New haplotypes for the non-coding region of mitochondrial DNA in cavity-nesting honey bees Apis koschevnikovi and Apis nuluensis Jun-Ichi Takahashi, Jun Nakamura, Masami Sasaki, Salim Tingek, Shin-Ichi Akimoto

To cite this version:

Jun-Ichi Takahashi, Jun Nakamura, Masami Sasaki, Salim Tingek, Shin-Ichi Akimoto. New haplo- types for the non-coding region of mitochondrial DNA in cavity-nesting honey bees Apis koschevnikovi and Apis nuluensis. Apidologie, Springer Verlag, 2002, 33 (1), pp.25-31. ￿10.1051/apido:2001002￿. ￿hal-00891904￿

HAL Id: hal-00891904 https://hal.archives-ouvertes.fr/hal-00891904 Submitted on 1 Jan 2002

HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Apidologie 33 (2002) 25–31 © INRA/DIB-AGIB/EDP Sciences, 2002 DOI: 10.1051/apido: 2001002 25

Original article

New haplotypes for the non-coding region of mitochondrial DNA in cavity-nesting honey bees Apis koschevnikovi and Apis nuluensis

Jun-ichi TAKAHASHIa, Jun NAKAMURAb, Masami SASAKIc*, Salim TINGEKd, Shin-ichi AKIMOTOa

a Department of Ecology and Systematics, Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589, Japan b Honeybee Science Research Center, Tamagawa University, Machida, Tokyo 194-8610, Japan c Laboratory of Entomology, Faculty of Agriculture, Tamagawa University, Machida, Tokyo, 194-8610, Japan d Agriculture Research Station Tenom, PO Box 197, 89908, Tenom, ,

(Received 23 April 2001; revised 29 August 2001; accepted 5 October 2001)

Abstract – The cavity-nesting honeybees, , A. koschevnikovi and A. nuluensis, are dis- tributed in Sabah, . We sequenced the non-coding intergenic region between the tRNA leu and COII mitochondrial genes of these three species. About 50 different haplotypes have been reported for the non-coding region of A. cerana, A. koschevnikovi and A. nigrocincta. This study detected new haplotypes in A. koschevnikovi and A. nuluensis, which we designated Sabah 1 and Sabah short, re- spectively. Sabah short is the first short sequence reported from Borneo.

Apis cerana / Apis koschevnikovi / Apis nuluensis / mitochondrial DNA

1. INTRODUCTION Smith (Hadisoesilo et al., 1996) and Apis nuluensis Tingek, Koeniger & Koeniger Recent taxonomic studies on cav- (Tingek et al., 1996), two newly described ity-nesting honey bees have reported three species. A. cerana distributed in East-Asia species in addition to Apis cerana Fabr. – ranging from Afghanistan east to far east- the recently rediscovered Apis koschevnikovi ern Russia and from north to Ja- v. Buttel-Reepen (Koeniger et al., 1988; pan. A. koschevnikovi is distributed in Maa, 1953; Tingek et al., 1988), and two Malaysia and Indonesia. A. nigrocincta is newly described species, distributed in Sulawesi and Mindanao.

* Correspondence and reprints E-mail: [email protected] 26 J.-I. Takahashi et al.

A. nuluensis is distributed in the highlands abundance of intraspecific polymorphism. of Sabah, Borneo. The distribution of these In A. cerana, polymorphism is often found honey bees are shown in Figure 1. These in a mitochondrial, non-coding intergenic three species are partially sympatric with A. region between tRNA leu and COII (Smith cerana. Three of the four Asian cavity-nest- and Hagen, 1996). At present about 50 dif- ing honey bees inhabit Borneo. Morpho- ferent haplotypes have been reported for logical comparisons of these three the non-coding region of A. cerana (De La sympatric species in Borneo indicate that A. Rua et al., 2000; Smith and Hagen, 1996; cerana shares more similarities with A. Smith et al., 2000). A total of seven nuluensis than with A. koschevnikovi haplotypes have been found for the (Fuchs et al., 1996). Similarly, an estimated intergenic region in A. cerana from Borneo phylogeny based on mitochondrial and nu- (Smith and Hagen, 1996). A. koschevnikovi cleotide DNA sequences indicates that A. from Borneo has a single known haplotype cerana and A. nuluensis are more closely (Smith and Hagen, 1996), and A. related (Arias et al., 1996; Tanaka et al., nigrocincta from Sulawesi and Sangihe 2001). have been shown to have two haplotypes Mitochondrial DNA sequences have (Smith et al., 2000). However, the sequence been used to determine intraspecific phylo- of A. nuluensis has not been studied. Thus, genetic relationships and the genetic struc- the present study aims to demonstrate the ture of species because of the haplotype variation of three species in

Figure 1. Map of the distribution of A. cerana (①), A. nuluensis (②), A. koschevnikovi (③) and A. nigrocincta (④) in Borneo and neighboring regions. New haplotypes in A. koschevnikovi and Apis nuluensis 27

Sabah based on sequence analysis of the beled cycle sequencing reaction was per- non-coding intergenic region and examines formed with a DNA Sequencing Kit FS whether the detected sequences match (Perkin Elmer) and primers E2 and H2, at a known sequences. temperature profile of 94 oC for 3 minutes followed by thirty 15-second cycles at 94 oC, 46 oC for 30 seconds and 70 oCfor 2. MATERIALS AND METHODS 2 minutes. The reaction products were ana- lyzed using an ABI373 automated se- We collected adult workers of A. cerana quencer (Perkin Elmer). and A. koschevnikovi from colonies at Tenom, Sabah, Malaysia (Borneo) and of A. nuluensis on flowers in the highlands of 3. RESULTS AND DISCUSSION Crocker Range near Tambunan, Sabah, in March 2000. The collected workers were The length of the PCR product of the transferred immediately to 95% ethanol for intergenic non-coding sequences ranged mitochondrial DNA analysis. Two adult from approximately 30 bp to 100 bp, depend- workers of each species were used for se- ing on the species. The sequences are shown quence analysis. Template DNA was ex- in Figure 2 and are available from DNA Data tracted from thoracic muscle tissue of each Bank of Japan (accession numbers individual by heating the homogenized tis- AB072435-AB072438 http://www.ddbj.nig. sue in 400 µl of lysis buffer (50 mM ac.jp). The sequences of the two samples of Tris-HCl [pH 8.0], 10 mM EDTA, 0.5% o A. cerana were identified and matched the SDS, 0.8 mg/ml Proteinase K) at 55 Cfor Borneo-2 haplotype described by Smith 24 h. The lysate was extracted twice with and Hagen (1996) (Fig. 2). The two individ- water-saturated phenol and once with chlo- uals of A. koschevnikovi had different se- roform. The DNA was recovered from the quences; one was a new haplotype, and the purified lysate by isopropanol precipita- other was identified as the haplotype de- tion, rinsed with 70% ethanol, dried, and scribed by Smith and Hagen (1996). The dissolved in 50 µl of TE solution (10 mM new haplotype, which we designated Tris-HCl [pH 8.0], 0.1 mM EDTA) and then o Sabah 1, has one G inserted between posi- stored at –20 C until the polymerase chain tions 27 and 28 of the haplotype from reaction (PCR) was performed. The total A. koschevnikovi of Smith and Hagen volume of the reaction mixture was 30 µl, (1996) (Fig. 2). The sequences from the two composed of 20 µl of distilled water, 3 µlof individuals of A. nuluensis were very short DNA extract, 200 pM of each primer (E2 and most of the intergenic non-coding se- forward: 5'-GGC AAG AAT AAG TGC quences were lost (Fig. 2). Short haplotypes ATT G-3', H2 reverse: 5'-CAA TAT CAT have been reported in from Tai- TGA TGA CC-3') (Cornuet and Garnery A. cerana wan and Philippines, and in A. nigrocincta 1991, 3 µl of dNTP mix (200 mM of each from Sulawesi and Sangihe, Indonesia but dNTP), 3 µl of 10 µl PCR buffer, 1.5 mM it has not been found in from Ma- MgCl , and 0.5 units of Taq polymerase A. cerana 2 laysia nor A. koschevnikovi (De La Rua et (Takara). The temperature profile was al., 2000; Smith and Hagen, 1996; Smith et 3 minutes at 94 oCfollowedbythirty o al., 2000). This Sabah-short sequence is the 30 cycles of seconds at 94 C,1minuteat first reported for and the first 46 oCand1minuteat70oC. The final A. nuluensis finding of a short haplotype from Apis in elongation step was increased to 10 min- Borneo. utes. The amplification products were purified using QIAquick PCR purification To summarize the short haplotypes of kits (QIAGEN). The dye-terminator la- cavity-nesting honey bees, the Sulawesi 28 J.-I. Takahashi et al. were first A. cerana, A. nigrocincta and and the Sabah-short sequence of A. koschevnikovi A. cerana, A. koschevnikovi . The sequences of A. nigrocincta and is first reported in this study. Gaps are shown by dashes. The underlined indicate the “stem” of the hairpin structure. Sequences of the non-coding intergenic region between tRNA leu and COII of mitochondrial DNA of Figure 2. A. koschevnikovi, A. nuluensis reported in a previous study (Smith and Hagen,A. nuluensis 1996). The Sabah-1 haplotype of New haplotypes in A. koschevnikovi and Apis nuluensis 29 A. cerana, . The four haplotypes sequences (Ce1, Ce2, Ce3 and Ce4) were reported by De la A. nigrocincta and Sequences of the non-coding intergenic region between tRNA leu and COII of mitochondrial DNA of A. koschevnikovi, A. nuluensis Rua et al. (2000). The common sequences are shown by dots. Figure 3. 30 J.-I. Takahashi et al. short is a very short sequence found in ACKNOWLEDGMENTS Sulawesi and Sangihe, Indonesia and is only known in A. nigrocincta (Smith et al., 2000). The Philippine short is the longest of We thank two anonymous referees for help- the short sequences and is limited to the ful criticism in earlier versions of the manu- Philippine islands of Negros and Panay script. We thank Professor Tadaharu Yoshida of (Smith et al., 2000). The Taiwan short is Tamagawa University and Professors Niko and Gudrun Koeniger of the Institut für only known from Taiwan (Smith and Bienenkunde for assistance and advice in col- Hagen, 1996). The sequence of the newly lecting samples in Sabah. We also thank Dr. discovered Sabah short is most similar to Eisuke Hasegawa of Hokkaido University for the Philippine short (Fig. 2). These advice on our experiments. haplotypes have limited utility in phylogen- etic analyses because the extremely short sequences are highly variable and conse- quently difficult to align. Therefore we sug- Résumé – Nouveaux haplotypes pour la gest that phylogenetic relationships among région non codante de l’ADN mitochon- extant populations of short haplotype indi- drial chez les abeilles qui nidifient dans viduals need to be constructed using data des cavités, Apis koschevnikovi et A. from other regions. Thus, we compared the nuluensis. Outre Apis cerana, trois autres number of substitutions in the nucleotide abeilles mellifères ont été décrites : Apis sequence of the 5'-end of the mtDNA CO II cerana, A. koschevnikovi et A. nuluensis. La gene in A. cerana and A. nuluensis from figure 1 donne leur répartition géographique. Sabah with previous data reported from A. La séquence de l’ADN mitochondrial d’A. cerana from the Philippines (De La Rua nuluensis n’a pas été étudiée jusqu’à et al., 2000). The nucleotide sequence of présent. Cette étude a donc pour but de the 5'-end of the mtDNA CO II gene was montrer la variation haplotypique des trois slightly more similar between A. cerana and espèces à Sabah, Bornéo, Malaisie, d’après A. nuluensis from Sabah (substitution num- la séquence entre l’ARNt et la région bers 14) than among from the A. cerana intergénique non codante COII.La Philippines or between A. cerana from the séquence de deux individus d’A. cerana Philippines and A. nuluensis (substitution concordait avec l’haplotype Bornéo-2 numbers 15–16) (Fig. 3). Short haplotypes décrit par Smith et Hagen (1996). L’analyse of A. cerana from Philippine and A. moléculaire précédente montrait que A. nuluensis did not appear to be closely related koschevnikovi de Bornéo n’avait qu’un seul and these data from the coding region are haplotype, mais cette étude a montré consistent with previous reports (Arias et al., l’existence d’un nouvel haplotype nommé 1996; Tanaka et al., 2001). Sabah-1. Parmi les trois espèces, la séquence de deux individus d’A. nuluensis It is likely that new haplotypes or addi- était la plus petite et ce nouvel haplotype a tional known haplotypes will be found in été nommé Sabah-short (= court). Il s’agit the A. cerana group from Borneo, because de la première description d’un haplotype only a few individuals have been surveyed court pour les abeilles de Bornéo nidifiant so far. If A. cerana individuals with the dans des cavités. La variation géographique short haplotype of the non-coding region de la région non codante sera utile pour were found in Borneo or A. nuluensis indi- reconstruire l’histoire évolutive d’A. viduals with normal haplotypes of the cerana et A. nuluensis. non-coding region were found, it may be useful in reconstructing the evolutionary Apis cerana / Apis koschevnikovi / Apis history of A. cerana and A. nuluensis. nuluensis / ADN mitochondrial / Bornéo New haplotypes in A. koschevnikovi and Apis nuluensis 31

Zusammenfassung – Ein neuer Haplo- sympatric species inferred from DNA sequences, Apidologie 27, 415–422. typ einer nichtcodierenden Region der Cornuet J.M., Garnery L. (1991) Putative origin of the mitochondrialen DNA bei den höhlen- intergenic region between COI and COII of Apis brütenden Honigbienen Apis koschevni- mellifera L. mitochondrial DNA, Genetics 128, kovi und Apis nuluensis. Die mitochondria- 393–403. le DNA von 3 Arten der Honigbienen, Apis de La Rua P., Simon U.E., Tilde A.C., Moritz R.F.A., , und Fuchs S. (2000) MtDNA variation in Apis cerana cerana Apis koschevnikovi Apis nulu- populations from the Philippines, Heredity 84, ensis von Borneo, Malaysia wurde zwi- 124–130. schen den Genloci tRNA leu und COII Fuchs S., Koeniger N., Tingek S. (1996) The sequenziert. Die Sequenzen von 2 Indivi- morphometric position of A. nuluensis (Tingek, duen von stimmte mit dem Bor- Koeniger and Koeniger) within cavity-nesting A. cerana honey bees, Apidologie 27, 397–406. neo-2 Haplotyp überein, der von Smith und Hadisoesilo S., Otis G.W., Meixner M. (1996) Two Hagen (1996) beschrieben wurde. Bisheri- distinct populations of cavity-nesting honey bees ge molekulare Analysen zeigten bei Apis (: ) in south Sulawesi, Indo- koschevnikovi von Borneo nur einen Haplo- nesia, J. Kans. Entomol. Soc. 68, 399–407. typ auf, aber bei dieser Untersuchung wur- Koeniger N., Koeniger G., Tingek S., Rinderer T.E. de ein neuer Haplotyp gefunden und als (1988) Reproductive isolation by different time of drone flight between Apis cerana Fabricius 1793 Sabah-1 bezeichnet. Die Sequenz der bei- and Apis vechti Maa 1953, Apidologie 19, den Bienen von Apis nuluensis war die kür- 103–106. zeste innerhalb der 3 Arten und dieser neue Maa T.C. (1953) An inquiry into the systematics of the tribus Apidini or honeybees (Hym), Treubia 21, Haplotyp wurde Sabah short (Sabah kurz) 525–640. genannt. Dies ist die erste Beschreibung ei- Smith D.R., Hagen R.H. (1996) The biogeography of nes kurzen Haplotyps bei den höhlenbrü- Apis cerana as revealed by mitochondrial DNA se- tenden Honigbienen von Borneo. Diese quence data, J. Kans. Entomol. Soc. 69, 294–310. geografische Variation in dieser nichtco- Smith D.R., Villafuerte L., Otis G., Palmer M.R. dierenden Region wird bei einer Rekon- (2000) Biogeography of Apis cerana F. and A. nigrocincta Smith: insights from mtDNA studies, struktion der Evolutionsgeschichte von Apidologie 31, 265–280. Apis cerana und Apis nuluensis nützlich Tanaka H., Roubik D.W., Kato M., Liew F., Gunsalam sein. G. (2001) Phylogenetic position of Apis nuluensis of northern Borneo and phylogeography of A. cerana as inferred from mitochondrial DNA se- Apis cerana / Apis koschevnikovi / Apis quences, Insectes Soc. 48, 44–51. nuluensis / mitochondriale DNA Tingek S., Mardan M., Rinderer G., Koeniger N., Koeniger G. (1988) Rediscovery of Apis vechti (Maa, 1953): the Sabah , Apidologie 19, 97–102. REFERENCES Tingek S., Koeniger N., Koeniger G. (1996) Descrip- tion of a new cavity-dwelling species of Apis (Apis nuluensis) from Sabah, Borneo with notes on its Arias M.S., Tingek S., Kelitu A., Sheppard W.S. occurrence and reproductive biology (Hyme- (1996) Apis nuluensis Tingek, Koeniger and noptera, Apoidea, Apini), Senckenbergiana Biol. Koeniger, 1996 and its genetic relationship with 76, 115–119.