Samples and DNA extraction

Hair and tissue were obtained from five wild boar individuals. Specifically, four females and one male (WBMoroc2) were sampled from the Middle Atlas in Morocco. Samples were collected during the 2014 and 2015 hunting seasons. Throughout the study area no special permits were required to legally hunt wild boars, only a general hunting licence. Animals were killed for other purposes and no authors were involved in hunting. Samples were obtained directly from licenced hunters.

Mitochondrial DNA was extracted from hair roots and tissue samples using the material and protocols for DNA isolation of the Invisorb® Spin Forensic Kit (STRATEC Biomedical AG, Berlin).

Mitochondrial DNA amplification and sequencing

Mitochondrial DNA was amplified using the primers and amplification profiles described by Alves et al. [29] (S1 Table). The thermocycling profile was the same for both cytochrome b and the control region: one cycle at 94°C for 2 min, followed by 30 cycles of 94°C for 45 seconds, 55°C for 45 seconds, 72°C for 1 min, and finally an extension step at 72 ºC for 10 min. PCR products were purified and sent to Macrogen (http://www.macrogen.com/eng/) for sequencing. We obtained two complementary fragments for each region, which were assembled using BioEdit 7.2.5 [30].

Y-chromosome amplification and sequencing

We analysed partial sequences of four Y-chromosome regions using the primers and amplification profiles described by Ramírez et al. [21]. These were carried out under the following conditions: 95°C for 10 min and 35 cycles of 94°C for 1 min, annealing temperature (Tm) (S1 Table) for 1 min, 72°C for 1 min, and finally an extension step at 72°C for 15 min [21,27]. PCR products were purified and sent to Macrogen for sequencing.

Mitochondrial DNA analyses

In all, 1,152 base pairs (bp) including the entire cytochrome b, were obtained for the five analysed wild boar samples (GenBank accession numbers: KU664546, KU6645407, KU66454, KU664553 and KU608293) and were aligned with the 358 wild boar sequences available in GenBank (Table A in S2 Table). Bearded pig (Sus barbatus), celebes wild boar (Sus celebensis), philippine warty pig (Sus philippensis) and common warthog (Phacochoerus africanus) were employed as the outgroups. The partial control region gene (995 bp) was amplified for the five analysed wild boar samples (GenBank accession numbers: KU664554—KU664558) and were aligned with the 1,210 sequences from GenBank (Table B in S2 Table). Celebes wild boar (S. celebensis) and common warthog (P. africanus) were employed as outgroups.

The sequences used for the analysis comprised wild boar with a wide geographical distribution, including Europe, Africa, Near East and Asia. These regions represent the geographic areas of interest for our study. From the available GenBank sequences, we selected those from wild boar. We ruled out most sequences from clones, feral pigs, mixed and archaeological specimens, as well as the sequences classified as unverified and predicted. The size of the final dataset to be used for the analyses varied after aligning all the selected sequences and removing the positions that contained gaps and missing data (N).

Sequences were aligned using BioEdit 7.2.5 and ClustalW alignment tool included in this software. The number of haplotypes was calculated using the DnaSP 5.10 software [31]. The cytochrome b haplotypes obtained here were given the code “CB”, and those obtained for the control region were coded as “CR”. The best nucleotide substitution models were selected using jModelTest 2.1.7 [32] under the Bayesian Information Criterion (BIC). The Pairwise genetic distances between sequences were calculated by MEGA6 [33] with 1,000 bootstraps replicates and gamma distribution (shape parameter = 0.5).

Time of divergence (T) was estimated using the molecular clock equation T = K/ (2r) [34], where T = divergence time in years, K = genetic distance and r = rate of nucleotide substitutions. The genetic distance (K) between P. africanus and genus Sus was calculated with the Tamura 3-parameter model and gamma distribution (shape parameter = 0.5) using MEGA6 in both cytochrome b and the control region. We assumed a substitution rate (r) of 1 x 10−⁸ per site per year for cytochrome b. This rate was previously estimated for complete mtDNA [35]. We used a higher substitution rate (r) of 1.37 x 10⁻⁸ per site per year, as estimated by Pesole et al. [36], for the control region in mammals [37,38].

Bayesian phylogenetic trees were constructed using BEAST 1.8.2 [39]. We assumed a strict clock and the coalescent prior with a constant size. Evolutionary parameters were given by jModeltest. At least two independent Markov chain Monte Carlo (MCMC) chains were run for 50 million generations, and parameter values were sampled every 1,000 generations. We examined the results using Tracer 1.6 [40]. We used TreeAnnotator 1.8.2 [39] to obtain the consensus trees. The first 10% of the sampling trees were ruled out as burn-in and the resulting trees were visualized in FigTree 1.4.2 (http://tree.bio.ed.ac.uk/software/figtree/). Two median-joining networks [41] were generated and visualized using Network 5.0.0.1 (http://www.fluxus-engineering.com). For the cythocrome b, with more complex data than the control region in the final dataset, we used the star contraction option and epsilon = 20.

Y-chromosome analyses

We obtained a partial sequence of 543 bp of the amelogenin (AMELY) gene, 421 bp intron 24 of the ubiquitin-specific protease 9 (USP9Y) gene, and two fragments that corresponded the ubiquitously transcribed tetratricopeptide repeat gene (UTY). These two fragments belong to 322 bp intron 1 (UTYin1) and 353 bp intron 9 (UTYin9) (GenBank accession numbers: KU664549—KU664552). These were aligned with 11 wild boar sequences from GenBank (Table C in S2 Table) using BioEdit 7.2.5 and ClustalW.

Cytochrome b analyses

Except for the network (1,030 bp), we used a 897 bp fragment for the analyses that corresponded to the cytochrome b gene. In all, 107 haplotypes were identified from 363 wild boars (S7 Table). The Moroccan and Tunisian samples are included in haplotypes CB9 and CB90 (Table A in S2 Table). CB9 is the commonest haplotype in Europe and is shared by some North African and 33 European (including Italian), four Asian and four wild boars from Near Eastern countries. When focusing on the Iberian Peninsula, we found that on the other side of the Strait of Gibraltar of the 13 Spanish wild boars, 8 were included in this haplotype.

For the phylogenetic analysis, the best model was the Generalised Time-Reversible Evolutionary Model with gamma distribution (GTR + G) [46] for cytochrome b. The Bayesian phylogenetic tree revealed the previously observed clades: E1 (European clade), E2 (Italian clade), NE (Near Eastern clade), A (Asian clade) (Fig 1A). The haplotypes with sequences from Morocco and Tunisia belonged to the European clade (E1).

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Fig 1. Bayesian phylogenetic trees based on mtDNA.

(A) The cytochrome b phylogenetic tree was constructed with 111 haplotypes (897 bp), which represent 363 wild boars and four outgroups. (B) The control region phylogenetic tree was constructed with 188 haplotypes (415 bp), which represented 1,215 wild boars and two outgroups. CEE is the abbreviation for Central and Eastern Europe. In both cases, the numbers above branches indicate posterior values as a percentage (%). Names of sequences correspond to those of the sequences used to represent each haplotype. The haplotype code assigned in this work has been added.

https://doi.org/10.1371/journal.pone.0181929.g001

The pairwise genetic distances between clades ranged from 0.0085 to 0.0138 (Table A in S3 Table). The mean distances between the haplotypes included in the European clade (E1) were calculated (Table B in S3 Table). The MoroccoA haplotype (CB90), exclusive of Africa, differs from the rest by more than CB9, which is the other haplotype with African sequences.

The cytochrome b gene contains six single nucleotide polymorphisms (SNPs) that allow the differentiation between European and Asian haplogroups [26,37,47]. The variable sites are located at positions 873/ 874/ 876/ 879/ 882 and 883 of the sequences from this study, which correspond to positions 15,035/ 15,036/ 15,038/ 15,041/ 15,044 and 15,045 of the pig mtDNA used as a reference (GenBank accession number AJ002189). The Moroccan and Tunisian cytochrome b sequences have European origin (GTGCAG).

In order to understand the genetic diversity in African wild boar populations, variable sites for their sequences were analysed (S5 Table). The cytochrome b sequences (1,047 bp) have seven nucleotide polymorphic sites in the analysed fragment. There are five transitions, one transversion and one deletion.

The time of divergence estimated between genus Sus and P. africanus was 7 million years (between 5.95 and 8.05) for cytochrome b (S6 Table). From the Bayesian phylogenetic tree, it was deduced that the time of isolation between the Asian and the other clades occurred approximately 818,300 years ago. The isolation of the European clade (E1) occurred some 429,500 years ago. The beginning of the isolation of haplotypes found in North Africa took place 63,800 years ago.

Networks were constructed to better visualize the relationships among clades (Fig 2A). We used fewer sequences with more base pairs to better understand existing relationships and to check for possible variations when using a larger segment size (1,030 bp). North African, Near Eastern and European wild boars clustered in the same way in both the network and the Bayesian phylogenetic tree.

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Fig 2. Median-joining networks based on mtDNA.

(A) The cytochrome b network was constructed with 308 wild boar sequences (1,030 bp). (B) The control region network was constructed with 328 wild boar sequences (534 bp).

https://doi.org/10.1371/journal.pone.0181929.g002

Control region analyses

We used a 415 bp fragment for the control region analyses, except for the network (534 bp). We obtained 187 haplotypes from 1,215 wild boar sequences (S7 Table) after aligning our sequences with others available in GenBank. Moroccan wild boar belongs to haplotypes CR1, CR140 and CR141 (Table B in S2 Table). CR1 is the commonest haplotype in Europe and is shared by some Moroccan and 274 European (including Italian), three Asian, one Australian (Oceania) and five wild boars from Near Eastern countries. In the Iberian Peninsula, 53 of the 121 wild boars from Portugal, and 24 of the 41 wild boars from Spain, are included in this haplotype. CR1 corresponds to the so-called CB9 in mtDNA cytochrome b. The Tunisian wild boar belongs to haplotypes CR181 and CR182, Egyptian to CR46 and the Sudanese wild boar to CR164.

For the Bayesian phylogenetic analysis, the best model was the Generalised Time-Reversible Evolutionary Model [46], with invariant sites and gamma distribution (GTR + I + G) for the control region. The Bayesian phylogenetic tree revealed the same clades seen in cytochrome b (Fig 1B). The sequences from Morocco and Tunisia clustered in the European clade (E1), except for the CR182 haplotype from Tunisia. The control region sequences from Egypt (CR46), Sudan (CR164) and Tunisia (CR182) belong to the Near Eastern clade (NE). Egypt shares a haplotype with some sequences from Iran.

The pairwise genetic distances between clades ranged from 0.0168 to 0.0293 (Table A in S4 Table). The mean distances between the haplotypes included in the European clade (E1) (Table B in S4 Table) showed that there was a longer distance between the haplotypes found in Morocco (between CR1 and CR 140/141) than between some Moroccan ones and the Tunisian one (between CR1 and CR181). The higher values in the mtDNA control region distances can be explained by it being a more hypervariable region than cytochrome b.

The analysed partial control region (406 bp) has 12 nucleotide polymorphic sites, including 11 transitions and one deletion (S5 Table).

The time of divergence estimated between genus Sus and P. africanus was 6.75 million years (between 5.51 and 7.99) for the control region. The time of the isolation between the Asian and the other clades occurred approximately 1,234,100 years ago (S6 Table). The time of divergence of the European clade (E1) was 696,000 years ago, and the beginning of the isolation of the haplotypes found in North Africa took place 116,600 years ago, according to the phylogenetic tree.

Finally, we generated the network (Fig 2B). In this case we included only the sequences that belonged to clades E1 (Europe), E2 (Italy) and NE (Near East) to focus on the connections of these groups. The relationships that we found were similar for both the network and the Bayesian phylogenetic tree.

Y-chromosome haplotype

For the Y-chromosome analysis, we sequenced the partial AMELY, USP9Y and UTY (UTYin1 and UTYin9) regions in one male from Morocco (WBMoroc2) in order to compare them with the published sequences of previous studies [21,48,49] and to identify their haplotype. There were three defined haplotypes, and our results showed that, according to Ramírez et al. [21], our sample belonged to the HY2 haplotype.

The role of the Strait of Gibraltar as a marine permeable barrier between Europe and Africa

The results are interesting because it would be more logical for North African wild boars to share their haplotypes with those of Egypt, Sudan, or even with those of the Near East, due to connectivity by land. However, all their haplotypes are European, except for CR182.

According to Manlius and Gautier [57], the so-called wild boars from Sudan are feral pigs, and the modern wild boars from Egypt were probably introduced by humans in the Neolithic period, like sheep and goats. Even so, presence of native wild boar at low densities in the past cannot be ruled out, and natural colonisation through Egypt is logical and supported by the existence of fossils found in North Africa. Accordingly, if the only contacts had been made by land through Near East countries, the African wild boar would form part of the Near Eastern clade, or would at least differ from European populations.

Therefore, isolation between populations from Egypt and North Africa in the past (in the Late Pleistocene) and contacts with the European wild boar across the Strait of Gibraltar, the most likely route, could have had a strong effect on the mtDNA of the African populations. Obviously, we cannot rule out previous contacts to the Late Pleistocene during former glacial periods. The absence of a genetic footprint could be due to the genetic flow not being enough to have endured [49]. The CR182 haplotype found in Tunisia should be the result of either genetic introgression with the Near East wild boar or a trace from the past. Indeed this haplotype is older than the rest by at least 81,000 years.

Contacts across the Strait of Gibraltar could have been possible during glacial periods when sea level fell and it was easier to cross. The Last Glacial Maximum, the maximum extent of glaciation during the Last Glacial period, occurred during the interval between 25,000 and 18,000 years ago [17,19], and the Last Glacial period finished 11,700 years ago. The Late Glacial, the beginning of the modern warm period, began approximately 13,000 years ago [58], but the rise in temperatures was gradual. During cold periods, the currents in the strait would have been minimal or inexistent, which would have facilitated crossing the strait [2].

As for the causes of movements, if they took place approximately from 90,000 onward, it is difficult to know if contacts were made by natural colonisations, by human action, or by a combination of both without having further data [48,49]. This is due to several events occurring at the same time. In addition, it would appear that the South region of the Iberian Peninsula and North Africa form part of a refugial subcentre denominated Atlanto-Mediterranean, from which species could have recolonised areas in North of Europe at the beginning of the post-glacial periods the [5,11,17].

In fact the existence of human contacts across the strait during the period between 12,000 and 10,500 years ago has been proven, which was when sea levels were still rising [13,59]. Since then human contacts have happened. The existence of cattle of one characteristic haplotype of African breeds in the Iberian Bronze Age suggests that contacts over the Strait of Gibraltar were caused by interactions among communities, their culture and livestock in prehistory [14]. Besides, other animals like genet (Genetta genetta), barbary ape (Macaca sylvanus) or egyptian mongoose (Herpestes ichneumon) are accepted as introductions between Europe and North Africa [24]. Movements of people between both continents, who took pigs with them from the 15th century onward on exploratory or commercial routes, and with the consequent genetic introgression, is another possible explanation for similarity [20,21]. In our case it is more likely that migrations occurred naturally. Accessible information suggests that people took domestic varieties of livestock, such as cattle or pigs, but no information on transporting wild boar is available.

Similarly, the arrival of the wild boar from mainland Asia to the Ryukyu islands in the Late Pleistocene seems to be a fact [51]. A much closer geographic proximity between the two regions when sea levels were lower could account for this dispersion.

Finally, a strong gene flow might have been persistent over time, and a population decline or a displacement of the original population, followed by the expansion of new haplotypes, could have occurred. As a result, the African wild boar forms part of the European clade, at least it does according to its mtDNA.

Therefore, we suggest that the Strait of Gibraltar acted as a bridge for wild boar (S. scrofa) to disperse in the Late Pleistocene. In this case, the dispersion of specimens accompanied by a strong gene flow would have been occurred from at least 90,000 years onward. Genetic analyses, the history of S. scrofa, and the fact that the Last Glacial period finished 11,700 years ago, all suggest natural dispersion, but we cannot rule out the contact by human action.