Ants of the Hengduan Mountains: a new altitudinal survey and updated checklist for Yunnan Province highlight an understudied insect biodiversity hotspot

Abstract China’s Hengduan Mountain region has been considered one of the most diverse regions in the northern hemisphere. Its stunning topography with many deep valleys and impassable mountain barriers has promoted an astonishing diversification in many groups of organisms including plants, birds, mammals, and amphibians. However, the insect biodiversity in this region is still poorly known. Here, the first checklist of ant species from the Southern Hengduan Mountain region is presented, generated by sampling ant diversity using a wide array of collection methods, including Winkler leaf litter extraction, vegetation beating, and hand collection. 130 species/morphospecies from nine subfamilies and 49 genera were identified. Among them, 17 species from 13 genera represent new records for Yunnan province, and eight species are newly recorded for China. Moreover, we believe 41 novel morphospecies (31% of the total collected taxa) will prove to be new to science. These results highlight the rich ant fauna of this region and strongly support its status as a biodiversity hotspot. The current ant species checklist for the whole of Yunnan Province was updated by recording 550 named species from 99 genera. Taken together, our results suggest that the Yunnan ant fauna still remains under-sampled, and future sampling will likely yield many more species, among them many undescribed ones.


introduction
The Hengduan Mountain region, located in the southeastern part of the Qinghai-Tibet Plateau, is one of the 35 recognized biodiversity hotspots in the world (Myers et al. 2000). The unique landscape, geomorphology, microhabitat differentiation and geographic isolation created by tectonic uplift during the last eight million years has promoted an astonishing diversification in many groups of organisms, making this region one of the most diverse temperate regions in the northern hemisphere (Boufford 2014;Price et al 2014;Xing and Ree 2017). For example, it harbors nearly 40 percent of China's vascular plant diversity (ca. 12,000 species), including more than 3,000 endemic species (Boufford 2014). However, aside from the well-documented plants and some vertebrates, the diversity of other groups, especially invertebrates in this region remains largely unknown. Insect taxonomic groups in particular have received limited attention, and our understanding of their diversity in the Hengduan Mountains is extremely fragmented.
Ants are an ecologically dominant component of many ecosystems in terms of their abundance, richness, and ecosystem function (Hölldobler and Wilson 1990). Globally, about 15,600 ant species and subspecies have been described (Bolton 2020), making them the most diverse group of social insects and one of the most diverse families of insects. Despite the fact that ant diversity is mainly concentrated within tropical regions (Dunn et al. 2009;Economo et al. 2018), the ant fauna of many other regions is still poorly known, especially in Asia (Guénard et al. 2010). Compiling and curating comprehensive and accurate ant species checklists for these regions is essential not only for insights into ant taxonomy and systematics, but also for long-term monitoring and conservation of these ecosystems (Guénard et al. 2017). The goal of this study is to provide a better understanding of the poorly known ant biodiversity in China's Hengduan Mountains. The ultra-variable topography of this region, ideal for creating numerous vicariance events, combined with its wide range of climatic zones has contributed to the exceptional richness of endemic species inhabiting this area. Nevertheless, the rough topography has also made access and exploration rather challenging in the past. Against the background of extraordinary levels of plant diversity harbored by the Hengduan Mountains, it remains unclear whether or not ants and other insects display similar patterns of high diversity and endemism in this region.
To address this gap, we here present the results of an ant biodiversity survey conducted in the Gaoligong Shan mountains (part of the Hengduan Mountains), Yunnan Province, southwest China undertaken in 2019. Our goal is to present a complete species checklist of ants from the Gaoligong Mountains, including new records, as well as to update the current ant species checklist for the whole of Yunnan Province.
The Gaoligong Shan mountains (lat. 24°560'-28°220'N, long. 98°080'-98°500'E) comprise the western-most part of the Hengduan Mountain Range, and are among the most biodiversity-rich areas in Yunnan (Li et al. 2008;Dumbecher et al. 2011;Lo and Bi 2019). The ant fauna in the Gaoligong Shan mountains remains poorly understood, despite several studies focusing on ant diversity patterns that have recorded 62 ant species from 31 genera (Xu 2001a, b), but lack a comprehensive list of species collected.
Yunnan province is the richest province of China in terms of ant diversity . The latest ant checklist of Yunnan was compiled almost 10 years ago and consisted of 462 ant species. Since then, new ant inventories have been conducted (e.g. Liu et al. 2015a), as well as new species descriptions (e.g., Guénard et al. 2013;Xu et al. 2014a, b;Liu et al. 2015b;Staab et al. 2018), and the identification of previously dubious records have sensibly modified our understanding of Yunnan's ant diversity and species composition. Therefore, in this study, we also provide an update to the ant species checklist of Yunnan province and discuss future trends.

Materials and methods
Ant specimens were collected from natural forests along an elevational gradient on both the eastern and western slopes of the Gaoligong Mountains in July 2019. We sampled leaf litter ants from 16 sites at roughly 150 m elevational intervals from 600 m to 3000 m, following the standardized sampling protocol developed in Liu et al. 2016. At each site, we established a 400 m 2 quadrat (20 m × 20 m) and collected leaf litter samples at the four corners of the quadrat (1 m 2 ). We also collected leaf litter within the quadrat to cover a variety of microhabitats. Finally, ants on the ground, lower vegetation, and tree branches were collected both by hand and using a beating sheet. Leaf litter samples were extracted using mini Winkler extractors for 72 hours using the shuffling method described in Guénard and Lucky (2011).
Ant specimens were first placed in 99% ethanol and later sorted into morphospecies and point mounted. Each mounted specimen was assigned a unique Museum of Comparative Zoology, Harvard University (MCZ) specimen code and collection labels. Extended depth of field specimen images were taken with a Leica DFC400 digital camera mounted on a Leica M205C stereomicroscope through the Leica Application Suite V4 software in the Ant Room at the MCZ. Specimens were identified to species / morphospecies using available keys, the digital resources on Antwiki (http:// www.antwiki.org) and AntWeb (http://www.antweb.org), as well as reference museum material. All mounted and alcohol-preserved ant specimens are currently deposited in the Ant Room of the MCZ.
Distribution maps of species were generated from records included within the Global Ant Biodiversity Informatics (GABI) database and available at https://antmaps. org (Janicki et al, 2016;Guénard et al. 2017). These maps are based on records reported at the country level, or at the first administrative division for the larger countries (China, India, Japan). For larger islands that form their own natural biogeographic units like Borneo, Sumatra, New Guinea, the distribution maps used the island boundary instead of political boundaries (see also .

Ants of the Hengduan Mountain region
More than 3000 specimens were collected during this survey, and 130 species and morphospecies in 49 genera and nine subfamilies were identified. After identification of 88 valid species from the 130 total collected species, a total of 17 new species records are presented for Yunnan province and eight represent new records for China (see Table 1). The newly recorded species belong to 13 genera from four subfamilies. Moreover, the 41 morphospecies that could not be identified are likely to represent new species.
Here, we present the list of ant species that were collected in the Gaoligong Shan mountains (Table 1), as well as images for each species (Figs 1-136).

Updated ant checklist in Yunnan
The ant species list of Yunnan Province was generated using records from GABI available at https://antmaps.org (Janicki et al. 2016;Guénard et al. 2017). In total, the Yunnan ant fauna is composed of 99 genera and 550 named species and subspecies. Among them, the ant genera Lasiomyrma, Lordomyrma, and Prionopelta are only known from unidentified morphospecies. Through our collection and the records from GABI, we have added 125 species and subspecies to the list of ants of Yunnan since the last ant checklist . We also excluded 26 species records from the previous list and explained our rationale in each case (Table 3).

Ants in the Hengduan Mountain region
Field inventories and data synthesis efforts are essential for our understanding of ant diversity in 'hotspots' that harbor most of Earth's biodiversity. Our study represents new survey data from an understudied region. We produce the first ant species checklist from China's Hengduan Mountains (130 species).
A majority of the ant species were only collected below 1500 m, consistent with the strong effect of elevation on ant diversity observed elsewhere (Suppl. material 1, Fig. S1). This also suggests that future sampling in low elevation areas may increase species detection. For example, the number of Strumigenys species recovered in this survey is relatively low compared to the overall richness of this genus. This could be because we have relatively few collection events at low elevations where many of these species are known to occur. Indeed, all six Strumigenys species were collected below 1000 m from only three independent Winkler sampling sites.
Many of the new records in our collection such as Aenictus brevinodus, Camponotus bellus leucodiscus, Cataulacus marginatus, Crematogaster quadriruga, Dilobocondyla eguchii, Gnamptogenys quadrutinodules, and Strumigenys taphra represent the northern-most records of their known distributional ranges. Species records such as Aenictus brevinodus, Camponotus bellus leucodiscus, Camponotus keihitoi, Cataulacus marginatus, Gnamptogenys quadrutinodules, and Strumigenys taphra show a disjunction from the rest of their known distributions. It is unclear whether those records represent true biogeographic disjunctions, or sampling / taxonomic artifacts. Another potential reason could be that they were collected in the past, but have not been reported due to the lack of taxonomic infrastructure and species check lists from this region (Guénard et al. 2017). Additional inventories of ant diversity and taxonomic treatments are needed to answer these questions.
Despite the comparatively small area of China's Hengduan Mountains that we explored for this inventory of myrmecofauna, we were able to collect 130 species, which accounts for more than 24 % of the total number of ant species (N = 550) for Yunnan province. Among them, more than 10% of the ant species that were collected in this survey represent new records for Yunnan province. Moreover, there are still more than 41 morphospecies (32% of the total collected) that we believe are undescribed and new to science. To date, three Myrmecina species (Figs 82-84) and one Gauromyrmex species (Fig. 75) are undergoing taxonomic revision, and species descriptions are being prepared.
Our sampling of the full ant diversity of the Hengduan mountain region is still relatively limited. For example, we only had one sampling site per elevation, which is insufficient to cover the complex topology of the Hengduan Mountains. We also only used leaf litter extraction and hand collection, which is unlikely to recover complete ant assemblages. The incorporation of additional sampling techniques into our methodology, such as pitfall trapping, soil baiting, twig sampling, light trapping and canopy fogging, will cover more strata and lifestyles, and thus significantly increase our rate of species discovery. Overall, our results highlight how little was previously known about the ant fauna in this region and emphasize the need for further collecting in order to better understand the hidden ant biodiversity in China's Hengduan Mountains, and Yunnan Province overall.

Ants in Yunnan
If the total species richness of ants in Yunnan, with 550 species, is still an underestimate of the full species numbers, the exceptional diversity of genera encountered in this region needs to be highlighted. With 99 genera, Yunnan generic diversity is only matched globally by a few regions in South East Asia, and Queensland, Australia. A major difference with other Asian regions lies in the composition of the genera retrieved and their origin. For instance, genera found in Borneo, Sumatra, Java, Vietnam, Thailand, Peninsular Malaysia are almost exclusively derived from tropical, Oriental origins. In contrast, the geographic location and topography of Yunnan province has promoted an intermixing of taxa from several biogeographic regions: the Oriental realm from the south, the Palearctic realm from the northwest, and the Sino-Japanese realm from the northeast. As a result, the composition of the Yunnan ant fauna includes both tropical, subtropical, and temperate elements. Such intermixed communities are evident even at a small scale. For instance, during previous fieldwork conducted in the Gaoligongshan Mountains in 2015 by two of the authors (BG and CL), for which specimens were unfortunately lost, the coexistence of tropical (Dorylus, Ectomomyrmex), subtropical (Temnothorax) and temperate genera (Formica, Lasius) was observed on a hillside at an elevation of about 1900 m on an ~ 250 m² patch of grassland. Interestingly, while this area exhibited a transition where fauna from distinctly different origins coexisted along a thin band of altitude, at lower elevations, tropical genera were dominant and at higher elevations, temperate genera became dominant. Overall, this generated an unexpectedly diverse faunal composition, with such mixed communities contemplated by Wheeler (1915) to explain the generic composition and diversity of fossil ants observed in Baltic amber. Possibly, the ant composition of genera now retrieved within Yunnan might represent the remains of a once more widespread assemblage found within Asia and Europe during the Miocene . This highlights the specific nature of the Yunnan ant fauna and its importance in studying ant biogeography within Asia. It also serves as an excellent example of the formation and stability of ant community assemblages over time. Other neighboring regions such as Myanmar, Bhutan, Nepal or northeast India are likely to exhibit similar features, but to this point, the myrmecological exploration of these regions has been largely fragmentary (Guénard et al. 2010. The diversity of Yunnan ants is also remarkable for particular ant genera for which their global peak of diversity is encountered in the region. While it is important to note that the global diversity of specific genera as well as their overall taxonomic descriptions remain incomplete, seven genera present their highest currently known global diversity in Yunnan (Cryptopone: 5 species, Ectomomyrmex: 8 species, Kartidris: 3 species, Perissomyrmex: 2 species, Ponera: 14 species, Prenolepis: 7 species, Stigmatomma: 11 species), while six others are remarkable by the level of global diversity there, among the highest observed globally (Aenictus: 19 species, Carebara: 19 species, Dilobocondyla: 3 species, Myrmecina: 7 species, Proceratium: 4 species, Recurvidris: 3 species).
In conclusion, the important topographic variation, with mountain ranges aligned along a north-south axis combined with the presence of multiple climatic zones, including tropical rainforest in the lowland areas of the southern part of Yunnan create a diversity of microhabitats for supporting a diverse ant fauna. Moreover, the geographic position of Yunnan at the confluence of three biogeographic realms may promote ant diversity in the region. The collection of these 16 new ant records for Yunnan together with our previous discovery of 40 new ant records for Yunnan (Liu et al. 2015a) suggest that the true ant diversity in Yunnan is significantly higher. Moreover, the species diversity of some ant genera in nearby regions (based on data from GABI) also suggests that some ant genera sampled will ultimately be much more diverse in Yunnan (Guénard et al. 2017). For example, Hong Kong has recorded a similar diversity of Strumigenys species while having an area nearly 350 times smaller and a much less diverse topography compare to Yunnan (Tang et al. 2019), perhaps because leaf litter extraction has not been widely used for sampling ants in Yunnan. Thus, our survey to date indicates that further intensive sampling focused on different ecological strata (arboreal, leaf litter, subterranean) and combining various methods of extraction in both tropical and mountain habitats should yield many additional records and new species discovery in this region.