SUMMARY Sexually dimorphic traits play key roles in animal evolution and behavior. Little is known, however, about the mechanisms governing their development and evolution. One recently evolved dimorphic trait is the male-specific abdominal pigmentation of Drosophila melanogaster, which is repressed in females by the Bric-à-brac (Bab) proteins. To understand the regulation and origin of this trait, we have identified and traced the evolution of the genetic switch controlling dimorphic bab expression. We show that the HOX protein Abdominal-B (ABD-B) and the sex-specific isoforms of Doublesex (DSX) directly regulate a bab cis-regulatory element (CRE). In females, ABD-B and DSXF activate bab expression whereas in males DSXM directly represses bab, which allows for pigmentation. A new domain of dimorphic bab expression evolved through multiple fine-scale changes within this CRE, whose ancestral role was to regulate other dimorphic features. These findings reveal how new dimorphic characters can emerge from genetic networks regulating pre-existing dimorphic traits.

Butterfly wings display pattern elements of many types and colors. To identify the molecular processes underlying the generation of these patterns, several butterfly cognates of Drosophila appendage patterning genes have been cloned and their expression patterns have been analyzed. Butterfly wing patterns are organized by two spatial coordinate systems. One system specifies positional information with respect to the entire wing field and is conserved between fruit flies and butterflies. A second system, superimposed on the general system and involving several of the same genes, operates within each wing subdivision to elaborate discrete pattern elements. Eyespots, which form from discrete developmental organizers, are marked by Distal-less gene expression. These circular pattern elements appear to be generated by a process similar to, and perhaps evolved from, proximodistal pattern formation in insect appendages.

The origin of new morphological characters is a long-standing problem in evolutionary biology. Novelties arise through changes in development, but the nature of these changes is largely unknown. In butterflies, eyespots have evolved as new pattern elements that develop from special organizers called foci. Formation of these foci is associated with novel expression patterns of the Hedgehog signaling protein, its receptor Patched, the transcription factor Cubitus interruptus, and the engrailed target gene that break the conserved compartmental restrictions on this regulatory circuit in insect wings. Redeployment of preexisting regulatory circuits may be a general mechanism underlying the evolution of novelties.

It has been proposed that the evolution of homeotic genes parallels, and to some degree directs, the evolution of segment diversity in the myriapod-insect lineage. But the discovery of discrete Antennapedia complex (ANT-C) and bithorax complex (BX-C) gene members in crustacea, chelicerates, annelids and various insects, as well as in vertebrates, indicates that the expansion and diversification of homeotic genes preceded the diversification of arthropods and insects. How, then, have these genes influenced the evolution of body plans? To address this question, we now examine homeotic gene expression and regulation in butterflies (Lepidoptera), which, unlike flies, possess larval abdominal limbs and two pairs of wings. We show that the difference in larval limb number between these insects results from striking changes in BX-C gene regulation in the butterfly abdomen, and we deduce that the wing-patterning genes regulated by Ultrabithorax have diverged in the course of butterfly and fly evolution. These findings have general implications for the role of homeotic genes in animal evolution.

SUMMARY The genetic origin of novel traits is a central but challenging puzzle in evolutionary biology. Among snakes, phospholipase A2 (PLA2)-related toxins have evolved in different lineages to function as potent neurotoxins, myotoxins, or hemotoxins. Here, we traced the genomic origin and evolution of PLA2 toxins by examining PLA2 gene number, organization, and expression in both neurotoxic and non-neurotoxic rattlesnakes. We found that even though most North American rattlesnakes do not produce neurotoxins, the genes of a specialized heterodimeric neurotoxin predate the origin of rattlesnakes and were present in their last common ancestor (~22 mya). The neurotoxin genes were then deleted independently in the lineages leading to the Western Diamondback (Crotalus atrox) and Eastern Diamondback (C. adamanteus) rattlesnakes (~6 mya), while a PLA2 myotoxin gene retained in C. atrox was deleted from the neurotoxic Mojave rattlesnake (C. scutulatus; ~4 mya). The rapid evolution of PLA2 gene number appears to be due to transposon invasion that provided a template for non-allelic homologous recombination.

The genetic origins of novelty are a central interest of evolutionary biology. Most new proteins evolve from preexisting proteins but the evolutionary path from ancestral gene to novel protein is challenging to trace, and therefore the requirements for and order of coding sequence changes, expression changes, or gene duplication are not clear. Snake venoms are important novel traits that are comprised of toxins derived from several distinct protein families, but the genomic and evolutionary origins of most venom components are not understood. Here, we have traced the origin and diversification of one prominent family, the snake venom metalloproteinases (SVMPs) that play key roles in subduing prey in many vipers. Genomic analyses of several rattlesnake (Crotalus) species revealed the SVMP family massively expanded from a single, deeply conserved adam28 disintegrin and metalloproteinase gene, to as many as 31 tandem genes in the Western Diamondback rattlesnake (Crotalus atrox) through a number of single gene and multigene duplication events. Furthermore, we identified a series of stepwise intragenic deletions that occurred at different times in the course of gene family expansion and gave rise to the three major classes of secreted SVMP toxins by sequential removal of a membrane-tethering domain, the cysteine-rich domain, and a disintegrin domain, respectively. Finally, we show that gene deletion has further shaped the SVMP complex within rattlesnakes, creating both fusion genes and substantially reduced gene complexes. These results indicate that gene duplication and intragenic deletion played essential roles in the origin and diversification of these novel biochemical weapons.

Autoradiography of restriction digests of DNA labeled in early S phase indicates that replication of the amplified dihydrofolate reductase (DHFR) domain of methotrexate-resistant CHOC 400 cells initiates within a 6.1-kilobase pair (kb) EcoRI-doublet located on the 3' side of the DHFR gene. To localize the DHFR origin fragment, synchronized CHOC 400 cells were either pulse labeled with[3H]thymidine in vivo or permeabilized and incubated with[32P]dATP under conditions that support limited chromosomal DNA replication. The temporal order of replication of amplified fragments was determined by hybridization of the in vivo or in vitro replication products to cloned fragments spanning the earliest-replicating portion of the DHFR domain. At the G1/S boundary, the labeled products derived from the replication of amplified sequences, either in whole or permeabilized cells, are distributed about an amplified 4.3-kb Xba I fragment that maps 14 kb downstream from the DHFR gene. As cells progress through the S phase, bidirectional replication away from this site is observed. These studies indicate that the 4.3-kb Xba I fragment contains the origin of replication associated with the amplified DHFR domain.To isolate DNA sequences that serve as initiation sites in mammalian chromosomes, we have studied the replication timing of restriction endonuclease fragments derived from the amplified dihydrofolate reductase (DHFR) domain of a methotrexate-resistant Chinese hamster ovary (CHO) cell line, CHOC 400 (1). The high copy number and homogeneity of the amplified DHFR domains (2) reduce the relative complexity of cellular replication studies several hundred times, thereby permitting functional delineation of the DHFR origin region in intact chromosomes prior to physical and biological characterization of isolated origin sequences.Pulse-labeling of synchronized cells has shown that replication of the amplified DHFR domains is initiated within a specific group of restriction fragments (3). These earlylabeled fragments (ELFs) have been isolated by molecular cloning (4) Ci/mmol; 1 Ci = 37 GBq) or [methyl-'4C]thymidine at 1 kiCi/ml (50-60 mCi/mmol, Amersham). DNA was prepared from lysed cells, digested with restriction enzymes, and blotted as described (4). DNA concentrations were determined fluorometrically (9). For fluorography, gels were impregnated with 5% (wt/vol) 2,5-diphenyloxazole (Sigma) in ethanol, rinsed in H20, dried, and exposed to preflashed x-ray film (XAR-5, Kodak) with an intensifying screen at -70'C for 3 wk. Digests labeled with ['4C]thymidine were separated by agarose gel electrophoresis, transferred to nitrocellulose, and exposed to x-ray film for 20-30 days.Restriction Mapping and Subcloning. Recombinant cosmids S13, S14, and S21 were mapped by agarose gel analysis of restriction digests performed separately or in combination. For subcloning, Xba I fragments from cosmids S13, S14, and S21 were purified from agarose gels by the method of Benson (10), and subcloned into pUC12 or pAN3 by standard ligation and t...

The homeotic genes have long been thought to play an important role in the diversification of arthropod appendages. Using recombinant Sindbis virus, we were able to investigate homeotic gene function in non-model arthropod species. We found that Ultrabithorax is sufficient to confer hindwing identity in butterflies and alter normal development of anterior structures in beetles. Recombinant Sindbis virus has broad potential as a tool for analyzing how the function of developmental genes has changed during the diversification of arthropods.