Comparing sampling protocols and results to better detect the amphibian fungal parasite Batrachochytrium dendrobatidis in the wild

73 Comparing sampling protocols and results to better detect the amphibian fungal parasite Batrachochytrium dendrobatidis in the wild Annette Taugbøl1 1 Norwegian Institute for Nature Research (NINA), Fakkelgården, 2624 Lillehammer, Norway Corresponding author: Annette Taugbøl ([email protected]) Copyright: © Annette Taugbøl. This is an open access article distributed under terms of the Creative Commons Attribution License (Attribution 4.0 International – CC BY 4.0). Research Article Abstract The pathogenic fungus Batrachochytrium dendrobatidis (Bd) causes chytridiomycosis in amphibians across the world, increasing the risk of population declines and species extinctions. Using a traditional sampling approach with traps, great crested newts (Triturus cristatus) and smooth newts (Lissotriton vulgaris) have been sampled in eight ponds for population estimates since 2013. The presence of Bd has been investigated by environmental DNA (eDNA) in the ponds since 2017, but results have often varied between sampling methods used during the same sampling period. By comparing results from pond water using two filter pore sizes (0.45 and 2.0 µm) and from amphibians (filtered bathwater, soft skin swabs, soft sandpaper rubbed on skin, and skin samples from between toes from toad carcasses), the results showed that filtered bathwater samples or gently rubbing amphibian skin with soft sandpaper are the most reliable methods for obtaining Bd DNA. Results from pond water filtered through the two pore sizes did not differ significantly across positive sites. Results also indicated that a variety of DNA concentrations should be tested in replicated qPCRs or ddPCRs to account for both potential inhibition and low levels of Bd DNA in the samples. Both crested newts and smooth newts were identified as infected with varying prevalence, but no trends in population declines were observed for any of the species during the sampling years 2013–2024. However, the number of crested newts in the monitoring ponds was altogether low and sometimes sporadic, and ponds with higher numbers of crested newts should be included in future studies on population trends in infected populations. Key words: Amphibians, Bd, eDNA, false negatives, method development, species detection Introduction Invasive alien species are regarded as one of the most important threats to native biodiversity because they can lead to local extinctions and the collapse of habitat types (Capinha et al. 2015; Early et al. 2016; Strand et al. 2019; Pysek et al. 2020). Invasive alien species are, however, not always easy to detect: most would recognize an elephant in the Arctic as an animal that does not belong there, but few, if any, would spot invasive microorganisms living in their garden – even though it is highly likely that some have already moved in (Lovett et al. 2016; Scott-Brown et al. 2018). The smaller the alien species, the less likely it is to be perceived by humans, but accurate recognition of pathogens is critical for wildlife disease research and conservation (Mörner et al. 2002; Buttke et al. 2015; Barroso et al. 2021; West et al. 2024). Academic editor: Andrew (Sandy) Liebhold Received: 26 May 2025 Accepted: 31 October 2025 Published: 19 November 2025 Citation: Taugbøl A (2025) Comparing sampling protocols and results to better detect the amphibian fungal parasite Batrachochytrium dendrobatidis in the wild. NeoBiota 104: 73–93. https://doi.org/10.3897/ neobiota.104.160230 NeoBiota 104: 73–93 (2025) DOI: 10.3897/neobiota.104.160230 Advancing research on alien species and biological invasions A peer-reviewed open-access journal NeoBiota 74 NeoBiota 104: 73–93 (2025), DOI: 10.3897/neobiota.104.160230 Annette Taugbøl: Method development within budget and time limitations Amphibians have been declining throughout the world since at least the 1950s (Houlahan et al. 2000; Finn et al. 2023), where the reasons for their disappearance are interactive and complex (Blaustein and Kiesecker 2002; Reid et al. 2019; Mi et al. 2023). Diseases caused by invasive alien pathogens are one of the factors driving these ever-lower numbers (Fisher et al. 2012; Martel et al. 2014; Fisher and Garner 2020; West et al. 2024). Invisible to the human eye, the generalist fungal amphibian skin pathogen Batrachochytrium dendrobatidis (hereafter referred to as Bd) (Longcore et al. 1999) has spread from its natural range in Asia (O’Hanlon et al. 2018; Sun et al. 2025) to every continent inhabited by amphibians (Greener et al. 2020), most likely through the pet and plant trade (Fu and Waldman 2022; Laorden-Romero et al. 2024). Bd was first discovered in Europe following a mass mortality event in 1997 (Bosch et al. 2001) and is now present in most European countries (Garner et al. 2005; Allain and Duffus 2019). In Norway, Bd was first detected from environmental DNA (eDNA) in 2017 from water collected from newt ponds (Taugbøl et al. 2021). Species detection from genetic traces in the environment is increasingly used in routine species surveys (Deiner et al. 2017; Fediajevaite et al. 2021; Sandercock et al. 2023) and is also a commonly used non-invasive method for detecting Bd (Chestnut et al. 2014; Brannelly et al. 2020a; Taugbøl et al. 2021; Everts et al. 2025). However, the application of eDNA species detection is not straightforward, and sampling from wild environments has a high chance of producing false negatives (Ficetola et al. 2015; Congram et al. 2022; Nogueira et al. 2025; Taugbøl et al. 2025). Bd lives as a sporangium in the keratinized skin of amphibians and spreads via aquatic zoospores that swim for about 2 cm (Piotrowski et al. 2004). Therefore, when targeting only water samples, the likelihood of detecting Bd DNA will depend on various factors, such as collecting water near infected hosts that are actively shedding the pathogen, the average infection load of hosts (Longo et al. 2023; Hartmann et al. 2024), and the quantity of zoospore release at the time of sampling. Swabbing has been the recommended (Hyatt et al. 2007) and most commonly used (Shin et al. 2014) sampling protocol for Bd since 2007. However, studies have since found the method to yield inconsistent results, especially for animals or sites with low infection intensity (Shin et al. 2014). For species negatively affected by Bd infection, valid inference of infection status is key to enabling rapid responses to prevent further pathogen pollution. Little is known about how Bd spreads from site to site once locally introduced (Johnson and Speare 2005; Liew et al. 2017; Prado et al. 2023) or what effect the parasite has once established, as not all amphibians respond equally to infection (Schloegel et al. 2006; Ryan et al. 2008; Crawford-Ash and Rowley 2021; Harmos et al. 2021). One method to measure potential effects of Bd in the wild is to collect data on both Bd-infected and Bd-free populations within the same geographic area (Pilliod et al. 2010; Taugbøl et al. 2021; West et al. 2024). The primary comparison between infected and non-infected ponds in Norway in 2017 concluded that there was little evidence for population decline of both newt species in Bd-infected ponds, but this finding may have been due to the inclusion of false-negative ponds in the “non-infected” group, thereby basing the model on incorrect input data. Furthermore, there were no data on how long Bd had been present in the ponds, which species were infected, or the prevalence among the newts occupying the Bd-infected ponds (Taugbøl et al. 2021). Further, the infected Norwegian ponds were scattered and intermingled with Bd-free ponds, and although this is com- 75 NeoBiota 104: 73–93 (2025), DOI: 10.3897/neobiota.104.160230 Annette Taugbøl: Method development within budget and time limitations monly observed in other survey areas (Kärvemo et al. 2018; Congram et al. 2022), suboptimal Bd detection methods could result in false negatives and an overall underestimation of the distribution range. When the Bd-positive Norwegian ponds were resampled in 2018, only one of the five ponds previously found to be infected in 2017 tested positive through filtered water samples (with more than two-thirds of qPCRs testing positive). Negative Bd results from three of the ponds conflicted with Bd-positive swabs collected from the same ponds, strengthening the assumption that the 2017 dataset included false negatives that may have confounded the population modeling results. This study compares how Bd detection varies with a range of sampling methods – including filtered water samples from pond water and amphibian bathwater and swabs from newts and toads (Bufo bufo) – from wild populations in southeastern Norway. The main objective was to identify a cost-effective sampling strategy that minimizes false negatives to the extent possible. As the funding sources and goals have varied across years, the direct comparisons across methods are kept within species and sampling years when more than one sample type was collected. Materials and methods Study area and limitations to sampling Sampling was conducted in the southeastern part of Norway (Fig. 1a) in two areas where Bd had previously been detected (Taugbøl et al. 2021; Strand et al. 2025). Newt samples were collected from eight ponds south of Oslo. Pond water was filtered in all sampling years for most ponds, as one of the original main aims was to check whether ponds with larger populations of newts also had higher levels of newt eDNA and if eDNA could thereby substitute monitoring with traditional trapping (Taugbøl et al. 2025). The same DNA extracts were also routinely tested for Bd, but as some ponds varied in infection status within and across years, additional alternative samples were needed to better determine the infection status of the ponds. Due to limited annual funding, comparable samples within a year were collected only during three of the eight sampling years from newt ponds: 2018, 2023, and 2024. Toad samples were collected from carcasses found at a road crossing in Nittedal (Fig. 1b). Obtaining sampling permits for tissue collection from live anurans (e.g., toes) is increasingly difficult. The original objective of the toad samples presented here was to assess toad DNA quality across several external sampling methodologies and to test whether the same samples could also yield reliable results for individual Bd infection status (Taugbøl 2024). As DNA from tissue would represent the relative DNA quality against which alternative samples were compared, all samples were collected from recently overrun toads from Nittedal, from a toad population found to have a Bd infection prevalence of 80% (Strand et al. 2025). Water filtration protocol from newt ponds Pond water samples were collected prior to trap placement during all sampling years. From each pond, 15 subsamples (collected 1–4 m apart) of 0.2 L surface-sampled water were pooled into a mixed sample (Fig. 2a). Subsampling increases the likelihood of filtering a sample that more closely represents the pond as a whole, as eDNA is likely to be unevenly distributed (Troth et al. 2021; Congram et al. 2022; 76 NeoBiota 104: 73–93 (2025), DOI: 10.3897/neobiota.104.160230 Annette Taugbøl: Method development within budget and time limitations Taugbøl et al. 2025). Due to high repeatability in eDNA results from newts filtered from the same mixed water source in 2018 (Taugbøl et al. 2025), only one sample of 0.5 L was filtered through a 0.45 µm cellulose nitrate filter (Fig. 2a; Nalgene CN 145-0045, Thermo Scientific) using a vacuum pump (Sartorius Microsart e.jet) connected to a three-place manifold (Pall filter manifold) in 2018. After filtration, each filter was preserved in a 2 mL tube containing 1440 µL ATL buffer (Qiagen). Based on previous findings of seasonal increases in Bd eDNA in one pond identified as positive in 2017 (Taugbøl et al. 2021), pond water was sampled and filtered four to five times from each pond in 2018: May 12 and 23, June 8, and July 3 and 13. Additionally, for a subset of ponds and sampling times in 2018 – as well as during later sampling years – water from the mixed sample was filtered through 2.0 µm glass fiber filters (Merck Millipore; Fig. 2a) using a self-priming peristaltic pump. These larger pore-size filters allowed filtration of a greater water volume (approximately 1–1.5 L) and were expected to increase the likelihood of detecting Bd DNA (Fossøy et al. 2020). In 2024, mixed pond water was filtered through duplicate sets of 0.45 µm and 2.0 µm filters during three time points in May: May 13–14, 22–23, and 27–28. For all sampling years, negative controls consisting of lake water and/or tap water filtered in the field were included to assess contamination risks (0.45 µm filters in 2018; 2.0 µm glass fiber filters in all other sampling years). Sampling newts with traps, bathwater, and soft swabs Great crested newts and smooth newts were caught using funnel traps originally designed for minnows. For each pond, 10 traps were systematically set along the shoreline during the peak breeding season each year and left in place for approximately 24 hours. Captured newts were placed in clean containers, unpacked at the site, and filled with pond water. In 2023 and 2024, 1–1.5 L of newt bathwater was filtered through a 2.0 µm glass fiber filter (Fig. 2b; Merck Millipore) using a self-priming peristaltic pump. In 2018, 2020, and 2023, newts were individually Figure 1. Sampling sites. a. The positions of the sampling sites in the southeastern part of Norway are shown as a green square; b. The sampling locations close to the capital of Norway (Oslo), where the square marked “toad crossing” indicates the site where the toad samples were collected, and the encircled numbers in the south indicate newt ponds: (1) Røer gård, (2) Garderenga, (3) Østre Glenne, (4) Ottarsrud, (5) Bellsjødammen, (6) Tokerud, (7) Solberg, and (8) Østre Støkken. Both maps were created with ArcGIS (Enterprise 11, 2022) in combination with Adobe Illustrator (Adobe Inc. 2019). Norway 1 2 4 5 78 6 3 Oslo Toad-crossing Newtponds ab 77 NeoBiota 104: 73–93 (2025), DOI: 10.3897/neobiota.104.160230 Annette Taugbøl: Method development within budget and time limitations selected and swabbed repeatedly over the feet and mouth with a soft cotton swab (Fig. 2b). All swab samples collected in 2018 and 2020 were stored individually in marked 1.5 mL Eppendorf tubes prefilled with 150 µL ATL buffer. In 2023, multiple swabs per pond (up to six) were stored in 5 mL Eppendorf tubes containing 4 mL ATL buffer as collective samples to test for Bd presence in five of the ponds. All captured individuals were released back into the ponds. For each pond, a catch per unit effort (CPUE) was calculated for each species as the number of animals divided by the product of trapping time and number of traps. Data on CPUE for the sampling years 2013–2016 were collected from Dervo et al. (2017). Sampling toad carcasses All toad samples were collected in April 2022. The toad sampling site (Fig. 1b) was chosen for two main reasons: first, toads from the site tested positive for Bd in 2021 (Strand et al. 2025); second, numerous roadkill casualties allowed for tissue sampling from carcasses as a comparative method for toad DNA. All observed live toads were carried across the road to the pond. A total of six fresh carcasses with minimal external damage were used to collect the following samples (Fig. 2b): soft cotton swabs – each of two randomly selected feet was swabbed 10 times; soft sandpaper (P400) – the remaining two feet were each swabbed 10 times; swim skin – a piece of webbed skin between the two longest toes from one of the hind legs; and toes from carcasses. The swab, sandpaper, and skin samples were stored in 150 µL lysis buffer. Replicates of soft skin swabs were also stored dry in Eppendorf tubes before DNA extraction. The bathwater sample was collected by filtering 0.5 L of pond water in which the carcass of each animal had spent 10–15 minutes (Fig. 2b). Filtered bathwater was always collected last to avoid influencing the results of the other sampling methods, in case exposure to water altered Bd detection. Toad bathwater was filtered through a 2.0 µm glass fiber filter with binder (Millipore) using a self-priming peristaltic pump (Makita USA Inc.) and Nalgene™ Single Use Analytical Filter Funnels (Thermo Scientific™). Each filter was cut in half, folded, and stored in two Eppendorf tubes prefilled with 1500 µL lysis buffer. All samples were kept at room temperature before being processed in the laboratory. Figure 2. Overview of samples. a. Water filtering: water was collected from several sites within each pond and mixed before being filtered through either (1) a 0.45 µm filter or (2) a 2.0 µm filter; b. Amphibian samples: (3) filtered bathwater filters pond water containing amphibians for 10–15 minutes (2.0 µL); (4) soft skin swabs; (5) toads only, soft sandpaper; (6) toads only, skin sample from between the two longest toes of one hind leg. All sampled toads were roadkill casualties. Illustrations were created using PowerPoint (Microsoft) and Adobe Illustrator (Adobe Inc. 2019). 1 2 Breeding pond 3 6 Holding bath 4 5 b a 78 NeoBiota 104: 73–93 (2025), DOI: 10.3897/neobiota.104.160230 Annette Taugbøl: Method development within budget and time limitations Molecular methods DNA from the 0.45 µm filters collected from the newt ponds, as well as from all swabs collected from newts, was extracted using the DNeasy Blood & Tissue Kit (Qiagen) following the modified protocol of Spens et al. (2017). DNA attached to the 2.0 µm glass fiber filters was extracted using a modified NucleoSpin Plant II Midi (Macherey-Nagel) protocol. DNA from all toad samples was extracted using the salt-extraction method developed by Aljanabi and Martinez (1997). After DNA extractions, the presence of Bd DNA was tested using duplicate digital droplet polymerase chain reactions (ddPCRs) in 2017 and 2018, with details as described in Taugbøl et al. (2021). Results were classified based on droplet counts: samples with three or more positive droplets were considered positive, while those with one to two droplets were considered weakly positive (Dobnik et al. 2015). Quantitative polymerase chain reaction (qPCR) was run in triplicate with species-specific primers for Bd, following the setup described by Boyle et al. (2004). qPCRs were conducted with 1 ng of input DNA across all years. Samples collected in 2024 (newts) and 2022 (toads) were also tested with 5 ng of input DNA. For all runs (ddPCR and qPCR), one or more negative controls were included as templates on each plate, using dH2O and fish DNA as templates, along with a positive sample of Bd DNA. None of the negative field samples or technical controls amplified Bd DNA. Statistical analysis All analyses were performed in R 4.0.1 (R Development Core Team 2021). Differences in individual Bd concentrations for each sampling method were tested using two-tailed t-tests and General Linear Models (GLMs) implemented in base R. Visualization of results was plotted using a combination of base R graphics and ggplot2 (Wickham 2016). Plots were further combined using Adobe Illustrator (Adobe Inc. 2019). All data are available in Suppl.material 1. Results Detection of Bd from newt ponds in relation to CPUE Trapping data (2013–2024) and Bd results (2017–2024, except 2021) are summarized for both newt species in Fig. 3. Apart from pond 8, no ponds tested consistently positive for Bd throughout the sampling years, and Bd results from mixed pond water filters and samples from bathwater and soft skin swabs were contradictory for ponds 1–4 and pond 7 for at least one sampling year (Fig. 3, heatmap). Due to the low number of ponds (n = 8) and the high likelihood of false-negative results, catch per unit effort (CPUE) for the newts is presented only as stacked bar plots (Fig. 3) and was not compared statistically based on infection status. Bd detection from pond water and newt swabs in 2022 In 2018, both mixed pond water and individual soft skin swabs were collected from the five ponds identified as Bd-infected in 2017. Great crested newts were identified as positive in four of the ponds (Fig. 4a). The prevalence of Bd DNA from the 79 NeoBiota 104: 73–93 (2025), DOI: 10.3897/neobiota.104.160230 Annette Taugbøl: Method development within budget and time limitations individual swabs ranged from 50% infected crested newts at pond 8 (based on the three-droplet cutoff) to 90% in the same pond when including samples with one to two positive droplets (Fig. 4a). Using three or more droplets as a cutoff, only pond 1 had positive samples for smooth newts, whereas all five ponds had ddPCRs with one to two positive droplets for this species (Fig. 4b). Filters from pond water identified two ponds as positive with three or more positive droplets – pond 7 and pond 8 – whereas pond 4 was positive with one to two droplets (Fig. 4c). Bd detection from pond water, newt swabs, and newt bathwater in 2023 A total of three sample types were collected and compared for Bd detection in 2023: mixed pond water (2.0 µm), newt bathwater (2.0 µm), and swab collections (more than one swab included in the same tube as a collective “pond sample,” collected only from ponds 6, 7, and 8). In total, six of the ponds tested positive for Bd in one or more sample types (Fig. 3). Mixed pond water identified three ponds as positive (Fig. 5): pond 6 (first time detected as Bd positive, with five of six positive qPCRs), pond 7 (one of six qPCRs positive from 1 ng of DNA input), and pond 8 (three of six qPCRs positive, all at 1 ng of DNA input). Figure 3. Bd infection status across years and estimates of newt populations per pond. The upper heatmap for each pond illustrates whether the pond was identified as infected (light pink), marginally infected (yellow; defined as fewer than three positive droplets in the ddPCRs or less than 66% positive qPCRs), or not infected by Bd (green), based on two sample types: (1) filters from mixed water samples and (2) samples collected from bathwater and/or soft skin swabs. The lower stacked bar plot illustrates catch per unit effort (CPUE) of smooth newt (light beige, upper part) and crested newt (light brown, lower part) for the eight ponds throughout the years 2013–2024. CPUE refers to the number of individuals caught in each pond, adjusted for trapping effort (number of traps × time in water). The horizontal blue line represents a CPUE of 0.2 to illustrate differences in population estimates between ponds. 2013 2014 2016 2017 2018 2019 2020 2021 2022 2023 2013 2014 2015 2016 2017 2018 2019 2020 2021 2022 2023 2024 2013 2014 2015 2016 2017 2018 2019 2020 2021 2022 2023 2024 2013 2014 2015 2016 2017 2018 2019 2020 2021 2022 2023 2024 2024 1 2 1 2 Pond 1 Pond 2 Pond 3 Pond 4 Pond 5 Pond 6 Pond 7 Pond 8 Smooth newt Crested newt Positive Low detectability No detection Bd eDNA No sampling CPUE 80 NeoBiota 104: 73–93 (2025), DOI: 10.3897/neobiota.104.160230 Annette Taugbøl: Method development within budget and time limitations Figure 4. Bd detection from swabs and pond water tested with ddPCR for the five sampled ponds in 2018. a. Results (in percentage) from soft skin swabs collected from great crested newts, with positive (orange) and negative (light blue) outcomes; b. Same as in (a), but for smooth newts; c. Results from the mixed pond water filter (0.45 µm), where green = negative Bd results, yellow = one to two positive droplets for one of the replicates, light orange = one replicate with three or more positive droplets, and dark orange = both replicates with three or more droplets. The DNA concentration in the sample was not found to influence Bd detection in the qPCR reactions (Fig. 5). When comparing the five sample types collected from ponds 6, 7, and 8, the average amount of DNA extracted for each sample type ranged from 33.94 ng, collected with swabs from great crested newts in pond 7, to 2,456 ng of DNA collected from bathwater from smooth newts in pond 6 (Fig. 5). Pond 8 had an overall higher concentration of Bd compared with ponds 6 and 7, where the sample type with the highest concentration had a mean Cq value of 29.9 and was extracted from swabs collected from smooth newts. Mixed pond water had the highest concentration of Bd in the samples collected from pond 8. Seasonal Bd detection from pond water and newt bathwater in 2024 In 2024, Bd was detected from mixed pond water in four of the ponds at all sampling times, while two ponds had one positive sample at one of the sampling times (ponds 1 and 2; Suppl. material 2: fig. S1). The 2.0 µm filter had slightly fewer Bd-positive qPCRs (77%) compared with the 0.45 µm filter (94%; Suppl. material 2: fig. S2), but this difference was not significant (t = 0.49, p = 0.62). The amount of DNA input (1 or 5 ng of DNA) produced significantly different results for the 81 NeoBiota 104: 73–93 (2025), DOI: 10.3897/neobiota.104.160230 Annette Taugbøl: Method development within budget and time limitations Figure 5. Cq values (duplicates, y-axis) of Bd in relation to the total DNA concentration of the sample (x-axis) from three ponds – pond 6 (dark green border), pond 7 (gray border), and pond 8 (black border) – where the results are divided into the five collected sample types (in blue, brown, coral, and gray) tested with 1 ng (diamond) and 5 ng (circle) of DNA input in the qPCRs. 0 500 1000 1500 2000 2500 25 30 35 40 45 Cq-value DNA-concentration Pond water Bathwater smooth Swabs smooth Bathwater great crested Swabs great crested Pond water Sample type DNA inputt 5 ng 1 ng Location pond 7 pond 8 Pond 6 Figure 6. Detection of Bd DNA from pond water sampled at three different times during spring 2024, where two replicated water samples were filtered through either 0.45 µm or 2.0 µm filters and the isolates were tested with both 1 and 5 ng of DNA input in triplicate. Sampling time 1 = May 13–14, sampling time 2 = May 22–23, and sampling time 3 = May 27–28. Data are shown only for ponds 6, 7, and 8, as these were the only ponds with consistent positive findings across all three sampling times. Pond 2 was positive at sampling time 2 (only for the 2.0 µm filter), and pond 1 was positive at sampling time 3 (only for the 0.45 µm filter). 0.45 34 36 38 40 42 44 2.0 0.45 2.0 0.45 2.0 0.45 2.0 Pond 5 Pond 6 Pond 7 Pond 8 0.45 2.0 0.45 2.0 0.45 2.0 0.45 2.0 0.45 2.0 0.45 2.0 0.45 2.0 0.45 2.0 Pond 5 Pond 6 Pond 7 Pond 8 Pond 5 Pond 6 Pond 7 Pond 8 Sampling time 1 Sampling time 2 Sampling time 3 5 ng DNA 1 ng DNA 0.45 µm filter, with 5 ng of input yielding a signal at 1.3 fewer cycles compared with 1 ng of input (t = 2.52, p = 0.013). When pond 8 was analyzed separately, the 2.0 µm filter gave positive signals at 2.04 fewer qPCR cycles compared with the 88 NeoBiota 104: 73–93 (2025), DOI: 10.3897/neobiota.104.160230 Annette Taugbøl: Method development within budget and time limitations Clavero M, García-Berthou E (2005) Invasive species are a leading cause of animal extinctions. 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The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/neobiota.104.160230.suppl2