Table 1. Presence or absence of diagnostic restriction sites in four regions of honeybee mtDNA, and Turkish Honeybees: Genetic structure of the noncoding intergenic region Variation and Evidence for a West Type 1, eastern Type 2, eastern Middle Fourth Lineage of Apis Gene Enzyme European Mediterranean Mediterranean African Eastern mellifera mtDNA Cytochrome b BglII 11 1 21 COI HincII 12 2 22 M. R. Palmer, D. R. Smith, and lsrRNA EcoRI 21 2 22 O. KaftanogÏlu COI XbaI 21 1 22 COI XbaI 22 1 22 Noncoding sequence PQ Q Q P0QP1Q The mtDNA of bees from 84 colonies of PQQ P0QQ PQQQ P QQQ Turkish honeybees (Apis mellifera) was 0 surveyed for variation at four diagnostic Western European, eastern Mediterranean, and African are three lineages of honeybee mtDNA characterized by restriction sites and the sequence of a restriction site and length differences. Type 1 and type 2 are two variants of the eastern Mediterranean lineage. noncoding intergenic region. These colo- ``Gene'' indicates the approximate location of restriction sites, ``Enzyme'' the restriction enzyme. The ``1'' sign indicates presence of a restriction site, ``2'' indicates its absence. A novel pattern of restriction sites was found nies came from 16 locations, ranging from in bees from Hatay, Turkey; here it is called ``Middle Eastern.'' Primers for cytochrome b reported in Crozier et al. European Turkey and the western Medi- (1991); others in Hall and Smith (1990). Terminology for structure of the noncoding region follows that of Cornuet terranean coast to the Caucasus Moun- et al. (1991). tains along the Georgian border, the east- ern Lake Van region, and the extreme ner et al. 1993; Moritz et al. 1994; Sheppard some Spanish honeybees, eastern Medi- south. Combined restriction site and se- et al. 1996; Smith 1991a,b; Smith and terranean mtDNA in A. m. ligustica, A. m. quence data revealed four haplotypes. Brown 1990; Smith et al. 1991) and se- carnica, and A. m. caucasica, and African Three haplotypes belonged to the eastern quence polymorphisms (Arias and Shep- mtDNA in A. m. scutellata and other bees Mediterranean mtDNA lineage. The fourth pard 1996; Garnery et al. 1992; Lee and from Africa. haplotype, which had a novel restriction Hall 1996) have revealed three main lin- The bees of Turkey are particularly rel- site pattern and noncoding sequence, eages of honeybee mtDNA: western Euro- evant for studies of honeybee biogeogra- was found in samples from the extreme pean, eastern Mediterranean, and African. phy. Turkey is located at the geographic south, near the Syrian border. We found The mitochondrial genome of honey- crossroads of Europe, Asia, and the Mid- two different noncoding sequences bees also contains a noncoding region lo- dle East and contains a wide range of cli- among the eastern Mediterranean haplo- cated between a leucine tRNA gene and mates and habitats within its borders. Not types. The ``Caucasian'' sequence match- the cytochrome oxidase II gene (COII; Cor- surprisingly, the honeybees of Turkey are es that described from A. m. caucasica, nuet et al. 1991). This noncoding sequence also quite diverse; based on morphomet- and the ``Anatolian'' sequence matches has a complex structure, which gives rise ric, behavioral, and ecological data, Rutt- that of A. m. carnica. The frequency of the to both sequence and length variation ner (1988) suggested that four subspecies ``Caucasian'' sequence was highest (98± (Hall and Smith 1991). Two basic ele- occur in Turkey: A. m. anatoliaca, A. m. 100%) in sites near the Georgian border ments, called ``P'' (or ``P0'') and ``Q'' are caucasica, A. m. meda, and A. m. syriaca. and decreased steeply to the south and found in the noncoding sequence; length According to Ruttner, A. m. caucasica oc- west. Elsewhere the Anatolian sequence variation stems from presence or absence curs in the extreme northeast of Anatolia was found. In European Turkey (Thrace) a of the P element and from tandem repeats (Asian Turkey), with bees resembling A. restriction site polymorphism previously of the Q element. The Q element itself con- m. caucasica occurring along the eastern reported from A. m. carnica in Austria and sists of three subunitsÐQ1, Q2, and Q3; Black Sea coast as far as Samsun. A. m. the Balkans was present at high frequen- Q3 is usually identical to the P element meda is found in the southeast, and A. m. cy. A novel mtDNA haplotype with a from the same genome (Cornuet et al. syriaca in the extreme south, near the bor- unique restriction site pattern and noncod- 1991). Sequence variation includes both der with Syria. A. m. anatoliaca occurs ing sequence was found among bees base substitutions and insertions/dele- throughout the rest of Turkey, including from Hatay, in the extreme south near the tions. The three mitochondrial lineages European Turkey. Syrian border. This haplotype differed from differ in length and sequence of the non- Genetic studies of the honeybees of Tur- the three previously known lineages of coding region: in the eastern Mediterra- key include allozyme studies (Asal et al. honeybee mtDNAÐAfrican, western Eu- nean lineage one ®nds a single Q element; 1995; Kandemir and Kence 1995) and a ropean, and eastern MediterraneanÐand in the western European and African lin- study of mtDNA restriction site polymor- may represent a fourth mitochondrial lin- eages one ®nds a P element followed by phisms (Smith et al. 1997). The restriction eage. one, two, or three repeats of the Q ele- site study showed that our Turkish sam- ment. The P element of the western Eu- ples possessed the eastern Mediterranean Honeybees (Apis mellifera) are geographi- ropean lineage has a 15-base deletion rel- lineage of honeybee mtDNA. Even though cally diverse, with as many as 25 subspe- ative to the African ``P0'' element (Cornuet two subspeciesÐA. m. anatoliaca and A. cies currently recognized (Ruttner 1988; et al. 1991). These lineage-speci®c differ- m. caucasicaÐwere believed to be repre- Sheppard et al. 1997). The honeybee mi- ences are summarized in Table 1 and Fig- sented in our samples, the only variation tochondrial genome has provided abun- ure 1. we found was among the bees of Thrace dant data for studies of honeybee phylog- The geographic distribution of these mi- (European Turkey), where we found an eny and biogeography. Studies of mitochon- tochondrial lineages corresponds roughly XbaI site previously known only from A. m. drial DNA (mtDNA) restriction site poly- with the distributions of honeybee sub- carnica (Meixner et al. 1993; Smith and morphisms (e.g., Crozier et al. 1991; Gar- species: western European mtDNA is Brown 1990). nery et al. 1993; Hall and Smith 1991; Meix- found primarily in A. m. mellifera and Here we examine mtDNA restriction site 42 and sequence variation in bees collected from 16 localities in Turkey: the 12 sites reported earlier (Smith et al. 1997) and 4 additional sites in southern and eastern Turkey. We survey the four restriction sites, which distinguish three major mt- DNA lineages within A. mellifera, and se- quence the noncoding intergenic region of the mitochondrial genome (Cornuet et al. 1991). Our restriction site survey and se- quence data reveal a novel mtDNA haplo- type, which may constitute a fourth mito- chondrial lineage in A. mellifera. Methods Collections Adult worker honeybees were collected from comb (or in one case from a swarm) and frozen in liquid nitrogen or preserved in 70% ethanol. Samples were collected from colonies in the following locations (see Figure 2). In 1994, samples were col- Figure 1. Structure and sequence of the noncoding region located between leucine tRNA and cytochrome oxidase lected from Bursa (one colony) and vil- II genes in the A. mellifera mitochondrial genome. The ``P'' and ``Q'' terminology follows the usage of Cornuet et al. (1991). (A) Structure of the noncoding region in three major lineages of honeybee mtDNA (W 5 western lages near Giresun (four colonies). In June European, E 5 eastern Mediterranean, A 5 African) and the novel mitochondrial haplotype found in Hatay (M 5 1995, samples were collected from Thrace Middle Eastern). In African and western European mtDNAs, the Q element may be repeated one to three times. (7 colonies from villages near TekirdagÏ); (B) Sequence of the noncoding region. Sequence of each element and subunit (P, Q1, Q2, Q3) shown separately. A 5 A. m. scutellata from African lineage; W 5 A. m. mellifera from western European lineage; M 5 Syrian sequence GoÈkcËeada (10 colonies); the Black Sea from the proposed Middle Eastern lineage; E-c 5 A. m. caucasica from the eastern Mediterranean lineage; E-l 5 A. coast near Bolu and YedigoÈller (4 colo- m. ligustica from the east Mediterranean lineage. A. m. ligustica and A. m. scutellata sequences from Garnery et al. nies); Menemen, Aegean Agricultural Re- (1992). search Institute (9 colonies); Beypazari (7 colonies); Erzurum (7 colonies); Ardahan (5 colonies); villages near ArdanucË (6 col- onies); villages near Artvin (5 colonies); and the villages of Posof, SËavsËat, and SuÈn- guÈlluÈ near the Georgian border (16 colo- nies). The bees from Bolu, SËavsËat, and Beypazari came from breeding colonies maintained by the Beekeeping Project of the Turkish Development Foundation (TKV); the rest were collected in situ. In 1998, samples were collected from Bitlis (9 colonies), MusË (2 colonies), Van (4 colo- nies), and Hatay (14 colonies). These collection sites fall within the ranges of A. m. anatoliaca, A. m. caucasica, A. m. meda, and A. m. syriaca as described by Ruttner (1988; see Figure 2). However, migratory bee-keeping is widely practiced in Turkey, and Caucasian bees (A. m. cau- casica) are highly prized by beekeepers. Both factors (especially the latter) can lead to transplantation and mixing of pop- ulations.