Heredity 61(1988) 73—84 The Genetical Society of Great Britain Received 30 September 1987

New African species in the obscura species group: genetic variation, differentiation and

Marie Louise Cariou,* Daniel Lachaise,* * Laboratoirede Biologie et Génétique Evolutives Leonidas Tsacas,* 91198, Gif-sur-Yvette, Cedex France. John Sourdis, tDepartmentof , Agricultural College of Athens, Greece. Costas Krimbast and University of Cambridge, Department of Genetics, Michael Ashburneri Downing Street, Cambridge CB2 3EH, U..

The Drosophila obscura species group was known in the Afrotropical region from a single species, . microlabis. This species was recently rediscovered in Kenya in addition to the discovery of three new obscura group species: D. kirumensis, D. cariouae and D. krimbasi. The patterns of allozyme variation in two African species, D. microlabis and D. kitumensis is compared to that of nine species of the obscura group. Genetic distances have been estimated from allelic frequencies at 35 loci and phylogenies inferred by methods assuming both constant as well as variable evolutionary rates. D. microlabis and D. kitumensis are evolutionary related and close to the Palearctic D. tristis and D. ambigua even if relatively distant. The data support Lakovaara and Saura' hypothesis recognising three species subgroups within the obscura group. In addition, we propose a fourth one for the new African species, the microlabis species subgroup.

INTRODUCTION recently described (Tsacas et al., 1985). Thus, there are at least four species of the obscura species TheDrosophila obscura species group is widely group in Kenya, and we suspect that many more distributed in the Palearctic and Nearctic regions. may occur in the East African highlands. All these In the Afrotropical region it was known from a species were collected at high altitudes, above 2000 single species, D. microlabis Seguy, described in meters. Neotropical species of the obscura species 1938 (Seguy, 1938) from a single male collected group (affinis subgroup), D. azteca and D. tolteca from Kenya (Marakwet, on the Elgeyo Escarp- as well as the Neotropical populations of D. ment) by the Omo expedition (Arambourg et al., pseudoobscura (Throckmorton, 1975) are also 1935). More recently, Tsacas (1974) confirmed that found only at high altitudes. Thus, there is strong this species was a member of the obscura group presumption that the obscura species group, which and assigned it to the obscura species subgroup. is basically Holarctic in its distribution (Wheeler, The Museum of Paris specimen remained, for half 1981), has extended its geographic range over both a century, the sole indication of the presence of the Afrotropical and Neotropical regions but only the obscura species group in the Afrotropical in the temperate climatic conditions of mountains. region. With the new African species the obscura During a recent field investigation in Kenya species group now includes 35 species. Although (D. Lachaise, . L. Cariou and M. Ashburner, this is one of the most extensively species groups Sept. 1984) D. microlabis was rediscovered from of Drosophila that have been studied (see, Buzzati- Mt Elgon, on the west side of the Kenya (or Traverso and Scossiroli, 1955; Lakovaara and Gregory) Rift Valley. In addition, three new Saura, 1982 for a review) many questions remain obscura group species were discovered: one also concerning the relationships between the species. from Mt Elgon, D. kitumensis Tsacas and two The division of the group into two species sub- sympatric species from Mt Kenya on the East side groups is itself still questionable. On the basis of of the Rift Valley, D. krimbasi Tsacas and D. morphological and allozymic characters, the cariouae Tsacas. All of these species have been obscura group has long been subdivided into two 74 M. L. CARIOU ET AL.

species subgroups: the affinis subgroup, with nine (4200 m) below Koitoboss peak. This track runs exclusively Nearctic species, and the widespread between the Komothon River (to the north) and Palearctic species D. helvetica—the obscura sub- the Kassowai River (to the south). It is generally group, which includes 14 Palearctic species and the same region as was covered by the Omo Expe- seven Nearctic species. Several of the Palearctic dition. D. microlabis was abundant at Mt Elgon species are widespread, D. subobscura, D. obscura, lodge and is apparently a widely distributed D. ambigua and D. bfasciata, which probably is species, it probably ranges from Mt Elgon to the the most widely distributed species within the Elgeyo Escarpment where it was collected in 1935. group. Lakovaara and Saura (1982) have argued The species can easily be grown on standard corn- that the division of the obscura group into two flour culture medium and a mass culture was estab- subgroups is not a "natural one" and suggested lished from 30 wild caught inseminated females. dividing the obscura species subgroup into two D. kitumensis was collected from the area in lineages: the pseudoobscura species subgroup and front of the Kitum Cave (2500 m) in submontane the obscura species subgroup to include the Nearc- with Diospyros abyssinica below the tic and Palearctic species, respectively. Podocarpus zone, 5 km from Main Gate of the Mt In the present work, we have the compared Elgon National Park (fig. 1). Kitum Cave is a large patterns of allozyme variation in two African and deep excavation, much visited by elephants species, D. microlabis and D. kitumensis, with that and buffaloes, located in valley of Rongai River. of nine species of the obscura group (sensu law). The area immediately in front of the cave is very Genetic distances have been estimated on the basis disturbed by and is a jumble of rocks of allelic frequencies at 35 loci, and phylogenies dominated by Discopodium penninervium (Hochst) have been inferred by methods assuming both con- (Solanaceae), the climbing plant Stephania stant as well as variable evolutionary rates. The abyssinica (Dillon and Rich) Woip. var. tomen- data support Lakovaara and Saura's hypothesis. tella (oliv.) Diels, Urtica massaica Mildbr. and In addition we propose a fourth species subgroup with much dead/rotting wood. The entire valley for the new African species, the microlabis species in front of the cave was wet and cooled by air from subgroup. the cave and was obviously different in its flora from the surrounding forest and from other cave valleys of the East slope of Mt Elgon (Makingeny MATERIAL AND METHODS Cave, Chepnyalil Cave) where D. kitumensis was The African species and their not observed. D. Kitumensis was only found at the east edge Drosophila cariouae and D. krimbasi were both of Cave entrance, in a sap rot on a large tree, collected from Mt Kenya (2470 m) at Main Gate Ekebergia rueppeliana (Fresen) A. . (Meliaceae). of Mt Kenya National Park, by banana traps Adult were associated with this sap rot and placed above a domestic rubbish dump just behind only D. kitumensis was collected there by sweep- gate hut. The former species was also collected by ing. This quite soft and wet substrate contained banana trap in Podocarpus/bamboo forest about larvae, presumably of the same species; it is likely 05km from the Main Gate. One strain of D. that the sap rot of . rueppeliana is the breeding cariouae failed to establish successfully after the site of D. kitumensis. Similar natural breeding sites, first generation, while D. krimbasi was described sap and slime fluxes of trees (Oak, sycamore, wil- from a single male. Therefore only two of the four low, fir and elm) have been reported for D. African species could be studied, D. microlabis and pseudoobscura, D. persimilis (Carson, 1951), D. D. kitumensis. ambigua (Prevosti, 1959), D. obscura (Gordon, D. microlabis was collected in banana traps 1942) and D. subobscura (Carson et al. 1985). and by sweeping at Mt Elgon Lodge (2200 m), D. kitumensis is difficult to breed, nevertheless, 15 km from Main Gate of Mt Elgon National Park a strain was established from 13 wild-caught (fig. 1). Most traps and sweep sites were in a females and kept on standard culture medium. domestic , i.e., garden with grass and shrubs but with small fruit dump (tomatoes). In spite of Other an intensive search, the species was not collected species in wild habitats in the Mt Elgon slope forest at ForD. subobscura we studied a natural population higher elevations. On Mt Elgon the collection sites from the French Pyrénées (Riberot locality), a followed the main track from the Main Gate of strain from Crete (Greece), another from Switzer- the National Park (2260 m) to the moorland land and two standard strains, Küstnacht NEW SPECIES OF DROSOPHILA 75

Mt KENyA 5199 tt

MIELGON ELGEYI)ESCARPMENT 4321,0 3370ttt

mic r

202 LAKE BARINGO

Se,:, E,,CO, Pod000teos Bptbpo Fotest 5 Aipt mV,tttcal bog-E t,.@us Sette.t,ptte Mootlaodo Sob_moottloto Fotest Otospytos ebyss!ttt0o ,genta Fotest — LobeIt gtbbetoa C Ole, kDjtttaOdsCflOt!CO Fotest

Figure 1 Localities of the four African species of the Drosophilaobscura speciesgroup in the highland on the eastern and western flanks of the Kenya (Gregory) Rift Valley (Altitudinal zonation of Mt Elgon and Mt Kenya after data in Schnell, 1977; Hamilton and Perrot, 1981; Geological map summarising the volcanic history of the Kenya Rift after King and Chapman, 1972).

(Lankinen and Pinsker,1977) and Chcu tory stocks for between species comparisons: three homogeneous for most enzyme loci. for D. bifasciata from Norway (strain 24), Italy Two natural populations collected in valleys in (Pavia) (strain 25) and U.S.S.R. (Alma Ata) and the French Pyrénées (Riberot and Estours) and only one for the following species: D. tristis (Swit- one strain from Switzerland represented D. zerand), D. ambigua (Mt Parnes, Greece), D. obscura. One population of D. helvetica from Gif rnadeirensis ( Island), D. guanche (Canary (France) was used. We also analysed some labora- Islands) and D. pseudoobscura (USA). 76 M. L. CARIOU ET AL.

Genetic assays some of these are rare and only detected in recently collected natural populations. The Amy locus was predominantly carried out on Electrophoresis appears to be highly variable, with 14 allozymes starch gel following the general procedures of identified. Shaw and Prasad (1970) and Ayala et a!. (1972) From the electrophoretic data, it follows that with some modifications. The c-amylase was the two African species, D. microlabis and D. analysed using a 5 per cent acrylamide gel as kitumensis are both clearly distinguished from the described in DaInou et a!. (1987). other species of the obscura group by different Electrophoresis was performed on adult flies alleles fixed at three loci, Pgm, Gdh and Amy. In except for Estl, Lapl and Acph2 (third instar addition they only share rare alleles with one or larvae) and Lap2 (pupae). Allozymes were desig- nated by their electrophoretic mobility relative to two species at three more loci, Mdh, Pgi and Est6. that of the most common allozyme of D. sub- Of the 35 loci analysed, eight are diagnostic obscura, numbered 100. For each locus, 30 to 85 between D. microlabis and D. kitumensis: 6Pgd, individuals were tested for D. microlabis, D. Xdh, Ao, Odh, Acphl, Acph2, Aiph and Lap 1. kitumensis and the natural populations of D. sub- Estimates of heterozygosity and percentage of obscura and D. obscura. The laboratory strains variable loci are given in table 2. The percentage were studied only as a reference for the between of polymorphic loci ranges from 3 per cent (labora- species comparisons and fewer individuals were tory strains) to 43 per cent (D. microlabis) with a scored, from 15 to 25. mean at 201. Drosophila kitumensis is polymor- Four kinds of Nei's genetic distances (D1) have phic at 26 per cent of its loci. The mean number been used (the usual standard, another when of alleles per polymorphic locus is 2 and the mean only the most frequent allele was considered, Dm, numbei of alleles per locus per species is respec- andtwo others) as well as two kinds of another tively 1 46 for D. microlabis and 1 26 for D. distance, D2 (depending on whether data of the kitumensis. The mean expected heterozygosity for most common allele or all alleles were used in each D. microlabis (0.094) and D. kitumensis (0.067) locus), all described in Krimbas and Sourdis comes within the range of variation found for other (1987). species of the obscura group (table 2). Four different methods for constructing trees D2 (D2 polymorphic) and Nei's standard (D1) were used: UPGMA (Sneath and Sokal, 1973), genetic distances based on allele frequencies are Farris (1972), Fitch and Margoliash (1967) and a presented in table 3. The genetic distances within modified version of Farris' method (Tateno et a!., the same species (D. subobscura, D. obscura and 1982). The three last methods were further D. bifasciata) are very small (from 0004 to 0.11) modified by us in such a way that trees with nega- indicating that strong differentiation does not exist tive distances were rejected (Sourdis and Krimbas, between either local or geographically distant populations of these species. This is in good agree- 1987). Concordance of reconstructed trees with the ment with previous studies (Lakovaara and Saura, original data could eventually indicate fidelity to 1971; Lakovaara et al., 1976; Loukas et al., 1979; the "real" phylogeny. The measure used was the Pinsker and Buruga, 1982). sum of the squares of the differences in distances Extensive isozyme divergence among species between the reconstructed and the original (experi- is further indicated by the great magnitude of mental) distances. These distances have been nor- genetic distances. Nei's distance ranges from 0269 malised in every matrix to one in order to get to 2416 and D2 from 0i05 to 0571. The two comparable data. least divergent species are the African D. microlabis and D. kitumensis, they are genetically closer to one another than any other couple of species known to have some degree of relatedness on the RESULTS basis of other critera, e.. D. tristis and D. ambigua (D1=0.404), or the triplet D. subobscura, D. Themost common allozymes of the 35 putative guanche, D. madeirensis (0.522< D1< 0.838). loci screened in the 19 populations and species Very high distances are obtained when D. he!- studied are presented in table 1. Variation is great vetica is paired with any other species; the highest between species and none of the 35 loci is strictly value is between this species and D. kitumensis monomorphic. Polymorphic loci are those already (D1, =2.416). known to be polymorphic in the Drosophila obscura Different trees can be constructed using either, group. The total number of alleles detected is 180, or both, different measurements of genetic distance mz Table I Electrophoreticmobilities (relative to D. subobscura) of the most common allozymes at 35 loci in African and other species of the D. obscura group. micr: D. D. D. 1: 2: 3: 4: 5: D. made: D. madeirensis; obs: D. Cl) microlabis; kitu: kitumensis; subo: subobscura, chcu, Küstnacht, Greece, France, Switzerland; guan: guanche; -u obscura, 1: Switzerland, 2: Riberot, France, 3: Estours, France; tris: D. tristis; ambi: D. ambigua; bifa: D. bifasciata; helv: D. helvetica; pseu: D. pseudoobscura m C-) bifa bifa Al. helv m Locus micr kitu subo 1 subo 2 subo 3 subo 4 subo 5 guan made obsc I obsc 2 obsc 3 tris ambi 24 bifa 25 pseu Cl) 0 Idh 97 97 100 100 100 100 100 99 102 100 100 100 97 97 94 94 94 104 104 -Il 6Pgd 106 102 100 100 102 100 100 101 104 102 102 102 102 102 102 102 102 100 105 0 Pgm 90 90 100 100 100 100 100 100 100 101 101 101 101 101 104 104 102 100 100 100 100 100 100 100 100 100 100 101 100 100 100 100 100 104 104 104 100 100 0 aGpdh C,) Fum I 97 97 100 100 100 100 100 100 100 97 97 97 97 97 97 97 97 102 97 0 Fum2 102 102 100 100 100 100 100 100 100 100 100 100 105 108 108 108 108 108 100 0 Xdh 100 101 100 100 100 100 100 101 100 100 100 100 101 100 100 99 99 98 101 Aldo 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 Hkl 103 103 100 100 100 100 100 98 98 101 101 101 100 101 103 103 103 104 104 Hk2 101 101 100 100 100 100 100 100 100 101 101 101 101 100 100 100 100 100 101 Hk3 103 103 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 99 Pgi 98 98 100 100 100 100 101 100 103 100 100 100 100 100 101 101 101 103 100 Mdh 103 103 100 100 100 100 100 100 100 102 102 102 104 106 106 106 106 114 102 Me 97 97 100 100 100 100 100 97 101 93 93 93 99 98 99 99 99 100 93 Ao 100 101 100 100 100 100 100 100 101 100 100 100 100 100 100 100 100 100 100 Odh 103 98 100 100 100 100 100 100 100 102 102 102 103 103 102 102 102 101 101 Adh 100 100 100 100 100 100 95 100 100 120 120 120 120 120 110 110 110 95 100 G6pdh 97 97 100 100 100 100 97 100 102 97 97 97 97 97 93 93 93 104 105 Ca 96 95 100 100 100 100 100 96 94 96 96 98 96 96 94 94 94 102 100 Es16 96 96 100 100 100 100 100 97 100 97 97 97 97 97 94 94 94 100 100 Esp 96 96 100 100 100 100 100 98 99 98 98 98 98 98 98 98 98 102 99 EstC 102 102 100 100 100 100 100 100 100 94 94 94 102 102 98 98 98 104 94 Sod 100 100 100 100 100 100 100 100 100 100 100 100 105 100 100 100 100 108 100 100 100 100 100 100 100 100 100 Gapdh 100 100 100 100 100 100 100 100 100 100 100 Gdh 92 92 100 100 100 100 100 102 103 97 97 97 97 97 102 102 102 105 105 Acphl 94 95 100 100 100 100 100 94 97 94 94 94 94 100 115 112 112 115 97 Sdh 95 95 100 100 100 100 100 101 101 100 100 100 100 101 101 101 101 101 100 Su 97 97 100 100 100 100 97 103 100 97 97 97 94 97 97 97 97 112 97 Got 102 102 100 100 100 100 100 102 102 102 102 102 102 102 103 103 103 103 100 102 102 97 90 93 105 101 Amy 118 118 100 100 100 100 100 90 95 102 97 90 105 100 102 94 98 102 Aiph 100 102 100 100 100 100 100 98 98 100 102 94 94 103 103 103 99 95 95 95 92 102 Acph2 94 97 100 100 100 100 100 100 100 99 Lapl 102 105 100 100 l00 100 100 100 101 100 100 100 104 100 99 99 99 102 102 Lap2 100 100 100 100 100 100 100 98 98 97 97 97 100 101 101 101 101 98 98 EsI 1 98 98 100 100 100 100 100 96 98 96 96 96 98 100 102 100 100 102 98

The following enzymes were run: malate dehydrogenase (Mdh), malic enzyme (Me), fumerase (Fum), oglycero-phosphatedehydrogenase (csGpdh), phosphoglucoisomerase (Pgi), glucose-6-phosphate dehydrogenase (G6pd), aldolase (Adh), glutamate oxalate transaminase (Got), isocitrate dehydrogenase(Idh), glutamate dehydrogenase (Gdh), octanol dehydrogenase (Odh), sorbitol dehydrogenase (Sdh), glyceraldehyde-3-phosphate dehydrogenase (Gapdh), succinate dehydrogenase (Su), superoxide dismutase as hexokinases (Sod), acid phosphatases (Acphl, Acph2), carbonic anhydrase (Ca), one esterase with naphtyl propionate substrate (Esp), (Hkl, Hk2, Hk3), xanthine dehydrogenase (Xdh), 6-phosphogluconatedehydrogenase (6Pgd), phosphoglucomutase(Pgm), alcohol dehydrogenase (Adh), aldehyde oxidase (Ao) and esterases (Estl, Est6, Estc), leucine aminopeptidases (Lapl, Lap2) alkaline phosphatase (Mph). 78 M. L. CARIOU ET AL.

Table 2 Amount of genetic varation in D. microlabis and D. kitumensis compared with different species analysed in our work and published data. The total number of loci studied is given within brackets

Present study Published data H exp. % var. joci (35) H exp. 0/var.loci References

D. microlabis 0094 43 — — D. kitumensis 0067 26 — — D. subobscura 1. Chcu 0023 3 0084 34(67) Cabrera et a!., 1983 2. Küstnacht 0065 20 — — 3. Greece 0022 11 — — 4. France (Riberot) 0089 26 0234 85 (20) Saura et a!., 1973 Canary Island — — 0082 57 (44) Cabrera et a!., 1980 Yugoslavia — 0114 75(28) Marinkovic eta!., 1978 Sweden—Italy — — 0142—0 118 94(17) Pinsker and Sperlich, 1979 Madeira—Azores Islands — — 0070—0081 32—35 (63) Larruga et a!., 1983 Finland — — 0076 47(32) Lakovaara and Saura, 1971b D. guanche 0110 29 0066 38(68) Cabrera eta!., 1983 D. madeirensis 0045 11 0103 57(67) Cabrera et al., 1983 D. obscura 1. France (Riberot) 0098 23 — — 2. France (Estours) 0077 17 — — 3. Switzerland 0072 17 — — Finland — — 0110 (33) Lakovaara and Saura, 1971a D. tristis (Switzer.) 0055 20 — — D. ambigua Greece 0071 26 — — Spain — — 0032 12(56) Cabrera et a!., 1983 D. bifasciata (Norway) 0080 20 0242 (21) Saura (1974) D. pseudoobscura (USA) 0043 9 0009 5 (63) Cabrera et a!., 1983 0150 (43) Prakash, 1977 D. persimilis (USA) — — 0108 (43) Prakash, 1977 D. miranda (USA) — — 0085 (43) Prakash, 1977 D. helvetica (France) 0012 3 — — or different algorithms. One way of evaluating It is worth emphasising that the five best trees different trees is by determining the tree that best all position the African species close to one another represents the data, although the "best fit" may and quite close to the European species-pair, D. not necessarily be the best hypothesis of evolution- tristis-D. ambigua, and also to some extent to the ary relationships. Therefore we have used several American D. pseudoobscura. other criteria based on extraneous information The unrooted tree obtained from D2 polymor- concerning group to judge tree reliabil- phic distance and the modified Farris clustering ity; these were, whether or not different popula- has the best fit (fig. 3). A phylogenetic interpreta- tions of the same species clustered, the relative tion of this unrooted tree is reasonably consistent positions of D. madeirensis and D. guanche close with the biochemical and chromosomal affinities to D. subobscura (Krimbas and Loukas, 1984) and previously inferred regarding the position of the the relative closeness of D. tristis and D. ambigua Palearctic species of the obscura group (see (Lakovaara et aL, 1972; Lakovaara and Keränen, Lakovaara and Saura, 1982, for a review). D. sub- 1980). obscura is close to its two endemic relatives D. Independently of branch lengths, the alternative guanche and D. madeirensis although the relative topologies depicted in fig. 2 differ in the hatched position of the these species differs from that found zone, that is in the branching of D. microlabis and by Cabrera et al. (1983) and Loukas et a!. (1984). D. kitumensis and, D. tristis, D. ambigua and D. This could result from the use of different sets of pseudoobscura. The trees are ranked according to foci, or the use here of a better method for tree their goodness-of-fit. They all satisfy the reliability construction, but may also be indicative of criteria, except number 3 where D. tristis and D. ambiguity regarding their relative genetic diver- ambigua are separated, and number 6, which is gence. rejected because D. madeirensis and D. guanche D. subobscura is significantly distant from D. do not cluster with D. subobscura. obscura, D. tristis and D. ambigua are obviously mz

(I) -a m C) m (1) 0 '1

0 0Cl) Table 3 D2 estimator of distance (above diagonal) and Nei's standard genetic distance, D1 (below diagonal) based upon 35 enzymatic loci between the African species and 17 populations of other species in the D. obscura group

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 microlabis 1 xxx 011 034 034 034 034 033 032 035 028 028 028 023 027 037 038 037 052 034 kitumensis 2 027 xxx 036 036 036 036 035 033 036 032 032 032 024 031 042 043 043 057 035 subobscura 1 3 134 1'63 xxx 002 001 002 003 015 016 027 024 025 027 022 034 034 034 036 024 2 4 131 164 001 xxx 002 003 0•04 016 0•17 027 025 0.25 027 022 035 035 034 037 0•25 3 5 135 156 0•01 002 xxx 002 003 015 015 027 024 024 026 021 034 0.34 033 037 024 4 6 135 156 001 001 002 xxx 004 015 016 028 025 025 0.27 0'22 035 035 034 037 024 5 7 125 151 009 010 010 Oil xxx 017 018 027 025 025 027 022 034 034 034 037 025 guanche 8 116 140 057 055 057 052 073 XXX 012 022 022 0.22 025 023 033 033 033 041 029 madeirensis 9 141 152 070 071 071 070 083 053 xxx 029 028 029 030 027 035 035 0.35 038 028 obscura 1 10 095 021 098 0•97 094 099 091 087 155 xxx 0•05 005 019 022 036 037 036 052 022 2 11 089 114 085 0•84 0•82 085 080 085 145 005 xxx 003 021 023 036 037 036 052 019 3 12 089 115 086 085 0•82 085 080 087 146 005 001 xxx 021 023 035 036 036 051 019 tristis 13 079 084 120 118 111 115 120 109 172 056 061 0•60 xxx 013 030 031 030 047 Oil ambigua 14 089 102 097 096 089 097 092 085 128 053 059 058 040 xxx 021 021 020 041 035 bifasciata 24 15 135 l47 146 146 139 148 1'30 120 144 113 100 102 129 078 xxx 003 003 042 045 25 16 149 151 149 149 142 146 133 125 153 125 111 1i2 131 080 004 xxx 001 042 046 Al 17 153 153 153 153 146 149 136 135 156 129 114 1i6 133 082 0'08 004 xxx 041 046 helvetica 18 180 242 130 132 135 126 116 123 122 200 187 188 190 141 133 140 146 xxx 0•42 pseudoobscura 19 122 111 097 098 097 095 104 111 104 088 081 083 107 130 168 173 l74 106 xxx 80 M. L. CARIOU ET AL.

subobscura bifasciata

guanchAmadeirens is helvetica obscura

D2pmod Farris tris mb 1 mkit 2 4 L.

D2m_ Farris D1st UPGMA 'Ii'' D1mUPGMA 6j;ei 89 Figure 2 Alternative topologies derived using different algorithms using D2 and Nei's genetic distances depict different relationships between species within the hatched zone. The trees are numbered according to their goodness-of-fit measures. mic: D. microlabis, kit: D. kitumensis, tris: D. tristis, amb: D. ambigua, obs: D. obscura, pseu: D. pseudoobscura, bet: D. helvetica.

ifa25 bifaai bifa 24

guan

made

obsc1

mi Cr

kit u

helv Figure 3 Evolutionary relationships of the African species to others of the Drosophila obscura group as deduced from allozyme divergences. The unrooted tree is constructed by the modified Farris method clustering of D2 polymorphic distances. NEW SPECIES OF DROSOPHILA 81 here related species. D. bfasciata appears geneti- Drosophila microlabis anq D. kitumensis from cally quite distinct from D. subobscura and the Mt Elgon are evolutionarily related. They always obscura relatives (D. obscura, D. tristis, D. branch together regardless of the genetic distance ambigua). D. helvetica is the most distant species or tree-building method used. This agrees with the and is not appreciably related to any of those morphological data (fig. 4): the two species have analysed here. very similar genitalia and long sex combs while The position of D. pseudoobscura is more prob- both D. cariouae and D. krimbasi have short sex lematic. Lakovaara and Saura (1982) found that combs and different male genitalia (Tsacas et at., the Nearctic obscura subgroup species were not 1985). These data support the vicariance appreciably related to the Palearctic species or to hypothesis. There are, however, two reservations, the Nearctic affinis subgroup. Our data show that first, there is no evidence for the relatedness of genetically, D. pseudoobscura is closest to D. D. cariouae and D. krimbasi; secondly, the single obscura (D1 =0.8)and is related to D. tristis and male assigned to a fifth African species, collected D. ambigua. The old laboratory strain of D. with D. cariouae in the Podocarpus/bamboo forest pseudoobscura used may not be representative of on Mt Kenya, is strikingly close in its genital mor- the species. However, Cabrera et al. (1983) also phology to D. kitumensis from Mt Elgon. found some relatedness between D. pseudoobscura Regarding the relationship of these species with and D. ambigua. Moreover, hybridizations provide its non-African obscura relatives, the most dis- some evidence relating D. pseudoobscura to some tantly related species is D. helvetica. Although Palearctic species: D. pseudoobscura and or D. included in the affinis subgroup D. helvetica is persimilis crossed to either D. bifasciata, D. tristis clearly not closely related to any of these Nearctic or D. ambigua yield either sterile hybrids or pupae species. However, a relationship of the African (Koske, 1953; Buzzati-Traverso and Scossiroli, species and the affinis subgroup cannot be discar- 1955) while the European species generally pro- ded. With respect to the obscura and pseudoobscura duce no interspecific hybrids when crossed to one lineages D. microlabis and D. kitumensis are closer another (except D. subobscura D. madeirensis to the Palearctic D. tristis and D. ambigua than to and D. madeirensis x D. guanche, Krimbas et at., the American D. pseudoobscura. However, the rel- 1984). evant genetic distances are large and any relation- ship is relatively distant. This conclusion is con- sistent with the observation that neither D. micro- DISCUSSION labis nor D. kitumensis will hybridise with any other obscura group species (all these studied in Thediscovery in the Kenya highlands of a complex this paper and also D. subsilvestris have been of Drosophila species belonging to the obscura tested) nor with each other (Tsacas et aL, 1985). group allows new insight into the evolutionary Electrophoretic evidence clearly shows strong history of this group. heterogeneity within what is generally admitted as Of the four African obscura relatives two were the obscura subgroup. Therefore, we concur with found on the Eastern slope of Mt Elgon to the Lakovaara and Saura (1982) in recognising three West of the Kenya Rift Valley and the others two species-subgroups instead of two, in the obscura on the western slope of Mt Kenya to the east of group and further propose to introduce a fourth the Rift. The four species were found at similar subgroup to include the African species, the micro- altitudes, between 2200 and 2500 metres and labis species subgroup (fig. 5). the question arises whether or not these two In summary, the extent of the newly discovered species pairs have resulted from a vicariant event, African lineage in the obscura species group pro- due to the aridification of the Rift as a climatic vides new insight into the origin and evolutionary barrier. development of the obscura species group. On the The Kenya (Gregory) Rift Valley is a sector of basis of a "symplesiomorphic" acrocentric X the Rift system of eastern Africa which has been chromosome, homologous to the X of D. marked by continuous volcanic activity throughout melanogaster, Patterson and Stone (1952) con- its history from Miocene times to the present day. sidered D. subobscura to be a representative of the The largest of the central volcanoes are on the "ancestral" lineage of the group. Assuming this, flanks of the Rift, but whereas those to the west Lakovaara and Saura (1982) stated that if such a (e.g., Mt Elgon) are Miocene in age, those to the X chromosome were to be found in D. microlabis, east (e.g., Mt Kenya) are mainly Pleistocene to this species might belong to an "ancestral" lineage Recent (King and Chapman, 1972). as well. Preliminary results indi"e that this is not 82 M. L. CARIOU ET AL.

PaIpbristle 2 1 1 1 Sex.comb teeth 6.8 16.8 6.716.7 810/912 7..11 I 7..11 can ouae kri mbasi microlabiskitumensis

_ I 0.94

x

0.44.>-

E 0.29' 0 C) 0 -0 0 .0

Figure 4 Cladogram (UPGMA method) based on morphological affinities between the four African species of the obscuraspecies group. Similarity was estimated foi each pair of species by the proportion of common characters on the total 17 characters considered, including sex combs and clasper combs, wing indices, oral bristles, ejaculatory bulbs and various features of both phallus and parameres. the case (Krimbas et al., in prep.) and that the two might have been "tristis-ambigua like" or a "bifas- African species have eight euchromatic arms more ciata like" species. or less similar to those of D. ambigua, D. tristis The origin of the obscura species group remains and D. bfasciata. Also, Marinkovic et a!. (1978) controversial, especially with respect to the suggested that the American obscura subgroup chronology of the splitting events of the major species diverged from the D. subobscura lineage. lineages. In view of affinities with the melanogaster Neither the present allozyme data nor those of species group, Throckmorton (1975) derived the Cabrera et a!. (1983) support this hypothesis but obscura species group from a primeval proto- rather suggest that the central taxon group might melanogaster/obscura trunk. On the basis of diver- well be the obscura-tristis-ambigua lineage, sity criteria the origin of the melanogaster species because both American (D. pseudoobscura) and group is assumed to be oriental (Bock and Wheeler, African species (D. microlabis and D. kitumensis) 1972; Bock, 1980; Tsacas, 1984; Lemeunier et a!., show some degree of relatedness to it. Species such 1986). A plausible scenario is to consider that from as D. bifasciata (and to some extent D. helvetica) that centre of origin the protoobscura stock first that have very large geographic ranges, from split into two ancestral lineages, one of them diver- Western Europe to Japan, have also been con- sifying in East Africa and the other extending to sidered to be potential evolutionary links between Europe, where it differentiated giving rise sub- the obscura and the affinis subgroups (Lakovaara sequently to both the New World pseudoobscura and Saura, 1982). Hence, the proto-obscura lineage and affinis lineages. NEW SPECIES OF DROSOPHILA 83

helv

UI x NEARCTIC SPECIES pseu

made PALEARCTIC SPECIES

1 guan obs2

24 ambi bifa25 4 tris al subo23

AFRICAN SPECIES m icr kitu Axis 1 Figure 5 Multivariate analysis (Centered data analysis, Lefebvre, 1976) based on allele frequencies at 35 loci. Plotting of populations and species on the plane defined by the 1st and 4th axes (37 per cent of total variability) clearly discriminate distinct lineages within the obscuraspeciesgroup.

Acknowledgements Thefield investigation in September 1984 AYALA, ., POWELL, . R., TRACEY, M. L., MOURAO, C. A. AND benefited from the Kenyan Government support. Collecting PEREZ-SALAS, s. 1972. Enzyme variability in the Drosophila permit in National Parks of Kenya was kindly made available willistoni group.III. Genie variation in natural populations by the Director of the Department of Wild Life in Nairobi. ofDrosophila willisioni. Genetics, 70, 113—139. The following persons from the National Museum of Kenya BOCK, I. R. 1980. Current status of the Drosophilamelanogaster in Nairobi generously provided assistance: Dr R. E. Leakey, speciesgroup (Diptera). Syst.Entomol., 5, 341—356. Director Chief Executive, Dr J. Mark Ritchie, Head of BOCK, I. R. AND WHEELER, M. R. 1972. The Drosophila Entomology Section; we are especially indebted to Miss C. melanogaster speciesgroup. Univ.Tex. Pub!.,7213,1-102. Kabuye, Head of East African Herborium, for plant iden- BUZZATI-TRAVERSO, A. A. AND SCOSSIROLI, R. E. 1955. The tification. Thanks are also due to Prof. G. K. Kinoti, Zoology "obscuragroup"of the genus Drosophila.Advances in Department, University of Nairobi and Dr T. Odhiambo, Direc- Genetics, 7, 47—92. tor of the International Centre of Physiology and CABRERA, . M., GONZALEZ, A. M. AND GULLON, A. 1980. (ICIPE). D.madeirensis andD.guanche werekindly provided Enzymatic in Drosophilasubobscura popu- by Prof. A. Prevosti. We thank Miss N. Groseille for expert lations from the . Evolution,34, 875—887. technical assistance in electrophoresis and maintaining strains CABRERA, V. M., GONZALEZ, A. M., LARRUGA, J. M. AND successfully, J. P. Gauthier for computer assistance. Financial GULLON, A. 1983. Genetic distance and evolutionary support for this research was provided by the Centre National relationships in the Drosophilaobscura group.Evolution, de Ia Recherche Scientifique and a Franco-Hellenic grant to 37, 675-689. C. K.rimbas and M. L. Cariou. CARSON, H. L. 1951. Breeding sites of Drosophilapseudoobscura andDrosophilapersimilis inthe transition zone of the Sierra Nevada. Evolution,5, 91-96. CARSON, H. L., KRIMBAS, C. B. AND LOUKAS, M. 1985. Slime fluxes, a larval niche of Drosophilasubobscura. Col. Biologia REFERENCES Gallo-Hellenica, 10, 319—321. DAINOU, 0., CARIOU. M. L., DAVID, J. R. AND HICKEY, 0. 1987. ARAMBOURG, C., CHAPPUIS, P. A. AND JEANNEL, R. 1935. Amylase gene duplication: an ancestral trait in the Mission scientifique de l'Omo: Itinéraire et liste des Drosophilamelanogaster speciessubgroup. Heredity,59, stations. Mém.Mus. Nat. Hist. Nat. (N.S.), 2, Fasc.1, 1-22. 245—251. 84 M. L. CARIOU ET AL.

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