From Least Concerned to Endangered? An integrated approach to determine the distribution, suitable habitat and future of Dryophytes immaculatus

From Least Concerned to Endangered? 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An integrated approach to determine the distribution, suitable habitat and future of Dryophytes immaculatus Amaël Borzée, Xiaoli Zhang, Vishal Kumar Prasad, Ruiyang Wang, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6711786/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 08 Jan, 2026 Read the published version in BMC Zoology → Version 1 posted 13 You are reading this latest preprint version Abstract Background The immaculate treefrogs, Dryophytes immaculatus , were reported to be abundant in the plains of southern Jiangsu, China, until the 1980s. However, the species has only scarcely been seen since the beginning of the century. Results First, we conducted surveys between 2017 and 2024 at more than 6000 independent sites to determine the occurrence of the species, conducted citizen science surveys resulting in more than 1300 entries to determine the occurrence of the species, and questioned farmers, when possible, about the presence of the species. We detected Dryophytes immaculatus at 207 independent sites, managed to confirm the presence of the species at an additional 11 independent sites based on citizen science data and confirmed its local extinction at eight additional sites. Next, based on the accumulated data, we developed a suite of ecological models, including some with putative absence, to determine the current suitable habitat for D. immaculatus. Finally, we also built ecological models based on climate change scenarios. The ecological models confirmed the habitat to be suitable in an area marginally broader than the one where the species was found, and the climate change scenarios highlighted a shift in the location of the suitable habitat for all scenarios and time periods tested, with a weak overlap with the current distribution of the species. Based on the data accumulated, we could also follow the categories and criteria of the IUCN Red List of Threatened Species and we suggested for the species to be listed as Endangered under both criteria B2ab(i,ii,iii,v) based on geographic range, and C2a(i) based on the small and declining population size. Conclusion The range of D. immaculatus has contracted over the last decades, at a speed high enough for people sharing their land with the species to remember them, and the habitat suitable for the species is further declining, and predicted to entirely collapse in all future climatic scenarios. While not Critically Endangered yet, D. immaculatus is in need of conservation actions, especially to prevent future decline in habitat quality. distribution surveys citizen science local extinction suitable habitat ecological niche modelling climate change scenarios IUCN Red List Figures Figure 1 Figure 2 Figure 3 Introduction “The one who knows the calls of the frog can do in one night the work of many years” [ 1 ]. While the decline and extinction of some species are documented and accounted for [ 2 – 4 ], other species that were once abundant become suddenly extirpated [ 5 , 6 ]. These rapid extinctions are not necessarily documented, and it is not always possible to retroactively understand the drivers of extinction [ 7 – 9 ]. One such large-scale extinction was that of amphibians in the late 1900s, later attributed to batrachochytrids, although for long ignored as it was expected to be a small and isolated event [ 10 , 11 ]. These silent extinctions are at the core of the biodiversity crisis [ 3 ], illustrating the loss of species and ecosystem functions before their documentation [ 12 – 16 ]. As the decline of abundant species is not considered a priority, resources to document these declines are not readily available, and the decline of abundant species is widespread and under-documented [ 17 – 19 ]. For instance, one of the most widespread amphibian species in northeast Asia, Dryophytes japonicus , is generally declining, but the decline is not perceived because of some very abundant subpopulations [ 20 ]. This pattern of silent declines is observed in most vertebrates, especially if they are not charismatic, which includes most amphibians in the eyes of a large part of the public [ 21 ]. It is therefore paramount to document the decline of species for their protection [ 22 ], and especially amphibians, as it is the most threatened group of vertebrates [ 23 , 24 ], but also the group with the lowest financial support for its conservation [ 21 ]. Among amphibians, a few dozen species are already officially extinct [ 25 ], although about a hundred more are already most likely extinct [ 26 ]. One of the baselines for the conservation of a species is to first understand its presence in terms of distribution and abundance [ 27 ], and therefore to conduct field surveys to document its presence. Hylids globally have been comparatively well studied, partially due to their charismatic nature, but also because of their general occurrence within countries with comparatively more abundant resources [ 28 ]. In comparison to the global threat to amphibians (41%; [ 23 ]), not many Hylid species are threatened, although this ratio is strongly biased for Northeast Asia, where three out of six species are threatened [ 28 ]. As only a few species in the region are threatened despite the general decline in habitat quality, and because most anurans are comparatively well understood [ 29 – 31 ], the Immaculate Treefrog ( Dryophytes immaculatus ) should be well-documented and non-threatened. However, this is not the case, despite the earlier documentation of large populations by older and knowledgeable herpetologists in the region [ 30 , 32 ]. Dryophytes immaculatus has been occurring over a relatively consistent area over the last 130,000 years, with a thermal refugium matching the current distribution of the species [ 33 ]. The species has adapted to changes in the landscape, and the transformation of natural wetlands into rice paddies. However, it has declined over the last decades because of habitat loss and agricultural practices [ 31 , 34 ]. While some of the background in the population dynamics of D. immaculatus is now documented, we highlight a surprising lack of knowledge on the distribution and ecology of the species, and a steeper decline over a broader area than expected, despite some populations at unexpected sites. Materials and methods Dryophytes immaculatus is not a “lost species”, and data are available in the literature, providing a wealth of information, despite the current questions regarding the species. We started by collecting data to document the past distribution of the species. Older references predicted the species to be present from Hunan [ 35 ] and Jiangxi Provinces [ 36 ] in the south, to northern Hebei in the north [ 37 ]. However, this represents an extent of 1500 km, and oddly, the species had not been consistently reported across most of this range. Therefore, we decided to also propagate online questionnaires to ask the broad public about the presence of the species and support and simplify the task of conducting field surveys. Citizen science inquiries To help guide the massive survey effort over more than 1500 km by 700 km, we also relied on the power of citizen science. We published online articles on social media calling for sightings of the species. On purpose, we did not provide specific guidelines about species identifications, focal regions, or specific reporting guidelines, to ensure we received as much information as possible. The reports could be anonymous, in Mandarin or in English, to any of the co-authors involved in the online campaign. The surveys were not structured and only consisted of a request for reports of sightings, supplemented by pictures and information about the area (GPS coordinates or address). The calls for data were published across WeChat, Red Note, Weibo, and other platforms where reposting was possible (e.g., https://weibo.com/2627373652/M3IjpCdhw ). Geographic extent of the surveys We complemented the online surveys with field surveys, first based on the most suitable landscapes based on habitat suitability models [ 33 ]. Within the area highlighted by the original models, we conducted aural surveys (details below) every 5 km within continuous and suitable landscapes, or at the closest patch of suitable landscape in all directions. However, the models of reference were only partially adequate as the area north of the Hui River was mostly planted with corn, and therefore, unable to sustain populations of D. immaculatus . Once reaching the generally longitudinal belt of corn plantations across Anhui and Henan, and to determine the northernmost populations of the species, we surveyed the most suitable habitat patch north of the northernmost known site for the species, within a 30 x 30 km cell, until reaching north of the “corn belt”. As one of the sister species manages to maintain populations along streams in generally unsuitable landscapes [ 38 ], we also conducted a transect with survey sites in every suitable habitat, with a maximum of 5 km between each site, going through the low-elevation plains north of the Huai River in Jiangsu, Anhui and Henan, west of the Shandong Peninsula and into Henan, and north of the Shandong Peninsula, in Hebei and reaching into Tianjin and Beijing, to ensure that we would not miss any population. We also conducted surveys around the site where a population was reported north of Beijing, in northern Hebei [ 37 ], but once the population was attributed to D. japonicus [ 31 ], we did not conduct additional surveys north of Beijing. The landscape above 300 m on the Shandong Peninsula was excluded from the surveys based on modelling results [ 33 ]. We also conducted surveys at all the sites where the species was reported through the citizen science surveys. These additional surveys were conducted independently of the geographic scheme described above, repeating some surveys very closely to the ones already conducted. These surveys were conducted at the focal sites, along with at least two sites 5 km apart in all four general cardinal directions, restricted to suitable habitats. As a result of the multi-sampling schemes, the sampling density was not consistent, and areas where the species was found to be present were more intensely surveyed (Fig. 1 ). This bias needs to be acknowledged, but does not impact the results as it provided datapoints at a better resolution when conducting downstream ecological models based on both presence and absence points. Field surveys protocol We conducted the field surveys starting from the breeding season of 2017, until the breeding season of 2024, targeting different geographic areas over the years due to the sheer surface of land to be investigated. Surveys were interrupted in 2020 because of the pandemic. The landscapes targeted for the surveys were alluvial wetlands, in their very large majority represented by rice paddies, as natural wetlands able to host the species are now rare in the area [ 39 ]. Days with very heavy rain were avoided, as well as days of new moon and full moon due to their depressing impact on the calling activity of the sister species with a similar ecology, D. suweonensis [ 40 ]. Surveys were conducted from mid-April, matching with the emergence of the species in Hefei and Jiangsu [ 31 ], and then expanded more broadly after confirmation of the beginning of the breeding season, to ensure the absence of false negatives. The latest surveys were conducted in July, as droughts delayed the calling activity of the species in 2023 [ 34 ]. The protocols for the field surveys strongly relied on the protocol developed and improved for D. suweonensis and D. flaviventris [ 38 , 41 , 42 ]. Broadly, surveys started from 10 minutes after sunset, although some individuals start calling about an hour before sunset, and finished before 3 am, as starting surveys too early or finishing too late might result in false negatives, and the investigators also need to sleep. Each year, we conducted additional ad-hoc partial ecological models based on all confirmed presence sites, supplementing the habitat suitability models of reference [ 33 ], to improve the quality of the surveys. At the beginning of the season, we started surveys from a known presence sites to ensure that the species had started breeding, and the species had to be found at a minimum of three sites where the species was present the year prior for the surveys to start for the year, and thus avoid false negatives. The sites selected for the year were not surveyed in any specific or geographic order to randomise the surveys, but most sites surveyed over the course of an evening were contiguous to ensure feasibility. The survey sites were most generally between 500 and 2000 m away from each other, including when located within the 5 and 10 km grid cells mentioned above, based on habitat suitability. However, when the species was detected, out of excitement and scientific curiosity, intermediary sites could be added every 100 m, until the species was not detected for 2000 m. The surveys were conducted by car. The surveyor(s) drove to the selected site, parked with the engine off, and waited for a minimum of 5 min silently, or until all expected non-focal species had restarted chorusing at the site ( Pelophylax nigromaculatus , Pelophylax plancyi , Microhyla fissipes and Fejervarya cf. kawamurai ). In case Hoplobatrachus chinensis was heard calling at the site, an additional minute was spent listening as the species was detected at every site where D. immaculatus was found, but not vice-versa. The 5 minutes spent waiting are expected to reach a probability of detection of 95% for D. immaculatus , based on accumulation curves plotted for D. suweonensis [ 43 ]. In case the species was detected, a short, low-quality recording of the calls (min 10 sec) was made in case of the need for data inspection, and uploaded to iNaturalist.com, and the surveys were continued to the next survey site. Visual confirmation was not done, and is unnecessary: “the one who knows the calls of the frog can do in one night the work of many years” [ 1 ]. The surveys were normalised in all aspects so that the results collected over the years could be integrated for analyses. To save time, and not take more than seven years, no covariates were collected. Field surveys conducted as an expansion of this project are recommended to follow the same protocol to ensure continuity in data collection. Modelling Next, we used ecological models to understand the distribution of the D. immaculatus through its suitable habitat, based on the different types of occurrence data we have collected, and to predict its distribution under several scenarios linked to climate change. To do so, we explored four primary categories of environmental variables, encompassing key factors potentially influencing species distribution: land use/land-cover (LUCC), topography (including elevation, slope, and aspect), human footprints, and bioclimatic. As climatic predictors, we specifically selected isothermality (bio3), temperature seasonality (bio4), max temperature of warmest month (bio5), mean temperature of wettest quarter (bio8), and precipitation of driest month (bio14), because of their demonstrated strong correlation with the distribution of the species [ 33 ]. Climatic variables and elevation were sourced from WorldClim v. 2.1 ( https://www.worldclim.org ). The digital elevation model (DEM) was downloaded from the Geospatial Data Cloud ( https://www.gscloud.cn/ ). Land use/land-cover data were obtained from Casearth ( www.data.casearth.cn ). The human footprint was acquired from the dataset available online [ 44 ]. Slope and aspect were derived from the DEM layer using the spatial analytical tools in ArcGIS Pro (v.3.0.2). Subsequently, all environmental layers were projected onto the WGS84 coordinate system using a 30 arc sec (circa 1 km 2 ) spatial resolution raster grid. None of the variables exhibited significant correlations with each other (|r| < 0.7; [ 45 ]). To project future data (2021–2100), we used four climate scenarios (SSP126, SSP245, SSP370, and SSP585), representing different socioeconomic development trajectories and greenhouse gas emission pathways [ 46 , 47 ]. This choice aimed to assess the most significant potential shifts in suitable habitat. We used the Maxent model to construct suitable distribution areas for D. immaculatus . As the prediction accuracy of MaxEnt models can be influenced by parameters such as feature class and beta-multiplier, we used the ENMeval package to determine the optimal combination of feature classes and regularisation multipliers [ 48 ]. The selection of the best feature class and regularisation multipliers was based on the lowest Akaike Information Criterion, corrected for small sample values (AICc; [ 49 ]). We ran different models based on the eight datasets of occurrence we collected to analyse the suitable habitat for the species under the future scenarios (Table 1 ). We verified the results using the area under the curve (AUC) values for the receiver operating characteristic curve and the true skill statistic (TSS) to evaluate the prediction accuracy of MaxEnt [ 50 – 52 ]. The AUC (0–1) quantifies the reliability of the predictions across four tiers: limited (0.6–0.7), moderate (0.7–0.8), good (0.8–0.9), and excellent (0.9-1.0; [ 53 ]). All models were run in R 4.3.3 with the “dismo” [ 54 ] and “terra” [ 55 ] packages. Table 1 Details of the datasets used to build the ecological niche models for Dryophytes immaculatus . Here, survey sites represent the sites where the species was found to be present during surveys. The credible citizen science sites were the sites we estimated to be correctly reporting the presence of the species. The extinct sites were the sites where the species was reported to be present in the past, but our surveys did not manage to detect the species. Putative absence sites were defined as the survey sites where the species was not detected, and background points were the 10,000 points randomly dropped by the software over the landscape as a proxy for absence. Dataset Occurrence data Variables Period A Survey sites LUCC, topography, human footprints, bioclimate Current B Survey sites topography, bioclimate Current C Survey sites + credible citizen science topography, bioclimate Current D Survey sites + credible citizen science + extinct topography, bioclimate Current E Survey sites + credible citizen science with putative absence points topography, bioclimate Current F Survey sites + credible citizen science + extinct with putative absence points topography, bioclimate Current G Survey sites topography, bioclimate Future H Survey sites with putative absence sites and background points topography, bioclimate Future Threat assessment Dryophytes immaculatus is a poorly researched species, mistakenly currently listed as “Least Concern” by the Red List of the International Union for the Conservation of Nature, despite acknowledging the decline [ 56 ]. The assessment conducted here follows the IUCN Red List categories and criteria [ 57 ], a generally robust and consistent method to assess threats to species globally [ 58 ]. Assessments for the threatened categories are conducted against quantitative thresholds for five criteria that determine whether a species is at risk of extinction: A, population size reduction; B, geographic range size; C, small population size and decline; D, very small population and/or restricted distribution; and E, quantitative analysis of extinction risk. All threats, habitats, uses and trades are presented following the IUCN Red List criteria and categories [ 57 ]. Results Occurrence data In total, we conducted 6,191 targeted surveys for this project and found Dryophytes immaculatus to be present at 207 independent sites (Supplementary Information Table 1 ). These details do not take into account the survey sites where no amphibian species were detected, as the landscape was different from the satellite view because of rapid development, which accounted for about 270 sites. Most of the D. immaculatus populations found during the surveys in this study were located between the Yangtze and Huai Rivers, with a small and isolated population in rice fields west of the Dabie Mountains. Most populations were found in central Anhui, with a few isolated populations further north and south in Anhui and Jiangsu (Fig. 1.1). The call for data resulted in the submission of more than 1300 datapoints, with pictures, explanations and other unrelated information. Due to the purposeful absence of a clear protocol to make reporting easier and diverse, and the inability to wade through this flow of data, we did not collect datapoints that could not be successfully traced back to the species. A wide diversity of (generally) green amphibians was sent to us, with most pictures likely representative of Zhangixalus dennysi and Hyla chinensis , two species clearly morphologically different from D. immaculatus , even as juveniles. Results were received from spring 2022, with the latest report received in October 2024, and resulted in 11 independent sites that could be unequivocally identified as D. immaculatus (Supplementary Information Table 1 ). The credible sites resulting from the citizen science surveys were spread across Jiangsu and Anhui, increasing the range and density of the occurrence of the species, with a few populations north of the Huai River (Fig. 1 .3). In addition, some of the putatively extinct populations were located south of the Yangtze River, as far south as southern Anhui and southern Hubei (Fig. 1 .4). All confirmed populations were found in low-elevation wetlands, such as alluvial floodplains, and even when present in mountainous areas in the Dabie Mountains, they were within the agricultural wetlands in the lowest valleys (Fig. 1 ). Finally, we collected eight independent sites for occurrence in the past, here defined as prior to 1980, but where the species could not be found again, and therefore were considered as putatively extinct (Supplementary Information Table 1 ). Non-confirmed reports that could not be categorically accepted or rejected due to the quality of the pictures and the absence of clear locality included areas north of Poyang Lake, Loudi in Hunan, and Nanchang in Jiangxi. Populations that need additional surveys to determine whether past reports were correct and they are now extinct, or the species was misidentified in the past, or the locality not recorded properly are the ones at the feet of Tianmu Mountain, for which museum vouchers morphologically match some of the characters present in D. immaculatus , individuals reported from Yangjifeng, Jiangxi Province [ 36 ] and individuals reported from Hunan Province [ 35 ]. Ecological niche modelling We ran the models using each of the eight occurrence datasets. The AUC values for all models were greater than 0.9, indicating an excellent overall prediction ability. The modelling results employing automatically generated background points revealed that high-suitability areas were predominantly distributed in low-lying regions, exhibiting significant spatial overlap with cultivated lands. These areas were exclusively located within Anhui and Jiangsu provinces, with notable concentrations in the cities of Chuzhou, Hefei, Lu’an, and Taizhou (Fig. 1 ). The land use/land-cover and anthropogenic activities had a negative effect on the suitable distribution areas of D. immaculatus (Fig. 1 .a-b). The inclusion of credible science citizen data and the putative extinction data in the model resulted in a significant expansion of the suitable distribution area (Fig. 1 .c-d). When we replaced the random background points with putative absence points, the highly suitable distribution area expanded (Fig. 2 ). Under these variables, the modelling results incorporating survey data and credible citizen science observations demonstrated that high-suitability areas were predominantly located in central and southwestern Anhui, as well as in proximity to the Yangtze River within southern Jiangsu, but also south of the Yangtze River where the species was not found during the surveys (Fig. 2 .a). Building upon these findings, the modelling approach incorporating extinct occurrence records revealed an expansion of high-suitability habitats into southeastern Hubei and southern Anhui (Fig. 2 .b). Climate change scenarios Projection of the current model onto future climate scenarios indicated significant changes in habitat suitability for D. immaculatus under different climate scenarios. The species was projected to lose a substantial portion of its high-suitability habitats, particularly those overlapping with cultivated lands, as temperatures rise (Fig. 3 ). Under the low-emission scenario (SSP126), high-suitability areas were primarily distributed in eastern Jiangsu Province, central Anhui Province, and the Dabie Mountain region during 2021–2040; however, these habitats are predicted to contract over time. In contrast, intermediate (SSP245) and high-emission scenarios (SSP370/585) predicted severe habitat loss for all future timelines, particularly in regions overlapping with croplands. By 2081–2100, under SSP585, high-suitability areas will persist only in isolated mountainous refugia within Anhui, while lowland agricultural areas will experience near-complete loss of suitable habitat. The Dabie Mountain region is projected to function as the only climatic refuge for D. immaculatus in response to future climate change. Threats and conservation Dryophytes immaculatus is impacted by habitat loss and degradation throughout its range and is not known to rely on natural habitat anywhere within its range to complete its life cycle. Instead, the species relies on artificial aquatic habitats, such as agricultural wetlands, for breeding. In addition, the species is impacted by global warming, as modelled above, and it has become locally extirpated at numerous sites, including its type locality. Within artificial wetlands, the species is predated by both native Pelophylax nigromaculatus (personal observation) and invasive American bullfrog ( Aquarana catesbeiana ; [ 59 ]). The species' generation time is 3 to 4 years ( sensu IUCN), and the life span in the wild is estimated from 5 to 6 years based on closely related species [ 39 ]. The species is not currently known to occur within any protected area. The result of the analyses following the guidelines of the IUCN Red List of Species [ 57 ] is presented below. In terms of population size, no data is available for the number of individuals despite the putative extirpations at eight independent sites (one location sensu IUCN due to the common threat of habitat conversion for rice agriculture). However, a decrease in the area of agricultural wetlands available will directly result in a decrease in population size. As of 2016, the area used for rice agriculture had decreased by 11% since 1980 [ 60 ], and has continued to decline since then, with a predicted continuing trend [ 61 ] due to the decrease in water availability [ 62 , 63 ], and the shift in diets and agricultural policies regulations [ 64 , 65 ]. The 11% decline in population size, even predicted, does not meet the threshold for a species to be listed as threatened under criteria A of the IUCN Red List of Species [ 57 ]. However, non-quantitative information resulting from the conversation with farmers would point to a 90% decline in population size at all the sites where the species used to be present before the 1980s. Regarding the geographic range, all individuals are included in one location as they are all under the single threat of agriculture. Based on the IUCN tool GEOCAT (geocat.iucnredlist.org) and the data resulting from the surveys, we determined that the EOO for the population surveyed (58,033 km 2 ) and determined through citizen science (24,714 km 2 ) resulted in a total of 104,501 km 2 , with a total of 193,550 km 2 when including populations putatively extinct since the 1980s. Similarly, for the AOO, the sites surveyed (296) and determined through citizen science (36) resulted in a total of 328 km 2 , with a total of 348 km 2 when including populations putatively extinct since the 1980s. Following this data, the species matches with an Endangered listing as the AOO is below the threshold for B2, and along with the information listed above, the species fulfils the criteria for B2ab(i,ii,iii,v). During the surveys, no specific data on population size was collected, but the number of calling individuals at most sites was well below 10. As a few sites in Anhui had larger populations, up to 30 individuals, we assume an average of 10 individuals per site and, therefore, a rough estimate of (207 + 11) x 10 = 2180 individuals. As the population size is observed to be declining and projected to keep on declining in the number of mature individuals in each subpopulation, the species reaches the threshold to be listed as Endangered under the criteria C2a(i). In terms of criteria D (very small or restricted population) and E (quantitative analysis), the number of mature individuals is estimated to be over the threshold, and there are no data available for a quantitative analysis. Discussion In this study, we showed the current range of Dryophytes immaculatus to be restricted between the Huai and Yangtze Rivers through field surveys that detected the presence of the species at 207 independent sites. In addition, these results were complemented with citizen science data, which reported the presence of the species at an additional 11 sites, generally extending the range of the species further east towards Shanghai urban area, and at one site south of the Yangtze River, towards the type locality of the species, where it is now locally extinct (Graphical Abstract). Local interviews also reported credible sightings of the species before the 1980s at an additional eight locations, all located further west and south. These extinctions show a contraction in the distribution of D. immaculatus , as the species was reported to be widespread between Nanjing and Shanghai a few decades ago. In addition, it is critical to note that these are the sites that could be tracked down, and most probably a small number compared to the real number of sites where the species when extinct. However, despite the likely presence of the species prior to the heavy development of the area, a critical perspective is needed due to the potential for errors in datasets originating from citizen science datasets [ 66 ]. For instance, sites outside the range of D. immaculatus have been reported, including some north to “Beijing area”, which are now attributable to “immaculate coloured” [ 31 ] D. japonicus individuals [ 37 ]. Other sites out of range have been reported (e.g., see AmphibiaChina.org), although the sites received from the citizen science surveys from these areas were all misidentifications, different enough to be confirmed through pictures (e.g., including Zhangixalus sp. and Odorrana sp.). Regarding the areas identified through citizen science, the risk of misidentification is comparatively low as there are no other Hylid species occurring in these areas, however noting the presence of Hyla dabieshanensis between the sites where D. immaculatus is recorded as locally extinct, although in a different habitat type [ 67 ]. The only potential for misidentification is the southernmost site in Zhejiang where D. immaculatus was recorded, as Hyla chinensis is also present in the area, despite clear morphological differences in adults [ 68 ]. The ecological models for habitat suitability (Fig. 1 ) all supported the same pattern, with high habitat suitability in the plains in central Anhui, with the area of suitable habitat expanding longitudinally when including the sites coming from citizen science data, and the sites where the species is now extinct. This variation in habitat suitability is likely resulting from the changes in the landscape because of human activities, while the ecological models taking into account the effects of human activities provided the lowest suitable area (Fig. 1 .a), clearly resulting from the widespread destruction of the habitat suitable for the species. These alterations are principally the decrease in area used for rice agriculture [ 60 , 61 ], but also the loss of habitat that can be used for overwintering in ditches, especially as a result of governmental policies for clean water, which indeed provide the results intended, but deprive the species from overwinter habitat. It is worth noting that, based on the ecological models, the habitat in the Dabie Mountains is not considered suitable, despite the presence of some individuals isolated in low-elevation agricultural wetlands. A new Hylid species was described in the area since the surveys, in early 2025, but clear morphological differences between D. immaculatus and H. dabieshanensis highlight the very improbable misidentification based on morphology [ 67 ]. However, call recordings of H. dabieshanensis are not available in the public domain, and misidentification during call surveys is a possibility, although unlikely due to the visual difference in the call spectrograms for the two species (comparison between [ 69 ] and [ 67 ]). If this were the case, and these sites would have to be removed from the models, then the suitable habitat would most likely contract as the remaining sites are found in a more homogeneous landscape. Another limitation of the models is that they include croplands as a variable, without differentiating between dry and flooded agriculture, despite the known impact on some species with higher dispersal abilities [ 70 ]. As the species cannot breed in corn and wheat agricultural areas, the differentiation might make a difference. However, dry crops are present in the north of Anhui and at the same latitude in Henan, where the habitat is not modelled as suitable, and therefore, we can expect the models to already correctly attribute suitable habitat in this regard. The two ecological models with the putative absence points were consistent with each other, with a larger suitable area for the model including the extinct populations. These results also agreed with the other models in terms of the general area of the suitable habitats (Fig. 2 ). The main difference was that when including the putative absence points, the whole range of the Dabie Mountain became suitable, despite field surveys having shown that the species was not present in most of the habitat. In addition, the lowland area south of Hubei (around Wuhan) was also considered to be suitable, and while the species was not found during the surveys, a site was provided by the citizen science data. The mountain range in southern Anhui was also considered suitable when including the putative absence points, here again, despite the absence of the species through field survey, but an extinct population with a preserved individual in a museum from Tianmu Mountain (collected in 1955 as Hyla arborea immaculata ). In terms of climate change, the four SSPs for the 2021–2040 time period showed a shift in suitable habitat both east and west of the current distribution of the species, with a weak overlap with the current populations (Fig. 3 ). The only increase in habitat suitability that matches the known D. immaculatus population was over the Dabie Mountains, which persisted for all four SSP for the 2041–2060 period, and for all time periods for the SSP 126 and 245. It is, however, important to note that this refugia against anthropogenically induced climatic variation does support the survival of the species as it is now, as only a very small proportion of the population would be maintained, and the species would likely become functionally extinct [ 71 ]. For D. immaculatus , all of the climatic scenarios resulted in a significant loss in suitable habitat, ranging from severe (SSP126 for 2021–2040) to an almost entire loss of overlap between presence and suitable habitat (SSP 245 and above for 2041–2060 and later). While these assessments are only models, they are in line with the global pattern for amphibians [ 72 ], and also for Dryophytes species in East Asia [ 73 , 74 ], highlighting the undeniable negative impact of anthropogenically induced climate change on the survival of the species. Based on the current extinction risk listed on the IUCN Red List of Threatened Species, D. immaculatus is listed as Least Concern [ 56 ]. However, when applying the Red List categories and criteria, D. immaculatus could be listed as Endangered due to both the geographic range in the form of the area of occupancy, matching with the criteria for B2ab(i,ii,iii,v), and the small and declining population size, under the criteria C2a(i). The suggestions reflect the situation on the ground as the species used to be common before the 1980s, based on the discussion with both elder farmers and researchers, but the species is currently difficult to find, and it has not been formally reported from any locality for about a decade prior to this study. This assessment might worsen once the impact of climate change can be quantified, due to its impact on both range and breeding phenology, and it is already known to have a negative impact [ 34 ]. Compared with other Hylids [ 28 ], D. immaculatus is comparatively more threatened due to its narrow range and low population size. Finally, likely due to the current increase in the presence of the species in the media (e.g., [ 75 ]), and a reintroduction project in Shanghai, the species is advertised on online pet shops, which might become a threat if the trade increases in volume. Compared with the two other species of the species complex, D. suweonensis and D. flaviventris , D. immaculatus is in a very similar conservation situation. The main threat to these three species is habitat loss, with the transformation of the natural wetlands where they used to thrive into agricultural wetlands, which have now lost in value and are being replaced by other crops or land use and are decreasing in area [ 65 ] and the area that is still planted being increasingly unhospitable to the species [ 76 ]. The variation in habitat available for the species is, however, not recent, having likely fluctuated with marine transgressions, and it is likely to have contracted over the last 21,000 years, following the increase in sea level and the flooding of the Yellow Sea [ 69 , 77 ]. The habitat available is also likely to have been impacted by the development of rice agriculture over the last 8000 years [ 78 – 81 ]. The species density is likely to have become more uniform in the landscape during the conversion of all habitats in the area into agricultural wetlands [ 82 ]. This change in habitat might have been positive for the species when wetlands increased in area and connectivity. However, this would need to be demonstrated through analyses based on population demographics, as done for other threatened species such as the condor [ 83 ]. More recently, changes in food preference in humans have also resulted in the decline in rice production, and the habitat available for D. immaculatus [ 64 , 84 ], as seen for the Korean sister species [ 65 ]. The population size of the species at all sites, except north of Hefei in Anhui, is now likely to be too low for the species to provide the ecosystem function it used to provide ([ 71 ] in relation to [ 85 ]). These functions likely included energy transfer from the aquatic to the terrestrial habitat during metamorphosis [ 85 ] and pest regulation as ambush predators in wetlands, including rice-eating pests [ 86 ]. The species is also likely to have stopped providing the ecosystem services that used to benefit humans, such as mosquito regulation as provided by amphibians in Central America [ 87 ], and it would be worth conducting surveys in areas where the species is still present in large numbers compared to areas where it is now known to be extinct to determine its impact on pests. Finally, additional surveys are needed to understand the exact distribution and population size of the species, and surveys in the Yancheng UNESCO-MAB Biosphere Reserve might result in the discovery of the species in an area that is still representative of the natural habitat of the species, as suggested by some of the models, and it would be the only area where the species is occurring in a protected area. Declarations Ethics approval and consent to participate This research was conducted under the Institutional Animal Care and Use Committee (IACUC) permit numbers 20-250-26, 20-240-13, 20-230-12 and 20-220-13 issued by Nanjing Forestry University. All humans interviewed agreed on sharing the information about the past presence of treefrogs on their land, and it did not include any information about any human being. We did not manage to ask the frogs for their consent. Consent for publication All authors have agreed to the publication of this manuscript. Availability of data and materials All the data used for this publication is available in the Supplementary Information. Competing interests None of the authors has competing interests to declare. Funding This project was funded by the Research Fund for International Scientists (RFIS) from the National Natural Science Foundation of China (NSFC; W2432021) and the Foreign Youth Talent Program of the Ministry of Science and Technology of the People’s Republic of China (QN2023014004L) awarded to AB. It was also funded by the Small Grants Program for New Records and Rediscoveries of Rare Species, initiated by Tencent Foundation and Shan Shui Conservation Center. Authors' contributions Conceptualisation: AB, YJ; Methodology: AB, ZZ, YJ; Formal analysis: AB, XZ, VKP, RW, SQ; Investigation: AB, XZ, VKP, RW, ZW, SQ, KRM, TG; Resources: AB, YJ, TG, JW; Writing - Original Draft: AB, VKP; Writing - Review & Editing: all authors. Acknowledgement The authors are grateful to Ningjing Wang for her help during the surveys, and for raising awareness about the threats to the species. 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The subset of maps at the bottom represents the suitable habitat based on (left to right), current with human impact; current with putative absence; climate change scenario for 2040 (SSP 126, i.e., best scenario) and climate change scenario for 2100 (SSP585, i.e., worse scenario). 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6711786","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":463143067,"identity":"60202730-d0cb-4e09-a5ed-8e954dfc2e9a","order_by":0,"name":"Amaël Borzée","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5ElEQVRIiWNgGAWjYLCChw1givEBkODhI0pLIkQLswFICxspWtgkwCQh1fzTDj98kLjDJl++vflY5dccOxk2BuaHj27g0SJxO83YIPFMmmVjz7G027LbkoEOYzM2zsFnze0EM4nEtsMGzBI5ZrcltzEDtfCwSePTIn87/fsPkBY2+fffiiW31RPWYnA7x4wBpIVHgoeN8eO2w4S1GN7OKZYA+sVAgifNWJpx23EeNmYCfpG7nb7xw8cdNgby7Ycffvy5rdqen7354WO83kcGzDxgkljlIMD4gxTVo2AUjIJRMGIAAC9ZRXrl++PAAAAAAElFTkSuQmCC","orcid":"","institution":"Nanjing Forestry University","correspondingAuthor":true,"prefix":"","firstName":"Amaël","middleName":"","lastName":"Borzée","suffix":""},{"id":463143070,"identity":"b0929957-1f58-4ba3-819f-f59587527266","order_by":1,"name":"Xiaoli Zhang","email":"","orcid":"","institution":"Nanjing Forestry University","correspondingAuthor":false,"prefix":"","firstName":"Xiaoli","middleName":"","lastName":"Zhang","suffix":""},{"id":463143071,"identity":"f882ae7a-7550-4b8d-9b14-74eabb184ec0","order_by":2,"name":"Vishal Kumar Prasad","email":"","orcid":"","institution":"Nanjing Forestry University","correspondingAuthor":false,"prefix":"","firstName":"Vishal","middleName":"Kumar","lastName":"Prasad","suffix":""},{"id":463143072,"identity":"ec5e45a1-af96-4053-81cb-60fb75ad0330","order_by":3,"name":"Ruiyang Wang","email":"","orcid":"","institution":"Jiangsu Province and Chinese Academy of Sciences, Nanjing Botanical Garden Mem. Sun Yat-Sen","correspondingAuthor":false,"prefix":"","firstName":"Ruiyang","middleName":"","lastName":"Wang","suffix":""},{"id":463143074,"identity":"686fc8ba-47ac-49e0-80f1-a5bac3e6bb78","order_by":4,"name":"Zhenqi Wang","email":"","orcid":"","institution":"The Co-Innovation Center for Sustainable Forestry in Southern China, Nanjing Forestry University","correspondingAuthor":false,"prefix":"","firstName":"Zhenqi","middleName":"","lastName":"Wang","suffix":""},{"id":463143076,"identity":"7ec23a7d-3b82-4bf1-9228-27d77090b998","order_by":5,"name":"Shujie Qin","email":"","orcid":"","institution":"Nanjing University","correspondingAuthor":false,"prefix":"","firstName":"Shujie","middleName":"","lastName":"Qin","suffix":""},{"id":463143077,"identity":"9b37c4ad-6317-456f-a49c-43ad633c8b36","order_by":6,"name":"Kevin R. Messenger","email":"","orcid":"","institution":"Nanjing Forestry University","correspondingAuthor":false,"prefix":"","firstName":"Kevin","middleName":"R.","lastName":"Messenger","suffix":""},{"id":463143079,"identity":"532343ac-81b3-4c8c-aebd-4b0cbd298938","order_by":7,"name":"Taoran Guo","email":"","orcid":"","institution":"Shanghai Tianyuan Ecological Technology Co","correspondingAuthor":false,"prefix":"","firstName":"Taoran","middleName":"","lastName":"Guo","suffix":""},{"id":463143081,"identity":"423e61a0-e2ab-4c0f-a0cc-ebf4ac76dcf8","order_by":8,"name":"Yikweon Jang","email":"","orcid":"","institution":"Ewha Womans University","correspondingAuthor":false,"prefix":"","firstName":"Yikweon","middleName":"","lastName":"Jang","suffix":""},{"id":463143083,"identity":"9e84b565-a6b6-42d6-904a-894d30cf744c","order_by":9,"name":"Jiechen Wang","email":"","orcid":"","institution":"Nanjing University","correspondingAuthor":false,"prefix":"","firstName":"Jiechen","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2025-05-21 02:38:08","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6711786/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6711786/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s40850-025-00248-w","type":"published","date":"2026-01-08T15:57:12+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":83661141,"identity":"cbf42d12-9c80-43b8-ba59-fff680fb98ca","added_by":"auto","created_at":"2025-05-30 10:05:30","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2330758,"visible":true,"origin":"","legend":"\u003cp\u003eSuitable habitat for \u003cem\u003eDryophytes immaculatus\u003c/em\u003eunder current climate scenarios. The details of the datasets are explained in Table 1. a. Results of the model run for dataset A (occurrences: survey; variables: land use/land-cover, topography, human footprints, bioclimate). b. Results of the model run for dataset B (occurrences: survey; variables: topography, bioclimate). c. Results of the model run for dataset C (occurrences: survey and credible; variables: topography, bioclimate). d. Results of the model run for dataset D (occurrences: survey, credible and extinct; variables: topography, bioclimate).\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-6711786/v1/01d38be6d56502fd7ca7d577.png"},{"id":83661144,"identity":"d925f29b-6f97-4281-ad62-52ab6a446f60","added_by":"auto","created_at":"2025-05-30 10:05:30","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3060862,"visible":true,"origin":"","legend":"\u003cp\u003eSuitable habitat for \u003cem\u003eDryophytes immaculatus\u003c/em\u003eunder current climate scenarios using putative absence points. The details of the datasets are explained on Table 1. a. Results of the model run for dataset E (occurrences: survey sites + credible citizen science with putative absence points; variables: topography, bioclimate). b. Results of the model run for dataset F (occurrences: survey sites + credible citizen science + extinct with putative absence points; variables: topography, bioclimate).\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-6711786/v1/2c48fa3ce384fdcd2df9c104.png"},{"id":83661146,"identity":"964e9762-1812-4f9f-9b11-baaa3185e363","added_by":"auto","created_at":"2025-05-30 10:05:30","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2305785,"visible":true,"origin":"","legend":"\u003cp\u003eEcological models for suitable habitat for \u003cem\u003eDryophytes immaculatus\u003c/em\u003e under climate change scenarios (SSP126, SSP245, SSP370, SSP585) for the periods 2021–2040, 2041–2060, 2061–2080 and 2081–2100.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-6711786/v1/9af36afb6951cbc58796897f.png"},{"id":100069212,"identity":"9765fdcc-2c32-404b-8c5d-8f9da12a8d5d","added_by":"auto","created_at":"2026-01-12 16:11:12","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":8075604,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6711786/v1/95473e8a-431e-4bec-8a8a-f7c6617cb313.pdf"},{"id":83661142,"identity":"e627e000-15d5-4a93-b9f4-0c172417025a","added_by":"auto","created_at":"2025-05-30 10:05:30","extension":"csv","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":15425,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable1occurence.csv","url":"https://assets-eu.researchsquare.com/files/rs-6711786/v1/59433a7d41914f5526db8ade.csv"},{"id":83661145,"identity":"1d8bd53e-7c9e-43b8-99bd-debe15d40029","added_by":"auto","created_at":"2025-05-30 10:05:30","extension":"jpeg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":968794,"visible":true,"origin":"","legend":"\u003cp\u003eGraphical abstract. Distribution and status of \u003cem\u003eDryophytes immaculatus\u003c/em\u003e populations in Jiangsu and Anhui, China. The subset of maps at the bottom represents the suitable habitat based on (left to right), current with human impact; current with putative absence; climate change scenario for 2040 (SSP 126, i.e., best scenario) and climate change scenario for 2100 (SSP585, i.e., worse scenario).\u003c/p\u003e","description":"","filename":"image1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6711786/v1/7875d269390bd2e3e8b77dfb.jpeg"}],"financialInterests":"No competing interests reported.","formattedTitle":"From Least Concerned to Endangered? An integrated approach to determine the distribution, suitable habitat and future of Dryophytes immaculatus","fulltext":[{"header":"Introduction","content":"\u003cp\u003e\u0026ldquo;The one who knows the calls of the frog can do in one night the work of many years\u0026rdquo; [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWhile the decline and extinction of some species are documented and accounted for [\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], other species that were once abundant become suddenly extirpated [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. These rapid extinctions are not necessarily documented, and it is not always possible to retroactively understand the drivers of extinction [\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. One such large-scale extinction was that of amphibians in the late 1900s, later attributed to batrachochytrids, although for long ignored as it was expected to be a small and isolated event [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. These silent extinctions are at the core of the biodiversity crisis [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], illustrating the loss of species and ecosystem functions before their documentation [\u003cspan additionalcitationids=\"CR13 CR14 CR15\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. As the decline of abundant species is not considered a priority, resources to document these declines are not readily available, and the decline of abundant species is widespread and under-documented [\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. For instance, one of the most widespread amphibian species in northeast Asia, \u003cem\u003eDryophytes japonicus\u003c/em\u003e, is generally declining, but the decline is not perceived because of some very abundant subpopulations [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThis pattern of silent declines is observed in most vertebrates, especially if they are not charismatic, which includes most amphibians in the eyes of a large part of the public [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. It is therefore paramount to document the decline of species for their protection [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], and especially amphibians, as it is the most threatened group of vertebrates [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], but also the group with the lowest financial support for its conservation [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Among amphibians, a few dozen species are already officially extinct [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], although about a hundred more are already most likely extinct [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. One of the baselines for the conservation of a species is to first understand its presence in terms of distribution and abundance [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], and therefore to conduct field surveys to document its presence.\u003c/p\u003e \u003cp\u003eHylids globally have been comparatively well studied, partially due to their charismatic nature, but also because of their general occurrence within countries with comparatively more abundant resources [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. In comparison to the global threat to amphibians (41%; [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]), not many Hylid species are threatened, although this ratio is strongly biased for Northeast Asia, where three out of six species are threatened [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. As only a few species in the region are threatened despite the general decline in habitat quality, and because most anurans are comparatively well understood [\u003cspan additionalcitationids=\"CR30\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], the Immaculate Treefrog (\u003cem\u003eDryophytes immaculatus\u003c/em\u003e) should be well-documented and non-threatened. However, this is not the case, despite the earlier documentation of large populations by older and knowledgeable herpetologists in the region [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cem\u003eDryophytes immaculatus\u003c/em\u003e has been occurring over a relatively consistent area over the last 130,000 years, with a thermal refugium matching the current distribution of the species [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. The species has adapted to changes in the landscape, and the transformation of natural wetlands into rice paddies. However, it has declined over the last decades because of habitat loss and agricultural practices [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. While some of the background in the population dynamics of \u003cem\u003eD. immaculatus\u003c/em\u003e is now documented, we highlight a surprising lack of knowledge on the distribution and ecology of the species, and a steeper decline over a broader area than expected, despite some populations at unexpected sites.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e \u003cem\u003eDryophytes immaculatus\u003c/em\u003e is not a \u0026ldquo;lost species\u0026rdquo;, and data are available in the literature, providing a wealth of information, despite the current questions regarding the species. We started by collecting data to document the past distribution of the species. Older references predicted the species to be present from Hunan [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e] and Jiangxi Provinces [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] in the south, to northern Hebei in the north [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. However, this represents an extent of 1500 km, and oddly, the species had not been consistently reported across most of this range. Therefore, we decided to also propagate online questionnaires to ask the broad public about the presence of the species and support and simplify the task of conducting field surveys.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCitizen science inquiries\u003c/h2\u003e \u003cp\u003eTo help guide the massive survey effort over more than 1500 km by 700 km, we also relied on the power of citizen science. We published online articles on social media calling for sightings of the species. On purpose, we did not provide specific guidelines about species identifications, focal regions, or specific reporting guidelines, to ensure we received as much information as possible. The reports could be anonymous, in Mandarin or in English, to any of the co-authors involved in the online campaign. The surveys were not structured and only consisted of a request for reports of sightings, supplemented by pictures and information about the area (GPS coordinates or address). The calls for data were published across WeChat, Red Note, Weibo, and other platforms where reposting was possible (e.g., \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://weibo.com/2627373652/M3IjpCdhw\u003c/span\u003e\u003cspan address=\"https://weibo.com/2627373652/M3IjpCdhw\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eGeographic extent of the surveys\u003c/h3\u003e\n\u003cp\u003eWe complemented the online surveys with field surveys, first based on the most suitable landscapes based on habitat suitability models [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Within the area highlighted by the original models, we conducted aural surveys (details below) every 5 km within continuous and suitable landscapes, or at the closest patch of suitable landscape in all directions.\u003c/p\u003e \u003cp\u003eHowever, the models of reference were only partially adequate as the area north of the Hui River was mostly planted with corn, and therefore, unable to sustain populations of \u003cem\u003eD. immaculatus\u003c/em\u003e. Once reaching the generally longitudinal belt of corn plantations across Anhui and Henan, and to determine the northernmost populations of the species, we surveyed the most suitable habitat patch north of the northernmost known site for the species, within a 30 x 30 km cell, until reaching north of the \u0026ldquo;corn belt\u0026rdquo;. As one of the sister species manages to maintain populations along streams in generally unsuitable landscapes [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e], we also conducted a transect with survey sites in every suitable habitat, with a maximum of 5 km between each site, going through the low-elevation plains north of the Huai River in Jiangsu, Anhui and Henan, west of the Shandong Peninsula and into Henan, and north of the Shandong Peninsula, in Hebei and reaching into Tianjin and Beijing, to ensure that we would not miss any population. We also conducted surveys around the site where a population was reported north of Beijing, in northern Hebei [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], but once the population was attributed to \u003cem\u003eD. japonicus\u003c/em\u003e [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], we did not conduct additional surveys north of Beijing. The landscape above 300 m on the Shandong Peninsula was excluded from the surveys based on modelling results [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWe also conducted surveys at all the sites where the species was reported through the citizen science surveys. These additional surveys were conducted independently of the geographic scheme described above, repeating some surveys very closely to the ones already conducted. These surveys were conducted at the focal sites, along with at least two sites 5 km apart in all four general cardinal directions, restricted to suitable habitats. As a result of the multi-sampling schemes, the sampling density was not consistent, and areas where the species was found to be present were more intensely surveyed (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). This bias needs to be acknowledged, but does not impact the results as it provided datapoints at a better resolution when conducting downstream ecological models based on both presence and absence points.\u003c/p\u003e\n\u003ch3\u003eField surveys protocol\u003c/h3\u003e\n\u003cp\u003eWe conducted the field surveys starting from the breeding season of 2017, until the breeding season of 2024, targeting different geographic areas over the years due to the sheer surface of land to be investigated. Surveys were interrupted in 2020 because of the pandemic. The landscapes targeted for the surveys were alluvial wetlands, in their very large majority represented by rice paddies, as natural wetlands able to host the species are now rare in the area [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Days with very heavy rain were avoided, as well as days of new moon and full moon due to their depressing impact on the calling activity of the sister species with a similar ecology, \u003cem\u003eD. suweonensis\u003c/em\u003e [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Surveys were conducted from mid-April, matching with the emergence of the species in Hefei and Jiangsu [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], and then expanded more broadly after confirmation of the beginning of the breeding season, to ensure the absence of false negatives. The latest surveys were conducted in July, as droughts delayed the calling activity of the species in 2023 [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe protocols for the field surveys strongly relied on the protocol developed and improved for \u003cem\u003eD. suweonensis\u003c/em\u003e and \u003cem\u003eD. flaviventris\u003c/em\u003e [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Broadly, surveys started from 10 minutes after sunset, although some individuals start calling about an hour before sunset, and finished before 3 am, as starting surveys too early or finishing too late might result in false negatives, and the investigators also need to sleep. Each year, we conducted additional ad-hoc partial ecological models based on all confirmed presence sites, supplementing the habitat suitability models of reference [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], to improve the quality of the surveys. At the beginning of the season, we started surveys from a known presence sites to ensure that the species had started breeding, and the species had to be found at a minimum of three sites where the species was present the year prior for the surveys to start for the year, and thus avoid false negatives. The sites selected for the year were not surveyed in any specific or geographic order to randomise the surveys, but most sites surveyed over the course of an evening were contiguous to ensure feasibility. The survey sites were most generally between 500 and 2000 m away from each other, including when located within the 5 and 10 km grid cells mentioned above, based on habitat suitability. However, when the species was detected, out of excitement and scientific curiosity, intermediary sites could be added every 100 m, until the species was not detected for 2000 m.\u003c/p\u003e \u003cp\u003eThe surveys were conducted by car. The surveyor(s) drove to the selected site, parked with the engine off, and waited for a minimum of 5 min silently, or until all expected non-focal species had restarted chorusing at the site (\u003cem\u003ePelophylax nigromaculatus\u003c/em\u003e, \u003cem\u003ePelophylax plancyi\u003c/em\u003e, \u003cem\u003eMicrohyla fissipes\u003c/em\u003e and \u003cem\u003eFejervarya\u003c/em\u003e cf. \u003cem\u003ekawamurai\u003c/em\u003e). In case \u003cem\u003eHoplobatrachus chinensis\u003c/em\u003e was heard calling at the site, an additional minute was spent listening as the species was detected at every site where \u003cem\u003eD. immaculatus\u003c/em\u003e was found, but not vice-versa. The 5 minutes spent waiting are expected to reach a probability of detection of 95% for \u003cem\u003eD. immaculatus\u003c/em\u003e, based on accumulation curves plotted for \u003cem\u003eD. suweonensis\u003c/em\u003e [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. In case the species was detected, a short, low-quality recording of the calls (min 10 sec) was made in case of the need for data inspection, and uploaded to iNaturalist.com, and the surveys were continued to the next survey site. Visual confirmation was not done, and is unnecessary: \u0026ldquo;the one who knows the calls of the frog can do in one night the work of many years\u0026rdquo; [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The surveys were normalised in all aspects so that the results collected over the years could be integrated for analyses. To save time, and not take more than seven years, no covariates were collected. Field surveys conducted as an expansion of this project are recommended to follow the same protocol to ensure continuity in data collection.\u003c/p\u003e\n\u003ch3\u003eModelling\u003c/h3\u003e\n\u003cp\u003eNext, we used ecological models to understand the distribution of the \u003cem\u003eD. immaculatus\u003c/em\u003e through its suitable habitat, based on the different types of occurrence data we have collected, and to predict its distribution under several scenarios linked to climate change. To do so, we explored four primary categories of environmental variables, encompassing key factors potentially influencing species distribution: land use/land-cover (LUCC), topography (including elevation, slope, and aspect), human footprints, and bioclimatic. As climatic predictors, we specifically selected isothermality (bio3), temperature seasonality (bio4), max temperature of warmest month (bio5), mean temperature of wettest quarter (bio8), and precipitation of driest month (bio14), because of their demonstrated strong correlation with the distribution of the species [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Climatic variables and elevation were sourced from WorldClim v. 2.1 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.worldclim.org\u003c/span\u003e\u003cspan address=\"https://www.worldclim.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The digital elevation model (DEM) was downloaded from the Geospatial Data Cloud (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.gscloud.cn/\u003c/span\u003e\u003cspan address=\"https://www.gscloud.cn/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Land use/land-cover data were obtained from Casearth (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ewww.data.casearth.cn\u003c/span\u003e\u003cspan address=\"http://www.data.casearth.cn\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The human footprint was acquired from the dataset available online [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Slope and aspect were derived from the DEM layer using the spatial analytical tools in ArcGIS Pro (v.3.0.2). Subsequently, all environmental layers were projected onto the WGS84 coordinate system using a 30 arc sec (circa 1 km\u003csup\u003e2\u003c/sup\u003e) spatial resolution raster grid. None of the variables exhibited significant correlations with each other (|r| \u0026lt; 0.7; [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]).\u003c/p\u003e \u003cp\u003eTo project future data (2021\u0026ndash;2100), we used four climate scenarios (SSP126, SSP245, SSP370, and SSP585), representing different socioeconomic development trajectories and greenhouse gas emission pathways [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. This choice aimed to assess the most significant potential shifts in suitable habitat.\u003c/p\u003e \u003cp\u003eWe used the Maxent model to construct suitable distribution areas for \u003cem\u003eD. immaculatus\u003c/em\u003e. As the prediction accuracy of MaxEnt models can be influenced by parameters such as feature class and beta-multiplier, we used the ENMeval package to determine the optimal combination of feature classes and regularisation multipliers [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. The selection of the best feature class and regularisation multipliers was based on the lowest Akaike Information Criterion, corrected for small sample values (AICc; [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]). We ran different models based on the eight datasets of occurrence we collected to analyse the suitable habitat for the species under the future scenarios (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). We verified the results using the area under the curve (AUC) values for the receiver operating characteristic curve and the true skill statistic (TSS) to evaluate the prediction accuracy of MaxEnt [\u003cspan additionalcitationids=\"CR51\" citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. The AUC (0\u0026ndash;1) quantifies the reliability of the predictions across four tiers: limited (0.6\u0026ndash;0.7), moderate (0.7\u0026ndash;0.8), good (0.8\u0026ndash;0.9), and excellent (0.9-1.0; [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]). All models were run in R 4.3.3 with the \u0026ldquo;dismo\u0026rdquo; [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e] and \u0026ldquo;terra\u0026rdquo; [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e] packages.\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\u003eDetails of the datasets used to build the ecological niche models for \u003cem\u003eDryophytes immaculatus\u003c/em\u003e. Here, survey sites represent the sites where the species was found to be present during surveys. The credible citizen science sites were the sites we estimated to be correctly reporting the presence of the species. The extinct sites were the sites where the species was reported to be present in the past, but our surveys did not manage to detect the species. Putative absence sites were defined as the survey sites where the species was not detected, and background points were the 10,000 points randomly dropped by the software over the landscape as a proxy for absence.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\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 \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDataset\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOccurrence data\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eVariables\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePeriod\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSurvey sites\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLUCC, topography, human footprints, bioclimate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCurrent\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSurvey sites\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003etopography, bioclimate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCurrent\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSurvey sites\u0026nbsp;+ credible citizen science\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003etopography, bioclimate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCurrent\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSurvey sites\u0026nbsp;+ credible citizen science\u0026nbsp;+ extinct\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003etopography, bioclimate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCurrent\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSurvey sites\u0026thinsp;+\u0026thinsp;credible citizen science with putative absence points\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003etopography, bioclimate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCurrent\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSurvey sites\u0026thinsp;+\u0026thinsp;credible citizen science\u0026thinsp;+\u0026thinsp;extinct with putative absence points\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003etopography, bioclimate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCurrent\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSurvey sites\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003etopography, bioclimate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFuture\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSurvey sites with putative absence sites and background points\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003etopography, bioclimate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFuture\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e\n\u003ch3\u003eThreat assessment\u003c/h3\u003e\n\u003cp\u003e \u003cem\u003eDryophytes immaculatus\u003c/em\u003e is a poorly researched species, mistakenly currently listed as \u0026ldquo;Least Concern\u0026rdquo; by the Red List of the International Union for the Conservation of Nature, despite acknowledging the decline [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. The assessment conducted here follows the IUCN Red List categories and criteria [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e], a generally robust and consistent method to assess threats to species globally [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. Assessments for the threatened categories are conducted against quantitative thresholds for five criteria that determine whether a species is at risk of extinction: A, population size reduction; B, geographic range size; C, small population size and decline; D, very small population and/or restricted distribution; and E, quantitative analysis of extinction risk. All threats, habitats, uses and trades are presented following the IUCN Red List criteria and categories [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e].\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eOccurrence data\u003c/h2\u003e \u003cp\u003eIn total, we conducted 6,191 targeted surveys for this project and found \u003cem\u003eDryophytes immaculatus\u003c/em\u003e to be present at 207 independent sites (Supplementary Information Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). These details do not take into account the survey sites where no amphibian species were detected, as the landscape was different from the satellite view because of rapid development, which accounted for about 270 sites. Most of the \u003cem\u003eD. immaculatus\u003c/em\u003e populations found during the surveys in this study were located between the Yangtze and Huai Rivers, with a small and isolated population in rice fields west of the Dabie Mountains. Most populations were found in central Anhui, with a few isolated populations further north and south in Anhui and Jiangsu (Fig.\u0026nbsp;1.1).\u003c/p\u003e \u003cp\u003eThe call for data resulted in the submission of more than 1300 datapoints, with pictures, explanations and other unrelated information. Due to the purposeful absence of a clear protocol to make reporting easier and diverse, and the inability to wade through this flow of data, we did not collect datapoints that could not be successfully traced back to the species. A wide diversity of (generally) green amphibians was sent to us, with most pictures likely representative of \u003cem\u003eZhangixalus dennysi\u003c/em\u003e and \u003cem\u003eHyla chinensis\u003c/em\u003e, two species clearly morphologically different from \u003cem\u003eD. immaculatus\u003c/em\u003e, even as juveniles. Results were received from spring 2022, with the latest report received in October 2024, and resulted in 11 independent sites that could be unequivocally identified as \u003cem\u003eD. immaculatus\u003c/em\u003e (Supplementary Information Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe credible sites resulting from the citizen science surveys were spread across Jiangsu and Anhui, increasing the range and density of the occurrence of the species, with a few populations north of the Huai River (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.3). In addition, some of the putatively extinct populations were located south of the Yangtze River, as far south as southern Anhui and southern Hubei (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.4). All confirmed populations were found in low-elevation wetlands, such as alluvial floodplains, and even when present in mountainous areas in the Dabie Mountains, they were within the agricultural wetlands in the lowest valleys (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFinally, we collected eight independent sites for occurrence in the past, here defined as prior to 1980, but where the species could not be found again, and therefore were considered as putatively extinct (Supplementary Information Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Non-confirmed reports that could not be categorically accepted or rejected due to the quality of the pictures and the absence of clear locality included areas north of Poyang Lake, Loudi in Hunan, and Nanchang in Jiangxi. Populations that need additional surveys to determine whether past reports were correct and they are now extinct, or the species was misidentified in the past, or the locality not recorded properly are the ones at the feet of Tianmu Mountain, for which museum vouchers morphologically match some of the characters present in \u003cem\u003eD. immaculatus\u003c/em\u003e, individuals reported from Yangjifeng, Jiangxi Province [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] and individuals reported from Hunan Province [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eEcological niche modelling\u003c/h3\u003e\n\u003cp\u003eWe ran the models using each of the eight occurrence datasets. The AUC values for all models were greater than 0.9, indicating an excellent overall prediction ability. The modelling results employing automatically generated background points revealed that high-suitability areas were predominantly distributed in low-lying regions, exhibiting significant spatial overlap with cultivated lands. These areas were exclusively located within Anhui and Jiangsu provinces, with notable concentrations in the cities of Chuzhou, Hefei, Lu\u0026rsquo;an, and Taizhou (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The land use/land-cover and anthropogenic activities had a negative effect on the suitable distribution areas of \u003cem\u003eD. immaculatus\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.a-b). The inclusion of credible science citizen data and the putative extinction data in the model resulted in a significant expansion of the suitable distribution area (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.c-d).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWhen we replaced the random background points with putative absence points, the highly suitable distribution area expanded (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Under these variables, the modelling results incorporating survey data and credible citizen science observations demonstrated that high-suitability areas were predominantly located in central and southwestern Anhui, as well as in proximity to the Yangtze River within southern Jiangsu, but also south of the Yangtze River where the species was not found during the surveys (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.a). Building upon these findings, the modelling approach incorporating extinct occurrence records revealed an expansion of high-suitability habitats into southeastern Hubei and southern Anhui (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.b).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eClimate change scenarios\u003c/h2\u003e \u003cp\u003eProjection of the current model onto future climate scenarios indicated significant changes in habitat suitability for \u003cem\u003eD. immaculatus\u003c/em\u003e under different climate scenarios. The species was projected to lose a substantial portion of its high-suitability habitats, particularly those overlapping with cultivated lands, as temperatures rise (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Under the low-emission scenario (SSP126), high-suitability areas were primarily distributed in eastern Jiangsu Province, central Anhui Province, and the Dabie Mountain region during 2021\u0026ndash;2040; however, these habitats are predicted to contract over time. In contrast, intermediate (SSP245) and high-emission scenarios (SSP370/585) predicted severe habitat loss for all future timelines, particularly in regions overlapping with croplands. By 2081\u0026ndash;2100, under SSP585, high-suitability areas will persist only in isolated mountainous refugia within Anhui, while lowland agricultural areas will experience near-complete loss of suitable habitat. The Dabie Mountain region is projected to function as the only climatic refuge for \u003cem\u003eD. immaculatus\u003c/em\u003e in response to future climate change.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eThreats and conservation\u003c/h2\u003e \u003cp\u003e \u003cem\u003eDryophytes immaculatus\u003c/em\u003e is impacted by habitat loss and degradation throughout its range and is not known to rely on natural habitat anywhere within its range to complete its life cycle. Instead, the species relies on artificial aquatic habitats, such as agricultural wetlands, for breeding. In addition, the species is impacted by global warming, as modelled above, and it has become locally extirpated at numerous sites, including its type locality. Within artificial wetlands, the species is predated by both native \u003cem\u003ePelophylax nigromaculatus\u003c/em\u003e (personal observation) and invasive American bullfrog (\u003cem\u003eAquarana catesbeiana\u003c/em\u003e; [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]). The species' generation time is 3 to 4 years (\u003cem\u003esensu\u003c/em\u003e IUCN), and the life span in the wild is estimated from 5 to 6 years based on closely related species [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. The species is not currently known to occur within any protected area. The result of the analyses following the guidelines of the IUCN Red List of Species [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e] is presented below.\u003c/p\u003e \u003cp\u003eIn terms of population size, no data is available for the number of individuals despite the putative extirpations at eight independent sites (one location \u003cem\u003esensu\u003c/em\u003e IUCN due to the common threat of habitat conversion for rice agriculture). However, a decrease in the area of agricultural wetlands available will directly result in a decrease in population size. As of 2016, the area used for rice agriculture had decreased by 11% since 1980 [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e], and has continued to decline since then, with a predicted continuing trend [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e] due to the decrease in water availability [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e, \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e], and the shift in diets and agricultural policies regulations [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e, \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]. The 11% decline in population size, even predicted, does not meet the threshold for a species to be listed as threatened under criteria A of the IUCN Red List of Species [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. However, non-quantitative information resulting from the conversation with farmers would point to a 90% decline in population size at all the sites where the species used to be present before the 1980s.\u003c/p\u003e \u003cp\u003eRegarding the geographic range, all individuals are included in one location as they are all under the single threat of agriculture. Based on the IUCN tool GEOCAT (geocat.iucnredlist.org) and the data resulting from the surveys, we determined that the EOO for the population surveyed (58,033 km\u003csup\u003e2\u003c/sup\u003e) and determined through citizen science (24,714 km\u003csup\u003e2\u003c/sup\u003e) resulted in a total of 104,501 km\u003csup\u003e2\u003c/sup\u003e, with a total of 193,550 km\u003csup\u003e2\u003c/sup\u003e when including populations putatively extinct since the 1980s. Similarly, for the AOO, the sites surveyed (296) and determined through citizen science (36) resulted in a total of 328 km\u003csup\u003e2\u003c/sup\u003e, with a total of 348 km\u003csup\u003e2\u003c/sup\u003e when including populations putatively extinct since the 1980s. Following this data, the species matches with an Endangered listing as the AOO is below the threshold for B2, and along with the information listed above, the species fulfils the criteria for B2ab(i,ii,iii,v).\u003c/p\u003e \u003cp\u003eDuring the surveys, no specific data on population size was collected, but the number of calling individuals at most sites was well below 10. As a few sites in Anhui had larger populations, up to 30 individuals, we assume an average of 10 individuals per site and, therefore, a rough estimate of (207\u0026thinsp;+\u0026thinsp;11) x 10\u0026thinsp;=\u0026thinsp;2180 individuals. As the population size is observed to be declining and projected to keep on declining in the number of mature individuals in each subpopulation, the species reaches the threshold to be listed as Endangered under the criteria C2a(i).\u003c/p\u003e \u003cp\u003eIn terms of criteria D (very small or restricted population) and E (quantitative analysis), the number of mature individuals is estimated to be over the threshold, and there are no data available for a quantitative analysis.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we showed the current range of \u003cem\u003eDryophytes immaculatus\u003c/em\u003e to be restricted between the Huai and Yangtze Rivers through field surveys that detected the presence of the species at 207 independent sites. In addition, these results were complemented with citizen science data, which reported the presence of the species at an additional 11 sites, generally extending the range of the species further east towards Shanghai urban area, and at one site south of the Yangtze River, towards the type locality of the species, where it is now locally extinct (Graphical Abstract). Local interviews also reported credible sightings of the species before the 1980s at an additional eight locations, all located further west and south. These extinctions show a contraction in the distribution of \u003cem\u003eD. immaculatus\u003c/em\u003e, as the species was reported to be widespread between Nanjing and Shanghai a few decades ago. In addition, it is critical to note that these are the sites that could be tracked down, and most probably a small number compared to the real number of sites where the species when extinct. However, despite the likely presence of the species prior to the heavy development of the area, a critical perspective is needed due to the potential for errors in datasets originating from citizen science datasets [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]. For instance, sites outside the range of \u003cem\u003eD. immaculatus\u003c/em\u003e have been reported, including some north to \u0026ldquo;Beijing area\u0026rdquo;, which are now attributable to \u0026ldquo;immaculate coloured\u0026rdquo; [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e] \u003cem\u003eD. japonicus\u003c/em\u003e individuals [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Other sites out of range have been reported (e.g., see AmphibiaChina.org), although the sites received from the citizen science surveys from these areas were all misidentifications, different enough to be confirmed through pictures (e.g., including \u003cem\u003eZhangixalus\u003c/em\u003e sp. and \u003cem\u003eOdorrana\u003c/em\u003e sp.). Regarding the areas identified through citizen science, the risk of misidentification is comparatively low as there are no other Hylid species occurring in these areas, however noting the presence of \u003cem\u003eHyla dabieshanensis\u003c/em\u003e between the sites where \u003cem\u003eD. immaculatus\u003c/em\u003e is recorded as locally extinct, although in a different habitat type [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e]. The only potential for misidentification is the southernmost site in Zhejiang where \u003cem\u003eD. immaculatus\u003c/em\u003e was recorded, as \u003cem\u003eHyla chinensis\u003c/em\u003e is also present in the area, despite clear morphological differences in adults [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe ecological models for habitat suitability (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) all supported the same pattern, with high habitat suitability in the plains in central Anhui, with the area of suitable habitat expanding longitudinally when including the sites coming from citizen science data, and the sites where the species is now extinct. This variation in habitat suitability is likely resulting from the changes in the landscape because of human activities, while the ecological models taking into account the effects of human activities provided the lowest suitable area (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.a), clearly resulting from the widespread destruction of the habitat suitable for the species. These alterations are principally the decrease in area used for rice agriculture [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e, \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e], but also the loss of habitat that can be used for overwintering in ditches, especially as a result of governmental policies for clean water, which indeed provide the results intended, but deprive the species from overwinter habitat.\u003c/p\u003e \u003cp\u003eIt is worth noting that, based on the ecological models, the habitat in the Dabie Mountains is not considered suitable, despite the presence of some individuals isolated in low-elevation agricultural wetlands. A new Hylid species was described in the area since the surveys, in early 2025, but clear morphological differences between \u003cem\u003eD. immaculatus\u003c/em\u003e and \u003cem\u003eH. dabieshanensis\u003c/em\u003e highlight the very improbable misidentification based on morphology [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e]. However, call recordings of \u003cem\u003eH. dabieshanensis\u003c/em\u003e are not available in the public domain, and misidentification during call surveys is a possibility, although unlikely due to the visual difference in the call spectrograms for the two species (comparison between [\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e] and [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e]). If this were the case, and these sites would have to be removed from the models, then the suitable habitat would most likely contract as the remaining sites are found in a more homogeneous landscape. Another limitation of the models is that they include croplands as a variable, without differentiating between dry and flooded agriculture, despite the known impact on some species with higher dispersal abilities [\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e]. As the species cannot breed in corn and wheat agricultural areas, the differentiation might make a difference. However, dry crops are present in the north of Anhui and at the same latitude in Henan, where the habitat is not modelled as suitable, and therefore, we can expect the models to already correctly attribute suitable habitat in this regard.\u003c/p\u003e \u003cp\u003eThe two ecological models with the putative absence points were consistent with each other, with a larger suitable area for the model including the extinct populations. These results also agreed with the other models in terms of the general area of the suitable habitats (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The main difference was that when including the putative absence points, the whole range of the Dabie Mountain became suitable, despite field surveys having shown that the species was not present in most of the habitat. In addition, the lowland area south of Hubei (around Wuhan) was also considered to be suitable, and while the species was not found during the surveys, a site was provided by the citizen science data. The mountain range in southern Anhui was also considered suitable when including the putative absence points, here again, despite the absence of the species through field survey, but an extinct population with a preserved individual in a museum from Tianmu Mountain (collected in 1955 as \u003cem\u003eHyla arborea immaculata\u003c/em\u003e).\u003c/p\u003e \u003cp\u003eIn terms of climate change, the four SSPs for the 2021\u0026ndash;2040 time period showed a shift in suitable habitat both east and west of the current distribution of the species, with a weak overlap with the current populations (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The only increase in habitat suitability that matches the known \u003cem\u003eD. immaculatus\u003c/em\u003e population was over the Dabie Mountains, which persisted for all four SSP for the 2041\u0026ndash;2060 period, and for all time periods for the SSP 126 and 245. It is, however, important to note that this refugia against anthropogenically induced climatic variation does support the survival of the species as it is now, as only a very small proportion of the population would be maintained, and the species would likely become functionally extinct [\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e]. For \u003cem\u003eD. immaculatus\u003c/em\u003e, all of the climatic scenarios resulted in a significant loss in suitable habitat, ranging from severe (SSP126 for 2021\u0026ndash;2040) to an almost entire loss of overlap between presence and suitable habitat (SSP 245 and above for 2041\u0026ndash;2060 and later). While these assessments are only models, they are in line with the global pattern for amphibians [\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e], and also for \u003cem\u003eDryophytes\u003c/em\u003e species in East Asia [\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e, \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e], highlighting the undeniable negative impact of anthropogenically induced climate change on the survival of the species.\u003c/p\u003e \u003cp\u003eBased on the current extinction risk listed on the IUCN Red List of Threatened Species, \u003cem\u003eD. immaculatus\u003c/em\u003e is listed as Least Concern [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. However, when applying the Red List categories and criteria, \u003cem\u003eD. immaculatus\u003c/em\u003e could be listed as Endangered due to both the geographic range in the form of the area of occupancy, matching with the criteria for B2ab(i,ii,iii,v), and the small and declining population size, under the criteria C2a(i). The suggestions reflect the situation on the ground as the species used to be common before the 1980s, based on the discussion with both elder farmers and researchers, but the species is currently difficult to find, and it has not been formally reported from any locality for about a decade prior to this study. This assessment might worsen once the impact of climate change can be quantified, due to its impact on both range and breeding phenology, and it is already known to have a negative impact [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Compared with other Hylids [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], \u003cem\u003eD. immaculatus\u003c/em\u003e is comparatively more threatened due to its narrow range and low population size. Finally, likely due to the current increase in the presence of the species in the media (e.g., [\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e]), and a reintroduction project in Shanghai, the species is advertised on online pet shops, which might become a threat if the trade increases in volume.\u003c/p\u003e \u003cp\u003eCompared with the two other species of the species complex, \u003cem\u003eD. suweonensis\u003c/em\u003e and \u003cem\u003eD. flaviventris\u003c/em\u003e, \u003cem\u003eD. immaculatus\u003c/em\u003e is in a very similar conservation situation. The main threat to these three species is habitat loss, with the transformation of the natural wetlands where they used to thrive into agricultural wetlands, which have now lost in value and are being replaced by other crops or land use and are decreasing in area [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e] and the area that is still planted being increasingly unhospitable to the species [\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e]. The variation in habitat available for the species is, however, not recent, having likely fluctuated with marine transgressions, and it is likely to have contracted over the last 21,000 years, following the increase in sea level and the flooding of the Yellow Sea [\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e, \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e]. The habitat available is also likely to have been impacted by the development of rice agriculture over the last 8000 years [\u003cspan additionalcitationids=\"CR79 CR80\" citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e]. The species density is likely to have become more uniform in the landscape during the conversion of all habitats in the area into agricultural wetlands [\u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e]. This change in habitat might have been positive for the species when wetlands increased in area and connectivity. However, this would need to be demonstrated through analyses based on population demographics, as done for other threatened species such as the condor [\u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e83\u003c/span\u003e]. More recently, changes in food preference in humans have also resulted in the decline in rice production, and the habitat available for \u003cem\u003eD. immaculatus\u003c/em\u003e [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e, \u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e], as seen for the Korean sister species [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe population size of the species at all sites, except north of Hefei in Anhui, is now likely to be too low for the species to provide the ecosystem function it used to provide ([\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e] in relation to [\u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e85\u003c/span\u003e]). These functions likely included energy transfer from the aquatic to the terrestrial habitat during metamorphosis [\u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e85\u003c/span\u003e] and pest regulation as ambush predators in wetlands, including rice-eating pests [\u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e86\u003c/span\u003e]. The species is also likely to have stopped providing the ecosystem services that used to benefit humans, such as mosquito regulation as provided by amphibians in Central America [\u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e87\u003c/span\u003e], and it would be worth conducting surveys in areas where the species is still present in large numbers compared to areas where it is now known to be extinct to determine its impact on pests. Finally, additional surveys are needed to understand the exact distribution and population size of the species, and surveys in the Yancheng UNESCO-MAB Biosphere Reserve might result in the discovery of the species in an area that is still representative of the natural habitat of the species, as suggested by some of the models, and it would be the only area where the species is occurring in a protected area.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eEthics approval and consent to participate\u003c/p\u003e\n\u003cp\u003eThis research was conducted under the Institutional Animal Care and Use Committee (IACUC) permit numbers 20-250-26, 20-240-13, 20-230-12 and 20-220-13 issued by Nanjing Forestry University. All humans interviewed agreed on sharing the information about the past presence of treefrogs on their land, and it did not include any information about any human being. We did not manage to ask the frogs for their consent.\u003c/p\u003e\n\u003cp\u003eConsent for publication\u003c/p\u003e\n\u003cp\u003eAll authors have agreed to the publication of this manuscript.\u003c/p\u003e\n\u003cp\u003eAvailability of data and materials\u003c/p\u003e\n\u003cp\u003eAll the data used for this publication is available in the Supplementary Information.\u003c/p\u003e\n\u003cp\u003eCompeting interests\u003c/p\u003e\n\u003cp\u003eNone of the authors has competing interests to declare.\u003c/p\u003e\n\u003cp\u003eFunding\u003c/p\u003e\n\u003cp\u003eThis project was funded by the Research Fund for International Scientists (RFIS) from the National Natural Science Foundation of China (NSFC; W2432021) and the Foreign Youth Talent Program of the Ministry of Science and Technology of the People\u0026rsquo;s Republic of China (QN2023014004L) awarded to AB. It was also funded by the Small Grants Program for New Records and Rediscoveries of Rare Species, initiated by Tencent Foundation and Shan Shui Conservation Center.\u003c/p\u003e\n\u003cp\u003eAuthors\u0026apos; contributions\u003c/p\u003e\n\u003cp\u003eConceptualisation: AB, YJ; Methodology: AB, ZZ, YJ; Formal analysis: AB, XZ, VKP, RW, SQ; Investigation: AB, XZ, VKP, RW, ZW, SQ, KRM, TG; Resources: AB, YJ, TG, JW; Writing - Original Draft: AB, VKP; Writing - Review \u0026amp; Editing: all authors.\u003c/p\u003e\n\u003cp\u003eAcknowledgement\u003c/p\u003e\n\u003cp\u003eThe authors are grateful to Ningjing Wang for her help during the surveys, and for raising awareness about the threats to the species. We are also most grateful to some of the citizen science participants who have provided data and agreed to be acknowledged, especially to Zeyang Liu, Shujun Xu and Jia Peng.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eWright AH, Wright AA. Handbook of Frogs and Toads of the United States and Canada. Comstock Publishing Associates, Ithaca, NY 1949(3rd edn).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWilson EO. The biological diversity crisis: a challenge to science. Issues Sci Technol. 1985;2(1):20\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEisenhauer N, Bonn A, Guerra AC. Recognizing the quiet extinction of invertebrates. Nat Commun. 2019;10:50.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCeballos G, Ehrlich PR, Raven PH. Vertebrates on the brink as indicators of biological annihilation and the sixth mass extinction. 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Environ Res Lett. 2022;17:104012.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-zoology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bzoo","sideBox":"Learn more about [BMC Zoology](http://bmczool.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bzoo/default.aspx","title":"BMC Zoology","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"distribution, surveys, citizen science, local extinction, suitable habitat, ecological niche modelling, climate change scenarios, IUCN Red List","lastPublishedDoi":"10.21203/rs.3.rs-6711786/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6711786/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe immaculate treefrogs, \u003cem\u003eDryophytes immaculatus\u003c/em\u003e, were reported to be abundant in the plains of southern Jiangsu, China, until the 1980s. However, the species has only scarcely been seen since the beginning of the century.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFirst, we conducted surveys between 2017 and 2024 at more than 6000 independent sites to determine the occurrence of the species, conducted citizen science surveys resulting in more than 1300 entries to determine the occurrence of the species, and questioned farmers, when possible, about the presence of the species. We detected \u003cem\u003eDryophytes immaculatus\u003c/em\u003e at 207 independent sites, managed to confirm the presence of the species at an additional 11 independent sites based on citizen science data and confirmed its local extinction at eight additional sites. Next, based on the accumulated data, we developed a suite of ecological models, including some with putative absence, to determine the current suitable habitat for \u003cem\u003eD. immaculatus.\u003c/em\u003e Finally, we also built ecological models based on climate change scenarios. The ecological models confirmed the habitat to be suitable in an area marginally broader than the one where the species was found, and the climate change scenarios highlighted a shift in the location of the suitable habitat for all scenarios and time periods tested, with a weak overlap with the current distribution of the species. Based on the data accumulated, we could also follow the categories and criteria of the IUCN Red List of Threatened Species and we suggested for the species to be listed as Endangered under both criteria B2ab(i,ii,iii,v) based on geographic range, and C2a(i) based on the small and declining population size.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe range of \u003cem\u003eD. immaculatus\u003c/em\u003e has contracted over the last decades, at a speed high enough for people sharing their land with the species to remember them, and the habitat suitable for the species is further declining, and predicted to entirely collapse in all future climatic scenarios. While not Critically Endangered yet, \u003cem\u003eD. immaculatus\u003c/em\u003e is in need of conservation actions, especially to prevent future decline in habitat quality.\u003c/p\u003e","manuscriptTitle":"From Least Concerned to Endangered? An integrated approach to determine the distribution, suitable habitat and future of Dryophytes immaculatus","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-30 10:05:25","doi":"10.21203/rs.3.rs-6711786/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-07-25T07:56:24+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-23T10:08:13+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-14T15:11:36+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-13T05:05:40+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"112484383331677462315694623536776352401","date":"2025-07-01T07:59:49+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"114268076189594639150720169493635233779","date":"2025-06-30T21:05:36+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"247347614039407107435428859175260896417","date":"2025-06-29T15:00:07+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"283427626678712472646371188856241066945","date":"2025-05-30T15:37:26+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"259046928766515788530654473681650208996","date":"2025-05-28T13:57:22+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-05-28T11:09:17+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-05-22T10:57:58+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-05-22T10:53:12+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Zoology","date":"2025-05-21T02:25:51+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-zoology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bzoo","sideBox":"Learn more about [BMC Zoology](http://bmczool.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bzoo/default.aspx","title":"BMC Zoology","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"fb607584-d3b5-4332-94c5-c3d9c55e75b4","owner":[],"postedDate":"May 30th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-01-12T16:02:18+00:00","versionOfRecord":{"articleIdentity":"rs-6711786","link":"https://doi.org/10.1186/s40850-025-00248-w","journal":{"identity":"bmc-zoology","isVorOnly":false,"title":"BMC Zoology"},"publishedOn":"2026-01-08 15:57:12","publishedOnDateReadable":"January 8th, 2026"},"versionCreatedAt":"2025-05-30 10:05:25","video":"","vorDoi":"10.1186/s40850-025-00248-w","vorDoiUrl":"https://doi.org/10.1186/s40850-025-00248-w","workflowStages":[]},"version":"v1","identity":"rs-6711786","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6711786","identity":"rs-6711786","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}