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Received Date : 03-Mar-2014 Revised Date : 22-May-2014 Accepted Date : 23-May-2014 Article type

: Original Article

Adaptive admixture in the West African bovine hybrid zone: insight from the Borgou population

Authors and Affiliations

Laurence FLORI1,2,*,§, Sophie THEVENON1, *,§, Guiguigbaza-Kossigan DAYO3,

Marcel

SENOU4, Souleymane SYLLA3, David BERTHIER1, Katayoun MOAZAMI-GOUDARZI2, Mathieu GAUTIER5 1

CIRAD, UMR INTERTRYP, F34398 Montpellier, France

2

INRA, UMR 1313 GABI, F78350 Jouy-en-Josas, France

3

CIRDES, BP 454 Bobo-Dioulasso, Burkina Faso

4

Faculté des Sciences Agronomiques, 06 BP 14 09 Cotonou, Bénin

5

INRA, UMR CBGP (INRA/CIRAD/IRD/Supagro), F34988 Montferrier-sur-Lez, France

*

These authors contributed equally to this work

§

Corresponding authors

This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/mec.12816 This article is protected by copyright. All rights reserved.

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Keywords: hybrid zone, adaptation, cattle, admixture, footprints of selection

Corresponding Authors: Laurence FLORI and Sophie THEVENON Address:

CIRAD, UMR INTERTRYP, TA A 17/G Campus International de Baillarguet F34398 Montpellier cedex 5, France

Fax : +33 (0)4 67 59 37 98 Emails: [email protected]

[email protected]

Running Title: Adaptive admixture in cattle

Abstract Understanding the adaptive response to environmental fluctuations represents a central issue in evolutionary biology. Population admixture between divergent ancestries has often been considered as an efficient short-term adaptation strategy. Cattle populations from the West African Bos taurus x Bos indicus hybrid zone represent a valuable resource to characterize the effect of such adaptive admixture at the genome level. We here provide a detailed assessment of the global and local genome ancestries of the Borgou breed, one of the most representative cattle of this hybrid zone. We analyzed a large data set

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consisting of 38,100 SNPs genotyped on 203 Borgou and 591 individuals representative of all the different cattle ancestries. At the global genomic level, we show that Borgou is a stabilized admixed breed whose origin (130 years ago) traces back to the great African rinderpest pandemic, several centuries after the last admixture events, the West-African zebus originate from (512 years ago). To identify footprints of adaptive admixture, we combined the identification of signatures of selection and the functional annotation of the underlying genes using systems biology tools. The detection of the SILV coat coloration gene likely under artificial selection may be viewed as a validation of our approach. Overall our results suggest that the long-term presence of pathogens and the intermediate environmental conditions are the main acting selective pressures. Our analytical framework can be extended to other model or non-model species to understand the process that shapes the patterns of genetic variability in hybrid zones.

Introduction

In a context of global warming, understanding the adaptive response to short-term climatic fluctuations represents a central issue in evolutionary biology. From the end 16th century onwards, West Africa experienced a well documented trend toward increasing aridity in Sahel and north savannah (Webb, 1995). Hence, today, several major ecological zones are characterized, in particular according to their underlying climatic conditions. The two extremes of the corresponding West African ecosystems consist of the arid and the humid Guinean ecosystems (OCDE, 2009). In West Africa, the distribution of cattle populations is tightly associated to their sub-species origin. Hence West African zebu (Bos indicus) cattle (AFZ), which are transhumant or semitranshumant livestock particularly adapted to hot and dry conditions are mainly found in arid, semi-arid and sub-humid dry zones (CIPEA, 1979). In contrast, West African taurine (Bos This article is protected by copyright. All rights reserved.

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taurus) cattle (AFT) generally belong to sedentary herds that predominate in hot and relatively humid and sub-humid zones. Finally, the so-called West African bovine hybrid zone, one of the four Bos taurus x Bos indicus hybrid zones in Africa (Freeman et al., 2006; Gautier et al., 2009), coincides with intermediate climatic zones (i.e. sub-humid dry and subhumid humid). The differences between AFT and AFZ distributions in West Africa may stem from their distinct history since domestication. Indeed, although the existence of a center of domestication of taurine cattle in Africa and a male-mediated influence of North-African aurochs are still debated (Gifford-Gonzalez, Hanotte, 2011; Stock, Gifford-Gonzalez, 2013), the current held view is that during Neolithic revolution (circa from 10,500 BP to 7,000 BP), taurine (humpless cattle) and zebus (ZEB, humped cattle) were domesticated in two different primary domestication centers located in the Fertile Crescent and in the Indus Valley, respectively (Loftus et al., 1994; Vigne, 2011). Then, successive migration waves of cattle followed movements of breeders at different period in the recent history and contributed to the settlement of cattle all around the world (Payne, Hodges, 1997). In West Africa, at least two main taurine introductions were proposed. Longhorn taurine were likely introduced at earlier period than Shorthorn taurine, present in West Africa by 4,000 BP as suggested by archeological evidences (Payne, Hodges, 1997). Although indicine breeds are supposed to have been introduced in West Africa by 700 AD via East Africa (Hanotte et al., 2002), historical records document a major wave of zebu introduction during the 19th century rinderpest epidemic outbreak (Epstein, 1971; Porter, 1991). The distinct abilities of their original ancestors together with various selective pressures (e.g. climate, food and water availability, pathogens) might have contributed to divergent local adaptation of current AFT and AFZ populations and their association to extreme ecosystems. For instance, the strong incidence of the Animal African Trypanosomoses that are caused by This article is protected by copyright. All rights reserved.

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tsetse-fly-transmitted protozoan parasites of the Trypanosoma genus present in the humid and sub-humid zones has likely promoted the survival of pure AFT which are trypanotolerant (i.e. able to survive and remain productive in enzootic areas) whereas AFZ die from the disease in the absence of treatment (Murray et al., 1984). Similarly, AFT are thought to be less susceptible than AFZ to other infectious (e.g. dermatophilosis) or tick-borne diseases endemic in sub-humid and humid areas (Mattioli et al., 2000). Conversely, AFZ are less susceptible than AFT to rinderpest (Halpin, 1975) and are generally more appreciated by breeders because of their higher zootechnical abilities (milk production, meat and draught power) which make them better suited to mixed crop farming system (Porter, 1991; Tano et al., 2003).

In transitional ecosystems, combining the advantages of both AFZ and AFT ancestral populations may early have represented a valuable strategy for breeders, e.g by making it possible to associate some disease resistance with production traits and size (Tano et al., 2003). Crossing AFT and AFZ has thus become a common practice among breeders in this area and the resulting products are often referred to as the generic Fulani word Méré. As a result, few cattle populations are well delimited as homogeneous populations, i.e. with stabilized ancestry proportions (Porter, 1991). The Borgou cattle breed represents a stabilized West African admixed cattle breeds assumed to be an hybrid between the White Fulani zebu and the (shorthorn) taurine Somba (Porter, 1991). Widespread in Benin where it represents 51% of the cattle population with 760,000 heads (Beninese Ministry of Agriculture, 2002) and in the neighboring regions of Nigeria and Togo, this breed displays intermediate traits between AFT shorthorn and AFZ. Studies based

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on molecular markers further revealed an AFTxAFZ admixture at the genome level (Dayo et al., 2009; Freeman et al., 2004; Gautier et al., 2009; Moazami-Goudarzi et al., 2001). Here, in order to characterize the origin of the West African bovine hybrid zone and the adaptive factors underlying its maintenance, we propose a detailed assessment of global and local ancestries of the admixed Borgou cattle breed using a large sample representative of two distinct regions and two time periods. We estimated the timing of admixture and both AFT and AFZ ancestry proportions in the Borgou breed and in two AFZ breeds using an approach accounting for the genetic drift occurring between the source populations and the current populations used as surrogates to the ancestral source populations (Loh et al., 2013; Patterson et al., 2012). At the local genomic level, we followed the approach proposed by Gautier & Naves (Gautier, Naves, 2011) to identify footprints of adaptive admixture within the Borgou genome. We further performed a detailed functional analysis of the underlying candidate genes under selection to identify putative physiological pathways affected by selective pressures in the West African hybrid zone. For this purpose, we sampled and genotyped 158 Borgou from two distinct locations in Benin on the Illumina BovineSNP50 (Matukumalli et al., 2009) and combined these genotypes to the ones already available for 45 Borgou sampled 12 years ago (Moazami-Goudarzi et al., 2001) and for 591 individuals from 20 European taurine (EUT), AFT, ZEB or admixed cattle breeds (Gautier et al., 2009; Gautier et al., 2010).

Materiel and methods Genotyping data

A total of 158 Borgou individuals selected on phenotypic criteria were newly genotyped on the Illumina BovineSNP50 chip assay v2 (Matukumalli et al., 2009) at INRA Labogena platform (Jouy-En-Josas, France) using standard procedures This article is protected by copyright. All rights reserved.

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(http://www.illumina.com/products/bovine_snp50_whole-genome_genotyping_kits.ilmn). These individuals originated from two Beninese National farms located in Bétécoucou (BOB, n=83) and Okpara (BOO, n=75) about 150 km apart (Figure 1).

We combined this newly generated data with genotypes available from previous studies using the Illumina BovineSNP50 chip assay v1 (Gautier et al., 2009; Gautier et al., 2010; Matukumalli et al., 2009). Details about sample composition, origin and breed name abbreviations are given in Table S1. Briefly, these included i) 45 Borgou (BOR) individuals which were sampled 12 years ago (Gautier et al., 2009; Moazami-Goudarzi et al., 2001) in Okpara (as for BOO), ii) 173 AFT individuals belonging to four different breeds, i.e. Baoulé (BAO), Somba (SOM), Lagune (LAG) and N’Dama (NDA separated in three populations ND1, ND2 and ND3 of different geographical origins), iii) 152 EUT (European Taurine) individuals belonging to six different breeds , i.e. Salers (SAL), Limousin (LMS), Holstein (HOL), Montbéliard (MON), Angus (ANG) and Hereford (HFD), iv)186 ZEB individuals from three Indian or Brazilian pure zebu, i.e. Nelore (NEL), Gir (GIR), Brahman (BRM) and from African zebu, i.e. Fulani zebu (ZFU), Bororo zebu (ZBO), Zebu from Madagascar (ZMA), and v) 80 individuals from four admixed breeds other than Borgou, i.e. Oulmès Zaër (OUL= EUTxAFT), Kuri (KUR=AFTxZEB), Sheko (SHK=AFTxZEB) and Santa Gertrudis (SGT=EUTxZEB). Overall, the two versions of the Illumina BovineSNP50 assay share 49,555 autosomal SNPs based on the bosTau6 UMD3.1 assembly (Liu et al., 2009). Among these, we discarded SNPs that i) displayed a MAF 3 (P 3 for one or both comparisons (AFZ/BORall and AFT/BORall). When several overlapping and contiguous windows were candidates, the chosen one contained the highest peak and the highest proportion of significant SNPs. The candidate regions were then annotated using the UCSC Genome browser (http://genome.ucsc.edu) and the UMD 3.1 assembly. A gene was considered as a candidate if the peak position was located up to 25 kb from its boundaries. Information on candidate genes under selection was collected via the Ensembl genome data basis (http://www.ensembl.org/index.html, Ensembl Biomart release 65 december 2011).

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Following an approach similar to the one described in (Flori et al., 2009; Gautier et al., 2009), functional and network analyses were carried out with Ingenuity Pathway Analysis (IPA) software (Ingenuity®Systems, www.ingenuity.com) using the Ingenuity Pathway Knowledge Base (IPKB). The candidate genes under selection were functionally annotated (Table S7). The top significant functions and diseases were obtained by comparing functions associated with eligible genes (i.e. genes associated with at least one functional annotation in Ingenuity Pathway Knowledge Base (IPKB)) against functions associated with all genes in the IPKB reference set using the right-tailed Fisher's exact test. A network analysis, which searches for interactions known from literature between candidate genes under selection and all other IPKB molecules (genes, gene products or small molecules), was also performed. For each network that contains at most 140 molecules (including candidate genes under selection), a score S was computed based on a right-tailed Fisher exact test for the overrepresentation of candidate genes under selection (S=-log(p-value)). A network was considered as significant when S>3. Because new molecules connected to the candidate genes under selection participate to networks, a whole network functional annotation is generally more informative than the candidate genes under selection annotation alone (Flori et al., 2009; Gautier et al., 2009). The top significant functions and diseases associated with significant networks were also reported (Table S8).

Results Genetic characterization of the Borgou breed As described above (Figure 1), we considered in our study three different Borgou populations representative of two time periods (BOR and BOO were sampled at a 12-years time interval in the Okpara Beninese district) and two different areas (BOB is contemporary with BOO but sampled more southern).

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To assess the genetic structure of the Borgou breed, we analyzed SNP genotyping data for the n=203 sampled Borgou animals (45 BOR, 75 BOO and 83 BOB) together with data available from previous studies for populations representative of the bovine diversity, leading to a large data set comprising 38,100 SNPs genotyped for 794 animals (corresponding to 25 populations from 21 different breeds). We first carried out an individual-based PCA (Figure 3A). The first two axes accounted respectively for 7.77% and 6.15% of the total variation. As already shown (Gautier et al., 2009; Gautier et al., 2010), this first factorial plan highlights the 2-Dimensional global structure of cattle genetic diversity that can be described as a triangle with apexes represented by the cattle breed types, AFT (in green), EUT (in red) and ZEB (in blue and purple). As expected, the newly sampled BOO and BOB were located close to the BOR individuals previously studied (Gautier et al., 2009; Moazami-Goudarzi et al., 2001), on the axis going from ZEB to AFT. However, three clearly outlier individuals were located within the AFZ (ZFU and ZBO West African zebu breeds) cloud.

To further quantify the different ancestry proportions of the Borgou breed, we carried out several unsupervised hierarchical clustering analyses using different number K of predefined clusters. In agreement with the above PCA and previously reported results (Gautier et al., 2009; Gautier et al., 2010), K = 3 distinguished three groups which might be interpreted as representative of EUT (cluster 1 in red regrouping the 6 European breeds with an average ancestry of 0.96), AFT (cluster 2 in green regrouping the 4 West African taurine breeds with an average ancestry of 0.96) and ZEB (cluster 3 in blue regrouping the 3 Indian or Brazilian indicine breeds with an average ancestry of 0.77 (Figure 3B and Table S2). The three Borgou populations BOR, BOO and BOB showed similar levels of individual average of ZEB ancestry (cluster 3), i.e. 0.41 (sd=0.07), 0.42 (sd=0.03) and 0.40 (sd=0.06) respectively, ranging from 0.29 to 0.68. Only the three individuals previously identified displayed clearly higher ZEB ancestry (>0.6) to the rest of the population, similar to the one observed for the ZBO and ZFU breeds (Table S2). These three Borgou animals were thus discarded from further analyses.

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Both PCA and Admixture results confirmed the previously reported admixed origin of the Borgou breed and suggested an overall homogeneity among the three different sampled Borgou populations. Accordingly, the BOR, BOO and BOB showed similar levels of heterozygosities (see Table S1) larger than the ones observed for AFT and ZEB breeds. However, they still remained smaller than the average heterozygosity of EUT breeds, due to the known bias of the BovineSNP50 chip assay designed from EUT (Gautier et al., 2009; Matukumalli et al., 2009). More interestingly, the three pairwise FST (Table S3) between BOO, BOB and BOR were all lower than 0.02 which was smaller than the values obtained for pairs involving other breeds. Only the ZBO-ZFU pair showed similarly low FST, these two populations being closely related and almost not differentiated (Gautier et al., 2009). Finally, the FIS was almost null within each of the BOR, BOO and BOB populations (Table S1) and combining all the Borgou individuals into a single population (BORall) did not suggest any clear pattern of population substructure (FIS =0.0068 for the BORall population).

Inference of the admixture history of the Borgou and West African zebu breeds In order to formally test for admixture in the Borgou population (BORall) and in AFZ breeds (ZFU and ZBO), we relied on the 3-population test (Patterson et al., 2012). This test allows identifying admixture events old as hundreds of generations ago and do not require accurate surrogates for the ancestral populations. The 12 tests which were performed with one AFT reference population (either SOM, BAO, LAG or ND3) and one ZEB reference population (either ZFU, ZBO or GIR) resulted in highly significant negative f3 values thus providing strong evidence in favor of the AFTxZEB admixed origin of the Borgou breed (Table S4). Highly significant negative f3 values were also obtained for both AFZ breeds, using one AFT reference population (SOM, BAO, LAG or ND3) and GIR as ZEB reference population, supporting an AFTxZEB admixture in ZFU and ZBO. We further used the f4 ratio estimation (Patterson et al., 2012) to infer the mixing proportions of the two ancestries. As shown in Figure 2 and Table S5, we considered several reference populations. The estimated proportion of AFT

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ancestry in Borgou breed varies between 0.37 (sd=5.10-3) and 0.64 (sd=4.10-3) (Table S5). Choosing the GIR among the purest available representative of ZEB ancestry, the proportion of AFT ancestry varied between 0.62 (sd=4.10-3) and 0.64 (sd=4.10-3). Moreover, the proportion of AFT ancestry was estimated between 0.39 (sd=6.10-3) and 0.40 (sd=6.10-3) for ZFU breed and between 0.38 (sd=5.10-3) and 0.39 (sd=5.10-3) for ZBO breed. Finally, we dated the Borgou and AFZ admixture using the 2-reference LD-decay test implemented in the ALDER software (Loh et al., 2013). Regarding Borgou breed, the estimated timing of admixture ranged from 18.9 (+/-1.2) to 21.7 (+/-1.3) generations ago, depending on the reference population used (Table 1 and S6). These estimations were thus relatively consistent whatever the AFT or ZEB populations chosen as references. For a given ZEB reference population, the amplitude of the tworeferences weighted LD curve was maximal for LAG and BAO (2.7 x 10-3 to 5.7 x 10-3), suggesting that both breeds are closer to the ancestral AFT population than ND3 and SOM breeds (Loh et al., 2013). Conversely, for a given AFT reference population, the amplitude of the two-references weighted LD curve was maximal for GIR (from 5.4 x 10-3 to 5.7 x 10-3), followed by ZBO (2.6 x 10-3 to 2.8 x 10-3) and ZFU (from 2.5 x 10-3 to 2.7 x 10-3), suggesting that GIR is closer to the ancestral ZEB population than AFZ. This result was consistent with the fact that GIR is one of the purest ZEB populations of our dataset (see PCA and Admixture results). Note that the estimated timing of admixture was equal to 21.7 (+/-1.3) generations ago when considering ND3 and GIR as reference populations (Table 1). In cattle, the generation time is usually assumed to vary from five to seven years (Gautier et al., 2007). Accordingly, the two BOR and BOO populations which were sampled twelve years apart might thus be separated by approximately two generations. When considering each of these two populations separately and ND3 and GIR as surrogates for the AFT and ZEB ancestries in the 2reference LD-decay test implemented in the ALDER (Table 1), the estimated timing of admixture was found equal to 17.8 (+/-1.6) for the BOR (sampled in 1998) and to 21.6 (+/-1.5) generations for the BOO (sampled in 2010). Hence, the shift in timing of admixture between these two populations as

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estimated by ALDER, i.e. 3.8 generations (+/- 2.2), was consistent with the assumed generation time of 5-7 years given the uncertainties associated to the short period of time separating the two samplings, the age of the sampled animals within each of the population and the expected variation associated with breeding practices or environmental conditions. In addition, the estimated timing of admixture from the BOO and the BOB sample was highly similar to the one derived from BORall sample. Overall, assuming a 6 years generation time for cattle and according to these results, the estimated Borgou admixture occurred about 130 years ago (+/- 8 years), corresponding to the late nineteenth century.

Regarding AFZ populations, a consistent estimation of timing of admixture between 81.8 (+/-6.3) and 83.3 (+/-6.4) generations ago for ZFU and between 86.1 (+/-4.4) and 89.9 (+/-4.5) generations ago for ZBO was obtained (Table 1 and S6) with a mean for both breeds around 85.3 +/- 5.5 generations ago. The amplitude of the 2-references weighted LD curves was similar whatever the AFT breed chosen as reference. The date of admixture event for both AFZ populations is then estimated to approximately 511 years ago (+/-33 years) coinciding with the late fifteenth century or the beginning of the sixteenth century.

Identification of footprints of selection and analysis of local ancestry

To identify footprints of selection in the admixed Borgou breed, we followed the approach described in Gautier and Naves relying on EHH-based tests (Gautier, Naves, 2011). We first computed iHS scores within the Borgou breed for each SNPs over the whole genome after pooling haplotypes from the BOR, BOO and BOB populations resulting in a total of n=400 BORall phased haplotypes for each bovine autosome. We further computed Rsb to compare the local extent of haplotype homozygoties

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between the Borgou breed and its different ancestries (AFT and AFZ). For each autosome, haplotypes of both SOM and BAO as representatives of AFT ancestry (n=132) and haplotypes of both ZFU and ZBO as representatives of AFZ (n=146) ancestry were pooled. The Manhattan plots corresponding to the four different tests (iHS within BORall and Rsb for BORallvsAFT, BORallvsAFZ and AFTvsAFZ pairwise comparisons) are given on Figure 4.

According to our somewhat stringent criteria (see Material and Methods), 19, 28, two and 12, candidate regions displayed significant footprints of selection for iHSBORall, RsbBORallvsAFT, Rsb BORallvsAFZ and RsbAFTvsAFZ respectively (see Table 2 and Figure 4). The average estimated SNP local ancestries over these different regions are also given in Table 2. Seven regions (#6, #10, #16, #25, #26, #39 and #40) were found significant both with iHSBORall and RsbBORallvsAFT (the two respective peaks being identical or less than 300kb apart). No other overlaps were found between the regions identified by the three different tests iHSBORall, RsbBORallvsAFT, RsbBORallvsAFZ. The partial overlap between iHS and Rsbbased tests might be explained by the specificities of the methods. Indeed, the iHS-based test has maximal power to detect loci under selection for which the favorable variant has not yet reached fixation (Voight et al., 2006) while the Rsb-based test is designed to detect selective sweeps that resulted in complete fixation of the beneficial allele in one population (Tang et al., 2007). In the present case, more regions were identified with the RsbBORallvsAFT test than with the RsbBORallvsAFZ. The loss of some Rsb signals in particular for the comparison between BORall and AFZ could be explained by the selection of the favorable haplotypes underlying the candidate regions in the AFZ population they originate from. Contributing to this hypothesis, regions #6, #9, #13 and #26 displayed significant RsbBORallvsAFT and RsbAFTvsAFZ scores and rather an indicine ancestry (positive mean ΔAFZ) provided by admixture analysis (Table 2).

Although no region displayed a significant signal associated to a local excess of AFT or AFZ contributions, the mean ΔAFZ suggested an excess or deficiency of AFZ ancestry for each region and contributed to the interpretation of the selection footprints. Hence, on the 42 identified regions, five

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showed mean ΔAFZ above 0.1 (#6, #26, #27, #29 and #39) and two below -0.1 (#11 and #23). Although not significant, these signals would sign a trend towards an excess of AFZ haplotypes. The five strongest signals were observed in regions #6 (RsbBORallvsAFT and iHSBORall), #26 (RsbBORallvsAFT), #27 (RsbBORallvsAFT) and #14 (RsbBORallvsAFT) with rather an excess of AFZ ancestry.

Functional annotation of the candidate regions

Among the 42 candidate regions identified above, 33 contained at least one known candidate genes under selection according to our criteria (see Material and Methods). For instance, the five strongest signals of the EHH-based tests obtained in four regions (#6, #26, #27 and #14) identified candidate genes under selection encoded i) the phosphodiesterase 1B, calmodulin-dependent (PDE1B), ii) the β-1,3-glucosyltransferase (B3GALTL), iii) the calcineurin B homologous protein 2 (CHP2) and iv) the uncharacterized protein KIAA1211 (Table 2).

To provide a more comprehensive picture of the functions underlying the candidate genes under selection, we performed a gene-network based annotation using the IPA software. Interestingly, 34 of 35 candidate genes under selection identified belonged to a single significant network (score=81; Figure 5) involved in three main groups of IPA function categories (i.e. Diseases and Disorders, Molecular and Cellular Functions and Physiological System Development and Functions). These functions are detailed in Table S8. Among the five most significant scores in each functional group, the presence of i) Developmental Disorder, Organismal Injury and abnormalities, Hereditary Disorder, Embryonic, Organ, Organismal and Tissue Development, ii) Immune Cell Trafficking, Inflammatory Response, Hematological System Development and Function, iii) Neurological Disease and Visual System Development and Function, iv) Reproductive System development and Function and v) Cell-to-Cell Signaling and Interaction, Gene Expression, RNA Damage and Repair, was emphasized.

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Discussion

In this study, we aimed at investigating in more details the origin of the West African AFTxZEB bovine hybrid zone and the environmental and adaptive factors underlying its maintenance. Among the different populations living in the area, the Borgou breed appeared as the most valuable population since its description as an admixed and homogeneous breed was early proposed based on phenotypic information (Epstein, 1971) and was further confirmed with molecular data (Dayo et al., 2009; Freeman et al., 2004; Gautier et al., 2009; Moazami-Goudarzi et al., 2001).

Here, we confirm and extend these previous results by analyzing a large sample (n=203) of Borgou individuals representative of two different areas and two different time periods and genotyped on about 40K SNPs. Importantly, the absence of any population substructure within the whole sample provides strong evidence in favor of the supposedly stabilized status of the Borgou population. Using f3 and f4-based approaches proposed by Patterson and colleagues that make explicit inferences about history by fitting phylogenetic tree-based models to genetic data (Patterson et al., 2012), we provide a clear evidence for the admixed status between two ancestral populations representative of AFT and ZEB ancestries of i) the Borgou breed (60% AFT and 40% ZEB) and of ii) the two AFZ breeds (ZFU and ZBO), which both share similar ancestry proportions (40% AFT and 60% ZEB).

Among the key questions that remained to be addressed, were the origins of these admixture events. Although, it is well documented that zebus moved southward during previous centuries due to the increasing aridity in West Africa (Webb, 1995), no study provided genetic evidence for the impact of this movement of populations on the origin of the West African hybrid zone. In addition, we actually do not know if pure zebus or zebus already admixed with AFT were among the source populations involved in the genesis of the Borgou breed. Based on the 2-reference LD weighted approach as implemented in ALDER, we here demonstrate that the admixture occurred about 130 years ago (i.e.

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at the late 1880s) in the Borgou breed. This period is concomitant to the great rinderpest pandemic that started in Erytrea in 1887, after the introduction of infected Indian zebus from Aden or Bombay by Italians and that rapidly spread over the whole continent over a ten-year period (Mack, 1970; Pastoret et al., 2006). Thereby, the plague was first reported in West Africa in the years 1890-1892 and caused over 90 % mortality in African cattle population leading to devastating effect for African agriculture. Nevertheless, differential mortality might have affected taurine and zebu cattle since the latter are considered as less susceptible to rinderpest (Halpin, 1975). Overall our results thus suggest that the rinderpest epidemic was a key determinant for the origin of the West African hybrid zone.

Interestingly, the admixture events leading to the formation of the ZFU and ZBO populations (West African zebus) were found older by several hundred years (about 500 years BP), thereby refining knowledge about the introgression of zebu in West Africa. Indeed, following the major influx of zebu after the Arab invasion of Egypt (3500 BP), it was previously proposed a gradual introduction of zebus in West Africa during the last 1600 years (Payne, Hodges, 1997). More importantly, our results suggest that admixed AFZ populations were already present before the rinderpest in West Africa although this event is sometimes considered as a major cause of zebu dispersal in West Africa (Gifford-Gonzalez, Hanotte, 2011). We can thus hypothesize that the ZEB ancestry in the Borgou likely originates from already admixed AFZ populations (rather than pure ZEB populations) living northern to the hybrid zone. This is in agreement by the concordance of ALDER results when choosing either an AFZ or a pure ZEB population as a surrogate to the ZEB ancestry. Conversely, the main AFT ancestry might originate from pure AFT populations living southern to the hybrid zone. These populations were presumably maintained (and probably protected from the rinderpest pandemic) thanks to their better ability to cope with environmental pressures such as the Trypanosoma sp. pressure which limited in turn their contact with zebu populations (the main vector of the rinderpest virus).

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To provide insights into the possible alternative pressures maintaining the bovine hybrid zones, we thus took a population functional genomics approach consisting beforehand in scanning the BOR genome for footprints of selection and functionally annotating the underlying candidate genes under selection.

Among the 35 known candidate genes under selection underlying the 42 footprints of selection that could be identified according to the defined criteria (see Material and Methods), 34 were involved in a single gene network with several main nodes (Figure 5). The main functions associated with this network (Table S8) are related to immune response and hematopoiesis (e.g. IPA functions “Immune Cell Trafficking”, “Inflammatory Response” and “Hematological System Development and Function”), tissue and organ development (e.g. IPA functions “Developmental Disorder”, “Organismal Injury and Abnormalities”, “ Embryonic Development”, “Organ development” and “Organismal Development”), neurology and vision (e.g. IPA functions “Neurological Disease” and “Visual System Development and Function”), hair and skin properties (e.g. IPA function “Hair and Skin Development and Function”) and reproduction (e.g. IPA function “Reproductive System Disease”). Some of these functions have already been highlighted in previous whole genome scan for footprints of selection signatures in tropical cattle (Gautier et al., 2009; Gautier, Naves, 2011). It is tempting to speculate that such functions might be directly associated with obvious selective pressures that have been exerted in the West African hybrid zone: in particular i) the long-term presence of pathogens such as Trypanosoma sp. or the occasional presence of some virulent pathogens such as Rinderpest virus, leading to the selection of genes involved in immune response and hematopoiesis, ii) the intermediate climatic and environmental conditions (in terms of temperature, thermal amplitude, solar radiation or feeding conditions) targeting genes involved in vision, neurology, reproduction and hair and skin properties. In addition to such natural selective pressures, artificial selection on coat color performed by breeders could also explained the “Hair and Skin development and Function” IPA annotation.

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Moreover, artificial selection on zootechnical performances (i.e. related to growth) could contribute to the selection of genes involved in organ and tissue development.

When looking more specifically at individual candidate genes under selection, it appears that some genes are involved in several physiological processes (e.g. PDE1B), in fundamental cellular processes (e.g. LRRK2, NEDD4, MAPKAPK2 ) or affect numerous tissues by their key role in embryonic development such as B3GALTL (Lesnik Oberstein et al., 2006), emphasizing the importance of multifunctionality in production under tropical environment. Likewise, some candidate genes under selection might deserve to be investigated in more details (e.g. via a reverse genetics approach).

First, some candidate genes under selection (i.e. PDE1B, BOLA-A, TICAM1, PRDM16 and the uncharacterized protein KIAA1211) are located in regions under selection previously detected in other studies focusing on West African cattle breeds (Gautier et al., 2009) and/or tropical cattle breeds derived from the same ancestral populations (Gautier, Naves, 2011) or European dairy breeds (Flori et al., 2009). The first gene, PDE1B (#6) contains the SNP showing the highest score for RsbBORallvsAFT and encodes a phosphodiesterase that catalyses hydrolyse of second messengers mediating intracellular signal transduction, i.e. cAMP and cGMP, which are thus key regulators of many physiological processes. Indeed, PDE1B is involved in neurological function (Siuciak et al., 2007) and immune response (Bender et al., 2005; Hurwitz et al., 1990). It may also play a role in metabolism as suggested by the PDE1B associations with carcass fat in taurine breeds (Stone et al., 2005). The second gene, BOLA-A (#38) encodes a protein belonging to the Major Histocompatibility Complex (MHC), involved in antigen presentation to T cells and in the initiation of adaptive immune response. The MHC region has been shown under balancing selection in several species and the action of pathogens has been identified as one of the major selective forces that shape MHC diversity (Hedrick, 2013). The third gene, TICAM1 (#16) encodes a Toll like receptor adapter that plays a role in innate immunity and possesses in particular a high type I IFN inducing activity during viral infection.

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Interestingly, TICAM1 is located within a trypanotolerance QTL mapping to BTA 7 chromosome which was identified on a Zebu Boran × N'Dama F2 cross (Hanotte et al., 2003). Further, some functional polymorphisms that could affect the response to Trypanosoma infection were also detected within this gene (Noyes et al., 2011), making it a strong candidate. Finally, PRDM16 (#34) encodes a zinc finger transcription factor mainly involved in hematopoiesis and metabolism (Aguilo et al., 2011). Polymorphisms in PRDM16 were in particular associated to body weight and average daily weight gain in Chinese cattle breeds (Han et al., 2012; Wang et al., 2012) while some results showed an involvement of PRDM16 in thermotolerance through its important role in determination of brown fat cells development (Fruhbeck et al., 2009; Smith et al., 2004).

Among the newly detected candidate genes under selection, NCOA2 (#31), which encodes the nuclear receptor coactivator 2, involved in the control of energy metabolism, is located within a QTL for residual feed intake and body weight in cattle (Pryce et al., 2012). In addition, it participates to a gene network controlling puberty in tropical cattle breeds (Fortes et al., 2011).

Finally, the identification of SILV (#8) might be viewed as a validation of our approach. Indeed SILV encodes a type I integral membrane protein in the pre-melanosome matrix (PMEL17), essential for melanosome development and is responsible for lightening or diluting the base color defined by the Extension locus (MC1R) in some cattle breeds (Kuhn, Weikard, 2007; Schmutz, Dreger, 2013). Accordingly, Borgou cattle are characterized by a uniform white to cream coat which is presumably the result of artificial selection by breeders. Given the color pattern of the current breeds representative of the various ancestries, the underlying variant might originate from the ZEB ancestry, which is in agreement with the positive ΔAFZ we observed, and might be fixed or close to fixation in Borgou breed.

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In the present study, we showed that Borgou breed is a stabilized AFTxAFZ admixed population and a relevant representative of the West African bovine hybrid zone. We also demonstrated that the origin of West African hybrid zone is intimately related to the great African rinderpest pandemic of the 1880s and proposed that its maintenance might be associated to several selective pressures such as the limited availability of food and water, the presence of pathogens one of them being Trypanosoma sp. and more generally the intermediate climatic and environmental conditions. Using systems biology tools, we identified several candidate genes under selection in which involvement in adaptive mechanisms needs to be deeply investigated and validated in model species through a reverse genetics approach. Such study paves the way towards identifying relevant phenotypes responding to adaptive constraints (e.g. immune parameters, coat color, daily weight gain, docility phenotypes and fertility) in experimental herds. More generally, our study that could be extended to other model or non model species illustrates how dense SNP genotyping data are relevant to better understand the process that shapes the patterns of both global and local genome variability in hybrid zones.

Acknowledgements The authors wish to thank Beninese breeders, Dr F.Z. Touré and Dr Saliou Alimy, directors of the two Beninese stations (Okpara and Bétécoucou) supported by the PAFILAV/Benin project and also Dr Faustin Fagbohoun (LABOVET, Bénin) for their participation to the present study. We also wish to thank Stephanie Lapeyre (CIRDES, Burkina Faso) and Isabelle Chantal (UMR INTERTRYP, CIRAD, France) for their contribution to field sampling and lab experiment and Guy Noë and Marie-Noël Rossignol (Labogena, INRA, France) for providing support in genotyping. This work was supported by the FRB AAP Innovative Projects grant 2009 (BORADMIX project AAP-IN-2009-010).

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Data accessibility

SNP genotyping data were deposited at DRYAD (doi:10.5061/dryad.281f2; Flori et al., 2014).

Author contributions MG conceived and designed the experiments. LF and MG performed genetic and statistical analyses. LF, ST and KMG performed functional analyses of candidate genes under selection. ST and DB organized sampling with GKD and MS, who were responsible for sampling in Beninese stations. SS participated to sampling and performed laboratory work. LF, ST and MG drafted the manuscript. All authors read and approved the final manuscript.

Figure Legends Figure 1. Map of the agro-climatic zones in West Africa The DIVA-GIS software version 7.5 (Hijmans et al., 2001) with the BIOCLIM system (Busby, 1991) was used to obtain the map. The different agro-climatic zones were defined based on the annual precipitation of the Worldclim database (http://www.worldclim.org) and according to the OECD 2009 criteria (OCDE, 2009). AFT breeds (ND3, BAO, SOM and LAG), AFZ breeds (ZFU and ZBO) and AFTxAFZ

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Borgou populations (BOR, BOO, BOB) used to infer admixture history of the Borgou breed are positioned on the map and are shown in red, grey and blue, respectively. Figure 2. Phylogeny used for the f4 ratio estimation according to (Patterson et al., 2012). The different cattle breeds used to estimate the f4-ratio are listed. Bison bison was chosen as outgroup. Figure 3. PCA and unsupervised hierarchical clustering results. A. PCA results. The individuals according to their coordinates are plotted on the first two principal components. Ellipses characterize the dispersion of each breed around its center of gravity. B. Unsupervised hierarchical clustering results of the 791 individuals genotyped for 38100 SNPs with an inferred number of clusters k=3 obtained with Admixture 1.04. For each individual, the proportions of each cluster (y axis) which were interpreted as representative of EUT, AFT and ZEB ancestries (see Results) are plotted in red, green and blue, respectively.

Figure 4. Plots over the genome of the BORall piHS(a) and BORallvsAFT (b), BORallvsAFZ (c), AFTvsAFZ (d) pRsb scores for each SNP (see Materials and methods).

Figure 5. Significant gene network obtained with IPA (S=54) including all the candidate genes under selection.

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Tables Table 1. Admixture dates estimated with ALDER software using ND3 and GIR as surrogates to the AFT and AFZ source populations. For each target population, the i) p-value estimated for admixture using the 2-reference data, ii) 2reference z-score for the amplitude and decay rate being significantly non zero, iii) 2-reference decay rate, corresponding to the number of generations since admixture and iv) 2-reference amplitude related to mixture proportions and branch lengths (Loh et al., 2013).

Target

2-reference

2-reference

2-reference amplitude

z-score

decay rate

(x10 )

p-value population

-3

BORall

7.7e-58

16.0

21.7 +/- 1.3

5.6 +/- 0 .2

BOR

3.1e-27

10.8

17.8 +/- 1.6

5.1 +/- 0.1

BOB

1.3e-32

11.9

24.4 +/- 2.0

5.8 +/- 0.2

BOO

4.3e-47

14.4

21.6 +/- 1.5

5.4 +/- 0.2

BOO+BOB

7.2e-54

15.4

23.0 +/- 1.4

5.7 +/- 0.2

ZFU

2.8e-38

12.0

82.7+/-6.3

6.5+/- 0.4

ZBO

9.1E-58

16.0

87.6+/- 4.7

6.9+/-0.4

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Table 2. Description of the regions under selection according to iHSBORall, RsbBORallvsAFT, RsbBORallvsAFZ and RsbAFTvsAFZ For each region, the position (in Mb) on the UMD3.1 bovine assembly with in brackets its total number of SNPs and the number of SNPs with a MAF>0.01 for which iHS was computed) are given. For iHS and Rsb tests, the peak position, the peak log (1/P) value where P represents the P-value of the corresponding score (see Material and Methods) and the number of SNP in brackets if any with log(1/P)>3. SNP average ΔAFZ (and min ΔAFZ/max ΔAFZ) were also reported. We indicate the closest gene (