Non-reproductive male cane toads (Rhinella marina) withhold sex-identifying information from their rivals

Abstract

A male cane toad (Rhinella marina) that mistakenly clasps another male (rather than a female) in a sexual embrace (amplexus) can be induced to dismount by a male-specific ‘release call'. Although that sex-identifying system can benefit both males in that interaction, our standardized tests showed that one-third of male cane toads did not emit release calls when grasped. Most of those silent males were small, had small testes relative to body mass, and had poorly developed secondary sexual characteristics. If emitting a release call is costly (e.g. by attracting predators), a non-reproductive male may benefit by remaining silent; other cues (such as skin rugosity) will soon induce the amplexing male to dismount, and the ‘opportunity cost’ to being amplexed (inability to search for and clasp a female) is minimal for non-reproductive males. Hence, male toads may inform other males about their sexual identity only when it is beneficial to do so.

1. Introduction

In many animal species, males exchange information with their rivals in the course of reproductive activities. For example, sexually divergent chromatic, morphological, acoustic and pheromonal traits can function to repel other males via displays or combat bouts [1]. In a small subset of taxa, some males abandon these male-specific traits and instead adopt female-like morphologies, physiologies or behaviours in ways that deceive rivals. For example, male gartersnakes (Thamnophis sirtalis parietalis) produce female-like skin lipids that induce courtship by other males, thereby providing the female-mimic with warmth, massaging and protection against predation [2]. In pied flycatchers (Ficedula hypoleuca), a female-like morphology may advantage males by confusing other males, and enabling the female-mimic to control the timing of battles [3].

Although rare, female mimicry has been reported in a diverse array of vertebrate lineages (e.g. fishes [4], reptiles [2,5,6], birds [7,8], mammals [9]). We provide the first example to our knowledge in anurans (but see [10,11], for examples in salamanders). Many anurans (frogs and toads) are sexually dimorphic not only in body size [12] but also in traits such as dorsal colouration and skin rugosity [13], and especially, in vocal communication [14]. Male frogs produce loud advertisement calls to attract reproductive females to oviposition sites, whereas females typically are mute [15]. When a reproductive female arrives, the male seizes her from above and behind, wrapping his forelegs around either her armpits or waist. This amplexus position places the male's vent close to the female's vent, increasing the probability that his sperm will fertilize her eggs as they are extruded [15–17].

Male anurans also produce another type of vocalization, less obvious than the advertisement call. Mate-searching male anurans are notoriously indiscriminate, clasping any suitably sized object that they encounter [18,19]. High concentrations of males around spawning sites means that many amplexus attempts are directed towards other males, thereby incurring costs to both males involved. A male that has seized another male cannot simultaneously clasp a female; and a male that has been seized cannot move about or feed [20]. That lose–lose outcome has favoured the evolution of species-specific ‘release calls' by the animal that has been seized, alerting an amplectant male to the fact that he has mistakenly grasped another male. Females of many anuran species lack vocal cords, rendering the ‘release call' a reliable indication of an individual's sex [21].

Although release calls have been experimentally demonstrated to terminate amplexus in cane toads [21], most literature on this phenomenon assumes that all male anurans emit release calls when seized by another male (as would be expected if sex identification benefits both participants). Indeed, researchers attempting to determine the sex of adult anurans often do so by holding the animal around the body, simulating an amplexing male, in an attempt to elicit the release call [15]. However, the assumption that all males emit release calls appears not to have been tested. During fieldwork on cane toads (Rhinella marina), we noted that some males refrained from calling even when seized. We conducted trials to clarify that variation, and to identify possible evolutionary advantages and disadvantages of this behaviour.

2. Material and methods

(a). Study species

Cane toads are large bufonids native to Latin America, but are now widespread in Australia [22]. Adult females grow larger than conspecific males, and they have relatively smooth brown skin and no nuptial pads on the fingers [22]. When seized in an amplexus clasp, females do not emit release calls (even at frequencies inaudible to humans [21]). Male cane toads tend to be more rugose than females, to be yellow rather than brown, and to develop nuptial pads on the thumbs when reproductive [22] (figure 1; electronic supplementary material, table S1).

Figure 1.

Examples of sexually dimorphic traits in cane toads, Rhinella marina. Panels (a) show blackness of nuptial pads. The toad on the left is given a score of 0, the toad in the centre is given a score of 1, and the toad on the right is given a score of 2. Panels (b) show skin colour. The toad on the left is given a score of 0, the toad in the centre is given a score of 1, and the toad on the right is given a score of 2.

(b). Location and timing of sampling

We collected cane toads from invasive populations within 50 km of the city of Darwin from October 2008 to February 2011 (see electronic supplementary material, table S2 for sample sizes). Our sample included both juvenile and adult toads (in this population, males mature at around 90 mm snout–urostyle length, SUL) [23]. We classified seasons into four categories: the wet (December–February, the time of monsoonal rains), the build-down (March–May, as the rains cease), the dry (June–August, with little rain) and the build-up (September–November, as storm clouds gather and produce occasional heavy downpours; see [24] for further descriptions).

(c). Collection of data

Toads were captured by hand at night and returned to our field laboratory in dampened cloth bags. The following day we picked up each toad with thumb and forefinger on either side of the armpits (mimicking the stimulus from an amplectant male) and recorded whether the animal remained silent, gave a soft call or gave a loud call (within the next 5 s). We also scored sexually dimorphic traits, as follows: rugosity of the skin, skin colour and elaboration of nuptial pads on the front feet (all on 3-point scales, see figure 1). The animals were then measured (SUL), weighed to 0.1 g, and killed humanely by interperitoneal injection of sodium pentobarbital. We dissected the animals via midventral incisions and the testes were removed, blotted dry, and weighed to 0.001 g (FX-200i WP, A&D Company, Tokyo, Japan).

(d). Statistical analyses

Using JMP 14 (SAS Institute, Cary, NC), we conducted a series of analyses combining continuous variables and ordinal variables. To quantify relative testis mass, we used residual scores from the linear regression of testis mass (combined) on total body mass. We used ANOVA to compare mean values of morphological and behavioural traits among seasons. We used ordinal logistic regression (with our release-call score as the dependent variable) to investigate links between release calls and other traits, both separately and in a regression with all of the morphological features as independent variables. To ensure that these results were not an artefact of combining juvenile and adult animals, we repeated the tests separately for each class (i.e. less than 90 mm versus greater than 90 mm SUL [23]). Almost all results yielded conclusions for both size classes identical to those for the combined sample (table 1).

Table 1.

Results of statistical tests of the association between morphological traits of male cane toads, and their propensity to emit a ‘release call' when seized. The table shows the likelihood ratio χ² values for 625 toads (96 juveniles and 529 adults). Degrees of freedom (d.f.) = 1 for continuous traits (snout–urostyle length and relative testis mass) and d.f. = 4 for ordinal traits (indices of nuptial pad development, skin rugosity and skin colour). All χ² values are significant at p < 0.001 except for body condition in juvenile toads (χ² = 0.20, 1 d.f., p = 0.65; italics).

morphological trait	all toads	juveniles	adults
snout–urostyle length (mm)	137.96	10.71	14.68
testes mass relative to body mass	67.77	14.10	56.06
nuptial pads	335.04	36.75	77.89
skin colour	189.18	38.78	176.55
skin rugosity	275.30	26.64	144.06
body condition	19.24	0.20	18.83

To assess the hypothesis that the development of sexually dimorphic traits is affected by testis size, we also conducted ordinal regressions with relative testis mass (see above) as the predictor variable. Preliminary analyses revealed similar patterns of covariation among variables across all sites and seasons, so for simplicity, we report the results of analyses based on the combined dataset.

3. Results

We obtained data on 625 male cane toads, of which we dissected 487 (132 to 184 per season; see electronic supplementary material, table S2). Mean body lengths did not differ significantly among seasons (F_3,649 = 1.73, p = 0.16), nor did the propensity to emit release calls (F_3,621 = 0.43, p = 0.73) or skin colour (F_3,637 = 0.75, p = 0.52). However, toads during the wet season were heavier (F_3,649 = 8.76, p < 0.0001), with larger nuptial pads (F_3,637 = 3.26, p < 0.025) and with larger testes relative to body mass (residual score: F_3,649 = 21.08, p < 0.0001). Overall, strong release calls were elicited from about half of the males that we tested (305 of 625, = 49%), with a further 18% (N = 115) producing a weak but detectable call. The remaining 205 males (33%) made no audible sound, but most vibrated the sides of the body in a way similar to that seen in female cane toads that are amplexed.

The propensity to give a release call was highly non-random. In an ordinal logistic regression, a male toad that was grasped was less likely to give the release call if it was smaller, had smaller testes relative to body mass, had poorly developed nuptial pads, had smooth skin and was brown (female-like) rather than yellow in colour (table 1). Those patterns were highly significant both among juvenile males and among adult males (table 1). In logistic regressions with each of these factors entered as the sole independent variable, all significantly predicted the probability of giving a release call (all p < 0.0001; figure 2). An adult toad's response was significantly affected by its body condition whereas this effect was not significant in juveniles but nonetheless, was significant overall (table 1). The link between body size and calling was not a simple consequence of a size threshold for reproduction: the range of male body sizes (SULs) over which we recorded release calls was 81.5–128.4 mm, whereas the range over which we failed to elicit calls was 51.1–131.5 mm. That is, many adult (greater than 90 mm SUL) toads failed to call, whereas some juveniles (less than 90 mm SUL) did so.

Figure 2.

Relationships between the propensity of a male cane toad to emit a release call when grasped (0 = no call, 1 = weak, 2 = strong) and morphological traits of the males involved. (a) Body size (snout–urostyle length, millimetres), (b) elaboration of nuptial pads, (c) dorsal colouration, (d) skin rugosity. See text §2c and figure 1 for explanation of scoring. Graphs show mean values and associated standard errors.

The relative testis mass (residual from linear regression of testis mass to body mass) was significantly positively associated with all of the reproductive-biology variables that we tested (versus call score χ² = 67.77, 1 d.f., p < 0.0001; versus nuptial pads χ² = 117.72, 1 d.f., p < 0.0001; versus skin colour χ² = 58.80, 1 d.f., p < 0.0001; versus skin rugosity χ² = 123.77, 1 d.f., p < 0.0001), consistent with endocrine control of secondary sexual characteristics.

4. Discussion

In many species of frogs and toads, males give specific ‘release calls' that terminate amplexus attempts by other males, whereas females are unable to give such calls [21] (but see [25]). Our data suggest that this dichotomy is an oversimplification: males do not necessarily emit release calls when seized, and their likelihood of doing so is linked to factors such as body size, relative testis size and the elaboration of sexually dimorphic morphological features. Thus, this sex-identifying call is facultative. Unfortunately for its use as a means of sex identification (by researchers), males that are the least likely to emit the release call are also the least likely to exhibit other sexually dimorphic traits.

Why did smaller, less reproductively active male toads fail to emit a release call when seized? Rapid termination of inappropriate amplexus can benefit not only the male that initiates this behaviour (clinging to a male rather than a female does not enable him to fertilize a clutch, and prevents him from amplexing any other toad) but also the male that is amplexed (carrying another toad impairs locomotor ability, feeding and probably other functions as well [20]). Under this scenario, we would expect all male anurans to emit the release call if they are seized by another male.

But male tactics may be more complex than suggested by this classical scenario. For a large male in peak reproductive condition, being clasped by a rival is a major cost because it prevents him from finding and clasping a female. For a smaller less reproductively active animal, the cost may be lower. He is unlikely to be engaged in reproductive behaviour and the amplexing male will release him fairly soon anyway, even if he fails to emit a release call (note that in [21], males that were experimentally muted (and thus could not give the release call) were nonetheless released sooner than were females). Ancillary costs of being amplexed, such as an inability to move around and feed [20], likely are trivial over a short period of time. If the sexual target is at risk of being drowned by an amplectant male toad [26], the clasped male can induce his suitor to dismount immediately by emitting the release call.

The energy cost of producing a release call is presumably trivial, so why would non-reproductive males tolerate rather than terminate amplexus? One possibility is that predators key in on acoustic signals from anurans to locate their prey. The toxicity of cane toads discourages attempts by eavesdropping predators [27] but some taxa nonetheless consume them. For example, both in their native range and their introduced range in Australia, cane toads are targeted by nocturnally active rodents [28]. In such a case, the benefit of early termination of amplexus may be outweighed by the risk of attracting a predator.

Our adaptationist hypothesis involves an unusual form of female mimicry by males, whereby the small male deceives his rivals not by adopting a female-specific trait, but instead by refraining from exhibiting a male-specific trait. By contrast, most examples of female mimicry in other organisms involve production of female-like pheromones [2,5] or adoption of female-like morphologies [4,7], postures, or behaviours [6]. Failing to exhibit a pre-existing response (such as the release call) may evolve more easily compared with elaborating a trait not usually present within males. Future work could usefully extend our study to the field, to see how male–male signalling operates within a mating aggregation of anurans.

Ethics

All procedures were approved by the University of Sydney Animal Care and Ethics Committee protocol 6705. No fieldwork permits were required, as this invasive species is not protected under Australian regulations.

Data accessibility

Data available from the Dryad Digital Repository at: https://doi.org/10.5061/dryad.q4t6h2b [29].

Authors' contributions

C.K. gathered data, R.S. analysed data, R.S. and C.K. drafted and revised the paper. Both authors approved the final version of the manuscript and agree to be held accountable for the content therein.

Competing interests

We declare we have no competing interests.

Funding

The work was supported by the Australian Research Council (grant no. FL120100074).