The Cyclically Seasonal Drosophila subobscura Inversion O7 Originated From Fragile Genomic Sites and Relocated Immunity and Metabolic Genes.

Chromosome inversions are important contributors to standing genetic variation in Drosophila subobscura. Presently, the species is experiencing a rapid replacement of high-latitude by low-latitude inversions associated with global warming. Yet not all low-latitude inversions are correlated with the ongoing warming trend. This is particularly unexpected in the case of O7 because it shows a regular seasonal cycle that peaks in summer and rose with a heatwave. The inconsistent behavior of O7 across components of the ambient temperature suggests that is causally more complex than simply due to temperature alone. In order to understand the dynamics of O7, high-quality genomic data are needed to determine both the breakpoints and the genetic content. To fill this gap, here we generated a PacBio long read-based chromosome-scale genome assembly, from a highly homozygous line made isogenic for an O3 + 4 + 7 chromosome. Then we isolated the complete continuous sequence of O7 by conserved synteny analysis with the available reference genome. Main findings include the following: (i) the assembled O7 inversion stretches 9.936 Mb, containing > 1,000 annotated genes; (ii) O7 had a complex origin, involving multiple breaks associated with non-B DNA-forming motifs, formation of a microinversion, and ectopic repair in trans with the two homologous chromosomes; (iii) the O7 breakpoints carry a pre-inversion record of fragility, including a sequence insertion, and transposition with later inverted duplication of an Attacin immunity gene; and (iv) the O7 inversion relocated the major insulin signaling forkhead box subgroup O (foxo) gene in tight linkage with its antagonistic regulatory partner serine/threonine-protein kinase B (Akt1) and disrupted concerted evolution of the two inverted Attacin duplicates, reattaching them to dFOXO metabolic enhancers. Our findings suggest that O7 exerts antagonistic pleiotropic effects on reproduction and immunity, setting a framework to understand its relationship with climate change. Furthermore, they are relevant for fragility in genome rearrangement evolution and for current views on the contribution of breakage versus repair in shaping inversion-breakpoint junctions.

Expression of thermal tolerance genes in two Drosophila species with different acclimation capacities.

Heat tolerance increases at higher acclimation temperatures in D. melanogaster, but not in D. subobscura. The two species represent separate lineages of the subgenus Sophophora of Drosophila with contrasting tropical African and temperate Palearctic evolutionary histories. D. melanogaster has five copies of the inducible hsp70 gene distributed in two clusters, named A (with two copies) and B (three copies), while D. subobscura has only two copies arranged similarly to cluster A of D. melanogaster. The hsp70s of the two species also differ in their cis-regulatory regions, with D. melanogaster exhibiting features of a faster and more productive promoter. We predicted that the interspecific variation in acclimation capacity of heat tolerance is explained by evolved variation in expression of the major group of heat shock proteins. To test this prediction, we compared basal levels of gene expression at different developmental temperatures within each of the two species. Furthermore, we explored the heat hardening dynamics by measuring the induction of gene expression during a ramping assay. The prediction of a stronger heat shock protein response in D. melanogaster as compared to D. subobscura was confirmed for both long-term acclimation and short-term hardening. For D. melanogaster the upregulation with temperature ramping ranged from less than two fold (hsp26) to 2500 fold (hsp70A) increase. In all cases induction in D. melanogaster exceeded that of D. subobscura homologs. These differences correlate with structural differences in the regulatory regions of hsp70, and might explain differences in acclimation capacity among species. Finally, in D. melanogaster we found an indication of an inverse relationship between basal and induced levels of hsp70A and hsp83 expression, suggesting a divergent role for thermal adaptation of these genes at benign and stressful temperatures, respectively.

Long-read based assembly and synteny analysis of a reference Drosophila subobscura genome reveals signatures of structural evolution driven by inversions recombination-suppression effects.

BACKGROUND: Drosophila subobscura has long been a central model in evolutionary genetics. Presently, its use is hindered by the lack of a reference genome. To bridge this gap, here we used PacBio long-read technology, together with the available wealth of genetic marker information, to assemble and annotate a high-quality nuclear and complete mitochondrial genome for the species. With the obtained assembly, we performed the first synteny analysis of genome structure evolution in the subobscura subgroup. RESULTS: We generated a highly-contiguous ~ 129 Mb-long nuclear genome, consisting of six pseudochromosomes corresponding to the six chromosomes of a female haploid set, and a complete 15,764 bp-long mitogenome, and provide an account of their numbers and distributions of codifying and repetitive content. All 12 identified paracentric inversion differences in the subobscura subgroup would have originated by chromosomal breakage and repair, with some associated duplications, but no evidence of direct gene disruptions by the breakpoints. Between lineages, inversion fixation rates were 10 times higher in continental D. subobscura than in the two small oceanic-island endemics D. guanche and D. madeirensis. Within D. subobscura, we found contrasting ratios of chromosomal divergence to polymorphism between the A sex chromosome and the autosomes. CONCLUSIONS: We present the first high-quality, long-read sequencing of a D. subobscura genome. Our findings generally support genome structure evolution in this species being driven indirectly, through the inversions' recombination-suppression effects in maintaining sets of adaptive alleles together in the face of gene flow. The resources developed will serve to further establish the subobscura subgroup as model for comparative genomics and evolutionary indicator of global change.

Chromosomal inversions promote genomic islands of concerted evolution of Hsp70 genes in the Drosophila subobscura species subgroup.

Heat-shock (HS) assays to understand the connection between standing inversion variation and evolutionary response to climate change in Drosophila subobscura found that "warm-climate" inversion O3+4 exhibits non-HS levels of Hsp70 protein like those of "cold-climate" OST after HS induction. This was unexpected, as overexpression of Hsp70 can incur multiple fitness costs. To understand the genetic basis of this finding, we have determined the genomic sequence organization of the Hsp70 family in four different inversions, including OST , O3+4 , O3+4+8 and O3+4+16 , using as outgroups the remainder of the subobscura species subgroup, namely Drosophila madeirensis and Drosophila guanche. We found (i) in all the assayed lines, the Hsp70 family resides in cytological locus 94A and consists of only two genes, each with four HS elements (HSEs) and three GAGA sites on its promoter. Yet, in OST, the family is comparatively more compact; (ii) the two Hsp70 copies evolve in concert through gene conversion, except in D. guanche; (iii) within D. subobscura, the rate of concerted evolution is strongly structured by inversion, being higher in OST than in O3+4 ; and (iv) in D. guanche, the two copies accumulated multiple differences, including a newly evolved "gap-type" HSE2. The absence of concerted evolution in this species may be related to a long-gone-unnoticed observation that it lacks Hsp70 HS response, perhaps because it has evolved within a narrow thermal range in an oceanic island. Our results point to a previously unrealized link between inversions and concerted evolution, with potentially major implications for understanding genome evolution.

Genome-wide evolutionary response to a heat wave in Drosophila.

Extreme climatic events can substantially affect organismal performance and Darwinian fitness. In April 2011, a strong heat wave struck extensive geographical areas of the world, including Western Europe. At that time, we happened to resume and extend a long-term time series of seasonal genetic data in the widespread fly Drosophila subobscura, which provided a unique opportunity to quantify the intensity of the genetic perturbation caused by the heat wave. We show that the spring 2011 genetic constitution of the populations transiently shifted to summer-like frequencies, and that the magnitude of the genetic anomaly quantitatively matched the temperature anomaly. The results provide compelling evidence that direct effects of rising temperature are driving adaptive evolutionary shifts, and also suggest a strong genetic resilience in this species.

'Costa da Morte' ataxia is spinocerebellar ataxia 36: clinical and genetic characterization.

Spinocerebellar ataxia 36 has been recently described in Japanese families as a new type of spinocerebellar ataxia with motor neuron signs. It is caused by a GGCCTG repeat expansion in intron 1 of NOP56. Family interview and document research allowed us to reconstruct two extensive, multigenerational kindreds stemming from the same village (Costa da Morte in Galicia, Spain), in the 17th century. We found the presence of the spinocerebellar ataxia 36 mutation co-segregating with disease in these families in whom we had previously identified an ~0.8 Mb linkage region to chromosome 20 p. Subsequent screening revealed the NOP56 expansion in eight additional Galician ataxia kindreds. While normal alleles contain 5-14 hexanucleotide repeats, expanded alleles range from ~650 to 2500 repeats, within a shared haplotype. Further expansion of repeat size was frequent, especially upon paternal transmission, while instances of allele contraction were observed in maternal transmissions. We found a total of 63 individuals carrying the mutation, 44 of whom were confirmed to be clinically affected; over 400 people are at risk. We describe here the detailed clinical picture, consisting of a late-onset, slowly progressive cerebellar syndrome with variable eye movement abnormalities and sensorineural hearing loss. There were signs of denervation in the tongue, as well as mild pyramidal signs, but otherwise no signs of classical amyotrophic lateral sclerosis. Magnetic resonance imaging findings were consistent with the clinical course, showing atrophy of the cerebellar vermis in initial stages, later evolving to a pattern of olivo-ponto-cerebellar atrophy. We estimated the origin of the founder mutation in Galicia to have occurred ~1275 years ago. Out of 160 Galician families with spinocerebellar ataxia, 10 (6.3%) were found to have spinocerebellar ataxia 36, while 15 (9.4%) showed other of the routinely tested dominant spinocerebellar ataxia types. Spinocerebellar ataxia 36 is thus, so far, the most frequent dominant spinocerebellar ataxia in this region, which may have implications for American countries associated with traditional Spanish emigration.

The Vein Patterning 1 (VEP1) gene family laterally spread through an ecological network.

Lateral gene transfer (LGT) is a major evolutionary mechanism in prokaryotes. Knowledge about LGT--particularly, multicellular--eukaryotes has only recently started to accumulate. A widespread assumption sees the gene as the unit of LGT, largely because little is yet known about how LGT chances are affected by structural/functional features at the subgenic level. Here we trace the evolutionary trajectory of VEin Patterning 1, a novel gene family known to be essential for plant development and defense. At the subgenic level VEP1 encodes a dinucleotide-binding Rossmann-fold domain, in common with members of the short-chain dehydrogenase/reductase (SDR) protein family. We found: i) VEP1 likely originated in an aerobic, mesophilic and chemoorganotrophic α-proteobacterium, and was laterally propagated through nets of ecological interactions, including multiple LGTs between phylogenetically distant green plant/fungi-associated bacteria, and five independent LGTs to eukaryotes. Of these latest five transfers, three are ancient LGTs, implicating an ancestral fungus, the last common ancestor of land plants and an ancestral trebouxiophyte green alga, and two are recent LGTs to modern embryophytes. ii) VEP1's rampant LGT behavior was enabled by the robustness and broad utility of the dinucleotide-binding Rossmann-fold, which provided a platform for the evolution of two unprecedented departures from the canonical SDR catalytic triad. iii) The fate of VEP1 in eukaryotes has been different in different lineages, being ubiquitous and highly conserved in land plants, whereas fungi underwent multiple losses. And iv) VEP1-harboring bacteria include non-phytopathogenic and phytopathogenic symbionts which are non-randomly distributed with respect to the type of harbored VEP1 gene. Our findings suggest that VEP1 may have been instrumental for the evolutionary transition of green plants to land, and point to a LGT-mediated 'Trojan Horse' mechanism for the evolution of bacterial pathogenesis against plants. VEP1 may serve as tool for revealing microbial interactions in plant/fungi-associated environments.

A novel MYH7 mutation links congenital fiber type disproportion and myosin storage myopathy.

This study aimed to identify the genetic defect in a multigenerational family presenting an autosomal dominant myopathy with histological features of congenital fiber type disproportion. Linkage analysis and genetic sequencing identified, in all affected members of the family, the c.5807A>G heterozygous mutation in MYH7, which encodes the slow/ß-cardiac myosin heavy chain. This mutation causes skeletal but not cardiac involvement. Myosin heavy chain expression pattern was also characterized by immunohistochemistry, western blot and q-PCR in muscle biopsies from two patients aged 25 and 62, respectively. While only congenital fiber type disproportion was observed in the younger patient, older patient's biopsy presented aggregates of slow myosin heavy chains, in fiber sub-sarcolemmal region. These clinico-pathologic findings suggest a novel phenotype within the emerging group of hereditary myosin myopathies, which in this family presents typical characteristics of congenital fiber type disproportion in early stages and later evolves to myosin storage myopathy.

Alternative splicing: a missing piece in the puzzle of intron gain.

Spliceosomal introns, a hallmark of eukaryotic gene organization, were an unexpected discovery. After three decades, crucial issues such as when and how introns first appeared in evolution remain unsettled. An issue yet to be answered is how intron positions arise de novo. Phylogenetic investigations concur that intron positions continue to emerge, at least in some lineages. Yet genomic scans for the sources of introns occupying new positions have been fruitless. Two alternative solutions to this paradox are: (i) formation of new intron positions halted before the recent past and (ii) it continues to occur, but through processes different from those generally assumed. One process generally dismissed is intron sliding--the relocation of a preexisting intron over short distances--because of supposed associated deleterious effects. The puzzle of intron gain arises owing to a pervasive operational definition of introns, which sees them as precisely demarcated segments of the genome separated from the neighboring nonintronic DNA by unmovable limits. Intron homology is defined as position homology. Recent studies of pre-mRNA processing indicate that this assumption needs to be revised. We incorporate recent advances on the evolutionarily frequent process of alternative splicing, by which exons of primary transcripts are spliced in different patterns, into a new model of intron sliding that accounts for the diversity of intron positions. We posit that intron positional diversity is driven by two overlapping processes: (i) background process of continuous relocation of preexisting introns by sliding and (ii) spurts of extensive gain/loss of new intron sequences.

Comment on "Global genetic change tracks global climate warming in Drosophila subobscura".

Balanyà et al. (Reports, 22 September 2006, p. 1773) build on earlier claims that chromosomal inversion polymorphisms of Drosophila subobscura are rapidly evolving in response to global warming. However, that conclusion is not adequately buttressed by their data, because they overlooked the lag between calendar and climatological dates created by the progressive lengthening of the growing season in their sampling approach.

Plant progesterone 5beta-reductase is not homologous to the animal enzyme. Molecular evolutionary characterization of P5betaR from Digitalis purpurea.

Plants of the genus Digitalis produce cardiac glycosides, i.e. digoxin, which are widely used for congestive heart failure. Progesterone 5beta-reductase (P5betaR) is a key enzyme in the biosynthesis of these natural products. Here, we have carried out the purification and partial amino acid sequencing of the native P5betaR from foxglove (Digitalis purpurea), and isolated a cDNA encoding this enzyme. Similarly to other steroid 5beta-reductases, the recombinant P5betaR catalyzes the stereospecific reduction of the Delta(4)-double bond of several steroids with a 3-oxo,Delta(4,5) structure. The gene encoding P5betaR is expressed in all plant organs, and maximally transcribed in leaves and mature flowers. P5betaR belongs to the short-chain dehydrogenase/reductase (SDR) superfamily, bearing no structural homology to its mammalian counterpart, which is a member of the aldo-keto reductase (AKR) superfamily. A similar situation occurs with 3beta-hydroxy-Delta(5)-steroid dehydrogenase (3betaHSD), the gene immediately preceding P5betaR in the cardenolide pathway, which suggests that the entire route has evolved independently in animals and plants. P5betaR is retained only in plants, where it is ubiquitous, and a few distantly related bacterial lineages after its diversification from the last universal common ancestor. Evolutionary conserved changes in its putative active site suggest that plant P5betaR is a member of a novel subfamily of extended SDRs, or a new SDR family.

Origins and evolution of spliceosomal introns.

Research into the origins of introns is at a critical juncture in the resolution of theories on the evolution of early life (which came first, RNA or DNA?), the identity of LUCA (the last universal common ancestor, was it prokaryotic- or eukaryotic-like?), and the significance of noncoding nucleotide variation. One early notion was that introns would have evolved as a component of an efficient mechanism for the origin of genes. But alternative theories emerged as well. From the debate between the "introns-early" and "introns-late" theories came the proposal that introns arose before the origin of genetically encoded proteins and DNA, and the more recent "introns-first" theory, which postulates the presence of introns at that early evolutionary stage from a reconstruction of the "RNA world." Here we review seminal and recent ideas about intron origins. Recent discoveries about the patterns and causes of intron evolution make this one of the most hotly debated and exciting topics in molecular evolutionary biology today.

Models of spliceosomal intron proliferation in the face of widespread ectopic expression.

It is now certain that today living organisms can acquire new spliceosomal introns in their genes. The proposed sources of spliceosomal introns are exons, transposons, and other introns, including spliceosomal and group II self-splicing introns. Spliceosomal introns are thought to be the most likely source, because the inserted sequence would immediately be endowed with the essential set of intron recognition sequences, thereby preventing the deleterious effects associated with incorrect splicing. The most obvious spliceosomal intron duplication pathways involve an RNA transcript intermediate step. Therefore, for a spliceosomal intron to be originated by duplication, either the source gene from which the novel intron is derived, or that gene and the recipient gene, which contains the novel intron, would need to be expressed in the germ line. Intron proliferation surveys indicate that putative intron duplicate-containing genes do not always match detectable expression in the germ line, which casts doubt on the generality of the duplication model. However, judging mechanisms of intron gain (or loss) from present-day gene expression profiles could be erroneous, if expression patterns were different at the time the introns arose. In fact, this may likely be so in most cases. Ectopic expression, i.e., the expression of genes at times and locations where the target gene is not known to have a function, is a much more common phenomenon than previously realized. We conclude with a speculation on a possible interplay between spliceosomal introns and ectopic expression at the origin of multicellularity.

Is ectopic expression caused by deregulatory mutations or due to gene-regulation leaks with evolutionary potential?

It has long been thought that gene expression is tightly regulated in multicellular eukaryotes, so that expression profiles match functional profiles. This conception emerged from the assumption that gene activity is synonymous with gene function. This paradigm was first challenged by comparative protein electrophoresis studies showing extensive differences in expression patterns among related species. The paradigm is now being challenged by evolutionary transcriptomics using microarray technologies. Most gene expression profiles display features that lack any obvious functional significance. The so-called "ectopic" expression refers to the expression of genes at times and locations where the target gene is not known to have a function. However, ectopic expression might be associated with genuine function even if this function is not essential or has yet to be ascertained. Alternatively, ectopic expression might come about as a superfluous by-product of regulatory systems, which would call for a revision of prevailing ideas about the specificity of gene regulation. We herein review available evidence for ectopic expression and the hypotheses proposed for its origin and evolution. We propose that ectopic expression must be regarded as part of an integrated phenotypic whole. It seems likely that ectopic expression represents a leak in the evolution of regulatory systems, but one that is endowed with considerable evolutionary possibilities.

Convergent neofunctionalization by positive Darwinian selection after ancient recurrent duplications of the xanthine dehydrogenase gene.

Gene duplication is a primary source of molecular substrate for the emergence of evolutionary novelties. The chances for redundant gene sequences to evolve new functions are small compared with the probability that the copies become disabled by deleterious mutations. Functional divergence after gene duplication can result in two alternative evolutionary fates: one copy acquires a novel function (neofunctionalization), or each copy adopts part of the tasks of their parental gene (subfunctionalization). The relative prevalence of each outcome is unknown. Similarly unknown is the relative importance of positive selection versus random fixation of neutral mutations. Aldehyde oxidase (Ao) and xanthine dehydrogenase (Xdh) genes encode two complex members of the xanthine oxidase family of molybdo-flavoenzymes that carry different functions. Ao is known to have originated from a duplicate of an Xdh gene in eukaryotes, before the origin of multicellularity. We show that (i) Ao evolved independently twice from two different Xdh paralogs, the second time in the chordates, before the diversification of vertebrates; (ii) after each duplication, the Ao duplicate underwent a period of rapid evolution during which identical sites across the two molecules, involving the flavin adenine dinucleotide and substrate-binding pockets, were subjected to intense positive Darwinian selection; and (iii) the second Ao gene likely endured two periods of redundancy, initially as a duplicate of Xdh and later as a functional equivalent of the old Ao, which is currently absent from the vertebrate genome. Caution is appropriate in structural genomics when using sequence similarity for assigning protein function.

Seasonal cycles of allozyme-by-chromosomal-inversion gametic disequilibrium in Drosophila subobscura.

Allozyme loci are frequently found non randomly associated to the chromosomal inversions in which they are included in Drosophila. Two opposite views compete to explain strong allozyme-by-inversion gametic disequilibria: they result from natural selection or, conversely, merely represent remnants of associations accidentally established at the origin of inversions. Empirical efforts aimed at deciding between adaptive and historical scenarios have focused on the spatial distribution of disequilibria. Yet, the evolutionary significance of these associations remains uncertain. I report here the results of a time-series analysis of the seasonal variation of alleles at six allozyme loci (Acph, Lap, Pept-1, Ao, Mpi, and Xdh) in connection with the O chromosomal polymorphisms of D. subobscura. The findings were: (1) in the segment I of the O chromosome, Lap and Pept-1 allozymes changed seasonally in a cyclical fashion within the ST gene arrangement, but they changed erratically within the 3 + 4 gene configuration; (2) the frequencies of Lap1.11 and Pept-1(0.40) within ST dropped to their lowest values in early and late summer, respectively, when the seasonal level of the ST arrangement is lowest. Furthermore, Lap1.11 and Pept-1(0.40) covary with ST only within these seasons, yet in a fashion inconsistent with these alleles having a major influence on the dynamics of the inversion; (3) seasonal cycling of alleles within inversions were not detected at Acph, Ao, Mpi, and Xdh, yet these loci are nearly monomorphic at the study population, and/or their sampled series were shorter than those for Lap and Pept-1; and (4) simply monitoring allozyme frequencies separately for each inversion proved to be superior, for evidencing the seasonal cycles of the disequilibria, to the use of the D' coefficient of association. Observed seasonal cycles of allozymes within inversions likely reflect natural selection.

A new Drosophila spliceosomal intron position is common in plants.

The 25-year-old debate about the origin of introns between proponents of "introns early" and "introns late" has yielded significant advances, yet important questions remain to be ascertained. One question concerns the density of introns in the last common ancestor of the three multicellular kingdoms. Approaches to this issue thus far have relied on counts of the numbers of identical intron positions across present-day taxa on the assumption that the introns at those sites are orthologous. However, dismissing parallel intron gain for those sites may be unwarranted, because various factors can potentially constrain the site of intron insertion. Demonstrating parallel intron gain is severely handicapped, because intron sequences often evolve exceedingly fast and intron phylogenetic distributions are usually ambiguous, such that alternative loss and gain scenarios cannot be clearly distinguished. We have identified an intron position that was gained independently in animals and plants in the xanthine dehydrogenase gene. The extremely disjointed phylogenetic distribution of the intron argues strongly for separate gain rather than recurrent loss. If the observed phylogenetic pattern had resulted from recurrent loss, all observational support previously gathered for the introns-late theory of intron origins based on the phylogenetic distribution of introns would be invalidated.

Evolution of cis-regulatory regions versus codifying regions.

Efforts to understand the genetic basis of evolutionary change have concentrated on proteins and their encoding DNA sequences. These studies have brought to light patterns and processes at the nucleotide level, yet the complex functional relationships between genetic variants and phenotypes remain poorly known. The realization that even a complete description of proteins and the effects of their activity will not suffice to understand the conditions under which they are time- and tissue-specifically expressed or repressed during development has refocused attention on cis-regulatory regions. In particular, promoter sequences are thought to hold the key for understanding the evolution of phenotypic differences between species. This is because of their complex organization into independent modules such that, unlike coding sequences in which mutations affect protein function every time the protein is expressed, mutations in cis-regulatory sequences may have minor or no pleiotropic effects. Complex information-encoding makes cis-regulatory regions poorly amenable to comparative methods designed for coding sequences. Some general conclusions are emerging as to how genetic variation is distributed across regulatory networks and the processes modulating the structure of this variation. We bring into this emerging scenario several recent findings pointing to different ways in which spliceosomal introns, pseudogenes and patterns of point mutation can be active players for the evolution of novel transcriptional profiles.

Ribonucleotide reductases: divergent evolution of an ancient enzyme.

Ribonucleotide reductases (RNRs) are uniquely responsible for converting nucleotides to deoxynucleotides in all dividing cells. The three known classes of RNRs operate through a free radical mechanism but differ in the way in which the protein radical is generated. Class I enzymes depend on oxygen for radical generation, class II uses adenosylcobalamin, and the anaerobic class III requires S-adenosylmethionine and an iron-sulfur cluster. Despite their metabolic prominence, the evolutionary origin and relationships between these enzymes remain elusive. This gap in RNR knowledge can, to a major extent, be attributed to the fact that different RNR classes exhibit greatly diverged polypeptide chains, rendering homology assessments inconclusive. Evolutionary studies of RNRs conducted until now have focused on comparison of the amino acid sequence of the proteins, without considering how they fold into space. The present study is an attempt to understand the evolutionary history of RNRs taking into account their three-dimensional structure. We first infer the structural alignment by superposing the equivalent stretches of the three-dimensional structures of representatives of each family. We then use the structural alignment to guide the alignment of all publicly available RNR sequences. Our results support the hypothesis that the three RNR classes diverged from a common ancestor currently represented by the anaerobic class III. Also, lateral transfer appears to have played a significant role in the evolution of this protein family.

A methodological bias toward overestimation of molecular evolutionary time scales.

There is presently a conflict between fossil- and molecular-based evolutionary time scales. Molecular approaches for dating the branches of the tree of life frequently lead to substantially deeper times of divergence than those inferred by paleontologists. The discrepancy between molecular and fossil estimates persists despite the booming growth of sequence data sets, which increasingly feeds the interpretation that molecular estimates are older than stratigraphic dates because of deficiencies in the fossil record. Here we show that molecular time estimates suffer from a methodological handicap, namely that they are asymmetrically bounded random variables, constrained by a nonelastic boundary at the lower end, but not at the higher end of the distribution. This introduces a bias toward an overestimation of time since divergence, which becomes greater as the length of the molecular sequence and the rate of evolution decrease.