Neolithic mitochondrial haplogroup H genomes and the genetic origins of Europeans

Abstract

Haplogroup H dominates present-day Western European mitochondrial DNA variability (>40%), yet was less common (~19%) among Early Neolithic farmers (~5450 BC) and virtually absent in Mesolithic hunter-gatherers. Here we investigate this major component of the maternal population history of modern Europeans and sequence 39 complete haplogroup H mitochondrial genomes from ancient human remains. We then compare this 'real-time' genetic data with cultural changes taking place between the Early Neolithic (~5450 BC) and Bronze Age (~2200 BC) in Central Europe. Our results reveal that the current diversity and distribution of haplogroup H were largely established by the Mid Neolithic (~4000 BC), but with substantial genetic contributions from subsequent pan-European cultures such as the Bell Beakers expanding out of Iberia in the Late Neolithic (~2800 BC). Dated haplogroup H genomes allow us to reconstruct the recent evolutionary history of haplogroup H and reveal a mutation rate 45% higher than current estimates for human mitochondria.

Figure 1. Mitochondrial haplogroup H sequence evolution

(a) Phylogenetic network of 39 prehistoric mitochondrial genomes sorted into two temporal groupings: Early Neolithic (left) and Mid-to-Late Neolithic (right). Node colours represent archaeological cultures. (b) A Bayesian skyride plot of 200 representative present-day and 39 ancient hg H mt genomes (the thick red line denotes the posterior median, thinner flanking lines denote the 95% credibility interval; note the logarithmic scale of the y-axis). Prehistoric samples (18 radiocarbon and 21 mean archaeological dates) served as internal calibration points (black bars). For comparison, census size estimates for the European population are shown as orange dots. Population density estimates from the archaeological record for key periods in Central Europe are plotted as blue squares in chronological order: LBK, Iron Age, Roman period, Merovingian, and Pre-industrial modern times (y-axis on the right).

Figure 2. Population affinities of select Neolithic cultures

(a) PCA biplot based on the frequencies of 15 hg H sub-haplogroups (component loadings) from 37 present-day Western Eurasian and three ancient populations (light blue: Western Europe; dark blue: Central and Eastern Europe; orange; Near East, Caucasus, and Anatolia; and pink: ancient samples). Populations are abbreviated as follows: GAL, Galicia; CNT, Cantabria; CAT, Catalonia; GAS, Galicia/Asturia; CAN, Cantabria2; POT, Potes; PAS, Pasiegos; VIZ, Vizcaya; GUI, Guipuzcoa; BMI, Basques; IPNE, Iberian Peninsula Northeast; TUR, Turkey; ARM, Armenia; GEO, Georgia; NWC, Northwest Caucasus; DAG, Dagestan; OSS, Ossetia; SYR, Syria; LBN, Lebanon; JOR, Jordan; ARB, Arabian Peninsula; ARE, Arabian Peninsula2; KBK, Karachay-Balkaria; MKD, Macedonia; VUR, Volga-Ural region; FIN, Finland; EST, Estonia; ESV, Eastern Slavs; SVK, Slovakia; FRA, France; BLK, Balkans; DEU, Germany; AUT, Austria, ROU, Romania; FRM, France Normandy; WIS, Western Isles; CZE, Czech Republic; LBK, Linear pottery culture; BBC, Bell Beaker culture; MNE, Middle Neolithic. (b) Procrustes analyses of geographic coordinates and PCA scores of the same dataset (similarity score t₀ = 0.733, p < 10⁻⁶, 100 000 permutations). (c) Ward clustering dendrogram of the three ancient groups and present-day populations (colour code as above and p values in % of approximately unbiased bootstrapping for the following three main clusters). (d) Results of the model-based test to identify the number of clusters by the model with the highest support (highest BIC; VEV= multivariate mixture model (ellipsoidal, equal shape)).

Figure 3. Schematic representation of experimental steps

(a) Probe DNA was prepared by amplifying a complete mitochondrial genome in two overlapping fragments by long-range PCR, followed by DNA fragmentation and biotinylation to form mtDNA ‘baits’ for targeted hybridisation. (b) Ancient DNA was enzymatically blunt-ended and phosphorylated, ligated to custom library adapters, followed by polymerase ‘fill-in’ to create ‘immortalised’ double-stranded DNA libraries. (c) Hybridisation-based DNA-capture using biotinylated probe bound to Streptavidin magnetic beads; following stringency washes, captured library constructs enriched in mtDNA sequences are eluted from the beads/probe via a novel polymerase strand-displacement reaction followed by PCR library reamplification. These steps can be carried out iteratively to maximise mtDNA content in enriched libraries (see Supplementary Methods for full details).