polyploid complex Odontophrynus (Anura: Odontophrynidae) inhabiting agroecosystems

Accepted Manuscript Evaluation in situ of genotoxic and cytotoxic response in the diploid/polyploid complex Odontophrynus (Anura: Odontophrynidae) inhabiting agroecosystems

Favio E. Pollo, Pablo R. Grenat, Manuel A. Otero, Selene Babini, Nancy E. Salas, Adolfo L. Martino PII:

S0045-6535(18)32010-1

DOI:

10.1016/j.chemosphere.2018.10.149

Reference:

CHEM 22411

To appear in:

Chemosphere

Received Date:

21 August 2018

Accepted Date:

21 October 2018

Please cite this article as: Favio E. Pollo, Pablo R. Grenat, Manuel A. Otero, Selene Babini, Nancy E. Salas, Adolfo L. Martino, Evaluation in situ of genotoxic and cytotoxic response in the diploid /polyploid complex Odontophrynus (Anura: Odontophrynidae) inhabiting agroecosystems, Chemosphere (2018), doi: 10.1016/j.chemosphere.2018.10.149

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ACCEPTED MANUSCRIPT Evaluation in situ of genotoxic and cytotoxic response in the diploid/polyploid complex Odontophrynus (Anura: Odontophrynidae) inhabiting agroecosystems

Favio E. Pollo a,b, Pablo R. Grenat a,b*, Manuel A. Otero a,b, Selene Babini a,b, Nancy E. Salasa, Adolfo L. Martinoa a-

Ecología-Educación Ambiental, Departamento de Ciencias Naturales, Facultad de

Ciencias Exactas, Físico-Químicas y Naturales, UNRC, ruta 36km 601, Río Cuarto, Córdoba, Argentina b-

Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Argentina

* Corresponding author; e-mails: [email protected]; [email protected]

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Abstract

2

Polyploidization has been documented across a wide range of vertebrates. Gene duplication

3

could promote better adaptation to environmental changes and to chronic injury or stress. We

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investigated if genotoxic and cytotoxic responses to agricultural impact are affected by ploidy.

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We evaluate syntopic populations of the cryptic diploid/polyploid complex Odontophrynus

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cordobae / O. americanus breeding in an agroecosystem from Central Argentina. The blood of

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72 adult anurans was analysed. We used erythrometry to distinguish Odontophrynus individuals

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with different ploidy levels. We calculated micronucleus frequencies (Mn) and erythrocytic

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nuclear abnormalities (ENAs) as genotoxic effects and enucleated, mitotic, pyknotic and

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immature erythrocytes as cytotoxic endpoints (CYT). Mn, ENAs and CYT frequencies were

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significantly different between diploid and polyploid organisms. The higher frequencies of Mn

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and CYT were recorded in polyploid organisms, and the higher frequency of ENAs was recorded

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in diploids. These results indicate that stress response, as indicated by most genotoxic and

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cytotoxic endpoints, was higher in polyploids respect to diploids. Polyploidy could provide

15

greater genetic flexibility increasing buffering against exogenous DNA-damaging agents and

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thus confer an advantage over diploids under certain environmental conditions.

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Keywords: Odontophrynus, polyploidy, diploid, agroecosystems, genotoxicity, cytotoxicity.

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1. Introduction

21

Polyploidization has been documented across a wide range of vertebrates (Le Comber and

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Smith, 2004; Gregory and Mable, 2005; Schmid et al., 2015) and is recognized as an important

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mechanism of evolution in several amphibian families (Evans et al., 2012; Schmid et al., 2015).

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Two documented advantages of gene duplication mediated by this mechanism are heterosis and

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gene redundancy which could promote better adaptation to environmental changes and to

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chronic injury or stress (Tymowska, 1991; Comai, 2005; Davoli and de Lange, 2011; Pandit et

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al., 2013). Potentially, polyploidization could protect cells against cytotoxic and genotoxic

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damage by increasing their gene copy numbers (Pandit et al., 2013). Thus, the allele loss in

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diploid cells could have extreme consequences while in polyploid cells the other homologs

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containing functional genes could maintain normal function without generating negative effects

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(Pandit et al., 2013). On the other hand, the probability that a polyploid cell is affected is greater

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the more molecular targets (sets of chromosomes) it contains. A greater genotoxic response

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associated with an increase in ploidy was reported by several authors in mammalian cells

34

(Uryvaeva and Delone, 1995; Hau et al., 2006). Similarly, laboratory assays using induced

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triploid fishes have shown greater sensitivity of polyploids to different endpoints compared to

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diploid individuals (Strunjak-Perovic et al., 2003; Karami et al., 2016). However, these

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relationships in naturally occurring polyploid species have not been proved.

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In central Argentina, the genus Odontophrynus includes two cryptic species with different ploidy

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levels: Odontophrynus cordobae (2n = 22) and O. americanus (4n = 44) (Martino and Sinsch,

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2002; Grenat et al., 2009b). Some of the southernmost populations of O. cordobae coexist in

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syntopy with populations of O. americanus in central-western Córdoba overlap with areas of

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intensive agriculture (Grenat et al., 2009b). Recently, triploid individuals have been found

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coexisting with diploids and tetraploids in the same reproductive sites, in all cases immersed in

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an agricultural matrix (Grenat et al., 2018). In this area, the agroecosystems occupy large

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portions of land and generate habitats that are usually used by amphibians to breed. In the last

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years the agricultural practices have incorporated an important variety of chemical products that

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improve the production of crops. Because of this, the health of amphibian using these habitats

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could be compromised by agricultural chemicals that by runoff reach these environments (Mann

49

et al., 2009). In recent years, numerous laboratory studies have evaluated cytotoxic and

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genotoxic effects of various agrochemicals on amphibian species (Lajmanovich et al., 2005;

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Josende et al., 2015; Soloneski et al., 2016; Gonçalves et al., 2017), even with O. cordobae

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(glyphosate - Bosch et al., 2011) and O. americanus (cypermethrin - Cabagna et al., 2006).

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However, few in situ studies examining amphibian populations inhabiting agroecosystems have

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been carried out, in which individuals are analysed in their natural environment (e.g. Caraffa et

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al., 2013; Pollo et al., 2015; Raghunath et al., 2017; Zhelev et al., 2018). In situ studies are

56

important because they provide environmental scenarios of actual exposure that are rarely

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replicated in the laboratory (Crane et al., 2007).

58

In our study, we investigated if the cytogenotoxic responses of anurans under the same

59

conditions of agricultural impact are affected by ploidy. For it, we studied syntopic populations

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of the cryptic diploid/polyploid complex Odontophrynus cordobae / O. americanus breeding in

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an agroecosystem from Central Argentina. This complex presents a unique opportunity to study

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in situ the potential differential responses of organisms with different ploidy level, coexisting in

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space and time, to the disturbances produced by agricultural practices.

64 65

2. Materials and Methods

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2.2 Study area

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The sampling area corresponds to a narrow contact zone between populations of O. cordobae

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and O. americanus located in the rural zone of Alcira Gigena, Córdoba, Argentina (32°45'S,

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64°20'W). This area constitutes the only contact and hybridization zone between these species

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reported to date and is mainly characterized by an agricultural landscape, which is fragmented by

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vegetated deep gullies with a typical dendritic drainage net. Here, O. americanus and O.

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cordobae are distributed as a mosaic of populations and coexist in some breeding sites (Martino

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and Sinsch, 2002; Grenat et al., 2009b; 2018). Particularly, the sampled breeding sites are

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located in an area of approximately 300m2 and correspond to flood depressions that receive

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water by runoff from agricultural fields. The species breed during austral spring-summer months

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(September–March) and daily reproductive activity takes place mainly between 2000 and 0500

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hours. Our surveys were conducted during the summer season of 2015 and 2017. In both periods,

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the breeding sites were surrounded by extensive soybean cultivation. Soybean crops account for

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80% of production in central Argentina (Butinof et al., 2015) and the main pesticides applied are

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glyphosate, cypermethrin, endosulfan and chlorpyrifos (Solis et al., 2017). High values of nitrate

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and phosphate in water are indicators of the presence of agrochemicals (Spear et al., 2009).

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Accordingly, studies conducted in the area have detected high concentrations of these

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compounds in water (Babini et al., 2015; 2016; 2018; Bionda et al., 2018).

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2.3 Sample collection

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We captured by hand 72 adult anurans from syntopic breeding sites. Each collected individual

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was sexed, using external secondary sexual characters, and total length (snout-vent length - SVL)

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was measured using a manual SometInox Extra Vernier caliper (0.01 mm). Immediately after

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capture, blood samples were obtained from the angularis vein puncture (Nöller, 1959; Martino

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and Sinsch, 2002). Smears of fresh blood were air-dried, fixed and stained using May Grunwald-

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Giemsa (Dacie and Lewis, 1995).

93 94

2.4 Erythrometric analysis: ploidy identification and deformability analysis

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Because amphibian blood cells conserve their nucleus, the erythrocyte and nuclear size are

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correlated with the DNA content (Stöck and Grosse, 1997). For this reason, the erythrometry is a

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technique commonly used for the distinction of Odontophrynus species with different ploidy

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levels (Martino and Sinsch, 2002; Grenat et al., 2009a, b; Otero et al., 2013). We measured

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length (Lcel and Lnuc) and width (Wcel and Wnuc) of 40 randomly chosen erythrocytes and their

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respective nuclei for all individuals using ImageJ software. Photographs were obtained by using

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Axiophot Microscope (Carl Zeiss) at magnification of 1000 X with Canon Power shot G6 Digital

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Camera and Zoom Browser EX, and saved in TIFF files. Cell (Acel) and nuclear (Anuc) areas

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were calculated assuming an ellipsoid shape (L*W*π/4). Ploidy level of each individual was

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determined using the limit values of cell and nuclear areas proposed by Grenat et al., (2018)

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using previously karyotyped individuals. A total of 29 diploids, 31 triploids and 12 tetraploids

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were identified.

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To analyse the erythrocyte deformation were calculated the cell and nuclear aspect ratio

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(CAR=Lcel/Wcel and NAR=Lnuc/Wnuc) and the nucleo cytoplasmic ratio (NR=Anuc/Acel).

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2.5 Cyto-genotoxicity analyses

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Two thousand erythrocytes per individual were examined using a microscope at 1000x

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magnification (Zeiss Primo Star iLED) and the results were expressed per 1000 cells (‰). Only

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mature and immature cells with nuclear membrane intact were counted. The coded and

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randomized slides were blind-scored by a single observer. Genotoxicity was tested using

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micronuclei (Mn) and erythrocytic nuclear abnormalities (ENAs), carried out in mature

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peripheral erythrocytes according to the procedures of Fenech (2000) and Carrasco et al., (1990)

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respectively. We used mature erythrocytes to establish the frequency of four nuclear lesions:

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notched, binucleated, lobed and blebbed (Pollo et al., 2015; Pollo et al., 2017). The results were

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expressed as ENA mean frequency (‰) of the sum of all abnormalities observed (Lajmanovich

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et al., 2014; Pollo et al., 2016). In addition, frequencies of enucleated erythrocytes, in mitotic

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division, pyknotic and immature erythrocytes were calculated as cytotoxicity effect (CYT) and

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the sum of these values was expressed as mean cytotoxicity frequency.

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2.6 Statistical Analysis

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Previous analyses showed no differences between triploids and tetraploids (Mn, p=0.5423; ENA,

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p=0.1441; CYT, p=0.1363; CAR, p=0.4606; NAR, p=0.5444; NR, p=0.0795) and between sexes

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(Mann–Whitney test, diploids: Mn, p=0.4721; ENA, p=0.7517; CYT, p=0.3753; CAR,

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p=0.2549; NAR, p=0.3545; NR, p=0.1649 – polyploids: Mn, p=0.4352; ENA, p=0.0763; CYT,

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p=0.4199; CAR, p=0.0522; NAR, p=0.2772; NR, p=0.6740) in the parameters examined.

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Consequently, we combined males and females and tested differences between diploid and

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polyploid individuals in subsequent analyses.

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Assumptions of normal distribution were tested with the Kolmogorov-Smirnov test. The NR and

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NAR presented normal distribution, so that parametric ANOVAs were performed for

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comparisons between ploidy levels. Mn, ENA and CYT frequencies and CAR were adjusted to a

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generalized linear mixed model (GLMM) to compare between diploid and polyploid organisms.

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CAR was adjusted to a Gamma distribution and inverse link function while Mn, ENA and CYT

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frequencies were adjusted to a binomial distribution and logit link function (Nelder and

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Wendderburn, 1972; Myers et al., 2002). The best model was selected using the Akaike

139

information criterion (AIC) and Bayesian information criterion (BIC) methods. Then, to test

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differences between means a post-hoc DGC test was used (Di Rienzo et al., 2002). This test uses

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the multivariate cluster analysis technique, mean chain or UPGMA (unweighted pair-group

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method using an arithmetic average) in a distance matrix obtained from the sampling means

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(Balzarini et al., 2008).

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Differences in proportion of each type of abnormality between diploid and polyploid individuals

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were examined for significance in pairs by the binomial test at p<0.05 significance. Furthermore,

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simple regressions to examine the possible relation between each parameter with body length,

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nuclear and cell areas of diploids and polyploids were performed.

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Principal Component Analysis (PCA) was performed based on four parameters (Mn, ENAs,

149

CYT and NR). Data set before plotting the PCA were standardized because the variables have

150

different units. All analyses were conducted using InfoStat (Di Rienzo et al., 2012), Statgraphic

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5.0 and R 3.3.2 (R Core Team, 2016).

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3. Results

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Mn, ENAs and CYT were detected in both species (Fig. 1). Frequencies of Mn, ENA and CYT

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are shown in Table 1. The proportion of individuals by ploidy level showing genotoxic (Mn and

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ENAs) or cytotoxic responses were only statistically different for Mn (Binomial test, Mn: Zdiploid-

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polyploid=-0.245,

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p=0.798).

159

A negative association between ENAs and nuclear area of diploid individuals was observed (r=-

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0.52; p<0.01) while no relation was observed between the remaining parameters with body

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length, nuclear and cell areas for diploids and polyploids (p>0.05).

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Mn, ENA and CYT frequencies were statistically different between diploid and polyploid

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organisms (GLMM Mn: F1, 69: 4.4, p<0.05; GLMM ENAs: F1, 69: 29.15p<0.0001; GLMM CYT:

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F1,

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organisms, and the highest frequency of ENAs was recorded in diploid organisms (Fig. 2).

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The NR was significantly different between diploid and polyploid organisms (ANOVA: F1,69:

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21.74, p<0.0001; Fig. 2). Polyploids showed the lowest values of NAR and CAR but no

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significant differences in these ratios between diploid and polyploid organisms were recorded

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(GLMM CAR: F1, 69: 0.67, p=0.4165; ANOVA NAR: F1, 69: 1.15, p=0.2881; Fig. 2).

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According to the results of PCA, the first two principal components were significant with

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eigenvalues higher than one (1.22 and 1.03 respectively). PC1 explained 30.6% of the variance

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and was defined by positive loadings for NR (0.61), CYT (0.52) and Mn (0.39), mainly

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associated to polyploid individuals and negative loading for ENAs (0.46), related to diploids.

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PC2 explained 25.8% of the variance and was characterized mainly by positively weighted Mn

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(0.74), associated to polyploid individuals and ENAs (0.67), associated with diploids organisms

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(Fig. 3).

69:

p=0.025; ENA: Zdiploid-polyploid=0.13, p=0.337; CYT: Zdiploid-polyploid=0.04,

4.04, p<0.05). The highest frequencies of Mn and CYT were recorded in polyploid

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4. Discussion

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Currently numerous ex situ toxicity assays are carried out to determine potential adverse effects

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on environment and species health. These studies are important for evaluating the sensitivity of

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different species to particular toxic chemicals, but often it is difficult to achieve ecological

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realism under controlled laboratory conditions (Preston and Shackelford, 2002). Furthermore,

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laboratory tests typically examine single compounds, whereas individuals inhabiting polluted

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environments are most often exposed to complex mixtures (Matson et al., 2009). Therefore, the

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extrapolation of these results to field situations can be in many cases inappropriate.

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Habitats that are immersed in an agricultural matrix are under intense pressure due to the impact

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of large quantities of pesticides. These commercial formulations contain complex mixtures

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which are used as insecticides, herbicides, fungicides and others (McLaughlin and Mineau,

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1995). Approximately 80-90% of applied pesticides reach nonspecific organisms as they are

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dispersed in the environment (Moses et al. 1993; www.epa.gov/pesticides). In complex

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environments such as agroecosystems, in situ studies are valuable because effects observed in

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the field are often the result of the net effects of stressors and can be directly examined (Preston

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and Shackelford, 2002). To our knowledge, this is the first in situ study comparing the incidence

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of genotoxic and cytotoxic damage in related diploid/polyploid species inhabiting human-

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perturbed landscapes.

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Previous data about the threshold level of abnormal cells and nuclei for natural populations of

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Odontophrynus cordobae and O. americanus have not been reported. Unfortunately, our study

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fails to compare disturbed and undisturbed environments because the syntopic condition of these

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species, which ensures similar habitat conditions, only occurs in agroecosystems. However,

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genotoxic effects of different agricultural formulations (glyphosate and cypermethrin) under

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laboratory conditions have showed that these species respond to these agrochemicals by

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producing greater Mn frequencies (Cabagna et al., 2006; Bosch et al., 2011). Our results were in

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accordance with values reported by Cabagna et al., (2006) in tadpoles of tetraploid O.

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americanus (1-3 Mn/1000 cells) while Bosch et al., (2011) observed a Mn frequency much

205

higher than that observed in our study for diploid O. cordobae. This higher response rate can be

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explained by the high concentrations of glyphosate that were used by these authors in the

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laboratory assays (100mg a.i./L), far higher than the expected environmental concentrations (2.6-

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3.7mg a.i./L; Giesy et al., 2000; Relyea, 2005). Thus, in this test the individuals could have

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shown extreme responses that could not be comparable with field situations.

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In any case, our field results confirm laboratory test data showing an increased production of

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micronucleated cells in both species and thus evidencing genotoxic damage. This could indicate

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that ponds present compounds capable of interacting with DNA (Omar et al., 2012). A positive

213

correlation between genotoxic damage and degradation of environmental quality by agricultural

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practices was found in other studies (Ossana and Salibián, 2013; Josende et al., 2015; Babini et

215

al., 2015). On the other hand, differences in Mn frequencies between ploidy levels were

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significant, with higher values in polyploids. These results agree with other investigations in

217

which the Mn frequency correlates with the number of chromosomes. Uryvaeva and Delone

218

(1995) found a positive relation between the percentage of micronucleated cells and the ploidy

219

level in mouse liver cells. Similarly, in fishes, induced triploids of rainbow trout Oncorhynchus

220

mykiss showed significantly higher Mn frequency than diploid individuals, possibly due to the

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genotoxic effect caused by the thermal shock used for polyploidy induction (Strunjak-Perović et

222

al., 2003). Thus, these results could indicate a greater susceptibility to genotoxic and mutagenic

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compounds of polyploids potentially related to the cellular size and amount of genetic material.

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Consequently, polyploid individuals could contain multiple molecular targets for genetic damage

225

in comparison with diploid cells during cell division, resulting in a greater number of Mn

226

(Strunjak-Perović et al., 2003). In fact, we found not only a higher Mn frequency, but also a

227

greater proportion of polyploid individuals in which this biomarker of genotoxic damage was

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detected. Some studies have also demonstrated differential genotoxic responses associated to

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ploidy level using other biomarkers. Hau et al., (2006) reported that polyploidization increases

230

the sensitivity to genotoxic stress in mammalian cells exposed to ionizing radiation. In other

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laboratory assays, Karami et al., (2016) observed differences in the responses to pesticide of

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diploid and induced triploid catfish Clarias gariepinus for morphometric, molecular and

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hormonal biomarkers. Lee et al., (2009) recognize that polyploidy could provide an advantage

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because the multiple gene copies may increase buffering against random, gene-inactivating

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mutations such as exogenous DNA-damaging agents. Spontaneous polyploidization arising

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during stress has been reported in mammalian cells (Celton-Morizur and Desdouets, 2010) and

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associated to a variety of pathological conditions, such as cancer and degenerative diseases

238

(Pandit et al., 2013). In example, hepatocytes and cancer cells increased their ploidy in response

239

to DNA damage whereas most other cells under go apoptosis, possibly indicating that

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polyploidization might be an alternative route to respond to genotoxic stress (Lee et al., 2009;

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Davoli and de Lange, 2011; Pandit et al., 2013).

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Some authors indicate that differences in genotoxicity in closely related species could be due

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toxicokinetic of the contaminant, hematopoietic cycle speed or incorrect or inefficient DNA

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repair (Palhares and Grisolia, 2002; Omar et al., 2012). The increased frequency of nuclear

245

abnormalities (ENAs) is an indicative of adverse cellular reactions and/or of control mechanisms

246

used to eliminate cells with damaged DNA (Fijan, 2002). The mechanisms responsible for ENAs

247

have not been fully understood. However, they have been interpreted as nuclear lesions

248

analogous to micronuclei (Ayllon and Garcia-Vazquez, 2000; Serrano Garcia and Montero

249

Montoya, 2001; Guilherme et al., 2008). Our results show that these abnormalities are induced

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by genotoxic compounds that exist in pond water, even if micronuclei are not induced, or appear

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at very low frequencies. Although the high frequency of ENAs recorded in our study would

252

indicate a greater sensitivity of diploids to compounds present in the water, some authors assert

253

that it is not a good indicator of genotoxic damage, since the cell viability is not reduced (de

254

Campos Ventura et al., 2008). Furthermore, values of ENAs were not directly correlated with

255

Mn frequency for the same species. This result could be related to the significant differences that

256

we found evaluating cytotoxic damage between ploidy conditions. Higher frequencies of

257

erythroblasts, reflecting an increased erythropoiesis, and pyknotic cells, associated with

258

apoptosis (Saquib et al., 2012; Peltzer et al., 2013), could indicate that polyploids would have a

259

greater ability to remove damaged cells before they enter into circulation, persisting only those

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that are not damaged (Natale et al., 2018). Furthermore, the increases in these frequencies have

261

been associated with a response to stress condition or cellular injury (Ray et al., 2005).

262

Variations in the shape of the red blood cells could provide a complementary approach for

263

detecting genotoxicity. Although polyploids showed less elongated erythrocytes than diploids,

264

we did not find significantly abnormal aspect ratios for nuclei (NAR) or cells (CAR).

265

Consequently, some research authors have reported that the proportion of abnormal erythrocyte

266

shapes in fishes and anurans increased with increasing ploidy levels (Gao et al., 2007; Lu et al.,

267

2009; Hermaniuk et al., 2013). However, polyploid individuals showed nucleocytoplasmic ratios

268

(NR) significantly higher than diploids. A conservative relationship in eukaryotic cells between

269

nuclear DNA content and cell size is well documented (Gregory, 2001). The relatively constant

270

NR could represent an optimization of the relationship between nuclear and cytoplasmic

271

compartments (Umen, 2005). In consequence, Grenat et al., (2009a) showed similar values of

272

NR in diploid O. cordobae (0.096) and polyploid O. americanus (0.101) adults. However, these

273

NR values were considerably lower than found in our study for Odontophrynus diploid and

274

polyploid individuals. Several studies in fishes and anurans have reported variations in NR of

275

individuals from polluted environments (Khuda Bukhsh et al., 2000; Zhelev et al., 2016; 2017).

276

Changes in NR may be due to variations in cell or nuclear size, or both. Comparing erythrocyte

277

and nuclear areas only of diploid and tetraploid individuals, we observed that nuclear size was

278

similar but cell size was notably lower in our study than in previous studies with these species,

279

mainly in tetraploids (Grenat et al., 2009a; Otero et al., 2013). Since smaller erythrocytes have

280

relatively larger surface areas, and therefore, exchange oxygen more efficiently, this result could

281

be associated to a response under hypoxic conditions of polluted ponds. Furthermore, in

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polyploids, higher frequencies of enucleated and mitotic cells were observed which may

283

represent a short-term mechanism for increasing oxygen carrying efficiency, particularly in

284

conditions of water contamination (Barni et al., 2007; Peltzer et al., 2013).

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In conclusion, the stress response, as indicated by most genotoxic and cytotoxic endpoints, was

286

higher in polyploid individuals respect to diploid organisms. Xenobiotics can selectively act on

287

sensitive phenotypes and consequently can reduce genetic diversity in the population

288

(Theodorakis et al., 2000; Johansson et al., 2005) leading to population reduction by the effects

289

of somatic and heritable mutations (Bickham et al., 2000). However, polyploidy should provide

290

greater genetic flexibility and thus confer an advantage over diploids under certain

291

environmental conditions. On the other hand, xenobiotics can not only cause severe toxicity, but

292

certain concentrations can damage processes such as growth, development, survival and

293

reproduction in amphibian species (Montalvão et al., 2017). Thus, future studies should be aimed

294

to evaluate survival, fecundity and reproductive value of diploid and polyploid populations

295

inhabiting polluted environments.

296 297

Acknowledgements. We thank the Secretary of Research and Technology of National

298

University of Río Cuarto (PPI 18/C448) and National Agency for Scientific and Technological

299

Promotion FONCYT (BID-PICT 0932-2012; BID-PICT 2533-2014) for provided funds. The

300

authors thank CONICET - Argentina (Argentinean National Research Council for Science and

301

Technology) for fellowships granted. Our study was authorized by Cordoba Environmental

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Agency (A.C.A.S.E.), Environmental Secretary of Córdoba Government.

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Figure captions

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Fig. 1 Erythrocytes in blood of diploid and polyploid Odontophrynus: Erythrocyte micronucleus

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in polyploid (A) and diploid (B) individuals. Blebbed (C), notched (D) and lobed (E) nuclei in

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Pyknotic erythrocytes in polyploid (I) and diploid (J) specimens. May Grünwald-Giemsa, 100 X.

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Fig. 2 Comparison between ploidy levels of mean values (± SE) of genotoxic, cytotoxic and

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test). Mn=micronucleus frequency; ENAs=nuclear abnormalities; CYT=cytotoxic effect;

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NR=nucleo cytoplasmic ratio; CAR=cell aspect ratio; NAR=nuclear aspect ratio.

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Fig. 3 PCA biplot based on indicators of genotoxicity, cytotoxicity and cell deformation showing

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the differentiation of diploid and polyploid individuals. Filled squares = diploids; empty squares

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= polyploids. Mn=micronucleus frequency; ENAs=erythrocytic nuclear abnormalities;

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CYT=cytotoxic effect; NR=nucleo cytoplasmic ratio.

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Table 1 Table 1. Frequencies (‰) of micronuclei (Mn), nuclear abnormalities (ENAs), mitotic erythroid, immature erythrocytes and enucleate cells in adults from cryptic diploid/polyploid complex Odontophrynus. ‰=frequency per one thousand; n=sample size. Ploidy level Diploid n = 29

Polyploid n = 43

47.41 ± 2.62

47.14 ± 2.76

‰ Mn ‰ ENAs

0.10 ± 0.31 8.45 ± 6.14

0.35 ± 0.53 5.06 ± 2.80

Cytotoxicity ‰ Enucleated ‰ Mitotic ‰ Pyknotic ‰ Immature

0.35 ± 0.79 0.17 ± 0.54 1.38 ± 1.88 6.31 ± 6.67

0.68 ± 1.03 0.47 ± 1.40 1.65 ± 1.42 6.12 ± 8.02

SVL (mm) Genotoxicity

552 553

ACCEPTED MANUSCRIPT Highlights 1. No differences were found in cytogenotoxic responses between 3n and 4n individuals 2. Diploids showed higher frequencies of nuclear abnormalities than polyploids 3. Polyploids showed higher incidence of Mn and cytotoxic effects than diploids 4. Nucleocytoplasmic ratio, but not cell and nuclear aspect, differed between ploidies