Aberystwyth University

Genetic diversity and phylogenetic analysis of the native mountain of Britain and Ireland reveal a novel rare population Winton, Clare Louise; Hegarty, Matthew John; McMahon, Robert; Slavov, Gancho Trifonu; McEwan, Neil Ross; Davies-Morel, Mina Clare Gwynne; Morgan, Charles Thomas; Powell, Wayne; Nash, Deborah Mary

Published in: Ecology and Evolution DOI: 10.1002/ece3.507 Publication date: 2013 Citation for published version (APA): Winton, C. L., Hegarty, M. J., McMahon, R., Slavov, G. T., McEwan, N. R., Davies-Morel, M. C. G., ... Nash, D. M. (2013). Genetic diversity and phylogenetic analysis of the native mountain ponies of Britain and Ireland reveal a novel rare population. Ecology and Evolution, 3(4), 934–947. https://doi.org/10.1002/ece3.507

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Download date: 03. Oct. 2019 1 2 Genetic diversity and phylogenetic analysis of native 3 4 mountain ponies of Britain and Ireland reveals a novel rare 5 population 6 7 Clare L. Winton*, Matthew J. Hegarty*, Robert McMahon, Gancho T. Slavov, Neil R. McEwan, Mina 8 C.G. Davies-Morel, Charly M. Morgan, Wayne Powell & Deborah M. Nash 9 IBERS, Aberystwyth University, Aberystwyth, Ceredigion, SY23 3DA, UK 10 11 12 13 Keywords Abstract 14 Conservation, microsatellite, mtDNA, 15 The conservation of unique populations of animals is critical in order to pre- phylogenetics, SNP, Welsh . CE: Vinoth 16 serve valuable genetic diversity and, where populations are free-living, maintain 17 Correspondence their irreplaceable influence upon habitat ecology. An accurate assessment of 18 Deborah Nash, Aberystwyth University, IBERS, genetic diversity and structure within and between populations is crucial in 19 Penglais, Aberystwyth, Ceredigion, UK, SY23 order to design and implement conservation strategies in natural and domesti- 20 3DA. cated species. Moreover, where it is possible to identify relic populations that Tel: +44 (0) 1970 622575, 21 are related to a structured breed an ideal opportunity presents itself to model Fax: +44 (0)1970 622350; 22 E-mail: [email protected] processes that reveal historical factors that have shaped genetic diversity. The 23 origins of native UK mountain and moorland ponies are uncertain, but they Dispatch: 6.2.13 Journal: ECE3 24 Received: 24 December 2012; Revised: 22 may have directly descended from prehistoric populations and potentially har- Author Received: No. of pages: 14 PE: Suganya 25 January 2013; Accepted: 23 January 2013 bour specific adaptations to the uplands of Britain and Ireland. To date, there 26 have been no studies of population structure and genetic diversity present 27 doi: 10.1002/ece3.507 within a free-living group of ponies in the Carneddau mountain range of North B 28 Funding Information . Herein, we describe the use of microsatellites and SNPs together with 29 Clare Winton was supported by an IBERS analysis of the mitochondrial control region to quantify the extent and magni- 30 PhD Scholarship (Aberystwyth University) and tude of genetic diversity present in the Carneddau pony and relate this to

31 a Royal Society Research Grant 2010/R2. several recognised British and Irish pony breeds. Our results establish that the 507 32 feral Carneddau ponies represent a unique and distinctive population that mer- 33 *Joint first authorship. its recognition as a defined population and conservation priority. We discuss 34 the implications for conservation of this population as a unique pool of genetic 35 diversity adapted to the British uplands and potentially of particular value in 36 maintaining the biodiversity of these habitats. 37 38 Introduction selecting from a sub-population within an existing breed, 39 to create a group of animals that contain a defined set of 40 An understanding of the genetic characteristics of a popu- desired traits (Goodwin et al. 2008).

41 lation is paramount in order to inform and implement Several definitions of what constitutes a breed exist, Journal Name Manuscript No. ECE3 42 conservation strategies that preserve and maintain genetic and the term is therefore flexible and potentially confus- 43 diversity (Hall and Bradley 1995). This principle applies ing dependent upon the context in which it is used. Clut- 44 to both domestic livestock breeds and wild species in situ. ton-Brock (1987) defines a breed as “a group of animals 45 Bruford et al. (2003) highlighted the complexity of that has been selected by humans to possess uniform 46 domestication and a need to better understand the popu- appearance that is inheritable and distinguishes it from 47 lation dynamics involved to guide livestock conservation other groups of animals within the same species.” Lush 48 and maintain biodiversity resources for the future. and Crow (1994) argues that the term “breed” was cre- 49 Domestication of livestock has resulted in the selection of ated by breeders of livestock prior to the recognition of 50 certain animals according to their suitability for specific genetic selection and is commonly used in lay-situations 51 tasks, leading to post-domestic polychotomy in pheno- according to popular consensus. In practice, certain breed 52 typic appearance within species. Breed development societies will not register an individual within a breed 53 occurs by the mixing of different populations, or by stud book without a pedigree demonstrating several

ª 2013 The Authors. Published by Blackwell Publishing Ltd. This is an open access article under the terms of the Creative 1 Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. Genetic Diversity in UK Native Mountain Ponies C. L. Winton et al.

1 generations of closed matings (Welsh Pony and Soci- 2 ety). Other societies register animals according to both 3 pedigree and morphological assessment of phenotype

4 based upon agreed guidelines (Connemara Society). COLOR 5 Nonetheless, the issue of whether or not a group of indi- 6 viduals can be designated as a certain breed should not 7 detract from the recognition that a particular population, 8 with a distinct genetic profile following substantial isola- 9 tion, may be worthy of conservation. 10 A better understanding of population origin and his- 11 tory of development gained through molecular genetic 12 analysis may provide information on relic populations. 13 Putative genetic links may then be made to ancestral 14 groups from which modern breeds have developed fol- Figure 1. The free-living Carneddau pony in its natural environment © 15 lowing periods of population management (Hall and in north Wales. Photo courtesy: 2012 Osian Rees. 16 Bradley 1995). Molecular approaches have begun to be 17 applied to the genetic examination of feral populations of 18 around the world in comparison to managed regis- morphology, <137.2 cm; Section C is a Welsh Pony of 19 tered breeds that show phenotypic and/or geographical Cob Type, of stocky morphology, <137.2 cm and Section 20 similarities (Vega-Pla et al. 2006; Plante et al. 2007; Ren- D, a Welsh Cob and >137.2 cm (Davies 1993). The term 21 do et al. 2012). The primary aims of these studies were to “Cob” refers to a generically bred animal of stocky mor- 22 identify conservation priorities and guide conservation phology, although in the case of the “Welsh Cob” it refers 23 management schemes. In Britain, there are only a limited to registered animals of a defined phenotype (according 24 number of free-living populations of domesticated live- to Society guidelines). All four sec- 25 stock species (Hall and Moore 1986; Goulding 2001; Wil- tions show phenotypic similarities and are assumed to 26 son 2003; Burthe et al. 2011), and to-date no molecular have arisen from a common ancestral stock similar to the 27 genetic analysis has been conducted upon semi-feral Brit- modern Section A. The Welsh Pony and Cob Society 28 ish native pony breeds with a view to better understand (WPCS) administer a stud book for pedigree documenta- 29 the structure of genetic diversity. tion of all four sections, which has been closed since 30 A free-living equine population known as Carneddau 1950, meaning that offspring resulting from breeding with 31 ponies exist in Snowdonia National Park (Wales) that unregistered animals thereafter may not themselves be 32 have been suggested to be direct descendants of the ances- registered (Davies 2001). 33 tral Welsh Mountain Pony (Fig. 1). The Carneddau No previous studies have examined Carneddau ponies 34 ponies are anecdotally thought to have existed during the in terms of effective population size, degree of inbreeding 35 Roman era and have been implicated as representative of or their molecular relationship to Welsh and other UK 36 the historical foundation of a modern, registered breed, and Ireland native pony breeds. The topography of Wales 37 the Welsh Section A pony. Carneddau ponies are one of the creates natural environmental barriers to gene flow and 38 last remaining collections of free-living ponies in Britain. population migration in the form of mountain ranges 39 Compared to other equine populations maintained on and expanses of water. More specifically, the Carneddau 40 hills or moors, Carneddau ponies are less intensively ponies are limited to the Carneddau mountain range and 41 managed in terms of reproduction, with intervention are believed to have maintained a relatively stable popula- 42 being limited to late autumn removal of a proportion of tion of around 300 breeding animals. Thus, the Carned- 43 offspring for sale. Access to grazing, water and shelter, dau may have evolved or maintained adaptations specific 44 social interactions and general maintenance of these to their upland habitat that have been lost from the 45 mares, and residual young stock are unhindered related ponies during breed formation. We present a 46 by human intervention. The geographically nearest group comprehensive analysis of the genetic diversity within the 47 of registered, traditionally managed animals to the Car- Carneddau ponies relative to other UK native pony 48 neddau population is the Welsh Pony and Cob, a breed breeds from Wales, Ireland and Scotland. The aim is to 49 indigenous to Wales. The Welsh Pony and Cob is sub- establish to what extent they represent a genetically 50 divided into four distinct sections whose defining criteria unique subpopulation formed as a result of long-term 51 are the animal’s adult height and conformation character- geographical isolation within Wales or whether this popu- 52 istics: Section A is a Welsh Mountain Pony, <121.9 cm; lation is a more recent separation from the existing Sec- 53 Section B is a Welsh , of fine-built tion A pony breed.

2 ª 2013 The Authors. Published by Blackwell Publishing Ltd. C. L. Winton et al. Genetic Diversity in UK Native Mountain Ponies ) 1 Materials and Methods 2

3 (SSR/SNP Sample collection and extraction of No. Mark- 4 genomic DNA 5 6 Studies were conducted using animals representative of No. Markers 7 different bloodlines for each of five British-Irish native MTCR haplotype diver- =

8 pony populations. Samples were collected from Welsh h standard deviation,

9 Section A and Section D animals, Connemara ponies = 0.003 17/48,715 0.037 17/48,715 0.029 17/48,716 À À À 0.001 17/48,703 0.004 17/48,710 / / / 10 (native to Ireland) and Highland ponies (native to Scot- /0.086 17/48,327 À À ** ** ** 11 land) from individuals registered with their respective ** 12 breed society stud book (see Table 1 for sample numbers IS (SSR/SNP) 13 and population identifier abbreviations). In each case, F 14 collection from siblings was avoided. Samples were also 15 collected from feral Carneddau ponies during a single

16 annual herd round-up in 2009, although the relationship number of MTCR haplotypes, inbreeding coefficient, SD = e (SSR/SNP) 17 between these source animals was not known. All samples = H H IS 18 were collected as tail hairs or buccal swabs. Total DNA F 19 extraction from hair was performed using the Qiagen â 20 DNeasy Blood and Tissue extraction kit (Qiagen, West 21 Sussex, UK) according to manufacturer’s instructions. o (SSR/SNP)

22 Total DNA from buccal samples were extracted using the H TM â 23 BuccalAmp DNA Extraction Kit (EPICENTRE Bio- 24 technologies, Cambio Ltd., UK). expected heterozygosity,

25 = E MNA (SSR/SNP) 26 H SSR genotyping and data analysis single nucleotide polymorphism, 27 = 28 Microsatellite (Simple Sequence Repeat; SSR) amplifica- â 0.0077 6.65/1.82 0.637/0.266 0.702/0.266 0.1015 0.0077 6.94/1.84 0.659/0.282 0.733/0.272 0.0868 0.0093 7.47/1.86 0.677/0.276 0.726/0.275 0.0536/ 0.0072 6.65/1.91 0.672/0.303 0.748/0.295 0.0922 0.0090 9.53/1.84 0.659/0.278 0.778/0.304 0.0749 29 tion by PCR was performed using the StockMarks 0.0075 6.41/1.80 0.650/0.263 0.723/0.262 0.0320/ Æ Æ Æ Æ Æ 30 equine genotyping kit (Applied Biosystems, Warrington, Æ

31 UK) comprising 17 multiplexed fluorescent dye-labelled SD Æ

32 primers for the microsatellite markers, recommended by p

33 the International Society of Animal Genetics. PCR ampli- observed heterozygosity, =

34 fication used 10 ng of template genomic DNA with O H 35 reagents at concentrations as provided; 2.5 lL Stockmarks simple sequence repeat, SNP 0.025 0.0190 0.0361 0.0146 0.017 0.0179 0.032 0.0137 0.0171 0.018 0.019 0.0142 â = Æ Æ Æ Æ Æ 36 PCR Buffer, 4 lL dNTP mix, 0.5 lL AmpliTaq Gold Æ SD calculations for each population. 37 DNA Polymerase, 4 lL multiplexed primer mix and 3 lL Æ IS

38 water. DNA was amplified in a G-Storm GS-1 thermal F

39 cycler (Gene Technologies Ltd., Essex, UK) and reactions and E Hh

40 incubated at 95°C for 10 min followed by 40 cycles of H ,

° ° ° O

41 95 C for 30 sec, 60 C for 30 sec and 72 C for 60 sec, fol- H mean number of alleles, =

° 0.01.

42 lowed by final extension at 72 C for 60 min. PCR prod- 0.05. <

43 ucts were diluted 1:20 with molecular grade water and P P< 44 subjected to capillary electrophoresis using an ABI 3730

45 DNA Analyzer (Applied Biosystems). Alleles were sized mitochondrial control region, SSR = (MTCR/SSR/SNP) TM 46 1 using the internal size standard GeneScan -500LIZ. Size N 47 analyses of separated DNA fragments were performed 48 with the GeneMapper software version 3.7 (Applied Bio- 49 systems). Alleles were scored based on PCR product size 50 and recorded manually. Allele frequencies were examined Summary statistics for MTCR, SSR and SNP genotyping for each of the five sample groups. MTCR nucleotide diversity, MNA

51 for significant deviation from Hardy-Weinberg expecta- = number of (SSR/SNP) markers used in p sample number, MTCR

52 tions using the exact test in Arlequin 3.5 (Excoffier et al. = Significant deviation from HWE at Significant deviation from HWE at sity, Section D (D) 46/46/12 26 0.950 Carneddau (S) 46/46/12 8 0.788 N= Section A (A) 47/48/12 17 0.932 ers Connemara (N) 46/46/12 19 0.878 Total 225/233/60 53 0.932 Highland (H) 45/39/12 14 0.916 * ** Table 1. 53 2005; Excoffier and Lischer 2010), with Markov chain Population (Identifier)

ª 2013 The Authors. Published by Blackwell Publishing Ltd. 3 Genetic Diversity in UK Native Mountain Ponies C. L. Winton et al.

1 length set to 100,000 following 10,000 dememorization similarity matrix used to establish the relationships 2 steps. Markers that demonstrated highly significant devia- between the horses using UPGMA clustering in MEGA4 3 tions in more than one population and/or contained large and by multidimensional scaling within PLINK. To 4 amounts of missing data values were removed to limit further investigate any higher order patterns within the 5 the influence of allele drop out. Loci with missing data SNP data, STRUCTURE analysis was performed on three 6 unevenly distributed across the populations (>5% per random subsamples of 5000 markers each analysed for 7 breed) were also excluded to limit ascertainment bias. values of K ranging from 2 to 10 (five iterations per K). 8 According to these criteria, five loci were removed from As Alpha values stabilised rapidly, burnin and MCMC 9 the analysis, including two that resulted in null alleles in values were set at 25,000 each to improve computational 10 other pony breeds (Rendo et al. 2012). Individual samples speed. Data from each subsample analysis were 11 with poor quality DNA with calls missing from >3 loci combined and DK statistics calculated using STRUC- 12 were also excluded. To elucidate the relationships between TURE Harvester as for the SSR analysis. Results from 13 the five populations and provide fine quantification of replicate runs at the optimal K were combined in 14 the different ancestral contributions, an unbiased Bayesian CLUMPP (Jakobsson and Rosenberg 2007) and the aver- 15 approach using Markov chain Monte Carlo (MCMC) age Q-table exported to DISTRUCT (Rosenberg 2004) 16 clustering of samples was conducted via the STRUCTURE for graphical presentation. 17 v2.2.3 software (Pritchard et al. 2000) using both SSR 18 and SNP loci. Allele calls were loaded into STRUCTURE Mitochondrial DNA sequencing and analysis 19 as diploid data for each individual and assessed for prior 20 values of K ranging from 2 to 9. Burn-in and MCMC Using a Welsh Cob sequence (GenBank AF072983), the 21 iteration settings were 50,000 and 10,000, respectively. primer pair (Forward - 5′-ATT TCT TCC CCT AAA 22 Allele frequencies were treated as correlated. For each CGA CAA C-3′) and (Reverse - 5′-CGT TCA ATT TAA 23 value of K, three replicate simulations were conducted. GTC CAG CTT C-3′) was designed to a 606 bp amplicon 24 The DK statistic (the second order rate of change in log of the mitochondrial control region (MTCR). PCR ampli- 25 probability between successive values of K) was calculated fications consisted of 10 ng template DNA, 7.5 lL of Im- 26 using STRUCTURE Harvester v0.6.7 (http://taylor0.biol moMix (Bioline, London, UK), 1 lmol/L forward and 27 ogy.ucla.edu/struct_harvest/) as per Evanno et al. (2005). reverse primer and water to a final volume of 15 lL. 28 Results from replicate runs at the optimal K were com- Amplifications were performed in a G-Storm GS-1 ther- 29 bined in CLUMPP (Jakobsson and Rosenberg 2007) and mal cycler at 95°C for 10 min, followed by 35 cycles of 30 the average Q-table exported to DISTRUCT (Rosenberg 95°C for 30 sec, 55°C for 30 sec and 72°C for 60 sec, 31 2004) for graphical presentation. Population summary with final extension at 72°C for 10 min. Reaction prod- â 32 statistics and analysis of molecular variance (AMOVA) ucts were purified using a Multiscreen 96-well filter 33 were calculated using Arlequin 3.5 (Excoffier and Lischer plate (Millipore, Durham, UK) to remove primers and 34 2010). dNTPs then diluted 1:20 with molecular grade water in 35 preparation for sequencing using an ABI 3730 DNA Ana- 36 lyzer (Applied Biosystems) and 1 lmol/L of the forward Single Nucleotide Polymorphism (SNP) 37 primer described above. To place the current data into a genotyping and data analysis 38 wider context against the major worldwide mtDNA 39 A subset of 12 individuals from each group was selected haplogroups, 83 reference horse and pony samples were 40 and 200 ng of genomic DNA was supplied to Central downloaded from GenBank based upon a recent whole 41 Biotechnology Services (Cardiff University) for genotyp- mitochondrial genome study (Achilli et al. 2012) and 42 ing using the Illumina Equine SNP50 assay. Data were included within the data file. Sequence data were trimmed 43 returned as binary allele calls and reformatted for to a common 519 bp sequence and aligned using BioEdit 44 STRUCTURE. Data were filtered for quality control pur- Sequence Alignment Editor v7.0.9.0 (Hall 1999) before 45 poses by removing any loci with >10% missing data and/ being exported in phylip (phy.) format. The package 46 or loci with extreme deviation from HW expectations NETWORK (http://www.fluxus-engineering.com) was 47 (P < 0.001) (determined using PLINK http://pngu.mgh. employed to produce a median-joining network based on 48 harvard.edu/purcell/plink/). After filtering observed het- maximum parsimony to reconstruct the phylogenetic

49 erozygosity (Ho), expected heterozygosity (He) and FIS relationships among haplotypes. Individuals showing 50 values were calculated using PLINK, using between 48,703 unusually long branch lengths as part of the network were 51 and 48,710 SNPs per population (see Table 1). The pro- examined more closely for poor sequence quality. 52 portion of shared alleles identical by state (IBS) between Sequence traces were checked for nucleotide call errors 53 individuals was calculated in PLINK and the resultant and either manually rectified or removed in the case of

4 ª 2013 The Authors. Published by Blackwell Publishing Ltd. C. L. Winton et al. Genetic Diversity in UK Native Mountain Ponies

1 overall low-quality sequence. Population allelic and possibility of allele drop out. Similar results were 2 molecular diversities were compared using Arlequin 3.5 obtained with SNP data again indicating that the Car- 3 (Excoffier and Lischer 2010). neddau had relatively high heterozygosity levels and no 4 evidence of significant inbreeding. 5 Results 6 Population structure and gene flow 7 Population genetic diversity parameters 8 To elucidate the relationships between the five popula- 9 Summary statistics for MTCR, SSR and SNP data are tions and provide finer quantification of the different 10 presented in Table 1. Analysis of MTCR sequence from ancestral contributions, STRUCTURE analysis was con- 11 230 individuals (n = 47 for Section A, n = 46 for Sec- ducted using both SSR and SNP loci. Initial analysis 12 tion D, n = 46 for Connemara, n = 45 for Highland and included a small number of two other Welsh pony 13 n = 46 for Carneddau) (Table 1) identified 54 haplo- groups, Section B and Section C. These two categories 14 types based on 60 variable nucleotide sites. Haplotype are known to be intermediate between Section A and D 15 diversity was high (0.932 Æ 0.0171) with relatively low ponies in terms of height and conformation and in fact 16 nucleotide diversity (0.018 Æ 0.0091). The Carneddau the direct offspring of a Section A crossed with a Section 17 possessed the lowest haplotype diversity of the five D may be registered as a Section C. This is clearly dem- 18 groups (but not the lowest nucleotide diversity), and onstrated within the STRUCTURE plots obtained (Fig. 19 also the lowest number of haplotypes (approximately A1). The partial assignment of most Section B/C animals 20 half that observed in the other groups). For SSRs, 225 to both Section A and Section D clusters confirmed the 21 individuals were successfully genotyped using the Stock- package was effectively detecting cases of known admix- 22 Marks kit (n = 48 for Section A, n = 46 for Section D, ture. The Section B and Section C animals were excluded 23 n = 46 for Connemara, n = 39 for Highland and n = 46 from the main analysis as the sample size available were 24 for Carneddau) (Table 1). The mean number of alleles much fewer than in the samples from the 5 populations 25 per locus (MNA) ranged from 6.4 to 7.5 for the 17 whose results are presented here. STRUCTURE analysis

26 SSRs, with average expected (HE) and observed (HO) of the log likelihood [Ln Pr(X/K)] of the posterior prob- 27 heterozygosity values of 0.778 and 0.659 respectively. In ability of the SSR data for a given K suggested the opti- 28 contrast to the MTCR results, the Carneddau showed mal value of K was 5. The five clusters that were 29 higher expected and observed heterozygosity than both assigned match closely to the populations from which 30 the Highland and the Section D. The Section D samples individuals had been sampled, though all populations

31 possessed the lowest number of alleles, the lowest HE contained individual animals that could not be unambig- 32 and HO and also the highest inbreeding coefficient (FIS), uously assigned to a single cluster (Fig. 2). Cluster analy- 33 demonstrating a clear reduction in heterozygosity proba- sis based on SNP data (Figs. 3, 4) also clearly 34 bly due to non-random mating. Section A showed the demonstrates the separation of individuals into groups

35 highest MNA although Connemara had equal HO and corresponding to their “breed”. The Section A and the 36 displayed higher HE. SNP results, based on 12 individu- Carneddau clusters are generally closest in multidimen- 37 als per population, show roughly similar patterns of sional space and in the hierarchical cluster “tree,” but are 38 diversity with no significant differences in heterozygosity consistently distinct, as demonstrated in the plot of mds 39 between the 5 groups, but with the Connemara ponies dimension 1 against dimension 4 (Fig. 4C). There are a 40 again showing the highest expected heterozygosity few Carneddau individuals who appear genetically dis-

41 (He = 0.342) and the Carneddau the highest observed tinct from the others Carneddau and cluster with the 42 (Ho = 0.338). Global exact tests of HWE performed Section A animals or in an intermediate position between 43 across all loci revealed all five groups deviated signifi- the two populations. Structure analysis of the SNPs 44 cantly (P < 0.01) from HWE indicating a significant favoured 6 clusters matching the five sampled popula- 45 level of structure between the groups. Molecular analysis tions, but with an additional split found mainly within 46 of variation demonstrated highly significant levels of the Section D cobs (Fig. 5A), a subdivision that is also 47 between group variation relative to within group for apparent, though less clearly so, in the mds plots 48 both SSR and MTCR results, with 9% and 85% of total (Fig. 4C). All populations showed significant divergence

49 variation between groups respectively. Individual HW from each other following pairwise FST analysis of SSR 50 tests indicate that consistently significant deviations were and SNP data (Table 2), with the closest relationship 51 found across several of the populations for a few of the found between the Section As and Carneddau. This is 52 SSR markers. These loci were removed from the data set also reflected in Fig. 3, where the Section A and Carned- 53 for later STRUCTURE analysis below, to exclude the dau form a sub grouping within the tree. Interestingly,

ª 2013 The Authors. Published by Blackwell Publishing Ltd. 5 Genetic Diversity in UK Native Mountain Ponies C. L. Winton et al.

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4 COLOR 5 6 7 8 9 10 11 12 13 Figure 2. The results of STRUCTURE analysis for SSR loci of each population for K = 5. STRUCTURE Harvester analysis as per Evanno et al. (2005) 14 suggests true value of K is 5. There is no real further subdivision beyond 5 clusters at K > 5. 15 16 17 for the SSRs, the Section As showed more divergence et al. 2012a), remains relatively high and are comparable 18 from the Section Ds (0.0711) compared to the relation- to values in other European pony breeds, including 19 ship of this group with the other pony breeds, including semi-feral ponies (Solis et al. 2005; Luis et al. 2007; 20 the Irish Connemara (0.0535) and Scottish Highland Rendo et al. 2012). Prystupa et al. (2012b) report lower 21 (0.0658). This finding is also supported by the data from numbers of mitochondrial haplotypes (approximately 22 the SNP analysis, where it is most clearly illustrated by half) and lower haplotype and nucleotide diversity in 23 relative positions of the Section A and Section D animals Canadian populations of the Connemara, Highland and 24 on dimension 1 in the mds plot of dimension 1 versus Welsh Pony than identified here. This perhaps reflects a 25 dimension 4 (Fig. 4C). sampling effect in the founding of those breeds in 26 Table 3 demonstrates that the maternal lineages in the Canada. 27 Section A and Section D animals cannot be distinguished, Reduced MTCR haplotype numbers and significant 28 but that the Carneddau are highly divergent both from population frequency divergence of the Carneddau could 29 these groups and from the Connemaras and Highlands. be explained by the effects of severe bottlenecking in a 30 The Carneddaus had fewer mitochondrial haplotypes small, recently established but geographically isolated 31 present (eight haplotypes within four major haplogroups) population. However, contrary to expectations for this 32 compared to other breeds, but contained a number of scenario, mtDNA haplotype diversity remained relatively 33 unique mitochondrial haplotypes present at high fre- high and analyses performed to detect evidence of recent 34 quency within the population (Fig. 6). Thus, Carneddaus potential population expansion demonstrate the Carned- 35 may have become isolated from the other groups quite daus have remained at a relatively constant size without 36 some time ago. any evidence of recent bottleneck contraction in allele 37 Analyses of potential population expansion based upon diversity. Similarly nuclear diversity is not significantly 38 Harpending’s raggedness score (Harpending 1994) indi- reduced in the Carneddau, despite the census number of 39 cated that the Carneddaus were the only population with the Carneddau being much lower than the other groups. 40 a highly significant deviation from a unimodal pattern of The Carneddau ponies exist as small, scattered harems 41 molecular diversity expected from a population expansion across their range and have had minimal human interven- 42 (raggedness index = 0.121, P < 0.001), although the Sec- tion. It appears that this breeding pattern has maintained 43 tion Ds also had a moderate disturbance (raggedness variation while the population has become genetically iso- 44 index = 0.022, P < 0.05). lated via drift or natural selection over an extended per- 45 iod, rather than as a consequence of a recent founder 46 Discussion effect. Other free-living ungulate populations have been 47 shown to avoid inbreeding, even in relatively small popu- 48 lations, with a social structure of female philopatry and Population genetic diversity parameters 49 male-biased dispersal patterns similar to the Carneddau 50 All groups displayed high levels of mitochondrial (Coltman et al. 2003; Lawson Handley et al. 2007). In 51 haplotype diversity (>0.788 Æ 0.0361). SSR/SNP analysis more intensively-managed breeds, popular stallions have 52 suggested that heterozygosity levels, although lower than artificially large harem sizes, resulting in high variance in 53 some existing reports (Leroy et al. 2009; Prystupa male reproductive success, as is apparent in equine

6 ª 2013 The Authors. Published by Blackwell Publishing Ltd. C. L. Winton et al. Genetic Diversity in UK Native Mountain Ponies

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4 COLOR 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 Figure 3. Clustering of genetic distance between individuals based on 1-proportion of alleles identical by state. The phylogeny was inferred using 47 the UPGMA method (Sneath and Sokal 1973). The optimal tree with the sum of branch length = 6.66,442,597 is shown. The tree is drawn to 48 scale, with branch lengths in the same units as those of the distances used to infer the phylogenetic tree. Phylogenetic analyses were conducted 49 in MEGA4 (Tamura et al. 2007). The identity of individual horses and the groups they belong to are shown on the right. 50 51 pedigree records. Population genetic theory predicts high in diversity in the Carneddau and other breeds, despite 52 reproductive variance will dramatically reduce the effec- the apparent differences in census numbers, may reflect 53 tive breeding size (Ne) of a population and the similarity this effect.

ª 2013 The Authors. Published by Blackwell Publishing Ltd. 7 Genetic Diversity in UK Native Mountain Ponies C. L. Winton et al.

1 (A) (A) 2 3

4 COLOR 5 6 7 COLOR 8 9 10 11 12 13 (B) 14 15 (B) 16 17 18 19 20 21 22 23 = 24 Figure 5. The results of STRUCTURE analysis for values of K 6 (Fig. 5A) and K=5 (Fig. 5B), averaged over three independent runs 25 of 5000 SNPs and 12 ponies per group. STRUCTURE Harvester 26 analysis suggests optimal cluster value of K is 6. 27 28

29 Table 2. H (on the diagonal for SSRs), Pairwise FST comparisons and 30 effective migration rates between groups based on SSR and SNP data. 31 Section Section 32 (C) A D Connemara Carneddau Highland 33 34 SSRs 35 Section A 2.013 3.27 4.42 5.44 3.55 36 Section D 0.0711 1.891 2.76 2.81 1.89 Connemara 0.0535 0.0831 2.150 2.68 3.26 37 Carneddau 0.0439 0.0817 0.0852 2.055 2.47 38 Highland 0.0658 0.1166 0.0711 0.0920 1.996 39 SNPs 40 Section A * 2.44 2.87 4.59 2.35 41 Section D 0.0931 * 2.69 2.47 2.18 42 Connemara 0.0802 0.0851 * 3.01 2.60 * 43 Carneddau 0.0517 0.0919 0.0766 2.47 Highland 0.0960 0.1030 0.0877 0.0918 * 44

45 Below diagonal: Pairwise FST based on number of different alleles. 46 Figure 4. A–C. Identity by State similarity scores were calculated in Above diagonal: effective number of migrants (Nm) per generation, = 2 47 PLINK from 48,721 diallelic markers. The resultant similarity between based on Slatkin 1995. Theta H estimates 4Nel (1/(1ÀHe) )À1. All F P < 48 individuals was subjected to classical metric multidimensional scaling ST values significant at 0.01. and the first four most significant dimensions extracted and used to 49 plot the three graphs shown above. Dimension 1 splits Section A/ 50 Carneddau from the rest, Dimension 2 seperates Section D and Population structure and gene flow 51 Highlands from the rest, Dimension 3 seperates Connemaras from the 52 rest and Dimension 4 seperates Carneddau and to a lesser extent The majority of genetic variation between domestic horses 53 Section A from the rest. and ponies can be attributed to differences between

8 ª 2013 The Authors. Published by Blackwell Publishing Ltd. C. L. Winton et al. Genetic Diversity in UK Native Mountain Ponies

1 Table 3. MtDNA population average pairwise differences. individuals within breeds, where only approximately 8% 2 of variation results from breed differences (Canon et al. Section Section 3 A D Carneddau Connemara Highland 2000; and also found here in this study). However, 4 despite evidence of admixture between these breeds, anal- 5 Section A 9.145 9.436 9.977*** 8.712*** 8.714** ysis of population structure using SSR data demonstrated À *** ** 6 Section D 0.072 9.871 10.588 8.874 9.027 that each population clustered individually according to Carneddau 1.540*** 1.788*** 7.729 10.810*** 10.986*** 7 = Connemara 0.672*** 0.471** 3.478*** 6.934 7.377* distinct genotype at the optimal value of K 5. A few 8 Highland 0.499** 0.450** 3.480*** 0.268* 7.284 Carneddau individuals showed approximately 25%, 50% 9 and 75% assignment to the Section As suggesting recent 10 Above diagonal: Average number of pairwise differences between admixture. This may have occurred if these animals were 11 populations (pXY). the first and second generation progeny of three Section Diagonal elements: Average number of pairwise differences within 12 A stallions released onto the mountain in 2007–2008, population (pX). 13 Below diagonal: Corrected average pairwise difference (pXYÀ rather than originating from retained similarity to the 14 (pX + pY)/2). Section A animals from a common ancestral population. 15 Probability of result: *<0.05, **<0.01, ***<0.001. Graziers released Section A stallions at this time to 16 17 18 19 20 21 22 23 24 25 26 27 28 29 POOR QUALITY COLOR FIG 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 Figure 6. A median-joining network for mtDNA haplotypes based on shared allele frequencies. Node size represents overall haplotype frequency 6 50 with pie charts within nodes showing frequency of that haplotype by population. Carneddau samples are displayed in red, Section A in yellow, 51 Section D in blue, Connemara in green and Highland in pink. Reference samples representing examples of each of the major haplogroups 52 identified by Achilli et al. (2012) are displayed in black and labelled according to sample number and haplogroup e.g. “1HapA” etc. Numerical 53 values indicate the number of nucleotide changes (>1 mutation) between primary nodes.

ª 2013 The Authors. Published by Blackwell Publishing Ltd. 9 Genetic Diversity in UK Native Mountain Ponies C. L. Winton et al.

1 attempt to maintain diversity, because of concerns about formation (Vila et al. 2001; McGahern et al. 2006a). 2 the small number of animals. Individuals S17 and S10 in However, breed-specific haplogroups were also found 3 particular cluster with the Section A horses, but do not (such as haplogroup B in Connemaras and haplogroup N 4 show a raised level of identity by state with each other, as in Highlands. The predominance of haplogroup I in the 5 would be expected if they were siblings by a single stal- Carneddau (76% of the population) associated with high 6 lion. Indeed, despite a lack of knowledge of relationships autosomal diversity is particularly noteworthy. This ha- 7 prior to sampling, the Carneddau do not demonstrate a plogroup has not been found above a frequency of 20% 8 higher degree of allele sharing than the individuals from in other populations (Achilli et al. 2012) (Fig. 7). Similar 9 the other breeds where siblings had been excluded over-representation of a particular mitochondrial haplo- 10 (Fig. 3). The central position of both the Section A and group and reduction in haplotype diversity have been 11 Carneddau animals in the plot of dimension 2 versus demonstrated for the native Irish (30– 12 dimension 3 (Fig. 4B) would be consistent with the 50% of population contain Achilli et al. (2012) haplo- 13 assumptions that these breeds have retained many ances- group D/Jansen et al. (2002) haplogroup E) where this 14 tral allele frequencies. In contrast the other groups have pattern was taken to indicate the archaic origin of the 15 diverged in different directions as a result of differential breed (McGahern et al. 2006b; Prystupa et al. 2012b). 16 selection and specialisation during breed formation. An The Kerry Bog also shows relatively high nuclear diversity

17 assessment of the SSR data revealed the presence of an (HO = 0.770, Prystupa et al. 2012a). However, the Ameri- 18 apparently private allele at locus HTG10 in the Carned- can population of this breed demonstrated low FIS values, 19 dau (unique alleles were also observed in the other combined with the lack of any breed-specific clustering 20 groups). The 97 bp allele was present in 26% of the Car- following Bayesian analysis. This suggests an effect of sig- 21 neddau samples, suggesting that lack of this allele in the nificant recent outbreeding and admixture with other 22 other groups is unlikely to be due to under-sampling. pony breeds (supported by historical records, McGahern 23 Several mitochondrial haplotypes were found to be et al. 2006b). This situation contrasts with the largely 24 unique to the Carneddau population, including one pre- population-specific SSR clustering pattern of the Carned- 25 dominant within this population. The diversity pattern of daus, demonstrating that the recent introduction of Sec- 26 the Carneddau showing restricted matrilines combined tion A stallions has not yet resulted in a predominant 27 with more comparable levels of nuclear diversity is unu- signature of admixture. 28 sual. It is possible that the recent admixture of the Sec- For the mitochondrial data, the Carneddau are the only 29 tion A stallions released into a small population of group consistently significantly different from the other 30 isolated Carneddau mares could also create the observed populations, either due to long isolation, or perhaps 31 diversity patterns. However, the significant differences in because they have not been exposed to the non-native 32 allele frequencies required to generate the SNP plots, and mares introduced to “improve” the other breeds. The 33 unique mitochondrial and SSR alleles present, suggests striking lack of distinction between the Section A and 34 this is not the case, or at least not the major cause. There- Section D populations in matrilinear diversity suggests 35 fore, the Carneddau have most likely been isolated for a that they originate from the same ancestral population of 36 relatively long period, with effective random breeding and mares. The alternative explanation of extensive inter- 37 low levels of variability in reproductive success maintain- change of mares is excluded by the clear contrast in the 38 ing allelic diversity while allowing change in allele nuclear data for these breeds; the distinct differentiation 39 frequencies. As such, the Carneddau appear to be a dis- in the SSR and SNP plots in combination with similarity 40 tinct population that shares an origin with the Welsh in mtDNA implies extensive male-biased line-breeding. 41 ponies in general and more particularly with Section A The Highland and Connemara matrilines were consis- 42 animals. However, they have been segregated for consid- tently more closely related to each other than to the 43 erable time to have acquired both allele frequency and Welsh ponies. This is again in contrast to the SSR results 44 mutational changes that are unique and perhaps adapted and dimension 3 of the SNP analysis, which suggest sig- 45 to their mountain habitat. The recent practice of intro- nificant gene flow between the Section As and Connemar- 46 ducing outside stallions potentially dilutes this distinctive as: nearly as great as between the Section As and 47 genetic signature. Carneddau. Taken together these results suggest that the 48 Mitochondrial sequencing shows extensive sharing of maternal and paternal histories of the British upland 49 haplotypes across all the populations analysed here and breeds studied here are quite distinct. 50 with those from other studies (Achilli et al. 2012; Pry- The division within the Section Ds may be consequent 51 stupa et al. 2012b). This is a likely legacy of extensive on two historic breeding events and subsequent stud 52 crossbreeding occurring historically between populations management decisions. In the 12th Century Spanish (Ibe- 53 of horses and ponies prior to recognised “closed” breed rian) stallions were imported and used to breed with

10 ª 2013 The Authors. Published by Blackwell Publishing Ltd. C. L. Winton et al. Genetic Diversity in UK Native Mountain Ponies

1 2 3 4 5 6 7 8 9 10 11 12 POOR QUALITY COLOR FIG 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 Figure 7. Haplogroup frequency (%) according to breed. The first row identifiers “A” to “R” represent the major worldwide mtDNA 7 28 haplogroups in horses: haplogroup classification is based upon control region motifs, as described by Achilli et al. (2012). Geographic regions* are 29 according to sequence data collated by Achilli et al. (2012) and comprises of 2401 reference sequences, including ancient (fossil) DNA data. 30 31 small mountain mares (Davies 1993, 1999). These crosses that is historically, geographically or reproductively iso- 32 produced the larger “Powys Horse” which may form one lated (has had little genetic influence from other breeds 33 of the founding types of the Section D ponies. In the for a long time period) could be considered to be a 34 19th century livestock drovers brought fashionable trot- unique population (Henson 1992). The Carneddau ponies 35 ters/Hackneys from the East of England to breed with may therefore represent an important source of uncharac- 36 local Welsh mares (Welsh Pony and Cob Society 2002). terized genetic diversity and adaptations restricted to less 37 Regional studs within Wales may have acquired different intensively managed upland populations. In this study we 38 levels of the incoming genes from these two periods and have detected more than 10 times the number of SNP 39 divergence in breeding target between studs maintained loci expected with extreme deviations from HW equilib- 40 the genetic dichotomy within the Welsh Section Ds, even rium after Bonferroni correction for the number of tested 41 after the closure of the stud book. loci. These thereby form a potential set of markers for 42 determining the genetic differentiation between the 43 groups as a consequence of drift or selection, and as a Implications for conservation of Carneddau 44 future target for understanding adaptations to the upland ponies 45 environment. Specific adaptations have been found in 46 Although clearly related to the Section A pony, the Car- upland and moorland populations of feral or free-living 47 neddau represent a genetically distinct population. For species previously, including parasite load tolerance (Colt- 48 many populations of domestic livestock the definition of man et al. 1999) and coat hair physiology (Wilson 2011). 49 uniqueness is relatively subjective and differences between The importance of preserving feral/wild populations as 50 two populations may only be a function of the relative valuable sources of genetic diversity for domestic relatives 51 frequencies of a few genes, often associated with a single has been highlighted in other species (Schoen and Brown 52 physical character or small group of specific characters 1993; Giovambattista et al. 2001; Tapio et al. 2005; Taber- 53 (Henson 1992; Groenveld et al. 2010). Any population let et al. 2008; Medugorac et al. 2009). The value of hardy

ª 2013 The Authors. Published by Blackwell Publishing Ltd. 11 Genetic Diversity in UK Native Mountain Ponies C. L. Winton et al.

1 native pony breeds for encouraging biodiversity and Consequently, the Carneddau are excluded from current 2 repopulation of rare species has also been recognized Rare Breeds Survival Trust conservation management 3 within conservation grazing schemes (Mitchell and Kirby schemes and are not considered for use within nationwide 4 1990; Kohyani et al. 2008). The semi-feral Carneddau conservation grazing projects utilising ponies such as 5 ponies play a vital role within the mountain ecology of Dartmoor, Exmoor and imported Polish Koniks. The cur- 6 the Snowdonia National Park, as they are involved in a rent lack of recognition surrounding the Carneddau 7 grazing scheme maintaining preferred habitat for an ponies contributes to the risk of losing a potentially use- 8 endangered bird, the red-billed chough (Pyrrhocorax pyr- ful, and distinct, native British breed. 9 rhocorax) (Murray 2006; Johnstone et al. 2010). Limited 10 funding was provided for the land owners to manage a Conclusions 11 subgroup of ponies upon the open mountain; however 12 the project has now ended. The genetic relationship of Carneddau ponies in relation 13 The Carneddau population is believed to be  300 to other native breeds of the UK and Ireland was revealed 14 individuals and at risk due to economic pressures on the by the integrated results of each genetic analysis. The 15 land they graze. A derogation has been granted so that Carneddau ponies have a shared ancestry with the recog- 16 only ponies sold at auction have to be passported and mi- nized Section A pony yet have remained in relative isola- 17 crochipped, rather than requiring that all new-born Car- tion, subjected to natural selective pressures for a 18 neddaus are issued with a passport (in accordance with significant period of time. This has been followed by 19 UK law for other equids). However, because the ponies more recent genetic introgression by Section A stallions 20 are unregistered their sales value is very low (typically a and round up practices. The genetic impact of human 21 meat value), and so the expense of microchipping and activities, particularly the selective use of crossing 22 passporting is unviable for their owners. Animals which upon the pony breeds, are evident in the comparison of 23 are sold have limited use; they have no pedigree and are the different genetic systems; both more recently in the 24 not registered as a recognised breed, preventing them effect upon SSR frequencies within the Carneddaus and 25 from competing in many show classes. From the perspec- historically in the SNP profile of the Section D cobs. 26 tive of population conservation, the removal of young fil- Despite the recent management practices, the Carneddau 27 lies for auction may compound the issue of reduced ponies maintain a distinct genetic signature that should 28 mitochondrial haplotype numbers. Other human selection be conserved. Given the restricted geographic distribution 29 practices are minimal in these animals, making them a and the low numbers of the Carneddau, in order to pro- 30 particularly important group. In the New Forest, Dart- vide some conservation protection measures, these ani- 31 moor and the Brecon Beacons, specially selected individ- mals should be classified as a rare pony population at 32 ual stallions are released every year into known, limited critical status. This study illustrates the rele- 33 geographical areas frequented by free-living mares and re- vance and potential power of combining autosomal and 34 captured at the end of the breeding season. With the maternally transmitted polymorphic assays to unravel 35 exception of three stallions released (but only one since genetic structure and relatedness in feral and intensively 36 removed) in 2007–2008, this does not occur with the Car- managed populations. Herein we provide new informa- 37 neddau population. tion and knowledge to support conservation strategies of 38 It is widely acknowledged that domestic pony and endangered breeds. 39 horse breeds have undergone varying degrees of admix- 40 ture and gene flow prior to establishment of defined, Acknowledgements 41 “closed” pedigrees (Thirstrup et al. 2008; Groenveld et al. 42 2010; Prystupa et al. 2012a). This is clearly evident in the We gratefully thank Susanne Schultz (The University of 43 genetic data presented here, for all of the populations Manchester) and Philip Watts (University of Liverpool), 44 considered. However, in terms of relative levels of admix- for development of the aims of this study in the initial 45 ture, the Carneddaus appear to show similar degrees to stages of the research. 46 that found between the recognised breeds included in this We also thank the photographer Osian Rees for allow- 47 analysis. In addition to this, the distinct genetic signatures ing the use of his photograph of a feral Carneddau pony 48 (mtDNA haplotypes, SSR frequencies, potential SNP within this manuscript. 49 adaptations) suggest that the Carneddau ponies are a References 50 defined, long-established population worthy of conserva- 51 tion. However, whilst the WPCS register Section A ponies Achilli, A., A. Olivieri, P. Soares, H. Lancioni, B. 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12 ª 2013 The Authors. Published by Blackwell Publishing Ltd. C. L. Winton et al. Genetic Diversity in UK Native Mountain Ponies

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1 Graphical Abstract 2 3 The contents of this page will be used as part of the graphical abstract 4 of html only. It will not be published as part of main. 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Herein, we describe the use of microsatellites and SNPs together with analysis of the mitochondrial control region to 21 quantify the extent and magnitude of genetic diversity present in the feral Carneddau pony and relate this to several 22 recognised British and Irish pony breeds. Our results establish that the feral Carneddau ponies represent a unique and dis- 23 tinctive population that merits recognition as a defined population and conservation priority. We discuss the implications 24 for conservation of this population as a unique pool of genetic diversity adapted to the British uplands and potentially of 25 particular value in maintaining the biodiversity of these habitats. 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53

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p i c t u r e d o p p o s i t e . W e ’ v e p i c k e d o u t s o m e o f t h e s e t o o l s e l o w :

b

S t r i k e s a l i n e t h r o u g h t e x t a n d o p e n s u p a t e x t S t r i k e s a r e d l i n e t h r o u g h t e x t t h a t i s t o e

b

o x w h e r e r e p l a c e m e n t t e x t c a n e e n t e r e d . d e l e t e d .

b b

i g h l i g h t a w o r d o r s e n t e n c e . i g h l i g h t a w o r d o r s e n t e n c e . H

‚ H ‚

R e p l a c e ( I n s ) i c o n i n t h e A n n o t a t i o n s S t r i k e t h r o u g h ( D e l ) i c o n i n t h e l i c k o n t h e l i c k o n t h e C

‚ C ‚

s e c t i o n . A n n o t a t i o n s s e c t i o n .

p e t h e r e p l a c e m e n t t e x t i n t o t h e l u e o x t h a t

b b y

‚ T

a p p e a r s .

i g h l i g h t s t e x t i n e l l o w a n d o p e n s u p a t e x t

a r k s a p o i n t i n t h e p r o o f w h e r e a c o m m e n t

M

H y

o x w h e r e c o m m e n t s c a n e e n t e r e d .

b b

n e e d s t o e h i g h l i g h t e d .

b

i g h l i g h t t h e r e l e v a n t s e c t i o n o f t e x t .

l i c k o n t h e A d d s t i c k n o t e i c o n i n t h e

H y

‚ ‚ C

A n n o t a t i o n s s e c t i o n .

l i c k o n t h e A d d n o t e t o t e x t i c o n i n t h e

‚ C

A n n o t a t i o n s s e c t i o n .

l i c k a t t h e p o i n t i n t h e p r o o f w h e r e t h e c o m m e n t

‚ C

s h o u l d e i n s e r t e d .

b

p e i n s t r u c t i o n o n w h a t s h o u l d e c h a n g e d

b y

‚ T

r e g a r d i n g t h e t e x t i n t o t h e e l l o w o x t h a t

b

p e t h e c o m m e n t i n t o t h e e l l o w o x t h a t

b

y

y y

‚ T

a p p e a r s .

a p p e a r s .

I n s e r t s a n i c o n l i n k i n g t o t h e a t t a c h e d f i l e i n t h e I n s e r t s a s e l e c t e d s t a m p o n t o a n a p p r o p r i a t e

a p p r o p r i a t e p a c e i n t h e t e x t . p l a c e i n t h e p r o o f .

l i c k o n t h e A t t a c h F i l e i c o n i n t h e A n n o t a t i o n s l i c k o n t h e A d d s t a m p i c o n i n t h e A n n o t a t i o n s C

‚ C ‚

s e c t i o n . s e c t i o n .

l i c k o n t h e p r o o f t o w h e r e y o u ’ d l i k e t h e a t t a c h e d e l e c t t h e s t a m p y o u w a n t t o u s e . ( T h e A p p r o v e d S

‚ C ‚

f i l e t o b e l i n k e d . s t a m p i s u s u a l l y a v a i l a b l e d i r e c t l y i n t h e m e n u t h a t

a p p e a r s ) .

e l e c t t h e f i l e t o b e a t t a c h e d f r o m y o u r c o m p u t e r

‚ S

o r n e t w o r k . l i c k o n t h e p r o o f w h e r e y o u ’ d l i k e t h e s t a m p t o

‚ C

a p p e a r . ( W h e r e a p r o o f i s t o b e a p p r o v e d a s i t i s ,

e l e c t t h e c o l o u r a n d t y p e o f i c o n t h a t w i l l a p p e a r

‚ S

t h i s w o u l d n o r m a l l y b e o n t h e f i r s t p a g e ) .

i n t h e p r o o f . l i c k O K .

C

A l l o w s s h a p e s , l i n e s a n d f r e e f o r m a n n o t a t i o n s t o b e d r a w n o n p r o o f s a n d f o r

c o m m e n t t o b e m a d e o n t h e s e m a r k s . .

l i c k o n o n e o f t h e s h a p e s i n t h e D r a w i n g

‚ C

s e c t i o n . M a r k u p s

l i c k o n t h e p r o o f a t t h e r e l e v a n t p o i n t a n d

‚ C

d r a w t h e s e l e c t e d s h a p e w i t h t h e c u r s o r .

o a d d a c o m m e n t t o t h e d r a w n s h a p e ,

‚ T

m o v e t h e c u r s o r o v e r t h e s h a p e u n t i l a n

a r r o w h e a d a p p e a r s .

o u b l e c l i c k o n t h e s h a p e a n d t y p e a n y

‚ D

t e x t i n t h e r e d b o x t h a t a p p e a r s .