Genetic structure in contemporary south Tyrolean isolated populations revealed by analysis of Y-chromosome, mtDNA, and Alu polymorphisms

Most of the inhabitants of South Tyrol in the eastern Italian Alps can be considered isolated populations because of their physical separation by mountain barriers and their sociocultural heritage. We analyzed the genetic structure of South Tyrolean populations using three types of genetic markers: Y-chromosome, mitochondrial DNA (mtDNA), and autosomal Alu markers. Using random samples taken from the populations of Val Venosta, Val Pusteria, Val Isarco, Val Badia, and Val Gardena, we calculated genetic diversity within and among the populations. Microsatellite diversity and unique event polymorphism diversity (on the Y chromosome) were substantially lower in the Ladin-speaking population of Val Badia compared to the neighboring German-speaking populations. In contrast, the genetic diversity of mtDNA haplotypes was lowest for the upper Val Venosta and Val Pusteria. These data suggest a low effective population size, or little admixture, for the gene pool of the Ladin-speaking population from Val Badia. Interestingly, this is more pronounced for Ladin males than for Ladin females. For the pattern of genetic Alu variation, both Ladin samples (Val Gardena and Val Badia) are among the samples with the lowest diversity. An admixture analysis of one German-speaking valley (Val Venosta) indicates a relatively high genetic contribution of Ladin origin. The reduced genetic diversity and a high genetic differentiation in the Rhaetoroman- and German-speaking South Tyrolean populations may constitute an important basis for future medical genetic research and gene mapping studies in South Tyrol.     

Discordant patterns of evolutionary differentiation in two Neotropical treefrogs

Comparative studies of codistributed taxa test the degree to which historical processes have shaped contemporary population structure. Discordant patterns of lineage divergence among taxa indicate that species differ in their response to common historical processes. The complex geologic landscape of the Isthmus of Central America provides an ideal setting to test the effects of vicariance and other biogeographic factors on population history. We compared divergence patterns between two codistributed Neotropical frogs ( Dendropsophus ebraccatus and Agalychnis callidryas ) that exhibit colour pattern polymorphisms among populations, and found significant differences between them in phenotypic and genetic divergence among populations. Colour pattern in D. ebraccatus did not vary with genetic or geographic distance, while colour pattern co-varied with patterns of gene flow in A. callidryas . In addition, we detected significant species differences in the phylogenetic history of populations, gene flow among them, and the extent to which historical diversification and recent gene flow have been restricted by five biogeographic barriers in Costa Rica and Panama. We inferred that alternate microevolutionary processes explain the unique patterns of diversification in each taxon. Our study underscores how differences in selective regimes and species-typical ecological and life-history traits maintain spatial patterns of diversification.     

Analysis of the region V mitochondrial marker in two Black communities of Ecuador,and in their parental populations

Data on the frequency of the mtDNA region V deletion were used to estimate the relative maternal contribution from the parental populations to the gene pools of the two Black communities of Rio Cayapas and Viche in northern Ecuador. Ethnohistorical records and nuclear DNA data indicate that these populations are hybrids of West African and Amerindian populations. The unique distribution of the DNA marker in these parental groups provided good admixture estimates. The fraction of mtDNA of Amerindian origin in the population of Rio Cayapas is quite small (8%±5%), whereas in the community of Viche the native Americans contributed the major portion of the gene pool (51%±15). The mtDNA estimate for Rio Cayapas is similar to that of some protein polymorphisms, which confirms the cultural and genetic isolation of this community from the neighboring native population. On the other hand, the admixture value obtained from nuclear genes in Viche is statistically different from the estimate obtained from mtDNA data. This supports the traditional belief, gathered from historical records and cultural data, that the contribution from Indian females was higher than that of Indian males, at least in the primary settlements of the African-American population of Esmeraldas.     

Genetic diversity and differentiation in a wide ranging anadromous fish,American shad (Alosa sapidissima), is correlated with latitude

Studies that span entire species ranges can provide insight into the relative roles of historical contingency and contemporary factors that influence population structure and can reveal patterns of genetic variation that might otherwise go undetected. American shad is a wide ranging anadromous clupeid fish that exhibits variation in demographic histories and reproductive strategies (both semelparity and iteroparity) and provides a unique perspective on the evolutionary processes that govern the genetic architecture of anadromous fishes. Using 13 microsatellite loci, we examined the magnitude and spatial distribution of genetic variation among 33 populations across the species' range to (i) determine whether signals of historical demography persist among contemporary populations and (ii) assess the effect of different reproductive strategies on population structure. Patterns of genetic diversity and differentiation among populations varied widely and reflect the differential influences of historical demography, microevolutionary processes and anthropogenic factors across the species' range. Sequential reductions of diversity with latitude among formerly glaciated rivers are consistent with stepwise postglacial colonization and successive population founder events. Weak differentiation among U.S. iteroparous populations may be a consequence of human‐mediated gene flow, while weak differentiation among semelparous populations probably reflects natural gene flow. Evidence for an effect of reproductive strategy on population structure suggests an important role for environmental variation and suggests that the factors that are responsible for shaping American shad life history patterns may also influence population genetic structure.     

The Genetics of Bene Israel from India Reveals Both Substantial Jewish and Indian Ancestry

The Bene Israel Jewish community from West India is a unique population whose history before the 18^th century remains largely unknown. Bene Israel members consider themselves as descendants of Jews, yet the identity of Jewish ancestors and their arrival time to India are unknown, with speculations on arrival time varying between the 8th century BCE and the 6th century CE. Here, we characterize the genetic history of Bene Israel by collecting and genotyping 18 Bene Israel individuals. Combining with 486 individuals from 41 other Jewish, Indian and Pakistani populations, and additional individuals from worldwide populations, we conducted comprehensive genome-wide analyses based on F_ST, principal component analysis, ADMIXTURE, identity-by-descent sharing, admixture linkage disequilibrium decay, haplotype sharing and allele sharing autocorrelation decay, as well as contrasted patterns between the X chromosome and the autosomes. The genetics of Bene Israel individuals resemble local Indian populations, while at the same time constituting a clearly separated and unique population in India. They are unique among Indian and Pakistani populations we analyzed in sharing considerable genetic ancestry with other Jewish populations. Putting together the results from all analyses point to Bene Israel being an admixed population with both Jewish and Indian ancestry, with the genetic contribution of each of these ancestral populations being substantial. The admixture took place in the last millennium, about 19–33 generations ago. It involved Middle-Eastern Jews and was sex-biased, with more male Jewish and local female contribution. It was followed by a population bottleneck and high endogamy, which can lead to increased prevalence of recessive diseases in this population. This study provides an example of how genetic analysis advances our knowledge of human history in cases where other disciplines lack the relevant data to do so.     

Shaping of genetic structure along Pleistocene and modern river systems in the hydrochorous riparian azalea, Rhododendron ripense (Ericaceae)

To determine the effects of hydrochory on the formation of the present range of a species and the spatial distribution of genetic variation, we assessed the rangewide genetic structure of a hydrochorous riparian Japanese species (Rhododendron ripense) using four nuclear microsatellite loci. The patterns of isolation by distance and Bayesian clustering analyses of 33 populations suggested that the present range, characterized by both localized and disjunct distributions across the sea, arose from two contrasting colonization events: (1) primary colonization along two Pleistocene rivers that have been submerged and become partly isolated by marine transgression by 6000 years ago, and (2) additional range expansions from these rivers into unconnected neighboring rivers as a result of river captures. Along the Pleistocene rivers, frequent gene flow by hydrochory resulted in the retention of considerable genetic diversity within each population and genetic homogenization among populations. Within unconnected neighboring rivers, genetic diversity was also retained by the simultaneous redistribution of many individuals as a result of river captures, whereas restricted gene flow within a river resulted in genetic divergence among the river populations. Thus, the evolutionary history of hydrochorous R. ripense appears to have been strongly shaped by both ancient and modern rivers.     

Ethiopian genetic diversity reveals linguistic stratification and complex influences on the Ethiopian gene pool

Humans and their ancestors have traversed the Ethiopian landscape for millions of years, and present-day Ethiopians show great cultural, linguistic, and historical diversity, which makes them essential for understanding African variability and human origins. We genotyped 235 individuals from ten Ethiopian and two neighboring (South Sudanese and Somali) populations on an Illumina Omni 1M chip. Genotypes were compared with published data from several African and non-African populations. Principal-component and STRUCTURE-like analyses confirmed substantial genetic diversity both within and between populations, and revealed a match between genetic data and linguistic affiliation. Using comparisons with African and non-African reference samples in 40-SNP genomic windows, we identified "African" and "non-African" haplotypic components for each Ethiopian individual. The non-African component, which includes the SLC24A5 allele associated with light skin pigmentation in Europeans, may represent gene flow into Africa, which we estimate to have occurred ~3 thousand years ago (kya). The non-African component was found to be more similar to populations inhabiting the Levant rather than the Arabian Peninsula, but the principal route for the expansion out of Africa ~60 kya remains unresolved. Linkage-disequilibrium decay with genomic distance was less rapid in both the whole genome and the African component than in southern African samples, suggesting a less ancient history for Ethiopian populations.     

Genomic Structure of the Immigrant Siddis of East Africa to Southern India: A Study of 20 Autosomal DNA Markers

The Siddis are a tribal group of African origin living in Karnataka, India. They have undergone considerable cultural change due to their proximity to neighboring population groups. To understand the biological consequences of these changes, we describe the genomic structure of the Siddis and the contribution from putative ancestral populations using 20 autosomal DNA markers. The distribution of Alu indel markers and a genetic distance analysis reveals their closer affinities with Africans. The levels of genomic diversity and heterozygosity are high in all the populations of southern India. Genetic admixture analysis reveals a predominant contribution from Africans, a lesser contribution from south Indians, and a slight one from Europeans. There is no evidence of gametic disequilibrium in the Siddis. The genetic homogeneity of the Siddis, in spite of its admixed origin, suggests the utility of this population for genetic epidemiological studies.     

Substitution of Hainan indigenous genetic lineage in the Utsat people,exiles of the Champa kingdom

The Utsat people do not belong to one of the recognized ethnic groups in Hainan, China. Some historical literature and linguistic classification confirm a close cultural relationship between the Utsat and Cham people; however, the genetic relationship between these two populations is not known. In the present study, we typed paternal Y chromosome and maternal mitochondrial (mt) DNA markers in 102 Utsat people to gain a better understanding of the genetic history of this population. High frequencies of the Y chromosome haplogroup O1a*-M119 and mtDNA lineages D4, F2a, F1b, F1a1, B5a, M8a, M*, D5, and B4a exhibit a pattern similar to that seen in neighboring indigenous populations. Cluster analyses (principal component analyses and networks) of the Utsat, Cham, and other ethnic groups in East Asia indicate that the Utsat are much closer to the Hainan indigenous ethnic groups than to the Cham and other mainland southeast Asian populations. These findings suggest that the origins of the Utsat likely involved massive assimilation of indigenous ethnic groups. During the assimilation process, the language of Utsat has been structurally changed to a tonal language; however, their Islamic beliefs may have helped to keep their culture and self-identification.     

Mitochondrial and Nuclear DNA Diversity in the Choco and Chibcha Amerinds of Panama

Mitochondrial and nuclear DNA diversities were determined for two Choco-speaking Amerind populations, the Embera and Wounan, sampled widely across their geographic range in eastern Panama. These data were compared with mitochondrial and nuclear diversities determined here and previously for neighboring Chibcha-speaking Ngobe and Kuna populations. Chocoan groups exhibited mitochondrial diversity levels typical for Amerind populations while Chibchan groups revealed reduced mitochondrial diversity. A slight reduction in autosomal levels of heterozygosity was determined for the Chibcha while X and Y variation appeared equivalent in all populations. Genetic distinctiveness of the two linguistic groups contradicts the anthropological theory that Paleoindians migrated repeatedly through the isthmian region and, instead, supports the idea of cultural adaptation by endogenous populations. Reduced genetic diversity in Chibchan populations has been proposed to represent a population bottleneck dating to Chibchan ethnogenesis. The relative sensitivities of haplotype pairwise difference distributions and Tajima's D to detect demographic events such as population bottlenecks are examined. Also, the potential impact of substitution rate heterogeneity, population subdivision, and genetic selection on pairwise difference distributions are discussed. Evidence is presented suggesting that a larger effective population size may obscure the historical signal obtained from nuclear genes while the single mitochondrial locus may provide a moderately strong signal.     

Dietary,Cultural, and Pathogens-Related Selective Pressures Shaped Differential Adaptive Evolution among Native Mexican Populations

Native American genetic ancestry has been remarkably implicated with increased risk of diverse health issues in several Mexican populations, especially in relation to the dramatic changes in environmental, dietary, and cultural settings they have recently undergone. In particular, the effects of these ecological transitions and Westernization of lifestyles have been investigated so far predominantly on Mestizo individuals. Nevertheless, indigenous groups, rather than admixed Mexicans, have plausibly retained the highest proportions of genetic components shaped by natural selection in response to the ancient milieu experienced by Mexican ancestors during their pre-Columbian evolutionary history. These formerly adaptive variants have the potential to represent the genetic determinants of some biological traits that are peculiar to Mexican people, as well as a reservoir of loci with possible biomedical relevance. To test such a hypothesis, we used genome-wide genotype data to infer the unique adaptive evolution of Native Mexican groups selected as reasonable descendants of the main pre-Columbian Mexican civilizations. A combination of haplotype-based and gene-network analyses enabled us to detect genomic signatures ascribable to polygenic adaptive traits plausibly evolved by the main genetic clusters of Mexican indigenous populations to cope with local environmental and/or cultural conditions. Some of these adaptations were found to play a role in modulating the susceptibility/resistance of these groups to certain pathological conditions, thus providing new evidence that diverse selective pressures have contributed to shape the current biological and disease-risk patterns of present-day Native and Mestizo Mexican populations.     

Determinants of Genetic Structure in a Nonequilibrium Metapopulation of the Plant Silene latifolia

Population genetic differentiation will be influenced by the demographic history of populations, opportunities for migration among neighboring demes and founder effects associated with repeated extinction and recolonization. In natural populations, these factors are expected to interact with each other and their magnitudes will vary depending on the spatial distribution and age structure of local demes. Although each of these effects has been individually identified as important in structuring genetic variance, their relative magnitude is seldom estimated in nature. We conducted a population genetic analysis in a metapopulation of the angiosperm, Silene latifolia, from which we had more than 20 years of data on the spatial distribution, demographic history, and extinction and colonization of demes. We used hierarchical Bayesian methods to disentangle which features of the populations contributed to among population variation in allele frequencies, including the magnitude and direction of their effects. We show that population age, long-term size and degree of connectivity all combine to affect the distribution of genetic variance; small, recently-founded, isolated populations contributed most to increase F _ST in the metapopulation. However, the effects of population size and population age are best understood as being modulated through the effects of connectivity to other extant populations, i.e. F _ST diminishes as populations age, but at a rate that depends how isolated the population is. These spatial and temporal correlates of population structure give insight into how migration, founder effect and within-deme genetic drift have combined to enhance and restrict genetic divergence in a natural metapopulation.     

Impacts of domestication on population genetics of a traditional Chinese medicinal herb,Atractylodes macrocephala (Asteraceae)

As a medicinal herb, Atractylodes macrocephala Koidz. experienced centuries of cultivation in China, and germplasm resources of all cultivated populations have degraded over time as a consequence of domestication processes. This study used chloroplast DNA and microsatellites to clarify not only the effects of domestication on population genetics, but also determine the geographic origins of landraces. The results revealed that cultivated populations (except the “Pingzhu” landrace) showed higher genetic diversity than their wild counterparts and low levels of genetic differentiation occurred between cultivated and wild groups. Furthermore, STRUCTURE and UPGMA analyses grouped all wild populations into three genotypic clusters, two of which (in Shaanxi and Hunan Province) shared the same gene pool with cultivated A. macrocephala, suggesting that wild populations in Central China have been involved in the origin of cultivated A. macrocephala. Moreover, the wild population from Qimen, Anhui Province and the cultivated “Pingzhu” landrace harbor unique gene pools and rare alleles that could be useful in future breeding efforts. This large‐scale analysis of population genetics on a medicinal herb that has a centuries‐long history of human‐mediated selection will facilitate utilization and conservation of the valuable genetic resources of medicinal species.     

Genetic Affinity of the Bhil,Kol and Gond Mentioned in Epic Ramayana

Kol, Bhil and Gond are some of the ancient tribal populations known from the Ramayana, one of the Great epics of India. Though there have been studies about their affinity based on classical and haploid genetic markers, the molecular insights of their relationship with other tribal and caste populations of extant India is expected to give more clarity about the the question of continuity vs. discontinuity. In this study, we scanned >97,000 of single nucleotide polymorphisms among three major ancient tribes mentioned in Ramayana, namely Bhil, Kol and Gond. The results obtained were then compared at inter and intra population levels with neighboring and other world populations. Using various statistical methods, our analysis suggested that the genetic architecture of these tribes (Kol and Gond) was largely similar to their surrounding tribal and caste populations, while Bhil showed closer affinity with Dravidian and Austroasiatic (Munda) speaking tribes. The haplotype based analysis revealed a massive amount of genome sharing among Bhil, Kol, Gond and with other ethnic groups of South Asian descent. On the basis of genetic component sharing among different populations, we anticipate their primary founding over the indigenous Ancestral South Indian (ASI) component has prevailed in the genepool over the last several thousand years.     

Population structure,seasonal genotypic differentiation,and clonal diversity of weedy dandelions in three Boston area populations (Taraxacum sp.)

Weedy dandelions have a worldwide distribution and thrive in urban environments despite a lack of sexual reproduction throughout most of its range. North American dandelions, introduced from Eurasia, are believed to be primarily, if not exclusively, apomictic triploids. In some European populations, apomicts co‐occur with diploid sexual individuals and hybridizations can create genetically unique apomicts, which may subsequently disperse and establish new populations globally. Using six nuclear microsatellite markers and a cpDNA intergenic spacer, we investigate the impact of this unusual natural history on population structure and diversity in three urban Boston area dandelion populations. Our results show high levels of genetic diversity within populations, spatial population structure, and seasonal genotypic differentiation in flowering times. We find evidence that sexual reproduction and recombination, presumably in Europe, and extensive gene flow drive these patterns of diversity and create the appearance of panmixia despite the lack of evidence for local sexual reproduction.     

Population structure of a parasitic plant and its perennial host

Characterization of host and parasite population genetic structure and estimation of gene flow among populations are essential for the understanding of parasite local adaptation and coevolutionary interactions between hosts and parasites. We examined two aspects of population structure in a parasitic plant, the greater dodder (Cuscuta europaea) and its host plant, the stinging nettle (Urtica dioica), using allozyme data from 12 host and eight parasite populations. First, we examined whether hosts exposed to parasitism in the past contain higher levels of genetic variation. Second, we examined whether host and parasite populations differ in terms of population structure and if their population structures are correlated. There was no evidence that host populations differed in terms of gene diversity or heterozygosity according to their history of parasitism. Host populations were genetically more differentiated (F(ST) = 0.032) than parasite populations (F(ST) = 0.009). Based on these F(ST) values, gene flow was high for both host and parasite. Such high levels of gene flow could counteract selection for local adaptation of the parasite. We found no significant correlation between geographic and genetic distance (estimated as pairwise F(ST)), either for the host or for the parasite. Furthermore, host and parasite genetic distance matrices were uncorrelated, suggesting that sites with genetically similar host populations are unlikely to have genetically similar parasite populations.     

Genetic variation in the enigmatic Altaian Kazakhs of South-Central Russia: insights into Turkic population history

The Altaian Kazakhs, a Turkic speaking group, now reside in the southern part of the Altai Republic in south-central Russia. According to historical accounts, they are one of several ethnic and geographical subdivisions of the Kazakh nomadic group that migrated from China and Western Mongolia into the Altai region during the 19th Century. However, their population history of the Altaian Kazakhs and the genetic relationships with other Kazakh groups and neighboring Turkic-speaking populations is not well understood. To begin elucidating their genetic history, we analyzed the mtDNAs from 237 Altaian Kazakhs through a combination of SNP analysis and HVS1 sequencing. This analysis revealed that their mtDNA gene pool was comprised of roughly equal proportions of East (A-G, M7, M13, Y and Z) and West (H, HV, pre-HV, R, IK, JT, X, U) Eurasian haplogroups, with the haplotypic diversity within haplogroups C, D, H, and U being particularly high. This pattern of diversity likely reflects the complex interactions of the Kazakhs with other Turkic groups, Mongolians, and indigenous Altaians. Overall, these data have important implications for Kazakh population history, the genetic prehistory of the Altai-Sayan region, and the phylogeography of major mitochondrial lineages in Eurasia.     

Historical sketch of Slovak Haban (Hutterite) population based on autosomal STR analysis

According to the Hutterite chronicles, the Habans arrived from Austrian Tyrol, Switzerland, and northernmost Italy and stayed in four regions of Slovakia (Soboti?te, Vel'ké Leváre, Moravsky Sv?ty Ján, Tren?ín). There are some communities in western Slovakia that retained their Haban cultural identity and still identify themselves as descendents of the Hutterite population with their own specific customs. Slovak Habans are typical founder population with significant social isolation for which high degree of inbreeding is typical. Present study investigated STR polymorphisms as a powerful genetic tool for population genetic studies. The aim was to perform a comparative, population genetic study based on 15 STR loci widely used in forensic genetics, of the Haban population, the Slovak majority population and the population of Tyrol. We analyzed allele frequencies and other statistical parameters in three selected populations in order to identify groups of specific ethnic origin and establish their genetic relationship. The data set included 110 unrelated Habans and 201 unrelated individuals from the Slovak majority population, as well as allelic frequencies for the population of Austrian Tyrol available in the literature. Population pairwise FST values used as a short term genetic distance between populations showed significant differentiation between the Habans and both reference populations (FST=0.0025 and 0.0042 for comparison with the Slovaks and Austrians, respectively; p<10(-3)). The Slovak Hutterites were demonstrated to be genetically distinct and more closely related to their geographic neighbors than to their historical ancestral population, which may be at least partially explained by gene flow between neighboring Haban and Slovak populations.     

Randomization Analyses: Mimicry, Geographic Variation and Cultural Evolution of Song in Brood-Parasitic Straw-Tailed Whydahs, Vidua fischeri

Although bird song has been an important model for investigating questions of behavior development, cultural evolution and population differentiation, the quantitative methods of analysis have been problematic. Here we develop and apply quantitative randomization methods to test hypotheses about these processes in a natural population of birds. Songs of the African brood-parasitic straw-tailed whydahs ( Vidua fischeri ) and songs of their host species, the purple grenadier ( Granatina ianthinogaster ), were compared in audiospectrograms for similarity to test the following hypotheses: Whydahs mimic the songs of their host species, they have local song dialects, neighboring males match their song themes, local males match the songs of local hosts, remote populations have different songs according to their geographic distance, and songs undergo cultural evolution over time across generations. Randomization analyses were completed using (1) Mantel matrix statistics and (2) tree-based measures employing Sankoff optimization of Manhattan matrices and approximate randomizations. Our results provide evidence for song mimicry, local song dialects, matching song themes between neighboring males, song matching of local whydah mimics and grenadier song models, correspondence of song differences and geographic distance, and cultural continuity with change in song traditions within a local population. These randomization methods may be useful in other studies of animal communication, and they are sufficiently general for use both with distance matrices derived either from naturalistic impressions of song similarity as in our example or from acoustic measurements.     

Demographic histories and genetic diversities of Fennoscandian marine and landlocked ringed seal subspecies

Island populations are on average smaller, genetically less diverse, and at a higher risk to go extinct than mainland populations. Low genetic diversity may elevate extinction probability, but the genetic component of the risk can be affected by the mode of diversity loss, which, in turn, is connected to the demographic history of the population. Here, we examined the history of genetic erosion in three Fennoscandian ringed seal subspecies, of which one inhabits the Baltic Sea ‘mainland’ and two the ‘aquatic islands’ composed of Lake Saimaa in Finland and Lake Ladoga in Russia. Both lakes were colonized by marine seals after their formation c. 9500 years ago, but Lake Ladoga is larger and more contiguous than Lake Saimaa. All three populations suffered dramatic declines during the 20th century, but the bottleneck was particularly severe in Lake Saimaa. Data from 17 microsatellite loci and mitochondrial control‐region sequences show that Saimaa ringed seals have lost most of the genetic diversity present in their Baltic ancestors, while the Ladoga population has experienced only minor reductions. Using Approximate Bayesian computing analyses, we show that the genetic uniformity of the Saimaa subspecies derives from an extended founder event and subsequent slow erosion, rather than from the recent bottleneck. This suggests that the population has persisted for nearly 10,000 years despite having low genetic variation. The relatively high diversity of the Ladoga population appears to result from a high number of initial colonizers and a high post‐colonization population size, but possibly also by a shorter isolation period and/or occasional gene flow from the Baltic Sea.