Russian ethnic history inferred from mitochondrial DNA diversity

With the aim of gaining insight into the genetic history of the Russians, we have studied mitochondrial DNA diversity among a number of modern Russian populations. Polymorphisms in mtDNA markers (HVS-I and restriction sites of the coding region) of populations from 14 regions within present-day European Russia were investigated. Based on analysis of the mitochondrial gene pool geographic structure, we have identified three different elements in it and a vast "intermediate" zone between them. The analysis of the genetic distances from these elements to the European ethnic groups revealed the main causes of the Russian mitochondrial gene pool differentiation. The investigation of this pattern in historic perspective showed that the structure of the mitochondrial gene pool of the present-day Russians largely conforms to the tribal structure of the medieval Slavs who laid the foundation of modern Russians. Our results indicate that the formation of the genetic diversity currently observed among Russians can be traced to the second half of the first millennium A.D., the time of the colonization of the East European Plain by the Slavic tribes. Patterns of diversity are explained by both the impact of the native population of the East European Plain and by genetic differences among the early Slavs.     

Origin of the Mongoloid component in the mitochondrial gene pool of Slavs

The data on mitochondrial DNA (mtDNA) restriction polymorphism in Czech population (n = 279) are presented. It was demonstrated that in terms of their structure, mitochondrial gene pools of Czechs and other Slavic populations (Russians, Poles, Slovenians, and Bosnians) were practically indistinguishable. In Czechs, the frequency of eastern-Eurasian (Mongoloid) mtDNA lineages constituted 1.8%. The spread of eastern-Eurasian mtDNA lineages belonging to different ethnolinguistic groups in the populations of Europe was examined. Frequency variations of these DNA lineages in different Slavic groups was observed, with the range from 1.2 and 1.6% in Southern and Western Slavs, respectively, to 1.3 to 5.2% in Eastern Slavs, the Russian population of Eastern Europe. The highest frequency of Mongoloid component was detected in the mitochondrial gene pools of Russian populations from the Russian North and the Northwestern region of Russia. This finding can be explained in terms of assimilation of northern-European Finno--Ugric populations during the formation of the Russian population of these regions. The origin of Mongoloid component in the gene pools of different groups of Slavs is discussed.     

Genetic Heritage of the Balto-Slavic Speaking Populations: A Synthesis of Autosomal,Mitochondrial and Y-Chromosomal Data

The Slavic branch of the Balto-Slavic sub-family of Indo-European languages underwent rapid divergence as a result of the spatial expansion of its speakers from Central-East Europe, in early medieval times. This expansion–mainly to East Europe and the northern Balkans–resulted in the incorporation of genetic components from numerous autochthonous populations into the Slavic gene pools. Here, we characterize genetic variation in all extant ethnic groups speaking Balto-Slavic languages by analyzing mitochondrial DNA (n = 6,876), Y-chromosomes (n = 6,079) and genome-wide SNP profiles (n = 296), within the context of other European populations. We also reassess the phylogeny of Slavic languages within the Balto-Slavic branch of Indo-European. We find that genetic distances among Balto-Slavic populations, based on autosomal and Y-chromosomal loci, show a high correlation (0.9) both with each other and with geography, but a slightly lower correlation (0.7) with mitochondrial DNA and linguistic affiliation. The data suggest that genetic diversity of the present-day Slavs was predominantly shaped in situ, and we detect two different substrata: ‘central-east European’ for West and East Slavs, and ‘south-east European’ for South Slavs. A pattern of distribution of segments identical by descent between groups of East-West and South Slavs suggests shared ancestry or a modest gene flow between those two groups, which might derive from the historic spread of Slavic people.     

The macrohaplogroup U structure in Russians

The structure and diversity of mitochondrial DNA (mtDNA) macrohaplogroup U lineages in Russians from Eastern Europe are studied on the basis of analysis of variation of nucleotide sequences of complete mitochondrial genomes. In total, 132 mitochondrial genomes belonging to haplogroups U1, U2e, U3, U4, U5, U7, U8a, and K are characterized. Results of phylogeographic analysis show that the mitochondrial gene pool of Russians contains mtDNA haplotypes belonging to subhaplogroups that are characteristic only of Russians and other Eastern Slavs (13.7%), Slavs in general (11.4%), Slavs and Germans (17.4%), and Slavs, Germans, and Baltic Finns (9.8%). Results of molecular dating show that ages of mtDNA subhaplogroups to which Russian mtDNA haplotypes belong vary in a wide range, from 600 to 17000 years. However, molecular dating results for Slavic and Slavic-Germanic mtDNA subhaplogroups demonstrate that their formation mainly occurred in the Bronze and Iron Ages (1000–5000 years ago). Only some instances (for subhaplogroups U5b1a1 and U5b1e1a) are characterized by a good agreement between molecular dating results and the chronology of Slavic ethnic history based on historical and archaeological data.     

On the origin of Mongoloid component in the mitochondrial gene pool of Slavs

The data on mitochondrial DNA (mtDNA) restriction polymorphism in Czech population (n = 279) are presented. It was demonstrated that in terms of their structure, mitochondrial gene pools of Czechs and other Slavic populations (Russians, Poles, Slovenians, and Bosnians) were practically indistinguishable. In Czechs, the frequency of eastern-Eurasian (Mongoloid) mtDNA lineages constituted 1.8%. The spread of eastern-Eurasian mtDNA lineages belonging to different ethnolinguistic groups in the populations of Europe was examined. Frequency variations of these DNA lineages in different Slavic groups was observed, with the range from 1.2 and 1.6% in Southern and Western Slavs, respectively, to 1.3 to 5.2% in Eastern Slavs, the Russian population of Eastern Europe. The highest frequency of Mongoloid component was detected in the mitochondrial gene pools of Russian populations from the Russian North and the Northwestern region of Russia. This finding can be explained in terms of assimilation of northern-European Finno-Ugric populations during the formation of the Russian population of these regions. The origin of Mongoloid component in the gene pools of different groups of Slavs is discussed.     

Ancient DNA Reveals Matrilineal Continuity in Present-Day Poland over the Last Two Millennia

While numerous ancient human DNA datasets from across Europe have been published till date, modern-day Poland in particular, remains uninvestigated. Besides application in the reconstruction of continent-wide human history, data from this region would also contribute towards our understanding of the history of the Slavs, whose origin is hypothesized to be in East or Central Europe. Here, we present the first population-scale ancient human DNA study from the region of modern-day Poland by establishing mitochondrial DNA profiles for 23 samples dated to 200 BC – 500 AD (Roman Iron Age) and for 20 samples dated to 1000–1400 AD (Medieval Age). Our results show that mitochondrial DNA sequences from both periods belong to haplogroups that are characteristic of contemporary West Eurasia. Haplotype sharing analysis indicates that majority of the ancient haplotypes are widespread in some modern Europeans, including Poles. Notably, the Roman Iron Age samples share more rare haplotypes with Central and Northeast Europeans, whereas the Medieval Age samples share more rare haplotypes with East-Central and South-East Europeans, primarily Slavic populations. Our data demonstrates genetic continuity of certain matrilineages (H5a1 and N1a1a2) in the area of present-day Poland from at least the Roman Iron Age until present. As such, the maternal gene pool of present-day Poles, Czechs and Slovaks, categorized as Western Slavs, is likely to have descended from inhabitants of East-Central Europe during the Roman Iron Age.     

Mitochondrial DNA perspective of Serbian genetic diversity

Although south‐Slavic populations have been studied to date from various aspects, the population of Serbia, occupying the central part of the Balkan Peninsula, is still genetically understudied at least at the level of mitochondrial DNA (mtDNA) variation. We analyzed polymorphisms of the first and the second mtDNA hypervariable segments (HVS‐I and HVS‐II) and informative coding‐region markers in 139 Serbians to shed more light on their mtDNA variability, and used available data on other Slavic and neighboring non‐Slavic populations to assess their interrelations in a broader European context. The contemporary Serbian mtDNA profile is consistent with the general European maternal landscape having a substantial proportion of shared haplotypes with eastern, central, and southern European populations. Serbian population was characterized as an important link between easternmost and westernmost south‐Slavic populations due to the observed lack of genetic differentiation with all other south‐Slavic populations and its geographical positioning within the Balkan Peninsula. An increased heterogeneity of south Slavs, most likely mirroring turbulent demographic events within the Balkan Peninsula over time (i.e., frequent admixture and differential introgression of various gene pools), and a marked geographical stratification of Slavs to south‐, east‐, and west‐Slavic groups, were also found. A phylogeographic analyses of 20 completely sequenced Serbian mitochondrial genomes revealed not only the presence of mtDNA lineages predominantly found within the Slavic gene pool (U4a2a*, U4a2a1, U4a2c, U4a2g, HV10), supporting a common Slavic origin, but also lineages that may have originated within the southern Europe (H5*, H5e1, H5a1v) and the Balkan Peninsula in particular (H6a2b and L2a1k). Am J Phys Anthropol 156:449–465, 2015. © 2014 Wiley Periodicals, Inc.     

Mitochondrial DNA variations in Russian and Belorussian populations

The sequence of the first hypervariable segment (HVS-I) of mitochondrial DNA (mtDNA) was determined in 251 individuals from three eastern Slavonic populations, two Russian and one Belorussian. Within HVS-I, 78 polymorphic positions were revealed. Within-population diversity of HVS-I varies slightly among three samples; its estimates do not differ strongly from those for European populations. Haplotype diversity for three populations calculated in this study is 0.949; mean pairwise differences estimate is 3.59. To assign mtDNA sequences to major phylogenetic clusters, haplogroup-specific restriction polymorphisms were selectively typed in most samples. The haplogroup distribution in the total Eastern Slavonic sample is similar to that reported for the European sample. However, the separate consideration of three Slavonic samples reveals the complicated structure of the mitochondrial gene pool in the Eastern European area. Data of this study support the proposed model of the origin of modern Eastern Slavs, which implies the admixture of ancient Slavonic tribes with pre-Slavonic populations of Eastern Europe. These data should contribute to general studies of mitochondrial DNA variations in Europe.     

Is there a Finno-Ugric component in the gene pool of Russians from Yaroslavl oblast? Evidence from Y-chromosome

The Upper Volga region was an area of contacts of Finno-Ugric, Slavic, and Scandinavian speaking populations in the 8th–10th centuries AD. However, their role in the formation of the contemporary gene pool of the Russian population of the region is largely unknown. To answer this question, we studied four populations of Yaroslavl oblast (N = 132) by a wide panel of STR and SNP markers of the Y-chromosome. Two of the studied populations appear to be genetically similar: the indigenous Russian population of Yaroslavl oblast and population of Katskari are characterized by the same major haplogroup, R-M198 (xM458). Haplogroup R-M458 composes more than half of Sitskari’s gene pool. The major haplogroup in the gene pool of the population of the ancient town of Mologa is N-M178. Subtyping N-M178 by newest “genomeera” Y-SNP markers showed different pathways of entering this haplogroup into the gene pools of Yaroslavl Volga region populations. The majority of Russian populations have subvariant N3a3-CTS10760; the regular sample of Yaroslavl oblast is equally represented by subvariants N3a3-CTS10760 and N3a4-Z1936, while subvariant N3a4-Z1936 predominates in the gene pool of population of Mologa. This N3a4-Z1936 haplogroup is common among the population of the north of Eastern Europe and the Volga-Ural region. The obtained results indicate preservation of the Finno-Ugric component in the gene pool of population of Mologa and a contribution of Slavic colonization in the formation of the gene pool of the Yaroslavl Volga region populations and make it possible to hypothesize the genetic contribution of the “downstream” (Rostov- Suzdal) rather than “upstream” (Novgorod) Slavic migration wave.     

Gene pool of the Novgorod population: Between the north and the south

We studied the Y-chromosome pool of the ethnic Russian population of Novgorod oblast (Russia) by 49 SNP and 17 STR markers. The total sample (N = 191) consists of four populations of the Novgorod region, including its southwestern (Shelon Pyatina) and eastern (Bezhetsk Pyatina) parts. Altogether, these four populations represent both the area of the Sopki archaeological culture (supposedly linked with the Novgorod Slovens tribe known from the chronicles) and the area of the Long Barrows culture (supposedly linked with the Krivichi Slavic tribe or with Balts). The pronounced genetic differences between southern and northern Russian populations are well known from previous studies; however, the Novgorod gene pool turned out to be neither northern nor southern, but a representative of the intermediate buffer zone. This zone was identified in this study and included a set of regional Russian populations from Pskov in the west to Kostroma in the east. All four studied populations of Novgorod region are genetically similar. The minor differences among them might represent the medieval Slavic migrations along the rivers, which survived despite the massive demographic shifts during the following history. Haplogroup N3 comprises one-fifth of the Novgorod pool of paternal lineages, with conditionally “Finnic” N3a4 and conditionally “East Baltic Sea Coast” N3a3 clades being almost equally frequent. The N3a3 phylogenetic network revealed the specific “Balto-Slavic” cluster of STR haplotypes, which is frequent in Baltic-speaking Lithuanians but infrequent in Finno-Ugric speaking Estonians. The Novgorod haplotypes lie outside this cluster, indicating that the Novgorod population received both N3a3 and N3a4 from Finno-Ugric speaking populations of the region, which, in turn, acquired the Mesolithic gene pool of the Northeastern Europe.     

Uniparental Genetic Heritage of Belarusians: Encounter of Rare Middle Eastern Matrilineages with a Central European Mitochondrial DNA Pool

Ethnic Belarusians make up more than 80% of the nine and half million people inhabiting the Republic of Belarus. Belarusians together with Ukrainians and Russians represent the East Slavic linguistic group, largest both in numbers and territory, inhabiting East Europe alongside Baltic-, Finno-Permic- and Turkic-speaking people. Till date, only a limited number of low resolution genetic studies have been performed on this population. Therefore, with the phylogeographic analysis of 565 Y-chromosomes and 267 mitochondrial DNAs from six well covered geographic sub-regions of Belarus we strove to complement the existing genetic profile of eastern Europeans. Our results reveal that around 80% of the paternal Belarusian gene pool is composed of R1a, I2a and N1c Y-chromosome haplogroups – a profile which is very similar to the two other eastern European populations – Ukrainians and Russians. The maternal Belarusian gene pool encompasses a full range of West Eurasian haplogroups and agrees well with the genetic structure of central-east European populations. Our data attest that latitudinal gradients characterize the variation of the uniparentally transmitted gene pools of modern Belarusians. In particular, the Y-chromosome reflects movements of people in central-east Europe, starting probably as early as the beginning of the Holocene. Furthermore, the matrilineal legacy of Belarusians retains two rare mitochondrial DNA haplogroups, N1a3 and N3, whose phylogeographies were explored in detail after de novo sequencing of 20 and 13 complete mitogenomes, respectively, from all over Eurasia. Our phylogeographic analyses reveal that two mitochondrial DNA lineages, N3 and N1a3, both of Middle Eastern origin, might mark distinct events of matrilineal gene flow to Europe: during the mid-Holocene period and around the Pleistocene-Holocene transition, respectively.     

Mitochondrial DNA variability in the Czech population, with application to the ethnic history of Slavs

Mitochondrial DNA (mtDNA) variability was studied in a sample of 179 individuals representing the Czech population of Western Bohemia. Sequencing of two hypervariable segments, HVS I and HVS II, in combination with screening of coding-region haplogroup-specific RFLP markers revealed that most Czech mtDNAs belong to the common West Eurasian mitochondrial haplogroups (H, pre-V HV*, J, T, U, N1, W, and X). However, about 3% of Czech mtDNAs encompass East Eurasian lineages (A, N9a, D4, M*). A comparative analysis with published data showed that different Slavonic populations in Central and Eastern Europe contain small but marked amounts of East Eurasian mtDNAs. We suggest that the presence of East Eurasian mtDNA haplotypes is not an original feature of the gene pool of the proto-Slavs but rather may be mostly a consequence of admixture with Central Asian nomadic tribes, who migrated into Central and Eastern Europe in the early Middle Ages.     

Phylogeography of mitochondrial DNA and Y-chromosome haplogroups reveal asymmetric gene flow in populations of Eastern India

Polymorphisms in mitochondrial (mt) DNA and Y-chromosomes of seven socially and linguistically diverse castes and tribes of Eastern India were examined to determine their genetic relationships, their origin, and the influence of demographic factors on population structure. Samples from the Orissa Brahmin, Karan, Khandayat, Gope, Juang, Saora, and Paroja were analyzed for mtDNA hypervariable sequence (HVS) I and II, eight Y-chromosome short tandem repeats (Y-STRs), and lineage-defining mutations diagnostic for Indian- and Eurasian-specific haplogroups. Our results reveal that haplotype diversity and mean pairwise differences (MPD) was higher in caste groups of the region (>0.998, for both systems) compared to tribes (0.917-0.996 for Y-STRs, and 0.958-0.988 for mtDNA haplotypes). The majority of paternal lineages belong to the R1a1, O2a, and H haplogroups (62.7%), while 73.2% of maternal lineages comprise the Indian-specific M*, M5, M30, and R* mtDNA haplogroups, with a sporadic occurrence of West Eurasian lineages. Our study reveals that Orissa Brahmins (a higher caste population) have a genetic affinity with Indo-European speakers of Eastern Europe, although the Y-chromosome data show that the genetic distances of populations are not correlated to their position in the caste hierarchy. The high frequency of the O2a haplogroup and absence of East Asian-specific mtDNA lineages in the Juang and Saora suggest that a migration of Austro-Asiatic tribes to mainland India was exclusively male-mediated which occurred during the demographic expansion of Neolithic farmers in southern China. The phylogeographic analysis of mtDNA and Y-chromosomes revealed varied ancestral sources for the diverse genetic components of the populations of Eastern India.     

East African pigs have a complex Indian,Far Eastern and Western ancestry

In this study, we have characterized the mitochondrial diversity of 81 swine from Uganda. Median‐joining network analysis of D‐loop sequences from these individuals and others characterized in previous studies allowed us to determine that Ugandan pigs cluster with populations from the West (Europe/North Africa), Far East and India. In addition, partial sequencing of the Y‐chromosome UTY locus in 18 Ugandan domestic pigs revealed the segregation of a single HY1 lineage that has a cosmopolitan distribution. A Western and Far Eastern ancestry for East African pigs had been already reported, but this is the first study demonstrating an additional contribution from the Indian porcine gene pool. This result is consistent with the high frequency of zebuine alleles in cattle from East Africa. The geographic coordinates of East Africa, at the crossroads of many trading routes that, through the ages, linked Europe, Africa and Asia, might explain the rich and complex genetic heritage of livestock native to this area.     

The russian gene pool: the gene geography of Alu insertions (ACE, APOA1, B65, PV92, TPA25)

For the first time, an analysis of five Alu insertion loci (ACE, APOA1, B65, PV92, TPA25) has been carried out in 10 Russian populations (1088 individuals) covering the whole historical area of the Russian ethnos. Depending on the locus, Russian populations exhibit similarity to their Western (European populations) or Eastern (populations of the Ural region) neighbors. Considering the frequencies of the studied Alu-insertions, the Russian gene pool exhibits low variation: average interpopulation variation (d) was 0.007, whereas on classical markers, mtDNA and Y chromosome, heterogeneity of the Russian gene pool is essentially higher (0.013, 0.033, 0.142, respectively). Therefore, on the intra-ethnic level, this set of five Alu insertions has low variation. However, a clear pattern was obtained in inter-ethnic comparison of 13 East European ethnic groups, which formed three clusters in accordance with their historical and geographical position: East Slavic, Caucasian and South Ural clusters. The obtained data confirm the efficiency of using Alu insertions for studying genetic differentiation and gene pool history of East European populations.     

Gene Pool Structure of Eastern Ukrainians as Inferred from the Y-Chromosome Haplogroups

Y chromosomes from representative sample of Eastern Ukrainians (94 individuals) were analyzed for composition and frequencies of haplogroups, defined by 11 biallelic loci located in non-recombining part of the chromosome (SRY1532, YAP, 92R7, DYF155S2, 12f2, Tat, M9, M17, M25,M89, andM56). In the Ukrainian gene, pool six haplogroups were revealed: E, F (including G and I), J, N3, P, and R1a1. These haplogroups were earlier detected in a study of Y-chromosome diversity on the territory of Europe as a whole. The major haplogroup in the Ukrainian gene pool, haplogroup R1a1 (earlier designated HG3), accounted for about 44% of all Y chromosomes in the sample examined. This haplogroup is thought to mark the migration patterns of the early Indo-Europeans and is associated with the distribution of the Kurgan archaeological culture. The second major haplogroup is haplogroup F (21.3%), which is a combination of the lineages differing by the time of appearance. Haplogroup P found with the frequency of 9.6%, represents the genetic contribution of the population originating from the ancient autochthonous population of Europe. Haplogroups J and E (11.7 and 4.2%, respectively) mark the migration patterns of the Middle-Eastern agriculturists during the Neolithic. The presence of the N3 lineage (9.6%) is likely explained by a contribution of the assimilated Finno–Ugric tribes. The data on the composition and frequencies of Y-chromosome haplogroups in the sample studied substantially supplement the existing picture of the male lineage distribution in the Eastern Slav population.     

Maternal Genetic Composition of a Medieval Population from a Hungarian-Slavic Contact Zone in Central Europe

The genetic composition of the medieval populations of Central Europe has been poorly investigated to date. In particular, the region of modern-day Slovakia is a blank spot in archaeogenetic research. This paper reports the study of mitochondrial DNA (mtDNA) in ancient samples from the 9^th–12^th centuries originating from the cemeteries discovered in Nitra-Šindolka and Čakajovce, located in western Slovakia (Central Europe). This geographical region is interesting to study because its medieval multi-ethnic population lived in the so-called contact zone of the territory of the Great Moravian and later Hungarian state formations. We described 16 different mtDNA haplotypes in 19 individuals, which belong to the most widespread European mtDNA haplogroups: H, J, T, U and R0. Using comparative statistical and population genetic analyses, we showed the differentiation of the European gene pool in the medieval period. We also demonstrated the heterogeneous genetic characteristics of the investigated population and its affinity to the populations of modern Europe.     

Mitochondrial DNA variation and language replacements in the Caucasus

Sequences of the first hypervariable segment of the mitochondrial DNA (mtDNA) control region were obtained from 353 individuals representing nine groups and four major linguistic families (Indo-European, Altaic and North and South Caucasian) of the Caucasus region. The diversity within and between Caucasus populations exceeded the diversity within Europe, but was less than that in the Near East. Caucasus populations occupy an intermediate position between European and Near Eastern populations in tree and principal coordinate analyses, suggesting that they are either ancestral to European populations or derived via admixture from European and Near Eastern populations. The genetic relationships among Caucasus populations reflect geographical rather than linguistic relationships. In particular, the Indo-European-speaking Armenians and Altaic-speaking Azerbaijanians are most closely related to their nearest geographical neighbours in the Caucasus, not their linguistic neighbours (i.e. other Indo-European or Altaic populations). The mtDNA evidence thus suggests that the Armenian and Azerbaijanian languages represent instances of language replacement that had little impact on the mtDNA gene pool.     

Differentiation of Slavs and their genetic position among Eurasian peoples from data on mitochondrial DNA diversity]

The distribution of identical and similar (phylogenetically related) types of hypervariable segment 1 (HVS1) of the mitochondrial DNA (mtDNA) was studied in human populations belonging to three Slavonic groups and nine ethnogeographic groups of Eurasia (total sample size 2772 people). The results testified to a common origin of West, South, and East Slavs and revealed a central place of West Slavs among all Slavonic ethnoses. Mixing was shown to play a substantial role in the formation of specific features of all three Slavonic gene pools. The mitochondrial gene pools of the Slavonic ethnoses proved to preserve features suggesting a common ancestor for these and South European populations (especially those of the Balkan Peninsula).     

Human Y‐chromosome short tandem repeats: A tale of acculturation and migrations as mechanisms for the diffusion of agriculture in the Balkan Peninsula

Southeastern Europe and, particularly, the Balkan Peninsula are especially useful when studying the mechanisms responsible for generating the current distribution of Paleolithic and Neolithic genetic signals observed throughout Europe. In this study, 404 individuals from Montenegro and 179 individuals from Serbia were typed for 17 Y‐STR loci and compared across 9 Y‐STR loci to geographically targeted previously published collections to ascertain the phylogenetic relationships of populations within the Balkan Peninsula and beyond. We aim to provide information on whether groups in the region represent an amalgamation of Paleolithic and Neolithic genetic substrata, or whether acculturation has played a critical role in the spread of agriculture. We have found genetic markers of Middle Eastern, south Asian and European descent in the area, however, admixture analyses indicate that over 80% of the Balkan gene pool is of European descent. Altogether, our data support the view that the diffusion of agriculture into the Balkan region was mostly a cultural phenomenon although some genetic infiltration from Africa, the Levant, the Caucasus, and the Near East has occurred. Am J Phys Anthropol, 2010. © 2010 Wiley‐Liss, Inc.