Obscura Species Group Research Articles

The length of the sperm nucleus in Drosophila obscura group species is depending on the total length of the sperm

Summary Males of the Drosophila obscura species group develop sperm of more than one length class. Also the lengths of the nuclei are different. When the total sperm length and the nucleus length were subject to a cluster analysis, it was shown that in D. subobscura and in D. affinis two classes of sperm exist. In D. azteca three classes were found. In the analyzed species the lengths of short sperm are more similar than the lengths of long ones. For example, the short sperm of D. subobscura and D. affinis have the same length while the long sperm of the two species are significantly different. This fact supports the idea that in evolution there is a tendency towards an increase in the length of sperm. But according to our data, in species with sperm heteromorphism preferentially the length of the long class is increased. In the two species D. subobscura and D. affinis, the size of the sperm nuclei corresponded to the total length of sperm. Long sperm had longer nuclei than short sperm. In D. azteca only for the length classes of short and medium sperm the same was found, but in the class of the longest sperm of D. azteca the lengths of nucleus and sperm were not correlated. In this class the total length of sperm varied while the length of the nuclei was rather constant with a mean value of 495 μm and a maximum of 631 μm. We assume therefore that the longest nuclei in this class have reached their upper limit of elongation.

IS THE PRODUCTION OF MULTIPLE SPERM TYPES ADAPTIVE?

Males of many species concurrently produce more than one sperm type, now called sperm heteromorphism. In the Drosophila obscura group, all species examined to date produce multiple sperm types that differ in sperm length. Short sperm types in at least three obscura group species do not participate in fertilization, leading to questions regarding the adaptive value of sperm heteromorphism. The common and pervasive inheritance of this trait in the obscura group, however, may indicate that sperm heteromorphism is phylogenetically constrained and therefore does not reflect an adaptive response to selection pressures. I measured interspecific variation in sperm length and determined the number of sperm types produced in 10 obscura group species. I subsequently tested if interspecific variation in sperm length is significantly associated with phylogeny by using an autoregressive comparative method. All obscura group species examined produce two visually distinct sperm lengths, short and long. Phylogenetic autoregression analyses indicated that 22% of the interspecific variation in long sperm is related to phylogeny, whereas short sperm are not significantly correlated with phylogeny. These results suggest different selection pressures on the two sperm length types; long sperm have evolved in response to fertilization demands and short sperm have been decoupled from these requirements.

Evolution of theDrosophila obscuraSpecies Group Inferred from theGpdhandSodGenes

TheDrosophila obscuragroup consists of several dozen Nearctic and Palearctic species, with a phylogeny that remains largely unresolved in spite of numerous morphological, cytogenetic, and molecular investigations. We have partially sequenced two genes,Gpdh(about 1000 bp) andSod(about 700 bp) in 12–13 species and 1 subspecies in order to settle the issues. Difficulties in resolving the phylogeny emanate from the rapid sequence of successive radiations. Nevertheless, the following conclusions are warranted: (1) The Palearctic species include two monophyletic subgroups,subobscura(which may be the most ancient clade of the whole group) andobscura,plus two other species with unresolved phylogenetic positions,D. bifasciataandD. subsilvestris;(2) the Nearctic species form a monophyletic group consisting of two monophyletic sister clades, theaffinissubgroup and thepseudoobscurasubgroup. The Palearctic radiation may have resulted from adaptation to expanding temperate forests in the Old World. A second radiation occurred during the colonization of the deciduous forests of the New World by the descendants of a single lineage that soon split into theaffinisandpseudoobscurasubgroups.

R1 and R2 retrotransposable elements of Drosophila evolve at rates similar to those of nuclear genes.

The non-long-terminal repeat retrotransposable elements, R1 and R2, insert at unique locations in the 28S ribosomal RNA genes of insects. Based on the nucleotide sequences of these elements in the eight members of the melanogaster species subgroup of the genus Drosophila, they have been maintained by vertical germline transmission for the 17-20 million year history of this subgroup. The stable inheritance of R1 and R2 within these species has enabled a determination of their nucleotide substitution rates. The sequence of the R1 and R2 elements from D. ambigua, a member of the obscura species group, has also been determined to enable an extrapolation of this rate over an estimated 45-60 million years. The mean rate of substitutions at synonymous sites (Ks) was 6.6 and 9.6 times the rate at replacement sites (Ka) in the R1 and R2 elements, respectively. Both elements appear to have been under selective pressure to maintain their open reading frames and thus their ability to retrotranspose for most of their evolution in these lineages. Using the rate of change at synonymous sites (Ks) as the best indicator of the nucleotide substitution rate, the mean Ks values for R1 and R2 were 2.3 and 2.2 times that of the alcohol dehydrogenase (Adh) genes. However, this faster rate is a result of the lower codon usage bias of R1 and R2 compared with that of Adh. When the Ks rates of R1 and R2 were compared with that of a larger number of nuclear genes available from at least two of the nine species under investigation, R1 and R2 were found to evolve in most lineages at rates similar to that of nuclear genes with low codon bias. The ability of R1 and R2 to maintain their presence in this species subgroup by retrotransposition while exhibiting rates of nucleotide evolution similar to nuclear genes suggests these transposition events are rare or not as error prone as that of retroviruses.

Phylogeny of the Drosophila obscura species group deduced from mitochondrial DNA sequences.

Approximately 2 kb corresponding to different regions of the mtDNA of 14 different species of the obscura group of Drosophila have been sequenced. In spite of the uncertainties arising in the phylogenetic reconstruction due to a restrictive selection toward a high mtDNA A+T content, all the phylogenetic analysis carried out clearly indicate that the obscura group is formed by, at least, four well-defined lineages that would have appeared as the consequence of a rapid phyletic radiation. Two of the lineages correspond to monophyletic subgroups (i.e., affinis and pseudoobscura), whereas the obscura subgroup remains heterogeneous assemblage that could be reasonably subdivided into at least two complexes (i.e., subobscura and obscura).

Characterization of the length polymorphism in the A+T-rich region of the Drosophila obscura group species

In the twelve Drosophila obscura group species studied, belonging to the affinis, obscura, and pseudoobscura subgroups, the mitochondrial DNA length ranges from 15.8 to 17.2 kb. This length polymorphism is mainly due to insertions/deletions in the variable region of the A + T-rich region. In addition, one species (D. tristis) possess a tandem duplication of a 470-bp fragment that contains the replication origin. The same duplication has occurred at least twice in the Drosophila evolutionary history due to the fact that the repetition is analogous to repetitions found in the four species of the D. melanogaster complex. By comparing the nucleotide sequence of the conserved region in D. ambigua, D. obscura, D. yakuba, D. teissieri, and D. virilis, we show the presence of a secondary structure, likely implied in the replication origin, which could favor the generation of this kind of duplications. Finally, we propose that the high A and T content in the variable region of the A + T-rich region favors the formation of less-stable secondary structures, which could explain the generation of minor insertion/deletions found in this region.

Heat shock proteins in three related Drosophila species belonging to the obscura group.

The effect of heat shock on protein synthesis in three related Drosophila species belonging to the obscura group was analyzed on SDS-acrylamide gels. Four major heat shock proteins (hsps) were found in these species, in which synthesis reaches a maximum at 34 degrees C. Although the higher molecular weight proteins are conserved, differences in size were found for the small hsps in these species. By means of in situ hybridization using D. melanogaster probes for the small hsp genes, it was inferred that the small hsp genes of the obscura group species are clustered at the 27A locus in all three species.

Genetic analysis of heat shock response in three Drosophila species of the obscura group.

Heat shock response was investigated in three species of the obscura group of the Drosophila genus (D. subobscura, D. guanche, and D. madeirensis) by chromosome cytology analysis and [3H]uridine labeling. A set of eight puffs (2C, 15DE, 18C, 27A, 31CD, 85AB, 89A, and 94A) were induced after heat treatments in each of the three species; 18C, 27A, 89A, and 94A were the most heavily labeled in the autoradiograms after the induced conditions. From the in situ results using the major heat shock genes of D. melanogaster as a probe, it was inferred that the 18C, 94A, 89A, and 27A loci of the three obscura group species are homologous to D. melanogaster loci, which contain, HSP82, HSP70, HSP68, and HSPs encoding for the small heat shock proteins, respectively. When this organization was compared with that of D. melanogaster, fewer evolutionary changes, mainly gene duplications, were found to have occurred in the obscura group species than in the D. melanogaster group. In the three species analyzed in this work, as well as in the other Drosophila species studied, the heat shock genes are distributed on D and E Muller's elements, behaving as single copy genes that do not move around the genome.

The Adh in Drosophila: Chromosomal location and restriction analysis in species with different phylogenetic relationships

Restriction analysis of the genomic region containing the Adh gene and in situ hybridization assays were performed in six Drosophila species belonging to three different subgenera: D. ambigua, D. subobscura, D. madeirensis and D. guanche (sg. Sophophora); D. immigrans (sg. Drosophila); and D. lebanonensis (sg. Pholadoris). In agreement with previous observations, comparison of restriction maps of the Adh region shows that D. subobscura and D. madeirensis are very closely related. Partial homology is also observed with the rest of the obscura group species. Nevertheless, no resemblance at the restriction map level is detected when more distantly related species are compared. In D. ambigua, D. immigrans and D. lebanonensis in situ hybridization assays reveal a single chromosomal location for Adh, which in D. lebanonensis appears to be sex linked. In contrast, in D. subobscura, D. madeirensis and D. guanche multiple sites of hybridization with homologous and heterologous probes are observed. For example, in D. subobscura and D. madeirensis the functional Adh gene is located on the U chromosome and additional homologous retrosequences are found on the E chromosome.

TheAdh genomic region ofDrosophila ambigua: Evolutionary trends in different species

The study of individual genes is essential to a comprehensive understanding of genome evolution. The wealth of information on alcohol dehydrogenase (Adh) in Drosophila makes this gene particularly suitable for such analysis. We have characterized more than 4 kb of the genomic Adh region in Drosophila ambigua and compared this region to Drosophila mauritiana and Drosophila pseudoobscura. The presence of two genes, Adh and 3'ORF (open reading frame), has been confirmed and some of their essential features have been inferred from primary structural analysis. Inter- and intraspecific comparisons have led us to support that both genes may have diverged from an ancient precursor. They appear to be evolving independently, and show a species-specific pattern. The Adh in the obscura group species lacks amino acids three and four when compared to the species of the melanogaster group and has accumulated most of its amino acid replacements in the third exon. Neither characteristic is observed when any other group species are compared, which suggests that these may be particular features of the evolution of the obscura group. The 3'ORF is highly conserved among the three species analyzed, although variability in the length of the third exon and the nucleotide substitution rate, which is much higher than in Adh, are worth noting. According to our data, both mutation/fixation rates and the distribution of mutations vary over time, which makes it difficult to predict the evolutionary dynamics of specific genome regions.

"Sex ratio" meiotic drive in Drosophila testacea.

We document the occurrence of "sex ratio" meiotic drive in natural populations of Drosophila testacea. "Sex ratio" males sire greater than 95% female offspring. Genetic analysis reveals that this effect is due to a meiotically driven X chromosome, as in other species of Drosophila in which "sex ratio" has been found. In contrast to other drosophilids, the "sex ratio" and standard chromosomes of D. testacea do not differ in gene arrangement, implying that the effect may be due to a single genetic factor in this species. In all likelihood, the "sex ratio" condition has evolved independently in D. testacea and in the Drosophila obscura species group, as the loci responsible for the effect occur on different chromosomal elements. An important ecological consequence of "sex ratio" is that natural populations of D. testacea exhibit a strong female bias. Because D. testacea mates, oviposits, and feeds as adults and larvae on mushrooms, this species provides an excellent opportunity to study the selective factors in nature that prevent "sex ratio" chromosomes from increasing to fixation and causing the extinction of the species.

The transposable portion of the genome of Drosophila algonquin is very different from that in D. melanogaster.

Four clones containing different transposable elements were isolated from a genomic library of Drosophila algonquin. Each clone was hybridized to salivary-gland chromosomes of three lines of D. algonquin and two lines of D. affinis. The estimated copy number in D. algonquin of the four element families varied from 59 to 333. The occupancy per site varied from 0.64 to 0.75. Thus the transposable portion of the D. algonquin genome is dominated by a few high-copy-number elements, each characterized by high occupancies. The copy number and occupancy values were very similar in D. affinis. This differs from the situation in D. melanogaster mobile middle-repetitive DNA, which has at least 30 and perhaps as many as 100 different families of mobile elements, with copy numbers ranging from 5 to 100. When several lines have been examined, elements in D. melanogaster are revealed to have very low occupancies. The four D. algonquin elements do not hybridize with D. melanogaster DNA, but they did hybridize with 15 obscura-group species, thereby revealing a pattern that is consistent with concerted evolution.

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

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

Nucleotide sequence of the Adh gene region of Drosophila pseudoobscura: evolutionary change and evidence for an ancient gene duplication.

The alcohol dehydrogenase (Adh) locus (ADH; alcohol: NAD+ oxidoreductase, EC 1.1.1.1) of Drosophila pseudoobscura was cloned and sequenced. Forty-five percent of the "effectively silent sites" have changed between Adh in D. pseudoobscura of the obscura species group and the homologous DNA sequence in D. mauritiana, the latter representing the melanogaster species group. The untranslated leader sequence of the adult transcript of D. pseudoobscura has two deletions relative to the D. mauritiana message. The ADH protein sequences of D. pseudoobscura is missing the third and fourth amino acids at the N-terminus relative to the D. mauritiana enzyme. Of the remaining 254 amino acid positions, 27 (10.64%) differ between the two species. Amino acid replacements are randomly distributed into hydrophilic and hydrophobic domains of ADH. However, replacement substitutions are distributed nonrandomly across the three exons among D. pseudoobscura and members of the melanogaster subgroup, suggesting that functional constraints across the exons are different. Surprisingly, silent substitutions are also nonrandomly distributed with the third exon being the most divergent. This pattern suggests possible selective constraints on supposedly neutral silent substitutions and/or variation in underlying mutation rates across the gene. The presence of transcriptional and translational signals at the beginning and end of conserved sequences 3' to Adh implies the existence of a previously undescribed gene. Codon usage and patterns of nucleotide divergence are consistent with a protein coding function for this gene. In addition, conservation of nucleotide and amino acid sequence and similarity in hydropathy plots suggests that the gene 3' to Adh represents an ancient duplication of the Adh gene.

Courtship feeding in Drosophila subobscura. I. The nutritional significance of courtship feeding

By feeding male Drosophila subobscura with stained yeast before courtship it was shown that the males transfer regurgitated crop contents to females during courtship. The female takes the drop of food with her proboscis directly from the male's extended proboscis and the male then attempts to copulate. The food passes into the female crop or ventriculus and females that take the drop have higher fecundity on a low-nutrient medium than those females denied access to the drop. ‘Starved’ females take the drop of food from the male more frequently than well-fed females and a comparison of crop sizes revealed that flies collected from the wild resemble the starved laboratory groups. Similar courtship feeding behaviours are described for other members of the obscura species group. Within the willistoni species group, male D. nebulosa deposit an anal drop containing gut contents on the substrate in front of the female during courtship and females consume this drop. A review of the literature suggests that various forms of courtship feeding may be widespread within the genus but that the extent of feeding by males of different species may vary.

Colonization of North America by the European Species, Drosophila subobscura and D. ambigua

Two European species, Drosophila subobscura and D. ambigua, have been found in the Pacific Northwest. Drosophila subobscura occurs from central California, N to southwestern British Columbia. Drosophila ambigua has been found only in the southwestern mainland of British Columbia. These species can be distinguished from the endemic obscura-subgroup species, D. pseudoobscura, D. persimilis and C. miranda, by sex comb, vaginal plate and wing bristle morphology, by allozymic differences and by differences in the polytene chromosomes. INTRODUCTION Students of the North American Drosophila obscura species group are not accustomed to viewing members of the group as cosmopolitan entities. The usual methods of species identification (Anderson et al., 1977; Crumpacker, 1973) simply provide means of distinguishing among the known endemic species. Thus difficulties arise when distinct species previously unknown from this continent begin appearing in collections. Two such species appeared in collections from the Pacific coastal region, from northern California to southern British Columbia. After ruling out the known North American representatives, analysis of metaphase chromosome configurations and hybridization results with D. pseudoobscura, D. miranda and D. persimilis suggested that the new residents are the widespread European species, D. subobscura and D. ambigua. Subsequent crosses to known European strains of these species verified the identifications. This colonization is not the first for Drosophila subobscura. In February 1978, it was found in Puerto Montt, Chile, and has since spread in South America to include a region from 29 ?-53 S lat (Budnik and Brncic, 1982). It has also spread eastward into Argentina (Prevosti et al., 1983). Its Old World range includes Europe from southern Scandinavia S to northern Africa and the Canary Islands, eastward to Iran, and westward to the Azores Islands (Prevosti, 1978). Drosophila ambigua occurs from southern Finland, S to the Iberian Peninsula and E to Greece (Lakovaara and Saura, 1982). The purpose of this paper is to report the apparently successful colonization by these two species in the Pacific Northwest. Morphological, allozymic and chromosomal means of distinguishing them from the indigenous Drosophila obscura subgroup species are presented, along with the presently known North American ranges of each. MATERIALS AND METHODS Collections were made at buckets of fermenting bananas. Females were immediately isolated individually into vials of standard medium of corn meal, yeast and sugars. In most collections, the species were identified by analyzing polytene chromosomes from third instar larvae from each female. The three indigenous species were identified by third and XL chromosomes (Anderson et al., 1977). The polytenes of Drosophila subobscura and D. ambigua bear little resemblance to one another or to those of any member of

Evolution of the obscura group drosophila species. I. Salivary chromosomes and quantitative characters in D. Subobscura and two closely related species

Three closely related Drosophila species of the “obscura” group have been examined: Dropophila madeirensis, D. guanche and D. subobscura. Some crosses are possible between them and F1 progeny were used in order to clarify chromosomal homologies. D. madeirensis has the following constitution in relation to D. subobscura: Am+1; JST; O3; U1+2 while D. guanche has: Am+g; Jg; Eg; O3+g; U1+2 (where m and g indicate inversions specific to those species). Some data are also presented on the inheritance of eight different quantitative characters used by some taxonomists to differentiate other palearctic obscura group species.

GENETIC DISTANCE AND EVOLUTIONARY RELATIONSHIPS IN THE DROSOPHILA OBSCURA GROUP.

The Drosophila obscura species group extends throughout the temperate zone of the Northern Hemisphere. Within it two subgroups are distinguished, affinis and obscura, with different numbers of representatives in the Old and New Worlds. Eight species of the affinis subgroup have been found in the Nearctic (D. affinis, D. algonquin, D. athabasca, D. azteca, D. narraganset, D. seminole, D. tolteca and D. dobzhanskii) and only one in the Palearctic (D. helvetica), while in the obscura subgroup 12 species are now known in the Old World (D. alpina, D. ambigua, D. bifasciata, D. eskoi, D. guanche, D. imaii, D. madeirensis, D. obscura, D. seguyi, D. subobscura, D. subsilvestris, and D. tristis) as compared with only five in the New World (D. frolovae, D. lowei, D. miranda, D. persimilis and D. pseudoobscura). This group, especially some species of the obscura subgroup, is one of the most comprehensively studied at all levels. Morphological, cytological, ethological and molecular, and phylogenetic relationships have been proposed within several clusters of species within the group (BuzzatiTraverso and Scossiroli, 1952; Clayton and Wheeler, 1975; Dobzhansky and Powell, 1975; Throckmorton, 1975 among others). However, a phylogeny including all or at least the great majority of the species of the group was not available until Lakovaara et al. (1972, 1976) constructed two phylogenetic hypotheses, first for the obscura subgroup and subsequently for the whole group. Based on genetic distances between species, these trees were constructed using enzymatic and other protein variants as taxonomic characters and assuming that the overall rate of evolutionary divergence is approximately homogeneous over all phyletic lines. Their results are, in general, in agreement with previous studies: the European species of the obscura subgroup were shown to be more closely related to each other than to their relatives in the New World, and similarly the species of the affinis subgroup of the New World were shown to be more closely related to each other than to those of the obscura subgroup, both of the Old as well as the New World. However, using the data of Lakovaara et al. (1972) but using methods which do not assume constant evolutionary rates for the species, Farris (1974) constructed a tree in which one of the Palearctic species, D. bifasciata, appears to be more closely related to the New World species of the subgroup than to those of the Old World. More recently, Marinkovic et al. (1978) established phylogenetic relationships among some of the American and European species of the obscura subgroup and found a European species, D. subobscura, to be apparently more closely related to American species of the subgroup than to other European species. It is of interest, therefore, to determine if these proposed close relationships between some American and European species of the obscura subgroup are consistent since it would greatly contribute to our understanding of the evolutionary history of the subgroup. The present work is an attempt to establish the relationships between a Nearctic species, D. pseudoobscura, and four Palearctic species, two of which have a wide geographical distribution, D. subobscura and D. ambigua. The other two are recently discovered species which are endemic to some of the Maccaronesian Islands, D. guanche Monclus (Monclus,

Photoresponse in Relation to Experimental Design in Sibling Sympatric Species of Drosophila

Photoresponses of isofemale lines of Drosophila pseudoobscura, D. persimilis and D. miranda captured in two locations were compared in two types of experimental apparatus, an open field and a Y-tube. Photoresponses were significantly more negative in the open field than in the Y-tube. Responses in one apparatus were unrelated to the responses in the other. The sibling sympatric species from each location could be distinguished by their light responses irrespective of the experimental design. The populations of D. pseudoobscura from the two locations were significantly different in both designs. These behavioral differences must be attributable to different genetic determinants. INTRODUCTION Rockwell and Seiger (1973a) in a review of photoresponse in the genus Drosophila noticed that in the past experimental designs tend to fall into one of three classes. In Class I, photoresponse is measured as the movement or distribution of the individuals relative to a directed light source. For example, in the very first paper that used fruit flies for research, Carpenter (1905) measured photoresponse in terms of movement toward a light source and distribution of flies in a light gradient. In Class II, photoresponse is measured by the distribution of flies in an open field in which areas of the field differ in quality or intensity of light. Movement of the flies is in a plane perpendicular to the light source (Rockwell and Seiger, 1973b; Rockwell et al., 1975). In Class III, photoresponse is measured by the frequency of choice flies make when they reach a juncture of two light intensities. The two lights may be provided by light directed at the choice point (Hirsch and Boudreau, 1958; Pittendrigh, 1958) or by a light shining perpendicular to the plane of movement (Lewontin, 1959; Dobzhansky et al., 1969; Seiger and Seiger, 1979). It is difficult to make generalizations about photoresponse using different experimental designs. It may be that different experimental designs elicit different photobehavior from any given population of Drosophila. This possibility was studied in the current research. MATERIALS AND METHODS Drosophila pseudoobscura, D. persimilis and D. miranda are sibling species of the obscura species group within the subgenus Sophophora of the genus Drosophila (Dobzhansky and Epling, 1944). The isofemale lines (each started from one female caught in the wild) used in this study were kindly provided by Dr. Francisco Ayala, University of California, Davis. This study of five isofemale lines each of D. pseudoobscura and D. persimilis from MacDonald Ranch, Calif., and D. pseudoobscura and D. miranda from Mather, Calif., began with the F2 generation and took somewhat less than a year to complete. All strains were cultured in half-pint milk bottles using a cream-of-wheat and raisin medium at a temperature of 20 zt 1C under constant illumination of 3.0 ergs/cm2-sec.

GENETIC POLYMORPHISM AND PHYLOGENY OF DROSOPHILA SUBOBSCURA.

The genus Drosophila consists of more than 1,200 species which have arisen through several evolutionary radiations. One major radiation includes the subgenus Sophophora which comprises the melanogaster lineage evolved in the Old World tropics, the saltans-willistoni lineages in the New World tropics, and the obscura group distributed throughout the temperate zone of the northern hemisphere. According to Throckmorton (1975) the obscura group may have originated from a protomelanogaster lineage of the Old World tropics. After adaptation to temperate habitats, the obscura group diversified and eventually spread to North America prior to midMiocene times (i.e., more than 15 million years ago). Several species of the group have been the subject of intensive evolutionary and genetic studies. Lakovaara et al. (1972a,b) have attempted to reconstruct the phylogeny of the group on the basis of electrophoretic studies of gene frequencies. Among the Old World species of the obscura group, D. subobscura is particularly interesting. The species exhibits a considerable degree of chromosomal polymorphism that has been the subject of numerous studies (Goldschmidt, 1956; Knight, 1961; Sperlich, 1961, 1964; Pentzos-Daponte, 1964; Krimbas, 1964, 1971; Prevosti, 1966; G6tz, 1967; Andjelkovic and Sperlich, 1973; Zouros and Krimbas, 1973; and others). Recently, genetic variation has been studied in D. subobscura using electrophoretic methods. Particularly noteworthy is the work of Lakovaara, Saura, and their co-workers, who examined 32 gene loci coding for proteins in one study of geographically marginal populations (Lakovaara and Saura, 1971a) and 20 gene loci in a study of some central as well as marginal populations (Saura et al., 1973). The present paper reports the results of a study of 28 gene loci coding for proteins in a large sample of D. subobscura flies from Fruska gora, Yugoslavia. This is the first extensive electrophoretic survey of a D. subobscura population from southeast Europe (a region extensively studied with respect to chromosomal polymorphism) and of a population which is continental (more than 100 km from the sea). We have also made direct comparisons in the same electrophoretic gels between D. subobscura and the North American species D. pseudoobscura and D. persimilis. Thus, we have established the equivalence between the allelic designations used for a variety of obscura group species by Lakovaara and co-workers, and the designations used by American researchers working on D. pseudoobscura and its close relatives (e.g. Prakash, Lewontin, and Hubby, 1969). This equivalence has made it possible to estimate the genetic distance among several species of the obscura group in which the same protein loci have been studied by different workers.