From Pampas to Patagonia: Human-Modified Environments Drive the Spread of the Argentine Ant Beyond Its Climatic Limits

Abstract

The Argentine ant (Linepithema humile) is a highly invasive species with a widespread global distribution. However, the dynamics of its recent expansion into southwestern Argentina remain unclear. We evaluated its spread, niche shifts, and genetic diversity using field surveys, distribution models, and mitochondrial DNA analyses. Our results revealed a strong expansion from Pampas into anthropogenic habitats (multiple urban and rural sites) in the Monte Desert, but not into natural habitats. The Argentine ant’s presence declined westward along the Río Negro Valley in the Monte Desert, and was virtually absent from the Patagonian Steppe, where it was found only in urban sites in Bariloche (ecotone with the Patagonian Forest). The distribution models identified isothermality and water balance as the key drivers of suitability. Thus, urbanization and irrigated agriculture seem to have facilitated its establishment in the Río Negro Valley by buffering climate extremes. Genetic analyses revealed widespread and novel haplotypes, which are consistent with multiple introductions and potential regional differentiation. This southward expansion underscores the critical role of urban areas and irrigated agriculture as refuges and stepping stones that facilitate Argentine ant survival in otherwise inhospitable environments. Continued monitoring of transitional zones in northern Patagonia is crucial to determining whether this front will remain stable or shift further south.

1. Introduction

Biological invasions are a major driver of global biodiversity loss, ecosystem degradation, and socio-economic disruption. Invasive alien species (IAS) can alter the structure of native communities and disrupt ecosystem processes, and in extreme cases, drive local extinctions, threatening both environmental integrity and human well-being [1,2,3,4]. IAS have contributed to nearly 60% of documented animal and plant extinctions and negatively impacted human welfare in over 85% of evaluated cases [3,4]. These impacts highlight the urgent need to improve our capacity to detect, understand, and manage IAS to safeguard biodiversity, sustain ecosystem services, and reduce long-term ecological and economic costs.

The geographic expansion of IAS typically follows a three-stage trajectory: introduction, establishment, and spread, each shaped by species traits, environmental conditions, and human-mediated pathways [5]. Blackburn et al. [6] conceptualize these stages as sequential barriers that species must overcome, including geographical, survival, reproduction, dispersal, and environmental barriers. Initial colonization often occurs in novel or disturbed habitats with low biotic resistance, while subsequent spread depends on factors such as physiological tolerances, landscape connectivity, and propagule pressure [7,8]. Among these factors, cities have emerged as key hubs of invasion. Urban areas facilitate species introductions through trade and transport, provide diverse novel disturbed habitats, and often exhibit reduced competition and predation pressures [9,10,11]. Furthermore, urban heat islands raise local temperatures compared to surrounding rural areas, potentially enabling thermally limited species to persist and spread beyond their climatic constraints [12,13]. As a result, cities frequently act as stepping-stones for biological invasions into adjacent semi-natural and natural ecosystems [14,15].

Ants are among the most successful invasive taxa, largely due to traits such as their small body size, social structure, generalist foraging, cryptic nesting habits, and high adaptability to disturbed environments [16,17]. Over 500 ant species have been recorded outside their native ranges, and many exert substantial ecological and economic impacts, displacing native fauna and altering ecosystem functions [16,18,19]. Urban areas and global transport networks have significantly facilitated their long-distance dispersal, while their local establishment is influenced by microhabitat conditions and temperature regimes often shaped by urbanization and climate change [20,21].

The Argentine ant, Linepithema humile, is recognized as one of the world’s most problematic invasive ant species [22], renowned for its ability to invade and dominate a wide range of ecosystems through numerical superiority [16,23]. Native to the temperate, humid regions of South America’s Río de la Plata Basin, including northeastern Argentina, it has also invaded drier areas beyond its native range within Argentina [24,25]. Native populations or invasive ones already established in Argentina, such as those reported by Vogel et al. [25] and Schulze-Sylvester et al. [26] in western regions of the country, may also serve as sources for secondary invasions, illustrating the bridgehead effect [27]. Bridgehead populations might even evolve traits that enhance invasiveness (e.g., higher tolerance to cold or desiccation), boosting their ability to establish and spread into more desert regions compared to native populations. The rapid evolution of thermal tolerance has only been documented in native ant populations, such as the acorn ant (Temnothorax curvispinosus), across an urban–rural temperature gradient in the United States. However, this highlights that invasive populations could also adapt quickly to temperature clines, enhancing their establishment and spread in regions previously considered climatically unsuitable [28].

The cold climate and pronounced annual seasonality of Patagonia are considered natural barriers to the spread of the species, as supported by previous species distribution models that predict very limited habitat suitability for L. humile in this region [29,30,31,32]. Nevertheless, highly invasive genetic variants widely distributed worldwide, or those previously introduced in western Argentina, could be expanding their niche southwestward within Argentina. Over the past 20 years, there were only a few unpublished and uncertain records of L. humile from anthropogenic environments in northern Río Negro (pers. obs.) and Chubut [24] provinces. This changed with the recent appearance of L. humile in San Carlos de Bariloche in western Patagonia [33], where the species appears to persist in close association with human activity [34].

This unexpected occurrence record suggests that urban microclimates, regional warming, and human-assisted dispersal may enable L. humile to colonize areas beyond its traditional climatic boundaries. For example, cities in the Río Negro Valley have experienced a temperature increase of approximately 1 °C since the 1960s [35,36], while urban heat island effects can elevate local temperatures by up to 4 °C above surrounding rural areas [37]. These combined thermal influences could be creating urban refuges that buffer L. humile against climatic constraints, thereby facilitating its introduction, persistence and expansion into southwestern Argentina. Although the impact of urbanization and climate change on invasion dynamics in the invasive range is well acknowledged, empirical knowledge on how L. humile is responding to these drivers in its native southern South America remains limited. In particular, little is known about whether expanding Argentine populations are shifting their ecological niche, whether they are genetically distinct or like other invasive populations worldwide, and if they are invading natural and semi-natural habitats beyond urban centers in northern Patagonia.

To address these gaps, this study integrates extensive field surveys, occurrence records, and genetic analysis to investigate L. humile’s ongoing expansion in Argentina. Specifically, we aim to: (1) assess the current distribution and spread patterns of L. humile over a 1000 km gradient from northern Buenos Aires to northern Patagonia; (2) detect their presence in the main cities of the Río Negro province (presumably invasive front) and the extent of colonization into semi-natural and natural habitats adjacent to these urban centers; (3) evaluate a potential niche shift of Argentine ant populations expanding into Patagonia; and (4) characterize mitochondrial haplotypes of populations expanding beyond the native range. This integrative approach will improve understanding of invasion dynamics in environments undergoing complex environmental changes and inform potential management strategies to mitigate impacts of one of the world’s most pervasive invasive ants.

2. Materials and Methods

2.1. Study Area

Field surveys were conducted during 2021 and 2022 in eight Argentine provinces in central Argentina (Buenos Aires, Santa Fe, Córdoba, San Luis, La Pampa, Mendoza, Neuquén, and Río Negro) to detect the presence of L. humile. A more intensive survey was then conducted in 2023 and 2024 in eight major cities in the province of Río Negro, in northern Patagonia, considered the potential southern expansion front of L. humile (Figure 1A). According to the 2022 Argentine National Population Census [38], the sampled cities in Río Negro Province ranged from 7429 inhabitants in General Conesa to 135,755 in San Carlos de Bariloche, with intermediate values such as 105,482 in Cipolletti, 108,680 in General Roca, 27,566 in Cinco Saltos, 33,034 in Villa Regina, 13,588 in Choele-Choel, and 59,993 in Viedma. Seven of these cities (Cinco Saltos, Cipolletti, General Roca, General Regina, Choele-Choel, General Conesa, and Viedma) are located along the Río Negro Valley within the Monte desert ecoregion. The eighth city, San Carlos de Bariloche, is the southernmost city where L. humile has been previously recorded [34] and is situated in the ecotone between the Patagonian Steppe and Forest ecoregions (Figure 1A). It is important to mention that due to the severe climatic conditions, the largest towns and cities in Patagonia (those with the highest populations) are situated near major roads and permanent water sources such as rivers, lakes, and lagoons. Consequently, much of Patagonia remains unpopulated, with few small towns or no human settlements.

2.2. Sampling Protocol

To detect the spread of the Argentine ant beyond its known native range (the Río de la Plata basin), surveys were conducted across natural (native), semi-natural (rural), and urban habitats in eight Argentine provinces, spanning four ecoregions: Pampa, Espinal, Monte, and Patagonian Steppe [39,40]. Collection sites were situated approximately 50 km apart along main and secondary roads (both paved and unpaved), as well as at town and city entrances. In urban areas, we surveyed public streets, parks, squares, vacant lots, and sidewalks. Sampling was not conducted within municipal, provincial, or national protected natural areas. At each visited location, we manually searched for foraging trails or colonies of L. humile on the ground, as well as the vegetation or nesting under or inside objects (e.g., stones, debris, wood, trash) for 20–30 min. Given the low tolerance to desiccation of this species [41], the nest search was focused on sites with permanent sources of water/humidity (sites with higher occurrence probability). Once found, a sample of 30–100 workers (and some occasional queens) was manually collected using soft forceps or hand-held aspirators. The samples were preserved in 2 mL vials containing 96% ethanol for further morphological examination under stereomicroscope at 60× and the genetic analysis.

To systematically assess the presence of invasive L. humile populations in the Río Negro province (presumed southern invasion front), nine sites were sampled within each habitat type (urban, rural, and natural) in each of the eight surveyed cities. The only exceptions were Viedma and Bariloche, where six sites per habitat type were sampled. To ensure independence, sites within each habitat and sampling period were separated by at least 200 m and sampled at three different times of day (morning, midday, and afternoon) to include much of the daily variation in temperature and humidity.

At each site, we employed two sampling methods:

• Baiting: Groups of five sugar-based baits (0.5–1 g peanut butter each) were placed on 7 cm diameter plastic cards on the ground at 10 m intervals to attract common or dominant ants, particularly highly invasive species such as L. humile [23]. The baits were left in place for 30 min. During this time, all ants attracted to the baits were collected with soft forceps or hand-held aspirators. The ants were then preserved together in a 2 mL vial containing 96% ethanol.
• Manual Collection: Manual searches for ants were conducted at each site, extending up to 100 m at distances greater than 10 m from the baited area (to avoid disturbing ant activity in bait locations). All ants found on the ground, in shrubby vegetation, and on buildings (e.g., house walls, perimeter walls, flowerbeds in urban habitats) were collected with a hand-held aspirator and soft forceps. The ants were also preserved in separate 2 mL vials with 96% ethanol.

In the laboratory, all samples were sorted and examined under a stereomicroscope to detect the presence of L. humile across cities, three habitat types (urban, rural, and natural), and sampling times (morning, midday, and afternoon).

Using the data from the systematic sampling, the relationship between the frequency of L. humile in each city (pooled bait and manual collection samples per site) and the population size of each city in the Río Negro Valley was analyzed using a non-parametric Spearman correlation. Here, we define the detection rate as the proportion of samples in which Argentine ants were found, compared to the total number of samples examined. A Firth logistic regression model was used to examine the influence of cities on detection rates (as the mean percent or frequency) across habitat types (natural, rural, and urban) in the cities surveyed along the Río Negro Valley. We chose this approach due to complete separation in the data, where some habitat categories (e.g., natural) had zero detections [42]. Standard maximum-likelihood logistic regression fails under such conditions, producing biased or infinite coefficient estimates [43]. Firth’s penalized likelihood logistic regression addresses this issue by shrinking coefficients to ensure stable and interpretable results [44]. Preliminary checks confirmed no perfect separation within cities but complete separation in natural habitats. There was also no multicollinearity (VIF < 2), an adequate sample size per city-habitat combination (minimum six sites), and a good model fit (Hosmer–Lemeshow p = 0.55; no overdispersion, p = 0.88).

All ant specimens were deposited in the biological collection of the Fundación para el Estudio de Especies Invasivas (FuEDEI), Buenos Aires. Except for those collected in San Carlos de Bariloche, which are stored at the Colección de Artrópodos del Centro Regional Universitario Bariloche (BACRU; Instituto de Investigaciones en Biodiversidad y Medioambiente–INIBIOMA, CONICET, Universidad Nacional Del Comahue, Bariloche).

2.3. Argentine Ant Distribution Models

To investigate whether the colonization of L. humile populations expanding into northern Patagonia could respond to a potential niche shift or urban areas facilitating the recent expansion into unsuitable regions, we applied ensemble species distribution models (SDMs). These ensemble species distribution models utilize species occurrence data and bioclimatic variables, combining multiple algorithms (including generalized linear and additive models, classification trees, and neural networks) to improve accuracy and minimize uncertainty. This approach predicts suitable habitats and detects potential niche shifts between native and introduced populations.

Occurrence data for L. humile were compiled, cleaned and validated from the Global Ant Biodiversity Informatics (GABI; [45]), the Global Biodiversity Information Facility (GBIF, [46]), and recent field surveys in Argentina (see Section 2.2), totaling 17,982 records (16,103 with coordinates). Newly occurrence records can be found in Table S2. After spatial thinning at 50 km to reduce sampling bias, 90 unique native-range records, 532 introduced-range records, and 622 combined records were retained. Two datasets were modeled independently: one with native-range occurrences and another including both native and introduced records. Environmental predictors included 19 bioclimatic variables from WorldClim v2 [47] and five from ENVIREM [48], cropped to South America. Eleven variables were kept after multicollinearity testing (using variable inflation factors), representing key climatic factors relevant to L. humile distribution.

Ensemble species distribution models were made, for the two datasets, using the ‘biomod2’ package, testing nine algorithms of machine learning, neural networks, decision trees, and regression models (see details in Supplementary Material) with four replicates of 1000 randomly generated pseudo-absence points and two cross-validation replicates (80% training data). Variable importance was evaluated across three random replicates to ensure robustness. Model performance was assessed via TSS, AUC, and Cohen’s kappa. Ensemble models were created by weighting individual models with TSS ≥ 0.7, selecting the weighted mean ensemble as the final robust prediction.

2.4. Genetic Analysis

Phylogenetic analyses were conducted on populations of L. humile collected in Argentina during 2021–2023. Following the protocols described by [49,50], specimens from both their putative native and introduced ranges within Argentina were genetically characterized using a 334 bp fragment of the mitochondrial Cytochrome c Oxidase subunit II (COII) gene, using the primers described in Vogel et al. [25]. Genomic DNA was extracted from one worker per colony per population. The COII fragment was amplified via polymerase chain reaction (PCR), purified, quantified, and subsequently sequenced by Macrogen Inc. In addition to our newly generated sequences, publicly available L. humile COII sequences from previous studies (e.g., [25,50]) were retrieved from GenBank, as well as closely related species like L. micans and L. oblongum. These sequences represent populations from other regions of southern South America as well as introduced populations worldwide, allowing for a comprehensive assessment of genetic relationships.

Sequence alignment was performed using Geneious Pro v4.8 (http://www.geneious.com/) with the Muscle algorithm [51]. Phylogenetic relationships among invasive L. humile populations from central Argentina and other invasive populations were inferred using IQ-TREE v2.2.2 [52], employing the best-fit substitution model selected by ModelFinder and branch support estimated with 1000 ultrafast bootstrap replicates. A haplotype network was subsequently constructed using the TCS algorithm [53] in PopArt v1.7 [54].

3. Results

3.1. Argentine Ant Spread

As a result of our surveys, we confirmed that L. humile has effectively extended its geographic distribution from southern Buenos Aires until northern Patagonia (Figure 1B–D), with the southwestern-most confirmed population established in San Carlos de Bariloche (Figure 1D). The occurrence pattern of L. humile over time and along latitudinal and longitudinal gradients (Figure 1B,C) seems to reveal a recent expansion of its geographical distribution range across the Monte, with a unique isolated small population in the ecotone between the Patagonian Steppe and Forest ecoregions (southward latitude ~39° S).

The Argentine ant has well-established populations in both natural and human-modified habitats within the Pampa and Espinal ecoregions. However, it maintains stable populations only in urban (cities) and semi-natural (rural and agricultural) anthropic habitats along the Río Negro Valley, which is part of the Monte Desert ecoregion (Figure 1A). Additionally, we found a few isolated populations in urban areas of some towns and cities in the southernmost portion of the Monte Desert ecoregion, such as San Antonio Oeste and Valcheta on the eastern coast of Río Negro, Villa El Chocón to the west, and the southernmost locations in the Patagonian Steppe/Forest ecotone in Bariloche. The absence of L. humile records in the natural habitats of the Monte and Patagonian Steppe/Forest ecoregions suggests that the species’ native range in southwestern Argentina ends approximately at the southwest limits of Espinal ecoregion (Figure 1A). Therefore, Argentine ant populations found in anthropogenic habitats in the Monte and in Bariloche (Patagonian Steppe/Forest ecotone) will correspond to the species’ potential invasive range.

In Bariloche, L. humile was only recorded in two urban sites located in public areas that were sampled at midday (Figure 2A). These are the same sites where the species was detected for the first time in 2019 [33]. It was not found in semi-natural habitats (e.g., fruit orchards) or in the native habitats of the Patagonian Steppe/Forest ecotone (Figure 2). In contrast, L. humile was common and abundant in anthropic habitats in the seven major cities surveyed across the Río Negro Valley (Figure 2B–G).

Figure 2. Map showing the presence (black points) and absence (grey points) of L. humile in urban (red), rural (dark green), and natural (light green) habitats in the eight cities surveyed in the province of Río Negro, Argentina. From west to east, a detailed view of each of the sampling sites: (A) Bariloche, (B) Cinco Saltos, (C) General Roca, (D) Villa Regina, (E) Choele-Choel, (F) General Conesa, and (G) Viedma.

Figure 2. Map showing the presence (black points) and absence (grey points) of L. humile in urban (red), rural (dark green), and natural (light green) habitats in the eight cities surveyed in the province of Río Negro, Argentina. From west to east, a detailed view of each of the sampling sites: (A) Bariloche, (B) Cinco Saltos, (C) General Roca, (D) Villa Regina, (E) Choele-Choel, (F) General Conesa, and (G) Viedma.

As expected, L. humile was detected exclusively in anthropogenic environments in Río Negro Province (Figure 2). In the Río Negro Valley, on average, the species occurred in 81% of urban sites and 55% of adjacent rural sites, including fruit orchards (e.g., apple and pear plantations). The Firth logistic regression model revealed a significant effect of habitat on detection rates (likelihood ratio test = 107.49, df = 9, p < 0.001). After adjusting for habitat type, city effects showed significantly increased detection odds in Choele-Choel (β = 2.46, 95% CI = 0.83–4.41, p = 0.002), Cipoletti (β = 2.21, 95% CI = 0.60–4.13, p = 0.006), and General Conesa (β = 2.21, 95% CI = 0.60–4.13, p = 0.006), General Roca (β = 1.97, 95% CI = 0.37–3.87, p = 0.015), Viedma (β = 3.67, 95% CI = 1.65–6.34, p < 0.001), and Villa Regina (β = 1.97, 95% CI = 0.37–3.87, p = 0.015). Cinco Saltos did not differ significantly from the reference city, Bariloche (β = 0.82, 95% CI = −0.82–2.71, p = 0.334). Overall, the predicted detection rates were highest in urban habitats in several cities, suggesting that the level of urbanization and city-specific factors strongly interact to shape detection rates. The between-cities difference in detection rates was evident in both urban and rural settings, with the most significant decline occurring in rural habitats (Figure 3A). The detection rate (mean percent or frequency) at urban cities of Río Negro Valley (excluding Bariloche, where it seems to survive only indoor) was strongly related with city population size (Spearman rank correlation test: r_s = 0.84, p = 0.017, Figure 3B).

3.2. Argentine Ant Distribution Models

The ensemble model for L. humile’s native range, built using a weighted mean approach based on TSS, showed high predictive accuracy (Table S1). It combined pseudo-absence data, cross-validation, and multiple algorithm outputs. Key metrics included ROC = 0.949, TSS = 0.841, sensitivity = 97.8%, and specificity > 86%. The moderate Kappa value (0.302) reflected conservative predictions, supporting the model’s reliability. The ensemble model for the combined native and introduced ranges also performed well, with ROC = 0.960, TSS = 0.784, sensitivity > 93.8%, and specificity > 84%. The higher Kappa (0.678) and specificity (96.4%) indicated stronger agreement but slightly reduced sensitivity, highlighting a trade-off favoring fewer false positives.

Isothermality (Bio03) consistently emerged as the most influential predictor in species distribution models for both the native dataset and the combined native-introduced dataset. In its native range, its mean importance was approximately 0.56 (Figure S6), while in the combined dataset it decreased to about 0.23 (Figure S6). In the native model, the next most important variables were the Thornthwaite aridity index, with a mean importance ≈ 0.23, and precipitation of the wettest month (Bio13), with an importance of ≈0.11. In contrast, for the combined dataset, the most influential variables after isothermality were evapotranspiration during the wettest quarter (PETWettestQ, importance ≈ 0.12) and evapotranspiration in the driest quarter (PETDriestQ, importance ≈ 0.09) (Figure S6). The aridity index and precipitation-related variables (Bio13 and Bio14) contributed minimally to the combined model, with importance values near 0.01–0.02.

Regarding the response curves, the ecological niche of L. humile in its native range appears to be primarily shaped by isothermality. Habitat suitability increases as isothermality decreases from 70% to 0% (Figure S4). The Thornthwaite aridity index also plays a significant role, showing a similar pattern in which suitability increases as aridity decreases from 50% to 0%. Additionally, precipitation during the wettest month (Bio13) negatively affects suitability, which increases as precipitation declines from 200 mm to 0 mm. Considering the combined native and introduced ranges, isothermality remains the dominant factor, with habitat suitability peaking around 50% (Figure S4). Evapotranspiration in the wettest quarter has two peaks in suitability: one higher at 50 mm and one slightly lower at 150 mm. Evapotranspiration in the driest quarter also influences suitability and peaks at 70 mm.

According to the species ensemble distribution model, based on 465 (90 after thinning) occurrence records from its known native range, the absence of L. humile records in natural habitats of the Monte, Patagonian Steppe, and Patagonian Steppe/Forest ecotone (its presumed invasive range) is consistent with suitability values lower than 250 (Figure 4A–C). By applying this threshold, we can infer that the most suitable areas for the native dataset extend beyond the Río de la Plata basin, reaching the ecotone between the Pampa and Espinal ecoregions (Figure 4B,C). Consequently, regions in South America located approximately at latitudes south of 20° S and north of approximately 29° S, and at longitudes west of 65° W and east of 40° W, are likely to correspond to introduced populations of the Argentine ant.

Considering the full dataset of 17,900 occurrence records (621 after spatial thinning) from both native and introduced ranges (Figure 4D), the ensemble model predicts areas with suitability values > 250 extending into the lower and middle Río Negro Valley, Mendoza Province (Monte ecoregion), the Chilean Espinal phytogeographic province, and the Patagonian Forest. Notably, these areas include the city of San Carlos de Bariloche, located in the ecotone between the Patagonian Forest and Steppe, as well as parts of the Patagonian Monte Desert near the Río Negro coast (e.g., Valcheta, San Antonio Oeste) and, further south, near Trelew in Chubut Province, where two unconfirmed occurrence sites have been reported (Figure 4E,F).

3.3. Invasive Haplotypes

The most widely distributed haplotype (H1, red dot in Figure 5), which was found in 14 sites in Santa Fe, Córdoba, south Buenos Aires and Río Negro had been previously recorded in invasive populations in Australia, Japan, United States, New Zealand, South Africa, and Spain [25]. The second haplotype (H2, brown dot) found in two sites in northern and southern Buenos Aires, had been previously detected in Spain. The haplotype H3, which had previously been recorded by Vogel et al. [25] in Argentina and Japan, was not detected in this study. The last haplotype (H4, green dot) found in six sites in Buenos Aires, Córdoba, Mendoza and Río Negro, could not be related with previously recorded haplotypes in the species’ invasive range.

Three haplotypes of another Linepithema species (L. micans) very similar to the Argentine ant, were found in our surveys. This species is closely related to L. humile, its sister species. One haplotype (HB, turquoise triangle) was found in two sites: one in southern Buenos Aires and the other over a thousand kilometers away in the city of San Martín de Los Andes, Neuquén province (Figure 5). Interestingly, this is the first and southernmost record of this species in Neuquén. A second haplotype (HD, blue triangle) was found at one site in central Buenos Aires. The last haplotype (HG, pink triangle) was found at one site in southwestern Buenos Aires. These haplotypes could not be associated with haplotypes present in other regions/countries due to the absence of available sequences for this species in repositories or publications.

4. Discussion

4.1. Argentine Ant Range Expansion

Our findings confirm a substantial expansion of the distributional range of the Argentine ant, L. humile, from southwestern Buenos Aires across the Pampa and Espinal ecoregions up to the Monte Desert ecoregion in northern Patagonia. The species occupies natural, rural (semi-natural), and urban habitats in the Pampa and Espinal ecoregion but it has colonized only anthropogenic environments into the Monte Desert and Patagonian Steppe ecoregions in Río Negro Province. This distribution pattern suggests longer-term establishment in the Pampa and Espinal, in contrast to the Monte Desert, where its more fragmented distribution (restricted to urban and semi-natural habitats) indicates a more recent colonization history. This spread likely occurred later, with the growth of cities and the subsequent development of surrounding agricultural areas about six decades ago. At that time, the region transitioned to a system of linear cities during the fruit boom, as well as the improvement of services and rural-industrial employment [55].

Detection rates were significantly higher in urban and rural habitats, whereas the species was absent from natural habitats. This finding is consistent with the pattern of anthropogenic facilitation of invasive species spread [9]. Our findings highlight cities as key drivers of heterogeneous L. humile detection rates. Urban habitats exhibit the highest odds of detection, consistent with the idea that L. humile benefit from human-modified landscapes. City-specific effects suggest that local factors, such as resource availability, connectivity, and human-mediated dispersal, influence the spread of L. humile beyond the effects of habitat [5]. Viedma and Choele-Choel exhibited the strongest effects, which is consistent with their potential roles as regional hubs or “stepping stones” in the spread of L. humile. In contrast, Cinco Saltos’s non-significant result suggests minimal deviation from the baseline (Bariloche).

The absence of L. humile in natural habitats within the Monte and Patagonian ecoregions suggest the idea that climatic constraints and sparse human settlements limit its further expansion. The lack of L. humile in natural habitats in the surveyed cities along the Río Negro Valley likely reflects a true absence or low detectability in less disturbed areas, resulting in nearly complete separation in logistic models [43]. Despite this constraint, Firth’s penalised likelihood method produced stable estimates and revealed that the presence of urbanisation significantly changes the probability of detection. While this approach reduces bias, future studies should investigate the underlying causes (e.g., land cover and human density) within cities. Limitations include possible unmeasured confounders (e.g., survey effort) and the coarse resolution of habitat categories.

These findings are consistent with patterns observed in other regions [56]. For example, L. humile rapidly colonized coastal and urban areas in California following its introduction. However, its expansion inland was limited by abiotic stressors, especially high summer temperatures and low soil moisture [16]. These climatic conditions are similar to those in Argentine Patagonia [57]. Similarly, L. humile has persisted mainly in urban areas since its introduction in central Chile in 1910, despite the region’s low climatic suitability [58]. This global pattern of L. humile thriving in climatically suitable areas and surviving in marginal zones via urban facilitation has been documented in the Mediterranean [30,31], the southeastern United States [59], South Africa [60], Australia [61], and New Zealand [62]. Human-mediated disturbance and dispersal are critical factors enabling its expansion in all these cases.

Our findings confirm that L. humile has effectively expanded its range in northern Patagonia but remains closely tied to human-modified environments. As in other regions of its introduced range [16,63,64], L. humile in Patagonia is strictly confined to urban and peri-urban or semi-natural (rural) habitats, with no detections in native environments across the surveyed gradient. The absence of L. humile in natural habitats, including the ecotone between the Patagonian Steppe and Forest in Bariloche and the Monte Desert in the Río Negro Valley, suggests that these areas currently act as biogeographic or climatic barriers to further natural spread. This is consistent with previous observations in marginal unsuitable climates [31,59].

The recent detection (since 2019) of L. humile in Bariloche, exclusively in highly disturbed urban locations, supports the assumption that its expansion into southern latitudes is ongoing and is likely facilitated by human-mediated jump dispersal. Similar patterns have been observed globally, where the species establishes satellite populations through human transport and thrives in disturbed environments [25,65]. Moreover, the positive relation between the detection rate of L. humile in urban habitats and city size along the Río Negro Valley supports the hypothesis that urban environments offer the necessary microclimatic buffering, resource availability, and connectivity to facilitate the establishment and persistence of this species [56,64].

In the Río Negro Valley, L. humile was widespread and abundant in urban areas, as well as in adjacent semi-natural areas, particularly in irrigated fruit plantations. Its presence in orchards but not in the native Monte habitat highlights the role of cultivated lands as corridors for invasion, facilitating the stepwise spread between urban centers. Similar associations between L. humile and agroecosystems have been reported in Mediterranean and subtropical climate regions [24]. Although prevalence remained high in fruit crops throughout most of the valley, there was a slight decline in presence in orchards compared to urban habitats as we move away from the native distribution area, especially in the upper Río Negro valley (Regina to Cinco Saltos). This trend could reflect the limiting effects of climatic gradients on its establishment as observed also in the model, which deserves further investigation.

4.2. Abiotic Constraints and Urban Facilitation

Our ensemble species distribution models for L. humile demonstrated high predictive performance, particularly for the native-range model, which highlights the robustness of our modeling framework. Our results are consistent with previous modeling efforts that have highlighted the response of L. humile to climatic variables, particularly thermal and moisture-related factors [30,59,66]. Isothermality (Bio03) rather than temperature seasonality, as reported recently by [67], emerged as the most influential predictor across all models. This also agrees with earlier findings that temperature variability constraints the distribution of the Argentine ant [59,66]. In our native-range model, lower isothermality, reflecting reduced diurnal variation relative to the annual range, was strongly associated with higher habitat suitability. The Monte and the Patagonian Steppe are both desert ecoregions with very low levels of humidity, which favors large temperature fluctuations between day and night. This finding supports the hypothesis that L. humile is adapted to thermally stable environments along the day. This condition likely explains the species’ ecological success in urbanized (anthropic) and coastal (natural) areas with more buffered temperature daily regimes [64,68].

The Thornthwaite aridity index and precipitation during the wettest month (Bio13) were also important factors in the native-range model, but their importance decreased when introduced-range data were included (global dataset). This shift could reflect a broader environmental tolerance in invasive haplotype populations [25,62], which may be due to niche expansion or simply adaptation to urban microhabitats that act as refuge mitigating climatic extremes [56]. Evapotranspiration variables (PETWettestQ and PETDriestQ) gained relevance in the global model instead, suggesting that water balance, rather than direct precipitation metrics (e.g., annual precipitation as reported by [67], better explain the distribution of invasive populations in semi-natural and natural environments. Relative ambient humidity is critical for this species, as it is highly susceptible to desiccation [41,69].

Response curves further illustrate the differences in the ecological niche of L. humile between its native and introduced ranges. In the native model, habitat suitability peaks at low values of isothermality, aridity, and precipitation. This aligns partially with the humid subtropical and temperate conditions of its core native range. In contrast, the global model shows peak suitability under moderate isothermality and bimodal responses to evapotranspiration. This also may reflect adaptations to novel climates or more likely human-modified environments that provide refuge [56,61].

According to the native-range model, the more suitable areas appear to extend southward through the Pampas into the Espinal–Pampas ecotone. Since~1987, the Espinal ecosystems have been drastically deforested, with 60% of natural forests (shrubland) converted to pastures or irrigated crops [70]. This habitat shift could have facilitated the spread and establishment of L. humile in this ecoregion. The absence of L. humile in natural areas of the Monte and Patagonian Steppe ecoregions, despite proximity to invaded urban and rural sites, supports the model-derived suitability threshold of 250. These regions consistently fall below this threshold, reinforcing their role as natural barriers to unassisted expansion. This aligns with observations that L. humile remains confined to anthropogenic habitats in these areas [25], highlighting the importance of human-mediated dispersal in its spread beyond climatically suitable zones. This pattern mirrors findings in Spain, where urban environments enabled L. humile to persist in otherwise unsuitable Mediterranean semi-arid colder habitats [71].

Wei et al. [67] identified the mean temperature of the coldest season as the main predictor of L. humile’s invasive range. The mean minimum temperature of the coldest month ranges in the entire Patagonian Steppe between −5 to 3 °C in July [72], whereas the mean temperature in the colder quarter in Bariloche is low 3 °C [72]. All these values are lower than minimum thermal tolerance (Critical Temperature minimum = 3.2 °C) reported by Muñoz [73] for a Buenos Aires population that was previously acclimated to low temperatures and had the same mitochondrial haplotype (H1) as the Bariloche population. This suggests that temperatures strongly affect the survival of L. humile during a long year period, and thus must limit its establishment in the natural coder habitat of the Patagonian Steppe, as observed for other highly invasive species [74]. However, recent findings indicate that a 2–4 °C of global warming could promote the naturalization of indoor L. humile populations in colder regions of the Northern Hemisphere (e.g., Europe) [75]. A similar process may occur in cold regions of the Southern Hemisphere, such as the Patagonian Steppe/Forest. This is particularly likely in cities such as Bariloche, where introduced populations that mostly remain indoors could colonize peri-urban semi-natural rural habitats. This could potentially amplify the species’ future economic and environmental impacts.

4.3. Mitochondrial Signatures of Expansion

The mitochondrial haplotypes detected in Argentine populations of L. humile provide novel insights into the invasion dynamics of this species in South America. The presence of three COII haplotypes previously documented in both native (South America) and invaded regions (e.g., Australia, Japan, United States, and Spain) support the hypothesis that some of the genetic diversity of the invasive population in Argentina reflects a global invasion lineage. This is consistent with patterns described by [25]. In particular, the widespread occurrence of haplotype H1 across five provinces (Santa Fe, Córdoba, Buenos Aires, CABA and Río Negro) suggests a dominant, and highly invasive mitochondrial lineage that has repeatedly succeeded in colonizing diverse habitats across continents.

The detection of haplotype H2 in two distinct sites within the province of Buenos Aires further supports the notion of multiple introductions or intra-regional dispersal events involving already invasive genotypes. The co-occurrence of H1 and H2 in Buenos Aires, a region previously identified as a key introduction hub in Argentina, aligns with the stepping-stone model of invasion [25,50]. According to this, probable secondary invasions (e.g., Bariloche or Valcheta) could originate from previously established invasive populations (e.g., Viedma or Choele-Choel in the Río Negro Valley) rather than directly from the native range (e.g., Pampa ecoregion).

The presence of the invasive haplotype H4 in populations from Córdoba, Mendoza and Río Negro was particularly intriguing. This lineage could not be associated with any other haplotype from the invasive range not detected, likely due to the smaller fragment size of our COII marker compared to that used in previous studies (e.g., those reported by [25] for La Rioja province in Argentina or Chile). This haplotype may represent a local invasive lineage that was previously undetected, or one haplotype from the native range that expanded to most desert regions in western Argentina. Unfortunately, we could not genetically analyze populations present in northwestern Argentina (La Rioja, Catamarca, Tucumán and Salta) to determine if they correspond to this same H4 haplotype. In any case, its presence raises important questions about the role of climatic and ecological factors, as well as cold- and desiccation-tolerant lineages, in facilitating the expansion of less globally dominant lineages, that are perhaps better adapted to drier regions. Future research integrating nuclear markers, whole-genome sequencing, or genome-wide association studies, together with ecological niche modeling, would provide valuable insights into the species evolutionary origin and mechanisms underlying its invasive potential.

The identification of three distinct haplotypes corresponding to L. micans is equally noteworthy. Linepithema micans is native to southeastern Brazil, eastern Paraguay and northeastern Argentina, but reaching southeast until Buenos Aires [76]. The presence of the species in San Martín de Los Andes (Neuquén province) and a few sites in Río Negro may reflect recent human-mediated dispersal of these Argentinean haplotypes or insufficient sampling in intermediate areas. Due to the close phylogenetic relationship and potential morphological misidentification between L. micans and L. humile [76], these findings highlight the importance of molecular tools for accurately identifying invasive species. This study highlights the importance of integrating genetic data with geographic, climatic, and ecological information to elucidate invasion pathways, thereby enhancing management strategies aimed at mitigating the impact of invasive ants.

5. Conclusions

Notably, L. humile invasions are not limited to long-distance dispersal via intercontinental ships. Human-facilitated terrestrial expansion also plays a significant role in the species’ transport from its native land. Our results confirm that the southern distribution limits of the Argentine ant have expanded from Buenos Aires across the Pampas and Espinal ecoregions (native range) to the Monte Desert ecoregion in northern Patagonia (invasive range). The species inhabits natural, rural, and urban habitats in the Pampas and Espinal ecoregions. However, in the Monte Desert and Patagonian Steppe, the species was only found in anthropogenic habitats. In cities of Río Negro, L. humile was present in urban and peri-urban (rural) areas, and in Bariloche in an urban area, but absent from nearby natural habitats, with the highest detection rates in urban habitats and a positive relation with the size of the city. This highlights the important role that cities and irrigated agriculture play in providing refuges and stepping stones that facilitate the spread of species into otherwise unsuitable landscapes. Species distribution models suggest that daily temperature variability and moisture loss restrict the establishment of species in Patagonia. However, no clear niche shift was identified since the species was not detected in natural habitats that were considered climatically unsuitable. Changes in land use and rising temperatures may encourage the naturalisation of species in semi-natural habitats, particularly in urbanised areas such as Bariloche. Genetic analyses revealed three L. humile haplotypes, including lineages that are widespread across the globe, which supports the idea that they are connected to broader invasion pathways. Haplotypes of a closely related species, L. micans, were also detected, highlighting the need for molecular tools to prevent misidentification. Our results suggest that urbanization and irrigated agriculture synergistically facilitate the southward expansion of L. humile from the Pampas to Patagonia, beyond its historical climatic southern boundaries, by providing refuges and stepping-stones in otherwise inhospitable landscapes.

Overall, our findings provide an integrated ecological, climatic and genetic perspective on the ongoing southward expansion of L. humile, emphasizing the importance of continued monitoring of transitional zones such as the ecotones between the Patagonian Steppe and the upper Río Negro Valley and between the Patagonian Steppe and Forest. It is essential to assess whether this expansion front remains stable or is shifting further south in response to human activity (e.g., climate change, commerce and tourism travel and urbanizations).