Stenopus hispidus (Crustacea: Decapoda: Stenopodidea) is a popular marine ornamental species with a circumtropical distribution. The planktonic larval stage lasts ∼120–253 days, indicating considerable dispersal potential, but few studies have investigated genetic connectivity on a global scale in marine invertebrates. To resolve patterns of divergence and phylogeography of S. hispidus, we surveyed 525 bp of mitochondrial cytochrome c oxidase subunit I (COI) from 198 individuals sampled at 10 locations across ∼27,000 km of the species range. Phylogenetic analyses reveal that S. hispidus has a Western Atlantic lineage and a widely distributed Indo-Pacific lineage, separated by sequence divergence of 2.1%. Genetic diversity is much higher in the Western Atlantic  relative to the Indo-Pacific , and coalescent analyses indicate that the Indo-Pacific population expanded more recently (95% HPD (highest posterior density) = 60,000–400,000 yr) than the Western Atlantic population . Divergence of the Western Atlantic and Pacific lineages is estimated at 710,000–1.8 million years ago, which does not readily align with commonly implicated colonization events between the ocean basins. The estimated age of populations contradicts the prevailing dispersal route for tropical marine biodiversity (Indo-Pacific to Atlantic) with the oldest and most diverse population in the Atlantic, and a recent population expansion with a single common haplotype shared throughout the vast Indian and Pacific oceans. In contrast to the circumtropical fishes, this diminutive reef shrimp challenges our understanding of conventional dispersal capabilities of marine species.The banded coral shrimp,  Despite the seemingly broad continuity of the world’s oceans, relatively few marine species achieve and maintain circumtropical distributions. To do so requires high dispersal potential to sustain gene flow across large geographic expanses, and a tolerance for a broad range of habitats and environmental conditions. In a recent survey of fishes, For benthic-associated taxa, there are additional challenges to maintaining a global distribution; only 7% (18 species) of the circumtropical fishes occupy shallow benthic habitats . AlthougThe accumulation of molecular data from a diversity of taxa has advanced the longstanding goal of understanding whether and where concordance exists between population separations within species, interspecific species divergences, and biogeographic barriers in the sea . ComparaStenopus hispidus  to examine the influence of biogeographic boundaries on this circumtropical species. Within the genus Stenopus (family Stenopodidae), there are 11 species, 10 of which are range-limited by biogeographic barriers. However, the family Stenopodidae is an understudied group without a detailed phylogenetic analysis of the current taxonomy. A recent phylogeny indicates that the family is non-monophyletic, and that Stenopus is a polyphyletic genus, with at least two species, S. earlei and S. goyi, likely to be reclassified into Juxtastenopus  were collected from ten locations across the >27,000 km species range (es range  (Papahānes range  or 95% ec oxidase subunit I (COI) gene was amplified by the polymerase chain reaction (PCR) using the primers LCO1490 and HCO2198  following manufacturer’s instructions or the ‘HotSHOT’ protocol  and was  HCO2198 . PCR ampMG819599–MG819631) while associated metadata can be found in the open access repository GeOMe . Amplification products were prepared for sequencing using 0.75 units of Exonuclease I and 0.5 units of Fast Alkaline Phosphatase  per 5 µl PCR product, and incubated at 37 °C for 30 min, followed by deactivation at 85 °C for 15 min. Purified DNA fragments were sequenced in the forward direction with fluorescently-labeled dideoxy terminators on an ABI 3730XL Genetic Analyzer  at the University of Hawai‘i Advanced Studies of Genomics, Proteomics and Bioinformatics sequencing facility. Unique or ambiguous sequences were sequenced in the reverse direction to assure correct base calls. Sequences were aligned, edited, and trimmed to a uniform length using GENEIOUS PRO 6.1.8 . There were no indels, frameshift mutations, or stop codons in the alignment. Sequences were deposited in Genbank , Stenopus zanzibaricus , and Stenopus scutellatus . For some reconstructions, we also used additional outgroups that are more distantly related, Synalpheus sp. , and Alpheus bellulus   .d) between lineages in DNASP 5  from a sister genus Alpheus . A maximum clade credibility tree was constructed using TREE ANNOTATOR 1.6.0. The program FIGTREE 1.4.2 (http://tree.bio.ed.ac.uk/software/figtree/) was used to visualize the phylogeny.Since divergence rates do not exist for the genus  Alpheus  to calcus ∼3 Mya , we usedh) and nucleotide diversity (π) were calculated using the program ARLEQUIN 3.5 (Haplotype (QUIN 3.5 . Region-QUIN 3.5  with defST) was calculated between each pair of sampling locations in ARLEQUIN using the TrN . Finally, we investigated the correlation between pairwise genetic (ΦST) and geographic (km) distance (log–log transformed), within ocean basins  using Mantel tests to test for significance  on the Isolation-by-Distance Web Service 3.16  was used to test for hierarchical population genetic structure among regions in ARLEQUIN using 50,000 permutations to test for significance. Locations where sample sizes were <5  were excluded from population-level analyses but included in overall diversity estimates. Pairwise genetic differentiation . The TMRCA for the Western Atlantic lineage was ∼500,000 years , while the Indo-Pacific lineage was ∼200,000 years .We resolved a 525 bp fragment of COI for 198 individuals at 10 locations across the circumtropical species range. Phylogenetic analyses revealed two lineages that corresponded to a Western Atlantic lineage observed at Curaçao, Belize, and Panama and an Indo-Pacific lineage consisting of all sample sites in the Pacific Ocean, Red Sea and Indian Ocean  . The aved = 2.1% . Coalesch) was 0.547 with a range from 0.055 to 0.962 (π) was 0.005 with a range from <0.001 to 0.006   and the Red Sea and Indian Ocean had the highest diversity  (Molecular indices are summarized in to 0.962 . Mean nuto 0.006 . Genetic< 0.001) . If we s N = 24) .ST = 0.868 (P < 0.001). A regional analysis of molecular variance (AMOVA) indicates significant genetic differentiation among the Red Sea/Indian, Pacific, and Western Atlantic Ocean Basins  data; however, we did not have enough sampling locations to test within ocean basins.Global population structure was ΦStenopus hispidus reveals two divergent and reciprocally monophyletic mitochondrial lineages: a diverse lineage in the Western Atlantic and a depauperate Indo-Pacific lineage, predominated by a single common haplotype observed at every sample site throughout the Indo-Pacific and Red Sea. As expected for reciprocally monophyletic lineages, genetic structure was high between the Western Atlantic and any site in the Indo-Pacific, with ΦST values ranging from 0.859 to 0.976  than in the Indo-Pacific populations (h = 0.165). This result is striking, because the expectation is that the habitats in the Indo-Pacific  should support a larger population size, and in turn, much greater genetic diversity than that of the more homogeneous and smaller  Western Atlantic. Lower diversity in the Indo-Pacific could result from such factors as abundance, population structuring, sex ratios, or mating architecture, but none of these would result in a single common haplotype shared across the Red Sea, Indian and Pacific Oceans. There are two classical explanations for the lack of genetic diversity in one population relative to another. First, the Indo-Pacific population may have experienced a recent bottleneck or selective sweep, reducing the genetic variation in that population , and the extant Indo-Pacific population ∼200,000 years old . All else being equal, given a split between Western Atlantic and Indo-Pacific lineages, we would expect lower rates of haplotype loss, and greater haplotype diversification, within the vast Indo-Pacific and Red Sea than the much smaller Atlantic over the same timescale. Additional studies with nuclear markers and expanded sampling throughout the Indo-Pacific may shed light on this unexpected finding.Unexpectedly, we observed much higher genetic diversity in the Western Atlantic pulation . In thispulation . This hypulation . Second,pulation . InsteadS. hispidus, we examined a few historical biogeographic scenarios that may explain the phylogeographic relationships observed in S. hispidus. Given our TMRCA dates of the lineage split   but has been regularly documented as far east in the Pacific Ocean as Easter Island, Chile , it is possible that initial colonists of the ETP are wiped out due to a loss of suitable habitat in that region.d, Chile . To datepedition . Rare inpedition . A more pedition . Easternpedition . Similarf Africa , despiteS. hispidus was estimated at 1.2 Mya   . Even th1.8 Mya) , eliminaS. hispidus lineages is a rare dispersal event via the currents around South Africa. Currents at the southern tip of Africa have facilitated species dispersal between the Atlantic and Indian Oceans   . If S. hHeteropriacanthus cruentatus is one of few examples of a non-pelagic tropical species that has dispersed through the Benguela Current from the Atlantic into the Indian Ocean, with two divergent lineages (a Caribbean lineage and a widely distributed Indo-Pacific lineage) separated by 10% at COI , where reports of S. hispidus are rare, perhaps for the same reasons as in the ETP. If this most common Indo-Pacific haplotype also occurs in the Eastern Atlantic, S. hispidus would be the only tropical invertebrate species reported to date to follow this surprising colonization route and disperse eastward through the cold Benguela Current.Colonizing in the opposite direction, from the Atlantic Ocean to the Indian Ocean is rare, particularly for tropical marine species without large pelagic adults. The glasseye fish, % at COI . HoweverS. hispidus, there is also significant population structure across our Indo-Pacific sampling locations, spanning from the Red Sea to French Polynesia and Hawai‘i. This broad region extends over approximately half the circumference of the globe, and contains six tropical biogeographic provinces , and the Red Sea and Palmyra Atoll . However, S. hispidus does not show significant structure between the Red Sea and French Polynesia, which included individuals from Marquesas , nor among any of the non-Red Sea comparisons throughout this broad geographic area , we observed both haplotypes that were private to the Red Sea, as well as a common haplotype that was shared across the breadth of the Indo-Pacific. This pattern may indicate incomplete lineage sorting in the Red Sea population (which would implicate the Red Sea as the refuge from which the Indo-Pacific was recolonized), or a more recent colonization by the Indo-Pacific lineage into a Red Sea population formerly isolated by Pleistocene sea level fluctuations. In either case, the significant population structure suggests there is likely limited gene flow occurring between these provinces, and additional study in this portion of the range is likely to offer fruitful insights.Our data is consistent with the growing database that supports the pattern of peripheral isolation among reef fishes and invertebrates distributed across the Indo-Pacific region . Provincne phyla , and manne phyla . The evone phyla . In the S. hispidus, extending across more than 27,000 km of the globe. Despite having a circumtropical distribution and a ∼253 day PLD, one of the longest mean larval durations in the marine realm, our data shows that gene flow is not sufficient to homogenize populations across the known species range. The recovery of two distinct lineages in the Western Atlantic and Indo-Pacific calls into question whether populations in these ocean basins are in fact a single taxonomic unit. Coalescence estimates indicate the closure of the Isthmus of Panama predated the divergence between extant lineages in the Western Atlantic and Indo-Pacific by more than a million years, eliminating closure of the Isthmus as a likely isolating mechanism. Although no explanation for the observed pattern is entirely satisfactory, the weight of available evidence points to the Indo-Pacific lineage arising by a rare dispersal event from the Atlantic around South Africa via the Benguela Current, thereby colonizing the Indian and Pacific Oceans, followed by dispersal across the Indo-Pacific in the last 200,000 years. Surprisingly, this benthic coral reef associated shrimp shows a single haplotype dominating the largest continuous tropical oceanic expanse on the planet—a result not seen in any other globally distributed marine species, including such highly mobile species as whale sharks and tunas  of Stenopus hispidus with sister species  and sister genera  as outgroups . The Indo-Pacific lineage of S. hispidus is shown in blue . Values above nodes are median node ages with 95% HPD intervals represented by blue node bars.A Bayesian time-calibrated phylogeny based on the cytochrome utgroups  (525 bp)Click here for additional data file.10.7717/peerj.4409/supp-2Figure S2(A) The Western Atlantic shows a relatively stable population whereas the (B) Overall Dataset with the Pacific population included shows evidence of a recent population expansion.Click here for additional data file.10.7717/peerj.4409/supp-3Supplemental Information 1Stenopus hispidus.FASTA file of unique sequences for Click here for additional data file.


Pure breeding of dogs has led to over 700 heritable disorders, of which almost 300 are Mendelian in nature. Seventy percent of the characterized mutations have an autosomal recessive mode of inheritance, indicative of positive selection during bouts of inbreeding primarily for new desired conformational traits. Samoyed suffer from several common complex genetic disorders, but up to this time only two X-linked and one autosomal dominant disorder have been identified. Previous studies based on pedigrees and SNP arrays have concluded that Samoyed breeders have done a good job in maintaining genetic diversity and avoiding excessive inbreeding. This may explain why autosomal recessive disorders have not occurred to the extent observed in many other breeds. However, an enamel hypoplasia analogous to a form of autosomal recessive amelogenesis imperfecta (ARAI) in humans has been recently characterized in Samoyed, although the causative mutation appears to have existed for three or more decades. The rise of such a mutation indicates that bouts of inbreeding for desired conformational traits are still occurring despite an old and well-defined breed standard. Therefore, the present study has two objectives: 1) measure genetic diversity in the breed using DNA and short tandem repeats (STR), and 2) identify the exact mutation responsible for enamel hypoplasia in the breed, possible explanations for its recent spread, and the effect of eliminating the mutation on existing genetic diversity.Canis familiaris autosome 8 (CFA8). The region contained a gene in the solute carrier 24 family (SCL24A4) that encodes a protein involved in potassium dependent sodium/calcium exchange and transport. Mutations in this gene were recently found to cause a similar type of enamel hypoplasia in people. Sequencing of this candidate gene revealed a 21 bp duplication in exon 17. A test for the duplication was in concordance with the disease phenotype. The exact incidence of affected dogs is unknown, but 12% of the 168 healthy dogs tested were heterozygous for the mutation. This population was biased toward close relatives, so a liberal estimate of the incidence of affected dogs in the breed would be around 3.6/1000. Theoretical calculations based on the comparison of the whole population with a population devoid of carriers indicated that eliminating the trait would not affect existing genetic diversity at this time.The recent discovery of an autosomal recessive amelogenesis imperfecta (ARAI) in Samoyed provides an opportunity to study the mutation as well as genetic factors that favored its occurrence and subsequent spread. The first step in the study was to use 33 short tandem repeat (STR) loci on 25/38 autosomes and seven STRs across the dog leukocyte antigen (DLA) class I and II regions on CFA12 to determine the DNA-based genetic profile of 182 individuals from North America, Europe and Australia. Samoyed from the three continents constituted a single breed with only slight genetic differences. Breed-wide genetic diversity was low, most likely from a small founder population and subsequent artificial genetic bottlenecks. Two alleles at each autosome locus occurred in 70–95% of the dogs and 54% of alleles were homozygous. The number of DLA class I and II haplotypes was also low and three class I and two class II haplotypes occurred in 80–90% of individuals. Therefore, most Samoyed belong to two lines, with most dogs possessing a minority of existing genetic diversity and a minority of dogs containing a majority of diversity. Although contemporary Samoyed lack genetic diversity, the bulk of parents are as unrelated as possible with smaller subpopulations either more inbred or outbred than the total population. A familial disorder manifested by hypocalcification of enamel has been recently identified. A genome wide association study (GWAS) on seven affected and five unrelated healthy dogs pointed to a region of extended homozygosity on The contemporary Samoyed, like many other breeds, has retained only a small portion of the genetic diversity that exists among all dogs. This limited genetic diversity along with positive genetic selection for desirable traits has led to at least three simple non-recessive genetic disorders and a low incidence of complex genetic traits such as autoimmune disease and hip dysplasia. Unlike many other pure breeds, the Samoyed has been spared the spate of deleterious autosomal recessive traits that have plagued many other pure breeds. However, ARAI due to a mutation in the SCL24A4 gene has apparently existed in the breed for several decades but is being increasingly diagnosed. The increase in diseased dogs is most likely due to a period of intensified positive selection for some desired conformational trait. A genetic test has been developed for identifying the mutation carriers which will enable the breeders to eliminate enamel hypoplasia in Samoyed by selective breeding and it appears that this mutation can be eliminated now without loss of genetic diversity. Their ancestry is from indigenous Laika of Siberia and Russia. Samoyed are now of average popularity but have seen significant population declines during WWI and a marked population increase in the 1980s and 1990s in the UK followed by an even more precipitous decline to present. Although previous pedigree and SNP-based testing suggest that the breed is not in any immediate danger, the present study indicates that genetic diversity may not be as great as presumed and that inbreeding is still a concern. Lack of genetic diversity coupled with bouts of inbreeding has led to a high incidence of complex genetic disorders and a high proportion of deleterious mutations with a recessive mode of inheritance in most dog breeds. Samoyed do suffer from several complex genetic disorders and two simple X-linked and one dominant conditions, but Samoyed have been surprisingly spared the autosomal recessive disorders that have plagued other breeds. However, a dental condition known as enamel hypoplasia has been recently recognized in the breed and although the causative mutation has apparently existed for many generations, the disorder is being diagnosed with increasing frequency. The incidence of carriers of the mutation appears to be low, which should allow breeders to eliminate the trait from the total population without further loss of genetic diversity. However, Samoyed breeders must take care that no further genetic diversity is lost and be prepared for other deleterious autosomal recessive traits that can result from conformation-driven bouts of strong human-directed positive selection. Pedigree derived breeding information should also be confirmed and augmented by DNA-based testing.Samoyed evolved in the later part of the 19,,,,,th century.8 Fig. The Samoyed is considered one of the basal breeds developed in the late Victorian era from indigenous Laika used by the Nenets (Samoyede) peoples of Northwest Russia and Siberia. Like many pure breeds, Samoyed have a convoluted history with several versions .1,2,1–5,9 However, it was the white dogs that he encountered in his travels that caught his fancy and led them to purchase a cream-colored bitch named Whitey Petchora [The rise of the Samoyed in America and other countries was preceded in the UK by the activities of Sir Ernest Kilburn-Scott and his wife Clara.7 Moustan was given to the countess as a gift and brought along with three other Samoyed to the USA in 1904. Moustan was shown extensively in America and the breed recognized by the AKC in 1906.The Kilburn-Scott’s showed their dogs in the foreign dog classes in the UK for several years and the breed was given conditional registration in 1902 and formal recognition under the original name Samoyede in 1912 by the UK kennel club. The name was later changed to Samoyed by both the AKC and UKC. The American connection was stimulated when a member of a famous European royal family, Rose de Mercy-Argenteau (Princess de Montglyon), was smitten with a large white champion Russian Samoyed named Moustan entered into the 1902 St. Petersburg dog show by Grand Duke Michael of Russia.4The Samoyed breed has retained an average popularity compared to other breeds over the century and registration numbers have been relatively steady in both the UK and USA except for a brief decline during WWI and a sharp rise after 1980 to a peak of 1200 registrants in the UK in 1995 and decline to pre-peak levels of 300 by 2014.,,11 There are only three simple deleterious genetic disorders in Samoyed with defined causes, X-linked glomerulopathy [Several diseases that appear to be heritable, but not genetically defined, have been observed at low frequency in the breed.ulopathy , X-linkeulopathy , and an ulopathy , 7.Except for two simple X-linked and dominant genetic disorders, Samoyed have been surprisingly free of deleterious autosomal recessive traits, which are frequent indicators of artificial genetic bottlenecks and popular sire effects and the bane of many other breeds. Over three hundred heritable disorders not linked to any breed standard were identified among the top 50 pure breeds of dogs as of 2010  and manyDeleterious autosomal recessive mutations occur with some frequency in large populations in nature, but they either remain at low frequency or eventually disappear. However, simple autosomal recessive mutations can rapidly amplify in small populations (such as dog breeds) if the defective allele is linked to regions of the genome under strong positive selection. In the case of many pure breeds of dogs, this strong positive selection is human directed towards conformation traits that are extolled by the show ring , 10. TheThe amount of genetic diversity in contemporary dog breeds reflects several factors. One factor is the number of founder animals that went into creation of the prototypic Samoyeds before the breed was recognized and its registry closed to outside dogs. If the founder population is large and genetically diverse, the prototypic dogs that are selected as breed founders will reflect that diversity. Once the registry is closed to outside dogs, the amount of genetic diversity of the new pure breed cannot theoretically increase except by rare natural mutation. Therefore, the goal of proper pure breeding is to select only the healthiest dogs and maintain the original genetic diversity and health for the rest of the breed’s existence. This requires random or careful selection of sires and dams. A second factor involves a loss of genetic diversity after the registry has been closed. It has been calculated that the average pure breed of dog retains only 87% of its original diversity , which iPositive selection for conformation traits has a much greater effect on genetic diversity than breeding for performance traits . PerformThere is no doubt that a show judge’s interpretation of ideal conformation affects how breeders select the parents of subsequent generations of puppies . HoweverSamoyed breeders have not recognized any deleterious traits caused by autosomal recessive mutations up to this time, which has been reassuring as most breeds suffer from several recessive disorders , 10. Hown = 32) and Australia (n = 6). Fourteen dogs were clinically affected, including 11 from the USA and three from Europe. Samples were solicited through web communications and owners/breeders wishing to submit DNA for testing were asked to make contact and request a DNA test kit that contained 2–3 cytology brushes for each dog. Further DNA was required from some cases in order to do extra investigations, so blood samples were collected by the veterinarian looking after those cases. Owners were encouraged to list each dogs’ registration number, registered name, call name of sire and dam, age, sex , coat color, whether affected or not, existence of other health conditions, and relationship if known to affected dogs. Digital photographs of teeth and pedigrees were also requested whenever possible.The 182 Samoyed in the study were from the North America (North America = 144), Europe  . Blood s13 and a second consisting of two di-STRs and 10/15 tetra-STRs validated for forensic testing [Thirty-three STR loci from across the canine genome were multiplexed into two panels, one consisting of 20/21 di-STRs recommended for canine parentage verification by the International Society of Animal Genetics (ISAG) testing . Ameloge testing . ExampleDLA88) and three STRs associated with DLA class II  were identified on Dogset.Four dinucleotide STRs from regions flanking the DLA class I  reflects the relationship of an individual’s parents as described by Amos et al.  and basePraw. The thresholds for final genome-wide significance was determined by MaxT permutation testing using 100,000 permutations with PLINK.Among the 182 Samoyed sampled, 7 dogs with enamel hypoplasia and 5 healthy dogs were selected for genome-wide association study (GWAS) based on clinical criteria described herein. SNP genotyping was performed at GeneSeek  with the Illumina  CanineHD Genotyping BeadChip containing 230 K markers placed on the CanFam3 reference sequence and the results were analyzed using PLINK . Data from GWAS was analyzed subjected to Bonferroni correction to account for multiple comparisons. The strongest signal from the unadjusted association analysis was termed SLC24A4 was conducted on genomic DNA from 4 Samoyed dogs (2 affected and 2 control). The complete sequence of SLC24A4 is publicly available and can be found on chromosome 8 at positions 1,507,274–1,679,208.The genetic analysis of 2+ at 95 °C for 5 min, and 85 °C for 5 min followed by 5 cycles of 94 °C × 1 min, 60 °C × 30 s, 72 °C × 30 s, and another 28 cycles of 94 °C × 45 s, 60 °C × 30 s, 72 °C × 30 s followed by 72 °C for 30 min using Taq DNA polymerase (Denville Scientific), and electrophoretically separated on an ABI DNA analyzer (Applied Biosystems). The predicted size of the wild type allele was 92 bp and 113 bp for the insertion mutant, which was verified using the software STRand [To confirm the identified mutation as causative, all 182 Samoyed, which included those used for GWAS, were genotyped for a 21 bp duplication in exon 17. A PCR reaction with SLC24A4-F (FAM labeled) SLC24A4-R primers  for the 182 dogs was 0.615, which was somewhat lower than the expected heterozygosity (He) of 0.641 that would be anticipated for a population that is in Hardy-Weinberg equilibrium . The difference in Ho and He led to a fixation index (F) (a measure of inbreeding) of +0.041. This indicated that a small subpopulation of the 182 dogs was more inbred (less heterogeneous) than the total population.Principal coordinate analysis was used to approximate the relatedness of individuals in a population to each other using the allele frequency data obtained from the 33 autosomal STRs Table . The actInternal relatedness (IR) was also calculated from allele and allele frequencies of 182 Samoyed obtained from analysis of the 33 autosomal STRs. Internal relatedness is an indirect measurement of how related a dog’s parents were to each other. Individual IR values can then be graphed to show the average IR values for the population  individual among the 182 dogs had an IR score of −0.252 and the most inbred (most related parents) dog had an IR score of +0.502 Table . The aveInternal relatedness scores can be used to approximate how much genetic diversity has been lost during a breed’s evolution by comparing their allele frequencies with the incidence of the same alleles in contemporary village  dogs. The assumption would be that contemporary free-roaming dogs and randomly breeding village from Lebanon, Iran, Taiwan, Thailand, Philippines, Brunei, Cook Islands and Bali  would po14Thirteen STR-associated DLA class I haplotypes were identified among the 182 Samoyed Table . Nine of14 Seventy-two percent of Samoyed possess the 2053 (shared) or 2095 (unique) DLA class II haplotypes.Twelve class II haplotypes were identified in Samoyed and three of these have not yet been identified in any other breed Table . The dom14 The total number of different DLA class I and II haplotypes differed among North American, European and Australian Samoyed as a reflection of population size. However, the most common haplotypes were shared in all three populations, while minor haplotypes were missing in in the smaller populations (data not shown).The number of class I and II haplotypes is about average for pure breeds that have been studied to date by our group.Although it appeared that Samoyed were being purposely selected for certain DLA class I and haplotypes, the comparative incidence of haplotypes does not reflect how randomly these specific haplotypes were segregating in the population. Therefore, a standard genetic assessment was made of the allele and allele frequencies of the seven loci associated with the DLA class I and II regions Table . The Na Fourteen affected dogs, 11 from the USA and 3 from Europe, were identified for this study. The disease was characterized by one of the authors (BS), and with pictures provided by some breeders and owners.The deciduous teeth in Samoyed enamel hypoplasia are normal, but abnormalities are apparent in adult teeth immediately upon eruption. An early sign of the disorder is bad breath. Figure −5, however the adjusted association was below significance (Pgenome = 0.256)  known to cause enamel hypoplasia in humans , an asynonymous change (C to T) in exon 12 changing amino acid in exon 12 from proline to leucine, and a 21 bp insertion in exon 17 were identified  dogs that were presumed to have enamel hypoplasia based on physical examination and DNA testing all were homozygous for the mutation. Twenty of 168 (12%) heathy dogs were found to be heterologous for the mutation and most were parents or known close relatives of affected dogs.A test was developed for detecting the 21 bp duplication in exon 17 of Enamel hypoplasia affected and carrier dogs were compared to the healthy Samoyed by PCoA Fig. . The anaBreeders have three options when faced with a deleterious autosomal recessive mutation: 1) do nothing, 2) eliminate the mutation from the entire breeding population through testing, and 3) avoid producing homozygous puppies by testing and breeding affected and carrier dogs only to normal dogs. The first option only applies to a large and genetically diverse population with a very low incidence of carriers that is under strict random breeding, thus keeping the number of affected dogs low. Option two is preferred when a test for the mutation is available, the carrier incidence is not high, and there is sufficient genetic diversity to avoid losing diversity in the process. The third option is preferable when genetic diversity is low, the population size is small, the incidence of the mutation is high, and the loss of diversity would be great. The best approach can be easily determined by measuring changes in basic genetic parameters when the affected and carriers are removed from the computation. Table 14 STRs are also economical and can work with limited amounts of DNA and even poor quality DNA. Unlike STRs, SNPs cannot interrogate highly polymorphic regions such as the DLA. Therefore, genetic diversity in the present study was determined with small panels of 33 STR loci on 25/38 autosomes and 7 STRs defining the DLA class I and II regions on CFA12.The objective of this study was to characterize an autosomal recessive mutation that appeared in Samoyed and to determine how genetic diversity and inbreeding may have contributed to its origin, spread, and potential effect on existing diversity. Therefore, genetic diversity in contemporary Samoyed was first evaluated. Genetic diversity can be measured from pedigrees, but if mating is non-random, their accuracy and quantity must be high . Pedigre18 Sorsa,It is uncertain how many dogs constituted the founding population for the Samoyed, although an analysis of pedigrees from dogs registered in the USA from 1990 to 1999 by Bell (2002)10 There were about 200 Samoyed registered in the UK in 1980, but this rapidly increased to a peak of over 1200 dogs by 1995. The breed appeared to lose popularity after this time and only about 300 Samoyed were registered in 2014. The population increase was closely linked to the number of puppies produced each year by a single sire.10 A single sire averaged 2.32 puppies in 1980, 10.56 offspring in 1994 and that dropped back to 7.05 in 2014. An observed and expected inbreeding coefficient was calculated and graphed for the period 1980–2014.10 The inbreeding coefficient is the probability that two alleles at a given gene locus in random members of a population are identical. In 1980, the observed inbreeding coefficient was 0.022, while the expected inbreeding coefficient was around 0.015.10 The difference was 0.007, or a 0.7% rise in identical alleles in the population prior to the population bubble starting in 1980. In contrast, the observed inbreeding coefficient at the peak of the population in 2001 was 0.11 and the expected inbreeding coefficient to 0.06, a calculated difference of 0.05. Therefore, allele sharing based on these figures increased by 4.3% (0.05–0.007) from 1989 to 2001, while the observed coefficient of inbreeding dropped to 0.08 in 2014 as the population numbers declined. Using the figures of this study,10 the expected coefficient of inbreeding at 2014 was around 0.06, a difference of 0.02, indicating a trend towards more random breeding. Overall, allele sharing among UK Samoyed increased from 0.7% in 1980 to 5% in 2001 and back to 0.2% in 2014. The author of UK kennel club survey concluded: “As with most breeds, the rate of inbreeding was at its highest in this breed in the 1980s and 1990s. This represents a ‘genetic bottleneck’, with genetic variation lost from the population. However, since 2000 the rate of inbreeding has slowed and even declined slightly, implying maintenance and even some replenishment of genetic diversity .”10 There are two problems with this conclusion. Inbreeding and genetic variation (diversity) are different things. It is possible to expand a portion of the population by rapid inbreeding without losing any original genetic diversity. Inbreeding can be reversed if even remnants of original diversity survive, but genetic diversity that is permanently lost through inbreeding can never be replaced from within the population. The conclusion of the UK Kennel Club study was also misleading because it implies that allele sharing at each locus is not a problem in the breed. Based on DNA testing, the average percentage of homozygous alleles across the 33 autosomal STR loci in contemporary Samoyed was 39% (SD = 9.5%), while the average percentage of homozygous alleles was 54% (SD = 38%) for the 7 STR loci in the DLA class I and II regions. Therefore, allele sharing in Samoyed is greater than the percentages indicated by this 1980–2014 population study by the UK Kennel Club. Given a high level of pre-existing allele sharing, the loss of even small amounts of heterozygosity in such population bubbles can have a dramatic effect. Such bouts of inbreeding are also times when new mutations occur and/or when existing deleterious mutations are amplified to clinical levels. It is possible, given the timelines that the causative mutation for ARAI in Samoyed occurred during this population bubble.The most extensive population and genetic study of the Samoyed was reported by the UK kennel club for the period between 1980 and 2014.The increased inbreeding observed during the 1980s and 1990s is typical of what occurs when a breed becomes popular and the need for puppies and their value greatly increases. Professional breeders usually blame commercial breeders for the great increase in inbred dogs during such periods, and there is a measure of truth to this belief. This is exemplified by what happens when popularity declines. Commercial breeders leave the market and professional breeders tend to return to more random breeding practices.10 Effective population size, founder equivalents and founder genome equivalents are related terms that usually refer to the “number of equally contributing (randomly breeding) founders that would be expected to produce (maintain) the same genetic diversity as in the population under study [The same UK Kennel club survey also provided an estimate of the effective population size for Samoyed of 64.7 dogs.er study .” The quer study . The “50er study . Nonethe14 The amount of genetic diversity found in contemporary Samoyed was greater than our group found in breeds such as Flat-coated retrievers, Doberman, and Bulldog; similar to the Alaskan Klee Kai, Biewer, and Black Russian Terrier; and less than Standard and Miniature Poodle, Havanese, Akita, Golden Retriever, and Italian Greyhound.14 There was evidence for some geographic differentiation of European and Australian dogs but the numbers, especially of Australian dogs were too small to confirm this finding. However, this differentiation was only slight and far less identifiable than between Japanese and American Akita as tested by our laboratoryThe present studies confirm and expand upon the findings of others regarding genetic diversity in the Samoyed. One hundred eighty-two dogs from different geographic regions of the world should provide a reliable measure, based on other breeds studied by our group, of the incidence of all major and most minor alleles present at each of the 33 autosomal STR loci and comparative incidence of DLA class I and II haplotypes.14 The major 2053 haplotype was also found in other breeds, while the Samoyed possess three unique class II haplotypes. These unique and/or major haplotypes are most certain to belong to founder dogs that had features strongly engrained in the breed standard and have been diligently maintained. A standard genetic assessment of the 7 STR loci associated with these haplotypes indicates that these haplotypes, although highly unbalanced in proportion, are largely in breed-wide equilibrium.Allele frequencies at each of the 33 autosomal STR loci indicated that 70–90% of the population was closely related to each other. Without accurate information on breed founders, it is not possible to say whether there were few founders at the start or that many founders were lost as the breed evolved over the last century. The DLA haplotypes confirmed that the contemporary Samoyed has been heavily selected for two lineages. The DLA region is in strong linkage disequilibrium and each class I and II haplotype is inherited largely unchanged over a long period of time from sire and dam. About 90% of all Samoyed tested shared three class I and four class II haplotypes. One class I (1152) and one class II (2053) haplotypes were found in over 50% of the dogs. The major 1152 haplotype along with three less common haplotypes were unique to the breed among those identified to date by our group.It is uncertain when the first case of enamel hypoplasia occurred in the breed, although some recall possible cases as far back as 1985. Based on the oldest age of affected dogs, the causative mutation has been present for several generations; the oldest affected dog was 11 years of age and assuming the parents were 4–5 years old at its birth. Both parents also had to be either heterozygous or homozygous for the mutation, pushing the timeline back several more generations. Although the mutation may be old, the occurrence of diseased dogs has been apparently increasing of late. This suggests that the mutation has been under recent positive selection, most likely in association with a desired trait. There are rumors that an affected popular sire may be involved, but this was not pursued in the present study. However, this is where pedigrees associated with DNA testing could be highly informative as to the precise origin of the mutation and the cause for its recent spread. Detective work of this type was used to trace the origins of sebaceous adenitis and Addison’s disease in the Standard Poodle . The exaOnline Mendelian Inheritance in Man as having an enamel phenotype, and of those, 71 have a known molecular etiology or linked genetic loci. The complexity of enamel formation allows for mutations in many different genes to cause a similar defect. Therefore, it is not surprising that enamel hypoplasia has been observed in many breeds, although only one other mutation causing a heritable enamel hypoplasia has been characterized to date, an ARAI involving the enamelin gene (ENAM) in Italian Greyhounds [Enamel formation is a complex process involving many genes working in unison and at various stages of tooth development. Wright and colleagues  listed 9eyhounds . One reaeyhounds . The enaeyhounds . An eveneyhounds . The comeyhounds .SCL24A on CFA8 and in strong linkage disequilibrium were ultimately identified in ARAI affected dogs, an asynonymous change (C to T) in exon 12 changing amino acid proline to leucine and a 21 bp duplication in exon 17. The leucine to proline change in exon 12 has been reported in GenBank in golden snub-nosed monkey  and camel  and was assumed to be a polymorphism present in normal animals and not deleterious. The insertion in exon 17 that caused a 21 bp insertion was in the terminal transmembrane region of SLC24A4 and would be more likely to inhibit function of the 4SCL24A4 protein, which transports one Ca++ and K+ ion in exchange for four Na + ions [Two potential deleterious mutations in a + ions . A dupliSCL24A4 mutations was highly conserved among all the affected Samoyed tested, indicating that the region possesses many genes and gene polymorphisms that define the breed phenotype in some manner. It is also likely that a polymorphism that was deemed favorable to the breed, most likely involving a conformational trait, also occurred in or near this same region and that it was amplified as well. The number of these selective sweeps varies greatly in number and size between breeds. However, sweeps become larger and more numerous as the phenotype deviates more and more from the ancestral dog. As such, the Bulldog and Bull Mastiff have numerous large selective sweeps [The large run of homozygosity (selective sweep) containing these e sweeps , 39, whie sweeps , 40. Thee sweeps . If the e sweeps , 14, 16.SLC24A4 has only been recently identified as a cause of amelogenesis imperfecta (AI) in humans. Parry and colleagues [SLC24A4 causing a leucine to arginine switch at position 436 and closely resembles the clinical appearance of the enamel hypoplasia in Samoyed [lleagues  identifilleagues  describeWhat should breeders do when a genetic mutation such as this occurs in their breed? The answer depends on how much genetic diversity will be lost in the attempt to eliminate it from the breed. In the case of Pug Dog Encephalitis, the recessive heritable associations to risk for the disease were present in one third of the dogs and the breed was limited in diversity . TherefoBased upon the analysis of autosomal and DLA-related STRs, Samoyed have a lower level of genetic diversity than estimated from prior pedigree or SNP-based studies. Eighty percent of the 182 dogs tested shared two alleles at each of 33 autosomal loci and three to four DLA class I and II haplotypes. This lack of genetic diversity, when coupled with bouts of human-directed artificial selection for favorable phenotypic traits, may have encouraged the appearance of a deleterious genetic disorder. A novel form of heritable enamel hypoplasia has been detected with increasing frequency in Samoyed and the causative autosomal recessive mutation has been characterized. The mutation appears to be many generations old, but recently amplified by positive selection for a linked desired trait. A genetic test has been developed for identifying the carriers, which will enable the breeders to eliminate the disorder by selective breeding.


Vigna unguiculata L. Walp) is an important legume crop due to its high protein content, adaptation to heat and drought and capacity to fix nitrogen. Europe has a deficit of cowpea production. Knowledge of genetic diversity among cowpea landraces is important for the preservation of local varieties and is the basis to obtain improved varieties. The aims of this study were to explore diversity and the genetic structure of a set of Iberian Peninsula cowpea accessions in comparison to a worldwide collection and to infer possible dispersion routes of cultivated cowpea.Cowpea  contains supplementary material, which is available to authorized users. Vigna unguiculata L. Walp., 2n = 2× = 22) is a member of the Fabaceae family and one of the most important grain legumes growing in tropical and subtropical regions [unguiculata while vegetable cowpea, commonly known as asparagus bean or ‘yardlong’ bean, belongs to subspecies sesquipedalis [Cowpea , and 53 accessions from genebanks at the National Institute for Agrarian and Veterinarian Research , the National Plant Genetic Resources Centre-National Institute for Agricultural and Food Technology Research , the Leibniz Institute of Plant Genetics and Crop Plant Research , the Botanic Garden Meise (Belgium), the University of Perugia , and the Brazilian Agricultural Research Corporation . These 53 accessions were chosen to represent worldwide cowpea diversity  agarose gel, stained with ethidium bromide. Equal amounts of the three DNA samples of each accession were bulked for genotyping to get a better estimation of diversity within each accession/bulk.Leaves from three individual plants of each accession were collected. Total genomic DNA from each plant was extracted from 50 mg of well-developed trifoliate leaves (two-weeks-old) with the NucleoSpin® Plant II kit  using the Lysis Buffer 1 (based on the CTAB method) and the standard protocol according to the manufacturer’s instructions. DNA concentrations were measured using a NanoDrop 1000 . In order to verify DNA integrity, 2 μL of DNA were subjected to gel electrophoresis on 1.0% . SNP can et al. . Qualityn et al. , 21, 22.K) between 1 and 8 using a burn-in period of 5000 iterations and a run length of 5000 Monte Carlo Markov Chain (MCMC) iterations. LnP(D) and ΔK values [K, a new run using a burn-in period of 100,000 and 100,000 MCMC was performed to assign accessions to subpopulations. Those accessions with a membership probability lower than 0.70 of belonging to one subpopulation were assigned to an ‘admixed’ group.Population structure was estimated using the Bayesian model-based approach implemented in the software STRUCTURE v2.3.4  and by PK values  were ploK values . After ePrincipal Component Analysis (PCA) was conducted in TASSEL v.5  on the sA neighbor-joining (NJ) tree was generated based on Manhattan distances using the R package “Phyclust” .He) and polymorphism information content (PIC) [V. unguiculata ssp. unguiculata accessions and then separately for Iberian Peninsula accessions and for the worldwide set of accessions as in Muñoz-Amatriaín et al. [Expected heterozygosity (nt (PIC)  were caln et al. .V. unguiculata ssp. unguiculata accessions from Iberian Peninsula based on simple matching coefficient .SNP data were used to generate a similarity matrix between A high-density genotyping array containing 51,128 SNPs  was usedsesquipedalis that were obtained from the National Plant Genetic Resources Centre-National Institute for Agricultural and Food Technology Research  genebank. This identity was also apparent at the phenotypic level .A total of 44,056 good-quality polymorphic SNPs and 91 samples were used for further analysis. Pairwise SNP comparisons among accessions showed that Ac39 and Ac43 were potentially duplicates . These two accessions are members of ssp. Genetic structure in the entire population of 91 accessions was evaluated using STRUCTURE v.2.3.5 , principK), from 1 to 8, reached a maximum at K = 4  consisted of ssp. unguiculata accessions  and the fact that grain-type cowpea (ssp. unguiculata) is the most cultivated and consumed in Europe, ssp. sesquipedalis accessions were not included in these analyses. Most of the 35 V. unguiculata ssp. unguiculata accessions from the Iberian Peninsula belonged to subpopulation 2, together with other Genebank accessions from Europe  and those from the world-wide collection . This indicates that genetic diversity in Iberian Peninsula ssp. unguiculata accessions is low compared to the diversity available in the world-wide sample of cultivated cowpeas. To better understand and compare accessions from the Iberian Peninsula at the genetic level, similarity matrix was generated based on comparisons between all 35 accessions  genebank, and their passport information is limited. Ac39 and Ac43 are both from Spain, but from two different regions: Ac1 is from Cordoba  and Ac43 from Ourense . A common cause of redundant accessions is the unwitting submission of the same accession to the genebank, then generating more than one name or designator. Identifying these redundant accessions is not possible using phenotype data alone [The SNP genotyping of these accessions enabled identification of one apparent duplication: Ac39 and Ac43, which are members of the subspecies ta alone . Duplicata alone .V. unguiculata ssp. sesquipedalis accessions (subpopulation 1). The aforementioned studies may not have identified those two populations because of a lack of accessions from these regions.The population structure analysis assigned the 91 accession to four subpopulations. In agreement with the results of Huynh et al.  and Xionunguiculata subpopulations enabled inference of possible dispersion routes of domesticated cowpea Additional file 2:K calculations for each number of K (right). (XLSX 12 kb)Raw STRUCTURE output for all runs (left) and ΔAdditional file 3:K) in the entire dataset. Plots were generated with Structure Harvester [K between 1 and 8. (B) ΔK values as a function of K. (TIFF 75 kb)Exploration of the optimal number of subpopulations (arvester . (A) EstAdditional file 4:Genetic structure information on the 91 accessions. The estimated membership of each accession in the four subpopulations is shown, as well as the PCA coordinates. (XLSX 17 kb)Additional file 5:Genomic location of unique alleles in Ac13, Ac5 and Ac38 on cowpea linkage groups (LGs). Genomic regions colored in red contain unique alleles in the corresponding accession, while regions containing non-unique alleles are represented in blue. For the figure, one marker per locus was kept, giving priority to unique alleles over non-unique ones. In white are represented regions lacking mapped SNPs. LG number and cM positions are based on the cowpea consensus genetic map available from Muñoz-Amatriaín et al. .


Micromeria from the Canary Islands by extensively studying their phylogeographic pattern based on 15 microsatellite loci and 945 samples. These results are interpreted according to the hypotheses outlined above.Especially on islands closer to the mainland, such as the Canary Islands, different lineages that originated by multiple colonization events could have merged by hybridization, which then could have promoted radiation events . This is an alternative to the scenario where evolution is mostly driven by drift . In the former case hybridization should be reflected in the genetic structure and diversity patterns of island species. In the present work we investigate Micromeria species and supported their current classification. We found that populations on younger islands were significantly more genetically diverse and less differentiated than those on older islands. Moreover, we found that genetic distance on younger islands was in accordance with an isolation-by-distance pattern, while on the older islands this was not the case. We also found evidence of introgression among species and islands.Genetic structure assessment allowed us to genetically differentiate most These results are congruent with a scenario of multiple colonizations during the expansion onto new islands. Hybridization contributes to the grouping of multiple lineages into highly diverse populations. Thus, in our case, islands receive several colonization events from different sources, which are combined into sink populations. This mechanism is in accordance with the surfing syngameon hypothesis. Contrary to the surfing syngameon current form, our results may reflect a slightly different effect: hybridization might always be related to colonization within the archipelago as well, making initial genetic diversity to be high to begin with. Thus the emergence of new islands promotes multiple colonization events, contributing to the establishment of hybrid swarms that may enhance adaptive ability and radiation events. With time, population sizes grow and niches start to fill. Consequently, gene-flow is not as effective at maintaining the species syngameon, which allows genetic differentiation and reproductive isolation to be established between species. This process contributes to an even further decrease in gene-flow between species.The online version of this article (doi:10.1186/s12862-017-1031-y) contains supplementary material, which is available to authorized users. Oceanic islands have always played an important role in evolutionary biology research . The higMicromeria (Lamiaceae) in Tenerife [The predictions of the surfing syngameon hypothesis are apparently in accordance with the distribution of genetic diversity across the Canary Islands, where genetic diversity is negatively related and differentiation positively related with island distance to the mainland when considering the overall genetic diversity per species per island . The CanTenerife , 23.Micromeria, covering the whole Canarian archipelago, to investigate how inter-island colonization influenced the distribution of population diversity and pair-wise population differentiation throughout the archipelago. If multiple colonization and hybridization do not occur with high frequency, we would expect a decrease in genetic diversity from the older islands to the most recently colonized islands. Since the older eastern islands are expected to have been colonized first, they would be significantly more diverse than the younger western islands. In addition, because the likelihood of gene-flow from the mainland is lower in younger islands, genetic differentiation should be higher. In an alternative scenario, hybrid swarm creation would be more recent in younger islands, leading to a high diversity but higher homogeneity of populations, and genetic differentiation should decrease from the older to the younger islands. In the latter case the formation of the syngameon would have a higher significance during the diversification within the archipelago. Moreover, if single colonization events prevail, we do not expect to find gene-flow between species on different islands.Here, we expand our previous microsatellite study  to incluMicromeria on the Canary Islands. We interpret these results in light of the surfing syngameon hypothesis. The expectations outlined by Caujapé-Castells [Here, these expectations are tested by investigating genetic structure, gene-flow among species on and between islands, and patterns of genetic diversity and differentiation of Castells  are not Micromeria Benth.  is composed of shrubs, subshrubs and herbs with monoecious flowers pollinated by insects. Seed dispersal happens mostly by wind, but ants and water also contribute to this process [Micromeria on the Canary Islands archipelago [M. lepida and M. gomerensis are part of the eastern lineage, while M. pedro-luisii is part of the western lineage [ process , 24. It  process  from whi process , 27. Thehipelago , 29. OneMicromeria species from the Canary Islands were previously described by Pérez de Paz [M. varia). These taxa occupy all islands except La Palma and have been recently separated based on molecular phylogenetic analyses [z de Paz  based onz de Paz , 27 usinanalyses , 27, 29 M. varia in Lanzarote and Gran Canaria as well as one of the subspecies from La Gomera form a monophyletic group, while the remaining ones are scattered in the phylogeny [M. varia s.s. on Tenerife is genetically and morphologically more similar to M. hyssopifolia [M. hyssopifolia and all species previously classified as M. varia (M. varia s.l.); (ii) by using only individuals from one lineage we compared the diversity among individuals belonging to the same monophyletic group; and (iii) by using the whole archipelago we assessed the diversity among all terminal branches of the Micromeria phylogeny.Populations from the species previously classified as hylogeny . It was opifolia . This vaMicromeria species, eight and seven respectively. La Gomera presents three species and the remaining islands one species each. All Micromeria species are single island endemics. In Tenerife, it has been suggested that the composition of species of Micromeria is linked to the geological history of the island [M. densiflora to Teno and M. rivas-martinezii and M. glomerata to Anaga. A fourth species, M. teneriffae, is also growing on the Anaga palaeo-island, but its range extends towards the southeast of the island up to Fasnia. Molecular studies suggest that these four species are older lineages that evolved before the connection of the three palaeo-islands by the formation of the central shield [Micromeria varia s.s. presents a disjoint distribution growing on the palaeo-islands of Anaga and Teno, though molecular studies suggest it has a more recent lineage [M. hyssopifolia growing throughout the island from the coast up to the pine forest, M. lachnophylla from the pine forest up to the high desert, and M. lasiophylla in the Las Cañadas cliffs at the skirts of the Teide volcano. On Gran Canaria on the other hand, species distribution is apparently not correlated to the island’s evolution [Tenerife and Gran Canaria are the largest islands in the archipelago and contain the highest number of e island , 23, 29.l shield . Microme lineage , 23. TheMicromeria present on the Canary Islands ; species . More specifically, genetic diversity and pairwise genetic differentiation were estimated for species and population groups, whereas migration rates were calculated only between island and species groups.In some of the analyses, individuals were put together into different groupings or levels: Meimberg , 27); anDNA isolation was done using the Macherey-Nagel Plant DNA Extraction Kit  using 20 mg of dried leaf material according to Puppo et al. .The 16 microsatellite markers described in Puppo et al.  were ampMain genetic structure patterns were evaluated through distance and clustering analyses. These were used to show that the species defined by Puppo & Meimberg , 27 are populations in PAUP v. 4.0 [http://biology-assets.anu.edu.au/GenAlEx/) considering each population as a sample point and using three different datasets: 1) all populations, 2) Gran Canaria populations, and 3) populations assigned to the western group in the first analysis. Gran Canaria was tested independently because it was clearly separated from the other islands in the whole archipelago analysis. All analyses were calculated using pairwise genotypic distance among populations calculated in GenAlEx. We used these two analyses to explore the maximum amount of information from the data. While the PCoA provides information regarding how individuals may be grouped, the UPGMA dendrogram provides hierarchical information that can be used to assess lineage content.To assess the relationships among species, an UPGMA dendrogram was constructed using uNeiD distance  among poP v. 4.0 . Princippopulation combinations was not possible computationally, this was done using both islands and species as groups. In the event that a clear division within species was observed in the STRUCTURE analyses, these were considered to be different groups. In La Gomera species the two highly divergent lineages were treated separately in the island comparison. For Migrate, due to computational limitations, migration between islands was estimated using only island groups and migration contained on one island using species groups. In BayesAss, the output values correspond to the portion of individuals originating from the population that it is being compared to, while for the Migrate they correspond to the average number of migrants per generation. For BayesAss, only results with a minimum 95% confident interval value above 0.001 were considered to represent significant migration rates. In the case of Migrate, only migration rates significantly higher than 0 were considered. BayesAss parameters, such as mixing parameters, number of iterations, and burn-in were optimized in several runs. An optimal run was considered to have migration, allele frequency, and inbreeding coefficient acceptance rates below 0.6 and an effective sample size above 100 for all parameters . The fio et al. . We usedspecies and population. We had a special focus on genetic diversity at the population level because it is not affected as much by sample size biases as the other groupings. Response variables for genetic diversity were assessed by calculating expected (HE) and observed heterozygosity (HO), allelic richness (Ar), and percentage of private alleles. Two private allele measures were calculated: one comparing each group (population or species) to the whole archipelago, the other comparing each group to the other groups from the same island. Response indices about genetic differentiation and quantification of genetic structure were calculated by estimating pairwise FST [http://biology-assets.anu.edu.au/GenAlEx/)To test how genetic diversity and differentiation varied throughout the archipelago and if it was in accordance with the predictions from the surfing syngameon hypothesis, several statistics were calculated according to two categorical explanatory variables: ise FST  and unbiise FST , while tspecies and population levels were used to perform statistical analyses for evaluating the differences in genetic diversity and differentiation among the three explanatory variables . The clear division of the Canary Islands by their age resulted in two age levels, namely young island species and old island species. Tenerife is a special case because formerly independent islands became later connected, and the so called palaeo-islands have had a more pronounced botanical singularity than the other islands. The species were grouped according to their position in the phylogenetic analysis to account for the possibility of recent range shift between old and young substrates. Thus the group of young island species were composed of species from the central area, M. lachnophylla, M. lasiophylla and M. hyssopifolia, and including the closely related species M. varia s.s., which expanded onto the palaeo-islands from the central part . When species were grouped according to their current main distributions, the results only changed slightly but were still significant. After the calculation of pairwise genetic differentiation, a third category was considered for this measure: differentiation between old and young areas. In summary, two age levels were defined for the genetic diversity measures (old and young) and three levels for the genetic differentiation measures .These measures on the M. varia /M.hyssopifolia species complex.Genetic differentiation results may reflect differences among lineages rather than age. This was overcome by doing analyses on genetic diversity and differentiation including only members of the western lineage (data subsets “within west”) that occupy both old and young islands. Age-related biases were assessed by performing a test including only the recently diverged taxa from the M. varia and M. hyssopifolia on western islands). According to histograms the response variables  appeared to be normally distributed. Variance homogeneity of the responses among the levels of categories at the species level  and at the population level  was not given in all cases according to Fligner Killeen Tests. Further, we found out that the explanatory variables  were highly related (i.e. collinear) to each other. Therefore it was not possible to include them in the same models , because this would lead to incorrect parameter estimation [Regarding statistical analysis, data exploration was done following Zuur et al.  for all timation . As a cospecies level were assessed for island and age while at the population level these differences were analyzed among island, age and species. However, in the data subset of M. varia and M. hyssopifolia, differences in genetic diversity were only assessed in terms of island age because of small sample size due to data aggregation. The same statistical framework was applied to genetic differentiation, but, at the species level, species was also included as an explanatory variable. We had to deal with an increased number of single tests for each response variable because the Kruskal Wallis Test can only account for differences in response variables among a single factor . Therefore, we corrected the initial significance level (α = 0.05) using the Sidak [Differences in genetic diversity on a he Sidak , 45 methv. 3.3.2 .FST with geographical distance in kilometers using the Isolation By Distance Web Service v. 3.23 [We performed an isolation by distance test (IBD) to evaluate if, alongside island age, the effect of geographical distance is another factor explaining genetic differentiation. To do this we compared populations pairwise  v. 3.23 . We ran M. gomerensis with the eastern islands; and M. lepida and M. pedro-luisii within the western islands). Genetic structure was congruent with species delimitation for both analyses. Interestingly, one M. teneriffae population (tetSC5) clusters within the other western islands, which may indicate gene-flow among these islands.Patterns of genetic distances (uNei among populations) were visualized using both dendrograms and PCoA analyses Fig. . They weM. gomerensis and M. lepida from La Gomera, together with M. mahanensis from Lanzarote; and 2) M. glomerata and M. rivas-martinezii from Tenerife. When a STRUCTURE analysis was conducted with each island independently, the only improvement was found for La Gomera, where three species  are separated , M. varia from Teno the highest HO (0.77), and M. helianthemifolia the highest Ar (8.9). Per population, HO varied between 0.14 and 0.68, HE between 0.22 and 0.78, and Ar between 1.91 and 14.33. Overall, genetic diversity seemed higher in the western islands, the island of Tenerife being the most diverse  and was consistently the highest between Lanzarote and M. glomerata . At the population level, pairwise FST ranged from 0.05 to 0.66 and uNei from 0.13 to 5.16.At the O in the western lineage, heterozygosity measures were significantly higher in younger islands than in older ones, which also corresponded to significant differences between island and species . In contrast M. rivas-martinezii shared all alleles with other Tenerife species. At the population level, the portion of private alleles varied from 0% for several populations of multiple species to 6.45% for one population from La Palma (M. herpyllomorpha). This population also presented the highest portion of private alleles from a single island (62.06%). Private alleles were not as informative when assessing the variation among the defined categories. Nevertheless, the number of private alleles per island was higher in younger islands when the dataset containing the whole archipelago was considered.The portion of private alleles per FST . When the analyses were made using either only populations from older regions or only populations from younger ones, it was possible to see that isolation by distance (IBD) was much more pronounced in younger regions  than in older ones . IBD better explained divergence for when just these two subsets of data were considered, rather than all individuals.Geographical distance was weakly correlated with We found that gene-flow through hybridization does not only exist on individual islands, but that it also seems to link different islands to one another. This is an important prerequisite that indicates that connectivity between islands might be higher than generally expected, creating a subsequent impact on colonization ability and diversification. With other types of markers, direct detection of gene-flow between islands was impossible (data not shown), mostly because of limited sampling and a lack of differentiation between taxa.Using the analyses of different programs, we found evidence for contemporary and historical gene-flow between islands. This was indicated by recent connectivity within the eastern and western lineages and by historical connectivity among the central species of Tenerife, Gran Canaria, and La Gomera. High historical migration rates are found between the two major lineages (east and west), but recent gene-flow between them is found to be sparse. Barriers to gene-flow might accumulate with time and can affect how genetic diversity is distributed. Nevertheless, introgression between these lineages is still found, especially through hybridization in La Gomera. This indicates that although highly diverged, these lineages are not yet completely isolated, which can be a consequence of continuous genetic exchange during the divergence of these lineages .M. varia group. We showed here that this is not only the case with Tenerife, but also on all islands where more than one species is found. Interestingly, Tenerife showed a larger amount of gene-flow than Gran Canaria when the total amount of migration is considered. Both islands are composed of old and young substrates from the Miocene and Pleistocene [In our previous study, we detected gene-flow between species within Tenerife, most pronouncedly between the species in the central part of Tenerife and members of the istocene , 48. Pupistocene , 23 founistocene , 48. TheThe current model of the species syngameon postulates that, within the archipelago, genetic diversity decreases with colonization steps. This is mostly a consequence of the islands closer to the mainland being more likely to work as allelic sink than the remaining ones. Our data supports rather a scenario where the younger islands receive a significant amount of genetic diversity, in which differentiation is lower than expected when considering the surfing syngameon hypothesis for the Canary Islands. The existence of gene-flow within and between islands may explain this pattern, where high levels of admixture increase genetic diversity and can ultimately have an adaptive effect. Here, we see that both distance to the source of colonization and time have an effect on diversity.E and HO) is a consequence of high number of alleles with low frequency in the populations. This indicates that these populations were previously highly diverse, but the frequency of many of their alleles decreased probably through drift, which is more pronounced in populations less connected through gene-flow.Within populations, genetic diversity was higher in younger islands, and genetic differentiation was always higher for older islands. Additionally, we observed a weak isolation by distance pattern (IBD), which may be a consequence of a stepwise colonization process from the oldest islands in the east to the younger islands in the west. Our data is composed of several species, and if they were completely reproductively isolated from each other, the differentiation pattern would be better explained by species segregation events that are not necessarily correlated with geographical distance. The fact that the IBD pattern is more pronounced in younger regions indicates that these species are genetically more homogeneous and more connected to gene-flow. This connectivity ultimately results in a species syngameon composed of highly genetically diverse and minimally differentiated units. For some tests, allelic richness results either did not differ between old and young islands, or showed a contradictory pattern when compared to the heterozygosity levels. This measure only considers the number of alleles per population . Thus, tOur results might be explained by the following mechanism: on younger islands the colonization process is still ongoing, so they contain a low number of taxa that may not have yet occupied all niches available, so they display a lower number of populations and individuals. Here, colonization may have a higher impact on the genepool of already established populations. On older islands, taxa had time to expand and are now represented by higher number of populations, individuals, and species. Thus, gene-flow onto these islands might have a lower effect and does not contribute much to changes in the genepool of established populations. Therefore, colonizers should have a lower effect on preventing population differentiation on the older islands, as well as a lower chance of increasing within-population diversity. This mechanism is similar to the one described by the niche-preemption hypothesis , 51 wherMicromeria, the syngameon is therefore not only expanding and shifting, but it is continuously being recreated at the colonization edge, which might thereby increase the potential for ecological speciation at the expansion front. A recent update of the surfing syngameon hypothesis incorporated the role of island ontogeny on shaping the species syngameon [Micromeria may lead to further refinements of this hypothesis.Considering a source-sink dynamics, the surfing syngameon hypothesis implies that the sink populations can have a cumulative effect on genetic diversity by maintaining their connection to their source through gene-flow. We can apply these assumptions to explain our results by presenting a scenario of inter-island colonization with continuous gene-flow. After the emergence of an island, individuals that can hybridize will colonize it from different sources. In this case the syngameon expands with the appearance of new islands and prevents the loss of genetic diversity at the colonization front by buffering founder effect. In the Canary Islands this expansion moves in an east-west direction. As the syngameon expands, populations on old islands might become isolated, speciate, and thus become disconnected with increasing reproductive isolation. This dynamic is similar to the model of species range shifts, in which new populations in the expansion front have a high connectivity to the source, and thus they receive higher amounts of gene-flow. Although differentiation between populations is low, the rear relict populations become increasingly isolated, showing low differentiation within populations and high differentiation between populations . In Micryngameon  and the We chose to use microsatellites, which are mostly used at the intraspecific level. Nevertheless, these also have successfully been used to evaluate species boundaries and test hypotheses related to the species concept , 55, supMicromeria species, supporting the last Micromeria species delimitation from Puppo & Meimberg [M. gomerensis and M. mahanensis, and M. glomerata and M. rivas-martinezii, all remaining species were differentiated in the analyses containing only individuals from the eastern and western groups. These species were differentiated in the other analyses (PCoA and UPGMA dendrogram) and in previous studies using nuclear markers [The genetic structure found in the present study was mostly congruent with previous phylogenetic studies. All species described in Tenerife had already been supported with microsatellite data . In the Meimberg , 27. STR markers , so this markers , making  markers . Thus, tMicromeria individuals from Fuerteventura were closely related to the ones from Lanzarote, indicating that they may constitute previously contiguous populations and thus should show similar genetic patterns. Besides sharing the same geological history, both islands present similar geomorphological features. They are mostly shaped by erosion that contributes to the loss of most of their previous altitudinal range, which resulted in a high extinction rate [Further tests of the hypotheses outlined here would benefit from a denser sampling of the eastern island of Lanzarote and the inclusion of samples from Fuerteventura. These are the oldest islands of the archipelago, and thus according to our expectations should be the least diverse and the more genetically differentiated. A higher sampling could result in an increase in genetic diversity, since it would be more likely to sample more alleles. However, given the results in other, older regions that were densely sampled, we do not expect this would be significant enough to change our observations. The island of Fuerteventura was connected to Lanzarote at several times in the past . In faction rate  and thusIn this study we decided to focus on the extension of the surfing syngameon predictions to colonization between islands within an archipelago. However, it would be interesting to measure the amount of gene-flow from the mainland and its influence on the genetic diversity of the archipelago taxa. At the moment it is still not known which mainland species are the closest relatives to the Canary Islands. The last phylogeny produced with mainland species showed that both Iberian and Moroccan species are the main candidates, but the analyses were not powerful enough to decide which one was more closely related to the Canary Islands species . We are Micromeria across the Canarian archipelago indicate the existence of a syngameon that may facilitate colonization and speciation on oceanic islands. The species syngameon seems to expand and shift in accordance with a range expansion scenario. Island colonization has many parallelisms with other evolutionary processes, and theoretically the model proposed here could be applied to scenarios where a range expansion is accompanied by adaptive speciation. We showed how genetic diversity is affected by gene-flow and hybridization, but we do not assess its adaptive implications. To do so, genomic approaches where both coding and non-coding variation is compared with ecological and morphological features might successfully be implemented.The results of our study of neutral genetic variation for Additional file 1: Table S1.Table S2. Summary statistic of the STRUCTURE analyses performed. This includes the K values tested and which ones were the best. It also includes the mean LnProb and similarity score of the 15 replicates obtained with CLUMPAK. Lastly we also include the results of the power analysis done with POWSIM, presented as percentage of runs that contradicted the null hypothesis of homogeneity of the data. Table S3. Migration rates calculated with BayesAss. Values correspond to percentage of individuals from the species (3.1) or island (3.2) in the upper line originated from species (3.1) or island (3.2) in the left column. The second value corresponds to the 95% confidence interval. Migration rates significantly higher than zero are marked in bold. Table S4. Migration rate calculated with Migrate. Values correspond to average number of individuals migrating per generation. Source species or islands are in the left column and sink species or islands in the upper line. Values with 95% intervals above zero (under brackets) are marked as bold. (XLSX 35 kb)List of all localities with information regarding the number of individuals sampled, species classification according to Puppo and Meimberg , 27, andAdditional file 2: Figure S1.Figure S2. Representation of expected heterozygosity (HE) and FST per population used as proxy of genetic diversity and differentiation. For each measure there is a graph summarizing it per island, per species, and per age class. From top to bottom the box-plots correspond to the different datasets used, including: all samples, only M. varia and M. hyssopifolia, and Western lineage . (DOCX 487 kb)Structure Harvester results for mean likelihood (above) and DeltaK values (below) per K. A) Analysis for the complete archipelago was conducted with 15 replicates of K = 2 to K = 30, resulting in the best K of 2, which is marked by the arrow. B) STRUCTURE analyses per island were conducted with different values of K (see Additional file