First documented record of the neotropical ant Brachymyrmex cordemoyi Forel, 1895 (Formicidae: Formicinae) in Germany

Brachymyrmex cordemoyi Forel, 1985 is a small ant species native to the Neotropics, but it has been introduced into Africa, Madagascar, the Arabian Peninsula and some tropical oceanic islands. We report on a non-native Brachymyrmex population that has been discovered indoors in the vicinity of Euskirchen (Germany). Specimens were identified morphologically and subjected to DNA barcoding; molecular data of the new population was analyzed jointly with morphologically verified GenBank entries. Morphological identification and phylogenetic inference based on a fragment of the mitochondrial gene Cytochrome Oxidase subunit 1 gave congruent results. The population was clearly assigned to B. cordemoyi and represents the second report of this species from Europe, with a recent record from the Netherlands. In Germany the species has only been found indoors, and the here reported population has been exterminated. This new record represents one of many indoor introductions of the genus Brachymyrmex, likely via tropical plants. We present a diagnosis of the species and additionally review all known introductions of the genus which have been identified to the species level.


Introduction
Global trade in general, and of plants and plant materials in particular, can lead to the anthropogenic dispersal of animals that upon establishment in new regions outside of their native range may constitute a first step towards biological invasion (Hulme 2009;Simberloff and Rejmanek 2011). Especially small and stress-resistant species may survive long-distance transport and may establish in new environments. Such new environments may have a special micro-climate, such as houses, greenhouses or swimming pools, where the invader may thrive locally, without escaping into natural environments (Kenis et al. 2007).
Insects and arachnids contain various species that are commonly dispersed via plants or plant products (Hulme et al. 2008;Roques 2010). The most publically known invaders are perhaps banana spiders (Blick et al. 2006), but a large number of small insects from various taxonomic groups are commonly imported (Malumphy 2012). Another group containing various common invaders are ants, which may become pests upon colonization (e.g. MacGown et al. 2007;Klotz et al. 2008;Gotzek et al. 2015). Whereas most non-native species cause little harm, some others may pose direct threats to biodiversity or economy and may be considered pests. Prominent examples are the fire ant Solenopsis invicta and the Argentinian ant Linepithema humile, which cause severe ecological problems as they replace the local fauna (Kenis et al. 2009). Another example is the pharaoh ant (Monomorium pharaonis), which represents an important household pest (Wetterer 2010).
In Germany 25 species of non-native ants have been recorded (Geiter et al. 2002). Several of these have become established and some are now considered to be pests, whereas others have a more restricted impact. Several species of the genus Brachymyrmex belong to such invaders. The genus Brachymyrmex is originally distributed in the Neotropics and consists of 40 valid species, subspecies and varieties (Ortiz-Sepulveda et al. 2019). Several species of Brachymyrmex have been recorded outside of their native ranges and B. patagonicus is even considered to be a pest (e.g. MacGown et al. 2007;Klotz et al. 2008;www.antmaps.org summarizes these records). In their native habitat most Brachymyrex species nest in soil or rotten wood, but in urban areas they also may nest in flower pots (Dash et al. 2005) and in this way they have invaded greenhouses (Forel 1907).
Three species of the genus have so far been recorded in Germany (B. longicornis, B. patagonicus and B. heeri). Here, we present the first record of B. cordemoyi from Germany. Specimens of this species were identified using diagnostic morphological features and the identification was subsequently verified with DNA barcoding. We also summarize all literature records of Brachymyrmex introductions where a species-level identification was made, although we have not been able to confirm the identifications for some of these records.

Materials and methods
Specimens were collected indoors by an insect exterminator in the vicinity of Euskirchen (50°40′02.3″N; 6°48′10.7″E) in Germany (the population was exterminated after sampling). Voucher specimens are deposited in the collection of the Zoological Museum Hamburg (ZMH) under accession number ZMH 2018/7. Individuals were morphologically identified by CMOS following the identification key of Ortiz-Sepulveda et al. (2019). Genomic DNA was extracted from one specimen using a standard Chelex protocol (Walsh et al. 1991). PCR was performed using the general barcoding primers (HCO-LCO, Folmer et al. 1994), DreamTaq Polymerase (Thermo Fisher Scientific, Bremen, Germany) and a standard protocol with an annealing temperature of 50 °C in a 10 μl volume. PCR products were checked on a 1% agarose gel stained with GelRed (Biotium, Fremont, Ca, USA), purified with a mix of Exonuclease and Shrimp Alkaline Phosphatase before sequencing by Macrogen Europe (Amsterdam, Netherlands).
The obtained sequence was inspected, aligned with species level data from public databases (see below) and subjected to phylogenetic analysis with Bayesian Inference (BI). We only included GenBank specimens for which voucher images are available that allow unambiguous identification (Table 2). In total, the analysis was performed with 28 specimens covering seven Brachymyrmex species and one specimen of Myrmelachista (the sister-  Table 2. Species identification, locality and NCBI Genbank accession numbers for the COI sequences of the taxa studied in the phylogenetic analysis. GenBank sequences generated by the International Barcode of Life consortium (iBoL) were only included if voucher images allowed unambiguous identification. One GenBank sequence from P.E. Hanish of a specimen of B. cordemoyi was also included in our phylogenetic analysis. Note that these sequences have been publicly deposited, but that the current study is their first report in the scientific literature.

Results and discussion
The Brachymyrmex population from Germany was morphologically identified as B. cordemoyi (Figure 1). Brachymyrmex cordemoyi strongly resembles B. obscurior and to lesser extent also B. patagonicus. These three species have scapes that touch the posterior cephalic margin, or extend beyond it, but by a distance that is smaller than the maximal diameter of the eye; in lateral view their mesonotum does not bulge out dorsally above the pronotum, and the metanotal groove is narrower than the diameter of the methathoracic spiracles. However, B. cordemoyi has a longer pronotum and mesonotum than B. obscurior, more ommatidia along the maximal diameter of the eye, and lighter-colored pubescence, which is denser on the dorsum of the entire body and appressed on the gaster (instead of decumbent in B. obscurior); the gaster also bears several scattered and sub-erect hairs mainly, but not exclusively along the edges of the segments. Brachymyrmex cordemoyi differs from B. patagonicus by having considerably denser pubescence on the gaster. It can also be confused with B. termitophilus, from which it differs in the body color (B. cordemoyi is dark brownish, whereas B. termitophilus is yellowish - Ortiz-Sepulveda et al. 2019). The phylogenetic analysis supports the morphological identification. The individual from Germany constitutes a well-supported monophyletic clade with other specimens of B. cordemoyi (BPP = 0.99, Figure 2). However, the deep split between the specimens of B. cordemoyi from the Comoros, Mauritius and Argentina and that from Germany, as well as the long branch-length towards the German specimen are noteworthy. In this respect it is important to highlight that B. cordemoyi has several diagnostic features, but also considerable intraspecific variation in various traits and that our current concept of B. cordemoyi may include several biological species (Ortiz-Sepulveda et al. 2019). Our data shows the value of DNA barcodes to identify species of Brachymyrmex as long as comparative sequences are available for well-identified individuals. Genetic resources are especially important considering the uncertain taxonomy of this genus and the ambiguity that long existed on putatively diagnostic morphological traits (Ortiz and Fernández 2014;Creighton 1950;Ortiz-Sepulveda et al. 2019). However, studies with additional genetic markers are required to better understand phylogenetic relationships within Brachymyrmex.
In Germany only Brachymyrmex heeri, B. patagonicus and B. longicornis have previously been reported (Table 1). The specimens of B. longicornis held at the ZMH are poorly preserved, however, they have been reported as syntypes in the past (Weidner 1972). Brachymyrmex longicornis is now considered to be a junior synonym of B. australis (Ortiz-Sepulveda et al. 2019).
Our new record of B. cordemoyi raises the number of exotic Brachymyrmex species in Germany to four, i.e. one species less than for Europe altogether (B. obscurior has been reported from the Netherlands). It is likely that additional species have been reported under other names or have just been overlooked or misidentified (Boer et al. 2018). Furthermore, considering the ease with which species of the genus Brachymyrmex can be transported, i.e. given their small size and their nesting habits, we can expect additional introductions in the future. The rise in global temperatures may even result in the establishment of perennial populations in natural environments in the future, at least in southern and central Europe.