Rumsaïs Blatrix
email: rumsais.blatrix@cefe.cnrs.fr

Théotime Colin

Philippe Wegnez

Christophe Galkowski

Philippe Geniez

Introduced ants (Hymenoptera, Formicidae) of mainland France and Belgium, with a focus on greenhouses Les fourmis (Hymenoptera, Formicidae) introduites en France métropolitaine et en Belgique, avec un focus sur les serres

Keywords: Formicidae, introduced species, tropical greenhouses, biological invasions Résumé Formicidae, espèce introduite, serre tropicale, invasions biologiques

The monitoring of introduced species is becoming more important as global trade intensifies. Although ants make up a larger proportion of species on the list of the most invasive species in the world compared with other groups, little is known about the occurrence of those introduced in France, especially inside heated buildings.

Here we review the literature available for mainland France and Belgium and report the results of a survey conducted with the help of tropical building managers between 2014 and 2016. We report for the first time in France the presence of Technomyrmex vitiensis and Plagiolepis alluaudi in multiple greenhouses.

Technomyrmex difficilis was also found in one greenhouse for the first time in Europe. The diversity of introduced ants in greenhouses is very low, and these buildings are most often dominated by one or two species. We compared the most recent data and those collected throughout the 20 th century, and showed that ant communities have changed substantially. Greenhouses could be responsible for the introduction of invasive species because they regularly import exotic plants, but we found no evidence that the three species of invasive ants present outdoors in France were introduced from greenhouses, where they rarely occur. We also report that introduced ants are pests in greenhouses because they disperse scale insects and kill biological control agents. The suppression of these ants could ease the maintenance of plants inside greenhouses.

Introduction

Intensifying global trade has increased the rate of exotic species introduction events [START_REF] Bertelsmeier | Recent human history governs global ant invasion dynamics[END_REF], especially for species originating from tropical habitats [START_REF] Westphal | The link between international trade and the global distribution of invasive alien species[END_REF]. In more temperate climate conditions, most of these species cannot survive in their area of introduction and are only intercepted at ports of entry [START_REF] Boer | Exotic ants in The Netherlands (Hymenoptera: Formicidae)[END_REF], but the few that survive and settle can rapidly become invasive. Tropical species are usually constrained by local climates, but infrastructures that are heated all year round provide shelter to many introduced species (e.g. [START_REF] Bauer | An analysis of weed floras in nurseries: Do polytunnels serve as ports of entry for alien plant species?[END_REF]. Comparing the species that can only survive in heated buildings with the invasive species living outdoor can help determine which traits are associated with invasiveness.

Nineteen ant species (Hymenoptera: Formicidae) are listed in the Global Invasive Species Database of the International Union for the Conservation of Nature (Global Invasive Species Database <www.iucngisd.org/gisd/>, accessed on 11.IX.2017), five of which are in the top one hundred world's worst invasive alien species list. Some members of the Formicidae family are experts at colonising new habitats and are well-adapted to human-mediated dispersion. In France, outdoor invasive species are well-studied and monitored by the public science project AntArea (www.antarea.fr), which maps all the ants of France living outdoors. However, ants introduced to the inside of buildings are poorly studied and there are no recent surveys available to this day. [START_REF] Rasplus | Hymenoptera. Chapter 12[END_REF] provide a list of Hymenoptera alien to Europe and list 16 ant species alien to France, which is likely an underestimation. The little data available are only rarely linked to a specific location or to a museum specimen. To fill this gap, we reviewed the literature and conducted a survey with the help of tropical building managers. We report this review here to assist those working in buildings with climate conditions similar to those in tropical environments to better know the introduced ant species that they may encounter.

Although plant trade is more likely to be the main point of entry for exotic ant species, exotic ants are rarely observed in plant shops (authors personal observations), possibly because of the high plant turnover or insecticide treatments.

We focused on public and private greenhouses open to the public because plants are often left undisturbed, allowing ant colonies to develop, which facilitates their detection. Greenhouses import a considerable amount of plants from the Neotropics, a region of the world considered as an important source of introduced ant species [START_REF] Miravete | How many and which ant species are being accidentally moved around the world?[END_REF]. Moreover, greenhouses are considered historical entry points for exotic ant species (e.g. [START_REF] Boer | Exotic ants in The Netherlands (Hymenoptera: Formicidae)[END_REF].

Materials and Methods

We considered data from: 1) the literature and personal observations, when species determination was verified by inspecting specimens (including those in museums) or judged unambiguous because the species has characteristic features that differentiate it clearly from other species (for example, the very particular color pattern of Tapinoma melanocephalum), and 2) data collected in greenhouses and tropical buildings in France and Belgium between 2014 and 2016. During this period, we solicited 26 organizations managing tropical greenhouses and obtained or collected samples from 11 of them. We also asked the employees of these structures to report any damage caused by the ants. We decided to focus on greenhouses because introduced ant species are particularly abundant and easy to detect in these structures. Species identification followed recent taxonomic work and specific keys (e.g. [START_REF] Bolton | Taxonomy of the dolichoderine ant genus Technomyrmex Mayr (Hymenoptera: Formicidae) based on the worker caste[END_REF], Bolton & Fischer 2011[START_REF] Seifert | Hypoponera ergatandria (Forel, 1893) -a cosmopolitan tramp species different from H. punctatissima (Roger, 1859) (Hymenoptera: Formicidae)[END_REF][START_REF] Sarnat | Introduced Pheidole of the world: taxonomy, biology and distribution[END_REF], and comparison with original descriptions and type specimens (we used high resolution images of type specimens provided by the AntWeb project, available from http://www.antweb.org). For most of the new records presented in this study, identification relied on tens of workers. Specimens are kept in the personal collections of the authors.

We classified the species based on [START_REF] Bolton | Bolton's catalogue of ants of the world: 1758-2005[END_REF] (We did not consider records of ants intercepted at entry points):

-Temporarily introduced ants: non-native ants that are found at the place of first record, and do not spread to other areas, forming populations that eventually go extinct.

-Maintained introduced ants: non-native ant populations for which the survival depends directly on human buildings, and that have spread to areas outside the place of first record. These species do not form stable populations outdoors.

-Permanently introduced ants: non-native ant species forming permanent populations outdoors. They can also be found indoors.

Categorization of ant species depends on the spatial extent and time period considered. Categorization in this paper was defined for the beginning of the 21 st century in France and Belgium (i.e. the same species may belong to a different category in another country). We decided not to report records of indigenous species, of species intercepted only at entry points, and of species that are indigenous to parts of France and Belgium but introduced in other areas, for example, Tapinoma magnum, Tapinoma darioi and Tetramorium immigrans. The first two species share traits with invasive species in many urban areas where they have been recently introduced [START_REF] Seifert | Four species within the supercolonial ants of the Tapinoma nigerrimum complex revealed by integrative taxonomy (Hymenoptera: Formicidae)[END_REF]), but there is some uncertainty regarding their origin and they may have been introduced from the French Mediterranean coast. The third species has most likely been introduced from Anatolia or Caucasia, but the date and place of introduction to Western Europe are unknown [START_REF] Wagner | Light at the end of the tunnel: integrative taxonomy delimits cryptic species in the Tetramorium caespitum complex (Hymenoptrea: Formicidae)[END_REF].

Results

We recorded 30 ant species that are either permanently introduced (three species), maintained introduced (15 species) or temporarily introduced (12 species) in mainland France or Belgium (Table 1). All occurrences of introduced species reported in this article are available online in the supplementary material section (Table S1).

List of records of the permanently introduced species

The following three species have established permanent and stable populations outdoor in France and/or Belgium. FRANCE -On the Atlantic coast, from Pyrénées-Atlantique to Morbihan, and more rarely away from the coast (Fig. 1c). This species is known from France since 1970 (Yeux island, J. Casevitz-Weulersse).

Lasius neglectus

List of records of the maintained introduced species found in mainland France and

Belgium

The following species, except for Hypoponera punctatissima, can only survive indoors in France and Belgium.

Brachymyrmex sp. (sub-family Formicinae)

BELGIUM -Tropical greenhouses of the botanical garden of Brussels [50°51.30'N 4°21.90'E], 1908, J. Bondroit [START_REF] Bondroit | Fourmis exotiques importées au jardin botanique de Brussels[END_REF] [START_REF] Bondroit | Les fourmis de France et de Belgique[END_REF]; Bonifacio [41°23.4'N 9°9.6'E], Ferton [START_REF] Bernard | Faune de l'Europe et du bassin méditerranéen. 3. Les fourmis (Hymenoptera Formicidae) d'Europe occidentale et septentrionale[END_REF]).

Nylanderia vividula (Nylander, 1846) (sub-family Formicinae) FRANCE -Reported to be inside heated greenhouses with no precise location [START_REF] Bernard | Faune de l'Europe et du bassin méditerranéen. 3. Les fourmis (Hymenoptera Formicidae) d'Europe occidentale et septentrionale[END_REF]. Wegnez [START_REF] Dekoninck | First record of infestation of the ghost ant Tapinoma melanocephalum (Fabricius, 1793) in Belgium (Hymenoptera, Formicidae)-A new indoor pest problem for the country[END_REF]. Tropical aquatic park of Oostduinkerke FRANCE -Mentioned inside greenhouses without any details [START_REF] Bondroit | Les fourmis de France et de Belgique[END_REF][START_REF] Bernard | Faune de l'Europe et du bassin méditerranéen. 3. Les fourmis (Hymenoptera Formicidae) d'Europe occidentale et septentrionale[END_REF]).

Tetramorium groupe simillimum (sub-family Myrmicinae) FRANCE -Mentioned inside greenhouses without any details [START_REF] Bernard | Faune de l'Europe et du bassin méditerranéen. 3. Les fourmis (Hymenoptera Formicidae) d'Europe occidentale et septentrionale[END_REF]).

List of records of the temporarily introduced species

Aphaenogaster gemella (Roger, 1862) (sub-family Myrmicinae). Outdoor in Saintes-Maries-de-la-mer [43°27.0'N 4°25.8'E], in Camargue and in Villefranche-sur-Mer, FRANCE [43°42.0'N 7°18.6'E], F. Bernard [START_REF] Bernard | Les fourmis et leur milieu en France méditerranéenne[END_REF]). These ants have not been found ever since. [START_REF] Dekoninck | An update of the checklist of the Belgian ant fauna with comments on new species for the country (Hymenoptera, Formicidae)[END_REF].

Camponotus barbaricus

Detection of the maintained introduced species

Our study in mainland France and Belgium revealed the presence of three permanently introduced and fifteen maintained ant species. We recorded a hundred occurrences of these fifteen maintained ants, and fifty-three of these occurrences had not been published before. The number of occurrences is highly variable between species (Fig. 2), ranging from one to twenty-one. Hypoponera punctatissima is the most commonly fund ant, mostly outdoors, but it is unsure whether this ant is an introduced species. The other most common species are Hypoponera ergatandria, Tapinoma melanocephalum, Monomorium pharaonis and Technomyrmex vitiensis (Fig. 2).

Forty-eight of one hundred occurrences came from twenty-two tropical greenhouses and tropical aquatic parks (among which eleven were surveyed specifically for this study) that were investigated to find ants. Between one and five species of maintained introduced ants live in each of these buildings (1.8 on average). The building where five species have been mentioned is the tropical greenhouse of the Jardin des Plantes de Paris, where occurrences span between 1878 and 2016. However, the five species have not been reported simultaneously. In this paper we reported occurrences dating from 1856 to 2017, 72% of which were collected after 1990.

Discussion

The main results of this study are the detection of exotic ants in every heated building we inspected and of Technomyrmex (T. vitiensis or T. difficilis) ants in five of the eleven buildings investigated between 2014 and 2016.

The first part of the discussion is dedicated to the description of the biology for the permanently (first three species) and maintained introduced ants, which are the most at risk of becoming invasive.

Lasius neglectus. Dark brown ant, about 3.5 mm long (Fig. 3a). In France, it is hardly distinguished from two indigenous species: Lasius alienus and Lasius lasioides. It is native to Western Asia and has invaded Europe recently [START_REF] Seifert | Rapid range expansion in Lasius neglectus (Hymenoptera, Formicidae) -an Asian invader swamps Europe[END_REF]. This species is well adapted to temperate climates. It is commonly found on roadsides [START_REF] Gippet | I'm not like everybody else: urbanization factors shaping spatial distribution of native and invasive ants are species-specific[END_REF]) and can live inside houses, where it is considered a pest. It has been found inside greenhouses in some countries [START_REF] Dekoninck | Records of a new pest ant species in Belgium, Technomyrmex vitiensis Mann[END_REF]) and protects sap-feeding hemipterans (especially scale insects) while feeding on their honeydew [START_REF] Van Loon | A new polygynous Lasius species (Hymenoptera: Formicidae) from central Europe. I. Description and general biology[END_REF]). Each colony is composed of several nests, each of which contains many queens [START_REF] Van Loon | A new polygynous Lasius species (Hymenoptera: Formicidae) from central Europe. I. Description and general biology[END_REF]). Lasius neglectus is the only strongly polygynous Lasius species in Europe. Colonies can extend and disperse by budding. Contrary to other Lasius species, virgin females do not seem to fly to mate or disperse [START_REF] Seifert | Rapid range expansion in Lasius neglectus (Hymenoptera, Formicidae) -an Asian invader swamps Europe[END_REF][START_REF] Espadaler | Biological constraints and colony founding in the polygynous invasive ant Lasius neglectus (Hymenoptera, Formicidae)[END_REF]. The rapid expansion of this species seems to result from accidental human-mediated dispersion that commonly occurs for polygynous species.

Linepithema humile. Brown ant, 2.5 to 3 mm long (Fig. 3b). Native to the watershed of the Paraná River in South America [START_REF] Wild | Taxonomy and distribution of the Argentine ant, Linepithema humile (Hymenoptera : Formicidae)[END_REF], it has been introduced outdoors in most of the regions of the world with a Mediterranean climate, at the end of the 19 th century [START_REF] Wetterer | Worldwide spread of the Argentine ant, Linepithema humile (Hymenoptera: Formicidae)[END_REF]). In cooler areas, it is only found in some buildings. It has a negative impact on biodiversity where it is introduced [START_REF] Cole | Effects of the Argentine ant on arthropod fauna of Hawaiian high-elevation shrubland[END_REF]). After the first phase of expansion, some populations began to decline [START_REF] Cooling | The widespread collapse of an invasive species: Argentine ants (Linepithema humile) in New Zealand[END_REF]. This ant can be found along the Mediterranean coast from Spain to Italy, in Corsica and on the Atlantic coast in Portugal, where it forms supercolonies across thousands of kilometres along the coast [START_REF] Giraud | Evolution of supercolonies: the argentine ants of Southern Europe[END_REF]): a series of small nests are exchanging queens and brood without any sign of aggression. Only three supercolonies have been described in Europe [START_REF] Blight | Variation in the level of aggression, chemical and genetic distance among three supercolonies of the Argentine ant in Europe[END_REF], suggesting a low number of independent introductions. It is a common ant in European greenhouses and is sometimes encountered in North-American greenhouses [START_REF] Wetterer | Worldwide spread of the Argentine ant, Linepithema humile (Hymenoptera: Formicidae)[END_REF]). It protects hemipterans and feeds on the honeydew they produce [START_REF] Vega | The Argentine ant -A significant invasive species in agricultural, urban and natural environments[END_REF]. Nests can be found in various ground cavities and they all contain multiple queens. New colonies are formed by budding. Outside of the buildings, this species goes through a hibernation period, which may also be the case in greenhouses with passive heating systems. It is hard to eradicate this ant and cooling down greenhouses during winter does not seem efficient. Other introduced species like Technomyrmex ants can outcompete Argentine ants inside greenhouses, but may be equally problematic for plants.

Monomorium carbonarium.

Entirely black and shiny, 2 mm long (Fig. 3c). This species is known from the Azores, Madeira and the Atlantic coast of Portugal, Spain, and France [START_REF] Espadaler | Transferred ants in the Iberian Peninsula[END_REF]. The Atlantic coast populations have most likely been introduced from Macaronesia. This species is mostly found outdoors [START_REF] Espadaler | Transferred ants in the Iberian Peninsula[END_REF][START_REF] Galkowski | Quelques fourmis nouvelles ou intéressantes pour la faune de France (Hymenoptera, Formicidae)[END_REF]), but they can also survive inside buildings (Odilon Redon High School, Pauillac, France, C. Galkowski).

They are mostly found in humid areas with loamy sandy soils on the coast. Apart from the coast, it is mostly found in urban areas around pools. These ants nest directly in the ground. Each nest is composed of many workers and queens. When introduced to a new area, they often become dominant and reduce the ant diversity in the area [START_REF] Galkowski | Quelques fourmis nouvelles ou intéressantes pour la faune de France (Hymenoptera, Formicidae)[END_REF].

Brachymyrmex sp. Light brown, 1.5 to 2 mm long. Brachymyrmex is a tropical genus that needs taxonomic revision. Different species have settled outside of their natural area and many were accidentally introduced inside greenhouses. Most species nest in the ground. Some of them protect root hemipterans.

Hypoponera ergatandria. Light brown, about 2.5 mm long (Fig. 3d). This species is morphologically like to Hypoponera punctatissima and is considered as a synonym species by some authors [START_REF] Bolton | Taxonomy of Afrotropical and West Palaearctic ants of the ponerine genus Hypoponera Santschi (Hymenoptera: Formicidae)[END_REF]. This tropical ant is probably native to Africa and was accidentally introduced outdoor everywhere in the World between the tropics. In Europe, it has been found inside heated buildings (greenhouses, plant stores, museums) in the following countries: Belgium, Germany, England, Spain, France, Holland, Poland, Czech Republic, Switzerland [START_REF] Bernard | Faune de l'Europe et du bassin méditerranéen. 3. Les fourmis (Hymenoptera Formicidae) d'Europe occidentale et septentrionale[END_REF][START_REF] Boer | Exotic ants in The Netherlands (Hymenoptera: Formicidae)[END_REF][START_REF] Seifert | Hypoponera ergatandria (Forel, 1893) -a cosmopolitan tramp species different from H. punctatissima (Roger, 1859) (Hymenoptera: Formicidae)[END_REF] and is most likely present in most tropical greenhouses in Europe. It is unsure yet whether it could survive outdoor like H. punctatissima. Hypoponera ergatandria lives exclusively underground and hunts small arthropods under leaf litter. It does not forage on plants and does not protect any hemipterans on the upper parts of the plants. Inside greenhouses, these ants dig galleries under flowerpots, stones, dead trees, and branches wherever the soil is humid. It can be hard to detect this ant that quickly runs away from the light, but was still found in four French greenhouses since 2015 and may be even more common.

Each colony contains multiple queens [START_REF] Seifert | Hypoponera ergatandria (Forel, 1893) -a cosmopolitan tramp species different from H. punctatissima (Roger, 1859) (Hymenoptera: Formicidae)[END_REF]. Queens are from two different types: some have wings and can fly, while some are "ergatoid" queens morphologically similar to workers. Mating occurs inside the nest [START_REF] Seifert | Hypoponera ergatandria (Forel, 1893) -a cosmopolitan tramp species different from H. punctatissima (Roger, 1859) (Hymenoptera: Formicidae)[END_REF]). This species is mostly dispersed by humans carrying plants or soil. Surprisingly, it is the only introduced Ponerinae species found inside buildings in France. Furthermore, none of the samples we collected inside buildings were identified as H. punctatissima that has very similar traits, even though this one is mentioned outdoor in France and Belgium [START_REF] Wegnez | Fourmis de Wallonie (2003-2011)[END_REF] <http://antarea.fr/fourmi/?repartition/repertition-especes.html?espece=5>).

Hypoponera punctatissima. Light brown, about 2.5 mm long (Fig. 3e). [START_REF] Bolton | Taxonomy of Afrotropical and West Palaearctic ants of the ponerine genus Hypoponera Santschi (Hymenoptera: Formicidae)[END_REF] describe the biology of this species precisely but it is not possible to determine whether they refer to H. punctatissima or H. ergatandria as they consider these species as synonyms. This species seems to be anthropophilic and may have been introduced by human activities in many parts of the world [START_REF] Delabie | The tramp ant Hypoponera punctatissima (Roger) (Hymenoptera: Formicidae: Ponerinae): new records from the southern hemisphere[END_REF]. [START_REF] Mcglynn | The worldwide transfer of ants: geographical distribution and ecological invasions[END_REF] consider this species as native to Europe, but other authors mention it as an introduced species [START_REF] Bolton | Taxonomy of Afrotropical and West Palaearctic ants of the ponerine genus Hypoponera Santschi (Hymenoptera: Formicidae)[END_REF][START_REF] Wegnez | Fourmis de Wallonie (2003-2011)[END_REF][START_REF] Seifert | Hypoponera ergatandria (Forel, 1893) -a cosmopolitan tramp species different from H. punctatissima (Roger, 1859) (Hymenoptera: Formicidae)[END_REF]. In France and Belgium, it is mostly found outdoors and rarely indoors (Belgium). Most of the specimens that were collected are swarming drones and reproductive females. In contrast to Hypoponera ergatandria, it is rarely found inside greenhouses. It is unsure yet whether there are stable outdoor populations of this species; the ants found outside buildings might just be forming temporary nests after introductions or come from colonies swarming from the buildings. This species lives exclusively in the ground and in the litter, where it preys on small arthropods. It does not forage on plants or protect hemipterans.

Monomorium pharaonis. Yellow with dark gaster, about 2 mm long (Fig. 3f). This tropical species, native to Asia (Wetterer 2010), has been introduced worldwide.

Between the tropics it is mostly found outdoor and indoor in urban areas. Under temperate climate it can only survive in heated buildings but has been mentioned outdoors multiple times [START_REF] Wetterer | Worldwide spread of the pharaoh ant, Monomorium pharaonis (Hymenoptera: Formicidae)[END_REF]. It seems to live in houses and hospitals [START_REF] Cornwell | The incidence of pest ants in Britain[END_REF][START_REF] Edwards | Distribution and importance of the Pharaoh's ant Monomorium pharaonis (L) in National Health Service Hospitals in England[END_REF]. In hospitals, it is a risk for public health as it can carry diseases [START_REF] Beatson | Pharaoh's ants as pathogen vectors in hospitals[END_REF]) and contaminate sterile equipment. This species is common in buildings [START_REF] Bondroit | Les fourmis de France et de Belgique[END_REF][START_REF] Bernard | Faune de l'Europe et du bassin méditerranéen. 3. Les fourmis (Hymenoptera Formicidae) d'Europe occidentale et septentrionale[END_REF]) and can be found in most European countries [START_REF] Wetterer | Worldwide spread of the pharaoh ant, Monomorium pharaonis (Hymenoptera: Formicidae)[END_REF]. It was the first introduced species reported in France, along with Paratrechina longicornis [START_REF] Nylander | Synopsis des formicides de France et d'Algérie[END_REF]. However, little is known about its occurrence in France and Belgium. A single colony can be made of multiple nests with multiple queens. Nests can be found in any type of gap, even those very narrow. Colonies migrate frequently and can disperse by budding, facilitating accidental dispersion by humans.

Nylanderia vividula. Brown, about 2 mm long (Fig. 3g). Head and gaster darker, with many thick erected hairs. The Nylanderia genus is found worldwide and has more than 130 species that can hardly be distinguished from one another.

Nylanderia vividula is native to North America. Despite many Nylanderia species being invasive, taxonomists started to pay attention to this genus only recently (LaPolla et al. 2011;[START_REF] Kallal | Monograph of Nylanderia (Hymenoptera: Formicidae) of the world, part II: Nylanderia in the Nearctic[END_REF]. For this reason, it is challenging to identify specimens that have been introduced outside of their natural range (LaPolla et al. 2011). Ants previously identified as Nylanderia vividula in the literature may belong to other species. Nylanderia ants have been found in many European countries, inside buildings but also outdoors in the extreme South of Europe.

Paratrechina longicornis. Dark brown, about 2.5 mm long. Body with many thick, long erected hairs, symmetrical along the bilateral body plan of the ant (Fig. 3h).

Very long antennae. Paratrechina longicornis is a tropical species probably native to South-East Asia [START_REF] Wetterer | Worldwide spread of the longhorn crazy ant, Paratrechina longicornis (Hymenoptera: Formicidae)[END_REF]. It has been introduced worldwide and has invaded the coasts and disturbed habitats of the tropics [START_REF] Wetterer | Worldwide spread of the longhorn crazy ant, Paratrechina longicornis (Hymenoptera: Formicidae)[END_REF]. Under temperate climates, it is only found in heated buildings and has been observed in most European countries. In France, it was reported from 1856 to 1968 inside greenhouses, especially in Paris [START_REF] Nylander | Synopsis des formicides de France et d'Algérie[END_REF][START_REF] Emery | Catalogo delle formiche esistenti nelle collezioni del Museo Civico de Genova. Parte seconda. Formice dell'Europa e delle regioni limitrofe in Africa e in Asia[END_REF][START_REF] André | Species des hyménoptères d'Europe et d'Algérie[END_REF][START_REF] Emery | Beiträge zur Monographie der Formiciden des paläarktischen Faunengebietes. (Hym.). Teil[END_REF][START_REF] Bondroit | Les fourmis de France et de Belgique[END_REF][START_REF] Bernard | Faune de l'Europe et du bassin méditerranéen. 3. Les fourmis (Hymenoptera Formicidae) d'Europe occidentale et septentrionale[END_REF]). We however did not detect this ant inside greenhouses during this study. This species has been observed protecting hemipterans and collecting honeydew [START_REF] Wetterer | Ecological dominance by Paratrechina longicornis (Hymenoptera: Formicidae), an invasive tramp ant, in biosphere 2[END_REF][START_REF] Wetterer | Worldwide spread of the longhorn crazy ant, Paratrechina longicornis (Hymenoptera: Formicidae)[END_REF]. A single colony can be made of many nests containing multiple queens [START_REF] Yamauchi | Social structure and reproductive systems of tramp versus endemic ants (Hymenoptera: Formicidae) of the Ryukyu Islands[END_REF]. Workers depend on sexual reproduction, but queens are genetically identical to their mothers and drones to their fathers [START_REF] Pearcy | Sib mating without inbreeding in the longhorn crazy ant[END_REF].

Pheidole anastasii. Yellow ant. The cuticle is punctuated on the whole body, resulting in a dull aspect. Just like for any Pheidole species, there is a strong dimorphism between the major workers that measure 3 to 3.5 mm and the minor workers that are only about 2 mm long. This tropical species comes from Central America. It has been seldom mentioned in North American and European greenhouses. The morphological differences between this species and Pheidole bilimeki have only been established recently [START_REF] Longino | Pheidole bilimeki reconsidered (Hymenoptera: Formicidae)[END_REF]. Some identifications in previous works might thus refer to Pheidole bilimeki [START_REF] Sarnat | Introduced Pheidole of the world: taxonomy, biology and distribution[END_REF]. Pheidole anastasii is omnivorous and workers forage on the ground and on the trees. They nest in any type of plant cavities [START_REF] Longino | Pheidole bilimeki reconsidered (Hymenoptera: Formicidae)[END_REF].

Pheidole bilimeki. Brown ant. The cuticle is punctuated on the whole body, resulting in a dull aspect (Fig. 3i). Just like Pheidole anastasii, there is a strong dimorphism between the major workers that measure 3 to 3.5 mm and the minor workers that are only about 2 mm long. This tropical species comes from Central America [START_REF] Sarnat | Introduced Pheidole of the world: taxonomy, biology and distribution[END_REF]. It has been seldom seen in greenhouses in North America and in the following European countries: Germany, United Kingdom, The Netherlands (identified as Pheidole anastasii according to [START_REF] Sarnat | Introduced Pheidole of the world: taxonomy, biology and distribution[END_REF], Ireland, Switzerland [START_REF] Forel | Fourmis de Costa-Rica, récoltées par M. Paul Biolley[END_REF][START_REF] Stelfox | A list of the Hymenoptera Aculeata (sensu lato) of Ireland[END_REF][START_REF] Boer | Exotic ants in The Netherlands (Hymenoptera: Formicidae)[END_REF]. In their natural area, these ants are considered as pests because they enter houses [START_REF] Longino | Pheidole bilimeki reconsidered (Hymenoptera: Formicidae)[END_REF]. They are omnivorous and nest in the ground.

Pheidole megacephala. Brown (Fig. 3j). Just like for any Pheidole species, there is a strong dimorphism between the major workers that measure 3 to 3.5 mm and the minor workers that are only about 2 mm long. This is a tropical species originating from Africa [START_REF] Wetterer | Worldwide spread of the African big-headed ant, Pheidole megacephala (Hymenoptera: Formicidae)[END_REF]. Following its accidental dispersal by humans, it is now widespread outdoors between the tropics and inside heated buildings in Europe and New-Zealand [START_REF] Wetterer | Worldwide spread of the African big-headed ant, Pheidole megacephala (Hymenoptera: Formicidae)[END_REF]. This highly invasive species has been seen in the buildings of the following European countries: England, The Netherlands, Germany, France and Italy [START_REF] Wetterer | Worldwide spread of the African big-headed ant, Pheidole megacephala (Hymenoptera: Formicidae)[END_REF]). It protects sap-feeding hemipterans and collects their honeydew [START_REF] Wheeler | The ants collected by the American Museum Congo Expedition[END_REF][START_REF] Beardsley | Field investigations on the interrelationships of the big-headed ant, the gray pineapple mealybug, and pineapple mealybug wilt disease in Hawaii[END_REF][START_REF] González-Hernández | Survey of the natural enemies of Dysmicoccus mealybugs on pineapple in Hawaii[END_REF]. It nests in the ground, under rocks or under the bark of dead trees and in various types of cavities on the floor. Each colony has multiple queens. New colonies are formed by budding [START_REF] Beardsley | Field investigations on the interrelationships of the big-headed ant, the gray pineapple mealybug, and pineapple mealybug wilt disease in Hawaii[END_REF]. Budding facilitates human-mediated dispersal because colonies can be split and translocated by humans and form new nests.

Plagiolepis alluaudi.

Yellow ants of about 1.5 mm long (Fig. 3k). This tropical species originates from Africa, probably in Madagascar where it is commonly found in forests [START_REF] Wetterer | Worldwide spread of Alluaud's little yellow ant, Plagiolepis alluaudi (Hymenoptera: Formicidae)[END_REF]. It has spread to Asia and Oceania, and is found on a few islands of the American Atlantic coast. In Europe, it can be found inside the greenhouses of several countries (England, Switzerland, Ireland, Scotland, Germany and The Netherlands) [START_REF] Wetterer | Worldwide spread of Alluaud's little yellow ant, Plagiolepis alluaudi (Hymenoptera: Formicidae)[END_REF]). It protects sap-feeding hemipterans and feed on their honeydew (Gonzalez-Hernandez et al. 1999). A colony can be made of many nests containing multiple queens. Outdoors, it nests in a variety of very small spaces: under bark of dead trees, in the holes made by wood-boring insects, under grass clumps, to name a few. Indoors, it nests in cavities formed between the stipes, petioles or leaves and plant stems. It nests preferentially above ground, but is commonly found under flowerpots. The nests are opportunistic, and the ants do not usually build structures or dig burrows. Each nest contains multiple queens, which facilitates their dispersal when materials or plants are exchanged. These ants are in addition very small and pale yellow, and can be hard to detect when they are present in small numbers.

Tapinoma melanocephalum. Bicolor ant (head and thorax dark brown, gaster light yellow) of about 1.5 mm (Fig. 3l). This tropical species is native to the Indo-Pacific region (Wetterer 2009a). It has been accidentally introduced everywhere between the tropics by human activities. Under temperate climate in the northern hemisphere, it has been observed inside the buildings (houses, restaurants, hospitals, tropical greenhouses) in the following countries: Canada, USA, England, Scotland, Germany, Finland, Sweden, Norway, France, Belgium, Switzerland, Italy, Spain, Austria, Denmark, The Netherlands, Romania, and Russia (Wetterer 2009a). In France, it is thought to be present in every tropical greenhouse, but has only been mentioned twice in the literature. This species protects various species of sapfeeding insects and feed on their honeydew [START_REF] Venkataramaiah | Ants associated with the mealybugs of coffee[END_REF]. It nests in a range of cavities, is very opportunistic and do not build or dig much. A whole nest can sometimes be found between two dead leaves. Colonies are made of many connected nests and usually contain many queens. Nests are regularly moved. These life traits can explain why it has often been dispersed by humans when translocating materials, plants or goods.

Technomyrmex difficilis. Black ant, 3.5 to 4 mm (Fig. 3m). This tropical species is probably native to Madagascar [START_REF] Wetterer | Worldwide spread of the difficult white-footed ant, Technomyrmex difficilis (Hymenoptera: Formicidae)[END_REF]. It has been introduced to the southern part of North America, in South-East Asia, and in Oceania [START_REF] Wetterer | Worldwide spread of the difficult white-footed ant, Technomyrmex difficilis (Hymenoptera: Formicidae)[END_REF]).

In the USA, it has been observed inside buildings in temperate climate. However, the actual distribution of this species is poorly understood since it was only distinguished from Technomyrmex albipes in 2007 [START_REF] Bolton | Taxonomy of the dolichoderine ant genus Technomyrmex Mayr (Hymenoptera: Formicidae) based on the worker caste[END_REF]. It had never been reported in Europe before. Even though we detected it only in one building in France, it was well established in this greenhouse and is at risk of being introduced to other places, especially if flower pots were to be translocated. We therefore decided to classify this species as a maintained introduced ant. This species protects sap-feeding hemipterans and feeds on their honeydew [START_REF] Warner | Bait preferences and toxicity of insecticides to white-footed ants Technomyrmex albipes (Hymenoptera: Formicidae)[END_REF]. Outdoors, nests are mostly found in the cavities formed between the stipes, petioles, and leaves when they are close to the stem, and more rarely in cavities at the ground level and in masonry cracks. As with other ants in the Technomyrmex genus, each colony is composed of many temporary nests containing very few queens but many egg-laying inter-caste (a morphological mix between queens and workers) [START_REF] Bolton | Taxonomy of the dolichoderine ant genus Technomyrmex Mayr (Hymenoptera: Formicidae) based on the worker caste[END_REF].

Colonies can disperse by budding.

Technomyrmex vitiensis. Black ant, 3.5 to 4 mm (Fig. 3n). This tropical species is probably native to South-East Asia. It has been introduced to many tropical greenhouses in Europe: Germany, Austria, Belgium, The Netherlands (identified as T. albipes), United Kingdom, Czech Republic [START_REF] Bolton | Taxonomy of the dolichoderine ant genus Technomyrmex Mayr (Hymenoptera: Formicidae) based on the worker caste[END_REF][START_REF] Boer | Exotic ants in The Netherlands (Hymenoptera: Formicidae)[END_REF][START_REF] Dekoninck | Records of a new pest ant species in Belgium, Technomyrmex vitiensis Mann[END_REF][START_REF] Pech | Ergatomorph wingless males in Technomyrmex vitiensis Mann, 1921 (Hymenoptera: Formicidae)[END_REF]. It has also been found in San Francisco in the USA [START_REF] Bolton | Taxonomy of the dolichoderine ant genus Technomyrmex Mayr (Hymenoptera: Formicidae) based on the worker caste[END_REF]. It had never been reported in mainland France before. This species protects sap-feeding hemipterans and feeds on their honeydew [START_REF] Bolton | Taxonomy of the dolichoderine ant genus Technomyrmex Mayr (Hymenoptera: Formicidae) based on the worker caste[END_REF]. Nests are mostly found in the natural cavities occurring in plants, for example between the stipes, petioles, and leaves when they are close to the stem. They can also be found in the litter, in pieces of dead wood and under the stones [START_REF] Bolton | Taxonomy of the dolichoderine ant genus Technomyrmex Mayr (Hymenoptera: Formicidae) based on the worker caste[END_REF]. They do not build or dig much, and they move their nests rapidly when disturbed. As for other Technomyrmex species, each colony is made of multiple nests that contain very few queens and many inter-caste individuals [START_REF] Oettler | Polymorphism of female reproductives in the tramp ant Technomyrmex vitiensis (Hymenoptera: Formicidae: Dolichoderinae)[END_REF]. Colonies can disperse by budding. With such traits, it seems likely that human activities can disperse this species, especially when relocating plants.

Tetramorium bicarinatum. Brown-orange with a darker gaster, about 4 mm long (Fig. 3o). The cuticle is highly sculpted, reticulated on the thorax and with longitudinal wrinkles on the head. This is a tropical species native to the Indo-Pacific.

It has been introduced to every tropical and subtropical regions of the world (Wetterer 2009b). However, it is uncommon in continental Africa and Western Asia.

In Europe, it is found inside greenhouses and heated buildings in many countries (Germany, England, Austria, Belgium, Scotland, Spain, France, Hungary, Northern Ireland, Italy, Monaco, Norway, The Netherlands) (Wetterer 2009b). This species is highly competitive. It maintains and protects sap-feeding hemipterans and feeds on their honeydew (Mann 192;[START_REF] Greenslade | Climate variability, biological control and an insect pest outbreak on Australia's Coral Sea islets: lessons for invertebrate conservation[END_REF]. It is omnivorous, with workers foraging on the ground and on plants. Nests are found in the ground.

Tetramorium groupe simillimum. Yellow to light brown, 2-2.5 mm long (Fig. 3p).

Cuticle with thin wrinkles. Species in the Tetramorium simillimum group are native to tropical Africa (Wetter & Hita Garcia 2015). Tetramorium simillimum (Smith, 1851) and Tetramorium caldarium (Roger, 1857) are invasive and have been mentioned in different greenhouses and heated buildings in Europe. Not long after they were described, these two species were considered synonyms, and then distinct species again in 1979 [START_REF] Bolton | The ant tribe Tetramoriini (Hymenoptera: Formicidae). The genus Tetramorium Mayr in the Malagasy region and in the New World[END_REF]. Tetramorium simillimum has only been mentioned by [START_REF] Bernard | Faune de l'Europe et du bassin méditerranéen. 3. Les fourmis (Hymenoptera Formicidae) d'Europe occidentale et septentrionale[END_REF] from "various greenhouses" in France. Since the two species were considered synonyms at this time, it is not possible to determine which species was mentioned. However, Tetramorium caldarium seem more common inside greenhouses and heated buildings in Europe [START_REF] Wetterer | Worldwide spread of Tetramorium caldarium (Hymenoptera: Formicidae)[END_REF].

Ants from the Technomyrmex albipes group were already known from most tropical greenhouses throughout Europe, but had never been reported in France so far.

These ants seem to have invaded European greenhouses recently. They are only commonly reported by the end of the 20 th century. It would be unlikely that they went unnoticed by the many famous European myrmecologists of the end of the 19 th and of the beginning of the 20 th century. For example, Paratrechina longicornis, Monomorium pharaonis and Tetramorium bicarinatum were already reported respectively by [START_REF] Nylander | Synopsis des formicides de France et d'Algérie[END_REF][START_REF] Nylander | Synopsis des formicides de France et d'Algérie[END_REF][START_REF] Bondroit | Les fourmis de France et de Belgique[END_REF]in French buildings (Nylander 1856;[START_REF] Bondroit | Les fourmis de France et de Belgique[END_REF]) and in Dutch buildings in 1909, 1877and 1909respectively (Boer & Vierbergen 2008), whereas Technomyrmex ants were first reported in France and

The Netherlands in 2014 and 1988 respectively. Paratrechina longicornis and Tetramorium bicarinatum were not detected during our 2014-2016 survey.

Monomorium pharaonis was not detected either and seem to only survive in houses and apartments. Our study shows that the composition of the community of introduced ants in heated buildings varies with time, similarly to reports from [START_REF] Boer | Exotic ants in The Netherlands (Hymenoptera: Formicidae)[END_REF]. Changes in plant trade and importations could partially explain this [START_REF] Bertelsmeier | Recent human history governs global ant invasion dynamics[END_REF], and the introduction of new species of ants may have led to other species disappearing as observed with other invasive species [START_REF] Lohrer | Interactins among aliens: apparent replacement of one exotic species by another[END_REF]. The growing awareness about various animal species living in the soil has also led to the adoption of new regulations restricting soil importations. These regulations may have discouraged the introduction of groundnesting species but not of arboreal ant species.

We investigated whether tropical greenhouses could be entry points for introduced species because they regularly import exotic plants. However, the three invasive ant species that survive outside greenhouses in France, Linepithema humile, Lasius neglectus and Monomorium carbonarium, have not been introduced through greenhouses. On the contrary, species like Paratrechina longicornis and Tetramorium bicarinatum, considered invasive in tropical and sub-tropical areas, have never been found outside buildings even though they are known from greenhouses for more than a hundred years. Tropical ants have narrower temperature ranges than ants from temperate climates [START_REF] Diamond | Who likes it hot? A global analysis of the climatic, ecological, and evolutionary determinants of warming tolerance in ants[END_REF], which limits the invasiveness of these species in France and Belgium. These results suggest that greenhouses are not currently the main source of invasive species, and highlight the need to collaborate with customs to investigate the role of plant or ant trade in the introduction of exotic species. It is unclear whether climate change will one day allow these species to spread from greenhouses and survive outdoors, although many of the ants we found inside heated buildings had shared features with known outdoor invasive ant species [START_REF] Bertelsmeier | Recent human history governs global ant invasion dynamics[END_REF]. The list we provide along with key biological features is a first step to assess risks of future invasions in mainland France and Belgium.

Presently, introduced ants that are found indoors are more problematic for the plants, since they protect and disperse sap-feeding hemipterans. When asked about inconveniences caused by the ants, the staff members did not report any for the buildings where Tapinoma melanocephalum was dominant. On the contrary, damages caused by Linepithema humile, Plagiolepis alluaudi and Technomyrmex ants were systematically reported. The staff members reported ants protecting and dispersing sap-feeding insects (especially scale insects) (Fig. 4b and4c) and sometimes killing biological control agents (parasitoid hymenopterans) (Fig. 4d). Some of them also reported that biological control agents were inefficient when ants were present. Ant baits were reported to decrease the number of ants, although they did not allow for the eradication of the colonies. Decreasing the temperature during several weeks in winter, a method used by the Naturospace of Honfleur, could help in reducing the number of ants, but cannot be applied to every kind of buildings. Some of these organizations also introduced natural predators, such as birds (Naturospace) or dendrobates (botanical garden of Meise, Belgium). The Amazonian greenhouse of the Lunaret zoo of Montpellier also has free flying birds, but this does not seem to have a significant impact on ant populations. A clear need for the development of environmentally friendly methods to control these ants was identified. We suggest that new plants should be quarantined in buckets surrounded by water and closely inspected to detect the presence of exotic insects before being moved inside heated buildings. Original soil should also be removed, heated above 60° C and replaced.

In temperate climates, tropical greenhouses are like islands for tropical ants, providing them with limited space and resources. We often observed a clear dominance of one species in each building. For example, Linepithema humile was dominant in the greenhouses of the botanical gardens of Meise (Belgium) and Tours Plagiolepis alluaudi (b) and Technomyrmex vitiensis (c) protecting sap-feeding hemipterans. Technomyrmex vitiensis workers colonizing a piece of cardboard (splitted in two to show the colony) used to release biological control agents against hemipterans (d). Photos: Théotime Colin (a, b) and Rumsaïs Blatrix (c, d).

Online supplementary material:

Table S1: Occurrence data for temporary, maintained and permanently introduced ants in France and Belgium.

  Van Loon, Boomsma & Andrasfalvy, 1990 (sub-family Formicinae) BELGIUM -Outdoor, in Ghent [51°2.4'N 3°43.2'E] since 1978 at least (Dekoninck et al. 2002), and in Flémalle [50°36.0'N 5°26.4'E], 2008, D. Ignace (Dekoninck et al. 2010). Indoor in the botanical garden of the University of Ghent [51°2.10'N 3°43.38'E] (Dekoninck et al. 2010). FRANCE -Outdoor in the Mediterranean area, in Corsica, on the Atlantic coast, along the Loire and Rhone rivers, more rarely in other areas (Fig. 2a). It can be found inside buildings, which is also the case for several indigenous species of Lasius, and has been found in the dry greenhouse of the botanical garden of Nantes [47°13.26'N 1°32.64'W] (Gouraud 2015) and in private houses in Saint-Désirat (Ardèche) [45°15'N 4°46.8'E]. Linepithema humile (Mayr, 1868) (sub-family Dolichoderinae) known as the « Argentine ant » BELGIUM -Greenhouses of the botanical gardens of Brussels [50°51.30'N 4°21.90'E], J. Bondroit (Bondroit 1911), of Ghent University [51°2.10'N 3°43.38'E], summer 2004, W. Dekoninck (Dekoninck, Maelfait, et al. 2006), and of Meise [50°55.50'N 4°19.80'E], 18.IV.2016, P. Wegnez. FRANCE -Outdoor it is mostly found along the Mediterranean coastline including Corsica, and is seldom found in the South-West of France (Fig. 1b). It can also survive in buildings like in the Paul Sabatier University in Toulouse [43°33.516'N 1°28.278'E], T. Colin and A. Dejean, or in the Eyzies biological station [44°56.4'N 1°1.2'E], B. Darchen (Galkowski 2008), the tropical greenhouses of the Jardin des Plantes de Paris [48°50.586'N 2°21.432'E], C. Galkowski (Galkowski 2008), and of Bordeaux [44°50.658'N 0°33.522'W], XI.2007, C. Galkowski (Galkowski 2008), the Amazonian greenhouse of the Lunaret zoo of Montpellier [43°38.358'N 3°52.440'E], 07.II.2014, R. Blatrix, and the tropical greenhouse of the botanical garden of Tours [47°23.364'N 0°40.020'E], 13.III.2015, R. Cézé et M. Froger. Monomorium carbonarium (Smith, 1858) (sub-family Myrmicinae)

  Paratrechina longicornis (Latreille, 1802) (sub-family Formicinae) BELGIQUE -Tropical aquatic park of De Haan [51°17.034'N 3°3.810'E], 11.XI.2010, W. Dekoninck and D. Brouckaert (Dekoninck & Brouckaert 2011). FRANCE -Tropical greenhouses in Paris [48°48'N 2°18'E] (Nylander 1856; Emery 1878; André 1883; Emery 1910; Bondroit 1918). Pheidole anastasii Emery, 1896 (sub-family Myrmicinae) FRANCE -Tropical greenhouse in Paris [48°48'N 2°18'E] (Bernard 1968). Pheidole bilimeki Mayr, 1870 (sub-family Myrmicinae) BELGIQUE -Tropical greenhouse of the botanical garden of Brussels [50°51.30'N 4°21.90'E], J. Bondroit[START_REF] Bondroit | Fourmis exotiques importées au jardin botanique de Brussels[END_REF].

FRANCE-

  Heated greenhouse in a plant store in Bordeaux [44°48'N 0°36'W], 2005, C. Galkowski (Casevitz-Weulersse & Galkowski 2009). Pheidole megacephala (Fabricius, 1793) (sub-family Myrmicinae) FRANCE -Heated buildings in Bordeaux [44°48'N 0°36'W], Lyon [45°48'N 4°48'E], and Paris [48°48'N 2°18'E] (Bernard 1968). Large tropical greenhouse of the Jardin des Plantes de Paris [48°50.586'N 2°21.432'E], 5.IV.2016, R. Blatrix et P. Wegnez. Plagiolepis alluaudi Emery, 1894 (sub-family Formicinae) BELGIQUE -Tropical greenhouse of the park Pairi Daiza à Brugelette [50°35.70'N 3°51.78'E], 28.IV.2016, P. Wegnez. Tropical greenhouse of the plant observatorium Sart Tilman in Liège [50°34.692'N 5°35.292'E], V.2016, P. Wegnez. FRANCE -Large tropical greenhouse of the Jardin des Plantes de Paris and associated buildings [48°50.58'N 2°21.42'E], 5.IV.2016, R. Blatrix et P. Wegnez. Tropical greenhouse of the botanical garden Henri Gaussen of Paul Sabatier University in Toulouse [43°35.6292'N 1°26.9628'E], VI.2016, N. Séjalon-Delmas. Tapinoma melanocephalum (Fabricius, 1793) (sub-family Dolichoderinae) BELGIQUE -House in Liège [50°37.716'N 5°34.908'E], 7.II.2006, 22.V.2006, P.

[

  51°7.92'N 2°42.72'E], 25.IV.2011, W. Dekoninck et D. Brouckaert (Dekoninck & Brouckaert 2011). FRANCE -Tropical greenhouse of Auteuil, Paris [48°50.778'N 2°15.126'E], 2002 (Casevitz-Weulersse & Galkowski 2009); VII.2008, H. Darras; V.2016, T. Colin. greenhouse of the botanical garden of Meise [50°55.50'N 4°19.80'E], 18.IV.2016, P. Wegnez. FRANCE -Amazonian greenhouse of the Lunaret zoo of Montpellier [43°38.358'N 3°52.440'E], 7.II.2014, 5.III.2015, 7.IX.2017, R. Blatrix. Acclimatisation greenhouse of the Lunaret zoo of Montpellier [43°38.346'N 3°52.620'E], 5.III.2015, R. Blatrix. Tropical greenhouse of the Naturospace of Honfleur [49°25.536'N 0°13.764'E], 24.III.2015, B. Mery. Tropical greenhouse of the botanical garden and conservatorium of Nancy [48°39.756'N 6°9.234'E], 24.VI.2015, L. Gardeux et A. Bour. Tropical greenhouse of Auteuil, Paris [48°50.778'N 2°15.126'E], V.2016, T. Colin. Tetramorium bicarinatum (Nylander, 1846) (sub-family Myrmicinae) BELGIQUE -Building in Herstal [50°40.2'N 5°37.8'E], V.2004, P. Wegnez (Dekoninck, Maelfait, et al. 2006). Tropical greenhouse of the park Pairi Daiza in Brugelette [50°35.70'N 3°51.78'E], 28.IV.2016, P. Wegnez.

  Emery, 1905 (sub-family Formicinae). Outdoor in Draguignan, FRANCE [43°32.4'N 6°27.6'E], H. Cagniant (Casevitz-Weulersse & Galkowski 2009). Camponotus nylanderi Emery, 1921 (sub-family Formicinae). Outdoor in Cannes, FRANCE [43°33.0'N 7°1.2'E], H. Cagniant. Cardiocondyla obscurior Wheeler, 1929 (sub-family Myrmicinae). Paris, FRANCE [48°48'N 2°18'E], 01.V.2009, A. Verfaillie. Dorylus fulvus (Westwood, 1839) (sub-family Dorylinae). Outdoor in Ajaccio, FRANCE [41°55.2'N 8°44.4'E], 1899, Dr. Joseph, and Rochefort-sur-mer, FRANCE [45°56.4'N 0°6.0'W], Giraud (Bondroit 1918). Lepisiota nigra (Dalla Torre, 1893) (sub-family Formicinae). Outdoor in Bourg-Achard, FRANCE [49°21.0'N 0°49.2'E], 2010, P. Guérin. Nylanderia fulva (Mayr, 1862) (sub-family Formicinae). Outdoor in Saint-Raphaël, FRANCE [43°24.6'N 6°46.8'E], 1939-1948, F. Bernard (Bernard 1949). Oecophylla sp. (sub-family Formicinae). Inside buildings in Carcassonne, FRANCE [43°12.6'N 2°21.0'E] (J. Casevitz-Weulersse, personal communication) ; Nantes, FRANCE [47°12'N 1°36'W], 2015, F. Diemerts (ERFA (group) 2017). Plagiolepis schmitzii Forel, 1895 (sub-family Formicinae). Outdoor in Port-Vendres, FRANCE [42°31.2'N 3°6.6'E], L. Passera (H. Cagniant, personal communication). Technomyrmex albipes (Smith, 1861) (sub-family Dolichoderinae). Outdoor in Narbonne, FRANCE [43°10.98'N 3°0.12'E], 10.XII.2015, C. Lebas. Tetramorium calidum Forel, 1907 (sub-family Myrmicinae). Inside a building in Toulouse, FRANCE [43°37.2'N 1°22.2'E], J.-L. Marrou (H. Cagniant, personal communication). Tetraponera allaborans (Walker, 1859) (sub-family Pseudomyrmecinae). Outdoor in Engis, BELGIQUE [50°34.8'N 5°24'E], 2003, M. Dethier

(

  France); Plagiolepis alluaudi in the large greenhouse of the Jardin des Plantes de Paris (France) (Fig. 4a) and in the greenhouse of the botanical garden of Paul Sabatier University in Toulouse (France); Tapinoma melanocephalum in the greenhouses of the arboretum of Chevreloup (France), in the zoo of the Jardin des Plantes de Paris (France) and in the greenhouses of the botanical gardens of Grenoble, Metz and Strasbourg (France); Technomyrmex difficilis in the greenhouse of the botanical garden of Lyon (France); and Technomyrmex vitiensis in the Amazonian greenhouse of the Lunaret zoo in Montpellier, the Naturospace of Honfleur (France) and in the greenhouses of the botanical garden of Ghent University (Belgium). The limited resources could explain the low number of introduced species living simultaneously in the same greenhouse. Invasive species can compete directly for the same food sources (Lach 2005). In 2016, two of us inspected the large tropical greenhouse of the Jardin des Plantes de Paris (built at the end of the 19 th century) for three hours and found only four introduced species (three ground nesting species: Pheidole megacephala, Brachymyrmex cordemoyi and Hypoponera ergatandria, and one arboreal species: Plagiolepis alluaudi). During the repeated surveys in 2014, 2015 and 2017 in the Amazonian greenhouse of the Lunaret zoo of Montpellier (built in 2006), only three introduced species were found (two ground nesting species: Linepithema humile and Hypoponera ergatandria, and one arboreal species: Technomyrmex vitiensis). Linepithema humile was already common in this area before the greenhouse was built and was already well established inside the greenhouse in 2014 (personal observation, R. Blatrix). It was however not detected in 2015 and 2017, possibly because of Technomyrmex vitiensis was then dominant in this building. Inside greenhouses, interspecific competition is probably high and genetic diversity extremely low, which may also cause rapid changes in the composition of ant communities. These factors make greenhouses candidates for the study of island biogeography and metapopulation dynamics.

Table 1 :

 1 List of the alien ant species introduced in mainland France and Belgium (except those species only intercepted at entry points). F: France, B: Belgium, O: outdoor, I: indoor, MI: maintained introduced, PI: permanently introduced, TI: temporarily introduced, -: no data.

Figure 1 :

 1 Figure legends:

Figure 2 :

 2 Figure 2: Occurrences of maintained introduced ants in mainland France andBelgium. Data are represented in dark grey if collected before 1990 and light grey if they were collected after. We used date of publication as the year of observation for published data that were not associated with a collection date.

Figure 3 :

 3 Figure 3: Photos of the main introduced ant species in mainland France and Belgium. Photos from AntWeb (www.antweb.org); photographers: April Nobile (e, g, k, m, n, o, p), Erin Prado (f), Michele Esposito (b, d), Ryan Perry (c), Shannon Hartman (j), Will Ericson (a, h), Zach Lieberman (i, l).

Figure 4 :

 4 Figure 4: Presence and impact of exotic ants in tropical greenhouses. Plagiolepis alluaudi colony under a flowerpot (the pot has been moved to show the colony) (a).

  Figure 1.

Figure 2 .

 2 Figure 2.

  , identified by Bondroit as

	FRANCE -Large greenhouse of the Jardin des Plantes de Paris [48°50.586'N BELGIQUE -Mentioned with no precise location (Dekoninck, Maelfait, et al. 2006).
	2°21.432'E], 05.IV.2016, R. Blatrix et P. Wegnez. The specimens found are Tropical greenhouse of the botanical garden of Liège [50°38.10'N 5°33.72'E], 2012,
	morphologically similar to the ones from the island of la Réunion, France, where P. Wegnez. Different houses in Liège [50°36'N 5°36'E], P. Wegnez.
	Brachymyrmex cordemoyi Forel, 1895 was first described, and might belong to this FRANCE -Houses in Paris [48°48'N 2°18'E] (Nylander 1856; André 1883; Bernard
	species. 1968), in Corsica (Emery 1916), in Bordeaux [44°48'N 0°36'W], in Lyon [45°48'N
	4°48'E], and in Marseille [43°18'N 5°24'E], C. Galkowski. Mentioned outdoor in
	Hypoponera ergatandria (Forel, 1983) (sub-family Ponerinae) Corsica
	BELGIUM -Tropical greenhouse of the botanical garden of Brussels [50°51.30'N
	4°21.90'E], XI.1909, J. Bondroit (Bondroit 1911). Heated buildings in Maarkedal
	[50°48'N 3°36'E], 10.XI.1995 and winter 1996, P. Grootaert (Dekoninck, Maelfait, et
	al. 2006). Tropical greenhouse of the botanical garden of Meise [50°55.50'N
	4°19.80'E], 18.IV.2016, P. Wegnez. Tropical greenhouse of the park Pairi Daiza in
	Brugelette [50°35.70'N 3°51.78'E], 28.IV.2016, P. Wegnez. Tropical greenhouse of
	the plant observatorium Sart Tilman in Liège [50°34.692'N 5°35.292'E], V.2016, P.
	Wegnez. Tropical greenhouse of the botanical garden of Liège [50°38.10'N
	5°33.72'E], 2012, P. Wegnez.
	FRANCE -Large tropical greenhouse of the Jardin des Plantes de Paris and
	associated buildings [48°50.58'N 2°21.42'E], J. Bondroit (Bernard 1968); 05.IV.2016,
	R. Blatrix et P. Wegnez. Acclimatisation and Amazonian greenhouses of the Lunaret
	zoo of Montpellier [43°38.346'N 3°52.620'E], 05.III.2015, 07.IX.2017, R. Blatrix.
	Tropical greenhouse of the botanical garden of Metz [49°6.264'N 6°9.642'E],
	VI.2015, A. Rotini. Zoo of the Jardin des Plantes de Paris [48°50.70'N 2°21.54'E],
	Brachymyrmex heeri Forel, 1874. 5.IV.2016, R. Blatrix et P. Wegnez. Tropical greenhouse of Auteuil, Paris

Table 1 .

 1

	46

Acknowledgements

We thank the staff members of the artificial tropical spaces that have allowed us to collect or directly sent us samples of ants, and helped us all along the study: botanical garden of Caen (N. Hubert); Chèvreloup arboretum (J.-M. Doremus) ; Museum of Natural History of Grenoble (S. Poletti) ; Naturospace of Honfleur (B. Damico, B. Mery) ; butterfly greenhouse of Hunawihr (Mr. Bueche) ; botanical garden of Liège ; botanical garden of Lyon (P. Avoscan, P. Boucheix, G. Cianfarani, G. Deparis, D. Deruaz, A. Donzet, F. Lardiere, F. Pautz, C Rimbaud, E. Rostaing, J.-M. Tete, F. Trescarte) ; botanical garden of Meise ; botanical garden of Metz (A. Rotini) ; Lunaret zoo of Montpellier (L. Gomel, D. Gomis, A. Françoise, R. Guicherd) ; botanical garden conservatory of Nancy (A. Bour , L. Gardeux, L. Peru, M. Remy) ; Pairi Daiza park of Brugelette ; Museum of Natural History of Paris (K. Ben Youb, E. Joly, S. Languille, F. Lemoine, J. Munier, R. Nattier, B. Noël, M. Saint Jalme, F. Saint-Hillier) ; Ecological station of Paris 7 University (O. Babiar) ; botanical garden of Strasbourg University (F. Tournay) ; botanical garden Henri Gaussen of Paul Sabatier University in Toulouse (N. Séjalon-Delmas) ; botanical garden of Tours (R. Cézé, M. Froger, R. Saulnier). We also would like to thank B. Kaufmann and J. Gippet for sharing all of their data on the occurrence of Lasius neglectus with us. We thank H. Cagniant, H. Callot, J. Ho-Huu and C. Lebas for the personal observations and ant samples they shared with us. Data from Corsica have

been obtained with the financial support of the Corsican Office for the Environnement and AntArea. We thank Duncan Jaroslow for his useful comments on the English version of the manuscript.


Vanessa Durrieu

Jean-Luc Putaux

Raphaël Passas

Alessandro Gandini

I N T R O D U C T I O N

The interest in aqueous-based resins for various applications such as coating or in the pharmaceutical field has recently increased due to the need for constant cost reductions and the necessity to control emissions of volatile organic compounds. As most polymers used in these applications are water insoluble, aqueous-based resins generally contain particles in suspension. The average particle size and size distribution are essential data on which the stability and properties of the dispersions depend [START_REF] Sudol | Emulsion Polymerization and Emulsion Polymers[END_REF]. Light scattering and cryo-transmission electron microscopy (cryo-TEM) have proved to be complementary techniques to characterise various natural [START_REF] Putaux | Ultrastructural aspects of phytoglycogen from cryo-TEM and quasi-elastic light scattering[END_REF] or synthetic [START_REF] Chalaye | Synthesis of composite latex particles filled with silica. Control of the composite particles composition[END_REF] polymer nanoparticles dispersed in water. Light scattering is non-destructive and provides fast measurements in a wide range of particle size. However, it cannot analyse double populations, large distributions, absorbent materials, or mixtures of substances with different densities [START_REF] Collins | Measurement of Particle Size and Particle Size Distribution[END_REF]. In this study, cryo-TEM and image analysis were combined to characterise a variety of aqueous polyurethane dispersions. The results were compared with those obtained from dynamic light scattering (DLS) experiments.

M AT E R I A L S A N D M E T H O D S

Aqueous polymer dispersions

Polyurethane-urea dispersions were prepared according to the method described by Lahtinen et al. [START_REF] Lahtinen | The chain extension of anionic prepolymers in the preparation of aqueous poly(urethane-urea) dispersions[END_REF]. A polyurethane pre-polymer with reactive isocyanate chain ends was synthesised and dispersed in water. Chain extension was carried out and nanoparticles formed. Several dispersions were studied, varying the nature and proportion of the raw materials (di-isocyanates, polyols and emulsifying agents).

Light scattering

A B S T R A C T

Light scattering is routinely used to measure the average particle size in aqueous polymer dispersions. However, difficulties arise for polymodal populations, nanometric particles, absorbent materials or mixtures of materials with different densities. A complementary approach consists in combining cryo-TEM and image analysis. Using these techniques, we studied various polyurethane aqueous dispersions. The nanoparticles were characterised in terms of morphology and size. Average diameters, calculated using a semiautomatic image analysis procedure, were compared to those obtained from dynamic light scattering.

K E Y W O R D S

polymer nanoparticles, cryo-TEM, image analysis, size distribution

A C K N O W L E D G E M E N T S

The authors thank SICPA S.A. (Annemasse, France) for their financial and technical support. an angle of 90˚. The Z-average mean diameter D Z(DLS) was calculated using the cumulant analysis (standard method ISO 13321). Dispersions exhibiting a polydispersity index (as defined by the spectrometer) higher than 0.25 were discarded from our comparative study.

A U T H O R D E TA I L S

Cryoelectron microscopy

According to the method described elsewhere [START_REF] Dubochet | Cryo-electron microscopy of vitrified specimens[END_REF][START_REF] Harris | Negative Staining and Cryoelectron Microscopy: the Thin Films Techniques[END_REF], thin liquid films of 1 mg/ml nanoparticle dispersions were prepared onto 'lacey' carbon films (Pelco, USA) and quench-frozen into liquefied ethane (Fig 1). Once transferred into a Gatan 626 cryoholder, the specimens were observed at -180˚C using a Philips CM200 cryomicroscope operated at 80 kV. Micrographs were recorded on Kodak SO163 films at magnifications of 11,500 and 20,000, with a defocus of 1-2 µm.

Image analysis and size distribution

The negatives were digitised using a Kodak Megaplus CCD camera. The magnification was chosen in order to include a large number of particles in the field of view, while maintaining a sufficient resolution. A sampling rate of 2 nm/pixel was considered to be a good compromise. Contrast enhancement and semiautomatic particle measurements were performed using routines from the Optimas 6.51 software (Media Cybernetics, Inc.) [START_REF]Optimas user guide[END_REF]. The number-average diameter D N , the weightaverage diameter D W , as well as a polydispersity index P D were defined using the following formulas:

where N i is the number of particles at the i th class in the size-distribution histogram and For each sample, size-distribution histograms were determined from a total of about 1000 particles accumulated from several cryo-TEM images.

W i = ∑D i N i ∑N i i i D w P D D w D n = = = ∑W i D i ∑W i i i ∑N i D i 4 ∑N i D i 3 i i ∑D i N i D 3 ∑N i D 3 i i =

R E S U LT S A N D D I S C U S S I O N

TEM observation

Figure 2a shows polyurethane nanoparticles after negative staining with uranyl acetate. Since the glass transition temperature (Tg) of the polymer was lower than room temperature (varying from -60 to 0˚C depending on the polymer composition), the soft particles could deform and flow during drying. Moreover, we could not decide whether the observed coalescence arose from a drying artifact or if such an aggregation existed in solution. The cryo-TEM image in Fig 2b clearly shows unaggregated spheroidal nanoparticles embedded in vitreous ice. More complex particles were also observed, as shown in Fig 2c ,where the larger objects exhibit a peculiar nano-encapsulated morphology, suggesting that the pre-polymer synthesis and the chain extension could not form homogeneous nanospheres. This is a typical example where the direct visualisation of the dispersion provided information about the mechanism of particle formation, whereas DLS would be insensitive to this type of morphological feature.

Image analysis

The contrast between the polymer particles and the embedding ice was generally low in cryo-TEM images, partly due to the strong inelastic electron scattering. Contrast enhancements were performed but they also increased the contribution of the background noise from the negatives. On the other hand, variations in the ice thickness induced background intensity modulations. Consequently, the grey-level histogram of the cryo-TEM micrographs generally exhibited a broad peak (Fig 3a) and it was difficult to define luminance classes corresponding to specific contributions from the supporting membrane, the particles and the embedding medium. To limit these problems, the treatments were performed on smaller regions of interest (ROI) selected by the user (Fig 3b). To get a better contrast, the TEM micrographs were recorded with a negative defocus, generating fresnel fringes around the ice-embedded objects.

Considering the high background noise, only a white fringe located outside of the particle surface could be seen. However, as we mostly used 1 to 2 mm defoci, we assumed that fresnel effects would not significantly influence the particle detection.

The main steps of the image processing procedure were: 6. Fitting of the individual objects with equivalent ellipses, the diameter being calculated as the mean between major and minor axes.

In order to validate our semi-automatic procedure, we checked that 'manual' measurements performed on the same images and numbers of particles yielded similar results. However, as expected, the semi-automatic image analysis generally ran four times faster. An example or size distribution histogram is presented in Fig 4b.

Surface tension effects may influence the particle distribution during sample preparation. Controlling the thickness of the vitreous ice film is generally difficult and depends on the diluent composition (salts, surfactants, organic solvents), the room humidity level, the blotting efficiency, etc. Thickness values that are proper for TEM imaging typically vary from 50 to 200 nm [START_REF] Harris | Negative Staining and Cryoelectron Microscopy: the Thin Films Techniques[END_REF]. The presence of a single layer of particles in the film is important, as illustrated in Fig 5a : the larger nanospheres are more or less isolated, while the smaller ones overlap along the observation axis. In that case, size-distribution analysis is hardly possible, although the operator still has a good general 'impression' on the suspension dispersity.

In addition, due to surface tension effects, the frozen film often exhibits a gradient of thickness. Consequently, the average background intensity in the image varies over the field of view. As explained earlier, binarisation can only be properly carried out after background fitting and correction. Another consequence is the fast reorganisation of the particles in the liquid film, just before freezing. The largest particles tend to accumulate near the edges of the carbon membrane where the water film is thicker. The smaller particles remain closer to the centre of the film. Depending on the local concentration and steepness of the gradient, regular distributions may be observed, the nanospheres reorganising along iso-thickness lines according to their size (Fig 5b). However, we cannot rule out the fact that some bigger objects may have migrated towards areas that are too thick for proper observation. These 'hidden' particles would thus be absent from the statistic treatment, which would yield an error in the mean diameter calculation. When With a Tg lower than room temperature, the suspended nanoparticles are soft in the liquid film and thus sensitive to the surface tension effects. If their size in the dispersion is larger than the local thickness of the liquid film, and provided that they cannot 'migrate' to thicker areas, the nanoparticles are squeezed within the thin film. Their apparent diameter thus increases, yielding an incorrect spread of the size-distribution histogram towards larger diameters. Indeed, it is difficult to visualise such an effect and judge if the particles are actually deformed. In our experiments, we assumed that the particle size might be unreliable above 100-120 nm.

After binarisation, the image analysis algorithm performs several erosions/dilatations which, depending on the particle shape, may generate an error on the contours. This error was estimated to be around 1 pixel, i.e. 2 nm, considering our sampling rate. Moreover, the successive erosions, carried out during the image 'cleaning', eliminated objects smaller than 5-6 pixels. Consequently, particles smaller than 10 nm were not properly taken into account in our size-distribution histograms. This is readily visible in Fig 4b although the effect was not detected in most samples.

Finally, several remarks can be made when comparing the mean diameters obtained by DLS and image analysis. Values corresponding to four different dispersions with increasing polydispersity index P D are given in Table 1. First of all, there is a significant difference between the values of D N and D Z(DLS) which increases at higher polydispersity. This could be expected, since the detection is based on different properties of the particles. In particular, DLS gives more statistical 'weight' to the larger particles which are more highly scattering objects. Moreover, DLS is sensitive to the ionic environment of the nanospheres that cryo-TEM cannot visualise since it has the same density as the embedding medium. The comparison of D Z(TEM) (calculated from the cryo-TEM data as a D(6,5) statistical mean) with D Z(DLS) is more relevant. D Z(DLS) always appears to be larger than D Z(TEM) . The values are close when P D is low, but the deviation becomes larger with increasing polydispersity. The fourth case, with P D =1.50, perfectly illustrates how cautious one has to be to interpret and compare the data obtained with techniques sensitive to different properties of the particles.

C O N C L U S I O N

In this work, several techniques were used concurrently to study aqueous dispersions of polyurethane nanoparticles. Light scattering was non-destructive and allowed us to characterise rapidly a large number of samples over a wide range of sizes. However, it showed some limitations in the case of polydisperse distributions and with particles smaller than 50 nm. The combined use of cryo-TEM and image analysis appeared to be complementary to DLS. In particular, the absence of aggregation as well as differences in nanoparticle morphology were important results provided by this direct visualisation. However, the size analysis of soft polymer particles only made sense for diameters below 100 nm. Moreover, the smaller objects (<10 nm) may not have been properly detected. These limits would obviously vary for different polymers, depending on factors such as particle density, crystallinity and Tg. In addition, the use of zeroloss energy-filtered imaging would certainly increase the contrast of cryo-TEM micrographs and consequently improve particle detection.

  DLS was carried out using a Malvern Autosizer 2c photon correlation spectrometer (Malvern Instruments, Orsay, France) equipped with a HeNe laser (10 mW, 632.8 nm) and operated at B I O G R A P H Y Dr Vanessa Durrieu received a PhD in materials science from the National Polytechnique Institute of Grenoble, France, in October 2002. She worked on the development and characterisation of aqueous polyurethane dispersions. She also has a master's degree in materials science and graduated as an engineer from the French Engineering School of Printing and Papermaking in Grenoble.

Figure 1 :

 1 Figure 1: (a) Production of thin vitrified films for cryo-TEM: 1. The excess of suspension was blotted with a filter paper; 2. The grid was quench-frozen into liquefied ethane at -171°C. (b) Schematic transverse view of a vitreous ice film containing polymer particles.

1 .

 1 Manual selection of a rectangular ROI (Fig 3b, step 1). 2. Local adaptive background smoothing to homogenise the luminance and improve object detection: this routine fits a polynomial surface to the sampled background and corrects the ROI luminance by subtracting this surface [8]. 3. Manual thresholding based on a visual control, followed by an image binarisation (Fig 3b, step 2). 4. Elimination of background heterogeneities using successive erosions/dilatations. 5. Edge detection of the particles (Fig 3b, step 3) and separation process.

Figure 2 :Figure 3 :

 23 Figure 2: (a) TEM image of polyurethane particles after negative staining. (b) Similar dispersion observed by cryo-TEM. (c) Cryo-TEM of a polymer dispersion with a different composition, showing particles with a complex inner morphology.

Figure 5 :

 5 Figure 5: Effect of the embedding liquid thickness on the distribution of polydisperse particles. (a) The thickness is more or less constant. Particles are mixed but the smaller ones overlap along the observation direction. (b) Regular reorganisation of the nanospheres in a thickness gradient. The larger particles are closer to the carbon membrane where the liquid film is thicker.

Figure 4 :

 4 Figure 4: (a) Cryo-TEM micrograph of a polyurethane suspension. (b) Corresponding size-distribution histogram obtained by semi-automatic image analysis, for a total of about 1000 particles. The shoulder in the low diameter region suggests a bimodal distribution.

Table 1 :

 1 Mean

diameters and polydispersity indexes calculated from cryo-TEM images and DLS experiments for four different polyurethane dispersions.