Author Correction: Epigenetic patterns associated with an ascidian invasion: a comparison of closely related clades in their native and introduced ranges.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

No evidence for an association between Clock gene allelic variation and migration timing in a long-distance migratory shorebird (Limosa lapponica baueri).

The gene Clock is a key part of the Core Circadian Oscillator, and the length of the polyglutamine (poly-Q) repeat sequence in Clock (ClkpolyQcds) has been proposed to be associated with the timing of annual cycle events in birds. We tested whether variation in ClkpolyQcds corresponds to variation in migration timing in the bar-tailed godwit (Limosa lapponica baueri), a species in which individuals show strong annual consistency in their migration timing despite the New Zealand population migrating across a 5-week period. We describe allelic variation of the ClkpolyQcds in 135 godwits over-wintering in New Zealand (N.Z.) and investigate whether polymorphism in this region is associated with northward migration timing (chronophenotype) from N.Z. or (for 32 birds tracked by geolocator) after the primary stopover in Asia. Six Clock alleles were detected (Q7‒Q12) and there was substantial variation between individuals (heterozygosity of 0.79). There was no association between ClkpolyQcds polymorphism and migration timing from N.Z. The length of the shorter Clock allele was related to migration timing from Asia, though this relationship arose largely from just a few northern-breeding birds with longer alleles. Other studies show no consistent associations between ClkpolyQcds and migration timing in birds, although Clock may be associated with breeding latitude in some species (as an adaptation to photoperiodic regime). Apparent relationships with migration timing could reflect latitude-related variation in migration timing, rather than Clock directly affecting migration timing. On current evidence, ClkpolyQcds is not a strong candidate for driving migration timing in migratory birds generally.

Epigenetic patterns associated with an ascidian invasion: a comparison of closely related clades in their native and introduced ranges.

Environmentally induced epigenetic modifications have been proposed as one mechanism underlying rapid adaptive evolution of invasive species. Didemnum vexillum is an invasive colonial ascidian that has established in many coastal waters worldwide. Phylogenetic analyses have revealed that D. vexillum populations consist of two distinct clades; clade B appears to be restricted to the native range (Japan), whereas clade A is found in many regions throughout the world, including New Zealand. The spread of D. vexillum clade A suggests that it might be intrinsically more invasive than clade B, despite low levels of genetic diversity compared to populations from the native region. This study investigated whether D. vexillum clade A exhibits epigenetic signatures (specifically differences in DNA methylation) associated with invasiveness. Global DNA methylation patterns were significantly different between introduced clade A colonies, and both clades A and B in the native range. Introduced colonies also showed a significant reduction in DNA methylation levels, which could be a mechanism for increasing phenotypic plasticity. High levels of DNA methylation diversity were maintained in the introduced population, despite reduced levels of genetic diversity, which may allow invasive populations to respond quickly to changes in new environments. Epigenetic changes induced during the invasion process could provide a means for rapid adaptation despite low levels of genetic variation in introduced populations.

Effects of temperature and salinity stress on DNA methylation in a highly invasive marine invertebrate, the colonial ascidian Didemnum vexillum.

Environmentally induced epigenetic changes may contribute to phenotypic plasticity, increase adaptive potential in changing environments, and play a key role in the establishment and spread of invasive species in new habitats. In this study, we used methylation-sensitive amplified polymorphism (MSAP) to assess environmentally induced DNA methylation changes in a globally invasive clonal ascidian, Didemnum vexillum. We tested the effect of increasing temperature (19, 25 and 27 °C) and decreasing salinity (34, 32, 30, 28 and 26 practical salinity units (PSU)) on global DNA methylation, growth and survival rates. Exposure to 27 °C resulted in significant changes in DNA methylation over time. Growth also decreased in colonies exposed to high temperatures, suggesting they were under thermal stress. In contrast, no differences in growth nor DNA methylation patterns were observed in colonies exposed to a decreasing salinity gradient, potentially due to prior adaptation. The results of this study show that environmental stress can induce significant global DNA methylation changes in an invasive marine invertebrate on very rapid timescales, and that this response varies depending on the type, magnitude, and duration of the stressor. Changes in genomic DNA methylation and the rate of growth may act to 'buy survival time' under stressful conditions, expanding the distribution limits of this globally invasive species.

Efficient dispersal and substrate acquisition traits in a marine invasive species via transient chimerism and colony mobility.

Over the past three decades the colonial ascidian Didemnum vexillum has been expanding its global range, significantly impacting marine habitats and aquaculture facilities. What biological features make D. vexillum so highly invasive? Here, we show that juxtaposed allogeneic D. vexillum colony fragments ('ramets') may, initially, form chimeric entities. Subsequently, zooids of the differing genotypes within such chimeras coordinately retreat away from fusion zones. A few days following such post-fusion retreat movements there is further ramet fission and the formation of zooid-depauperate tunic zones. Using polymorphic microsatellite loci to distinguish between genotypes, we found that they were sectorial at the fusion zones and the subsequent ramet movements resulted in further spatial separation of the paired-genotypes indicating that the fusion events observed did not lead to formation of long-term, stable chimeras. Thus, movements of D. vexillum colony ramets from initial fusion zones lead to progressive segregation of genotypes probably minimizing potential somatic/germ-cell competition/parasitism. We speculate that relatively fast (≤10 mm/day) movement of D. vexillum colonies on substrates along with frequent, and perhaps unrestrained, transient allogeneic fusions play significant roles in this species' striking invasiveness and capacity to colonize new substrates.

Geographically conserved microbiomes of four temperate water tunicates.

Tunicates are useful models for exploring microbiomes because they have an innate immune system resembling that of chordates. Automated ribosomal RNA intergenic spacer analysis and High-Throughput Sequencing were used to compare the tunic microbiomes of Ciona robusta (formerly Ciona intestinalis type A), Ciona savignyi, Botrylloides leachi and Botryllus schlosseri sampled from three distinct locations with limited genetic connectivity. Bacterial phylotype profiles were conserved within each species, and there were no detectable differences between tunic and tunic + cuticle subsamples from an individual. Bacterial operational taxonomic unit (OTU) diversity was lowest for C. savignyi (320 ± 190 OTUs) and highest for B. schlosseri (1260 ± 190 OTUs). Each species had a distinct set of bacterial OTUs (pseudo-F = 3.0, p > 0.001), with the exception of B. leachi and B. schlosseri from one sampling location (t = 1.2, p = 0.09). Of note were OTUs assigned to Alphaproteobacteria from C. robusta plus Phyllobacteriaceae and Endozoicomonas from C. savignyi. These OTUs contributed 51, 22 and 10% of sequence reads, respectively, and are related to known bacterial symbionts. The within-species conservation of core OTUs across three distinct and co-occurring populations of tunicates provides compelling evidence that these tunicates foster defined microbiomes.

Tunicate pregnane X receptor (PXR) orthologs: transcript characterization and natural variation.

Vertebrate pregnane X receptor (PXR, NR1I2), a ligand-activated nuclear receptor (NR), regulates expression of detoxification genes. Vertebrate PXR orthologs may adaptively evolve to bind deleterious/toxic xenobiotics typically encountered by organisms from their diet. Tunicates (phylum Chordata) are marine filter-feeders that form a sister clade to the Vertebrata. Genomes of two tunicate taxa, Ciona intestinalis and Botryllus schlosseri, encode at least two PXR orthologs (abbreviated VDR/PXRα and ß). Here we report characterization of the transcript structures and sequence variation of three tunicate PXR orthologs: C. intestinalis VDR/PXRα and ß, and B. schlosseri VDR/PXRα. The three predicted proteins consist of both DNA-binding (DBD) and ligand-binding (LBD) domains typical of NRs. The C. intestinalis VDR/PXRß LBD may be significantly larger than that of the VDR/PXRα orthologs. In both tunicate taxa, the mRNAs were characterized by high frequencies of single nucleotide polymorphisms (SNPs, ca. 3 SNPs/100 base pairs). The majority of SNPs were synonymous and standard tests (Tajima's D, dN/dS ratios) indicated strong purifying selection. However, one base pair frameshift allelic variants were found in the C. intestinalis VDR/PXRα and ß genes. The predicted proteins consisted of a DBD but lacked an LBD. The persistence of these variants may possibly reflect constitutive expression of detoxification genes as a selective advantage in the marine environment. These results provide a foundation for further investigations into the molecular evolution, population genetics and functioning of tunicate receptors involved in detection of marine bioactive compounds.

Detection of marine microalgal biotoxins using bioassays based on functional expression of tunicate xenobiotic receptors in yeast.

Marine microalgae can produce biotoxins that cause widespread poisoning in marine ecosystems and may also affect human health. While established microalgal biotoxins are detectable using chemical methods, a need remains for robust, inexpensive bioassays. Ligand-binding domains (LBDs) from a tunicate nuclear receptor, VDR/PXRα, which is orthologous to both the vertebrate pregnane X receptor (PXR) and the vitamin D receptor (VDR), can be activated by microalgal biotoxins when expressed in mammalian cell lines. Building on this observation, we developed a generic recombinant yeast bioassay platform that expresses chimeric proteins containing tunicate VDR/PXRα LBDs which mediate ligand-dependent transcription of a reporter gene (lacZ) encoding an easily assayed enzyme (ß-galactosidase). Recombinant yeast strains expressing VDR/PXRα LBDs from two tunicate species, Ciona intestinalis and Botryllus schlosseri, were exposed to both synthetic and natural toxins. Structurally simple synthetic chemicals (n-butyl-p-aminobenzoate, carbamazepine, p-aminobenzoic acid, and bisphenol-A) generated EC50 values in the µM range, while more structurally complex marine biotoxins (okadaic acid, pectenotoxin-11, and portimine) activated the assays in the nM range. Given the large number of tunicate species, we propose that tunicate VDR/PXR LBDs may be used as 'sensor elements' in similar yeast-based high-throughput bioassays for detection of established microalgal biotoxins and uncharacterised marine bioactive compounds.

Comparison of whole mitochondrial genome sequences from two clades of the invasive ascidian, Didemnum vexillum.

The mitochondria are the main source of cellular energy production and have an important role in development, fertility, and thermal limitations. Adaptive mitochondrial DNA mutations have the potential to be of great importance in determining aspects of the life history of an organism. Phylogenetic analyses of the globally invasive marine ascidian Didemnum vexillum using the mitochondrial cytochrome c oxidase 1 (COX1) coding region, revealed two distinct clades. Representatives of one clade (denoted by 'B') are geographically restricted to D. vexillum's native region (north-west Pacific Ocean, including Japan), whereas members of the other clade (denoted by 'A') have been introduced and become invasive in temperate coastal areas around the world. Persistence of clade B's restricted distribution may reflect it being inherently less invasive than clade A. To investigate this we sought to determine if the two clades differ significantly in other mitochondrial genes of functional significance, specifically, alterations in amino acids encoded in mitochondrial enzyme subunits. Differences in functional mitochondrial genes could indicate an increased ability for clade A colonies to tolerate a wider range of environmental temperature. Full mitochondrial genomic sequences from D. vexillum clades A and B were obtained and they predict significant sequence differences in genes encoding for enzymes involved in oxidative phosphorylation. Diversity levels were relatively high and showed divergence across almost all genes, with p-distance values between the two clades indicating recent divergence. Both clades showed an excess of rare variants, which is consistent with balancing selection or a recent population expansion. Results presented here will inform future research focusing on examining the functional properties of the corresponding mitochondrial respiration enzymes, of A and B clade enzymes. By comparing closely related taxa that have differing distributions it is possible to identify genes and phenotypes suited to particular environments. The examination of mitochondrial genotypes, and associated enzyme functioning, across populations may aid in our understanding of thermal tolerance and environmental adaptation.

Marine invertebrate xenobiotic-activated nuclear receptors: their application as sensor elements in high-throughput bioassays for marine bioactive compounds.

Developing high-throughput assays to screen marine extracts for bioactive compounds presents both conceptual and technical challenges. One major challenge is to develop assays that have well-grounded ecological and evolutionary rationales. In this review we propose that a specific group of ligand-activated transcription factors are particularly well-suited to act as sensors in such bioassays. More specifically, xenobiotic-activated nuclear receptors (XANRs) regulate transcription of genes involved in xenobiotic detoxification. XANR ligand-binding domains (LBDs) may adaptively evolve to bind those bioactive, and potentially toxic, compounds to which organisms are normally exposed to through their specific diets. A brief overview of the function and taxonomic distribution of both vertebrate and invertebrate XANRs is first provided. Proof-of-concept experiments are then described which confirm that a filter-feeding marine invertebrate XANR LBD is activated by marine bioactive compounds. We speculate that increasing access to marine invertebrate genome sequence data, in combination with the expression of functional recombinant marine invertebrate XANR LBDs, will facilitate the generation of high-throughput bioassays/biosensors of widely differing specificities, but all based on activation of XANR LBDs. Such assays may find application in screening marine extracts for bioactive compounds that could act as drug lead compounds.

Increased inter-colony fusion rates are associated with reduced COI haplotype diversity in an invasive colonial ascidian Didemnum vexillum.

Considerable progress in our understanding of the population genetic changes associated with biological invasions has been made over the past decade. Using selectively neutral loci, it has been established that reductions in genetic diversity, reflecting founder effects, have occurred during the establishment of some invasive populations. However, some colonial organisms may actually gain an ecological advantage from reduced genetic diversity because of the associated reduction in inter-colony conflict. Here we report population genetic analyses, along with colony fusion experiments, for a highly invasive colonial ascidian, Didemnum vexillum. Analyses based on mitochondrial cytochrome oxidase I (COI) partial coding sequences revealed two distinct D. vexillum clades. One COI clade appears to be restricted to the probable native region (i.e., north-west Pacific Ocean), while the other clade is present in widely dispersed temperate coastal waters around the world. This clade structure was supported by 18S ribosomal DNA (rDNA) sequence data, which revealed a one base-pair difference between the two clades. Recently established populations of D. vexillum in New Zealand displayed greatly reduced COI genetic diversity when compared with D. vexillum in Japan. In association with this reduction in genetic diversity was a significantly higher inter-colony fusion rate between randomly paired New Zealand D. vexillum colonies (80%, standard deviation ±18%) when compared with colonies found in Japan (27%, standard deviation ±15%). The results of this study add to growing evidence that for colonial organisms reductions in population level genetic diversity may alter colony interaction dynamics and enhance the invasive potential of newly colonizing species.

Activation of a tunicate (Ciona intestinalis) xenobiotic receptor orthologue by both natural toxins and synthetic toxicants.

Vertebrate xenobiotic receptors are ligand-activated nuclear receptors (NRs) that bind exogenous biologically active chemicals before activating the transcription of genes involved in xenobiotic metabolism and excretion. Typically, xenobiotic receptors have ligand binding domains (LBDs) that can accommodate a structurally diverse array of molecules and in addition display high levels of inter-taxa sequence diversity suggestive of positive selection. Pursuing the idea that xenobiotic receptors may adaptively evolve to bind toxic chemicals commonly present in an organism's environment/diet, we examined ligand binding by a xenobiotic receptor orthologue of a marine filter-feeding organism. The solitary tunicate Ciona intestinalis (Phylum Chordata) genome encodes an orthologue of the vertebrate pregnane X receptor (PXR) and vitamin D receptor (VDR), here denoted CiVDR/PXRα. In a luciferase reporter assay the CiVDR/PXRα was activated, at nanomolar concentrations, by two of four natural marine microalgal biotoxins tested (okadaic acid, EC50 = 18.2 ± 0.9 nM and pectenotoxin-2, EC50 = 37.0 ± 3.5 nM) along with 1 of 11 synthetic toxicants (esfenvalerate: EC50 = 0.59 ± 0.7 µM). Two related C. intestinalis NRs, orthologous to vertebrate farnesoid X receptor and liver X receptors, respectively, along with the PXR of a freshwater fish (zebrafish, Danio rerio), were not activated by any of the 15 chemicals tested. In contrast, human PXR was activated by okadaic acid at similar concentrations to CiVDR/PXRα (EC50 = 7.2 ± 1.1 nM) but not by pectenotoxin-2. A common features pharmacophore developed for the CiVDR/PXRα ligand consisted of an off-center hydrogen bond acceptor flanked by two hydrophobic regions. The results of this study are consistent with the original hypothesis that natural toxins, present in the diet of filter-feeding marine invertebrates, may have acted as selective agents in the molecular evolution of tunicate xenobiotic receptors. Bioassays based on tunicate xenobiotic receptor activation may find application in marine environmental monitoring and bioprospecting.

Evidence for adaptive evolution of olfactory receptor genes in 9 bird species.

It has been suggested that positive selection, in particular selection favoring a change in the protein sequence, plays a role in the evolution of olfactory receptor (OR) gene repertoires in fish and mammals. ORs are 7-transmembrane domain (TM) proteins, members of the G-protein-coupled receptor superfamily in vertebrate genomes, and responsible for odorant binding and discrimination. OR gene repertoires in birds are surprisingly large and diverse, suggesting that birds have a keen olfactory sense. The aim of this study is to investigate signatures of positive selection in an expanded OR clade (group-gamma-c) that seems to be a characteristic of avian genomes. Using maximum-likelihood methods that estimate the d(N)/d(S) ratios and account for the effects of recombination, we show here that there is evidence for positive selection in group-gamma-c partial OR coding sequences of 9 bird species that are likely to have different olfactory abilities: the blue tit (Cyanistes caeruleus), the black coucal (Centropus grillii), the brown kiwi (Apteryx australis), the canary (Serinus canaria), the galah (Eolophus roseicapillus), the kakapo (Strigops habroptilus), the mallard (Anas platyrhynchos), the red jungle fowl (Gallus gallus), and the snow petrel (Pagodroma nivea). Positively selected codons were predominantly located in TMs, which in other vertebrates are involved in odorant binding. Our data suggest that 1) at least some avian OR genes have been subjected to adaptive evolution, 2) the extent of such adaptive evolution differs between bird species, and 3) positive selective pressures may have been stronger on the group-gamma-c OR genes of species that have well-developed olfactory abilities.

Evidence for increased olfactory receptor gene repertoire size in two nocturnal bird species with well-developed olfactory ability.

BACKGROUND: In vertebrates, the molecular basis of the sense of smell is encoded by members of a large gene family, namely olfactory receptor (OR) genes. Both the total number of OR genes and the proportion of intact OR genes in a genome may indicate the importance of the sense of smell for an animal. There is behavioral, physiological, and anatomical evidence that some bird species, in particular nocturnal birds, have a well developed sense of smell. Therefore, we hypothesized that nocturnal birds with good olfactory abilities have evolved (i) more OR genes and (ii) more intact OR genes than closely related and presumably less 'olfaction-dependent' day-active avian taxa. RESULTS: We used both non-radioactive Southern hybridization and PCR with degenerate primers to investigate whether two nocturnal bird species that are known to rely on olfactory cues, the brown kiwi (Apteryx australis) and the kakapo (Strigops habroptilus), have evolved a larger OR gene repertoire than their day-active, closest living relatives (for kiwi the emu Dromaius novaehollandiae, rhea Rhea americana, and ostrich Struthio camelus and for kakapo the kaka Nestor meridionalis and kea Nestor notabilis). We show that the nocturnal birds did not have a significantly higher proportion of intact OR genes. However, the estimated total number of OR genes was larger in the two nocturnal birds than in their relatives. CONCLUSION: Our results suggest that ecological niche adaptations such as daily activity patterns may have shaped avian OR gene repertoires.

Avian olfactory receptor gene repertoires: evidence for a well-developed sense of smell in birds?

Among vertebrates, the sense of smell is mediated by olfactory receptors (ORs) expressed in sensory neurons within the olfactory epithelium. Comparative genomic studies suggest that the olfactory acuity of mammalian species correlates positively with both the total number and the proportion of functional OR genes encoded in their genomes. In contrast to mammals, avian olfaction is poorly understood, with birds widely regarded as relying primarily on visual and auditory inputs. Here, we show that in nine bird species from seven orders (blue tit, Cyanistes caeruleus; black coucal, Centropus grillii; brown kiwi, Apteryx australis; canary, Serinus canaria; galah, Eolophus roseicapillus; red jungle fowl, Gallus gallus; kakapo, Strigops habroptilus; mallard, Anas platyrhynchos; snow petrel, Pagodroma nivea), the majority of amplified OR sequences are predicted to be from potentially functional genes. This finding is somewhat surprising as one previous report suggested that the majority of OR genes in an avian (red jungle fowl) genomic sequence are non-functional pseudogenes. We also show that it is not the estimated proportion of potentially functional OR genes, but rather the estimated total number of OR genes that correlates positively with relative olfactory bulb size, an anatomical correlate of olfactory capability. We further demonstrate that all the nine bird genomes examined encode OR genes belonging to a large gene clade, termed gamma-c, the expansion of which appears to be a shared characteristic of class Aves. In summary, our findings suggest that olfaction in birds may be a more important sense than generally believed.

Detection of olfactory receptor transcripts in bird testes.

The sense of smell is mediated through olfactory receptors (ORs) expressed in olfactory sensory neurons of the olfactory epithelium. Interestingly, some OR genes also function in another context: they are expressed in nonolfactory testicular tissue and in sperm of mammals and fish where they mediate sperm flagellar motility. The presence of OR transcripts in testicular tissue of both mammals and fish suggests that this is a conserved trait among vertebrates. In birds, sperm competition is widespread and its outcome depends, in part, on sperm motility. Thus, avian testicular OR gene expression might be particularly interesting to study, especially in the context of current ideas on postcopulatory sexual selection. Using reverse transcription-polymerase chain reaction with degenerate primers specific for OR genes and subsequent cloning, we here demonstrate that multiple OR gene transcripts are present in chicken (Gallus gallus domesticus) testes. Moreover, we show that they belong to the class-gamma OR gene clade. We discuss the potential significance and evolutionary implications of avian testicular OR gene expression.

Biogenic Trace Amine-Associated Receptors (TAARS) are encoded in avian genomes: evidence and possible implications.

Recent studies of mammals and fish indicate that most trace amine-associated receptors (TAARs) may be involved in the detection of volatile biogenic compounds. It has therefore been suggested that this new class of "olfactory" receptors could be highly relevant for social communication and individual recognition. To determine if TAAR orthologues are encoded in avian genomes, we initiated BLAST searches of the Gallus gallus genome and public avian expressed sequence tags databases and performed associated phylogenetic analyses of the TAAR homologues identified. Our results suggest that a minimum of 3 TAAR paralogues are encoded in the G. gallus genome and that these are putative orthologues of the human/mouse genes TAAR1, TAAR2, and TAAR5. It is noteworthy that TAAR5 is activated by compounds that have been found in avian feces. We tentatively suggest that avian TAARs may compensate for the lack of an avian equivalent of the mammalian vomeronasal system and therefore may be important mediators of socially important avian chemical cues.

Drd4 gene polymorphisms are associated with personality variation in a passerine bird.

Polymorphisms in several neurotransmitter-associated genes have been associated with variation in human personality traits. Among the more promising of such associations is that between the human dopamine receptor D4 gene (Drd4) variants and novelty-seeking behaviour. However, genetic epistasis, genotype-environment interactions and confounding environmental factors all act to obscure genotype-personality relationships. Such problems can be addressed by measuring personality under standardized conditions and by selection experiments, with both approaches only feasible with non-human animals. Looking for similar Drd4 genotype-personality associations in a free-living bird, the great tit (Parus major), we detected 73 polymorphisms (66 SNPs, 7 indels) in the P. major Drd4 orthologue. Two of the P. major Drd4 gene polymorphisms were investigated for evidence of association with novelty-seeking behaviour: a coding region synonymous single nucleotide polymorphism (SNP830) and a 15bp indel (ID15) located 5' to the putative transcription initiation site. Frequencies of the three Drd4 SNP830 genotypes, but not the ID15 genotypes, differed significantly between two P. major lines selected over four generations for divergent levels of 'early exploratory behaviour' (EEB). Strong corroborating evidence for the significance of this finding comes from the analysis of free-living, unselected birds where we found a significant association between SNP830 genotypes and differing mean EEB levels. These findings suggest that an association between Drd4 gene polymorphisms and animal personality variation predates the divergence of the avian and mammalian lineages. Furthermore, this work heralds the possibility of following microevolutionary changes in frequencies of behaviourally relevant Drd4 polymorphisms within populations where natural selection acts differentially on different personality types.

Molecular analysis of clock gene expression in the avian brain.

Birds are equipped with a complex circadian pacemaking system that regulates the rhythmicity of physiology and behavior. As with all organisms, transcriptional and translational feedback loops of clock genes represent the basic molecular mechanism of rhythm generation in birds. To investigate avian clock gene expression, partial cDNA sequences of six mammalian clock gene homologs (Bmal1, Clock, Per2, Per3, Cry1, and Cry2) and a novel avian cryptochrome gene (Cry4) were cloned from the house sparrow, a model system in circadian research. Expression patterns were analyzed by semi-quantitative RT-PCR and RNase protection assays using total RNA extracted from adult male house sparrow brains. With the exception of Cry4, pronounced rhythmic mRNA expression of all the clock genes analyzed was encountered, with mRNA levels varying considerably between the various genes. Although some basic features of the molecular circadian feedback loop appear to be similar between mammals and birds, the precise phase relationships of the clock gene mRNA rhythms relative to each other and to the light zeitgeber differ significantly between the house sparrow and mammals. Our results point to the existence of differences in the organization of avian and mammalian circadian clock mechanisms.

Convergent evolution of strigiform and caprimulgiform dark-activity is supported by phylogenetic analysis using the arylalkylamine N-acetyltransferase (Aanat) gene.

Alternative hypotheses propose the sister order of owls (Strigiformes) to be either day-active raptors (Falconiformes) or dark-active nightjars and allies (Caprimulgiformes). In an effort to identify molecular characters distinguishing between these hypotheses we examined a gene, arylalkylamine N-acetyltransferase (Aanat), potentially associated with the evolution of avian dark-activity. Partial Aanat coding sequences, and two introns, were obtained from the genomic DNA of 16 species: Strigiformes (four species), Falconiformes (four species), Caprimulgiformes (five species), with outgroups: Ciconiiformes (one species), Passeriformes (one species), and Apterygiformes (one species). Phylogenetic trees derived from aligned, evolutionarily conserved Aanat regions did not consistently recover clades corresponding to orders Strigiformes and Falconiformes but did place a caprimulgiform clade more distant from the strigiform and falconiform species than the latter two groups are to each other. This finding was supported by spectral analysis. The taxonomic distribution of seven intronic indels is consistent with the Aanat derived phylogenetic trees and supports conventional family-level groupings within both Strigiformes and Caprimulgiformes. The phylogenetic analyses also indicate that Caprimulgiformes is a polyphyletic grouping. In conclusion the data support, but do not conclusively prove, the proposal that Falconiformes is the sister order to Strigiformes and therefore, that the dark-activity characteristic of Strigiformes and Caprimulgiformes arose by convergent evolution.