Adaptive intraspecific divergence: An example using the animal cytochrome <Emphasis Type="Italic">b</Emphasis> gene

The topologies of phylogenetic trees characterized by a high level of intraspecific divergence between the phylogenetic DNA groups (clades) are often explained in terms of the theory of Pleistocene refugia. To elucidate the issue of the adaptive role of intraspecific divergence, the changes in the physicochemical properties of amino acids in the course of cladogenesis (MM01 model of the TreeSAAP 3.2 package) were analyzed in this work using as an example the nucleotide sequences of the cytochrome b gene in some species of northern animals (lemmings, redbacked voles, chipmunks, flying squirrels, ermines). It was shown that the process of intraspecific divergence was rarely accompanied by radical amino acid substitutions in cytochrome b caused by adaptation (directional selection). In connection with this, the hypothesis is discussed according to which the adaptive variants formed in the species at the peak of cold were lost with climatic warming due to the drift or selection against individuals adapted to cold.     

Evolutionary divergence of the cytochrome b5 gene of Drosophila

Cytochrome proteins perform a broad spectrum of biological functions ranging from oxidative metabolism to electron transport and are thus essential to all organisms. The b-type cytochrome proteins bind heme noncovalently, are expressed in many different forms and are localized to various cellular compartments. We report the characterization of the cytochrome b₅ (Cyt-b) gene of Drosophila virilis and compare its structure to the Cyt-b gene of Drosophila melanogaster. As in D. melanogaster, the D. virilis gene is nuclear encoded and single copy. Although the intron/exon structures of these homologues differ, the Cyt-b proteins of D. melanogaster and D. virilis are approximately 75% identical and share the same size coding regions (1,242 nucleotides) and protein products (414 amino acids). The Drosophila Cyt-b proteins show sequence similarity to other b-type cytochromes, especially in the N-terminal heme-binding domain, and may be targeted to the mitochondrial membrane. The greatest levels of similarity are observed in areas of potential importance for protein structure and function. The exon sequences of the D. virilis Cyt-b gene differ by a total of 292 base changes. However, 62% of these changes are silent. The high degree of conservation between species separated by 60 million years of evolution in both the DNA and amino acid sequences suggests this nuclear cytochrome b₅ locus encodes an essential product of the Drosophila system.Correspondence to: C.E. Rozek     

Nuclear and cytoplasmic contributions to intraspecific divergence in an annual legume

The genetic architecture of trait differentiation was evaluated between two ecologically distinct populations of Chamaecrista fasciculata. Individuals from Maryland and Illinois populations were crossed to create 10 types of seed: Maryland and Illinois parents, reciprocal F1 and F2 hybrids, and backcrosses to Maryland and to Illinois on reciprocal F1 hybrids. Reciprocal crosses created hybrid generation seeds with both Maryland and Illinois cytoplasmic backgrounds. Experimental individuals were grown in a common garden near the site of the Maryland population. In the garden, plants from the Illinois population flowered, set fruit, and died earlier than those from Maryland, likely reflecting adaptations to differences in growing season length between the two populations. Although reproductive components at the flower and whole plant level differed between the two populations, reproductive output as measured by fruit and seed production was similar. Cytoplasmic genes had a subtle but pervasive effect on population differentiation; hybrids with Maryland cytoplasm were significantly differentiated from those with Illinois cytoplasm when all characters were evaluated jointly. The nuclear genetic architecture of population differentiation was evaluated with joint scaling tests. Depending on the trait, both additive and nonadditive genetic effects contributed to population differentiation. Intraspecific genetic differentiation in this wild plant species appears to reflect a complex genetic architecture that includes the contribution of additive, dominance, and epistatic components in addition to subtle cytoplasmic effects.     

Adaptive gene expression divergence inferred from population genomics

Detailed studies of individual genes have shown that gene expression divergence often results from adaptive evolution of regulatory sequence. Genome-wide analyses, however, have yet to unite patterns of gene expression with polymorphism and divergence to infer population genetic mechanisms underlying expression evolution. Here, we combined genomic expression data—analyzed in a phylogenetic context—with whole genome light-shotgun sequence data from six Drosophila simulans lines and reference sequences from D. melanogaster and D. yakuba. These data allowed us to use molecular population genetics to test for neutral versus adaptive gene expression divergence on a genomic scale. We identified recent and recurrent adaptive evolution along the D. simulans lineage by contrasting sequence polymorphism within D. simulans to divergence from D. melanogaster and D. yakuba. Genes that evolved higher levels of expression in D. simulans have experienced adaptive evolution of the associated 3′ flanking and amino acid sequence. Concomitantly, these genes are also decelerating in their rates of protein evolution, which is in agreement with the finding that highly expressed genes evolve slowly. Interestingly, adaptive evolution in 5′ cis-regulatory regions did not correspond strongly with expression evolution. Our results provide a genomic view of the intimate link between selection acting on a phenotype and associated genic evolution.     

Aphid species identification using cuticular hydrocarbons and cytochrome b gene sequences

Abstract:  In Tunisia, four major aphid species have been identified based on adult female's morphological characters: Aphis gossypii Glover, Aphis craccivora Koch, Myzus persicae Sluzer and Macrosiphum euphorbiae Thomas. Species identification at individual collection sites is often difficult because adults are much fewer in number than larvae which are not so easy to distinguish morphologically. We therefore set up an experiment to determine if cuticular hydrocarbon phenotypes and mitochondrial DNA haplotypes could be used to distinguish such sympatric species. Results showed that each species had an unique cuticular hydrocarbon phenotype and mitochondrial cytochrome b sequence. Cytochrome b restriction fragment-length polymorphism markers, especially Dde I, identified in this sudy constitute a relatively simple and useful approach to distinguish the four species even at the nymphal stage.     

Identification of mosquito bloodmeals using mitochondrial cytochrome oxidase subunit I and cytochrome b gene sequences

Abstract.  Primer pairs were designed and protocols developed to selectively amplify segments of vertebrate mitochondrial cytochrome oxidase subunit 1 (COI) and cytochrome b (Cyt b ) mtDNA from the bloodmeals of mosquitoes (Diptera: Culicidae). The protocols use two pairs of nested COI primers and one pair of Cyt b primers to amplify short segments of DNA. Resultant sequences are then compared with sequences in GenBank, using the BLAST function, for putative host identification. Vertebrate DNA was amplified from 88% of our sample of 162 wild-caught, blood-fed mosquitoes from Oregon, U.S.A. and GenBank BLAST searches putatively identified 98% of the amplified sequences, including one amphibian, seven mammalian and 14 avian species. Criteria and caveats for putative identification of bloodmeals are discussed.     

Detection and quantification of Pfiesteria piscicida by using the mitochondrial cytochrome b gene

Mitochondrial cytochrome b was isolated from the dinoflagellate Pfiesteria piscicida, and the utility of the gene for species identification was examined. One of the primer sets designed was shown to be highly specific for P. piscicida. A time step PCR protocol was used to demonstrate the potential of this primer set for quantification of this species.     

Influence of gene position on the expression divergence of oxidative response genes in intraspecific yeast

Phenotypic variation can arise from differences in the protein coding sequence and in the regulatory elements. However, little is known about the contribution of regulatory difference to the expression divergence, especially the cis and trans regulatory variation to the expression divergence in intraspecific populations. In this study, we used two different yeast strains, BY4743 and RM11‐1a/α, to study the regulatory variation to the expression divergence between BY and RM under oxidative stress condition. Our results indicated that the expression divergence of BY and RM is mainly due to trans regulatory variations under both normal and oxidative stress conditions. However, cis regulatory variation seems to play a very important role in oxidative stress response in yeast because 36% of genes showed an increase in cis regulatory variation effect compared with 13% of genes that showed an increase in trans regulatory variation effect after oxidative stress. Our data also indicated that genes located on the longer arm of the chromosomes are more susceptible to cis variation effect under oxidative stress than genes on the shorter arm of the chromosomes.     

Molecular divergence and phylogeny: rates and patterns of cytochrome b evolution in cranes

Analyses of complete cytochrome b sequences from all species of cranes (Aves: Gruidae) reveal aspects of sequence evolution in the early stages of divergence. These DNA sequences are > or = 89% identical, but expected departures from random substitution are evident. Silent, third- position pyrimidine transitions are the dominant substitution type, with transversion comprising only a small fraction of sequence differences. Substitution patterns are not clearly manifested until divergence has reached a moderate level (> 3%), as expected for a stochastic process. Variation in the frequency of mismatch types among lineages decreases at larger divergences, but the level of bias does not decay. Divergence varies up to fivefold among gene regions but is not correlated with structural domain. All protein structural domains except extramembrane 4 display < 20% variable residues. Regions corresponding to putative functional domains show the excepted conservation of amino acids, although the C-terminal portion of the Q0 reaction center displays several nonconservative replacements. Phylogenetic analyses incorporating substitution asymmetries produced mixed results. Distances estimated with multiple parameters (transition, codon-position, composition, and pyrimidine-transition biases) yielded identical additive tree topologies with comparable bootstrap values, all consistent with uncontroversial species relationships. Maximum likelihood analysis incorporating these biases, as well as equally weighted parsimony analysis, produced similar results. Static, differential weighting for parsimony did not improve the phylogenetic signal but produced unusual trees with low bootstraps. The overall rate of nucleotide substitution varies slightly but significantly among cranes, and calibration of distances against fossil dates suggests divergence rates of 0.7%-1.7% per million years.      

The largemouth bass cytochrome b gene

The cytochrome b gene is one of the protein-coding genes of the mitochondrial genome that has gained importance because of the ease with which molecular techniques can be applied to the analysis of its structure. The nucleotide sequence of the largemouth bass ( Microplerus salmoides ) cytochrome b gene was determined and the inferred amino acid structure is presented in the form of a structural model derived originally from rat cytochrome b . The inferred amino acid sequences from divergent animal species are aligned and compared in the context of this model. The data suggest that regions of the gene may be evolving at different rates due to different selection pressures associated with functional constraints. Conserved and variable regions of cytochrome b have been identified and can be targeted for species identification, the examination of intraspeciflc variation, and phylogenetic reconstructions in future research.     

Adaptive divergence and the evolution of reproductive isolation in the wild: an empirical demonstration using introduced sockeye salmon

Populations exposed to different ecological environments should diverge for phenotypic traits that influence survival and reproduction. This adaptive divergence should reduce gene flow between populations because immigrants become less fit than residents and because hybrids perform poorly in either environment (i.e., ecologically-dependent reproductive isolation). Here I demonstrate adaptive divergence and the evolution of reproductive isolation in populations of sockeye salmon (Oncorhynchus nerka) introduced from a common ancestral source into a new lake system (Lake Washington, Washington). The introduced fish founded several new populations, two of which experience very different environments during breeding and early development (Cedar River v.s. Pleasure Point beach). Over 13 generations, the two populations diverged for adult traits (female body size, male body depth; measured in the wild) and embryo traits (survival to hatching, development rate, size at emergence; measured in a common environment). The rates of divergence for these characters were similar to those observed in other examples of rapid evolution, and can best be attributed to natural selection. Partial reproductive isolation has evolved in concert with adaptive divergence: the rate of exchange of adults between the populations (determined using natural tags) is higher than the rate of gene flow (determined using DNA microsatellites). The demonstration that adaptive divergence can initiate reproductive isolation in less than 13 generations suggests that the first signs of ecological speciation may appear soon after new environments are first colonized.     

Potentials for monitoring gene level biodiversity: using Sweden as an example

Programs for monitoring biological diversity over time are needed to detect changes that can constitute threats to biological resources. The convention on biological diversity regards effective monitoring as necessary to halt the ongoing erosion of biological variation, and such programs at the ecosystem and species levels are enforced in several countries. However, at the level of genetic biodiversity, little has been accomplished, and monitoring programs need to be developed. We define “conservation genetic monitoring” to imply the systematic, temporal study of genetic variation within particular species/populations with the aim to detect changes that indicate compromise or loss of such diversity. We also (i) identify basic starting points for conservation genetic monitoring, (ii) review the availability of such information using Sweden as an example, (iii) suggest categories of species for pilot monitoring programs, and (iv) identify some scientific and logistic issues that need to be addressed in the context of conservation genetic monitoring. We suggest that such programs are particularly warranted for species subject to large scale enhancement and harvest—operations that are known to potentially alter the genetic composition and reduce the variability of populations.     

Variation in Capsella (shepherd's purse): an example of intraspecific functional diversity

The extent of functional trait diversity is quantified for 157 different Capsella bursa-pastoris (L.) medic (shepherd's purse) accessions. These individuals encompass replicate progeny generated from seed of 53 different Capsella 'maternal lines' that were isolated at random as they emerged from soil cores (used to estimate baseline seed bank numbers and weed diversity) at 34 different arable sites across the United Kingdom. The replicate progeny were subject to ex situ characterisation for traits determining life history and architecture. Seven leaf-type classes were identified and representative parents of each leaf type were distinguishable using four different simple sequence repeat markers. Life-history traits were only loosely associated with leaf shape, and cluster analysis grouped the accessions into three broad types: small plants that flowered early with intermediate reproductive output; large plants, with intermediate time to flowering and a high reproductive output; late-flowering plants, of intermediate size and low reproductive output. The most common leaf-type variants (83% of accessions) demonstrated a short time to flowering (ca. 70 days), while rarer variants included those that flowered after 140 days, accumulated more nitrogen and produced less seed: possibly representing advantageous and disadvantageous traits (respectively), in modern arable rotations. A wide trait variation was therefore found in Capsella bursa-pastoris despite decades of agricultural intensification, the range of time-to-flowering for C. bursa-pastoris being as broad the mean flowering times of the commoner annual and winter annual arable species. We propose the use of traits, rather than species, as the accounting unit to quantify functional biodiversity in arable systems.     

Divergence of the cytochrome b gene in grouse species

A 720-bp cytochrome b gene fragment of the mitochondrial genome was sequenced in ten species and seven potential subspecies of grouse. Of 187 variable sites detected, 140 were parsimony informative. The distribution of nucleotides in three positions of codons had a similar pattern with and did not fundamentally differ from that of the nucleotide composition of this gene in other animals, including birds. The nucleotides found at the first codon position were shown to be uniformly distributed, whereas the third position was characterized by a significant decrease in the amount of guanine and, to a lesser extent, thymine. Based on the data on the nucleotide sequences of the cytochrome b gene fragment, a phylogenetic tree was constructed. This tree fits well the morphological and ecological differentiation of the species studied.     

Yeast cytochrome b2 gene: isolation with antibody probes

An efficient technique was used to clone the gene for yeast cytochrome b2, (a nuclear encoded mitochondrial protein) using the expression vector, lambda gt11 (lac 5 nin 5 c1857 S100). This enables the insertion of yeast DNA into the beta-galactosidase structural gene (lacZ) and promotes synthesis of hybrid proteins. Screening of antigen producing clones in the lambda gt11 recombinant genomic library was achieved using antiserum against cytochrome b2 according to Young and Davis (1983) Two recombinants containing part of the gene coding for cytochrome b2 were isolated and characterized as follows: by their expression in Escherichia coli cells, examined by immuno-blotting with antibodies to pure cytochrome b2. by DNA sequence analysis. One recombinant carries a 3 Kb yeast DNA insert which contains the whole nucleotide sequence encoding cytochrome b2 and a few amino acids of the amino terminal presequence.     

Mutational analysis of the mouse mitochondrial cytochrome b gene

The protonmotive cytochrome b protein of the mitochondrial bc1 respiratory chain complex contains two reactions centers, designated Qo and Qi, which can be distinguished by the effects of different inhibitors. The nucleotide sequences have been determined of the mitochondrial cytochrome b genes from a series of mouse cell mutants selected for increased inhibitor resistance. Each mutant contains a single nucleotide change which results in an amino acid substitution. When the proximity of the altered amino acid residues to the histidines involved in heme ligation is considered, the results support a model for cytochrome b folding in which there are eight transmembrane domains rather than the nine of the Widger-Saraste model. Replacement of the Gly38 residue by valine results in resistance to the Qi inhibitors antimycin A and funiculosin but not 2-n-heptyl-hydroxyquinoline-N-oxide. Based upon sequence comparisons of mitochondrial and bacterial cytochrome b and chloroplast b6 proteins, the region of the molecule involved in antimycin binding is as highly conserved as those domains involved in heme ligation. It is suggested that the antimycin binding domain of cytochrome b is involved in forming the Qi reaction center. Alterations of the Gly142 and Thr147 residues result in resistance to myxothiazol and stimatellin, respectively. While both inhibitors block the Qo reaction center, the two mutations do not confer cross-resistance to each other. This region of cytochrome b is the most highly conserved during evolution and these inhibitor binding sites probably occur within the protein domain constituting the Qo reaction center. In addition, there is a less conserved region of the protein, defined by the Leu294 residue, which may function in binding the hydrophobic portions of Qo inhibitors.     

Rapid identification of the Leptosphaeria maculans avirulence gene AvrLm2 using an intraspecific comparative genomics approach

Five avirulence genes from Leptosphaeria maculans, the causal agent of blackleg of canola (Brassica napus), have been identified previously through map‐based cloning. In this study, a comparative genomic approach was used to clone the previously mapped AvrLm2. Given the lack of a presence–absence gene polymorphism coincident with the AvrLm2 phenotype, 36 L. maculans isolates were resequenced and analysed for single‐nucleotide polymorphisms (SNPs) in predicted small secreted protein‐encoding genes present within the map interval. Three SNPs coincident with the AvrLm2 phenotype were identified within LmCys1, previously identified as a putative effector‐coding gene. Complementation of a virulent isolate with LmCys1, as the candidate AvrLm2 allele, restored the avirulent phenotype on Rlm2‐containing B. napus lines. AvrLm2 encodes a small cysteine‐rich protein with low similarity to other proteins in the public databases. Unlike other avirulence genes, AvrLm2 resides in a small GC island within an AT‐rich isochore of the genome, and was never found to be deleted completely in virulent isolates.     

From sensor data to animal behaviour: an oystercatcher example

Animal-borne sensors enable researchers to remotely track animals, their physiological state and body movements. Accelerometers, for example, have been used in several studies to measure body movement, posture, and energy expenditure, although predominantly in marine animals. In many studies, behaviour is often inferred from expert interpretation of sensor data and not validated with direct observations of the animal. The aim of this study was to derive models that could be used to classify oystercatcher (Haematopus ostralegus) behaviour based on sensor data. We measured the location, speed, and tri-axial acceleration of three oystercatchers using a flexible GPS tracking system and conducted simultaneous visual observations of the behaviour of these birds in their natural environment. We then used these data to develop three supervised classification trees of behaviour and finally applied one of the models to calculate time-activity budgets. The model based on accelerometer data developed to classify three behaviours (fly, terrestrial locomotion, and no movement) was much more accurate (cross-validation error?=?0.14) than the model based on GPS-speed alone (cross-validation error?=?0.35). The most parsimonious acceleration model designed to classify eight behaviours could distinguish five: fly, forage, body care, stand, and sit (cross-validation error?=?0.28); other behaviours that were observed, such as aggression or handling of prey, could not be distinguished. Model limitations and potential improvements are discussed. The workflow design presented in this study can facilitate model development, be adapted to a wide range of species, and together with the appropriate measurements, can foster the study of behaviour and habitat use of free living animals throughout their annual routine.     

Rooting phylogenies using gene duplications: an empirical example from the bees (Apoidea)

The placement of the root node in a phylogeny is fundamental to characterizing evolutionary relationships. The root node of bee phylogeny remains unclear despite considerable previous attention. In order to test alternative hypotheses for the location of the root node in bees, we used the F1 and F2 paralogs of elongation factor 1-alpha (EF-1α) to compare the tree topologies that result when using outgroup versus paralogous rooting. Fifty-two taxa representing each of the seven bee families were sequenced for both copies of EF-1α. Two datasets were analyzed. In the first (the "concatenated" dataset), the F1 and F2 copies for each species were concatenated and the tree was rooted using appropriate outgroups (sphecid and crabronid wasps). In the second dataset (the "duplicated" dataset), the F1 and F2 copies were aligned to each another and each copy for all taxa were treated as separate terminals. In this dataset, the root was placed between the F1 and F2 copies (e.g., paralog rooting). Bayesian analyses demonstrate that the outgroup rooting approach outperforms paralog rooting, recovering deeper clades and showing stronger support for groups well established by both morphological and other molecular data. Sequence characteristics of the two copies were compared at the amino acid level, but little evidence was found to suggest that one copy is more functionally conserved. Although neither approach yields an unambiguous root to the tree, both approaches strongly indicate that the root of bee phylogeny does not fall near Colletidae, as has been previously proposed. We discuss paralog rooting as a general strategy and why this approach performs relatively poorly with our particular dataset.