Neutral and Non-Neutral Evolution of Drosophila Mitochondrial DNA

To test hypotheses of neutral evolution of mitochondrial DNA (mtDNA), nucleotide sequences were determined for 1515 base pairs of the NADH dehydrogenase subunit 5 (ND5) gene in the mitochondrial DNA of 29 lines of Drosophila melanogaster and 9 lines of its sibling species Drosophila simulans. In contrast to the patterns for nuclear genes, where D. melanogaster generally exhibits much less nucleotide polymorphism, the number of segregating sites was slightly higher in a global sample of nine ND5 sequences in D. melanogaster (s = 8) than in the nine lines of D. simulans (s = 6). When compared to variation at nuclear loci, the mtDNA variation in D. melanogaster does not depart from neutral expectations. The ND5 sequences in D. simulans, however, show fewer than half the number of variable sites expected under neutrality when compared to sequences from the period locus. While this reduction in variation is not significant at the 5% level, HKA tests with published restriction data for mtDNA in D. simulans do show a significant reduction of variation suggesting a selective sweep of variation in the mtDNA in this species. Tests of neutral evolution based on the ratios of synonymous and replacement polymorphism and divergence are generally consistent with neutral expectations, although a significant excess of amino acid polymorphism within both species is localized in one region of the protein. The rate of mtDNA evolution has been faster in D. melanogaster than in D. simulans and the population structure of mtDNA is distinct in these species. The data reveal how different rates of mtDNA evolution between species and different histories of neutral and adaptive evolution within species can compromise historical inferences in population and evolutionary biology.     

Excess amino acid polymorphism in mitochondrial DNA: contrasts among genes from Drosophila, mice, and humans

Recent studies of mitochondrial DNA (mtDNA) variation in mammals and Drosophila have shown an excess of amino acid variation within species (replacement polymorphism) relative to the number of silent and replacement differences fixed between species. To examine further this pattern of nonneutral mtDNA evolution, we present sequence data for the ND3 and ND5 genes from 59 lines of Drosophila melanogaster and 29 lines of D. simulans. Of interest are the frequency spectra of silent and replacement polymorphisms, and potential variation among genes and taxa in the departures from neutral expectations. The Drosophila ND3 and ND5 data show no significant excess of replacement polymorphism using the McDonald-Kreitman test. These data are in contrast to significant departures from neutrality for the ND3 gene in mammals and other genes in Drosophila mtDNA (cytochrome b and ATPase 6). Pooled across genes, however, both Drosophila and human mtDNA show very significant excesses of amino acid polymorphism. Silent polymorphisms at ND5 show a significantly higher variance in frequency than replacement polymorphisms, and the latter show a significant skew toward low frequencies (Tajima's D = -1.954). These patterns are interpreted in light of the nearly neutral theory where mildly deleterious amino acid haplotypes are observed as ephemeral variants within species but do not contribute to divergence. The patterns of polymorphism and divergence at charge-altering amino acid sites are presented for the Drosophila ND5 gene to examine the evolution of functionally distinct mutations. Excess charge-altering polymorphism is observed at the carboxyl terminal and excess charge-altering divergence is detected at the amino terminal. While the mildly deleterious model fits as a net effect in the evolution of nonrecombining mitochondrial genomes, these data suggest that opposing evolutionary pressures may act on different regions of mitochondrial genes and genomes.      

Shared evolutionary origin of major histocompatibility complex polymorphism in sympatric lemurs

Genes of the major histocompatibility complex (MHC) play a central role in adaptive immune responses of vertebrates. They exhibit remarkable polymorphism, often crossing species boundaries with similar alleles or allelic motifs shared across species. This pattern may reflect parallel parasite‐mediated selective pressures, either favouring the long maintenance of ancestral MHC allelic lineages across successive speciation events by balancing selection (“trans‐species polymorphism”), or alternatively favouring the independent emergence of functionally similar alleles post‐speciation via convergent evolution. Here, we investigate the origins of MHC similarity across several species of dwarf and mouse lemurs (Cheirogaleidae). We examined MHC class II variation in two highly polymorphic loci (DRB, DQB) and evaluated the overlap of gut–parasite communities in four sympatric lemurs. We tested for parasite‐MHC associations across species to determine whether similar parasite pressures may select for similar MHC alleles in different species. Next, we integrated our MHC data with those previously obtained from other Cheirogaleidae to investigate the relative contribution of convergent evolution and co‐ancestry to shared MHC polymorphism by contrasting patterns of codon usage at functional vs. neutral sites. Our results indicate that parasites shared across species may select for functionally similar MHC alleles, implying that the dynamics of MHC‐parasite co‐evolution should be envisaged at the community level. We further show that balancing selection maintaining trans‐species polymorphism, rather than convergent evolution, is the primary mechanism explaining shared MHC sequence motifs between species that diverged up to 30 million years ago.     

A conserved supergene locus controls colour pattern diversity in Heliconius butterflies

We studied whether similar developmental genetic mechanisms are involved in both convergent and divergent evolution. Mimetic insects are known for their diversity of patterns as well as their remarkable evolutionary convergence, and they have played an important role in controversies over the respective roles of selection and constraints in adaptive evolution. Here we contrast three butterfly species, all classic examples of Müllerian mimicry. We used a genetic linkage map to show that a locus, Yb, which controls the presence of a yellow band in geographic races of Heliconius melpomene, maps precisely to the same location as the locus Cr, which has very similar phenotypic effects in its co-mimic H. erato. Furthermore, the same genomic location acts as a "supergene", determining multiple sympatric morphs in a third species, H. numata. H. numata is a species with a very different phenotypic appearance, whose many forms mimic different unrelated ithomiine butterflies in the genus Melinaea. Other unlinked colour pattern loci map to a homologous linkage group in the co-mimics H. melpomene and H. erato, but they are not involved in mimetic polymorphism in H. numata. Hence, a single region from the multilocus colour pattern architecture of H. melpomene and H. erato appears to have gained control of the entire wing-pattern variability in H. numata, presumably as a result of selection for mimetic "supergene" polymorphism without intermediates. Although we cannot at this stage confirm the homology of the loci segregating in the three species, our results imply that a conserved yet relatively unconstrained mechanism underlying pattern switching can affect mimicry in radically different ways. We also show that adaptive evolution, both convergent and diversifying, can occur by the repeated involvement of the same genomic regions.     

Heterorhabditis spp., Neoaplectana spp., and Steinernema kraussei: Interspecific and intraspecific differences in infectivity for insects

The effectiveness of various dosages of different species/strains of nematodes was compared for Galleria mellonella and various pest insects that live in or pupate in soil. Neoaplectana feltiae (= carpocapsae), the only nematode species tested by most other workers, was never the most infective for any of the insect species tested and was least infective for two. All species/strains of nematode were able to kill insects of each species. The degree of infectivity of each of the nematode species/strains for different hosts varied considerably, and no one species/strain of nematode was the most infective for all insect species. This indicates the importance of testing a number of nematode species against any particular insect before commencing field evaluations for biological control.     

Colour constancy in insects

Colour constancy is the perceptual phenomenon that the colour of an object appears largely unchanged, even if the spectral composition of the illuminating light changes. Colour constancy has been found in all insect species so far tested. Especially the pollinating insects offer a remarkable opportunity to study the ecological significance of colour constancy since they spend much of their adult lives identifying and choosing between colour targets (flowers) under continuously changing ambient lighting conditions. In bees, whose colour vision is best studied among the insects, the compensation provided by colour constancy is only partial and its efficiency depends on the area of colour space. There is no evidence for complete ‘discounting’ of the illuminant in bees, and the spectral composition of the light can itself be used as adaptive information. In patchy illumination, bees adjust their spatial foraging to minimise transitions between variously illuminated zones. Modelling allows the quantification of the adaptive benefits of various colour constancy mechanisms in the economy of nature. We also discuss the neural mechanisms and cognitive operations that might underpin colour constancy in insects.     

Estimating the Rate of Adaptive Molecular Evolution When the Evolutionary Divergence Between Species is Small

We investigate the extent by which the estimates of the rate of adaptive molecular evolution obtained by extending the McDonald-Kreitman test are biased if the species, subjected to analysis, diverged recently. We show that estimates can be biased if the nucleotide divergence between the species is low relative to within species variation, and that the magnitude of the bias depends on the rate of adaptive evolution and the distribution of fitness effects of new mutations. Bias appears to be because of three factors: (1) misattribution of polymorphism to divergence; (2) the contribution of ancestral polymorphism to divergence; and (3) different rates of fixation of neutral and advantageous mutations. If there is little adaptive molecular evolution, then slightly deleterious mutations inflate estimates of the rate of adaptive evolution, because these contribute proportionately more to polymorphism than to nucleotide divergence than neutral mutations. However, if there is substantial adaptive evolution, polymorphism contributing to apparent divergence may downwardly bias estimates. We propose a simple method for correcting the different contributions of slightly deleterious and neutral mutations to polymorphism and divergence, and apply it to datasets from several species. We find that estimates of the rate of adaptive molecular evolution from closely related species may be underestimates by ~10% or more. However, after the contribution of polymorphism to divergence is removed, the rate of adaptive evolution may still be overestimated as a consequence of ancestral polymorphism and time for fixation effects. This bias may be substantial if branch lengths are less than 10N (e) generations.     

Increased divergence but reduced variation on the Z chromosome relative to autosomes in Ficedula flycatchers: differential introgression or the faster-Z effect?

Recent multilocus studies of congeneric birds have shown a pattern of elevated interspecific divergence on the Z chromosome compared to the autosomes. In contrast, intraspecifically, birds exhibit less polymorphism on the Z chromosome relative to the autosomes. We show that the four black-and-white Ficedula flycatcher species show greater genetic divergence on the Z chromosome than on the autosomes, and that the ratios of intraspecific polymorphism at Z-linked versus autosomal markers are below the neutral expectation of 75%. In all species pairs, we found more fixed substitutions and fewer shared polymorphisms on the Z chromosome than on the autosomes. Finally, using isolation with migration (IMa) models we estimated gene flow among the four closely related flycatcher species. The results suggest that different pattern of evolution of Z chromosomes and autosomes is best explained by the faster-Z hypothesis, since the estimated long-term gene flow parameters were close to zero in all comparisons.     

Molecular and ecological signs of mitochondrial adaptation: consequences for introgression?

The evolution of the mitochondrial genome and its potential adaptive impact still generates vital debates. Even if mitochondria have a crucial functional role, as they are the main cellular energy suppliers, mitochondrial DNA (mtDNA) introgression is common in nature, introducing variation in populations upon which selection may act. Here we evaluated whether the evolution of mtDNA in a rodent species affected by mtDNA introgression is explained by neutral expectations alone. Variation in one mitochondrial and six nuclear markers in Myodes glareolus voles was examined, including populations that show mtDNA introgression from its close relative, Myodes rutilus. In addition, we modelled protein structures of the mtDNA marker (cytochrome b) and estimated the environmental envelopes of mitotypes. We found that massive mtDNA introgression occurred without any trace of introgression in the analysed nuclear genes. The results show that the native glareolus mtDNA evolved under past positive selection, suggesting that mtDNA in this system has selective relevance. The environmental models indicate that the rutilus mitotype inhabits colder and drier habitats than the glareolus one that can result from local adaptation or from the geographic context of introgression. Finally, homology models of the cytochrome b protein revealed a substitution in rutilus mtDNA in the vicinity of the catalytic fraction, suggesting that differences between mitotypes may result in functional changes. These results suggest that the evolution of mtDNA in Myodes may have functional, ecological and adaptive significance. This work opens perspective onto future experimental tests of the role of natural selection in mtDNA introgression in this system.     

Hybridization between mtDNA-defined phylogeographic lineages of black ratsnakes (Pantherophis sp.)

Phylogeographic analyses using mitochondrial DNA (mtDNA) have revealed many examples of apparently deep historical subdivisions ('phylogroups') within many vertebrates. It remains unclear whether these phylogroups represent independently evolving, adaptively differentiated lineages or groups that show little functional differentiation and, hence, will merge on contact. Here, we use mtDNA sequence data to evaluate the phylogeographic relationships between two of the northernmost populations of black ratsnakes (Pantherophis obsoletus complex) in Ontario, Canada and previously analysed populations in the United States. We then use population-level analyses to evaluate the level of adaptive divergence between previously established mtDNA phylogroups. Phylogenetic analyses show that southern Ontario snakes have mtDNA haplotypes that fall within the Central mtDNA phylogroup, as designated by Burbrink et al. (2000). In contrast, snakes in eastern Ontario carry either Central or Eastern-specific haplotypes. Within the hybrid region, we found highly variable frequencies of mtDNA haplotypes among isolated sub-populations, no association between variation in cytonuclear (mtDNA) and nuclear (microsatellite DNA) markers, no difference in survival or reproductive success among snakes with different mtDNA haplotypes, and no effect of mate similarity in mtDNA on female clutch size. These results argue that the Eastern and Central phylogroups have merged in this region, likely due to a lack of adaptive differentiation between individuals in each lineage. Hence, in these snakes, phylogeographic structure in mtDNA is more a reflection of historical isolation rather than adaptive divergence. The observed reticulation between lineages and lack of evidence for hybrid disgenesis also bears on the classification of these lineages as distinct species.     

Large extent of mitochondrial DNA transfer from Oreochromis aureus to O. niloticus in West Africa

Introgressive hybridization has an important evolutionary significance in terms of gene diversity and speciation. Among the major groups of vertebrates, fish show a strong propensity to hybridize. In order to highlight the possible occurrence of gene flow between two tilapia species, Oreochromis niloticus and O. aureus, a comparison of allozyme and mitochondrial DNA (mtDNA) polymorphism was performed on sympatric and allopatric populations of these two species. Nuclear data were congruent with the morphological identification of O. niloticus and O. aureus populations. In opposition, the mtDNA analysis resulted in two strictly differentiated groups which did not follow the morphological and nuclear DNA classification. The first group consisted of East African O. niloticus populations and the second included all the O. aureus populations and the West African O. niloticus populations. Moreover, in some cases, the same sequences were detected in both species. These data strongly support a differential introgression of mtDNA from O. aureus to O. niloticus involving all the West African area. This work points out the risk of misinterpretation of mtDNA or nuclear DNA data when only one single class of marker is used.     

How and When Do Insects Rely on Endogenous Protein and Lipid Resources during Lethal Bouts of Starvation? A New Application for 13C-Breath testing

Most of our understanding about the physiology of fasting and starvation comes from studies of vertebrates; however, for ethical reasons, studies that monitor vertebrates through the lethal endpoint are scant. Insects are convenient models to characterize the comparative strategies used to cope with starvation because they have diverse life histories and have evolved under the omnipresent challenge of food limitation. Moreover, we can study the physiology of starvation through its natural endpoint. In this study we raised populations of five species of insects (adult grasshoppers, crickets, cockroaches, and larval beetles and moths) on diets labeled with either ¹³C-palmitic acid or ¹³C-leucine to isotopically enrich the lipids or the proteins in their bodies, respectively. The insects were allowed to become postabsorptive and then starved. We periodically measured the δ¹³C of the exhaled breath to characterize how each species adjusted their reliance on endogenous lipids and proteins as energy sources. We found that starving insects employ a wide range of strategies for regulating lipid and protein oxidation. All of the insects except for the beetle larvae were capable of sharply reducing reliance on protein oxidation; however, this protein sparing strategy was usually unsustainable during the entire starvation period. All insects increased their reliance on lipid oxidation, but while some species (grasshoppers, cockroaches, and beetle larvae) were still relying extensively on lipids at the time of death, other species (crickets and moth larvae) allowed rates of lipid oxidation to return to prestarvation levels. Although lipids and proteins are critical metabolic fuels for both vertebrates and insects, insects apparently exhibit a much wider range of strategies for rationing these limited resources during starvation.     

Low neutral and immunogenetic diversity in northern fringe populations of the green toad Bufotes viridis: implications for conservation

Genetic variation is often lower at high latitudes, which may compromise the adaptability and hence survival of organisms. Here we show that genetic variability is negatively correlated with northern latitude in European green toads (Bufotes viridis). The result holds true for both putatively neutral microsatellite variation and supposedly adaptive MHC Class IIB variation. In particular, our findings have bearing on the conservation status of this species in Sweden, on the northern limit of its distribution where local populations are small and fragmented. These genetically impoverished populations are closely related to other populations found around the Baltic Sea basin. The low neutral and adaptive variation in these fringe populations compared to population at central ranges confirms a pattern shared across all other amphibians so far studied. In Sweden, the situation of green toads is of concern as the remaining populations may not have the evolutionary potential to cope with present and future environmental challenges.

     

Deep sympatric mitochondrial divergence without reproductive isolation in the common redstart Phoenicurus phoenicurus

Mitochondrial DNA usually shows low sequence variation within and high sequence divergence among species, which makes it a useful marker for phylogenetic inference and DNA barcoding. A previous study on the common redstart (Phoenicurus phoenicurus) revealed two very different mtDNA haplogroups (5% K2P distance). This divergence is comparable to that among many sister species; however, both haplogroups coexist and interbreed in Europe today. Herein, we describe the phylogeographic pattern of these lineages and test hypotheses for how such high diversity in mtDNA has evolved. We found no evidence for mitochondrial pseudogenes confirming that both haplotypes are of mitochondrial origin. When testing for possible reproductive barriers, we found no evidence for lineage‐specific assortative mating and no difference in sperm morphology, indicating that they are not examples of cryptic species, nor likely to reflect the early stages of speciation. A gene tree based on a short fragment of cytochrome c oxidase subunit 1 from the common redstart and 10 other Phoenicurus species, showed no introgression from any of the extant congenerics. However, introgression from an extinct congeneric cannot be excluded. Sequences from two nuclear introns did not show a similar differentiation into two distinct groups. Mismatch distributions indicated that the lineages have undergone similar demographic changes. Taken together, these results confirm that deeply divergent mitochondrial lineages can coexist in biological species. Sympatric mtDNA divergences are relatively rare in birds, but the fact that they occur argues against the use of threshold mtDNA divergences in species delineation.     

Evolutionary loss of ascorbic acid synthesizing ability

The ability to synthesize ascorbic acid has been found only in terrestrial vertebrates. The ability is not present in certain passerine birds, in fruit-eating bats, in guinea-pigs and in Anthropoidea. We postulate that these species lost this ability by a neutral evolutionary change that occurred sporadically by mutation. The change was adopted in the genetic make-up of a few groups of birds and mammals that are widely-scattered in phylogeny. Many herbivorous vertebrate species which consume diets high in ascorbic acid have retained the ability to synthesize it, so that its loss does not appear to be adaptive.     

Population genomics of eusocial insects: the costs of a vertebrate‐like effective population size

The evolution of reproductive division of labour and social life in social insects has lead to the emergence of several life‐history traits and adaptations typical of larger organisms: social insect colonies can reach masses of several kilograms, they start reproducing only when they are several years old, and can live for decades. These features and the monopolization of reproduction by only one or few individuals in a colony should affect molecular evolution by reducing the effective population size. We tested this prediction by analysing genome‐wide patterns of coding sequence polymorphism and divergence in eusocial vs. noneusocial insects based on newly generated RNA‐seq data. We report very low amounts of genetic polymorphism and an elevated ratio of nonsynonymous to synonymous changes – a marker of the effective population size – in four distinct species of eusocial insects, which were more similar to vertebrates than to solitary insects regarding molecular evolutionary processes. Moreover, the ratio of nonsynonymous to synonymous substitutions was positively correlated with the level of social complexity across ant species. These results are fully consistent with the hypothesis of a reduced effective population size and an increased genetic load in eusocial insects, indicating that the evolution of social life has important consequences at both the genomic and population levels.     

Biotope prioritisation in the Central Apennines (Italy): species rarity and cross-taxon congruence

The conservation status of invertebrates is usually lesser known than that of vertebrates, and strategies to identify biotopes to preserve invertebrate diversity are typically based on a single surrogate taxon, or even on the use of vertebrates as surrogates. Aim of this research is to illustrate a method for biotope prioritisation that can be easily adapted to different animal groups and geographical contexts. A two-step protocol for biotope prioritisation is proposed on the basis of a multidimensional characterisation of species vulnerability. Firstly, species vulnerability is estimated from rarity measures which include geographical range, abundance and biotope specialisation. Then, these values of vulnerability are used to rank biotopes. The method was applied here to the tenebrionid beetles, the butterflies, the birds and the mammals of the Central Apennines, a montane area of high conservation concern for South Europe. This study provides evidence for the importance of including insects in conservation decisions, because vertebrates are poor surrogates for insects. Conservation efforts in the reserves included in the study area are mostly focused on vertebrates, for which woodlands are considered particularly important. However high altitude open biotopes are crucial for both tenebrionids and butterflies, and preservation of such kind of biotopes would be beneficial also for vertebrates. The approach applied here demonstrates that (1) vertebrates are poor surrogates for insects, and (2) measures of species rarity, typically used in vertebrate conservation, can be obtained also for insects, for which a veritable amount of data are hidden in specialised literature and museum collections.     

Isolation by adaptation in Neochlamisus leaf beetles: host-related selection promotes neutral genomic divergence

This study tests how divergent natural selection promotes genomic differentiation during ecological speciation. Specifically, we use adaptive ecological divergence (here, population divergence in host plant use and preference) as a proxy for selection strength and evaluate the correlation between levels of adaptive and genetic differentiation across pairwise population comparisons. Positive correlations would reveal the pattern predicted by our hypothesis, that of 'isolation by adaptation' (IBA). Notably, IBA is predicted not only for selected loci but also for neutral loci. This may reflect the effects of divergent selection on neutral loci that are 'loosely linked' to divergently selected loci or on geneflow restriction that facilitates genetic drift at all loci, including neutral loci that are completely unlinked to those evolving under divergent selection. Here, we evaluate IBA in maple- and willow-associated populations of Neochlamisus bebbianae leaf beetles. To do so, we collected host preference data to construct adaptive divergence indices and used AFLPs (amplified fragment length polymorphisms) and mitochondrial sequences to quantify genetic differentiation. Partial Mantel tests showed significant IBA in 'pooled' analyses of putatively neutral and of putatively selected ('outlier') AFLP loci. This pattern was also recovered in 12% of 'locus-specific' analyses that separately evaluated genetic differentiation at individual neutral loci. These results provided evidence for widespread effects of selection on neutral genomic divergence. Our collective findings indicate that host-related selection may play important roles in the population genomic differentiation of both neutral and selected gene regions in herbivorous insects.     

Widespread recombination in published animal mtDNA sequences

Mitochondrial DNA (mtDNA) recombination has been observed in several animal species, but there are doubts as to whether it is common or only occurs under special circumstances. Animal mtDNA sequences retrieved from public databases were unambiguously aligned and rigorously tested for evidence of recombination. At least 30 recombination events were detected among 186 alignments examined. Recombinant sequences were found in invertebrates and vertebrates, including primates. It appears that mtDNA recombination may occur regularly in the animal cell but rarely produces new haplotypes because of homoplasmy. Common animal mtDNA recombination would necessitate a reexamination of phylogenetic and biohistorical inference based on the assumption of clonal mtDNA transmission. Recombination may also have an important role in producing and purging mtDNA mutations and thus in mtDNA-based diseases and senescence.