Hsp70 Duplication in the Drosophila melanogaster Species Group: How and When Did Two Become Five?

Brian R. Bettencourt and Martin E. Feder

Department of Organismal Biology and Anatomy
Committee on Evolutionary Biology, University of Chicago


    Abstract
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Supplementary Material
 Acknowledgements
 References
 
To determine how the modern copy number (5) of hsp70 genes in Drosophila melanogaster evolved, we localized the duplication events that created the genes in the phylogeny of the melanogaster group, examined D. melanogaster genomic sequence to investigate the mechanisms of duplication, and analyzed the hsp70 gene sequences of Drosophila orena and Drosophila mauritiana. The initial two-to-four hsp70 duplication occurred 10–15 MYA, according to fixed in situ hybridization to polytene chromosomes, before the origin and divergence of the melanogaster and five other species subgroups of the melanogaster group. Analysis of more than 30 kb of flanking sequence surrounding the hsp70 gene clusters suggested that this duplication was likely a retrotransposition. For the melanogaster subgroup, Southern hybridization and an hsp70 restriction map confirmed the conserved number (4) and arrangement of hsp70 genes in the seven species other than D. melanogaster. Drosophila melanogaster is unique; tandem duplication and gene conversion at the derived cluster yielded a fifth hsp70 gene. The four D. orena hsp70 genes are highly similar and concertedly evolving. In contrast, the D. mauritiana hsp70 genes are divergent, and many alleles are nonfunctional. The proliferation, concerted evolution, and maintenance of functionality in the D. melanogaster hsp70 genes is consistent with the action of natural selection in this species.


    Introduction
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Supplementary Material
 Acknowledgements
 References
 
Five hsp70 genes evolved from two in Drosophila melanogaster's recent evolutionary past (Leigh Brown and Ish-Horowicz 1981Citation ). A key step in this process was the duplication of an ancient, stable, and highly conserved two-copy hsp70 gene cluster (fig. 1 ). Feder and Krebs (1998)Citation hypothesized that the hsp70 cluster duplication was a recent, derived feature of the eight-species melanogaster subgroup that may have facilitated the evolution of thermotolerance and niche expansion in D. melanogaster and Drosophila simulans and that the hsp70 gene/protein is of key adaptive significance to these species. However, as the phylogenetic relatedness, hsp70 gene number, and hsp70 nucleotide sequence(s) of several allied species and species subgroups remain unknown, these hypotheses were untested. Here, we localize the hsp70 cluster duplication in the phylogeny of the large melanogaster species group to determine when two genes became four, utilize D. melanogaster genomic sequence to determine how various mechanisms of duplication and gene conversion created the five genes, and, finally, examine hsp70 nucleotide sequences of related Drosophila species to determine the adaptive significance of the gene family's molecular evolution.



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Fig. 1.—Simplified Drosophila phylogeny (left) with hsp70 genome schematic (right) of selected groups. Question marks indicate gaps in knowledge at the outset of this study. Dark arrows represent the number and distribution of hsp70 coding sequences, compiled from (1) Gubenko and Baricheva (1979)Citation ; (2) Peters, Lubsen, and Sondermeijer (1980)Citation ; (3) Bonorino et al. (1993)Citation ; (4) Molto et al. (1992)Citation ; (5) Konstantopoulou, Nikolaidis, and Scouras (1998)Citation ; and (6) Leigh Brown and Ish-Horowicz (1981)Citation

 
Localizing the phylogenetic placement of the initial two-to-four duplication is challenging because the phylogenetic relationships of many species subgroups within the melanogaster group are enigmatic. Only three main lineages are well-supported: the ananassae and montium subgroups form separate lineages, while the melanogaster subgroup clusters with the suzukii, takahashii, ficusphila, eugracilis, and elegans subgroups (Ashburner 1989Citation a; see fig. 1 ). Larger-scale relationships within Drosophila, however, are now well supported (fig 1 ).

Hsp70 gene number/organization is now known for many of these taxa. The primitive condition for the genus Drosophila is two hsp70 genes at one cluster in a characteristic inverted-pair arrangement (fig. 1 ). In species groups other than melanogaster, the hsp70-containing cytological locus is single and resides in Muller's E element, according to analyses of heat-induced transcriptional puffing (in repleta, virilis, willistoni, and obscura spp.; see Peters, Lubsen, and Sondermeijer [1980Citation ]; Gubenko and Baricheva [1979Citation ]; Bonorino et al. [1993Citation ]; and Molto et al. [1992Citation ], respectively). Within the melanogaster group, Konstantopoulou, Nikolaidis, and Scouras (1998)Citation recently characterized the hsp70 genes of Drosophila auraria, a member of the large montium species subgroup. This species retains the ancestral condition, with two hsp70 genes arranged as an inverted pair on the E element. The montium subgroup is basal to an as-yet-unresolved cluster of six species subgroups (see above), including the eight-species melanogaster subgroup (Pelandakis and Solignac 1993Citation ; Russo, Takezaki, and Nei 1995Citation ). Within the melanogaster subgroup, Leigh Brown and Ish-Horowicz (1981)Citation established that the hsp70 cluster is duplicated in Drosophila yakuba, Drosophila teissieri, Drosophila simulans, Drosophila mauritiana (four genes each), and D. melanogaster (the only species with five genes; see fig. 1 ).

At the outset of this research, Drosophila orena and Drosophila erecta, basal members of the subgroup, remained unexamined (fig. 1 ). Thus, the two-to-four hsp70 duplication clearly postdates the origin of the melanogaster group, but could predate, coincide with, or have occurred after the radiation of the melanogaster subgroup. Here, we resolve this uncertainty by determining both the number of hsp70-containing chromosomal loci in representative species from all species subgroups within the melanogaster group and hsp70 endonuclease data for previously unexamined members of the melanogaster subgroup.

How did the two-to-four duplication event occur? Simple duplication via unequal crossing over seems unlikely, given that the two hsp70 clusters are some 500 kb apart and no classical or molecular genetic studies have yet identified additional duplicated genes in either the intervening or the flanking regions. However, unequal crossing over followed by a large-scale chromosomal rearrangement could yield the long distance between the gene clusters. Chromosomal arrangements differ extensively among the ananassae, montium, and melanogaster subgroups (Hinton and Downs 1975Citation ; Lemeunier and Ashburner 1984Citation ; Drosopoulou et al. 1997Citation ). The polytene chromosomes of the other subgroups remain too poorly mapped, however, to determine whether these subgroups share a rearrangement containing both hsp70 clusters. Alternatively, the additional hsp70 cluster could be inserted elsewhere via double crossing over with homologous flanking sequences (e.g., simple or inverted repeats). Finally, the lack of introns in the hsp70 coding sequences, the presence of polyA tails, and numerous simple repetitive sequences within and around the hsp70 clusters are all consistent with a transposition-mediated mechanism (see GenBank accession number X78403; Mason et al. 1982Citation ; Konstantopoulou et al. 1995Citation ).

Apparently, simple tandem duplication gave rise to the fifth hsp70 gene at 87C1 in D. melanogaster (Leigh Brown and Ish-Horowicz 1981Citation ). However, contrasting patterns of 5'- and 3'-flanking sequence homology among the three hsp70 genes at 87C1 suggest a more complex, mosaic history (Torok et al. 1982Citation ; Bettencourt 2001Citation ). Here, we analyze the genomic sequence around and between the hsp70 clusters of D. melanogaster to determine which mechanisms created first four, and then five hsp70 genes.

Finally, the D. melanogaster hsp70 genes were created and are maintained as functional copies by concerted evolution, vary and respond to laboratory and natural selection (Bettencourt 2001)Citation , and play a critical role in inducible thermotolerance, a phenotype with demonstrable fitness effects in nature (Feder and Krebs 1997Citation ). These facts suggest that this species exploited "preadaptive" duplications for adaptive evolution. Other species possessing duplicate hsp70 genes need not have done the same; accordingly, here we examine hsp70 sequences of D. mauritiana, a closely related species that inhabits a different ecological niche (David et al. 1989Citation ).


    Materials and Methods
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Supplementary Material
 Acknowledgements
 References
 
Fly Stocks
Drosophila ananassae, Drosophila eugracilis, Drosophila takahashii, Drosophila ficusphila, Drosophila lucipennis, Drosophila elegans, and D. yakuba were obtained from the National Drosophila Species Center (Bowling Green, Mich.; stock numbers 0371.0, 0451.0, 0311.0, 0441.0, 0331.0, 0461.0, and 0261.0, respectively). Drosophila auraria, D. teissieri, D. mauritiana, Drosophila sechellia, and D. erecta stocks were obtained from C.-I. Wu (University of Chicago). Drosophila orena was provided by C. Laurie (Duke University). Drosophila simulans (strain DSRT) was provided by T. Karr (University of Chicago). All stocks were maintained at 22°C, 12 h : 12 h light : dark, on cornmeal-based medium in standard rearing vials.

DNA Probes
Probes for both fixed in situ hybridization (FISH) and genomic Southern blotting were prepared via PCR using the plasmid pDM300 as template (a gift of S. L. Lindquist). pDM300 contains one D. melanogaster hsp70 gene from the 87C1 cluster (McGarry and Lindquist 1986Citation ). For FISH to polytene chromosomes, a biotin-labeled probe of 542 bp (approximately the third quartile of the ~2-kb hsp70 coding sequence) was amplified with biotin-labeled dUTP (Boehringer Mannheim) and the primers HSP70-U (5'-AAAGTAAGCCGTGCCAGGTT-3') and D-L (5'-ACTTCTATCTGGGGCACACC-3'). For Southern blotting, a DIG-labeled probe of 1,022 bp (the 3' half of the coding sequence) was amplified with DIG-labeled dUTP (Boehringer Mannheim) and the primers HSP70-U (see above) and A-L (5'-CCAGAGTAGCCGCCAAATCC-3'). This probe is highly specific for hsp70 genes (for details and amplification conditions, see Bettencourt, Feder, and Cavicchi 1999Citation ).

FISH
To obtain larvae for dissection, 5–15 mated females of each species were allowed to oviposit on a soft banana-based medium for 3 days at 18°C. Adults were then cleared and larvae were allowed to develop at 18°C, with dry yeast added approximately 1 week later. Salivary glands of wandering third-instar larvae were dissected, and polytene chromosomes were prepared according to Ashburner (1989b). After overnight hybridization at 65°C with the biotin-labeled probe, chromosomes were reacted with biotin-specific antibodies (Boehringer Mannheim) and mounted in the presence of FITC-conjugated DAPI. Chromosomes were visualized with a Nikon fluorescence microscope and a digital CCD camera.

Southern Blotting
Genomic DNA was extracted from ~75 adult flies of each species with 1:1 phenol-chloroform and resuspended in 50 µl of TE buffer. Ten micrograms of DNA was digested with appropriate restriction enzymes (Promega), electrophoresed on 0.5%–1% agarose gels, and transferred to Nytran nylon membranes (Schliecher and Schuell). Hybridization to the DIG-labeled probe was conducted overnight at 65°C according to the Boehringer-Mannheim DIG/Genius User's Guide (http://biochem.boehringer-mannheim.com/). Blots were visualized with CSPD chemiluminescent substrate (Boehringer Mannheim). Exposures to Fuji X-ray film varied from 15 to 45 min. After development, films were scanned on an Agfa flatbed scanner and images were processed in Adobe Photoshop.

Mapping of the D. orena and D. erecta hsp70 genes followed the restriction maps for D. melanogaster, D. simulans, and D. mauritiana generated by Leigh Brown and Ish-Horowicz (1981)Citation . Figure 2A summarizes these map data.



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Fig. 2.—Hsp70 restriction maps. CDS = hsp70 coding sequence. Arrows indicate direction of transcription. Restriction site symbols are identical in both maps. A, Map of sites conserved in Drosophila melanogaster (fifth hsp70 gene, at 87C1, not shown), Drosophila simulans, and Drosophila mauritiana, according to data of Leigh Brown and Ish-Horowicz (1981)Citation . The map not to scale; arrows on either side of selected sites indicate that their positions may change. A star indicates a polymorphic D. mauritiana Xho1 site. B, Restriction map of Drosophila orena hsp70 genes generated by this study. The map is drawn to scale (indicated by 1 kb bar)

 
Analysis of D. melanogaster Genomic Sequence
We utilized the draft sequence of the right arm of the third chromosome of D. melanogaster (Celera Genomics Inc.) to estimate the extent of homologous (duplicated) sequence in the 87A7 and 87C1 clusters. Specifically, we examined the 33.5 kb both upstream and downstream of both gene clusters via BLAST (http://www.ncbi.nlm.nih.gov/BLAST/) to determine what (if any) sequences were duplicated along with the hsp70 genes. For reference, the stop codons of hsp70Aa, hsp70Ab, hsp70Ba, and hsp70Bc lie at positions 7746934, 7753245, 8258188, and 8267143 in the "3R.fas" draft sequence, respectively. As most of the hsp70 coding sequences are partially or entirely unresolved in the draft sequence, these positions are noted strictly for convenience and are not reflective of the actual distances between the hsp70 clusters. We compared the sequences beginning 350 bp past the stop codons to exclude the 3' untranslated regions (UTRs), which display paralogy (Torok et al. 1982Citation ).

Isolation and Characterization of the D. orena and D. mauritiana hsp70 Genes
Using both the Celera genomic sequence and predicted transcripts, we designed primers specific for exons in the predicted genes most closely flanking both the 87A7 and the 87C1 hsp70 clusters (accession information: 87A7—upstream, CG18546, downstream, CG3281; 87C1—upstream, CG5608, downstream, CG7078). All four primers—87A7-U, 87A7-L, 87C1-U, and 87C1-L—were used previously, along with the primer START-U (5'-ATGCCTGC(C/T)ATTGGAATCGA-3'), to PCR amplify the hsp70 genes of D. simulans, as described elsewhere (Bettencourt 2001)Citation . PCR was conducted with the MasterAmp kit (Epicentre), which utilizes a proofreader enzyme for fidelity. PCR products were purified, cloned, and sequenced with internal primers and an ABI automated sequencer as described in Bettencourt (2001)Citation . Sequences were aligned using CLUSTALX and analyzed with DNASP (J. Rozas) and SITES (J. Hey).


    Results
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Supplementary Material
 Acknowledgements
 References
 
Duplication of the hsp70 Gene Cluster
The hsp70 gene probe hybridized strongly to single bands on polytene chromosomes of both D. auraria and D. ananassae (fig. 3 ). On D. elegans, D. ficusphila, D. eugracilis, D. lucipennis, D. takahashii, D. orena, and D. erecta chromosomes, two bands yielded equally strong signals (fig. 3 ). Comparison with identically prepared D. melanogaster chromosomes controlled for the intensity, location, and spacing of signals relative to one another (not shown). An additional control for probe specificity examined chromosomes of a D. melanogaster strain bearing an additional transgenic hsp70 gene cluster on the second chromosome (see Welte et al. 1993Citation ); the probe hybridized to the transgenes strongly, in addition to the two wild-type hsp70 clusters on the third chromosome (fig. 4 ).



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Fig. 3.—Hsp70-containing loci (marked by arrows) visualized by FISH on polytene chromosomes of Drosophila species, and localization of locus duplication on a phylogenetic tree. Color originals (available upon request) clearly distinguish between the FISH signal and chromosomal bands (data not shown)

 


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Fig. 4.—Hsp70-containing loci visualized by FISH on polytene chromosomes of a D. melanogaster strain bearing three hsp70 transgenes on the second chromosome. Wild-type hsp70 loci are marked by their cytological positions, transgenes by the arrow

 
In all species, including melanogaster, additional hybridization signals of much lower intensity were evident at other chromosomal loci. In melanogaster, these signals mapped to the other hsp70 family members (hsp68 and the hsc70s; see Holmgren et al. 1979Citation ; Perkins et al. 1990Citation ). The positions of these faint signals were often conserved across species. For example, hsc70-2 lies at 87D11 in melanogaster, distal to the hsp70 genes; in many other species, a faint signal was evident at the same approximate distance from the strongly hybridizing hsp70 clusters (see D. erecta in fig. 3 ; see also Drosopoulou, Konstantopoulou, and Scouras 1996Citation ). This is not surprising given the conserved hsp70, hsp68, and hsc placement on the E element (Drosopoulou et al. 1997Citation ).

Hsp70 Gene Organization in the melanogaster Subgroup
Southern blots performed as positive controls confirmed the map generated by Leigh Brown and Ish-Horowicz (1981)Citation (figs. 2 and 5 ). Digestion of D. simulans and D. mauritiana DNA with Xba1 and Bgl2 yielded four hybridization signals (simulans, ~9, 4, 3, and 2.5 kb; mauritiana ST strain, ~9, 4, 3.5, and 3 kb), each containing one hsp70 gene (fig. 5 ). Different strains of D. mauritiana varied in the sizes of the four hsp70-containing fragments; for example, the G105 strain lacked the ~3.5-kb band but gained a ~6-kb band (data not shown). Beyond this prominent polymorphism, all species display some restriction site polymorphism in the 75-fly genomic preparations and sporadic faint signals. Drosophila sechellia, unmapped by Leigh Brown and Ish-Horowicz (1981)Citation , displays a banding pattern identical to its sister species D. simulans (fig. 5 ). Drosophila yakuba and D. teissieri also possessed four hsp70 genes in the conserved arrangement (fig. 5 ; see also Leigh Brown and Ish-Horowicz 1981). Drosophila yakuba displayed significant restriction site polymorphism; the most informative digest was Xba1/Bgl1, which yielded four signals of ~10, 3, 2.5, and 2.3 kb (fig. 5 ). Digestion of D. teissieri DNA with Xba1/Bgl2 yielded four signals, of ~5, 4, 2.8, and 2.6 kb (fig. 5 ).



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Fig. 5.—Genomic Southern blots of Drosophila species. Each signal-producing fragment bears a single hsp70 gene. Multiple blots are illustrated; lanes were scaled electronically to size standards (run in each blot) illustrated in lanes 1 and 9. Lanes 1 and 9, size standards of 21, 9, 6.5, 4.3, 2.3, and 2 kb. Lane 2, Drosophila orena Bgl1/EcoR1. Lane 3, Drosophila erecta EcoR1. Lane 4, Drosophila yakuba Xba1/Bgl1. Lanes 5–8, Xba1/Bgl2 digests of Drosophila teissieri, Drosophila sechellia, Drosophila simulans, and Drosophila mauritiana, respectively

 
Drosophila orena and D. erecta, basal members of the melanogaster subgroup (Cariou 1987Citation ; Caccone et al. 1996Citation ), both exhibited a restriction pattern consistent with four hsp70 genes in two clusters. Many of the restriction sites conserved in the other melanogaster subgroup species, typically those lying in or near the coding region, were similarly conserved in these species. Flanking (noncoding) sites varied often in position and occasionally in order. The most informative digests for D. orena were Xho1, Xho1/Bgl2, Xho1/Xba1, Xba1/Bgl2, Bgl1, EcoR1, and Bgl1/EcoR1. Digestion with Xho1 yielded four signals: ~18 kb, ~9 kb (double intensity), and ~4 kb. Xho1/Bgl2 yielded four: ~9.5, 9, 7, and 4 kb. Xho1/Xba1 yielded four: ~9, 4.5, 4.3, and 3 kb. Bgl1 yielded four: ~6.5, 5, 4, and 2.5 kb (blots not shown). EcoRI yielded just one band, of very high intensity, of ~8 kb (not shown). Bgl1/EcoR1 yielded four distinct bands of equal intensity, of ~3.5, 2.5, 2.3, and 1.7 kb (fig. 5 ). These results are consistent with the map illustrated in figure 2B. For D. erecta, the most informative digestions were EcoR1, Xba1/EcoR1, Xba1/Bgl2, and Bgl1. Digestion with EcoR1 yielded four signals, of ~10, 7, 6, and 4 kb (fig. 5 ). Xba1/EcoR1 yielded four: ~10, 6, 5, and 4.5 kb. Bgl1 yielded three: ~4 kb (double intensity), 2.5, and 2 kb (not shown). Clearly, this species has diverged from its sister species orena in hsp70 restriction sites, which is not surprising considering their divergence in chromosome arrangement (Lemeunier and Ashburner 1984Citation ; Ashburner 1989Citation a). Nonetheless, in combination with the FISH results (see above), the observed Southern hybridization patterns are consistent with four hsp70 genes being present in two clusters. Drosophila orena and D. erecta, the basal members of the melanogaster subgroup, have four hsp70s in two clusters: the two-to-four hsp70 duplication predates the divergence of the subgroup.

Unit of Duplication in the Two-to-Four Event
In all four pairwise comparisons (87A7 upstream vs. 87C1 upstream, 87A7 downstream vs. 87C1 downstream, 87A7 upstream vs. 87C1 downstream, 87A7 downstream vs. 87C1 upstream), basic filtered BLAST (http://www.ncbi.nlm.nih.gov/BLAST) failed to reveal any homologous sequences in the ~33.5 kb flanking the hsp70 genes. Only one unfiltered BLAST revealed a putatively homologous run of T nucleotides (TTTTTTTTTTTTTTTTTTTGCT) ~29.3 kb and ~28.3 kb upstream of 87A7 and 87C1, respectively (in reverse orientation, upstream of 87C1).

Using the GeneScene application (http://www.fruitfly.org/annot/genescene-launch-static.html), we identified the four predicted genes that most closely flanked the 87A7 and 87C1 hsp70 clusters. The four genes, listed in order of increasing distance from the centromere and labeled with accession and distance from the nearby hsp70 gene (measured in base pairs between the beginning or end of the closest predicted exon and hsp70 stop codon), were 87A7 (CG18546, 442 bp upstream; CG3281, 165 bp downstream) and 87C1 (CG5608, 333 bp upstream; CG7078, 1,569 bp downstream). All four of the genes are completely nonhomologous with respect to one another. For example, CG3281 encodes a 1,854-bp zinc-finger-containing transcription factor, while CG7078, whose transcript is 2,090 bp, encodes a G protein-linked receptor. Neither is repeated at either 87A7 or 87C1, further suggesting that the sequences flanking both of the hsp70 clusters are novel and completely unrelated. Only the hsp70 genes are duplicated.

The intergenic regions of 87A7 and 87C1 (between the hsp70 genes) were not compared. Previous workers found that these regions were no longer similar: at 87C1, a large (>40 kb) amount of repetitive sequence lay between the proximal and distal hsp70 genes, as compared with the 1–2 kb at 87A7 (see GenBank entries J01103 and K01293). Both regions were unresolved (N's) in the Celera sequence.

Origin and Molecular Evolution of a Fifth hsp70 Gene at 87C1 in D. melanogaster
The Celera genomic sequence reveals that the hsp70Bb/hsp70Bc region is of mosaic origin, with both gene conversion and duplication likely contributing (fig. 6 ). As noted by previous authors, the 3'-flanking region of hsp70Bb is nearly identical to that of hsp70Ba for ~350 bp (Torok et al. 1982Citation ; Bettencourt 2001Citation ). In the genomic sequence, this homologous region extends for 545 bp past the polyA addition signal of hsp70Bb and includes 234 bp of CG5608-derived sequence (in reverse orientation, bearing a 39-bp deletion; see fig. 6 ). Continuing in the 3' orientation from hsp70Bb, 38 bp of unique sequence precedes 209 bp of hsp70Bbhsp70Bc intergenic sequence that is 93% identical (excluding a 26-bp indel) to that found 5' of hsp70Bb (fig. 6 ). The hsp70Bc 5' UTR immediately follows, differing from that of hsp70Bb at only 3 of 330 sites (fig. 6 ). Thus, duplicated sequence flanks hsp70Bb on both sides, consistent with tandem duplication. The portion of hsp70Bb's 3' flank that is homologous to hsp70Ba and CG5608 likely arose via gene conversion (fig. 6 ). Conversion between hsp70Ba and hsp70Bb also occurs in the coding regions (Bettencourt 2001)Citation .



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Fig. 6.—Schematic of the 87C1 locus of Drosophila melanogaster, with alignment of sequences involved in the evolution of the hsp70Bb/hsp70Bc region. Solid colors indicate coding regions (or sequences derived from coding regions); hatched regions are noncoding. Shared hatch mark type indicates high (>90%) sequence similarity. Hatching for alignment is identical to schematic. C.R. = converted region. Triangles mark deletions. Ba3'Rc = reverse complement of hsp70Ba 3'-flanking sequence. BbBcIG = hsp70Bb/hsp70Bc intergenic region. Ba 5', Bb 5', Bc 5' = 5'-flanking regions of hsp70Ba, hsp70Bb, and hsp70Bc, respectively

 
The D. orena hsp70 Gene Family: Homogenized and Nondegenerate, Like Those of D. simulans and D. melanogaster
The four hsp70 genes of D. orena are 96.6% identical in their 1,929–1,932-bp coding regions, echoing the pattern observed in D. simulans and D. melanogaster, whose hsp70 genes are highly homogenized (Bettencourt 2001)Citation . Beginning at position 283 and continuing through the stop codon, the hsp70Ab allele in our sample resembled hsp70Ba, consistent with intercluster conversion (fig. 7 ). This type of homogenizing intercluster conversion among coding sequences is common in the hsp70 gene family (Leigh Brown and Ish-Horowicz 1981Citation ; Bettencourt 2001Citation ). Most differences among genes are silent (63 silent, 19 replacement, 1 indel codon), and all four genes encode full-length Hsp70 proteins of 642–643 amino acids.



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Fig. 7.—Nucleotide polymorphism in the hsp70 coding sequences of Drosophila orena. Nucleotide numbering ("Pos") begins at the first base of the start codon. Synonymous and replacement substitutions are labeled S and R, respectively. Nucleotides identical to the first sequence are indicated by dots; deletions are indicated by dashes. Gray bars indicate putative intercluster conversion tracts

 
The D. mauritiana hsp70 Gene Family: Divergence and Concerted Degeneration
In contrast to the D. orena hsp70 gene family, whose members are all apparently functional and highly similar to one another, many D. mauritiana hsp70 alleles are likely nonfunctional and/or divergent (fig. 8 ). Of the eight D. mauritiana hsp70 sequences analyzed (two alleles of each gene), five are likely pseudogenes. For example, both hsp70Aa{Psi} alleles bear a 5-bp deletion at positions 555–560 that takes the coding sequence out of frame and introduces numerous premature stop codons. These alleles thus encode a frameshifted and truncated Hsp70 protein of 186 amino acids. Hsp70Ab1 encodes a full-length Hsp70 protein, although it differs from all other genes/alleles by a 15-bp insertion at positions 1850–1865. This region of the hsp70 gene is evolutionarily labile and contains similar indels in D. simulans and D. melanogaster (Bettencourt 2001)Citation . The hsp70Ab{Psi} allele is a pseudogene; a 5-bp deletion at positions 889–893 takes it out of frame. Similar to the D. orena hsp70Ab sequence above, hsp70Ab{Psi} likely results from intercluster conversion: it differs from the two hsp70Ba{Psi} alleles at only three sites. Another likely intercluster conversion tract marks positions 557–565 on the hsp70Ab1 allele. Both hsp70Ba{Psi} alleles in the sample are also pseudogenes, sharing the same deletion at positions 889–893. The hsp70Ab{Psi} and hsp70Ba{Psi} pseudogenes encode a frameshifted and truncated Hsp70 protein of 399 amino acids. Hsp70Ab4 and both hsp70Ba{Psi} alleles also share a 9-bp deletion in the labile region (positions 1842–1849). Both hsp70Bb alleles encode full-length Hsp70 proteins bearing one deleted codon in the labile region (positions 1847–1849).



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Fig. 8.—Nucleotide polymorphism in the hsp70 coding sequences of Drosophila mauritiana. Nucleotide numbering ("Pos") begins at the first base of the start codon. Synonymous and replacement substitutions are labeled "S" and "R," respectively. Nucleotides identical to the first sequence are indicated by dots; deletions are indicated by dashes. Gray bars indicate putative intercluster conversion tracts

 

    Discussion
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Supplementary Material
 Acknowledgements
 References
 
Both in situ and Southern hybridization data are consistent with duplication of the hsp70 gene cluster having occurred before the divergence of the melanogaster, ficusphila, elegans, takahashii, suzukii, and eugracilis species subgroups from the ananassae and montium subgroups. This result agrees with analyses of other traits, which divided the melanogaster group into three main lineages; the ananassae subgroup, the montium subgroup, and the remaining six subgroups (Lemeunier, David, and Tsacas 1986Citation ; Ashburner 1989Citation a; Clark, Maddison, and Kidwell 1994Citation ). That all six subgroups share the hsp70 cluster duplication does not resolve this polytomy. According to Russo, Takezaki, and Nei's (1995)Citation estimate of the divergence time of the montium and melanogaster subgroups, the hsp70 cluster duplication occurred <=12.7 ± 1.88 MYA. Thus, while the initial duplication event that created the hsp70 inverted pair is ancient and stable (as old as the Diptera; see Benedict, Cockburn, and Seawright 1993Citation ), the two-to-four hsp70 duplication is far younger. The subgroups sharing a duplicated hsp70 cluster—melanogaster, ficusphila, elegans, takahashii, suzukii, and eugracilis—originated, diverged, and radiated into >=40 species in a relatively short time (Lemeunier, David, and Tsacas 1986Citation ).

The two-to-four duplication event was likely a retrotransposition: no other duplicated sequences flank the 87A7 and 87C1 gene clusters of D. melanogaster. In addition, the hsp70 genes have no introns, are surrounded by simple repetitive DNA (Mason et al. 1982Citation ), and bear polyA tails (at least in the case of the ancestral two-copy hsp70 cluster of D. auraria described in Konstantopoulou et al. [1995Citation ]). Whether the intergenic region at either gene cluster retains any mobile element–derived sequence remains to be investigated. The origin of the fifth hsp70 gene in D. melanogaster was more complex: tandem duplication and remodeling via gene conversion likely formed the mosaic hsp70Bb/hsp70Bc region.

Duplicated genes often either diverge toward new functions or degenerate toward non- or subfunctionality (Nei and Hughes 1992Citation ; Lynch and Force 2000Citation ). Instead, the hsp70 genes of D. melanogaster persist as functional duplicate copies, consistent with one adaptive role of Hsp70 expression, inducible thermotolerance (Feder and Krebs 1997, 1998Citation ). Drosophila melanogaster exploits its five hsp70 genes to achieve efficient, extremely high Hsp70 protein expression (Bettencourt, Feder, and Cavicchi 1999Citation ). The high inducible thermotolerance conferred by this expression affords survival of a wide range of thermal challenges across the species' broad distribution (Feder, Blair, and Figueras 1997Citation ; Roberts and Feder 1999Citation ). Drosophila simulans expresses nearly as much Hsp70 and is often as thermotolerant with its four hsp70s (Krebs 1999Citation ), which are all functional gene copies (Bettencourt 2001)Citation . Duplication is a relatively simple and effective way to increase expression of a selected gene (Spofford 1972Citation ), and evolution may have used this mechanism (among others) to increase Hsp70 expression in these species. Sequence analysis of D. melanogaster and D. simulans is consistent with this assertion: both species' hsp70 genes display rapid concerted evolution and strong purifying selection on polymorphisms that are shared among genes (Leigh Brown and Ish-Horowicz 1981Citation ; Bettencourt and Feder 1998, 1999Citation ; Bettencourt 2001Citation ).

In contrast, the hsp70 gene family of D. mauritiana is degenerate. Null alleles of three of the four hsp70 genes are at high frequencies; four or five functional hsp70 genes are apparently not necessary for survival. Purifying selection alone is thus perhaps not likely to be responsible for the maintenance of duplicate functional hsp70 gene copies in D. simulans and D. melanogaster; natural selection may favor their function. In the absence of data directly linking the presence of null alleles with reduced exposure to thermal stress and/or functional consequences for Hsp70 expression and thermotolerance in any of these species, this is an untested but exciting hypothesis for future study.

Duplication of the hsp70 cluster may have been a key step in the evolutionary pathway of D. melanogaster and D. simulans toward increased Hsp70 expression, inducible thermotolerance, niche breadth, and geographic range (Krebs and Feder 1997Citation ). This pathway, however, is but one of several available for populations evolving environmental stress tolerance. Additionally, the two-to-four hsp70 duplication is not the only step, considerably predated the escape of D. melanogaster and D. simulans from their ancestral niche and range, and has not conferred a similar ecological/geographic breadth on the other species that share it. Other species in the melanogaster subgroup, all with four hsp70 genes, exhibit narrower Hsp70 "expression curves" sensu Bettencourt, Feder, and Cavicchi (1999)Citation , are less inducibly thermotolerant, and have far more restricted ranges than D. melanogaster (Stanley et al. 1980Citation ; Lachaise et al. 1988Citation ; Benson 1998Citation ). In some of these species, such as D. mauritiana, selection on the duplicated hsp70 sequences themselves appears to be relaxed due to a reduced functional role, and thus alleles at both 87A and 87C are null. However, D. orena, also restricted in range and thermotolerance, possesses four apparently functional hsp70 genes. Clearly, whether selection acts on the hsp70 genes themselves can vary in a lineage- and gene-specific fashion. Furthermore, while hsp70 duplication does not necessitate an evolutionary increase in Hsp70 expression and thermotolerance, the converse is true: several other drosophilid groups have achieved high Hsp70 expression, thermotolerance, and broad distributions with single hsp70 clusters (Krebs 1999Citation ; Krebs and Bettencourt 1999Citation ). Numerous regulatory mechanisms can increase Hsp70 expression given a constant number of hsp70 copies (Lindquist, DiDomenico, and Bugaisky 1982Citation ; Petersen and Lindquist 1989Citation ; Morimoto et al. 1994Citation ). Analysis of hsp70 sequence in these single-cluster species could elucidate the evolutionary acquisition of high Hsp70 expression and thermotolerance via regulation rather than duplication.

In conclusion, the hsp70 genome of D. melanogaster has undergone amplification during its evolutionary past in a pattern consistent with an adaptive role. This provides further support for the hypothesis that hsp70 is a target of strong natural selection in D. melanogaster, a hypothesis now supported by multiple lines of evidence (e.g., variation in nature [Krebs and Feder 1997Citation ], response to laboratory selection [Bettencourt, Feder, and Cavicchi 1999Citation ], link to fitness in the wild [Roberts and Feder 1999Citation ], and concerted evolution via gene conversion and natural selection [Bettencourt 2001Citation ]). However, in other species, both closely and distantly related, different evolutionary means have been used to achieve similar ends.


    Supplementary Material
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Supplementary Material
 Acknowledgements
 References
 
GenBank accession numbers for hsp70 sequences are as follows: D. orena, AF302410–AF302413; D. mauritiana, AF302414–AF302421.


    Acknowledgements
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Supplementary Material
 Acknowledgements
 References
 
We thank W. Wang and M. Long (University of Chicago) for their invaluable assistance with FISH, and J. Gladstone for sequencing. Earlier versions of this manuscript were greatly improved by comments from D. Rand and one anonymous reviewer. This work was supported by a National Science Foundation grant (IBN-9723298) to M.E.F. and a Howard Hughes Medical Institute Predoctoral Fellowship to B.R.B.


    Footnotes
 
David M. Rand, Reviewing Editor

1 Keywords: Drosophila hsp70, gene duplication concerted evolution Back

2 Address for correspondence and reprints: Brian R. Bettencourt, Department of Organismal Biology and Anatomy, University of Chicago, 1027 East 57th Street, Chicago, Illinois 60637. E-mail: b-bettencourt{at}uchicago.edu Back


    References
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 Materials and Methods
 Results
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 Acknowledgements
 References
 

    Ashburner M., 1989a. Drosophila: a laboratory handbook Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y

    ———. 1989b. Drosophila: a laboratory manual Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y

    Benedict M. Q., A. F. Cockburn, J. A. Seawright, 1993 The Hsp70 heat-shock gene family of the mosquito Anopheles albimanus. Insect Mol. Biol 2:93-102[Medline]

    Benson D. A., 1998 The evolution of Hsp70-mediated thermotolerance in Drosophila. Undergraduate honors thesis, University of Chicago

    Bettencourt B. R., 2001 Molecular and phenotypic adaptation of hsp70 and thermotolerance Ph.D. thesis, University of Chicago

    Bettencourt B. R., M. E. Feder, 1998 Molecular evolution of the Drosophila hsp70 gene family Am. Zool 38:159A.

    ———. 1999 Gene conversion and natural selection drive hsp70 molecular evolution in Drosophila. Am. Zool 39:15A.

    Bettencourt B. R., M. E. Feder, S. Cavicchi, 1999 Experimental evolution of Hsp70 expression and thermotolerance in Drosophila melanogaster. Evolution 53:484-492[ISI]

    Bonorino C. B. C., M. Pereira, C. E. V. Alonso, V. L. S. Valente, E. Abdelhay, 1993 In situ mapping of the hsp70 locus in seven species of the willistoni group of Drosophila. Rev. Bras. Genet 16:561-571[ISI]

    Caccone A., E. N. Moriyama, J. M. Gleason, L. Nigro, J. R. Powell, 1996 A molecular phylogeny for the Drosophila melanogaster subgroup and the problem of polymorphism data Mol. Biol. Evol 13:1224-1232[Abstract]

    Cariou M. L., 1987 Biochemical phylogeny of the eight species in the Drosophila melanogaster subgroup, including D. sechellia and D. orena. Genet. Res 50:181-186[ISI][Medline]

    Clark J. B., W. P. Maddison, M. G. Kidwell, 1994 Phylogenetic analysis supports horizontal transfer of P transposable elements Mol. Biol. Evol 11:40-50[Abstract]

    David J. R., S. F. Mcevey, M. Solignac, L. Tsacas, 1989 Drosophila communities on Mauritius and the ecological niche of Drosophila mauritiana Diptera Drosophilidae Rev. Zool. Afr 103:107-116

    Drosopoulou E., I. Konstantopoulou, Z. G. Scouras, 1996 The heat shock genes in the Drosophila montium subgroup: chromosomal localization and evolutionary implications Chromosoma 105:104-110[ISI][Medline]

    Drosopoulou E., M. Tsiafouli, P. Mavragani-Tsipidou, Z. G. Scouras, 1997 The glutamate dehydrogenase, E74 and putative actin gene loci in the Drosophila montium subgroup. Chromosomal homologies among the montium species and D. melanogaster. Chromosoma 106:20-28[ISI][Medline]

    Feder M. E., N. Blair, H. Figueras, 1997 Natural thermal stress and heat-shock protein expression in Drosophila larvae and pupae Funct. Ecol 11:90-100[ISI]

    Feder M. E., R. A. Krebs, 1997 Ecological and evolutionary physiology of heat-shock proteins and the stress response in Drosophila: complementary insights from genetic engineering and natural variation Pp. 155–173 in R. Bijlsma and V. Loeschcke, eds. Stress, adaptation, and evolution. Birkhäuser Verlag, Basel, Switzerland

    ———. 1998 Natural and genetic engineering of thermotolerance in Drosophila melanogaster. Am. Zool 38:503-517[ISI]

    Gubenko I. S., E. M. Baricheva, 1979 Drosophila virilis puffs induced by temperature and other environmental factors Genetika 15:1399-1414[ISI]

    Hinton C. W., J. E. Downs, 1975 The mitotic, polytene, and meiotic chromosomes of Drosophila ananassae. J. Hered 66:353-361[ISI][Medline]

    Holmgren R., K. Livak, R. Morimoto, R. Freund, M. Meselson, 1979 Studies of cloned sequences from four Drosophila heat shock loci Cell 18:1359-1370[ISI][Medline]

    Konstantopoulou I., N. Nikolaidis, Z. G. Scouras, 1998 The hsp70 locus of Drosophila auraria (montium subgroup) is single and contains copies in a conserved arrangement Chromosoma 107:577-586[ISI][Medline]

    Konstantopoulou I., C. A. Ouzounis, E. Drosopoulou, M. Yiangou, P. Sideras, C. Sander, Z. G. Scouras, 1995 A Drosophila hsp70 gene contains long, antiparallel, coupled open reading frames (LAC ORFs) conserved in homologous loci J. Mol. Evol 41:414-420[ISI][Medline]

    Krebs R. A., 1999 A comparison of Hsp70 expression and thermotolerance in adults and larvae of three Drosophila species Cell Stress Chap 4:243-249[ISI][Medline]

    Krebs R. A., B. R. Bettencourt, 1999 Evolution of thermotolerance and variation in the heat shock protein, HSP70 Am. Zool 39:910-919[ISI]

    Krebs R. A., M. E. Feder, 1997 Natural variation in the expression of the heat-shock protein Hsp70 in a population of Drosophila melanogaster, and its correlation with tolerance of ecologically relevant thermal stress Evolution 51:173-179[ISI]

    Lachaise D., M. L. Cariou, J. R. David, F. Lemeunier, L. Tsacas, M. Ashburner, 1988 Historical biogeography of the Drosophila melanogaster species subgroup Evol. Biol 22:159-225[ISI]

    Leigh Brown A. J., D. Ish-Horowicz, 1981 Evolution of the 87A and 87C heat-shock loci in Drosophila. Nature 290:677-682[ISI][Medline]

    Lemeunier F., M. Ashburner, 1984 Relationships within the Drosophila melanogaster species subgroup of the genus Drosophila (Sophophora): 4. The chromosomes of 2 new species Chromosoma 89:343-351[ISI]

    Lemeunier F., J. R. David, L. Tsacas, 1986 The melanogaster species group Pp. 148–256 in M. Ashburner, H. L. Carson, and J. N. Thompson, eds. The genetics and biology of Drosophila. Vol. 3e. Academic Press, London

    Lindquist S. L., B. J. DiDomenico, G. Bugaisky, 1982 Regulation of the heat shock response in Drosophila and yeast Pp. 167–176 in M. J. Schlesinger, M. Ashburner, and A. Tissieres, eds. Heat shock from bacteria to man. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y

    Lynch M., A. Force, 2000 The probability of duplicate gene preservation by subfunctionalization Genetics 154:459-473[Abstract/Free Full Text]

    McGarry T. J., S. Lindquist, 1986 Inhibition of heat shock protein synthesis by heat-inducible antisense RNA Proc. Natl. Acad. Sci. USA 83:399-403[Abstract]

    Mason P. I., I. Torok, I. Kiss, F. Karch, A. Udvardy, 1982 Evolutionary implications of a complex pattern of DNA sequence homology extending far upstream of the hsp70 genes at loci 87A7 and 87C1 in Drosophila melanogaster. J. Mol. Biol 156:21-35[ISI][Medline]

    Molto M. D., L. Pascual, M. J. Martinez-Sebastian, R. De Frutos, 1992 Genetic analysis of heat shock response in three Drosophila species of the obscura group Genome 35:870-880[ISI][Medline]

    Morimoto R. I., D. A. Jurivich, P. E. Kroeger, S. K. Mathur, S. P. Murphy, A. Nakai, K. Sarge, K. Abravaya, L. T. Sistonen, 1994 Regulation of heat shock gene transcription by a family of heat shock factors Pp. 417–456 in R. I. Morimoto, A. Tissieres, and C. Georgopoulos, eds. The biology of heat shock proteins and molecular chaperones. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y

    Nei M., A. L. Hughes, 1992 Balanced polymorphism and evolution by the birth-and-death process in the MHC loci Pp. 27–38 in K. Tsuji, M. Aizawa, and T. Sasazuki, eds. Eleventh Histocompatibility Workshop and Conference. Vol. 2. Oxford University Press, Oxford, England

    Pelandakis M., M. Solignac, 1993 Molecular phylogeny of Drosophila based on ribosomal RNA sequences J. Mol. Evol 37:525-543[ISI][Medline]

    Perkins L. A., J. S. Doctor, K. Zhang, L. Stinson, N. Perrimon, E. A. Craig, 1990 Molecular and developmental characterization of the heat shock cognate 4 gene of Drosophila melanogaster. Mol. Cell. Biol 10:3232-3238[ISI][Medline]

    Peters F. P., N. H. Lubsen, P. J. Sondermeijer, 1980 Rapid sequence divergence in a heat shock locus of Drosophila. Chromosoma 81:271-280[ISI][Medline]

    Petersen R. B., S. Lindquist, 1989 Regulation of HSP70 synthesis by messenger RNA degradation Cell. Regul 1:135-149[ISI][Medline]

    Roberts S. P., M. E. Feder, 1999 Natural hyperthermia and expression of the heat shock protein Hsp70 affect developmental abnormalities in Drosophila melanogaster. Oecologia 121:323-329[ISI]

    Russo C. A. M., N. Takezaki, M. Nei, 1995 Molecular phylogeny and divergence times of drosophilid species Mol. Biol. Evol 12:391-404[Abstract]

    Spofford J. B., 1972 A heterotic model for the evolution of duplications Brookhaven Symp. Biol 23:121-143[Medline]

    Stanley S. M., P. A. Parsons, G. E. Spence, L. Weber, 1980 Resistance of species of the Drosophila melanogaster subgroup to environmental extremes Aust. J. Zool 28:413-421[ISI]

    Torok I., P. J. Mason, F. Karch, I. Kiss, A. Udvardy, 1982 Extensive regions of homology associated with heat-induced genes at loci 87A7 and 87C1 in Drosophila melanogaster. Pp. 19–25 in M. J. Schlesinger, M. Ashburner, and A. Tissieres, eds. Heat shock from bacteria to man. Cold Spring Harbor Laboratory Press, New York

    Welte M. A., J. M. Tetrault, R. P. Dellavalle, S. L. Lindquist, 1993 A new method for manipulating transgenes: engineering heat tolerance in a complex, multicellular organism Curr. Biol 3:842-853[ISI]

Accepted for publication March 13, 2001.