related to body condition and notype are integrated, a question of fundamental importance to two important resource-related physiological processes vital to evolutionary ecology (also see Irschick et al., 2007, 2008; Hill, survival and thus evolutionary fitness, especially in stressful 2011, 2014; Armbruster et al., 2014; Morehouse, 2014). supralittoral habitats: that is the rates of metabolism and evapo- The horned isopod Deto echinata of South Africa (Fig. 1) rative water loss. represents a potentially useful model system for studying the physiological ecology of sexual dimorphism because the rela- Materials and methods tive size of its sexually dimorphic dorsal spine-like horns (‘coniform protuberances’, as described by Coleman & Leis- Source and maintenance of animals tikow, 2001) is easily quantified as horn length. Furthermore, this species lives in harsh, species-poor, supralittoral (upper Individuals of the oniscidean isopod Deto echinata (family shore splash zone) habitats where possible effects of body con- Detonidae) were collected on 10 and 23 October 2012 along dition and various resource-related physiological processes on the rocky shoreline of Cape Hangklip between Pringle Bay the expression of environmentally sensitive secondary sexual and Betty’s Bay of the Western Cape Province of South traits should be readily discernable. In supralittoral habitats ani- Africa. They were especially abundant in moist rock crevices mals must endure repeated bouts of desiccation and interrupted where they co-occur with another common supralittoral onis- feeding due to exposure to widely varying physical conditions cidean isopod Ligia dilatata (also see Coleman & Leistikow, (including broad ranges of temperature, moisture and salinity: 2001). In the laboratory, the isopods were maintained in plastic Raffaelli & Hawkins, 1999; Hurtado, Lee & Mateos, 2013). containers with moist algal wrack (collected at Cape Hangklip) The pronounced sexual dimorphism of the dorsal horns of as food in a MRC LE-509 environmental control chamber D. echinata – longer in males than females – has already been (Holon, Israel) set at 20 Æ 0.1°C and a 12D:12N light cycle. reported (Chilton, 1915; Barnard, 1932; Branch et al., 1994), Morphometric measurements were made on 34 adult male, 16 but has not yet been quantified or explained. Nor has horn adult female and 10 juvenile isopods within 2 weeks of their length been related quantitatively to body size or condition or capture; and respirometry trials were carried out using a sepa- any kind of resource-related physiological process. Many rate group of 30 adult males after 1–3 months acclimation in behavioral and evolutionary studies of sexually dimorphic an environmental control chamber. Although some animals ‘horns’ have been carried out in numerous kinds of animals died or became lethargic during the acclimation period, most

(a)

(b) (c)

Figure 1 Photographs of adult males of the horned isopod Deto echinata. (a) Close-up view of seven pairs of incurved dorsal horns with rounded tips. Black line with arrows at both ends shows distance measured for the length of the longest (5th) horn on the right-hand side. (b) Male with relatively short horns. (c) Male with relatively long horns.

100 Journal of Zoology 300 (2016) 99–110 ª 2016 The Zoological Society of London D. S. Glazier, S. Clusella-Trullas and J. S. Terblanche Sexually dimorphic isopod horns of them appeared to remain healthy, as indicated by their rapid First, atmospheric air was pumped through two scrubber col- movement when disturbed. No lethargic animals were used in umns, one containing soda lime (Merck, Gauteng, RSA) and the respirometry trials, and to be sure that there was no poten- the other silica gel/Drierite (1:1 ratio) (WA Hammond Drierite tial time bias males with different horn lengths were selected Company, Xenia, OH, USA), to remove CO2 and H2O vapor, randomly during the acclimation period. respectively. Second, the air passed through a Side-Trak Model 840 flow control valve (Sierra Instruments, Monterey, fl Morphometric and body condition CA, USA) connected to a MFC-2 mass ow control unit measurements (Sable Systems International, Las Vegas, NV, USA), which controlled the air flow at a constant rate of 100 ml minÀ1. Æ Horn length and body length were measured ( 0.1 mm) using Third, the air flowed through the zero channel of the CO2/ fi a clear photographic lm displaying a metric ruler placed H2O analyzer, then through a 5-mL plastic syringe containing under each specimen and viewed with an Olympus SZ2-ILTS an isopod, finally looping back to another channel of the 9 fi stereoscopic microscope (Tokyo, Japan) at 10 magni cation. CO2/H2O analyzer. Data output from the CO2/H2O analyzer – Each animal had several (usually 6 7) lateral pairs of dorsal (differences between the concentrations of CO2 and H2Oin horns of varying length (Fig. 1; also see Barnard, 1932). The the air before and after passing through a syringe containing mean total number of horns per individual (13.4 Æ 0.3 95% the animal) was recorded on a computer using LiCor Li-7000 confidence limits; n = 38) did not vary significantly with body software. length (r = 0.101; P = 0.544), and was not significantly differ- During each trial [mean duration (h) = 16.6 Æ 0.7 SEM, start- ent between males (13.4; n = 29; identified by their genitalia) ing in the morning or early afternoon], the plastic syringe con- and females (13.2; N = 9; t = 0.641; P = 0.525; identified by taining a respiring isopod was partially enclosed in a large their oostegites€ or brood plates). In addition, the longest horns plastic bag kept inside a high precision thermostatic bath of males were somewhat curved (Fig. 1). Therefore, to stan- (Huber CC-410 Ethanol Bath Compatible Control unit, Offen- dardize comparisons among individuals, each dorsoventrally burg, Germany) set at 20 Æ 0.02°C. The syringe contained a flattened specimen was pinned with one lateral side facing up, loose rod-shaped piece of plastic to which an isopod could thus allowing the length of the longest horn (either the 3rd,4th cling, thus minimizing its activity. However, short periods of or 5th horn from the anterior end on the right-hand side) to be activity (involving postural adjustments and routine exploration estimated as the straight line distance between the tip of a horn of the chamber) occurred in virtually all individuals, thus and its base where it was attached to a dorsal pereon (thoracic) allowing active metabolic rates to be estimated. Activity of segment. Body length was measured from the anterior end of individual isopods was identified using an infrared electronic the cephalon to the posterior tip of the telson. Body mass of activity detector (AD-1 Sable Systems), which provides a sim- live animals and those dried in an oven (50°C) for 60 h was ple qualitative (binary) estimate of activity versus inactivity by measured (Æ0.1 mg) using a Mettler Toledo New Classic MF optical detection of movement, recorded as a voltage signal (MS104S) electronic microbalance (Greifensee, Switzerland). (0–5 V range). Baseline levels of CO2 and H2O were deter- Effects of body condition (body mass adjusted to body length) mined for an empty syringe, both before and after each trial to on horn length were estimated by employing general linear determine analyzer drift, which was usually non-existent. Iso- models (GLM) to examine how horn length (response variable) pod body wet mass (Æ0.1 mg) was measured before and after varied in relation to both body length and body mass (explana- each trial. tory variables). Although there is some controversy about using Respirometry and activity data were analyzed using Expe- _ _ body mass relative to body length as an estimate of body con- Data (version 1.1.25, Sable Systems). Data on VCO2 and VH2O dition (see e.g. Garcıa-Berthou, 2001; Green, 2001; Peig & were corrected for potential baseline drift at standard tempera- Green, 2010; Labocha, Schutz & Hayes, 2014), previous work ture and pressure and converted to ml hÀ1 and mg hÀ1, respec- _ _ on other crustaceans has shown that it is strongly correlated tively. Resting VCO2 and VH2O were estimated as the average with body fat content (Glazier, 2000) and food availability or values calculated from the first relatively stable (flat) sequence quality (Basset & Glazier, 1995; Glazier, 1998; Vignes et al., of readings (1 secÀ1) extending over a time period of at least 2012; Larranaga,~ Basaguren & Pozo, 2014). 6 min (>350 readings) during each trial run (mean number of _ _ readings per trial for VCO2 and VH2O were 1245 and 2362, respectively; n = 30). For the 20 trials where the activity Respirometry detector was used (based on availability in our laboratory), the _ _ _ _ Rates of metabolism and water loss (VCO2 and VH2O, respec- estimates of resting VCO2 and VH2O coincided with periods of _ tively) were estimated using a calibrated infrared LiCor little or no activity. Active VCO2 was calculated as the average nd Li-7000 CO2/H2O analyzer (Lincoln, NE, USA) in an open value of readings occurring during the 2 h of a trial run. The flow-through respirometry system, plumbed in differential 2nd h was chosen because during this period an isopod showed mode [i.e. setup to read a reference (scrubbed) gas sample in several bouts of activity in a relatively undisturbed state (pre- the first channel of the analyzer and to subtract this back- sumably resulting from 1 h of prior acclimation to being ground reading from the animal’sCO2 production recorded in secluded in a syringe with minimal external stimuli). Thus, the second channel]. A Hailea Model ACO-9610 air pump during this time period the activity was assumed to be rela- (RaoPing County, GuangDong Province, China) propelled air tively natural (endogenously produced) and not artificially throughout the system via hydrophobic Bev-A-line tubing. stimulated, as was more likely to have occurred during the

Journal of Zoology 300 (2016) 99–110 ª 2016 The Zoological Society of London 101 Sexually dimorphic isopod horns D. S. Glazier, S. Clusella-Trullas and J. S. Terblanche

1st h after an animal had been handled and then placed in a horn length adjusted to body length did not differ significantly respirometry chamber. The rate of cuticular water loss (total between females and juveniles (ANCOVA: F1,23 = 0.561; water loss minus that due to respiration) was calculated as the P = 0.462). In addition, the scaling slopes of log10 horn length _ _ fi intercept of a linear regression of VH2O versus VCO2 estimated in relation to log10 body length did not differ signi cantly from readings taken over a 6 h interval (from the beginning of between females (1.28 Æ 1.29 95% CI) and juveniles the 2nd h to the end of the 7th h) of each trial run (following (1.36 Æ 1.93) or with the collective scaling slope the method described in Gibbs & Johnson, 2004; Clusella- (1.76 Æ 0.58), as seen by the overlapping 95% confidence _ Trullas & Chown, 2008). Calculations of cuticular VH2O by the intervals (Table 1). intercept method have been shown to be precise and repeatable Both males and females/juveniles showed significantly posi- in insects and comparable to those of other methods (Gray & tive relationships between body dry mass and body length, but Chown, 2008; Groenewald et al., 2013). For each respirometry the scaling slope was significantly higher for females/juveniles trial where the activity detector was used (n = 20), two mea- (2.89) than for males (1.79) (Table 1; Fig. 3). sures of activity were estimated: duration of active period (i.e. Relative horn length was significantly positively correlated the total time during which an isopod showed activity) and with body condition (i.e. body dry mass independent of body percent time active during the 2nd h of a trial run (one trial length) for both males and females/juveniles each analyzed was omitted because it showed 0% activity, which could not separately, as revealed by GLM analyses of log10 horn length be graphed on a log scale in Fig. 4). in relation to log10 body dry mass and log10 body length (Table 2). Statistical analyses Physiological correlates of relative horn Least squares linear regression analysis was used to calculate length in male isopods scaling relationships between various traits and body length. This method was preferred over alternative methods, such as These analyses focus only on the 30 adult male isopods used reduced major axis analysis, because the natural and measure- in the respirometry trials. Male horn length showed strong pos- ment variation in several traits (Y variables) appeared to be itive allometry with body length (Table 1; Fig. 4a), which more than that of body length (X variable) (Smith, 2009), and agrees with the data for a separate collection of male isopods we wished to predict the value of a trait based upon its rela- (Table 1; Fig. 2) reported in the previous section. In addition, tionship with body length (following Sokal & Rohlf, 1995). relative horn length was significantly positively correlated with Logarithmic transformation was used to permit easy detection two measures of body condition: log10 body wet mass relative of proportional relationships between Y and X values (see Gla- to log10 body length, and log10 body dry mass relative to log10 zier, 2013). Residual values of various traits were calculated body length (GLM results in Table 2), as depicted in (Fig. 5a, using log-linear regressions with body length. Since there is b). some controversy about the statistical use of residual analyses Other morphometric, physiological and behavioral traits (e.g. Garcıa-Berthou, 2001; Freckleton, 2002), the results of showed highly significant correlations with body length these analyses were used only to illustrate data patterns graphi- (Table 1), including body wet mass (Fig. 4a), body dry mass cally (Fig. 5). For statistical testing, GLM were employed to (Table 1), resting and active metabolic (CO2 release) rates examine how horn length (response variable) varied in relation (Fig. 4b), whole body and cuticular H2O loss rates (Fig. 4c), to body length (first explanatory variable) and various sec- and duration of the active period during a respirometry trial ondary explanatory (physiological and behavioral) variables. (Fig. 4d). In addition, we observed highly significant, nearly _ Statistical results were considered significant if P ≤ 0.05. All isometric correlations between log10 resting VCO2 and log10 statistical analyses were carried out using SYSTAT 10 (Systat body wet mass (Y = 0.942 Æ 0.188 95% CI (X) – 2 Software, San Jose, CA, USA). 3.937 Æ 0.421; r = 0.791; P < 0.00001; n = 30) and log10 body dry mass (Y = 0.898 Æ 0.170 (X) – 3.272 Æ 0.277; 2 Results r = 0.804; P < 0.00001; n = 30). However, percent time active during the 2nd h of a respirometry trial was not signifi- Sexual dimorphism of horn length and body cantly correlated with body length (Table 1). Relative horn length was significantly positively correlated condition _ with log10 resting VCO2 adjusted to log10 body length (Table 2; Horn length showed significant positive allometry (scaling Fig. 5c), but only weakly, not significantly correlated with fi > _ exponent signi cantly 1) in both males and females/juveniles log10 active VCO2 adjusted to log10 body length (Table 2; (Table 1; Fig. 2). However, the scaling exponent was about Fig. 5d). Furthermore, relative horn length was not signifi- twice as high for males (3.34) than for females/juveniles cantly correlated with the percent time active (Table 2; (1.76). Therefore, not only do males exhibit longer horns than Fig. 5h), the relative duration of the active period (Table 2; females/juveniles, but also this difference becomes dispropor- Fig. 5g) or the relative whole body and cuticular water loss tionately greater with increasing body size (Fig. 2). Females rates (Table 2; Fig. 5e,f). These non-significant patterns _ and juveniles of uncertain sex were grouped together to maxi- were observed, even though log10 resting whole VCO2 was fi _ = mize the body size range examined, and thus the strength of signi cantly correlated with resting VCO2 (r 0.570, fi = = _ the horn length scaling relationship. This was justi ed because P 0.001, n 30), and the active VCO2 metabolic rate was

102 Journal of Zoology 300 (2016) 99–110 ª 2016 The Zoological Society of London D. S. Glazier, S. Clusella-Trullas and J. S. Terblanche Sexually dimorphic isopod horns

Table 1 Least squares regression (LSR) equations for log10-transformed values of various morphometric, physiological and behavioral traits (Y) in relation to log10 body length (X) of two study groups of horned isopods (Deto echinata) Group Dependent variable (Y) LSR equation (Æ95% C.I.) r2 Pn #1 M Horn length Y = 3.34 Æ 0.40(X) – 3.75 Æ 0.46 0.90 < 0.00001 34 #1 F/J Horn length Y = 1.76 Æ 0.58(X) – 2.74 Æ 0.58 0.62 < 0.00001 26 #1 M Body dry mass Y = 1.78 Æ 0.28(X) – 0.47 Æ 0.33 0.84 < 0.00001 34 #1 F/J Body dry mass Y = 2.89 Æ 0.40(X) – 1.63 Æ 0.41 0.90 < 0.00001 26 #2 M Horn length Y = 3.81 Æ 0.51(X) – 4.41 Æ 0.62 0.90 < 0.00001 30 #2 M Body wet mass Y = 2.37 Æ 0.31(X) – 0.67 Æ 0.38 0.90 < 0.00001 30 #2 M Body dry mass Y = 2.39 Æ 0.46(X) – 1.31 Æ 0.56 0.81 < 0.00001 30 #2 M RMR Y = 2.18 Æ 0.58(X) – 4.50 Æ 0.71 0.68 < 0.00001 30 #2 M AMR Y = 1.27 Æ 0.85(X) – 3.15 Æ 1.05 0.25 0.0050 30

#2 M RH2OY= 1.75 Æ 0.71(X) – 1.47 Æ 0.87 0.48 0.00002 30

#2 M CH2OY= 2.26 Æ 0.74(X) – 2.19 Æ 0.91 0.59 < 0.00001 30 #2 M DAP Y = 1.44 Æ 0.70(X) – 0.72 Æ 0.84 0.51 0.00039 20 #2 M PTA NS 0.14 0.117 19

#1 group, adult males (M), adult females (F) and juveniles (J) used for allometric scaling analyses of horn length and body mass in relation to body length. #2, a separate set of adult males (M) used in the respirometry analyses. Horn and body length measured in mm, and body mass in -1 À1 À1 mg. RMR, resting metabolic rate (ml CO2 h ); AMR, active metabolic rate (ml CO2 h ); RH2O, resting water loss rate (mg H2Oh ); À1 2 CH2O, cuticular water loss rate (mg H2Oh ); DAP, duration of active period (h); PTA, percent time active; r , coefficient of determination; P, significance level, n, sample size; NS, non-significant LSR not shown. 95% confidence intervals (C.I.) are given for each LSR parameter.

1.0 2.0 Slope = 3.337 ± 0.396 Slope = 1.788 ± 0.279 Slope = 1.761 ± 0.582 Slope = 2.888 ± 0.404 0.5 1.6 Males Males 0.0 1.2 –0.5 body mass (mg) horn length (mm) 10

10 0.8 –1.0 Juveniles & Females Log

Log Juveniles & Females –1.5 0.4 0.8 0.9 1.0 1.1 1.2 1.3 1.4 0.8 1.0 1.2 1.4

Log10 body length (mm) Log10 body length (mm)

Figure 2 Allometric scaling of log10 dorsal horn length in relation to Figure 3 Allometric scaling of log10 body dry mass in relation to log10 log10 body length of adult males (●), juveniles (○) and adult females body length of adult males (●), juveniles (○) and adult females (□)of (□) of the horned isopod Deto echinata. The diagonal lines represent the horned isopod Deto echinata. The diagonal lines represent the the least squares regression equations for group #1 given in Table 1. least squares regression equations for group #1 given in Table 1. The The slopes (Æ95% confidence intervals) differ significantly from one slopes (Æ95% confidence intervals) differ significantly from one another and from 1. another and from 1. significantly correlated with percent activity (r = 0.789, (Table 2; Fig. 5a–c). However, relative horn length is only P = 0.00004, n = 19). weakly (not significantly) correlated with body length adjusted active metabolic rate (Fig. 5d), and is not significantly corre- Discussion lated with the adjusted rates of whole body or cuticular water loss rates (Fig. 5e,f), or with two measures of activity level Two major findings of this study are (1) the lengths of the (Fig. 5g,h) (also see Table 2). dorsal horns of the horned isopod Deto echinata are not only The strong positive allometry of male horn size in D. echi- much larger in males than females (and juveniles), but their nata is consistent with similar growth patterns seen for many allometric relationship with body size is also significantly dif- kinds of sexually dimorphic, exaggerated traits in a variety of ferent: during body growth the horns increase in length dispro- animals (Gould, 1974; Emlen & Nijhout, 2000; Emlen, 2008; portionately more in males than in females (Table 1; Fig. 2); Shingleton & Frankino, 2013; Lavine et al., 2015). However, (2) male horn length relative to body length is significantly the positive allometry observed for male D. echinata is among correlated with body condition (i.e. body length adjusted body the steepest ever reported. The slopes of 3.3 to 3.8 (Table 1, mass) and resting metabolic rate adjusted to body length Figs 2 and 4a) are considerably higher than the slopes of

Journal of Zoology 300 (2016) 99–110 ª 2016 The Zoological Society of London 103 Sexually dimorphic isopod horns D. S. Glazier, S. Clusella-Trullas and J. S. Terblanche

Table 2 General linear model (GLM) analyses of log10 horn length (mm) in relation to log10 body length (mm) and an additional log10 morphometric, physiological or behavioral trait of two study groups of horned isopods (Deto echinata). Statistical significance (P) of the independent effect of each explanatory variable is shown (t-tests). Significant t values (P ≤ 0.05) are in bold

Group Response variable 1st explanatory variable t (P)2nd explanatory variable t (P) rn #1 M Horn length Body length 5.09 (0.00002) Body dry mass 2.50 (0.018) 0.96 34 #1 F/J Horn length Body length À0.49 (0.63) Body dry mass 2.88 (0.0084) 0.85 26 #2 M Horn length Body length 1.78 (0.087) Body dry mass 5.45 (0.00001) 0.98 30 #2 M Horn length Body length 4.92 (0.00004) Body dry mass 3.47 (0.0018) 0.96 30 #2 M Horn length Body length 7.54 (<0.00001) RMR 2.07 (0.048) 0.95 30 #2 M Horn length Body length 12.96 (<0.00001) AMR 1.70 (0.10) 0.95 30

#2 M Horn length Body length 10.43 (<0.00001) RH2O 1.16 (0.26) 0.95 30

#2 M Horn length Body length 9.83 (<0.00001) CH2O -0.88 (0.93) 0.95 30 #2 M Horn length Body length 7.42 (<0.00001) DAP 0.37 (0.72) 0.94 20 #2 M Horn length Body length 7.89 (<0.00001) PTA 0.25 (0.98) 0.91 19

#1 group, adult males (M), adult females (F) and juveniles (J) used for allometric scaling analyses of horn length and body mass in relation to body length. #2, a separate set of adult males (M) used in the respirometry analyses. Body mass measured in mg. RMR, resting metabolic À1 À1 À1 rate (mL CO2 h ); AMR, active metabolic rate (mL CO2 h ); RH2O, resting water loss rate (mg H2Oh ); CH2O, cuticular water loss À1 rate (mg H2Oh ); DAP, duration of active period (h); PTA, percent time active; r, correlation coefficient for overall GLM relationship, n, sample size.

(a) (b) 2.6 1.2 –1.0 Body wet mass –1.2 Active metabolic rate 1.0 2.4 Horn length Resting metabolic rate –1.2 0.8 –1.6 2.2 0.6 –1.4

2.0 0.4 –2.0 –1.6 0.2 1.8 –1.8 0.0 horn length (mm) –2.4 active metabolic rate Body wet mass (mg) resting metabolic rate 1.6 –0.2 10 –2.0 10 10 10

–0.4 Log –2.8 –2.2 Log Log

1.4 Log –0.6 1.0 1.1 1.2 1.3 1.0 1.1 1.2 1.3 (c) (d) 2.0 2.0 Resting water loss rate 1.4 1.0 Cuticular water loss rate 1.2 1.6 1.5 0.8

1.0 O loss rate O loss rate 2 2 0.6 0.8 1.2 1.0

0.4 0.6 0.8 percent time active 10 resting H 0.5 duration of active period cuticular H Duration of active period

10 0.4 10 0.2 10 Percent time active Log Log

0.2 Log Log 0.4 0.0 0.0 1.0 1.1 1.2 1.3 1.4 1.0 1.1 1.2 1.3 Log10 body length (mm) Log10 body length (mm)

Figure 4 Scaling relationships of various traits (Y) in relation to log10 body length (X) of adult males of the horned isopod Deto echinata used in the respirometry trials. The diagonal lines represent the least squares regression equations for group #2 given in Table 1. (a) Y = log10 body wet À1 mass (○) or log10 horn length (●). (b) Y = log10 resting (●) or active (○) metabolic rate (ml CO2 h ). Active metabolic rate was the mean rate of nd CO2 release recorded during the 2 h of a respirometry trial when an isopod showed considerable activity. (c) Y = log10 resting rates of whole À1 body (○) and cuticular (●) water loss (mg H2Oh ). (d) Y = log10 duration of active period (●) = log10 total period (h) of a respirometry trial nd during which an isopod showed activity, or log10 percent time active during the 2 h of a respirometry trial (○).

104 Journal of Zoology 300 (2016) 99–110 ª 2016 The Zoological Society of London D. S. Glazier, S. Clusella-Trullas and J. S. Terblanche Sexually dimorphic isopod horns

(a) (b) t = 5.45 t = 3.47 0.3 0.3 P = 0.00001 P = 0.0018 n = 30 n = 30 0.2 0.2

0.1 0.1

0.0 0.0

Residual horn length –0.1 –0.1

–0.1 0.0 0.1 –0.1 0.0 0.1 0.2 Residual body wet mass Residual body dry mass (c) (d) t = 2.07 t 0.3 0.3 = 1.70 P = 0.048 P = 0.101 n = 30 n = 30 0.2 0.2

0.1 0.1

0.0 0.0

–0.1 Residual horn length –0.1

–0.3 –0.2 –0.1 0.0 0.1 0.2 –0.4 –0.2 0.0 0.2 Residual resting metabolic rate Residual active metabolic rate (e) (f) t t 0.3 = 1.16 0.3 = –0.88 P = 0.256 P = 0.946 n = 30 n = 30 0.2 0.2

0.1 0.1

0.0 0.0

Residual horn length –0.1 –0.1

–0.3 –0.2 –0.1 0.0 0.1 0.2 –0.3 –0.2 –0.1 0.0 0.1 0.2

Residual resting H2O loss rate Residual cuticular H2O loss rate (g) (h) t t 0.3 = 0.37 0.3 = 0.25 P = 0.715 P = 0.980 n = 20 n = 19 0.2 0.2

0.1 0.1

0.0 0.0

Residual horn length –0.1 –0.1

–0.2 –0.1 0.0 0.1 0.0 0.4 0.8 1.2 1.6 2.0

Residual duration of active period Log10 percent time active

Figure 5 Relationships between the residuals of horn length in adult males of the horned isopod Deto echinata and the residuals of (a) body À1 À1 wet mass, (b) body dry mass, (c) resting metabolic rate (mL CO2 h ), (d) active metabolic rate (mL CO2 h ), (e) resting rate of water loss (mg À1 À1 H2Oh ), (f) rate of cuticular water loss (mg H2Oh ) and (g) duration of active period (h), and the absolute values of the log10 percent time active. The residuals were all based on the linear regressions with body length given for group #2 in Table 1, as depicted in Fig. 4. The samples size (n) and significance of these relationships (t and P values), based on GLM analyses (see Table 2), are shown.

1.5–2.5 typically observed for sexually selected ornaments and high because of a biased focus on species showing exaggerated weapons in relation to body length, according to the datasets sexual traits. Many sexual traits in birds and flies show isomet- analyzed by Kodric-Brown, Sibly & Brown (2006). Moreover, ric (scaling slope ~ 1) or even negatively allometric relation- according to Bonduriansky (2007a), the range claimed to be ships (scaling slopes < 1) with body size (see Bonduriansky, typical by Kodric-Brown et al. (2006) may itself be unusually 2007a). An extensive, recently published meta-analysis, showing

Journal of Zoology 300 (2016) 99–110 ª 2016 The Zoological Society of London 105 Sexually dimorphic isopod horns D. S. Glazier, S. Clusella-Trullas and J. S. Terblanche that the scaling exponent for morphological secondary sexual However, why is active metabolic rate adjusted to body length traits in diverse animals averages 1.30 Æ 0.10 (95% C.I.; not quite significantly correlated with relative horn length n = 90) and ranges from 0.3 to 3.0 (Voje, 2016), further sup- (Table 2; Fig. 5d)? Perhaps, this is because the active metabo- ports our claim that the positive allometry of horn length in lism that we measured in our captive experimental animals that D. echinata is extraordinarily steep. had no access to food was influenced by various behavioral The apparently strong condition-dependence of relative horn activities unrelated to resource uptake and processing. Stronger length in D. echinata is also similar to that seen for a variety correlations may have been observed if we had measured the of other sexually dimorphic traits, including weapons used in active metabolism of freely foraging males or their sustained male–male competition and ornamental structures, behaviors maximal metabolic rate during peak performance, hypotheses and color patterns attractive to the opposite sex (Andersson, requiring testing. 1994; Johnstone, 1995; Bonduriansky, 2007b; Johnstone et al., In any case, the above observations are consistent with sex- 2009; Emlen et al., 2012; Warren et al., 2013; Johns et al., ual selection theory that posits that females prefer to mate with 2014; Morehouse, 2014; Lavine et al., 2015). However, the males with exaggerated sexually dimorphic traits because their correlation between relative horn size and our measure of body expression is positively correlated with fitness, i.e. ‘good condition – body mass relative to length – may be criticized as genes’ that can be passed onto a female’s offspring (Zahavi, being the result of an autocorrelation between these two vari- 1975; Kodric-Brown & Brown, 1984; Andersson, 1994; John- ables. If larger horns add significant mass to an animal, then a stone, 1995; Johnstone et al., 2009). It is also possible that higher body mass per unit length may simply be the result of exaggerated sexually dimorphic traits may signal superior fight- larger horns, rather than being an independent indicator of ing ability, and thus be favored by sexual selection resulting body condition. However, three observations suggest that this from male–male competition (Andersson, 1994; Emlen, 2008, is an unlikely explanation of the patterns reported here. First, 2014; also see further discussion below). Accordingly, we females with much smaller, less variable horns (length ranging hypothesize that the relatively large horns observed in male only from 0.1 to 0.2 mm; see Fig. 2) show a stronger correla- D. echinata are the result of sexual selection because females tion (higher r2) and steeper slope between relative horn length prefer to mate with, and (or) rival males are less successful and body mass per length than do males with much larger, competing with, large-horned males that can acquire and pro- more variable horns (length ranging from 0.2 to 3.7 mm: see cess resources better than small-horned males. Fig. 2) (Table 1; also see below). Second, although the slope This hypothesis raises four major questions. First, are the of horn length in relation to body length is steeper for males large dorsal horns of male D. echinata the result of sexual than for females and juveniles (Table 1; Fig. 2), the opposite selection, natural selection or both? A plausible hypothesis is seen for body mass in relation to length (Table 1; Fig. 3a). based on natural selection, rather than on sexual selection is Third, body wet mass (which includes non-structural body that the horns of D. echinata have been favored because they water reserves) is more strongly correlated with relative horn aid in predator avoidance. Both male and female D. echinata length than is body dry mass (Table 2; Fig. 5a,b), which is the have horns, which in other species indicates their use in preda- opposite of that expected if the mass of the largely exoskeletal tor defense (Emlen, 2008). However, this hypothesis is weak- horns was autocorrelated with body mass per length. However, ened by four observations. First, female horns are mere stubs, future studies should examine other measures of body condi- and thus would not appear to be effective in deterring preda- tion, as recommended by several researchers working on sexu- tors. Perhaps, the existence of horns in females represents a ally dimorphic traits (e.g. Lailvaux & Irschick, 2006; Irschick non-adaptive genetic correlation (like nipples in male mam- et al., 2008; Hill, 2011). mals; but see below). Second, male horns are blunt and some- Relative horn length in males is not only significantly corre- what curved (not sharply pointed), and thus not effective for lated with body condition but also with resting metabolic rate puncturing the skin of potential predators (as the senior author adjusted to body length (Table 2; Fig. 5c). This finding is the has observed by poking his own skin with the horns of male first showing a correlation between resting metabolic rate and D. echinata). Third, horned isopods can see predators at large the naturally varying size of a sexually dimorphic structure in distances away, and if necessary rapidly escape into protective any kind of organism. Previous studies have reported that male rock crevices (D.S. Glazier, pers. obs.). Fourth, a similar look- fiddler crabs with sexually selected enlarged claws have higher ing isopod Ligia dilatata occurs in great abundance in the metabolic rates than those whose claws were removed (Allen same microhabitats as D. echinata, and thus is presumably & Levinton, 2007); and male swordtail fish also show an asso- subject to similar predation, but it has no dorsal horns. It is ciation between active metabolic rate and artificially manipu- also possible that the dorsal horns may help horned isopods lated sword length (Basolo & Alcaraz, 2003). Our finding that avoid predation, not by acting as weapons, but by mechani- relative horn length is correlated with both body condition and cally inhibiting ingestion by small-gaped predators, by enabling resting metabolic rate is important, because it suggests that an them to better resist attempts of predators to pull them out exaggerated trait – in this case horn length – may be associ- from rock crevices, or by giving them a tactile sense of nearby ated with the ability of an individual to acquire and process enemies. The horns of D. echinata are covered with setae resources (assuming that body condition, as estimated by body (D.S. Glazier, pers. obs.), and crustacean setae generally have mass per length, reflects an ability to acquire and store mechanical or sensory functions (Watling, 1989; Garm, 2004). resources, whereas metabolic rate likely reflects resource However, these alternative possible roles of the dorsal horns in processing; see Glazier, 2000, 2015; Brown et al., 2004). predator avoidance are not consistent with the observations that

106 Journal of Zoology 300 (2016) 99–110 ª 2016 The Zoological Society of London D. S. Glazier, S. Clusella-Trullas and J. S. Terblanche Sexually dimorphic isopod horns females of D. echinata have very small horns and the coexist- A fourth major question is why do the length and shape of ing isopod L. dilatata has no horns at all. dorsal horns vary among different species of Deto: from ‘long Although sexual selection has probably played a greater role spines’ in D. echinata to relatively short ‘tubercles’ in D. aci- in the evolution of large dorsal horns in male D. echinata than nosa (Chilton, 1915)? In addition, some Deto species display has natural selection (e.g. for predator avoidance), a second different sexually dimorphic structures, such as elongated uro- major question is whether the horns serve as ‘ornaments’ for pods in male D. marina (Chilton, 1917) and large lateral glob- sexual attraction of females, or as ‘weapons’ used in male– ular extensions of the first pereon segment in male male competition for mates. Three observations support the D. bucculenta (Chilton, 1915). Similar extensive interspecific view that the dorsal horns of male horned isopods more likely variation in the size and shape of sexually dimorphic traits is evolved as sexually attractive ornaments or as intimidating sig- also seen in dung beetles (superfamily Scarabaeoidea): some nals to rival males, rather than as actively used weapons. First, species have horns, whereas others do not, even within the it is difficult to imagine how the dorsal horns could be used same genus (Emlen, Lavine & Ewen-Campen, 2007; Emlen, directly to fight other males. Horns used for fighting in other 2014). Although the causes of this variation among Deto spe- animals tend to occur on the head where attacks are more cies remain unknown, the apparently ornamental nature of their easily performed (Emlen, 2008, 2014; Kim et al., 2011; sexually dimorphic structures suggests that their relative size McCullough & Emlen, 2013; To€ıgo et al., 2013). Other possi- probably acts more as a signal of fitness to potential mates or bilities that require testing are that the horns are used as of fighting ability to rival males, rather than being directly obstructions to prevent other males from gaining access to involved in male–male competition (in contrast to dung beetles females in rock crevices, or to dislodge rival males already where the horns are weapons directly used in male–male com- coupled with females. Second, the size of the dorsal horns in petition: Emlen et al., 2007; Simmons & Ridsdill-Smith, 2011; males is related to body condition, which is consistent with the Emlen, 2014). view that they serve as ornamental indicators of fitness to prospective female mates, though this hypothesis requires test- Conclusions ing (see Barnett et al., 2015). Whether female horned isopods actually prefer males with longer horns and better ‘genes’ is The dorsal horns of the isopod Deto echinata are strongly sex- still unknown. Furthermore, the question remains why relative ually dimorphic: males not only have much longer horns than female horn length is also correlated with body condition. Is females, but also their horns show much stronger positive this the result of a genetic correlation across the sexes or is allometry with body growth. Positive correlations of relative female horn length also involved in mate choice by males (e.g. horn length with body condition and relative metabolic rate, as ‘mutual sexual selection’: see Tobias, Montgomerie & Lyon, well as other observations, are consistent with our tentative 2012; Stuart-Fox & Goode, 2014), or in conferring other social hypothesis that the sexual dimorphism of horns in D. echinata advantages (e.g. ‘social selection’: see Tobias et al., 2012; is the result of sexual selection. If larger horns signal higher Clutton-Brock & Huchard, 2013; Cain & Rosvall, 2014)? Per- fitness related to a better ability to acquire and process haps, males prefer females with conspicuous horns that indi- resources, large-horned males may be preferred more often by cate good body condition, but that are not too large so as to females and (or) have greater competitive success with rival interfere with mating activity. Third, strong positive allometry males than small-horned males. However, this hypothesis of sexually dimorphic structures, as occurs for the dorsal horns assumes that body condition and metabolic rate are positively of male D. echinata, is thought by some (but not all) investi- correlated with fitness, which has not yet been tested. gators to be more likely for sexually selected ornaments versus Although the horns of D. echinata are unlikely the result of weapons (Gould, 1974; Bonduriansky, 2007a; but see Kodric- selection for predator defense, other explanations for their sex- Brown et al., 2006). Nevertheless, it is also possible that male ual dimorphism are also possible and should be tested. It horn size may indicate relative ability to compete with other would also be useful to examine not only the benefits of large males (because of an association with level of ‘health’, horns in terms of mating success, but also possible energetic ‘strength’ and fitness; also see Andersson, 1994; Emlen, 2008, and survival costs related to resource allocation away from 2014). If so, the horns may be indirect ‘assessment signals’ other vital functions or structures. Large horns in some other (Emlen, 2008), and thus may play a role in both intersexual animal species appear to have low fitness costs (e.g. McCul- attraction and intrasexual competition. However, it remains lough & Emlen, 2013; To€ıgo et al., 2013), but this has not yet unknown whether male D. echinata fight with other males for been examined in horned isopods. access to females. In our study, we tested the idea that sexual trait variation A third major question is why Ligia dilatata, a very simi- may be linked to various vital processes (Hill, 2011, 2014). lar looking isopod that scavenges similar food (mainly algal We examined two vital physiological processes: rates of meta- wrack) in the same rocky supralittoral microhabitats, does not bolism and evaporative water loss. However, only relative rest- exhibit sexually dimorphic horns like D. echinata? Both spe- ing metabolic rate was significantly correlated with relative cies show sexual dimorphism in body size and shape (Bar- horn length among males of D. echinata. In addition, relative nard, 1932), but only D. echinata displays horns – perhaps, active metabolic rate was only weakly (not significantly) corre- other interspecific differences in reproductive behavior or life- lated with relative horn length, which was not surprising given history strategy have favored horns in D. echinata, but not the lack of correlation with two measures of activity level. L. dilatata. These kinds of analyses are valuable because they increase our

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