Copyright 0 1996 by the Genetics Society of America

Differentiation of Muller’s Chromosomal Elements D and E in the Obscura Group of Drosophila

Carmen Segarra,* Griselda Ribot and Montserrat AguadC*

*Departament de Genitica, Facultat de Biologia, Universitat de Barcelona, 08071 Barcelona, Spain and Centre d ’Znvestigacio’ i Desenvolupament, CSIC, 08036 Barcelona, Spain

Manuscript received January 2, 1996 Accepted for publication May 20, 1996

ABSTRACT Twenty-two markers located on Muller’s elements D or E have been mapped by in situ hybridization

in six species of the obscura group of Drosophila and in D. melanogaster. The obscura species can be grouped into a Palearctic cluster (D. subobscura, D. madeirensis and D. guanche) and a Nearctic one (D. pseudoobscura, D. persirnilis and D. miranda) . Eleven of the probes contain known genes: E74, Acp70A, Est- 5, hsp28/23, hsp83, emc, hsp70, Xdh, Acph-I, Cec and rp49. The remaining probes are recombinant phages isolated from a D. subobscura genomic library. All these markers hybridize to the putative homologous chromosome or chromosomal arm of elements D and E. Thus, these elements have conserved their genic content during species divergence. Chromosomal homologies proposed previously for each ele- ment among the species of the same cluster have been compared with the present results. The distribu- tion of markers within each element has changed considerably as inferred from pairwise comparisons of obscura species included in the two different clusters. Only chromosomal segments defined by closely linked markers have been conserved: one such segment has been detected in element D and three in element E between D. subobscura and D. pseudoobscura.

P ARTICULAR probes can be accurately mapped by in situ hybridization on polytene chromosomes

of Drosophila. This technique is sensitive enough to give positive results in cross-hybridizations and thus can be applied in evolutionary studies. Initially, it was used to recognize homologous chromosomal ele- ments among different Drosophila species (STEINE- MANN 1982; STEINEMANN et al. 1984; WHITING et al. 1989; PAPACEIT and JUAN 1993; among others). An- other more detailed approach has been to identify homologous segments within a particular chromo- somal element among different species (SEGARRA and AGUADE 1992; SEGARRA et al. 1995). These later studies indicate extensive reshuffling within Muller’s A ele- ment (MULLER 1940) according to comparisons of, for instance, D. melanogaster with D. pseudoobscura or with D. subobscura, and even from comparisons of the two obscura group species.

In the present study, this latter approach is extended to Muller’s elements D and E. Twenty-two probes that hybridize to these elements in D. melanogaster have been mapped in six species of the obscura group. The obsc- ura group species studied are included in two different clusters. The first cluster is the D. subobscura cluster with the three Palearctic and closely related species: D. subobscura, D. madeirensis and D. guanche. The other cluster is the D. pseudoobscura cluster that includes the three Nearctic and closely related species: D. pseudoobsc-

Corresponding authur; Carmen Segarra, Departament de Gen&ica, Facultat de Biologia, Universitat de Barcelona, Diagonal 645, 08071 Barcelona, Spain. E-mail: carme@porthos.bio.ub.es

Genetics 144: 139-146 (September, 1996)

ura, D, persirnilis and D. miranda. As indicated in Table 1, Muller’s element E is autosomal in the seven species, while element D is autosomal in D. subobscura, D. ma- dezrensis and D. guanche and corresponds to the right arm (XR) of the metacentric sex chromosome in D. pseudoobscura, D. persimilis and D. miranda. Muller’s ele- ments D and E are fused to form the 3L and 3R chromo- somal arms, respectively, of the metacentric autosome 3 of D. melanogaster (BUZZATI-TRAVERSO 1955).

The accurate mapping of these markers in the ob- scura group species contributes to their genetic char- acterization. As a second aim, this study attempts to corroborate previously proposed chromosomal ho- mologies among the species in the same obscura clus- ter. Third, it tries to identify homologous chromosomal segments between the species of different clusters. Fourth, the extent of reorganization within D and E elements during the divergence of the species can be analyzed. In fact, the rate of evolution of the different chromosomal elements may be affected by their autoso- mal or sex chromosomal character. CHAFUESWORTH et al. (1 987) propose that the fixation rate of underdomi- nant chromosomal rearrangements is higher with sex linkage than for autosomes and indicate that in the genus Drosophila there are slightly more X-linked than autosomal-fixed paracentric inversions. If sex chromo- somes indeed evolve faster, one would expect a @on‘ that the distribution of markers would be more con- served in element E, which is autosomal in the seven species studied.

C. Segarra, G. Rib6 and M. AguadC 140

TABLE 1

Homologies between Muller’s elements and the chromosomes or chromosomal arms of D. melunogmter

and the obscura group species

D. subobscura D. pseudoobscura Muller’s D. madeirensis D. persimilis element D. melanogaster D. guanche D. miranda

D ?L (autosomal) J (autosomal) XR (sexual) E ?R (autosomal) 0 (autosomal) 2 (autosomal)

MATERIALS AND METHODS

Two strains of D. subobscura were used: ch cu, which has J , and chromosomal arrangements, and an isochromoso- mal line with the O,, arrangement. D. madeirensis and D. guan- che strains were available in our laboratory. D. pseudoobscura, D. persimilis and D. miranda strains were kindly provided by R. C. LEWONTIN.

Preparations of polytene chromosomes suitable for in situ hybridization were obtained following MONTGOMERY et al. (1987). The 22 probes used in the present study are summa- rized in Table 2. Some probes correspond to recombinant DNA plasmids or phages including totally or partially known genes cloned in D. melanogaster, D. subobscura or D. pseudoobsc- ura. Additionally, some anonymous phages from a random genomic library of D. subobscura (AGUADE 1988) that gave a single hybridization signal in this species were also used as markers; the name of those phages begins with ‘“hub”. Probes were labeled with lGbiotin dUTP (Boehringer Mann- heim) by nick translation. Prehybridization, hybridization and detection conditions were as described in SEGARRA and ACU- ADE (1992).

The following polytene chromosome photomaps were used

to identify the location of hybridization sites: D. madeirensis (PAPACEIT and PREVOSTI 1991), D. guanche (MOLTO et al. 1987), D. pseudoobscura (STOCKER and KASTRITSIS 1972), D. persimilis (MOORE and TAYLOR 1986), D. miranda (DM et al. 1982) and D. melanogaster (LEFEVRE 1976). In the case of D. subobscura, the KUNZE-MUHL and MULLER (1958) cytological map has been used. This map was taken as a reference to establish chromosomal sections in D. madeirensis and D. guan- che photomaps. Unfortunately, sections of the photomaps of D. pseudoobscura and D. pasimilis are not in complete agree- ment, and sections of the D. miranda photomap were newly established by DAS et al. (1982) without considering the pre- viously established subdivisions by DOBZHANSKY and TAN (1936).

RESULTS

All probes that map on the ?L chromosomal arm of D. melanogaster hybridize on chromosomeJof the three obscura Palearctic species and on the X R chromosomal arm of the obscura Nearctic representatives. Similarly, all probes hybridizing to the ?R chromosomal arm of D. melanogaster are located on chromosome 0 of the Palearctic species and on chromosome 2of the Nearctic representatives of the obscura group species. Photo- graphs showing the hybridization signals of the 22 probes in the different species are available from the authors on request.

Figure 1 shows a linear representation of the location of markers along element D in the different species. Only the close linkage between Acp70A and ADsubS40 is conserved in all the six obscura species. Comparing

TABLE 2

List of the 22 probes used in the present study

Chromosomal Probe name (species) Symbol element Reference

Recombinant phage Fa (D. sub) kDsubFa D A. MUNTE (personal communication) Recombinant phage F62 (D. sub) xDsubF62 D J. M. MART~N-CAMPOS (personal communication) Ecdysone-inducible E74 (D. mel) E 74 D BURTIS et al. (1990) Recombinant phage 5’30 (D. sub) xDsubS30 D L. SANCHEZ (personal communication) Recombinant phage S40 (D. sub) kDsubS40 D S. CIRERA (personal communication) Accessoly male protein 70A (D. mel) Acp 70A D S. CIRERA (personal communication) Esterase-5 (U. pseudo) Est-5 D BRADY et al. (1990) Recombinant phage Fy (D. sub) kDsubFy D Present study Heat shock proteins 28/23 (D. mel) hsp28/ 23 D CORCES et al. (1980) Recombinant phage S25 (D. sub) kDsubS25 D L. SANCHEZ (personal communication) Heat shock protein 83 (D. mel) hsp83 D HOLMCREN et al. (1979) Extra-macrochaetae (D. mel) emc D GARRELL and MODOLLEL (1990) Recombinant phage FC4 (D. sub) Recombinant phage S14 (D. sub) Heat shock protein 70 (D. mel) Xanthine dehydrogenase (D. sub) Recombinant phage S9 (D. sub) Recombinant phage G2 (D. sub) Recombinant phage S6 (D. sub) Acid phosphatase-1 (D. sub) Ribosomal protein 49 (D. pseudo) Cecropin (D. sub)

kDsubFC4 kDsubSl4 hsp 70 Xdh kDsubS9 kDsubG2 kDsubS6

r - 4 9 Cec

Acph-1

E E E E E E E E E E

S. RAMOS-ONSINS (personal communication) Present study LWAK et al. (1978) J. M. COMERON and M. AGUAD~ (unpublished) Present study J. M. MARTIN-CAMPOS (personal communication) Present study A. NAVARRO-SABATE (personal communication) SEGARRA and AGUADE (1993) S. RAMOS-ONSINS (Dersonal communication)

D. sub, D. subobscura; D. mel, D. melanogaster; D. pseudo, D. pseudoobscura.

D . subobscura

D . madeirensis

D . guanche

D . pseudoobscura

D . persimi 1 i s

D . miranda

D . melanogaster

Evolution of D and E Elements 141

h s p 8 3 A c p 7 l J A A D s u b F 6 2 A D s u b F y E s t -5 l D s u b S 4 l J

h s p 2 8 / 2 3 A D s u b F a k D s u b S 2 5

ernc E74 ADsubS3lJ

0 I 1 I1 1 I I I I I I 1 I , I 1 I I I I I I , I

hsp83 Acp7UA ADsubF62 ADsubFy E s t - 5 A D s u b S 4 0 ADsubS25

h s p 2 8 / 2 3 ADsubFa emc ADsubS30 0 I 1 I 1 I I I I I I I 1

I , I I I I I

h s p 8 3 A c p l V A A D s u b F G 2 A D s u b F y E s t - 5 A D s u b S 4 0 ADsubS25

h s p 2 8 / 2 3 A D s u b F a ~ 7 4 ernc ADsub.530 0 I 1 I 1 I I I I I I I 1

I , , I I I I I I I ,

ADsubFa E74 Acp7UA E s t - 5 h s p 8 3 A D s u b S d V

ADsubS30 ADsubFG2 emc APsubS25 I I I1 I I I I

ADsubFy h s p 2 8 / 2 3

m I I I 1

E74 ADsubFa ADsubSQO ADs u b F y h s p 2 U / 2 3 h s p 8 3 E s t - 5 A c p 7 U A A D s u b . 5 3 0 A D s u b F 6 2 A D s u b S 2 5 emc

0 I 1 I I I 1 I 1 I , I I 1 I 1 1 1 I I I I 1 I

Acp7VA ADsubFa ADsubF52 E74 ADsubS3LJ ADsubS40 Es t -5 ADsubFy ADsubS25

h s p 2 8 / 2 3 h s p 8 3 emc

I) I 1 I I I 1 I I 1 I I I I 1 I I I , I I , I I I

FIGURE 1.-Schematic representation of the location of markers along element D of the studied species. The different elements have been given the same length to show the relative position of markers among them. Centromere is placed on the left and telomere on the right of the elements. See Table 2 for notation of markers.

any of the species of the obscura group with D. melano- gaster, the segment delimited by Acp70A and ADsubS40 also seems to be conserved, but the two markers are more tightly linked in the obscura group species than in D. melanogaster. Although the segment between ADsubFy ADsubF62 probes is also maintained among the Palearc- tic and Nearctic representatives except for D. pusimilis, the banding pattern of this segment when comparing, for instance, D. subobscura and D. pseudoobscura, is differ- ent enough for it not to be considered a true homolo- gous segment. Moreover, the three Palearctic species and D. mlanogasteralso share the segment Est-5-Acp70A, although the banding pattern of this segment between D. melanogasterand the obscura representatives does not show a good correspondence.

D. subobscura and D. madeirensis share the same linear array of element D markers with similar relative dis- tances (Figure 1). D. subobscura and D. guanche differ by the inversion of the E74 and emc loci. D. pseudoobscura and D. persimilis also differ by the inversion of two mark- ers (ADsubFy and ADsubS30). D. pseudoobscura and D. miranda differ by the inverted orientation of the three more distal markers (emc-hsp28/23-ADsubS25) and also of the four more proximal ones (ADsubFa-Est-5-E74- hsp83).

Figure 2 shows the location of markers along element E in the different species. The order of markers in D. subobscura is given according to the Os, gene arrange- ment that corresponds with that in the cytological map (KUNZE-MOHL and MULLER 1958). The figure also in- cludes the location of the Antennapedia (Antp) gene that

had been mapped previously by TEROL et al. (1991). All probes except the hsp70 hybridize to a single site in the different species. In D. melanogaster, hsp70 genes are organized in two clusters localized at sections 87A and 87C, but in situ hybridization of hsp70 probes in this species also give weak signals at positions 95D and 870 ( HOLMGREN et al, 1979). In D. subobscura and D. guanche, only two rather strong hybridization signals were de- tected. However, in D. madeirensis and the three obsc- ura Nearctic species, in addition to these two strong signals, some very weak signals were also detected at other locations on the same chromosomal arm (result not shown).

Between the obscura group species and D. mlanogas- tu, only the close linkage between the rp49 and Acph-1 loci has been maintained. In addition, the six obscura group species share the nearly telomeric position of Cec. In D. melanogaster, although Cec is also the most distal locus of those analyzed, the distance between Cec and the telomere is longer and so delimits a different seg- ment. The close linkage between hDsubG2and one of the signals from hsp70 is also maintained in the six obscura species. This result allowed us to differentiate in these species the two signals detected with the hsp70probe (as hsp70/1 and hsp70/2). Another segment delimited by ADsubS9 and hsp70/1 seems to be conserved in five of the six obscura species (except for D. miranda). Although the length of the segment is fairly conserved, the band- ing pattern differs when comparing, for instance, D. su- bobscura and D. pseudoobscura. Finally, the AntpWsubFC4- ADsubS6 segment is also maintained in the obscura spe-

142 C. Segarra, G. Rib6 and M. AguadC

hsp70/1 Acph-1 Antp ADsubFC4 ADsubS6 Xdh ADsubG2 ADsubS9 rp49 hsp70/2 ADsubSl4

Ce c

D . subobscura 0 I I 1 I I I I I1 I I I I 1 I , I I 1 8 I

hsp70/l Antp A ~ s u b ~ ~ 4 A ~ s u b s 6 Xdh ADsubG2 ADsubS9 hsp70/2 rp49 AcPh-A ADsubS14

Cec

D . madeirensis - I I 1 I I 1 I I I I I I I 1 I , I I I I

hsp70/1 h t P ADsubFC4 ADsubSG ADsubS9 bsubG2 x& hsp70/2 rp49 AcPh-1 ADsubS14

I I 8 I 1 I I I I I I I 1 I I I I I I

CeC

D . guanche - ADsubS6 ADsubFC4 Antp ADsubGZ ADsubS9 X&

ADsubS14 hsp70/1 rp49

D . pseudoobscura - hsp70/2 A d - 1 Cec I I 1 I I 1 I 1 I II I I I I I , , I I

hsp70/l ADsubSll ADsubS6 ADsubFC4 Antp

rp49

D . p e r s i r n i l i s X& ADsubS9 ADsubG2 hsp70/2 Acph-l Cec

0 I I I I 1 I I I I I1 I I I I 1 , I I I

ADsubSl4 hsp70/1 ADsubSG ADsubFC4 Antp ADsubG2 Xdh ADsubS9 hsp70/2 Acph-l Cec

rp49

D . miranda 0 I I I I I I I I II I I I I I 1 I I I

ADsubFC4 Antp ADsubSl4 hsp70 ADsubG2

hsp70 Xdh ADsubS9 ADsubSG Acph-l Cec rp4 9

D . melanogaster - ; ; I I I 1 I 1 I 1 1 1

FIGURE 2.-Schematic representation of the location of markers along element E of the studied species. The different elements have been given the same length to show the relative position of markers among them. Centromere is placed on the left and telomere on the right of the elements. See Table 2 for notation of markers.

I I I I 1 I I O

cies. However, the different length of AntpXDsubFC4 and hDsubFC4ADsubSG between the Palearctic and Nearctic species clearly indicates that these two segments are not homologous among the obscura species. D. subobscura and D. madeirensis differ by the inverted

orientation of Acph-l-~p49-hsp70/2. The order of these loci in D. guanche is in agreement with that of D. ma- deirensis. However, in D. guunche there is an additional inversion including Xdh-hDsubG2-hsp70/1-ADsubS9. D. pseudoobscura and D. persirnilis show an inverted orienta- tion of Xdh-hDsubS9-hsp70/1-ADsubG2-ADsubSl4. D. pseu- doobscuru and D. miranda differ by the inverted position

Tables 3 and 4 show the location of D and E elements markers, respectively, in the different species according to their photographic or cytological maps (see UTERI- a s AND METHODS). There is a discrepancy in the loca- tion of the E74 gene in D. pseudoobscura with that pre-

of Xdh-ADsubS9.

viously reported by JONES et al. (1991) that can be attributed to a different interpretation of the polytene chromosome map. Otherwise, the locations of hsp28/ 23 and hsp70 in the three species of the D. subobscura cluster are in agreement with those previously reported (MOLTO et al. 1993a,b).

DISCUSSION

The established chromosomal homologies ( BUZZATI- TRAVER~O 1955) of element D ( J chromosome in the Palearctic species and X R chromosomal arm in the Nearctic species), and of element E (0 chromosome of the Palearctic species and chromosome 2 of the Nearc- tic species) are completely supported by the present results. Moreover, no instance of exchange of genetic material among different elements has been detected, which indicates that elements D and E have largely con-

TABLE 3

Cytological location of the 12 probes on Muller's element D of the different species studied

Species emc hsp83 ADsubS25 hsp28/23 kDsubFy Est-5 Acp70A ADsubS40 ADsubS30 E74 ADsubF62 ADsubFa

D. subobscura 31B 18C 35B 2 7A 22C 19A 20A 2OB 35C 34E 20E 28B D. madeirensis 31B 18C 35B 2 7A 22C 19A 20A 2OB 35C 34E 2OE 28B D. guanche 31B 18C 35B 2 7A 220 19A 2OA 20B 35C 34E 20E 28B D. pseudoobscura 38 23 40 40 35 21 25 25 35 23 37 21 D. persirnilis 39 24 40 40 35 23 26 26 35 24 26 23 D. miranda 9 c - 1 0A 9F 13A - 18B 18A 13A - I1E D. melanogaster 610 63BC 66B 6 7B 67B 69A 70A 70DE 72F 74F 76A 7 7B

~~

-

See Table 2 for notation of markers.

143 Evolution of D and E Elements

TABLE 4

Cytological location of the 11 probes on Muller’s element E of the different species studied

Species xDsubFC4 ADsubS14 A n y hsp70 Xdh ADsubS9 ADsubG2 xDsubS6 Acph-1 $49 Cec

D. subobscura 83B 98D 80C 89A-94A 86C 90D 89A 83C 91C 91C 99C D. madeirasis 83B 980 80C 89A-94A 86C 90D 89A 83C 91C 91C 99C D. guanche 820 980 80C 89A-94A 86C 90D 89A 83B 91C 91C 99C D. pseudoobscura 49 52 50 58-53 55 54 53 48 62 62 62 D. persirnilis 48 52 50 58-53 55 54 53 47 62 62 62 D. miranda 49 46B 48 45-38 44c 43c 45 50 35 35 35 D. melanogaster 82F 83D 84A 87A-87C 870 95C 95E 96E 99D 99D 99E

See Table 2 for notation of markers.

semed their genic content during the divergence of the species studied.

This conclusion is in agreement with previous stud- ies on the location of other single-copy probes in spe- cies of the obscura group and in D. melanogaster (STEINEMANN 1982; STEINEMANN et al. 1984; KRISHNAN

et al. 1991). However, in contrast with our results, some exchanges between elements have also been de- scribed. These cases are generally associated with mul- tiple-copy genes like 5SRNA genes, the histone genes (STEINEMANN 1982; STEINEMANN et al. 1984; FELGER and PINSKER 1987) or tRNA genes (TONZETICH et al. 1990). Exchange of multiple-copy genes between dif- ferent elements has been discussed as a result of the action of transposable elements (FELGER and PINSKER 1987; TONZETICH et al. 1990). An exchange between elements A and D in the D.

pseudoobscura species cluster has also been reported ( SE- GARRA and AGUADB 1992; SEGARRA et al. 1995). How- ever, these elements are fused in this cluster, and thus this exchange can be easily explained by a pericentric inversion after the centric fusion. This mechanism may also act in other species with chromosomes generated by centric fusions, e.g., chromosome 3 of D. melanogaster resulting from a centric fusion of D and E elements. In the present study, however, no exchanges between element D (3L chromosomal arm) and element E (3R chromosomal arm) of D. melanogaster have been de- tected. This is consistent with the low probability of fixation of pencentric inversions, due to the production of aneuploid gametes that leads to low fitness when heterozygous (CHARLESWORTH et al. 1987).

The present results allow us to test the homologies proposed previously within elements D and E in these species. Studies on interspecific hybrids between D. su- bobscura and D. madeirensis (KRIMBAS and LOUKAS 1984; PAPACEIT and PREVOSTI 1991) indicate that both are homosequential for the J chromosome ( D element), which agrees with the identical distribution of markers in those species (Figure 1). Moreover, interspecific hy- brids indicate that element E differs between them by the O3 inversion (KRIMBAS and LOUKAS 1984; PAPACEIT and PREVOSTI 1991) consistent with our observation of an inverted orientation of Acph-l-rp49hsp70/2 (Figure

2). Additionally, the mapping of Acph-I at the most proximal band of section 9lCindicates that this marker is located very close to the breakpoint 91BC of the 0, inversion.

D. subobscura and D. guanche differ with respect to element D by an inversion located at sections 3OA-34E (MOLTO et al. 1987), which causes the inverted orienta- tion of E74 and emc genes between the two species (Fig- ure 1). The inverted position of E74 (section 34E) indi- cates that this gene is located within the inversion and very close to its distal breakpoint. In fact, no chromo- some band can be distinguished between the E74 hy- bridization signal and the proposed inversion break- point. In addition, the location of E74 allows definitive rejection of the distal breakpoint 34A previously sug- gested by KRIMBAS and LOUKAS (1984) for this inversion since it could not explain the inverted orientation of E74. The E elements of D. subobscura and D. guanche also differ by the 0, inversion and by an additional overlapping inversion with breakpoints mapped at sec- tions 84D/85A-94B/94A by MOLTO et al. (1991), consis- tent with the inverted orientation of ADsubS9-hsp70/ I-ADsubG2-Xdh (Figure 2). hsP70/2 that maps at section 94A, though not included in this inversion, must be located very near to its breakpoint 94B/94A. The differ- ent location of ADsubFy and ADsubFC4 in D. subobscura and D. madeirensis with respect to D. guanche (Tables 3 and 4) is due to a slightly different interpretation of the location of subsections in the photographic map of the latter species with respect to KUNZE-MUHL and MULLER’S (1958) cytological map.

Element D in D. pseudoobscura and D. persimilis differs by an inversion, revealed by the study of interspecific hybrids. MOORE and TAYLOR (1986) considered that this inversion involves sections 26and ?7, modifying the pre- vious suggestion (sections 30 and 38) of DOBZHANSKY and TAN (1936). This inversion accounts for the in- verted orientation of ADsubFy and xDsubS30 between these two species (Figure 1). Moreover, xDsubF62 (sec- tion 37 in D. pseudoobscura), though not included in this inversion, must be located very near to its distal breakpoint. In fact, the ADsubF62 marker allows us to map more accurately this breakpoint within section 37 (Figure 3). This is in contrast with MOORE and TAYLOR’S

144 C. Segarra, G. Rib6

FIGURE 3. -In situ hybridization of hDsubF62 on D. pseudoob s c u m (A) and D. persirnilis (B) polytene chromosomes. Hybrid- ization signals are arrowheaded. Comparison of both photo- graphs clearly indicates that hDsubF62 is not affected by the inversion that differentiates element D of these species. The corrected distal breakpoint of this inversion is indicated by an arrow. The location of the breakpoint previously proposed by MOORE and TAYLOR (1986) is shown by a line. Micrographs were obtained at -8OOX.

(1986) map, where this inversion affects the whole of section 37. Thus, part of section 26 in their map corre- sponds to the distal part of section 37 of the D. pseudoob scua map (STOCJXER and KASTRITSIS 1972). This misin- terpretation accounts for the different location of hDsubF62 in D. pseudoobscura (section 37) and D. pmim- ilk (section 26) (Table 3). The E elements of these two species differ by an inversion of sections 52-56 (DOBZ- HANSKY and TAN 1936; MOORE and TAYLOR 1986), ac- counting for the inverted orientation of Xdh-hDsubS9- hsp7O/l-~subGZ-hDsubSl4 between the species (Figure 2). The slightly different locations of some of the mark- ers when comparing both species (Tables 3 and 4) is probably due to the partial disagreement of section lim- its between D. pseudoobscura and D. perximilis maps.

D. pseudoobscura and D. miranda differ by a small inver- sion in the distal part of element D. According to the cytological map of DOBZHANSKY and TAN (1936), this inversion includes sections 40-42. The inverted orien- tation of emc-hsp28/23-hDsub25 is due to this inversion (Figure 1). Emc, which maps at section 38 of the photo- graphic map by STOCKER and KASTRITSIS (1972), is af- fected by the inversion that thus reveals a disagreement

and M. agUad6

between the cytological and photographic maps of D. psmdoobscura in these sections. Moreover, the species differ by an additional inversion of hsp83-E74Est-5- hDsubFu in the proximal part of the chromosomal arm (Figure 1). This inversion was not previously reported by DOBZHANSKY and TAN (1936). One possible explana- tion is that the strain of D. miranda used in this study has a different gene arrangement for the proximal end than that used by DOBZHANSKY and TAN (1936). In this case, the gene arrangement we have detected should be polymorphic in D. miranda. The presence of this inversion makes it difficult to accurately localize on the DAS et al. (1982) photographic map the markers in- cluded in it (Table 3), since the stock used by these authors is unknown and its arrangement is difficult to ascertain by simple inspection of the banding pattern in their map. D. pseudoobscura and D. miranda differ with respect to element E only by a small inversion that affects section 54 (DoBZHANSKYand TAN 1936) and that explains the inverted position of Xdh and hDsubS9when comparing those species (Figure 2).

In addition to checking previously established in- trachromosomal homologies, the present results can be used to study the homologies between D. melanogaster and the obscura group species. Markers are distributed rather evenly along element D of D. melanogaster, the smallest segment being that delimited by XDsubFy and hsp28/23. None of the studied element D segments can be considered homologous between D. melanogaster and the obscura group species. However, markers are dis- tributed in a more clustered fashion on element E of D. melanogaster. The cluster formed by the most closely linked loci is Acph-l+p49-Cec. Of these loci, only the close linkage between q49 and A@h-1 is maintained in the obscura species. Thus, only one conserved segment has been detected on element E when comparing D. mehnogasterand D. pseudoobscura, for example. Only one conserved segment (z-Pgd) was also detected on ele- ment A of these two species after mapping a total of 32 markers (SEGARRA d al. 1995). These conserved seg- ments are among the smallest chromosomal segments studied. The presence of conserved segments shared by D. melanogaster and the obscura group species allows inference of the ancestral relative order of some loci before the split of the melanogaster and obscura groups of Drosophila. According to the most parsimonious ex- planation, the close linkage between $49 and Acph-1 (element E ) must be ancestral to the divergence of the species studied. The same argument can be applied to element A conserved segments (SEGARRA et al. 1995): z-Pgd (disrupted in the D. subobscura lineage) and c-DSO1481 (disrupted in the D. pseudoobscura lineage).

Moreover, the present results also allow inference of chromosomal homologies of elements D and E be- tween, for instance, D. subobscura (as representative of the Palearctic obscura group species cluster) and D. pseudoobscura (as representative of the Nearctic cluster).

Evolution of D and E Elements 145

ELEMENT D

hsp83 Acp7OA ADsubF62 ADsubFy Est-5 , k k u b S l O

hsp28 /23 ADsubFa ADsubS25

emc E74 LDsubS30 D. subobscura

D. gseudoobscura ADsubFa E 7 4 Acp7OA ADsubS30 ADsubF62 emC LDsubS25

Est-5 hsp83 ADsubS4O asubFy hsp28 i23

ELEMENT E

D. subobscura

D. pseudoobscura

Antp ADsubFC4 ADsubS6 xdh LDsubGZ ADsubS9 rp49 hsp7OiZ aDsubS1 4 hsp7Oi1 Acph-l Cec

LDsubS6 ADsubFC4 Antp ADsubGZ ADsubS9 h s p 7 0 / 2 Acph-l Cec

ADsubS14 hsp70/1 Xdh rp4 9

FIGURE 4.-Schematic comparison of the distribution of markers along elements D (top) and E (bottom) between D. subobscura and D. pseudoobscura. Identical markers from both species are connected by thin lines. See Table 2 for notation of markers.

Despite conservation of the genic content of elements D and E, considerable reorganization within each ele- ment is detected between these species (Figure 4). In- spection of this figure seems to indicate that reshuffling has affected element D more than E. Nine of the E element markers map on the distal half of this element in both species. In addition, in element D there are markers, such as E74, that move more drastically from one part of the chromosome to the other. Apart from this qualitative observation, three conserved segments (Cec-telomere, Acph-I-rp49 and ADsubG2-hsp70/1) were detected in element E, but only one (Acp70A-ADsubS40) in element D, all delimited by closely linked markers. Although the detection of a higher number of con- served segments in element E may be partly attributable to our happening to have studied a higher number of tightly linked markers in this element, this cannot be the only explanation. In fact, the segments E74-hsp83 and hsp28/23ADsubS25 on element D are also delimited by closely linked markers in D. pseudoobscura, and these segments have clearly been disrupted in D. subobscura. Thus, element E seems to be more conserved than D. However, a larger number of markers is required to estimate the number of inversions differentiating ele- ments D and E of D. subobscura and D. pseudoobscura. Even if this number is higher for element D, estimates of the evolutionary rates of both chromosomal elements

between the obscura species and an outgroup species (D. melanogaster) would be required to detect in which lineage (D. subobscura or D. pseudoobscura) element D has evolved faster. Only this relative rate test would allow us to contrast the hypothesis (CHARLESWORTH et al. 1987) of a higher rate of fixation of chromosomal inversions in the D. pseudoobscura lineage, given the sex chromosomal character of element D in this species.

We thank authors cited in Table 2 for providing the different probes and R. J. MACINTYRE for sharing unpublished results and clones of Acph-I in D. melanogaster. We also thank J. BRAVERMAN and J. ROZAS for critically reading the manuscript. This work was s u p ported by grants PB91-0245 and PB940923 from DirecciBn General de InvestigaciBn Cientifica y Tkcnica, Ministerin de EducaciBn y Cien- cia, Spain and by grant GRQ93-1100 from Comissi6 Interde- partamental de Recerca i Innovaci6 Tecnologica, Generalitat de Cata- lunya.

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