Sharp decline of the Mediterranean tree frog (Hyla meridionalis) over 40 years in the southeast of Iberia: causes, and possible solutions to an extinction

Sharp decline of the Mediterranean tree frog (Hyla meridionalis) over 40 years in the southeast of Iberia: causes, and possible solutions to an extinction | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Sharp decline of the Mediterranean tree frog (Hyla meridionalis) over 40 years in the southeast of Iberia: causes, and possible solutions to an extinction Mariano Paracuellos, Emilio Rodríguez-Caballero, Enrique Villanueva, and 20 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4700075/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Water-dependent amphibians often present marginal populations in arid and semi-arid regions of the world. This is the case of the Mediterranean tree frog ( Hyla meridionalis ) in the southeast of Iberia, where the species is currently threatened. Here we provide an update of the distribution of Mediterranean tree frog in the region by surveying in localities with appropriate characteristics for the species. In part of these localities, there is historical (1980s-1990s) occurrence data, which allows studying temporal changes in the presence of the species. Furthermore, for the Adra river basin, where human-induced environmental changes and frog persistence over the last 40 years are known, we study how habitat changes and level of protection relate to local frog extinction. The Mediterranean tree frog currently appears in low numbers in 17 sampled points, and has disappeared in 27 of the 43 sampled points where it was present 40 years ago. Sampled points with nowadays frog presence have almost three times surface of suitable and legally protected habitats than those without frog. In addition, sampled points where the frog have disappeared experienced a more pronounced reduction in suitable habitat, and have three times less protected area than those where the frog remained. In the current global change scenario, it is urgent to restore and protect areas that include masses of fresh or brackish water with shallow shores covered by thick marsh vegetation and to reinforce populations to prevent the probable extinction of the Mediterranean tree frog in the southeast of Iberia. Agricultural impact conservation amphibian species distribution greenhouses temporal evolution Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction The negative effects of global change are notoriously evident for the most vulnerable aquatic-dependent amphibians, especially at the edges of their distributional ranges (Duellman 1999 ; Collins and Crump 2009 ). In this context, many species of the family Hylidae are expected to experience sharp declines because they are particularly sensitive to human induced environmental alterations (e.g., Dufresnes et al. 2013 ; Hoskin et al. 2013 ; Vasconcelos and do Nascimento 2016 ). Indeed, hylids are often used as sentinels of the conservation status of the habitats they inhabit (Semlitsch et al. 2000 ; AbuBakr and Crupper 2010 ; Zazeri et al. 2010 ; Gonçalves et al. 2013 ; Sillero 2014 ; Santos et al. 2015 ; Valdespino et al. 2015 ). The Mediterranean tree frog ( Hyla meridionalis ) was most likely introduced during prehistoric or historical periods in the Iberian Peninsula (Dufresnes and Alard 2020 ), where it is mostly abundant. The impacts of global change on this species are expected bigger in arid and semi-arid regions where the lack of water is the main life constraint. However, several authors suggest the species to be threatened and at risk of extinction in the Iberian southeastern edge, the driest region in Europe (e.g., 175–300 mm/year in a large part of the territory; United Nations Environment Programme 1992 ), due to reduction and fragmentation of their populations ( e.g ., Tejedo and Reques 2002 ; Torralva et al. 2005 ; González-Miras and Nevado 2008 ; Alaminos 2013 ). In fact, the Mediterranean tree frog is currently included in the Spanish List of Wild Species needing Special Protection, which implies a periodic re-assessment of its conservation status (Ministry of the Environment and Rural and Marine Affairs 2011 ). So far, only one study has assessed the conservation status of Mediterranean tree frog populations in the SE of the Iberian Peninsula (Paracuellos et al. 2017 ). That work showed evidence of decline in Almería and Murcia provinces since 1980s-1990s (Paracuellos et al. 2017 ). However, recent frog presence could have been underestimated based on reporting frog-singing activity in a single day. Activities and hence detection of amphibians are highly dependent on changing environmental conditions (Heyer et al. 1994 ; Duellman, 1999 ). Therefore, re-sampling in different years encompassing a huge range of environmental conditions would be necessary to avoid false absences (Gómez-Rodríguez et al. 2012 ) and verifying the decline of the Mediterranean tree frog in the Iberian southeastern. The first aim of the present study is to re-evaluate the conservation status of the Mediterranean tree frog in the Iberian southeastern based on updated and multi-replicated records of singing activity in localities with appropriate characteristics for the species. In a portion of these localities, there was historical (1980s-1990s) presence data, which allows studying temporal changes in the presence of the species. Furthermore, we aim to identify the potential drivers that triggered changes in the geographical distribution of the species in the Iberian southeastern and to evaluate the effectiveness of potential conservation measures for planning future conservation actions. These last two objectives are addressed by studying how human induced habitat changes and level of protection at the local level relate with frog persistence at the Adra river basin. Material and methods Study area The study was conducted in the Iberian southeastern, at the provinces of Almeria, Murcia and Alicante (Fig. 1 ). There is data on the historical presence (years 1980s and 1990s) of Mediterranean tree frog in different localities within this study area that were compiled in a previous work by Paracuellos et al. ( 2017 ). Localities with historical presence data were sampled in this study together with other localities selected on maps having suitable environmental conditions for the species. These included coastal and inland temporary or permanent waterbodies with abundant marsh vegetation such as waterlogged meadows, low-flowing streams, ponds and wetlands (Sillero 2014 ). Overall, sampling points were located in backwater zones of permanent and ephemeral rivers and wetlands in six regions (Fig. 1 ). These are the (i) West and (ii) East of Almería, the (iii) Andarax river basin and (iv) Los Vélez in Almería province, the (v) high plateau in Murcia province, and the (vi) south in Alicante province (Fig. 1 ). Sample points range from the sea level in the coast to 1,081 m a.s.l. (Table 1 in Appendix). Table 1 in Appendix . Paracuellos et al. Region/locality Geographical coordinate Altitude (m a. s. l.) Current seasonality Sampled points with frog presence Sampled points Total num. of visits Sampling periods (i) West of Almería : 36°45'27"N 2°49'9"W 70 Permanent and temporary 13 78 332 06/04–14/07 Adra river and lagoons * 36°46'538"N 3°0'6"W 64 Permanent and temporary 12 38 186 06/04–16/06 Cañada de las Norias 36°45'43"N 2°44'13"W 25 Permanent 0 6 24 29/04–19/06 Punta Entinas-Sabinar 36°41'34"N 2°41'58"W 0 Permanent and temporary 0 10 40 11/05–19/06 Ribera de la Algaida 36°47'13"N 2°35'36"W 2 Temporary 0 2 8 05/05–13/06 Playa Serena golf course 36°43'28"N 2°38'8"W 1 Permanent 0 4 16 28/04–11/07 Almerimar golf course 36°42'12"N 2°47'34"W 4 Permanent 0 4 16 30/04–14/07 Guardias Viejas lagoons 36°42'33"N 2°49'12"W 4 Permanent 0 3 12 22/04–12/07 Sotomontes pond 36°42'51"N 2°50'28"W 16 Permanent 0 1 4 22/04–12/07 La Almunya del Sur garden 36°45'27"N 2°51'47"W 84 Permanent 1 1 2 26/04–25/05 El Toril 36°45'14"N 2°52'7"W 84 Permanent 0 1 2 26/04–25/05 Cañada de Ugíjar 36°44'44"N 2°48'55"W 43 Permanent 0 1 2 26/04–25/05 Cañada de Onáyar 36°44'55"N 2°50'1"W 40 Permanent 0 1 2 26/04–25/05 El Loco stream 36°43'58"N 2°51'49"W 29 Permanent 0 2 4 26/04–25/05 El Cura ravine 36°50’12”N 2°38’21”W 300 Permanent and temporary 0 4 14 06/04–12/06 (ii) East of Almería : 37° 7'51"N 1°55'55"W 172 Permanent and temporary 4 39 174 26/03–18/06 Alías river 36°59'36"N 1°58'24"W 98 Permanent 0 4 16 15/04–10/06 Aguas river * 37°8'16"N 1°57'49"W 104 Permanent 3 24 104 26/03–16/06 Antas river * 37°12'2"N 1°48'54"W 6 Permanent 0 4 20 26/03–14/06 Almanzora river * 37°17'34"N 1°52'15"W 81 Permanent and temporary 1 5 24 26/03–18/06 Los Canos salt marsh * 37°13'20"N 1°48'37"W 4 Permanent 0 2 10 26/03–09/06 (iii) Andarax river basin : 37°03’32”N 2°38’00”W 449 Permanent 0 10 38 07/04–09/06 Nacimiento river 37°03’32”N 2°38’00”W 449 Permanent 0 10 38 07/04–09/06 (iv) Los Vélez : 37°41'27"N 2° 5'42"W 1.069 Permanent 0 5 20 09/05–07/07 La Canastera ravine 37°41'23"N 2°5'45"W 1.064 Permanent 0 2 6 10/05–22/06 La Fuente ravine 37°41'32"N 2° 5'38"W 1.041 Permanent 0 3 14 09/05–07/07 (v) High plateau : 38°36'8"N 1°14'10"W 782 Permanent and temporary 0 4 17 18/04–30/06 Tobarrillas stream * 38°43'59"N 1°8'42"W 831 Temporary 0 2 7 18/04–30/06 El Pino source * 38°32’17”N 1°17’43”W 617 Temporary 0 1 5 18/04–30/06 El Zorro pool * 38°28’52”N 1°20’12”W 509 Permanent 0 1 5 18/04–30/06 (vi) South of Alicante : 38°12'43"N 0°39'54"W 18 Permanent and temporary 0 8 32 08/04–11/06 El Hondo 38°10'56"N 0°45'16"W 0 Permanent and temporary 0 4 16 08/04–11/06 Clot de Galvany 38°14'44"N 0°31'54"W 2 Permanent and temporary 0 4 16 15/04–10/06 In addition, we conducted a more detailed analysis of the factors determining the historical presence of the Mediterranean tree frog in the Adra river basin (Fig. 1 F’). The Adra basin is included within West of Almería region and is a hydrological unit with connected waterbodies and a high number of sampled points (N = 38) covering a huge range of environmental conditions. Changes in the degree of agricultural intensification and habitat suitability for the frog in the basin can be tracked on aerial photographs taken in the 80s and today. Hence, for each sample point we quantified the historical and current extent of five features of habitat relevant for the Mediterranean tree frog (Lizana and Barbadillo 1997 ; Sillero 2014 ; Márquez and Lizana 2002 ; Smith and Sutherland 2014 ; Maceda-Veiga et al. 2016 ), namely: (a) distance to nearest roads or paths, as indicator of human presence and activity, and, within a circle with a radius of 500 m centered on each sampling point, the surface occupied by (b) suitable habitat, measured as the total surface of permanent or temporal waterbodies with shallow shores covered by thick marsh vegetation ( sensu Sillero 2014 ), (c) open-air crops, (d) intensive greenhouse crops, and (6) the invasive alien species giant reed ( Arundo donax ). The giant reed is one of the world’s worst invasive plants (Lowe et al. 2000 ), especially in river bed areas, where it modifies many ecosystem functions (Dudley 2000 ; Aguiar and Ferreira 2013 ), impoverishing the invertebrate fauna and hence very likely the habitat suitability for the frog (Paracuellos 1997 , Maceda-Veiga et al. 2016 ). Assessment of current presence based on calling activity To detect the presence of frogs, nocturnal listening of the calling activity of males and intensive visual searches of adults or larvae were carried out in several points at each locality in the years 2016, 2018 and 2019. This methodology is widely used to assess anuran presence at the local level (e.g., Heyer et al. 1994 ). Sampling was performed in the frame of a citizen science program where volunteers were coordinated by expert herpetologists at each locality. All volunteers were familiar with the songs of the species and previously trained with recordings of their calls (see a similar case in Paracuellos et al. 2022 ). To maximize male detection, samplings were conducted on the date of maximum calling activity in each sampled point. These dates are known from monthly censuses of male calling activity carried out through the year in a previous work (Paracuellos et al. 2022 ). To minimize false absences, we discarded sampling the nights ambient temperature dropped below 14º C (Paracuellos et al. 2022 ), since frog vocalizations decrease a lot (Llusia et al. 2013a , b , c ;rquez et al. 2014). In addition, we tried sampling in dates there was surface water in all the points (Paracuellos et al. 2022 ). Each point was sampled at night during the two hours closest to sunset (the time from which nuptial chorus activity increases, e.g., Márquez et al. 2014 ). It was avoided sampling whenever strong wind, rain or anthropogenic noise could have interfered with call detection, and nights with clear, moonlit skies with more than 50% brightness, as their light may influence reproduction and probability of visual detection (Dorcas et al. 2009 ; Vignoli and Luiselli 2013 ; Onorati and Vignoli 2017 ). Once at the point, we spent about 15–30 minutes searching for adults or larvae based on auditory and visual cues at the water points and surrounding marsh vegetation. In case no individuals were detected during this first sampling, we reproduced during 15 minutes via Bluetooth for speakers or smartphones sound calls of male Mediterranean tree frog (extracted from Márquez and Matheu 2004 ) to stimulate calling and detect nuptial males in their response (e.g., Heyer et al. 1994 ). After 30–45 minutes of no frog detection it was considered that the species was absent in the sampled point. In whole, the 144 sample points in 27 localities were monitored during 2016, 2018 and 2019 (Table 1 in Appendix) during 613 different surveys. Number of sample points were proportional to the area sampled in each locality and spaced more than 50 m apart (Table 1 , Fig. 1 ). Sampling dates extended from March 26th to July 14th in 56 census days. 87% of the points were sampled four or five days (usually one in 2016 and two more both in 2018 and 2019). The remaining 13% were visited between one and three days during the same years. Statistical analyses To determine the magnitude of change in the distribution of the frog in the Iberian southeastern we targeted on the 54 sampled points in three regions (East and West of Almeria and Murcia high plateau, Fig. 1 , Table 1 in Appendix) with information on the presence-absence of the species in historical (1980s-1990s) and nowadays (2016–2019) times. We analysed changes in the percentage of sampling points with the presence of the species between these periods on East and West of Almeria regions using binomial generalized linear models. In the Murcia high plateau, only visual data comparison between historical and current periods was done due to small sample size. Secondly, we aimed to identify the potential drivers of Mediterranean tree frog decline in the Adra river basin by relying in historical and nowadays information on frog presence and key environmental variables for the frog. We first analyse temporal patterns in habitat features in the sampled points by using dependent sample T-tests. Then we used independent sample T-tests to study if the current values of habitat variables differ between the sampled points where the Mediterranean tree frog is currently found and missing. In addition, we calculated the difference between historical and current values of environmental variables for each sampled point, and we analyze whether these vary between points where the frog has been lost and where it remained by using independent samples T-tests. Finally, using independent sample T-tests, we evaluate the importance of protecting the habitat for the frog by analyzing whether the surface of ​​legally protected habitats within a circle with a radius of 500 ms centered on each sampling point varies: 1) between sampled points with current presence and absence of the frog; 2) between sampled points where the species have been lost or not during the last 40 years. Analyses were performed using the lme (Bates et al. 2015 ) and stats packages in R Statistical Software (v. 4.1.2.; R Core Team 2021 ). Standard model validation graphs (Zuur et al. 2009 ) revealed that model assumptions of homogeneity of variance and normality of residuals were fulfilled. Results Current distribution of Mediterranean tree frog in the Iberian southeastern The Mediterranean tree frog has been recorded in 17 out of the 144 sampled points during 2016–2019 (Table 1 in Appendix). Size of choruses at these points is strikingly small, with four or less singing males at most points (Table 2 in Appendix). Nowadays the Mediterranean tree frog appears in the East and the West of Almería province, but in none of sampled points at Murcia or Alicante provinces (Table 1 in Appendix, Fig. 1 ). In the West of Almería, most of records came from the Adra river basin (12 sampling points, Table 1 in Appendix), while in the East of Almería they came from the Aguas and Almanzora rivers (4 sampling points, Table 1 in Appendix). Table 2 in Appendix . Paracuellos et al. Sampled point code Nº of individuals AA02 1 AA03 1 AA05 2 AA06 20 AA07 30 AA08 4 AA09 4 AA12 10 AG01 1 AG02 1 AG03 1 AS01 1 DT08 1 DT10 1 MZ01 3 RA02 1 RA06 1 Average 4.88 SE 1.96 Figure 1 about here Temporal changes in the Mediterranean tree frog occurrence Globally, the species has become rare in the Iberian southeastern, going from being present in 43 (i.e. 79.63%) of the 54 sampled points 40 years ago to only 16 (i.e. 29.6%) (χ 2 1 = 27.23, P < 0.001) (Fig. 1 ). Separate analyses for the three regions with historical data show: 1) that the species has disappeared from the three sampled points where it appeared in the Murcia high plateau (Fig. 1 A); that the species went from being present in 11 ( i.e. 84.6%) of 13 sampled points to only 4 ( i.e. 30.7%) in the East of Almería (χ 2 1 = 5.67, P = 0.02) (Fig. 1 E), and from being present in 29 ( i.e. 76.3%) of 38 sampled points to only 12 ( i.e. 31.6%) in the West of Almería (χ 2 1 = 13.56, P < 0.001) (Fig. 1 F). Drivers of Mediterranean tree frog decline in the Adra basin The Adra river basin has undergone pronounced environmental changes during the last 40 years (Fig. 1 in Appendix). Specifically, the distance to roads or paths from each sampling point has been reduced by 260 m (Fig. 1 in Appendix, t 36 = 2.63, P = 0.012) and the suitable habitat for the Mediterranean tree frog has been reduced by more than 57% (Fig. 1 in Appendix, t 36 = 4.91, P = 0.0001). This translates into an increase in the area of open-air crops of more than 29% ( t 36 = 4.37, P = 0.0001) and greenhouse crops of almost 600% ( t 36 = 11.40, P < 0.0001), but not in significant changes in the area occupied by giant reed ( t 36 = 1.83, P = 0.075). Nowadays, the sampled points where the frog is present cover three times more appropriate ( t 35 = 4.19, P = 0.0001), and legally protected habitat ( t 35 = 3.77, P = 0.0005) than those where the frog has not been found (Fig. 2 ). However, there were no differences between sampled points with frogs and without frogs in the distance to roads or paths ( t 35 = 0.17, P = 0.86), or area occupied by greenhouse crops ( t 35 = 1.28, P = 0.28), open-air crops ( t 35 = 0.94, P = 0.35), or giant reed ( t 35 = 1.02, P = 0.31) (Fig. 2 ). Figure 2 about here Moreover, the magnitude of environmental changes since 1980s-1990s differed between points where the frog has disappeared and where it persisted since the former have almost four times greater reduction in the suitable habitat for the frog ( t 27 = 2.39, P = 0.002), and, a nearly significant greater increase in the area of open-air crops ( t 27 = 1.84, P = 0.07) (Fig. 3 ). Points where the frog have disappeared and where it did not, however, showed similar shortening of the distance to roads or paths ( t 27 = 0.63, P = 0.53), increase in surface of greenhouse crops ( t 27 = 0.32, P = 0.74) and reduction in surface of giant reed ( t 27 = 0.93, P = 0.36) (Fig. 3 ). On the other hand, the points where the frog became extinct had a protected area around three times less than those where it persisted ( t 27 = 3.80, P = 0.0007, Fig. 4 ). Figures 3 and about here Discussion The extensive multi-replicated sampling at the regional level and the disappearance of the Mediterranean tree frog from 63% of sampled points with presence 40 years ago confirms that the anuran is at risk of extinction in the Iberian southeastern, such as already suggested by several authors (Tejedo and Reques 2002 ; Torralva et al. 2005 ; González-Miras and Nevado 2008 ; Alaminos 2013 ; Paracuellos et al. 2017 ). The species has become rare in the Adra, Aguas and Almanzora rivers and has disappeared in all other sampled points. The decline scenario of the frog is exacerbated by the reduced number of individuals detected at each sampled point and a high population fragmentation, as points with frog presence were isolated from each other (minimum distance of more than 7 km) and from the nearest populations in Granada or Albacete (minimum distance of more than 52 km; Tejedo and Reques 2002 ; Fig. 1 , Table 1 in Appendix). The general decline in the Iberian southeastern could explain that the Mediterranean tree frog has not colonised apparent suitable localities that we have now sampled but were not in 1980s-1990s. The only exception to this pattern is a garden where the species was recently detected (Table 1 in Appendix), most likely due to an unintentional introduction associated with the transport of ornamental plants (see nearby case in Paracuellos et al. 2018 ). The detailed analysis in the Adra river basin suggests that the key factor in the decline of the Mediterranean tree frog would be the loss of suitable habitats for the frog. The loss of ponds and sheets of water surrounded by marsh vegetation has been accentuated in the Adra basin, while intensive open-air and greenhouse crops have proliferated, and more roads or paths have been built over the last 40 years (see Results). Considering high sensitivity of hylids to changes in their environment (Semlitsch et al. 2000 ; AbuBakr and Crupper 2010 ; Zazeri et al. 2010 ; Gonçalves et al. 2013 ; Sillero 2014 ; Santos et al. 2015 ; Valdespino et al. 2015 ), our results suggest, in agreement with Paracuellos et al. ( 2017 ), that the natural habitats where Mediterranean tree frog inhabits in the Iberian southeastern are being strongly degraded since the 1980s due to an intensification of agriculture practices that affects the viability of local populations. Although the intensification of agriculture in the Iberian southeastern was not measured in this work, it is associated with a massive extraction and pollution of waterbodies (streams and aquifers) by biocides, fertilisers and other agricultural waste (Villegas and Sánchez 1995 ; Nevado and Paracuellos 2002 ; Casas et al. 2003 ; Paracuellos 2006 ; Robledano et al. 2006 ; Torralva et al. 2006). Moreover, the ever-increasing colonisation and proximity of greenhouse crops, buildings and roads or paths could increase the risk of microplastic pollution. These factors may impair habitat quality by their cascading adverse effects on frogs and tadpoles, including impaired growth and body condition, altered pigmentation, histopathological, behavioural and morphological changes, weakened immune responses, toxicity and, therefore, loss of survival and breeding success (Szkudlarek et al. 2023 ). Beyond the changes derived from agricultural intensification, suitable habitat loss may be also amplified by increasing air temperature and altered precipitation in the region due to ongoing climate change (MedECC 2019 ), which further limits water availability and quality in the last refuges for the species. It is also worth mentioning that remaining frog populations in the Iberian southeastern are at the geographical limits of the species' distribution, which may aggravate the environmental and genetic stresses, making them more susceptible and less able to respond to diseases, toxins and major habitat fluctuations (Shafer 1990 ; Duellman 1999 ; Dufresnes et al. 2013 ). Conservation implications Our results conclusively show the importance of maintaining protected areas in the Iberian southeastern to preserve populations of the Mediterranean tree frog and to avoid its extinction. Within the Adra river basin, sampled points with more protected area around are more likely to have the frog. Protected areas include more surface area of ​​suitable habitats for the frog, which are areas of the Adra river and lagoons (Regional Government of Andalusia 1989 , 2015 ) where other aquatic species considered as globally «endangered» (IUCN 2024 ), such as the plant Zannichellia contorta (Ortega et al. 2004 ), the endemic Spanish killifish ( Apricaphanius iberus ; Paracuellos and Nevado 1994 ), or the white headed duck ( Oxyura leucocephala ; Paracuellos et al. 2007 ), coexist. However, current protected areas are very isolated and represent far oasis in West of Almería, where environmental and socio-economic conditions have favoured the proliferation of intensive greenhouse crops all over (Caparrós-Martínez et al, 2020 ), with associated negative environmental impacts (López-Gálvez and Salinas 1999 ; Palomar Oviedo 1999; Martínez-Valderrama et al. 2024). Therefore, we urge to intensify conservation actions in the protected areas and surroundings where Mediterranean tree frog still survives, improving conditions for it and the rest of the associated biodiversity. Also, it is urgent to declare new protected areas that include masses of fresh or brackish water with shallow shores covered by thick marsh vegetation to increase connectivity and prevent the probable extinction of the Mediterranean tree frog in the Iberian southeastern. Moreover, given the small size of current populations, we advocate translocations of frog specimens from the same genetic population still existing in the Iberian southeastern, according to conventional procedures that minimise risks of biosafety and the spread of diseases (Martínez-Silvestre et al. 2023 ). Finally, future monitoring of the species in the area should continue to obtain information of the status of its populations and distribution and the environmental quality in real time. Declarations Acknowledgements This study was carried out with the help of José A. Alguazas, Abel Amat, Francisco M. Benavides, Juan J. Benavides, Nerea Benavides, Jesús Cabrera, Alicia Caler, Soledad Callejón, César Caparrós, Juan A. Caparrós, Alfonso Carrasco, Hugo Cayuela, Esther Cerezo, Jorge Chaves, Carlos Collado, Antonio Cruz, Mercedes Cuenca, Pilar Díaz, M. Dolores Escobar, Daniel Espín, Enrique Fernández, Trino Ferrández, Januario Formoso, Modesta Galdeano, F. Javier García, Agustín Gómez, César Gómez, Lidia Gómez, José M. Gómez, Pedro Guerrero, Leonardo Gutiérrez, Jorge Heredia, Fátima El Jabari, Piotr Jankowski, Florencia Jaurena, Philip Kramer, Antonia López, M. Dolores López, Pablo López, Pedro Lozano, Nadia Manroua, Tobías Martín, Ana B. Martínez, Elena Martínez, Pablo Martínez, Yolanda Martínez, Cesaltina Mendes, Daniel Moreno, Daniel Motos, Alberto Navarro, Faminio Nente, Andrés F. Ochoa, Lorena Olmo, Juan F. Parrilla, M. Dolores Peramo, Yolanda Pérez, Swann Perrachon, Yolanda Pérez, Alejandro Ramírez, Ángeles Ramírez, María Ramos, Pedro Rascón, José Rodríguez, M. Paz Romero, Lidia del Rosal, Francisco Rubio, Rubén Salvador, Encarnación Samblás, José G. Sánchez, M. Carmen Sanz, Mariana Silva, Antonio Tapia, Erik Torralvo, M. Trinidad Torralvo, Alfredo Valdivia, F. Javier Valenzuela, José A. Villegas and Rubén Vives who volunteered for the citizen science project Hyla del Sureste (https://proyecto-hyla-del-sureste.webnode.es/). We are grateful to Rafael Márquez for his advice and review. We thanks to Institute of Almerian Studies and Council of Almería for grants awarded to Enrique Villanueva. RCE was supported by the CRUST R-Forze (PID2021-127631NA-I00) project funded by FEDER / Ministerio de Ciencia e Inovacion-Agencia Estatal de Investigación; the project TED2021-132332B-C21 funded by MCIN/AEI/10.13039/501100011033 and European Unión “Next Generation EU”/PRTR and the Ramon y Cajal fellowship (RYC2020-030762-I). The Government of Andalucía provided authorisations to sample frog presence. References AbuBakr S, Crupper SS (2010) Prevalence of cadmium resistance in Staphylococcus sciuri isolated from the Gray Treefrog, Hyla chrysoscelis (Anura: Hylidae). Phyllomedusa 9:141-146. http://doi.org/10.11606/issn.2316-9079.v9i2p141-146 Aguiar FCF, Ferreira MT (2013) Plant invasions in the rivers of the Iberian Peninsula, South-Western Europe–a review. Plant Biosyst 147:1107-1119. https://doi.org/10.1080/11263504.2013.861539 Alaminos E (2013) Ranita meridional Hyla meridionalis Boettger, 1874. In: Parrondo P (coord) Guía de los anfibios del sureste ibérico. Proyecto Amphibia. 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Zazeri P, Cristina T, Margarido S, de Lima D, de Cerqueira D, Alves E (2010) Esterase inhibition in tadpoles of Scinax fuscovarius (Anura, Hylidae) as a biomarker for exposure to organophosphate pesticides. Environ Sci Pollut Res 17:1411-1421. http:// doi.org/10.1007/s11356-010-0326-y Zuur AF, Ieno EN, Walker N, Saveliev AA, Smith GM (2009) Mixed effects models and extensions in ecology with R. Springer-Verlag, New York. http:// doi.org/10.18637/jss.v032.b01 Additional Declarations No competing interests reported. Supplementary Files Appendix.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Avilés","email":"","orcid":"","institution":"Estación Experimental de Zonas Áridas (EEZA-CSIC)","correspondingAuthor":false,"prefix":"","firstName":"Jesús","middleName":"M.","lastName":"Avilés","suffix":""}],"badges":[],"createdAt":"2024-07-07 11:57:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4700075/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4700075/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":62136306,"identity":"556cd3cc-6529-4fcf-914e-38b812b2b761","added_by":"auto","created_at":"2024-08-09 16:25:14","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2252839,"visible":true,"origin":"","legend":"\u003cp\u003eGeographic distribution of the study area, sampling points and the historical presence of the Mediterranean tree frog in southeastern Iberia. The squares indicate the different regions where the sampling was carried out and the panels correspond to the high plateau of Murcia (A), south of Alicante (B), Los Vélez (C), Andarax river basin (D), and the East (E) and West (F) of Almería, respectively. The Adra river basin within the West of Almería is shown in a separate panel (F'). The coloured circles show the temporal changes in the presence of the species.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-4700075/v1/406a17e6bfacdb7026e99462.png"},{"id":62136303,"identity":"984f241a-6bda-4fd7-b132-64d3935887b6","added_by":"auto","created_at":"2024-08-09 16:25:14","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":305778,"visible":true,"origin":"","legend":"\u003cp\u003eAverage (SE) habitat values in relation to Mediterranean tree frog presence in 2016-2019 for the 38 sampled points within the Adra river basin. (a) Distance to roads or paths, (b) surface of suitable habitat, (c) surface of open-air crops, (d) surface of greenhouse crops, (e) surface of great reed (\u003cem\u003eArundo donax\u003c/em\u003e), and (f) surface of legally protected area.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-4700075/v1/11e4c10e03d48d32c7fce259.png"},{"id":62137442,"identity":"ee724e58-8404-40f6-a8ac-e912768907ff","added_by":"auto","created_at":"2024-08-09 16:33:14","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":255111,"visible":true,"origin":"","legend":"\u003cp\u003eAverage (SE) differences (historical-current) in habitat values for the 29 sampled points within the Adra river basin where the Mediterranean tree frog have loss and persisted during the last 40 years. (a) Distance to roads or paths, (b) surface of suitable habitat, (c) surface of open-air crops, (d) surface of greenhouse crops, and (c) surface of great reed (Arundo donax).\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-4700075/v1/d4e613bdad016765d0651853.png"},{"id":62137444,"identity":"ce69f836-6e7c-46b7-ac64-a13a0e32c091","added_by":"auto","created_at":"2024-08-09 16:33:15","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":203188,"visible":true,"origin":"","legend":"\u003cp\u003eAverage (SE) legally protected area for the 29 sampled points within the Adra river basin where the Mediterranean tree frog have loss and persisted during the last 40 years.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-4700075/v1/1ed1d484b592effd6db4fc32.png"},{"id":64175105,"identity":"62e7c8f8-e13c-4b45-a03f-887dc1b0590c","added_by":"auto","created_at":"2024-09-09 12:37:38","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5315885,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4700075/v1/a153c13e-4a66-4e60-8d64-6ba835c6e9e2.pdf"},{"id":62136304,"identity":"92bdb3c1-1b04-4a0c-b72f-87e8cf972da5","added_by":"auto","created_at":"2024-08-09 16:25:14","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":151877,"visible":true,"origin":"","legend":"","description":"","filename":"Appendix.docx","url":"https://assets-eu.researchsquare.com/files/rs-4700075/v1/24e7b1eab71ad9b2e53f0968.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Sharp decline of the Mediterranean tree frog (Hyla meridionalis) over 40 years in the southeast of Iberia: causes, and possible solutions to an extinction","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe negative effects of global change are notoriously evident for the most vulnerable aquatic-dependent amphibians, especially at the edges of their distributional ranges (Duellman \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Collins and Crump \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). In this context, many species of the family \u003cem\u003eHylidae\u003c/em\u003e are expected to experience sharp declines because they are particularly sensitive to human induced environmental alterations (e.g., Dufresnes et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Hoskin et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Vasconcelos and do Nascimento \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Indeed, hylids are often used as sentinels of the conservation status of the habitats they inhabit (Semlitsch et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; AbuBakr and Crupper \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Zazeri et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Gon\u0026ccedil;alves et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Sillero \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Santos et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Valdespino et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe Mediterranean tree frog (\u003cem\u003eHyla meridionalis\u003c/em\u003e) was most likely introduced during prehistoric or historical periods in the Iberian Peninsula (Dufresnes and Alard \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), where it is mostly abundant. The impacts of global change on this species are expected bigger in arid and semi-arid regions where the lack of water is the main life constraint. However, several authors suggest the species to be threatened and at risk of extinction in the Iberian southeastern edge, the driest region in Europe (e.g., 175\u0026ndash;300 mm/year in a large part of the territory; United Nations Environment Programme \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e1992\u003c/span\u003e), due to reduction and fragmentation of their populations (\u003cem\u003ee.g\u003c/em\u003e., Tejedo and Reques \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Torralva et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Gonz\u0026aacute;lez-Miras and Nevado \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Alaminos \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). In fact, the Mediterranean tree frog is currently included in the Spanish List of Wild Species needing Special Protection, which implies a periodic re-assessment of its conservation status (Ministry of the Environment and Rural and Marine Affairs \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSo far, only one study has assessed the conservation status of Mediterranean tree frog populations in the SE of the Iberian Peninsula (Paracuellos et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). That work showed evidence of decline in Almer\u0026iacute;a and Murcia provinces since 1980s-1990s (Paracuellos et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). However, recent frog presence could have been underestimated based on reporting frog-singing activity in a single day. Activities and hence detection of amphibians are highly dependent on changing environmental conditions (Heyer et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1994\u003c/span\u003e; Duellman, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). Therefore, re-sampling in different years encompassing a huge range of environmental conditions would be necessary to avoid false absences (G\u0026oacute;mez-Rodr\u0026iacute;guez et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) and verifying the decline of the Mediterranean tree frog in the Iberian southeastern.\u003c/p\u003e \u003cp\u003eThe first aim of the present study is to re-evaluate the conservation status of the Mediterranean tree frog in the Iberian southeastern based on updated and multi-replicated records of singing activity in localities with appropriate characteristics for the species. In a portion of these localities, there was historical (1980s-1990s) presence data, which allows studying temporal changes in the presence of the species. Furthermore, we aim to identify the potential drivers that triggered changes in the geographical distribution of the species in the Iberian southeastern and to evaluate the effectiveness of potential conservation measures for planning future conservation actions. These last two objectives are addressed by studying how human induced habitat changes and level of protection at the local level relate with frog persistence at the Adra river basin.\u003c/p\u003e"},{"header":"Material and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy area\u003c/h2\u003e \u003cp\u003eThe study was conducted in the Iberian southeastern, at the provinces of Almeria, Murcia and Alicante (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e1\u003c/span\u003e). There is data on the historical presence (years 1980s and 1990s) of Mediterranean tree frog in different localities within this study area that were compiled in a previous work by Paracuellos et al. (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Localities with historical presence data were sampled in this study together with other localities selected on maps having suitable environmental conditions for the species. These included coastal and inland temporary or permanent waterbodies with abundant marsh vegetation such as waterlogged meadows, low-flowing streams, ponds and wetlands (Sillero \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Overall, sampling points were located in backwater zones of permanent and ephemeral rivers and wetlands in six regions (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e1\u003c/span\u003e). These are the (i) West and (ii) East of Almer\u0026iacute;a, the (iii) Andarax river basin and (iv) Los V\u0026eacute;lez in Almer\u0026iacute;a province, the (v) high plateau in Murcia province, and the (vi) south in Alicante province (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Sample points range from the sea level in the coast to 1,081 m a.s.l. (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e in Appendix).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cb\u003ein Appendix\u003c/b\u003e. Paracuellos et al.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRegion/locality\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGeographical coordinate\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAltitude\u003c/p\u003e \u003cp\u003e(m a. s. l.)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCurrent seasonality\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSampled points with frog presence\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSampled points\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eTotal num. of visits\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eSampling periods\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e(i) West of Almer\u0026iacute;a\u003c/b\u003e:\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e36\u0026deg;45'27\"N 2\u0026deg;49'9\"W\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e70\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003ePermanent and temporary\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e13\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e78\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e332\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003e06/04\u0026ndash;14/07\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAdra river and lagoons *\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e36\u0026deg;46'538\"N 3\u0026deg;0'6\"W\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePermanent and temporary\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e186\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e06/04\u0026ndash;16/06\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCa\u0026ntilde;ada de las Norias\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e36\u0026deg;45'43\"N 2\u0026deg;44'13\"W\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePermanent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e29/04\u0026ndash;19/06\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePunta Entinas-Sabinar\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e36\u0026deg;41'34\"N 2\u0026deg;41'58\"W\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePermanent and temporary\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e11/05\u0026ndash;19/06\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRibera de la Algaida\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e36\u0026deg;47'13\"N 2\u0026deg;35'36\"W\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTemporary\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e05/05\u0026ndash;13/06\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePlaya Serena golf course\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e36\u0026deg;43'28\"N 2\u0026deg;38'8\"W\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePermanent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e28/04\u0026ndash;11/07\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAlmerimar golf course\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e36\u0026deg;42'12\"N 2\u0026deg;47'34\"W\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePermanent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e30/04\u0026ndash;14/07\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGuardias Viejas lagoons\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e36\u0026deg;42'33\"N 2\u0026deg;49'12\"W\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePermanent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e22/04\u0026ndash;12/07\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSotomontes pond\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e36\u0026deg;42'51\"N 2\u0026deg;50'28\"W\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePermanent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e22/04\u0026ndash;12/07\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLa Almunya del Sur garden\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e36\u0026deg;45'27\"N 2\u0026deg;51'47\"W\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePermanent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e26/04\u0026ndash;25/05\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEl Toril\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e36\u0026deg;45'14\"N 2\u0026deg;52'7\"W\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePermanent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e26/04\u0026ndash;25/05\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCa\u0026ntilde;ada de Ug\u0026iacute;jar\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e36\u0026deg;44'44\"N 2\u0026deg;48'55\"W\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePermanent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e26/04\u0026ndash;25/05\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCa\u0026ntilde;ada de On\u0026aacute;yar\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e36\u0026deg;44'55\"N 2\u0026deg;50'1\"W\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePermanent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e26/04\u0026ndash;25/05\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEl Loco stream\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e36\u0026deg;43'58\"N 2\u0026deg;51'49\"W\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePermanent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e26/04\u0026ndash;25/05\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEl Cura ravine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e36\u0026deg;50\u0026rsquo;12\u0026rdquo;N 2\u0026deg;38\u0026rsquo;21\u0026rdquo;W\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e300\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePermanent and temporary\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e06/04\u0026ndash;12/06\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e(ii) East of Almer\u0026iacute;a\u003c/b\u003e:\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e37\u0026deg; 7'51\"N 1\u0026deg;55'55\"W\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e172\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003ePermanent and temporary\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e4\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e39\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e174\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003e26/03\u0026ndash;18/06\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAl\u0026iacute;as river\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e36\u0026deg;59'36\"N 1\u0026deg;58'24\"W\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePermanent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e15/04\u0026ndash;10/06\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAguas river *\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e37\u0026deg;8'16\"N 1\u0026deg;57'49\"W\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e104\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePermanent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e104\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e26/03\u0026ndash;16/06\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAntas river *\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e37\u0026deg;12'2\"N 1\u0026deg;48'54\"W\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePermanent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e26/03\u0026ndash;14/06\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAlmanzora river *\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e37\u0026deg;17'34\"N 1\u0026deg;52'15\"W\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePermanent and temporary\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e26/03\u0026ndash;18/06\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLos Canos salt marsh *\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e37\u0026deg;13'20\"N 1\u0026deg;48'37\"W\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePermanent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e26/03\u0026ndash;09/06\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e(iii) Andarax river basin\u003c/b\u003e:\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e37\u0026deg;03\u0026rsquo;32\u0026rdquo;N 2\u0026deg;38\u0026rsquo;00\u0026rdquo;W\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e449\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003ePermanent\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e0\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e10\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e38\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003e07/04\u0026ndash;09/06\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNacimiento river\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e37\u0026deg;03\u0026rsquo;32\u0026rdquo;N 2\u0026deg;38\u0026rsquo;00\u0026rdquo;W\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e449\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePermanent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e07/04\u0026ndash;09/06\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e(iv) Los V\u0026eacute;lez\u003c/b\u003e:\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e37\u0026deg;41'27\"N 2\u0026deg; 5'42\"W\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e1.069\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003ePermanent\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e0\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e5\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e20\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003e09/05\u0026ndash;07/07\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLa Canastera ravine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e37\u0026deg;41'23\"N 2\u0026deg;5'45\"W\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.064\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePermanent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e10/05\u0026ndash;22/06\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLa Fuente ravine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e37\u0026deg;41'32\"N 2\u0026deg; 5'38\"W\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.041\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePermanent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e09/05\u0026ndash;07/07\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e(v) High plateau\u003c/b\u003e:\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e38\u0026deg;36'8\"N 1\u0026deg;14'10\"W\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e782\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003ePermanent and temporary\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e0\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e4\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e17\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003e18/04\u0026ndash;30/06\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTobarrillas stream *\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e38\u0026deg;43'59\"N 1\u0026deg;8'42\"W\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e831\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTemporary\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e18/04\u0026ndash;30/06\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEl Pino source *\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e38\u0026deg;32\u0026rsquo;17\u0026rdquo;N 1\u0026deg;17\u0026rsquo;43\u0026rdquo;W\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e617\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTemporary\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e18/04\u0026ndash;30/06\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEl Zorro pool *\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e38\u0026deg;28\u0026rsquo;52\u0026rdquo;N 1\u0026deg;20\u0026rsquo;12\u0026rdquo;W\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e509\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePermanent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e18/04\u0026ndash;30/06\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e(vi) South of Alicante\u003c/b\u003e:\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e38\u0026deg;12'43\"N 0\u0026deg;39'54\"W\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e18\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003ePermanent and temporary\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e0\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e8\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e32\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003e08/04\u0026ndash;11/06\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEl Hondo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e38\u0026deg;10'56\"N 0\u0026deg;45'16\"W\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePermanent and temporary\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e08/04\u0026ndash;11/06\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eClot de Galvany\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e38\u0026deg;14'44\"N 0\u0026deg;31'54\"W\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePermanent and temporary\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e15/04\u0026ndash;10/06\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eIn addition, we conducted a more detailed analysis of the factors determining the historical presence of the Mediterranean tree frog in the Adra river basin (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e1\u003c/span\u003eF\u0026rsquo;). The Adra basin is included within West of Almer\u0026iacute;a region and is a hydrological unit with connected waterbodies and a high number of sampled points (N\u0026thinsp;=\u0026thinsp;38) covering a huge range of environmental conditions. Changes in the degree of agricultural intensification and habitat suitability for the frog in the basin can be tracked on aerial photographs taken in the 80s and today. Hence, for each sample point we quantified the historical and current extent of five features of habitat relevant for the Mediterranean tree frog (Lizana and Barbadillo \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e1997\u003c/span\u003e; Sillero \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; M\u0026aacute;rquez and Lizana \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Smith and Sutherland \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Maceda-Veiga et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), namely: (a) distance to nearest roads or paths, as indicator of human presence and activity, and, within a circle with a radius of 500 m centered on each sampling point, the surface occupied by (b) suitable habitat, measured as the total surface of permanent or temporal waterbodies with shallow shores covered by thick marsh vegetation (\u003cem\u003esensu\u003c/em\u003e Sillero \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), (c) open-air crops, (d) intensive greenhouse crops, and (6) the invasive alien species giant reed (\u003cem\u003eArundo donax\u003c/em\u003e). The giant reed is one of the world\u0026rsquo;s worst invasive plants (Lowe et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2000\u003c/span\u003e), especially in river bed areas, where it modifies many ecosystem functions (Dudley \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Aguiar and Ferreira \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), impoverishing the invertebrate fauna and hence very likely the habitat suitability for the frog (Paracuellos \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e1997\u003c/span\u003e, Maceda-Veiga et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eAssessment of current presence based on calling activity\u003c/h2\u003e \u003cp\u003eTo detect the presence of frogs, nocturnal listening of the calling activity of males and intensive visual searches of adults or larvae were carried out in several points at each locality in the years 2016, 2018 and 2019. This methodology is widely used to assess anuran presence at the local level (e.g., Heyer et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1994\u003c/span\u003e). Sampling was performed in the frame of a citizen science program where volunteers were coordinated by expert herpetologists at each locality. All volunteers were familiar with the songs of the species and previously trained with recordings of their calls (see a similar case in Paracuellos et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTo maximize male detection, samplings were conducted on the date of maximum calling activity in each sampled point. These dates are known from monthly censuses of male calling activity carried out through the year in a previous work (Paracuellos et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). To minimize false absences, we discarded sampling the nights ambient temperature dropped below 14\u0026ordm; C (Paracuellos et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), since frog vocalizations decrease a lot (Llusia et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2013a\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003eb\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003ec\u003c/span\u003e;rquez et al. 2014). In addition, we tried sampling in dates there was surface water in all the points (Paracuellos et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Each point was sampled at night during the two hours closest to sunset (the time from which nuptial chorus activity increases, e.g., M\u0026aacute;rquez et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). It was avoided sampling whenever strong wind, rain or anthropogenic noise could have interfered with call detection, and nights with clear, moonlit skies with more than 50% brightness, as their light may influence reproduction and probability of visual detection (Dorcas et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Vignoli and Luiselli \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Onorati and Vignoli \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Once at the point, we spent about 15\u0026ndash;30 minutes searching for adults or larvae based on auditory and visual cues at the water points and surrounding marsh vegetation. In case no individuals were detected during this first sampling, we reproduced during 15 minutes via Bluetooth for speakers or smartphones sound calls of male Mediterranean tree frog (extracted from M\u0026aacute;rquez and Matheu \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2004\u003c/span\u003e) to stimulate calling and detect nuptial males in their response (e.g., Heyer et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1994\u003c/span\u003e). After 30\u0026ndash;45 minutes of no frog detection it was considered that the species was absent in the sampled point.\u003c/p\u003e \u003cp\u003eIn whole, the 144 sample points in 27 localities were monitored during 2016, 2018 and 2019 (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e in Appendix) during 613 different surveys. Number of sample points were proportional to the area sampled in each locality and spaced more than 50 m apart (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Sampling dates extended from March 26th to July 14th in 56 census days. 87% of the points were sampled four or five days (usually one in 2016 and two more both in 2018 and 2019). The remaining 13% were visited between one and three days during the same years.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analyses\u003c/h2\u003e \u003cp\u003eTo determine the magnitude of change in the distribution of the frog in the Iberian southeastern we targeted on the 54 sampled points in three regions (East and West of Almeria and Murcia high plateau, Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e in Appendix) with information on the presence-absence of the species in historical (1980s-1990s) and nowadays (2016\u0026ndash;2019) times. We analysed changes in the percentage of sampling points with the presence of the species between these periods on East and West of Almeria regions using binomial generalized linear models. In the Murcia high plateau, only visual data comparison between historical and current periods was done due to small sample size.\u003c/p\u003e \u003cp\u003eSecondly, we aimed to identify the potential drivers of Mediterranean tree frog decline in the Adra river basin by relying in historical and nowadays information on frog presence and key environmental variables for the frog. We first analyse temporal patterns in habitat features in the sampled points by using dependent sample T-tests. Then we used independent sample T-tests to study if the current values of habitat variables differ between the sampled points where the Mediterranean tree frog is currently found and missing.\u003c/p\u003e \u003cp\u003eIn addition, we calculated the difference between historical and current values of environmental variables for each sampled point, and we analyze whether these vary between points where the frog has been lost and where it remained by using independent samples T-tests. Finally, using independent sample T-tests, we evaluate the importance of protecting the habitat for the frog by analyzing whether the surface of ​​legally protected habitats within a circle with a radius of 500 ms centered on each sampling point varies: 1) between sampled points with current presence and absence of the frog; 2) between sampled points where the species have been lost or not during the last 40 years.\u003c/p\u003e \u003cp\u003eAnalyses were performed using the lme (Bates et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) and stats packages in R Statistical Software (v. 4.1.2.; R Core Team \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Standard model validation graphs (Zuur et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2009\u003c/span\u003e) revealed that model assumptions of homogeneity of variance and normality of residuals were fulfilled.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n \u003ch2\u003eCurrent distribution of Mediterranean tree frog in the Iberian southeastern\u003c/h2\u003e\n \u003cp\u003eThe Mediterranean tree frog has been recorded in 17 out of the 144 sampled points during 2016\u0026ndash;2019 (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e in Appendix). Size of choruses at these points is strikingly small, with four or less singing males at most points (Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e in Appendix). Nowadays the Mediterranean tree frog appears in the East and the West of Almer\u0026iacute;a province, but in none of sampled points at Murcia or Alicante provinces (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e in Appendix, Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). In the West of Almer\u0026iacute;a, most of records came from the Adra river basin (12 sampling points, Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e in Appendix), while in the East of Almer\u0026iacute;a they came from the Aguas and Almanzora rivers (4 sampling points, Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e in Appendix).\u0026nbsp;\u003c/p\u003e\u0026nbsp;\u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003e\u003cstrong\u003ein Appendix\u003c/strong\u003e. Paracuellos et al.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSampled point code\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eN\u0026ordm; of individuals\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAA02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAA03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAA05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAA06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAA07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAA08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAA09\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAA12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAG01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAG02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAG03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAS01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDT08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDT10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMZ01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRA02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRA06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eAverage\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e4.88\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eSE\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e1.96\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003c/p\u003e\n \u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e \u003cstrong\u003eabout here\u003c/strong\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003eTemporal changes in the Mediterranean tree frog occurrence\u003c/h2\u003e\n \u003cp\u003eGlobally, the species has become rare in the Iberian southeastern, going from being present in 43 (i.e. 79.63%) of the 54 sampled points 40 years ago to only 16 (i.e. 29.6%) (\u0026chi;\u003csup\u003e2\u003c/sup\u003e\u003csub\u003e1\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;27.23, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). Separate analyses for the three regions with historical data show: 1) that the species has disappeared from the three sampled points where it appeared in the Murcia high plateau (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA); that the species went from being present in 11 (\u003cem\u003ei.e.\u003c/em\u003e 84.6%) of 13 sampled points to only 4 (\u003cem\u003ei.e.\u003c/em\u003e 30.7%) in the East of Almer\u0026iacute;a (\u0026chi;\u003csup\u003e2\u003c/sup\u003e\u003csub\u003e1\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;5.67, \u003cem\u003eP\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.02) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eE), and from being present in 29 (\u003cem\u003ei.e.\u003c/em\u003e 76.3%) of 38 sampled points to only 12 (\u003cem\u003ei.e.\u003c/em\u003e 31.6%) in the West of Almer\u0026iacute;a (\u0026chi;\u003csup\u003e2\u003c/sup\u003e\u003csub\u003e1\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;13.56, \u003cem\u003eP\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eF).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n \u003ch2\u003eDrivers of Mediterranean tree frog decline in the Adra basin\u003c/h2\u003e\n \u003cp\u003eThe Adra river basin has undergone pronounced environmental changes during the last 40 years (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e in Appendix). Specifically, the distance to roads or paths from each sampling point has been reduced by 260 m (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e in Appendix, \u003cem\u003et\u003c/em\u003e\u003csub\u003e\u003cem\u003e36\u003c/em\u003e\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;2.63, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.012) and the suitable habitat for the Mediterranean tree frog has been reduced by more than 57% (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e in Appendix, \u003cem\u003et\u003c/em\u003e\u003csub\u003e\u003cem\u003e36\u003c/em\u003e\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;4.91, P\u0026thinsp;=\u0026thinsp;0.0001). This translates into an increase in the area of open-air crops of more than 29% (\u003cem\u003et\u003c/em\u003e\u003csub\u003e\u003cem\u003e36\u003c/em\u003e\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;4.37, P\u0026thinsp;=\u0026thinsp;0.0001) and greenhouse crops of almost 600% (\u003cem\u003et\u003c/em\u003e\u003csub\u003e\u003cem\u003e36\u003c/em\u003e\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;11.40, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), but not in significant changes in the area occupied by giant reed (\u003cem\u003et\u003c/em\u003e\u003csub\u003e\u003cem\u003e36\u003c/em\u003e\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;1.83, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.075).\u003c/p\u003e\n \u003cp\u003eNowadays, the sampled points where the frog is present cover three times more appropriate (\u003cem\u003et\u003c/em\u003e\u003csub\u003e\u003cem\u003e35\u003c/em\u003e\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;4.19, P\u0026thinsp;=\u0026thinsp;0.0001), and legally protected habitat (\u003cem\u003et\u003c/em\u003e\u003csub\u003e\u003cem\u003e35\u003c/em\u003e\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;3.77, P\u0026thinsp;=\u0026thinsp;0.0005) than those where the frog has not been found (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). However, there were no differences between sampled points with frogs and without frogs in the distance to roads or paths (\u003cem\u003et\u003c/em\u003e\u003csub\u003e\u003cem\u003e35\u003c/em\u003e\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.17, P\u0026thinsp;=\u0026thinsp;0.86), or area occupied by greenhouse crops (\u003cem\u003et\u003c/em\u003e\u003csub\u003e\u003cem\u003e35\u003c/em\u003e\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;1.28, P\u0026thinsp;=\u0026thinsp;0.28), open-air crops (\u003cem\u003et\u003c/em\u003e\u003csub\u003e\u003cem\u003e35\u003c/em\u003e\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.94, P\u0026thinsp;=\u0026thinsp;0.35), or giant reed (\u003cem\u003et\u003c/em\u003e\u003csub\u003e\u003cem\u003e35\u003c/em\u003e\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;1.02, P\u0026thinsp;=\u0026thinsp;0.31) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e \u003cstrong\u003eabout here\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eMoreover, the magnitude of environmental changes since 1980s-1990s differed between points where the frog has disappeared and where it persisted since the former have almost four times greater reduction in the suitable habitat for the frog (\u003cem\u003et\u003c/em\u003e\u003csub\u003e27\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;2.39, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.002), and, a nearly significant greater increase in the area of open-air crops (\u003cem\u003et\u003c/em\u003e\u003csub\u003e27\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;1.84, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.07) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). Points where the frog have disappeared and where it did not, however, showed similar shortening of the distance to roads or paths (\u003cem\u003et\u003c/em\u003e\u003csub\u003e27\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.63, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.53), increase in surface of greenhouse crops (\u003cem\u003et\u003c/em\u003e\u003csub\u003e27\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.32, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.74) and reduction in surface of giant reed (\u003cem\u003et\u003c/em\u003e\u003csub\u003e27\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.93, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.36) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). On the other hand, the points where the frog became extinct had a protected area around three times less than those where it persisted (\u003cem\u003et\u003c/em\u003e\u003csub\u003e27\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;3.80, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0007, Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eFigures 3 and about here\u003c/h3\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe extensive multi-replicated sampling at the regional level and the disappearance of the Mediterranean tree frog from 63% of sampled points with presence 40 years ago confirms that the anuran is at risk of extinction in the Iberian southeastern, such as already suggested by several authors (Tejedo and Reques \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Torralva et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Gonz\u0026aacute;lez-Miras and Nevado \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Alaminos \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Paracuellos et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The species has become rare in the Adra, Aguas and Almanzora rivers and has disappeared in all other sampled points. The decline scenario of the frog is exacerbated by the reduced number of individuals detected at each sampled point and a high population fragmentation, as points with frog presence were isolated from each other (minimum distance of more than 7 km) and from the nearest populations in Granada or Albacete (minimum distance of more than 52 km; Tejedo and Reques \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e in Appendix). The general decline in the Iberian southeastern could explain that the Mediterranean tree frog has not colonised apparent suitable localities that we have now sampled but were not in 1980s-1990s. The only exception to this pattern is a garden where the species was recently detected (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e in Appendix), most likely due to an unintentional introduction associated with the transport of ornamental plants (see nearby case in Paracuellos et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe detailed analysis in the Adra river basin suggests that the key factor in the decline of the Mediterranean tree frog would be the loss of suitable habitats for the frog. The loss of ponds and sheets of water surrounded by marsh vegetation has been accentuated in the Adra basin, while intensive open-air and greenhouse crops have proliferated, and more roads or paths have been built over the last 40 years (see Results). Considering high sensitivity of hylids to changes in their environment (Semlitsch et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; AbuBakr and Crupper \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Zazeri et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Gon\u0026ccedil;alves et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Sillero \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Santos et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Valdespino et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), our results suggest, in agreement with Paracuellos et al. (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), that the natural habitats where Mediterranean tree frog inhabits in the Iberian southeastern are being strongly degraded since the 1980s due to an intensification of agriculture practices that affects the viability of local populations.\u003c/p\u003e \u003cp\u003eAlthough the intensification of agriculture in the Iberian southeastern was not measured in this work, it is associated with a massive extraction and pollution of waterbodies (streams and aquifers) by biocides, fertilisers and other agricultural waste (Villegas and S\u0026aacute;nchez \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; Nevado and Paracuellos \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Casas et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Paracuellos \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Robledano et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Torralva et al. 2006). Moreover, the ever-increasing colonisation and proximity of greenhouse crops, buildings and roads or paths could increase the risk of microplastic pollution. These factors may impair habitat quality by their cascading adverse effects on frogs and tadpoles, including impaired growth and body condition, altered pigmentation, histopathological, behavioural and morphological changes, weakened immune responses, toxicity and, therefore, loss of survival and breeding success (Szkudlarek et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBeyond the changes derived from agricultural intensification, suitable habitat loss may be also amplified by increasing air temperature and altered precipitation in the region due to ongoing climate change (MedECC \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), which further limits water availability and quality in the last refuges for the species. It is also worth mentioning that remaining frog populations in the Iberian southeastern are at the geographical limits of the species' distribution, which may aggravate the environmental and genetic stresses, making them more susceptible and less able to respond to diseases, toxins and major habitat fluctuations (Shafer \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e1990\u003c/span\u003e; Duellman \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Dufresnes et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eConservation implications\u003c/p\u003e \u003cp\u003eOur results conclusively show the importance of maintaining protected areas in the Iberian southeastern to preserve populations of the Mediterranean tree frog and to avoid its extinction. Within the Adra river basin, sampled points with more protected area around are more likely to have the frog. Protected areas include more surface area of ​​suitable habitats for the frog, which are areas of the Adra river and lagoons (Regional Government of Andalusia \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e1989\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) where other aquatic species considered as globally \u0026laquo;endangered\u0026raquo; (IUCN \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), such as the plant \u003cem\u003eZannichellia contorta\u003c/em\u003e (Ortega et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2004\u003c/span\u003e), the endemic Spanish killifish (\u003cem\u003eApricaphanius iberus\u003c/em\u003e; Paracuellos and Nevado \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e1994\u003c/span\u003e), or the white headed duck (\u003cem\u003eOxyura leucocephala\u003c/em\u003e; Paracuellos et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2007\u003c/span\u003e), coexist. However, current protected areas are very isolated and represent far oasis in West of Almer\u0026iacute;a, where environmental and socio-economic conditions have favoured the proliferation of intensive greenhouse crops all over (Caparr\u0026oacute;s-Mart\u0026iacute;nez et al, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), with associated negative environmental impacts (L\u0026oacute;pez-G\u0026aacute;lvez and Salinas \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Palomar Oviedo 1999; Mart\u0026iacute;nez-Valderrama et al. 2024). Therefore, we urge to intensify conservation actions in the protected areas and surroundings where Mediterranean tree frog still survives, improving conditions for it and the rest of the associated biodiversity. Also, it is urgent to declare new protected areas that include masses of fresh or brackish water with shallow shores covered by thick marsh vegetation to increase connectivity and prevent the probable extinction of the Mediterranean tree frog in the Iberian southeastern. Moreover, given the small size of current populations, we advocate translocations of frog specimens from the same genetic population still existing in the Iberian southeastern, according to conventional procedures that minimise risks of biosafety and the spread of diseases (Mart\u0026iacute;nez-Silvestre et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Finally, future monitoring of the species in the area should continue to obtain information of the status of its populations and distribution and the environmental quality in real time.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e This study was carried out with the help of Jos\u0026eacute; A. Alguazas, Abel Amat, Francisco M. Benavides, Juan J. Benavides, Nerea Benavides, Jes\u0026uacute;s Cabrera, Alicia Caler, Soledad Callej\u0026oacute;n, C\u0026eacute;sar Caparr\u0026oacute;s, Juan A. Caparr\u0026oacute;s, Alfonso Carrasco, Hugo Cayuela, Esther Cerezo, Jorge Chaves, Carlos Collado, Antonio Cruz, Mercedes Cuenca, Pilar D\u0026iacute;az, M. Dolores Escobar, Daniel Esp\u0026iacute;n, Enrique Fern\u0026aacute;ndez, Trino Ferr\u0026aacute;ndez, Januario Formoso, Modesta Galdeano, F. Javier Garc\u0026iacute;a, Agust\u0026iacute;n G\u0026oacute;mez, C\u0026eacute;sar G\u0026oacute;mez, Lidia G\u0026oacute;mez, Jos\u0026eacute; M. G\u0026oacute;mez, Pedro Guerrero, Leonardo Guti\u0026eacute;rrez, Jorge Heredia, F\u0026aacute;tima El Jabari, Piotr Jankowski, Florencia Jaurena, Philip Kramer, Antonia L\u0026oacute;pez, M. Dolores L\u0026oacute;pez, Pablo L\u0026oacute;pez, Pedro Lozano, Nadia Manroua, Tob\u0026iacute;as Mart\u0026iacute;n, Ana B. Mart\u0026iacute;nez, Elena Mart\u0026iacute;nez, Pablo Mart\u0026iacute;nez, Yolanda Mart\u0026iacute;nez, Cesaltina Mendes, Daniel Moreno, Daniel Motos, Alberto Navarro, Faminio Nente, Andr\u0026eacute;s F. Ochoa, Lorena Olmo, Juan F. Parrilla, M. Dolores Peramo, Yolanda P\u0026eacute;rez, Swann Perrachon, Yolanda P\u0026eacute;rez, Alejandro Ram\u0026iacute;rez, \u0026Aacute;ngeles Ram\u0026iacute;rez, Mar\u0026iacute;a Ramos, Pedro Rasc\u0026oacute;n, Jos\u0026eacute; Rodr\u0026iacute;guez, M. Paz Romero, Lidia del Rosal, Francisco Rubio, Rub\u0026eacute;n Salvador, Encarnaci\u0026oacute;n Sambl\u0026aacute;s, Jos\u0026eacute; G. S\u0026aacute;nchez, M. Carmen Sanz, Mariana Silva, Antonio Tapia, Erik Torralvo, M. Trinidad Torralvo, Alfredo Valdivia, F. Javier Valenzuela, Jos\u0026eacute; A. Villegas and Rub\u0026eacute;n Vives who volunteered for the citizen science project Hyla del Sureste (https://proyecto-hyla-del-sureste.webnode.es/). We are grateful to Rafael M\u0026aacute;rquez for his advice and review. We thanks to Institute of Almerian Studies and Council of Almer\u0026iacute;a for grants awarded to Enrique Villanueva. RCE was supported by the CRUST R-Forze (PID2021-127631NA-I00) project funded by FEDER / Ministerio de Ciencia e Inovacion-Agencia Estatal de Investigaci\u0026oacute;n; the project TED2021-132332B-C21 funded by MCIN/AEI/10.13039/501100011033 and European Uni\u0026oacute;n \u0026ldquo;Next Generation EU\u0026rdquo;/PRTR and the Ramon y Cajal fellowship (RYC2020-030762-I). The Government of Andaluc\u0026iacute;a provided authorisations to sample frog presence.\u003c/p\u003e\n"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAbuBakr S, Crupper SS (2010) Prevalence of cadmium resistance in \u003cem\u003eStaphylococcus sciuri\u003c/em\u003e isolated from the Gray Treefrog, \u003cem\u003eHyla chrysoscelis\u003c/em\u003e (Anura: Hylidae). Phyllomedusa 9:141-146. http://doi.org/10.11606/issn.2316-9079.v9i2p141-146\u003c/li\u003e\n\u003cli\u003eAguiar FCF, Ferreira MT (2013) Plant invasions in the rivers of the Iberian Peninsula, South-Western Europe\u0026ndash;a review. 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Springer-Verlag, New York. http:// doi.org/10.18637/jss.v032.b01\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Agricultural impact, conservation, amphibian species, distribution, greenhouses, temporal evolution","lastPublishedDoi":"10.21203/rs.3.rs-4700075/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4700075/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eWater-dependent amphibians often present marginal populations in arid and semi-arid regions of the world. This is the case of the Mediterranean tree frog (\u003cem\u003eHyla meridionalis\u003c/em\u003e) in the southeast of Iberia, where the species is currently threatened. Here we provide an update of the distribution of Mediterranean tree frog in the region by surveying in localities with appropriate characteristics for the species. In part of these localities, there is historical (1980s-1990s) occurrence data, which allows studying temporal changes in the presence of the species. Furthermore, for the Adra river basin, where human-induced environmental changes and frog persistence over the last 40 years are known, we study how habitat changes and level of protection relate to local frog extinction. The Mediterranean tree frog currently appears in low numbers in 17 sampled points, and has disappeared in 27 of the 43 sampled points where it was present 40 years ago. Sampled points with nowadays frog presence have almost three times surface of suitable and legally protected habitats than those without frog. In addition, sampled points where the frog have disappeared experienced a more pronounced reduction in suitable habitat, and have three times less protected area than those where the frog remained. In the current global change scenario, it is urgent to restore and protect areas that include masses of fresh or brackish water with shallow shores covered by thick marsh vegetation and to reinforce populations to prevent the probable extinction of the Mediterranean tree frog in the southeast of Iberia.\u003c/p\u003e","manuscriptTitle":"Sharp decline of the Mediterranean tree frog (Hyla meridionalis) over 40 years in the southeast of Iberia: causes, and possible solutions to an extinction","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-09 16:25:10","doi":"10.21203/rs.3.rs-4700075/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"67db26fa-ca35-44a9-b140-378ac1b5d934","owner":[],"postedDate":"August 9th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-09-09T12:29:29+00:00","versionOfRecord":[],"versionCreatedAt":"2024-08-09 16:25:10","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4700075","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4700075","identity":"rs-4700075","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}