DOI 10.2478/JAS-2019-0008 J. APIC. SCI. VOL. 63 NO. 1 2019J. APIC. SCI. Vol. 63 No. 1 2019 Original Article PHYLOGENETIC ANALYSIS OF TETRAGONULA MINANGKABAU AND OTHER SPECIES USING CYTOCHROME B GENE Buti Y. Christy* Dewi I. Roesma Dahelmi Biology Department, Faculty of Mathematic and Natural Science, Andalas University, Padang, Indonesia *corresponding author: [email protected] Received: 01 August 2018;accepted: 03 March 2019 Abstract Tetragonula a genus in the Meliponini tribe (), is difficult to identify due to the many cryptic species. As technology develops, molecular taxonomic studies are used to help identify species with limited morphological characteristics. This study presents an analysis of the phylogenetic relationship between several species in the Tetragonula ge- nus based on the sequences of the cytochrome b gene. Maximum parsimony, neighbor joining, maximum likelihood and minimum evolution methods were used to construct phylogenetic trees. The sequence divergence between T. minangkabau collected from Limau Manis and Ulu Gadut is 0.8%, while between T. minangkabau with T. minangkabau forma darek 5.5%. The low sequence divergence indicated that T. minangkabau and T. minangkabau forma darek have a close phylogenetic relationship. The analysis showed that Tetragonula (T. minangkabau, T. minangkabau forma darek, T. laeviceps, T. drescheri and T. fuscobalteata) is monophyletic. A sequence divergence of 5.5% supports the sepa- ration of Tetragonula minangkabau and T. minangkabau forma darek.

Keywords: cytochrome b, phylogenetic analysis, Tetragonula, Tetragonula minangkabau

INTRODUCTION Inoue (1985) revealed one species in Sumatra belonging to the Tetragonula laeviceps group Stingless bees are included in the family Apidae, with a smaller size and paler body parts compared Meliponini tribe and represent a group of cos- to the typical T. laeviceps. Due to these mor- mopolitan bees that can be found in the tropics phological differences, this species was named (O’Toole & Raw, 1991). From the taxonomic Tetragonula minangkabau (Sakagami & Inoue, aspect, various publications reported the dif- 1985), named after Minangkabau, which is the ferences in the number of species in Meliponini name of the ethnic community that inhabits the tribe. This is due to the large number of West Sumatra region. In the same year, Sakagami taxonomic revisions and cryptic species. There and Inoue also found two groups in the collected are over 374 species of stingless bees which T. minangkabau specimens that differed in head are divided into twenty-six genera (Michener, width and hind tibia length. The data obtained 2007). Another source states that members showed that the size of specimens from Lubuk of the Meliponini tribe are more than thirty- Minturun and other locations in West Sumatra three genera and 397 species (Moure, Urban, (generally from the highlands) differed sig- & Melo, 2007). Cortopassi-Laurino et al. (2006) nificantly. Specimens found in the highlands state that more than 600 species were larger and possessed bigger hind legs belonging to fifty-six genera have been found and paler colored metasomas. This group of worldwide. Among them, 400 species are found specimens was named Tetragonula minangka- in the Neotropic region and at least forty-five bau forma darek. According to Braby, Eastwood species are found in Southeast Asia. & Murray (2012) forma is one of intraspesific In Indonesia and especially Sumatra, there has unit (beside varieties and races) which is not been research on the etho-ecological aspects formally recognized under ICZN (International of stingless bee since 1980. Sakagami and Code of Zoological Nomenclature). The name 117 Christy et AL. Phylogenetic analysis of Tetragonula minangkabau

“darek” was proposed for the larger form as it bee) genus. means “highlands”, used especially in reference From the differences in body length and to the Bukittinggi area (Sakagami & Inoue, 1985; coloration, we hypothesised that T. minangkabau Sakagami, Inoue, & Salmah, 1990). Neverthe- forma darek can be classified as a different less, the taxonomic position of this form is still species from T. minangkabau. The purpose of unclear. this study was to analyze the phylogenetic Molecular markers have been used in many relationship among Tetragonula minangkabau, studies related to the stingless bee, including T. minangkabau forma darek and several species population, taxonomy and phylogenetic in the Tetragonula genus. inference. The use of molecular markers to determine the status of a taxa has been carried METHODS out in Liotrigona spp. using the COI gene (Koch, 2010) and nine Amazonian stingless bees using Stingless bee samples were collected directly 16S rRNA and COI with the PCR-SSCP technique with the use of nets and tweezers at (Souza & Carvalho-Zilse, 2014). Thummajit- the nest entrance. Ten individuals from each sakul et al. (2014) have studied the population colony were collected and preserved in alcohol. structure of Tetragonula pagdeni in Thailand Such specific characteristics from samples using the cytochrome b gene and ATPase6- obtained as body coloration, shape and color tRNAASP. Rasmussen and Cameron (2007) of the nest entrance were noted and pho- used mitochondrial genes (16S rRNA) and core tographed as supporting data for identifica- genes (nuclear long-wavelength rhodopsin copy tion. In sample collection and identification we I, elongation factor-1α copy F2 and arginine followed Sakagami, Inoue and Salmah’s method- kinase) to prove the non-monophyletic nature ology (1990). The locations of the Tetragonula of the genus Trigona sensu lato. Ramirez et al. sample collection is shown in Tab. 1. All the (2010) have conducted phylogenetic relation- samples obtained were used for the identifica- ship studies of the stingless bee for the genus tion process but only one individual from each Melipona using different fragments including colony was analyzed molecularly. mitochondrial CO1, ribosomal 16S, nuclear EF1-a, DNA isolation was performed according to the ArgK and Pol-II. Among examples of molecular INVITRO GEN PureLinkTM Genomic DNA Mini Kit studies on bees other than the Meliponini, protocol. Isolated DNA was visualized through Koulianos and Schmid-Hempel (2000) conducted the electrophoresis process on 1.2% agarose a study that used the cytochrome b and COI gel. Electrophoresis was performed at a voltage genes to determine the phylogenetic relation- of 100 mV for thirty-five minutes. The gel was ship of nineteen species in the Bombus (bumble then placed on top of the UV illuminator (GelDoc) Table 1. Location of Tetragonula sample collection

Species Location Altitude (meters above sea level) Limau Manis 210 Tetragonula minangkabau Ulu Gadut 500 T. minangkabau forma darek Solok 1500 Limau Manis 255 T. fuscobalteata Sijunjung 220 T. laeviceps Malaysia 54 T. drescheri Malaysia 54 T. minangkabau Malaysia 54

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Table 2. List of species from NCBI GenBank used for construction of phylogenetic trees based on cytochrome b gene

Accession No. Species Location number 1 Trigona clypearis AY575083 Australia (a) 2 Trigona laeviceps AY575080 Thailand (a) 3 Trigona collina AY575081 Thailand (a) 4 Tetragonula pagdeni FJ012146 Thailand (b)

Annotation: (a) Franck et al. (2004); (b) Thummajitsakul et al. (2008) and the visualization results were documented. of the sequences obtained with sequences of DNA amplification was performed using the genes listed in the public database using the SensoQuest PCR machine. The PCR solution BLAST (Basic Local Alignment Search Tool) contained 1x reaction buffer (GoTaq® Green program which is done online via the NCBI’s Master Mix, Promega), 0.8 μM forward primer, National Center of Biotechnology Information 0.8 μM reverse primer, < 250 ng DNA template website: http://www.ncbi.nlm.nih.gov/BLAST. and nuclease freewater (DD H2O), which were Sequences of cytochrome b genes were aligned added to the total volume of 25 μl inside the PCR with data from GenBank (Tab. 2) with the use tube. The process of cytochrome gene amplifi- of the ClustalX program version 2.1 (Larkin et cation resulted in 600 bp fragment using cytb-F al., 2007). The alignment results were edited and cytb-R primers: TTCACTATATTATAAAA- manually using the BioEdit program (Hall, 1999). GATGTAAGTTC and: GGCAAAAAGAAAATAT- Furthermore, the sequences were converted CATTCAGG, respectively (Thummajitsakul et into the FASTA format (.fas) for the analysis of al., 2014). The PCR thermal profile consisted of the nucleotide variation. initial denaturation at 95°C for three minutes, Phylogenetic analysis based on DNA sequences followed by five cycles at 94°C for 30 seconds, of the partial cytochrome b gene was performed 42°C for 30 seconds and 72°C for 50 seconds. using the MEGA6 program (Tamura et al., 2013). Thirty-five cycles were then performed at 94°C Phylogenetic trees were constructed separately for thirty seconds, 58°C for thirty seconds and for cytochrome b genes using the Neighbour 72°C for fifty seconds. Post-elongation was Joining (NJ) analysis. Phylogenetic tree construc- done at 72°C for seven minutes. tion was also done for Maximum Likelihood (ML), The amplified DNA was visualized through the Minimum Evolution (ME) and Maximum Parsimony electrophoresis process using 2% agarose gel. (MP) methods with bootstrap 1000 replications. The Thermo Scientific GeneRuler 100 bp DNA The value of the divergence sequence was Ladder was used for sizing the DNA. Electro- obtained using the Kimura 2-Parameter model. phoresis was performed at a voltage of 100 mV The ingroup used in this research can be seen for 35 min. The gel was then placed on top of in Tab. 2. The outgroup used in this research the UV illuminator (GelDoc) and the visualization were Apis cerana (EF467437) and Bombus hy- results were documented. The DNA amplifica- perboreus (AF066968). tion results were sent to the sequence service companies (Lab MacroGen USA DNA Sequencing) RESULTS in South Korea. Forward and reverse DNA sequences were The GenBank accession numbers for these assembled to obtain DNA sequences with the sequences are as follows: T. fuscobaltea- use of the STAR DNA program (Seqman). The ta – MH449661, MH449662; T. drescheri – analysis then proceeds with the comparison MH449658; T. minangkabau – MH449659, 119 Christy et AL. Phylogenetic analysis of Tetragonula minangkabau

Fig. 1. Tetragonula Phylogenetic Tree based on primer for cytochrome b gene with Neighbour Joining analysis (NJ). Annotation: The number listed on tree branch shows the bootstrap value (ML/NJ/ME/MP). Bold text showed the samples from this research.

MH449660, T. minangkabau forma darek – the Malaysia Genom Institute (MGI). MH449663. All sequences used in this study The second subcluster contained only one showed a higher A-T than G-C composition. The species, Trigona clypearis. The sequence composition of A-T for the partial DNA sequence divergence between this species and members of cytochrome b was 73.27% while G-C com- of the first subcluster ranged from 25.1% to position was 26.73%. The aligned sequences 30%. Between the second and third subcluster, were 488 base pairs with 238 conserved sites the sequence divergence ranged from 24.9% to (48.77%), 245 variable sites (50.20%), 174 26.1%. The high sequence divergence between parsimony sites (71.02%) and 71 singleton sites the first, second and third subclusters supports (28.97%). the separation of these three subclusters. For phylogenetic analysis, four methods were The third subcluster was a sister clade of the applied to construct the phylogenetic tree but first and second subclusters and consisted of the results were identical. Thus we show only T. fuscobalteata collected from Limau Manis and one phylogenetic tree obtained with Neighbour Sijunjung. The sequence divergence between Joining analysis. The results of the Tetragonula T. fuscobalteata collected in Sijunjung and Limau phylogenetic analysis as shown in Fig. 1 formed Manis and the first subcluster species ranged two main clusters. Cluster 1 consisted of from 31.7-35.9% and 30.8-33% respectively. three subclusters, namely the T. minangkabau These values supported the separation of - T. aeviceps - T. drescheri group - T. clypearis T. fuscobalteata from the previous two subclus- group and T. fuscobalteata group. The first ters and their placement in the third subcluster. species group of the first subcluster had Cluster 2 (or the fourth subcluster) consisted sequence divergence values ranging from 0.6% of three species from Thailand, which were to 25.5% with bootstrap values of 97/99/98/97 T.collina, T. pagdeni and T. laviceps, and their (ML/NJ/ME/MP). The first subcluster’s second sequence data was obtained from GenBank. group consisted of T. laeviceps, T. drescheri The bootstrap value in this cluster was quite low, and T. minangkabau with bootstrap values ​​as at 33/43/43/41 (ML/NJ/ME/MP). The sequence 100/100/100/100 (ML/NJ/ME/MP). The three divergence between T. collina and T. laeviceps species in this cluster were collected in the was 26.1% and between T. collina and T. pagdeni same location, the stingless bee repository at was 25.1%. The lower sequence divergence 120 J. APIC. SCI. Vol. 63 No. 1 2019 value 9.4% was obtained between Tetragonula according to genetic structure of population. pagdeni and T. laeviceps. A sequence divergence between species within a genus is stated by Kartavtsev & Lee (2006) DISCUSSION and Kartavtsev (2011) for 10.69 ± 1.34 and 10.31 ± 0.93, respectively. A higher sequence The conclusion from the phylogenetic analysis divergence according to Ming et al. (2015) is was that T. minangkabau and T. minangkabau when different species in the same genus are forma darek can be classified as the same species, indicated by a sequence divergence over 25.3%. but differ at the ‘forma’ level. This condition was Other research by Franck et al. (2004) found proved from low sequence divergence between that the sequence divergence of four Trigona the two samples, which shows the close phy- species (Trigona (Tetragonula) carbonaria, logenetic relationship. In the first subcluster’s T. hockingsi, T. davenporti and T. (Tetragonilla) first group, T. minangkabau forma darek was collina) ranged from 18 to 24%. The similarity separated from the T. minangkabau cluster of of all this research is the cytochrome b that Limau Manis and Ulu Gadut. This separation used to measuring the divergence value. But the ocurred due to the variation in the genes within divergence value obtained by Kartavtsev & Lee the species, which is known as forma. (2006) and Kartavtsev (2011) is generalized Morphologically, the differences between these from more than 20,000 vertebrate and inverte- two groups can be observed by the body length brate species, where there was no mentions of and coloration of T. minangkabau and T. minang- cytochrome b use in invertebrate taxon. As for kabau forma darek. The sequence divergence Ming et al. (2015), the value obtained was only between T. minangkabau collected from Limau for Tribolium and Franck et al. (2004) research Manis and Ulu Gadut was 0.8%, while between was focused on Trigona. T. minangkabau with T. minangkabau forma Members of the first subcluster’s second darek 5.5%. The low sequence divergence​​ group had a low sequence divergence. The indicated that T. minangkabau and T. minang- sequence divergence between T. minangkabau kabau forma darek are the same species. This and T. drescheri was 0.6%, and ​​the sequence result is in line with Kartavtsev & Lee (2006) divergences between T. drescheri - T. laeviceps and Kartavtsev (2011) who reported that the and T. minangkabau - T. laeviceps were 1.4% divergence between taxa of such rank as and 2.0%, respectively. All three species in subspecies, sibling species and semi-species this group were collected in the same location were 5.52 ± 1.34 and 5.10 ± 0.91, respectively. where all of the stingless bee colonies were put In these studies these literatures, T. minangka- in a box labeled by species name. After the iden- bau forma darek is referred as a subspecies, tification process based on the morphological but in the current one we prefer to call them character, it was discovered that T. minangka- ‘forma’ (forms) since the information about this bau and T. laeviceps individuals collected in this group is still limited. So far we only know the location were bigger than those in the other morphology and molecular aspects (obtained location (body length 3.9 - 5 mm). Sakagami & from this study) and have not obtained data Inoue (1985) stated that the T. drescheri body about population genetics, behavior or ecology. length ranged between 4.8 and 4.9 mm, but a Braby, Eastwood, & Murray (2012) defined large variety of sizes can be found in individuals subspecies to be restricted to an extant from different locations. Based on this informa- group that comprises evolving populations rep- tion, these samples were allegedly as putative resenting partially isolated lineages of species T. drescheri. that are allopatric, phenotypically distinct, have The sequence divergence between T. minang- at least one fixed diagnosable character state, kabau collected from Malaysia T. minangkabau and these character differences are assumed from Limau Manis, Ulu Gadut and T. minang- to be correlated with evolutionary process kabau forma darek were 24.4%, 23.8% and

121 Christy et AL. Phylogenetic analysis of Tetragonula minangkabau

23.8%, respectively. Samples of T. laeviceps Agricultural University for their help, assistance, collected from Malaysia and the T. minangkabau and advice during this research and data analysis. group had sequence divergence ranging from Finally, we thank the anonymous referees for 23.8% to 24.4%. A high sequence divergence their constructive comments on the manuscript. among members in the first and second groups also support the hypothesis that they are of REFERENCES different species. Furthermore, T. laeviceps was also found in two different clusters (Clusters 1 Barley, A.J., White, J., Diesmos, A.C., & Brown, R.M. and 2) with a sequence divergence of 35.3%. (2013). The Challenge of Species Delimitation at In the third subcluster, two populations of the Extremes: Diversification Without Morphologi- T. fuscobalteata from Sijunjung and Limau Manis cal Change in Philippine Sun Skinks. Evolution, 67-12, showed genetic variation of 13.2% of sequence 3556-3572. http://doi.org/10.1111/evo.12219 divergence and bootstrap value of 99/99/98/99 (ML/NJ/ME/MP). Although molecularly T. fusco- Braby, M.F., Eastwood, R., & Murray, N. (2012). The balteata from the two populations has a high Subspecies Concept in Butterflies: Has Its Applica- sequence divergence, morphologically there tion in Taxonomy and Conservation Biology Outlived were no significant differences. According to Its Usefulness? Biological Journal of Linnean Society, Schönrogge et al. (2002) and Barley et al. (2013) 106, 699-716. in the process of evolution, genetic differentia- tion can occur even without any changes in mor- Cortopassi-Laurino, M.V.L., Imperatriz-Fonseca, D.W., phological characteristics. Selective pressures Roubik, A., Dollin, T., Heard, I., Aguilar, G.C., … Nogueira- on important adaptive characteristics can lead Neto, P. (2006). Global Meliponiculture: Challenges to conservation of certain external morphologi- and Opportunities. Apidologie, 37(2), 275-292. cal characteristics. http://doi.org/10.1051/apido:2006027 The results of this study support the use of the name Tetragonula for Trigona in the tropics Crozier, R.H., & Crozier, Y.Z. (1993). The Mitochondrial region. Rasmussen & Cameron (2007) stated Genome of the Honeybee Apis mellifera: Complete that Trigona only be used for the Neotropic Sequence and Genome Organization. Genetics, 133, taxa. From this research, it can be concluded 97-117. that the phylogenetic relationship of Tetragonu- la (T. minangkabau, T. minangkabau forma darek, Franck, P., Cameron, E, Good, G., Rasplus, J.Y., Oldroyd, T. laeviceps, T. drescheri and T. fuscobalteata) B.P. (2004). Nest Architecture and Genetic Differen- is monophyletic. The sequence divergence of tiation in a Species Complex of Australian Stingless 5.5% supports the separation of Tetragonula Bees. Molecular Eology, 13(8), 2317-2331. https:// minangkabau and T. minangkabau forma darek doi.org/10.1111/j.1365-294X.2004.02236.x but proves that it is still the same species. To ensure the taxonomic rank of T. minangkabau Engel, M.S., Michener, C. D., & Boontop, Y. (2017). forma darek, further research on morphologi- Notes on Southeast Asian Stingless Bees of the cal analysis (especially regarding the shape of Genus Tetragonula (: Apidae), with the male genitalia), nest architecture, behavior and Description of a New Species from Thailand. Ameri- population genetic are needed. can Museum Novitates. No. 3886, 17 p.

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