Molecular heterochrony in the early development of Drosophila

Heterochrony, the relative change of developmental timing, is one of the major modes of macroevolutionary change; it identifies temporally disassociated units of developmental evolution. Here, we report the results of a fine-scale temporal study for the expression of the developmental gene hairy and morphological development in three species of Drosophila, D. melanogaster, D. simulans, and D. pseudoobscura. The results suggest that between and among closely related species, temporal displacement of ontogenetic trajectory is detected even at the earliest stage of development. Overall, D. simulans shows the earliest expression, followed by D. melanogaster, and then by D. pseudoobscura. Setting D. melanogaster as the standard, we find the approximate time to full expression is accelerated by 13 min, 48 s in D. simulans and retarded by 24 min in D. pseudoobscura. Morphologically, again with D. melanogaster setting the standard, initiation of cellularization is faster in D. simulans by 15 min, 42 s; and initiation of morphogenesis is faster in D. simulans by 18 min, 7 s. These results seem to be consistent with the finding that the approximate time to full expression of hairy is accelerated by 13 min, 48 s in D. simulans. On the other hand, the same morphological events are delayed by 5 min, 32 s, and by 11 min, 32 s, respectively, in D. pseudoobscura. These delays are small, compared with the 24-min delay in full expression. The timing changes, in total, seem consistent with continuous phyletic evolution of temporal trajectories. Finally, we speculate that epigenetic interactions of hairy expression timing and cell-cycle timing may have led to morphological differences in the terminal system of the larvae.

Molecular studies suggest that cartilaginous fishes have a terminal position in the piscine tree

The Chondrichthyes (cartilaginous fishes) are commonly accepted as being sister group to the other extant Gnathostomata (jawed vertebrates). To clarify gnathostome relationships and to aid in resolving and dating the major piscine divergences, we have sequenced the complete mtDNA of the starry skate and have included it in phylogenetic analysis along with three squalomorph chondrichthyans—the common dogfish, the spiny dogfish, and the star spotted dogfish—and a number of bony fishes and amniotes. The direction of evolution within the gnathostome tree was established by rooting it with the most closely related non-gnathostome outgroup, the sea lamprey, as well as with some more distantly related taxa. The analyses placed the chondrichthyans in a terminal position in the piscine tree. These findings, which also suggest that the origin of the amniote lineage is older than the age of the oldest extant bony fishes (the lungfishes), challenge the evolutionary direction of several morphological characters that have been used in reconstructing gnathostome relationships. Applying as a calibration point the age of the oldest lungfish fossils, 400 million years, the molecular estimate placed the squalomorph/batomorph divergence at ≈190 million years before present. This dating is consistent with the occurrence of the earliest batomorph (skates and rays) fossils in the paleontological record. The split between gnathostome fishes and the amniote lineage was dated at ≈420 million years before present.

Molecular phylogenetic analysis of evolutionary trends in stonefly wing structure and locomotor behavior

Insects in the order Plecoptera (stoneflies) use a form of two-dimensional aerodynamic locomotion called surface skimming to move across water surfaces. Because their weight is supported by water, skimmers can achieve effective aerodynamic locomotion even with small wings and weak flight muscles. These mechanical features stimulated the hypothesis that surface skimming may have been an intermediate stage in the evolution of insect flight, which has perhaps been retained in certain modern stoneflies. Here we present a phylogeny of Plecoptera based on nucleotide sequence data from the small subunit rRNA (18S) gene. By mapping locomotor behavior and wing structural data onto the phylogeny, we distinguish between the competing hypotheses that skimming is a retained ancestral trait or, alternatively, a relatively recent loss of flight. Our results show that basal stoneflies are surface skimmers, and that various forms of surface skimming are distributed widely across the plecopteran phylogeny. Stonefly wings show evolutionary trends in the number of cross veins and the thickness of the cuticle of the longitudinal veins that are consistent with elaboration and diversification of flight-related traits. These data support the hypothesis that the first stoneflies were surface skimmers, and that wing structures important for aerial flight have become elaborated and more diverse during the radiation of modern stoneflies.

Evidence that ultraviolet markings are associated with patterns of molecular gene flow

Recent studies have shown UV vision and markings to be important in vertebrates, particularly birds, where behavioral experiments have demonstrated its potential importance in sexual selection. However, there has been no genetic evidence that UV markings determine patterns of evolution among natural populations. Here we report molecular evidence that UV markings are associated with the pattern of gene flow in the Tenerife lizard (Gallotia galloti). This species has vicariance-induced, approximate east–west lineages in Tenerife closely congruent with the primary lineages of the sympatric gecko species. Against expectations, these molecular phylogeographic lineages (representing geological history) and isolation-by-distance do not appear to influence gene flow. Sexually mature males from populations either side of a latitudinal ecotone have different UV markings and gene flow appears to be linked to this difference in UV markings. It may be that these groups with different UV sexual markings mate assortatively, restricting the gene flow between them. This has implications for debate on the relative importance of vicariance and biotopes in influencing biodiversity, with this evidence supporting the latter.

Evolution of genome–phenome diversity under environmental stress

The genomic era revolutionized evolutionary biology. The enigma of genotypic-phenotypic diversity and biodiversity evolution of genes, genomes, phenomes, and biomes, reviewed here, was central in the research program of the Institute of Evolution, University of Haifa, since 1975. We explored the following questions. (i) How much of the genomic and phenomic diversity in nature is adaptive and processed by natural selection? (ii) What is the origin and evolution of adaptation and speciation processes under spatiotemporal variables and stressful macrogeographic and microgeographic environments? We advanced ecological genetics into ecological genomics and analyzed globally ecological, demographic, and life history variables in 1,200 diverse species across life, thousands of populations, and tens of thousands of individuals tested mostly for allozyme and partly for DNA diversity. Likewise, we tested thermal, chemical, climatic, and biotic stresses in several model organisms. Recently, we introduced genetic maps and quantitative trait loci to elucidate the genetic basis of adaptation and speciation. The genome–phenome holistic model was deciphered by the global regressive, progressive, and convergent evolution of subterranean mammals. Our results indicate abundant genotypic and phenotypic diversity in nature. The organization and evolution of molecular and organismal diversity in nature at global, regional, and local scales are nonrandom and structured; display regularities across life; and are positively correlated with, and partly predictable by, abiotic and biotic environmental heterogeneity and stress. Biodiversity evolution, even in small isolated populations, is primarily driven by natural selection, including diversifying, balancing, cyclical, and purifying selective regimes, interacting with, but ultimately overriding, the effects of mutation, migration, and stochasticity.

Integrated fossil and molecular data reconstruct bat echolocation

Molecular and morphological data have important roles in illuminating evolutionary history. DNA data often yield well resolved phylogenies for living taxa, but are generally unattainable for fossils. A distinct advantage of morphology is that some types of morphological data may be collected for extinct and extant taxa. Fossils provide a unique window on evolutionary history and may preserve combinations of primitive and derived characters that are not found in extant taxa. Given their unique character complexes, fossils are critical in documenting sequences of character transformation over geologic time and may elucidate otherwise ambiguous patterns of evolution that are not revealed by molecular data alone. Here, we employ a methodological approach that allows for the integration of molecular and paleontological data in deciphering one of the most innovative features in the evolutionary history of mammals—laryngeal echolocation in bats. Molecular data alone, including an expanded data set that includes new sequences for the A2AB gene, suggest that microbats are paraphyletic but do not resolve whether laryngeal echolocation evolved independently in different microbat lineages or evolved in the common ancestor of bats and was subsequently lost in megabats. When scaffolds from molecular phylogenies are incorporated into parsimony analyses of morphological characters, including morphological characters for the Eocene taxa Icaronycteris, Archaeonycteris, Hassianycteris, and Palaeochiropteryx, the resulting trees suggest that laryngeal echolocation evolved in the common ancestor of fossil and extant bats and was subsequently lost in megabats. Molecular dating suggests that crown-group bats last shared a common ancestor 52 to 54 million years ago.

Vagaries of the molecular clock

The hypothesis of the molecular evolutionary clock asserts that informational macromolecules (i.e., proteins and nucleic acids) evolve at rates that are constant through time and for different lineages. The clock hypothesis has been extremely powerful for determining evolutionary events of the remote past for which the fossil and other evidence is lacking or insufficient. I review the evolution of two genes, Gpdh and Sod. In fruit flies, the encoded glycerol-3-phosphate dehydrogenase (GPDH) protein evolves at a rate of 1.1 × 10−10 amino acid replacements per site per year when Drosophila species are compared that diverged within the last 55 million years (My), but a much faster rate of ≈4.5 × 10−10 replacements per site per year when comparisons are made between mammals (≈70 My) or Dipteran families (≈100 My), animal phyla (≈650 My), or multicellular kingdoms (≈1100 My). The rate of superoxide dismutase (SOD) evolution is very fast between Drosophila species (16.2 × 10−10 replacements per site per year) and remains the same between mammals (17.2) or Dipteran families (15.9), but it becomes much slower between animal phyla (5.3) and still slower between the three kingdoms (3.3). If we assume a molecular clock and use the Drosophila rate for estimating the divergence of remote organisms, GPDH yields estimates of 2,500 My for the divergence between the animal phyla (occurred ≈650 My) and 3,990 My for the divergence of the kingdoms (occurred ≈1,100 My). At the other extreme, SOD yields divergence times of 211 My and 224 My for the animal phyla and the kingdoms, respectively. It remains unsettled how often proteins evolve in such erratic fashion as GPDH and SOD.

The evolution of plant nuclear genes

We analyze the evolutionary dynamics of three of the best-studied plant nuclear multigene families. The data analyzed derive from the genes that encode the small subunit of ribulose-1,5-bisphosphate carboxylase (rbcS), the gene family that encodes the enzyme chalcone synthase (Chs), and the gene family that encodes alcohol dehydrogenases (Adh). In addition, we consider the limited evolutionary data available on plant transposable elements. New Chs and rbcS genes appear to be recruited at about 10 times the rate estimated for Adh genes, and this is correlated with a much smaller average gene family size for Adh genes. In addition, duplication and divergence in function appears to be relatively common for Chs genes in flowering plant evolution. Analyses of synonymous nucleotide substitution rates for Adh genes in monocots reject a linear relationship with clock time. Replacement substitution rates vary with time in a complex fashion, which suggests that adaptive evolution has played an important role in driving divergence following gene duplication events. Molecular population genetic studies of Adh and Chs genes reveal high levels of molecular diversity within species. These studies also reveal that inter- and intralocus recombination are important forces in the generation allelic novelties. Moreover, illegitimate recombination events appear to be an important factor in transposable element loss in plants. When we consider the recruitment and loss of new gene copies, the generation of allelic diversity within plant species, and ectopic exchange among transposable elements, we conclude that recombination is a pervasive force at all levels of plant evolution.

Biochemical evolution III: Polymerization on organophilic silica-rich surfaces, crystal–chemical modeling, formation of first cells, and geological clues

Catalysis at organophilic silica-rich surfaces of zeolites and feldspars might generate replicating biopolymers from simple chemicals supplied by meteorites, volcanic gases, and other geological sources. Crystal–chemical modeling yielded packings for amino acids neatly encapsulated in 10-ring channels of the molecular sieve silicalite-ZSM-5-(mutinaite). Calculation of binding and activation energies for catalytic assembly into polymers is progressing for a chemical composition with one catalytic Al–OH site per 25 neutral Si tetrahedral sites. Internal channel intersections and external terminations provide special stereochemical features suitable for complex organic species. Polymer migration along nano/micrometer channels of ancient weathered feldspars, plus exploitation of phosphorus and various transition metals in entrapped apatite and other microminerals, might have generated complexes of replicating catalytic biomolecules, leading to primitive cellular organisms. The first cell wall might have been an internal mineral surface, from which the cell developed a protective biological cap emerging into a nutrient-rich “soup.” Ultimately, the biological cap might have expanded into a complete cell wall, allowing mobility and colonization of energy-rich challenging environments. Electron microscopy of honeycomb channels inside weathered feldspars of the Shap granite (northwest England) has revealed modern bacteria, perhaps indicative of Archean ones. All known early rocks were metamorphosed too highly during geologic time to permit simple survival of large-pore zeolites, honeycombed feldspar, and encapsulated species. Possible microscopic clues to the proposed mineral adsorbents/catalysts are discussed for planning of systematic study of black cherts from weakly metamorphosed Archaean sediments.

Flower color variation: A model for the experimental study of evolution

We review the study of flower color polymorphisms in the morning glory as a model for the analysis of adaptation. The pathway involved in the determination of flower color phenotype is traced from the molecular and genetic levels to the phenotypic level. Many of the genes that determine the enzymatic components of flavonoid biosynthesis are redundant, but, despite this complexity, it is possible to associate discrete floral phenotypes with individual genes. An important finding is that almost all of the mutations that determine phenotypic differences are the result of transposon insertions. Thus, the flower color diversity seized on by early human domesticators of this plant is a consequence of the rich variety of mobile elements that reside in the morning glory genome. We then consider a long history of research aimed at uncovering the ecological fate of these various flower phenotypes in the southeastern U.S. A large body of work has shown that insect pollinators discriminate against white phenotypes when white flowers are rare in populations. Because the plant is self-compatible, pollinator bias causes an increase in self-fertilization in white maternal plants, which should lead to an increase in the frequency of white genes, according to modifier gene theory. Studies of geographical distributions indicate other, as yet undiscovered, disadvantages associated with the white phenotype. The ultimate goal of connecting ecology to molecular genetics through the medium of phenotype is yet to be attained, but this approach may represent a model for analyzing the translation between these two levels of biological organization.

Evolution of C4 Photosynthesis in Flaveria Species1 : Isoforms of NADP-Malic Enzyme

NADP-malic enzyme (NADP-ME, EC 1.1.1.40), a key enzyme in C4 photosynthesis, provides CO2 to the bundle-sheath chloroplasts, where it is fixed by ribulose-1,5-bisphosphate carboxylase/oxygenase. We characterized the isoform pattern of NADP-ME in different photosynthetic species of Flaveria (C3, C3-C4 intermediate, C4-like, C4) based on sucrose density gradient centrifugation and isoelectric focusing of the native protein, western-blot analysis of the denatured protein, and in situ immunolocalization with antibody against the 62-kD C4 isoform of maize. A 72-kD isoform, present to varying degrees in all species examined, is predominant in leaves of C3 Flaveria spp. and is also present in stem and root tissue. By immunolabeling, NADP-ME was found to be mostly localized in the upper palisade mesophyll chloroplasts of C3 photosynthetic tissue. Two other isoforms of the enzyme, with molecular masses of 62 and 64 kD, occur in leaves of certain intermediates having C4 cycle activity. The 62-kD isoform, which is the predominant highly active form in the C4 species, is localized in bundle-sheath chloroplasts. Among Flaveria spp. there is a 72-kD constitutive form, a 64-kD form that may have appeared during evolution of C4 metabolism, and a 62-kD form that is necessary for the complete functioning of C4 photosynthesis.

Crystal structure of D-amino acid oxidase: a case of active site mirror-image convergent evolution with flavocytochrome b2.

D-amino acid oxidase is the prototype of the FAD-dependent oxidases. It catalyses the oxidation of D-amino acids to the corresponding alpha-ketoacids. The reducing equivalents are transferred to molecular oxygen with production of hydrogen peroxide. We have solved the crystal structure of the complex of D-amino acid oxidase with benzoate, a competitive inhibitor of the substrate, by single isomorphous replacement and eightfold averaging. Each monomer is formed by two domains with an overall topology similar to that of p-hydroxybenzoate hydroxylase. The benzoate molecule lays parallel to the flavin ring and is held in position by a salt bridge with Arg-283. Analysis of the active site shows that no side chains are properly positioned to act as the postulated base required for the catalytic carboanion mechanism. On the contrary, the benzoate binding mode suggests a direct transfer of the substrate alpha-hydrogen to the flavin during the enzyme reductive half-reaction.The active site Of D-amino acid oxidase exhibits a striking similarity with that of flavocytochrome b2, a structurally unrelated FMN-dependent flavoenzyme. The active site groups (if these two enzymes are in fact superimposable once the mirror-image of the flavocytochrome b2 active site is generated with respect to the flavin plane. Therefore, the catalytic sites of D-amino acid oxidase and flavocytochrome b2 appear to have converged to a highly similar but enantiomeric architecture in order to catalvze similar reactions (oxidation of alpha-amino acids or alpha-hydroxy acids), although with opposite stereochemistry.

Molecular cloning of violaxanthin de-epoxidase from romaine lettuce and expression in Escherichia coli.

Plants need to avoid or dissipate excess light energy to protect photosystem II (PSII) from photoinhibitory damage. Higher plants have a conserved system that dissipates excess energy as heat in the light-harvesting complexes of PSII that depends on the transthylakoid delta pH and violaxanthin de-epoxidase (VDE) activity. To our knowledge, we report the first cloning of a cDNA encoding VDE and expression of functional enzyme in Escherichia coli. VDE is nuclear encoded and has a transit peptide with characteristic features of other lumen-localized proteins. The cDNA encodes a putative polypeptide of 473 aa with a calculated molecular mass of 54,447 Da. Cleavage of the transit peptide results in a mature putative polypeptide of 348 aa with a calculated molecular mass of 39,929 Da, close to the apparent mass of the purified enzyme (43 kDa). The protein has three interesting domains including (i) a cysteine-rich region, (ii) a lipocalin signature, and (iii) a highly charged region. The E. coli expressed enzyme de-epoxidizes violaxanthin sequentially to antheraxanthin and zeaxanthin, and is inhibited by dithiothreitol, similar to VDE purified from chloroplasts. This confirms that the cDNA encodes an authentic VDE of a higher plant and is unequivocal evidence that the same enzyme catalyzes the two-step mono de-epoxidation reaction. The cloning of VDE opens new opportunities for examining the function and evolution of the xanthophyll cycle, and possibly enhancing light-stress tolerance of plants.

A new high molecular weight immunoglobulin class from the carcharhine shark: implications for the properties of the primordial immunoglobulin.

All immunoglobulins and T-cell receptors throughout phylogeny share regions of highly conserved amino acid sequence. To identify possible primitive immunoglobulins and immunoglobulin-like molecules, we utilized 3' RACE (rapid amplification of cDNA ends) and a highly conserved constant region consensus amino acid sequence to isolate a new immunoglobulin class from the sandbar shark Carcharhinus plumbeus. The immunoglobulin, termed IgW, in its secreted form consists of 782 amino acids and is expressed in both the thymus and the spleen. The molecule overall most closely resembles mu chains of the skate and human and a new putative antigen binding molecule isolated from the nurse shark (NAR). The full-length IgW chain has a variable region resembling human and shark heavy-chain (VH) sequences and a novel joining segment containing the WGXGT motif characteristic of H chains. However, unlike any other H-chain-type molecule, it contains six constant (C) domains. The first C domain contains the cysteine residue characteristic of C mu1 that would allow dimerization with a light (L) chain. The fourth and sixth domains also contain comparable cysteines that would enable dimerization with other H chains or homodimerization. Comparison of the sequences of IgW V and C domains shows homology greater than that found in comparisons among VH and C mu or VL, or CL thereby suggesting that IgW may retain features of the primordial immunoglobulin in evolution.

The molecular biology of apoptosis.

All multicellular organisms have mechanisms for killing their own cells, and use physiological cell death for defence, development, homeostasis, and aging. Apoptosis is a morphologically recognizable form of cell death that is implemented by a mechanism that has been conserved throughout evolution from nematode to man. Thus homologs of the genes that implement cell death in nematodes also do so in mammals, but in mammals the process is considerably more complex, involving multiple isoforms of the components of the cell death machinery. In some circumstances this allows independent regulation of pathways that converge upon a common end point. A molecular understanding of this mechanism may allow design of therapies that either enhance or block cell death at will.