BACKGROUND
the behavioral and morphological diversity present in modern purebred dogs is remarkable.
for at least  <dig> – <dig>  years, dogs have existed in a variety of sizes and shapes, but the majority of the currently recognized  <dig> breeds have only existed since around  <dig> or later, when dog shows became popular, kennel clubs were founded and stud books were established  <cit> .
in parallel to phenotypic selection, genetic differentiation between breeds has increased and is today very large  <cit> .
the reasons for this high degree of genetic differentiation are the reproductive isolation among breeds, founder effects and bottlenecks experienced during the time of breed creation or later, extreme selection and use of popular sires  <cit> .
the isolation and small effective population size of modern breeds has resulted in widespread inbreeding and the expression of a large number of genetic diseases.
many of these are also among the most frequently occurring diseases in humans, such as cancer, heart problems, deafness, blindness and joint diseases  <cit> .
the genetic disorders in the dog often mimic human diseases closely in physiology, disease presentation and clinical response.
more than  <dig> genetic diseases that are found in humans have also been described in dogs, about 46% occurring mainly in either one or a few breeds  <cit> .
the fact that purebred dogs are separated into genetically differentiated breeds and that they live in the same environment as humans, makes them an ideal model to map genes for human disorders  <cit> .
another advantage is that linkage disequilibrium  in dog breeds extends over 20– <dig> times longer genomic regions than in humans, which means that a smaller number of markers are required for genome-wide scans in dogs.
this, together with the low haplotype diversity within ld regions and high degree of haplotype sharing among breeds  <cit> , facilitates the identification of chromosomal regions where candidate genes are located.
association mapping of phenotypic traits or diseases is a population-based approach  and depends highly on the level of ld  <cit> , which in dogs is related to the genetic uniformity of breeds.
dogs are today divided into different breeds based mainly on morphological appearance and/or behavior.
breeds are defined as "intraspecies groups that have relatively uniform physical characteristics developed under controlled conditions by man  <cit> ."
however, homogeneity within breeds may not always be the case.
for example, the official standards for many breeds allow a number of color types and patterns, while other combinations are unacceptable  <cit> .
variation in size can lead to the identification of different types within a breed.
additionally, breeders are increasingly interested in specializing in either dogs for show, or dogs for working purposes or as pets.
these divergent selective forces within breeds are a form of disruptive selection: extreme forms are favored while intermediate forms are selected against  <cit> .
since human preferences play the same role of assortative mating in keeping the breeding lines separate, these are likely to lead to the formation of genetically separated groups, thus violating the assumption of uniformity within breeds.
one example of this breed fragmentation is the case of the poodle.
according to the world canine organization, fci   <cit> , poodles are grouped in four different sizes: standard , medium sized , miniature  and toy .
at the same time, five uniform colors are allowed: black, brown, white, silver and apricot  <cit> .
despite the different sizes and colors, all poodles are currently recognized as a single breed.
disruptive selection for very specific phenotypes and assortative mating might have led to fragmentation, and to genetically differentiated types within this breed.
in this study we used purebred poodles from sweden as an example to study how genetic diversity at  <dig> microsatellite loci is partitioned within a single dog breed, and then compare this pattern to variation within and among eight other purebred dog breeds.
this knowledge can contribute to a better understanding of the history and relationship between breeds, exemplifies the genetic diversification of populations as a result of disruptive selection, and it is also vital to the design of optimal search strategies for association mapping of diseases or phenotypic traits  <cit> .
RESULTS
the study of  <dig> swedish poodles revealed an average of  <dig>  alleles per locus, a significant  heterozygote deficit across all  <dig> markers , and positive inbreeding coefficient .
this heterozygote deficit was consistently found in  <dig> of  <dig> markers, indicating that the deviation from hardy-weinberg equilibrium affects the whole genome rather than being a local, locus-specific phenomenon such as selection or genotyping problems .
heterozygote deficit could indicate a preferential mating with relatives within poodles, or the presence of population structure within the breed   <cit> .
to assess whether the heterozygote deficit was due to the fragmentation of poodles into the four discrete size classes acknowledged by fci, we measured the degree of population differentiation using fst  <cit>  .
standard poodles were very well separated from the smaller poodles, with pairwise fst values in the range of  <dig>  to  <dig>  , which suggests very limited genetic exchange between them.
on the other hand, the differentiation between the three smaller size classes  is much lower, with fst values at least  <dig>  times smaller than the smallest value observed between any of them and standard poodles.
among them, miniature and toy poodles were indistinguishable  .
measured as pairwise fst , based on variation at  <dig> microsatellite loci.
*p <  <dig> ; **p <  <dig> ; ***p <  <dig> .
we used the program structure to identify the groups into which poodles are divided.
we determined the number of existing clusters in two ways, by considering the log 'probability of data' lnp for different numbers of k, and by using the statistic Δk  <cit> , which considers the rate of change in lnp among successive k values.
the latter approach indicated that the optimal number of groups was k =  <dig>  however, lnp continued to increase with increasing k until k =  <dig>  and thereafter lnp decreased.
at k =  <dig>  standard poodles appeared separated from the smaller poodles , and they remained separated with increasing k values.
larger k values failed to separate the other sizes .
to corroborate that the genetic differentiation observed between the poodle size classes is inversely related to the current gene flow between them, we examined pedigree information from the swedish kennel club  <cit> .
we recorded the size registered for the parents, grandparents and great-grandparents for all  <dig> poodles studied.
the pedigree information indicated that the gene flow estimates between the different poodle size classes are almost identical considering parents, grandparents  or great-grandparents, suggesting that the patterns of gene exchange have remained relatively constant over recent times .
the pedigree information confirms the complete isolation of standard poodles .
similarly, about 87% of the ancestors of the medium sized poodles belonged to the medium size size-class, while the corresponding proportions were only around 63% for miniature and around 36% for toy.
the genetic exchange between the two smallest sizes thus appeared to be very large and 52–61% of the ancestors  of toy were in fact miniature poodles.
the patterns of genetic exchange suggested by the pedigree closely parallel the genetic differentiation between the size classes , as confirmed by a mantel correlation test .
since the analysis with structure suggested that the likelihood of the clustering within poodles was highest with k =  <dig>  we examined more closely the characteristics for each dog available at the registry.
this revealed that the five groups at k =  <dig> could be clearly defined by a combination of size and coat-color.
multiple runs of the program consistently provided the same results.
after rearranging the results of structure shown in figure 1a according to size and color criteria, the division between groups could be seen more clearly .
as indicated above, at k =  <dig>  the first group to separate from the rest corresponds to the standard poodles.
at k =  <dig>  a new group is formed within the smaller size classes of poodles by separating individuals with black and brown coat colors from those with the other three colors .
at k =  <dig> the group of black and brown small sized poodles is further subdivided and medium sized form a separate group from miniature and toy.
finally, at k =  <dig> the group containing the small  poodles of pale colors, is separated into two groups: one containing silver  and white dogs and the other dogs with apricot color.
consequently, our results indicate that the genetic diversity within poodles is clearly separated into two groups which differ by size  and that actually five separate clusters can be discerned based on a combination of color and size.
using fst to measure the degree of differentiation between the five groups  confirms that standard poodles are very different from the three smaller-bodied groups of poodles .
however, the differentiation among the four groups of smaller poodles, defined on the basis of color and size, is about four times larger than the largest difference observed between the size classes for the three small-sized poodles, reaching fst values of up to  <dig>  .
measured as pairwise fst , between five size and color groups found in poodles  and eight additional breeds, for  <dig> loci.
all results are highly significant .
<dig> medium size poodles with brown or black color
<dig> miniature poodles and toy poodles with brown or black color
<dig> medium size poodles, miniature poodles and toy poodles with silver  or white color
<dig> medium size poodles, miniature poodles and toy poodles with apricot color
a reanalysis of the pedigree information revealed that the clusters shown by the structure analysis matched better the breeding practices for poodles.
for each one of the five groups defined, more than 80% of the parents and great-grandparents originated from the same group , as compared to the much lower values for the size classes .
our analysis of pedigree and phenotype data thus confirmed that the genetic clusters identified within poodles are the result of specific breeding preferences of dog owners, and not just originating from mating related dogs.
to our knowledge, these groups have not been identified or suggested before, thus our genetic analysis revealed hidden population substructure within poodles.
these five groups of poodles were also genetically compared to  <dig> dogs from eight other breeds .
standard poodles were as differentiated from smaller poodles as different breeds are from each other .
for instance, the pairwise fst value between standard poodles and black and brown medium sized poodles was  <dig> , larger than 40% of the comparisons between the five groups of poodles and the other  <dig> breeds included in this study.
for example, the fst value between giant schnauzer and small white and silver poodles is just  <dig> .
a neighbor-joining analysis  based on another measure of genetic differentiation, nei's  <cit>  standard genetic distance , shows standard poodles at the end of a long branch, indicating genetic uniqueness and separation from all other poodles.
one possible explanation for the large difference between standard and small sized poodles could be a past bottleneck that reduced the diversity in the first compared to the latter.
this bottleneck could translate into a strong differentiation by founder effects and drift.
however, this does not seem to be the main reason for their uniqueness, because their allelic richness was similar to that observed in other groups of poodles after correcting for differences in sample size .
number of dogs tested, average sample size per locus, inbreeding coefficient , expected and observed heterozygosity , and allelic richness corrected for sample size .
significance refers to the difference between he and ho, *p <  <dig> ; **p <  <dig> ; ***p <  <dig> .
<dig> medium sized poodles, miniature poodles and toy poodles
<dig> medium sized poodles with brown or black coat-color
<dig> miniature poodles and toy poodles with brown or black coat-color
<dig> medium sized poodles, miniature poodles and toy poodles with silver  or white coat-color
<dig> medium sized poodles, miniature poodles and toy poodles with apricot coat-color
<dig> without size correction
in order to compare the uniqueness of the five poodle groups in relation to the differentiation between recognized dog breeds we conducted assignment tests.
if breeds  are well differentiated, most of the individuals should be correctly assigned to their nominal group.
in a first test we divided all poodles into two groups , and compared them to dogs from  <dig> other breeds.
only  <dig> out of  <dig> dogs were incorrectly assigned and none of these mis-assignments corresponded to poodles.
this indicates that established dog breeds are well-isolated populations with unique microsatellite allele frequencies , and that a similar degree of differentiation exists between standard poodles and the group of small sized poodles.
a second assignment test was made examining only the four coat-color/size groups of small poodles.
this resulted in a high self-assignment, but  <dig> out of  <dig> were matched to a different color/size combination, showing a lower degree of differentiation between groups within small poodles.
poodles could represent a unique case among dogs if they developed from the mixing of separate dog lineages.
if this was the case, we would expect to find a larger genetic diversity in poodles than that observed in other breeds and, more importantly, a larger inbreeding coefficient fis due to population fragmentation .
when considering all poodles as members of a single group, they show high genetic diversity  and a large inbreeding coefficient fis, further evidenced by a significant heterozygote deficit.
however, several other dog breeds also had high levels of microsatellite diversity he , fis values as large as those in poodles  or significant heterozygote deficits .
although the allelic richness corrected for sample size was larger when all poodles were considered as one single group, the differences compared to the other breeds were small .
it could be argued that differences in genetic diversity between breeds are the result of differences in population size.
however, this does not seem to be the case.
genetic diversity  was not correlated to breed population size as inferred by the number of registries at the swedish kennel club during  <dig> .
similarly, the inbreeding coefficient fis was not correlated with breed population size either .
since genetic diversity and inbreeding coefficient are not related to population size, the large differences in diversity and positive inbreeding coefficient in some breeds could be indicative of some degree of genetic fragmentation within these breeds, similar to that observed in poodles.
discussion
about  <dig> dog breeds exist today.
all of these breeds are characterized by unique morphology, characteristic behavior and often also by a suite of genetic diseases.
the morphological differences between breeds are so large that they easily exceed the differentiation between all species in the family canidae  <cit> , and probably no other vertebrate has comparable phenotypic diversity.
our results suggest that currently the number of genetically differentiated groups of purebred dogs might be even larger than the number of breeds: some breeds, such as the poodle, are likely to encompass multiple genetically divergent subgroups.
selection patterns within breeds might lead to an increase in the number of genetically differentiated dog breeds.
poodles represent a breed encompassing apparent phenotypic diversity, which translates in the identification of four classes with regard to body size.
however, our analyses revealed that this is not the only reason for the poodle intra-breed structure.
only a limited set of coat colors are accepted in purebred poodles, as for most modern breeds, and coats are required to be uniform  <cit> .
since other colors and coat patterns are not acceptable in registered purebred poodles, this results in disruptive selection by which some extreme combinations are favored while intermediate types are rejected.
as a consequence of this, the genetic diversity of poodles is fragmented into five distinct groups defined by a combination of size and color .
one of the groups, standard poodles, appears as different from the other poodle groups as recognized dog breeds are different from each other .
this result emphasizes how strong the intra-breed differentiation can be.
historically, standard poodles were used as working dogs, while the three smaller poodle classes were mostly bred for company.
standard poodles have been bred separately from the smaller sizes for more than  <dig> years in europe .
in the us, however, some degree of interbreeding among the recognized poodle size classes occurs.
this could result in different patterns of differentiation in american poodles.
our results show a strong differentiation between standard poodles and the smaller sizes .
due to these different breeding practices, it is possible that standard poodles are not as differentiated from small sized poodles in the us as they are in europe.
this study is based on poodles born in sweden; population structure in other countries may be similarly affected by local regulations and breeding practices.
the long isolation between standard poodles and the smaller sizes in europe has resulted in a differentiation as pronounced as that between well defined breeds.
founder effects and subsequent bottlenecks during the history of breeds, together with highly selective breeding practices  <cit> , have led to the differentiation between breeds and probably have also led to the strong separation between standard poodles and the group of small sized poodles.
this process results in a rapid random genetic drift and unique allele frequencies.
consequently, attempts to reconstruct the relationship between breeds based on their allelic composition  <cit>  are likely to have been heavily influenced by such random effects  and may not necessarily reflect true breed history.
for example, our results suggest that, for neutral genetic markers, standard poodles are as differentiated from black and brown medium sized poodles as from giant schnauzers or siberian huskies .
this indicates that genetic distance based on neutral allelic frequencies might actually be a poor indicator of breed relatedness.
the four groups within the small sized poodles show lower levels of differentiation, below the values commonly observed between the other breeds or between them and standard poodles .
nevertheless, our results confirm that these dogs do not constitute a panmictic breeding population where all individuals randomly breed to each other.
the situation observed in this study for the poodles, intra-breed structure, may be present in other breeds as well, as suggested by the large inbreeding coefficient in some of the breeds , consistent with population fragmentation  <cit> .
similarly, schelling, gaillard and dolf  <cit>  observed that while longhaired and smooth dachshunds were genetically similar, wirehaired dachshunds clustered separately in a phylogenetic tree.
for some breeds such as german shepherds, siberian huskies and labrador retrievers, two different lines of selection are maintained within the breed, separating animals intended for competition in dog shows for their appearance, and animals selected for work.
in these cases the morphology of the two types is becoming more and more divergent over time, which will likely result in an intra-breed population structure comparable to that observed in poodles.
the existence of separated lines of selective breeding within the same breed is likely to lead to the establishment of distinct genetic clusters within recognized dog breeds.
thus, an ongoing process of selective breeding is leading to a progressive increase in the number of dog breeds, and this increase will continue as long as the popularity of purebred dogs and dog shows continues.
CONCLUSIONS
the partitioning of genetic diversity into discrete classes  has made the dog an exceptional model for the study of the association between genotype and phenotype and for the identification of genes involved in phenotypic and behavioral traits or diseases  <cit> .
however, the use of dogs as a model is determined by the uniformity within breeds.
our results suggest that genetically divergent groups of dogs can exist within the same breed.
the implications of this for association mapping studies are two-fold.
first, the number of genetically identifiable breeds may be even larger than suspected , increasing the value of dogs as a model organism for association studies.
since these genetically differentiated groups are likely to include only a small part of the haplotype diversity attributed to the breed and to represent more inbred lines, ld is expected to be larger  <cit> .
this would facilitate the identification of markers linked to the trait under study.
second, this intra-breed structure violates the assumption of uniformity within breeds.
such cryptic  population structure, if ignored, is likely to confound association studies.
association studies should target genetically homogeneous groups within breeds, which may often be phenotypically and/or behaviorally separated.
assortative mating is leading to the creation of strong population structure within dog breeds and has contributed to the extreme plasticity of dogs and domestic animals under selection.
domestic animals are invaluable models to understand evolution, and in the same way that they were a major source of inspiration for darwin  <cit> , they continue to help us understand the origin of biodiversity.

 the behavioral and morphological diversity present in modern purebred dogs is remarkable. for at least – years, dogs have existed in a variety of sizes and shapes, but the majority of the currently recognized breeds have only existed since around or later, when dog shows became popular, kennel clubs were founded and stud books were established. in parallel to phenotypic selection, genetic differentiation between breeds has increased and is today very large. the reasons for this high degree of genetic differentiation are the reproductive isolation among breeds, founder effects and bottlenecks experienced during the time of breed creation or later, extreme selection and use of popular sires. the isolation and small effective population size of modern breeds has resulted in widespread inbreeding and the expression of a large number of genetic diseases. many of these are also among the most frequently occurring diseases in humans, such as cancer, heart problems, deafness, blindness and joint diseases. the genetic disorders in the dog often mimic human diseases closely in physiology, disease presentation and clinical response. more than genetic diseases that are found in humans have also been described in dogs, about 46% occurring mainly in either one or a few breeds. the fact that purebred dogs are separated into genetically differentiated breeds and that they live in the same environment as humans, makes them an ideal model to map genes for human disorders. another advantage is that linkage disequilibrium in dog breeds extends over 20– times longer genomic regions than in humans, which means that a smaller number of markers are required for genome-wide scans in dogs. this, together with the low haplotype diversity within ld regions and high degree of haplotype sharing among breeds, facilitates the identification of chromosomal regions where candidate genes are located. association mapping of phenotypic traits or diseases is a population-based approach and depends highly on the level of ld, which in dogs is related to the genetic uniformity of breeds. dogs are today divided into different breeds based mainly on morphological appearance and/or behavior. breeds are defined as "intraspecies groups that have relatively uniform physical characteristics developed under controlled conditions by man." however, homogeneity within breeds may not always be the case. for example, the official standards for many breeds allow a number of color types and patterns, while other combinations are unacceptable. variation in size can lead to the identification of different types within a breed. additionally, breeders are increasingly interested in specializing in either dogs for show, or dogs for working purposes or as pets. these divergent selective forces within breeds are a form of disruptive selection: extreme forms are favored while intermediate forms are selected against. since human preferences play the same role of assortative mating in keeping the breeding lines separate, these are likely to lead to the formation of genetically separated groups, thus violating the assumption of uniformity within breeds. one example of this breed fragmentation is the case of the poodle. according to the world canine organization, fci, poodles are grouped in four different sizes: standard, medium sized, miniature and toy. at the same time, five uniform colors are allowed: black, brown, white, silver and apricot. despite the different sizes and colors, all poodles are currently recognized as a single breed. disruptive selection for very specific phenotypes and assortative mating might have led to fragmentation, and to genetically differentiated types within this breed. in this study we used purebred poodles from sweden as an example to study how genetic diversity at microsatellite loci is partitioned within a single dog breed, and then compare this pattern to variation within and among eight other purebred dog breeds. this knowledge can contribute to a better understanding of the history and relationship between breeds, exemplifies the genetic diversification of populations as a result of disruptive selection, and it is also vital to the design of optimal search strategies for association mapping of diseases or phenotypic traits.  the study of swedish poodles revealed an average of alleles per locus, a significant heterozygote deficit across all markers, and positive inbreeding coefficient. this heterozygote deficit was consistently found in of markers, indicating that the deviation from hardy-weinberg equilibrium affects the whole genome rather than being a local, locus-specific phenomenon such as selection or genotyping problems. heterozygote deficit could indicate a preferential mating with relatives within poodles, or the presence of population structure within the breed. to assess whether the heterozygote deficit was due to the fragmentation of poodles into the four discrete size classes acknowledged by fci, we measured the degree of population differentiation using fst. standard poodles were very well separated from the smaller poodles, with pairwise fst values in the range of to, which suggests very limited genetic exchange between them. on the other hand, the differentiation between the three smaller size classes is much lower, with fst values at least times smaller than the smallest value observed between any of them and standard poodles. among them, miniature and toy poodles were indistinguishable. measured as pairwise fst, based on variation at microsatellite loci. *p <; **p <; ***p <. we used the program structure to identify the groups into which poodles are divided. we determined the number of existing clusters in two ways, by considering the log'probability of data' lnp for different numbers of k, and by using the statistic Δk, which considers the rate of change in lnp among successive k values. the latter approach indicated that the optimal number of groups was k = however, lnp continued to increase with increasing k until k = and thereafter lnp decreased. at k = standard poodles appeared separated from the smaller poodles, and they remained separated with increasing k values. larger k values failed to separate the other sizes. to corroborate that the genetic differentiation observed between the poodle size classes is inversely related to the current gene flow between them, we examined pedigree information from the swedish kennel club. we recorded the size registered for the parents, grandparents and great-grandparents for all poodles studied. the pedigree information indicated that the gene flow estimates between the different poodle size classes are almost identical considering parents, grandparents or great-grandparents, suggesting that the patterns of gene exchange have remained relatively constant over recent times. the pedigree information confirms the complete isolation of standard poodles. similarly, about 87% of the ancestors of the medium sized poodles belonged to the medium size size-class, while the corresponding proportions were only around 63% for miniature and around 36% for toy. the genetic exchange between the two smallest sizes thus appeared to be very large and 52–61% of the ancestors of toy were in fact miniature poodles. the patterns of genetic exchange suggested by the pedigree closely parallel the genetic differentiation between the size classes, as confirmed by a mantel correlation test. since the analysis with structure suggested that the likelihood of the clustering within poodles was highest with k = we examined more closely the characteristics for each dog available at the registry. this revealed that the five groups at k = could be clearly defined by a combination of size and coat-color. multiple runs of the program consistently provided the same results. after rearranging the results of structure shown in figure 1a according to size and color criteria, the division between groups could be seen more clearly. as indicated above, at k = the first group to separate from the rest corresponds to the standard poodles. at k = a new group is formed within the smaller size classes of poodles by separating individuals with black and brown coat colors from those with the other three colors. at k = the group of black and brown small sized poodles is further subdivided and medium sized form a separate group from miniature and toy. finally, at k = the group containing the small poodles of pale colors, is separated into two groups: one containing silver and white dogs and the other dogs with apricot color. consequently, our results indicate that the genetic diversity within poodles is clearly separated into two groups which differ by size and that actually five separate clusters can be discerned based on a combination of color and size. using fst to measure the degree of differentiation between the five groups confirms that standard poodles are very different from the three smaller-bodied groups of poodles. however, the differentiation among the four groups of smaller poodles, defined on the basis of color and size, is about four times larger than the largest difference observed between the size classes for the three small-sized poodles, reaching fst values of up to. measured as pairwise fst, between five size and color groups found in poodles and eight additional breeds, for loci. all results are highly significant. medium size poodles with brown or black color miniature poodles and toy poodles with brown or black color medium size poodles, miniature poodles and toy poodles with silver or white color medium size poodles, miniature poodles and toy poodles with apricot color a reanalysis of the pedigree information revealed that the clusters shown by the structure analysis matched better the breeding practices for poodles. for each one of the five groups defined, more than 80% of the parents and great-grandparents originated from the same group, as compared to the much lower values for the size classes. our analysis of pedigree and phenotype data thus confirmed that the genetic clusters identified within poodles are the result of specific breeding preferences of dog owners, and not just originating from mating related dogs. to our knowledge, these groups have not been identified or suggested before, thus our genetic analysis revealed hidden population substructure within poodles. these five groups of poodles were also genetically compared to dogs from eight other breeds. standard poodles were as differentiated from smaller poodles as different breeds are from each other. for instance, the pairwise fst value between standard poodles and black and brown medium sized poodles was, larger than 40% of the comparisons between the five groups of poodles and the other breeds included in this study. for example, the fst value between giant schnauzer and small white and silver poodles is just. a neighbor-joining analysis based on another measure of genetic differentiation, nei's standard genetic distance, shows standard poodles at the end of a long branch, indicating genetic uniqueness and separation from all other poodles. one possible explanation for the large difference between standard and small sized poodles could be a past bottleneck that reduced the diversity in the first compared to the latter. this bottleneck could translate into a strong differentiation by founder effects and drift. however, this does not seem to be the main reason for their uniqueness, because their allelic richness was similar to that observed in other groups of poodles after correcting for differences in sample size. number of dogs tested, average sample size per locus, inbreeding coefficient, expected and observed heterozygosity, and allelic richness corrected for sample size. significance refers to the difference between he and ho, *p <; **p <; ***p <. medium sized poodles, miniature poodles and toy poodles medium sized poodles with brown or black coat-color miniature poodles and toy poodles with brown or black coat-color medium sized poodles, miniature poodles and toy poodles with silver or white coat-color medium sized poodles, miniature poodles and toy poodles with apricot coat-color without size correction in order to compare the uniqueness of the five poodle groups in relation to the differentiation between recognized dog breeds we conducted assignment tests. if breeds are well differentiated, most of the individuals should be correctly assigned to their nominal group. in a first test we divided all poodles into two groups, and compared them to dogs from other breeds. only out of dogs were incorrectly assigned and none of these mis-assignments corresponded to poodles. this indicates that established dog breeds are well-isolated populations with unique microsatellite allele frequencies, and that a similar degree of differentiation exists between standard poodles and the group of small sized poodles. a second assignment test was made examining only the four coat-color/size groups of small poodles. this resulted in a high self-assignment, but out of were matched to a different color/size combination, showing a lower degree of differentiation between groups within small poodles. poodles could represent a unique case among dogs if they developed from the mixing of separate dog lineages. if this was the case, we would expect to find a larger genetic diversity in poodles than that observed in other breeds and, more importantly, a larger inbreeding coefficient fis due to population fragmentation. when considering all poodles as members of a single group, they show high genetic diversity and a large inbreeding coefficient fis, further evidenced by a significant heterozygote deficit. however, several other dog breeds also had high levels of microsatellite diversity he, fis values as large as those in poodles or significant heterozygote deficits. although the allelic richness corrected for sample size was larger when all poodles were considered as one single group, the differences compared to the other breeds were small. it could be argued that differences in genetic diversity between breeds are the result of differences in population size. however, this does not seem to be the case. genetic diversity was not correlated to breed population size as inferred by the number of registries at the swedish kennel club during. similarly, the inbreeding coefficient fis was not correlated with breed population size either. since genetic diversity and inbreeding coefficient are not related to population size, the large differences in diversity and positive inbreeding coefficient in some breeds could be indicative of some degree of genetic fragmentation within these breeds, similar to that observed in poodles. discussion about dog breeds exist today. all of these breeds are characterized by unique morphology, characteristic behavior and often also by a suite of genetic diseases. the morphological differences between breeds are so large that they easily exceed the differentiation between all species in the family canidae, and probably no other vertebrate has comparable phenotypic diversity. our results suggest that currently the number of genetically differentiated groups of purebred dogs might be even larger than the number of breeds: some breeds, such as the poodle, are likely to encompass multiple genetically divergent subgroups. selection patterns within breeds might lead to an increase in the number of genetically differentiated dog breeds. poodles represent a breed encompassing apparent phenotypic diversity, which translates in the identification of four classes with regard to body size. however, our analyses revealed that this is not the only reason for the poodle intra-breed structure. only a limited set of coat colors are accepted in purebred poodles, as for most modern breeds, and coats are required to be uniform. since other colors and coat patterns are not acceptable in registered purebred poodles, this results in disruptive selection by which some extreme combinations are favored while intermediate types are rejected. as a consequence of this, the genetic diversity of poodles is fragmented into five distinct groups defined by a combination of size and color. one of the groups, standard poodles, appears as different from the other poodle groups as recognized dog breeds are different from each other. this result emphasizes how strong the intra-breed differentiation can be. historically, standard poodles were used as working dogs, while the three smaller poodle classes were mostly bred for company. standard poodles have been bred separately from the smaller sizes for more than years in europe. in the us, however, some degree of interbreeding among the recognized poodle size classes occurs. this could result in different patterns of differentiation in american poodles. our results show a strong differentiation between standard poodles and the smaller sizes. due to these different breeding practices, it is possible that standard poodles are not as differentiated from small sized poodles in the us as they are in europe. this study is based on poodles born in sweden; population structure in other countries may be similarly affected by local regulations and breeding practices. the long isolation between standard poodles and the smaller sizes in europe has resulted in a differentiation as pronounced as that between well defined breeds. founder effects and subsequent bottlenecks during the history of breeds, together with highly selective breeding practices, have led to the differentiation between breeds and probably have also led to the strong separation between standard poodles and the group of small sized poodles. this process results in a rapid random genetic drift and unique allele frequencies. consequently, attempts to reconstruct the relationship between breeds based on their allelic composition are likely to have been heavily influenced by such random effects and may not necessarily reflect true breed history. for example, our results suggest that, for neutral genetic markers, standard poodles are as differentiated from black and brown medium sized poodles as from giant schnauzers or siberian huskies. this indicates that genetic distance based on neutral allelic frequencies might actually be a poor indicator of breed relatedness. the four groups within the small sized poodles show lower levels of differentiation, below the values commonly observed between the other breeds or between them and standard poodles. nevertheless, our results confirm that these dogs do not constitute a panmictic breeding population where all individuals randomly breed to each other. the situation observed in this study for the poodles, intra-breed structure, may be present in other breeds as well, as suggested by the large inbreeding coefficient in some of the breeds, consistent with population fragmentation. similarly, schelling, gaillard and dolf observed that while longhaired and smooth dachshunds were genetically similar, wirehaired dachshunds clustered separately in a phylogenetic tree. for some breeds such as german shepherds, siberian huskies and labrador retrievers, two different lines of selection are maintained within the breed, separating animals intended for competition in dog shows for their appearance, and animals selected for work. in these cases the morphology of the two types is becoming more and more divergent over time, which will likely result in an intra-breed population structure comparable to that observed in poodles. the existence of separated lines of selective breeding within the same breed is likely to lead to the establishment of distinct genetic clusters within recognized dog breeds. thus, an ongoing process of selective breeding is leading to a progressive increase in the number of dog breeds, and this increase will continue as long as the popularity of purebred dogs and dog shows continues.  the partitioning of genetic diversity into discrete classes has made the dog an exceptional model for the study of the association between genotype and phenotype and for the identification of genes involved in phenotypic and behavioral traits or diseases. however, the use of dogs as a model is determined by the uniformity within breeds. our results suggest that genetically divergent groups of dogs can exist within the same breed. the implications of this for association mapping studies are two-fold. first, the number of genetically identifiable breeds may be even larger than suspected, increasing the value of dogs as a model organism for association studies. since these genetically differentiated groups are likely to include only a small part of the haplotype diversity attributed to the breed and to represent more inbred lines, ld is expected to be larger. this would facilitate the identification of markers linked to the trait under study. second, this intra-breed structure violates the assumption of uniformity within breeds. such cryptic population structure, if ignored, is likely to confound association studies. association studies should target genetically homogeneous groups within breeds, which may often be phenotypically and/or behaviorally separated. assortative mating is leading to the creation of strong population structure within dog breeds and has contributed to the extreme plasticity of dogs and domestic animals under selection. domestic animals are invaluable models to understand evolution, and in the same way that they were a major source of inspiration for darwin, they continue to help us understand the origin of biodiversity. 
the genetic structure observed in poodles is the result of local mating patterns, implying that breed fragmentation may be different in different countries. we studied patterns of genetic diversity within poodles and compared it to dogs from eight other breeds. patterns of genetic diversity in other breeds suggest that assortative mating leading to fragmentation may be a common feature within many dog breeds. breeds are considered to be uniform groups with similar physical characteristics, shaped by selection rooted in human preferences. these characteristics are important for association mapping of candidate genes for diseases and therefore make dogs ideal models for gene mapping of human disorders. this has led to a large genetic difference between breeds and a large extent of linkage disequilibrium within breeds. our analyses revealed strong population structure within poodles, with differences among some poodle groups as pronounced as those among other well-recognized breeds. instead, a novel set of five poodle groups was identified, defined by combinations of size and color, which is not officially recognized by the kennel clubs. there are around internationally recognized dog breeds in the world today, with a remarkable diversity in size, shape, color and behavior. such pronounced structuring within dog breeds can increase the power of association mapping studies, but also represents a serious problem if ignored. the same processes which have historically created dog breeds are still ongoing, and create further subdivision within current dog breeds. matings have not taken place at random or within traditionally identified size classes in poodles. however, genetic uniformity within breeds may not always be the case.
 there are around internationally recognized dog breeds in the world today, with a remarkable diversity in size, shape, color and behavior. breeds are considered to be uniform groups with similar physical characteristics, shaped by selection rooted in human preferences. this has led to a large genetic difference between breeds and a large extent of linkage disequilibrium within breeds. these characteristics are important for association mapping of candidate genes for diseases and therefore make dogs ideal models for gene mapping of human disorders. however, genetic uniformity within breeds may not always be the case. we studied patterns of genetic diversity within poodles and compared it to dogs from eight other breeds.  our analyses revealed strong population structure within poodles, with differences among some poodle groups as pronounced as those among other well-recognized breeds. pedigree analysis going three generations back in time confirmed that subgroups within poodles result from assortative mating imposed by breed standards as well as breeder preferences. matings have not taken place at random or within traditionally identified size classes in poodles. instead, a novel set of five poodle groups was identified, defined by combinations of size and color, which is not officially recognized by the kennel clubs. patterns of genetic diversity in other breeds suggest that assortative mating leading to fragmentation may be a common feature within many dog breeds.  the genetic structure observed in poodles is the result of local mating patterns, implying that breed fragmentation may be different in different countries. such pronounced structuring within dog breeds can increase the power of association mapping studies, but also represents a serious problem if ignored. in dog breeding, individuals are selected on the basis of morphology, behaviour, working or show purposes, as well as geographic population structure. the same processes which have historically created dog breeds are still ongoing, and create further subdivision within current dog breeds. 