Tetramorium caespitum and T. immigrans are both palearctic species ( Wagner et al., 2017 ). However, while T. caespitum is common across all of Europe ( Seifert, 2007 ), T. immigrans is primarily found in the Mediterranean, Western Europe, Central Europe, the Balkans, Eastern Europe, Anatolia and Caucasus ( Wagner et al., 2017 ), is more thermophilic and has a more southern and urban distribution than T. caespitum ( Seifert, 2018 ; Wagner et al., 2017 ). Using climatic niche modelling, we compare the climatic niche and habitat use in Europe for these two taxa, in order to determine potential differences in their ecological preferences. Finally, we compare the distance to cities and harbours along with sampled habitat types for data collected through the Ant Hunt with data collected by scientists from 1990 to 2015 to determine the extent to which data collection by citizens is or is not poised to help document introductions and shifting distributions.

Here we report on the discovery of a newly established ant taxon for Denmark through the citizen science project ‘the Ant Hunt,’ Tetramorium immigrans (Santschi, 1927). T. immigrans has recently been raised to the level of a cryptic sister species of the morphologically similar T. caespitum ( Wagner et al., 2017 ). An even more recent study has demonstrated that T. immigrans and T. caespitum hybridize ( Cordonnier et al., 2019 ), making identification difficult.

In 2017 and 2018, with a pilot study in 2015, children, in schools or with their families, collected ants for a project called the Ant Hunt (In Danish, ‘Myrejagten’) at 792 sites in Denmark. Although not the main aim of the project, we predicted that due to the expected amount of sampling in urban areas, new species for Denmark were likely to be discovered.

The introduction and establishment of new species outside of their native range, which then go on to become invasive, threaten biodiversity ( IPBES, 2019 ). Monitoring introduced and invasive species (species that have been introduced to an area outside of their range by humans and as invasives have a detrimental impact on nature, economy or human health) has been challenging in general, but has become increasingly problematic and important with accelerating trade, commerce ( Meurisse et al., 2019 ) and climate change ( Bellard et al., 2013 ). Recently, citizen science is being called on as a potential tool for successfully monitoring biodiversity on a large scale ( Tulloch et al., 2013 ; Theobald et al., 2015 ; Sauer et al., 2017 ). Citizen science can help to document introduced species in general, but particularly in some of the habitats typically missed by traditional surveys.

Materials and Methods

Biological data During the Ant Hunt, families and schools across Denmark collected ants by conducting baiting experiments at a site of their choosing. Participants ranged across all ages, but the average participants were children aged 5–11 years accompanied by a grown-up aged 31–50. They set out six different resources (saltwater, sugar water, oil, dissolved protein powder, a cookie and water) on bait cards and waited for two hours before collecting all the ants that were foraging on the cards. Ants were then frozen and counted before they were placed in 96% ethanol and sent to the Natural History Museum of Denmark for identification. All experiments were registered in an online database with date and GPS-coordinates (see Supplemental Material S1 for detailed protocol). In total, families and schools completed 792 experiments, of which 566 contained ants. The ants were identified using a variety of taxonomic keys (Collingwood, 1979; Seifert, 2007; Douwes et al., 2012; Lebas et al., 2016; Wagner et al., 2017). In total, participants had collected 16,985 specimens from 29 species (Table 1). Of these, specimens from two experiments could not be identified to species level due to missing body parts and specimens from two experiments were flagged as potentially new taxa for Denmark. These were T. immigrans and Technomyrmex albipes. The establishment of Technomyrmex albipes could not be confirmed. However, after the original discovery of T. immigrans during the Ant Hunt, trained scientists resurveyed the location several times throughout 2015–2019. The presences of T. immigrans was confirmed during every survey and the species was seen to expand to an area of approx. 40 m, with more than 20 nest entrances along the pavement. Species Experiments Formica cinerea 11 Formica exsecta 1 Formica fusca 89 Formica lugubris 1 Formica picea 1 Formica polyctena 43 Formica pratensis 1 Formica pressilabris 1 Formica rufa 7 Formica rufibarbis 6 Formica sanguinea 2 Formica sp.2 1 Formica truncorum 2 Hypoponera punctatissima 1 Lasius flavus 23 Lasius fuliginosus 12 Lasius niger 354 Lasius platythorax 47 Lasius psammophilus 2 Lasius umbratus 1 Myrmicinae sp.2 1 Myrmica lobicornis 2 Myrmica rubra 37 Myrmica ruginodis 35 Myrmica rugulosa 6 Myrmica sabuleti 4 Myrmica scabrinodis 4 Myrmica schencki 3 Technomyrmex albipes1 1 Tetramorium caespitum 26 Tetramorium immigrans1 1 DOI: 10.7717/peerj.8850/table-1 Tetramorium immigrans (Figs. 1A and 1B) tends to have a larger overall body size, denser striation/sculpturation of the head, thorax and petiolar nodes, as well as a more pronounced microscopic scale pattern on the first gastral tergite than T. caespitum (Wagner et al., 2017). Because of the difficulty in distinguishing T. immigrans from other species in the T. caespitum complex, we visually inspected all specimens found of T. caespitum in the Ant Hunt and randomly selected 10 samples from a broad range of localities (Fig. 1C). These were then compared to the 67 specimens of Tetramorium workers from the Botanical garden in Copenhagen, which is part of the Natural History Museum of Denmark (Fig. 1D), which were examined visually for morphological characters distinguishing typical forms of T. immigrans and T. caespitum. Voucher specimens were stored at the Natural History Museum of Denmark (NHMD 0000188537). Figure 1: Tetramorium immigrans in Denmark. (A) Photo of T. immigrans specimen from the Botanical Garden of Copenhagen. Taken by Rasmus S. Larsen and edited to remove background. Scale: 1 mm. (B) CT-scan of T. immigrans by Carsten Gundlach, 3D Imaging Center, DTU. Scale 0.7 mm. A video of the scan is available as Video S1. (C) Map of Denmark showing analysed samples of T. caespitum (filled orange crosses, 10 localities), observed localities of T. caespitum (open orange crosses, 83 localities), the location of T. immigrans (blue star) and localities of Ant Hunt experiments where neither T. caespitum nor T. immigrans was found (open red circles, 735 experiments). (D) Zoom in of the Botanical Garden at the Natural History Museum of Denmark in Copenhagen from Google Maps. Red circles indicate locations of T. immigrans. One specimen was chosen to be inspected by X-ray micro computed tomography (MicroCT). The specimen was placed on a designed holder and placed inside a Zeiss Xradia 410 versa system. The system was operated with a high voltage of 40 kV and a power of 10 W. The data acquisition consisted of 3,201 images while rotating 360 degrees, each image had a pixel size of 4.14 µm. All data was reconstructed into a 4 mm by 4 mm by 4 mm 3D volume with a voxel size of 4.14 µm. The reconstructed image is shown in Fig. 1B and raw data is available through Figshare (Gundlach et al., 2020).

DNA analysis For the DNA analysis of T. immigrans we selected two specimens from the confirmed find in the Botanical Gardens of Copenhagen, one from 2015 and one from 2018 and further selected 14 specimens of presumed T. caespitum, of which 10 had known coordinates and the remaining four were known to be from somewhere in Denmark. Up until DNA extraction, all samples from the Ant Hunt were kept in 96% ethanol in a freezer, while samples from the Natural History Museum of Denmark had been kept as pinned specimens. We extracted DNA by cutting off a small piece of the middle leg of each specimen, to which we added 100 µl of 10% Chelex in Tris-HCI buffer. This was mixed and centrifuged for 10 min, after which the solution was heated to 99 °C for 15 min and centrifuged again. The supernatant was used as a template for PCR reactions. We used primers LCO1490 and HCO2198 (Folmer et al., 1994) to amplify mitochondrial COI gene and primer D2B and D3A-r (Saux, Fisher & Spicer, 2004) to amplify nuclear 28S rDNA gene. PCR reactions were carried out using RedTaq ReadyMix PCR Reaction Mix with 100 µg/mL Bovine serum albumin. The PCR reaction conditions consisted of an initial denaturing step of 94 °C for 5 min, followed by 35 cycles of 94 °C for 40 s, 48 °C (LCO1490/HCO2198) or 56 °C (D2B/D3A-r) for 40 s, and 72 °C for 60 s, and finally an extension step at 72 °C for 5 min. PCR products were purified using Invitek PCR clean-up MSB spin PCRapace kit and Sanger sequenced in both directions using the Mix2Seq from Eurofins. Molecular identification was carried out by comparing samples to reference sequences from Wagner et al. (2017) (COI) and Schär et al. (2018) (28S). Raw sequences were edited and aligned using the software geneious v. 2019.0.4. A maximum likelihood tree with the best-fit model automatically selected by modelfinder and 1,000 rapid bootstrap replications was created for the alignment of COI sequences, using the program ‘IQ-TREE’ v. 1.6.1 (Nguyen et al., 2015). Tetramorium immigrans has previously been found to be distinguished from similar species (including T. caespitum) by having a one base insertion (C) at site 438 of the 28S rDNA fragment amplified by the primers D2B and D3A-r (Saux, Fisher & Spicer, 2004; Schär et al., 2018). We aligned the sequence of the specimen from Copenhagen to the reference sequences mentioned above to see if the characteristic insertion of T. immigrans is present. All genetic data referred to in this publication are available via the European Nucleotide Archive project PRJEB36036.

Habitat differences We used the CORINE 100 × 100 m land cover raster dataset (Copernicus, 2012) and extracted land cover values for all data points for both species using the spatial analysis tool ‘extract values to points’ in ArcGIS (ESRI, 2010). The CORINE land cover dataset consists of 48 land cover types. For this analysis we excluded all data points that were labelled with no data or one of the water based land cover types (‘water bodies,’ ‘water courses,’ ‘sea and ocean’). The remaining 39 land cover types were reduced to six major classes (‘Artificial surface,’ ‘Agriculture,’ ‘Forest,’ ‘Scrub and/or herbaceous vegetation associations,’ ‘Open spaces with little or no vegetation’ and ‘Wetlands’) following the CORINE land cover nomenclature (Copernicus, 2015). To test whether sampling of T. immigrans and T. caespitum were spatially biased, we compared the fraction of samples within each of the six land cover classes with the fraction of these land cover types in Europe using chi-square tests. We then did the same comparing the two species to each other to determine if T. immigrans and T. caespitum were being sampled in different habitats.