Introducción

The stingless bees (Apidae: Meliponini) are a cosmopolitan group of bees in the tropics and subtropics. Stingless bees are distributed in tropical regions of the world across the Neotropical, Afrotropical, and Australasian realms (^{Michener, 2000}) and are the highest diversity among bees consist of more than 600 species (^{Rasmussen, 2008}; ^{Rasmussen & Cameron, 2007}). Indonesia, as part of the Indo-Malayan and Australasian ecoregions, has 46 recorded stingless bee species across the islands of Sumatra, Java, Borneo, Timor, Sulawesi, Ambon, Maluku, and Papua (^{Kahono et al., 2018}). The species of this group are small to medium sized with a vestigial stinger and live in colonies (^{Michener, 1974}). These beehives can be found under the soil surface, in tree and wood cavities, house wall cavities, or hollows of bamboo trees (^{Dollin et al., 1997}; ^{Engel et al., 2019}; ^{Michener, 1974}; ^{Sakagami et al., 1983}). Stingless bees have a high prospect as a pollinator of agricultural crops related to its small body size, have a high foraging activity, and high adaptation to environmental stress (^{Kahono, 2015}). Stingless bees play an important role in pollinating of various plant species (^{Michener, 2007}), include in mustard (^{Atmowidi et al., 2007}), Tetragonula laeviceps in strawberry (Fragaria x annanassa) and Heterotrigona itama in melon (Cucumis melo) in the greenhouse (^{Atmowidi et al., 2022}). Nesting preference and nest characteristics, such as the morphology of nest entrance, nest architecture, and nest height from the ground are supporting characters that can be used to identify stingless bee species. Nest entrances vary in shape, texture, length, ornamentation, and color according to each species (^{Anaktototy et al., 2021}; ^{Kelly et al., 2014}). For example, the nest entrance of Heterotrigona itama (Cockerell, 1918) is funnel-shaped and in Geniotrigona thoracica (Smith, 1857) is rounded (^{Kelly et al., 2014}). The internal nest architecture consists of brood cells, honey and pollen pots (^{Efin et al., 2019}; ^{Franck et al., 2004}; ^{Michener, 1974}; ^{Sayusti et al., 2021}).

The Cycloops Mountains Nature Reserve (CMNR) located in the Jayapura region of Papua Province, Indonesia. It is a tropical forest area with high biodiversity and ecological functions that are important for human life. Human activities in this area have occurred for a long time that impact the biodiversity, including stingless bees. Forest disturbance in CMNR is mainly caused by community socio-economic activity (^{Ngutra, 2017}). Limited information and knowledge are a limiting factor for the local community. People in Papua haven't cultivated yet the stingless bees for producing honey and propolis. Community knowledge is key factor affecting stingless bee conservation. Until now, poor knowledge of taxonomy and phylogeny of these bees in the studied región are based for conducting this research. Therefore, studying the diversity and nests characteristics of stingless bees in the CMNR area is needed. This study aims to determine the species richness, characteristics of the nesting sites, and nest architecture of stingless bees in the CMNR, Papua, Indoesia.

Materials and methods

Observation of nest characteristic of stingless bees: The observations of stingless bees in the CMNR were conducted at three locations, namely Maribu village (including Mount Batu, Dumukribun, Dawari, and Maribu Kampung), Dosay village (including Kausei and Dansari), and Kemiri village (including Kemiri-2 of Forestry Residential) (Table 1). Nest of stingless bee was documented using a Nikon D5300 camera. The nest characteristics at Mount Batu and Dawari were observed by a road sampling method (^{Garton et al., 2004}). While in Maribu Kampung, Dosay village, and Kemiri village, sampling was based on information from local people. The nest features observed were nesting site, nest entrance measurements (length, diameter, height from ground), shape, and texture (^{Dollin et al., 1997}; ^{Kelly et al., 2014}). The coordinates of nesting sites were determined using GPS (Garmin etrex 10).

Collection, preservation, and identification of stingless bee specimens: Five individuals of worker bees from each colony were collected. The samples were then preserved using the dry preservation method (^{Borror et al., 1989}). Samples were stored in bottles and labeled. Identification of stingless bee specimens were based on ^{Sakagami et al. (1990}), ^{Dollin et al. (1997}), ^{Rasmussen et al. (2017}), ^{Engel et al. (2019}), and ^{Engel (2019}). The identification was carried out at the Laboratory of Animal Biosystematics and Ecology, Department of Biology, IPB University, Bogor, Indonesia. Then, the specimens were verified and deposited at the Research Center for Ecology and Ethnobiology, Research and Innovation Agency (BRIN), Cibinong, West Java, Indonesia.

Data analysis: The t-test analysis was used to compare the nest entrance variation within species of T. sapiens, consisting of the width and height of the nest entrance, funnel length, and the height from the ground using Paleontological Statistics version 4.09 (^{Hammer et al. 2001}).

Results

Richness of stingless bee: Two species of stingless bees were found in the three villages, namely Tetragonula sapiens and Heterotrigona (Platytrigona) planifrons (Fig. 1). A total of twenty-two colonies were found, i.e., one in Maribu Village including Gunung Batu (Dumukribun), four in Dawari, seven in Maribu Kampung, five in Dosay village and Kausei, one in Dansari, and five in Kemiri Village includes the Kemiri-2 of forestry residential (Table 1). Tetragonula sapiens was the dominant species (21 colonies; 95 %) found in all observation sites, while, Heterotrigona planifrons (one colony; 5 %) was only found in Kausei village.

Fig. 1 Worker of two species of stingless bees found in Cycloop Mountains Nature Reserve. A. Tetragonula sapiens and B. Heterotrigona planifrons. 

Nesting sites: The dominant nesting site of T. sapiens was in house foundations (nine colonies, 43 %), followed by coconut tree cavities (four colonies, 19 %), house walls (three colonies, 14 %), dry wood (two colonies, 10 %), fern tree (two colonies, 10 %), and in the soil (one colony, 5 %). The nest of H. planifrons (one colony) was found in a coconut tree cavity.

Nest entrance characteristics: The nest entrance tunnel of T. sapiens varied, being horizontally elongated (4 colonies), oval (6 colonies), round (2 colonies), irregular (2 colonies), ellipses (4 colonies), and vertically elongated (2 colonies), while in H. planifrons it was vertically elongated (1 coloni). The color of T. sapiens entrances was brown, dark brown, blackish, light brown with grayish on the tube-end, brownish-gray, dark brown, blackish gray with blackish on the tube-end, with hard to soft textures. In H. planifrons, the color of the nest entrance tunnel was light brown with a hard texture (Fig. 2). The nest entrance opening of H. planifrons was 5.5 cm in height and 7.5 cm in wide, while in T. sapiens ranged 0.6-3 cm in height and 0-3.5 cm in wide (Table 2). The differences in the diameter of the nest entrance opening in T. sapiens and H. planifrons may be due to differences in body size, colony size, and nest age. In Dosay-Kausei villages, we found the vertically elongated nest entrance of H. planifrons with characteristics was 2.0 cm in width and 5.5 cm in height, light brown color, 7.5 cm of funnel length, and no ornamentation (Fig. 2) with a thick and hard-texture in tube-end, respectively and the nest height from ground was 162 cm (0-325 cm) (Table 2).

Fig. 2 Types of nesting entrance opening of T. sapiens in Maribu, Dosay, and Kemiri villages. A.-D. elongated horizontal with dark brown, blackish and slightly blackish gray on tube-end. E.-J. oval with brown, blackish brown, light brown on tube end, blackish gray, and brownish black. K.-L. round with dark brown. M.-N. irregular with dark brown and brownish gray. O.-R. ellipses with brown, dark brown, and blackish gray. S. Elongated vertically with dark brown. T. elongated vertical with light brown hard texture in H. planifrons. Scale = 1 cm). 

Nest architecture: The brood cells of T. sapiens in three villages (four colonies) were a vertically cluster, horizontally cluster, and semi-cluster. The brood cells were connected to other cells by lamellate pillar structures (Fig. 3, Table 3). The brood cells were oval in shape and light brown in color (brood cells with adult larvae) and dark brown (brood cells with young larvae). The pollen pots of T. sapiens were irregular (0.5-1.1 cm in width and 7.5-12.3 cm in length) with brown, dark brown, and black colors. The honey pots had an irregular shape (0.6-1.2 cm in width) and were dark brown, blackish, and black. The nest heights of T. sapiens from ground level ranged from 15 to 290 cm (Table 3).

Fig. 3 The internal nest architecture of four colonies of T. sapiens in Maribu, Dosay, and Kemiri villages, Papau: A.-B. vertical cluster, C. semi-cluster, D. horizontal cluster, and E. layered vertical comb in H. planifrons (insert for detail). ne=nest entrance, bc=brood cells, pc=pollen cells, hc=honey cells, lp=lamellate pillars. Scale=1cm. 

In Dosay-Kausei village, the brood cells of H. planifrons had a vertically elongated with a layered vertical comb with a lamellate pillar structure that connect it to the tree trunk. The brood cells of the species was oval in shape, light brown (old cells) and dark brown (young cells), with height ranging from 1.58-2.22 cm and a width of 0.55-0.62 cm (Fig. 3). The pollen pots were irregular, while the honey pots were an irregular shape with blackish (Table 3).

Discussion

Morphologically, stingless bees are difficult to identify (^{Sakagami & Inoue, 1985}). The current study identified two species as Tetragonula sapiens (Australasian stingless bee) and Heterotrigona (Platytrigona) planifrons (Papuasian stingless bee). This study added a new distribution record for T. sapiens and H. planifrons in Papua, specifically in the CMNR area of Jayapura Regency. Previous studies reported T. sapiens was distributed in the Solomon islands and Papua New Guinea (^{Dollin et al., 1997}), while H. planifrons has been reported from the Central Mamberamo District, Bovendigul District, Merauke District, Manokwari of Papua, and in the Bulolo District, Morobe, and Oro Provinces of Papua New Guinea (^{Engel, 2019}). Previous studies in Papua reported four species of Tetragonula, namely T. fuscobalteata (Cameron, 1908), T. sapiens (Cockerell, 1911) (^{Dollin et al., 1997}), T. clypearis (Friese, 1908), and T. biroi (Friese, 1898) (The Papua Insect Foundation, 2008), and four species of Heterotrigona, namely H. planifrons (Smith, 1865), H. lamingtonia (Cockerell, 1929), H. flaviventris (Friese, 1908) and H. keyensis (Friese, 1901) (^{Engel et al., 2019}; ^{Engel, 2019}).

The nesting sites of stingless bee are usually in a tree cavity, soil, and hollow walls (^{Slaa et al., 2000}). Some species, such as of the genera Tetragonula, Tetragonisca, Partamona, Paratrigona, and Plebeia build nests on tree trunks or solid substrates (^{Roubik, 2006}). In this study, we found that the nesting site of T. sapiens was dominatly in house foundation (9 colonies), followed by coconut trees (4 colonies), house walls (3 colonies), dry wood (2 colonies), fern trees (2 colonies), and soil (1 colony). In contrast, the nesting site of H. planifrons was in a coconut tree cavities. The limited nesting sites of H. planifrons (1 colony found) may be a result of burning the nest due to their aggressive biting.

The nests of aggressive stingless bees are usually found individually in specific tree trunks, such as palm trees (^{Roubik, 2006}). Meanwhile, the nests of non-aggressive stingless bee colonies are generally close to other stingless bee species (^{Roubik, 1983}). The current study found more colonies building nests close-together in sap-producing trees, such as Psidium or cashew (Anacardium occidentale: Anacardiaceae), merbau (Intsia bijuga: Fabaceae), mango (Mangifera indica: Anacardiaceae), jackfruit (Artocarpus heterophyllus: Moraceae), and wood milk (Alstonia scholaris: Apocynaceae). Resin in the nest entrance protects the colony from predators (^{Roubik, 2006}). The texture of the nest entrance is related to the various resins collected from gummy plants. Variation in the nest entrance is influenced by nest age, genetics, predators, parasites, symbionts, rain, wind, and light (^{Sakagami et al., 1983}; ^{Wille & Michener, 1973}). This study found a pronged and a branched nest entrance of T. sapiens in Maribu village. A branching nest entrance has also been found in Lepidotrigona ventralis (Smith, 1857) (in Thailand), Lepidotrigona, Plebeia, Scaptotrigona, and Tetragona (^{Roubik, 2006}).

The results showed that the nest entrance of T. sapiens was a short funnel, whereas in H. planifrons it was an elongated. Unfortunatley, we only found one colony of the species and no variation of nest entrance characteristics was shown. We suppost that there are variation of the nest entrance characteristics in the species. So, more exploration in a wide areas in Papua should be done in the future. Nest entrances with a short funnel are usually found in stingless bees of the Neotropics (T. cilipes) and Asia (T. collina). Similarly, an elongated nest entrance was also found in two Asian stingless bees with aggressive behavior, namely H. canifrons and H. itama (^{Sakagami et al., 1983}). The color and texture of the nest entrances of T. sapiens varied, such as brown, dark brown, blackish, cream (grayish on the tube-end), brownish, brownish-black, blackish gray (blackish on the tube-end), with a hard and soft texture. In H. planifrons (Smith, 1865), the color of nest entrance is light brown with a hard texture. The color variation of the nest entrance may be due to the different resin sources (^{Sakagami et al., 1983}; ^{Wille & Michener, 1973}). Nest entrance variation in T. sapiens may be related to polyphenism, in which two or more phenotypes result from the same genotype (^{Mayr, 1963}). Polyphenism is influenced by environmental conditions that cause phenotypic preferences in neurochemical and hormonal pathways related to nest-building behavior (^{Simpson et al., 2011}).

The brood cells of T. sapiens are connected by a columnar lamellate pillar (Fig. 3, Table 3), oval shape and light- and dark-brown, while the pollen and honey pots were irregular, probably an adaptation to substrate types. The modification of nest architecture in stingless bees is closely related to the adaptability of a species to its environment. Species with high intraspecific and phenotypic variations are usually more adaptive to environmental changes than endemic species with low intraspecific variations and limited distribution in certain geographic area (^{Cohet et al., 1980}; ^{Lavergne & Molofsky, 2007}). In this study, T. sapiens found in all sampling sites with various nest entrance characters and able to live at varying altitudes (107-364 m.a.s.l). In contrast, H. planifrons showed a limited distribution, at just one sampling site at an altitude of 127 m.a.s.l. The observations and interviews with local communities at West Sentani to Sentani districts showed H. planifrons had a limited distribution and was only found in the Dosay village.

Ethical statement: the authors declare that they all agree with this publication and made significant contributions; that there is no conflict of interest of any kind; and that we followed all pertinent ethical and legal procedures and requirements. All financial sources are fully and clearly stated in the acknowledgements section. A signed document has been filed in the journal archives.