Monitoring the influx of new species through citizen science: the first introduced ant in Denmark

Climate change and invasive species threaten biodiversity, yet rigorous monitoring of their impact can be costly. Citizen science is increasingly used as a tool for monitoring exotic species, because citizens are geographically and temporally dispersed, whereas scientists tend to cluster in museums and at universities. Here we report on the establishment of the first exotic ant taxon (Tetramorium immigrans) in Denmark, which was discovered by children participating in The Ant Hunt. The Ant Hunt is a citizen science project for children that we ran in 2017 and 2018, with a pilot study in 2015. T. immigrans was discovered in the Botanical Garden of the Natural History Museum of Denmark in 2015 and confirmed as established in 2018. This finding extends the northern range boundary of T. immigrans by almost 460 km. Using climatic niche modelling, we compared the climatic niche of T. immigrans in Europe with that of T. caespitum based on confirmed observations from 2006 to 2019. T. immigrans and T. caespitum had a 13% niche overlap, with T. immigrans showing stronger occurrence in warmer and drier areas compared to T. caespitum. Mapping the environmental niches onto geographic space identified several, currently uninhabited, areas as climatically suitable for the establishment of T. immigrans. Tetramorium immigrans was sampled almost three times as often in areas with artificial surfaces compared to T. caespitum, suggesting that T. immigrans may not be native to all of Europe and is being accidentally introduced by humans. Overall, citizen scientists collected data on ants closer to cities and harbours than scientists did and had a stronger bias towards areas of human disturbance. This increased sampling effort in areas of likely introduction of exotic species naturally increases the likelihood of discovering species sooner, making citizen science an excellent tool for exotic species monitoring, as long as trained scientists are involved in the identification process.

81 immigrans is primarily found in the Mediterranean, Western Europe, Central Europe, the 82 Balkans, Eastern Europe, Anatolia and Caucasus , is more thermophilic and 83 has a more southern and urban distribution than T. caespitum (Seifert, 2018;Wagner et al., 84 2017). Using climatic niche modelling, we compare the climatic niche and habitat use in Europe 85 for these two taxa, in order to determine potential differences in their ecological preferences. 86 Finally, we compare the distance to cities and harbours along with sampled habitat types for data 87 collected through the Ant Hunt with data collected by scientists from 1990-2015 to determine the 88 extent to which data collection by citizens is or is not poised to help document introductions and 89 shifting distributions. 90 Materials & Methods 91 Biological data 92 During the Ant Hunt, families and schools across Denmark collected ants by conducting baiting 93 experiments at a site of their choosing. Participants ranged across all ages, but the average 94 participants were children aged 5-11 years accompanied by a grown-up aged 31-50. They set out 95 six different resources (saltwater, sugar water, oil, dissolved protein powder, a cookie and water) 96 on bait cards and waited for two hours before collecting all the ants that were foraging on the 97 cards. Ants were then frozen and counted before they were placed in 96 % ethanol and sent to 98 the Natural History Museum of Denmark for identification. All experiments were registered in 99 an online database with date and GPS-coordinates (see supplemental material S1 for detailed 100 protocol). In total, families and schools completed 792 experiments, of which 566 contained 101 ants. 102 The ants were identified using a variety of taxonomic keys (Collingwood, 1979;Seifert, 103 2007; Douwes et al., 2012;Lebas et al., 2016;Wagner et al., 2017). In total, participants had 104 collected 16,985 specimens from 29 species (Table 1). Of these, specimens from two 105 experiments could not be identified to species level due to missing body parts and specimens 106 from two experiments were flagged as potentially new taxa for Denmark. These were T. 107 immigrans and Technomyrmex albipes. The establishment of Technomyrmex albipes could not 108 be confirmed. However, after the original discovery of T. immigrans during the Ant Hunt, 109 trained scientists resurveyed the location several times throughout 2015-2019. The presences of 110 T. immigrans was confirmed during every survey and the species was seen to expand to an area 111 of approx. 40 metres, with more than 20 nest entrances along the pavement.

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Tetramorium immigrans ( Fig. 1a and Fig. 1b) tends to have a larger overall body size, 113 denser striation/sculpturation of the head, thorax and petiolar nodes, as well as a more 114 pronounced microscopic scale pattern on the first gastral tergite than T. caespitum (Wagner et 115 al., 2017). Because of the difficulty in distinguishing T. immigrans from other species in the T. 116 caespitum complex, we visually inspected all specimens found of T. caespitum in the Ant Hunt 117 and randomly selected 10 samples from a broad range of localities (Fig. 1c). These were then 118 compared to the sixty-seven specimens of Tetramorium workers from the Botanical garden of 119 Copenhagen (Fig. 1d), which were examined visually for morphological characters 120 distinguishing typical forms of T. immigrans and T. caespitum. Voucher specimens were stored 121 at the Natural History Museum of Denmark (NHMD 0000188537).

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One specimen was chosen to be inspected by X-ray micro computed tomography 123 (MicroCT). The specimen was placed on a designed holder and placed inside a Zeiss Xradia 410 124 versa system. The system was operated with a high voltage of 40kV and a power of 10W. The 125 data acquisition consisted of 3201 images while rotating 360 degrees, each image had a pixel 126 size of 4.14µm. All data was reconstructed into a 4mm by 4mm by 4mm 3D volume with a voxel 127 size of 4.14µm. The reconstructed image is shown in figure 1b and raw data is available through 128 Figshare (Gundlach, 2019). 129 DNA analysis 130 For the DNA analysis of T. immigrans we selected two specimens from the confirmed find in the 131 Botanical Gardens of Copenhagen, one from 2015 and one from 2018 and further selected 14 132 specimens of presumed T. caespitum, of which 10 had known coordinates and the remaining four 133 were known to be from somewhere in Denmark.

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Up until DNA extraction, all samples from the Ant Hunt were kept in 96 % ethanol in a 135 freezer, while samples from the Natural History Museum of Denmark had been kept as pinned 136 specimens. We extracted DNA by cutting off a small piece of the middle leg of each specimen, 137 to which we added 100 µl of 10 % Chelex in Tris solution. This was mixed and centrifuged for 138 10 minutes, after which the solution was heated to 99° C for 15 minutes and centrifuged again. 139 The supernatant was used as a template for PCR reactions. We used primers LCO1490 and 140 HCO2198 (Folmer et al., 1994) to amplify mitochondrial COI gene and primer D2B and D3A-r 141 (Saux et al., 2004) to amplify nuclear 28S rDNA gene. PCR reactions were carried out using 142 REDTaq ReadyMix PCR Reaction Mix with 10 µg/mL Bovine serum albumin. The PCR 143 reaction conditions consisted of an initial denaturing step of 94°C for 5 minutes, followed by 35 144 cycles of 94°C for 40 seconds, 48°C (LCO1490/HCO2198) or 56°C (D2B/D3A-r) for 40 145 seconds, and 72°C for 60 seconds, and finally an extension step at 72°C for 5 minutes. PCR 146 products were purified using Invitek PCR clean-up MSB spin PCRapace kit and Sanger 147 sequenced in both directions using the Mix2Seq from Eurofins.

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To test for niche equivalency and niche similarity, we compared the observed D metric 199 with 1000 simulated values of D. The niche equivalency test determines whether the overlap 200 between the niches of the two species is higher than two random niches drawn from the same 201 data pool. The niche similarity test determines whether the overlap between the two niches is 202 higher than when one species' niche is randomly drawn in the study area (Broennimann et al., 203 2012). If the observed value of D falls within the density of 95% of the simulated values, there is 204 no detectable difference in the climatic niche of T. immigrans and T. caespitum. Finally, we 205 projected the environmental niche of T. immigrans and T. caespitum onto Europe to visually 206 compared the two species and pinpoint areas of suitable climate for T. immigrans to establish. 207 Habitat differences 208 We used the CORINE 100x100 m land cover raster dataset (Copernicus, 2012) and 209 extracted land cover values for all data points for both species using the spatial analysis tool 210 'extract values to points' in ArcGIS (ESRI, 2010). The CORINE land cover dataset consists of 211 48 land cover types. For this analysis we excluded all data points that were labelled with no data 212 or one of the water based land cover types ("water bodies", "water courses", "sea and ocean"). 213 The remaining 39 land cover types were reduced to six major classes ("Artificial surface", 214 "Agriculture", "Forest", "Scrub and/or herbaceous vegetation associations", "Open spaces with 215 little or no vegetation" and "Wetlands") following the CORINE land cover nomenclature 216 (Copernicus, 2015).

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To test whether sampling of T. immigrans and T. caespitum were spatially biased, we 218 compared the fraction of samples within each of the six land cover classes with the fraction of 219 these land cover types in Europe using chi-square tests. We then did the same comparing the two 220 species to each other to determine if T. immigrans and T. caespitum were being sampled in 221 different habitats. 222 Monitoring for new species 223 In order to determine the suitability of citizen science for early detection and monitoring of 224 introduced species, we compared the citizen scientist collected dataset of the Ant Hunt with a 225 dataset collated from the Natural History Museum of Denmark, a personal collection by Sämi 226 Schär and the Ph.D. course EuroAnts, which was collected in Denmark from 1990-2015, with 227 2015 being the most recent year with available data. We compared the datasets based on two 228 measures, 1) distance to likely introduction sites and 2) sampling effort in different land cover 229 types. We calculated the average distance from data points in each dataset to Denmark's seven 230 major cities (cities of 50.000+ inhabitants) and 31 major harbours (harbours with a yearly goods 231 turnover of one million ton) in ArcGIS (ESRI, 2010) and compared the two datasets using a 232 Mann Whitney U-test.
For the land cover analysis, we used the above-mentioned major categories, but also 234 included the category Water as an approximation of samples collected close to the shore of water 235 bodies (Copernicus, 2015). We summarised the number of samples collected in these seven 236 major land cover classes and compared the observed values with expected values based on the 237 availability of each land cover class using Chi-square tests. Based on availability of each land 238 cover class we calculated the ratio of observed samples to expected samples to determine how 239 much more or less a specific land cover type was sampled than what would be expected by 240 chance.  Although we did not collect traditional morphometric data, we regard the find of T. 247 immigrans in Denmark to be verified. We base this determination on a maximum likelihood tree 248 of the mitochondrial COI gene (Fig. 2a), the existence of a characteristic one base insertion (C) 249 in the 28S rDNA fragment (Fig. 2b), and visual examination of diagnostic characters. This Environmental niche overlap between T. immigrans and T. caespitum, measured as D, 255 which compares the frequency of observations for each species within the chosen climatic 256 categories, was 13 % with only a slight difference of the niche centroid in environmental space 257 (Fig. 3a). Based on the contribution of the five climatic variables along the two axes, T. 258 caespitum is present in colder and wetter areas, compared to T. immigrans, which prefers warmer 259 and drier conditions with stable temperatures (Fig. 3b). Tetramorium immigrans and T. 260 caespitum differed in mean climatic values for eight of the original ten climatic variables (Table 261 S1 and Fig. S2), with no difference in precipitation seasonality and precipitation of coldest 262 quarter. Despite these slight differences, the two species' niches were more similar to each other 263 than two randomly drawn niches within the same data pool (niche equivalency test, p = 1) and 264 more similar than when a random niche of either species was drawn within the available climatic 265 space (niche similarity test, p = 0.22).

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Projection of the climatic niche into geographical space shows Eastern and Central 267 Europe to be the most climatically suitable for both species along with the northern part of 268 Southern Europe and the southern part of Northern Europe (Fig. 3cd), although T. immigrans 269 may have a slightly more southern distribution than T. caespitum. 270 Habitat differences 271 The current available data does not allow for an exact determination of the land use of T. 272 caespitum and T. immigrans, but we can determine in which land cover types the two species are 273 currently observed. Tetramorium caespitum was mostly observed in forest and agricultural land 274 use types (34.1 % and 34.96 % of observations, respectively, Table 2). Tetramorium immigrans 275 was mostly found in land types with artificial surfaces (48.89 %, Table 2). Both species were 276 rarely found in wetland areas (T. immigrans: 1.11 % and T. caespitum 0.14 % of observations). 277 Observations of both species differed significantly from what would be expected if land use 278 reflected availability of the six land cover types in Europe (Chi square tests, χ 2 = 322.71, df = 5, 279 p < 0.001 for T. immigrans and χ 2 = 243.95, df = 5, p < 0.001 for T. caespitum). 280 The two species also showed significant difference in the number of observations in each 281 land cover type compared to each other (Chi-square test, χ 2 = 82.66, df = 5, p < 0.001). 282 Tetramorium immigrans was found almost three times more often in land use types with artificial 283 surfaces than T. caespitum (ratio: 0.35; Table 2). On the other hand, T. caespitum was sampled 284 over twice as often in forests than T. immigrans (ratio 2.56; Table 2). 285 Monitoring for new species 286 Overall, data collected by citizen scientists during the Ant Hunt was significantly closer to cities 287 than data collected by scientists from 1990-2019 (mean 31.27 km ± 29.72 SD and 34.52 km ± 288 23.21 SD, respectively, W = 127623, p < 0.001). For the Ant Hunt, 101 of 667 observations (15 289 %) were within Denmark's major cities compared to only 4 out of 448 (0.9 %) scientist-collected 290 samples. Data from the Ant Hunt was also significantly closer to major harbours than data from 291 scientists (20.66 km ± 14.90 SD and 28.25 km ± 12.62 SD, respectively, W = 98154, p < 0.001). 292 Only six of the major harbours in Denmark were outside of cities with more than 5000 residents 293 (Fig. 4), suggesting a high correlation between industrial harbours and residential areas.

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Both scientists and citizen scientists were significantly biased regarding which land 295 cover classes they sampled within (Chi-square test, χ 2 = 653.75, df = 6, p < 0.001 and χ 2 = 296 3094.4, df = 6, p < 0.001, respectively). However, although both datasets were biased towards 297 areas with artificial surfaces, the effect was far more pronounced among citizen scientists, who 298 sampled artificially surfaced areas eight times more than expected. Scientists only sampled 299 artificial surface areas three times more than expected by random sampling. Both citizens and 300 scientists avoided agricultural areas, but scientists sampled forests, scrub, coastal areas and 301 wetlands 2-3 times more than expected by random sampling (Table 3).  308 The climatic niche of the two species overlapped by 13 % and our study confirms previous 309 assessments that T. immigrans prefers warmer and drier climates than T. caespitum (Seifert, 310 2018). Mean temperature of warmest quarter (°C) was 20.27 ± 2.06 SD for T. immigrans and 311 16.77 ± 2.43 SD for T. caespitum. This is in accordance with previously recorded standard air 312 temperatures (°C) for May-August for both species (19.9 ± 2.5 and 16.1 ± 2.0 SD, respectively; 313 Wagner et al., 2017).

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There was a large discrepancy between identified climatically suitable areas based on the 315 climatic niche model and current known distribution of T. immigrans, in accordance with 316 previous models of the climatic niche for T. immigrans (Steiner et al., 2008). Coupled with the 317 knowledge that northern observations of T. immigrans have been largely within cities and the 318 large gap in the known distribution from Poland to Denmark, we deem it likely that T. immigrans 319 is not native to northern Europe and is being accidentally introduced by humans.

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Others have also hypothesized that T. immigrans may not be native in most of Europe. 321 Specifically, the species is thought to be introduced in France, Germany and Poland (Gippet et   Whether T. immigrans will be able to further establish and spread in Central, 326 Northeastern and Northern Europe will depend on a number of factors common for 327 establishment success, including propagule pressure (Lockwood et al., 2005) and competition 328 with pre-established species (Menke et al., 2007). It may well be, that T. immigrans will not be 329 able to spread beyond cities in its' northern range, which is a common pattern for introduced 330 species ( King & Porter, 2007). Certainly, places identified as climatically suitable by the species 331 distribution model should be monitored closely (Fig. 3d) and further studies on the species' 332 distribution, competitive ability and climatic tolerances would be of high value to determine the 333 risk of spread.

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While not a main goal of the Ant Hunt, the finding of this species shows how the 335 engagement of untrained volunteers, even children, can be a great asset to the monitoring of 336 biodiversity, especially when it comes to detecting newly introduced species. This is evident 337 from the sample bias of citizen scientists towards cities, harbours and areas of high human 338 disturbance. On the other hand, scientists are more prone to sampling in natural areas. We argue 339 the case that engaging the aid of amateur participants; even as young as 6-10 year-old children, 340 can be a valuable tool for biodiversity monitoring. Citizen scientists are best able to search for 341 species where scientists are most likely to miss them and where introductions are most likely. 342 Conclusions 343 We hypothesized that, although not necessarily the main goal of citizen science projects, these 344 projects have a high likelihood of turning up new species, due to the large amount of sampling 345 being carried out in areas of likely introduction, such as harbours and cities.

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During the Ant Hunt, a citizen science project, where children set out baiting experiments 347 to help understand the community composition and resource requirements of ants under different 348 environmental conditions, two new species were discovered. One species, Tetramorium 349 immigrans, was determined to be established in the Botanical Garden of the Natural History 350 Museum of Denmark in Copenhagen.

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Our findings push the distribution of T. immigrans north by almost 460 km. Our 352 subsequent analysis of the climatic niche and potential geographical distribution of T. immigrans 353 adds some support to the current speculation that this species may not be native to all of Europe 354 and is spreading through introduction to cities, with many currently uninhabited locations 355 identified as climatically suitable. A systematic survey of the land use preferences of T. 356 immigrans along with a genetic mapping is needed to fully map and understand the dispersal of 357 T. immigrans across Europe.  Manuscript to be reviewed