Hindawi Scientifica Volume 2020, Article ID 1609473, 8 pages https://doi.org/10.1155/2020/1609473 Research Article Assessment of Genetic Variation in Apis nigrocincta (Hymenoptera: Apidae) in Sulawesi Revealed by Partial Mitochondrial Cytochrome Oxidase I Gene Sequences Christian A. Lombogia,1,2 Jimmy Posangi,3 Hard N. Pollo,4 Max Tulung,1 and Trina E. Tallei 5 1Entomology Study Program, Postgraduate Program, Universitas Sam Ratulangi, Manado, North Sulawesi, Indonesia 2Nursing Study Program, Faculty of Nursing, Universitas Katolik De La Salle, Manado, North Sulawesi, Indonesia 3Public Health Study Program, Faculty of Public Health, Universitas Sam Ratulangi, Manado, North Sulawesi, Indonesia 4Forestry Science Study Program, Faculty of Agriculture, Universitas Sam Ratulangi, Manado, North Sulawesi, Indonesia 5Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas Sam Ratulangi, Manado, North Sulawesi, Indonesia Correspondence should be addressed to Trina E. Tallei; [email protected] Received 12 December 2019; Accepted 18 March 2020; Published 7 April 2020 Academic Editor: Yang Cao Copyright © 2020 Christian A. Lombogia et al. (is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Asian cavity-nesting honey bee Apis nigrocincta, a native bee species from Sulawesi and the Philippines, plays a vital role in pollinating flowering plants in local ecosystem and agriculture. In this study, we assessed the intraspecific genetic variation of A. nigrocincta using the sequence of cytochrome c oxidase subunit I (COI). Molecular phylogenetic analysis showed that there were three main clades in A. nigrocincta specimens from Sulawesi based on their respective locations (North, Central, and South Sulawesi). Genetic distance analysis using the Kimura 2-parameter (K2P) model showed that the intraspecific genetic distance in Sulawesi specimens ranged from 0.000 to 0.055. (ere are 26 nucleotide polymorphic sites within Sulawesi A. nigrocincta. (e variation was dominated by transition T ↔ C. (e molecular identification result was supported by morphological identification. (e results of the two methods agree that the specimen under study was A. nigrocincta. (e result of genetic distance calculation indicated that although the tested specimens were derived from remote locations, the genetic variation was still within the range of intraspecific variation. 1. Introduction Indonesia, and Mindanao, the Philippines), and A. nuluensis (Kalimantan, Indonesia), with approximately 44 subspecies (ere are about 30,000 bee species distributed worldwide. [4]. About 17,000 of them have been described [1]. Of them, (e honey bees are prone to extinction due to their about 20,000 species belong to the superfamily Apoidea [2]. haplodiploid sex-determining mechanism, limited pop- Apidae, the largest family within this superfamily, contains ulation because of anthropogenic activities [5], and low at least 5,700 species of bees [3]. Honey bees are included in intraspecific genetic variation. (e intraspecific genetic the genus Apis. (ese insects play a significant role in the variation in A. nigrocincta has been poorly studied. In recent pollination of important crops. Currently, there are nine years, molecular methods have been applied to study in- species of honey bees known to inhabit the world: Apis traspecific genetic variation, species identification, and dorsata, A. laboriosa, A. mellifera, A. florea, A. andreni- phylogenetic relationship among close-related taxa [6]. One formis, A. cerana, A. koschevnikovi, A. nigrocincta (Sulawesi, technique used for this purpose is DNA barcoding. (is 2 Scientifica method has been applied to conservation biology field be- 3. Results cause it provides information relevant to wildlife conser- vation management [7]. For animals, the area used for DNA 3.1. Morphometric Analysis. (e photograph of honey bee barcoding is the cytochrome c oxidase I (COI) gene. In this specimen obtained from Manado Mahakeret is shown in study, we examined the intraspecific variation in A. nigro- Figure 1. (e specimen hereinafter referred to as CAL01. (e cincta to facilitate species identification and analyze their results of the nine major morphological characteristics of the genetic distances. honey bee depicted by acronyms as length of head (HD), antenna (AT), proboscis (PB), thorax (TO), abdomen (AB), 2. Materials and Methods fore-wing (FW), hind-wing (FL), midleg (ML), and hind-leg (HL) are presented in Table 1. (ese characters were 2.1. Sample Collection. (e adult worker bees were captured compared with the characters of A. nigrocincta provided by using a swingnet from an abandoned local garden in Hadisoesilo et al. [12]. (e morphometric analysis has been Mahakeret village, city of Manado, North Sulawesi province, used previously to study genetic variability in honey bees Indonesia (1°28’53”N and 124°50’21”E). (e location was [13]. situated at an altitude of about 40 meters above sea level. (e average temperature at sample collection place (March 2019) was between 29°C and 32°C with humidity of 85–89%. (e 3.2. Molecular Analysis. (e cytochrome oxidase I (COI) percentage of vegetation cover was 80%. (e vegetation sequence of A. nigrocincta CAL01 has been deposited in consisted mainly of bushes, grasses, and wild flowers such as GenBank with accession number MK880239. (e complete Sphagneticola trilobata (L.) Pruski, Tachytarpheta jamai- BLAST search is presented in Table 2. (e location of all censis (L.) Vahl., and Mikania micrantha Kunth. Sulawesi specimens is shown in Figure 2. Because many sequences have 72% query cover, the sequences of some specimens were cut to match the length of other specimens. 2.2. Morphometric Analysis. (e observed morphological Even so, percent identity ranged between 92.24% and 100%. characters included length of following parts: head (HD), With a 72% query cover, the specimen CAL01 had a 100% antenna (AT), proboscis (PB), thorax (TO), abdomen (AB), identity with the DQ020233 specimen from Bogani Nani fore-wing (FW), hind-wing (FL), midleg (ML), and hind-leg Wartabone National Park, Gorontalo, Indonesia. With a (HL) using a caliper [8]. 100% cover query, the specimen CAL01 had a 92.24% identity with the KY834222 specimen from Islamabad, 2.3. Molecular Identification Pakistan. (e molecular phylogenetic analysis by the ML method 2.3.1. Sample Preparation, PCR, and Sequencing. Total DNA based on the K2P model is shown in Figure 3. (e tree was extracted from coxa connected to the abdomen using ZR reveals that there were three main clades in Sulawesi A. Tissue & Insect DNA MiniPrep™ (Zymo Research). (e nigrocincta specimens. Estimation of genetic distance genomic DNA was cleaned with DNA Clean & Concen- amongst A. nigrocincta is shown in Table 3. Analysis was trator™-5 (DCC™-5) (Zymo Research) for the generation of performed using the K2P model [10] integrated in MEGA high-quality DNA for PCR. (e DNA barcode of COI region v10.0.4 [11]. (e intraspecific genetic distance ranged from was amplified using Toyobo KOD FX Neo PCR Master Mix 0.000 to 0.055 (excluding specimen from Pakistan). Table 4 with primer pairs LCO1490 and HCO2198 [6]. (e con- shows the polymorphic nucleotides of the COI. Twenty-six dition of PCR included 2 min of initial denaturation at 95°C polymorphic sites were detected within A. nigrocincta. followed by 35 cycles of denaturation at 98°C for 10 sec, Variation of intraspecific COI gene showed that transition annealing at 54°C for 30 sec, elongation at 68°C for 45 sec and T ↔ C dominated the polymorphic pattern. additional extension for 5 min at 68°C. (e PCR products Estimation of substitution using the maximum com- were sequenced bidirectionally using the same PCR primer posite likelihood method can be seen in Table 5. Different pairs at 1st BASE DNA Sequencing Services Malaysia. transition substitution rates are shown in bold and trans- version substitution shown in italics. (e sum of the values 2.3.2. Analysis of COI Data. Chromatograms were subjected of v was equal to 100. (e number of transition substitution to the procedure as was done previously [9]. (e clean was 86.89, while the number of substitution was 13.11. COI sequence of the specimen was deposited in GenBank (erefore, the transition/transversion ratio (ti/tv) was 6.63. (http://www.ncbi.nlm.nih.gov). Identification was per- However, sampling size strongly influences the ti/tv. (e formed using BLAST identity search provided by the same estimated maximum likelihood ti/tv bias (R) was 9.05 under platform. (e clean sequences were aligned using Clustal O the K2P model [10]. (1.2.1) multiple sequence alignment (http://www.ebi.ac.uk/ Tools/msa/clustalo) with other allied honey bee COI se- 4. Discussion quences from different parts in Sulawesi retrieved from GenBank. (e evolutionary history was inferred by the Referring to the morphometric analysis conducted by Maximum Likelihood (ML) method based on the Kimura 2- Hadisoesilo et al. [12], as well as other physical character- parameter (K2P) model [10]. Evolutionary analyses were istics, it is believed that the honey bee being studied was Apis conducted in MEGA v10.0.4 [11]. nigrocincta (Figure 1). Nevertheless, this method still has Scientifica 3 Figure 1: Apis nigrocincta CAL01captured in Mahakeret. Table 1: Morphometric analysis of Apis nigrocincta CAL01. Characters Length (mm) Length (mm) [12] Head (HD) 3.57 ± 0.08 N/A Antenna (AT)