Advertisement call and genetic structure conservatism: good news for an endangered Neotropical frog

Introduction

The greatest threat to endangered amphibians is habitat change caused by anthropogenic activities, which alters resource availability, environmental quality, and ecological processes (Metzger, 2001; Stuart et al., 2004). Such negative impacts have important implications for organisms that face new selective pressures exerted by habitat conversion (Forman, 1995). In addition, habitat fragmentation causes isolation of populations, and places them at risk of extinction towing to demographic stochasticity, genetic depression, social dysfunction, and exogenous factors such as strong climatic variations and disasters (Simberloff, 1986). Therefore, it is predicted that species affected by these changes would (1) migrate to appropriate adjacent areas; (2) undergo local decline and extinction; or (3) undergo local adaptation.

Surprisingly, several species thrive in modified sites even after profound anthropogenic transformation. Because these landscapes often exhibit physical, climatic, and biological (e.g., species assemblage) shifts, the ability of the remaining species to persist is likely a consequence of phenotypic plasticity in traits such as behavior, morphology, and reproduction (Mayr, 1963; Pulido & Berthold, 2004; Merckx & Dyck, 2006).

One of the most important phenotypic traits in evolutionary studies of anurans is the male advertisement call, because components of these calls are fundamental to species recognition and mate choice, and are thus under sexual selection (Ryan, 1991; Wycherley, Doran & Beebee, 2002; Smith, Osborne & Hunter, 2003; Kaefer & Lima, 2012; Grenat, Valetti & Martino, 2013). Moreover, calls are subject to natural selection over larger geographic ranges, mainly when populations are isolated by physical barriers (Simões et al., 2008; Kaefer, Tsuji-Nishikido & Lima, 2012; Tsuji-Nishikido et al., 2012). Because anurans tend to not disperse over long distances (Blaustein, Wake & Sousa, 1994; Tozetti & Toledo, 2005; Lougheed et al., 2006), sexual phenotypic traits in anurans are likely influenced by local environmental conditions (Bosch & De la Riva, 2004; Ey & Fischer, 2009).

The anuran advertisement call is a multidimensional signal that can be viewed as a collection of spectral and temporal acoustic traits that are influenced, for example, by body size, air temperature, and social context (Wells & Taigen, 1986; Gerhardt, 1991; Bee, 2002; Gerhardt & Huber, 2002; Wong et al., 2004; Toledo et al., 2015a). Therefore, sexual selection, habitat structure, and climatic conditions might all cause variation in call traits among populations (Jang et al., 2011; Faria et al., 2009; Kaefer & Lima, 2012; Kaefer, Tsuji-Nishikido & Lima, 2012; Narins & Meenderink, 2014). In addition, other biotic processes, such as interspecific acoustic interactions, which generate distinctive background noise, can affect call variation among populations (Littlejohn, 1976; Höbel & Gerhardt, 2003).

Different traits of advertisement calls may have distinct roles in anuran communication and, therefore, may evolve by distinct selective pressures (Cocroft & Ryan, 1995; Erdtmann & Amézquita, 2009; Goicoechea, De La Riva & Padial, 2010). As a result, call traits should vary in unique and predictable ways. Gerhardt (1991) classified these traits as static or dynamic acoustic traits. Typically, spectral acoustic traits show low variability (static) and are related to conspecific recognition. Consequently, static traits are subject to stabilizing or weakly directional selection by female choice. On the other hand, most temporal acoustic traits show higher variability (dynamic) and are thought to indicate a male’s investment in reproduction; these may be subject to directional selection by females for values above species means (Gerhardt, 1991; Gerhardt & Bee, 2007).

However, acoustic variation is not always related to genetic variation at the population level (Heyer & Reid, 2003; Lougheed et al., 2006; Kaefer et al., 2013). In these cases, despite some phylogenetic signal being recorded in vocalizations (Erdtmann & Amézquita, 2009; Goicoechea, De La Riva & Padial, 2010; Tobias, Evans & Kelley, 2011; Gingras et al., 2013), the evolution of genotypes and phenotypes (as acoustic traits) may be decoupled (Lougheed et al., 2006) or asynchronous (Kaefer et al., 2013).

Herein, we speculated that historical modification of landscapes by agricultural crops has created barriers among persistent populations, and affected phenotypic and genetic traits in an endangered (Assembleia Legislativa do Estado de São Paulo, 2014) Neotropical frog, Proceratophrys moratoi. We tested the hypothesis that unique selective pressures among these remaining populations have led to divergence in acoustic traits and increased genetic structure.

Methods

Sites

We studied six populations of P. moratoi, which represent almost its entire known geographic distribution (Martins & Giaretta, 2012). We sampled the populations in two southeastern Brazilian states (Fig. 1): São Paulo (Avaré, Bauru, Itirapina and São Carlos) and Minas Gerais (Ituiutaba and Uberlândia). These regions represent an important center of agricultural and livestock production (Ministério do Meio Ambiente, 2005) with a remarkable history of land use modification and natural habitat devastation (Dean, 1995). With the exception of the populations from Itirapina, Bauru, and Uberlândia, which were in protected natural reserves, all populations inhabited modified landscapes.

Results

Acoustic similarities and variability

The structure of calls from all individuals presented the same pattern of a single periodic pulse train (Fig. 2A). However, we found slight differences in spectral and temporal traits among calls from distinct localities (Table 1). Male calls from Avaré had the lowest frequencies, whereas calls from Ituiutaba had the highest frequencies. The individual from São Carlos presented the longest calls, whereas the individual from Bauru emitted the shortest calls. Males from Itirapina emitted calls with the highest pulse rates; the call from the male from Avaré had the lowest pulse rate. Temporal traits of the advertisement call presented high coefficients of variation among males (above 12%), and were considered dynamic (Fig. 2B). Among the spectral traits, frequency range showed the highest coefficient of variation among males, whereas the other three spectral traits presented an intermediate variation (between 5 and 12%; Fig. 2B). All call traits presented low variation within males, with CVs of lower than 8% (Fig. 2C), and the majority was considered static, with < 5% variation.

Table 1:

Acoustic traits (mean ± SD, range) of seven populations of Proceratophrys moratoi from southeastern Brazil and two close species as outgroups.

Data from the population of Botucatu were extracted from Brasileiro, Martins & Jim (2008).

Groups	Population (n = calls, M = males)	Frequency range (Hz)	Minimum frequency (Hz)	Peak of dominant frequency (Hz)	Maximum frequency (Hz)	Call duration (s)	Pulses per note	Pulses rate (p/s)
Proceratophrys moratoi	Avaré (n = 8; M = 1)	291 ± 20 (258–301)	980 ± 20 (947–990)	1,184 ± 23 (1,163–1,206)	1,270 ± 23 (1,249–1,292)	0.297 ± 0.01 (0.277–0.315)	20 ± 0.9 (19–22)	69 ± 1.6 (66–71)
	Bauru (n = 29; M = 1)	423 ± 23 (387–474)	1,029 ± 13 (990–1,034)	1,314 ± 73 (1,077–1,378)	1,452 ± 20 (1,421–1,464)	0.227 ± 0.02 (0.160–0.260)	21 ± 2.2 (15–24)	92 ± 2.8 (81–96)
	Botucatu (n = 59; M = 2)	730	928	1,348 ± 86.6 (1,153–1,420)	1,659	0.207 ± 17.6 (146–238)	17.5 ± 1.5 (12–20)	–
	Itirapina (n = 78; M = 3)	353 ± 32 (281–388)	1,092 ± 34 (1,077–1,206)	1,317 ± 38 (1,265–1,421)	1,445 ± 27 (1,406–1,507)	0.245 ± 0.02 (0.183–0.288)	23 ± 2.4 (17–27)	94 ± 3.2 (85–103)
	Ituiutaba (n = 54; M = 2)	433 ± 66 (301–517)	1,129 ± 57 (1,077–1,249)	1,440 ± 26 (1,378–1,464)	1,562 ± 19 (1,550–1,593)	0.240 ± 0.01 (0.196–0.263)	19 ± 2 (14–22)	81 ± 4.9 (67–87)
	São Carlos (n = 26; M = 1)	288 ± 20 (258–301)	1,206 ± 0 (1,206)	1,386 ± 24 (1,335–1,464)	1,494 ± 20 (1,464–1,507)	0.307 ± 0.02 (0.274–0.382)	25 ± 1.4 (23–28)	83 ± 4.5 (71–89)
	Uberlândia (n = 141; M = 7)	343 ± 95 (215–474)	1,054 ± 70 (947–1,206)	1,286 ± 90 (1,120–1,464)	1,397 ± 92 (1,249–1,550)	0.262 ± 0.03 (0.186–0.316)	18 ± 1.3 (15–22)	71 ± 9.5 (60–97)
Outgroup	Proceratophrys boiei (n = 5; M = 1)	577 ± 38 (517–603)	474 ± 0 (474)	637 ± 19 (603–646)	1,051 ± 38 (990–1,077)	0.743 ± 0.05 (0.666–0.795)	32 ± 1.4 (30–34)	43.1 ± 1.1 (42–45)
	Odontophrynus americanus (n = 15; M = 2)	287 ± 24 (234–328)	681 ± 24 (656–703)	825 ± 43 (750–890)	968 ± 38 (937–1,031)	0.664 ± 0.10 (0.508–0.816)	57 ± 4.6 (49–65)	86 ± 9.5 (79–103)

DOI: 10.7717/peerj.2014/table-1

Cluster analysis placed O. americanus and P. boiei as outgroups in relation to the focal P. moratoi individuals (Fig. 3). Individuals were not grouped by population, except for the males from Itirapina. The male from Avaré had the most distinct call, and was grouped with two individuals from Uberlândia. However, other males from Uberlândia were placed in the other two major groups, and individuals were not organized according to geographical distance among populations. This was confirmed by the absence of a correlation between geographical distance and the acoustic distance between populations (r = −0.23; p = 0.73).

Haplotype network and genetic distance

We found 7 haplotypes in the 26 partial sequences of the mitochondrial 16S gene (Fig. 4). Most haplotypes (H1–H4) were shared among multiple populations, but three haplotypes (H5–H7) were found in one individual each, and were limited to Uberlândia (H5, H6) or Ituiutaba (H7). Genetic distances of P. moratoi averaged 0.2% (0.0–0.4) between populations (Table 2) and 0.25% (0.0–0.5) within populations. Acoustic and geographical distance between populations was not correlated with genetic distance (r = −0.32; p = 0.86; and r = −0.32; p = 0.87).

Table 2:

Genetic distances (p-distance) based on 16S mitochondrial genes between individuals of six Proceratophrys moratoi populations in the upper matrix and the respective geographic distance (in km) in the lower matrix.

Interpopulation variation (%)							Intrapopulation variation (%)
	Itirapina	São Carlos	Bauru	Avaré	Uberlândia	Ituiutaba
Itirapina	–	0.2	0.4	0.2	0.2	0.0	Itirapina	0.0
São Carlos	21.65	–	0.3	0.3	0.2	0.3	São Carlos	0.1
Bauru	114.54	116.59	–	0.2	0.4	0.4	Bauru	0.2
Avaré	129.93	141.36	59	–	0.4	0.3	Avaré	0.5
Uberlândia	354.97	331.88	375.50	437.27	–	0.3	Uberlândia	0.3
Ituiutaba	392.80	372.92	375.45	437.25	119.44	–	Ituiutaba	0.1

DOI: 10.7717/peerj.2014/table-2

Discussion

In the present study, we found a common structural pattern (a single periodic pulse train; Fig. 1) for all individuals, which is consistent with the findings of previous reports on the advertisement call of P. moratoi (Brasileiro, Martins & Jim, 2008; Martins & Giaretta, 2012).

The variability in the advertisement call of P. moratoi follows a general pattern among anurans; spectral traits exhibit lower variation than do temporal ones, with the exception of frequency range (Gerhardt, 1991; Gerhardt & Huber, 2002). The low coefficients of variation in call traits we observed among males (showing highly stereotyped signals) could be attributed to stabilizing selection (Kaefer & Lima, 2012; Kaefer, Tsuji-Nishikido & Lima, 2012), which is usually a result of generalized female choice (Jennions & Petrie, 1997). Alternatively, it could reflect the absence of selection and the presence of neutral/stochastic processes (Erdtmann & Amézquita, 2009; Kaefer et al., 2013; Toledo et al., 2015b). Because sexual signals carry important information for mate recognition (Ryan, 1991), spectral traits would not be expected to diverge rapidly. Temporal traits, which are generally dynamic, have been shown to vary with social or environmental conditions (Bosch & de la Riva, 2004; Ey & Fischer, 2009). For example, some species respond to vocal interactions and chorus composition with rapid temporal adjustments in their calling behavior (Schwartz, 2001). Therefore, temporal traits such as pulse rate and call duration may be affected by social context. In species wherein females make choices based on temporal traits alone (Littlejohn, 1965), these temporal traits would be predicted to minimize the patterns observed in spectral traits. However, we observed that mate choice by acoustic properties remains to be tested, since we do not know how female P. moratoi individuals select males.

Habitat structure, background noise, and other environmental differences are pivotal in the evolution of acoustic communication in frogs (Goutte, Dubois & Legendre, 2013; Schwartz & Bee, 2013). These factors certainly contribute to regional divergence in call traits among individuals (Amézquita et al., 2006). Each reproductive environment could present distinct species composition and considerably different acoustic qualities. Based on this ecophenotypic hypothesis, we expected that local pressures would modulate call features in P. moratoi males from distinct localities, because populations of this threatened species surrounded by human-transformed landscapes could show low connectivity. In contrast, cluster analyses failed to group individuals by population (geographical context). Taken together, these results indicate minimal pressure for signal divergence, which could be explained by the following, non-exclusive hypotheses: (1) the reproductive environment in the sampled localities is similar with respect to habitat structure and background noise; (2) the female choice drives stable selection, which equalizes the general acoustic features of males from different localities; (3) the populations were recently connected, presenting traces of recent genetic flow; and (4) random evolutionary processes act on the calls (Toledo et al., 2015b). We did not test these hypotheses, but it is unlikely that populations were recently connected, as genetic distance was not correlated with acoustic distance among populations. Furthermore, although some studies have reported a correlation between genetic and acoustic distances (Smith, Osborne & Hunter, 2003; Amézquita et al., 2009), many others have shown that geographical variation in sexual signals and genetic distances among populations do not co-vary (Heyer & Reid, 2003; Lougheed et al., 2006; Pröhl et al., 2007). In these cases, it is possible that evolution has been decoupled for genotypic and phenotypic features (Lougheed et al., 2006). Cluster analysis demonstrated that a phylogenetic signal in anuran advertisement call (as a phenotypic trait) might not evolve as rapidly as DNA differences appear, a finding corroborated by Kaefer et al. (2013). Consequently, our results suggest that a phylogenetic signal would be apparent only when higher taxonomic levels are compared, for example, different species, genera, or families. This finding could be explained, in part, by the conservative nature of the molecular marker used—the 16S gene—which is commonly employed to separate different species (Fouquet et al., 2007; Brusquetti et al., 2014; Yang et al., 2014; Lourenço et al., 2015).

Although our dataset is limited, we observed that acoustic and genetic variation appears to be conserved among individuals distributed across human-altered landscapes. Our preliminary results showed similar call types and genotypes (also presenting low genetic divergence) among different populations. Such reduced acoustic and possible genetic structure could be considered in future conservation actions; for example, these findings suggest that communication barriers (prezygotic) would pose no obstacle to reproduction (Dobzhansky, 1951; Tucker & Gerhardt, 2011) if a reintroduction program were initiated for this species. Nevertheless, playback experiments are required for testing this hypothesis first. From the genetic point of view, if the actual molecular marker used in the present study could represent the genomes of all individuals, the genetic barrier (postzygotic) would pose no obstacle to reproduction too (Dobzhansky, 1951; Tucker & Gerhardt, 2011). The type locality of this species (Botucatu) is still preserved, and a reintroduction could be considered after complementary genetic, natural history, and experimental research. The causes of the decline of this population are unknown, and therefore, a reintroduction initiative could also aid in understanding past decline (if the cause is still active) and help to prevent further decline here (for example, of Bokermannohyla izecksohni—another species that could be threatened in Botucatu; L. F. Toledo & C. Z. Torres, 2015, unpublished data) and elsewhere. Thus, the findings of the present study add to the growing body of literature supporting P. moratoi as a potential candidate for conservation actions, although additional work is necessary before an action plan could be initiated.