Mitochondrial Haplogroup U5b3: A Distant Echo of the Epipaleolithic in Italy and the Legacy of the Early Sardinians

There are extensive data indicating that some glacial refuge zones of southern Europe (Franco-Cantabria, Balkans, and Ukraine) were major genetic sources for the human recolonization of the continent at the beginning of the Holocene. Intriguingly, there is no genetic evidence that the refuge area located in the Italian Peninsula contributed to this process. Here we show, through phylogeographic analyses of mitochondrial DNA (mtDNA) variation performed at the highest level of molecular resolution (52 entire mitochondrial genomes), that the most likely homeland for U5b3—a haplogroup present at a very low frequency across Europe—was the Italian Peninsula. In contrast to mtDNA haplogroups that expanded from other refugia, the Holocene expansion of haplogroup U5b3 toward the North was restricted by the Alps and occurred only along the Mediterranean coasts, mainly toward nearby Provence (southern France). From there, ∼7,000–9,000 years ago, a subclade of this haplogroup moved to Sardinia, possibly as a result of the obsidian trade that linked the two regions, leaving a distinctive signature in the modern people of the island. This scenario strikingly matches the age, distribution, and postulated geographic source of a Sardinian Y chromosome haplogroup (I2a2-M26), a paradigmatic case in the European context of a founder event marking both female and male lineages.     

Origin, diffusion, and differentiation of Y-chromosome haplogroups E and J: inferences on the neolithization of Europe and later migratory events in the Mediterranean area

The phylogeography of Y-chromosome haplogroups E (Hg E) and J (Hg J) was investigated in >2400 subjects from 29 populations, mainly from Europe and the Mediterranean area but also from Africa and Asia. The observed 501 Hg E and 445 Hg J samples were subtyped using 36 binary markers and eight microsatellite loci. Spatial patterns reveal that (1). the two sister clades, J-M267 and J-M172, are distributed differentially within the Near East, North Africa, and Europe; (2). J-M267 was spread by two temporally distinct migratory episodes, the most recent one probably associated with the diffusion of Arab people; (3). E-M81 is typical of Berbers, and its presence in Iberia and Sicily is due to recent gene flow from North Africa; (4). J-M172(xM12) distribution is consistent with a Levantine/Anatolian dispersal route to southeastern Europe and may reflect the spread of Anatolian farmers; and (5). E-M78 (for which microsatellite data suggest an eastern African origin) and, to a lesser extent, J-M12(M102) lineages would trace the subsequent diffusion of people from the southern Balkans to the west. A 7%-22% contribution of Y chromosomes from Greece to southern Italy was estimated by admixture analysis.     

Dissecting the molecular architecture and origin of Bayash Romani patrilineages: genetic influences from South-Asia and the Balkans

The Bayash are a branch of Romanian speaking Roma living dispersedly in Central, Eastern, and Southeastern Europe. To better understand the molecular architecture and origin of the Croatian Bayash paternal gene pool, 151 Bayash Y chromosomes were analyzed for 16 SNPs and 17 STRs and compared with European Romani and non-Romani majority populations from Europe, Turkey, and South Asia. Two main layers of Bayash paternal gene pool were identified: ancestral (Indian) and recent (European). The reduced diversity and expansion signals of H1a patrilineages imply descent from closely related paternal ancestors who could have settled in the Indian subcontinent, possibly as early as between the eighth and tenth centuries AD. The recent layer of the Bayash paternal pool is dominated by a specific subset of E1b1b1a lineages that are not found in the Balkan majority populations. At least two private mutational events occurred in the Bayash during their migrations from the southern Balkans toward Romania. Additional admixture, evident in the low frequencies of typical European haplogroups, J2, R1a, I1, R1b1b2, G, and I2a, took place primarily during the early Bayash settlement in the Balkans and the Romani bondage in Romania. Our results indicate two phenomena in the Bayash and analyzed Roma: a significant preservation of ancestral H1a haplotypes as a result of considerable, but variable level of endogamy and isolation and differential distribution of less frequent, but typical European lineages due to different patterns of the early demographic history in Europe marked by differential admixture and genetic drift.     

Increased resolution of Y chromosome haplogroup T defines relationships among populations of the Near East, Europe, and Africa

Increasing phylogenetic resolution of the Y chromosome haplogroup tree has led to finer temporal and spatial resolution for studies of human migration. Haplogroup T, initially known as K2 and defined by mutation M70, is found at variable frequencies across West Asia, Africa, and Europe. While several SNPs were recently discovered that extended the length of the branch leading to haplogroup T, only two SNPs are known to mark internal branches of haplogroup T. This low level of phylogenetic resolution has hindered studies of the origin and dispersal of this interesting haplogroup, which is found in Near Eastern non-Jewish populations, Jewish populations from several communities, and in the patrilineage of President Thomas Jefferson. Here we map 10 new SNPs that, together with the previously known SNPs, mark 11 lineages and two large subclades (T1a and T1b) of haplogroup T. We also report a new SNP that links haplogroups T and L within the major framework of Y chromosome evolution. Estimates of the timing of the branching events within haplogroup T, along with a comprehensive geographic survey of the major T subclades, suggest that this haplogroup began to diversify in the Near East -25 kya. Our survey also points to a complex history of dispersal of this rare and informative haplogroup within the Near East and from the Near East to Europe and sub-Saharan Africa. The presence of T1a2 chromosomes in Near Eastern Jewish and non-Jewish populations may reflect early exiles between the ancient lands of Israel and Babylon. The presence of different subclades of T chromosomes in Europe may be explained by both the spread of Neolithic farmers and the later dispersal of Jews from the Near East. Finally, the moderately high frequency (-18%) of T1b* chromosomes in the Lemba of southern Africa supports the hypothesis of a Near Eastern, but not necessarily a Jewish, origin for their paternal line.     

Phylogeographic analysis of mitochondrial DNA in northern Asian populations

To elucidate the human colonization process of northern Asia and human dispersals to the Americas, a diverse subset of 71 mitochondrial DNA (mtDNA) lineages was chosen for complete genome sequencing from the collection of 1,432 control-region sequences sampled from 18 autochthonous populations of northern, central, eastern, and southwestern Asia. On the basis of complete mtDNA sequencing, we have revised the classification of haplogroups A, D2, G1, M7, and I; identified six new subhaplogroups (I4, N1e, G1c, M7d, M7e, and J1b2a); and fully characterized haplogroups N1a and G1b, which were previously described only by the first hypervariable segment (HVS1) sequencing and coding-region restriction-fragment-length polymorphism analysis. Our findings indicate that the southern Siberian mtDNA pool harbors several lineages associated with the Late Upper Paleolithic and/or early Neolithic dispersals from both eastern Asia and southwestern Asia/southern Caucasus. Moreover, the phylogeography of the D2 lineages suggests that southern Siberia is likely to be a geographical source for the last postglacial maximum spread of this subhaplogroup to northern Siberia and that the expansion of the D2b branch occurred in Beringia ~7,000 years ago. In general, a detailed analysis of mtDNA gene pools of northern Asians provides the additional evidence to rule out the existence of a northern Asian route for the initial human colonization of Asia.     

Genetic Evidence of an East Asian Origin and Paleolithic Northward Migration of Y-chromosome Haplogroup N

The Y-chromosome haplogroup N-M231 (Hg N) is distributed widely in eastern and central Asia, Siberia, as well as in eastern and northern Europe. Previous studies suggested a counterclockwise prehistoric migration of Hg N from eastern Asia to eastern and northern Europe. However, the root of this Y chromosome lineage and its detailed dispersal pattern across eastern Asia are still unclear. We analyzed haplogroup profiles and phylogeographic patterns of 1,570 Hg N individuals from 20,826 males in 359 populations across Eurasia. We first genotyped 6,371 males from 169 populations in China and Cambodia, and generated data of 360 Hg N individuals, and then combined published data on 1,210 Hg N individuals from Japanese, Southeast Asian, Siberian, European and Central Asian populations. The results showed that the sub-haplogroups of Hg N have a distinct geographical distribution. The highest Y-STR diversity of the ancestral Hg N sub-haplogroups was observed in the southern part of mainland East Asia, and further phylogeographic analyses supports an origin of Hg N in southern China. Combined with previous data, we propose that the early northward dispersal of Hg N started from southern China about 21 thousand years ago (kya), expanding into northern China 12–18 kya, and reaching further north to Siberia about 12–14 kya before a population expansion and westward migration into Central Asia and eastern/northern Europe around 8.0–10.0 kya. This northward migration of Hg N likewise coincides with retreating ice sheets after the Last Glacial Maximum (22–18 kya) in mainland East Asia.     

The Peopling of Europe from the Mitochondrial Haplogroup U5 Perspective

It is generally accepted that the most ancient European mitochondrial haplogroup, U5, has evolved essentially in Europe. To resolve the phylogeny of this haplogroup, we completely sequenced 113 mitochondrial genomes (79 U5a and 34 U5b) of central and eastern Europeans (Czechs, Slovaks, Poles, Russians and Belorussians), and reconstructed a detailed phylogenetic tree, that incorporates previously published data. Molecular dating suggests that the coalescence time estimate for the U5 is ∼25–30 thousand years (ky), and ∼16–20 and ∼20–24 ky for its subhaplogroups U5a and U5b, respectively. Phylogeographic analysis reveals that expansions of U5 subclusters started earlier in central and southern Europe, than in eastern Europe. In addition, during the Last Glacial Maximum central Europe (probably, the Carpathian Basin) apparently represented the area of intermingling between human flows from refugial zones in the Balkans, the Mediterranean coastline and the Pyrenees. Age estimations amounting for many U5 subclusters in eastern Europeans to ∼15 ky ago and less are consistent with the view that during the Ice Age eastern Europe was an inhospitable place for modern humans.     

Phylogeny and biogeography of the alpine newt Mesotriton alpestris (Salamandridae, Caudata), inferred from mtDNA sequences

In this paper, we performed phylogenetic analyses of Mesotriton alpestris populations from the entire range of species distribution, using fragments of two mtDNA genes, cytochrome b (309bp) and 16S rRNA ( approximately 500bp). Sequence diversity patterns and phylogenetic analyses reveal the existence of a relict lineage (Clade A) of late Miocene origin, comprising populations from south-eastern Serbia. This lineage is proposed to be ancestor to a western and an eastern lineage, which diverged during the middle Pliocene. The western lineage is further divided in two clades (Clades B, C) of middle Pliocene origin that represent populations from Italy (B) and populations from central Europe and Iberia (C). Further subdivision, dated back to the middle-late Pliocene, was found within the eastern lineage, representing southern (Clade D) and central-northern (Clade E) Balkan populations, respectively. Extensive sequence divergence, implying greater isolation in multiple refugia, is found within eastern clades, while the western clades seem to have been involved in the colonization of central, western and north-eastern Europe from a hypothetical refugium in central Europe. The extent of divergence does not support the current taxonomy indicating cryptic speciation in the Balkans, while paedomorphic lineages were found to have been evolved during early-middle Pleistocene probably as a response to the ongoing dramatic climatic oscillations.     

High-resolution phylogenetic analysis of southeastern Europe traces major episodes of paternal gene flow among Slavic populations

The extent and nature of southeastern Europe (SEE) paternal genetic contribution to the European genetic landscape were explored based on a high-resolution Y chromosome analysis involving 681 males from seven populations in the region. Paternal lineages present in SEE were compared with previously published data from 81 western Eurasian populations and 5,017 Y chromosome samples. The finding that five major haplogroups (E3b1, I1b* (xM26), J2, R1a, and R1b) comprise more than 70% of SEE total genetic variation is consistent with the typical European Y chromosome gene pool. However, distribution of major Y chromosomal lineages and estimated expansion signals clarify the specific role of this region in structuring of European, and particularly Slavic, paternal genetic heritage. Contemporary Slavic paternal gene pool, mostly characterized by the predominance of R1a and I1b* (xM26) and scarcity of E3b1 lineages, is a result of two major prehistoric gene flows with opposite directions: the post-Last Glacial Maximum R1a expansion from east to west, the Younger Dryas-Holocene I1b* (xM26) diffusion out of SEE in addition to subsequent R1a and I1b* (xM26) putative gene flows between eastern Europe and SEE, and a rather weak extent of E3b1 diffusion toward regions nowadays occupied by Slavic-speaking populations.     

Land Snails as a Diet Diversification Proxy during the Early Upper Palaeolithic in Europe

Despite the ubiquity of terrestrial gastropods in the Late Pleistocene and Holocene archaeological record, it is still unknown when and how this type of invertebrate resource was incorporated into human diets. In this paper, we report the oldest evidence of land snail exploitation as a food resource in Europe dated to 31.3-26.9 ka yr cal BP from the recently discovered site of Cova de la Barriada (eastern Iberian Peninsula). Mono-specific accumulations of large Iberus alonensis land snails (Ferussac 1821) were found in three different archaeological levels in association with combustion structures, along with lithic and faunal assemblages. Using a new analytical protocol based on taphonomic, microX-Ray Diffractometer (DXR) and biometric analyses, we investigated the patterns of selection, consumption and accumulation of land snails at the site. The results display a strong mono-specific gathering of adult individuals, most of them older than 55 weeks, which were roasted in ambers of pine and juniper under 375°C. This case study uncovers new patterns of invertebrate exploitation during the Gravettian in southwestern Europe without known precedents in the Middle Palaeolithic nor the Aurignacian. In the Mediterranean context, such an early occurrence contrasts with the neighbouring areas of Morocco, France, Italy and the Balkans, where the systematic nutritional use of land snails appears approximately 10,000 years later during the Iberomaurisian and the Late Epigravettian. The appearance of this new subsistence activity in the eastern and southern regions of Spain was coeval to other demographically driven transformations in the archaeological record, suggesting different chronological patterns of resource intensification and diet broadening along the Upper Palaeolithic in the Mediterranean basin.     

Mitogenomes from Two Uncommon Haplogroups Mark Late Glacial/Postglacial Expansions from the Near East and Neolithic Dispersals within Europe

The current human mitochondrial (mtDNA) phylogeny does not equally represent all human populations but is biased in favour of representatives originally from north and central Europe. This especially affects the phylogeny of some uncommon West Eurasian haplogroups, including I and W, whose southern European and Near Eastern components are very poorly represented, suggesting that extensive hidden phylogenetic substructure remains to be uncovered. This study expanded and re-analysed the available datasets of I and W complete mtDNA genomes, reaching a comprehensive 419 mitogenomes, and searched for precise correlations between the ages and geographical distributions of their numerous newly identified subclades with events of human dispersal which contributed to the genetic formation of modern Europeans. Our results showed that haplogroups I (within N1a1b) and W originated in the Near East during the Last Glacial Maximum or pre-warming period (the period of gradual warming between the end of the LGM, ∼19 ky ago, and the beginning of the first main warming phase, ∼15 ky ago) and, like the much more common haplogroups J and T, may have been involved in Late Glacial expansions starting from the Near East. Thus our data contribute to a better definition of the Late and postglacial re-peopling of Europe, providing further evidence for the scenario that major population expansions started after the Last Glacial Maximum but before Neolithic times, but also evidencing traces of diffusion events in several I and W subclades dating to the European Neolithic and restricted to Europe.     

Molecular biogeography of red deer Cervus elaphus from eastern Europe: insights from mitochondrial DNA sequences

European red deer are known to show a conspicuous phylogeographic pattern with three distinct mtDNA lineages (western, eastern and North-African/Sardinian). The western lineage, believed to be indicative of a southwestern glacial refuge in Iberia and southern France, nowadays covers large areas of the continent including the British Isles, Scandinavia and parts of central Europe, while the eastern lineage is primarily found in southeast-central Europe, the Carpathians and the Balkans. However, large parts of central Europe and the whole northeast of the continent were not covered by previous analyses. To close this gap, we produced mtDNA control region sequences from more than 500 red deer from Denmark, Germany, Poland, Lithuania, Belarus, Ukraine and western Russia and combined our data with sequences available from earlier studies to an overall sample size of almost 1,100. Our results show that the western lineage extends far into the European east and is prominent in all eastern countries except for the Polish Carpathians, Ukraine and Russia where only eastern haplotypes occurred. While the latter may actually reflect the natural northward expansion of the eastern lineage after the last ice age, the present distribution of the western lineage in eastern Europe may in large parts be artificial and a result of translocations and reintroduction of red deer into areas where the species became extinct in historical times.     

A new cytochrome b phylogroup of the common vole (Microtus arvalis) endemic to the Balkans and its implications for the evolutionary history of the species

The phylogeographic architecture of the common vole, Microtus arvalis, has been well‐studied using mitochondrial DNA and used to test hypotheses relating to glacial refugia. The distribution of the five described cytochrome b (cyt b) lineages in Europe west of Russia has been interpreted as a consequence of postglacial expansion from both southern and central European refugia. A recently proposed competing model suggests that the ‘cradle’ of the M. arvalis lineages is in western central Europe from where they dispersed in different directions after the Last Glacial Maximum. In the present study, we report a new cyt b lineage of the common vole from the Balkans that is not closely related to any other lineage and whose presence might help resolve these issues of glacial refugia. The Balkan phylogroup occurs along the southern distributional border of M. arvalis in central and eastern Bosnia and Herzegovina, Montenegro, and eastern Serbia. Further north and west in Slovenia, Bosnia and Herzegovina, and Serbia, common voles belong to the previously‐described Eastern lineage, whereas both lineages are sympatric in one site in Bosnia and Herzegovina. The Balkan phylogroup most reasonably occupied a glacial refugium already known for various Balkan endemic species, in contrast to the recently proposed model. South‐east Europe is an absolutely crucial area for understanding the postglacial colonization history of small mammals in Europe and the present study adds to the very few previous detailed phylogeographic studies of this region. © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 100 , 788–796.     

Phylogeography,population genetics and conservation of the European red deer Cervus elaphus

• 1 During the Last Glacial Maximum, European red deer Cervus elaphus occurred in refugia in Iberia/southern France, Italy, the Balkans and the Carpathians. Most of Europe, including large parts of the east and north‐east, is now inhabited by red deer from the western lineage. The eastern lineage is largely confined to south‐eastern Europe; a third lineage comprises Sardo‐Corsican and Barbary red deer.
• 2 Sardo‐Corsican, Barbary and Mesola red deer are genetically unique units. They exhibit low levels of genetic diversity and deserve particular protection, since conservation strategies should target genetic information.
• 3 Hybridization between sika Cervus nippon and red deer occurs rarely, but may lead to extensive introgression, particularly in parts of the British Isles. Further expansion of both species may lead to increased hybridization in continental Europe.
• 4 Although hunting has an impact on red deer gene pools, the main threat today is habitat fragmentation in human‐dominated landscapes. The resulting increase in genetic drift and inbreeding reduces variability in isolated populations and may lead to inbreeding depression. To support vital meta‐populations, migration corridors should be established.

     

Origin of a new Reticulitermes termite (Isoptera, Rhinotermitidae) inferred from mitochondrial and nuclear DNA data

The Holoarctic termite genus Reticulitermes is widely distributed in Europe. A new Reticulitermes species, R. sp. nov, was recently found in France and Italy. Its phylogenetic position was investigated using a 743-bp fragment of mitochondrial 16S rRNA-ND1 genes and 382-bp of the nuclear ITS2 region. Phylogenies for these sequences were estimated by neighbor-joining, maximum-parsimony and maximum-likelihood analysis. The results strongly supported a relationship between R. sp. nov. and the termite species from the eastern Mediterranean area including Reticulitermes balkanensis from the Balkans, Reticulitermes lucifugus from Turkey and Reticulitermes clypeatus from Israel. The hypothesis of a relationship between R. sp. nov. and the Japanese Reticulitermes speratus was rejected by parametric bootstrap. The current distribution of R. sp. nov. could be linked to postglacial colonization routes between Balkan refuge and northern regions.     

Phylogeography of the barbel (Barbus barbus) assessed by mitochondrial DNA variation

Using the phylogeographic framework, we assessed the DNA sequence variation at the mitochondrial cytochrome b gene across the distribution range of the barbel Barbus barbus, a widely distributed European cyprinid. Reciprocal monophyly of non-Mediterranean European and Balkan/Anatolian populations is taken as evidence for a long-term barrier to gene flow, and interpreted as a consequence of survival of the species in two separate refugia during several later glacial cycles. Lack of profound genealogical divergence across Europe from western France to the northwestern Black Sea basin is consistent with recent colonization of this area from a single glacial refuge, which was probably located in the Danube River basin. This may have occurred in two steps: into the Western European river basins during the last interglacial, and throughout the Central European river basins after the last glacial. The populations from the Balkans and Anatolia apparently did not contribute mitochondrial DNA to the post-Pleistocene colonization of non-Mediterranean Europe. Lack of detectable variation within the Balkans/Anatolia is attributed mainly to recent expansion throughout these regions, facilitated by the freshwater conditions and seashore regression in the Black Sea during the Late Pleistocene and Early Holocene.     

Origin and post-glacial dispersal of mitochondrial DNA haplogroups C and D in northern Asia

More than a half of the northern Asian pool of human mitochondrial DNA (mtDNA) is fragmented into a number of subclades of haplogroups C and D, two of the most frequent haplogroups throughout northern, eastern, central Asia and America. While there has been considerable recent progress in studying mitochondrial variation in eastern Asia and America at the complete genome resolution, little comparable data is available for regions such as southern Siberia--the area where most of northern Asian haplogroups, including C and D, likely diversified. This gap in our knowledge causes a serious barrier for progress in understanding the demographic pre-history of northern Eurasia in general. Here we describe the phylogeography of haplogroups C and D in the populations of northern and eastern Asia. We have analyzed 770 samples from haplogroups C and D (174 and 596, respectively) at high resolution, including 182 novel complete mtDNA sequences representing haplogroups C and D (83 and 99, respectively). The present-day variation of haplogroups C and D suggests that these mtDNA clades expanded before the Last Glacial Maximum (LGM), with their oldest lineages being present in the eastern Asia. Unlike in eastern Asia, most of the northern Asian variants of haplogroups C and D began the expansion after the LGM, thus pointing to post-glacial re-colonization of northern Asia. Our results show that both haplogroups were involved in migrations, from eastern Asia and southern Siberia to eastern and northeastern Europe, likely during the middle Holocene.     

Glacial history and colonization of Europe by the blue tit Parus caeruleus

Mitochondrial control region sequences from European populations of the blue tit Parus caeruleus were used to reveal the Pleistocene history and the post-glacial recolonization of Europe by the species. The southern subspecies, P. c. ogliastrae was found to represent a stable population with isolation-by-distance structure harboring a lot of genetic variation, and the northern subspecies P. c. caeruleus a recently bottlenecked and expanded population. We suggest that after the last Ice Ages, the subspecies have colonized Europe from two different southern refuges following previously proposed general recolonization routes from the Balkans to northern and Central Europe, and from the Iberian Peninsula north- and eastwards. The two subspecies form a wide secondary contact zone extending from southern Spain to southern France.     

Genetic structure of the paternal lineage of the Roma people

According to written sources, Roma (Romanies, Gypsies) arrived in the Balkans around 1,000 years ago from India and have subsequently spread through several parts of Europe. Genetic data, particularly from the Y chromosome, have supported this model, and can potentially refine it. We now provide an analysis of Y-chromosomal markers from five Roma and two non-Roma populations (N = 787) in order to investigate the genetic relatedness of the Roma population groups to one another, and to gain further understanding of their likely Indian origins, the genetic contribution of non-Roma males to the Roma populations, and the early history of their splits and migrations in Europe. The two main sources of the Roma paternal gene pool were identified as South Asian and European. The reduced diversity and expansion of H1a-M82 lineages in all Roma groups imply shared descent from a single paternal ancestor in the Indian subcontinent. The Roma paternal gene pool also contains a specific subset of E1b1b1a-M78 and J2a2-M67 lineages, implying admixture during early settlement in the Balkans and the subsequent influx into the Carpathian Basin. Additional admixture, evident in the low and moderate frequencies of typical European haplogroups I1-M253, I2a-P37.2, I2b-M223, R1b1-P25, and R1a1-M198, has occurred in a more population-specific manner.     

Phylogeography and Pleistocene refugia of the Little Owl Athene noctua inferred from mtDNA sequence data

Pleistocene glaciations greatly affected the distribution of genetic diversity in animal populations. The Little Owl is widely distributed in temperate regions and could have survived the last glaciations in southern refugia. To describe the phylogeographical structure of European populations, we sequenced the mitochondrial cytochrome c oxidase I (COI) and control region (CR1) in 326 individuals sampled from 22 locations. Phylogenetic analyses of COI identified two deeply divergent clades: a western haplogroup distributed in western and northwestern Europe, and an eastern haplogroup distributed in southeastern Europe. Faster evolving CR1 sequences supported the divergence between these two main clades, and identified three subgroups within the eastern clade: Balkan, southern Italian and Sardinian. Divergence times estimated from COI with fossil calibrations indicate that the western and eastern haplogroups split 2.01–1.71 Mya. Slightly different times for splits were found using the standard 2% rate and 7.3% mtDNA neutral substitution rate. CR1 sequences dated the origin of endemic Sardinian haplotypes at 1.04–0.26 Mya and the split between southern Italian and Balkan haplogroups at 0.72–0.21 Mya, coincident with the onset of two Pleistocene glaciations. Admixture of mtDNA haplotypes was detected in northern Italy and in central Europe. These findings support a model of southern Mediterranean and Balkan refugia, with postglacial expansion and secondary contacts for Little Owl populations. Central and northern Europe was predominantly recolonized by Little Owls from Iberia, whereas expansion out of the Balkans was more limited. Northward expansion of the Italian haplogroup was probably prevented by the Alps, and the Sardinian haplotypes remained confined to the island. Results showed a clear genetic pattern differentiating putative subspecies. Genetic distances between haplogroups were comparable with those recorded between different avian species.