Expanded distribution range of nine orchid bee species (Hymenoptera, Apidae, Euglossini) in Costa Rica

The Monteverde region of Costa Rica is a hotspot of endemism and biodiversity. The region is however disturbed by human activities such as agriculture and urbanization. This study provides a list of orchid bees (Hymenoptera: Euglossini) compiled from field surveys conducted during January-October 2019 in the premontane wet forest of San Luis, Monteverde, Costa Rica. We collected 36 species of Euglossine bees across four genera. We provide new geographic distribution and elevation data for nine species in two genera. Due to their critical role in the pollination of orchids and other plants, the distribution and abundance of Euglossine bees has relevance to plant biodiversity and conservation efforts. This is especially important in a region with a high diversity of difficult to study epiphytic orchids such as in the Monteverde region.
 A total of 2,742 Euglossine male individuals across four genera (Eufriesea, Eulaema, Euglossa, and Exaerete) were collected in this study. Updated geographic distributions and elevation ranges were established for nine species of Euglossini in two genera: Eufriesea concava (Friese, 1899), Eufriesea mussitans (Fabricius, 1787), Eufriesea rufocauda (Kimsey, 1977), Euglossa dodsoni (Moure, 1965), Euglossa dressleri (Moure, 1968), Euglossa hansoni (Moure, 1965), Euglossa ignita (Smith, 1874), Euglossa tridentata (Moure, 1970), Euglossa turbinifex (Dressler, 1978). For all species except Eufriesea concava (Friese), which had one prior recording in 1993, these are the first recorded occurrences in the Monteverde region of Costa Rica, according to the Global Biodiversity Information Facility (GBIF) database (https://doi.org/10.15468/9f9kgp). Expanded elevation ranges are also provided for Euglossa allosticta, Euglossa bursigera, Euglossa mixta, Euglossa heterosticta, and Euglossa maculabris, though these 5 species have been previously recorded in the Monteverde region and thus are not described in detail here.


Introduction
Hymenoptera in the tribe Euglossini (orchid bees) are a diverse group of New World tropical pollinators accounting for up to 25% of total bee communities in the Neotropics ( McCravy 2016, Roubik andHanson 2004). Orchid bees have a geographical range spanning from Mexico to Argentina, with one species known in the southern United States. There are 268 documented species with 66 known to inhabit Costa Rica from four genera: Eufriesea, Euglossa, Eulaema, and Exaerete. Orchid bees play a vital role in the pollination of orchids (Orchidacea) and other plant groups (Dressler 1982, Botsch et al. 2017. Euglossine bees fly distances of up to 23 km per day, and thus are important pollinators of plants with low population densities such as many orchid species (Janzen 1971, Tonhasca Jr et al. 2003, Wikelski et al. 2010. Nearly 700 orchid species rely exclusively on pollination by male orchid bees (Roubik and Hanson 2004). The male bees visit orchids to collect odorous compounds to attract mates. The chemicals are stored in specialized setae-lined organs on the hind tibia of the males, a physical characteristic found nowhere else in the insect world (Eltz et al. 2005). Female orchid bees are polylectic and will collect pollen from a wide diversity of plant species in tropical forests to provision their offspring, and thus, contribute to reproductive success of many tropical plant species (Ferreira-Caliman et al. 2018).
The global decline of bees has been widely observed, and are especially pronounced in areas of intense agriculture, urbanization, and pesticide use (Biesmeijer et al. 2006, Potts et al. 2010, Goulson et al. 2015, Koh et al. 2016, Sánchez-Bayo and Wyckhuys 2019. Though documented population trends of tropical bees are consistent with these global trends, studies on bees in the tropics are vastly underrepresented, and their status largely unknown (Prado et al. 2017). The few long-term studies that exist document Euglossine population declines to varying degrees (Vega-Hidalgo et al. 2020a). Populations of some other wild bee groups have appeared largely stable in protected areas such as nature preserves (Frankie et al. 1998, Cairns et al. 2005, Roubik and Villanueva-Gutierrez 2009, Archer 2013, Herrera 2019, however declines in Euglossine populations have been recorded even in natural areas that have not been modified (Nemésio 2013, Nemésio et al. 2016, Hallmann et al. 2017, Storck-Tonon and Peres 2017, Rada et al. 2019, Vega-Hidalgo et al. 2020b). More multi-year, year-round studies are needed to fully elucidate the status of Euglossine and other tropical bees.
Euglossini vary in abundance with season, with some species (e.g. many Eufriesea spp.) only being active for a few months of the year (Nemésio 2011). In the lowland forests of Costa Rica, Euglossine bees exhibit seasonal variation in overall abundance and species diversity (Janzen et al. 1982). Hence, sampling throughout the year during the wet and dry seasons is necessary to fully assess the species diversity of orchid bees in an area. Another study examined fragmentation of habitat types at elevations of 900-1,400 meters in southern Costa Rica (Brosi 2009). We sampled orchid bees in Monteverde in Costa Rica using baited traps, and our study expanded on reported elevation ranges for Euglossa allosticta, Euglossa bursigera, Euglossa mixta, Euglossa heterosticta, and Euglossa maculabris. We also provide new data on an additional nine species of orchid bees previously undocumented at this elevation range or the Monteverde region: Eufriesea concava, Eufriesea mussitans, Eufriesea rufocauda, Euglossa dodsoni, Euglossa hansoni, Euglossa ignita, Euglossa tridentata, Euglossa turbinifex, Euglossa dressleri. Most tropical bee studies are conducted in the understory due to dense forests using pan traps and timed observations, making our study unique in both the sampling of the canopy and the trap type.

General description
Purpose: Euglossine bees were sampled in the Monteverde region of Costa Rica at an elevation immediately below the cloud forest (1,100-1,170 meters above sea level), what is considered premontane wet forest. To our knowledge, this area has not been previously sampled for orchid bees. This area was chosen as it provided the opportunity to sample understory, canopy, and open areas to capture a range of diversity in the orchid bee community. In this way, it is more representative of the general state of orchid bee diversity in the region; much of Central America's forests are degraded, secondary forests and the remaining primary forests are predominantly in reserves, in a patchwork of human managed and semi-natural environments (personal communication, Jacob Podesta). It is therefore necessary to understand how Euglossine diversity responds to the anthropogenic changes in their geographic range to inform management strategies to promots and maintain orchid bee diversity. We collected a total of 2,742 specimens, represented by 36 species across four genera, all of which were adult males. We provide expanded geographic distribution and elevation ranges for nine bee species.

Sampling methods
Study extent: Three areas of the 66 hectare Universityof Georgia Costa Rica campus were sampled: Forest A (trap locations F1-F5), Forest B (F6-F10), and the open area through the developed part of campus (O1-O10) ( Table 1). At each forest trap location, one each of understory and canopy traps were positioned, so that Forests A and B each contained 10 traps. Sampling occurred for five consecutive days each month January-October 2019. As summarized in Table 2 and detailed below, Forest A was sampled January-October, Forest B was sampled in April and October, and the open area was sampled in June-October.
Sampling description: Male Euglossine bees are attracted to baited traps using artificial compounds that mimic those of desired orchids, a method employed regularly by researchers (Hedström et al. 2006, Sandino 2004, Velez and Pulido-Barrios 2005. Cineole is used in this study as it is considered a universal attractant for male orchid bees ( Buchmann 2019). Traps used to sample bees were made using a 3.5L container with lid, with an opening of 4 cm located on one side of the container. A single metal screw hook was attached to the top of the container lid and used to secure the trap. On the inside of the lid, a galvanized wire was used to secure a cotton ball wrapped in wire mesh which served as the attractant site for the cineole.
The dry season is typically marked by high wind speeds and little rainfall. Dry season sampling occurred January to May 2019. During this time, the five sites and ten traps in Forest A were sampled each month, with Forest B being sampled during the peak dry season in April.
The wet season sampling took place June to October 2019. In addition to the ten traps in forest A, ten trap sites in the open area were added during the wet season. In October, the peak wet season, Forest B was added, for a total of 30 operational traps.The Forest B site was sampled during the historical peak dry and wet seasons to provide additional data for comparison. We selected trapping sites where the elevation range along a transect was no more than 100 m.
At each of the habitat types, traps were established along a transect, with each sub-unit being 100 m apart (Fig. 1). In the two forested transects, five sub-units were selected with a canopy and understory trap. Understory traps were installed at a height of 1.5 m above ground. In the open area site, ten traps 1.5 m above the ground were established at different sub-units, each 100 m apart. Sampling the canopy in addition to the understory allowed us to expand the potential biodiversity in our samples, as canopy sampling is rarely included in most Eugolssine trapping studies, and canopy specialist groups are potentially under-represented (Sobek et al. 2009, Ulyshen et al. 2010, Urban-Mead et al. 2021).
Quality control: Individuals collected at each trap were placed in an 8 oz. Whirlpack plastic pouch. The bags were padded with tissue paper to prevent the movement of live bees from damaging themselves or other specimens. Each collection bag was labeled with the name of the sampling site, date collected, name of the individual collecting the sample, and the trap number. The bees were returned to the laboratory and placed in the freezer for at least 24 hours to ensure that all of the specimens were dead before identification. Specimens were mounted or preserved according to standard protocols as follows: 1.
The bags were removed from the freezer and placed in a jar containing 70% ethanol.

2.
The bees were removed from the ethanol and placed on a sheet of tissue paper to dry and were identified while still flexible.

3.
The bees were mounted, taking into consideration the visibility of the most important diagnostic features of their anatomy. Fig. 2 is an example of a mounted orchid bee. Note that the midlegs are positioned to ensure that the middle tibial and velvet area are clearly visible and that the forelegs and antenna do not obscure the mandibles or ivory bands, respectively. 4.
Mounted specimens were dried at approximately 65 degrees celcius for at least seven days before being placed in long term storage cabinets.

5.
A minimum of 50 individuals per species were mounted, and the remaining specimens were preserved in ethanol.
Standard procedures were followed during specimen sorting and identification in the laboratory. All Euglossine specimens were identified to species level using physical characteristics as described in Roubik and Hanson's dichotomous key in "Orchid bees of tropical America Biology and fileld guide" (Roubik and Hanson 2004). Each specimen was assigned a unique specimen code consistent with the collection database and labeled in the reference collection at the CIEE campus The resulting specimen collection will be used to inform future research and as an education tool. Pollinaria attached to bees at the time of collection were preserved on the specimens for future identification. Specimens of non-target taxa were mounted or preserved in ethanol for future identification. ; 2) open low-intensity agricultural areas; and 3) areas considered highly modified with roads, multiuse recreation areas and buildings/structures within a 40 m elevation range (Fig. 1). Fifty ha are secondary forest, and the remaining 12 ha consisted of open pasture or facilities. This area exhibits weather patterns consistent with a Central American premontane wet forest, with the driest months being January-April, and the wettest months being May-October. Peak dry season is April, corresponding to the highest average temperatures.

Geographic coverage
October is the month with the most rainfall and the lowest average temperatures (World Weather & Climate Information 2019). The average annual temperatures range between 15.6 and 29.4 Celsius. The Monteverde region in Costa Rica is largely covered by montane forest, also classified as cloud forest. The area sampled in this study is premontane wet forest, which has elevation of (1,100-1,500 m) and receives an average of 2.4 m of rainfall annually. Premontane wet forest is slightly lower in elevation than cloud forest. Physical descriptions and baseline distribution and elevation data was based on Roubik and Hanson's "Orchid bees of tropical America Biology and field guide" (Roubik and Hanson 2004) and cross-referenced with GBIF databases to check for any updates ( GBIF.org 2021a, GBIF.org 2021b, GBIF.org 2021c, GBIF.org 2021d, GBIF.org 2021f, GBIF.org 2021g, GBIF.org 2021h, GBIF.org 2021i). Table 3 includes catch numbers of each species. The dataset includes 36 species across four genera of Euglossini (Eufriesea, Euglossa, Eulaema, and Exaerete), and includes new records for the Monteverde region and expanded elevation range of the following taxa:

Eufriesea concava (Friese, 1899) -123 individuals
Identification. Body 17-19 mm long. The clypeus has two strong sublateral ridges and the area between these ridges is concave and polished.
Remarks. This species has been documented "Mexico to Southeastern Brazil; lowlands up to 1,100 m" (Roubik and Hanson 2004). In Costa Rica, this species has been documented throughout the country in the provinces of Alajuela, Guanacaste, Limon, Puntarenas, and San Jose (GBIF.org 2021a). There is one prior occurrence of a single specimen of E. concava in the Monteverde region, documented in 1993 ( Fuentes 1993). This study provides expanded elevation data for E. concava at 1,081-1,168 m.

Eufriesea mussitans (Fabricius, 1787) -1025 individuals
Identification. Body 17-21 mm long. The clypeus has two strong sublateral ridges and a medial ridge. Medial ridge distinguishes between E. mussitans from E. concava, the latter of which exhibits a concave area between the sublateral ridges.
Remarks. This species has been documented "Mexico to southeastern Brazil; lowlands up to 1,000 m" (Roubik and Hanson 2004). In Costa Rica, E. mussitans have been documented in the northern provinces of Guanacaste and Alajuela (GBIF.org 2021b). This is the first known collection of E. concava in the Puntarenas province, or at the elevation of 1,041-1,168 m.

Eufriesea rufocauda (Kimsey, 1977) -4 individuals
Identification. Body ~14 mm long. Clypeus does not possess sublateral ridges. Face bronze below and green above in males. The anterior part of tergum II is dark, and the posterior part of tergum II is reddish copper with yellow hairs. In lateral view, male labrum has a square outline; when viewed dorsally, a pair of prominent conical points is visible.
Differs from E. chrysopyga as: 1) E. chrysopyga has a uniformly purple tergum II; and 2) lateral view of male labrum of E. chrysopyga has a triangular outline and dorsal view reveals barely visible pair of conical points.

Remarks:
In Costa Rica, E. rufocauda has been previously documented in Limon, green. Broad longitudinal depression present in the middle of the scutellum. Two tufts on the middle tibia are touching, possibly appearing to be one tuft. Distal tuft is larger. A pair of widely separated diagonal slits is present on scutellum II, each with a dense row of setae.

Remarks. Euglossa ignita has been documented in all seven provinces in Costa Rica:
Guanacaste, Alajuela, Heredia, Cartago, Limon, and southern Puntarenas on the Osa Peninsula (GBIF.org 2021g). This study documents occurrences of E. ignita in the Monteverde region and at an elevation of 1,083-1,137 m.

Euglossa tridentata (Moure, 1970) -74 individuals
Identification. Body is green and 11-12 mm long; tongue is much shorter than the body. Ivory eye bands are complete, and clypeus is green. Three teeth on mandibles. Middle tibia has two tufts with distal tuft having a shallow notch. Similar to E. deceptrix and E. variabilis, though E. deceptrix and E. variabilis both have two teeth on mandibles.
Remarks. In Costa Rica, E. tridentata has been recorded in Guanacaste, Alajuela, Heredia, Limon, San Jose and Puntarenas, outside of the Monteverde region (GBIF.org 2021h). This study provides expanded occurrence data for E. tridentata at 1,041-1,168 m and in the Monteverde region.

Euglossa turbinifex (Dressler, 1978) -1 individual
Identification. Body is 11 mm long and mostly green with a bluish green mesosoma. Tongue is the length of the body. Mandibles have two teeth. Two tufts on the middle tibia, basal tuft larger than distal one. Sternum II has a pair of small semicircular depressions containing rows of setae. Similar to E. bursigera, but E. bursigera often has a more bronzish body and has three teeth on the mandible.

Remarks.
In Costa Rica. E. turbinifex has been recorded in Alajuela, Heredia, and Limon. In the GBIF database, less than 40 total specimens are documented, 20 of them being in Costa Rica (GBIF.org 2021i). This study documents one occurrence of E. turbinifex in the Monteverde region of the Puntarenas province at an elevation of 1,154 m.

Additional information
Due to changing climatic conditions and other anthropogenic effects, consistent sampling at various elevations is needed to track distributional changes in fauna over time. Shifting ranges are predicted as Euglossine bees respond to climate change (Silva et al. 2015), and thus a complete understanding of their current range will be critical for tracking future shifts. Given the co-dependent and intimate relationships between orchids and Eulossine bees, it is important to monitor populations and species of both the groups to detect future declines or recovery under restoration practices. Our 10-month study sampled across wet and dry seasons, in both forest and open areas, as well as canopy and understory, and was therefore, able to yield a more complete species-list in this region, including detecting highly seasonal species such as Eufriesea mussitans. This demonstrates that a 12-month study would be useful in detecting other ephemeral species that occur in November and December, and we suggest that continued monitoring of specialist bees are needed for their protection and conservation efforts in such hyper-diverse and endemic tropical ecosystems.
campus, formerly University of Georgia, Costa Rica campus. Funding was provided by the D.B. Warnell School of Forestry and Natural Resources, University of Georgia, and the Benjamin A. Gilman International Scholarship. Thank you to Kris Irwin (University of Georgia) for making this study possible by initiating contact with the University of Georgia Costa Rica faculty, and approving and supporting the project. Thank you to Brandon Horn, Nathan Lasala, Alfonso Tascón, other resident naturalists and CIEE staff for moral, logistic, language and occasional field support. Special thanks to Ernest Minnema and Fabricio Camacho for helping reset canopy traps -without your help, field work would have been cut short. Neil Cobb (Northern Arizona University) and Joe McHugh (University of Georgia) assisted with data management.