*Article* **High Diversity in Urban Areas: How Comprehensive Sampling Reveals High Ant Species Richness within One of the Most Urbanized Regions of the World**

**François Brassard 1,2,\*, Chi-Man Leong 1,3, Hoi-Hou Chan <sup>4</sup> and Benoit Guénard <sup>1</sup>**


**Abstract:** The continuous increase in urbanization has been perceived as a major threat for biodiversity, particularly within tropical regions. Urban areas, however, may still provide opportunities for conservation. In this study focused on Macao (China), one of the most densely populated regions on Earth, we used a comprehensive approach, targeting all the vertical strata inhabited by ants, to document the diversity of both native and exotic species, and to produce an updated checklist. We then compared these results with 112 studies on urban ants to illustrate the dual roles of cities in sustaining ant diversity and supporting the spread of exotic species. Our study provides the first assessment on the vertical distribution of urban ant communities, allowing the detection of 55 new records in Macao, for a total of 155 ant species (11.5% being exotic); one of the highest species counts reported for a city globally. Overall, our results contrast with the dominant paradigm that urban landscapes have limited conservation value but supports the hypothesis that cities act as gateways for exotic species. Ultimately, we argue for a more comprehensive understanding of ants within cities around the world to understand native and exotic patterns of diversity.

**Keywords:** biological invasions; biodiversity; species checklist; urban ecology; conservation

#### **1. Introduction**

Over the past century, urbanization has increased drastically in most regions around the world [1–3]. This increase threatens biodiversity [4–6], with pollution [7–9], habitat loss [10], and the spread of invasive species [11] being major causes of local species extinction or population decline. As such, urban habitats have historically been considered as species-poor concrete jungles [12]. However, urban environments are not necessarily depauperate ecosystems, and may, in fact, have some degree of conservation value by harboring native species [13]. Recent studies suggest that urban habitats can harbor a high diversity of both native (including endemic species) and non-native species, and even, sometimes, surpass surrounding rural areas in terms of species richness [12–16]. The survey and monitoring of biodiversity within cities may thus allow the identification of novel habitats and species worthy of protection within urban matrices. In particular, urban habitats including large and high quality patches of green spaces and forest fragments may still support high species diversity [17–20]. How much biodiversity these areas can contain is still open for debate, but it is paramount to understand the potential conservation value of urban centers.

**Citation:** Brassard, F.; Leong, C.-M.; Chan, H.-H.; Guénard, B. High Diversity in Urban Areas: How Comprehensive Sampling Reveals High Ant Species Richness within One of the Most Urbanized Regions of the World. *Diversity* **2021**, *13*, 358. https://doi.org/10.3390/d13080358

Academic Editor: Michael Wink

Received: 2 July 2021 Accepted: 29 July 2021 Published: 4 August 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Beyond assessing the number of species in cities, another crucial component to consider is the composition and identity of the species present. Indeed, cities can facilitate invasions by non-native species in part due to their high level of disturbance, which provides ecological niches suitable for many exotic species [21–23]. Moreover, the constant flux of merchandise in and out of urban centers through airports, harbors, and train stations make them the ideal gateways for exotic species introductions with the common arrival of new propagules [24–26]. Consequently, surveying urban centers and their surroundings to detect new arrivals is essential to limit their spread and mitigate their potential impact on native biodiversity. This is especially true for coastal regions, which host the highest richness of exotic species [27], and, in the case of China, may represent a source of spread towards more inland regions [28].

To evaluate the biodiversity value of urban environments, surveying all flora and fauna would be ideal, but unrealistic, especially for tropical and subtropical regions where there is limited data and taxonomic knowledge. As such, ecological surveys must select a subset of taxa representing useful biodiversity proxies. For conservation monitoring purposes, ants represent an ideal taxon [29]. Indeed, their taxonomy is relatively well-resolved in comparison with most other diverse insect groups, and they can be sampled through the use of standardized and replicable protocols [30]. They are also ecologically and taxonomically diverse, abundant, and ubiquitous [31]. Moreover, they are adequate bioindicators [32,33], play key ecological roles as predators, scavengers, and herbivores [31,32,34,35], with some species acting as ecosystem engineers by modifying soil properties [34].

Ants also include some of the most damaging invasive species, impacting native ants [35–37], non-ant invertebrates [38], vertebrates [39], and plant communities [40], ultimately causing ecosystem disruptions [41]. Invasive ant species can also negatively impact human socioeconomic activities such as farming or education [42], whereas others are considered household pests [43,44], with some exotic species even acting as vectors of pathogens in hospitals [45,46]. Thus, surveying the ant fauna of urban regions and producing ant species checklists should be an important tool not only to evaluate the conservation value of cities, but also to record the worldwide spread of exotic species.

To date, the majority of studies have been limited to ecological studies (Table 1), limited in time and space, and using only a subset of sampling methods to characterize urban ant communities. However, establishing an exhaustive list of a region's ant fauna presents multiple concerns and challenges. For instance, within a specific habitat, and especially within tropical regions, distinct ant species are stratified along the vertical strata (i.e., from underground to the canopy) [47–51]. Generally, ants can be classified into three broad categories: arboreal, epigeic (i.e., ground surface-dwelling, including ants living in leaf litter), and hypogeic (i.e., subterranean). To perform a complete inventory of ants within a region, surveys should, thus, include the different dimensions of this vertical stratification by using methods targeting species from each microhabitat. Unfortunately, most studies in urban habitats use sampling methods focusing mainly or solely on epigeic ants [52], thereby potentially under-estimating the species richness and composition of local communities, as well as the magnitude of invasions. Additionally, this may misrepresent the diversity of hypogeic and arboreal ants within urban habitats and overlook the potential discovery of undescribed species [53–55].

Considering the current expansion of urban habitats [1], describing patterns in urban ant diversity is of great urgency. This is essential to foster biodiversity in cities, but also to gain a better understanding of which factors may facilitate the spread of exotic species. To the best of our knowledge, however, few cities have comprehensive ant species checklists, especially in tropical Asia (Figure 1, Table 1), which makes meaningful comparisons in urban biodiversity challenging at best. More biodiversity surveys and checklists are, thus, required to address this issue.

**Figure 1.** World map showcasing the locations of studies compiled in Table 1. Points show urban locations for which ant species richness estimates were available. Color indicates the type of study (i.e., ecological survey or checklist, see legend).

One such region is the Guangdong–Hong Kong–Macao Greater Bay Area, a major Asian megalopolis [56]. Located in subtropical China, it covers an area of 56,000 km2 and has a combined population of 68 million people [56]. Within the Greater Bay Area, Macao can be distinguished by its human density, which exceeds 20,000 hab./km2, making it the most densely populated region on the planet [57,58]. Macao also represents an historical global hotspot in its role within the global trade exchange, first within the Portuguese network and then within China [59,60], making it particularly vulnerable to biological invasions. Finally, the development of Macao has led to a complex matrix of habitats with various levels of disturbance. As such, Macao represents a unique opportunity to better understand how much biodiversity a city characterized by extreme urban development may contain and assess the role of cities as gateways for exotic species.

Following the publication of a preliminary checklist of the ant species of Macao [53] and the discovery of a new subterranean ant species [61], a new survey was conducted across Coloane Island, in the southern half of Macao. To our knowledge, our survey is the first to use an exhaustive sampling approach covering all vertical strata inhabited by ants within an urban area (i.e., arboreal, ground-dwelling, and subterranean). We hypothesized that this sampling coverage would uncover a substantial amount of new species records, give a fair representation of the ant species richness of Macao, and detect new introductions of exotic species. Moreoever, we expected that this methodology, which could be replicated across cities around the world, would be particularly useful for finding cryptic and potentially undescribed species. Finally, we compare our results with previous published studies on urban ants to illustrate the potential that cities may represent for ant diversity, but also more broadly for other insect groups.

**Table 1.** Ecological and taxonomic studies that produced ant species richness values for a city. Studies are classified by a function of their study region and ranked by the function of the overall species richness retrieved. Studies not providing a complete species list or without enough information on habitats sampled were not included. (\*) Complete list of species not provided in article, (\*\*) a combination of 3 articles by the same author and in the same city, and (\*\*\*), richness value is a combination of more than one urban region.



**Table 1.** *Cont.*


**Table 1.** *Cont.*


**Table 1.** *Cont.*


**Table 1.** *Cont.*


**Table 1.** *Cont.*

#### **2. Materials and Methods**

#### *2.1. Geographic and Climatic Characteristics of Macao*

Macao is a special administrative region on the southern coast of China. It is located 60 km south-west of the Hong Kong special administrative region, separated from it by the pearl river delta. Macao's climate is characterized by dry winters and hot summers [176], with an average daily temperature of 22.8 ◦C and an annual rainfall of 1967 mm [177].

In the 19th century, Macao's land surface was only 10.28 km<sup>2</sup> but, following numerous reclamation projects, it now covers around 32.9 km2 [58,178]. Despite its high urbanization, Macao still retains several nature parks consisting of young secondary forests, most of which are on Coloane Island. Macao's government started managing these forest patches

in 1980, protecting them from wild-fires and establishing restauration plantations of *Pinus massoniana* and *Acacia confusa* [179].

#### *2.2. Sampling Effort and Collection Methods*

Most ant specimens examined were collected during a survey conducted in 2019, from March to October, across 21 plots in Coloane Island, Macao (Figures S1 and S2, Table S1). The survey focused on collecting ants within Coloane's nature parks, which consist of secondary forests, but also covered two golf courses and a mangrove site. To extensively sample the hypogeic, epigeic, and arboreal ants of Macao, we used a range of sampling methods during the 2019 survey. Across the 21 sites, we used 225 ground baits, hand collection, and 42 leaf litter extractions with Winkler bags. Half the Winkler extractions consisted of combining the leaf litter of 4 × 1 m2 quadrats taken at each corner of a plot of 20 × 20 m (i.e., standard area method), and half consisted of combining a few handfuls of leaf litter taken at 12 random locations within the same plot (i.e., species pool method). For a subset of 16 sites, we used 256 subterranean and 320 arboreal baits, and 1024 artificial nests. For more details on traps, baits, and nest design, see Brassard et al. (2020) [54]. Note that the nests were built following Booher et al. (2017) [180], and were mainly used to obtain sociometric data for the species collected (i.e., colony size and composition). The remainder of the specimens included are from collections made by hand or leaf litter extractions between 2015 and 2020 from different locations across Macao, with detailed collection information presented in the species accounts section.

#### *2.3. Sample Processing*

We processed samples by first sorting specimens to morphospecies, which we then stored in ethanol 70%. For each morphospecies, we point-mounted at least one individual and labeled it with a locality and collection label. All specimens are currently located in the Insect Biodiversity and Biogeography Laboratory (IBBL) at The University of Hong Kong.

#### *2.4. Imaging*

We used a Leica DFC450 camera mounted on a Leica M205 C dissecting microscope to image mounted specimens of each species and morphospecies. We used the Leica Application suite v. 4.5 to take, stack, and enhance image montages. When necessary, we used Adobe Photoshop Lightroom to make final color corrections and diminish ghosting effects.

#### *2.5. Mapping Species Distributions and Urban Studies*

We used *R* to produce all maps [181]. The maps shown at the south-east Asia scale use records at the country level, or the administration level for larger countries (e.g., China, India, and Japan). Following previous work [182,183], we used island boundaries instead of political boundaries for large islands. For maps centered on Macao, we used the GPS coordinates associated with each specimen to add their collection localities.

#### *2.6. Analyses*

We produced maps, bar graphs, heatmaps, species accumulation curves, and Venn diagrams using *ggplot2* [184], whereas we used Adobe Illustrator to assemble the species account figures (Figures A1–A158). We produced species accumulation curves and diversity estimates using the package *iNEXT* [185].

#### *2.7. Literature Search*

To compile studies that produced a species checklist for cities, we performed a Scopus search using the following formula on the 9th of February 2021: "formicidae" AND "checklist" AND "city" OR "urban". We then pruned the resulting dataset manually by reading the abstracts and only keeping the studies that produced a total number of species for a city. We further added appropriate studies known by authors that were not present within the Scopus search. In particular, we use the literature information combined in GABI [186] to identify suitable articles on urban ants. We classified studies as either ecological or checklists. If a study was primarily hypotheses driven, with limited sampling efforts in time, habitats, or in the methods used, it was classified as ecological, whereas studies solely producing a species checklist, including records from previous published studies, were classified as checklists. Studies including other non-urban habitats outside the main city area, and for which detailed information did not allow to separate species composition and richness, were not considered.

#### *2.8. Notes on Invasion Status*

An understanding of the native and introduced ranges of species represents a fundamental step in the detection and management of biological invasions. However, for many species of ants, clear geographic boundaries between those ranges remain undetermined, either at global (e.g., uncertainty in the realm of origin) or regional scales (e.g., native vs. introduced range within a particular realm). Here, we thus distinguish three categories between native, exotic, and tramp species. For species that we could establish with some confidence whether or not they were introduced, we used the exotic and native status, respectively. We used the tramp status for species whose biogeographic origins were more uncertain in Macau or south China. Note that all species here labelled as tramps have been previously transported in other regions of the world and have established populations in non-native habitats. This demonstrates their potential to colonize new regions. Furthermore, these tramp species often occur within anthropogenic habitats. As such, tramp species, regardless of their potential non-native status, are important to consider from a management perspective, as they have the potential to invade non-native localities. The establishment of the native and exotic ranges for each species was based on the maps available on antmaps.org [186,187].

#### *2.9. Notes on Records*

Since the last publication of a species checklist for Macao [53], two studies with a focus on specific genera (i.e., *Polyrhachis* and *Strumigenys*) published new species records for the region [54,55]. Since all but one species record—*Polyrhachis tyrannica* [55]—are from specimens collected during our 2019 sampling, we here report these specimens, with the exception of *P. tyrannica*, as new records for Macao.

#### *2.10. Notes on Taxonomy*

To identify our specimens at species-level, we used the Insect Biodiversity and Biogeography Lab (IBBL) ant collection as a reference. For the especially challenging species, we relied on the taxonomic knowledge of Dr. Benoit Guénard. To verify our identification of the genera *Nylanderia* and *Carebara*, we shared stacked images with specialists familiar with their taxonomy, Dr. Jason L. Williams and Dr. Georg Fischer, respectively. When a species could not be identified at species level with certainty, we labelled it as "nr." the morphologically closest known species (e.g., *Colobopsis* nr. *nipponica*). If the unknown species was morphologically distinct but not identifiable, we used the unique morphospecies code their collector used to label it (e.g., *Camponotus* sp.1 FB).

#### **3. Results**

Our 2019 survey collected a total of 112 species and morphospecies from 46 genera and nine subfamilies. Among these, 51 species (46%), 10 genera (22%), and one subfamily (11%) represented new records for Macao. We also found four additional species and one new genus record among the specimens opportunistically collected between 2017 and 2020. In total, the new records reported here include one subfamily, 11 genera, and 55 species and morphospecies (Figure 2, Table 2). The new genera reported for Macao are: *Brachymyrmex* (exotic), *Buniapone*, *Dilobocondyla*, *Gesomyrmex*, *Iridomyrmex*, *Mayriella*, *Probolomyrmex*, *Proceratium*, *Pseudolasius*, *Rotastruma* and *Vollenhovia*, while the Proceratiinae subfamily is here recorded for the first time. The overall number of ant species known

from Macao thus increases by 55%, from 100 to 155 species and morphospecies (Figure 2, Table 2), which represents the third highest urban ant diversity out of 123 entries (see Figure 1, Table 1). For images of these species and maps of their distribution in Macao and SE Asia, see Appendix A (Figures A1–A158).

**Figure 2.** Number of new records per year in Macao. Bar plots showing (**a**) the number of new ant species record for Macao based on literature records and this study and (**b**) bar plots showing the accumulation of ant species found in Macao based on literature records and this study. The proportion of native species (blue), tramp species (light orange), and exotic species (dark red) are denoted within bar plots.



The sampling methods varied in their overlap for the species they collected (Figure 3). Of the 112 species collected during the 2019 survey, 10 species were only found in leaf litter extractions, five in ground baits, 10 in subterranean baits, 13 in hand collections, and eight in arboreal baits, whereas the other 66 were collected with more than one method. Artificial nests did not collect new species records nor unique species, but they did provide

sociometric data for 15 species from a total of 913 nests recovered, for a colonization rate of 3% (Table S2).

**Figure 3.** Heatmap showing the number of shared species between collection methods used in the 2019 survey. Color illustrates the strength of the correlation between sampling methods in the shared species they collect. The diagonal represents the total number of species collected for each method. Abbreviations are for hand collection (Hand. Col.), subterranean trap (Sub. Trap.), ground nest (G. Nest), ground bait (G. Bait), arboreal bait (Arb. Bait), leaf litter extraction with the species pool technique (LLSP), and leaf litter extraction with the standard area technique (LLSA).

We found few habitat generalists, with only seven species collected in all strata (Table 3, Figure 4)—*Monomorium intrudens*, *M. floricola*, *Nylanderia sharpii*, *Pheidole megacephala*, *P. tumida*, *Tapinoma indicum*, and *T. melanocephalum*—three of which (43%) are exotics. Ten species were only collected within the subterranean stratum and 14 species only in the arboreal stratum (Table 3, Figure 4). In contrast, most species were ground-dwelling (*n* = 52) or collected within two strata (*n* = 36). The highest proportion of new records found solely within one stratum were in the subterranean (9/10 species: 90%), arboreal (9/14 species: 64%), and then ground (25/52 species: 48%) strata (Figure 4).

**Table 3.** Summary checklist of the species recorded during previous studies and the current study in Macao. Status of species are mentioned as native, tramp, or exotic. The asterisk symbol (\*) denotes new records. The dagger symbol (†) denotes morphospecies collected in 2019 that probably belong to previous records but could not be assigned a species name due to incomplete taxonomic descriptions (as such, they were not counted in the total number of species). The diesis symbol (‡) denotes a species collected previously, but with a mislabeled status. An "X" under the column Arboreal, Ground, or Subterranean indicates that this species was collected within this stratum during the 2019 survey. We left blanks for species not collected during the 2019 survey. For images of species and maps of their distribution in Macao and SE Asia, see Figures A1–A158. For detailed accounts of the material examined, see the species account section of the supplementary material.



**Table 3.** *Cont.*


**Table 3.** *Cont.*


**Figure 4.** Venn diagram showing the total number of species (112 spp.) and new species records (51 n.r.) collected in each stratum during the 2019 survey. Numbers in parentheses represent the proportion of species collected within a stratum that are new records from the last 2017 checklist (Leong et al. 2017). Note that this figure does not include the four records added from specimens collected besides the main 2019 survey.

At least five of the species collected during our 2019 survey, which belong to the genera *Strumigenys*, *Syllophopsis*, *Tetramorium*, and *Vollenhovia*, were considered potentially novel to science at the time of collection. We found three of the undescribed species in subterranean traps (i.e., *Strumigenys subterranea*, *Syllophopsis* nr. *Cryptobia*, and *Tetramorium* sp. 9 JF), one in leaf litter samples (i.e., *Vollenhovia* sp. 2 BG), and one in arboreal traps (i.e., *Tetramorium* sp. 2 JF).

Several of the new records have rarely been reported in the literature and represent extensions of their known range. First, we found workers of *Dilobocondyla propotriangulata*, an arboreal species described from Vietnam [197], at two different sites in Macao, which represents the third and fourth records of the species worldwide. Second, we found workers and a queen of *Mayriella granulata*, also described from Vietnam [198], which represents the first record of this species in China. Lastly, we found a worker of *Probolomyrmex* (*P. dammermani*), a pantropical but rarely collected genus [199–201], which represents the first record of this species in China.

Before our survey, 14 tramp and 14 exotic species were known to occur in Macao. Here, we report four additional exotic species records: *Brachymyrmex patagonicus*, *Plagiolepis alluaudi*, *Tetramorium insolens*, and *T. tonganum*. We also report three additional tramp species records: *Cardiocondyla wroughtonii*, *Iridomyrmex* sp. *anceps* complex, and *Monomorium intrudens*. Moreover, we report new localities in Macao for several exotic species: *Anoplolepis gracilipes*, *Cardiocondyla minutior*, *Monomorium pharaonis*, *Ooceraea biroi*, *Paratrechina longicornis*, *Pheidole megacephala*, *Solenopsis invicta*, *Strumigenys emmae*, *S. membranifera*, *S. nepalensis*, and *Tetramorium lanuginosum*.

Nevertheless, despite achieving a high sampling coverage (i.e., between 80 to 98% depending on the method), species accumulation curves indicate that further sampling should uncover several more species on Coloane Island (Figure 5, Table 4). Indeed, estimates predict that each sampling method could collect from 2 to 25 additional species each.


**Table 4.** Summary of the species richness collected, the sampling completeness, and the richness estimates for each sampling method used during the sampling done in Coloane in 2019.

We identified 112 studies, representing 109 cities, that focused on ants within urban environments (Figure 1, Table 1). Among those, 23 studies provided species checklists, while 88 represented ecological surveys. The studies were unevenly distributed across biogeographic regions. The highest numbers were from North America (*n* = 41), Asia (*n* = 34), Europe (*n* = 24), and Central and South America (*n* = 21), whereas Australia (*n* = 6) and Africa (*n* = 5) had the lowest number of studies. Although Asia had the second highest number of studies, most of these originated from temperate regions, with only eight studies (23.5%) conducted within tropical or subtropical regions.

**Figure 5.** Species diversity in relation to sampling coverage for each standardized technique used in Macao in 2019. Values in the legend represent the number of species collected, and, in brackets, the estimated number of species that would be collected by this method if sampling coverage would reach 100% (asymptotic estimates of order q = 0 obtained using the function ChaoRichness in the package iNEXT). The dotted line shows the extrapolation for the predicted number of new records if sampling completeness would reach 100% (i.e., a value of 1.00 on the graph). Shaded areas represent the 95% confidence intervals of each curve. Calculation method used species incidence frequency. Leaf litter samples were considered as four units of 1 m2 per Winkler sack (which pooled 4 m2 of leaf litter). Abbreviations are (LLSP) leaf litter extraction with the species pool technique and (LLSA) leaf litter extraction with the standard area technique.

#### **4. Discussion**

A common perception of urban biodiversity is that it is characterized by low species richness and dominated by exotic species [12]. This perception may be induced by the excess of local scale studies (α diversity) compared to the limited number of studies at larger scale (γ diversity) encompassing the full diversity encountered within of a city (Table 1). Here, our results contrast with the former assumption but agree with the latter. Indeed, we found that Macao hosts a diverse ant fauna, but that a high number of that fauna consists of exotic and tramp species.

Macao's ant fauna presents one the highest known ant richness reported for an urban region (Table 1). Our results indicate that, while there are few comprehensive studies for tropical regions—most studies on urban ants have been conducted within temperate regions where species diversity is usually much lower than in tropical and subtropical regions [202]— several cities, including Macao, offer potential conservation values for ants. For instance, a study limited to the botanical garden of Bogor (Java), a small green oasis within an urban area, captured 216 ant species [69]. Similarly, ecological studies in Abidjan (Ivory Coast, 176 species) and Uberlândia (Brazil, 143 species), among others, also presented high ant species richness [62,155]. Altogether, these results highlight the potential conservation value of urban habitats, but also their potential to increase the biogeographic and taxonomic knowledge on ants. Indeed, contrary to most natural habitats, urban habitats are characterized by their easy access, which facilitates continuous and thorough sampling. As for tropical forests, the vertical stratification of ants within cities does exist, and, as a result, researchers should consider diversifying their sampling approach to include subterranean and arboreal communities as well as epigeic ants.

Ant assemblages are known to be highly structured along a vertical gradient ranging from the top soil layer (first 50 cm) to the tree canopy [203,204], but such stratification had not been shown for urban environments prior to this study. Our results show that ignoring these strata may lead to an underestimation of species richness estimates. Indeed, although most of the new species records were collected within the ground stratum, we found several previously unrecorded arboreal and subterranean specialists. In particular, of the 35 species collected with our subterranean trapping, 10 were found only within that stratum, nine of which were new records, and three represented undescribed species. Remarkably, this parallels the results of previous surveys focusing on multiple strata but conducted within natural ecosystems [205]. For instance, in Ecuador, Wilkie and collaborators collected 47 species in subterranean probes, nine of which were exclusively subterranean, and two were undescribed [205]. It is also worth noting that 14 species collected during the 2019 survey were unique to the arboreal stratum, nine of which were new records, and one was an undescribed species. This shows the importance of using sampling methods targeting the ant communities of all vertical strata, instead of focusing solely on ants found within a single stratum as is commonly done in urban studies [52].

Other methods captured fewer novel records, but, nonetheless, provided ecological and biogeographic information for a wide range of species. For instance, ground baits often collected large series of workers, including multiple worker castes for polymorphic species, which is often essential for their identification. As for ground nests, they had the lowest rate of capture, but provided important and rarely collected sociometric data, including new colony size information for seven species (Guénard, unpublished). Since we still lack information on the sociometry of most ant species [206,207], ground nests proved especially useful in collecting this valuable data. However, it is worth noting that the colonization rate of the ground nests, with 3%, was neatly inferior to the rate observed in previous studies using similar devices (e.g., 8% in [180]), or from other urban areas [141]. While the type of nests may be suboptimal for the ant community present in Macao, it is surprising that they were not more heavily exploited by ants, especially because urban habitats are usually characterized by limited nesting resources [141]. Perhaps the subtropical conditions characterized by heavy rains prevented the establishment of ants. Indeed, several nests had their openings clogged with mud and some had their inner cavities filled with fungal growths. Just as with temperate regions [141], the testing and deployment of different artificial nest apparatuses may represent an interesting opportunity to census urban ants and their sociometric characteristics.

Even though we used an exhaustive sampling approach, and our results substantially increased our knowledge on Macanese ants, species accumulation curves indicate a substantial number of unrecorded species to be collected using a similar methodology (Figure 4). This is supported by the several species previously recorded within Macao, but not collected in this study (Table 2). Previous studies conducted within more natural or relatively undisturbed habitats showed that achieving an exhaustive sampling of local diversity represents a challenging task [125,208,209]. After two separate survey programs ([53], this study), our results confirm this is similar for urban areas. Furthermore, other sampling methods not used here could have been added, such as pitfall traps to collect larger ground-dwelling ants [210], canopy fogging to collect arboreal species [211], Malaise traps to collect alates [212], and multiple soil sampling methods to collect subterranean ants [50]. Thus, while our study contributes to a better understanding of ant species richness and composition in Macao, it represents a steppingstone and not a final outcome, with future sampling likely to provide additional new records, and potentially more undescribed species.

Despite the geographic limitations of our sampling, being restricted to Coloane island, this allowed us to considerably increase the list of Macanese ant species. These new records highlight the potential for urban forest fragments and other urban habitats to maintain a significant portion of ant diversity. Similar examples can be found in Asia, such as in in Bogor and Singapore [69,102]. Likewise, Hong Kong harbors a high ant richness (Lee et al. in press, Guénard unpublished), with numerous new records and species recently reported (e.g., [55,192]). Additionally, comparative studies focusing on old growth and secondary forests in both cities also reported no significant difference in ant richness between the types of forests ([213,214], Nooten et al. submitted). As such, past studies and the current one should motivate the conservation of forest patches in and around urban matrices in Asia, whether they are primary or secondary.

Nevertheless, a key difference between disturbed and undisturbed habitats lies within their species composition instead of in the number of species they harbor, and a significant part of Macao's fauna consists of non-native species. The previous survey reported several new records of exotic species in Macao [53], which are here completed with the additions of four exotic and three tramp species (Figure 1), totaling in 18 exotic species and representing 11.5% of the Macanese ant fauna. The most notable newly reported exotic is *Brachymyrmex patagonicus*, a major pest in south-east USA [215]. This represents the second record of *B. patagonicus* in continental Asia, with the first report from Hong Kong [216]. Its presence in Macao is worrisome, and clear plans to determine the extent of its distribution, with programs to destroy established populations, should be developed quickly. We also report the spread of several alien and potentially harmful species. We found the three exotic species *Anoplolepis gracilipes, Monomorium pharaonis*, and *Paratrechina longicornis* in five, seven, and six forested sites, respectively. They can, thus, establish populations and persist in forested habitats, where their effects on native ants are unknown. Through our standardized sampling, we also found the notorious invasive species *Solenopsis invicta* and *Pheidole megacephala* but, interestingly, rarely found it in Coloane's forested sites. Despite finding several workers and queens of *S. invicta* through hand collection in open and urban habitats, we found this species in only one forested site near a hiking trail: one individual was found in a leaf litter sample and several workers colonized two ground baits at that site. This reflects previous findings that the distribution of *S. invicta* is mainly restricted to disturbed habitats [217–219]. Similarly, while we found a high abundance of workers of *P. megacephala* on one golf course, we collected only two individuals in forested sites. For both of these species, notorious for their destructive impacts on native communities [220–222], this would indicate that, for now, they are mainly restricted to more open and disturbed habitats. This suggests that forested habitats may help preserve local biodiversity while simultaneously limiting the distribution of some invasive ant species, reflecting results from Hong Kong and Hainan, where less disturbed habitats harbored relatively fewer exotic species than did more disturbed ones [223].

Nonetheless, the occurrence of exotic species in Macao is high. In comparison, the checklist of the ants of Yunnan reports that only 2% of its species are introduced [224], a proportion 5.75 times lower than in Macao. Based on current knowledge, Macao appears to have one of the highest numbers and proportions of exotic species encountered within cities (Table 1). Alas, we also report the presence of 17 tramp species, some of which may very well be introduced, as our biogeographic understanding and the region of origin for several species remains limited; thus, the overall number may be even higher. The high proportion of exotic and tramp species in Macao supports the hypothesis that coastal cities act as gateways for the introduction of exotic species through high propagule pressure [225]. In addition, since the occurrence of non-native species is an indicator of reduced ecological integrity [226], the high proportion of non-native species we found in Macao suggests these habitats have suffered substantial damage. It is, thus, crucial to make periodical biodiversity surveys in Macao and other cities to monitor their habitats' health through time, as well as to identify new arrivals of exotics and prevent their further spread.

An historic baseline is lacking for Macao's ant assemblages, but we suspect there may be substantial differences compared to the ant assemblages before urbanization. Indeed, several genera known to occur in southern China and within neighboring cities of the Greater Bay Area, such as Hong Kong, are missing from Macao. These include *Aenictus* and *Dorylus* army ants, *Discothyrea*, *Odontomachus*, *Ponera*, and weaver ants (*Oecophylla smaragdina*). The absence of army ants in Coloane (except *O. biroi*, an exotic species), such as species of *Aenictus* and *Dorylus*, may be explained by the morphology of the queen caste in these genera: they lack wings and, thus, cannot disperse by flight [227]. As such, if these ants disappeared from Coloane during a disturbance event, we may expect that it would be arduous for these species to recolonize the island. Of course, native army ant species may have been absent from Coloane throughout Macao's urbanization history due to its insularity or of its small size ([228], but see [229]). However, the presence of native army ants on several of Hong Kong' islands—for example, the *Aenictus* species found on Lamma Island (13.55 km2, François Brassard pers. obs.)—suggests they could have been extirpated from Coloane Island and then failed to recolonize. More puzzling is the absence of genera such as *Discothyrea, Odontomachus, Oecophylla*, and *Ponera*. Small and relatively uncommon species of *Discothyrea* and *Ponera* may have escaped our sampling or may be especially sensitive to disturbance. However, for unknown reasons, large and conspicuous species such as *Odontomachus* and especially *Oecophylla*, frequently encountered on forest edges or in disturbed habitats within Hong Kong, are probably truly absent from the island.

#### **5. Conclusions**

In summary, this study highlights the importance of conducting holistic biodiversity surveys in cities to discover new records as well as potential new species for science, and to monitor the introduction of new exotic species. Our results suggest that forest patches in cities can harbor a diverse ant fauna and may have a significant conservation value. However, exhaustive ant diversity surveys in cities are rare, and are often based on incomplete sampling approaches. Thus, until the completion of several more surveys in cities around the world, particularly within tropical regions, a clear understanding of how urban environments may act as biodiversity refuges and gateways for exotic species will be lacking. As such, we advocate that conservation management practices should implement regular biodiversity surveys using an exhaustive sampling approach in urban regions worldwide. Whenever possible, we also recommend that urban biodiversity assessments be combined with surveys done in less urbanized habitats nearby to compare the diversity and composition of each habitat.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10 .3390/d13080358/s1. Figure S1: Map of Coloane showing the 21 sites sampled in the 2019 survey. White dots mark sites where the full protocol was done (i.e., standardized and species pool leaf litter extractions, ground baiting, ground nests, subterranean traps, and arboreal traps), whereas grey dots mark preliminary sites where only ground baiting and leaf litter extractions were done. Hand

collection was also opportunistically used at each site. Figure S2: Design of a 20 × 20 m sampling plot. Each of the 1 × 1 m quadrats where subterranean traps and leaf litter extraction (standardized area) was performed were placed at a corner of the plot. Black dots show the emplacement of nest bundles. For the species pool leaf litter extraction, 12 microhabitats were sampled within the light gray area. For the arboreal baiting protocol, four trees measuring a minimum of 5 m height were sampled within the grey area. Table S1: List of the localities of each sampling sites, their associated number, and their geolocation. The date refers to the first sampling event made at a site, which corresponded to the leaf litter extraction and placement of subterranean traps. Sampling protocols are defined as follows: the letter (P) signifies a partial sampling protocol (i.e., leaf litter extraction, ground baiting, and hand collection), whereas the letter (F) signifies a full protocol (i.e., leaf litter extraction, ground baiting, ground nests, subterranean traps, arboreal traps, and hand collection). Table S2: Sociometry data collected using ground nests. Macao species: material examined.

**Author Contributions:** Conceptualization, F.B. and B.G.; methodology, F.B. and B.G.; validation, F.B. and B.G.; formal analysis, F.B.; investigation, F.B.; resources, B.G., H.-H.C., and C.-M.L.; data curation, F.B. and B.G.; writing—original draft preparation, F.B.; writing—review and editing, F.B., B.G., C.-M.L., and H.-H.C.; visualization, F.B.; supervision, B.G.; project administration, B.G.; funding acquisition, B.G. All authors have read and agreed to the published version of the manuscript.

**Funding:** F.B. was supported by the Instituto para os Assuntos Municipais, Macao SAR, China, CML was supported by the Macao Foundation and the Direcção dos Serviços do Ensino Superior, Macao SAR, China, and CML and FB were supported by The University of Hong Kong, Hong Kong SAR, China.

**Institutional Review Board Statement:** Ethical review and approval were waived for this study, due to our work being conducted on invertebrates.

**Data Availability Statement:** The data presented in this study are available in tables within the main text and within the supplementary material section.

**Acknowledgments:** We thank Jason L. Williams and Georg Fischer for their help with the identification of species within the genera *Nylanderia* and *Carebara*, respectively. We acknowledge that the images we shared with these specialists are not optimal for identification and, as such, if some misidentifications are later revealed, these errors should be considered our own, not theirs. We thank Siu Yiu for her help conducting fieldwork, as well as with sorting, mounting, and imaging specimens. We thank Carly McGregor for her help conducting fieldwork. We thank Sabine Nooten for her help producing species accumulation curves. We also thank the Environmental Protection Bureau, Caesars Golf Macau, and the Macau Golf and Country Club for allowing us to sample on their premises.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

#### **Appendix A**

The appendix shows all species for which we had specimens within the IBBL collection. These are shown in lateral, dorsal, and face view. Within the figure, we include a map of south-east Asia showing the distribution of the species. We colored polygons according to whether a species was recorded as a native or exotic species, or unrecorded for a specific region. Note that we could not use the label tramp because most studies do not distinguish beyond native or exotic. We also include an inset map showing where the species was found in Macao. Black triangles indicate collection locations made during the 2019 survey, whereas white dots indicate collection locations not done during the survey.

#### *Appendix A.1 AMBLYOPONINAE*

**Figure A1.** *Stigmatomma rothneyi* Forel, 1900 worker (MAC\_S11\_LLSA\_Sp.2, IBBL).

#### *Appendix A.2 DOLICHODERINAE*

**Figure A2.** *Chronoxenus morpho 1* worker (MAC\_S14\_LLSP, IBBL).

**Figure A3.** *Chronoxenus morpho 2* worker (MAC\_S21\_q2\_50\_sp.2, IBBL).

**Figure A4.** *Dolichoderus* nr. *sibiricus* Emery, 1889 worker (FB19279, IBBL).

**Figure A5.** *Iridomyrmex anceps* grp. Roger, 1863 worker (FB19166, IBBL).

**Figure A6.** *Ochetellus glaber* Mayr, 1862 worker (MAC\_S19\_LLSP\_sp.5, IBBL).

**Figure A7.** *Tapinoma indicum* Forel, 1895 worker (MAC\_S01\_LLSP\_Sp.9, IBBL).

**Figure A8.** *Tapinoma melanocephalum* Fabricius, 1793 worker (MAC\_S01\_LLSP\_Sp.6, IBBL).

**Figure A9.** *Tapinoma* sp. 1 FB worker (MAC\_S11\_T4\_1m\_sp.2, IBBL).

**Figure A10.** *Technomyrmex brunneus* Forel, 1895 worker (FB19281, IBBL).

**Figure A11.** *Technomyrmex horni* Forel, 1912 worker (MAC\_S01\_LLSP\_Sp.2, IBBL).

#### *Appendix A.3 DORYLINAE*

**Figure A12.** *Ooceraea biroi* Forel, 1907 worker (MAC\_S02\_LLSP\_Sp.4, IBBL).

#### *Appendix A.4 FORMICINAE*

**Figure A13.** *Acropyga acutiventris* Roger, 1962 worker (*Acropyga acutiventris*, CML collection).

**Figure A14.** *Acropyga* sp. mo02 gyne (*Acropyga* sp. mo02, CML collection).

**Figure A15.** *Anoplolepis gracilipes* Smith, 1857 worker (MAC\_S14\_LLSA\_Sp.14, IBBL).

**Figure A16.** *Brachymyrmex patagonicus* Mayr, 1868 worker (FB19202, IBBL).

**Figure A17.** *Camponotus carin* Emery, 1889 worker (*Camponotus carin*, CML collection).

**Figure A18.** *Camponotus* nr. *irritans* Smith, F., 1857 worker (MAC\_S20\_B08, IBBL).

**Figure A19.** *Camponotus* nr. *irritans* Smith, F., 1857 major (MAC\_S20\_B08, IBBL).

**Figure A20.** *Camponotus lighti* Wheeler, 1927 worker (*Camponotus lighti*, CML collection).

**Figure A21.** *Camponotus lighti* Wheeler, 1927 major (*Camponotus lighti*, CML collection).

**Figure A22.** *Camponotus mitis* Smith, 1858 worker (MAC\_S07\_LLSA\_sp.1, IBBL).

**Figure A23.** *Camponotus mitis* Smith, 1858 major (MAC\_S11\_LLSP\_Sp.12, IBBL).

**Figure A24.** *Camponotus nicobarensis* Mayr, 1865 worker (MAC\_S21\_LLSP, IBBL).

**Figure A25.** *Camponotus nicobarensis* Mayr, 1865 major (MAC\_S16\_T1\_5m\_sp.3, IBBL).

**Figure A26.** *Camponotus parius* Emery, 1889 worker (*Camponotus parius,* CML collection).

**Figure A27.** *Camponotus variegatus* dulcis Dalla Torre, 1893 worker (*Camponotus variegatus* dulcis, CML collection).

**Figure A28.** *Camponotus vitiosus* Smith, 1874 worker (MAC\_S20\_T4\_2m\_Sp.1, IBBL).

**Figure A29.** *Camponotus vitiosus* Smith, 1874 major (MAC\_S14\_T1\_3m\_Sp.1, IBBL).

**Figure A30.** *Camponotus* sp.1 FB worker (MAC\_FB19182, IBBL).

**Figure A31.** *Colobopsis* nr. *nipponica* Wheeler, 1928 worker (*Colobopsis* nr. *nipponica*, CML collection).

**Figure A32.** *Colobopsis* nr. *vitrea* Smith, 1860 worker (*Colobopsis* nr. *vitrea*, CML collection).

**Figure A33.** *Colobopsis* nr. *vitrea* Smith, 1860 major (FB19268, IBBL).

**Figure A34.** *Gesomyrmex howardi* Wheeler, W. M., 1921 worker (MWong\_MaiPo\_7viii2018, IBBL).

**Figure A35.** *Gesomyrmex howardi* Wheeler, W. M., 1921 supermajor (MWong\_MaiPo\_7viii2018\_ Colony4\_6\_2.6x12.5, IBBL).

**Figure A36.** *Lepisiota rothneyi* Forel, 1894 worker (MAC\_S06\_B08\_Sp.1\_top, IBBL).

**Figure A37.** *Nylanderia amia* Forel, 1913 worker (ANTWEB1016677, IBBL).

**Figure A38.** *Nylanderia bourbonica* Forel, 1886 worker (MAC\_S20\_LLSP\_Sp.4, IBBL).

**Figure A39.** *Nylanderia indica* Forel, 1894 worker (MAC\_S12\_LLSP\_sp.4, IBBL).

**Figure A40.** *Nylanderia sharpii* Forel, 1899 worker (MAC\_S19\_LLSA\_Sp.5, IBBL).

**Figure A41.** *Nylanderia taylori* Forel, 1894 worker (MAC\_S21\_GN1\_H4\_n1\_bottom, IBBL).

**Figure A42.** *Nylanderia* sp. 3 BG worker (MAC\_S15\_B03\_sp.3, IBBL).

**Figure A43.** *Nylanderia* sp. 6 BG worker (MAC\_S15\_LLSP\_sp.10, IBBL).

**Figure A44.** *Paraparatrechina* sp.1 BG Forel, 1913 worker (MAC\_S18\_q2\_37.5\_Sp.2, IBBL).

**Figure A45.** *Paratrechina longicornis* Latreille, 1802 worker (MAC\_S11\_LLSP\_Sp.6, IBBL).

**Figure A46.** *Plagiolepis alluaudi* Emery, 1894 worker (MAC\_S14\_T2\_2m\_Sp.1, IBBL).

**Figure A47.** *Polyrhachis confusa* Emery, 1893 worker (FB19152, IBBL).

**Figure A48.** *Polyrhachis demangei* Santschi, 1910 worker (*Polyrhachis demangei*, CML collection).

**Figure A49.** *Polyrhachis dives* Smith, 1857 worker (MAC\_S03\_HC\_01\_Sp.2, IBBL).

**Figure A50.** *Polyrhachis illaudata* Walker, 1859 worker (MAC\_S03\_HC\_01\_Sp.1, IBBL).

**Figure A51.** *Polyrhachis latona* Wheeler, 1909 worker (MAC\_S03\_LLSA\_Sp.5, IBBL).

**Figure A52.** *Polyrhachis tyrannica* Smith, 1858 worker (K6558(2)). Species images taken from Wong and Guénard 2020 [55] with permission.

**Figure A53.** *Pseudolasius risii* Forel, 1894 worker (*Pseudolasius risii*, CML collection).

#### *Appendix A.5 LEPTANILLINAE*

**Figure A54.** *Leptanilla macaoensis* Leong, Yamane & Guénard, 2018 worker (LCM00039, IBBL). Species images taken from Leong, Yamane & Guénard, 2018 [61] with permission.

#### *Appendix A.6 MYRMICINAE*

**Figure A55.** *Cardicondyla minutior* Forel, 1899 worker (MAC\_S04\_LLSP\_sp.6, IBBL).

**Figure A56.** *Cardiocondyla wroughtonii* Forel, 1890 worker (MAC\_S11\_T3\_3m\_sp.4, IBBL).

**Figure A57.** *Carebara affinis* Jerdon, 1851 worker (MAC\_S9\_37.5\_q2\_sp.1, IBBL).

**Figure A58.** *Carebara affinis* Jerdon, 1851 major (MAC\_S8\_25\_q1\_sp.1, IBBL).

**Figure A59.** *Carebara* nr. *diversa* Jerdon, 1851 worker (MAC\_S18\_q2\_37.5\_sp.3, IBBL).

**Figure A60.** *Carebara* nr. *diversa* Jerdon, 1851 major (MAC\_S18\_q2\_37.5\_sp.3, IBBL).

**Figure A61.** *Carebara diversa laotina*, Santschi, 1921 worker (*Carebara diversa laotina*, CML collection).

**Figure A62.** *Carebara diversa laotina*, Santschi, 1921 major (*Carebara diversa laotina*, CML collection).

**Figure A63.** *Carebara melasolena* Zhou & Zheng, 1997 worker (MAC\_S12\_LLSA\_sp.1, IBBL).

**Figure A64.** *Carebara melasolena* Zhou & Zheng, 1997 major (MAC\_S12\_LLSA\_sp.1, IBBL).

**Figure A65.** *Carebara sangi* Eguchi & Bui, 2007 worker (MAC\_S13\_q1\_25\_Sp.2, IBBL).

**Figure A66.** *Carebara zengchengensis* Zhou, Zhao & Jia, 2006 worker (MAC\_S12\_q3\_37.5\_Sp.4, IBBL).

**Figure A67.** *Carebara zengchengensis* Zhou, Zhao & Jia, 2006 worker (MAC\_S12\_q3\_37.5\_Sp.4, IBBL).

**Figure A68.** *Crematogaster binghamii* Forel, 1904 worker (MAC\_S21\_B08\_sp.1, IBBL).

**Figure A69.** *Crematogaster ferrarii* Emery, 1888 worker (MAC\_S01\_LLSP\_Sp.7, IBBL).

**Figure A70.** *Crematogaster quadriruga* Forel, 1911 worker (MAC\_S06\_LLSA\_sp.1, IBBL).

**Figure A71.** *Crematogaster quadriruga* Forel, 1911 intercaste (MAC\_S17\_LLSA\_sp.3, IBBL).

**Figure A72.** *Crematogaster rogenhoferi* Mayr, 1879 worker (MAC\_S19\_T4\_1m\_sp.1, IBBL).

**Figure A73.** *Dilobocondyla propotriangulata*, Bharti & Kumar, 2013 worker (FB19145, IBBL).

**Figure A74.** *Mayriella granulata*, Dlussky & Radchenko, 1990 worker (MAC\_S18\_LLSA\_Sp.4, IBBL).

**Figure A75.** *Meranoplus* sp. mo01 nr. *bicolor* Guérin-Méneville, 1844 worker (*Meranoplus* sp. mo01 nr. *Bicolor*, CML collection).

**Figure A76.** *Monomorium chinense* Santschi, 1925 worker (MAC\_S20\_LLSP\_sp.12, IBBL).

**Figure A77.** *Monomorium floricola* Jerdon, 1851 worker (MAC\_S03\_LLSA\_Sp.8, IBBL).

**Figure A78.** *Monomorium intrudens* Smith, 1874 worker (MAC\_S18\_LLSP\_sp.3, IBBL).

**Figure A79.** *Monomorium pharaonis* Linnaeus, 1758 worker (MAC\_S09\_LLSA\_sp.9, IBBL).

**Figure A80.** *Monomorium* sp. psw-cn01 worker (MAC\_S21\_LLSA\_bottom\_sp.2, IBBL).

**Figure A81.** *Myrmecina nomurai* Okido, Ogata & Hosoishi, 2020 worker (MAC\_S05\_LLSA\_ Sp.1, IBBL).

**Figure A82.** *Myrmecina sinensis* Wheeler, W. M., 1921 worker (*Myrmecina sinensis*, CML collection).

**Figure A83.** *Pheidole elongicephala* Eguchi, 2008 worker (MAC\_S09\_q2\_25\_sp.2, IBBL).

**Figure A84.** *Pheidole fervens* Smith, 1858 worker (MAC\_S19\_q4\_GL\_03\_Sp.2, IBBL).

**Figure A85.** *Pheidole fervens* Smith, 1858 major (MAC\_S19\_q4\_GL\_03\_Sp.2, IBBL).

**Figure A86.** *Pheidole hongkongensis* Wheeler, 1928 worker (MAC\_S21\_LLSP\_Sp.9, IBBL).

**Figure A87.** *Pheidole hongkongensis* Wheeler, 1928 major (MAC\_S07\_B08\_sp.1, IBBL).

**Figure A88.** *Pheidole megacephala* Fabricius, 1793 worker (MAC\_S13\_LLSP\_Sp.1, IBBL).

**Figure A89.** *Pheidole megacephala* Fabricius, 1793 major (MAC\_S13\_LLSP\_Sp.1, IBBL).

**Figure A90.** *Pheidole nodus* Smith, 1874 worker (MAC\_S02\_B09\_sp.1\_top, IBBL).

**Figure A91.** *Pheidole ochracea* Eguchi, 2008 worker (MAC\_S03\_B03\_sp.1, IBBL).

**Figure A92.** *Pheidole ochracea* Eguchi, 2008 major (MAC\_S03\_B03\_sp.1, IBBL).

**Figure A93.** *Pheidole parva* Mayr, 1865 worker (MAC\_S20\_LLSA\_sp.4, IBBL).

**Figure A94.** *Pheidole parva* Mayr, 1865 major (MAC\_S20\_LLSA\_sp.4, IBBL).

**Figure A95.** *Pheidole pieli* Santschi, 1925 worker (MAC\_S17\_LLSA\_Sp.4, IBBL).

**Figure A96.** *Pheidole* nr. *ryukyuensis* Ogata, 1982 worker (MAC\_S20\_12.5\_q4\_sp.2, IBBL).

**Figure A97.** *Pheidole taipoana* Wheeler, 1928 worker (MAC\_S04\_LLSA\_Sp.4, IBBL).

**Figure A98.** *Pheidole taipoana* Wheeler, 1928 major (MAC\_S04\_B07\_sp.1\_top, IBBL).

**Figure A99.** *Pheidole tumida* Eguchi, 2008 worker (MAC\_S04\_B06\_sp.2\_top, IBBL).

**Figure A100.** *Pheidole tumida* Eguchi, 2008 major (MAC\_S7\_GN1\_H4\_n1, IBBL).

**Figure A101.** *Pheidole vulgaris* Eguchi, 2006 worker (MAC\_S12\_B04\_sp.2\_bottom, IBBL).

**Figure A102.** *Pheidole zoceana* Santschi, 1925 worker (MAC\_S11\_LLSA\_sp.6, IBBL).

**Figure A103.** *Pheidole zoceana* Santschi, 1925 major (MAC\_S17\_LLSA\_sp.1, IBBL).

**Figure A104.** *Recurvidris recurvispinosa* Forel, 1890 (MAC\_S12\_LLSA\_Sp.9, IBBL).

**Figure A105.** *Rotastruma stenoceps* Bolton, 1991 worker (MAC\_S15\_LLSA\_\_sp.6, IBBL).

**Figure A106.** *Solenopsis geminata* Fabricius, 1804 worker (*Solenopsis geminata*, IBBL).

**Figure A107.** *Solenopsis geminata* Fabricius, 1804 major (*Solenopsis geminata*, IBBL).

**Figure A108.** *Solenopsis invicta* Buren, 1972 worker (MAC\_S09\_LLSP\_Sp.5, IBBL).

**Figure A109.** *Solenopsis invicta* Buren, 1972 major (MAC\_S09\_B05\_sp.1\_bottom, IBBL).

**Figure A110.** *Solenopsis jacoti* Wheeler, 1923 worker (MAC\_S12\_q3\_37.5\_sp.2\_top, IBBL).

**Figure A111.** *Strumigenys elegantula* Terayama & Kubota, 1989 worker (MAC\_S04\_LLSP\_sp.9, IBBL).

**Figure A112.** *Strumigenys emmae* Emery, 1890 worker (MAC\_S20\_LLSP\_Sp.7, IBBL).

**Figure A113.** *Strumigenys exilirhina* Bolton, 2000 worker (MAC\_S01\_LLSA\_Sp.3, IBBL).

**Figure A114.** *Strumigenys feae* Emery, 1895 worker (MAC\_S15\_LLSP\_Sp.8, IBBL).

**Figure A115.** *Strumigenys membranifera* Emery, 1869 worker (MAC\_S15\_GN3\_H3\_n1\_top, IBBL).

**Figure A116.** *Strumigenys minutula* Terayama & Kubota, 1989 worker (MAC\_S14\_LLSP\_Sp.4, IBBL).

**Figure A117.** *Strumigenys nepalensis* Baroni Urbani & De Andrade, 1994 worker (MAC\_S19\_ LLSP\_Sp.3, IBBL).

**Figure A118.** *Strumigenys sauteri* Forel, 1912 worker (MAC\_S04\_LLSP\_sp.2, IBBL).

**Figure A119.** *Strumigenys subterranea* Brassard, Leong & Guénard, 2020 worker (MAC\_S12\_q4\_ 12.5\_sp.2, IBBL).

**Figure A120.** *Syllophopsis* nr. *cryptobia* worker (MAC\_S12\_q3\_37.5\_sp.3, IBBL).

**Figure A121.** *Syllophopsis* sp. mo01 nr. *sechellensis* Emery, 1894 worker (*Syllophopsis* sp. mo01 nr. *Sechellensis*, CML collection).

**Figure A122.** *Syllophopsis* sp. 1 FB worker (MAC\_S18\_q3\_12.5\_sp.4, IBBL).

**Figure A123.** *Syllophopsis* sp. 2 FB worker (MAC\_S20\_LLSP\_sp.10\_top, IBBL).

**Figure A124.** *Tetramorium bicarinatum* Nylander, 1846 worker (MAC\_S19\_LLSP\_Sp.9, IBBL).

**Figure A125.** *Tetramorium indicum* Forel, 1913 worker (MAC\_S08\_T3\_1m\_sp.1, IBBL).

**Figure A126.** *Tetramorium insolens* Smith, 1861 worker (MAC\_S17\_LLSA\_Sp.3, IBBL).

**Figure A127.** *Tetramorium kraepelini* Forel, 1905 worker (MAC\_S8\_GN1\_H2\_n1\_sp.1, IBBL).

**Figure A128.** *Tetramorium lanuginosum* Mayr, 1870 worker (MAC\_S17\_LLSA\_Sp.2, IBBL).

**Figure A129.** *Tetramorium nipponense* Wheeler, 1928 worker (MAC\_S7\_GN2\_H4\_n1, IBBL).

**Figure A130.** *Tetramorium parvispinum* Emery, 1893 worker (*Tetramorium parvispinum,* CML collection).

**Figure A131.** Tetramorium simillinum Smith, 1851 worker (Tetramorium simillinum, IBBL).

**Figure A132.** *Tetramorium tonganum* Mayr, 1870 worker (MAC\_S10\_T2\_1m\_sp.2, IBBL). Note that we changed the location of the map of Macao to show the localities where this species has been recorded as an exotic species in Southeast Asia.

**Figure A133.** *Tetramorium wroughtonii* Forel, 1902 worker (MAC\_S10\_B03\_sp.1\_top, IBBL).

**Figure A134.** *Tetramorium* nr. *elisabethae* Forel, 1904 worker (MAC\_S18\_q1\_25\_Sp.2, IBBL).

**Figure A135.** *Tetramorium* sp. 1 BG (*obesum* group Bolton, 1976) worker (MAC\_S04\_LLSP\_Sp.3, IBBL).

**Figure A136.** *Tetramorium* sp. 2 JF worker (MAC\_S15\_T1\_3m\_sp.2, IBBL).

**Figure A137.** *Tetramorium* sp. 9 JF worker (MAC\_S18\_q2\_25\_sp.1, IBBL).

**Figure A138.** *Vollenhovia* sp. 1 BG queen (MAC\_S06\_GN3\_H3\_n1, IBBL).

**Figure A139.** *Vollenhovia* sp. 2 BG worker (MAC\_S02\_LLSP\_Sp.5, IBBL).

#### *Appendix A.7 PONERINAE*

**Figure A140.** *Anochetus risii* Forel, 1900 worker (MAC\_FB19180, IBBL).

**Figure A141.** *Brachyponera obscurans* Mayr, 1862 worker (MAC\_S19\_LLSA, IBBL).

**Figure A142.** *Buniapone amblyops* Emery, 1887 worker (MAC\_S12\_q4\_50\_sp.2\_top, IBBL).

**Figure A143.** *Diacamma* sp. 1 worker (MAC\_S15\_LLSA\_sp.1, IBBL).

**Figure A144.** *Ectomomyrmex annamitus* André, 1892 worker (MAC\_S18\_q2\_37.5\_Sp.4, IBBL).

**Figure A145.** *Ectomomyrmex leeuwenhoecki* Forel, 1886 worker (MAC\_S18\_GN5\_H4\_n1, IBBL).

**Figure A146.** *Euponera pilosior* Wheeler, 1928 worker (MAC\_S12\_q3\_50\_Sp.1, IBBL).

**Figure A147.** *Harpegnathos venator* Smith, 1858 worker (MAC\_ZOO\_HC07\_Sp.1, IBBL).

**Figure A148.** *Hypoponera exoecata* Wheeler, 1928 worker (*Hypoponera exoecata*, CML collection).

**Figure A149.** *Hypoponera* sp. psw-cn01 worker (*Hypoponera* sp. psw-cn01, CML collection).

**Figure A150.** *Leptogenys chinensis* Mayr, 1870 worker (RHL00861, IBBL).

**Figure A151.** *Leptogenys peuqueti* André, 1887 worker (*Leptogenys peuqueti,* CML collection).

**Figure A152.** *Odontoponera denticulata* Smith, 1858 worker (MAC\_S09\_LLSA\_Sp.3, IBBL).

**Figure A153.** *Pseudoneoponera rufipes* Jerdon, 1851 worker (MAC\_S03\_LLSA\_Sp.1, IBBL).

#### *Appendix A.8 PROCERATIINAE*

**Figure A154.** *Probolomyrmex dammermani* Wheeler, W. M., 1928 worker (*Probolomyrmex dammermani*, IBBL).

**Figure A155.** *Proceratium* sp. cf. *bruelheidei* Staab, Xu & Hita Garcia, 2018 queen (MAC\_S05\_ LLSP\_Sp.1, IBBL).

#### *Appendix A.9 PSEUDOMYRMICINAE*

**Figure A156.** *Tetraponera allaborans* Walker, 1859 worker (*Tetraponera allaborans*, CML collection).

**Figure A157.** *Tetraponera binghami* Forel, 1902 worker (FB19140, IBBL).

**Figure A158.** *Tetraponera nitida* Smith, 1860 worker (MAC\_GOV\_Workshop, IBBL).

#### **References**


### *Article* **Biogeography of Iberian Ants (Hymenoptera: Formicidae)**

**Alberto Tinaut and Francisca Ruano \***

Department of Zoology, University of Granada, Campus de Fuentenueva s/n, 18071 Granada, Spain; hormiga@ugr.es

**\*** Correspondence: fruano@ugr.es

**Abstract:** Ants are highly diverse in the Iberian Peninsula (IP), both in species richness (299 cited species) and in number of endemic species (72). The Iberian ant fauna is one of the richest in the broader Mediterranean region, it is similar to the Balkan Peninsula but lower than Greece or Israel, when species richness is controlled by the surface area. In this first general study on the biogeography of Iberian ants, we propose seven chorological categories for grouping thems. Moreover, we also propose eight biogeographic refugium areas, based on the criteria of "refugia-within-refugium" in the IP. We analysed species richness, occurrence and endemism in all these refugium areas, which we found to be significantly different as far as ant similarity was concerned. Finally, we collected published evidence of biological traits, molecular phylogenies, fossil deposits and geological processes to be able to infer the most probable centre of origin and dispersal routes followed for the most noteworthy ants in the IP. As a result, we have divided the Iberian myrmecofauna into four biogeographical groups: relict, Asian-IP disjunct, Baetic-Rifan and Alpine. To sum up, our results support biogeography as being a significant factor for determining the current structure of ant communities, especially in the very complex and heterogenous IP. Moreover, the taxonomic diversity and distribution patterns we describe in this study highlight the utility of Iberian ants for understanding the complex evolutionary history and biogeography of the Iberian Peninsula.

**Citation:** Tinaut, A.; Ruano, F. Biogeography of Iberian Ants (Hymenoptera: Formicidae). *Diversity* **2021**, *13*, 88. https://doi.org/ 10.3390/d13020088

Academic Editor: Luc Legal Received: 6 December 2020 Accepted: 12 February 2021 Published: 19 February 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

**Keywords:** species richness; species occurrence; endemic species; distribution ranges; dispersal routes; centre of origin; refugium areas

#### **1. Introduction**

Ants bring together a number of traits that make up an interesting subject for biogeographical studies: they are conspicuous and one of the most well-known taxa of the terrestrial invertebrates [1], near-ubiquitous in terrestrial ecosystems [2]. They are an ecologically dominant faunal group [3] and highly diverse but still accessible [3] and show an extreme diversity of dispersal strategies although they are sessile superorganisms [4].

Local ant species richness is strongly correlated with temperature, which is the most important factor determining the structure of ant communities [1]. Ants are highly diverse in hot and dry habitats [5] and in low-elevation, low-latitude forests [6]. The latitudinal diversity gradient [6] with an asymmetric northern hemispheric pattern has been detected for ants, which is impossible to explain by only contemporary biotic and abiotic variables [5]. None of the current factors sufficiently correlate to explain current ant diversity [2,5, 7]. Moreover, modern ant assemblages suggest a climate-driven past reorganization of the Palaearctic ant fauna [7]. Many ant lineages which previously lived in a warmer Europe, were not able to survive through long-term cooling (Pliocene) and glacial cycles (Pleistocene) but some of them persisted in Indomalayan and Australian regions [7]. Thus, the high extinction pattern in the northern hemisphere appears to have conditioned its marked asymmetric latitudinal diversity gradient [5,7].

Knowledge of the ants found in the Iberian Peninsula (the IP including Andorra, Gibraltar, Portugal and Spain) is still fragmentary [8]. Moreover, there are very few publications treating data regarding their biogeography, and they are limited to some

genera, species or zones [8–13]. The biogeography or philogeography of other Iberian arthropods have been addressed for example for the coleopteran genera *Berberomeloe* [14], *Pimelia* [15], *Blaps* [16], *Cephalota* [17], *Hydraena* [18] and some others cited throughout this article.

The IP forms part of the Mediterranean biodiversity hotspot, unique in the Palaearctic region. It is a threatened area with elevated species richness and a high level of endemism in all the taxa [19–21]. Hewitt [22] established that the IP has an intermediate position as far as species richness and the number of paleoendemic species are concerned, compared to the other two Mediterranean peninsulas (Italy and the Balkans). This high level of diversity within the IP was attributed mainly to its environmental heterogeneity conditioned by its complex orography. The IP mountain ranges are at different latitudes and altitudes with an east-western orientation. These ranges are sometimes permeable, but others act as geographic barriers producing isolated valleys, plateaus and plains. The eastwestern orientation also permits the establishment of particular and markedly different microhabitats on the southern and northern slopes, which vary depending on the altitude and latitude.

On the other hand, changing paleogeography and paleoclimate in the region favoured the convergence of different lineages, sharing evolutionary history for a long period [23]. Thus, in the early Paleogene (60 mya), the small Iberian plate, fragmented during the Mesozoic (100 mya) and united with the European plate, had a semitropical climate until the end of the Miocene (23-5.3 mya) [24,25] defining similar biomes to the current African savannah. Moreover, in the southern territories a succession of separations and unifications with the African plate continued until the end of the Miocene, closing the IP with Africa during the evaporitic Messiniense [26]. These semi-tropical communities from the Miocene were composed of species of Asiatic and African origin [27]. Later, successive climatic and orographic changes in the Pliocene and Pleistocene produced migrations towards the IP [25,28–30] which led to the formation of a mosaic of fragmented populations that persisted in small refugia throughout the entire Iberian refugium (the "refugia-withinrefugium" scenario) [22,31–33] which is very important for the understanding of the diversity and phylogeography of some Iberian taxa [31,32,34]. After the glacial periods the IP played an important role when the Palaearctic peninsulas acted as providers of re-colonizing species to the northern territories [35–37]. These relatively recent biogeographical processes have greatly conditioned the current faunistic composition of the IP.

Thus, to the best of our knowledge, this is the first biogeographic global study on the richness and distribution of the Iberian formicids. We have undertaken this challenge well aware that the knowledge about ant distribution and endemic species in the IP is still incomplete. Our goals have been (1) to analyse the number of ant genera and species in the IP but also to compare them with other Mediterranean countries, especially France (linking the IP with the European Palaearctic) and Morocco (with the African Palaearctic), (2) to take into account the putative refugia within the Iberian refugium, and we propose a subdivision of the IP in refugium areas and attribute a chorological category to each species, and (3) we have defined four different groups of species depending on the different dispersal patterns (centre of origin and dispersal routes) based on biological traits, molecular phylogenies, fossil deposits and geological processes, when available.

#### **2. Materials and Methods**

#### *2.1. Species Included in the Study*

In this study we have included known species in the IP (Andorra, Gibraltar, Portugal and Spain) and compared them with the species found in nearby countries such as France and Morocco. We have tried to use the most updated published lists in the four countries closely involved adding new citations such as some general reviews [38–40]. Only described and named species are included in our study (those species appearing as "sp.", species 1, 2, species under description, etc. are not included). Moreover, we have only considered mainland species, avoiding the species which only appear on the islands

belonging to the four countries. In general, we have not undertaken taxonomic problems such as synonyms or misidentifications of species included in the different faunistic lists, assuming as correct the most recent reference. For the nomenclature of the social parasite species of *Temnothorax* and *Tetramorium* we have adopted the criterion of Seifert et al. [41]. With this method of managing citations our final results might show slight differences with some published lists, but this has not affected our main goals.

For the Portuguese species we focused on Salgueiro [42], taking into account previous publications of Collingwood & Prince [43] and later ones such as Boeiro et al. [44] and Gonçalves et al. [45]. We have excluded from our lists *Temnothorax caparica* Henin, Paiva and Collingwood, 2001, which resulted to be a misidentification of *Cardiocondyla mauritanica* Forel, 1890 [46].

For the Spanish species we focused on the list included in Sánchez-García et al. [47] and added some recently described species [48] and some new findings [49].

In the case of Andorra we have used the list of species of Bernadou et al. [50]. For Gibraltar there is no up-to-date catalogue and citations appear in general articles for the Iberian ants, except for some recent records that have been taken into account [51–53].

For the French species we used Casevitz-Weulersse & Galkowski [54] adding some of the variations included in Monnin et al. [55], and Seifert [56] to clarify the status and current distribution of the European *Lasius* species.

The faunistic list of the Moroccan ant species was constructed from the results of Cagniant [57] and also included the additions provided in different publications [53,58–66].

We have also used the available data on the myrmecofauna of most of the other Mediterranean countries (Table 1) permitting us to put the IP ant species into a Mediterranean context.


**Table 1.** List of Mediterranean countries and number of cited species.

#### *2.2. Biogeographical Analysis*

We undertook three different biogeographical aspects of the Iberian biogeography: 1. Analysis of the chorology of all the species included in the Iberian list; 2. Assessment of the distribution range of these species in the IP, emphasizing the endemic, rare and relict species; 3. To analyse the colonization history of the IP and propose four different ant groups, taking into account their most probable centres of origin and dispersal routes.

#### 2.2.1. Chorology

According to Ribera [78] we established similar chorological categories (categories one to four) but adding three more (five to seven). Specifically, the seven chorological categories we have used are: 1. southern species (S) present in northern Africa and in some areas of the IP but never extending their distribution beyond the north of the Pyrenees; 2. northern species (N), mostly distributed in the north of Europe, northern Pyrenees and some other northern areas in the IP; 3. Iberian endemic species present only in the IP (X) or extending their distribution to the northern slopes of the Pyrenees or some areas in southeastern France (XS); 4. trans-Iberian species (T) present in the northern Pyrenees, the IP and northern Africa; 5. species living only in the northern Mediterranean basin (MN); 6. species distributed over all the Mediterranean basin (M); 7. introduced species (I).

#### 2.2.2. Distribution Range

We have also aimed to portray the distribution of all the listed species for the IP in as much detail as possible, although the ant distribution and endemic species is still incomplete and the methods and effort sampling were heterogenous (pitfall traps, visual inspection, etc.) and sometimes unknown to us, such as those taken from bibliography which only report the species occurrence. Therefore, we have defined biogeographical subdivisions following the criteria established in other studies. Arnan et al. [79] established only two subdivisions in the IP, the Atlantic and Mediterranean, but we have subdivided the two biogeographic subregions into eight refugium areas (Figure 1), following Gómez and Lunt's criteria [32] based on general biota occurrence (plants, vertebrate and invertebrates animals). We have limited the refugium areas to mountain ranges which act as borders because ants prefer low altitudes [6]. We considered an Atlantic and a Mediterranean coast refugia, which have been defined as biogeographical areas for butterflies [80]) and birds [81]. Some of these areas are supported as refugia such as the southern plateau, Atlantic and south-eastern Mediterranean for *Pimelia* species [15] or the entire Mediterranean during the Pleistocene for *Polyommatus* species [37]. Thus, we have finally defined the following refugium areas (Figure 1): 1. the Cantabric including the Cantabric and Basque ranges and a part of the eastern Galaic mountains; 2. the Pyrenean including all the southern slopes of the Pyrenean range from Navarra to Catalonia; 3. the Mediterranean, including the eastern and south-eastern coasts; 4. the Atlantic including the western Iberian coasts; 5. the northern plateau, bordered to the south by the northern slopes of the Central and Iberic ranges, and to the north by the Cantabric range; 6. the southern plateau, bordered to the north by the southern slopes of the Central and Iberic ranges and to the south by the Sierra Morena range; 7. the Guadalquivir Valley; and 8. the Ebro Valley. The geographical position of the citations has been obtained from the AntMaps.org web page [82], and confirmed with our own data and recent publications and reviews (see Material and Methods 1.1). Moreover, we have analysed the occurrence and the number of species found in every refugium area, as well as the number of refugium areas occupied by each species. We also analysed the endemic species distribution highlighting the rare species and the refugium areas which they inhabit. Finally, we tested the similarity of each of the refugium areas by mean of hierarchical clustering analysis based on Jaccard's index as the association estimate and the paired group algorithm UPGMA (Unweighted Pair Grouping method with Arithmetic Means) procedure [80,83,84] as the agglomeration criterion using PAST program V. 4.04 [85] and excluding ubiquitous species. The significant differences in similarity (Jaccard's Index) have been calculated among all the branches of the cluster [80,86,87].

**Figure 1.** Aprioristic refugium areas considered in the Iberian Peninsula. 1. Cantabric; 2. Pyrenean; 3. Mediterranean coast; 4. Atlantic; 5. Northern plateau; 6. Southern plateau; 7. Guadalquivir Valley; 8. Ebro Valley.

#### 2.2.3. Origin and Dispersal Routes

We collected biogeographical evidence from the bibliography about biological traits, dated molecular phylogenies (with different estimation methods) and fossil deposits of the different ant subfamilies, genera and species when available. We inferred from these data the origin and the most plausible dispersal routes up to the current distribution range, taking into account the most probable geology and climate distribution admitted for each epoch in the bibliography and the comparison results obtained from other taxa.

#### **3. Results**

#### *3.1. Taxonomic Richness*

The Iberian Peninsula (IP) has 299 ant species, the highest number compared to the French (211) and the Moroccan (233) ant faunistic lists (see Table S1 in Supplementary Material). The Iberian ant richness is only comparable with the 259 ant species of Greece and the 286 of Turkey. To the north the ant richness followed the latitudinal diversity gradient (Belgium 85 species [88], Norway 57 species [89]). Nevertheless, the ratio species richness/surface in the three countries revealed a similar ratio for the IP and Morocco (5.1 × <sup>10</sup>−<sup>4</sup> and 5.2 × <sup>10</sup>−<sup>4</sup> species/km<sup>2</sup> respectively) and much lower for France (3.2 × <sup>10</sup>−<sup>4</sup> species/km2). Even in a wide view of the Mediterranean context, the ant fauna of the IP is the second in species richness, with only a few species less than the Balkan peninsula but not when the ratio of species/surface is taken into account, where the IP has a higher ratio than France, similar to Morocco, but surpassed by Greece and Israel. The Iberian ant species appear grouped in seven subfamilies (Amblyoponinae, Ponerinae, Proceratiinae, Leptanillinae, Dolichoderinae, Formicinae and Myrmicinae), but Morocco has two more subfamilies (Cerapachyinae and Dorylinae) and France one subfamily less than the IP (Amblyoponinae). The subfamilies Amblyoponinae, Cerapachyinae and Dorylinae, which are widely considered as tropical [90], are absent from France and only the subfamily Amblyoponinae is present in the IP.

The subfamily Myrmicinae had the highest species richness for the three studied areas, 114 species (54% of the total ants) in France, 171 (57%) in the IP and 147 (63%) in Morocco. The subfamily Formicinae has a lower number of species than Myrmicinae in all the three areas: 77 in France (36.5%), 95 species (33%) in the IP and 61 (26%) in Morocco. The subfamily Dolichoderinae appears far below, followed by Dorylinae and Cerapachyinae (see Supplementary material Table S1).

The most diverse genus in the IP is *Temnothorax* (47 species and 16% of the total species richness), followed by *Lasius* (25 species, 8%), *Formica* (23 sp., 8%) and *Camponotus* (20 species, 7%). *Temnothorax* is also highly diverse in France (28 sp., 13%) and Morocco (39 sp., 17%) but represents a slightly higher percentage of the total species in the latter country. The other most abundant genera in France are *Formica* (13%), *Lasius* (12%) and *Myrmica* (10%) and in Morocco *Messor* (12%), *Aphaenogaster* (11%) and *Cataglyphis* (9%).

Although the total number of endemic species in the IP (72 sp., 24 %) is the highest in the Mediterranean countries (21 endemic ant species in Greece [74]; 25 in Israel [76]; and 56 in Morocco [57]), but again the ratio of endemic species/surface is similar to Morocco (1.25 × <sup>10</sup>−<sup>4</sup> endemic sp./km2) and surpassed by Greece (1.6 × <sup>10</sup>−<sup>4</sup> endemic sp./km2) and Israel (12.0 × <sup>10</sup>−<sup>4</sup> endemic sp./km2). In the IP, the highest number of endemic ants belong to Myrmicinae (41 species) followed by the subfamily Formicinae (25 species, Figure 2). Amongst the Myrmicinae, the genus *Temnothorax* includes the most endemic species (16 species, 22% of the total endemic species and 30% of the full *Temnothorax* species). Nevertheless, the genus *Cataglyphis* is the most proportionally rich in endemic species, all of its 10 Iberian species are endemic. Although a positive correlation exists between the species richness and the number of endemic species within a genus (R = 0.73; F(1,28) = 31.81; *p* < 0.0001), some genera are especially above the expected number of endemic species, such as *Cataglyphis*, *Temnothorax* or *Goniomma*, and other genera below, such as *Lasius*, *Tetramorium* and *Formica* (Figure 3).

Moreover, the presence of an endemic genus in the IP, *Iberoformica,* with only one living species in the world, *I. subrufa,* is very remarkable. This species extends its distribution to some southern French locations. The exceptional appearance of the genus *Rossomyrmex* in the IP is also noteworthy. This genus has three different species in Asia (the Anatolian plateau, the plains of the Caspian Sea and other regions of Central Asia), which are different to the one in the IP, *R. minuchae* [30,58], the only species of this genus which exists in the full western Palaearctic.

Another interesting group of species because of their natural history are the social parasites of which 43 species appear in the IP, including temporal and permanent parasitism (22 and 21 species). Together they account for 14.4% of the total Iberian species. The number and percentage of parasite species decreased in France (20 temporal + 13 permanent which represent 15.7%), and many more in Morocco (5 temporal + 7 permanent which constitutes 5.2%).

Finally, comparing the shared species belonging to every subfamily amongst the IP, France and Morocco, (Table 2; Table S1) we obtained the highest percentage of overlapping species between the French and the Iberian ant lists (84%). Nevertheless, the IP only shared 59% of its species with the French list. Morocco shared the lowest number of species with France 21%. and the highest with the IP 37% (Table 2). A cluster based on the similarity (Jaccard's Index, Cophenetic correlation index = 0.98) amongst the three areas, grouped France and the IP together (Figure 4) without significant differences between them (Jaccard's Index = 0.52, p > 0.05) and showed significant differences between Morocco with France and the IP (Jaccard's Index = 0.12 and 0.19 respectively, p < 0.01). Moreover, the replacement of species is more frequent between Morocco and the IP, but the overlapping is mainly between France and the IP (Table S1). For instance, France has three *Bothriomyrmex* species shared with the IP, but only one of the six *Bothriomyrmex* from Morocco appear amongst the four species in the IP. Even more striking is the case of the genus *Cataglyphis*, none of the 22 species in Morocco appear amongst the 10 species in the IP, which moreover are Iberian endemic species. Something similar occurred in other genera, such as *Aphaenogaster, Oxyopomyrmex* and *Temnothorax,* although to a lesser extent.

**Figure 2.** Number of species (orange) and endemic species (blue) belonging to each ant subfamily in the Iberian Peninsula (IP).

**Figure 3.** Positive correlation between species richness and the number of endemic species in each genera, including only native species (R = 0.73; *p* < 0.0001; F(1,28) = 33.32).



**Figure 4.** Cluster analysis based on the similarity (Jaccard's Index) of the ant species found in the three adjacent areas (IP, France and Morocco).

#### *3.2. Biogeographical Analysis*

#### 3.2.1. Chorology

The most frequent chorological category in the IP was the northern species (N, 30.8%), which together with the trans-Iberian species (T, 11.7%) are 42.5% (Figure 5). On the other hand, the Mediterranean species, including the northern Mediterranean (MN, 6.7%), all the Mediterranean basin (M, 6.7%), the southern Mediterranean (S, 12%), and the Iberian endemic species (X + XS, 24.1%) represented 49.5% of the total species. The lacking percentage corresponded to the introduced species (I, 8%) (Figure 5). Amongst the ant subfamilies, the northern species (N) are clearly more abundant in Formicinae and Myrmicinae (Figure 6). In the latter subfamily the "endemic species" is the next most abundant chorological category (Figure 6). Nevertheless, the percentage of endemic species is similar for Formicinae and Myrmicinae (26% and 25%). The next most abundant category in Myrmicinae is the southern species (S), contrarily under-represented amongst the Formicinae ants (Figure 6).

**Figure 5.** Percentage of species pertaining to the different chorological categories (S: Southern; N: Northern; X + XS: Endemic; T: Trans-Iberian; MN: Northern Mediterranean; M: Mediterranean; I: Introduced.

**Figure 6.** Chorological composition of the three more abundant subfamilies. (S: southern species, N: northern species, X + XS: endemic species, T: Trans-Iberian species, MN: north Mediterranean species, M: Mediterranean and I: introduced species).

#### 3.2.2. Distribution Range

We have evaluated the importance of every refugium area harbouring ant species richness (see Supplementary material Table S2). We found that all the aprioristic defined refugium areas presented significant differences in similarity (Jaccard's index, *p* < 0.01) with respect to the others (Table 3). Those refugia with the high similarity index were the northern plateau, the Guadalquivir Valley and the southern plateau (Jaccard's index = 0.4; Table 3, Figure 7).

The refugium area with the highest number of ant species is the Mediterranean (144 species), followed by the Guadalquivir Valley (118 species) (Figure 8). These two refugium areas also contain the highest number of endemic species, followed by the northern plateau and the Pyrenees. The Atlantic and the southern plateau refugia contain an intermediate number of species, and the Cantabric together with the Ebro Valley present a similarly low number. The number of ubiquitous species, occurring in all the eight refugium areas was relatively low with only 46 species.

Many of the Iberian ant species appeared exclusively in one of the Iberian refugium areas (86 species, 29%) and may be considered as rare species, although they are not always endemic. Progressively a lower number of species occupied more refugium areas, with only a final increase for ubiquitous species (Table 4). The 21 endemic species which appear in only one refugium area are shown in Table 5.

Analysing the most diversified or peculiar ant genera (see Annex S2, Supplementary Material), we found the genus *Lasius* (25 species) to be the most widely distributed across all the IP and, when local, generally appear in the northern half of the IP and frequently in the Pyrenees, the Ebro Valley and the Mediterranean refugia. There are only two endemic species in this genus that are widely distributed (*L. cinereus* and *L. piliferus*). The genus *Formica* is composed of 23 species, the majority appearing in the Pyrenees (14 species) or the Mediterranean (9 species) refugia. Twelve *Formica* species of the IP belong to the *rufa* group; again, the majority of these latter species (7 of them) were present in the Pyrenean refugium and only 3 in the Guadalquivir Valley. The genus *Camponotus* is composed of 20 species, of which 16 are widely distributed in the IP. Nevertheless, the only two strictly endemic species (X chorological category) belonging to this genus (*C. haroi* and *C. amaurus*) presented a narrow distribution linked to the Mediterranean coast and the northern plateau. In the case of the genus *Cataglyphis*, 10 species are represented in the Iberian myrmecofauna, all of which are endemic (nine strictly X and one shared with France XS). Species from this genus have never been found in the most northern zones (Cantabric or Pyrenees) and principally were present in the southern areas (southern plateau, the Guadalquivir Valley and on the Mediterranean coast). Four of the endemic *Cataglyphis* species (*C. floricola, C. gadeai*, *C. humeya and C. tartessica*) are exclusively distributed in either the Guadalquivir Valley or the Mediterranean refugium areas. The genus *Themnothorax*, includes 47 species in the IP, 16 of which are endemic. Twenty-five percent of the *Temnothorax* species are distributed in only one refugium area, and the areas with the most *Temnothorax* species are the northern plateau, the Mediterranean coast and the Guadalquivir Valley. The refugium area with the lowest number of *Temnothorax* species is the Cantabric. Finally, the genus *Stigmatomma* is composed of only three species, with a narrow distribution, in only one or two refugium areas, thus in the Guadalquivir Valley we found the three species and one of them also on the Mediterranean and Atlantic coasts. In fact, the Guadalquivir Valley is the refugium containing the highest number of ant species linked with tropical environments, such as *Stigmatomma emeryii*, *S. impressifrons*, and the Ponerinae *Anochetus ghilianii* and *Chryptopone ochracea*.


**Table 3.** Jaccard's Index (black numbers) and probability associated to significant differences (blue numbers) amongst the occurrence of species in paired refugium areas of the IP.

**Figure 7.** Cluster analysis based on the similarity of ant species occurrence (Jaccard's Index) in the eight refugium areas of the IP, all of them showing significant differences (*p* ≤ 0.01; Refugium areas 1. Cantabric; 2. Pyrenean; 3. Mediterranean coast; 4. Atlantic; 5. Northern plateau; 6. Southern plateau; 7. Guadalquivir Valley; 8. Ebro Valley).

**Figure 8.** Number of species (orange) and endemism (blue) in the different refugium areas of the IP. **Table 4.** Number of ant species occupying an increasing number of refugium areas.



**Table 5.** List of 21 endemic species appearing in only one refugium area in the IP.

#### **4. Discussion**

#### *4.1. Taxonomic Richness*

Ants showed a higher number of species in the IP (299 species), compared with the adjacent Mediterranean countries, with only a few species less than the Balkan Peninsula. A similar number of species is found in Greece and Turkey [74,75] (Table 1). Following the expected latitudinal diversity gradient there is a decrease in species richness to the north [88,89]. Although the IP has a higher ant species richness than Morocco [5,91,92], the last has two more subfamilies than the IP and three more than France, which follows the latitudinal diversity gradient. These results varied when the area surface is taken into account, the ratio is similar between the IP and Morocco, being higher than in France and much lower than other Mediterranean countries such as Israel.

With respect to the richness of the endemic species the same pattern occurred. The IP shows the highest number (72 endemic species), compared with other Mediterranean countries such as Greece, (21 species [74]); Israel (25 species [76]) or Morocco (56 species [57]), but when the surface areas are considered, a similar ratio between the IP and Morocco again occurred, but this was much higher in the eastern Mediterranean.

Nevertheless, not only the number of species and endemic taxa in the IP are remarkable but also the singularity of the Iberian myrmecofauna including one endemic genus (*Iberoformica*), with only one endemic species *I. subrufa* and the fossil of its probable ancestor *I. horrida* [93]. Moreover, the IP is the only place in Europe where the genus *Rossomyrmex* is located outside Asia. The presence of these two exclusive genera highlights the importance of the IP as a refugium. At least for *Rossomyrmex*, there is evidence that have been several episodes of extinction of this genus between the nearest geographical point (Turkey) and the IP. This genus has survived in the IP and not in other nearby countries, as have other emblematic animals such as the *Lynx* or the Azure-winged magpie [94].

The presence of these two ant genera as well as the low number of Iberian ant species shared with Morocco (29%) and with France (59%), while France shares with the IP 84%, and Morocco 37% with the IP, indicates that the IP harbours a unique and fairly exclusive ant fauna and functions as a "cul de sac" of ant species [94].

Several factors are responsible for the high ant biodiversity in the IP, as mentioned in the introduction, derived from the highly complex Iberian orography and convulse geological and climatic history. Another factor affecting the Iberian ant diversity is the peninsular effect, which explains the increase in diversity near the isthmus [95,96], assessed in the IP for birds [97] and butterflies [98]. Moreover, the role of the Palaearctic peninsulas during the Quaternary glaciations, as refugia for biota and centres of speciation [36], together with the Iberian complex orography producing "refugia-within-refugium" effect that has been assessed for different animal groups, contributed to increase the Iberian biodiversity [14,99].

Analysing the contribution in the IP ant diversity of the most relevant worldwide genera according to Wilson (1976 in Pie & Feitosa, [100]), only *Camponotus* make an important contribution to Iberian biodiversity, whilst *Pheidole* and *Crematogaster* had few species represented. This phenomenon is common to all Europe [69]. In the IP, the genus that we can consider "to have conquered the world" following Wilson's criteria must be *Temnothorax,* the most diverse Iberian genus (47 Iberian species) with the highest number of Iberian endemic species (16). This is an abundant genus in all the IP habitats, except in the high mountains above 2,700 m a.s.l., reflecting a high plasticity and adaptation ability. Probably its small size and its mode of mate location behaviour, by means of female pheromones, act as a speciation driver in this genus. Nevertheless, other diverse genera such as *Lasius*, *Formica* or *Camponotus* have a much lower number of endemic species than expected. Again, the mating behaviour by mass nuptial flights, which facilitates the populations mixing at a landscape level, might influence the lower rates of speciation in these genera. Moreover, other genera, such as *Cataglyphis* or *Goniomma*, showed a higher number of endemic species than expected. Other factors, probably linked with the biogeography, biotic traits and plasticity of the genera, may be responsible for this high level of endemicity.

All these data contribute to highlight the singularity of the Iberian myrmecofauna and its relevance in the Mediterranean region, placing the IP in a similar position to the Balkan Peninsula as a centre of ant diversification.

#### *4.2. Biogeographical Analysis*

#### 4.2.1. Chorology

The chorological analysis showed two groups of ants, one widely distributed in the northern half of Iberia, sharing species with the western Palaearctic region but sometimes extending up to northern Africa, (N + T chorological categories), and the other group, mainly including species belonging to the Mediterranean basin and northern Africa (S + MN + M + X + XS chorological categories). This result is congruent with those obtained for aquatic coleopterans [78] and showed the IP acts as a transitional element between Europe (north Palaearctic) and north Africa (south Palaearctic).

This north-south distribution pattern is shown at different taxonomic levels (ant genera and species), thus we can find genera preferentially distributed in the north of the IP, such as *Formica*, *Lasius, Myrmica* and *Leptothorax* and others in the southern half, such as *Cataglyphis*, *Proformica*, *Stigmatomma*, *Anochetus*, *Messor*, etc. At species level, we can find the same pattern, with some typically northern species such as *Lasius fuliginosus*, most of the *Formica rufa* group, *Camponotus herculeanus*, *C. ligniperda*, *Tapinoma pygmaeum*, the three *Leptothorax* species, and more examples included in the Supplementary Material (Annex S2). In the same way, a similar number of species could be considered as southern, such as some of those included in the genus *Cataglyphis* (*C. humeya*, *C. floricola*, *C. tartessica*), *Goniomma* (*G. collingwoodi*, *G. compressiquama*), *Messor timidus*, *Aphaenogaster striativentris*, the three species of the genus *Stigmatomma*, or *Anochetus ghilianii,* amongst others (see Table S2).

#### 4.2.2. Distribution Ranges

The complex orography of the IP produced not only a north-south division of species distribution, but even in the eight proposed refugium areas, we can find a significantly different ant fauna. This result supports the fact that similar processes affected all the biota, with common patterns emerging, although some slight differences in shape probably depend on biotic traits of the studied species such as dispersal ability [32,80,81,84,101]. Most of the proposed biogeographical subdivisions of the IP established between five (Mollusca: Pulmonata [101]) to eleven zones (birds [81]). Our proposal is closer to that of vascular plants and vertebrates [32,81,84]. The social character of ants does not appear to alter the biogeographical pattern followed for the general biota, except for their presence in the high mountains. Amongst the IP refugia the most similar for occurrence of ants (high Jaccard's diversity Index) are the northern plateau, the Guadalquivir Valley and the southern plateau. From the analysis of the species occurrence within each refugium areas, we have proved that the Mediterranean and Guadalquivir Valley refugia show the highest species richness and number of endemic species, highlighting their importance for ant conservation. In fact, Andalusia is considered the Iberian region with the highest general biodiversity [21] where both refugium areas are represented. In the Guadalquivir Valley refugium, we can find some special habitats such as the prequaternary forest with relict plant species located near the Straits of Gibraltar [102]. In this habitat we can also find the very few semitropical ant species appearing in the IP, which belong to the subfamilies Amblyoponinae together with some Ponerinae such as *Anochetus ghilianii*.

The Pyrenean and the northern plateau are the following refugium areas in biodiversity importance. The Pyrenean range is in the isthmus, concentrating the peninsular effect, which together with the high heterogeneity derived from the altitudinal variation and its west-east position, produced an important climatic variability, responsible for the species richness in this refugium. Nevertheless, the number of endemic species is not very high (10 species), pointing to the possibility of gene flow between the Pyrenean and the European populations, diluting speciation possibilities. The northern plateau is the oldest region of the IP, including the Hercynian plate; its age may be the cause of the increment in biodiversity in this refugium.

Most of the species are distributed in more than one refugium area, but about a third is found in only one IP refugium (86 species). On the other hand, only 46 species are ubiquitous in all the IP.

Similarly, from analysing the distribution of endemic species in the different refugium areas, we have been unable to find a clear pattern, finding 21 endemic species limited to only one refugium and only five endemic species being considered ubiquitous. These results are congruent with those found by different authors [78,99] pointing to different causes for the absence of clear patterns of endemicity in the IP, including sampling artifacts.

On the other hand, although mountain ranges promoted species richness and endemicity [103], this pattern is not detectable for the Iberian and Palaearctic formicids. Only one ant species is exclusive to high mountains: *Proformica longiseta* (between 1800 and

3000 m a.s.l. in the Sierra Nevada mountains [104] and the Baetic range [13]). According to these findings, the majority of the Iberian ant endemic species are related with semiarid habitats with scarce vegetation at low altitudes, such as some of the species pertaining to the genus *Temnothorax*, as *T. ansei*, *T. blascoi*, *T. caesari*, *T. crepuscularis* [105]. Another important number of endemic species are found in medium altitude mountain ranges, such as *Teleutomyrmex kutteri* (1700–2250 m a.s.l. in different Baetic ranges [106,107]), *Temnothorax gredosi* or *T. conatensis* (900–1500 m a.s.l. [108,109]).

Whereas the rare species, i.e., those found in only one or two refugium areas, include for instance the social parasite ant species, where their way of life makes them difficult to find. These are known in very few and sometimes very distant areas, such as *Rossomyrmex minuchae* [12,110], *Myrmoxenus bernardi* [111] or *Anergates atratulus* [112]. Another example of a rare species is *Aphaenogaster cardenai*, an Iberian endemic species of relatively wide distribution [113], but living in scarcely sampled habitats (the mesovoid shallow substratum, shallow caves and mines [114]).

#### 4.2.3. Origin and Dispersal Routes

Other different causes responsible for the high ant biodiversity in the IP are related with the centre of origin and dispersal routes of the ancestors of the current ant fauna. In agreement with Blaimer et al. [115] the origin of formicids probably occurred during late Cretaceous (104–117 mya), but the rise of the modern ant fauna probably occurred during the early Cenozoic and continued until the present day. There are signs that all the genus-level taxa appeared from the first 50–60 mya are now extinct in the western Palaearctic [7]. Blaimer et al. [115] gave the estimated time of divergence for several genera from the subfamily Formicinae, thus, the clade giving place to the tribe Formicini, appeared 60 mya, and the most probable ancestral range was the Palaearctic region. These data are congruent with the estimated age of *Cataglyphoides constrictus,* a probable ancestor of the genus *Cataglyphis* (but see [116]), or *Formica horrida*, probably a species belonging to the current genus *Iberoformica* [93]. Both fossils were found in the Baltic amber (middle to late Eocene 37–42 mya) [117]. According to Blaimer et al. [115] the *Formica*/*Iberoformica* clade appearance is dated to 50 mya, the clade of *Rossomyrmex*, *Cataglyphis*/*Bajcaridris*, *Proformica* diverged in 45 mya, *Bajcaridris*/*Proformica* in 20 mya and finally *Rossomyrmex*/*Cataglyphis* in 15 mya, which are in agreement with the results of Guenard et al. [7].

The genus *Temnothorax*, which belongs to the subfamily Myrmicinae, showed its centre of origin in the Palaearctic region during the Eocene-Oligocene transition (38–33 mya) [118]. The genus *Myrmica* appeared to diversify following drastic climatic cooling in the same epoch (34 mya), and its most probable centre of origin is central or south-eastern Asia [119]. The genus *Stenamma* shared its time of origin with the previous genera but the centre of origin is estimated in the Nearctic region, and two dispersal waves to the Palaearctic were detected, the first in early Oligocene (30 mya), leaving in the Iberian myrmecofauna the species *S. striatulum,* and the second at late Pliocene (about 3 mya) with *S. debile* [120]. Undoubtedly posterior dispersals have occurred, as has been demonstrated for other insects such as the butterfly *Parnassius apollo* with a recent (late Pleistocene) colonization of most of their range [121].

With this retrospective view, we know that most of the current extant ant genera inhabited the Palaearctic region 15 mya (Middle Miocene), but the question is whether they inhabited the IP at the same time.

From the Cenozoic (60 mya), the Iberian plate was fused with the European plate [95]. Unfortunately, the ant fossil data from this time are scarce and, in some cases, too ancient to resolve the question [122,123]. We only have some scarce data from recent periods, thus, the existence of the genera *Camponotus*, *Dolichoderus*, *Iridomyrmex*, *Lasius* or *Formica*, *Liometopum* and *Messor*, in the Middle and Late Miocene of the IP has been reported (13 to 7 mya) [124,125]. The paleoclimate and paleoenvironments of the Miocene (23 mya) have been recreated more accurately through the coral reef and mammal fossils [24,29,30,126]. During this period the IP climate was semitropical and the biota was equivalent to the

current African savannah [127], including important Asian and African components [27]. Nevertheless, from the Miocene until the Pliocene (5.33 mya) the biogeography of the IP was quite convulsed, especially on the southern extreme, frequently joined and separated with the African plate [26]. Moreover, the climate was cooling which finally provoked the substitution of the semitropical fauna for others better adapted to temperate or cool climates [7].

In this situation, some ant genera may have inhabited the Iberian savannah from the Miocene and later the steppes from the Pliocene and Pleistocene [7,115]. This dynamic process of sequenced colonization, extinction and diversification did not stop. Thus, during the transition Pliocene-Pleistocene, the effect of Quaternary glaciations on these Asian and African fauna dispersed to the IP, giving way to the current myrmecofauna. This view is reinforced with data on radiation of the *Formica* genus and the *Formica rufa* group species, dated in the Middle Pliocene and Pleistocene [128,129], and this was similar for other insects, as has been proved by means of molecular analyses in different coleoptera [14–18]) and in plants [130,131], pointing to a general process for all the biota of the IP.

In our analysis of the origin and dispersal routes which shaped the Iberian ant myrmecofauna, we can differentiate four groups of dispersal patterns:

#### Relict Species

From the middle to late Eocene (34–42 mya) [117] the climate was semi-tropical and the ant fauna known from the Baltic amber was very diverse including extant genera or their ancestors [132,133]. Some of them, such as *Formica* or *Aphaenogaster*, which nowadays have reached a high diversity, must have previously adapted to semi-tropical warm environments that were predominant when they were trapped by the amber. Later they were able to adapt to the cooling produced from the end of the Miocene and have survived until now. Nevertheless, both genera do so in different ways, one adapting to temperate or cool habitats (*Formica*, now more diversified to the north of the IP) and the other to temperate or warm habitats (*Aphaenogaster*, currently more diversified in the south of the IP). Nevertheless, some other genera were unable to adapt to the changing conditions and disappeared from the western Palaearctic [7], such as the fossils of the genera *Pachycondyla* from Denmark, preceding the Paleocene-Eocene transition (55 mya) [134]. In some rare cases, semitropical ant species such as *Anochetus* and *Stigmatomma,* found in Santo Domingo amber (16–19 mya) [117,135] and the Baltic amber [133] respectively, and currently well distributed in tropical and semitropical regions, were able to remain in some restricted locations of the IP surrounding the Straits of Gibraltar [136,137], an exceptional zone also inhabited by paleoendemic plant species [102,138]. Although Jowers et al. [9] concluded that *Anochetus ghilianii* is a recent invader of the IP, we think that the coincidence in this area of botanical paleoendemic species together with different semitropical ants (*Anochetus* and *Stigmatomma* species), it must be the result of a similar biogeographical process acting on them. Anyway, *A. ghilianii* should be considered as a relict Moroccan species and its presence in the IP is noteworthy.

#### Asian-IP Disjunct Species

In the Pliocene (5.33 mya) the Mediterranean Sea reopened through the Straits of Gibraltar and the climate was cooling, favouring the substitution of the existing semitropical fauna for one better adapted to temperate climates. This temperate fauna proceeded principally from Central Asia, reaching the IP as confirmed by fossil deposits [25,28–30]. The Asian colonizing waves have occurred repeatedly during the transition Pliocene-Pleistocene (2.6, 2.5 and 1.7 mya) [25] even during the late Pleistocene [139] and later. The migrant mega-fauna must have been dispersing together with invertebrates, among them obviously ants, sharing the same origin and similar ecological requirements. These Asiatic and African faunas from the later Pliocene, evolved under glacial and interglacial periods during the Pleistocene, and many of these species became extinct or remained isolated in some refugia throughout the entire Iberian refugium, setting up the current

myrmecological fauna [22,31–33]. Some of these species continued isolated and now have a narrow distribution range, as is the case of the slave-making ant *R. minuchae* in Spain, and the other three species of the genus, which are dispersed, although with a wider distribution range in Asia. The genus *Rossomyrmex* can be considered as a relict in the IP. On the other hand, the species of the genus *Proformica*, the host of *Rossomyrmex*, with a probable centre of origin in Central Asia [11], were able to recolonize wide distribution ranges both before and after glacial periods, occupying all the southern Palaearctic from Central Asia to the IP. This genus, more diversified in the western Mediterranean, specifically in the IP, is not distributed in Morocco, where it has been replaced by the close genus *Bajcaridris* [11,115,140]. This fact suggests the hypothesis of a frequent dispersal route on the two sides of the Mediterranean (Figure 9) with speciation concluding in different genera (*Proformica* and *Bajcaridris*). An opposite direction of the dispersal route (from western Mediterranean to Asia) has been suggested for *Rossomyrmex* [11] as occurred, for example, with the plant genus *Odontites* [130].

**Figure 9.** Suggested dispersal routes affecting the myrmecofauna of the IP. (Base map obtained from https://laboratoriorediam.cica.es/VisorRediam/ (Accessed on 2 December 2020)). Junta de Andalucía. Consejería de Agricultura, Ganadería, Pesca y Desarrollo Sostenible (ámbito de Desarrollo Sostenible).

Another genus offering interesting disjunct distributions is *Cataglyphis*, specifically the species group *altisquamis* shows two different and discontinuous distribution ranges: one group of five species distributed from Central Asia to the western Mediterranean and the other nine species distributed in the western Mediterranean (IP and Morocco) [141] (Figure 11). This second group of species is absent from Turkey to France, by the northern route, and from Egypt and Libya, by the southern route, due to extinction processes in the intermediate points. The age of the entire genus and its high diversity in Asia point to a Central Asian origin. Three different dispersal routes are possible from Asia to the western Mediterranean: 1. the simultaneous dispersal route via the two sides of the Mediterranean, 2. the dispersal by the northern Mediterranean side across to the Balkan region, and 3. the dispersal only by the southern Mediterranean side (Figure 9). The three are possible but until now only the southern route (3) has been suggested for the *Cataglyphis albicans* group [10]. Nevertheless, the three dispersal routes from Asia have also been proposed for the genus *Cephalota* (Coleoptera: Cicindelidae) [17] and may be more frequent dispersal patterns than had generally been considered. An extension of the phylogenetic study on *Cataglyphis* including middle-west distributed species should produce a wide and accurate view for the biogeography and dispersal patterns of these species.

The genera *Goniomma* and *Oxyopomyrmex*, both endemic of the Mediterranean basin, also show disjunction between the eastern and western Mediterranean distributions [142, 143] (Figure 10). Their centre of origin is unknown, although they probably appeared in the Miocene (12 mya) [90]. A similar distribution pattern is found in the coleopteran species of the genus *Pimelia* [15], *Berberomeloe* [14] and the plant genus *Odontites* [130].

Another example of current Iberian fauna showing Asian-IP disjunct distribution is the Iberian azure-winged magpie [144] or amongst the insects, the lepidopteran *Pseudochazara wiliamsi* or the coleopteran of the subgenus *Parentius* (see [145]), and together with these, we find many other species adapted to arid or semi-arid environments such as Monegros (Zaragoza) or Guadix-Baza (Granada) [146,147] or to mountain ranges [148]. Thus, all these examples reinforce the importance of Asia as a centre of origin for the Iberian fauna.

**Figure 10.** Disjunct distribution range of the species belonging to genus *Goniomma*. Note the presence of one species in Israel (citations from [71] are included; base map as in Figure 9).

**Figure 11.** Disjunct distribution of the species belonging to the *Cataglyphis altisquamis* group (modified from [149]; base map as in Figure 9).

#### Baetic-Rifan Species

This dispersal pattern includes the shared species between the south of the IP and the north of Africa. Within this group, the ants with apterous or brachypterous females, such as *Monomorium algiricum, A. ghilianii*, *Stigmatomma* species or *Aphaenogaster senilis* group, gain biogeographical relevance, due to their handicap for flight dispersal across the Mediterranean Sea. One plausible explanation for this kind of distribution comes from the geological history of this territory. South-western Iberia and the north of Morocco share stratigraphic deposits, forming the Baetic-Rifan territories [150] derived from the Miocenic island of Alborán, emerged in the western Mediterranean 16 mya [126]. This island was united with Morocco until 8 mya when it again became disconnected and isolated until 5.9 mya. After this, it was again partially united with the IP until 5.3 mya. At this time, the Straits of Gibraltar reopened, dividing the territories of the Miocenic island of Alborán between the IP and Morocco. The existence of this island may explain the relatively high presence of apterous species in this zone, because apterism is one of the known island syndromes. This effect is known in the Baetic-Rifan territories not only for ants [151] but also for *Pimelia* and *Blaps* Tenebrionid [15,17] and Meloid (*Berberomeloe* group) coleoptera [14], both of them apterous. The existence of this island is more extended in geological time than the habitually invoked desiccation of the Mediterranean during the Messiniense (late Miocene) to explain the shared presence of species between the IP and northern Africa.

#### Alpine Species

Another important and more recent dispersal route is related with the Pleistocene glaciations, the last occurring 10,000 years ago. Many northern species survived and took refuge in the southern Palaearctic peninsulas, and during interglacial periods returned to the north and/or the populations climbed the slopes of the mountains [37]. One good example of this groupis the pair of Iberian endemic species *Formica frontalis/F. dusmeti*, very close phylogenetically to *F. truncorum*, distributed in northern Europe ([129], unpublished data). Probably the presence of *T. kutterii* in the south of the IP and *T. schneideri* in the northern IP and in the Alps is likely to have had this similar origin. Some species of *Myrmica*, *Temnothorax*, *Lasius*, etc, may belong to this group of alpine species, but this should be confirmed by phylogenetic molecular studies on the Mediterranean species.

#### **5. Conclusions**

The taxonomic diversity and distribution patterns we have presented in this study highlight the importance of Iberian ants for a better understanding of the complex evolutionary history and biogeography of the IP. Moreover, we have tried to show the importance of biogeography driving the structure of the current ant communities, ecological interactions, such as hierarchic competence and biotic factors, only explain a part of their structure [152]. Ours results on ant biogeography support the hypothesis that the centre of origin of the species and their dynamic processes (dispersal, vicariance, speciation and extinction) are the missing link to fully explain and understand the evolution and the structure of these ant communities [2,5,7]. We hope the hypothesis and proposals put forward in this study will promote new biogeographical, phylogenetic and evolutionary studies about the ant fauna of the Mediterranean Basin and produce a wide and accurate view of the centre of origin and the dispersal patterns of Iberian ants.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/1424-281 8/13/2/88/s1, Table S1. Ant species list of France, Morocco and the IP. Table S2. List of Iberian ant species and their occurrence in the refugium areas.

**Author Contributions:** Conceptualization, A.T. and F.R.; methodology, A.T. and F.R.; formal analysis, A.T. and F.R.; investigation, A.T. and F.R; writing—original draft preparation, A.T. and F.R.; writing review and editing, A.T. and F.R.; project administration, F.R.; funding acquisition, F.R. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was partially funded by the project RTA2015-00012-C02-02 (Ministry of Science and Innovation, INIA and FEDER funds).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available in supplementary material.

**Acknowledgments:** We thank José M. Martín, Alfonso Arribas and Elvira Martín who provided some references and exchange of ideas and Pedro Sandoval for his help with the figures. Angela Tate reviewed the English edition. We are also grateful to the three anonymous reviewers who made a careful and significant improvement to the manuscript.

**Conflicts of Interest:** The authors declare no conflict of interest.

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