Is there evidence for a trade-off between sperm competition traits and forelimb musculature in the western grey kangaroo?

Is there evidence for a trade-off between sperm competition traits and forelimb musculature in... Abstract Males may use tactics before, during and after mating to increase their reproductive success. With finite energy resources available, theory predicts that there should be a trade-off between investment in pre-copulatory traits (e.g. body size, armaments) and post-copulatory traits (e.g. testes size, spermatogenic efficiency). Western grey kangaroos (Macropus fuliginosus) are found in large, labile mixed-sex groups, in which the males show a dominance hierarchy. Males show indeterminate growth, and will reach up to six times the body mass of females. While the largest males use their size as a reproductive advantage, forelimb musculature further aids male–male contest, female attraction and/or female coercion. Under a trade-off scenario, we therefore predicted that larger, more muscular males would show less investment in sperm competitive traits. Consistent with this prediction, more muscular males showed decreased spermatozoa velocity. However, muscularity was also positively correlated with mass of two pairs of bulbourethral accessory glands, as well as mass of the penis and its muscles of erection. Seasonal changes in muscularity and accessory gland masses were also evident. Male kangaroos therefore invest in multiple reproductive traits on which selection can work. INTRODUCTION Males may invest in many traits (e.g. increased body size, muscularity, weapons) as a means of securing reproductive opportunities through male–male competition, display and/or female coercion (Clutton-Brock & Parker, 1995). However, the race is not won by simply mating with a female, because in species where females mate promiscuously, sperm from different males also need to compete for access to an ovum (Parker, 1970, 1998; Rose, Nevison & Dixson, 1997). High-quality males may be able to invest in both pre- and post-copulatory competitive traits, but when males have finite resources, then trade-offs between investing in pre-copulatory and post-copulatory traits are likely (Preston et al., 2001; Pizzari, Cornwallis & Froman, 2007; Perry & Rowe, 2010). Trade-offs in energy investment can be indicated by different growth patterns (Box 1). Several traits used for display or securing matings show positive allometry (e.g. Eberhard, 1985; Lüpold et al., 2014; Morris & Brandt, 2014; Buzatto, Roberts & Simmons, 2015; Rico-Guevara & Araya-Salas, 2015), including the forelimb muscles of male kangaroos (Warburton, Bateman & Fleming, 2013). Differential investment can also be evident in anatomical traits that increase post-copulatory competitiveness, such as genital size (e.g. Lüpold, McElligott & Hosken, 2004; Kinahan et al., 2007). Finally, various traits can increase competitiveness for a male’s ejaculate, such as spermatozoa number (e.g. Tourmente, Gomendio & Roldan, 2011), spermatozoa speed (Fitzpatrick et al., 2009; Lüpold et al., 2009; Ramón et al., 2013), spermatozoa longevity (see Snook, 2005 for a review) or impeding access for a competitor’s sperm (Harcourt, 1991; Ramm, Parker & Stockley, 2005). Box 1. Sexual selection and allometric growth Differential investment in morphological traits that reflect trade-offs in energy investment can be indicated by different allometric patterns, where the size of a trait increases disproportionately with increasing body size (Huxley, 1924; Eberhard, Rodriguez & Polihronakis, 2009; Voje & Hansen, 2013). Static allometry (sensuHuxley, 1924) is narrowly defined by the specific power law relation Y = aXb, between a trait Y and body size X, which then yields the standard linear allometric equation log(Y) = log(a) + b log(X), where b (β) is the allometric slope (Voje & Hansen, 2013). A trait that increases proportionally with body mass shows isometry (β = 1); for example, an isometric trait could remain 10% of the animal’s body mass over a range of adult body sizes. Positive allometry is where there is disproportionately greater growth of a particular trait, while negative allometry is where a particular trait shows little or no growth, despite increasing body size. Polymorphisms corresponding to sigmoidal scaling are also possible (Bonduriansky & Day, 2003), where investment in a particular trait is evident only for larger (generally) individuals. Interpreting these sometimes complex patterns requires an understanding of the potential underlying processes that contribute to selection acting on each trait: natural vs. sexual selection, and directional vs. stabilizing selection. Sexual selection can be inferred for a trait where there is a role for the trait in dominance contests or courtship displays, such as display feathers of birds (e.g. Owens & Hartley, 1998) or horns and antlers of mammals (e.g. Lincoln, 1994). If males and females of the same species have different traits, then that suggests that sexual selection may be involved. Similarly, if the two sexes have the same trait but it differs in relative size or development, then one can tentatively assume that sexual selection is involved (e.g. Mitchell, Van Sittert & Skinner, 2009; Simmons & Scheepers, 1996). Directional selection can act to increase elaboration of traits due to greater reproductive success of individuals possessing this trait. Ornaments and weapons, for example, consistently show positive allometries (Kodric-Brown, Sibly & Brown, 2006). For traits that are used for sexual display, developing and/or maintaining the structure is likely to have differential costs with respect to body size (Bonduriansky & Day, 2003), ensuring that the trait can be used as an honest aid for female choice or male competitor assessment (Petrie, 1992). Positive allometry resulting in dimorphism has also been invoked to indicate sexual selection for functional traits present in both sexes (Eberhard, 1985; Warburton et al., 2013; Lüpold et al., 2014; Morris & Brandt, 2014; Buzatto et al., 2015; Rico-Guevara & Araya-Salas, 2015). Finally, sexual selection may also act to stabilize particular traits where an increase in their size would confer a disadvantage (Bonduriansky, 2007); for example, the sclerotized genitalia of arthropods require that male intromittent organs do not become too large or too small for most female genitals, and stabilizing sexual selection around average-sized genitalia therefore acts to maintain constant male traits over a range of body sizes (Bonduriansky, 2007; Eberhard et al., 2009). Kangaroos (Macropodidae) tend to be found in large, labile, mixed-sex and mixed-age groups (mobs), with group size influenced by population density, season and available forage (Jarman & Coulson, 1989; Jarman, 1991). Many kangaroo species display hierarchical promiscuity, with males of high rank – established through body size, male–male combat and display – achieving greater mating success (Croft, 1989; Glanslosser, 1989; Jarman, 1989; Miller et al., 2010). Fighting to establish a dominance hierarchy in kangaroos can end in death for some males (Toni, 2017). In kangaroos, both sexes show indeterminate body growth, continuing to grow throughout life (Jarman, 1989, 1991). Males show disproportionate investment in forelimb musculature as they increase in body size: while female forelimb muscles show isometric growth, the muscles of males demonstrate positive allometry, with muscles growing 2–2.4× heavier than predicted from their body mass (Jarman, 1989; Warburton et al., 2013). Larger body size and greater muscularity would aid in male–male competition, display and potentially also female coercion (Croft, 1989), but there are clearly other tactics used by kangaroos to secure matings. For eastern grey kangaroos (Macropus giganteus), despite a wide range in body sizes, where the smallest fathers weighed 40% less than the heaviest ones, there was very little reproductive skew (Rioux-Paquette et al., 2015). Males can maximize their chances of contacting and inspecting females by spending time at resources frequented by females (e.g. waterholes, food patches), with the largest males having larger home-ranges (Croft, 1989). In most seasonally breeding marsupials, there are also seasonal changes in testis morphology and function and in the size of the male reproductive accessory glands (e.g. Inns, 1982; Todhunter & Gemmell, 1987; Paris et al., 2005; Taggart et al., 2005; Hogan et al., 2010). Marked sexual dimorphism, indeterminate growth and seasonal breeding patterns (Poole, 1975) make kangaroos interesting models to compare potential trade-offs in energy allocation between pre- and post-copulatory competitive traits, which are potentially costly, mutually exclusive reproductive strategies. The mating system of western grey kangaroos (Macropus fuliginosus, see Supporting Information, Fig. S1) has not been explicitly studied, although they are believed to establish dominance hierarchies (Paplinska et al., 2010; Richards, Grueter & Milne, 2015). Macropus fuliginosus males on average are twice as heavy as females but can be up to six times heavier (Coulson, 2008; Jarman, 1991) and this skew in body mass suggests a possibility for different mating tactics in this species. An equal sex ratio is evident at birth (Arnold et al., 1991; Mayberry et al., 2010), but male-biased mortality results in skew towards females, with 1.4 to 3 adult females per male recorded for various populations (Norbury, Coulson & Walters, 1988; Arnold et al., 1991; Arnold, Steven & Weeldenberg, 1994). In a study of a captive population, most M. fuliginosus young were sired by a few males (Poole, 1976), suggesting that competition among males for access to females is likely, providing opportunities for both pre- and post-copulatory competition (Paplinska et al., 2010; Richards et al., 2015). If there was a trade-off in reproductive strategy evident in M. fuliginosus, then we predicted that males that have invested in pre-copulatory traits to secure mating opportunities (i.e. larger muscles and body size) would invest less in sperm competitive traits (i.e. testes size, accessory gland size, sperm velocity). By contrast, males excluded from regular access to fertile females through pre-copulatory male–male competition are more likely to invest more in sperm competition traits so that, if they do gain access to a female, they improve their chance of fertilizing her. MATERIAL AND METHODS Specimens of adult male western grey kangaroo from various locations around Pinjarra, south-west Western Australia, were purchased from professionals who culled at night under commercial licence for pet food products. To capture variation in the traits examined, we sampled adult males (adult status confirmed by the presence of spermatozoa in the epididymis) across as wide range of body mass as possible (30–87 kg) during March–August (non-breeding season) and October–January (breeding season) (Mayberry et al., 2010). Whole carcasses were weighed in the field, reproductive organs were collected in situ, and the carcasses were labelled for identification and retrieval of forelimbs upon removal at an abattoir. We collected 72 males (53 ± 19 kg, body mass range 20–93 kg) for assessment of forelimb musculature, and 51 of these individuals were also analysed for their reproductive organ characteristics. Animals collected in the breeding (60 ± 14 kg kg, range 39–87 kg, N = 25) or non-breeding seasons (55 ± 19 kg, range 31–85 kg, N = 26) did not significantly differ in average body mass (F1,49 = 2.02, P = 0.162). Testes and sperm competition traits As sperm competitiveness can be influenced by ‘quality’ (morphology and locomotion) (Fitzpatrick et al., 2009; Lüpold et al., 2009; Ramón et al., 2013) the testes of each animal were collected and immediately refrigerated at 4°C for processing as soon as logistically possible the next morning, i.e. within 12 h of death. All samples were handled in the same way to enable comparison within our dataset. We dissected out the testes and epididymides and weighed each. The vas deferens and the cauda epididymis of one testis was flushed using a modification of published methods (Cary et al., 2004; Martinez-Pastor et al., 2006) where EquiPro (Equine semen extender, Minitube Australia Pty Ltd, Smythesdale, Victoria, Australia) was flushed through the epididymis into a 10-mL centrifuge tube using a pulsing technique, and the resultant fluid diluted to a concentration of approximately 25 million spermatozoa/mL for further assessment (‘flushed solution’). The flushed solution was incubated at 37 °C for 30 min and then a 20 μL sample was placed onto a microscope slide for video recording (Motic Image Plus Version 2.0) at 10× power using a phase contrast microscope (Olympus CX41) with warming stage at 37 °C (Tokai Hit Olympus). Visual assessments of total motility (percentage of spermatozoa moving) and progressive motility (percentage of spermatozoa moving that were progressive/moving forward) were carried out by a single observer (M.L.M.), and analysis of straight-line velocity (µm/s) of a sample of spermatozoa was calculated by timing the path of each across a set dimension on a computer screen (sample size range 15–35 individual spermatozoa with a stopping rule that there was no correlation between the number measured and velocity; coefficient of variation 39 ± 11%). One sample slide per individual was prepared for assessment of spermatozoa morphology; 15 µL of the flushed solution was spread on a microscope slide and air-dried, and then stained using SpermBlue fixative and stain (Van der Horst et al., 2009; Van der Horst & Maree, 2010) (Microptic SL, Barcelona, Spain) and 100 spermatozoa were examined. Photographs of individual spermatozoa at 40× power (using Moticam 2300 3.0MP Live resolution, Digital Microscopy) were used to measure spermatozoa head and midpiece + tail lengths (μm) (Motic Image Plus Version 2.0). The heads of linear spermatozoa were measured tip to midpiece using a straight line; the head length of T-shaped spermatozoa was measured tip to tip. Midpiece and tail were measured separately and total length was calculated as the sum of head, midpiece and tail lengths. Testes histology and immunohistochemistry One approach to studying testicular function in vertebrates is the analysis of reproductive hormone receptors in gonadal tissue, i.e. luteinizing hormone receptor (LH-R) and follicle-stimulating hormone receptor (FSH-R). Vertebrate LH and FSH are involved in sex steroid production and gametogenesis. FSH is important for quantitatively normal spermatogenesis, and FSH-R levels change in the testes during the cycle of the seminiferous epithelium (Heckert & Griswold, 2014). Testosterone, secreted by the Leydig cells upon stimulation by LH, is also involved in spermatogenesis (Tanaka et al., 2016). We analysed testes histology and expression of gonadotropin-receptor immunostaining using the second testis from each individual as a marker of the sensitivity of the testes to FSH and LH. Subsamples of the testes were taken immediately after collection from the central part of each testis, cut into 10-mm cubes and immersion-fixed in normal buffered formalin (NBF) for 24 h. Samples were then rinsed and stored in 70% alcohol before processing and embedding in paraffin wax. Sections (5 μm) were cut and mounted on polylysine-coated glass slides and dried overnight at 42 °C prior to histological and immunohistochemical analysis. Testis microstructure was quantified for the negative control slides and analysed with computer-aided image analysis using an Olympus BX51microscope at 10× magnification and an Olympus DP70 camera connected to a computer running Image-Pro Plus software (Media Cybernetics, Rockville, MD, USA). Using this setup, the percentage area of interstitial tissue per total tissue area in the field of view (FOV) and the percentage area of seminiferous tubule epithelium per total tissue area in the FOV were estimated. The interstitial tissue contains the Leydig cells that possess LH-R, and the seminiferous tubule epithelium tissue contains the Sertoli cells that possess FSH-R. The immunohistochemistry methods used here were as described by Miller et al. (2005). Briefly, a Vectastain Elite ABC kit (Vector Laboratories, Burlingame, CA, USA) was used for immunohistochemical staining according to the protocol recommended by the manufacturer. The primary anti-FSH receptor antibody (N-terminal ab150557: Abcam, Sapphire Bioscience, Redfern, NSW, Australia) and the primary anti-LH receptor antibody (sc-25828: Santa Cruz Biotechnology, VWR International, Murarrie, QLD, Australia) were used at a dilution of 1:400. Control slides were incubated with a pre-absorbed serum instead of primary antibody. To remove possible variation between sections arising from manual staining techniques, and thereby allowing quantification for comparisons between animals, strict adherence to timing was followed at each step so that each section was treated identically. A control slide for each sample was similarly processed, but no antibody was applied. Tissue density of immunostaining of FSH-R and LH-R was analysed using computer-aided image analysis. The system was composed of an Olympus BX51 microscope, at 10× magnification, and an Olympus DP70 camera connected to a computer running Image-Pro Plus software. Five randomly chosen FOV per section were quantified for the number of positively stained cells (brown – determined as a constant colour intensity grading between sections) and expressed as a sum of pixels; data for the control were used to correct the sample tissue. Total relevant tissue area (Sertoli cell area for FSH-R and insterstitial area for LH-R) was then quantified so that the positively immunostained cells could be expressed as a percentage of the total relevant tissue area. Five random FOV were selected from each testis as this was sufficient replication to stabilize the mean and standard errors, as described previously (Murray et al., 2000). Reproductive organs and accessory glands We recorded the mass of reproductive organs and accessory glands (Fig. 1) as these may all have a role in increasing reproductive success. We cleared the reproductive organs and accessory glands of connective tissue and then isolated and separately weighed the prostate gland, each of the bulbourethral (Cowper’s) glands, and the paired urethral bulb (m. bulbospongiosus) and crural bulb (m. ischiocavernosus) muscles. We then measured the length of the penis, from the base of the crural bulbs to the terminal tip, and recorded the mass of the penis. Figure 1. View largeDownload slide The reproductive tract of the male western grey kangaroo dissected to reveal the arrangement of the organs. In situ the prostate gland lies within the pelvic cavity, while the retracted penis and associated glands and muscles lie within the perineal region adjacent to the cloaca. Figure 1. View largeDownload slide The reproductive tract of the male western grey kangaroo dissected to reveal the arrangement of the organs. In situ the prostate gland lies within the pelvic cavity, while the retracted penis and associated glands and muscles lie within the perineal region adjacent to the cloaca. Forelimb muscles Carcasses for dissection were skinned and excess connective tissue was removed immediately before dissection to avoid dehydration of tissues. Ten intrinsic forelimb muscles/muscle groups were isolated and removed and then weighed immediately following the methods of Warburton et al. (2013). Body mass (mb) In addition to the whole animal body mass measurements, we also estimated body mass from femur shaft circumference (Helgen et al., 2006). Circumference of the cleaned and air-dried femur was measured using a tape-measure at the narrowest point (approximately mid-shaft), immediately below the posterior tubercle. This measure showed high repeatability (coefficient of variation of three repeated measures on each bone averaged 0.48 ± 0.34%). We calculated the relationship between femur shaft circumference and body mass as  log10(mb)=0.9273⋅log10(femur shaft circumference)+0.1274 (1) These estimated mb values were strongly correlated with field body mass measurements (R2 = 0.9007; N = 59 individuals). Body mass estimated from femur circumference was used in preference to field body mass measurement for statistical analyses because it allows estimation of body size independent of digestive contents at the time of death. Data analyses All mass measurements were log10-transformed and anatomical traits were compared against log10(mb) (estimated from the femur shaft circumference) by reduced major axis regression (Bonduriansky, 2007) in the program PAST (Hammer, Harper & Ryan, 2001). Slopes of these lines were tested against the hypothesis that β ≠ 1 (Hammer et al., 2001). We calculated an ‘index of muscularity’ as the average of the residuals (proportion of expected value) of each of the masses of ten muscle groups against log10(mb); this index was BoxCox-transformed to normalize the data distribution (Shapiro–Wilk test). Anatomical measures and sperm traits were tested against the index of muscularity by multiple regression analyses (Statistica; StatSoft Inc., 2007), including breeding season (dummy variable: breeding vs. non-breeding) and body mass (covariate) to control for these factors. In addition to the mass measurements, we similarly analysed penis length (BoxCox-transformed to normalize the data distribution; Shapiro–Wilk test). RESULTS Allometric relationships in anatomical traits All the forelimb muscles showed a strong positive allometry, with slopes for the relationship between log10(muscle mass) and log10(mb) of 1.92–2.39 (Table 1). Mass of the genitalia (penis, urethral and crural bulb muscles, testes, epididymides) and length of the penis were also significantly correlated with body mass (Table 2). Prostate mass showed no correlation with body mass, even when analysed for breeding and non-breeding seasons separately. Percentage area of testicular interstitial tissue was positively correlated with body mass, but the tissue density of LH-R (LH-R/% interstitium) was negatively correlated with body mass (Fig. 2). Table 1. Reduced major axis regression analyses for the masses of forelimb muscles, testes, epididymides, penis and accessory glands compared against log10-body mass for 51 male western grey kangaroos   Slope (β)  SE β  P (β = 1)  R  P(R)  Forelimb muscle mass (g)   m. deltoideus  2.21  0.09  ***  0.93  ***   m. supraspinatus  2.08  0.08  ***  0.95  ***   m. intraspinatus & m. teres minor  2.25  0.09  ***  0.94  ***   m. teres major  2.39  0.11  ***  0.93  ***   m. subscapularis  2.28  0.09  ***  0.94  ***   m. biceps brachii  2.37  0.10  ***  0.94  ***   m. brachialis  2.07  0.10  ***  0.92  ***   mm. triceps group  1.92  0.08  ***  0.94  ***   Extensors (lateral)  2.17  0.08  ***  0.95  ***   Flexors (medial)  2.36  0.10  ***  0.94  ***    Slope (β)  SE β  P (β = 1)  R  P(R)  Forelimb muscle mass (g)   m. deltoideus  2.21  0.09  ***  0.93  ***   m. supraspinatus  2.08  0.08  ***  0.95  ***   m. intraspinatus & m. teres minor  2.25  0.09  ***  0.94  ***   m. teres major  2.39  0.11  ***  0.93  ***   m. subscapularis  2.28  0.09  ***  0.94  ***   m. biceps brachii  2.37  0.10  ***  0.94  ***   m. brachialis  2.07  0.10  ***  0.92  ***   mm. triceps group  1.92  0.08  ***  0.94  ***   Extensors (lateral)  2.17  0.08  ***  0.95  ***   Flexors (medial)  2.36  0.10  ***  0.94  ***  ***P < 0.001. Data for both breeding and non-breeding season were pooled for these analyses. View Large Table 2. Summary of multiple regression analyses comparing each dependent measure against body mass (estimated from femur shaft circumference), season (breeding or non-breeding seasons) and muscularity (BoxCox-transformed average residual of ten muscles/muscle groups) in western grey kangaroos   Log-mb  Season  Index of muscularity†  R  P(R)  Anatomical traits   Index of muscularity†  −0.25    −0.28  *  –    0.13  *   Penis length†  0.83  ***  0.06    0.06    0.83  ***   Log-penis mass  0.82  ***  0.14    0.21  *  0.84  ***   Log-average mass urethral bulb  0.78  ***  0.24  **  0.33  ***  0.86  ***   Log-average mass crural bulb (N = 43)  0.84  ***  0.18  *  0.34  ***  0.91  ***   Log-testes mass  0.78  ***  −0.02    0.12    0.76  ***   Log-epididymis mass  0.82  ***  0.11    0.01    0.84  ***   Log-BG1 average mass (N = 50)  0.57  ***  0.32  **  0.18    0.69  ***   Log-BG2 average mass (N = 50)  0.64  ***  0.33  **  0.35  ***  0.79  ***   Log-BG3 average mass  0.44  ***  0.42  ***  0.29  *  0.71  ***   Log-prostate mass (N = 43)  0.16    0.80  ***  0.11    0.87  ***  Sperm traits   Total motility (% moving) †  0.13    −0.04    0.17    0.18     Progressive motility (% moving forward)†  0.02    −0.22    0.15    0.22     Average velocity  0.14    0.18    −0.35  *  0.39  *   Average head length†  −0.13    −0.38  **  0.37  *  0.49  **   Average midpiece length  −0.04    0.28    −0.24    0.29     Average tail length  −0.23    −0.18    −0.01    0.31     Average total length  −0.25    −0.17    −0.01    0.32    Morphologically normal spermatozoa †‡  −0.07    0.74  ***  −0.02    0.73  ***   Testes histology   Density of FSH-R (FSH-R/% tubule wall)  0.10    0.07    0.42  **  0.39  *   Total amount of FSH-R [density × √(testes mass)]  −0.04    0.11    0.40  **  0.40  *   Density of LH-R (LH-R/% interstitium)  −0.38  **  −0.27  *  0.21    0.55  ***   Total amount of LH-R [density × √(testes mass)]  −0.43  **  −0.25    0.16    0.54  ***    Log-mb  Season  Index of muscularity†  R  P(R)  Anatomical traits   Index of muscularity†  −0.25    −0.28  *  –    0.13  *   Penis length†  0.83  ***  0.06    0.06    0.83  ***   Log-penis mass  0.82  ***  0.14    0.21  *  0.84  ***   Log-average mass urethral bulb  0.78  ***  0.24  **  0.33  ***  0.86  ***   Log-average mass crural bulb (N = 43)  0.84  ***  0.18  *  0.34  ***  0.91  ***   Log-testes mass  0.78  ***  −0.02    0.12    0.76  ***   Log-epididymis mass  0.82  ***  0.11    0.01    0.84  ***   Log-BG1 average mass (N = 50)  0.57  ***  0.32  **  0.18    0.69  ***   Log-BG2 average mass (N = 50)  0.64  ***  0.33  **  0.35  ***  0.79  ***   Log-BG3 average mass  0.44  ***  0.42  ***  0.29  *  0.71  ***   Log-prostate mass (N = 43)  0.16    0.80  ***  0.11    0.87  ***  Sperm traits   Total motility (% moving) †  0.13    −0.04    0.17    0.18     Progressive motility (% moving forward)†  0.02    −0.22    0.15    0.22     Average velocity  0.14    0.18    −0.35  *  0.39  *   Average head length†  −0.13    −0.38  **  0.37  *  0.49  **   Average midpiece length  −0.04    0.28    −0.24    0.29     Average tail length  −0.23    −0.18    −0.01    0.31     Average total length  −0.25    −0.17    −0.01    0.32    Morphologically normal spermatozoa †‡  −0.07    0.74  ***  −0.02    0.73  ***   Testes histology   Density of FSH-R (FSH-R/% tubule wall)  0.10    0.07    0.42  **  0.39  *   Total amount of FSH-R [density × √(testes mass)]  −0.04    0.11    0.40  **  0.40  *   Density of LH-R (LH-R/% interstitium)  −0.38  **  −0.27  *  0.21    0.55  ***   Total amount of LH-R [density × √(testes mass)]  −0.43  **  −0.25    0.16    0.54  ***  The final column shows the regression coefficient for each model. For each measure, the standardized beta values (std-β) are presented, with statistical significance indicated as *P < 0.05, **P < 0.01 and ***P < 0.001. For some individuals, the muscles of the penis, bulbourethral glands and prostate were damaged during removal and therefore their mass values were not accurate; these were treated as missing values. Sample sizes were N = 51 individuals except where indicated otherwise. BG, bulbourethral gland; LH-R, luteinizing hormone receptor; FSH-R, follicle-stimulating hormone receptor. †BoxCox-transformed values were used where indicated. ‡The most frequent spermatozoa defects were breeding season: tail defect (middle third of the tail defected; 4.31%), head defect (3.28%), midpiece defect (3.00%); non-breeding season: head defect (5.68%), detached head (3.45%), midpiece defect (2.64%). View Large Figure 2. View largeDownload slide Relationships between (A) follicle-stimulating hormone receptor (FSH-R) density with muscularity, and (B) luteinizing hormone receptor (LH-R) density with body mass in western grey kangaroos. Figure 2. View largeDownload slide Relationships between (A) follicle-stimulating hormone receptor (FSH-R) density with muscularity, and (B) luteinizing hormone receptor (LH-R) density with body mass in western grey kangaroos. Breeding season effects The index of muscularity showed a significant influence of breeding season (Table 2) with greater muscularity evident for males sampled within the breeding season (Fig. 3). There was no breeding season effect on mass of the genitalia (testes, epididymides, penis), but there was for the muscular bulbs of the penis and the accessory glands, in particular for the prostate gland (Table 2), which was 2.5 times heavier during the breeding season (Fig. 4A). Seasonal differences were also found for sperm traits, with significantly shorter spermatozoa head length (Fig. 4B) and fewer abnormalities (Fig. 4C) for animals sampled in the breeding season. There was a significant effect of breeding season on the tissue density of LH-R, with greater densities during the breeding season. Figure 3. View largeDownload slide Relationship between the index of muscularity (average residuals of this relationship for all ten muscles/muscle groups) and body mass for western grey kangaroos sampled within or out of the breeding season. Figure 3. View largeDownload slide Relationship between the index of muscularity (average residuals of this relationship for all ten muscles/muscle groups) and body mass for western grey kangaroos sampled within or out of the breeding season. Figure 4. View largeDownload slide Three traits that showed a breeding season effect in western grey kangaroos. The raw values are shown for clarity; transformed values were used for statistical analyses. Figure 4. View largeDownload slide Three traits that showed a breeding season effect in western grey kangaroos. The raw values are shown for clarity; transformed values were used for statistical analyses. Relationships between muscularity and sperm competition traits There were positive correlations between muscularity and the masses of the penis, urethral bulb, crural bulb, and bulbourethral glands 2 and 3 (Fig. 5; Table 2). A negative relationship between spermatozoa head length and velocity indicated spermatozoa with shorter head length moved faster (Fig. 6A). Males that were more muscular had slower spermatozoa with longer head lengths (Fig. 6B, C; Table 2). There were no correlations between muscularity and testes or epididymis mass. Figure 5. View largeDownload slide Traits that showed a correlation with the index of muscularity in western grey kangaroos. BG, bulbourethral gland; UB, urethral bulb; CB, crural bulb. Figure 5. View largeDownload slide Traits that showed a correlation with the index of muscularity in western grey kangaroos. BG, bulbourethral gland; UB, urethral bulb; CB, crural bulb. Figure 6. View largeDownload slide Relationships between (A) sperm velocity and head length and the correlation between these sperm traits with the index of muscularity (B and C) in western grey kangaroos. Figure 6. View largeDownload slide Relationships between (A) sperm velocity and head length and the correlation between these sperm traits with the index of muscularity (B and C) in western grey kangaroos. Other sperm traits (motility, progressive motility, average midpiece length, average tail length, average total length) showed no relationships with season, body mass or muscularity (Table 2). There were no significant correlations between tissue density of FSH-R (FSH-R/% seminiferous tubule epithelium) and testes mass but there was a negative relationship between LH-R and testes mass (P = 0.035). FSH-R density was not significantly correlated with any of the sperm traits, while LH-R density was positively correlated with average spermatozoa head length. DISCUSSION There was no clear support for trade-offs in investment between pre- and post-copulatory traits by male western grey kangaroos. Kangaroos that were more muscular had spermatozoa with longer head lengths that were ultimately slower, which would make them less competitive. However, there were also correlations between muscularity and the masses of bulbourethral glands 2 and 3, the penis and muscles of erection (urethral bulb and crural bulb), but these were positive relationships, and therefore did not indicate a trade-off but rather a similar direction of selection acting on these traits. The forelimb musculature of western grey kangaroos shows marked development in males but not females (Jarman, 1983, 1989, 1991; Warburton et al., 2013), with positive allometry (β = 1.92–2.39) of these muscles in males. The role of these muscles is assumed to be in ritualized fighting, displaying to females and coercing females to mate, and therefore the muscles are subject to sexual selection mechanisms (Clutton-Brock & Parker, 1995). There was an apparent trade-off in muscle development with body size; it was not the heaviest males that showed the greatest muscularity, but rather intermediate-mass males (Fig. 3). Body mass also plays an important role in securing matings, with larger males dominating access to receptive females (Croft, 1989; Miller et al., 2010). The heaviest males can therefore afford to rely on their size alone to impress females, out-compete rivals or coerce females (or all three tactics), while the lighter adult males may still need to engage in combat to ensure access to females (Rioux-Paquette et al., 2015). Interestingly, we found a significant inverse relationship between LH-R density and body mass, where the largest males had the lowest density of LH-R. Because testosterone is (mainly) produced by testicular Leydig cells in response to LH, the lower LH-R for larger males suggests they also have lower circulating testosterone levels. Alternatively, studies have shown a loss of testicular LH receptors in rats after systemic administration of LH (Hsueh, Dafau & Catt, 1976, 1977), suggesting that the larger male kangaroos may have higher LH concentrations and that testosterone secretion was being homeostatically regulated at the level of the target (Leydig) cell through a reduction in testicular LH receptors. Unfortunately, we could not collect blood samples to measure LH or testosterone concentrations for logistical reasons due to the method of culling, but a study with male eastern grey kangaroos found that males that sire offspring have significantly higher testosterone concentrations than non-sire males (Miller et al., 2010). The role of the three pairs of bulbourethral glands in marsupials (Rodger & Hughes, 1973) has not yet been clarified, but it is suspected that they contribute to formation of a post-copulatory ‘plug’. The semen of macropodids coagulates rapidly after ejaculations, and forms a plug in the female genital tract (Taggart et al., 1998; Paris et al., 2005). The plug could act to prevent spermatozoa draining from the female, keep the ejaculate near the cervix, or act as a sperm reservoir (Taggart et al., 1998). In many taxa, plugs act as barriers to the sperm of subsequent males, which may assist in providing a first male advantage (e.g. Munroe & Koprowski, 2012), reducing selective pressure on investment in sperm competition traits (Lemaître et al., 2011). We found shorter spermatozoa head length and fewer abnormalities for animals sampled in the breeding season, which may reflect seasonal variation in sperm maturation and storage. We did not record correlations with muscularity for the other spermatozoan measurements. This may reflect greater variation in our data due to specimen handling, as we could not control for the time between death of the animal and collection of spermatozoa due to our opportunistic acquisition of cadavers. We did not flush epididymides consistently with known volumes, which would have been required to allow accurate sperm counts. Sperm counts may have revealed more informative relationships with muscularity measures. In addition to the seasonal changes in sperm morphology, we found marked seasonal changes in prostate mass, which was 2.5 times greater during the breeding season (but showed no relationship with body mass). The prostate produces the seminal plasma that supports and nourishes the spermatozoa (Taggart et al., 2005). Enlargement of the prostate during the breeding season would therefore facilitate semen production or maintenance of a fertile ejaculate. CONCLUSION Increased body size in kangaroos increases nutritional requirements and will therefore require larger home-ranges (Norbury, Sanson & Lee, 1989). Male kangaroos that invest in costly tactics such as increased body size and muscularity ‘thereby put themselves at risk if environmental conditions deteriorate’ (Dawson, 2002: 87) and are more vulnerable to stochastic environmental events (Robertson, 1986; Norbury et al., 1988; Coulson, 2006). Food restriction will therefore limit the benefits of increased muscularity in larger males. Competing natural and sexual selective forces are therefore likely to be evident for species such as kangaroos, modifying differential investment in various morphological traits. While we found some evidence of trade-offs relating to male muscularity, there are evidently complex interactions between morphological adaptations, sperm competition and non-sperm components of the ejaculate at play. Our results emphasize the importance of simultaneously studying a range of pre- and post-copulatory mechanisms by which sexual selection could act in order to capture the relative importance of different traits and understand their interplay (Parker, Lessells & Simmons, 2013). Perhaps this finding highlights the complex relationship between the efficacy of pre- and post-copulatory reproductive tactics, and warns against simply assuming that obvious physical traits will necessarily translate into improved reproductive success. SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article at the publisher’s web-site: Figure S1. Photographs of the western grey kangaroo, Macropus fuliginosus. ACKNOWLEDGEMENTS We thank D. Nottle, L. Boston, J. Hong and M. Gibberd for specimen acquisition and technical support, and D. Supreme, K. Napier, V. Lenard and C. Flandrin for help with dissections. Financial support was provided by Murdoch University to all authors; we have no competing interests to declare. We thank G. Coulson and the anonymous reviewers for their constructive comments on the manuscript. The project was conducted under the approval of Murdoch University Animal Ethics Committee (Cadaver Notification) and a Department of Parks and Wildlife Regulation 17 licence SF009843. 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Is there evidence for a trade-off between sperm competition traits and forelimb musculature in the western grey kangaroo?

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Abstract

Abstract Males may use tactics before, during and after mating to increase their reproductive success. With finite energy resources available, theory predicts that there should be a trade-off between investment in pre-copulatory traits (e.g. body size, armaments) and post-copulatory traits (e.g. testes size, spermatogenic efficiency). Western grey kangaroos (Macropus fuliginosus) are found in large, labile mixed-sex groups, in which the males show a dominance hierarchy. Males show indeterminate growth, and will reach up to six times the body mass of females. While the largest males use their size as a reproductive advantage, forelimb musculature further aids male–male contest, female attraction and/or female coercion. Under a trade-off scenario, we therefore predicted that larger, more muscular males would show less investment in sperm competitive traits. Consistent with this prediction, more muscular males showed decreased spermatozoa velocity. However, muscularity was also positively correlated with mass of two pairs of bulbourethral accessory glands, as well as mass of the penis and its muscles of erection. Seasonal changes in muscularity and accessory gland masses were also evident. Male kangaroos therefore invest in multiple reproductive traits on which selection can work. INTRODUCTION Males may invest in many traits (e.g. increased body size, muscularity, weapons) as a means of securing reproductive opportunities through male–male competition, display and/or female coercion (Clutton-Brock & Parker, 1995). However, the race is not won by simply mating with a female, because in species where females mate promiscuously, sperm from different males also need to compete for access to an ovum (Parker, 1970, 1998; Rose, Nevison & Dixson, 1997). High-quality males may be able to invest in both pre- and post-copulatory competitive traits, but when males have finite resources, then trade-offs between investing in pre-copulatory and post-copulatory traits are likely (Preston et al., 2001; Pizzari, Cornwallis & Froman, 2007; Perry & Rowe, 2010). Trade-offs in energy investment can be indicated by different growth patterns (Box 1). Several traits used for display or securing matings show positive allometry (e.g. Eberhard, 1985; Lüpold et al., 2014; Morris & Brandt, 2014; Buzatto, Roberts & Simmons, 2015; Rico-Guevara & Araya-Salas, 2015), including the forelimb muscles of male kangaroos (Warburton, Bateman & Fleming, 2013). Differential investment can also be evident in anatomical traits that increase post-copulatory competitiveness, such as genital size (e.g. Lüpold, McElligott & Hosken, 2004; Kinahan et al., 2007). Finally, various traits can increase competitiveness for a male’s ejaculate, such as spermatozoa number (e.g. Tourmente, Gomendio & Roldan, 2011), spermatozoa speed (Fitzpatrick et al., 2009; Lüpold et al., 2009; Ramón et al., 2013), spermatozoa longevity (see Snook, 2005 for a review) or impeding access for a competitor’s sperm (Harcourt, 1991; Ramm, Parker & Stockley, 2005). Box 1. Sexual selection and allometric growth Differential investment in morphological traits that reflect trade-offs in energy investment can be indicated by different allometric patterns, where the size of a trait increases disproportionately with increasing body size (Huxley, 1924; Eberhard, Rodriguez & Polihronakis, 2009; Voje & Hansen, 2013). Static allometry (sensuHuxley, 1924) is narrowly defined by the specific power law relation Y = aXb, between a trait Y and body size X, which then yields the standard linear allometric equation log(Y) = log(a) + b log(X), where b (β) is the allometric slope (Voje & Hansen, 2013). A trait that increases proportionally with body mass shows isometry (β = 1); for example, an isometric trait could remain 10% of the animal’s body mass over a range of adult body sizes. Positive allometry is where there is disproportionately greater growth of a particular trait, while negative allometry is where a particular trait shows little or no growth, despite increasing body size. Polymorphisms corresponding to sigmoidal scaling are also possible (Bonduriansky & Day, 2003), where investment in a particular trait is evident only for larger (generally) individuals. Interpreting these sometimes complex patterns requires an understanding of the potential underlying processes that contribute to selection acting on each trait: natural vs. sexual selection, and directional vs. stabilizing selection. Sexual selection can be inferred for a trait where there is a role for the trait in dominance contests or courtship displays, such as display feathers of birds (e.g. Owens & Hartley, 1998) or horns and antlers of mammals (e.g. Lincoln, 1994). If males and females of the same species have different traits, then that suggests that sexual selection may be involved. Similarly, if the two sexes have the same trait but it differs in relative size or development, then one can tentatively assume that sexual selection is involved (e.g. Mitchell, Van Sittert & Skinner, 2009; Simmons & Scheepers, 1996). Directional selection can act to increase elaboration of traits due to greater reproductive success of individuals possessing this trait. Ornaments and weapons, for example, consistently show positive allometries (Kodric-Brown, Sibly & Brown, 2006). For traits that are used for sexual display, developing and/or maintaining the structure is likely to have differential costs with respect to body size (Bonduriansky & Day, 2003), ensuring that the trait can be used as an honest aid for female choice or male competitor assessment (Petrie, 1992). Positive allometry resulting in dimorphism has also been invoked to indicate sexual selection for functional traits present in both sexes (Eberhard, 1985; Warburton et al., 2013; Lüpold et al., 2014; Morris & Brandt, 2014; Buzatto et al., 2015; Rico-Guevara & Araya-Salas, 2015). Finally, sexual selection may also act to stabilize particular traits where an increase in their size would confer a disadvantage (Bonduriansky, 2007); for example, the sclerotized genitalia of arthropods require that male intromittent organs do not become too large or too small for most female genitals, and stabilizing sexual selection around average-sized genitalia therefore acts to maintain constant male traits over a range of body sizes (Bonduriansky, 2007; Eberhard et al., 2009). Kangaroos (Macropodidae) tend to be found in large, labile, mixed-sex and mixed-age groups (mobs), with group size influenced by population density, season and available forage (Jarman & Coulson, 1989; Jarman, 1991). Many kangaroo species display hierarchical promiscuity, with males of high rank – established through body size, male–male combat and display – achieving greater mating success (Croft, 1989; Glanslosser, 1989; Jarman, 1989; Miller et al., 2010). Fighting to establish a dominance hierarchy in kangaroos can end in death for some males (Toni, 2017). In kangaroos, both sexes show indeterminate body growth, continuing to grow throughout life (Jarman, 1989, 1991). Males show disproportionate investment in forelimb musculature as they increase in body size: while female forelimb muscles show isometric growth, the muscles of males demonstrate positive allometry, with muscles growing 2–2.4× heavier than predicted from their body mass (Jarman, 1989; Warburton et al., 2013). Larger body size and greater muscularity would aid in male–male competition, display and potentially also female coercion (Croft, 1989), but there are clearly other tactics used by kangaroos to secure matings. For eastern grey kangaroos (Macropus giganteus), despite a wide range in body sizes, where the smallest fathers weighed 40% less than the heaviest ones, there was very little reproductive skew (Rioux-Paquette et al., 2015). Males can maximize their chances of contacting and inspecting females by spending time at resources frequented by females (e.g. waterholes, food patches), with the largest males having larger home-ranges (Croft, 1989). In most seasonally breeding marsupials, there are also seasonal changes in testis morphology and function and in the size of the male reproductive accessory glands (e.g. Inns, 1982; Todhunter & Gemmell, 1987; Paris et al., 2005; Taggart et al., 2005; Hogan et al., 2010). Marked sexual dimorphism, indeterminate growth and seasonal breeding patterns (Poole, 1975) make kangaroos interesting models to compare potential trade-offs in energy allocation between pre- and post-copulatory competitive traits, which are potentially costly, mutually exclusive reproductive strategies. The mating system of western grey kangaroos (Macropus fuliginosus, see Supporting Information, Fig. S1) has not been explicitly studied, although they are believed to establish dominance hierarchies (Paplinska et al., 2010; Richards, Grueter & Milne, 2015). Macropus fuliginosus males on average are twice as heavy as females but can be up to six times heavier (Coulson, 2008; Jarman, 1991) and this skew in body mass suggests a possibility for different mating tactics in this species. An equal sex ratio is evident at birth (Arnold et al., 1991; Mayberry et al., 2010), but male-biased mortality results in skew towards females, with 1.4 to 3 adult females per male recorded for various populations (Norbury, Coulson & Walters, 1988; Arnold et al., 1991; Arnold, Steven & Weeldenberg, 1994). In a study of a captive population, most M. fuliginosus young were sired by a few males (Poole, 1976), suggesting that competition among males for access to females is likely, providing opportunities for both pre- and post-copulatory competition (Paplinska et al., 2010; Richards et al., 2015). If there was a trade-off in reproductive strategy evident in M. fuliginosus, then we predicted that males that have invested in pre-copulatory traits to secure mating opportunities (i.e. larger muscles and body size) would invest less in sperm competitive traits (i.e. testes size, accessory gland size, sperm velocity). By contrast, males excluded from regular access to fertile females through pre-copulatory male–male competition are more likely to invest more in sperm competition traits so that, if they do gain access to a female, they improve their chance of fertilizing her. MATERIAL AND METHODS Specimens of adult male western grey kangaroo from various locations around Pinjarra, south-west Western Australia, were purchased from professionals who culled at night under commercial licence for pet food products. To capture variation in the traits examined, we sampled adult males (adult status confirmed by the presence of spermatozoa in the epididymis) across as wide range of body mass as possible (30–87 kg) during March–August (non-breeding season) and October–January (breeding season) (Mayberry et al., 2010). Whole carcasses were weighed in the field, reproductive organs were collected in situ, and the carcasses were labelled for identification and retrieval of forelimbs upon removal at an abattoir. We collected 72 males (53 ± 19 kg, body mass range 20–93 kg) for assessment of forelimb musculature, and 51 of these individuals were also analysed for their reproductive organ characteristics. Animals collected in the breeding (60 ± 14 kg kg, range 39–87 kg, N = 25) or non-breeding seasons (55 ± 19 kg, range 31–85 kg, N = 26) did not significantly differ in average body mass (F1,49 = 2.02, P = 0.162). Testes and sperm competition traits As sperm competitiveness can be influenced by ‘quality’ (morphology and locomotion) (Fitzpatrick et al., 2009; Lüpold et al., 2009; Ramón et al., 2013) the testes of each animal were collected and immediately refrigerated at 4°C for processing as soon as logistically possible the next morning, i.e. within 12 h of death. All samples were handled in the same way to enable comparison within our dataset. We dissected out the testes and epididymides and weighed each. The vas deferens and the cauda epididymis of one testis was flushed using a modification of published methods (Cary et al., 2004; Martinez-Pastor et al., 2006) where EquiPro (Equine semen extender, Minitube Australia Pty Ltd, Smythesdale, Victoria, Australia) was flushed through the epididymis into a 10-mL centrifuge tube using a pulsing technique, and the resultant fluid diluted to a concentration of approximately 25 million spermatozoa/mL for further assessment (‘flushed solution’). The flushed solution was incubated at 37 °C for 30 min and then a 20 μL sample was placed onto a microscope slide for video recording (Motic Image Plus Version 2.0) at 10× power using a phase contrast microscope (Olympus CX41) with warming stage at 37 °C (Tokai Hit Olympus). Visual assessments of total motility (percentage of spermatozoa moving) and progressive motility (percentage of spermatozoa moving that were progressive/moving forward) were carried out by a single observer (M.L.M.), and analysis of straight-line velocity (µm/s) of a sample of spermatozoa was calculated by timing the path of each across a set dimension on a computer screen (sample size range 15–35 individual spermatozoa with a stopping rule that there was no correlation between the number measured and velocity; coefficient of variation 39 ± 11%). One sample slide per individual was prepared for assessment of spermatozoa morphology; 15 µL of the flushed solution was spread on a microscope slide and air-dried, and then stained using SpermBlue fixative and stain (Van der Horst et al., 2009; Van der Horst & Maree, 2010) (Microptic SL, Barcelona, Spain) and 100 spermatozoa were examined. Photographs of individual spermatozoa at 40× power (using Moticam 2300 3.0MP Live resolution, Digital Microscopy) were used to measure spermatozoa head and midpiece + tail lengths (μm) (Motic Image Plus Version 2.0). The heads of linear spermatozoa were measured tip to midpiece using a straight line; the head length of T-shaped spermatozoa was measured tip to tip. Midpiece and tail were measured separately and total length was calculated as the sum of head, midpiece and tail lengths. Testes histology and immunohistochemistry One approach to studying testicular function in vertebrates is the analysis of reproductive hormone receptors in gonadal tissue, i.e. luteinizing hormone receptor (LH-R) and follicle-stimulating hormone receptor (FSH-R). Vertebrate LH and FSH are involved in sex steroid production and gametogenesis. FSH is important for quantitatively normal spermatogenesis, and FSH-R levels change in the testes during the cycle of the seminiferous epithelium (Heckert & Griswold, 2014). Testosterone, secreted by the Leydig cells upon stimulation by LH, is also involved in spermatogenesis (Tanaka et al., 2016). We analysed testes histology and expression of gonadotropin-receptor immunostaining using the second testis from each individual as a marker of the sensitivity of the testes to FSH and LH. Subsamples of the testes were taken immediately after collection from the central part of each testis, cut into 10-mm cubes and immersion-fixed in normal buffered formalin (NBF) for 24 h. Samples were then rinsed and stored in 70% alcohol before processing and embedding in paraffin wax. Sections (5 μm) were cut and mounted on polylysine-coated glass slides and dried overnight at 42 °C prior to histological and immunohistochemical analysis. Testis microstructure was quantified for the negative control slides and analysed with computer-aided image analysis using an Olympus BX51microscope at 10× magnification and an Olympus DP70 camera connected to a computer running Image-Pro Plus software (Media Cybernetics, Rockville, MD, USA). Using this setup, the percentage area of interstitial tissue per total tissue area in the field of view (FOV) and the percentage area of seminiferous tubule epithelium per total tissue area in the FOV were estimated. The interstitial tissue contains the Leydig cells that possess LH-R, and the seminiferous tubule epithelium tissue contains the Sertoli cells that possess FSH-R. The immunohistochemistry methods used here were as described by Miller et al. (2005). Briefly, a Vectastain Elite ABC kit (Vector Laboratories, Burlingame, CA, USA) was used for immunohistochemical staining according to the protocol recommended by the manufacturer. The primary anti-FSH receptor antibody (N-terminal ab150557: Abcam, Sapphire Bioscience, Redfern, NSW, Australia) and the primary anti-LH receptor antibody (sc-25828: Santa Cruz Biotechnology, VWR International, Murarrie, QLD, Australia) were used at a dilution of 1:400. Control slides were incubated with a pre-absorbed serum instead of primary antibody. To remove possible variation between sections arising from manual staining techniques, and thereby allowing quantification for comparisons between animals, strict adherence to timing was followed at each step so that each section was treated identically. A control slide for each sample was similarly processed, but no antibody was applied. Tissue density of immunostaining of FSH-R and LH-R was analysed using computer-aided image analysis. The system was composed of an Olympus BX51 microscope, at 10× magnification, and an Olympus DP70 camera connected to a computer running Image-Pro Plus software. Five randomly chosen FOV per section were quantified for the number of positively stained cells (brown – determined as a constant colour intensity grading between sections) and expressed as a sum of pixels; data for the control were used to correct the sample tissue. Total relevant tissue area (Sertoli cell area for FSH-R and insterstitial area for LH-R) was then quantified so that the positively immunostained cells could be expressed as a percentage of the total relevant tissue area. Five random FOV were selected from each testis as this was sufficient replication to stabilize the mean and standard errors, as described previously (Murray et al., 2000). Reproductive organs and accessory glands We recorded the mass of reproductive organs and accessory glands (Fig. 1) as these may all have a role in increasing reproductive success. We cleared the reproductive organs and accessory glands of connective tissue and then isolated and separately weighed the prostate gland, each of the bulbourethral (Cowper’s) glands, and the paired urethral bulb (m. bulbospongiosus) and crural bulb (m. ischiocavernosus) muscles. We then measured the length of the penis, from the base of the crural bulbs to the terminal tip, and recorded the mass of the penis. Figure 1. View largeDownload slide The reproductive tract of the male western grey kangaroo dissected to reveal the arrangement of the organs. In situ the prostate gland lies within the pelvic cavity, while the retracted penis and associated glands and muscles lie within the perineal region adjacent to the cloaca. Figure 1. View largeDownload slide The reproductive tract of the male western grey kangaroo dissected to reveal the arrangement of the organs. In situ the prostate gland lies within the pelvic cavity, while the retracted penis and associated glands and muscles lie within the perineal region adjacent to the cloaca. Forelimb muscles Carcasses for dissection were skinned and excess connective tissue was removed immediately before dissection to avoid dehydration of tissues. Ten intrinsic forelimb muscles/muscle groups were isolated and removed and then weighed immediately following the methods of Warburton et al. (2013). Body mass (mb) In addition to the whole animal body mass measurements, we also estimated body mass from femur shaft circumference (Helgen et al., 2006). Circumference of the cleaned and air-dried femur was measured using a tape-measure at the narrowest point (approximately mid-shaft), immediately below the posterior tubercle. This measure showed high repeatability (coefficient of variation of three repeated measures on each bone averaged 0.48 ± 0.34%). We calculated the relationship between femur shaft circumference and body mass as  log10(mb)=0.9273⋅log10(femur shaft circumference)+0.1274 (1) These estimated mb values were strongly correlated with field body mass measurements (R2 = 0.9007; N = 59 individuals). Body mass estimated from femur circumference was used in preference to field body mass measurement for statistical analyses because it allows estimation of body size independent of digestive contents at the time of death. Data analyses All mass measurements were log10-transformed and anatomical traits were compared against log10(mb) (estimated from the femur shaft circumference) by reduced major axis regression (Bonduriansky, 2007) in the program PAST (Hammer, Harper & Ryan, 2001). Slopes of these lines were tested against the hypothesis that β ≠ 1 (Hammer et al., 2001). We calculated an ‘index of muscularity’ as the average of the residuals (proportion of expected value) of each of the masses of ten muscle groups against log10(mb); this index was BoxCox-transformed to normalize the data distribution (Shapiro–Wilk test). Anatomical measures and sperm traits were tested against the index of muscularity by multiple regression analyses (Statistica; StatSoft Inc., 2007), including breeding season (dummy variable: breeding vs. non-breeding) and body mass (covariate) to control for these factors. In addition to the mass measurements, we similarly analysed penis length (BoxCox-transformed to normalize the data distribution; Shapiro–Wilk test). RESULTS Allometric relationships in anatomical traits All the forelimb muscles showed a strong positive allometry, with slopes for the relationship between log10(muscle mass) and log10(mb) of 1.92–2.39 (Table 1). Mass of the genitalia (penis, urethral and crural bulb muscles, testes, epididymides) and length of the penis were also significantly correlated with body mass (Table 2). Prostate mass showed no correlation with body mass, even when analysed for breeding and non-breeding seasons separately. Percentage area of testicular interstitial tissue was positively correlated with body mass, but the tissue density of LH-R (LH-R/% interstitium) was negatively correlated with body mass (Fig. 2). Table 1. Reduced major axis regression analyses for the masses of forelimb muscles, testes, epididymides, penis and accessory glands compared against log10-body mass for 51 male western grey kangaroos   Slope (β)  SE β  P (β = 1)  R  P(R)  Forelimb muscle mass (g)   m. deltoideus  2.21  0.09  ***  0.93  ***   m. supraspinatus  2.08  0.08  ***  0.95  ***   m. intraspinatus & m. teres minor  2.25  0.09  ***  0.94  ***   m. teres major  2.39  0.11  ***  0.93  ***   m. subscapularis  2.28  0.09  ***  0.94  ***   m. biceps brachii  2.37  0.10  ***  0.94  ***   m. brachialis  2.07  0.10  ***  0.92  ***   mm. triceps group  1.92  0.08  ***  0.94  ***   Extensors (lateral)  2.17  0.08  ***  0.95  ***   Flexors (medial)  2.36  0.10  ***  0.94  ***    Slope (β)  SE β  P (β = 1)  R  P(R)  Forelimb muscle mass (g)   m. deltoideus  2.21  0.09  ***  0.93  ***   m. supraspinatus  2.08  0.08  ***  0.95  ***   m. intraspinatus & m. teres minor  2.25  0.09  ***  0.94  ***   m. teres major  2.39  0.11  ***  0.93  ***   m. subscapularis  2.28  0.09  ***  0.94  ***   m. biceps brachii  2.37  0.10  ***  0.94  ***   m. brachialis  2.07  0.10  ***  0.92  ***   mm. triceps group  1.92  0.08  ***  0.94  ***   Extensors (lateral)  2.17  0.08  ***  0.95  ***   Flexors (medial)  2.36  0.10  ***  0.94  ***  ***P < 0.001. Data for both breeding and non-breeding season were pooled for these analyses. View Large Table 2. Summary of multiple regression analyses comparing each dependent measure against body mass (estimated from femur shaft circumference), season (breeding or non-breeding seasons) and muscularity (BoxCox-transformed average residual of ten muscles/muscle groups) in western grey kangaroos   Log-mb  Season  Index of muscularity†  R  P(R)  Anatomical traits   Index of muscularity†  −0.25    −0.28  *  –    0.13  *   Penis length†  0.83  ***  0.06    0.06    0.83  ***   Log-penis mass  0.82  ***  0.14    0.21  *  0.84  ***   Log-average mass urethral bulb  0.78  ***  0.24  **  0.33  ***  0.86  ***   Log-average mass crural bulb (N = 43)  0.84  ***  0.18  *  0.34  ***  0.91  ***   Log-testes mass  0.78  ***  −0.02    0.12    0.76  ***   Log-epididymis mass  0.82  ***  0.11    0.01    0.84  ***   Log-BG1 average mass (N = 50)  0.57  ***  0.32  **  0.18    0.69  ***   Log-BG2 average mass (N = 50)  0.64  ***  0.33  **  0.35  ***  0.79  ***   Log-BG3 average mass  0.44  ***  0.42  ***  0.29  *  0.71  ***   Log-prostate mass (N = 43)  0.16    0.80  ***  0.11    0.87  ***  Sperm traits   Total motility (% moving) †  0.13    −0.04    0.17    0.18     Progressive motility (% moving forward)†  0.02    −0.22    0.15    0.22     Average velocity  0.14    0.18    −0.35  *  0.39  *   Average head length†  −0.13    −0.38  **  0.37  *  0.49  **   Average midpiece length  −0.04    0.28    −0.24    0.29     Average tail length  −0.23    −0.18    −0.01    0.31     Average total length  −0.25    −0.17    −0.01    0.32    Morphologically normal spermatozoa †‡  −0.07    0.74  ***  −0.02    0.73  ***   Testes histology   Density of FSH-R (FSH-R/% tubule wall)  0.10    0.07    0.42  **  0.39  *   Total amount of FSH-R [density × √(testes mass)]  −0.04    0.11    0.40  **  0.40  *   Density of LH-R (LH-R/% interstitium)  −0.38  **  −0.27  *  0.21    0.55  ***   Total amount of LH-R [density × √(testes mass)]  −0.43  **  −0.25    0.16    0.54  ***    Log-mb  Season  Index of muscularity†  R  P(R)  Anatomical traits   Index of muscularity†  −0.25    −0.28  *  –    0.13  *   Penis length†  0.83  ***  0.06    0.06    0.83  ***   Log-penis mass  0.82  ***  0.14    0.21  *  0.84  ***   Log-average mass urethral bulb  0.78  ***  0.24  **  0.33  ***  0.86  ***   Log-average mass crural bulb (N = 43)  0.84  ***  0.18  *  0.34  ***  0.91  ***   Log-testes mass  0.78  ***  −0.02    0.12    0.76  ***   Log-epididymis mass  0.82  ***  0.11    0.01    0.84  ***   Log-BG1 average mass (N = 50)  0.57  ***  0.32  **  0.18    0.69  ***   Log-BG2 average mass (N = 50)  0.64  ***  0.33  **  0.35  ***  0.79  ***   Log-BG3 average mass  0.44  ***  0.42  ***  0.29  *  0.71  ***   Log-prostate mass (N = 43)  0.16    0.80  ***  0.11    0.87  ***  Sperm traits   Total motility (% moving) †  0.13    −0.04    0.17    0.18     Progressive motility (% moving forward)†  0.02    −0.22    0.15    0.22     Average velocity  0.14    0.18    −0.35  *  0.39  *   Average head length†  −0.13    −0.38  **  0.37  *  0.49  **   Average midpiece length  −0.04    0.28    −0.24    0.29     Average tail length  −0.23    −0.18    −0.01    0.31     Average total length  −0.25    −0.17    −0.01    0.32    Morphologically normal spermatozoa †‡  −0.07    0.74  ***  −0.02    0.73  ***   Testes histology   Density of FSH-R (FSH-R/% tubule wall)  0.10    0.07    0.42  **  0.39  *   Total amount of FSH-R [density × √(testes mass)]  −0.04    0.11    0.40  **  0.40  *   Density of LH-R (LH-R/% interstitium)  −0.38  **  −0.27  *  0.21    0.55  ***   Total amount of LH-R [density × √(testes mass)]  −0.43  **  −0.25    0.16    0.54  ***  The final column shows the regression coefficient for each model. For each measure, the standardized beta values (std-β) are presented, with statistical significance indicated as *P < 0.05, **P < 0.01 and ***P < 0.001. For some individuals, the muscles of the penis, bulbourethral glands and prostate were damaged during removal and therefore their mass values were not accurate; these were treated as missing values. Sample sizes were N = 51 individuals except where indicated otherwise. BG, bulbourethral gland; LH-R, luteinizing hormone receptor; FSH-R, follicle-stimulating hormone receptor. †BoxCox-transformed values were used where indicated. ‡The most frequent spermatozoa defects were breeding season: tail defect (middle third of the tail defected; 4.31%), head defect (3.28%), midpiece defect (3.00%); non-breeding season: head defect (5.68%), detached head (3.45%), midpiece defect (2.64%). View Large Figure 2. View largeDownload slide Relationships between (A) follicle-stimulating hormone receptor (FSH-R) density with muscularity, and (B) luteinizing hormone receptor (LH-R) density with body mass in western grey kangaroos. Figure 2. View largeDownload slide Relationships between (A) follicle-stimulating hormone receptor (FSH-R) density with muscularity, and (B) luteinizing hormone receptor (LH-R) density with body mass in western grey kangaroos. Breeding season effects The index of muscularity showed a significant influence of breeding season (Table 2) with greater muscularity evident for males sampled within the breeding season (Fig. 3). There was no breeding season effect on mass of the genitalia (testes, epididymides, penis), but there was for the muscular bulbs of the penis and the accessory glands, in particular for the prostate gland (Table 2), which was 2.5 times heavier during the breeding season (Fig. 4A). Seasonal differences were also found for sperm traits, with significantly shorter spermatozoa head length (Fig. 4B) and fewer abnormalities (Fig. 4C) for animals sampled in the breeding season. There was a significant effect of breeding season on the tissue density of LH-R, with greater densities during the breeding season. Figure 3. View largeDownload slide Relationship between the index of muscularity (average residuals of this relationship for all ten muscles/muscle groups) and body mass for western grey kangaroos sampled within or out of the breeding season. Figure 3. View largeDownload slide Relationship between the index of muscularity (average residuals of this relationship for all ten muscles/muscle groups) and body mass for western grey kangaroos sampled within or out of the breeding season. Figure 4. View largeDownload slide Three traits that showed a breeding season effect in western grey kangaroos. The raw values are shown for clarity; transformed values were used for statistical analyses. Figure 4. View largeDownload slide Three traits that showed a breeding season effect in western grey kangaroos. The raw values are shown for clarity; transformed values were used for statistical analyses. Relationships between muscularity and sperm competition traits There were positive correlations between muscularity and the masses of the penis, urethral bulb, crural bulb, and bulbourethral glands 2 and 3 (Fig. 5; Table 2). A negative relationship between spermatozoa head length and velocity indicated spermatozoa with shorter head length moved faster (Fig. 6A). Males that were more muscular had slower spermatozoa with longer head lengths (Fig. 6B, C; Table 2). There were no correlations between muscularity and testes or epididymis mass. Figure 5. View largeDownload slide Traits that showed a correlation with the index of muscularity in western grey kangaroos. BG, bulbourethral gland; UB, urethral bulb; CB, crural bulb. Figure 5. View largeDownload slide Traits that showed a correlation with the index of muscularity in western grey kangaroos. BG, bulbourethral gland; UB, urethral bulb; CB, crural bulb. Figure 6. View largeDownload slide Relationships between (A) sperm velocity and head length and the correlation between these sperm traits with the index of muscularity (B and C) in western grey kangaroos. Figure 6. View largeDownload slide Relationships between (A) sperm velocity and head length and the correlation between these sperm traits with the index of muscularity (B and C) in western grey kangaroos. Other sperm traits (motility, progressive motility, average midpiece length, average tail length, average total length) showed no relationships with season, body mass or muscularity (Table 2). There were no significant correlations between tissue density of FSH-R (FSH-R/% seminiferous tubule epithelium) and testes mass but there was a negative relationship between LH-R and testes mass (P = 0.035). FSH-R density was not significantly correlated with any of the sperm traits, while LH-R density was positively correlated with average spermatozoa head length. DISCUSSION There was no clear support for trade-offs in investment between pre- and post-copulatory traits by male western grey kangaroos. Kangaroos that were more muscular had spermatozoa with longer head lengths that were ultimately slower, which would make them less competitive. However, there were also correlations between muscularity and the masses of bulbourethral glands 2 and 3, the penis and muscles of erection (urethral bulb and crural bulb), but these were positive relationships, and therefore did not indicate a trade-off but rather a similar direction of selection acting on these traits. The forelimb musculature of western grey kangaroos shows marked development in males but not females (Jarman, 1983, 1989, 1991; Warburton et al., 2013), with positive allometry (β = 1.92–2.39) of these muscles in males. The role of these muscles is assumed to be in ritualized fighting, displaying to females and coercing females to mate, and therefore the muscles are subject to sexual selection mechanisms (Clutton-Brock & Parker, 1995). There was an apparent trade-off in muscle development with body size; it was not the heaviest males that showed the greatest muscularity, but rather intermediate-mass males (Fig. 3). Body mass also plays an important role in securing matings, with larger males dominating access to receptive females (Croft, 1989; Miller et al., 2010). The heaviest males can therefore afford to rely on their size alone to impress females, out-compete rivals or coerce females (or all three tactics), while the lighter adult males may still need to engage in combat to ensure access to females (Rioux-Paquette et al., 2015). Interestingly, we found a significant inverse relationship between LH-R density and body mass, where the largest males had the lowest density of LH-R. Because testosterone is (mainly) produced by testicular Leydig cells in response to LH, the lower LH-R for larger males suggests they also have lower circulating testosterone levels. Alternatively, studies have shown a loss of testicular LH receptors in rats after systemic administration of LH (Hsueh, Dafau & Catt, 1976, 1977), suggesting that the larger male kangaroos may have higher LH concentrations and that testosterone secretion was being homeostatically regulated at the level of the target (Leydig) cell through a reduction in testicular LH receptors. Unfortunately, we could not collect blood samples to measure LH or testosterone concentrations for logistical reasons due to the method of culling, but a study with male eastern grey kangaroos found that males that sire offspring have significantly higher testosterone concentrations than non-sire males (Miller et al., 2010). The role of the three pairs of bulbourethral glands in marsupials (Rodger & Hughes, 1973) has not yet been clarified, but it is suspected that they contribute to formation of a post-copulatory ‘plug’. The semen of macropodids coagulates rapidly after ejaculations, and forms a plug in the female genital tract (Taggart et al., 1998; Paris et al., 2005). The plug could act to prevent spermatozoa draining from the female, keep the ejaculate near the cervix, or act as a sperm reservoir (Taggart et al., 1998). In many taxa, plugs act as barriers to the sperm of subsequent males, which may assist in providing a first male advantage (e.g. Munroe & Koprowski, 2012), reducing selective pressure on investment in sperm competition traits (Lemaître et al., 2011). We found shorter spermatozoa head length and fewer abnormalities for animals sampled in the breeding season, which may reflect seasonal variation in sperm maturation and storage. We did not record correlations with muscularity for the other spermatozoan measurements. This may reflect greater variation in our data due to specimen handling, as we could not control for the time between death of the animal and collection of spermatozoa due to our opportunistic acquisition of cadavers. We did not flush epididymides consistently with known volumes, which would have been required to allow accurate sperm counts. Sperm counts may have revealed more informative relationships with muscularity measures. In addition to the seasonal changes in sperm morphology, we found marked seasonal changes in prostate mass, which was 2.5 times greater during the breeding season (but showed no relationship with body mass). The prostate produces the seminal plasma that supports and nourishes the spermatozoa (Taggart et al., 2005). Enlargement of the prostate during the breeding season would therefore facilitate semen production or maintenance of a fertile ejaculate. CONCLUSION Increased body size in kangaroos increases nutritional requirements and will therefore require larger home-ranges (Norbury, Sanson & Lee, 1989). Male kangaroos that invest in costly tactics such as increased body size and muscularity ‘thereby put themselves at risk if environmental conditions deteriorate’ (Dawson, 2002: 87) and are more vulnerable to stochastic environmental events (Robertson, 1986; Norbury et al., 1988; Coulson, 2006). Food restriction will therefore limit the benefits of increased muscularity in larger males. Competing natural and sexual selective forces are therefore likely to be evident for species such as kangaroos, modifying differential investment in various morphological traits. While we found some evidence of trade-offs relating to male muscularity, there are evidently complex interactions between morphological adaptations, sperm competition and non-sperm components of the ejaculate at play. Our results emphasize the importance of simultaneously studying a range of pre- and post-copulatory mechanisms by which sexual selection could act in order to capture the relative importance of different traits and understand their interplay (Parker, Lessells & Simmons, 2013). Perhaps this finding highlights the complex relationship between the efficacy of pre- and post-copulatory reproductive tactics, and warns against simply assuming that obvious physical traits will necessarily translate into improved reproductive success. SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article at the publisher’s web-site: Figure S1. Photographs of the western grey kangaroo, Macropus fuliginosus. ACKNOWLEDGEMENTS We thank D. Nottle, L. Boston, J. Hong and M. Gibberd for specimen acquisition and technical support, and D. Supreme, K. Napier, V. Lenard and C. Flandrin for help with dissections. Financial support was provided by Murdoch University to all authors; we have no competing interests to declare. We thank G. Coulson and the anonymous reviewers for their constructive comments on the manuscript. The project was conducted under the approval of Murdoch University Animal Ethics Committee (Cadaver Notification) and a Department of Parks and Wildlife Regulation 17 licence SF009843. 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