Arboreal Calling Behaviour Across the Range of the Spring Peeper: Potential Consequences and Benefits

Adaptation is a central pillar of speciation as it is a direct outcome of natural selection. Traits that increase the fitness of individuals by enhancing growth, reproductive output, or survival capability are considered adaptive and are often under directional selection. Rates of evolutionary change in these traits may be modulated by other micro-evolutionary forces such as genetic drift and gene flow, which may constrain adaptive divergence if they are antagonistic to the direction of selection (Garant et al. 2007, Wright 1951). However, adaptive divergence of populations may occur despite significant influences of genetic drift (Funk et al. 2016) and gene flow (Fitzpatrick et al. 2015). Such strong selective forces may cause demographic differences among populations to arise and, with sufficient time, even reproductive isolation and population diversification (Carnicer et al. 2012). The evolution of traits involved in mating systems has been a subject of study since Darwin noted the striking ornamentation and mating displays that are evident in nature and that may be under sexual selection (Darwin 1871). Since then, myriad examples of traits under sexual selection have been documented in nature (see Ryan and Keddy-Hector 1992 and references therein), and many empirical examples of sexually selected attributes contributing to reproductive isolation have prompted the wide acceptance of sexual selection as having a major role in speciation (Andersson 1994, Coyne and Orr 2004, Panhuis et al. 2001). In many mating systems, the choosy sex (often the female) imposes selection on traits implicated in mating displays that are expressed in the opposite sex, resulting in it becoming more costly and elaborate (Ryan and KeddyHector 1992, Andersson 1994). This relationship between quality/intensity of the mating display trait and female choice has been hypothesized to be a consequence of natural selection on mate preference that produces a tangible benefit, such as nuptial gifts (Kirkpatrick and Ryan 1991), or may lead to offspring of better genetic quality (Zahavi 1975). Such a relationship may also be a manifestation of runaway selection, where female preference and male trait (for example) are genetically linked (Fisher 1958). Selection on the male trait imposes indirect selection on female preference for that trait, and the two co-evolve in this fashion (Fisher 1958). Last, sensory exploitation has also been proposed as an explanation for the relationship between preferences for male traits (Ryan and Rand 1990, Ryan et al. 1990). This hypothesis requires that females having a sensory preference for some facet of a mating display, where sexual selection favours male traits that exploit this preference (Ryan and Rand 1990). Under this hypothesis, male traits can only evolve within the bounds of the existing female sensory system (Ryan and Rand 1990). These alternative hypotheses are not mutually exclusive and patterns of trait evolution that are consistent with the predictions of one hypothesis may also be explicable, at least in part, by other causal factors (Ryan and Keddy-Hector 1992).

Functional Traits

In addition to traits that directly influence reproductive success and survivability, those that indirectly enhance reproductive success or chance of survival may also be under selection (Arnold 1983, Violle et al. 2007). These traits are considered functional traits and were first recognized through Arnold’s (1983) “performance paradigm”. According to this framework, traits that directly relate to fitness are considered performance traits (Arnold 1983). Traits that modulate the effectiveness of performance traits (therefore only indirectly influencing fitness) are considered functional traits (Arnold 1983). For example, colouration in some species of moths serves as camouflage and thus directly influences fitness by enabling individuals to evade predators (Endler 1984, Poulton 1980). After landing on tree bark, moths will alter their position to enhance crypticity (Kang et al. 2012). In this example, the moth’s colouration is the performance trait, and the behaviour that enhances the performance trait is the functional trait. Functional traits involved in mating displays may be under indirect sexual selection as they influence traits involved in reproduction. For example, male gliding lizards (Draco sumatranus) exhibit body-modifying behaviour in response to sun position to enhance the visibility of their colourful dewlap (Klomp et al. 2017). This behaviour increases the conspicuousness of the signal by enhancing its luminance, which may lead to greater conspecific recognition and potential mate detectability (Klomp et al. 2017). Similarly, male tree-hole frogs (Metaphrynella sundana) actively alter the pitch of their call according to the amount of water in the tree cavity they call from to enhance the attractiveness of their mating signal (Lardner and bin Lakim 2002). These studies suggest that successful mating may partially depend on traits associated with the mate recognition system that are ancillary to the main display. It is therefore important to consider the evolution of potential functional traits as well as performance traits when investigating adaptive trait evolution.

Trade-offs

Many selected traits involved in reproduction may be subject to trade-offs influenced by other aspects of a species’ ecology (Ryan 1986, 1988, Simmons and Emlen 2006, Evans 2010, Engqvist 2011). Such factors may not act exclusively and many disparate factors could shape variation in the same trait. Gamete quantity and size is an iconic example of a trade-off that was first modelled by Smith and Fretwell (1974). Smith and Fretwell (1974) determined that the optimal size and number off eggs were determined by offspring fitness as it related to initial investment. Since this seminal work, studies finding evidence for antagonistic selective pressures on traits causing trade-offs have increased (e.g. Ghalambor et al. 2004, Scales and Butler 2015, Blanchard and Moreau 2016). Trade-offs associated with mating displays have also been documented, many of them leading to the notion that traits under sexual selection may be considered honest indicators of fitness. A key example of this is carotenoid-based colouration in birds where the trade-off is hypothesized to be the cost of maintaining bill colouration and immune system functioning. Carotenoids are typically obtained and sequestered from an individual’s diet and it was originally proposed that variation in diet accounted for variation in carotenoid colouration (Endler 1980, Hill 1999). However, carotenoids also serve as an immunostimulant in birds (Chew 1993) and have been found to slow the replication rates of parasites (Baeta et al. 2008). There is thus a trade-off between allocating carotenoids to colour to attract a mate versus allocating them towards combating parasitism; indeed it has been suggested that only males of high quality can both allocate carotenoids to plumage colour and sustain the heavy parasite load associated with it (Blount et al. 2003, Baeta et al. 2008).

Trade-offs in Anuran Calling

As mentioned above, some traits may be subject to multiple trade-offs acting on them at once. Anuran calling is subject to several antagonistic forces of selection leading to trade-offs. Many anuran species form large choruses where males call to attract females and mate choice is primarily determined by male call quality (reviewed in Gerhardt and Huber 2002). Producing and maintaining calls is exceptionally energetically costly for males, with some estimates for gray treefrogs (Hyla versicolor) reaching around 25 times their resting metabolic rate (Taigen and Wells 1985). Therefore, call evolution may be limited by metabolic rate and individuals may experience trade-offs between call performance and maintenance of calling (Reichert and Gerhardt 2012). Like many traits that increase conspicuousness, male calls may also increase the risk of predation or parasitism (Tuttle and Ryan 1981, Bernal et al. 2006, Page and Ryan 2008). Studies on the calls of túngara frogs (Engystomops pustulosus) have shown that predators, such as bats, and blood-sucking flies actually preferentially predate/parasitize calling individuals (Tuttle and Ryan 1981, Bernal et al. 2006, Page and Ryan 2008). The bat predators have even been shown to use the advertisement calls of the males to locate them within a chorus (Tuttle and Ryan 1981, Page and Ryan 2008). Last, signal efficacy relies on receiver’s ability to discriminate; therefore, receiver morphology/ neurophysiology may constrain the evolution of particular call traits (Akre et al. 2011, Schrode et al. 2014). These studies collectively demonstrate the need to consider multiple facets of trait evolution and the forces that may affect it (Gerhardt 1994).

Study Species

The spring peeper (Pseudacris crucifer) is a small tree frog species (Family: Hylidae) that is broadly distributed across eastern North America from the Gulf Coast north to the James Bay lowlands, and from the Atlantic coast inland to west of the Mississippi River (see Figure 1). During the breeding season, males congregate in wetlands, small vernal pools, swamps, marshes, and lake margins to begin calling to attract females (Conant and Collins 1998, Zimmitti 1999). Because of varying seasonality across latitudes, males begin chorusing in late December and early January in the southern parts of the species’ range (e.g. Florida through west Texas), to April and May in the northern extent. Calls are comprised of a single tone “peep” (Taigen et al. 1985) that are on average 87 decibels in amplitude, based on measurements in four populations from New York (Brenowitz et al. 1984). Although female attendance at spring peeper choruses is generally poorly quantified, males remain calling throughout the night and may return to call for several nights (Rosen and Lemon 1974). Male spring peepers are territorial and protect their calling site from competing males using both aggressive vocalizations and physical contact (Rosen and Lemon, 1974). Spring peepers, like many anuran species, may adopt satellite behaviour as an alternative mating tactic where they do not call, but position themselves close to calling males to intercept females (Lance and Wells 1993, Stewart et al. 2017). Figure 1. A: Picture of a calling male spring peeper. Photo taken by N. Cairns. B: Range map of the spring peeper showing the six mitochondrial lineages' range limits. Map taken from Stewart and Lougheed (2013). A B Eastern Interior West Southwest Southeast Texas Spring peeper males can vary their calling behaviour to maximize their calling performance. For example, auditory cues including conspecific advertisement calls, but also aggressive trill calls, and traffic noise, can cause males to alter aspects of the mating call (e.g. call duration), or to respond with an aggressive trill call (Rosen and Lemon 1974, Schwartz 1989, Marshall et al. 2003, Humfeld et al. 2009, Zimmitti 2009). The presence of an “intruder” on a male’s territory, tested using mating call playback, has been shown to cause individuals to respond with aggressive trill calls (Rosen and Lemon 1974, Schwartz 1989, Marshall et al. 2003, Humfeld et al. 2009). Playback calls with longer trill durations elicited the most aggressive calling behaviour (Schwart 1989). Aggressive calls themselves may be plastic, their frequency and structure determined by chorus density and inter-male spacing (Marshall et al. 2003), but whether a male switches from advertisement calling to aggressive calling depends on the social context (Humfeld et al. 2009). Humfield et al. (2009) performed a series of studies to test what intruder male behaviours would elicit aggressive calls. They found that males were more likely to respond aggressively if the intruder male began with advertisement calls, then switched to aggressive calls, rather than the calling aggressively right after the intrusion (Humfeld et al. 2009). Hanna et al. (2014) tested whether traffic noise would cause male spring peepers to modify their calling behaviour and found that background masking noise cause spring peepers to produce calls with shorter duration. They also found that, if the traffic noise masked the high frequencies of the advertisement call, males would produce lower frequency calls. This relationship has been reported for other species of frogs and may indicate a response to urbanization and anthropogenic noise (Cunnington and Fahrig 2010). These studies demonstrate that, although variation in anuran advertisement calls has a genetic basis (Welch et al. 2014), environmental and social cues can elicit plastic vocal responses and associated behaviours. The spring peeper shows marked genetic structure across its geographic range with six mitochondrial lineages (Figure 1) of varying ages (Austin et al. 2002, 2004, Stewart and Lougheed 2013). These mitochondrial lineages are thought to have diverged in geographic isolation beginning in the Pliocene, and while their distributions will have waxed and waned over the ensuing years, they are now in secondary contact in many areas across the species range (Austin et al. 2004, Stewart and Lougheed 2013). Recent work investigating the contact zone dynamics between the two most closely related mitochondrial lineages found evidence of call differences and reproductive character displacement, potentially reinforced by female choice for their natal lineage male advertisement calls (Stewart et al. 2015). There is also evidence of reduced hybrid survival at the larval stage in this contact zone (Stewart and Lougheed 2013), implying a cost to mating with non-natal individuals and an important potential role for calls in identifying appropriate mates. More broadly, advertisement call differences among all mitochondrial lineages, while subtle, are statistically significant (Cicchino et al. unpubl.), again suggesting that advertisement call might be implicated in population divergence. The male calling behaviour of this species has been well-documented (e.g. Rosen and Lemon 1974, Brenowitz et al. 1984, Taigen et al. 1985, Sullivan and Hinshaw 1990, Zimmitti 1999, Marshall et al. 2003); however, studies and field guides provide divergent accounts of preferred or typical calling sites. Some articles and field guides suggest that the species is exclusively terrestrial (e.g. IUCN 2014, Rosen and Lemon 1974, Conant and Collins 1998), calling exclusively from the ground or from floating vegetation. Others report that male spring peepers call both terrestrially and from perches above or near water bodies (e.g. Wygoda 1984, Brenowitz et al. 1984, Dodd 2013). Our combined range-wide observations suggest that this variability in the literature is a product of geographic variation in calling behaviour, with males in southern populations often calling from bushes or tree branches, while their northern counterparts call almost exclusively from the ground (Cicchino, Cairns and Lougheed pers. obs.). Previous empirical studies on spring peeper calls have demonstrated the benefits of calling from elevated perches as it increases call transmission distance thereby presumably increasing the changes of attracting a female (Brenowitz et al. 1984; Parris, 2002). As arboreal calling would appear to provide obvious benefit to males, it is reasonable to ask the question as to why all males do not undertake it.

Objectives and Hypotheses

In this study, I first quantify perching behaviour across the spring peeper’s range and test for differences in prevalence and proportion of arboreal calling behaviour among populations. Finding marked population-level differences, I then investigate factors that may cause or limit this potentially adaptive behaviour. Although past studies have indicated a sound transmission advantage to calling from elevated perches in this species (Brenowitz et al. 1984, Parris 2002), this benefit may not be ubiquitous across its range. For example, perching behaviour may be a way to circumvent increased call degradation in habitats with dense leaf litter, but in habitats with low leaf litter density or sparse vegetation, sound propagation from elevated perches may not differ from ground-level propagation. I thus hypothesize that perching behaviour arises because of local selection for enhanced sound transmission. I predict that, in habitats with greater leaf litter and understory density, perching behaviour will be more prevalent; but in habitats with less leaf litter and sparse understory, this behaviour will not be common. To test this, I performed playback experiments at peak breeding times for local populations across the spring peeper’s range with source representative calls projected at different heights to quantify sound degradation in each habitat. Environmental conditions, such as temperature and humidity, influence many aspects of anuran ecology, including physiological processes, locomotor performance, and behaviour (Rome et al. 1992, Shoemaker et al. 1992). Temperature is also implicated in anuran reproductive success as it is positively correlated with many temporal characteristics of advertisement calls, which females often use to discriminate between males (Gerhardt and Huber 2002). Maximizing call transmission efficacy is critical for individual males realizing mating opportunities, and fine-scale temperature differences between arboreal and terrestrial micro-habitats can drive calling site selection (Höbel and Barta 2014). However, arboreal behaviour may expose individuals to higher wind levels as well as decrease contact with moist vegetation or water, therefore increasing the risk of desiccation. I thus also hypothesize that there can be a physiological cost to arboreal calling in some environments, which may be mitigated by higher air temperatures and humidity in some parts of the range or at different parts of the breeding season, or some combination thereof. I thus expect to find increased prevalence in perching behaviour in populations that typically breed at higher air temperatures and humidity. To address desiccation risk, I performed a study measuring the effect of perch height on evaporative water loss and body temperatures in different environmental conditions using model frogs. In addition to testing for spatial variation in arboreality, I investigate morphological variation that may also be related to arboreal behaviour. Over time, morphological adaptations to enhance arboreal behaviour may have been selected for, which would result in morphological differences among populations. Certain traits may be better suited to arboreal habitats, such as longer forelimbs typical of hylids, although these have been asserted to be for increased dexterity in manipulating prey (Gray et al. 1997). Alternatively, morphological differences may have arisen and thereafter allowed populations to exploit an arboreal niche, which also would result in morphological differences between populations. As the evolution of ecological niche and morphology is often linked (Scharf et al. 2000, Ortiz-Medrano et al. 2016), I hypothesize that there will be observable phenotypic differences among populations that have diverged in their arboreal behaviour.