Genetic diversity within and between British and Irish breeds: The maternal and paternal history of native ponies

Abstract The UK and Ireland have many native pony breeds with historical and cultural importance as well as being a source of uncharacterized genetic diversity. However, there is a lack of comprehensive research investigating their genetic diversity and phylogenetic interrelationships. Many studies contain a limited number of pony breeds or small sample sizes for these breeds. This may result in erroneous grouping of pony breeds that otherwise have intricate interrelationships with each other and are not evaluated correctly when placed as a token subset of a larger dataset. This is the first study that specifically investigates the genetic diversity within and between British and Irish native pony breeds using large sample numbers from locations of their native origin. This study used a panel of microsatellite markers and sequence analysis of the mitochondrial control region to analyze the genetic diversity within and between 11 pony breeds from Britain and Ireland. A large dataset was collected (a total of 485 animals were used for mtDNA analysis and 450 for microsatellite analysis), and previously published data were used to place the British and Irish ponies in a global context. The native ponies of Britain and Ireland were found to have had a complex history, and the interrelationships between the breeds were revealed. Overall, high levels of genetic diversity were maintained in native breeds, although some reduction was evident in small or isolated populations (Shetland, Carneddau, and Section C). Unusual mitochondrial diversity distribution patterns were apparent for the Carneddau and Dartmoor, although among breeds and global haplogroups there was a high degree of haplotype sharing evident, well‐represented within British and Irish ponies. Ancestral maternal diversity was maintained by most populations, particularly the Fells and Welsh ponies, which exhibited rare and ancient lineages. The maternal and paternal histories of the breeds are distinct, with male‐biased crossings between native breeds, and other shared influences, likely Arabs and Thoroughbreds, are apparent. The data generated herein provide valuable information to guide and implement the conservation of increasingly rare native genetic resources.


| INTRODUC TI ON
Evidence has been found of horses present in Britain as early as the Middle Pleistocene, but horse and human populations seem to have become established following the last ice age during the middle Devensian period (Currant & Jacobi, 2001;Hosfield, 2011;Schreve, 2001  There have been limited studies of the native British and Irish pony breeds that specifically investigated the genetic diversity within and between these populations in relation to each other. One study examined Irish breed maternal origins and mitochondrial diversity , and three further works investigated genetic diversity in British and Irish breeds present in North America (Prystupa, Hind, Cothran, & Plante, 2012;Prystupa, Juras, Cothran, Buchanan, & Plante, 2012;Winton et al., 2015). Bower et al. (2011) investigated the maternal origins of the Thoroughbred in context with a number of British and Irish pony breeds. Others have incorporated subsets of British and Irish pony breeds as part of a wider dataset for microsatellite or mitochondrial analysis, although the sample sizes were small and sample origins were often not stated or were from daughter export populations (Jansen et al., 2002;Luis, Juras, Oom, & Cothran, 2007;Leroy et al., 2009;Georgescu, Manea, Dudu, & Costache, 2011;van de Goor, Haeringen, & Lenstra, 2011).
This study determines the genetic diversity and history of British and Irish pony breeds, by examining them as populations in their own right and in relation to each other.
Native pony breeds are part of British and Irish history and culture and represent an important source of uncharacterized genetic diversity. The adaptations to local conditions present within native ponies may provide an inimitable pool of genotypic characteristics of potential value to breeders of modern domestic horses. However, many of these breeds have suffered population decline and several are categorized as being "at risk" by the Rare Breeds Survival Trust (https ://www.rbst.org.uk/rare-breeds-watch list). Worldwide, pony populations that have historically undergone population reduction but have been subject to carefully managed conservation programs have shown success in improving their genetic viability. For example, following a 10-year breeding management and conservation plan, the previously endangered Pottoka breed now displays high genetic diversity (Cañon et al., 2000;Rendo, Iriondo, Manzano, & Estomba, 2012;Solis et al., 2005). The Irish Kerry Bog Pony and the Scottish Eriskay pony have only been recognized as distinct breeds relatively recently (Beck, 1990;. These are now considered to have ancient regional ancestry and as such are currently subject to breeding management and conservation efforts. Therefore, understanding the diversity within and between the British and Irish native pony breeds is essential to enact appropriate, informed conservation plans. The aim of this study was to assess the genetic diversity, population structure, and gene flow of native ponies of Britain and Ireland using mitochondrial DNA and a panel of microsatellite markers. The dataset generated herein was also placed in context with other comparable datasets, including , Achilli et al. (2012), Prystupa, Hind, et al. (2012) and Prystupa, Juras, et al., 2012).

| Sample collection and genomic DNA extraction
Studies were undertaken using animals representative of different bloodlines for each of eleven British and Irish native pony breed populations ( Figure 1). DNA samples were collected as previously described for UK and Irish sources (Winton et al., 2013). A total of 485 animals were used for the mtDNA analysis and 450 for the microsatellite analysis (see Summary Statistic tables in relevant sections for population sample numbers).

| Microsatellite genotyping
SSR genotyping of samples was performed for 17 SSR loci using commercially available kits and protocols as described previously (Winton et al., 2013). PCR products were sized using an ABI 3730 Genetic Analyzer (Applied Biosystems) with the internal 600-Liz size standard. Genotypes were determined using Genemapper ® software v3.0 (Applied Biosystems).

| Microsatellite analysis
Allele frequencies were examined for significant deviation (p < .05) from Hardy-Weinberg expectations using the exact test in Arlequin 3.5 (Excoffier, Laval, & Schneider, 2005;Excoffier & Lischer, 2010), with the Markov chain length set to 100,000 following 10,000 dememorization steps. Markers displaying highly significant deviations in more than one population and/or containing large amounts of missing data values were removed to limit the influence of allele dropout.
Loci with missing data unevenly distributed across the populations (>5% per breed) were also excluded resulting in seven loci being removed from the analysis, including two that had demonstrated null alleles in another study (Rendo et al., 2012). Individual samples with poor quality DNA with calls missing from >3 loci were also excluded.
An unbiased Bayesian approach using Markov chain Monte Carlo (MCMC) clustering of samples was conducted via the STRUCTURE v2.2.3 software (Pritchard, Stephens, & Donnelly, 2000). Parameters were set as for diploid data for each individual and assessed for values of K ranging from 1 to 14. Burn-in and MCMC iteration settings were 50,000 and 100,000, respectively. Allele frequencies were treated as correlated. For each value of K, six replicate simulations were conducted. The ΔK statistic (the second order rate of change in log probability [Ln Pr(X/K)] between successive values of K) was calculated using STRUCTURE Harvester v0.6.7 (http://taylo r0.biolo gy.ucla.edu/ struct_harve st/) as per Evanno, Regnaut, and Goudet (2005). Results from replicate runs at the optimal K were combined in CLUMPP (Jakobsson & Rosenberg, 2007)  org/~tryph on/popul ation s/). This matrix was visualized by drawing an unrooted neighbor-joining phylogenetic tree with MEGA 5.1 (Tamura et al., 2011). An additional distance matrix was calculated based on Reynold's unweighted genetic distance (Reynolds, Weir, & Cockerham, 1983) using Populations 1.2.31 (Olivier Langella; http:// bioin forma tics.org/~tryph on/popul ation s/) and visualized by drawing a NeighborNet in SplitsTree 4.13.1 (Huson & Bryant, 2006). Population summary statistics, pairwise F ST comparisons, and analysis of molecular variance (AMOVA) were calculated using Arlequin 3.5 (Excoffier & Lischer, 2010). Pairwise F ST comparisons were performed using two methods to calculate distance: one based on the number of different alleles and the other based on the sum of squared size difference, similar to the R ST measure of (Slatkin, 1995).
The R ST calculation is a genetic distance method designed for SSR data, a measure of pairs of populations based on the stepwise mutation model (SMM) calculating the average number of allelic size differences between populations.
In order to account for difference in sample size, allelic richness (AR) was calculated for each locus and population based on a minimum sample size of 21 individuals using FSTAT v2.9.3.2 (Goudet, 1995).

| mtDNA genotyping
UK samples were sequenced for a 606bp mtDNA fragment of the mitochondrial control region (MTCR) as described previously (Winton et al., 2013). Sequence data were trimmed to a common 519bp sequence and aligned using BioEdit Sequence Alignment Editor v7.0.9.0 (Hall, 1999) Xu and Arnason (1994).
The 83 reference sequences from Achilli et al. (2012) were downloaded to create a skeleton network of the globally defined horse haplogroups. Sequence traces were checked for nucleotide call errors and either manually rectified or removed in the case of overall low-quality sequence.

| Population genetic diversity parameters
Summary statistics for microsatellite (SSR) data analysis are pre-

| Population structure and gene flow
Molecular analysis of variation demonstrated highly significant genetic structure within the dataset (p < .001), with 6.94% of the total variation existing between populations. Variation within individuals was 91.51%. STRUCTURE Harvester analysis suggested the uppermost hierarchical level of structure was three clusters, with probable subclusters at K = 5 and K = 8 (Figures 2 and 3; Evanno et al., 2005).
In general, this analysis indicated no single clear structure in the data reflecting a complex history of population interactions rather than a simple history of population bifurcation. All populations contained individual animals that could not be unambiguously assigned to a single cluster ( Figure 3). There were also misassigned individuals in populations; both Carneddau and Section D populations had animals with near 100% probability of assignment to the Shetland cluster; and a Connemara were similarly assigned to the Section D cluster. At K = 5 the feral Carneddau population, the Connemara and the Shetland showed distinct breed clusters, while the Sections C and D, and the Fell and Highland ponies clustered into two groups, respectively. When K was increased to 8, the Fell and Highland breeds remained in a single cluster and a high number of Section Bs showed high probability of assignment to the New Forest cluster. While for most other populations, specific genotypic signatures were evident, there was a relatively high degree of admixture between many of the pony breeds, particularly the Section A.
The unrooted neighbor-joining phylogenetic tree based on Nei's standard distance measure (D s ) (Nei, 1972) between all individual ponies demonstrated a variable degree of clustering according to breed ( Figure 4). As also seen in the STRUCTURE plots, Shetlands showed the greatest breed-specific clustering (Figures 3 and 4). The main Fell pony cluster contained a number of Highland pony samples, echoing the admixture between these breeds evident in the STRUCTURE plots. The Carneddau ponies split into two main clusters, also containing several Section As or Section Ds in each. Overall, the tree showed relatively high admixture between the pony breeds, a lack of deep structure in the phylogeny and most variation occurring between individuals.
All NeighborNet networks produced by SplitsTree using the different genetic distance matrices showed similar topologies, with a high degree of unresolved reticulation at the center of the network. This display of many alternative phylogenies indicates the relatively recent between-breed admixture between British and Irish pony breeds. Many of the splits confirmed the clustering found according to Bayesian analysis ( Figure 3). The Shetlands were the most diverged from the other breeds ( Figure 5), while the New Forest and the Section B showed lower divergence from many populations. The Carneddau population also diverged considerably from any other breed. The Section C and Section D clearly showed a close relationship for all networks, as did the Fell and Highland ponies. Correspondingly, the estimated effective number of migrants (N m ) was highest between the Section C and Section D, while the Highland shared the highest N m with the Fell (

| Population genetic diversity parameters
Summary statistics are presented in Table 3. Analysis of mitochondrial control region sequence from 485 individuals identified 91 haplotypes based on 83 variable nucleotide sites. Overall, haplotype diversity was high (0.965 ± 0.003) with relatively low nucleotide

F I G U R E 2
The ΔK plot, describing the rate of change in the log probability of the data between successive K values from 1 to 14.
The modal value of this distribution is the true K, or the uppermost level of hierarchical structure, as per Evanno et al. (2005) F I G U R E 3 Consensus plots for six independent STRUCTURE analysis runs for 12 SSR loci of 11 populations for (

| Population structure and gene flow
Significant genetic structure was apparent (p < .001), with 14% of the total variation found between populations. Table 4 demonstrates that the maternal lineages in the Section A, Section C, and Section D animals could not be distinguished, with the lowest average number of pairwise nucleotide differences for all populations examined.
In contrast, the Welsh Section B population showed significant divergence (p < .001) from the other Welsh populations. The Section B group was not found to be significantly differentiated from the Irish Connemara pony, with a very low value for the average pairwise

F I G U R E 4
The unrooted individual neighbor-joining phylogenetic tree based on Nei's standard distance (Nei, 1972 within, or missing from, the nearest "shared" haplotype node. In contrast, the Section A showed few breed-specific haplotypes yet also had representative animals within most haplogroups (all except B F I G U R E 5 (a) NeighborNet drawn using a Reynold's unweighted genetic distance (Reynolds et al., 1983)

| D ISCUSS I ON
The results revealed lower autosomal genetic differentiation according to breed (8%) compared to previously reported data of other horse breeds worldwide, in which 10%-17% of the genetic diversity could be partitioned this way (Aberle, Hamann, Drögemüller, & Distl, 2004;Glowatzki-Mullis et al., 2006;Plante et al., 2007;Prystupa, Juras, et al., 2012). In part, this may be because this study consists solely of pony breeds originating from Britain and Ireland and recent interbreeding must be considered more widely during their histori- Heterozygosity levels for British and Irish ponies, although lower than some existing reports (Leroy et al., 2009;Prystupa, Juras, et al., 2012), remain relatively high and are comparable to values in other European pony breeds, including semiferal ponies (Luis et al., 2007;Rendo et al., 2012;Solis et al., 2005). Mitochondrial diversity was very high in British and Irish ponies, with ponies representing almost all of the global horse haplotypes (Jansen et al., 2002). Mitochondrial sequencing shows extensive sharing of haplotypes across all the populations analyzed here and with those from other studies (Achilli et al., 2012;Prystupa, Hind, et al., 2012). This is a likely legacy of of horses and ponies prior to recognized "closed" breed formation The Shetland pony showed evidence of more restricted diversity for mitochondrial analyses than other breeds. It is highly diverged from other pony breeds of Britain and Ireland for both autosomal and mitochondrial analyses and has been found to cluster with Nordic breeds, indicating a predominantly Scandinavian origin or influence (Bjornstad & Roed, 2001;Petersen et al., 2013;Prystupa, Hind, et al., 2012;Prystupa, Juras, et al., 2012). However, it is notably more closely related maternally to the Fell than to any other British or Irish breed analyzed here, with reduced pairwise differences and a high number of shared haplotypes. It is possible that the isolated Fell ponies of northern England shared and maintained ancestral links due to an ancient migration of ponies through Britain northwards to the Shetland Isles (Russell, 1996). Alternatively, a shared ancestral TA B L E 4 MtDNA population average pairwise nucleotide differences, within-population pairwise differences, and estimated effective number of migrants Note: Above diagonal: The effective number of migrants (horses) per generation (N m ), according to Slatkin and Hudson (1991), inf = infinity. The  (Bowden et al., 2007;Goodacre et al., 2005;Richardson, 1990). This relationship has since diverged for the more rapidly evolving SSR markers. The drift shown here corresponds to the geographical location of the origin of these ponies; the Shetland Isles are a collection of remote islands located some 100 miles north of Scotland and 200 miles west of Norway. Many pony breeds suffered a population decline during the World Wars and economic depression, including the Shetland pony. However, the demand for these ponies may have remained comparatively more stable than other breeds due to the continuation of subsistence agriculture on the Shetland Islands, use within mines, and steady export to America and the Netherlands (Beck, 1990;Dent & Goodall, 1988;Russell, 1996). The restrictions in mitochondrial haplotype diversity appear to be the result of limited ancestral founding mares rather than any recent decline, and nuclear diversity is comparable to other British and Irish breeds. The census number of Shetlands is high within the UK as they are a well-known and popular breed and are not considered to be at risk. If a wide range of mares and stallions continue to be used for breeding to ensure conservation of ancestral lines and continuity of gene flow, there is no risk to the genetic integrity of the Shetland pony breed.
The Dartmoor ponies demonstrated low mtDNA diversity but moderate to high nuclear diversity. This pattern is unusual in horse breeding within a closed population (Prystupa, Hind, et al., 2012;Prystupa, Juras, et al., 2012) (Edwards, 1992). These introductions largely coincided with the increased popularity of the sport of Polo, which resulted in crossing of Thoroughbred and Arab sires with native pony mares (Palmer, 1990). Other studies have also found that outcrossing usually occurs through the male (Pirault, Sophy Danvy, Verrier, & Leroy, 2013). The mating of mares from limited lineages may have occurred during the following population expansion, resulting in the overrepresentation of haplogroup L seen in the modern Dartmoor pony. It is also possible that Dartmoor mares descend from the ancestral population of modern Iberian stock, given the similarity in haplogroup frequency patterns and that Exeter has been an established port town since Roman times (Edwards, 1992). Theta H values, as analyzed for the SSR dataset, were reduced for the Dartmoor, indicating a lower number of effective breeders than the other pony populations. The Dartmoor ponies would benefit from careful breeding management F I G U R E 6 It shows a median-joining network for mtDNA haplotypes based on shared mitochondrial haplotype frequencies. Node size represents overall haplotype frequency. The pie charts within nodes show frequency of that haplotype according to population (see population color legend). Reference samples representing examples of each of the major haplogroups identified by Achilli et al. (2012) are displayed in black and labeled according to sample number and haplogroup, for example, "1HapA." Numerical values indicate the number of nucleotide changes (>1 mutation) between primary nodes/haplotypes. Branch lengths are proportional to the distance between nodes in order to preserve the limited mitochondrial diversity and rare matrilines. These individuals might be identified through comparison of mitochondrial results with genealogical records to allow for breeding selection accordingly.
The striking lack of distinction between the Section A, Section C, and Section D populations in matrilineal diversity suggests that they originate from the same ancestral population of mares. Extensive interchange of mares between the populations is excluded by the clear contrast in the nuclear data for the Section A and Section D. The distinct differentiation in the SSR plots in combination with similarity in mtDNA implies extensive male-biased line-breeding. There is historical evidence of multiple importations of foreign stallions to breed with local Welsh mares and that this influenced the development of the Section D (Davies, 1993(Davies, , 1999Welsh Pony & Cob Society, 2002).
The central position of the Section A animals in the NeighborNet plots would be consistent with the assumptions that this breed has retained many ancestral allele frequencies. Alternatively, it may well reflect the influence that Welsh ponies have had on the development of many other breeds, resulting in maintained shared allele frequencies between these populations and the Section A. In contrast, the other native breeds have diverged in different directions to each other because of differential selection and specialization during breed formation. The high degree of admixture shown within the STRUCTURE plots and the N-J tree according to the SSR data substantiate this. It is known that Arab and Thoroughbred stallions were introduced to Welsh hill pony mares in the 18th and 19th centuries and some of the individual variation within the Section As may reflect the presence of these alleles (Edwards, 1992). The census population for Welsh Ponies and Cobs is high both within the UK and globally, so as a breed they are not at risk. However, they do show lower observed heterozygosity and a higher inbreeding coefficient than other native breeds, which is likely to be a reflection of use of fewer sires in more recent times. Increasing the pool of sires for breeding (rather than limiting it according to popular champion animals) would be better for the genetic viability, particularly for the rarer Section C animals. The practice of breeding from high numbers of mares should continue to preserve the high ancestral diversity and rare matrilines found in the Welsh Ponies and Cobs. The

F I G U R E 7
Haplogroup frequency (%) according to breed. Segments represent proportion of individuals belonging to labeled haplogroup genetic signature shown by the Section A pony may also be a result of Wahlund effect, whereby the formation and divergence of unrecognized subpopulations result in an overall reduction in observed heterozygosity compared to that predicted from allele frequencies calculated overall (Allendorf, Luikart, & Aitken, 2013). The Section A pony group shows high allelic richness and mean numbers of alleles, but has a heterozygote deficit (lower H O and moderately high F IS ). This phenomenon has been seen in other species such as goats, chickens, and wild sheep (Barker et al., 2001;Pham et al., 2013;Worley et al., 2004). STRUCTURE plots showed a variable probability of assignment of the Section A with a number of other populations, but no obvious within-population subclusters. However, there were some small subclusters of Section A individuals present within mixed groups of individuals for the N-J tree. It is possible that cryptic population substructure within the Section A has resulted in lower total heterozygosity for the population. This may result from selec- It is particularly interesting that the Welsh Section B is not mitochondrially distinct from the Dartmoor pony or the Connemara, yet is highly divergent from the other Welsh populations. There is some maintained relationship with the Dartmoor for the autosomal data, but no more than the relationship with the Section A and Section Cs.
The Section B has clearly had a different maternal history relative to the other Welsh pony populations. The modern Section B Welsh Pony was developed into a taller, "more refined" riding pony from the Section A Mountain Pony and included considerable influence from Arab and Barb horses (Edwards, 1992). Both Arabs and Welsh ponies have had a significant influence on the development of the New Forest breed (Edwards, 1992); thus, the genotype sharing between the Section B and the New Forest displayed in the STRUCTURE plot at K = 8 is not surprising.
Despite considerable demographic changes over the last 135 years, the New Forest breed has high mitochondrial diversity.
It is possible that this is due to the traditional husbandry meth-  Note: Haplogroup frequency (%) according to geographical region, as described by Achilli et al. (2012). The first row identifiers "A" to "R" represent the major worldwide mtDNA haplogroups in horses.
Haplogroup classification is based on control region motifs. Geographic regions are designated according to sequence data collated by Achilli et al. (2012) and in total comprise of 2,166 reference sequences, including ancient (fossil) DNA data.
divergence also confirms the mixed heritage of the New Forest ponies for the autosomal data. Prystupa, Juras, et al. (2012) and Van de Goor et al. (2011) found similar genetic patterns for the New Forest breed. Petersen et al. (2013) (Edwards, 1992).
Fell ponies displayed very high maternal diversity, with a broad distribution of haplogroups, and moderately high nuclear diversity.
Fell ponies, like many other native breeds, suffered population declines following the increased use of motor vehicles and mechanized farming in the early 1900s. Despite this, traditional shepherding of the fell uplands continued to some extent, as did limited keeping of free-roaming herds on the mountains (Richardson, 1990). The Fell has shared maternal lineages with Sections A, C, and D, and to a lesser extent a relationship with the New Forest and Shetland. This is in contrast to the SSR data, where the Fells show the closest relationship to the Highland. According to Richardson (1990), up until the late 19th century the name "Galloway" was used indiscriminately for members of both an old Scottish breed (now extinct) and the Fell ponies prior to their official breed name (decided in 1898), and a shared connection between the two populations previously theorized (Beck, 1990;Dent & Goodall, 1988;Richardson, 1990 (Lewis, 1988;Richardson, 1990

| CON CLUS IONS
This is the first study that specifically investigates the genetic di- Genetic studies that assess diversity and characterize origins of population are thus valuable tools in the development of conservation strategy. In this regard, the data herein indicate that for many of the native breeds, the breeding programmes should be maintained according to their current strategies as they are successfully sustaining their inherent genetic diversity. The Fell ponies face the challenge of reducing the proportion of FIS carriers in the population, but great care must be taken to ensure unique ancestral maternal haplotypes are not lost in the process. The Welsh A, C, and D also have a number of rare ancestral matrilines; however, it is important that as many quality sires as possible are included to maximize their nuclear genetic diversity. Additionally, this study recommends that Dartmoor breeding programmes should be carefully managed to preserve diversity and limited matrilines. This study has afforded valuable insight into the genetic diversity, the origins, and intricate interrelationships of most of the native pony breeds of Britain and Ireland.

ACK N OWLED G M ENTS
We are grateful for funding provided by IBERS PhD Scholarship (Aberystwyth University) and a Royal Society Research Grant 2010/ R2, which supported Clare Winton.

CO N FLI C T O F I NTE R E S T
There are no conflicts of interest between authors, and permission from each author has been granted. No other institutions hold copyright over this work.

AUTH O R CO NTR I B UTI O N S
Clare Winton conducted sample collection, laboratory and data analyses, and interpretation as well as writing of the manuscript. Robert McMahon provided invaluable input to data analysis approach and conducted some of those analyses, advising on their interpretation.
Mathew Hegarty supervised sample analyses in the laboratory, data analyses, and interpretation. Neil McEwan oversaw the experimental setup (including research grant writing), cosupervised the student, and proof-read and refined the manuscript. Mina Davies Morel provided historical context and interpretation of the data. Charly Morgan supported all laboratory analyses. Deborah Nash devised the study, wrote grant-funding applications, supervised the student, and assisted with writing the manuscript.

DATA AVA I L A B I L I T Y S TAT E M E N T
Mitochondrial sequence data generated as part of this project are stored in FASTA format and will be uploaded to the NCBI/GenBank nucleotide sequence repository. Other mtDNA sequences incorporated into the analysis were downloaded from this source and can be retrieved as per the relevant citations. Raw and processed SSR data trace files, and allele calls have been uploaded to DRYAD (https :// doi.org/10.5061/dryad.63xsj 3tzk). These data were converted into several formats for use in packages such as STRUCTURE; should access to each individual format be deemed necessary, they will also be stored on the DRYAD repository.