The Pelophylax esculentus complex in North-Western Romania: distribution of the population systems

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1 North-Western Journal of Zoology Vol. 6, No. 2, 2010, pp P-ISSN: , E-ISSN: Article No.: The Pelophylax esculentus complex in North-Western Romania: distribution of the population systems István SAS University of Oradea, Faculty of Sciences, Department of Biology, Universitatii str. 1, , Oradea, Romania, Abstract. The aim of this study was to clarify the distribution of the Pelophylax esculentus complex population systems in north-western Romania. The study area is represented by north-western Romania including the administrative territories delineated by Arad, Bihor and Satu-Mare counties. Surveys were conducted between September 2004 and May There were herpetofaunistically surveyed 884 geographic localities in north-western Romania. In 666 of these localities it was identified/reconfirmed the presence of water frogs from the P. esculentus complex (666-ridibundus, 97- esculentus, 33-lessonae). There were identified 39 distinct populations of P. lessonae and 127 populations of P. kl. esculentus respectively. In areas where all the three forms of water frogs or at least two of them are present, there were identified 127 population systems: R-E (83), L-R-E (39) and E (5). In all populations of water frogs from north-western Romania, the parental species (P. ridibundus, P. lessonae) are represented both by males and females. Concerning the hybrid P. kl. esculentus we could not identify males of this form in every population, the females being able to reproduce with the males of parental species. In the case of two populations of P. kl. esculentus we have not identified males (all-females E system). It is possible that these populations are included in a parthenogenetic cycle. During the study, mixed pairs in amplexus were identified exclusively between males of P. lessonae and females of P. kl. esculentus, and between males of P. kl. esculentus and females of P. ridibundus, being also identified several spawns. Most of the L-R-E system populations are found in Carei Plain and Livada Plain (the Tur Valley). Conservation in time of these populations is also ensured by their placement in Natura 2000 Habitats Directive sites. Key words: water frogs, Pelophylax esculentus complex, population systems, E system, all-females, parthenogenetic, north-western Romania Introduction Palearctic water frogs include several species (Dubois & Ohler 1996) characterized by their widespread distribution and frequent occurrence of interspecific hybridization (Graf & Polls-Pelaz 1989). The Palearctic water frogs comprise three hybridogenetic systems (Polls- Pellaz 1989, also see the references cited in: Schmeller et al. 2005, Marracci & Ragghianti 2008), which are geographically separated. Each complex consists of the largest water frog species, Pelophylax ridibundus, a small parental species and their medium-sized hybrids. In Romania, there are two valid species from the water frogs group {Pelophylax lessonae (Camerano, [1882]) and Pelophylax ridibundus (Pallas, 1771)} and the hybrid form between them {Pelophylax kl. esculentus (Linnaeus, 1758)}. In nature, there were described several ways of coexistence of the esculentus complex s forms, the hybrid being found in various habitats specific to the two parental species. Therefore, within the common distribution area of the P. esculentus complex s forms, besides pure populations of P. lessonae (L-L) and P. ridibundus (R-R) there are known L-E (lessonaeesculentus), R-E (ridibundus-esculen-tus), L-R-E (lessonae-ridibundus-esculentus) and E-E (esculentus) population systems (see reviewed in Plöt- NwjZ, Oradea, Romania, Oradea, Romania

2 The Pelophylax esculentus complex in North-Western Romania 295 ner 2005). Throughout the distribution area of these three forms, the L-E and R-E systems are considered to be the most common. There were frequently identified L-R-E systems also (e.g. Borkin et al. 1986, Günther 1983, Gubányi 1996, Tunner & Heppich-Tunner 1991, Gubányi & Korsós 1992, Krizmanić 2008a,b, Paunović et al. 2010); even so they are regarded as being much rarer. The parental species and the hybrid between them, in many situations are found in pure populations (Günther 1997a,b,c, Graf & Polls-Pellaz 1989, Arioli et al. 2010). The knowledge on the distribution of water frogs from the P. esculentus complex in Romania is not elucidated. This is primarily due to the fact that many Romanian herpetologists do not distinguish among the three forms of water frogs (e.g. Cogălniceanu et al. 2006, Demeter et al. 2006, Hartel et al. 2006, 2010, Ghiurcă et al. 2009, Székely et al. 2009), because morphological identification in the field requires experience. Data on the distribution of the three forms of water frogs in Romania are available only for the following regions: north-western Romania (e.g. Covaciu-Marcov 2004, Covaciu- Marcov et al. 2007a, 2008a, and see the material and methods), southern and south-western Romania (e.g. Covaciu-Marcov et al. 2009a,b, 2010a, Sas et al. 2010), north-eastern Romania (e.g. Covaciu-Marcov et al. 2008b, Gherghel et al. 2008) and Dobruja, respectively (e.g. Covaciu-Marcov et al. 2006a, Strugariu et al. 2008). However, these herpetofaunistic studies only bring data on the presence/absence of the water frogs close to some localities. Informations about integration of these in population systems are relatively few. In Romania, besides pure populations of P. ridibundus which are the most common, there were recorded mainly R-E system populations (e.g. Vancea et al 1989, Covaciu-Marcov 2004, Gherghel et al. 2008, Covaciu-Marcov et al. 2008c, 2009c), respectively there are a few references to L-R-E system populations also (e.g. Covaciu-Marcov et al. 2009b,d). Recently, in north-western Romania there was reported an E system population (Sas et al. 2009a). The highly complex hybridogenetic system of P. esculentus complex is very interesting for evolutionary ecology due to the various impacts hybridogenesis has on the ecology and genetics of the involved species (see in: Schmeller 2004). But in order to determine the status of any amphibian species, first it needs to be clarified its spatial distribution (see in Sas 2009, Strugariu et al. 2009, Covaciu-Marcov et al. 2010b). Thus, the aim of this study was to clarify the distribution situation of the population systems of water frogs in north-western Romania, to create a basis for future studies on the populations of the hybrido-genetic P. esculentus complex from this region. For P. lessonae species and the hybrid P. esculentus we also intended to identify the detailed distribution on reproductive populations. Materials and methods The study area is represented by north-western Romania including the administrative territories delineated by Arad, Bihor and Satu-Mare counties (Fig.1). Surveys were conducted between September 2004 and May During the study we used the ARVES method (amphibian and reptile visual encounter surveys), this being the most common and standardized method for herpetofauna studies and inventories (Campbell & Christian 1982, Crump & Scott 1994). Each locality from the study area was investigated, primarily being sought aquatic breeding habitats. Localities with water frogs, already known from the literature (Covaciu-Marcov 2004, and also see the reviewed reference list in Sas 2009), were examined in the first place. During the study there were identified several new localities with water frogs. These new data were part of herpetofaunistic studies conducted and published in collaboration with the members of the Herpetological Club of Oradea (for details see in Sas 2009). These published faunistic data refer only to the spatial distribution of species in localities (presence/absence) and are not based on displaying the distribution on reproductive populations, on integration in population systems of the water frogs. Identification of water frog individuals was based on their biometric attributes, indicated in the literature (e.g. Berger 1966, Wijnands & Van Gelder 1976). The simplest

3 296 Sas, I. method to identify water frogs from the P. esculentus complex (especially for determining the parental species) is according to the shape of the callus internus (e.g. Berger 1966, Günter et al. 1991) and depending on the presence or absence of the yellow coloration on the flanks (also see in: Krizmanić 2008a); these morphological characters being used regularly by many authors (Reyer & Bättig 2004, Ragghianti et al., 2007; Plötner et al., 2008, Neveu 2009, Arioli et al. 2010,). The biometric characters, for the studied populations of water frogs, were measured randomly, at about twothree individuals per population. Although morphological determination can be used successfully even in cases apparently more difficult (see in: Tunner 2000), data were confirmed based on the sound emissions of the studied frogs (this being considered the most accurate method to distinguish among water frogs e.g. Schneider et al. 1984, Wycherley et al. 2002). The obtained quantitative biometric and bioacoustic data (for details see Sas 2009) will not be mentioned on the pages of this paper. Results During the past ten yeas ( ), in collaboration with the members of the Herpetological Club of Oradea, there were herpetofaunistically surveyed 884 geographic localities in north-western Romania (see the faunistic works reviewed in Sas 2009). In 666 of these localities it was identified / reconfirmed the presence of water frogs from the P. esculentus complex (666-ridibundus, 97-esculentus, 33- lessonae). Since P. ridibundus is the most common amphibian species in north-western Romania (Covaciu-Marcov 2004, also see in Sas 2009) detailed information will be presented exclusively for P. lessonae and P. esculentus. Consequently, there were identified 39 distinct populations of P. lessonae and 127 populations of P. kl. esculentus respectively. In north-western Romania the three forms of water frogs from the P. esculentus complex occur in different population systems (Table 1, Fig.1). Most of the populations are composed exclusively of P. ridibundus species (R-R). Generally, in areas with populations of type R-R, the other two forms of water frogs (lessonae and esculentus) are missing. In areas where all the three forms of water frogs or at least two of them are present, there were identified 127 population systems: R-E (83), L-R-E (39) and E (5) (Table1, Fig.1). L-system populations, composed exclusively of P. lessonae, were not identified. If the species P. lessonae is present in a population, it always occurs with the other two forms (L-R-E system). In all populations of water frogs from north-western Romania, the parental species (P. ridibundus, P. lessonae) are represented both by males and females (Table 1). Concerning the hybrid P. kl. esculentus we could not identify males of this form in every population, the females being able to reproduce with the males of parental species. Even though males of esculentus are present in a population, their number is always small. In the case of two E system populations we identified only female individuals. Discussion In north-western Romania, populations of water frogs composed exclusively of the parental species P. ridibundus are prevailing. This species occur both in pure populations (R-R) and together with the other two forms of water frogs (R-E, L-R-E) (excepting 5 populations of E-E system). Beside the R-R system populations, in north-western Romania there are frequently found R-E system populations (83), those from the L-R-E system being much rarer (39). Distribution pattern, herpetofaunistical co-occurrence P. ridibundus is the best represented species of the herpetofauna from north-western Romania (Covaciu-Marcov 2004, Sas 2009). Here, it is a eurytopic species, inhabiting any type of aquatic habitat and is found up to an altitude of 700m (Covaciu-Marcov 2004) with species of amphibians and reptiles common to plain and

4 The Pelophylax esculentus complex in North-Western Romania 297 Population Cod Arad county Table 1. Locality and population record of the P. esculentus complex in NW Romania. (AR- Arad county, BH- Bihor county, SM- Satu-Mare county, R- ridibundus, L- lessonae, E- esculentus, Syst.- population system, pr.- predominate, m- males, f- females, X- presence) UTM (10x10 km) Locality Syst. pr. R L E m f m f m f POP 001 FS 12 Aciuţa (AR) RE R X X - - X X POP 002 ES 56/66 Apateu (AR) RE R X X - - X X POP 003 ES 74/75 Beliu (AR) RE R X X X POP 004 ES 74 Bocsig (AR) RE R X X X POP 005 ER 99 Căprioara (AR) RE R X X X POP 006 ES 70 Căpruţa (AR) RE R X X - - X X POP 007 ES 75 Chişlaca (AR) RE R X X X POP 008 ES 41 Cuvin (AR) E E X POP 009 ES 62 Dud (AR) RE R X X X POP 010 ES 80 Dumbrăviţa (AR) RE R X X X POP 011 ES 80 Dumbrăviţa (AR) RE R X X - - X X POP 012 FS 02 Gurahonţ (AR) RE R X X - - X X POP 013 ES 84 Igneşti (AR) RE R X X - - X X POP 014 ES 84 Igneşti (AR) LRE E X X X X X X POP 015 ES 64 Ineu de Arad (AR) RE R X X X POP 016 FS 02 Iosaş RE R X X X POP 017 ES 75 Lunca Teuzului (AR) RE R X X - - X X POP 018 ES 63 Mocrea (AR) RE R X X X POP 019 FS 02 Pescari (AR) RE R X X X POP 020 ES 84 Prunişor (AR) LRE E X X X X X X POP 021 ES 84 Prunişor (AR) RE E X X - - X X POP 022 ES 84 Prunişor (AR) RE R X X - - X X POP 023 ES 56 Satu Nou (AR) RE R X X X POP 024 ER 99 Săvîrşin (AR) RE R X X X POP 025 ES 83 Sebiş (AR) RE R X X - - X X POP 026 FS 13 Vidra (AR) RE R X X X Bihor county POP 027 ES 57 Arpăşel (BH) RE R X X - - X X POP 028 ET 86/96 Curtuişeni (BH) RE R X X - - X X POP 029 ET 86/96 Curtuişeni (BH) LRE R X X X X X X POP 030 ES 48/58 Salonta (BH) RE R X X X POP 031 ET 84 Săcuieni Bihor (BH) RE R X X X POP 032 ET 85/86 Şimian (BH) LRE E X X X X X X POP 033 ET 85/86 Şimian (BH) RE R X X - - X X POP 034 ET 86 Valea lui Mihai (BH) RE R X X - - X X POP 035 ET 86 Valea lui Mihai (BH) RE R X X - - X X POP 036 ET 75 Voivozi (V. lui Mihai) (BH) RE E X X - - X X POP 037 FT 03 Voivozi (V. lui Mihai) (BH) RE R X X - - X X Satu-Mare county POP 038 FT 48 Amaţ (SM) RE R X X X POP 039 ET96/FT06 Andrid (SM) RE R X X - - X X POP 040 FT 69 Apa (SM) RE R X X - - X X POP 041 FU 50 Agrij (SM) LRE E POP 042 FT59 Băbăşeşti (SM) RE E X X - - X X POP 043 FT 59 Băbăşeşti (SM) RE R X X - - X X

5 298 Sas, I. Table 1. (Continued) Population UTM R L E Locality Syst. pr. Cod (10x10 km) m f m f m f POP 044 FU 51 Băbeşti (SM) LRE R X X X X X X POP 045 FU 40 Bercu (SM) RE R X X - - X X POP 046 FT 08 Berea (SM) RE E X X - - X X POP 047 FT 08 Berea (SM) LRE E X X X X X X POP 048 FT 29 Boghiş (SM) RE R X X X POP 049 FT07/08/18 Carei (SM) RE R X X X POP 050 FT 07 Ciumeşti (SM) LRE E X X X X X X POP 051 FU 50 Ciuperceni (SM) LRE E X X X X X X POP 052 FU 62 Comlăuşa (SM) RE R X X X POP 053 FT 68 Crucişor (SM) RE R X X - - X X POP 054 FT 28 Dacia (SM) RE R X X - - X X POP 055 FT 39 Dara (SM) E E X X POP 056 FT 39 Dara (SM) LRE E X X X X X X POP 057 FT 39 Decebal (SM) LRE E X X X X X X POP 058 FT 28/29 Doba (SM) RE R X X - - X X POP 059 FU 30 Dorolţ (SM) RE E X X - - X X POP 060 FU 30 Dorolţ (SM) LRE E X X POP 061 FU 50 Drăguşeni (SM) LRE E X X X X X X POP 062 FU 50 Dumbrava (SM) RE R X X X POP 063 FT 08 Foieni (SM) E E X POP 064 FT 08 Foieni (SM) LRE E X X X X X X POP 065 FT 08 Foieni (SM) LRE E X X X X X X POP 066 FT 08 Foieni (SM) RE R X X - - X X POP 067 FT 38 Gelu (SM) RE R X X X POP 068 FU 61 Gherţa Mică (SM) RE E X X - - X X POP 069 FU 61 Gherţa Mică (SM) RE R X X - - X X POP 070 FU 61 Gherţa Mică (SM) RE R X X - - X X POP 071 FU 61 Gherţa Mică (SM) LRE E POP 072 FU 41/51 Halmeu (SM) RE R X X - - X X POP 073 ET 98 Horea (SM) LRE E X X X X X X POP 074 ET 98 Horea (SM) LRE E X X X X X X POP 075 FT 48 Hrip (SM) RE R X X X POP 076 FT 07 Ianculeşti (SM) RE R X X - - X X POP 077 FT 69 Iojib (SM) RE R X X - - X X POP 078 FU 50/60 Livada (SM) LRE E X X X X X X POP 079 FU 50/60 Livada (SM) LRE E X X X X X X POP 080 FU 50/60 Livada (SM) LRE E X X X X X X POP 081 FU 50/60 Livada (SM) E E X X POP 082 FU 50/60 Livada (SM) RE E X X - - X X POP 083 FU 50 Livada Mică (SM) LRE E X X X X X X POP 084 FT 48 Mădăras (SM) RE R X X X POP 085 FT 06 Mănăstirea Portăriţa (SM) RE R X X - - X X POP 086 FT 68 Măriuş (SM) RE R/E X X - - X X POP 087 FT 57 Mediaşa (SM) RE R X X X POP 088 FT 69 Medieşi Vii (SM) LRE E X X X X X X POP 089 FT 59/69 Medieşu Aurit (SM) RE R X X X POP 090 FT 69 Medieş Râturi (SM) RE R X X X POP 091 FU 51 Mesteacănu (SM) RE R X X X

6 The Pelophylax esculentus complex in North-Western Romania 299 Table 1. (Continued) Population Cod UTM (10x10 km) Locality Syst. pr. R L E m f m f m f POP 092 FU 40/41 Micula (SM) RE E X X - - X X POP 093 FU 40/41 Micula (SM) LRE E X X X X X X POP 094 FT 28 Moftinu Mare (SM) RE R X X - - X X POP 095 FU 40 Nisipeni (SM) RE E X X - - X X POP 096 FU 40 Noroieni (SM) RE E X X - - X X POP 097 FT79/FU70 Oraşu Nou (SM) RE E X X - - X X POP 098 FU 60 Păşunea Mare (SM) LRE E X X X X X X POP 099 FU 30 Petea (SM) E E X X POP 100 ET 96/97 Pişcolt (SM) RE E X X - - X X POP 101 ET 96/97 Pişcolt (SM) LRE E X X X X X X POP 102 FT 59 Potău (SM) RE R X X X POP 103 FU 41 Porumbeşti (SM) RE R X X - - X X POP 104 FU 41 Porumbeşti (SM) LRE E X X X X X X POP 105 ET 97 Resighea (SM) LRE E X X X X X X POP 106 ET 97 Resighea (SM) RE E X X - - X X POP 107 FT 48 Ruşeni (SM) RE R X X X POP 108 ET97/FT07 Sanislău (SM) LRE E X X X X X X POP 109 FT 38 SătmăLRE (SM) RE E X X - - X X POP 110 FT 39/49 Satu Mare (SM) LRE R X X X X X X POP 111 ET 97 Scărişoara Nouă (SM) LRE L X X X X X X POP 112 ET 97 Scărişoara Nouă (SM) LRE E X X X X X X POP 113 ET 97 Scărişoara Nouă (SM) LRE E X X X X X X POP 114 FT69 Someşeni (SM) RE R X X - - X X POP 115 FU 62/63 Tarna Mare (SM) RE R X X - - X X POP 116 FT 58 Tătăreşti (SM) RE R X X X POP 117 FT 29 Traian (SM) RE R X X - - X X POP 118 FU 61 Turţ (SM) LRE E X X X X X X POP 119 FU 51 Turu Lung (SM) LRE E X X X X X X POP 120 FU 61 Turu Lung Vii (SM) LRE E X X X X X X POP 121 FT 08 Urziceni (SM) LRE E X X X X X X POP 122 FT 08 Urziceni (SM) RE E X X - - X X POP 123 FT 08 Urziceni de Pădure (SM) LRE E X X X X X X POP 124 FU 62 Valea Seacă (SM) RE R X X X POP 125 FU 70/80 Vama (SM) RE R X X - - X X POP 126 FT 39 Vetiş (SM) LRE E X X X X X X POP 127 FT 47/48 Viile Satu Mare (SM) RE R X X X hilly-mountainous areas. P. ridibundus is the only water frog that occurs in thermal habitats and forms non-hibernating populations in north-western Romania (see in Sas et al. 2009b,c). Instead, the P. lessonae species occurs solely in lowlands (below 204m) and the hybrid P. esculentus does not exceed either the altitudinal limit of 300m. In Romanian Moldavia the P. lessonae species reaches higher altitudes, primarily because of the particular landscape of this region (see in Sas 2009). Populations of P. lessonae at altitudes higher than m have been identified only in the central area of Romania (inside the Carpathian Basin at Reci 546m Csata & Csata 1996; outside the Carpathian Basin at Râul Doamnei 683m Covaciu- Marcov et al. 2010a). Analyzing Fig.1, one can observe that in

7 300 Sas, I. north-western Romania towards the southern areas there are R-E system populations, in the northern regions being already present all the three forms of the hybridogenetic complex (L- R-E). The only exception is represented by an isolated area in the Teuz River Valley (see in Covaciu-Marcov et al. 2006b) where the presence of P. lessonae species has also been recently recorded, in an L-R-E system. It is obvious that the establishment of hybridogenetic populations depends exclusively on the distribution features of P. lessonae species as the other two forms have a much wider distribution in north-western Romania. In the study area P. lessonae occurs with glacial relict species (such as Rana arvalis, Zootoca vivipara, Vipera berus) and with low altitude populations of some hilly-mountainous species. Most of the P. lessonae populations and also the largest ones (implicitly the L-R-E system) are found in Carei Plain and Livada Plain (the Tur Valley) where they are integrated into metapopulations (see in Sas 2009). The long-term persistence of these populations is assured also by the integration of the two above mentioned areas in the Natura 2000 network (Carei Plain - ROSCI0020, Tur River ROSCI0214). Beside P. lessonae there are important populations of R. arvalis (Sas et al. 2008) and Z. vivipara (Covaciu-Marcov et al. 2008d) with which it occurs together. An exception exists in the case of the southern population, from Teuz Valley. The most plausible explanation is that the two species, R. arvalis and Z. vivipara are related to a much colder climate (the 10ºC isotherm, also see in Covaciu-Marcov 2004) compared with P. lessonae species, which is better adapted to drought. In Teuz Valley, but in Livada Plain too, P. lessonae occurs, even in common habitats, along with another glacial relict species, V. berus (Covaciu-Marcov et al. 2006b, 2008a). In the Livada Plain there are found low altitude populations of Bombina variegata ( m - Covaciu-Marcov et al. 2009e), Rana temporaria (160m - Covaciu- Marcov & Ferenti 2008), Salamandra salamandra ( m - Covaciu-Marcov et al. 2007b), also. Similar situations are encountered in Teuz Valley as well, where at an altitude of 135m reside species like S. salamandra and R. temporaria (Covaciu-Marcov et al. 2008e). It should also be mentioned the fact that around Turţ locality, although they do not occur together with P. lessonae (located at a distance of about 10 km), there are several low altitude populations of Lissotriton montandoni (see in: Covaciu-Marcov et al. 2007c, 2010c). In Carei Plain, beside the two relict elements, P. lessonae is also found along with the lizard Podarcis tauricus, a Balkan steppe species with completely opposite ecological requirements and postglacial history which reaches here the northern limit of its range (see discussion in Covaciu-Marcov et al. 2009d). A similar situation is found in southwestern Romania also, where P. lessonae occurs with southern species such as Testudo hermanni, Podarcis muralis or Vipera ammodytes (see in: Covaciu-Marcov et al. 2009b). Population systems Beside the biometric and bioacoustic confirmations it is very important that we identified in every population of P. ridibundus both females and males. Matings between hybrids (ExE matings) can lead to offspring of P. ridibundus (eg. Hotz et al. 1992, Pagano et al. 1997), but these usually die during metamorphosis (Christiansen et al. 2005, Arioli 2007 cited in Arioli et al. 2010) due to the deleterious alleles accumulated on the clonally transmitted R genome (e.g. Berger 1977, 1983, Graf & Muller 1979, Uzzel et al. 1980), excluding the chances of forming populations of Pelophylax ridibundus. Exception can occur only when the parents possess and transmit different clonal R genomes (Vorburger 2001, Guex et al. 2002, also see in Som & Reyer 2006), but in such cases result all female offspring (e.g. Hotz et al. 1992). Only matings between LLR-triploid hybrids can produce male offspring, but these have

8 The Pelophylax esculentus complex in North-Western Romania 301 morphological and biometric characters typical to hybrids, primarily due to the double lessonae chromosomes (LL) (Tunner 2000). Although RRL-triploids exhibit a more ridibundus-like phenotype, these are females and they can be easily defined morphologically (Günther 1975, Berger et al. 1978). Hence, we can exclude the possibility that in any R-R, R-E or L-R-E system populations from north-western Romania, the individuals of P. ridibundus proceed from matings between hybrids. It has to be noted that in many aquatic habitats P. lessonae seems to occur exclusively with P. kl. esculentus making up L-E system population. However, we consider inaccurate to speak about L-E populations because close to each aquatic habitat with L-E there are aquatic habitats with P. ridibundus species. Thus, although P. ridibundus seems to be missing from a habitat, during the breeding period it migrates toward ponds with P. lessonae (see in Rybacki & Berger 1994) and P. kl. esculentus converting the apparently L-E system into a L- R-E hybridogenetic system. The assessment of population systems is properly made if it is done solely during the reproductive periods and not after breeding (when water frogs have already occupied other environments, frequently not being linked anymore). Nevertheless, it has to be mentioned that in most populations of water frogs from north-western Romania, the three forms occur together in the post-reproductive period also, the only condition being the persistence of some aquatic habitats (because P. ridibundus is more aquatic). During the study, mixed pairs in amplexus were identified exclusively between males of P. lessonae and females of P. kl. esculentus (Fig.2), and between males of P. kl. esculentus and females of P. ridibundus, being also identified several spawns (Fig.3). We did not record pairs in amplexus between lessonae and ridibundus. In the study area mixed pairs in amplexus can already be seen in April, while pairs of malesfemales P. lessonae only from May. This seems to be related to the sexual activity of these Figure 1. Distribution draft of the population systems of Pelophylax esculentus complex in Arad (AR), Bihor (BH) and Satu-Mare (SM) counties [R- P. ridibundus; E- P. esculentus; L- P. lessonae]

9 302 Sas, I. forms also, which begins earlier in P. kl. esculentus than in P. lessonae (Heym 1974), thus the rep-roductive period has two stages (also see in: Gubányi 1996). Lessonae males have higher sexual activity than esculentus males (Blankenhorn 1977). Esculentus males do not account for the proportion of heterospecific pairs as they are always under-represented and mostly esculentus females are found in amplexus with lessonae males than vice versa (Lengagne et al. 2006). In addition, P. esculentus is a superior larval competitor, and adult females are about three times more fecund (Berger & Uzzell 1980), and has higher survival rate than P. lessonae (also see in Anholt et al. 2003). On the other hand, P. lessonae males using the biggeris-better strategy (Blankenhorn 1977) would reproduce almost exclusively with females of P. esculentus. Thus we can consider that without the reproduction in two stages, esculentus would rapidly displace its sexual host (P. lessonae) and lacking a viable sexual partner would then go extinct from these populations. Identifying R-E and E-E system populations is of great theoretical importance. In the common hybridogenesis model of the P. esculentus complex (e.g. Schultz 1969, Uzzel & Berger 1975) the hybrid can produce viable offspring through sexual parasitism. Because in hybridogenesis P. esculentus prior to meiosis exclude the L parental genome, only the R genome being replicated and transmitted clonally (R-hybridogenesis sensu Polls-Pelaz 1994), it needs mating with its sexual host, represented by P. lessonae. But, in the R-E system the hybrid coexists with P. ridibundus and in the E-E system appears alone without the parental species. Sometimes the hybrids exclude their ridibundus genome and produce sperm with a lessonae genome (Anti-R-hybridogenesis, sensu Polls-Pelaz 1994) (or a mixture of both kinds of sperm Vinogradov et al. 1991, and also see the discussion in Ragghianti et al. 2007). After Ragghianti et al. (2007), a particular combination of R-E system lessonae and R-E system ridibundus genomes is necessary to lead to the R-E system type of hybridogenetic gametogenesis. Another possibility for maintaining hybrids in these populations would be the presence of triploid individuals (LRR or LLR) beside diploid ones (LR) in the R-E and E-E system populations (see fig.1 with gametogenesis and mating combination, in Arioli et al or in Christiansen et al. 2010). In the northern part of the study area, in the vicinity of some R-E or E-E system populations there are important P. lessonae populations too. Hence, gene flow between hybrids and their sexual host (P. lessonae) cannot be excluded. But in the southern area (see Fig.1) the absence of P. lessonae excludes this possibility (excepting the Teuz River population). It is very important that in north-western Romania there were identified five cases of reproductive populations consisting solely of the hybrid P. kl. esculentus (E-E system). Although P. kl. esculentus is a hybrid, many times appears alone as well, in the absence of parental species in all-hybrid populations (e.g. Graf & Polls-Pellaz 1989, Günther 1997a, Plötner 2005, and also see in Aroli 2007, Jacob 2007 cited in Christiansen et al. 2010), because it has become reproductively independent of the parental forms (Graf & Polls-Pelaz 1989). Practically the all-hybrid populations secondarily acquired sexual reproduction, have transformed from clonal to sexual hybrids, having a great evolutionary potential (Christiansen & Reyer 2009). All-hybrid populations (E-E system) mainly occur in the northern area of their distribution range as a consequence of polyploidization (RRL or LLR triploids) (Günther et al. 1979, Christiansen & Reyer 2009, Christiansen et al. 2010, and see in Arioli et al and the references cited therein). In the case of two populations of P. kl. esculentus we have not identified males (all-females E system). One of these all-females E system populations (POP063 Table 1) has been recently described (Sas et al. 2009a). The second

10 The Pelophylax esculentus complex in North-Western Romania 303 Figure 2. Amplex between P. lessonae male and P. esculentus female (April 2009, POP111 see Table 1) Figure 3. P. lessonae male and several lessonae x esculentus spawn (April 2009, the Vermes marsh at POP111 see Table 1)

11 304 Sas, I. population is located in northern Livada Plain (POP081 Table 1), inhabiting a habitat made by a water accumulation in an abandoned quarry. Although in the case of these two E system populations there would be possible an influx of males of parental species in the breeding period, during the study we have encountered only female individuals of P. esculentus. In 1993, Schmidt worked out a pattern which considers that water frogs can reproduce both sexually and asexually (asexuality characterizes hybridogenesis - see in: Schmidt 1993). Hotz and collaborators (1992) consider that P. kl. esculentus has a reproductive system analogous to the parthenogenetic cycles. It is possible that these populations are included in a parthenogenetic cycle, but in order to prove this, beside further field observations, experimental laboratory studies are needed in the future. Concerning the population from Livada Plain, although repeatedly there were captured exclusively female hybrids, taking account of the large surface of the habitat and its unapproachable sections we cannot assert for certain a parthenogenetic status of this population. It is possible that these populations had in the past male individuals also. But following the isolation of these populations from those of the parental species, reproduction could have been achieved only by matings between hybrids. Thus it is quite possible that after males disappeared from the population, the frogs have entered into a parthenogenetic cycle to preserve the population over time. It remains to be seen whether males will appear in the future in these populations which could indicate a population consisting of triploid individuals or of triploid individuals also. Perspectives By clarifying the distribution of water frogs in north-western Romania, this area becomes the only one from the country for which detailed information on distribution of the three forms and the integration of these in various population systems are available. Identification of numerous L-R-E (L-E) system populations is very important with regard to the hybridogenetic complex. Conservation in time of these populations is also ensured by their placement in Natura 2000 Habitats Directive sites: Carei Plain - ROSCI0020, Tur River - ROSCI0214 (the importance of N2K sites see reviewed in Gagyi-Pallfy 2007, Badea 2007). Many R-E and E-E system populations are also found in these areas. The southern populations from the study area (R-E, E-E and L-R-E systems from Teuz Valley) are almost entirely located outside of any protected areas. The only exceptions are some R-E populations that occur in Natura 2000 sites (Drocea - ROSCI0070, Cermei Plain ROSPA0014). Although P. ridibundus species and the hybrid P. esculentus are not protected (OUG 57/2007), R-E, E-E system populations (all-female E as well) have a remarkable theoretical importance, particularly in terms of evolutionary biology. All these bring into question by what mechanisms these populations are maintaining over time (clonal or/and sexual reproduction) and what is the ploidy of hybrids from these populations. References Anholt, B.R., Hotz, H., Guex, G.D., Semlitsch, R.D. (2003): Overwinter survival of Rana lessonae and its hemiclonal associate Rana esculenta. Ecology 84(2): Arioli, M. (2007): Reproductive patterns and population genetics in pure hybridogenetic water frog populations of Rana esculenta. PhD Thesis 2007, University of Zurich, Switzerland. Arioli, M., Jakob, C., Reyer, H.U. (2010): Genetic diversity in water frog hybrids (Pelophylax esculentus) varies with population structure and geographic location. Molecular Ecology 19: Badea, A.B. (2008): Protected areas as instrument for nature protection. AES Bioflux 2008 (pilot b): [In Romanian].

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