First Record of the Introduced Ant Myrmica specioides in the Eastern United States

The ant Myrmica specioides Bondroit, 1918 (Hymenoptera: Formicidae) is reported for the first time from the Eastern United States. This species is native to the West Palearctic region and has previously been known as an introduced species in Northwestern North America. It was found in 2013 in the Boston metropolitan area. The species was identified by morphometric comparison to type specimens and DNA-barcoding. The distribution and invasive potential of M. specioides are discussed.


Introduction
Invasive alien species are considered one of the main causes of the current biodiversity crisis (Pyšek et al., 2020).At least 200 ant species (Hymenoptera: Formicidae) have been introduced outside their natural range by humans, and some of them have become invasive (Wong et al., 2021).Myrmica Latreille, 1804, is a diverse ant genus distributed throughout the Holarctic.A total of three species of Myrmica are so far known to have established major populations outside their native range: M. rubra (Wheeler, 1908), M. scabrinodis (Ellison et al., 2012;Seifert, 2018) and M. specioides (Jansen & Radchenko, 2008), all of which are of Palearctic origin and have been introduced to the Nearctic region.However, only M. rubra seems to have become widespread yet (Janicki et al., 2016).This is the only one of the three species known to establish supercolonies (van der Hammen et al., 2002) and is now considered invasive in North America (Naumann & Higgins, 2015).Most Myrmica species inhabit rather moist and cool habitats, and their colonies are typically mediumsized compared to other ants.They possess a powerful sting and prey on small arthropods.To a lesser extent, they also engage in symbioses with plant sap-feeding insects such as aphids (Hemiptera).
Ants of the genus Myrmica are notably difficult to determine, and some groups are still taxonomically unresolved, especially in the Nearctic region (Jansen et al., 2009).The morphological traits used for species delimitation in this genus are predominantly measures of body shape, although cuticular sculpture may also differ between taxa.The shape and relative size of the antennal scapes and of ridges (carinae) and processes thereof are of particular taxonomical importance.In addition, the distance between the frontal carinae, the shape of the petiole, and the length of the propodeal spines are also diagnostic characters.Not rarely, there is gradual variation in such traits within populations of the same taxon or between closely related taxa, complicating morphological species delimitation.For example, the taxonomically relevant  (Seifert, 2018).It is also not well studied if these phenotypic traits may be under selection by the environment or the result of phenotypic plasticity.In previous studies, the concordance between morphology and mitochondrial DNA trees was found to be rather low in Myrmica ants (Blatrix et al., 2020;Seifert et al., 2018).Therefore, future work is required to clarify this genus's taxonomy and systematics.For now, morphology aided by DNA-barcoding appears to be the best strategy to identify Myrmica specimens.The aim of this work is to identify an ant species from the Palearctic Myrmica scabrinodis-group found in the Boston metropolitan area and to clarify if it is an alien or invasive species. .A total of 11 specimens were collected in 95% ethanol and preserved at -20ºC.A total of three workers were pinned for morphological studies.The specimens were visually inspected and measured using a Leica EZ4 binocular with graticule at 16-70 × magnification.Multiple specimen images were created by using an Apple iPhone 12 mini through a Leica EZ4 binocular with changing focus.The images were aligned using align image_stack v. 2019.0.0 and stacked using GIMP v. 2.10.A total of 12 measurements which can be obtained from photographs taken in standard view (full face, lateral and dorsal view) were taken from a photographed specimen of interest, as well as AntWeb (2022)-images of 15 type and 3 non-type specimens representing 18 species of the M. scabrinodis-group for comparison: CW, Cwb, CL, SL, SW, PEW, ML, MW, EL (Seifert, 2018), FW, FLW and ESL (Radchenko & Elmes, 2010).Allometric effects were removed from all these raw measurements using the method of Lleonart et al. (2000) as implemented in the R package "allomr" (Schär, 2022), by scaling all specimens to the mean head size (CS=(CW+CL)/2) of 1.09 mm.A 13 th morphometric variable (pcSLd) was defined de novo: the proportion of the antennal scape (SL) being reached by a basal dorsal carina.Measured in dorsal view along SL, from the proximal anterior corner of the antennal scape to the distal end of the dorsal carina at the scape base, divided by SL (Fig 1b, pcSLd=a/SL).Equal to 0 if a dorsal carina is lacking.Dendrogram clustering of the 13 obtained morphometric variables was performed using the R package "cluster" (Maechler et al., 2021)

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DNA-barcoding of a specimen from Sommerville was performed by the Centre for Biodiversity Genomics, University of Guelph, Canada, using the primers LepF1 and LepR1 (deWaard et al., 2008).The resulting sequence was compared to a set of 154 previously published reference sequences from the M. scabrinodis-group (Blatrix et al., 2020;Jansen et al., 2010).Only unique sequences (mitotypes) were subsequently used (n=89).DNA sequences were aligned using the R package "msa" (Bodenhofer et al., 2015), and a maximum likelihood tree with 1000 ultrafast bootstrap replications was created in IQTREE (Minh et al., 2020).The nucleotide substitution model (TIM2+F+I+G4) was chosen using ModelFinder (Kalyaanamoorthy et al., 2017).The resulting dendrogram was visualized using the R-package "ggtree" (Yu et al., 2017).

Results
The specimens are stored at the research collection of R. Vila (voucher MM21B014a1) and the private collection of S. Schär (collection code SS13B020).The habitat of the ants reported here was a small urban hedgerow with some tree stumps surrounded by asphalt and concrete.These Myrmica ants have an antennal scape that is sharply bent near its insertion and with a well expressed caudal carina (Fig 1).This antennal morphology is not found among native Myrmica ants of New England (Ellison et al., 2012).However, it is present in the Palearctic M. scabrinodis-group.Morphometric data is given in Table S1.Morphometric comparison to type and non-type worker specimens of the Myrmica scabrinodisgroup suggests that these specimens from Sommerville are morphometrically most similar to the type of M. specioides (Fig 3).There are a few morphological traits that further support this hypothesis: The antennal scape has a very short dorsal carina, but a well visible caudal carina (Fig 1a, b).However, the latter is not extended into a large posterior lobe (Fig 1a).The frontal lobes are rather distant in their middle section (mean FI (Radchenko & Elmes, 2010): 0.39, n=3) and not strongly diverging anteriorly (mean FLI (Radchenko & Elmes, 2010) : 1.28, n=3) (Fig 1a).Typically for M. specioides, the propodeal spines of are relatively short (Fig 1c, d).In lateral view, the petiole lacks a dorsal plane.Instead, it is more or less gradually sloping downwards posteriorly (Fig 1c).The petiole is rather narrow, as seen in dorsal view, but not as narrow as in M. constricta or M. rugulosa (mean PW/CS: 0.27, n=3).Finally, the antennal scape is sharply bent near its insertion, as seen in the caudal view (Fig 1d).
The complete DNA-barcode (658 bp of the mitochondrial gene COI) could be obtained for a specimen from the colony of Sommerville (BOLD Process ID: ANTEU038-21, GenBank accession number OY397299).The phylogenetic tree based on a set of 89 COI mitotypes belonging to 12 taxa of the M. scabrinodis-group (Fig 4), recovers the sequenced specimen from Sommerville as most closely related (0.5%-0.7% divergence) to two M. specioides (Fig 4), both of which collected from Canada.A comparison with COI sequences on BOLD (December 2022) revealed 70 closely related sequences (<0.5% divergence), 67 from Europe and 3 from North America.Of those, 64 (91%) were identified as M. specioides too.The remaining sequences were either not identified to species level (n=1), identified as M. scabrinodis (n=3), M. constricta (n=1), or M. hellenica (n=1).The latter sample is interesting, as it was also found in the Eastern United States (New York state).A 100% match was found with two specimens, one M. specioides from Switzerland and an unidentified Myrmica from Germany.

Discussion
This article reports the first record of the introduced ant Myrmica specioides in the Eastern United States.However, it cannot be ruled out that it has been recorded under other names in this region before (e.g., M. hellenica or M. scabrinodis).
For example, Clark et al. (2011) reported a morphologically similar M. scabrinodis from the Boston Harbor Islands.Ellison et al. (2012) mentioned the same species from Porter Square, Cambridge, which is located <1 mile away from our finding of M. specioides (Fig 2b).We encourage future DNA-barcoding of these and other specimens of the M. scabrinodis-group from Eastern North America in order to rule out earlier confusion of M. specioides with other taxa and to better understand the introduced range of this species.Myrmica specioides has been known as an introduced species in Northwestern North America for some time, namely in the state of Washington (Jansen & Radchenko, 2008) and Southwestern British Columbia (Naumann & Higgins, 2015) (Fig 2).Given the results here published, this species is now also recognized as present in New England and the area of Boston in particular.Not much is known about the ecology and behavior of M. specioides in the introduced range.It is known to be an aggressive, polygynous, and locally abundant species, traits that suggest it could potentially become invasive (Jansen & Radchenko, 2008).In both the native and introduced range, it is a species found in urban areas.Myrmica specioides is also known to prey on other species of ants (Seifert, 2018).However, evidence that it behaves as an invasive species in North America has not been published so far.The population in Sommerville in 2013 appeared to be rather inconspicuous.
Nevertheless, we suggest that the ecological impact and spread of M. specioides in North America should be monitored.

Fig 2 .
Fig 2. Distribution of M. specioides in both native (yellow) and introduced (red) range (a) (data from AntMaps 2022, Guénard et al., 2017; Kass et al., 2022).The precise locality of the new record (b) is represented by a red star.

Fig 3 .Fig 4 .
Fig 3. Dendrogram clustering of 13 morphometric measurements and indices (see main text) of 13 reference (type) specimens of the Myrmica scabrinodis group, and a specimen of the sample to be identified collected in Sommerville (labeled "XXX", photographed in Fig 1).The type status of the reference specimens is indicated by colored symbols.