## 1 Introduction
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; 48; 49; 50; 51]. Generally, ants can be classified into three broad categories: arboreal, epigeci (i.e., ground surface-dwelling, including ants living in leaf litter), and hypogeei (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 epigeci 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 hypogeei and arboreal ants within urban habitats and overlook the potential discovery of undescribed species [53; 54; 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. 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 km\({}^{2}\) 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./km\({}^{2}\), 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. 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).