Acta Herpetologica 13(2): 125-140, 2018 ISSN 1827-9635 (print) © Firenze University Press ISSN 1827-9643 (online) www.fupress.com/ah DOI: 10.13128/Acta_Herpetol-22611 Batrachochytrium dendrobatidis in Hungary: an overview of recent and historical occurrence Judit Vörös1,2, Dávid Herczeg3,4,*, Attila Fülöp5, Tünde Júlia Gál1, Ádám Dán6, Krisztián Harmos7, Jaime Bosch8,9 1 Department of Zoology, Hungarian Natural History Museum, 1088 Budapest, Hungary 2 Laboratory for Molecular Taxonomy, Hungarian Natural History Museum, 1083 Budapest, Hungary 3 Lendület Evolutionary Ecology Research Group, Plant Protection Institute, Centre for Agricultural Research, Hungarian Academy of Sciences, 1022 Budapest, Hungary. *Corresponding author. E-mail herczegdavid88@gmail.com 4 Institute for Veterinary Medical Research, Centre for Agricultural Research, Hungarian Academy of Sciences, 1143 Budapest, Hungary 5 MTA–DE Behavioural Ecology Research Group, Department of Evolutionary Zoology and Human Biology, University of Debrecen, 4032 Debrecen, Hungary 6 Molecular Biology Department, Veterinary Diagnostic Directorate, National Food Chain Safety Office, 1143 Budapest, Hungary 7 Bükk National Park Directorate, 3304 Eger, Hungary 8 Museo Nacional de Ciencias Naturales, CSIC, 28006 Madrid, Spain 9 Centro de Investigación, Seguimiento y Evaluación, Parque Nacional de la Sierra de Guadarrama, 28740 Rascafría, Spain 1, 2, 3, 4 These authors contributed equally to this work Submitted on: 2018, 5th February; revised on: 2018, 5th May; accepted on: 2018, 28th August Editor: Marcello Mezzasalma Abstract. Batrachochytrium dendrobatidis (Bd) is a fungal pathogen which causes the emerging infectious disease chytridiomycosis. Bd presents low host specificity and threatens amphibians worldwide, thus systematic inventory is the key in order to detect and mitigate the effects of the disease. Extensive data collection was conducted in Hungary in 2009-2015 from fourteen different areas. Combined data – recent field sampling on sixteen taxa and the examina- tion of archived Bombina spp. specimens – from 1360 individuals were analysed with qPCR. Two sentinel taxa, Bom- bina variegata and the members of the Pelophylax esculentus complex were marked to monitor the occurrence of Bd in two core areas (Bakony Mts and Hortobágy National Park, respectively) of sampling. Climatic variables were also examined in core areas to test their effect on prevalence and infection intensity. Among the sixteen sampled amphib- ian taxa seven tested positive for Bd and the overall prevalence in Hungary was 7.46%. Among the ethanol-fixed Bom- bina spp. individuals Bd was not detected. In the first core area (Bakony Mts) the overall prevalence in B. variegata was 10.32% and juvenile individuals showed significantly higher prevalence than adults. On the other hand there was a significant negative relationship between infection prevalence and monthly mean air temperature. Finally, in the other core area (Hortobágy National Park) the overall prevalence in P. esculentus complex was 13.00%, and no differ- ences were found in prevalence or infection intensity between sexes, sampling years or age classes. Keywords. Chytridiomycosis, emerging infectious diseases, Pelophylax esculentus complex, Bombina variegata, inventory, Central-Europe. INTRODUCTION Over the past decades several epidemics – caused by emerging infectious diseases – resulted in the large- scale decline of numerous animal species globally (Dob- son and Foufopoulos, 2001). One such emerging disease is chytridiomycosis in amphibians caused by the fungal pathogen Batrachochytrium dendrobatidis [hereafter, Bd 126 Judit Vörös et alii (Longcore et al., 1999)]. Bd is a highly generalist, water- borne pathogen which is primarily transmitted through direct contact with aquatic zoospores or infected individ- uals (Fisher et al., 2009). Bd is responsible for population declines, mass mortalities and even extinction of species, and presents one of the greatest threats to amphibians worldwide (Berger et al., 1998; Skerratt et al., 2007; Fisher et al., 2009). Bd is widespread on all continents where amphibians occur (Olson et al., 2013), but the heaviest disease out- breaks were observed in the American Neotropics, Aus- tralia, North-America and Western Europe (Fisher et al., 2009). In Europe, the first detection of Bd related mass mortalities dates back to 1997 when the first recorded population decline as a result of mass die-off after the emergence of chytridiomycosis was observed in Central Spain, in the Guadarrama Mountain National Park, and targeted the Common midwife toad, Alytes obstetricans (Bosch et al., 2001). Though, as a result of the increased attention in the subsequent years, studies performed in the same region revealed that other species are highly susceptible to the disease as well (e.g. Salamandra sala- mandra, Bufo spinosus; Bosch and Martínez-Solano, 2006; Bosch et al., 2007). Moreover, the evidenced strong population declines of A. obstetricans, A. muletensis and A. dickhilleni in the Iberian Peninsula (Bosch et al., 2001; Walker et al., 2010; Bosch et al., 2013; Doddington et al., 2013; Rosa et al., 2013), and the high susceptibility of these species made the midwife toads the “flagship” spe- cies of European chytridiomycosis threat. Central Europe harbours several amphibian species that might be susceptible to chytridiomycosis, such as S. salamandra, B. bufo, Bombina bombina or Bombina var- iegata (Baláž et al., 2014a,b). In the recent years Bd infec- tion was detected in various areas of the Czech Republic, as a result of a systematic inventory (Civiš et al., 2012). Furthermore, the presence of the fungus was recently reported in low prevalence from Luxembourg (Wood et al., 2009), Poland (Sura et al., 2010; Kolenda et al., 2017), Germany (Ohst et al., 2013), Austria (Sztatecsny and Gla- ser, 2011), Slovakia (Baláž et al., 2014b) and Italy (Feder- ici et al., 2008; Tessa et al., 2013). New data indicate that the fungus is present also in the Balkans, e.g., in Serbia (Mali et al., 2017), Albania, Montenegro and Macedo- nia (Vojar et al., 2017). Though, interestingly, no nega- tive effects or Bd-linked population declines have been detected from Central-Eastern-Europe so far (Vörös et al., 2014). Some aspects of chytridiomycosis epizootics show environmental correlates (Olson et al., 2013). Bd presents a reasonably wide environmental tolerance under a vari- ety of temperature and precipitation regimes (Ron, 2005), but previous studies postulated that climate (Berger et al., 2004; Bosch et al., 2007; Murray et al., 2009; Blaustein et al., 2010; Rohr et al., 2010; Rödder et al., 2010) and elevation (Lips et al., 2008; Walker et al., 2010; Becker and Zamudio, 2011) can significantly influence Bd out- breaks. Furthermore, large intra- and interspecific vari- ations exist, especially in the prevalence (Gründler et al., 2012; Böll et al., 2014; Spitzen-Van Der Sluijs et al., 2014), but also in the intensity of infection (Van Sluys and Hero, 2009; Baláž et al., 2014a; Spitzen-Van Der Slui- js et al., 2014). In addition, behavioural differences influ- ence the susceptibility to Bd which is further affected by the intraspecific variability related to sex and life stages (Blaustein et al., 2005, Garcia et al., 2006, Williams and Groves, 2014). Hungary is situated in the Carpathian Basin, a region with high amphibian diversity due to different climatic and zoogeographical influences (Vörös et al., 2014). Pre- vious findings about the occurrence of Bd in Hungary are restricted to a few areas and species where the presence was initially detected (Gál et al., 2012; Baláž et al., 2014b, Vörös et al., 2014, Drexler et al., 2017). Therefore, no large-scale distribution data on Bd presence is available to date from the country. Our study displays multiple goals. First, we present a general overview on the occurrence of Bd in Hungary summarising data collected between the years 2009-2015. The data set includes the general occurrence of Bd on six- teen amphibian taxa with a special focus on the yellow- bellied toad Bombina variegata and water frogs belonging to the Pelophylax esculentus complex. We selected these two target taxa because these species may present high levels of infection intensity in Europe and so they may also act as sentinel taxa (Baláž et al., 2014b); in addition, they can play a role in the spread and the persistence of the disease (Baláž et al., 2014a). Second, by studying B. variegata populations in Hun- gary we assessed whether distinct phylogenetic lineages – Alpine (West of the Danube) and Carpathian, occurring in the North Hungarian Range East of the Danube (Vörös et al., 2006) – express differences in prevalence and infec- tion intensity. Moreover, to explore the historical distri- bution of Bd in Hungary field surveys were complement- ed with available archived samples of Bombina spp. from museum collections which comprise a dataset covering a 70 years’ time frame (1936-2005) prior to our field sam- pling. Third, in order to further monitor Bd infection lev- els of amphibians in Hungary, we selected one popula- tion of two of the most susceptible taxa in Central-East- ern Europe, B. variegata and the P. esculentus complex (Baláž et al., 2014b), and extensively sampled these 127Occurrence of Bd in Hungary populations for three consecutive years in two core areas. Finally, we aimed to use climatic data (monthly mean precipitation and monthly mean air temperature) in these core areas to test if there is any correlation between the previously mentioned climatic variables and the occurrence of Bd. MATERIALS AND METHODS Data collection Altogether 1233 specimens belonging to sixteen amphib- ian taxa were studied in the field between 2009-2015. Sam- pling was conducted in fourteen different regions in 45 distinct sampling points throughout Hungary, covering a great vari- ety of wetland habitats (i.e. irrigation canals, streams, marsh- lands, ponds, fishponds, water reservoirs and temporary wet- land habitats) and elevations ranging between 84 and 734 m a.s.l. (Fig. 1, Table 1). Bombina variegata was surveyed in five regions from Transdanubia (Region 1, 2, 3, 5 and 8 in Table 1 and Fig. 1) representing the Alpine (Western) genetic lineage, and in three regions from the North Hungarian Mountains (Region 10, 12 and 13 in Table 1 and Fig. 1) representing the Carpathian (Eastern) genetic lineage, covering the distribution of the species in Hungary (Vörös et al., 2006). Identification of the two Bombina species and their hybrids was performed considering morphological characters plus genetic information provided by previous researches in Hungary (Vörös et al., 2006, 2007). Members of the Pelophylax esculentus complex were sampled in eight regions (Region 1, 3, 4, 7, 8, 9, 10 and 14 in Table 1 and Fig. 1). Age classes were characterized as tadpoles, juveniles and adults based on the external features of each spe- cies examined in the field. In those cases when we couldn’t distinguish between age and sex of an individual we discarded the sample for further analysis. Additionally, 127 ethanol-fixed specimens of Bombina spp., deposited in the Hungarian Natu- ral History Museum (Budapest, Hungary) and Savaria Museum (Szombathely, Hungary), collected between 1936 and 2005 from regions matching the current distribution of the species were swabbed (Supplementary table S1). Fig.1. Map of Hungary showing sampling locations of Bd negative (black filled circles), Bd positive (red triangles) and archived (white cir- cles) samples. Pie charts indicate Bd prevalence of the fourteen studied geographic regions. Numbers of regions correspond to Table 1. The two core areas are marked with asterisks (Region 3 and 14). Drawing of Bombina variegata and Pelophylax ridibundus are the courtesy of Márton Zsoldos. 128 Judit Vörös et alii Ta bl e 1. S um m ar y of r eg io ns , s am pl in g lo ca tio ns , c oo rd in at es a nd s am pl ed s pe ci es in o ur in ve nt or y. m tD N A li ne ag es w er e in di ca te d as A lp in e (A lp ) or C ar pa th ia n (C ar p) in t he c as e of B . v ar ie ga ta . L at = L at itu de ; L on g = Lo ng itu de ; N = N um be r of in di vi du al s sa m pl ed ; P re v = Pr ev al en ce ; G E = G en om ic e qu iv al en ts . N r. of r eg io n A lt La t Lo ng Sp ec ie s m tD N A lin ea ge B . va ri eg at a N Po si tiv e/ Sa m pl ed Pr ev ( % ) Pr ev 9 5% C I (% ) G E m ea n G E m ed ia n G E SD G E ra ng e 1- Ő rs ég 31 5. 0 46 .8 7 16 .1 3 Bo m bi na v ar ie ga ta A lp 2 16 / 6 8 23 .5 3 14 .0 9- 35 .3 8 34 .4 5 5. 01 58 .3 2 0. 20 -1 82 .7 8 26 4. 0 46 .8 7 16 .4 5 Bo m bi na v ar ie ga ta A lp 7 25 3. 0 46 .8 9 16 .4 3 H yl a ar bo re a 1 25 3. 0 46 .8 9 16 .4 3 Li ss ot ri to n vu lg ar is 1 25 3. 0 46 .8 9 16 .4 3 R an a ar va lis 1 25 3. 0 46 .8 9 16 .4 3 R an a da lm at in a 4 31 5. 0 46 .9 0 16 .2 4 Bo m bi na v ar ie ga ta A lp 48 31 5. 0 46 .9 0 16 .2 4 Ic ht hy os au ra a lp es tr is 1 26 7. 0 46 .9 1 16 .2 3 Pe lo ph yl ax e sc ul en tu s 1 31 5. 0 46 .9 0 16 .2 4 R an a te m po ra ri a 2 2- So pr on i M ts 49 3. 0 47 .6 5 16 .4 8 Bo m bi na v ar ie ga ta A lp 14 4 / 14 28 .5 7 8. 38 -5 8. 10 2. 05 2. 40 1. 13 0. 48 -2 .9 0 3- B ak on y M ts 45 5. 0 47 .0 6 17 .6 7 Bo m bi na b om bi na 2 37 / 6 06 6. 11 4. 33 -8 .3 2 21 .1 5 5. 19 45 .5 8 0. 16 -2 10 .3 0 31 6. 0 47 .2 3 17 .7 4 Bo m bi na v ar ie ga ta A lp 3 32 7. 0 47 .2 7 17 .6 9 Bo m bi na v ar ie ga ta A lp 15 32 7. 0 47 .2 7 17 .6 9 Bu fo b uf o 2 32 7. 0 47 .2 7 17 .6 9 Ic ht hy os au ra a lp es tr is 12 32 7. 0 47 .2 7 17 .6 9 Li ss ot ri to n vu lg ar is 19 32 7. 0 47 .2 7 17 .6 9 R an a da lm at in a 25 34 8. 0 47 .2 3 17 .6 4 Bo m bi na b om bi na 2 34 8. 0 47 .2 3 17 .6 4 Bo m bi na v ar ie ga ta A lp 31 0 35 6. 0 47 .2 3 17 .6 5 Bu fo b uf o 61 35 6. 0 47 .2 3 17 .6 5 Bu fo v ir id is 39 34 8. 0 47 .2 3 17 .6 4 Li ss ot ri to n vu lg ar is 5 35 6. 0 47 .2 3 17 .6 5 Pe lo ph yl ax r id ib un du s 24 34 8. 0 47 .2 3 17 .6 4 Pe lo ph yl ax s p. 4 34 8. 0 47 .2 3 17 .6 4 R an a da lm at in a 83 4- H an sá g 11 3. 0 47 .6 6 16 .7 4 Bo m bi na b om bi na 4 3 / 33 9. 09 1. 92 -2 4. 33 0. 56 0. 16 0. 70 0. 15 -1 .3 7 11 6. 0 47 .6 3 17 .0 8 Pe lo ph yl ax r id ib un du s 29 5- M ec se k M ts 38 1. 0 46 .2 2 18 .3 3 Bo m bi na v ar ie ga ta A lp 12 0 / 23 0. 00 0. 00 -1 4. 82 23 2. 0 46 .1 6 18 .2 4 Bo m bi na v ar ie ga ta A lp 8 41 5. 0 46 .2 0 18 .3 3 Bo m bi na v ar ie ga ta A lp 3 6- K is ku ns ág 89 .0 46 .6 1 19 .1 2 Tr itu ru s do br og ic us 13 0 / 13 0. 00 0. 00 -2 4. 71 7- Bu da pe st 10 0. 0 47 .1 8 18 .5 3 Bo m bi na b om bi na 4 2 / 18 11 .1 1 1. 38 -3 4. 71 36 .7 7 36 .7 7 50 .1 1 1. 34 -7 2. 20 11 1. 0 47 .4 2 19 .1 4 Bu fo v ir id is 4 15 6. 0 47 .5 3 19 .2 2 Pe lo ph yl ax r id ib un du s 10 129Occurrence of Bd in Hungary N r. of r eg io n A lt La t Lo ng Sp ec ie s m tD N A lin ea ge B . va ri eg at a N Po si tiv e/ Sa m pl ed Pr ev ( % ) Pr ev 9 5% C I (% ) G E m ea n G E m ed ia n G E SD G E ra ng e 8- Pi lis -V is eg rá di M ts 16 8. 0 47 .7 8 19 .0 4 Bo m bi na b om bi na 1 0 / 78 0. 00 0. 00 – 4 .6 2 41 8. 0 47 .7 8 19 .0 0 R an a da lm at in a 5 26 1. 0 47 .5 7 18 .9 4 Bu fo b uf o 1 26 1. 0 47 .5 7 18 .9 4 Sa la m an dr a sa la m an dr a 35 21 6. 0 47 .6 4 18 .7 8 Bo m bi na b om bi na 2 32 9. 0 47 .7 6 18 .8 5 R an a te m po ra ri a 2 18 3. 0 47 .7 6 18 .9 1 Sa la m an dr a sa la m an dr a 7 23 4. 0 47 .6 1 18 .8 8 H yl a ar bo re a 1 23 4. 0 47 .6 1 18 .8 8 Pe lo ph yl ax s p. 3 20 8. 0 47 .8 5 19 .1 2 R an a te m po ra ri a 1 20 9. 0 47 .8 5 19 .1 1 Sa la m an dr a sa la m an dr a 1 10 7. 0 47 .7 7 19 .0 9 H yl a ar bo re a 2 10 7. 0 47 .7 7 19 .0 9 Pe lo ph yl ax s p. 4 35 8. 0 47 .7 2 19 .0 6 Bo m bi na b om bi na x va ri eg at a 1 35 8. 0 47 .7 2 19 .0 6 Bo m bi na v ar ie ga ta A lp 2 30 1. 0 47 .7 8 18 .9 9 Pe lo ph yl ax r id ib un du s 8 30 1. 0 47 .7 8 18 .9 9 R an a te m po ra ri a 2 9- G öd öl lő H ill s 22 4. 0 47 .6 3 19 .3 8 Li ss ot ri to n vu lg ar is 20 0 / 56 0. 00 0. 00 -6 .3 8 15 6. 0 47 .5 3 19 .2 2 Pe lo ph yl ax r id ib un du s 1 11 1. 0 47 .7 6 17 .3 4 R an a ar va lis 1 96 .0 47 .2 6 19 .2 3 R an a ar va lis 17 96 .0 47 .2 6 19 .2 3 R an a da lm at in a 3 96 .0 47 .2 6 19 .2 3 Tr itu ru s do br og ic us 14 10 -M át ra M ts 49 2. 0 47 .9 0 19 .9 8 Bo m bi na v ar ie ga ta C ar p 2 7 / 10 3 6. 80 2. 78 -1 3. 50 6. 93 2. 13 9. 19 0. 61 -2 3. 55 64 8. 0 47 .9 3 19 .8 9 Bo m bi na v ar ie ga ta C ar p 2 64 8. 0 47 .9 3 19 .8 9 Sa la m an dr a sa la m an dr a 6 59 8. 0 47 .9 0 19 .9 7 Bo m bi na b om bi na 2 58 7. 0 47 .8 5 19 .9 6 Bo m bi na v ar ie ga ta C ar p 3 31 6. 0 47 .9 7 19 .5 2 Sa la m an dr a sa la m an dr a 1 72 0. 0 47 .9 0 19 .9 3 Bo m bi na v ar ie ga ta C ar p 4 40 3. 0 47 .9 2 19 .9 7 Bo m bi na b om bi na 2 30 4. 0 47 .9 3 19 .9 8 Bo m bi na b om bi na x va ri eg at a 1 63 6. 0 47 .8 7 19 .9 7 Bo m bi na v ar ie ga ta C ar p 32 72 7. 0 47 .8 8 20 .0 1 Bu fo b uf o 1 72 7. 0 47 .8 8 20 .0 1 Ic ht hy os au ra a lp es tr is 11 130 Judit Vörös et alii N r. of r eg io n A lt La t Lo ng Sp ec ie s m tD N A lin ea ge B . va ri eg at a N Po si tiv e/ Sa m pl ed Pr ev ( % ) Pr ev 9 5% C I (% ) G E m ea n G E m ed ia n G E SD G E ra ng e 41 1. 0 47 .9 3 19 .9 6 Pe lo ph yl ax e sc ul en tu s 1 72 7. 0 47 .8 8 20 .0 1 R an a te m po ra ri a 1 72 7. 0 47 .8 8 20 .0 1 Sa la m an dr a sa la m an dr a 3 36 4. 0 47 .9 0 19 .7 4 Bo m bi na b om bi na 3 36 2. 0 47 .9 3 19 .7 6 Bo m bi na v ar ie ga ta C ar p 1 52 2. 0 47 .8 9 20 .1 0 Bo m bi na b om bi na 6 27 4. 0 47 .9 1 20 .1 4 Bo m bi na b om bi na x va ri eg at a 1 63 3. 0 47 .8 9 20 .1 1 Bo m bi na v ar ie ga ta C ar p 12 63 6. 0 47 .9 3 19 .9 3 Bo m bi na b om bi na x va ri eg at a 5 41 1. 0 47 .9 3 19 .9 6 Bo m bi na v ar ie ga ta C ar p 2 41 1. 0 47 .9 3 19 .9 6 Pe lo ph yl ax e sc ul en tu s 1 11 -B ük k M ts 24 9. 0 48 .1 2 20 .2 4 Bu fo b uf o 1 1 / 9 11 .1 1 0. 28 -4 8. 25 8. 10 8. 10 N A N A 32 0. 0 48 .1 5 20 .1 0 R an a te m po ra ri a 1 44 3. 0 48 .0 4 20 .5 6 Ic ht hy os au ra a lp es tr is 6 33 0. 0 48 .1 5 20 .0 8 R an a te m po ra ri a 1 12 -A gg te le k K ar st 28 6. 0 48 .5 4 20 .6 6 Bo m bi na v ar ie ga ta C ar p 6 0 / 12 0. 00 0. 00 -2 6. 46 23 8. 0 48 .5 3 20 .6 4 Sa la m an dr a sa la m an dr a 6 13 -Z em pl én M ts 46 8. 0 48 .2 7 21 .2 9 Bo m bi na v ar ie ga ta C ar p 10 6 / 22 27 .2 7 10 .7 3- 50 .2 2 24 4. 00 10 1. 15 32 8. 43 13 .0 3- 88 2. 54 28 1. 0 48 .4 8 21 .3 3 Bo m bi na v ar ie ga ta C ar p 6 34 1. 0 48 .4 8 21 .3 2 R an a te m po ra ri a 1 34 1. 0 48 .4 8 21 .3 2 Sa la m an dr a sa la m an dr a 4 44 9. 0 48 .4 0 21 .4 5 Bo m bi na v ar ie ga ta C ar p 1 14 -H or to bá gy 86 .0 47 .5 7 20 .9 4 Pe lo ph yl ax e sc ul en tu s 18 16 / 1 78 8. 99 5. 23 -1 4. 19 10 .4 8 1. 48 17 .9 8 0. 64 -5 7. 91 84 .0 47 .6 0 20 .8 8 Pe lo ph yl ax le ss on ae 1 86 .0 47 .5 7 20 .9 4 Pe lo ph yl ax r id ib un du s 2 85 .0 47 .6 2 21 .0 8 Pe lo ph yl ax e sc ul en tu s 25 86 .0 47 .6 1 21 .0 7 Pe lo ph yl ax r id ib un du s 56 86 .0 47 .6 3 21 .0 8 Pe lo ph yl ax s p. 12 85 .0 47 .4 4 21 .1 4 Pe lo ph yl ax e sc ul en tu s 20 85 .0 47 .4 4 21 .1 4 Pe lo ph yl ax r id ib un du s 42   84 .0 47 .4 5 21 .1 7 Pe lo ph yl ax s p.   2               To ta l 12 33 131Occurrence of Bd in Hungary Systematic sampling of sentinel taxa in two core areas Core areas were selected based on the prevalence found previously or in the first year of sampling (Gál et al., 2012; Baláž et al., 2014b). In Bakony Mts, B. variegata was systemati- cally sampled in 2010-2012. Data of 2010 were published previ- ously (Gál et al., 2012), thus our analyses includes a comparison of data from 2010 and new data from 2011 and 2012. Surveys were completed between March and September in 2010, April and September in 2011, May and July in 2012. The assigned locality, Iharkút (see asterisk on Fig. 1), is an old open baux- ite mine, where human activities are common due to being a famous paleontological research site (Ősi et al., 2012). In Iharkút we were able to locate only two water bodies: a small lake and a nearby stream. Because of the close proximity (ca. 50 meters) and the presumed connection of the two habitats, all the toads belonged to the same population. Members of the P. esculentus complex were screened for Bd in the Hortobágy National Park (HNP; see asterisk on Fig. 1). HNP is the largest continuous alkaline steppe in Europe cover- ing 80.000 hectares. This natural reserve is abundant in wetland habitats like alkaline marshes, fishponds, wet grasslands and wet meadows (Ecsedi, 2004). Pelophylax species were sampled in three sites at HNP – Nádudvar-Kösély canal near the city Nádud- var, a fish pond system located eastwards to Hortobágy village and a marshland system at Egyek-Pusztakócs village – between April and October during three consecutive years (2012-2014). Taxonomic identification of Pelophylax esculentus complex Water frog taxon identification was determined using the technique described by Hauswaldt et al. (2012), and is based on allele-size polymorphism in intron-1 of the serum albumin gene (SAI-1; Plötner et al., 2009) named RanaCR1, was identi- fied in the serum albumin intron-1 (SAI-1, with a slight modi- fication in PCR protocol (Herczeg et al., 2017). To verify SAI- 1 fragments we sequenced representative alleles on a Hitachi 3130 Genetic Analyzer (Applied Biosystems, UK). Consensus sequences were compiled using BioEdit version 7.0.9.0 (Hall, 1999) and aligned manually. If genetic samples were not avail- able we referred to the individuals as Pelophylax sp. Sampling protocol We collected Bd samples following Hyatt et al. (2007) by either swabbing the skin of the individuals or clipping one of the toes. According to Hyatt et al. (2007) skin swabbing and toe clipping show similar performances in detectability of Bd. Skin swabbing was performed using two types of sterile swabs (SWA90006; Biolab, Budapest, Hungary, 5 mm diameter; and MW100-100; Medical Wire and Equipment, Wiltshire, England, 3 mm diameter). We collected each sample in a standardized way with three strokes on each side of the abdominal midline, the inner thighs, hands and feet. Toe clipping was performed using sterilized scissors and toe clips were stored in 70% EtOH in a freezer at -80 ˚C. Skin swabs were stored dry in individu- ally labelled vials and transferred to a freezer for longer storage throughout the field season. For both sampling procedures we used a new pair of disposable gloves per individual, and after each sampling event we sterilized all the used sampling equip- ment in order to avoid cross-contamination. Mouthpart (oral disc) of larvae were swabbed following Hyatt et al. (2007). Eth- anol-fixed specimens of Bombina spp. were screened by skin swabbing following methodology presented above. Genetic analysis of Bd samples DNA was extracted using PrepMan Ultra Sample Prepa- ration Reagent (Thermo Fisher Scientific, Waltham, Massa- chussetts, USA) following the recommendations of Boyle et al. (2004). Because of size differences between swabs (i.e. 3 mm vs. 5 mm; see above), only the top 3 mm of the larger swabs was used in all cases. Extracted DNA was analysed using real-time quantitative polymerase chain reaction (qPCR) following the amplification methodology of Boyle et al. (2004) and Hyatt et al. (2007) targeting the partial ITS-1 – 5.8S rRNA regions. Sam- ples were run in triplicate and an internal positive control was included (TaqMan exogenous internal positive control reagents; 4308323; Thermo Fisher Scientific, Waltham, Massachussetts, USA) to detect potential inhibitors present in the DNA extrac- tions. We considered evidence of infection if genomic equiva- lents (GE) were ≥ 0.1 and we considered a sample positive if all three wells returned a positive reaction. When a sample returned an equivocal result, it was re-run. If it again returned an equivocal result, it was considered negative (N = 17, 1.3% of total samples). The templates were run on a Rotor-Gene 6000 real-time rotary analyser (Corbett Life Science, Sydney, Aus- tralia). GE were estimated from standard curves based on posi- tive controls of 100, 10, 1, 0.1 developed from the Bd isolate IA 2011, from Acherito Lake, Spain. Finally, GE values of the three positive replicates were averaged. In order to identify lineages of Bd found on amphibians in Hungary, 2 µl of DNA extract from three individuals (one juve- nile P. ridibundus plus one juvenile B. variegata from Bakony Mts, and one adult B. variegata from Őrség) were selected as template for amplification of a partial fragment of ITS-1 rRNA. Nested PCR approach described by Gaertner et al. (2009) was performed. The amplified fragments were sequenced on an Applied Biosystems/Hitachi 3130 Genetic Analyser (Thermo Fisher Scientific, Waltham, Massachussets, USA). Sequences were aligned manually using BioEdit version 7.0.9.0. (Hall, 1999) and were blasted against available sequences from Gen- Bank for identification. Climatic data Climatic data were provided by the Hungarian Meteoro- logical Service (OMSZ). For the core areas of B. variegata and P. esculentus complex climatic data were obtained from the closest meteorological station of each sampling site: Pápa city (47.29, 17.37), 135.5 m a.s.l, 21.5 km distance from Iharkút (Bakony Mts), and Kunmadaras village (47.46, 20.89), 88.8 m a.s.l. 12.5 132 Judit Vörös et alii km distance from Egyek-Pusztakócs (HNP), which is the closest sampling point to the station. We used monthly mean precipi- tation and monthly mean air temperature data for the period 2010-2014 to test if any relationship between climate and preva- lence or infection intensity exists. Statistical analyses Statistical analyses were performed in R (version 3.4.4; R Core Team, 2018). Prevalence was expressed as a discrete bino- mial variable (uninfected vs. infected). Infection intensity was expressed through GE value. First, we calculated infection prev- alence (%) of different amphibian species together with their 95% Clopper-Pearson confidence intervals (95% CI) as follows. Prevalence values were obtained by dividing the cumulative number of positive samples with the total number of samples per species and multiplied with 100 to obtain percentile values, while 95% CI values were calculated using the R package ‘Prop- CIs’ (function ‘exactci’; Scherer, 2018). In Bd infected species we calculated the mean, median, SD and range of GE values as well. Second, we tested whether prevalence and infection inten- sity differed between phylogenetic lineages of B. variegata, and in the two sentinel taxa (i.e. B. variegata and P. esculentus com- plex) we also tested for differences between study years, sexes and age classes. Prevalence values were compared with Chi- square tests, while infection intensities were compared using Mood’s median test, as implemented in the R package ‘RVAide- Memoire’ (function ‘mood medtest’; Hervé, 2018). Finally, in the two sentinel taxa we tested the relationship between climatic variables and prevalence and infection inten- sity. We note here that the data set of the P. esculentus complex was restrained only on P. ridibundus, as the Bd infection of P. esculentus was very low (i.e. two infected individuals in total) and the sample size of P. lessonae was also not representative (N = 1). The relationship between the climatic factors and infection prevalence was tested using generalized linear mixed models (GLMMs) with binomial error distribution term and the rela- tionship between the climatic factors and infection intensity was analysed using linear mixed models with Gaussian distribution (LMMs). Prevalence and infection intensity, respectively, were entered as dependent variables in the models, while the focal climatic variable (i.e., air temperature or precipitation) was set as continuous predictor. In all models sampling year was entered as a random effect to control for the interannual vari- ations in infection prevalence or intensity. Additionally, in the case of P. ridibundus, collection site ID within the HNP was entered also as a random factor to account for the variations in prevalence and intensity between collection sites. To assure the adequate distribution of model residuals, for the LMMs GE val- ues were log(x+1)-transformed. Prior entering into the models, log(x+1)-transformed GE values and the continuous predictors (i.e. climatic variables) were scaled to mean = 0 and SD = 1 to improve model convergence (see also Schielzeth 2010). Model fits were checked visually by plot diagnosis. In all cases for the statistical comparison of infection intensities only infected indi- viduals were used. Mixed models were constructed using the ‘lme4’ package for R (Bates et al., 2015), and P-values for the linear mixed models were obtained using the function ‘Anova’ (type III) from the R package ‘car’ (Fox and Weisberg, 2011). We used a significance level of P ≤ 0.05 throughout. RESULTS Bd occurrence in Hungary In Hungary, nine regions were infected with Bd and the overall prevalence was 7.46% (95% CI: 6.05-9.07), indi- cating a low presence of the fungus in the country (Table 1). Among the sixteen sampled amphibian taxa seven were found infected with Bd, including one unidentified Pelophylax individual (Table 2). Details on prevalence and summary statistics of GE values are presented in Table 2; while the geographic distribution of the sampling sites with the site-specific prevalence is shown in Fig. 1. Bd occurrence in Bombina variegata In B. variegata the overall prevalence was 12.69% (95% CI: 9.91-15.92). Details on prevalence and sum- mary statistics of GE values for the different regions are presented in Table 3. We found no significant difference between the two lineages of B. variegata in infection prevalence (NAlpine = 422, NCarpathian = 82; χ2 = 0.155, df = 1, P = 0.693) and intensity (NAlpine = 52, NCarpathian = 12, P = 0.750). Bd was not detected among the ethanol-fixed B. variegata specimens. In Bakony Mts between 2010 and 2012 we sampled 310 individuals of B. variegata, among which 32 individ- uals were found to be infected with Bd. Here the overall prevalence was 10.32 % (95% CI: 7.16–14.25), and the mean, median, SD and range of GE values were 15.92, 5.09, 38.60 and 0.159–210.3, respectively. There was no significant difference in infection prevalence (N2010 = 80, N2011 = 144, N2012 = 86; χ2 = 4.980, df = 2, P = 0.082) nor in intensity between the three study years (N2010 = 13, N2011 = 14, N2012 = 5, P = 0.201), and we found no sig- nificant difference in prevalence (Nmales = 113, Nfemales = 90; χ2 = 0.241, df = 1, P = 0.623) and infection intensity between sexes (Nmales = 8, Nfemales = 2, P = 0.545). How- ever, there was a significant difference in prevalence between the two age classes (Njuveniles = 105, Nadults = 204; χ2 = 11.563, df = 1, P < 0.001), with juveniles being more infected than adults (proportion of individuals infected: 19.04% versus 5.88%). Differences in infection intensity between the two age classes were not significant (Nju- veniles = 20, Nadults = 12, P = 0.273). There was significant negative relationship between infection prevalence and monthly mean air temperature (χ2 = 4.482 df = 1, P = 133Occurrence of Bd in Hungary 0.034), and a marginally significant positive relationship between prevalence and monthly mean precipitation (χ2 = 3.611, df = 1, P = 0.057). There was no significant rela- tionship between infection intensity and monthly mean air temperature (χ2 = 0.180, df = 1, P = 0.671). Howev- er, there was a significant positive relationship between Table 2. Batrachochytrium dendrobatidis (Bd) infection in amphibian species sampled in Hungary between the years 2009 and 2015. Prev = prevalence; GE = genomic equivalents of zoospores. Species Positive/ Sampled Prev (%) Prev 95% CI (%) GE mean GE median GE SD GE range Order Anura Family Bombinatoridae Bombina bombina 1 / 29 3.45 0.09-17.76 16.41 16.41 NA NA Bombina variegata 64 / 504 12.70 9.92-15.92 40.08 4.96 120.76 0.16-882.54 Bombina bombina x variegata 0 / 8 0.00 0.00-36.94 Family Bufonidae Bufo bufo 0 / 66 0.00 0.00-5.44 Bufo viridis 2 / 43 4.65 0.57-15.81 36.77 36.77 50.11 1.34-72.20 Family Hylidae Hyla arborea 0 / 4 0.00 0.00-60.24 Family Ranidae Pelophylax esculentus 2 / 66 3.03 0.37-10.52 1.07 1.07 0.41 0.78-1.36 Pelophylax lessonae 0 / 1 0.00 0.00-97.5 Pelophylax ridibundus 21 / 164 12.80 8.10-18.91 20.21 1.59 41.72 0.15-164.30 Pelophylax sp. 1 / 33 3.03 0.08-15.76 15.75 15.75 NA NA Rana dalmatina 0 / 120 0.00 0.00-3.03 Rana arvalis 0 / 19 0.00 0.00-17.65 Rana temporaria 0 / 11 0.00 0.00-28.49 Order Caudata Family Salamandridae Salamandra salamandra 0 / 63 0.00 0.00-5.69 Triturus dobrogicus 0 / 27 0.00 0.00-12.77 Lissotriton vulgaris 0 / 45 0.00 0.00-7.87 Ichthyosaura alpestris 1 / 30 3.33 0.08-17.22 Total 92 / 1233 7.46 6.05-9.07 Table 3. Batrachochytrium dendrobatidis (Bd) detection in regions representing the surveyed local populations of B. variegata in Hungary. Prev = prevalence; GE = genomic equivalents of zoospores. Genetic lineage Region Positive/ Sampled Prev (%) Prev 95% CI (%) GE mean GE median GE SD GE range Alpine Őrség 16 / 57 28.07 16.97-41.54 34.45 5.01 58.32 0.20-182.78 Soproni Mts 4 / 14 28.57 8.39-58.10 2.05 2.40 1.13 0.48-2.90 Bakony Mts 32 / 328 9.76 6.77-13.49 15.93 5.09 38.61 0.16-210.30 Mecsek Mts 0 / 23 0.00 0.00-14.82 Pilis-Visegrádi Mts 0 / 2 0.00 0.00-84.19 Carpathian Mátra Mts 6 / 58 10.34 3.89-21.17 5.36 1.86 8.97 0.61-23.55 Aggtelek Karst 0 / 6 0.00 0.00-45.93   Zemplén Mts 6 / 16 37.50 15.20-64.57 244.00 101.15 328.43 13.03-882.54 Total 64 / 504 12.59 9.83-15.80 134 Judit Vörös et alii infection intensity and monthly mean precipitation (χ2 = 4.227, df = 1, P = 0.039); though, this significant rela- tionship disappeared after removing one outlier GE value from the data set (χ2 = 1.510, df = 1, P = 0.219). All the three sequences (i.e. sequences obtained from juvenile P. ridibundus and B. variegata from Bakony Mts, and one adult B. variegata from Őrség) were identi- fied as ITS-1 rRNA of Bd, belonging to the globally dis- persed Bd-GPL lineage (GenBank accession numbers: MH745069-71). One sequence showed 100% identity with Bd from Cape Cod (GenBank accession number: FQ176489.1, FQ176492.1), South Africa (JQ582903- 4, 15, 37), and Italy (FJ010547). The second sequence was 100% identical with a sequence of Bd from Equa- dor (FJ232009.1), and the third sequence represented a unique haplotype. Genetic distance (p-distance) among sequences ranged between 0.005-0.035. Bd occurrence in Pelophylax ridibundus In Hortobágy between 2012 and 2014 we sampled 100 individuals of P. ridibundus, among which 13 were found to be infected with Bd. Here the overall prevalence was 13.00% (7.10-21.20), and the mean, median, SD and range of GE values were 11.52, 1.59, 19.63 and 0.635– 57.905, respectively. We found a significant difference in infection prevalence between years (N2012 = 35, N2013 = 48, N2014 = 17; χ2 = 27.750, df = 2, P < 0.001); all the infected individuals being captured in 2012 (prevalence: 37.14%), while no infected individuals being found in 2013-2014. We found no significant difference in prevalence (Nmales = 42, Nfemales = 30; χ2 = 0.002, df = 1, P = 0.958) and infec- tion intensity between sexes (Nmales = 7, Nfemales = 6, P = 1.000). Age classes did not differ in infection prevalence (Njuveniles = 9, Nadults = 72; χ2 = 0.827, df = 1, P = 0.363). Infection intensities of the different age classes cannot be compared because no infected juveniles were cap- tured. We found no significant relationship between infection prevalence and monthly mean air temperature (χ2 = 2.375, df = 1, P = 0.123), and between prevalence and monthly mean precipitation (χ2 = 0.010, df = 1, P = 0.920). Since infection prevalence was relatively low in the P. esculentus complex and infected individuals were captured in the same month and year, the relationship between climatic variables and infection intensity could not be tested in this taxa. DISCUSSION Low Batrachochytrium dendrobatidis prevalence was experienced throughout the country (Table 1, Table 2), with similar or slightly lower values than in neighbouring countries e.g. Czech Republic (Baláž et al., 2014a; 19% average at country level), Austria (Sztatecsny and Gla- ser, 2011; 5.9-45% at country level) or Poland (Kolenda et al., 2017; 18% average at country level). Overall, seven taxa carried the infection: Bombina bombina, Bombina variegata, Bufo viridis, Pelophylax ridibundus, Pelophy- lax esculentus, Pelophylax sp. and Ichthyosaura alpestris. In accordance with previous studies in Central Europe (Ohst et al., 2013; Baláž et al., 2014a,b; Kolenda et al., 2017), B. variegata and the members of the P. esculentus complex showed the highest prevalence and Bd infec- tion intensity in Hungary. On the other hand, there was no difference in prevalence and infection intensity was detected between the two ancient phylogenetic lineages of B. variegata. Bd was present in nine of the fourteen studied regions. The highest prevalence was experienced in the Alpine foothills at Őrség (Region 1), Soproni Mts (Region 2), and in the Zemplén Mts (Region 13). These three regions represent the margins of the Alps and Car- pathians (respectively) hosting populations with continu- ous distribution towards the higher regions. On the other hand, the remnant mountain regions, where prevalence was much lower (Regions 3, 10 and 11), are geographi- cally isolated from other higher elevations. In contrast, amphibians from five regions (Regions 5, 6, 8, 9 and 12) seemed to not carry Bd. This either indicates that Bd has not reached these parts of the country yet, or more com- prehensive sampling would be needed to locate its pres- ence. The Carpathian Basin combines the characteristics of the neighbouring regions. Despite the relatively small extent of Hungary, the climatic elements have distinct temporal and spatial characters (Mezősi, 2017). Although the majority of the country has an elevation of less than 300 m a.s.l., Hungary has several moderately high ranges of mountains and the highest peak located in the Mátra Mts at 1014 m a.s.l. (Table 1, Region 10). Overall, our results rather supporting the relationship between the measured climatic variables and prevalence or infection intensity. We found significant relationship regarding B. variegata individuals in the Bakony Mts core area, where prevalence was negatively affected by monthly mean tem- perature. Furthermore, the monthly mean precipitation positively affected the Bd infection intensity. Nonetheless, the robustness of the latter result is questionable, since the relationship disappeared when we excluded an out- lier value from the analysis. This substantial effect of one outlier value could have on the outcomes of this analy- sis suggests the need for an extensive sampling in order to test whether this result is a statistical artefact or a real biological phenomenon. 135Occurrence of Bd in Hungary To determine the time and location of the emergence or introduction of Bd in different regions worldwide, it is important to study archived specimens deposited to museum collections. To examine the historical pres- ence of the fungus in Hungary we screened archived specimens of Bombina spp. collected in the regions 1, 2, 3, 8, 10, 12, 13 and the Kőszeg Mts (archived data only) between 1936 and 2005. In total 127 specimens were analysed and all of the samples were Bd negative. Both for field and for museum samples we used the same detection methodology, following Hyatt et al. (2007). The detection probability with qPCR is more sensitive and accurate compared to conventional PCR or histol- ogy (Annis et al., 2004; Boyle et al., 2004; Kriger et al., 2006). There is no difference in regard of Bd detectability between sample collection techniques (i.e., skin swabbing, brushing or scraping). Nonetheless, preservation meth- odology and storage history may have influence on the results (Soto-Azat et al., 2009). The Amphibian Collec- tion of the Hungarian Natural History Museum is stored in ethanol, but no record is available about the mode of initial preparation. As formaldehyde is known to inhib- it PCR reaction, there is therefore a slight chance that qPCR reactions failed to detect Bd in our archived sam- ples; however, this may be an unlikely possibility. Although with testing archived specimens we did not find evidence on when Bd might have been introduced into the country, our genetic analyses showed that the fungus found on amphibians in Hungary is a member of the Bd-GPL lineage. This was confirmed by a recent study tracking the origin of Bd using a full genome approach, which detected Bd-GPL lineage in Hungary (from Iharkút, Bakony Mts; O’Hanlon et al., 2018) and is in line with previous findings reporting that this lineage has a widespread distribution in Europe (Farrer et al., 2007). During the surveys in the core area of Bakony Mts (Region 3, Table 1) juvenile B. variegata individuals showed a significantly higher prevalence compared to adults. The same pattern was observed for two B. var- iegata populations in a seven-year period study in the Netherlands, which the authors explained by the less developed immune responses, or immunosupression, following the stress of metamorphosis (Spitzen-van der Sluijs et al., 2017). Quite surprisingly, during our study, two juveniles changed infection state once (recovered from Bd positive). It is a relatively common phenomenon in the field, when infected adult frogs lose and regain the infection which may be caused by overwintering tadpoles or larvae acting as reservoirs (Briggs et al., 2010, Spitzen- van der Sluijs et al., 2017). In contrast, it is less frequent with juvenile individuals as it was experienced in our study. Similar pattern was observed for Epidalea calamita in Spain, where juveniles changed infection state towards the end of metamorphosis, possibly mediated by the increasing water temperature in permanent ponds (Bosch et al., unpublished). In Iharkút (Bakony Mts), during our study period the environmental conditions changed unexpectedly. The lake which hosted most of the amphibian species – including B. variegata – dried out after the first season of sample collection. In the second year only four individuals of B. variegata were captured around this locality, however the rest of the specimens (N = 181) found shelter in a nearby stream unsuitable for breeding. During the third year the lake kept dry and only seven out of 87 individu- als were found in or around the lake. Even though there was no difference in prevalence between the three years, they showed a downward trend towards significance. Already low prevalence (23%) dropped down to 11% in the second and to 5% in the third year. This trend could be associated with the differences in habitat type, as it was observed for Salamandra salamandra in the Guadar- rama National Park, Spain (Medina et al., 2015). Here, Bd infection was greater in salamander larvae from per- manent ponds, while it was absent or weak in tempo- rary water bodies and permanent streams. Also, infec- tion intensity in larval cohorts was reduced when water was flowing rather than standing. Same authors suggested that increased water flow rate reduce the likelihood of successful pathogen transmission. Chytridiomycosis is limited to the keratinized tissues of the host individual, therefore tadpoles and post-met- amorphic amphibians are mostly affected by the disease (Rachowicz and Vredenburg, 2004). Our dataset cov- ered all life stages of amphibians and the presence of the infection was not detected in tadpoles of B. bufo and R. dalmatina (N = 39). On the other hand, post-metamor- phic and juvenile individuals were found infected in the regions 1, 3, 10 and 13 of B. variegata and the members of the P. esculentus complex, even though all sampled individuals apparently didn’t display any clinical sign of chytridiomycosis. In Central Europe the P. esculentus complex is formed by two sexual species, the P. ridibundus and the P. lessonae and their interspecific mating produces the hybridogenetic P. esculentus. Overall, our results in the core area of Hortobágy National Park showed higher Bd prevalence in P. ridibundus compared to the hybrid P. esculentus (Table 2) which is related to the fact that the hybrids have more effective peptide defence system against Bd and have a richer peptide repertoire than both parental species (Daum et al., 2012). Further, contrary to what was observed in B. variegata in the Bakony Mts core area, we did not find differences in Bd infection between 136 Judit Vörös et alii life stages and sexes in P. ridibundus individuals. Our results fit into the general pattern showing sig- nificant variability in the effects of chytridiomycosis across Europe. The marked difference in species sus- ceptibility between amphibian species/communities of Western and Central-Eastern Europe might be deter- mined by multiple linked factors, e.g. virulence of dif- ferent Bd strains (Farrer et al., 2007), genotype (Savage and Zamudio, 2011), behaviour (Williams and Groves, 2014), microbial skin community compound of host spe- cies (Bletz et al., 2013), or structure of amphibian com- munities (Becker et al., 2014). In the Iberian Peninsula – that received the most attention due to mass amphib- ian mortalities caused by chytridiomycosis – infection was clustered within high-altitude areas, where environ- mental conditions are the most optimal for growth of Bd (Piotrowski et al., 2004). In contrast, Hungary harbours only low-elevation mountains, where environmental con- ditions might be less favourable for Bd-linked epidemics. Differences in elevation might explain the relatively lower impact and infection values of amphibians in Hungary, than it was reported for surrounding countries in Central and Eastern Europe (e.g., Austria, Sztatecsny and Gla- ser, 2011; Czech Republic, Baláž et al., 2014a or Poland, Kolenda et al., 2017). Since Bd-related disease outbreak have been proven to be climate-driven (Bosch et al., 2007), amphibians of Central-Eastern Europe might be heavily impacted in the future due to global climate change. Changes in the climate might alter Bd diffusion and make it’s spreading less predictable, thus areas not yet affected by epidemics require particular attention and constant monitoring. ACKNOWLEDGEMENTS We thank B. Halpern, K. Szabó, I. Kiss, E. Jáger, K. Suri, R. Dankovics, B. Velekei, A. Ősi, G. Deák, Cs. Tóth, A. Bérczes, G. Magos, L. Urbán, B. Bándli, M. García-París, P. Mikulíček, C. Gabor, C. Serrano-Lagu- na, Zs. Végvári, D. R. Brooks, E. Vörös, L. Vörös, E. Kovács and F. Hock for providing samples or helping in the field. We are grateful to T. Garner (Zoological Society of London) for important initial and then con- tinuous help with Bd research in Hungary. T. Papp and M. Benkő (Institute for Veterinary Medical Research, Budapest) kindly provided facility and reagents for qPCR. We gratefully acknowledge the excellent tech- nical assistance of Á. Juhász and E. Ottinger (National Food Chain Safety Office), as well as M. Tuschek and V. Krízsik (HNHM). Savaria Museum Szombathely pro- vided toad specimens for sampling. During the project JV was supported by the Hungarian Scientific Research Fund (OTKA K77841) and by the Bolyai János Research Scholarship of the Hungarian Academy of Sciences (BO/00579/14/8). DH was supported by the European Union and co-financed by the European Social Fund through the Social Renewal Operational Programme under the projects TÁMOP–4.2.2/B–10/1–2010–0024 and SROP-4.2.2.B-15/1/KONV-2015-0001. AF was sup- ported by the Hungarian National Research, Develop- ment and Innovation Office (OTKA grant no. K112527). Research permit was issued by the National Inspectorate of Environment, Nature Conservation and Water Man- agement (14/3535/2/2010) and the Tisza Region Inspec- torate of Environment, Nature Conservation and Water Management (4633/OH/2012). SUPPLEMENTARY MATERIAL Supplementary material associated with this article can be found at manuscript number 22611. REFERENCES Annis, S.L., Dastoor, F.P., Ziel, H., Daszak, P., Longcore, J.E. (2004): A DNA-based assay identifies Batra- chochytrium dendrobatidis in amphibians. J. Wildl. Dis. 40: 420-428. Baláž, V., Vojar, J., Civiš, P., Andera, M., Rozínek, R. (2014a): Chytridiomycosis risk among Central Euro- pean amphibians based on surveillance data. Dis. Aquat. Organ. 112: 1-8. 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