present in the five specimens analyzed miranda was found and thus we could not Analysis of the variability of Drosophila azteca and Dro- sophila athabasca populations revealed by random am- and absent in six D. persimilis and seven increase the sample size of that species. plified polymorphic DNA. J Zool Syst Evol Res 35:159– D. pseudoobscura of different origin. The Here we describe species-specific bands 164. number of specimens analyzed is small for of D. pseudoobscura, D. persimilis, and D. Pascual M, Balanya` J, Latorre A, and Serra L, 1997b. D. miranda, but since the lines used came miranda that discriminate between these Diagnosis of sibling species of Drosophila involved in the colonization of North America by Drosophila subob- from different localities (see Materials and three sibling species. These results, along scura. Mol Ecol 6:293–296. Methods), it can be assumed that these with those of a previous study using D. Powell JR, 1983. Interspecific cytoplasmic gene flow in bands are diagnostic. We estimated the azteca and D. athabasca (Pascual et al. the absence of nuclear gene flow: evidence from Dro- size of species-specific bands observed by 1997b) permit classification of all the spec- sophila. Proc Natl Acad Sci USA 80:492–495. Pascual et al. (1997b) in D. athabasca and imens collected in all samples of the dis- Prakash S, 1977. Genetic divergence in closely related D. azteca. A total of nine bands clearly tribution range of these five Nearctic spe- sibling species Drosophila pseudoobscura, D. persimilis and D. miranda. Evolution 31:14–23. identified D. athabasca and seven bands cies of the obscura group. We may thus be Prevosti A, Serra L, Aguade´M, Ribo´G, Mestres F, Bal- identified D. azteca (Table 1). able to study their population dynamics an˜a` J, and Monclu´s MG, 1989. Colonization and estab- The amount of variation between D. and assess its influence on the colonizing lishment of the palearctic species D. subobscura in pseudoobscura and D. persimilis was esti- success of D. subobscura. North and South America. In: Evolutionary biology of transient unstable populations (Fontdevila A, ed). Ber- mated using the analysis of molecular From the Departament de Gene`tica, Universitat de Bar- lin: Springer-Verlag; 114–129. variance. D. miranda was not included in celona, Av. Diagonal 645, 08028 Barcelona, Spain. Ad- Wang RL and Hey J, 1996. The speciation history of the analysis due to the low number of in- dress correspondence to Marta Pascual at the address Drosophila pseudoobscura and close relatives: infer- dividuals. Only polymorphic bands were above or e-mail: [email protected]. This ences from DNA sequence variation at the period lo- work was supported by grant PB96-0793-C04-03 from cus. Genetics 144:1113–1126. used in the analysis. Of the overall phe- the DGES, Spain. We thank E. Haring, R. Huey, M. Noor, notypic variation, 58.38% was due to vari- C. Segarra, and D. Sperlich for providing us with flies Received February 18, 1999 Accepted May 31, 1999 ation between species and 41.62% to intra- of different origin. We thank R. J. MacIntyre for his help- ful comments and suggestions. Corresponding Editor: Ross MacIntyre specific variation. Population structure was not analyzed because the number of ᭧ 1999 The American Genetic Association individuals was very low in some locali- ties. Significant genetic differentiation be- tween species was detected (␾ ϭ 0.584, References ST Reproductive and Mate p Ͻ .001). Tests of significance for vari- ance components are based on 1,000 ran- Anderson WW, Ayala FJ, and Michod RE, 1977. Chro- Choice Strategies in the dom permutations. mosomal and allozymic diagnosis of three species of Drosophila: D. pseudoobscura, D. persimilis, and D. mir- Hermaphroditic Flatworm A total of 39 males and females from Bel- anda. J Hered 68:71–74. Echinostoma caproni lingham (WA) and Salem (OR) kept in 70% Armstrong J, Gibbs A, Peakall R, and Weiller G, 1996. ethanol were amplified with some of the RAPDistance programs: version 1.04 for the analysis of S. Trouve´, F. Renaud, P. Durand, diagnostic primers. The aim of this analy- patterns of RAPD fragments. Canberra, Australia: Aus- tralian National University. and J. Jourdane sis was to increase the number of D. mir- Barrio E, Latorre A, Moya A, and Ayala FJ, 1992. Phy- anda in the sample. Males were morpho- logenetic reconstruction of the Drosophila obscura Due to the important role that mating sys- logically classified to the subgroup level group, on the basis of mitochondrial DNA. Mol Biol tems play in the evolution of species, we ahead of time and then the result of the Evol 9:621–635. investigate the selfing rate and mate RAPD amplification was cross-checked Beckenbach AT and Prevosti A, 1986. Colonization of choice in the simultaneous hermaphroditic North America by the European species D. subobscura with the previous identification in order to and D. ambigua. Am Midl Nat 115:10–18. parasite Echinostoma caproni (Tremato- assess the reliability of the system. A blind Beckenbach AT, Wei YW, and Liu H, 1993. Relationships da). The echinostomes were maintained in experiment, previously carried out with D. in the Drosophila obscura species group, inferred from two situations in mice: (1) double infec- azteca and D. athabasca males using a mitochondrial cytochrome oxidase II sequences. Mol tions where the two individuals do or do Biol Evol 10:619–634. morphological character and comparing not belong to the same geographic area Buzzati-Traverso AA and Scossiroli R, 1955. The ‘‘ob- the results with RAPD banding pattern has scura group’’ of the genus Drosophila. Adv Genet 7:47– isolate, and (2) triple infections where two also shown the value of the technique 92. of the three individuals originate from the (Pascual et al. 1997b). Bellingham (WA) in- Dobzhansky TH and Epling C, 1944. Contributions to same isolate and the third one originates dividuals were classified as follows: 4 af- the genetics, taxonomy and ecology of Drosophila pseu- from a different isolate. This experimental doobscura and its relatives. Publications of the Carne- finis subgroup males as D. athabasca, 16 gie Institute of Washington 554:1–183. design permits analysis of intra- and inter- subgroup males as isolate selfing rates and of mate prefer- pseudoobscura D. pseu- Excoffier L, 1995. WINAMOVA version 1.55. Genetics doobscura, and 8 obscura group females as and biometry. Geneva, Switzerland: University of Ge- ence. We predict, in the first experiment, 7 D. athabasca and 1 D. pseudoobscura. Sa- neva. no difference between intra- and interiso- lem (OR) males, from the pseudoobscura Latorre A, Barrio E, Moya A, and Ayala FJ, 1988. Mito- late selfing rates. In the second experi- subgroup, were classified as 7 chondrial DNA evolution in the Drosophila obscura ment we expect a preferential outcrossing D. pseu- group. Mol Biol Evol 5:717–728. doobscura and 4 D. persimilis. All individ- between individuals originating from the Palacios C and Gonza´lez-Candelas F, 1997. Analysis of uals were amplified with at least two prim- population genetic structure and variability using same isolate in order to avoid hybrid ers, those producing species-specific RAPD markers in the endemic and endangered Limon- breakdown. The results obtained corrob- bands. In each case opa-4 yielded a scor- ium dufourii (Plumbaginaceae). Mol Ecol 6:1107–1121. orate our predictions and emphasize the able set of bands for D. athabasca, opa-7 Pascual M, Ayala FJ, Prevosti A, and Serra L, 1993. Col- important and synergistic roles of selfing, onization of North America by D. subobscura: ecologi- for D. pseudoobscura, opa-9 and opa-16 for cal analysis of three communities of drosophilids in inbreeding depression, and hybrid break- D. persimilis and opb-8 for D. miranda California. J Zool Syst Evol Res 32:44–50. down in the evolution of echinostome re- (Table 1, Figure 1). Unfortunately no D. Pascual M, Balanya` J, Latorre A, and Serra L, 1997a. productive strategies. 582 The Journal of Heredity 1999:90(5) Reproductive strategies constitute a major reproduction of the echinostomes, which, timate the selfing rate involved within an factor shaping the evolution of organisms. like many parasites, often evolve in low- isolate. Twenty days postinfection the in- Indeed, mating systems influence the ge- density populations (Charnov et al. 1976; testines of the mice were opened and the netic variability as well as the genetic Ghiselin 1974; Tomlinson 1966). Second, worms collected. The uterus of each adult structure of populations (Charlesworth inbreeding depression decreases the fit- was torn to collect the eggs. Thirty larvae and Charlesworth 1987; Jarne 1995). In ness of self-fertilized offspring. Third, after hatched from the eggs of each worm were this context, hermaphrodites offer the op- a period of isolated evolution, hybrid individually brought into contact with one portunity to investigate the evolution of breakdown should reduce genetic ex- mollusc. Genetic exchanges were assessed sexual reproductive modes since a single change between different isolates. from a ‘‘progeny-array analysis’’ by com- individual can self- or cross-fertilize. Com- Consequently, in the light of these pre- paring the mother’s genotype to that of pared to plants, few studies have been dictions and our knowledge of echino- her progeny at the rediae stage (larval conducted on mating systems in animals. stomes, we present a study on individuals stage in the first intermediate host). Elec- These works have been mostly carried out originating from a new natural population, trophoretic analyses were performed ac- on molluscs [see Jarne et al. (1993) for a discovered in May 1996 in Mali, showing cording to the procedures described in review; Doums et al. 1996; Sta¨dler et al.