Functional evolution of Erg potassium channel gating reveals an ancient origin for IKr

Mammalian Ether-a-go-go related gene (Erg) family voltage-gated K⁺ channels possess an unusual gating phenotype that specializes them for a role in delayed repolarization. Mammalian Erg currents rectify during depolarization due to rapid, voltage-dependent inactivation, but rebound during repolarization due to a combination of rapid recovery from inactivation and slow deactivation. This is exemplified by the mammalian Erg1 channel, which is responsible for I_Kr, a current that repolarizes cardiac action potential plateaus. The Drosophila Erg channel does not inactivate and closes rapidly upon repolarization. The dramatically different properties observed in mammalian and Drosophila Erg homologs bring into question the evolutionary origins of distinct Erg K⁺ channel functions. Erg channels are highly conserved in eumetazoans and first evolved in a common ancestor of the placozoans, cnidarians, and bilaterians. To address the ancestral function of Erg channels, we identified and characterized Erg channel paralogs in the sea anemone Nematostella vectensis. N. vectensis Erg1 (NvErg1) is highly conserved with respect to bilaterian homologs and shares the I_Kr-like gating phenotype with mammalian Erg channels. Thus, the I_Kr phenotype predates the divergence of cnidarians and bilaterians. NvErg4 and Caenorhabditis elegans Erg (unc-103) share the divergent Drosophila Erg gating phenotype. Phylogenetic and sequence analysis surprisingly indicates that this alternate gating phenotype arose independently in protosomes and cnidarians. Conversion from an ancestral I_Kr-like gating phenotype to a Drosophila Erg-like phenotype correlates with loss of the cytoplasmic Ether-a-go-go domain. This domain is required for slow deactivation in mammalian Erg1 channels, and thus its loss may partially explain the change in gating phenotype.Voltage-gated ion channel families are highly conserved across the Eumetazoa (cnidarians and bilaterians) (1, 2). Vertebrates recently expanded the number of ion channel genes within each of the conserved families because of vertebrate-specific gene duplications. Additionally, phylogenetically restricted duplications of ion channel genes appear common throughout the Eumetazoa (1, 3–5). Thus, there is little 1:1 gene orthology between the eumetazoan phyla (1). However, numerous studies show extremely high functional conservation, including family-specific gating properties. For example, Shaker-related voltage-gated K⁺ channels first cloned in Drosophila show a high fidelity of gating phenotype to their mammalian counterparts (6). Subsequent studies have shown this functional conservation extends to cnidarians (4, 7–10), which separated from bilaterians near the base of the eumetazoan tree over 500 Mya (11). One exception to this pattern of high conservation is the Ether-a-go-go related gene (Erg) family (or Kv11) of voltage-gated K⁺ channels. The three mammalian Erg orthologs show striking gating differences compared with Drosophila Erg (seizure, DmErg).The mammalian Erg gating phenotype is typified by human Erg1 (HsErg1), which underlies I_Kr, a K⁺ current that repolarizes the late plateau phase of ventricular action potentials (12, 13). HsErg1 loss-of-function mutations prolong the QT interval in ECG recordings, indicating impaired action potential repolarization (14). Several key gating features adapt Erg1 for ventricular action potential plateau repolarization. First, Erg1 channels inactivate rapidly in response to depolarization (Fig. 1 A–C). Second, recovery from inactivation through the open state is extremely rapid (Fig. 1B), whereas channel deactivation is slow (Fig. 1D); the combination produces a jump in Erg1 current in response to repolarization (15). The net effect is that peak Erg1 current flow is delayed and specifically accelerates cardiac action potential plateau repolarization (15), and the length of the plateau is dependent on Erg1 current density (16). The physiological role of mammalian Erg2 and Erg3 channels has not been extensively characterized, but they share an I_Kr-like gating phenotype (17).Open in a separate windowFig. 1.Comparison of HsErg1 and DmErg gating phenotypes. (A) Families of outward currents recorded from Xenopus oocytes expressing HsErg1 (Left) and DmErg + DAO (Right) in response to depolarizations (Inset). Scale bars indicate time and current amplitude. Currents elicited by a step to +60 mV are highlighted, and arrows indicate (1) rectification of HsErg1 during depolarization by inactivation, (2) rebound in HsErg1 current in response to repolarization due to rapid recovery and slow deactivation, and (3) rapid DmErg deactivation. (B) Comparison of HsErg1 (black) and DmErg (red) currents during a protocol in which channels were first activated by a 1 s step to +60 mV, returned to –100 mV for 10 ms, and then returned to +60 mV. Currents are normalized in peak amplitude for comparison. HsErg1 is inactivated at the end of the first depolarization, recovers to the open state at −100 mV, and inactivates rapidly from a high peak during the second pulse. DmErg1 remains active throughout the first +60 mV pulse, closes at –100 mV, and reactivates during the second +60 mV pulse. (C) Peak HsErg1 current during an initial depolarization (* in B) normalized to peak current after recovery from inactivation (# in B): inactivation reduces the HsErg1 current >20-fold during the first step. Data show mean ± SEM, n = 6 cells. (D) Time constant of deactivation (Tau_DEACT) measured from tail currents recorded at the indicated voltages for HsErg1 (black) and DmErg (red). Data show mean ± SEM, n = 6–7 cells. (E) Normalized GV curves for HsErg1 and DmErg fit with a single Boltzmann distribution (parameters in SI Methods. Scale bar indicates that time and current amplitudes have been normalized.In contrast, DmErg does not inactivate during depolarization (Fig. 1 A and B) and deactivates rapidly upon repolarization (Fig. 1D) (18). The voltage-activation curve (GV) of DmErg is shifted to hyperpolarized potentials, suggesting influence on subthreshold excitability (Fig. 1E). Modeled HsErg1 and DmErg responses to a crude plateau action potential waveform (Fig. 1F and Fig. S1) point to distinct physiological roles. HsErg1 current is attenuated during the plateau by inactivation and rebounds sharply as the plateau decays. These features allow HsErg1 to accelerate late repolarization without blocking the plateau itself (15). Peak DmErg current flows during the plateau, and the current decays rapidly during repolarization. DmErg would therefore directly combat plateau formation. Loss of HsErg1 inactivation in humans indeed leads to a shortened QT interval based on premature action potential repolarization (16). The specific contribution of DmErg to firing patterns in native cells is unknown, but its gating features are consistent with regulation of subthreshold excitability or rapid action potential repolarization. Temperature-sensitive mutations in the seizure locus that encodes DmErg cause bursts of uncoordinated motor output (19) suggestive of changes in subthreshold excitability. The Caenorhabditis elegans Erg ortholog (CeErg, encoded by unc-103) has not been functionally expressed, but genetic analysis demonstrates that it regulates the excitation threshold of vulva muscles in females and protractor muscles in males (20–23).The Erg, Ether-a-go-go (Eag), and Elk gene families comprise the EAG superfamily of voltage-gated K⁺ channels. These gene families are highly conserved in eumetazoan genomes, and Eag channels display a high functional conservation in the bilaterians. Given the distinct gating phenotypes of the Erg genes in Drosophila and mammals, we decided to explore the functional evolution of the Erg gene family to determine the origins of the distinct I_Kr-like and DmErg gating phenotypes in the Erg gene family. We functionally characterized CeErg and Erg paralogs from the starlet sea anemone Nematostella vectensis. We examined CeErg to determine whether the DmErg gating phenotype was present in multiple protostome invertebrate phyla. We reasoned that comparison of bilaterian and Nematostella Erg channels would provide insight into ancestral Erg gating phenotypes present before the cnidarian/bilaterian divergence. Functional and phylogenetic analysis presented here supports an I_Kr-like phenotype as the ancestral gating pattern. An alternate DmErg-like gating phenotype has emerged independently at least twice during metazoan evolution (once in cnidarians and at least once in protostomes) and correlates with loss of the cytoplasmic eag gating domain.     

FRIGIDA-related genes are required for the winter-annual habit in Arabidopsis

In temperate climates, the prolonged cold temperature of winter serves as a seasonal landmark for winter-annual and biennial plants. In these plants, flowering is blocked before winter. In Arabidopsis thaliana, natural variation in the FRIGIDA (FRI) gene is a major determinate of the rapid-cycling vs. winter-annual flowering habits. In winter-annual accessions of Arabidopsis, FRI activity blocks flowering through the up-regulation of the floral inhibitor FLOWERING LOCUS C (FLC). Most rapid-flowering accessions, in contrast, contain null alleles of FRI. By performing a mutant screen in a winter-annual strain, we have identified a locus, FRIGIDA LIKE 1 (FRL1), that is specifically required for the up-regulation of FLC by FRI. Cloning of FRL1 revealed a gene with a predicted protein sequence that is 23% identical to FRI. Despite sequence similarity, FRI and FRL1 do not have redundant functions. FRI and FRL1 belong to a seven-member gene family in Arabidopsis, and FRI, FRL1, and at least one additional family member, FRIGIDA LIKE 2 (FRL2), are in a clade of this family that is required for the winter-annual habit in Arabidopsis.     

Fine structure mapping of a gene-rich region of wheat carrying Ph1, a suppressor of crossing over between homoeologous chromosomes

The wheat gene-rich region (GRR) 5L0.5 contains many important genes, including Ph1, the principal regulator of chromosome pairing. Comparative marker analysis identified 32 genes for the GRR controlling important agronomic traits. Detailed characterization of this region was accomplished by first physically localizing 213 wheat group 5L-specific markers, using group 5 nulli-tetrasomics, three Ph1 gene deletion/insertion mutants, and nine terminal deletion lines with their breakpoints around the 5L0.5 region. The Ph1 gene was localized to a much smaller region within the GRR (Ph1 gene region). Of the 61 markers that mapped in the four subregions of the GRR, 9 mapped in the Ph1 gene region. High stringency sequence comparison (e < 1 x10(-25)) of 157 group 5L-specific wheat ESTs identified orthologs for 80% sequences in rice and 71% in Arabidopsis. Rice orthologs were present on all rice chromosomes, although most (34%) were on rice chromosome 9 (R9). No single collinear region was identified in Arabidopsis even for a smaller region, such as the Ph1 gene region. Seven of the nine Ph1 gene region markers mapped within a 450-kb region on R9 with the same gene order. Detailed domain/motif analysis of the 91 putative genes present in the 450-kb region identified 26 candidates for the Ph1 gene, including genes involved in chromatin reorganization, microtubule attachment, acetyltransferases, methyltransferases, DNA binding, and meiosis/anther specific proteins. Five of these genes shared common domains/motifs with the meiosis specific genes Zip1, Scp1, Cor1, RAD50, RAD51, and RAD57. Wheat and Arabidopsis homologs for these rice genes were identified.     

Establishing the validity of domestication genes using DNA from ancient chickens

Modern domestic plants and animals are subject to human-driven selection for desired phenotypic traits and behavior. Large-scale genetic studies of modern domestic populations and their wild relatives have revealed not only the genetic mechanisms underlying specific phenotypic traits, but also allowed for the identification of candidate domestication genes. Our understanding of the importance of these genes during the initial stages of the domestication process traditionally rests on the assumption that robust inferences about the past can be made on the basis of modern genetic datasets. A growing body of evidence from ancient DNA studies, however, has revealed that ancient and even historic populations often bear little resemblance to their modern counterparts. Here, we test the temporal context of selection on specific genetic loci known to differentiate modern domestic chickens from their extant wild ancestors. We extracted DNA from 80 ancient chickens excavated from 12 European archaeological sites, dated from ∼280 B.C. to the 18th century A.D. We targeted three unlinked genetic loci: the mitochondrial control region, a gene associated with yellow skin color (β-carotene dioxygenase 2), and a putative domestication gene thought to be linked to photoperiod and reproduction (thyroid-stimulating hormone receptor, TSHR). Our results reveal significant variability in both nuclear genes, suggesting that the commonality of yellow skin in Western breeds and the near fixation of TSHR in all modern chickens took place only in the past 500 y. In addition, mitochondrial variation has increased as a result of recent admixture with exotic breeds. We conclude by emphasizing the perils of inferring the past from modern genetic data alone.The resolution afforded by multiple genetic loci and—more recently—complete genomes has led to an increased understanding of the pattern and process of plant and animal domestication (1, 2). More specifically, genetic analyses have uncovered selective sweeps, quantitative trait loci, and even causative mutations underlying a wide range of behavioral and morphological traits, some of which define specific breeds, and others that differentiate domestic plants and animals from their wild ancestors (1, 3, 4).Because many of these traits are present in either single or relatively few closely related modern breeds, the earliest occurrences of specific phenotypes (and the underlying causative mutations) are presumed to have occurred well after the initial domestication process. These phenotypes are referred to (at least in the plant genetic literature) as “improvement genes” (2). In animals, these traits include hairlessness in Mexican and Peruvian dogs (5), dorsal hair ridges in Vietnamese, Thai, and Rhodesian Ridgebacks (6), excessive skin folds in western Shar-Peis (7), double muscling in two cattle breeds (8), and a curly coat mutation found in Selkirk Rex cats (9), none of which are thought to have been present during early domestication.Some causative mutations, however, underlie traits found in numerous, distantly related breeds. Alleles that are fixed in domestic variants—and often presumed to have been under selection at the outset of domestication—are referred to in both the plant (2) and animal (3) domestication literature as “domestication loci” (or domestication genes). In some cases, including gray coloring (10) and altered gaits in horse breeds (11), brachycephaly in dogs (12), and muscle growth in pigs (13), no hypotheses have been proposed for the time-frame of first appearance of these traits. In others, however, the commonality of both small size (14, 15) and chondrodysplasia (16) across modern dog breeds and the widespread occurrence of pea-combs in chickens (17), led the authors of these studies to suggest that the genetic mutations underlying these characteristics were selected for during the early stages of the domestication process. More recently, a whole-genome resequencing study that compared variation in 14 unrelated dog breeds and wolves identified 36 regions potentially targeted during early domestication and included 10 genes that allowed dogs to better digest starches (18). Because increased amylase activity was ubiquitous in dogs but absent in wolves, the authors concluded that this change must have occurred when early dogs began adapting to a starch-rich diet provided by early farmers.Recent genetic and archaeological research has also shed light on domestic chickens and their primary ancestor, the Red Junglefowl (Gallus gallus) (19). Based on archaeological bones identified from Neolithic sites in the Yellow River basin, chickens were thought to have been domesticated as early as 6000 B.C. (20). This conclusion has recently been questioned, however, because bones presumed to originate from chickens in the original faunal analysis (21, 22) have since been shown to be pheasants (23, 24). As a result, a reevaluation of all of the early finds is necessary to establish the true chronology and geography of chicken domestication.Genes that differentiate modern domestic chickens from Red Junglefowl include those that underlie the yellow skin phenotype present in the vast majority of Western, commercial chicken breeds, as well as numerous geographically restricted and fancy breeds. Yellow skin is caused by a recessive allele of the BCDO2 (β-carotene dioxygenase 2) gene (25). BCDO2 encodes the β-carotene dioxygenase 2 enzyme that cleaves colorful carotenoids into colorless apocarotenoids (26). Although the expression of the dominant allele in skin tissue results in white skin color, the recessive allele possesses one or more cis-acting and tissue-specific regulatory mutations that inhibit expression of BCDO2 in skin tissue. Provided that sufficient carotenoids are available in the diet, the recessive allele reduces carotenoid cleavage and allows them to be deposited in skin tissue, leading to yellow skin (25). This recessive BCDO2 allele is thought to have been acquired through hybridization with the Gray Junglefowl (Gallus sonneratii) in South Asia (25). Red and Gray Junglefowl are known to hybridize in contact zones in the Indian subcontinent (27, 28), and it is possible that domestic poultry engaged in the same behavior after they were introduced from Southeast Asia. Given the ubiquity and genomic signatures of strong human-driven selection of the yellow skin trait in modern, Western commercial chickens (29), Eriksson et al. (25) suggested that this trait was favored by humans after chickens acquired the trait in South Asia, but before the first wave of domesticated chickens arrived in Europe between 900 and 700 B.C. (30, 31).In addition, a recent analysis of pooled wild and domestic chicken samples revealed strong selection signatures across a number of loci, as well as a missense mutation in the thyroid-stimulating hormone receptor (TSHR), a locus possibly linked to shifts in seasonal mating (29). Given its ubiquity in domestic breeds (264 of 271 birds representing 36 global populations were homozygous for the sweep allele; the remaining 7 were heterozygous) and the general absence of the derived allele in Red Junglefowl, the authors of that study concluded that the TSHR locus may have played a crucial role during chicken domestication (29).Here, we investigate whether the TSHR gene was selected for during the early stages of chicken domestication (29), and if early poultry keepers favored the BCDO2 gene that underlies yellow skin in chickens soon after it was acquired from the Gray Junglefowl (25, 29). To do so, we genotyped SNPs linked with the sweep alleles in both TSHR and BCDO2 in 80 ancient European chickens dating from ∼280 B.C. to the 18th century A.D. (Table S1 and SI Materials and Methods). If TSHR played a critical role during the domestication process, all of the samples analyzed here should have been fixed for the derived TSHR allele, as has been demonstrated in worldwide modern chicken populations (29). Similarly, if BCDO2 and the yellow skin phenotype was favored and maintained soon after its introgression from Gray Junglefowl, a significant proportion of the ancient European individuals should also possess this phenotype. Finally, we assess the hypothesis that the presence of mitochondrial DNA (mtDNA) control region (CR) haplogroups A–D has resulted from the recent introduction of East Asian chickens into the European gene pool, and that haplogroup E is historically associated with European chickens (32).     

A guardian of grasses: specific origin and conservation of a unique disease-resistance gene in the grass lineage

The maize Hm1 gene provides protection against a lethal leaf blight and ear mold disease caused by Cochliobolus carbonum race 1 (CCR1). Although it was the first disease-resistance (DR) gene to be cloned, it remains a novelty because, instead of participating in the plant recognition and response system as most DR genes do, Hm1 disarms the pathogen directly. It does so by encoding an NADPH-dependent reductase, whose function is to inactivate Helminthosporium carbonum (HC) toxin, an epoxide-containing cyclic tetrapeptide, which the pathogen produces as a key virulence factor to colonize maize. Although CCR1 is strictly a pathogen of maize, orthologs of Hm1 and the HC-toxin reductase activity are present in the grass family, suggesting an ancient and evolutionarily conserved role of this DR trait in plants. Here, we provide proof for such a role by demonstrating its involvement in nonhost resistance of barley to CCR1. Barley leaves in which expression of the Hm1 homologue was silenced became susceptible to infection by CCR1, but only if the pathogen was able to produce HC toxin. Phylogenetic analysis indicated that Hm1 evolved exclusively and early in the grass lineage. Given the devastating ability of CCR1 to kill maize, these findings imply that the evolution and/or geographical distribution of grasses may have been constrained if Hm1 did not emerge.     

Interspecific introgressive origin of genomic diversity in the house mouse

We report on a genome-wide scan for introgression between the house mouse (Mus musculus domesticus) and the Algerian mouse (Mus spretus), using samples from the ranges of sympatry and allopatry in Africa and Europe. Our analysis reveals wide variability in introgression signatures along the genomes, as well as across the samples. We find that fewer than half of the autosomes in each genome harbor all detectable introgression, whereas the X chromosome has none. Further, European mice carry more M. spretus alleles than the sympatric African ones. Using the length distribution and sharing patterns of introgressed genomic tracts across the samples, we infer, first, that at least three distinct hybridization events involving M. spretus have occurred, one of which is ancient, and the other two are recent (one presumably due to warfarin rodenticide selection). Second, several of the inferred introgressed tracts contain genes that are likely to confer adaptive advantage. Third, introgressed tracts might contain driver genes that determine the evolutionary fate of those tracts. Further, functional analysis revealed introgressed genes that are essential to fitness, including the Vkorc1 gene, which is implicated in rodenticide resistance, and olfactory receptor genes. Our findings highlight the extent and role of introgression in nature and call for careful analysis and interpretation of house mouse data in evolutionary and genetic studies.Classical laboratory mouse strains, as well as newly established wild-derived ones, are widely used by geneticists for answering a diverse array of questions (1). Understanding the genome contents and architecture of these strains is important for studies of natural variation and complex traits, as well as evolutionary studies in general (2). Mus spretus, a sister species of Mus musculus, impacts the findings in M. musculus investigations for at least two reasons. First, it was deliberately interbred with laboratory M. musculus strains to introduce genetic variation (3). Second, Mus musculus domesticus is partially sympatric (naturally cooccurring) with M. spretus (Fig. 1).Open in a separate windowFig. 1.Species ranges and samples used in our study. The species range of M. spretus is shown in green (4), and the species range of M. m. domesticus includes the blue regions, the range of M. spretus, and beyond (1). M. m. domesticus and M. spretus samples were obtained from locations marked with red circles and purple diamonds, respectively. The samples originated from within and outside the area of sympatry between the two species. (SI Appendix, Table S1, provides additional details about the samples used in our study.)Recent studies have examined admixture between subspecies of house mice (5–8), but have not studied introgression with M. spretus. In at least one case (5), the introgressive descent of the mouse genome was hidden due to data postprocessing that masked introgressed genomic regions as missing data. In another study reporting whole-genome sequencing of 17 classical laboratory strains (6), M. spretus was used as an outgroup for phylogenetic analysis. The authors were surprised to find that 12.1% of loci failed to place M. spretus as an outgroup to the M. musculus clade. The authors concluded that M. spretus was not a reliable outgroup but did not pursue their observation further. On the other hand, in a 2002 study (9), Orth et al. compiled data on allozyme, microsatellite, and mitochondrial variation in house mice from Spain (sympatry) and nearby countries in western and central Europe. Interestingly, allele sharing between the species was observed in the range of sympatry but not outside in the range of allopatry. The studies demonstrated the possibility of natural hybridization between these two sister species. Further, the study of Song et al. (10) demonstrated a recent adaptive introgression from M. spretus into some M. m. domesticus populations in the wild, involving the vitamin K epoxide reductase subcomponent 1 (Vkorc1) gene, which was later shown to be more widespread in Europe, albeit geographically restricted to parts of southwestern and central Europe (11).Major, unanswered questions arise from these studies. First, is the vicinity around the Vkorc1 gene an isolated case of adaptive introgression in the house mouse genome, or do many other such regions exist? Second, is introgression between M. spretus and M. m. domesticus common outside the range of sympatry? Third, have there been other hybridization events, and, in particular, more ancient ones? Fourth, what role do introgressed genes, and, more generally, genomic regions, play?To investigate these open questions, we used genome-wide variation data from 20 M. m. domesticus samples (wild and wild-derived) from the ranges of sympatry and allopatry, as well as two M. spretus samples. For detecting introgression, we used PhyloNet-HMM (12), a newly developed method for statistical inference of introgression in genomes while accounting for other evolutionary processes, most notably incomplete lineage sorting (ILS).Our analysis provides answers to the questions posed above. First, we find signatures of introgression between M. spretus and each of the M. m. domesticus samples. The amount of introgression varies across the autosomes of each genome, with a few chromosomes harboring all detectable introgression, and most of the chromosomes have none. We detected no introgression on the X chromosome. Further, the amount of introgression varied widely across the samples. Our analyses demonstrate introgression outside the range of sympatry. In fact, our results show more signatures of introgression in the genomes of allopatric samples from Europe than in sympatric samples from Africa. For the third question, we used the length distribution and sharing patterns of introgressed regions across the samples to show support for at least three hybridization events: an ancient hybridization event that predates the colonization of Europe by M. m. domesticus and two more recent events, one of which presumably occurred about 50 y ago and is related to warfarin resistance selection (10). For the fourth question, our functional analysis of the introgressed genes shows enrichment for certain categories, most notably olfaction—an essential trait for the fitness of rodents. Understanding the genomic architecture and evolutionary history of the house mouse has broad implications on various aspects of evolutionary, genetic, and biomedical research endeavors that use this model organism. The PhyloNet-HMM method (12) can be used to detect introgression in other eukaryotic species, further broadening the impact of this work.     

Mosaic genome of endobacteria in arbuscular mycorrhizal fungi: Transkingdom gene transfer in an ancient mycoplasma-fungus association

For more than 450 million years, arbuscular mycorrhizal fungi (AMF) have formed intimate, mutualistic symbioses with the vast majority of land plants and are major drivers in almost all terrestrial ecosystems. The obligate plant-symbiotic AMF host additional symbionts, so-called Mollicutes-related endobacteria (MRE). To uncover putative functional roles of these widespread but yet enigmatic MRE, we sequenced the genome of DhMRE living in the AMF Dentiscutata heterogama. Multilocus phylogenetic analyses showed that MRE form a previously unidentified lineage sister to the hominis group of Mycoplasma species. DhMRE possesses a strongly reduced metabolic capacity with 55% of the proteins having unknown function, which reflects unique adaptations to an intracellular lifestyle. We found evidence for transkingdom gene transfer between MRE and their AMF host. At least 27 annotated DhMRE proteins show similarities to nuclear-encoded proteins of the AMF Rhizophagus irregularis, which itself lacks MRE. Nuclear-encoded homologs could moreover be identified for another AMF, Gigaspora margarita, and surprisingly, also the non-AMF Mortierella verticillata. Our data indicate a possible origin of the MRE-fungus association in ancestors of the Glomeromycota and Mucoromycotina. The DhMRE genome encodes an arsenal of putative regulatory proteins with eukaryotic-like domains, some of them encoded in putative genomic islands. MRE are highly interesting candidates to study the evolution and interactions between an ancient, obligate endosymbiotic prokaryote with its obligate plant-symbiotic fungal host. Our data moreover may be used for further targeted searches for ancient effector-like proteins that may be key components in the regulation of the arbuscular mycorrhiza symbiosis.Soil fungi from the Glomeromycota, which form arbuscular mycorrhiza (AM) symbioses with the vast majority of land plants, are major players in terrestrial ecosystems (1). This symbiosis originated more than 450 million years ago (2) and represents an impressive example of an ancient and stable mutualistic association: AM fungi (AMF) efficiently explore the soil with their fine mycelium and supply plants with inorganic nutrients and water, whereas the plants provide carbohydrates derived from photosynthesis in above-ground organs.Interestingly, AMF, as biotrophic and obligate plant symbionts, themselves host additional endosymbionts in their cytoplasm, biotrophic endobacteria (3, 4). These endobacteria were electron-microscopically described in the 1970s as bacterium-like organisms (5). Two types were later defined, the first one being found only in members of the family Gigasporaceae. This Gram-negative bacterium was named Candidatus Glomeribacter gigasporarum (CaGg) and is related to Burkholderia (4). The second and much more widespread type represents the only known fungal endobacteria belonging to the Mollicutes (“Mollicutes-related endobacteria”; MRE), although related endobacteria (e.g., Mycoplasma and Phytoplasma species) are widespread as biotrophic parasites of humans, mammals, reptiles, fishes, arthropods, or plants. MRE were frequently detected in the intraradical and extraradical mycelium and in spores of AMF; however, they could never be detected free-living (3). Strikingly, MRE have recently been demonstrated to also occur in several non-AMF species from the genus Endogone (Mucoromycotina), where some members are also plant symbionts (6).MRE are associated with all major phylogenetic lineages of AMF studied so far and, thus, indirectly also with more than 80% of all land plants. They are coccoid, located in the cytoplasm without a surrounding host-membrane, and appear to possess a Gram-positive cell wall, which is surprising because of the phylogenetic relationship with cell wall-lacking Mollicutes (3). During their long-lasting coevolution, MRE have formed distinct, monophyletic evolutionary lineages within their fungal hosts, with a 16S rRNA gene (16S) sequence divergence of up to 20% (3, 7).We hypothesized that MRE play an important biological role in AM, consistent with the observation that they have been maintained as ancient endosymbionts in major evolutionary AMF lineages that separated hundreds of million years ago. To obtain hints for such roles and to shed light on MRE evolution, we analyzed the genome of a MRE colonizing the AMF Dentiscutata heterogama FL654 (syn.: Scutellospora heterogama; Gigasporaceae), termed DhMRE. Only three genomes of fungal endobacteria have been published so far: from CaGg, which colonize some AMF from the Gigasporaceae (8); from Burkholderia rhizoxinica, the endobacterium of Rhizopus microsporus, which participates in the production of rhizoxin (a potent antimitotic agent that acts as a virulence factor) (9); and from a betaproteobacterial endosymbiont associated with Mortierella elongata (10).We present the annotation of a MRE genome draft, the phylogenetic placement of MRE based on a multilocus analysis, the evolutionary implications about the origin of MRE-fungus (-plant) associations, and evidence for transkingdom horizontal gene transfer (HGT). Moreover, the genome presents genes coding for proteins with likely regulatory functions in the interaction between this obligate symbiotic prokaryote and its obligate plant-symbiotic fungal host.     

Polymorphisms in the 3'-untranslated region of the CDH1 gene are a risk factor for primary gastric diffuse large B-cell lymphoma

Background

Primary gastric B-cell lymphomas arise from mucosa-associated lymphatic tissue (MALT) in patients with chronic Helicobacter pylori infection. We investigated whether germline variants in the CDH1 gene, coding for E-cadherin, genetically predispose patients to primary gastric B-cell lymphoma.

Design and Methods

Single marker analyses of the CDH1 gene were conducted in patients with primary gastric B-cell lymphoma (n=144), in patients with primary gastric high-grade lymphoma (n=61), and in healthy blood donors (n=361). Twelve single nucleotide polymorphisms were genotyped by TaqMan^® technology. Allelic imbalance was tested by pyrosequencing and clone direct sequencing of heterozygote genomic and cDNA. Mutation detection was conducted around the poly-A signal of the CDH1 3′-untranslated region. The influence of the 3′-untranslated region on protein translation was determined by a luciferase reporter assay.

Results

Single marker analyses identified two single nucleotide polymorphisms in strong linkage disequilibrium located in the CDH1 3′-untranslated region. One of them was significantly associated with primary gastric diffuse large B-cell lymphomas after correction for multiple testing and this association was confirmed in an independent sample set. Patients homozygous for the rare T allele (rs1801026) had a 4.9-fold increased risk (95% CI: 1.5–15.9) of developing primary gastric diffuse large B-cell lymphoma. Allelic imbalance and reporter gene assays indicated a putative influence on mRNA stability and/or translational efficacy.

Conclusions

We identified variants in CDH1 as the first potential genetic risk factors for the development of primary gastric diffuse large B-cell lymphomas. One of the potentially causative variants affects allelic CDH1 expression. These findings support the hypothesis that besides somatic alterations of B-cells, germline variants in the CDH1 gene contribute to a predisposition to the development of primary gastric diffuse large B-cell lymphomas.     

Evolutionary origin of the amnioserosa in cyclorrhaphan flies correlates with spatial and temporal expression changes of zen

Higher cyclorrhaphan flies including Drosophila develop a single extraembryonic epithelium (amnioserosa), which closes the germband dorsally. In most other insects two extraembryonic epithelia, serosa and amnion, line the inner eggshell and the ventral germband, respectively. How the two extraembryonic epithelia evolved into one is unclear. Recent studies have shown that, in the flour beetle Tribolium and in the milkweed bug Oncopeltus, the homeobox gene zerknüllt (zen) controls the fusion of the amnion with the serosa before dorsal closure. To understand the origin of the amnioserosa in evolution, we examined the expression and function of zen in the extraembryonic tissue of lower Cyclorrhapha. We show that Megaselia abdita (Phoridae) and Episyrphus balteatus (Syrphidae) develop a serosa and a dorsal amnion, suggesting that a dorsal amnion preceded the origin of the amnioserosa in evolution. Using Krüppel (Kr) and pannier (pnr) homologues of Megaselia as markers for serosal and amniotic tissue, respectively, we show that after zen RNAi all extraembryonic tissue becomes indistinguishable from amniotic cells, like in Tribolium but unlike in Drosophila, in which zen controls all aspects of extraembryonic development. Compared with Megaselia and Episyrphus, zen expression in Drosophila is extended to cells that form the amnion in lower Cyclorrhapha and is down-regulated at the developmental stage, when serosa cells in lower Cyclorrhapha begin to expand. These expression differences between species with distinct extraembryonic tissue organizations and the conserved requirement of zen for serosa development suggest that the origin of an amnioserosa-like epithelium was accompanied by expression changes of zen.     

Codon 104 variation of p53 gene provides adaptive apoptotic responses to extreme environments in mammals of the Tibet plateau

Mutational changes in p53 correlate well with tumorigenesis. Remarkably, however, relatively little is known about the role that p53 variations may play in environmental adaptation. Here we report that codon asparagine-104 (104N) and glutamic acid-104 (104E), respectively, of the p53 gene in the wild zokor (Myospalax baileyi) and root vole (Microtus oeconomus) are adaptively variable, meeting the environmental stresses of the Tibetan plateau. They differ from serine-104 (104S) seen in other rodents, including the lowland subterranean zokor Myospalax cansus, and from serine 106 (106S) in humans. Based on site-directed mutational analysis in human cell lines, the codon 104N variation in M. baileyi is responsible for the adaptive balance of the transactivation of apoptotic genes under hypoxia, cold, and acidic stresses. The 104E p53 variant in Microtus oeconomus suppresses apoptotic gene transactivation and cell apoptosis. Neither 104N nor 104E affects the cell-cycle genes. We propose that these variations in p53 codon 104 are an outcome of environmental adaptation and evolutionary selection that enhance cellular strategies for surviving the environmental stresses of hypoxia and cold (in M. baileyi and M. oeconomus) and hypercapnia (in M. baileyi) in the stressful environments of the Qinghai-Tibet plateau.The regulatory mechanisms of p53 mutation related to tumorigenesis have been widely studied and elucidated (1, 2). Notably, however, p53 evolution and adaptation to environmental stresses have not attracted as much attention. Current studies show that p53 is a master sensor and regulator in response to various stressors, such as DNA damage and hypoxia (3–6). Activation of p53 by stresses results in cell-cycle arrest, DNA repair, senescence, or apoptosis in which a series of p53 target genes are involved to maintain genomic integrity (2). The p53 variations associated with environmental stresses have been described in the Mexican salamander axolotl Ambystoma mexicanum and the Israeli blind subterranean mole rat (Spalax judaei; hereafter, S.j.) (7–9).For animals existing on high plateaus, hypoxia and cold serve as strong environmental selective pressures generating adaptive complexes to cope with these stresses. Animals that have evolved on plateaus adopt various strategies involving multiple variations to regulate a series of genes (3, 7). The zokor (Myospalax baileyi, Thomas, 1911; hereafter M.b.) and root vole (Microtus oeconomus, Pallas, 1776; hereafter M.o.) are the dominant native mammals living on the alpine meadow of the Qinghai-Tibet Plateau of China at altitudes of 3,000–4,500 m (equivalent to 11.0–13.0% O₂ at sea level). M.b. is genetically close to Myospalax cansus (Lyon, 1907; hereafter, M.c.), which lives in subterranean burrows at a lower altitude of about 800 m in the lowland of western China. M.b. and M.c. spend their entire life cycle at 70–250 cm underground with significantly low O₂ and high CO₂ levels in their burrows (10). Since the collision of the Indian and the Eurasian plates during the Tertiary (40–50 Mya) formed the Tibet plateau (11), small mammals living in this region have been geographically and ecologically isolated from other species and have adapted to the stressful plateau environment, contributing to the East Asian biodiversity (12–15). Our previous work demonstrated that mammals of the Qinghai-Tibet plateau are well adapted to the hypoxic environment (16–19), with particular expression patterns of HIF-1α and IGF-I and its binding protein (IGFBP-1), which mediate protection against hypoxia (20–23). Cells exposed to hypoxia succumb to p53-dependent apoptosis (24–27); thus mutations in p53 are required for cell survival under selective pressures. We examined the hypothesis that plateau mammals are adapted to this environment with p53 alterations linked to hypoxia, hypercapnia (high CO₂), and cold.Here we report that the variations of p53 codon 104 in three rodent species during long-term evolution and adaptation at the Qinghai-Tibet plateau reflect diverse survival strategies. The present study provides insights into the contribution of p53 variations to native mammals’ adaptation to the diverse and extreme environmental stresses of their habitats.     

Evolutionary origin of cAMP-based chemoattraction in the social amoebae

Phenotypic novelties can arise if integrated developmental pathways are expressed at new developmental stages and then recruited to serve new functions. We analyze the origin of a novel developmental trait of Dictyostelid amoebae: the evolution of cAMP as a developmental chemoattractant. We show that cAMP's role of attracting starving amoebae arose through recruitment of a pathway that originally evolved to coordinate fruiting body morphogenesis. Orthologues of the high-affinity cAMP receptor (cAR), cAR1, were identified in a selection of species that span the Dictyostelid phylogeny. The cAR1 orthologue from the basal species Dictyostelium minutum restored aggregation and development when expressed in an aggregation-defective mutant of the derived species Dictyostelium discoideum that lacks high-affinity cARs, thus demonstrating that the D. minutum cAR is a fully functional cAR. cAR1 orthologues from basal species are expressed during fruiting body formation, and only this process, and not aggregation, was disrupted by abrogation of cAR1 function. This is in contrast to derived species, where cAR1 is also expressed during aggregation and critically regulates this process. Our data show that coordination of fruiting body formation is the ancestral function of extracellular cAMP signaling, whereas its derived role in aggregation evolved by recruitment of a preexisting pathway to an earlier stage of development. This most likely occurred by addition of distal cis-regulatory regions to existing cAMP signaling genes.     

Mechanism of staphylococcal multiresistance plasmid replication origin assembly by the RepA protein

The staphylococcal multiresistance plasmids are key contributors to the alarming rise in bacterial multidrug resistance. A conserved replication initiator, RepA, encoded on these plasmids is essential for their propagation. RepA proteins consist of flexibly linked N-terminal (NTD) and C-terminal (CTD) domains. Despite their essential role in replication, the molecular basis for RepA function is unknown. Here we describe a complete structural and functional dissection of RepA proteins. Unexpectedly, both the RepA NTD and CTD show similarity to the corresponding domains of the bacterial primosome protein, DnaD. Although the RepA and DnaD NTD both contain winged helix-turn-helices, the DnaD NTD self-assembles into large scaffolds whereas the tetrameric RepA NTD binds DNA iterons using a newly described DNA binding mode. Strikingly, structural and atomic force microscopy data reveal that the NTD tetramer mediates DNA bridging, suggesting a molecular mechanism for origin handcuffing. Finally, data show that the RepA CTD interacts with the host DnaG primase, which binds the replicative helicase. Thus, these combined data reveal the molecular mechanism by which RepA mediates the specific replicon assembly of staphylococcal multiresistant plasmids.The emergence of multidrug-resistant bacteria is a mounting global health crisis. In particular, multidrug-resistant Staphylococcus aureus is a major cause of nosocomial and community-acquired infections and is resistant to most antibiotics commonly used for patient treatment (1). Hospital intensive care units in many countries, including the United States, now report methicillin-resistant S. aureus infection rates exceeding 50% (2, 3). Antibiotic resistance in contemporary infectious S. aureus strains, such as in hospitals, is often encoded by plasmids that can be transmitted between strains via horizontal DNA transfer mechanisms. These plasmids are typically classified as small (<5 kb) multicopy plasmids, which usually encode only a single resistance gene; medium-sized (8–40 kb) multirestance plasmids that confer resistance to multiple antibiotics, disinfectants, and/or heavy metals; and large (>40 kb) conjugative multiresistance plasmids that additionally encode a conjugative DNA transfer mechanism (4–6). Importantly, sequence analyses have shown that most staphylococcal conjugative and nonconjugative multiresistance plasmids encode a highly conserved replication initiation protein, denoted RepA_N (5–15). RepA_N proteins are also encoded by plasmids from other Gram-positive bacteria as well as by some phage, underscoring their ubiquitous nature (10). These RepA proteins are essential for replication of multiresistance plasmids, and hence plasmid carriage and dissemination, yet the mechanisms by which these proteins function in replication are currently unknown.The DNA replication cycle can be divided into three stages: initiation, elongation, and termination. Replication initiation proteins (RepA) mediate the crucial first step of initiation. Bacterial chromosome replication is initiated by the chromosomal replication initiator protein, DnaA, which binds the origin and recruits the replication components known as the primosome (16). In Gram-negative bacteria the primosome includes DnaG primase, the replicative helicase (DnaB), and DnaC (17). Replication initiation in Gram-positive bacteria involves DnaG primase and helicase (DnaC) and the proteins DnaD, DnaI, and DnaB (18–22). DnaD binds first to DnaA at the origin. This is followed by binding of DnaI/DnaB and DnaG, which together recruit the replicative helicase (23, 24). Instead of DnaA, plasmids encode and use their own specific replication initiator binding protein. Structures are only available for RepA proteins (F, R6K, and pPS10 Rep) harbored in Gram-negative bacteria. These proteins contain winged helix-turn-helix (winged HTH) domains and bind iteron DNA as monomers to, in some still unclear manner, drive replicon assembly (25–27).Replication mechanisms used by plasmids harbored in Gram-positive bacteria are less well understood and are distinct from their Gram-negative counterparts. Indeed, most plasmid RepA proteins in Gram-negative and Gram-positive bacteria show no sequence homology and seem to be unrelated. The multiresistance RepA proteins are arguably among the most abundant of plasmid Rep proteins, yet how they function is not known. Data suggest that these proteins are composed of three main regions: an N-terminal domain (NTD) consisting of ∼120 aa, a long and variable linker region (∼30–50 residues), and a C-terminal domain (CTD) of ∼120 residues (28–31). The NTD and CTD are both essential for replication. The NTD exhibits the highest level of sequence conservation, which has resulted in the designation of plasmids that encode these proteins as the RepA_N replicon family (10). Although not as well conserved as the NTD, RepA CTD regions show homology between plasmids found in genus-specific clusters, suggesting that this domain may perform a host-specific role (28–32). Although the function of the RepA CTD remains enigmatic, recent studies have indicated that the NTD mediates DNA binding and interacts with iterons that reside within the plasmid origin (30). The essential roles played by RepA proteins in multiresistance plasmid retention marks them as attractive targets for the development of specific chemotherapeutics. However, the successful design of such compounds necessitates structural and mechanistic insight. Here, we describe a detailed dissection of the RepA proteins from the multiresistance plasmids pSK41 and pTZ2126. The combined data reveal the molecular underpinnings of a minimalist replication assembly mediated by multiresistance RepA proteins.     

From the Cover: Earliest economic exploitation of chicken outside East Asia: Evidence from the Hellenistic Southern Levant

Chicken (Gallus gallus domesticus) is today one of the most widespread domesticated species and is a main source of protein in the human diet. However, for thousands of years exploitation of chickens was confined to symbolic and social domains such as cockfighting. The question of when and where chickens were first used for economic purposes remains unresolved. The results of our faunal analysis demonstrate that the Hellenistic (fourth–second centuries B.C.E.) site of Maresha, Israel, is the earliest site known today where economic exploitation of chickens was widely practiced. We base our claim on the exceptionally high frequency of chicken bones at that site, the majority of which belong to adult individuals, and on the observed 2:1 ratio of female to male bones. These results are supported further by an extensive survey of faunal remains from 234 sites in the Southern Levant, spanning more than three millennia, which shows a sharp increase in the frequency of chicken during the Hellenistic period. We further argue that the earliest secure evidence for economic exploitation of chickens in Europe dates to the first century B.C.E. and therefore is predated by the finds in the Southern Levant by at least a century. We suggest that the gradual acclimatization of chickens in the Southern Levant and its gradual integration into the local economy, the latter fully accomplished in the Hellenistic period, was a crucial step in the adoption of this species in European husbandry some 100 y later.In the modern world, the chicken (Gallus gallus domesticus) is one of the most widespread livestock species and is a major source of animal protein in the human diet. The ancestor of the domestic chicken is the red jungle fowl (Gallus gallus), originating in Southeast Asia, with possible genetic contributions from closely related species through hybridization (1–5). Intensive hybridization between the modern chicken and its wild ancestor caused a loss of the wild progenitor genes (6, 7). Consequently, recent studies usually have focused either on the genetics of the chicken progenitor (8–12) or on zooarchaeological evidence for the domestication of chickens (13–15).The dispersal trajectory of chickens to West Asia, to the Mediterranean, and to Europe following its initial domestication in Southeast Asia remains largely unknown. Moreover, there are only very partial data, and thus there is great uncertainty regarding the place and time of the earliest economic exploitation of chickens: When and where did chickens move from being an exotic species, used only sporadically for symbolic and ritual purposes, to an important livestock species in the Mediterranean and European economies (16, 17)? Our study of chicken remains from the Southern Levant (Israel, the Palestinian Authority, and Jordan) and particularly from the Hellenistic site of Maresha in Southern Israel sheds new light on these issues.We define three main phases in the cultural history of chicken use, based on archaeological, historical, and iconographic evidence (Fig. 1). The early phase (Fig. 1, phase A) may have already begun around the sixth millennium B.C.E. when the chicken was initially domesticated during several independent domestication events in Southeast Asia and China (1, 2, 4, 11, 12). On the Indian subcontinent, which also constitutes a part of the natural dispersal range of the jungle fowl, chicken remains were recorded at a few second millennium B.C.E. sites, and it is commonly assumed that domestication occurred there independently (1, 14, 15, 18, 19). The second phase took place in the third–second millennia B.C.E. and includes the dispersal of the chicken out of its natural distribution range to West Asia (Fig. 1, phase B). The earliest chicken remains in the Near East were retrieved in Iran, Anatolia, and Syria and dated to the third millennium B.C.E. or slightly earlier (20). In Egypt, the oldest known chicken remains are possibly even earlier (16). At this early phase, chicken remains in archaeological sites are very sparse and often are not associated with domestic contexts. Historical and iconographic records demonstrate an acquaintance with the chicken from the mid-second millennium B.C.E. in Egypt, Mesopotamia, and the Levant (21). All these sources relate to chickens (almost exclusively cocks) as an exotic bird, used inter alia for cockfighting and displayed as exotica in royal zoos. The third phase includes its introduction to Europe (Fig. 1, phase C1) and the intensification of its use mainly on this continent (Fig. 1, phase C2).Open in a separate windowFig. 1.The dispersal of chickens in the Old World: the area marked “A” is the geographical range of the jungle fowl in South Asia and its initial domestication, which already may have begun around the sixth millennium B.C.E. in Southeast Asia and possibly in China; The area marked “B” maps the dispersal of chickens to West Asia during the third and second millennia B.C.E. C1 represents the first wave of chicken dispersal into Europe: introduction to Europe during the eighth century B.C.E. (chicken remains have low representation in sites). C2 represents the second wave of chicken dispersal into Europe and other regions from the first century B.C.E. (chicken remains have higher representation in sites). The location of Maresha is marked in the enlarged map (Inset).Archaeologically, chicken remains are first observed in Europe only in late ninth and eighth century B.C.E. contexts. The introduction of chickens to this region usually is attributed to the Phoenicians who brought chickens from their homeland to their colonies in the West (17, 22). This hypothesis is based on the fact that the earliest chicken remains in Europe were retrieved from Phoenician sites, mostly (although not only) in Iberia (23–25). The oldest reliable dated remains of chickens from central Europe (in the Czech Republic) are from the eighth century B.C.E. (26). The continued presence of chickens has been confirmed in Iberia (27, 28), as well as in southern France and Greece (24, 29), during the second half of the first millennium B.C.E. (Fig. 1, phase C1). However, a survey of the zooarchaeological literature of Europe demonstrates that before the first century B.C.E. the proportion of chicken remains in archaeological sites was extremely low and hardly ever exceeded 3% of the total faunal remains (25, 30, 31).The historical evidence also marks the eighth century B.C.E. (or even slightly later) as the arrival date of chickens in Europe. The arrival of chickens in Greece likely postdates Homer (around the eighth century B.C.E.), because the Greek poet does not mention this bird, but chickens are mentioned by Theognis of Megara in the sixth century (32). From the seventh century B.C.E., cocks are depicted on Greek coins and vases (28). In the fifth century B.C.E., the Greek playwright Aristophanes refers to the chicken as the “Persian bird” or “Median bird” (33), possibly indicating that in this period chickens were imported to Greece from Persia (14, 34). By the third century cocks became portrayed more frequently in Egypt (14, 22, 35 and references therein), but in Ptolemaic papyri chickens are hardly mentioned compared with other domesticated species (36). The symbolic role of cocks is well demonstrated by the Roman writer Cicero in his De Divinatione (37), where he mentions that cocks accompanied the Roman armies in 249 B.C.E. and that their behavior was observed carefully before battle as a sign of defeat or victory. Finally, fighting cocks are mentioned by Roman writers such as Varro (38) and Columella (39) (see also refs. 14 and 17).Returning to faunal data, from the first century B.C.E., more sites with chicken remains are known in Europe, and the proportions of chickens at these sites are higher (Fig. 1, phase C2). This increase is apparent in Roman sites in Italy (40) and later in Southern Britain (13) and Sweden (41, 42). Significant proportions of chicken remains are observed in some first century B.C.E. locations in the Near East, such as in Sagalassos in Anatolia (43, 44) and Petra in Jordan (45, 46), and at Berenike (47) and Mons Claudianus (48) in Egypt. Indeed, the relative number of chicken remains in Berenike during Roman times is almost threefold that of the Ptolemaic period (49).Unlike chicken bones, chicken egg shells often are overlooked during excavation (50). The first archaeological evidence for chicken eggs in the Mediterranean is from the first century B.C.E. This evidence includes some examples from Mons Claudianus and a high percentage of medullary bones from Berenike, indicative of females during laying time (47).Although the faunal evidence points to the first century B.C.E. as a turning point in patterns of chicken exploitation in the Mediterranean, the historical and iconographic records imply a slightly earlier date for its economic utilization. For example, a Roman law in the Lex Faunia (161 B.C.E.) banned the consumption of more than a single chicken per meal. Other remarkable testimonies for the integration of the chicken into European livestock in the first century B.C.E. are provided by the Greek historian Diodorus Siculus, who described the sophisticated technique of artificial incubation of chicken eggs in Ptolemaic Egypt (51), and by the Roman historian Varro, who offered advice on how to treat hens during laying time (38). Subsequently, in the first century C.E. the Roman writer Columella and the Roman culinary Apicius mention chicken eggs among the ingredients in culinary recipes (39, 52).We propose that the intensification in chicken exploitation in Europe during phase C2, as reflected by the archaeological and historical records, is related to our new data regarding chicken husbandry in the Southern Levant. The main new data we present here are from the site of Maresha, a national park situated in the Judean foothills in Southern Israel (Fig. 1 and Fig. S1) and dated to the Hellenistic period (fourth–second centuries B.C.E.). Located on an important trading route, Maresha flourished as a leading city of the region of Idumea, and its population comprised a complex ethnic mosaic (53). The town was in ruins by the late second century B.C.E. and was never resettled. In Hellenistic Maresha we note that, in addition to the symbolic cock painted in the so-called “Sidonian” tomb there (54), unisex chicken figurines are more common than any other animal figurines except for riders on horses (55, 56).Open in a separate windowFig. S1.(A) Plan of Maresha in the Hellenistic period with analyzed subterranean complexes 1, 169, 89, 147, and 57 indicated. Image courtesy of ref. 76. (B) Examples of subterranean complexes at Maresha. (Upper) Olive press, subterranean complex no. 44 in Maresha. (Lower) Columbarium, subterranean complex no. 30 in Maresha. Image courtesy of Boaz Zissu.The unprecedented amount of chicken remains revealed at Maresha, far outside the original distribution of the domestic fowl, coupled with the clear chronology of the findings and the excellent preservation of the chicken bones, render Hellenistic Maresha a key site for understanding the new role of the chicken in the Mediterranean during this period. The study of the faunal evidence at Maresha is followed by a comparative chronological and regional study, based on the frequency of chicken remains as presented in 234 faunal reports from the Southern Levant, spanning all periods until early modern times. This study provides diachronic data on the process of introduction and subsequent widespread adoption of the chicken in Levantine economies. We offer suggestions based on these data regarding the time and mode of expansion of chickens from Southwest Asia to Europe and throughout the Mediterranean.