Integrative taxonomy reveals cryptic diversity in North American Lasius ants, and an overlooked introduced species Author Schär, Sämi Institut de Biologia Evolutiva (CSIC-UPF), Passeig Marítim de la Barceloneta, 37, 08003 Barcelona, Catalonia, Spain saemi.schaer@gmail.com Author Talavera, Gerard Institut Botànic de Barcelona (IBB, CSIC-Ajuntament de Barcelona), Passeig del Migdia s / n, 08038 Barcelona, Catalonia, Spain Author Rana, Jignasha D. Department of Biological Sciences, The George Washington University, 800 22 nd Street, NW, Suite 6000, Washington, DC 20052, USA Author Espadaler, Xavier CREAF and Unitat d’Ecologia, Facultat de Ciències, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain Author Cover, Stefan P. Museum of Comparative Zoology, Harvard University, 26 Oxford Street, Cambridge, MA 02138, USA Author Shattuck, Steven O. Museum of Comparative Zoology, Harvard University, 26 Oxford Street, Cambridge, MA 02138, USA Author Vila, Roger Institut de Biologia Evolutiva (CSIC-UPF), Passeig Marítim de la Barceloneta, 37, 08003 Barcelona, Catalonia, Spain text Scientific Reports 2022 5970 2022-04-08 12 1 1 12 http://dx.doi.org/10.1038/s41598-022-10047-9 journal article 10.1038/s41598-022-10047-9 511e61d0-c5f9-4952-b270-bfe31f64e7f6 PMC8993915 35396496 6987323 Description of Lasius ponderosae sp. nov. Lasius ponderosae Schär, Talavera, Rana, Espadaler, Cover, Shattuck and Vila. ZooBank LSID : urn:lsid:zoobank.org:act: 22E2743A-2F1C-4870-B318-A1F2DF2B464C Etymology : ponderosae alludes to the ponderosa pine tree ( Pinus ponderosa ) that is at the centre of occurrence in the ponderosa pine—gambel oak communities in the western Rocky Mountains and northern Arizona . Type material : located at the Museum of Comparative Zoology, Cambridge, USA . Two paratype workers each will be deposited at the collections of University of California Davis ( UCDC ), the University of Utah ( JTLC ) and the Natural History Museum of Los Angeles County ( LACM ). Holotype : worker , Fig. 3a–c . Type locality: USA , Utah : Uintah Co. , Uintah Mtns. , 2408 m . 18.6 mi N . Jct. Rt. 40 on Rt. 191, 40.66378°N , − 109.47918°E , leg. 15.VII.2013 , S. P. Cover ; J. D. Rana , collection code SPC 8571 . Measurements [mm]: HL: 0.899, HW: 0.823, SL: 0.821, EL: 0.239, EW: 0.189, ProW: 0.56, ML: 1.069, HTL: 0.863, CI: 92, SI: 100. Paratypes : 15 workers , two gynes ( Fig. 3d–f ), two males ( Fig. 3g –i ) from the same series as the holotype , morphometric data is given in the Appendix, Table S 5 and Table S 6. CO 1 mitotype h17: Genbank Accession no. LT 977508 . Figure 1. Molecular phylogeny of 26 Holarctic ant taxa belonging to the subgenus Lasius sensu Wilson (1955) and two outgroup taxa ( L. pallitarsis and L. mixtus ). The phylogeny was calculated under the coalescent model and incorporates data from 9 genes (mtDNA: COI, COII, 16S, nuDNA: Defensin, H3, LR, Wg, Top1 & 28S). Names of species native to the Nearctic are shown in red and those of species native to the Palearctic in blue. Node labels show posterior probability (Bayesian inference) followed by bootstrap support (Maximum likelihood). The scale bar indicates the length of 0.01 substitutions/site. Description of the worker caste: A member of a complex of cryptic species resembling L. niger . Intermediate in overall body size, antennal scape length and eye size and comparable to related species ( Table 1 ). Terminal segment of maxillary palps and torulo-clypeal distance relative to head size shorter than in related Palearctic species ( Table 1 ). Mandibles with 8 or rarely 7 or 9 regular denticles and lacking offset teeth at their basal angle. Penultimate and terminal basal mandibular teeth of subequal size, and the gap in between with subequal area than the basal tooth. Anterior margin of clypeus evenly rounded. Dorsofrontal profile of pronotum slightly angular ( Fig. 4a ). Propodeal dome short and flat, usually lower than mesonotum ( Fig. 4a ). Body with abundant and long pilosity, especially lateral propodeum, genae, hind margin and underside of head. Pilosity of tibiae and antennal scapes variable, ranging from almost no setae (" L. alienus "-like phenotype) to very hairy (" L. niger "-like phenotype). Microscopic pubescent hairs on forehead between frontal carinae long and fine. Clypeus typically with only few scattered pubescent hairs ( Figs. 3 , 4c ). Coloration of body dark brown, occasionally yellowish- or reddish-brown or slightly bicolored with head and thorax lighter than abdomen. Femora and antennal scapes brown. Mandibles and distal parts of legs yellowish to dark brown. Specimens of all 3 castes are shown in Fig. 3a–i and morphometric data are summarized in Table 1 and raw measurements are available in Table S 5 and S 6. Figure 2. Mitotype tree and distribution maps for 98 DNA-barcodes belonging to 7 mitotypes of the ant Lasius niger (blue, n= 70) and 15 mitotypes of L. ponderosae sp. nov. (red, n = 28). The red dashed line delimits the expected natural range of L. ponderosae sp. nov. 53 Maps have been created using the free R-package “ggmap” v3.0.0 (https://github.com/dkahle/ggmap) in R v4.1.1. Map tiles by Stamen Design, under CC BY 3.0. Diagnosis: Lasius ponderosae sp. nov. workers key out to " L. niger " using Wilson’s 1955 key to the Nearctic Lasius species. However, some populations with reduced pilosity may also be identified as " L. alienus " using this key. Lasius alienus is a Eurasian species not known from North America 33 . The Nearctic " L. alienus " sensu Wilson (1955) includes both, L. americanus as well as populations of L. ponderosae sp. nov. with sparse setae counts on tibia and/or scapes. Lasius ponderosae sp. nov. can be distinguished from L. americanus by the presence of abundant, long setae surpassing the sides of the head in full face view (nGen> 5 and nOcc> 10 vs. nGen <5 and nOcc < 10 in L. americanus ). Distinguishing Lasius ponderosae sp. nov. from related Eurasian species (e.g., L. niger or L. platythorax ) by subjective eye inspection is difficult because there are no easily visible morphological traits allowing a separation of Lasius ponderosae sp. nov. from all these taxa. Lasius ponderosae sp. nov. is therefore a cryptic species. For L. niger , introduced to North America, nest samples can ofen be distinguished from L. ponderosae sp. nov. using the average mesosoma profile ( Fig. 4a,b ) and by fewer pubescent hairs on the clypeus ( Fig. 4c,d ). A distinction between single workers of L. ponderosae sp. nov. and L. niger , L. platythorax and related Palearctic species can be achieved by calculating the following discriminant function with measurements taken in mm: Figure 3. Frontal, lateral and dorsal view of the holotype worker ( a–c ), a paratype gyne ( d–f ) and a paratype male of Lasius ponderosae sp. nov. ( g–i ). D = − 43.792* GUHL + 113.436*dCLAN + 75.68* MP 6 − 0.431*nSt − 10.075. Negative values of D indicate L. ponderosae sp. nov. ( L. ponderosae sp. nov. , n = 39, median, range: − 3.18 [− 5.83, − 0.43]; L. niger , L. platythorax , L. grandis , L. japonicus and L. cinereus , n = 49, median, range: 2.67 [0.18, 4.61]). A principal component analysis plot for the four most diagnostic variables ( GUHL , dCLAN, MP 6 and nSt) is shown in Fig. 5 . For a definition of the variables used in the function, see Supplementary Table S 4 and Supplementary Fig. S1 . All morphometric data are available in Table S 6. Distribution: Western North America: Alberta , Arizona , Baja California , California , Colorado , Idaho , Montana , Nevada , Oregon , Utah and Washington 53 . Habitat : Occurring in a broad range of habitats and elevations, from 20–3220 m a. s. l. (mean: 1997 m ) 53 . Typical for mid-elevations in the mountains, whose primary habitat is ponderosa pine forest and its associated communities (e.g., dry meadows, sagebrush, gambel oak woodland) or at higher elevations in meadows bordered by aspen-spruce or alpine scree slopes, but also in other habitats. Nests in and under dead wood, and under stones in soil 53 .