Methyl Jasmonate Induces Gums and Stimulates Anthocyanin Accumulation in Peach Shoots

The effect of methyl jasmonate (JA-Me) on the induction of gum was studied in relation to the action of ethylene in peach (Prunus persica Batsch cv. Benishimizu) shoots. JA-Me applied at concentrations of 0.1–2.5% (w/w) in lanolin paste to current growing or older shoots substantially induced gums 3 days after treatment. The amount of gums exuded increased depending on the dose of JA-Me. Ethephon (2-chloroethyl- phosphonic acid) at 1 or 2% (w/w) in lanolin induced gum and strongly enhanced the promoting effect of JA-Me on gum formation. JA-Me also induced anthocyanin accumulation in current growing shoots, but ethephon did not. Anthocyanin accumulation in response to JA-Me at a concentration of 10 mg/liter or higher was observed also in the cut shoots of peach. Ethephon (100 mg/liter) substantially inhibited anthocyanin accumulation induced by JA-Me. These facts suggest that JA-Me plays an important role in gum formation as well as ethylene and in anthocyanin accumulation and that these processes are not necessarily accompanied by each other in peach shoots. Received January 26, 1998; accepted March 4, 1998     

Gum Formation by Methyl Jasmonate in Tulip Shoots is Stimulated by Ethylene

The promotive effect of methyl jasmonate (JA-Me) on the induction of gum in tulip shoots (Tulipa gesneriana L. cvs. Gudoshnik and Apeldoorn) was studied in the presence of ethylene. Gum formation in the stem and the basal part of the leaves was induced by JA-Me (1% w/w in lanolin) and stimulated strongly by the simultaneous application of 1 or 5 mm 1-aminocyclopropane-1-carboxylic acid (ACC). JA-Me at a concentration of 0.1% did not induce gum, but that together with ACC at a concentration of 1 or 5 mm induced it substantially. Although JA-Me stimulated ethylene production substantially in the stem of intact tulips, ethephon (1% w/w) or ACC (1 or 5 mm) did not induce gum formation in tulip shoots. JA-Me induced gum formation in tulip shoots even in the presence of aminooxyacetic acid or cobalt ions. Moreover, gum formation was also observed in the cut shoot applied with JA-Me as a solution at concentrations of 0.23 mm or more. These results strongly suggest that JA-Me is required for gum formation in tulip shoots, and ethylene probably makes the tissues of shoots sensitive to JA-Me. Received March 23, 1998; accepted June 10, 1998     

Inhibitory Effects of Methyl Jasmonate on the Germination and Ethylene Production in Cocklebur Seeds

Methyl jasmonate (JA-Me) inhibited the germination of cocklebur (Xanthium pennsylvanicum Wallr.) seeds. The inhibition of the germination of cocklebur seeds treated with JA-Me at concentrations less than 300 μm was nullified by ethylene applied exogenously, although the inhibitory effect of 1,000 μm JA-Me was not recovered completely even by high concentrations of ethylene (10,000 μL/liter). JA-Me inhibited ethylene production before seed germination. The level of 1-aminocyclopropane-1-carboxylic acid (ACC) in the cotyledonary tissues treated with JA-Me decreased but not the level of 1-(malonylamino)cyclopropane-1-carboxylic acid (MACC). JA-Me inhibited the conversion of ACC to ethylene in the tissues. These results suggested that JA-Me inhibits ethylene production by prevention of ACC oxidation in addition to ACC synthesis. We believe that the inhibition of ethylene production by JA-Me results in the retardation of the germination of cocklebur seeds. Received June 4, 1997; accepted October 23, 1997     

Senescence of Flag Leaves and Ears of Wheat Hastened by Methyl Jasmonate

Treatment of flag leaves and ears of wheat plants with MJ (jasmonic acid methylester) (10⁻⁵ and 10⁻⁴ m) did not increase ethylene production, but it did accelerate senescence as indicated by the loss of chlorophyll. MJ also caused the closure of stomata, and consequently the rates of transpiration and photosynthesis decreased. Early maturity shortened the grain filling period, so the thousand grain weight was lower. Although ethylene elicited the same physiologic effects, the syndrome of senescence by MJ is independent of the former. We conclude that senescence and death in wheat are far from being elucidated; however, MJ and ethylene seem to participate in the phenomenon. Received July 10, 1997; accepted January 5, 1998     

Methyl Jasmonate Reduces Water Stress in Strawberry

The effect of methyl jasmonate (MJ) on changes of oxygen-scavenging enzyme activities and membrane lipid composition was studied in strawberry leaves under water stress. Under water stress, MJ treatment reduced the increase of peroxidase (EC 1.11.1.7; POD) activity, maintained higher catalase (EC 1.11.1.6; CAT) and superoxide dismutase (EC 1.15.1.1; SOD) activities, and ascorbic acid content. In addition, MJ treatment reduced transpiration and membrane-lipid peroxidation as expressed by malondialdehyde (MDA) content, lessened the reduction of membrane lipids, glycolipids [monogalactosyl diglyceride (MGDG), digalactosyl diglyceride (DGDG)], and phospholipids [phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), and phosphatidylinositol (PI)]. In water-deficit conditions, MJ treatment also alleviated the decline in the degree of fatty acid unsaturation and the ratio of linolenic (18:3) to linoleic acid (18:2). These results indicate that MJ treatment appears to alter the metabolism of strawberry plants rendering the tissue better able to withstand water stress. Received June 16, 1999; accepted October 1, 1999     

Drought Stress Syndrome in Wheat Is Provoked by Ethylene Evolution Imbalance and Reversed by Rewatering, Aminoethoxyvinylglycine, or Sodium Benzoate

In this work we present evidence that the drought stress syndrome in the flag leaves and ears of wheat plants, provoked by the production of ethylene (shortening the grain filling period and lowering the grain weight) is reversed by the application of a free radical scavenger, sodium benzoate or the ethylene synthesis inhibitor, aminoethoxyvinylglycine. Rehydration by watering also attenuated the detrimental effect of the water deficit. Consequently, the grain filling period was longer, the grain weight increased, and the total protein content was higher than that in plants watered regularly. Received June 11, 1998; accepted March 10, 1999     

Changes of Respiration Rate, Ethylene Evolution, and Abscisic Acid Content in Developing Inflorescence and Young Fruit of Olive (Olea europaea L. cv. Konservolia)

Simultaneous measurements of respiration, ethylene production, and abscisic acid (ABA) concentrations, as well as the growth parameters length, fresh weight (FW), and dry weight (DW) of olive (Olea europaea L. cv. Konservolia) inflorescence were carried out at short intervals (3–7 days) during the period from bud burst until the 3rd week after full bloom (AFB), when young fruit reached 8 mm in length. The axis of inflorescence elongated remarkably during the 3rd week after bud burst (ABB), massive bract shedding occurred during the 4th week ABB, full bloom (FB) was observed 7 weeks ABB, and massive floral organ abscission 1 week AFB. The results showed a continuous increase in inflorescence FW and DW from bud burst until 4 days before FB. Respiration rate, ethylene production, and levels of ABA were relatively high during the first 3 weeks ABB. After this period, respiration and ethylene followed a similar pattern of changes, inversely to that of ABA concentration. An accumulation of inflorescence ABA 6 and 4 days before FB was associated with the minimum values of respiration and ethylene production on the same dates. The sharp decrease in the ABA concentration during FB and 3 days later was followed by a high rise in ethylene and an increase in respiration rate, which both rose further 1 week AFB. The results suggest a possible correlation of ABA with the early stage of floral abscission, whereas ethylene production seems to be correlated with the terminal separatory activity in olive inflorescence abscission processes. Received May 28, 1998; accepted November 17, 1998     

Soaking Summer Squash Seeds in Low Concentrations of Cobalt Solution Before Sowing Increased Plant Growth, Femaleness, and Fruit Yield via Increasing Plant Ethylene Level

Soaking summer squash (Cucurbita pepo cv. Eskandarany) seeds in continuously aerated solutions of 0.25, 0.50, and 1.00 ppm Co²⁺ for 48 h before sowing strongly increased plant growth, femaleness, and fruit yield compared with those of water- (control) or 0.5 mm AOA (aminooxyacetic acid)-soaked seeds. Following the same pattern, plants of Co²⁺-soaked seeds produced significantly higher ethylene levels as early as the seedling stage (14 days after seed sowing) up to the onset of flower initiation (OFI) stage (30 days after seed sowing), with more pronounced levels of all measured parameters for plants of 1.00 ppm Co²⁺-soaked seeds. Plants of AOA-soaked seeds, however, behaved in a pattern similar to those of control in all measured parameters. The only exception was the significant ethylene inhibition noticed only at the plant seedling stage with AOA-seed soaking, which indicated the short term of AOA inhibition to ethylene when applied as early as the seed germination stage. When AOA was applied foliarly before and at the OFI stage, the increased plant femaleness obtained with Co²⁺ seed soaking was arrested. It is indicated, therefore, that summer squash plant femaleness is more responsive to plant ethylene-modulated levels before or at the OFI stage than earlier stages. Furthermore, all seed soaking treatments had no effect on plant leaf number or plant and fruit Co²⁺ content, which strongly indicated that the positive impact of Co²⁺ on summer squash plant growth and femaleness was mainly the result of the so-called ``low Co²⁺ level-induced ethylene.' The percentage of fruit yield increase reached about 26, 40, and 56% over the control by 0.25, 0.50, and 1.00 ppm Co²⁺ seed soaking, respectively, whereas AOA seed soaking resulted in only a 4.5% yield reduction. To ensure the permanent positive impact of Co²⁺ on ethylene production as well as the short period of AOA inhibition, seeds were resoaked in water or AOA for 8 h after being soaked in 1.00 ppm Co²⁺ solution for 40 h. The results obtained emphasized the previous findings because AOA inhibition was restricted on ethylene production only at the seedling stage. Meanwhile, all Co²⁺ positive effects were obtained 2 weeks later, even with AOA seed resoaking. These data strongly suggested that the positive impact of Co²⁺ on ethylene production is more permanent than the negative impact of AOA. Hence its application, in low concentrations, as early as the seed germination stage, strongly increased summer squash plant growth, femaleness, and fruit yield by increasing the plant ethylene level. Received July 21, 1997; accepted November 10, 1997     

Evolution of the PP2C Family in Caenorhabditis: Rapid Divergence of the Sex-Determining Protein FEM-2

To investigate the causes and functional significance of rapid sex-determining protein evolution we compared three Caenorhabditis elegans genes encoding members of the protein phosphatase 2C (PP2C) family with their orthologs from another Caenorhabditis species (strain CB5161). One of the genes encodes FEM-2, a sex-determining protein, while the others have no known sex-determining role. FEM-2's PP2C domain was found to be more diverged than the other PP2C domains, supporting the notion that sex-determining proteins are subjected to selective pressures that allow for or cause rapid divergence. Comparison of the positions of amino acid substitutions in FEM-2 with a solved three-dimensional structure suggests that the catalytic face of the protein is highly conserved among C. elegans, CB5161, and another closely related species C. briggsae. However, the non-conserved regions of FEM-2 cannot be said to lack functional importance, since fem-2 transgenes from the other species were unable to rescue the germ-line defect caused by a C. elegans fem-2 mutation. To test whether fem-2 functions as a sex-determining gene in the other Caenorhabditis species we used RNA-mediated interference (RNAi). fem-2 (RNAi) in C. elegans and C. briggsae caused germ-line feminization, but had no noticeable effect in CB5161. Thus the function of fem-2 in CB5161 remains uncertain. Received: 11 April 2001 / Accepted: 6 August 2001     

Fast Evolution of Interleukin-2 in Mammals and Positive Selection in Ruminants

Interleukin-2 (IL-2) is a cytokine involved in induction and regulation of the immune response in mammals. There have been numerous reports about the search for IL-2 in species other than mammals, and recently an IL-2-like gene has been isolated in chicken. Using PCR, we searched for IL-2 gene sequences in a wide variety of mammals, including marsupials and monotremes, as well as in birds. Although we can readily amplify IL-2 gene fragments in placental mammals, no amplification was obtained in other species. This is best explained by very high substitution rates. This suggest that strategies to isolate IL-2 homologous genes outside mammals should involve functional assays, as for the chicken gene, and not hybridization-based techniques. Nonsynonymous substitution rates are especially high in ruminants, due to positive selection acting on regions important in term of structure-function. We suggest that, although globally similar, the immune response of various mammals is not identical, mainly at the level of cytokine-mediated regulations. Received: 27 July 1999 / Accepted: 15 April 2000     

Comments on the Role of H2S in the Chemistry of Earth's Early Atmosphere and in Prebiotic Synthesis

Free energy calculations and experimental measurements have been used to show that H₂S/CO₂ mixtures outgassing from a prebiotic Earth's crust would have produced a reducing gas mixture containing CO, H₂, H₂O, and S _x as principal components. Due to rapid recombination of H₂, CO, and S _x to H₂S and CO₂ on cooling from a high temperature to ambient conditions, reducing components would have been retained only if efficient quenching of the reduced gas mixture had been possible. Consequently, subsea vents or vents with efficient infusion of water would have been ideal sites for retention of reduced species and for prebiotic organic synthesis. It is suggested that C/H/O/S ratios are important factors in controlling the degree of prebiotic organic synthesis and, hence, the emergence of life, since if oxygen is abundant, CO₂ and SO₂ would have been dominant species. Received: 5 March 1997 / Accepted: 15 December 1997     

Short Retroposons of the B2 Superfamily: Evolution and Application for the Study of Rodent Phylogeny

Short retroposons can be used as natural phylogenetic markers. By means of hybridization and PCR analysis, we demonstrate that B2 retroposon copies are present only in the three rodent families: Muridae, Cricetidae, and Spalacidae. This observation highlights the close phylogenetic relation between these families. Two novel B2-related retroposon families, named DIP and MEN elements, are described. DIP elements are found only in the genomes of jerboas (family Dipodidae) and birch mice (family Zapodidae), demonstrating the close relationship between these rodents. MEN element copies were isolated from the squirrel, Menetes berdmorei, but were not detected in three other species from the family Sciuridae. The MEN element has an unusual dimeric structure: the left and right monomers are B2- and B1-related sequences, respectively. Comparison of the B2, DIP, MEN, and 4.5S₁ RNA elements revealed an 80-bp core sequence located at the beginning of the B2 superfamily retroposons. This observation suggests that these retroposon families descended from a common progenitor. A likely candidate for this direct progenitor could be the ID retroposon. Received: 20 December 1996 / Accepted: 17 June 1997     

Characterization of a Novel Class of Interspersed LTR Elements in Primate Genomes: Structure, Genomic Distribution, and Evolution

Retrovirus-like sequences and their solitary (solo) long terminal repeats (LTRs) are common repetitive elements in eukaryotic genomes. We reported previously that the tandemly arrayed genes encoding U2 snRNA (the RNU2 locus) in humans and apes contain a solo LTR (U2-LTR) which was presumably generated by homologous recombination between the two LTRs of an ancestral provirus that is retained in the orthologous baboon RNU2 locus. We have now sequenced the orthologous U2-LTRs in human, chimpanzee, gorilla, orangutan, and baboon and examined numerous homologs of the U2-LTR that are dispersed throughout the human genome. Although these U2-LTR homologs have been collectively referred to as LTR13 in the literature, they do not display sequence similarity to any known retroviral LTRs; however, the structure of LTR13 closely resembles that of other retroviral LTRs with a putative promoter, polyadenylation signal, and a tandemly repeated 53-bp enhancer-like element. Genomic blotting indicates that LTR13 is primate-specific; based on sequence analysis, we estimate there are about 2,500 LTR13 elements in the human genome. Comparison of the primate U2-LTR sequences suggests that the homologous recombination event that gave rise to the solo U2-LTR occurred soon after insertion of the ancestral provirus into the ancestral U2 tandem array. Phylogenetic analysis of the LTR13 family confirms that it is diverse, but the orthologous U2-LTRs form a coherent group in which chimpanzee is closest to the humans; orangutan is a clear outgroup of human, chimpanzee, and gorilla; and baboon is a distant relative of human, chimpanzee, gorilla, and orangutan. We compare the LTR13 family with other known LTRs and consider whether these LTRs might play a role in concerted evolution of the primate RNU2 locus. Received: 29 September 1997 / Accepted: 16 January 1998     

Evolution of Substrate Specificities in the P-Type ATPase Superfamily

P-type ATPases make up a large superfamily of ATP-driven pumps involved in the transmembrane transport of charged substrates. We have performed an analysis of conserved core sequences in 159 P-type ATPases. The various ATPases group together in five major branches according to substrate specificity, and not according to the evolutionary relationship of the parental species, indicating that invention of new substrate specificities is accompanied by abrupt changes in the rate of sequence evolution. A hitherto-unrecognized family of P-type ATPases has been identified that is expected to be represented in all the major phyla of eukarya. Received: 21 May 1997 / Accepted: 1 August 1997     

Effects of Waterlogging on Nitrogen Accumulation and Alleviation of Waterlogging Damage by Application of Nitrogen Fertilizer and Mixtalol in Winter Rape (Brassica napus L.)

A study on the physiological and yield effects of waterlogging and the alleviation of waterlogging damage by the application of nitrogen fertilizers and mixtalol in winter rape was conducted in experimental tanks especially designed for controlling soil moisture content. The results showed that waterlogging at the seedling and stem elongation stages causes a significant decrease in nitrogen content and rate of nitrogen accumulation. Leaf chlorophyll content, superoxide dismutase and catalase activities, and root oxidizability (capacity for root oxidation) and root exudate were also reduced by waterlogging. The experiments confirmed that the physiological function of rape plants was retarded during the time of waterlogging at the seedling stage, and its adverse effects remained. Plant height, stem width, and the number of primary branches per plant were decreased significantly by waterlogging at the seedling and stem elongation stages. Pods per plant and seeds per pod were also reduced significantly, giving a 21.3 and 12.5% decrease of seed yield from the control for treatments at the seedling and stem elongation stages, respectively. Foliar sprays of nitrogen fertilizers at the seedling stage or mixtalol at the flowering stage alleviated plant damage caused by waterlogging by retarding chlorophyll and nitrogen degradation, increasing superoxide dismutase and catalase activities and root oxidizability, and improving yield components and seed yield of waterlogged plants. Therefore, besides draining off water, alleviation of waterlogging damage may be controlled by applying nitrogen fertilizer and a suitable plant growth regulator at appropriate growth stages. Received July 3, 1996; accepted December 26, 1996     

Selection and Methionine Accumulation in the Fat Body Protein 2 Gene (FBP2), a Duplicate of the Drosophila Alcohol Dehydrogenase (ADH) Gene

The Drosophila fat body protein 2 gene (Fbp2) is an ancient duplication of the alcohol dehydrogenase gene (Adh) which encodes a protein that differs substantially from ADH in its methionine content. In D. melanogaster, there is one methionine in ADH, while there are 51 (20% of all amino acids) in FBP2. Methionine is involved in 46% of amino acid replacements when Fbp2 DNA sequences are compared between D. melanogaster and D. pseudoobscura. Methionine accumulation does not affect conserved residues of the ADH-ADH^r-FBP2 multigene family. The multigene family has evolved by replacement of mildly hydrophobic amino acids by methionine with no apparent reversion. Its short-term evolution was compared between two Drosophila species, while its long-term evolution was compared between two genera belonging respectively to acalyptrate and calyptrate Diptera, Drosophila and Sarcophaga. The pattern of nucleotide substitution was consistent with an independent accumulation of methionines at the Fbp2 locus in each lineage. Under a steady-state model, the rate of methionine accumulation was constant in the lineage leading to Drosophila, and was twice as fast as that in the calyptrate lineage. Substitution rates were consistent with a slight positive selective advantage for each methionine change in about one-half of amino acid sites in Drosophila. This shows that selection can potentially account for a large proportion of amino acid replacements in the molecular evolution of proteins. Received: 12 December 1994 / Accepted: 15 April 1996     

Effects of blue light deficiency on acclimation of light energy partitioning in PSII and CO2 assimilation capacity to high irradiance in spinach leaves

Blue light effects on the acclimation of energy partitioningcharacteristics in PSII and CO₂ assimilation capacity in spinachto high growth irradiance were investigated. Plants were grownhydroponically in different light treatments that were a combinationof two light qualities and two irradiances, i.e. white lightand blue-deficient light at photosynthetic photon flux densities(PPFDs) of 100 and 500 µmol m^–2 s^–1. The CO₂assimilation rate, the quantum efficiency of PSII (PSII) andthermal dissipation activity / in young, fully expanded leaves were measured under 1,600 µmol m^–2 s^–1white light. The CO₂ assimilation rate and PSII were higher,while / was lower in plants grown under high irradiancethan in plants grown under low irradiance. These responses wereobserved irrespective of the presence or absence of blue lightduring growth. The extent of the increase in the CO₂ assimilationrate and PSII and the decrease in / by high growth irradiance was smaller under blue light-deficient conditions. These resultsindicate that blue light helps to boost the acclimation responsesof energy partitioning in PSII and CO₂ assimilation to highirradiance. Similarly, leaf N, Cyt f and Chl contents per unitleaf area increased by high growth irradiance, and the extentof the increment in leaf N, Cyt f and Chl was smaller underblue light-deficient conditions. Regression analysis showedthat the differences in energy partitioning in PSII and CO₂assimilation between plants grown under high white light andhigh blue-deficient light were closely related to the differencein leaf N.     

Extensive Amplification and Transposition of a Novel Repetitive Element, Xstir, Together with Its Terminal Inverted Repeat in the Evolution of Xenopus

A DNA fragment containing short tandem repeat sequences (approximately 86-bp repeat) was isolated from a Xenopus laevis cDNA library. Southern blot and in situ hybridization analyses revealed that the repeat was highly dispersed in the genome and was present at approximately 1 million copies per haploid genome. We named this element Xstir (Xenopus short tandemly and invertedly repeating element) after its arrangement in the genome. The majority of the genomic Xstir sequences were digested to monomer and dimer sizes with several restriction enzymes. Their sequences were found to be highly homogeneous and organized into tandem arrays in the genome. Alignment analyses of several known sequences showed that some of the Xstir-like sequences were also organized into interspersed inverted repeats. The inverted repeats consisted of an inverted pair of two differently modified Xstirs separated by a short insert. In addition, these were framed by another novel inverted repeat (Xstir-TIR). The Xstir-TIR sequence was also found at the ends of tandem Xstir arrays. Furthermore, we found that Xstir-TIR was linked to a motif characterizing the T2 family which belonged to a vertebrate MITE (miniature inverted-repeat transposable element) family, suggesting the importance of Xstir-TIR for their amplification and transposition. The present study of 11 anuran and 2 urodele species revealed that Xstir or Xstir-like sequences were extensively amplified in the three Xenopus species. Genomic Xstir populations of X. borealis and X. laevis were mutually indistinguishable but significantly different from that of X. tropicalis. Received: 5 April 2000 / Accepted: 3 August 2000     

The Effects of Social Structure, Geographical Structure, and Population Size on the Evolution of Mitochondrial DNA: II. Molecular Clocks and the Lineage Sorting Period

Evolutionary geneticists have increasingly used sequence variation in mitochondrial DNA (mtDNA) as a source of historical information. However, conclusions based on these data remain tentative because a sufficiently clear understanding of the evolutionary dynamics of mtDNA has yet to be developed. In this paper we present the results of computer simulations designed to illustrate the effects of social structure, geographical structure, and population size on the rate of nucleotide substitution and lineage sorting of mtDNA. The model is based in part on the social structure of macaque monkeys. Simulated populations of females were divided into 25 social groups; the animals in each were distributed in a hierarchy of four dominance rank categories. The probabilities for offspring survivorship were varied among dominance ranks to reflect the fitness consequences of social structure. Population size was varied across runs from 100 to 300 females. The pattern of female migration was also varied to mimic either the island model or the stepping-stone model. All these variables are shown to affect the lineage sorting period (LSP), and certain combinations of parameter values can cause the retention of mtDNA polymorphisms for a very long time. In addition, the simulations exhibited a negative relationship between the LSP and substitution rate over a modest and realistic range of LSP values. An important implication of these results is that estimates of time since isolation based on the assumption of a constant molecular clock may be biased and unreliable.     

Erratic Evolution of Glycerol-3-Phosphate Dehydrogenase in Drosophila,Chymomyza, and Ceratitis

We have studied the evolution of Gpdh in 18 fruitfly species by sequencing 1,077 nucleotides per species on average. The region sequenced includes four exons coding for 277 amino acids and three variable-length introns. Phylogenies derived by a variety of methods confirm that the nominal genus Zaprionus belongs within the genus Drosophila, whereas Scaptodrosophila and Chymomyza are outside. The rate of GPDH evolution is erratic. The rate of amino acid replacements in a lineage appears to be 1.0 × 10⁻¹⁰/site/year when Drosophila species are considered (diverged up to 55 million years ago), but becomes 2.3 × 10⁻¹⁰ when they are compared to Chymomyza species (divergence around 60 My ago), and 4.6 × 10⁻¹⁰ when species of those two genera are compared with the medfly Ceratitis capitata (divergence around 100 My ago). In order to account for these observations, the rate of amino acid replacement must have been 15 or more times greater in some lineages and at some times than in others. At the nucleotide level, however, Gpdh evolves in a fairly clockwise fashion. Received: 13 June 1996 / Accepted: 16 August 1996