Old wild wolves: ancient DNA survey unveils population dynamics in Late Pleistocene and Holocene Italian remains

Abstract

Background: The contemporary Italian wolf (Canis lupus italicus) represents a case of morphological and genetic uniqueness. Today, Italian wolves are also the only documented population to fall exclusively within the mitochondrial haplogroup 2, which was the most diffused across Eurasian and North American wolves during the Late Pleistocene. However, the dynamics leading to such distinctiveness are still debated.

Methods: In order to shed light on the ancient genetic variability of this wolf population and on the origin of its current diversity, we collected 19 Late Pleistocene-Holocene samples from northern Italy, which we analyzed at a short portion of the hypervariable region 1 of the mitochondrial DNA, highly informative for wolf and dog phylogenetic analyses.

Results: Four out of the six detected haplotypes matched the ones found in ancient wolves from northern Europe and Beringia, or in modern European and Chinese wolves, and appeared closely related to the two haplotypes currently found in Italian wolves. The haplotype of two Late Pleistocene samples matched with primitive and contemporary dog sequences from the canine mitochondrial clade A. All these haplotypes belonged to haplogroup 2. The only exception was a Holocene sample dated 3,250 years ago, affiliated to haplogroup 1.

Discussion: In this study we describe the genetic variability of the most ancient wolf specimens from Italy analyzed so far, providing a preliminary overview of the genetic make-up of the population that inhabited this area from the last glacial maximum to the Middle Age period. Our results endorsed that the genetic diversity carried by the Pleistocene wolves here analyzed showed a strong continuity with other northern Eurasian wolf specimens from the same chronological period. Contrarily, the Holocene samples showed a greater similarity only with modern sequences from Europe and Asia, and the occurrence of an haplogroup 1 haplotype allowed to date back previous finding about its presence in this area. Moreover, the unexpected discovery of a 24,700-year-old sample carrying a haplotype that, from the fragment here obtained, falls within the canine clade A, could represent the oldest evidence in Europe of such dog-rich clade. All these findings suggest complex population dynamics that deserve to be further investigated based on mitochondrial or whole genome sequencing.

Keywords: Ancient DNA; Canid; Canis lupus; Control region; HVR1 variability; Italian wolf; Population genetics; Wolf; mtDNA.

Figure 1. Median-Joining network based on Alignment A (57 bp sequences obtained in this study and modern Eurasian wolf populations).

White circles represent median vectors; black circles correspond to nucleotide mutations. Haplotypes (circles) are colored according to their geographical provenience while the Italian (ancient and modern) haplotypes are represented by two different colors to discern them from the other European haplotypes. Hg2 is represented by the green area on the left, whereas Hg1 is encompassed by the shaded cyan area on the right. The date of each sample is reported in brackets and is intended in years before present (BP).

Figure 2. Median-Joining network based on Alignment B (57 bp) including ancient wolf and dog haplotypes plus the two extant Italian wolf haplotypes.

When not specified the haplotypes depicted are referred to wolves. Diamonds highlight exclusive dog haplotypes; asterisks indicate the haplotypes shared between dogs and wolves and triangles designate the three haplotypes that present sequences with uncertain attribution to dogs or wolves. White circles represent median vectors, black circles correspond to nucleotide mutations. Italian samples analyzed by Verginelli et al. (2005) are also shown in the figure using the pink color. Haplotypes (circles) are colored according to their geographical provenience. Hg1 is represented by the cyan area on the right whereas Hg2 is represented by the green area on the top-left. The date of each sample is reported in brackets and is intended in years before present (BP).

Figure 3. Maximum-likelihood tree based on the Alignment C (330 bp).

Modern wolf haplotypes are represented by the letter W followed by a number and, in some cases, the letter D is placed next to the W to represent a terminal node where there is a shared haplotype between dogs and wolves. Ancient wolf samples are represented in the figure using their country of provenance and the reported age (in italics, BP). Dog Clades are highlighted as: Clade A (red); Clade B (turquoise); Clade C (green); Clade D (purple). Asterisks highlight statistical support when bootstrap values are found in >50% of 1,000 replicates. See Tables S2 and S3 for the list of samples.