# Acoustics of parental and hybridogenetic water frogs (Pelophylax: Amphibia: Ranidae): a tool for monitoring an invasive species

Filipe Gomes De Almeida, François Ciavatti, Angélica Pulido, Thomas Gendre, Philippe Geniez, Aimeric Eble, Elise Leblanc, Yoann Mansier, Ivain Martinossi-Allibert, Blaise Raymond, et al.

► To cite this version:

Filipe Gomes De Almeida, François Ciavatti, Angélica Pulido, Thomas Gendre, Philippe Geniez, et al. Acoustics of parental and hybridogenetic water frogs (Pelophylax: Amphibia: Ranidae): a tool for monitoring an invasive species. Amphibia-Reptilia, 2024, 45 (3), pp.333-348. 10.1163/15685381-bja10192. hal-04959451

HAL Id: hal-04959451

https://cnrs.hal.science/hal-04959451v1

Submitted on 24 Feb 2025

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
<figure><img src="image_2.png" /></figure>

<figure><img src="image_3.png" /></figure>

# Acoustics of parental and hybridogenetic water frogs (*Pelophylax*: Amphibia: Ranidae): a tool for monitoring an invasive species

Filipe Gomes de Almeida¹,²,*; François Ciavatti¹,²; Angélica Pulido¹,²,⁴; Thomas Gendre²;

Philippe Geniez³; Aimeric Eble¹,²; Elise Leblanc¹,²; Yoann Mansier¹,²;

Ivain Martinossi-Allibert¹,²; Blaise Raymond¹,²; Adrien Pineau¹,²; Jérémie Demay²;

Pierre-André Crochet¹

1. CEFE, CNRS, Univ Montpellier, EPHE, IRD, 34293 Montpellier, France

2. Conservatoire d'espaces naturels d'Occitanie, 34000 Montpellier, France

3. CEFE, Univ Montpellier, CNRS, EPHE-PSL, IRD, Biogéographie et Ecologie des Vertébrés, 34090 Montpellier, France

4. Department of Ecology and Evolution, University of Lausanne, 1015 Lausanne, Switzerland

*Corresponding author; e-mail: filipegmsalmeida@gmail.com

ORCID iDs: Gomes de Almeida: 0000-0002-6877-7014; Pulido: 0000-0003-0862-4582;

Geniez: 0000-0002-9255-6174; Martinossi-Allibert: 0000-0002-8332-8620; Crochet: 0000-0002-0422-3960

Received 15 January 2024; final revision received 4 July 2024; accepted 22 July 2024;
published online 9 August 2024; published in print 5 September 2024

Associate Editor: Gentile Francesco Ficetola

**Abstract.** The *Pelophylax* genus comprises two main hybridogenetic systems resulting from hybridization between *Pelophylax ridibundus* and other species: the *P. lessonae-esculentus* (LE) system and the *P. perezi-grafi* (PG) system, whose distribution ranges lie partly (LE) or entirely (PG) outside the natural distribution of *P. ridibundus*. The introductions and subsequent expansions of *P. ridibundus* in many areas naturally inhabited by these hybridogenetic systems have led to their decrease or local extinction. One of the major impediments to efficient population surveys and conservation measures for these systems is the difficulties of reliable species identification due to their similar morphology and poorly documented vocalizations, especially for the PG system. Our aim in this work was to design a reliable method of acoustic identification to distinguish the taxa composing the hybridogenetic systems from *P. ridibundus* and to assess the possibility of separating the PG and LE systems by their vocalizations. Our acoustic identification methods proved highly accurate to separate the PG and LE systems from *P. ridibundus* but only a fraction of the PG and LE calls can be identified.

**Keywords:** acoustic identification, bioacoustics, conservation, hybridogenesis, PeloCall_ID-shiny app.

## Introduction

European Water frogs of the genus *Pelophylax* generate several hybridogenetic systems (Günther, 1990; Bülbül et al., 2011; Ualiyeva et al., 2022) via hybridization between *P. ridibundus* and another species. They are maintained by a hemiclonal reproduction where, during gametogenesis, the taxon of hybrid origin (“hybrid taxon”) suppresses one of the parental genomes, commonly the genome of the species it lives in sympatry with (Dufresnes and Mazepa, 2020);

recombination usually does not occur in the hybrid taxon (but see Christiansen and Reyer, 2009). The genome of the gametic cells is thus transmitted clonally, and only the somatic cells contain both genetic backgrounds. Crossings between the hybrid taxon and the co-occurring parental species generate hybrid progeny, but crossings between hybrids typically fail to produce viable individuals as they transmit a clonal genome that has accumulated deleterious mutations (Dufresnes and Mazepa, 2020). Therefore, to be maintained in nature, the hybrid taxon
relies on mating with the parental species whose genome is eliminated during gametogenesis.

In the *P. lessonae-esculentus* system (LE), distributed from Western Europe to the Volga-Urals region of Russia (Graf and Polls Pelaz, 1989), the hybrid taxon *P. kl. esculentus* (*P. esculentus* hereafter, originating from past hybridization between *P. ridibundus* and *P. lessonae*; we follow Speybroeck et al., 2020 here for taxonomy) reproduces by mating with *P. lessonae* (Graf and Mueller, 1979). In the *P. perezi-grafi* (PG) system, limited to southern and south-western France and northwestern Spain, the hybrid taxon *P. kl. grafi* (*P. grafi* hereafter) reproduces with *P. perezi* (Graf and Polls Pelaz, 1989; Crochet et al., 1995; Pagano et al., 2001a,b; Demay et al., 2023). Other systems involving *P. ridibundus* and *P. lessonae* as sexually reproducing parental species are known (see Dufresnes and Mazepa, 2020, for a detailed overview), but they have more restricted distributions. Last, in the EE system, *P. esculentus* maintains all-hybrid populations thanks to a combination of individuals of various ploidy levels producing gametes transmitting the L and R genomes (Günther, 1990; Christiansen and Reyer, 2009).

Within the distribution of the LE and PG systems, parental species always coexist with the associated hybrid taxa. In these areas, conservation of the species involved in these systems thus relies on preserving the mixed populations of the hybridogenetic systems. Although it is important to differentiate hybrids and parental species – for example, to identify how habitat preferences influence population composition (Mikulicek et al., 2015) – the main purpose of this paper is to find ways to monitor *P. ridibundus* invasion in the native distribution areas of PG and LE systems. In light of that, we thus treat mixed populations of the PG and LE systems as our main taxonomic unit for identification purposes, since distinguishing *P. esculentus* and *P. grafi* from the parental species

they coexist with is not essential for these hybridogenetic systems’ conservation and distribution mapping. The EE system is an exception since *P. esculentus* maintains all-hybrid populations (Günther, 1990; Christiansen and Reyer, 2009), but this system is not evaluated in this paper.

Although individuals of *Pelophylax grafi* and *P. esculentus* carry at least one set of *P. ridibundus* chromosomes, they usually do not originate from matings involving *P. ridibundus*. In the PG system, *P. grafi* individuals typically originate from matings between *grafi* and *perezi*, while in the LE system, *P. esculentus* individuals originate from matings between *P. esculentus* and *P. lessonae*. Indeed, *P. ridibundus* was naturally absent from large areas inhabited by the LE system in Italy, the Benelux, and most of France and Switzerland (Pagano et al., 2001a; Pagano et al., 2003; Domeneghetti et al., 2013; Laghi et al., 2013; Dubey et al., 2014; Dufresnes et al., 2018; Bellati et al., 2019) while the natural distribution of the PG system and *P. ridibundus* apparently did not overlap. As a consequence, it has been suggested that the *P. ridibundus* genome in PG may have originated from hybridization events between *P. perezi* and *P. esculentus* in areas where the two species co-occur (Hotz et al., 1994; Pagano et al., 2001a).

In recent years, *P. ridibundus* has expanded its distribution (Pagano et al., 2001b; Vorburger and Reyer, 2003; Plötner, 2005) by a combination of anthropogenic translocations and dispersal from translocated populations (e.g., Dubois, 1983, 1985, 2002; Holsbeek and Jooris, 2010; Dufresnes et al., 2017). As a consequence, new opportunities for hybridization between *P. perezi* or *P. lessonae*, and *P. ridibundus* have arisen (Holsbeek and Jooris, 2010). In the PG and LE systems, crossing between *P. ridibundus* and the hybrid taxon often produces new *P. ridibundus* individuals, while crossings between *P. ridibundus* and the other parental species will generate new hybrids that no longer carry deleterious mutations, allowing the production of viable *P. ridibundus* via back-crossing
(Dufresnes and Mazepa, 2020). This is not the case in the *P. ridibundus-esculentus* (RE) system, since crossing between *P. ridibundus* and *P. esculentus* can produce offspring of both taxa in different proportions (Uzzell and Berger, 1975; Uzzell et al., 1977). Additionally, *P. ridibundus* may have a significant impact on the local populations through competition (Cherry and Francillon-Vieillot, 1992; Luquet et al., 2011). *Pelophylax ridibundus* has thus the potential to replace the hybridogenetic system in just a few generations (Vorburger and Reyer, 2003; Holsteek and Jooris, 2010; Leuenberger et al., 2014) by a combination of genetic replacement of the indigenous genome and direct competition, to the point where the expansion of *P. ridibundus* currently constitutes the main threat to the LE and PG system's survival in many areas.

Difficulties in correct species identification of European water frogs are one of the major challenges to enlarge our knowledge of the ecology, evolution, and conservation of these species (Günther, 1990; Pagano and Joly, 1999; Plötner, 2005). *Pelophylax lessonae* and *P. ridibundus* are easy to distinguish morphologically but identification of the *P. esculentus* is complicated by its intermediate morphology and by the existence of triploid hybrids (Dufresnes and Mazepa, 2020) that can be phenotypically similar to either parental species (Plötner, 2010; Hoffmann and Reyer, 2013). This problem is exacerbated in the PG system where *P. perezi* is morphologically very similar to *P. ridibundus*. Nuclear genetic markers allow reliable identification (reviewed in Cuevas et al., 2022) but are expensive, time-consuming, and rely on the capture of individuals. A reliable acoustic identification method, as developed for other groups of amphibians (Padial et al., 2008; Funk et al., 2009), would thus represent a great opportunity to monitor the invasion of *P. ridibundus* into native populations of the LE and PG systems.

Schneider and Steinwarz (1990) showed that there are clear differences between the mating calls of *P. perezi* and *P. ridibundus*, with longer intervals between pulse groups, higher

pulse repetition rate, and number of pulses in *P. ridibundus*. The vocalizations of *P. grafi* are poorly documented and no previous study has attempted to establish diagnostic characters to discriminate between *P. ridibundus* and the two taxa of the PG system. Acoustic differences between *P. ridibundus* and *P. lessonae* are better documented and Wycherley et al. (2002) showed that, by using call subunits and Fourier transformation, it is possible to discriminate individuals of *P. ridibundus*, *P. lessonae*, and *P. esculentus*, however, their approach is difficult to replicate (pers. obs.). Schneider and Steinwarz (1990) also suggested that, despite many similarities, *P. perezi* subtly differs in call duration and number of pulse groups from *P. lessonae* and *P. esculentus* but they have not tested if these mean differences allow safe separation of the LE and PG systems.

The main goal of this study is to design a method to separate *P. ridibundus* from the LE and PG systems based on sound recordings of their mating calls. Given that the distributions of the PG and LE systems in the west of France are still imperfectly known, we also examine whether these two systems can be separated by acoustics traits. Although our study is based on recordings obtained mostly in France, we have no information to suggest that our method should not be applicable in other areas inhabited by the PG and LE systems. We did not attempt to separate *P. perezi* from *P. grafi* or *P. lessonae* from *P. esculentus* as this is less crucial for mapping geographic distributions and conservation of the PG and LE systems.

## Material and methods

### Data collection

We used recordings of water frogs' mating calls obtained during diurnal and nocturnal field works between 2001 and 2021. Recordings obtained by the co-authors were made using either a Marantz PMD 220 portable cassette recorder, a Marantz PMD661 solid state recorder or a Zoom H5 Handy recorder coupled to a Sennheiser directional microphone, or a Zoom H1n Handy recorder with built-in microphones. Recordings obtained from amateur naturalists were
<figure><img src="image_4.png" /><figcaption>**Figure 1.** Locations of the recordings used in this study. PG = *Pelophylax perezi-grafi* system; LE = *Pelophylax lessonae-esculentus* system; RR = *Pelophylax ridibundus*.</figcaption></figure>

collected with a diversity of materials including mobile phones and digital cameras. Recordings were obtained in France (N = 296), Greece (N = 3), and Spain (N = 1, see fig. 1).

Recordings of individuals with genetic identification were obtained as follows: calling males were located at night and sound-recorded before being captured by hand or with a hand net. Samples (buccal swabs, toe clip, or muscle tissue for vouchered specimens) were identified by sequencing or RFLP genotyping of at least two nuclear markers. In the PG system, a combination of the gene/restriction enzymes RAG1/DraII and TYR1/BalI ensures dual-locus support for identifications. Similarly, the LE system employs RAG1/DraII and CMYC2/SspI combinations for the same purpose. When all five species are possible, the initial step involves double digestion of POMC by HmcII and of RAG1 by DraII, which yields a species-specific pattern. This pattern indicates the system of origin (LE or PG). Confirmation involves a second step using TYR1/BalI if PG is suggested initially, or CMYC2/SspI if LE is suggested (see Cuevas et al., 2022, for more details). Information on the origins of the sound recordings, vouchers, and tissue samples can be found in supplementary table S1, and characteristic calls for each species can be found in supplementary audio S1.

Some *Pelophylax* acoustic variables can vary with temperature (Schneider and Steinwarz, 1990; Schneider and Sinsch, 1992) but we recorded mating calls between March and July, covering a wide range of temperature variations. Typically, temperature affects the pace of delivery of the notes within a call (faster pace and smaller internote intervals with higher temperature (Schneider and Steinwarz, 1990; Schneider and Sinsch, 1992; Schneider et al., 1993)),

but the effects of temperature on the note (= pulse group according to Schneider and Sinsch (2007) terminology) parameters themselves have not been documented. Most of the variables included in our analyses describe the structure of the notes themselves; combined with the wide range of temperatures where each taxon was recorded, it allowed us to propose identification indexes that are not dependent upon temperature.

We analyzed 100 individuals of each group of interest (PG, LE, and R). Among these, 13 individuals of *P. perezi*, 22 of *P. grafi*, 2 of *P. esculentus*, and 14 of *P. ridibundus* have been sound recorded, captured, and identified genetically by the PCR-RFLP method of Cuevas et al. (2022). The other individuals have been classified "a priori" by a combination of their acoustic features (*P. ridibundus* is often identifiable due to its distinctive mating call in comparison to other taxa; fig. 2) and locality (the LE and PG systems have mostly non-overlapping distributions in France and selecting recordings originating from areas of allopatry allowed to obtain reference samples for these two systems). A series of principal component analyses (PCA) adding genetically identified individuals was performed to check the validity of our a priori identification. Crucially, none of our results depended on our initial identification: this initial identification was only used to select recordings to measure and ensure we reached our target sample size of 100 individuals per group. Yet, as can be seen from the Results section, these initial identifications were largely accurate.

Mating calls of *P. ridibundus* vary with geography and two main types of calls occur in Europe (e.g., Schneider and Sinsch, 1992), corresponding to the subspecies *kurtmuelleri* (southern Balkans) and *ridibundus* (most of Europe,
<figure><img src="image_5.png" /><figcaption>**Figure 2.** Oscillogram (left) and spectrogram (right) of mating calls of individuals genetically identified as (a) *Pelophylax ridibundus*, (b) *Pelophylax perezi*, (c) *Pelophylax grafi*, (d) *Pelophylax esculentus* and (e) *Pelophylax lessonae*.</figcaption></figure>

taxonomy based on Speybroeck et al., 2020). Our sample of recordings from France includes the two types of calls based on mtDNA barcoding results (Dufresnes et al., 2017; unpubl. data) so our results on identification should be robust to this variation in Europe.

## Acoustic variables

Mating calls (also called advertisement calls) of water frogs consist of a series of notes each made of many pulses. They

are typically delivered in series starting with short calls and ending with long, excited calls. Long calls can be recognized by the increased number of notes, faster delivery (smaller interval between notes), and louder volume, and typically occur towards the end of a call series. For measuring call parameters, we discarded the short calls from the beginnings of the series but also the very last calls when they differed significantly from the average ones because acoustic variables tend to be less typical in these extreme
calls. Note that call-centered and note-centered approaches provide the same terminology for *Pelophylax* calls (see Fig. 7B in Kohler et al., 2017). Furthermore, we follow Hoffmann and Reyer (2013) to name the acoustic components of *Pelophylax* calls, except that we replace “pulse group” with “note” to agree with the terminologies defined by Kohler et al. (2017).

For each individual, we measured 12 acoustic variables from oscillograms and 3 from spectrograms of the mating calls (fig. 3) produced with the default options of the software Audacity (version 3.2.5; available from https://audacityteam.org/). These 15 variables were chosen based on previous work on the acoustics of the genus (see Introduction) and by visually comparing oscillograms of songs identified by genetics or geography. Although we did not anticipate that all 15 variables would be useful or necessary for acoustic identification, the lack of previous work on the identification of the PG system led us to select a

large number of variables for initial screening in the hope to find enough informative variables for reliable identification. All variables were measured by the first author to avoid observer bias; for each individual, they were measured from several calls (typically 2-3) and the mean of the measurements was used. Two additional variables (nrr and prr) were computed from the measured variables for illustrative and identification purposes but were not included in PCA or other statistical analyses since they are derived for other variables, introducing redundant information. The table with the measure for each recording used here is available in supplementary table S2. Note that recordings typically need to be cleaned before analysis (see the Indexes Tutorial in supplementary text S1 for details).

The acoustic variables used were (see fig. 3, taken from the oscillograms unless stated otherwise): number of notes per call (nbn); call duration (cd, duration of call in seconds

<figure><img src="image_6.png" /><figcaption>**Figure 3.** View of the acoustic variables. (a) Acoustic variables measured from oscillograms – nbn = the number of notes per call; cd = call duration in seconds; p80 = first note which reaches 80% of the height of the maximum energy note; pn = the number of notes to reach the one with the highest peak; pt = time to reach the highest peak; nbp = the number of pulses per note; nd = note duration in seconds; op = the number of oscillations; pd = pulse duration in seconds; ndy = note dynamic; lp = last pulse separate or not; fa = full amortization; (b) Acoustic variables measured from spectrograms – ifr = initial frequency; mfr = maximum frequency; efr = end frequency.</figcaption></figure>
from the middle of the first note to the middle of the last note); note repetition rate (nrr, = (nbn - 1)/(cd); peak note (pn, the number of notes within a call to reach the highest energy peak); peak time (pt, the time in seconds from the beginning of the first note to the maximum energy peak in the call); number of pulses (= oscillations pulses) per note (nbp, measured from at least 2 notes per call, selecting notes from the middle of the call); note duration (nd, the duration in seconds of each note for which the number of pulses has been measured, measured from the start of the first pulse to the start of the last pulse of the note); pulse repetition rate (prr = (nbp - 1)/(nd); number of oscillations per pulse (op, the average of the number of oscillations per pulse in one pulse per note, in two different notes, excluding the first and last pulses and counting oscillations between two maximal oscillations); pulse duration (pd, the average duration of one pulse in seconds); note dynamics (ndy, the average number of pulses after the most powerful one within a note, using two notes from each of the selected calls); last pulse (lp, "yes" if the last pulse in a note is separated from the rest of the note, otherwise "no", choose "yes" if at least part of the recording presents this characteristic); full amortization (fa, "yes" if, in some part of the pulse, oscillations disappear between two pulses, "no" otherwise); 80% of the peak note (p80, counting how many notes are necessary to reach 80% of the height of the maximum energy note); initial frequency (ifr, initial frequency of the main harmonic in Hz, using spectrograms); maximum frequency (mfr, highest frequency of the main harmonic in Hz, using spectrograms); end frequency (efr, frequency of the main harmonic at the end of a call in Hz, using spectrograms). A table with a description of all these variables is available in supplementary table S3.

## Index classification

To establish the range of each acoustic variable in *P. perezi* and *P. grafi*, we used only the individuals with genetic identification (see table 1). To test for acoustic differences between the taxa and to check the validity of our a priori identifications, we performed a series of principal component analyses (PCA) using the R package stats 3.6.2 (R Core Team, 2023). Since both hybridogenetic systems sound similar (to a human ear), but differ from *P. ridibundus*, the first identification index (R-H index) aimed at separating *P. ridibundus* from the two groups, PG and LE system. We first established the range of each acoustic variable for each group (RR, PG, and LE) and selected the acoustic variables that overlapped less between these two groups. We then scored these variables in all individuals, giving a score of 0 if its value was within the range of *P. ridibundus* or 1 if it was within the range of PG and LE (table 1). Summing all variable scores provided a final identification index score. To establish the range of values of the identification index that permits a reliable taxon assignment, all recordings in our samples were scored and the amount of overlap of the index values was assessed. Taxon assignment was based on the position of the individuals in the PCA scatterplot and compared with their genetic identification when available. Our workflow is summarized in supplementary

fig. S1. A tutorial on how to measure the acoustic variables and use the R-H index is available in supplementary text S1.

We built a similar index, the PG-LE index, to separate the PG and LE systems, using variables that present a reduced overlap between the two systems. Density plots for each system and each acoustic variable (supplementary fig. S2) were used to define intervals for scoring variables (table 2) as -1 if their value is more typical for PG, 1 if it is more typical for LE, or 0 if it is in the overlap zone. To check the method, we used the same approach as for the R-H index above, only using specimens originating from areas of allopatry between the PG and LE systems. A tutorial on how to measure the acoustic variables and use the PG-LE index is available in supplementary text S1.

## Random forest identification

The random forest algorithm can use continuous and categorical variables and build multiple decision trees to evaluate if a particular combination of traits can predict the response variable (Liaw and Wiener, 2002; Biau, 2012). A random forest classification model was created using the "randomForest" v.4.7-1.1 package (Liaw and Wiener, 2002) and the 15 acoustic variables to identify PG, LE, and RR. The data was divided into 70% for training and 30% for testing. To evaluate the model performance, we employed the out-of-bag (OOB) error rate, which serves as an internal validation measure. During the construction of each tree, approximately one-third of the data is not used for training, and the OOB error rate is calculated by assessing the prediction accuracy on this out-of-bag subset. This provides a robust estimate of the model's generalization error. The area under the curve (AUC) of the receiver operating characteristic curve (ROC) was also used to evaluate the model. The ROC curve is a graphical representation of the trade-off between sensitivity and 1-specificity across different classification thresholds. The AUC quantifies the area under this curve, providing a comprehensive measure of the model's ability to distinguish between different classes, with higher values indicating better overall performance. In addition to OOB error and AUC, the study employed mean decrease accuracy (MDA) and the Boruta test from the "Boruta" v.8 package (Kursa and Rudnicki, 2010) to gauge the importance of each variable in the model. These techniques contribute to a comprehensive evaluation of the model's predictive performance and offer insights into the significance of individual features in making accurate classifications.

## Results

### The advertisement calls of *Pelophylax grafi*

We had access to recordings of 22 *Pelophylax grafi* and 13 *P. perezi* that were genetically identified after being recorded in the field (see methods). As can be seen from fig. 2b and heard
<table><caption>**Table 1.** Ranges of acoustic variables (Var) for *P. perezi*, *P. grafi*, PG, LE and RR (see Material and methods and supplementary table S3 for variable names and definitions). Values for *P. perezi* and *P. grafi* are based on genetically identified specimens only. “R-H index value” provides the values used for scoring each variable used in the R-H index. nbn = number of notes per call; cd = call duration (sec); nrr = note repetition rate; nbp = number of pulses per note; nd = note duration; prr = pulse repetition rate; op = number of oscillations per pulse; pd = pulse duration (sec); pn = number of notes to reach the one with the highest peak; pt = time to reach the highest peak; ndy = note dynamic; p80 = first note which reaches 80% of the height of the maximum energy note; lp = last pulse; fa = full amortization; ifr = initial frequency (Hz), mfr = maximum frequency (Hz); efr = end frequency (Hz).</caption><thead><tr><th>Var</th><th><i>P. perezi</i></th><th><i>P. grafi</i></th><th>RR</th><th>LE</th><th>PG</th><th>R-H Index value</th></tr></thead><tbody><tr><td>nbn</td><td>10-21</td><td>7-16</td><td>5-9</td><td>12-31</td><td>7-21</td><td>≤9 = 0/≥10 = 1</td></tr><tr><td>cd</td><td>0.345-0.676</td><td>0.405-1.107</td><td>0.323-0.861</td><td>0.633-1.823</td><td>0.345-0.676</td><td></td></tr><tr><td>nrr</td><td>21.40-39.71</td><td>12.15-24.60</td><td>9.91-16.84</td><td>10.83-34.19</td><td>12.15-39.71</td><td></td></tr><tr><td>nbp</td><td>4-7</td><td>5-10</td><td>13-22</td><td>4-7</td><td>4-10</td><td>≥11 = 0/≤10 = 1</td></tr><tr><td>nd</td><td>0.013-0.033</td><td>0.02-0.036</td><td>0.029-0.05</td><td>0.014-0.043</td><td>0.013-0.036</td><td></td></tr><tr><td>prr</td><td>130-235</td><td>137-363</td><td>300-517</td><td>81-300</td><td>130-363</td><td>≥300 = 0/≤300 = 1</td></tr><tr><td>op</td><td>9-13</td><td>8-13</td><td>4-6</td><td>8-21</td><td>8-13</td><td>≤7 = 0/≥8 = 1</td></tr><tr><td>pd</td><td>0.003-0.005</td><td>0.002-0.005</td><td>0.002-0.004</td><td>0.003-0.011</td><td>0.002-0.005</td><td></td></tr><tr><td>pn</td><td>4-18</td><td>1-13</td><td>1-7</td><td>4-28</td><td>1-18</td><td></td></tr><tr><td>pt</td><td>0.156-0.644</td><td>0.024-0.731</td><td>0.003-0.874</td><td>0.182-1.634</td><td>0.014-0.731</td><td></td></tr><tr><td>p80</td><td>1-8</td><td>1-10</td><td>1-5</td><td>3-23</td><td>0-10</td><td></td></tr><tr><td>ndy</td><td>0-1</td><td>0-1</td><td>3-10</td><td>0-3</td><td>0-1</td><td>≥3 = 0/≤2 = 1</td></tr><tr><td>lp</td><td>yes</td><td>yes</td><td>no</td><td>yes</td><td>yes</td><td>no = 0/yes = 1</td></tr><tr><td>fa</td><td>yes</td><td>yes</td><td>no</td><td>yes</td><td>yes</td><td>no = 0/yes = 1</td></tr><tr><td>ifr</td><td>1969-3232</td><td>2007-3370</td><td>1728-3471</td><td>1294-3022</td><td>1969-3437</td><td></td></tr><tr><td>mfr</td><td>2345-3515</td><td>2348-3647</td><td>1952-3884</td><td>2074-3568</td><td>2345-3966</td><td></td></tr><tr><td>efr</td><td>2255-3164</td><td>2141-3217</td><td>1504-3694</td><td>1777-3321</td><td>1984-3559</td><td></td></tr></tbody></table>

<table><caption>**Table 2.** Scoring system for the PG-LE index. nbn = number of notes per call; cd = call duration (sec); pd = pulse duration (sec); pn = number of notes to reach the one with the highest peak; pt = time to reach the highest peak; ndy = note dynamic; p80 = first note which reaches 80% of the height of the maximum energy note; ifr = initial frequency (Hz).</caption><thead><tr><th rowspan="2">Acoustic variables</th><th colspan="3">PG-LE Index</th></tr><tr><th>-1</th><th>0</th><th>1</th></tr></thead><tbody><tr><td>nbn</td><td>≤9</td><td>>9 and <25</td><td>≥25</td></tr><tr><td>cd</td><td>≤0.4</td><td>>0.4 and <1</td><td>≥1</td></tr><tr><td>pd</td><td>≤0.004</td><td>>0.004 and <0.0055</td><td>≥0.0055</td></tr><tr><td>pn</td><td>≤8</td><td>>8 and <16</td><td>≥16</td></tr><tr><td>pt</td><td>≤0.3</td><td>>0.3 and <0.8</td><td>≥0.8</td></tr><tr><td>ndy</td><td>≤0</td><td>=1</td><td>≥2</td></tr><tr><td>p80</td><td>≤3</td><td>>3 and <10</td><td>≥11</td></tr><tr><td>ifr</td><td>≥2500</td><td><2500 and >2000</td><td>≤2000</td></tr></tbody></table>

from supplementary audio S1, mating calls of *P. grafi* differ from *P. perezi* by having more clearly separated notes within the call. The separation of *P. grafi* from *P. perezi* is nevertheless complicated by variation within both species as some *P. perezi* individuals can sound similar to some *P. grafi* while some individuals of *P. grafi* can sound similar to *P. perezi* while others have well-separated notes resembling *P. ridibundus* (pers. obs.). This renders identification of these three species difficult to the human ear, even if

*P. perezi* and *P. ridibundus* can easily be identified with a bit of experience. We thus used variables measured from oscillograms and spectrograms to investigate if these three groups can be separated.

Using our 17 variables, *P. perezi* and *P. grafi* present overlapping clusters and ranges for all acoustic variables measured (table 1, fig. 4a), but both clusters are separate from *P. ridibundus* (fig. 4b). Given that *P. perezi* and *P. grafi* co-occur as the PG system in nature (except in
<figure><img src="image_7.png" /><figcaption>**Figure 4.** (a) PCA between *Pelophylax grafi* and *P. perezi* using only specimens with genetic identification, (b) PCA between PG and RR. PG = *Pelophylax perezi-grafi* system; RR = *Pelophylax ridibundus*.</figcaption></figure>

parts of Iberia where *P. grafi* is absent), they were grouped to create the index identification.

*Separating P. ridibundus from the PG and LE systems*

The PCA revealed that RR forms a clearly distinct cluster that does not overlap with LE or PG (figs. 4b and 5a,b). All a priori and genetically identified individuals fell in their expected cluster (but for LE we had only 2 genetically identified individuals). Yet, the acoustics features of individuals identified here as LE and RR agree with previous findings (Wycherley et al., 2002; Hoffmann and Reyer, 2013), confirming that the two clusters in fig. 5a indeed correspond with the RR and LE groups.

Seven acoustic variables whose range overlaps least between RR and LE+PG were used to build the identification method (table 1). Based

on the distribution of the R-H index (fig. 6a), we suggest that recordings with a score value ≥ 5 can be identified as PG or LE, and those with a score value ≤2 as *P. ridibundus*. Although there is a single recording with a score of 4 (of the PG system according to its position in the PCA, result not shown), we suggest leaving as indeterminate recordings with an index score of 3 or 4, based on the distribution of the R-H index score.

*Separating LE from PG*

Visual examination of oscillograms and spectrograms revealed some differences between LE and PG. Notably, LE has a higher number of notes per call, resulting in longer calls with a different shape on oscillograms, characterized by energy peaks near the end of the call, while calls from the PG system are more rectangular
<figure><img src="image_8.png" /><figcaption>**Figure 5.** (a) PCA between LE and RR. (b) PCA between PG and LE together and RR. (c) PCA between the hybridogenetic systems PG and LE. PG = *Pelophylax perezi-grafi* system; LE = *Pelophylax lessonae-esculentus* system (using specimens from allopatry areas); RR = *Pelophylax ridibundus*.</figcaption></figure>

(energy more evenly spread; fig. 2B-C). However, all variables overlap between PG and LE (table 1), and the PCA comparing LE and PG shows a large overlap between the two groups (fig. 5c). Density plots of individual variables (supplementary fig. S2) revealed eight variables with reduced overlap between PG and LE (table 2), which were used to generate the PG-LE index score.

The PG-LE index scores range from −6 to 8. Comparing the PG-LE index score with the individual identifications (based on distribution range, see above) suggested that values between −6 and −3 can be safely linked to the PG system, while values between 2 and 8 are diagnostic of the LE system (fig. 6b). However, only 65% of the specimens (69 LE and 59 PG) could be accurately identified by their PG-LE index
<figure><img src="image_9.png" /><figcaption>**Figure 6.** (a) Distribution of the R-H index score for RR versus PG and LE; (b) Distribution of the PG-LE index score for PG and LE.</figcaption></figure>

scores, as many individuals exhibited scores in overlapping ranges (from -2 to 1).

## Random forest classification

The random forest classification model had an OOB error of 6.31%. All variables were relevant to the model according to the Boruta analysis. Furthermore, according to the MDA estimations, cd, pt, and p80 were the most important variables for the model. The model presented an accuracy of 96% when tested, an AUC of 0.994 for PG, 0.995 for LE, and 1 for RR (supplementary fig. S3). To conclude, the model reliably separated RR from LE and PG but was unable to fully distinguish between LE and PG; its performance was better for this than our index score,

but since the model does not return a reliability measure for a given identification (probability of error), it cannot be used to obtain a safe identification between PG and LE for a single individual. However, in the context of analyzing recordings of many individuals in a population, this method can help to have insights about the species composition of that area since it has a high accuracy of 95%.

## Acoustic identification application

We developed an application called PeloCall_ID using “shiny” v.1.7.4 (Chang et al., 2023), which automates the three identification methods proposed here (R-H and PG-LE indexes, and the random forest identification). The app is available at (https://filipegmsalmeida.shinyapps.io/PeloCall_ID/). Users simply need
to measure and compile the acoustic variables analyzed in this research in an Excel table and use it as input. These data will generate three tables as outputs, presenting the identification according to each of the methods described here. First, the R-H index is calculated for all individuals imputed, and then the individuals identified as PG or LE are selected to be scored according to the PG-LE index and have a new identification. Finally, the app identified the individuals according to the random forest model, designed using the data set that was used in this study. All tables can be downloaded, including the score for each variable, the final score for each individual, and the identification according to the criteria of each method. A tutorial on how to measure the acoustic variables and an Excel file demonstrating the input format is available in the app.

## Discussion

### Acoustic identification

The acoustic identification method designed in this study has proven highly reliable in distinguishing the two hybridogenetic systems PG and LE from *P. ridibundus*. The vast majority of specimens have a R-H identification index value of 0,1 or 2 (*ridibundus*) or 6 or 7 (LE and PG) and individuals with R-H index scores of 3-4 (unidentified) represent a small minority. This identification method will thus constitute a convenient tool for monitoring water frog populations. Note that the computation of the R-H identification index is not always necessary: many recordings can be identified by ear or by checking oscillograms against values in table 1. Only ambiguous recordings that do not exhibit the full suite of characters typical of one or the other category (RR or PG+LE) will need to be scored.

Despite an in-depth examination of oscillograms, we could not find any diagnostic combination of characters to safely distinguish the PG from the LE system due to the extensive

overlap between them in all variables. The PG-LE identification index only identifies 64% of individuals with the most typical call characteristics. The random forest model provides higher accuracy (96%) but lacks individual identification support. To achieve reliable identification between PG and LE at the population level with the random forest model (available in the PeloCall_ID app), we recommend using at least 5 individuals identified as the same taxon. To establish the occurrence of LE or PG in areas of sympatry we would thus recommend having several recordings identified per taxon by the random forest model, or at least one individual with a PG-LE index score value diagnostic of one taxon. Alternatively, since the PG system differs morphologically from the LE system in several characters, a combination of photos and sound recordings should allow reliable identification of PG and LE populations in potential areas of sympatry (western France), where the geographical distribution of water frogs is still imperfectly known (Pagano et al., 2001a).

### Why are the hybrid taxa more similar to one of the parental species?

As can be seen from fig. 4b and supplementary fig. S4, *Pelophylax grafi* has acoustics parameters closer to *P. perezi* than to *P. ridibundus*. The same pattern has already been reported from the LE system, where *P. esculentus* is also closer to *P. lessonae* than *P. ridibundus* for several call parameters (Hoffmann and Reyer, 2013; see also fig. 5a).

In amphibians, mating calls are important for attracting females (Gerhardt et al., 2000). Given that hybridogenetic species often depend on reproducing with one of their parental species to persist (Dufresnes and Mazepa, 2020), sexual selection would be expected to displace the acoustic parameters of the hybrid taxa towards the parental species they need to mate with (*P. perezi* for *P. grafi* and *P. lessonae* for *P. esculentus* in the LE system). Of course, other hypotheses could explain this pattern though (genetic dominance, phenotypic matching of
call parameters to the calls of co-occurring taxa) and dedicated studies would be necessary to test the sexual selection hypothesis.

Note that Lodé (2001) also claimed that hybridogenetic individuals generally resemble the indigenous species with which they live in sympatry, but the acoustic variables, spectrograms, and oscillograms that he presented for *P. perezi* suggest that his “*P. perezi*” individuals were in fact *P. ridibundus*. As a result, the conclusions of Lodé (2001) are difficult to uphold.

## Conservation of taxa of hybrid origin

Theoretical population models and empirical surveys (Vorburger and Reyer, 2003; Holsbeek et al., 2008; Holsbeek and Jooris, 2010; Quilodran et al., 2015; Dufresnes et al., 2018; Demay et al., 2023) indicate a negative impact on native water frogs by the expansion of *P. ridibundus* in areas where it was naturally absent. As a result, there is an increasing interest in developing conservation measures for the native systems. Quilodran et al. (2015) suggest increasing the habitat size favorable to the native systems, for example, ponds with submerged vegetation and low levels of dissolved oxygen where tadpoles of *P. ridibundus* have lower performance than tadpoles of the LE system (Plénet et al., 2000; Holenweg Peter et al., 2002). Habitat management has proven successful in increasing the abundance of other threatened amphibians (Rannap et al., 2009), even when impacted by another invasive frog species (Adams and Pearl, 2007). However, ecological factors favoring *P. perezi* and *P. grafi* over *P. ridibundus* are currently poorly known, but Schmeller et al. (2007) suggested that *P. ridibundus* is rare in aquatic habitats with medium to high salinity, low pH, and low oxygen concentration. This could explain the persistence of the PG system in coastal marshes in areas that have been colonized by *P. ridibundus* (Demay et al., 2023).

In many areas, we still have an imprecise knowledge of the distribution and habitat use of the native and invasive water frog taxa. One

of the main impediments to clarify these questions has long been the lack of usable identification tools for naturalists and stakeholders. For example, most records of water frogs in biodiversity databases in France are still identified as genus only (pers. obs.). Much information can be gained by mobilizing the community of amateur and professional naturalists that provide most of the biodiversity occurrence records in Europe. Cheap and light digital sound recordings are increasingly used by naturalists in Europe, and with this study, we verified that correct identification of water frogs can be achieved from sound recordings obtained with digital cameras or even mobile phones. Our bioacoustics tools should thus help document the distribution of water frogs and identify the ecological variables driving the composition of water frog communities. Sound recordings provide an easier and quicker way to sample the composition of populations compared to the previous approaches (genetics, or morphology in the case of the LE system) that rely on capturing individuals for identification and are not accessible to most naturalists.

We wish to add one word of caution: our field experience suggests that the various species present in a given habitat do not necessarily call at the same time. It is thus possible to record only one of the species that are present during any given visit, and several visits in slightly different conditions (time of day/time of year) can be necessary to detect the acoustic activity of all the species that make up a given population.

Hybridogenetic lineages, although formed naturally, are sometimes not included in protected/endangered species lists as they are not categorized as independent evolutionary lineages (Kraus, 1995). The hybridogenetic *P. grafi* and *P. esculentus* are indeed currently not recognized as distinct species by the Amphibian Species of the World (Frost, 2023) and consequently they are not evaluated by the International Union for Conservation of Nature (IUCN). On the contrary, others have argued
that lineages of hybrid origin formed by natural evolutionary processes should be considered eligible for conservation (Kraus, 1995; Christiansen and Reyer, 2009), even if they need another species to persist and reproduce. The accumulation of deleterious mutations in the *P. ridibundus* genomes transmitted through *P. esculentus* and *P. grafi* (see Introduction) demonstrates that they are evolving independently. In addition, pure hybrid populations of *P. esculentus*, maintained by a combination of diploid and triploid individuals transmitting different genomes, have been documented (the EE system, see Christiansen and Reyer, 2009, for example). Therefore, we agree with Christiansen and Reyer (2009, see their concluding paragraph) that *P. esculentus* from the EE system should be treated as a species of hybrid origin for conservation purposes. We furthermore argue that the *P. esculentus* populations formed by *P. lessonae* or *P. ridibundus* clones evolving in the other systems should be preserved as well. Even if pure hybrid populations have not been reported to date in the PG system, *P. grafi* also represents a self-perpetuating lineage that cannot be replaced by new hybridizations between *P. perezi* and *P. ridibundus*. Hybridization and polyploidy have been shown to be important in the evolution of several plants and cannot be neglected from animal evolutionary history (Soltis and Soltis, 1999; Choleva et al., 2012). We argue that conservation efforts for *Pelophylax* hybridogenetic systems should be promoted, especially in light of the significant threat they face from the human-mediated invasions of *P. ridibundus* into their natural habitats. In this context, our study represents an initial step toward implementing conservation measures, as it introduces a reliable and accessible method for identifying *P. ridibundus* within these hybridogenetic systems.

**Acknowledgements.** FGA and AP thank the Erasmus Mundus Programme in Evolutionary Biology (MEME). We thank the naturalists who shared recordings of *Pelophylax*

with us for this study; their names can be found in supplementary table S1. This study was supported by a REPERE grant awarded to the Conservatoire des espaces naturels Languedoc-Roussillon and the CEFE and coordinated by John Thompson (CNRS, CEFE). This work also benefited from recurrent funding from the CNRS to PAC.

**Conflict of interest.** The authors have no conflict of interest to declare.

**Supplementary material.** Supplementary material is available online at: https://doi.org/10.6084/m9.figshare.26349256

## References

Adams, M.J., Pearl, C.A. (2007): Problems and opportunities managing invasive bullfrogs: is there any hope? In: Biological invaders in inland waters: Profiles, distribution, threats, p. 679-693. Gherardi, F., Ed., Springer, Dordrecht.

Bellati, A., Bassu, L., Nulchis, V., Corti, C. (2019): Detection of alien *Pelophylax* species in Sardinia (western Mediterranean, Italy). BioInvasions Rec. **8**: 8-25.

Biau, G. (2012): Analysis of a random forests model. J. Mach. Learn. Technol. **13**: 1063-1095.

Bülbül, U., Matsui, M., Kutrup, B., Eto, K. (2011): Taxonomic relationships among Turkish water frogs as revealed by phylogenetic analyses using mtDNA gene sequences. Zool. Sci. **28**: 930-936.

Chang, W., Cheng, J., Allaire, J., Xie, Y., McPherson, J. (2023): shiny: Web Application Framework for R.

Cherry, M.I., Francillon-Vieillot, H. (2009): Body size, age and reproduction in the leopard toad, *Bufo pardalis*. J. Zool. **228**: 41-50.

Choleva, L., Janko, K., De Gelas, K., Bohlen, J., Šlechtová, V., Rábová, M., Ráb, P. (2012): Synthesis of clonality and polyploidy in vertebrate animals by hybridization between two sexual species. Evolution **66**: 2191-2203.

Christiansen, D.G., Reyer, H.U. (2009): From clonal to sexual hybrids: genetic recombination via triploids in all-hybrid populations of water frogs. Evolution **63**: 1754-1768.

Crochet, P.-A., Dubois, A., Tunner, H. (1995): *Rana (Pelophylax) ridibunda* Pallas, 1771, *Rana (Pelophylax) perezi* Seoane, 1885 and their associated klepston (Amphibia, Anura): morphological diagnoses and description of a new taxon. Bull. Mus. natl. hist. nat., Sect. A Zool. biol. écol. anim. **17**: 11-30.

Cuevas, A., Patrelle, C., Ciavatti, F., Gendre, T., Sourrouille, P., Geniez, P., Doniol-Valcroze, P., Crochet, P.-A. (2022): A new PCR-RFLP method for the identification of parental and hybridogenetic western European water frogs including the *Pelophylax perezi-grafi* system. Salamandra **58**: 218-230.
Demay, J., Ciavatti, F., Cuevas, A., Doniol-Valcroze, P., Eble, A., Leblanc, E., Mansier, Y., Martinossi-Allibert, I., Nicolas, J., Pineau, A. (2023): Distribution des grenouilles vertes du système perezi-grafi et des autres espèces du genre *Pelophylax* (Amphibia: Ranidae) dans leur aire méditerranéenne française à l'ouest du Rhône. Bull. Soc. Herp. Fr. **182**: 1-11.

Domeneghetti, D., Bruni, G., Fasola, M., Bellati, A. (2013): Discovery of alien water frogs (gen. *Pelophylax*) in Umbria, with first report of *P. shqipericus* for Italy. Acta Herpetol. **8**: 171-176.

Dubey, S., Leuenberger, J., Perrin, N. (2014): Multiple origins of invasive and 'native' water frogs (*Pelophylax* spp.) in Switzerland. Biol. J. Linnean Soc. **112**: 442-449.

Dubois, A. (1983): A propos de cuisses de grenouilles. Protection des amphibiens, arrêtés ministériels, projets d'élevage, gestion des populations naturelles, enquêtes de répartition, production, importations et consommation: une équation difficile à résoudre. Les propositions de la Société Batrachologique de France, Alytes **2**: 69-111.

Dubois, A. (1985): A nouveau sur les cuisses de grenouilles. Circalytes **1**: 1-21.

Dubois, A. (2002): Les Amphibiens et les introductions d'espèces allogènes dans les milieux. Gestion et protection des Amphibiens: de la connaissance à la prise en compte dans les aménagements. Association française des ingénieurs écologues, Paris.

Dufresnes, C., Denoël, M., Di Santo, L., Dubey, S. (2017): Multiple uprising invasions of *Pelophylax* water frogs, potentially inducing a new hybridogenetic complex. Sci. Rep. **7**: 6506.

Dufresnes, C., Leuenberger, J., Amrhein, V., Bühler, C., Thiébaud, J., Bohnenstengel, T., Dubey, S. (2018): Invasión genetics of marsh frogs (*Pelophylax ridibundus* sensu lato) in Switzerland. Biol. J. Linnean Soc. **123**: 402-410.

Dufresnes, C., Mazepa, G. (2020): Hybridogenesis in water frogs. eLS **1**: 718-726.

Frost, D.R. (2023): Amphibian Species of the World: an online reference. https://amphibiansoftheworld.amnh.org/index.php. American Museum of Natural History, New York.

Funk, W.C., Cannatella, D.C., Ryan, M.J. (2009): Genetic divergence is more tightly related to call variation than landscape features in the Amazonian frogs *Physalaemus petersi* and *P. freibergi*. J. Evol. Biol. **22**: 1839-1853.

Gerhardt, H.C., Tanner, S.D., Corrigan, C.M., Walton, H.C. (2000): Female preference functions based on call duration in the gray tree frog (*Hyla versicolor*). Behav. Ecol. **11**: 663-669.

Graf, J.D., Mueller, W.P. (1979): Experimental gynogenesis provides evidence of hybridogenetic reproduction in the *Rana esculenta* complex. Experientia **35**: 1574-1576.

Graf, J.D., Polls Pelaz, M. (1989): Evolutionary genetics of the *Rana esculenta* complex. In: Evolution Ecology of Unisexual Vertebrates, p. 289-301. Dawley, R.M., Bogart, J.P., Eds, New York State Museum, New York.

Günther, R. (1990): Die Wasserfrösche Europas. A. Ziemsen Verlag, Wittenberg.

Hoffmann, A., Reyer, H.-U. (2013): Genomic effects on advertisement call structure in diploid and triploid hybrid waterfrogs (Anura, *Pelophylax esculentus*). BMC Ecol. **13**: 1-17.

Holenweg Peter, A.-K., Reyer, H.-U., Abt Tietje, G. (2002): Species and sex ratio differences in mixed populations of hybridogenetic water frogs: The influence of pond features. Ecoscience **9**: 1-11.

Holsbeek, G., Jooris, R. (2010): Potential impact of genome exclusion by alien species in the hybridogenetic water frogs (*Pelophylax esculentus* complex). Biol. Invasions **12**: 1-13.

Holsbeek, G., Mergeay, J., Hotz, H., Plötner, J., Volck-aert, F.A.M., De Meester, L. (2008): A cryptic invasion within an invasion and widespread introgression in the European water frog complex: consequences of uncontrolled commercial trade and weak international legislation. Mol. Ecol. **17**: 5023-5035.

Hotz, H., Uzzell, T., Berger, L. (1994): Hemiclonal hybrid water frogs associated with the sexual host species *Rana perezi*. Zool. Pol. **39**.

Koehler, J., Jansen, M., Rodriguez, A., Kok, P.J., Toledo, L.F., Emmrich, M., Glaw, F., Haddad, C.F., Roedel, M.-O., Vences, M. (2017): The use of bioacoustics in anuran taxonomy: theory, terminology, methods and recommendations for best practice. Zootaxa **4251**: 1-124.

Kraus, F. (1995): The conservation of unisexual vertebrate populations. Conserv. Biol. **9**: 956-959.

Kursa, M.B., Rudnicki, W.R. (2010): Feature selection with the Boruta package. J. Stat. Softw. **36**: 1-13.

Laghi, P., Miserocchi, D., Valli, M. (2013): Determinazione genetica della presenza delle rane verdi alloc tone *Pelophylax ridibundus* e *Pelophylax kurtmuelleri* (Amphibia, Anura, Ranidae) in due località della Romagna. Quaderni del Museo di Storia Naturale di Fer rara **1**: 75-78.

Liaw, A., Wiener, M. (2002): Classification and regression by randomForest. R News **2**: 18-22.

Lodé, T. (2001): Character convergence in advertisement call and mate choice in two genetically distinct water frog hybridogenetic lineages (*Rana kl esculenta*, *Rana kl grafi*). J. Zool. Syst. Evol. Res. **39**: 91-96.

Luquet, E., Vorburger, C., Hervant, F., Joly, P., Kaufmann, B., Schmeller, D.S., Léna, J.P., Grolet, O., Konecny, L., Plénet, S. (2011): Invasiveness of an introduced species: the role of hybridization and ecological constraints. Biol. Invasions **13**: 1901-1915.

Mikulíček, P., Kautman, M., Kautman, J., Pruvost, N.B. (2015): Mode of hybridogenesis and habitat preferences influence population composition of water frogs (*Pelophylax esculentus* complex, Anura: Ranidae) in a region of sympatric occurrence (western Slovakia). J. Zool. Syst. Evol. Res. **53**: 124-132.

Padial, J.M., Koehler, J., Munoz, A., De la Riva, I. (2008): Assessing the taxonomic status of tropical frogs through bioacoustics: geographical variation in the advertisement calls in the *Eleutherodactylus discoidalis* species group (Anura). Zool. J. Linn. Soc. **152**: 353-365.
Pagano, A., Crochet, P.A., Graf, J.D., Joly, P., Lodé, T. (2001a): Distribution and habitat use of water frog hybrid complexes in France. *Glob. Ecol. Biogeogr.* **10**: 433-441.

Pagano, A., Dubois, A., Lesbarrères, D., Lodé, T. (2003): Frog alien species: a way for genetic invasion? *C. R. Biol.* **326**: 85-92.

Pagano, A., Joly, P. (1999): Limits of the morphometric method for field identification of water frogs. *Alytes* **16**: 130-138.

Pagano, A., Lodé, T., Crochet, P.-A. (2001b): New contact zone and assemblages among water frogs of Southern France. *J. Zool. Syst. Evol. Res.* **39**: 63-68.

Plenet, S., Pagano, A., Joly, P., Fouillet, P. (2000): Variation of plastic responses to oxygen availability within the hybridogenetic *Rana esculenta* complex. *J. Evol. Biol.* **13**: 20-28.

Plötner, J. (2005): Die westpaläarktischen Wasserfrösche: von Märtyrern der Wissenschaft zur biologischen Sensation. *Laurenti Bielefeld.*

Plötner, J. (2010): Möglichkeiten und Grenzen morphologischer Methoden zur Artbestimmung bei europäischen Wasserfröschen (*Pelophylax esculentus*-Komplex). *Zeitschrift für Feldherpetologie* **17**: 129-146.

Plötner, J., Uzzell, T., Beerli, P., Spolsky, C., Ohst, T., Litvinchuk, S.N., Guex, G.D., Reyer, H.U., Hotz, H. (2008): Widespread unidirectional transfer of mitochondrial DNA: a case in western Palaearctic water frogs. *J. Evol. Biol.* **21**: 668-681.

Quilodran, C.S., Montoya-Burgos, J.I., Currat, M. (2015): Modelling interspecific hybridization with genome exclusion to identify conservation actions: the case of native and invasive *Pelophylax* waterfrogs. *Evol. Appl.* **8**: 199-210.

R Core Team (2023): R: A language and environment for statistical computing, version 4.2.3. R Foundation for Statistical Computing, Vienna, Austria.

Rannap, R., Lohmus, A., Briggs, L. (2009): Restoring ponds for amphibians: a success story. *Hydrobiologia* **634**: 87-95. DOI:10.1007/s10750-009-9884-8.

Schmeller, D.S., Pagano, A., Plenet, S., Veith, M. (2007): Introducing water frogs – is there a risk for indigenous species in France? *C R Biol* **330**: 684-690.

Schneider, H., Sinsch, U. (1992): Mating call variation in lake frogs referred to as *Rana ridibunda* Pallas, 1771: taxonomic implications. *J. Zool. Syst. Evol. Res.* **30**: 297-315.

Schneider, H., Sinsch, U., Sofianidou, T.S. (1993): The water frogs of Greece: Bioacoustic evidence for a new species. *J. Zool. Syst. Evol. Res.* **31**: 47-63.

Schneider, H., Steinwarz, D. (1990): Mating call and terrestrial calls of the spanish lake frog, *Rana perezi* (Ranidae, Amphibia). *Zool. Anz.* **225**: 265-277.

Soltis, D.E., Soltis, P.S. (1999): Polyploidy: recurrent formation and genome evolution. *Trends Ecol. Evol.* **14**: 348-352.

Speybroeck, J., Beukema, W., Dufresnes, C., Fritz, U., Jablonski, D., Lymberakis, P., Martínez-Solano, I., Razzetti, E., Vamberger, M., Vences, M. (2020): Species list of the European herpetofauna-2020 update by the Taxonomic Committee of the Societas Europaea Herpetologica. *Amphib.-Reptil.* **41**: 139-189.

Ualiyeva, D., Ermakov, O.A., Litvinchuk, S.N., Guo, X., Ivanov, A.Y., Xu, R., Li, J., Xu, F., Arifulova, I.I., Kaptyonkina, A.G., Khromov, V.A., Krainyuk, V.N., Sarzhanov, F., Dujsebayeva, T.N. (2022): Diversity, Phylogenetic Relationships and Distribution of Marsh Frogs (the *Pelophylax ridibundus* complex) from Kazakhstan and Northwest China. *Diversity* **14**.

Uzzell, T., Berger, L. (1975): Electrophoretic phenotypes of *Rana ridibunda*, *Rana lessonae*, and their hybridogenetic associate, *Rana esculenta*. *Proc. Acad. Nat. Sci. Philadelphia* **127**: 13-24.

Uzzell, T., Günther, R., Berger, L. (1976): *Rana ridibunda* and *Rana esculenta*: a leaky hybridogenetic system (Amphibia Salientia). *Proc. Acad. Nat. Sci. Philadelphia* **128**: 147-171.

Vorburger, C., Reyer, H.-U. (2003): A genetic mechanism of species replacement in European waterfrogs? *Conserv. Genet.* **4**: 141-155.

Wycherley, J., Doran, S., Beebee, T.J. (2002): Male advertisement call characters as phylogeographical indicators in European water frogs. *Biol. J. Linnean Soc.* **77**: 355-365.