The advancements made in tissue culture techniques has made it possible to regenerate various horticultural species in vitro as micropropagation protocols for commercial scale multiplication are available for a wide range of crops. Clonal propagation and preservation of elite genotypes, selected for their superior characteristics, require high degree of genetic uniformity amongst the regenerated plants. However, plant tissue culture may generate genetic variability, i.e., somaclonal variations as a result of gene mutation or changes in epigenetic marks. The occurrence of subtle somaclonal variation is a drawback for both in vitro cloning as well as germplasm preservation. Therefore, it is of immense significance to assure the genetic uniformity of in vitro raised plants at an early stage. Several strategies have been followed to ascertain the genetic fidelity of the in vitro raised progenies comprising morpho-physiological, biochemical, cytological and DNA-based molecular markers approaches. Somaclonal variation can pose a serious problem in any micropropagation program, where it is highly desirable to produce true-to-type plant material. On the other hand, somaclonal variation has provided a new and alternative tool to the breeders for obtaining genetic variability relatively rapidly and without sophisticated technology in horticultural crops, which are either difficult to breed or have narrow genetic base. In the present paper, sources of variations induced during tissue culture cycle and strategies to ascertain and confirm genetic fidelity in a variety of in vitro raised plantlets and potential application of variants in horticultural crop improvement are reviewed. Plant tissue culture techniques proffer a substitute method of vegetative propagation of horticultural crops , naphthalene acetic acid (NAA) and BAP (6-benzylaminopurine), synthetic phenylurea derivatives  have been most frequently considered to be responsible for genetic variability  callus cultures. Matsuda et al.  it is cheaper than other methods of genetic manipulation and does not require ‘containment’ procedures. (2) Tissue culture systems are available for more plant species than can be manipulated by somatic hybridization and transformation at the present time. (3) It is not necessary to have identified the genetic basis of the trait, or indeed, in the case of transformation, to have isolated and cloned it. (4) Novel variants have been reported among somaclones, and evidences indicate that both the frequency and distribution of genetic recombination events can be altered by passage though tissue culture. This implies that variation may be generated from different locations of the genome than those, which are accessible to conventional and mutation breeding Karp . (5) TheSomaclonal variation has been most successful in crops with limited genetic systems  and/or narrow genetic bases. In ornamental plants, for instance, the exploitation of in vitro-generated variability has become part of the routine breeding practice of many commercial enterprises.1 segregate. Therefore for quantitative traits such as yield, it is virtually impossible to select individuals with improvements in the R1 generation. Though techniques for selection of somaclones resistant to various biotic and abiotic stresses had been worked out in many horticultural crops, unfortunately, no in vitro selection methods exist for complicated traits such as yield, soluble solids, sweetness, texture or shelf life  with X-rays with LD50 close to 20 Gy. Among plants regenerated from leaves with 20 Gy, one was phenotypically different, and was subsequently isolated and cloned. This somaclone was extremely dwarfed and was stable in both greenhouse and field tests. Employing more than one mutagen results in further improvement in recovery of somaclones in vitro. Murti et al. (Fusarium oxysporum f.sp. cubense) culture filtrate (EMS + CF line) and (4) EMS-treated embryogenic calli, followed by in vitro selection on fusaric acid. The Foc resistance abaca variants were successfully identified from four tested abaca variant lines, although with different frequencies. However, more Foc resistance abaca plants were identified from EMS + CF line than the others. Earlier, Bidabadi et al.  with all our previous knowledge and experience should be followed to achieve the desideratum.


Genetic biodiversity contributes to individual fitness, species' evolutionary potential, and ecosystem stability. Temporal monitoring of the genetic status and trends of wild populations' genetic diversity can provide vital data to inform policy decisions and management actions. However, there is a lack of knowledge regarding which genetic metrics, temporal sampling protocols, and genetic markers are sufficiently sensitive and robust, on conservation-relevant timescales. Here, we tested six genetic metrics and various sampling protocols  for monitoring genetic erosion following demographic decline. To do so, we utilized individual-based simulations featuring an array of different initial population sizes, types and severity of demographic decline, and DNA markers [single nucleotide polymorphisms (SNPs) and microsatellites] as well as decline followed by recovery. Number of alleles markedly outperformed other indicators across all situations. The type and severity of demographic decline strongly affected power, while the number and arrangement of temporal samples had small effect. Sampling 50 individuals at as few as two time points with 20 microsatellites performed well (good power), and could detect genetic erosion while 80–90% of diversity remained. This sampling and genotyping effort should often be affordable. Power increased substantially with more samples or markers, and we observe that power of 2500 SNPs was nearly equivalent to 250 microsatellites, a result of theoretical and practical interest. Our results suggest high potential for using historic collections in monitoring programs, and demonstrate the need to monitor genetic as well as other levels of biodiversity. A major tool in conservation biology is the temporal monitoring of biodiversity with indicators such as the number of species or size of populations. Monitoring indicators across time periods can often identify negative trends, with the ultimate goal being the detection of a decrease early enough to signal a conservation need Noss . For exahttps://www.cbd.int/sp/) aims to ‘minimize genetic erosion’ and ‘safeguard genetic diversity’ in species of agricultural, socio-economic and cultural importance. National and international resource management agencies, especially forestry, fisheries, and agriculture, are also seeking to monitor and preserve genetic diversity of the wild populations they utilize . These indicators are here tested under different population decline models, sampling efforts, and sampling schemes. Recognizing that many species of concern now have sufficient genomic resources available, we also test the power of moderate and large numbers of loci. We answer the following questions: (i) which indicator is most sensitive to the decline, (ii) how many temporal samples are needed, (iii) what temporal sampling pattern  is most appropriate, (iv) is power of an indicator dependent on the type of population decline, and (v) what is the effect of sample size and marker number on power? We also test the ability to detect genetic change after population demographic recovery. We evaluate two types of genetic markers: microsatellites, which are currently the most common markers in ecology and conservation and for which baseline data are available from many endangered species, and single nucleotide polymorphisms (SNPs), which are rapidly emerging as an affordable, high-throughput, high genome-coverage marker.Our goal was to determine what genetic metrics are most sensitive and robust, what temporal sampling protocols are appropriate, and what genetic markers show sufficient resolution, on conservation-relevant time scales. Here, we use realistic, individual-based simulations to evaluate the power of six potential indicators and various sampling designs to detect genetic erosion after demographic decline. The evaluated indicators, which are common summaries of a population's genetic status  undergoing exponential decline for three primary situations: decline to N = 200, N = 50, and N = 20 . We designate these (relatively) as weak, moderate, and strong declines. Decline occurs over 10 generations, thus a 97.5% exponential decline gives population sizes at each generation of: 2000, 1383, 956, 661, 457, 316, 219, 151, 105, 72, and 50 . Note that forward-in-time individual-based simulations, including Nemo, generally model  census population size Nc. Of course, effective population size (Ne) is the parameter directly influencing loss of genetic diversity  below this (Table S1). We first simulated exponential decline to represent gradual habitat loss or climate-induced decline, which is the most common shape exhibited by animal population declines . We simulated a single population with census size N = 10 000 to N = 300, 100, and 50 . Here, the 99% decline allows direct comparison of magnitude to the 99% situation described above, while the decline to 50 allows direct comparison to final population size of 50 described above. Additionally, we simulated populations that instantly recover  to original size after periods of 2, 10, and 20 generations of low population size to determine whether increases in genetic metrics are detectable. We performed 100 replicates of every scenario (a complete list of scenarios is presented in Table S2). Each replicate was run for 10 000 generations to reach equilibrium, so each simulation replicate has an independent starting condition.To determine if results depend on initial population size we performed additional simulations from initial In general, 50 samples were taken every generation and genotyped at 20 microsatellite DNA loci. In the 99% decline only 20 individuals remained and all were sampled. Twenty microsatellites provide a level of resolution that is consistent with many ongoing studies. We also tested additional sampling approaches: all individuals in the population (thus no sampling error), and 50 samples but 250 microsatellites. For two scenarios , we tested also a scheme of 50 samples at 2500 SNPs.K), allelic size range for microsatellites (Kr), observed and expected heterozygosity , the Garza-Williamson M-ratio bottleneck statistic for microsatellites (GW), and Wright's inbreeding coefficient (Fis). An important caution for real monitoring programs is that if sample sizes are not equal for all time points sampled, and sampling is not exhaustive, allelic richness determined by rarefaction should be substituted for number of alleles, K (see Discussion). Sampling and analysis were performed with the custom-made software ConvFstat (available at sites.google.com/site/hoban3/scripts), which samples, converts file formats and runs arlecore, a command-line version of Arlequin  over replicates, for each pair-wise comparison and each indicator. This sum, divided by 100 (the number of replicates), represents the power to conclude whether the genetic indicator is significantly different between any two generations.Samples from each generation were compared pairwise to samples from every other generation. Paired tests, with loci as replicates, have been used previously to compare modern and historical samples  a conclusion as to whether a significant difference was observed. Summed over replicates and divided by 100, this represents power of a given sampling scheme to conclude that genetic diversity is declining over time in a given situation.We tested 20 specific temporal sampling schemes, for example, different combinations of generations to be sampled. These schemes vary in terms of different number of samples, different temporal ‘clumping’ of samples, and whether or not samples are available before decline begins  and heterozygosity . Theoretical expectations for heterozygosity for a population of Ne = 2000 is 0.667  shows high power and outperformed the other indicators, which typically show <0.50 power, often much less  for comparing the three most recent generations (when populations size is lowest) to previous generations. In the case of moderate (97.5%) decline, substantial power was obtained only when comparing the most recent generation to previous generations; all other inter-generational comparisons showed low power. For weak (90%) decline, power never exceeds 0.20 for any indicator for any intergenerational comparison.Overall, less Fig., Data S1K, power for instant declines was >0.90. On the other hand, a response was still nearly undetectable for weak cases, with power reaching >0.50 only after eight generations and never exceeding 0.62. Again, K and Kr perform best, but other indicators  performed well in some cases . For example, sampling generations 1-2-8-9 , had <0.30 power to detect differences, while sampling generations 1-2-10-11 had power of 0.80 , and poorly for weak ones .Again, power was much higher under instant than exponential decline Table. SimilarK . Unsurprisingly, greater power was achieved for a decline from N = 10 000 to 50 than from N = 2000 to 50. When considering a decline of equivalent percentage, the 97% and 99% declines from N = 10 000 showed less power than the 97% and 99% declines from N = 2000.In all additional simulations the strong effects of decline type and weaker effects of particular sampling strategy were apparent, and the best indicator remained As expected, genotyping 250 microsatellites achieved higher power than 20. Nonetheless, there was still low power early in the exponential decline – reasonable power was not achieved until seven generations after decline or later . Nonetheless, we observed that recovery after two generations resulted in a population that lost approximately one-third of the heterozygosity and number of alleles that would be lost in a more delayed recovery  showed the clearest response and highest power for monitoring genetic decline across all scenarios  in simulations parameterized to black grouse, as did Pinsky and Palumbi  if samples are extremely close in time. This is consistent with theoretical expectations that population size, time between samples, and time since start of demographic decline will affect genetic erosion. Under instant decline, on the other hand, using K, decreased genetic diversity can be identified with good power within one or two generations after decline, for severe (99%) or moderate (97.5%) declines, and notably even while population size remains constant. However, weak (90%) declines yielded lower power, in agreement with Pinsky and Palumbi . Our results should not be interpreted that weak declines are unimportant, only that the resulting genetic loss may be difficult to detect in the short term.Principally, we showed that power critically depends on the type and severity of demographic decline. All genetic indicators show markedly lower power under exponential population decline Figs, Table2. Palumbi , who tesK) when declines were moderate or severe and when sampling spans the period of decline. For exponential declines, power is largely unaffected by the spacing of samples in time, and whether two, four, six, or more samples are taken. For instant declines, power is usually improved by approximately 0.20 if samples are available before the onset of the decline as compared to when sampling started multiple generations into the decline. This result emphasizes the importance of ancient or archived/museum samples , results show that as few as two temporal samples can reveal genetic diversity loss (with N ∼ 100), genetic diversity loss should be readily identifiable before severe erosion occurs. Specifically, by examining the mean values of K at each generation .With reasonable numbers of markers and samples, once a population is small N ∼ 00, genetM-ratio) that we evaluated can be calculated for any previously popular  or future  marker types. Individual SNP loci are generally less polymorphic than microsatellite loci. In our study, 2500 SNPs were comparable to 250 microsatellites, suggesting that ability to detect genetic losses may depend on the total number of alleles available. One issue with SNPs and microsatellites is ascertainment bias- during marker development, loci are usually chosen to be polymorphic in the current population, thus genetic losses may be underestimated. Our simulations did not replicate such bias. Monitoring adaptive genetic biodiversity is also emerging as a complement to neutral markers , and when population decline was instantaneous the genetic response was linear Figs also obsNew variation is only introduced via mutation in an isolated population (significant increase of genetic variation can occur via migration), so once diversity is lost, it is very slowly restored even if population size fully recovers. Thus, monitoring genetic increase after population size recovery has essentially no power. However, if population decline was recent, rapid population demographic recovery can instantly halt loss of remaining diversity, as illustrated in Fig.M-ratio and Fis are ‘compound’ indicators that are often interpreted, when computed on a single time sample, as evidence of recent bottlenecks or excess homozygotes in small populations, respectively. Both estimators, perhaps due to their variance, perform poorly. It is unsurprising that Fis shows little response as our populations are modeled with random mating. It may be surprising that M-ratio performs more poorly than the statistics on which it is based , though this statistic has high variance and moreover was designed to detect extremely severe declines. It is currently unclear how other ‘synthetic’  indicators perform relative to basic metrics such as number of alleles. If ‘compound’ statistics in general show high variance, direct and more easily interpretable genetic measures may be most promising. Work to develop further metrics is required. Possibilities include use of temporal FST or the allele frequency spectrum to monitor another aspect of genetic erosion ; these could complement the indicators we investigated  while heterozygosity is a measure of evenness. In both disciplines, the two aspects are typically correlated. The most responsive indicator, allelic diversity, is notable because it directly represents extinction of genetic variants, with direct consequences for alleles with present or future functional/adaptive importance. The G-W Our study focused on genetic erosion, but temporal monitoring may be desired for other reasons such as changes in genetic connectivity due to fragmentation or to monitor the response of a population to genetic restoration or ‘genetic rescue’, such as via translocation , and plans of appropriate actions to implement after thresholds are observed  exceeds several hundreds or the decline is very recent (a few generations), even when using many genetic markers. Monitoring will likely be most effective once population size is small (Ne < 100), and sampling should increase in frequency after this size threshold. Small but constant-size or slowly recovering populations are also in need of intense genetic monitoring. This concurs with Lindenmayer et al. . This is analogous to species disappearance following habitat loss, which may be small at initial levels of habitat loss, but then accelerates, with potential for further species loss even after habitat loss ceases (Krauss et al. Lastly, we emphasize an observation which might not be intuitive for non-geneticists such as most policy makers: genetic loss may sometimes be slight even if a population is declining, while substantial genetic loss can occur quickly in populations of small, stable size (e.g.,


Notorynchus cepedianus, a common coastal species in the eastern North Pacific, was sampled during routine capture and tagging operations conducted from 2005–2012. One hundred and thirty three biopsy samples were taken during these research operations in Willapa Bay, Washington and in San Francisco Bay, California. Genotypic data from seven polymorphic microsatellites  were used to describe N. cepedianus genetic diversity, population structure and relatedness. Diversity within N. cepedianus was found to be low to moderate with an average observed heterozygosity of 0.41, expected heterozygosity of 0.53, and an average of 5.1 alleles per microsatellite locus. There was no evidence of a recent population bottleneck based on genetic data. Analyses of genetic differences between the two sampled estuaries suggest two distinct populations with some genetic mixing of sharks sampled during 2005–2006. Relatedness within sampled populations was high, with percent relatedness among sharks caught in the same area indicating 42.30% first-order relative relationships . Estuary-specific familial relationships suggest that management of N. cepedianus on the U.S. West Coast should incorporate stock-specific management goals to conserve this ecologically important predator.The broadnose sevengill shark,  Sharks are predators found in every ocean of the world. They tend to mature slowly and have low reproductive rates, making many species extremely vulnerable to exploitation –5. Of thNotorynchus cepedianus Peron, 1807, (Chondrichthyes: Hexanchidae) is a relatively common, large coastal species in many temperate marine ecosystems. It frequently inhabits bays and estuaries throughout the eastern North Pacific and many aspects of it’s biology and ecology has been studied .–28.HO =selected . However. (2009)  reported. (2004)  reported species .N. cepedianus compared to other shark species, suggests potential past population bottlenecks that may now be affecting diversity. There was a targeted fishery for N. cepedianus in San Francisco Bay from the 1930’s to the 1980’s [N. cepedianus did not experience a significant loss of diversity due to take from targeted fishing between 1930–1980. However there is evidence that the effectiveness of detecting bottlenecks using this software depends on the number of generations post bottleneck [N. cepedianus is unknown, but is thought to be relatively long (between 10–15 years) [N. cepedianus diversity using more molecular markers.The relatively low genetic variation reported here within e 1980’s . We emplttleneck . For exattleneck . The gen5 years) . The sevN. cepedianus revealed a moderate to high degree of genetic structure  in Willapa Bay and Humboldt Bay in California where they were found the rest of the year [Population comparisons between California and Washington fidelity . Althougfidelity . For exathe year . In addithe year .N. cepedianus it is thought that these two groups may have separate breeding grounds. This is the case in other species of sharks that exhibit relatively high stock structure, such as Carcharhinus limbatus in the Gulf of Mexico and Atlantic [C. limbatus did not disperse randomly between regions during migrations to nursery areas and related females tended to return to the same region. A similar pattern may hold true for female N. cepedianus sampled here: perhaps these sharks travel in sexually segregated and related groups to separate nursery and breeding grounds. As within C. limbatus there is most likely migration of females and males between regions resulting in limited gene flow, as suggested by some of the SF-1 N. cepedianus intermixing with the WA population in the PCoA and the assignment plot (Based on the genetic structuring found between the California and Washington populations of Atlantic . In thisent plot . The mixN. cepedianus caught in each area and during similar time periods (Negaprion brevirostris and Hexanchus griseus [N. cepedianus [One of the most interesting findings of this preliminary study was the high degree of relatedness among  periods . The rel griseus , 37 One  griseus . Such aspedianus .Embiotoca jacksoni [Dascyllus aruanus [Paralabrax clathratus [Salmo salar [E. jacksoni, or have known migration routes such as S. salar, and may facilitate effective dispersal of the genes shared within the related group by increasing the odds that any single individual survives [Several recent studies have documented kin association in the aquatic environment , 39–44.  aruanus , kelp baathratus , and Atlmo salar . This misurvives , 44. Thesurvives –47.Notorynchus cepedianus occupies shallow, near shore estuaries such as San Francisco Bay in California and Willapa Bay in Washington during at least part of the year and has historically been targeted by fisheries [N. cepedianus on the U.S. West Coast should incorporate geographically explicit, population-specific goals to ensure the long-term persistence of this ecologically important predator.isheries , 10. Thi


Campylopterus curvipennis) is correlated with genetic divergence, and examined processes that explain best the origin of intraspecific song variation. We contrasted estimates of genetic differentiation, genetic structure, and gene flow across leks from microsatellite loci of wedge-tailed sabrewings with measures for acoustic signals involved in mating derived from recordings of males singing at leks throughout eastern Mexico. We found a strong acoustic structure across leks and geography, where lek members had an exclusive assemblage of syllable types, differed in spectral and temporal measurements of song, and song sharing decreased with geographic distance. However, neutral genetic and song divergence were not correlated, and measures of genetic differentiation and migration estimates indicated gene flow across leks. The persistence of acoustic structuring in wedge-tailed sabrewings may thus best be explained by stochastic processes across leks, in which intraspecific vocal variation is maintained in the absence of genetic differentiation by postdispersal learning and social conditions, and by geographical isolation due to the accumulation of small differences, producing most dramatic changes between populations further apart.Hummingbirds have developed a remarkable diversity of learned vocalizations, from single-note songs to phonologically and syntactically complex songs. In this study we evaluated if geographic song variation of wedge-tailed sabrewings ( Understanding the processes and mechanisms by which phenotypic diversity arises is fundamental in evolutionary biology. Although most of research has focused in the role that natural selection can play in the processes of population divergence, an interest to study the role of stochastic processes involved in phenotypic and population divergence has raised lately e.g., e.g., Song divergence among populations can be driven by three non-mutually exclusive processes: (1) selection of particular acoustic traits due to environmental or habitat characteristics, where different frequencies of sound travel best in different environments or because ambient noise differences between environments Campylopterus curvipennis , is a sexually monochromatic, size dimorphic hummingbird with a wide distribution through the cloud forest and humid tropical forest of eastern Mexico The wedge-tailed sabrewing, Campylopterus curvipennis is a species complex composed of three allopatrically distributed lineages corresponding to subspecies C. c. curvipennis along the Sierra Madre Oriental, C. c. excellens in the Sierra de los Tuxtlas, and C. c. pampa in the Yucatan Peninsula. Second, morphological and acoustic data along with information about the habitat (climate and topography) indicated that these subspecies are morphologically and acoustically divergent, independently of habitat-related variation, and that the genetic divergence among subspecies was coupled with song divergence. Third, a coalescent analysis to evaluate whether song divergence could be attributed to drift or selection, suggested that the fixation of song types, assumed to be encoded by nuclear genes, has occurred faster than expected by genetic drift, suggesting that selection may have played a role in driving song evolution in the past. However, the assumption that fixation of song types is encoded by nuclear genes needs to be taken with caution in such a dynamic cultural system. Furthermore, the observed positive relationship between genetic and acoustic distances in that study, independent of geographic distances, is expected under a drift model of song evolution, where song divergence is higher between allopatric populations that have been isolated for the longest time (i.e. subspecies) with low potential for hybridization  for the field study described. This collecting permit specifically allowed for the collection of tail feathers from the birds. Manipulation of birds in the field was minimal. Birds were captured at leks with mist nets and their two outermost tail feathers were removed for genetic analyses before they were released. All procedures with birds were carried out in accordance with the Guidelines for the Use of Wild Birds in Research proposed by the North American Ornithological Council and the ethics of experimental procedures were revised and authorized by the Animal Care and Use Committee under the Graduate Studies Committee (Doctorado en Ciencias Biomédicas) of the Universidad Nacional Autónoma de México (UNAM), the Instituto de Ecología, A.C. (INECOL), and CONACyT (61710). While the field studies involve a non-threatened or protected species, no specific permits are required for song recording or field studies as the one described here.Table S1), with 3–27 individuals sampled per lek. Sampled leks along the Sierra Madre Oriental (SMO) were categorized in three geographic areas: (i) northern limit of their distribution, from southern Tamaulipas and north of San Luis Potosí ; (ii) central, from south of San Luis Potosí ; and (iii) southern limit of the distribution, from Puebla and Veracruz  (Table S1).Feather samples were collected from a total of 105 wedge-tailed sabrewings captured during the 2006–2008 breeding seasons  according to Morin et al. STST parameters of genetic differentiation due to genetic structure in RSTCALC v2.2 v2.9.3 K) based on microsatellite data. We ran two independent STRUCTURE analyses, with the LOCPRIOR or the default mode, both under the admixture model with correlated allele frequencies K, from K = 1 to K = 12. The length of the burn-in was 500,000 and the number of Markov chain Monte Carlo (MCMC) replications after the burn-in was 1,000,000. To determine an accurate number of clusters we calculated the statistic ΔK based on the rate of change in the log probability of data between successive K values following Evanno et al. To investigate lek genetic structure we calculated global and pairwise RemN) between leks and between groups of leks  was estimated with a maximum likelihood coalescent approach using genotypic data and MIGRATE v3.0 ST calculations. We ran five short and three long chains  or 20 short and six long chains  for estimation between leks and between groups of leks, respectively, after discarding the first 5,000 genealogies as a burn-in.Gene flow (4c. 9 recordings per bird). Some of these recordings were used in previous studies www.birds.cornell.edu/raven).Songs were recorded from territory-holding males at the same leks in which feather samples were taken. Between 3 and 13 birds were recorded at each lek, with 5 to 19 song recordings obtained from each of 56 males . For the introductory syllable we quantified the total number of elements, and the number of different elements. For the introductory and the shared syllable, we measured the duration (s), minimum frequency (kHz), bandwidth , and peak frequency . Because the shared syllable has several elements (notes), we took the same measures on two of their elements  and the duration of another element . In total six measures were taken on the introductory and 13 on the shared syllable.Based on the digitized songs we constructed three different data sets for acoustic analyses: (1) presence/absence of syllable types, (2) relative frequency of each syllable per song, and (3) acoustic traits of two comparable syllables. For the first two data sets, we used syllabic units throughout the wedge-tailed sabrewing repertoire. Syllables were visually classified by CG based on the structure observed on the printed spectrograms, and assigned a letter or combination of letters for further identification. A syllable was defined as an element or several elements always grouped together in a fixed composition. The visual classification of syllable types was not difficult, as syllables are discrete units composed of one or more elements that in general differ consistently in their acoustic structure in obvious ways (for details see K). The ploidy option was set at 1, and ten independent chains were run for each K, from K = 1 to K = 12. Length of the burn-in was 250,000 and the number of Markov chain Monte Carlo (MCMC) replications after the burn-in was 500,000. To determine the accurate number of clusters, ΔK was also calculated.To evaluate song divergence among leks, we performed a hierarchical cluster analysis using the unweight pair-group method with arithmetic mean (UPGMA) and the Euclidean distances to construct a dendrogram in SPSS (SPSS Inc.) based on the binary presence/absence matrix of syllable types. We additionally ran STRUCTURE v2.2.3 We used a principal components analysis (PCA) with the measurements of the introductory and the shared syllable to reduce the number and intercorrelation of the variables. The resulting PC scores were tested for significant differences among leks and between groups of leks (see Results) performing multivariate analyses of variance (MANOVA) followed by one-way ANOVAs. Mensural data and counts were log- and square root transformed, respectively, to produce normality in the data. Finally, we used the relative frequency of syllables, and the measurements of the introductory and shared syllable as predictors in a discriminant function analysis (DFA) for each of the three data sets to examine whether individuals could be classified according to their lek of origin.ST and FST distances. These relationships were controlled for potential effects of geographic distance through partial Mantel tests. To test for the effects of isolation by distance we looked at the relationships between acoustic and geographic distance, controlling for genetic distance. Finally, we tested the relationships between genetic and geographic distances.Acoustic distance matrices regarding song sharing were built by estimating the pairwise Jaccard similarity coefficient between individuals among leks. This coefficient was calculated in ESTIMATES v8.0.0 The number of alleles per locus varied from 3 to 15, and the observed heterozygosity values indicate no consistent deviations from H-W equilibrium. After Bonferroni corrections, only four leks deviated from H-W equilibrium in locus CACU13-2, two in locus CACU5-7, and one lek in locus CACU13-7 Table 1.ST estimate ± SE, –0.032±0.0017, p = 0.455; global FST estimate ± SE, 0.021±0.002, p = 0.972). Pairwise RST among leks ranged from –0.053 to 0.082, and pairwise FST ranged from –0.0138 to 0.249. Results of STRUCTURE analysis showed no genetic clustering when population information was not provided, whereas a weak genetic structure among leks was revealed when using the LOCPRIOR function. When ΔK was calculated the break in slope of the distribution of L(K) was at K = 2 . In addition, we observed a geographic pattern in song sharing: leks from the northern , central  and southern limit of the distribution  are clustered together in different groups, except three individuals from the Orduña lek, which are basal in the dendrogram. On average, the proportion of syllables shared between individuals within leks was 0.60±0.18 (mean±SD), whereas the proportion of syllables shared among leks was 0.15±0.04, these correspond to 20.4 and 5.1 syllables respectively  was at K = 3 when ΔK is calculated  and among groups of leks along the Sierra Madre Oriental . Regarding measurements of the shared syllable, the 13 variables were reduced to three principal components that accounted for 81% of the variation. The first PC (52.1%) was mainly explained by the peak frequency of the syllable, PC2 (18.8%) by the peak frequency of the second element, and PC3 (10%) was explained by duration, minimum frequency, and bandwidth of the syllable and their elements. A MANOVA with the PC scores also showed significant variation among leks  but the variation among groups of leks was not significant .The six measurements of the introductory syllable were reduced to a single principal component that accounted for 85.7% of the total variation. A one-way ANOVA with the PC scores showed that spectral and temporal measurements of the introductory syllables varied significantly among leks . Likewise, the correlations between FST values and acoustic measures were no statistically significant . In contrast, the correlation between acoustic and geographic distances was positive and significant when using the song sharing distance matrix  or the shared syllable . Removing the effects of genetic distance did not significantly alter the positive relationship between geographic and song sharing distances . Finally, genetic and geographic distances were not significantly correlated .We found that the correlation between Rcurvipennis subspecies studied here are not genetically structured along the Sierra Madre Oriental. Measures of migration rates, genetic differentiation and population structure indicated gene flow among leks and low levels of genetic structure. The relationships between acoustic and genetic distances were sharply discordant with previous results comparing acoustic and genetic data of all three wedge-tailed sabrewing subspecies. González et al. ST and RST values) and acoustic distances (song sharing and common syllable), and the significance of these relationships was not altered when the effects of geographic distance were removed. These analyses indicate that genetically divergent subspecies shared fewer syllable types, and differed acoustically in the common syllable, independently of distance, which might be the result of long isolation after divergence in allopatry. However, the relationships between acoustic and genetic distances were not statistically significant within the curvipennis subspecies studied here . Although song traits play a potential role in increasing genetic polymorphisms and generating reproductive isolation and speciation in parapatry  were more divergent between than within leks.Our study revealed that lek members of the curvipennis subspecies yielded a non-significant correlation between genetic and acoustic distance. Studies using rapidly evolving genetic markers in a wide variety of song learners, mainly songbirds and parrots, have shown similar patterns of genetic differentiation (FST), and have also failed to find genetic divergence coupled with song divergence Both the genetic adaptation and epiphenomenon hypotheses predict that dialects do affect genetic structure  showed that individuals changed their song-type at leks during a four-year period, and that all song replacements occurred after a crystallized song was already produced, which indicates that song learning may well extend throughout their lifespan The decoupled patterns between the neutral genetic and acoustic distance highlights the role that learning might play in the formation of lek-level vocal structure in wedge-tailed sabrewings. As in songbirds and parrots, song learning in hummingbirds is culturally transmitted through imitation Table S2), indicating that the observed geographic structure of curvipennis songs can not be explained by founder effects after colonization of northern areas K = 3; curvipennis songs along the Sierra Madre Oriental lend support to the idea that song sharing between lek members , could be a selective force operating to promote convergence within wedge-tailed sabrewings leks, where song sharing among neighbors confers social benefits that could possibly increase their reproductive success The decreased syllable sharing with increased geographic distance (isolation-by-distance) suggests that song divergence is also a result of neutral processes, where individuals within leks copy syllables randomly from each other and small differences in song accumulate along a geographical gradient due to the reduced probability of interaction among individuals from more distant leks (i.e. between geographic areas with no apparent barriers to gene flow). Then, the accumulated syllabic differences in song are probably distributed within a lek by behavioral matching between its members, which would explain why lek members share a high proportion of syllables. Regardless of the distance between leks, the song of each lek had a characteristic introductory syllable with strong differences in structure. This finding is consistent with previous observations in which members of song neighborhoods within leks shared the introductory syllable In summary, our study reveals independence between acoustic and neutral genetic divergence in wedge-tailed sabrewings, contrary to predictions of neutral evolution hypotheses. Along the continuous distribution of this subspecies, with no apparent geographic barriers, genetic divergence appears to be restricted by homogenizing gene flow, and geographical structure of vocal variation corresponds with restricted south-to-north gene flow between geographic areas of the Sierra Madre Oriental. Finally, the highly structured and marked song divergence between leks suggests that the evolution of song elaboration in wedge-tailed sabrewing resulted from a combination of processes probably linked to postdispersal learning, isolation by distance, and social factors associated with male-male interactions. As these birds are seemly open-ended learners, we interpret our results as that cultural variation appears more plausible than genetic variation in modifying songs differently at different leks and that social selection is involved in inducing this divergence. With negligible geographic barriers and no behavioral checks on dispersal, little genetic differentiation has developed in wedge-tailed sabrewings along the Sierra Madre Oriental despite the apparent intense social  selection acting on the elaborate acoustic traits see also . AlthougFigure S1Measurements taken from the (A) introductory, and (B) shared syllable emitted by every recorded individual. Numbers in (B) refer to three elements where the same measures as the complete syllable were taken. A fragment of a song bout is shown in (C) indicating the introductory syllable with an asterisk and the shared syllable with arrows.(TIF)Click here for additional data file.Table S1Localities, geographic location and altitude of wedge-tailed sabrewing sampled leks. Regions correspond to north, central and south of the Sierra Madre Oriental .(DOC)Click here for additional data file.Table S2Mean ± SD of spectral and temporal measurements of introductory syllables across leks of wedge-tailed sabrewings. N = number of individuals, n = number of songs.(DOC)Click here for additional data file.Table S3Mean ± SD of spectral and temporal measurements of the shared syllable and three of its elements, across leks of wedge-tailed sabrewings see . N = num(DOC)Click here for additional data file.Audio S1Song of El Cielo lek.(WAV)Click here for additional data file.Audio S2Song of Gómez Farías lek.(WAV)Click here for additional data file.Audio S3Song of El Naranjo lek.(WAV)Click here for additional data file.Audio S4Song of Aquismón lek.(WAV)Click here for additional data file.Audio S5Song of Xilitla lek.(WAV)Click here for additional data file.Audio S6Song of Cuetzalan lek.(WAV)Click here for additional data file.Audio S7Song of Macuiltépetl lek.(WAV)Click here for additional data file.Audio S8Song of La Orduña lek.(WAV)Click here for additional data file.Audio S9Song of Ursulo Galván lek.(WAV)Click here for additional data file.


Ficedula hypoleuca) breeding in two habitat patches between which dispersal and subsequent reproductive performance is influenced by phenotype (body size). For this, we conducted a cross-fostering experiment in a two-patch system: an oakwood and a conifer plantation separated by only 1 km.Habitat selection may have profound evolutionary consequences, but they strongly depend on the underlying preference mechanism, including genetically-determined, natal habitat and phenotype-dependent preferences. It is known that different mechanisms may operate at the same time, yet their relative contribution to population differentiation remains largely unexplored empirically mainly because of the difficulty of finding suitable study systems. Here, we investigate the role of early experience and genetic background in determining the outcome of settlement by pied flycatchers  moved away from the rearing habitat and, as previously observed in unmanipulated individuals, dispersal between habitats was phenotype-dependent. Pied flycatchers breeding in the oak and the pine forests are differentiated by body size, and analyses of genetic variation at microsatellite loci now provide evidence of subtle genetic differentiation between the two populations. This suggests that phenotype-dependent dispersal may contribute to population structure despite the short distance and widespread exchange of birds between the study plots.Taken together, the current and previous findings that pied flycatchers do not always settle in the habitat to which they are best suited suggest that their strong tendency to return to the natal patch regardless of their body size might lead to maladaptive settlement decisions and thus constrain the potential of phenotype-dependent dispersal to promote microgeographic adaptation. Selection of breeding environments is an important determinant of individual fitness and has therefore been a topic of considerable study in the realm of the ecology and evolution of dispersal –5. RecenNumerous theoretical studies suggest that individual variation in habitat preferences can play an important role in population differentiation and ultimately also sympatric speciation (reviewed by ). NevertDifferent mechanisms generating habitat preference and nonrandom dispersal have been well characterized from a theoretical standpoint , 7, 11. Ficedula hypoleuca) breeding in two contrasting habitats: a coniferous forest and a nearby deciduous forest separated by only 1 km. Males, and to a lesser degree, females breeding in the deciduous forest are morphologically different from those breeding in the nearby coniferous forest, the latter being smaller in size [In this study, we tested the role of early life experience and genetic background in the settlement patterns of pied flycatchers  forest of 9.3 ha, and a nearby (1.1 km) mixed pine (mostly Pinus sylvestris) plantation of 4.8 ha separated by unsuitable breeding habitat for pied flycatchers (i.e. a mixture of rock outcrops and riverside vegetation holding few suitable holes). Many aspects of the ecology of pied flycatchers have been studied in the deciduous and the coniferous forest since wooden nest-boxes (156 and 81) were provided in 1984 and 1988, respectively (e.g. [Fieldwork was conducted between 2006 and 2015 in a two-patch system: a mature oak (ly (e.g. ). Pied fly  using a nest box trap, and measured for tarsus length (to the nearest 0.05 mm) and body mass (to the nearest 0.1 g). Fledgling mass and tarsus length were measured on day 13, when nestlings have already attained the definitive adult size , 40. A sCross-fostering was conducted in 2006–2009  and 2012–2013 . Note that recruits stemming from complete and half clutches or broods swapped between nests are equally suitable for the purpose of this study. We cross-fostered birds both within and between habitats in order to test, on one hand, how likely it is for a bird raised in its habitat of origin to return there to breed (i.e. control group) and, on the other hand, how likely it is for a bird experimentally raised in a habitat other than its habitat of origin to move back to its habitat of origin . Cross-fostering was carried out 2–3 days after the onset of incubation (clutches) or on day 2–3 after hatching (broods). All transfers were performed between nests of similar phenology . Nestlings were individually marked for individual identification with a unique combination of colour markings on underparts (legs and wings) made with non-toxic felt pens and repainted every 2–3 days. To avoid skewing the masses of experimental nestlings in any direction during partial cross-fostering and thus minimize non-deliberate effects of competition between native and foster birds, all nestlings were weighed, ranked for weight, and then sequentially assigned to be either cross-fostered or remaining in the source nest.Experimental manipulations had no apparent effect on recruitment rates (see ‘results’). For all recruits stemming from cross-fostering experiments that were subsequently captured as first-time breeders, we recorded whether they returned to breed in their rearing patch or, conversely, moved into the adjacent one. It should be noted that, because the two habitat patches sampled in this study are isolated from other patches by unsuitable habitat, pied flycatchers returning to breed in the study area must choose between settling in their natal or the adjacent plot, or risk not breeding at all .To examine genetic differences between pied flycatchers breeding in the two plots we followed the rationale described by . Our datFST) between study plots , with twenty replicates for each K, were performed with a 105 burning period followed by 106 MCMC repeats after burning. To obtain the true value of K based on the method described by [We used GENETIX to estimate the extent of genetic differentiation . Furthetations;  to inferribed by  we used ribed by .n = 43), results were exactly the same (details not shown), suggesting that the age at first breeding does not affect the settlement pattern of pied flycatchers.To investigate the roles of early experience and genetic variation in determining selection of either habitat by adult pied flycatchers in the two-patch system, we fitted a generalized linear mixed model  including birds’ propensity to return to the rearing patch  as the response variable. As fixed effects, we included sex, the type of experimental treatment (cross-fostering within or between patches) as a control term, and the life stage of cross-fostering (egg or nestling) to account for possible ontogenetic effects on imprinting. Laying date at first reproduction, which is an accurate proxy for the arrival date in our study population , was alsIt is possible that some fledglings had visited (and possibly imprinted on) the adjacent forest prior to departure to the wintering areas, since post-fledging explorations may exceed the minimum distance separating the two plots .Migratory birds may rely on a host of environmental cues and constraints to guide their settlement decisions after arrival, such as nest-site  and foodFinally, we tested the interaction between tarsus length and rearing patch according to the notion by  that, whhttp://www.R-project.org) using the function lmer in the package ‘lme4’ [GLMMs were fitted in R 2.14.0 . Recruitment rates did not differ between cross-fostered and non-manipulated fledglings either  and 11.7 % (n = 1658); χ2 = 1.003, d.f. = 1, P = 0.32), indicating that cross-fostering had no effect on the natural recruitment rates of pied flycatchers. Nests subjected to egg or nestling translocations showed similar recruitments rates  and 13.7 % (n = 293 nestlings); χ2 = 0.030, d.f. = 1, P = 0.83), indicating that the life stage at which the experiment was carried out did not bias our estimates. Overall recruitment rates did not differ either between cross-fostered males and females .Between 2006 and 2013, a total of 445 individuals from 116 nests (58 dyads) and 496 individuals from 120 nests (60 dyads) were cross-fostered between and within patches, respectively. Overall recruitment rates of cross-fostered young from experimental and control nests were, respectively, 14.7 % (57 recruits from 389 confirmed fledged offspring) and 11.8 % (48 recruits from 406 confirmed fledged offspring), the difference being not statistically significant  or between  patches , which was corroborated by the exact test of genotypic differentiation . However, unlike in the coniferous habitat , the deciduous forest population showed a deviation from H-W equilibrium  due to an excess of homozygotes in three markers , which might affect the FST estimates. Further analyses showed that the excess of homozygotes in those loci was due to rare, private alleles appearing at low frequencies. After removing from the analyses the individuals carrying rare alleles in the deciduous patch, the two populations showed no evidence for H-W disequilibrium , had similar genetic diversity  and, although marginally non-significant, FST values were of similar magnitude as that obtained from the entire data set . These results suggest that rare alleles in some individuals from the deciduous forest likely contributed to, but were not fully responsible for, the genetic differentiation of pied flycatchers between the study plots. Bayesian clustering approach did not provide support for two genetic clusters. This is not surprising since, as shown by simulations [n = 2) and low genetic differentiation, as occurs in our system.ulations , STRUCTUBy following individuals that had been cross-fostered to the same or a different habitat patch type, we provided evidence that pied flycatchers returned mostly to breed as adults in the area where they had fledged, regardless of their sex and origin. This is consistent with the hypothesis that individuals’ decisions on where to settle are based on the environment they experienced at an early age, rather than on their genetic background. Birds breeding in the coniferous and the deciduous forest differ in body size , 29 and,Our study supports earlier evidence from experimental , 62 and It is known that experience in the natal site can strongly influence later habitat choice in the pied flycatcher , 62, butBesides the duration of the imprinting process, the spatial scale of post-fledging exploration may affect settlement patterns in the subsequent years. Male pied flycatchers commonly settle closer to their natal sites than females, although both sexes seem to imprint on a similarly small area . Mean dSome individuals could have visited the study area in one or more seasons before reproducing for the first time and explored future territories away from the area they explored as a fledgling . But if d = 0.52) showed that, for α = 0.05, an overall sample size of n = 120 would be required to have 80 % power, which means that, according to the actual recruitment rate (13.2 %) and proportion of recruits moving between habitats (27.6 %), over 3200 individuals would need to be cross-fostered. Still, taken together, the results of this experiment and our previous studies [So far, few studies have attempted to tease apart the relative effects of natal patch preferences and phenotype-dependent dispersal on population differentiation.  also rep studies , 29 give studies  that pheFST = 0.008; [Passer domesticus)   but, unlike in the stickleback populations, phenotype-dependent dispersal and natal habitat preferences of pied flycatchers do not appear to act synergistically but partially cancel out each other.  showed t= 0.008; ), and alFinland; ) or the  Europe; ). Accord Europe; , 33.Early experience in the natal patch may play a crucial role in determining subsequent dispersal and settlement in the pied flycatcher (, 61, 69,


This study examines genetic diversity among 102 registered English Bulldogs used for breeding based on maternal and paternal haplotypes, allele frequencies in 33 highly polymorphic short tandem repeat (STR) loci on 25 chromosomes, STR-linked dog leukocyte antigen (DLA) class I and II haplotypes, and the number and size of genome-wide runs of homozygosity (ROH) determined from high density SNP arrays. The objective was to assess whether the breed retains enough genetic diversity to correct the genotypic and phenotypic abnormalities associated with poor health, to allow for the elimination of deleterious recessive mutations, or to make further phenotypic changes in body structure or coat. An additional 37 English bulldogs presented to the UC Davis Veterinary Clinical Services for health problems were also genetically compared with the 102 registered dogs based on the perception that sickly English bulldogs are products of commercial breeders or puppy-mills and genetically different and inferior.Four paternal haplotypes, with one occurring in 93 % of dogs, were identified using six Y-short tandem repeat (STR) markers. Three major and two minor matrilines were identified by mitochondrial D-loop sequencing. Heterozygosity was determined from allele frequencies at genomic loci; the average number of alleles per locus was 6.45, with only 2.7 accounting for a majority of the diversity. However, observed and expected heterozygosity values were nearly identical, indicating that the population as a whole was in Hardy-Weinberg equilibrium (HWE). However, internal relatedness (IR) and adjusted IR (IRVD) values demonstrated that a number of individuals were the offspring of parents that were either more inbred or outbred than the population as a whole. The diversity of DLA class I and II haplotypes was low, with only 11 identified DLA class I and nine class II haplotypes. Forty one percent of the breed shared a single DLA class I and 62 % a single class II haplotype. Nineteen percent of the dogs were homozygous for the dominant DLA class I haplotype and 42 % for the dominant DLA class II haplotype. The extensive loss of genetic diversity is most likely the result of a small founder population and artificial genetic bottlenecks occurring in the past. The prominent phenotypic changes characteristic of the breed have also resulted in numerous large runs of homozygosity (ROH) throughout the genome compared to Standard Poodles, which were phenotypically more similar to indigenous-type dogs.English bulldogs have very low genetic diversity resulting from a small founder population and artificial genetic bottlenecks. Although some phenotypic and genotypic diversity still exists within the breed, whether it is sufficient to use reverse selection to improve health, select against simple recessive deleterious traits, and/or to accommodate further genotypic/phenotypic manipulations without further decreasing existing genetic diversity is questionable. The English bulldog is one of the most popular breeds in the world because of its child-like appearance and demeanor. The alterations in body type and behavior needed to create the breed have required physical changes well beyond its village dog ancestors. These changes have occurred over hundreds of years but have become particularly rapid over the last decades. Unfortunately, popularity does not equate to health and there have been increasing pressures on breeders to moderate the extreme physical changes that now affect the breed and its health. Improving health through genetic manipulations presumes that enough diversity still exists to improve the breed from within, and if not, to add diversity by outcrossing to other breeds. The present study was an assessment of genetic diversity that still exists in a representative number of individual English bulldogs using DNA rather than pedigrees. The results confirm that the breed has lost considerable genetic diversity through such things as small founder population and artificial genetic bottlenecks resulting from highly focused selection for specific desired physical traits. This is manifested by a narrowing of allele diversity in many parts of the genome, and the creation of numerous large regions of the genome that are essentially identical within the breed, which are significantly different from other dogs. Loss of genetic diversity is also pronounced in the region of the genome that contains many of the genes that regulate normal immune responses. The loss of genetic diversity and extreme changes in various regions of the genome will make it very difficult to improve breed health from within the existing gene pool. Loss of present genetic diversity is further threatened by rapid integration of new coat color mutations, increased wrinkling of the coat, and attempts to create a more compact body type. Contrary to current beliefs, brachycephaly and the resulting breathing problems in the breed are the result of complex changes in head structure, and cannot be corrected by merely lengthening the face. Furthermore, other issues in English bulldogs need to be addressed, including many serious health problems that are not associated with brachycephaly, but are intrinsic to inbreeding.th to 18th centuries [The first mention of what might be a contemporary type bulldog was reported in 1632 [Attempts to legislate against bull baiting began in the UK in 1802 and the sport was finally abolished by an Act of Parliament in 1835, which led the breed to the brink of extinction [2A number of modern breeds use “bull” or “bulldog” in their names and all have evolved from the original Bulldogs and Mastiff-type dogs , 2. Some,7,8The outward appearance of many dog breeds change with time, and this is also true of the English bulldog.4th century also had its share of health problems and critics.5 Therefore, the English bulldog was not initially popular in the UK, although its popularity has greatly increased over the last decades in many affluent countries [5Although much has been said about the ill-health of contemporary English Bulldogs, the English bulldog of the late 19ountries . Englishountries . Health 5,10,11The health problems of the English bulldog have been well documented and start with conception, fetal development and parturition. Severe conformational changes have necessitated a high rate of artificial insemination and Caesarean sections and litter sizes tend to be small.The brachycephalic syndrome is a leading cause of ill-health and death in the breed. However, the syndrome is not caused by brachycephaly per se, as brachycephalic breeds such as the boxer do not suffer the syndrome to the same degree. The bulldog tongue is excessively large at the base, the palate is large and easily obstructed by the base of the tongue, the lower jaw is pushed forward (prognathous), and the nares are frequently stenotic and the trachea hypoplastic. This leads to loud panting during physical exercise, stridor during rest and slobbering; sleep apnea, hypercapnia and hypochloremia/hypomagnesemia; exercise intolerance, cyanosis and collapse; and choking fits manifested by gagging, retching, vomiting, aerophagia/flatulence and aspiration pneumonia –12. The Chondrodysplasia, a heritable skeletal disorder that has been incorporated into the phenotype of many dog breeds , predisp12, a strange argument given the continuous phenotypic changes that have occurred within the breed over many decades.6,7,8 Groups in The Netherlands have called for a ban on English bulldogs based on a belief that the breed can no longer be returned to a health, while supporters believe that the breed can be returned to health from within and are petitioning the government against the ban.14Severe health problems in English bulldogs in the USA and the UK have prompted articles  such as th to 4th in popularity between 2013 and 2015 in AKC registrations [6,7,8, bulldogs that reproduce without assistance, move freely, walk or run for long distances, and breathe normally even at rest are the exception.English bulldogs have risen from 5Efforts are underway in the UK to improve the health of bulldogs from within the breed by making modest changes on what is acceptable in a show dog. However, there is very little knowledge about the actual genetic status of the breed and whether enough genetic and phenotypic diversity still exists to significantly improve health without further reducing existing genetic diversity or relying on outcrossing. Breeders still rely heavily on pedigrees and coefficients of inbreeding (COI). The problem is that pedigrees emphasize ancestors and inheritance by descent but not actual genetic makeup. They are also subject to parentage errors and COIs based on a few recent generations are of limited value in a breed that started with few founders subjected to numerous artificial bottlenecks that occurred decades and even centuries earlier. Therefore, we endeavored to conduct a broad-based genetic assessment of the breed using DNA rather than pedigrees. The findings of this study indicate that English bulldogs may have insufficient genetic diversity to significantly improve gross physical abnormalities associated with poor health, to eliminate simple recessive deleterious traits, or to use inbreeding to rapidly integrate new coat colors and to breed for a smaller and more compact shape without further decreasing genetic diversity in individuals and adding to their health problems.The Veterinary Genetics Laboratory (VGL)  provided DNA samples of 102 registered English Bulldogs; 87 of the dogs were from the USA, six from Finland, three each from Canada and Austria, and one each from Czechoslovakia, Hungary, and Argentina. DNA from these dogs was submitted mainly for coat color or hyperuricosuria mutation testing and used in breeding programs. As such, they were presumed to be of adequate health and therefore listed as “controls”. Thirty-seven DNA samples were collected from whole blood of English bulldogs submitted for various diagnostic tests at the UC Davis Veterinary Medical Teaching Hospital (VMTH). These dogs were seen for a variety of health problems ranging from breathing problems, eye problems, skin disorders, orthopedic problems, or cancer and were therefore listed as “case”. DNA was extracted using established procedures .650.79.2, 990.35.4, MS34A, MS34B, MS41A, and MS41B [Maternal haplotypes were determined by sequencing 655 bp of the mitochondrial D-loop (nucleotide 15453–16107) in 48 English Bulldogs as described . Dogs wend MS41B , 22.Thirty-three STR loci across 25 chromosomes were used to assess genomic diversity, while four STR loci were used to determine DLA class I haplotypes and three STR loci were used for DLA class II haplotypes. The primer sequences, dye markers, conditions for amplification and analysis of these STR markers have been published .A genetic assessment using allele frequencies was conducted using GenAlEx 6.5 software . The popInternal relatedness (IR) is a statistical estimate of how closely an individual dog’s parents were related to each other . It also–homozyg-group. The analysis yielded the number, size range of ROH, and the portion of ROH that was shared (consensus ROH) by all individuals interrogated. This information was extended by looking for the consensus ROH that were shared by 6–9 of the 10 individuals tested.Illumina 170 K CanineHD datasets for English bulldog and Standard poodle were obtained from other GWAS studies , 29 and Paternal haplotypes were determined for 44 male English Bulldogs in the study population using six Y-specific STRS. Haplotype 1 was dominant in 93.1 % of the dogs, while haplotype 2–4 were observed in 2.3 % of dogs each (Table EBU-A (GenBank:KP665923), EBU-C (GenBank:KP665928), EBU-J (GenBank:KP665924), EBU-K (GenBank:KP665914), and EBU-7 (GenBank:KP665930). Maternal haplotype frequencies and variations of base pair positions are listed in Table Five maternal haplotypes were identified in the control population based on mitochondrial sequences. The sequences of the haplotypes observed in English bulldogs corresponded to GenBank Accession numbers as follows: VGL1165), and the lowest was three (INRA21). Most of the loci had one or two alleles that dominated in frequency. Nineteen of 33 loci had single alleles with a frequency ≥ 50 %, which are highlighted in Table REN162C04 was virtually fixed with a frequency of 0.99 amongst the 102 dogs studied  to +0.304 (most inbred) Fig. . This suThe adjusted IR value (IR-village dog or IRVD) for the population gave a more accurate measure of just how inbred the parents of modern English bulldogs were compared to village- or landrace- type dogs from which the breed evolved. IRVD values ranged from 0.043 to 0.64 with a mean of 0.335  was 51.35 Mb on five of the six male dogs in the group. The number of consensus ROH that were detected on autosomes increased as the number of dogs that shared larger consensus ROH decreased. Nine of ten of the dogs shared eight additional ROH compared to 10/10 dogs; 8/10 dogs an additional 16 ROH compared to 10/10 and 9/10 dogs; 7/10 dogs had 39 more ROH compared to 8/10, 9/10 and 10/10 dogs; and 6/10 dogs had 53 more ROH than 7/10, 8/10, 9/10 and 10/10 dogs (Table The number and size of ROH were much lower in Standard Poodles than English bulldogs Table . No consn = 87) and several other countries (n = 15). Thirty-seven pet English bulldogs seen for health problems at UC Davis Veterinary Clinical Services were also included in the study. The populations that were tested evolved from one major and three minor paternal haplotypes and three major and two minor maternal haplotypes. One paternal haplotype, which was found only in a one dog, appeared to be distinct. The two remaining minor paternal haplotypes, also in single dogs, differed from the dominant paternal haplotype by a simple mutation. This pattern of a single dominant paternal haplotype and a small number of maternal haplotypes has been observed in most pure breeds and is therefore not unique to English bulldogs [This study examined genetic diversity among contemporary English bulldogs used for breeding purposes from the USA  as well as small Mastiff-type dogs , while EBU-K has been found in Asian Mastiffs such as the Shiba Inu and Shikoku. Therefore, the maternal haplotypes seen in English bulldogs support the general belief that English bulldogs evolved from Mastiff-type dogs crossed with brachycephalic breeds such as the Pug . It is tREN162C04 was homozygous in 101/102 dogs studied. Loss of genetic diversity as a result of pure-breeding, especially when those breeds undergo selection for conformation, is well documented [Evidence obtained from the 33 genomic STR loci provides additional evidence for the small founder population and artificial genetic bottlenecks that led to the modern English bulldog. The average number of alleles per locus was similar to other pure breeds that have been diversity tested.cumented . The varcumented –35. The Measurements of observed and expected heterozygosity (Ho and He) and the inbreeding coefficient F are also useful in looking at the genetic makeup of a population. Observed and expected heterozygosity were similar for the English bulldogs tested, yielding an inbreeding coefficient F that was close to zero (0.001). This indicated that the population as a whole was in HWE despite a limited gene pool and that English bulldog breeders were doing a reasonable job of identifying more distantly related dogs for mating. However, IR values indicated that He, Ho and F values were misleading, as many individual dogs in the study were actually products of parents that were much more related to each other than assumed from the population-wide fixation indices.Internal relatedness has been widely used as an indicator of population fitness –42, implThe low number of different DLA class I and II haplotypes in English bulldogs was associated with an imbalance in the relative frequency of each haplotype. Four of 11 class I and 3/9 class II haplotypes were found in over 90 % of the individual English bulldogs tested. Moreover, there was a high level of homozygosity among the dominant DLA class I (19 %) and II 42 %) haplotypes. Although not balanced in frequency, genetic assessment of the seven STR alleles associated with the DLA class I and II regions showed them to be randomly segregating at this time. This indicated that the over-representation of certain haplotypes occurred at the onset of breed creation as a result of small founder numbers, and/or that it was associated with artificial genetic bottlenecks that were subsequently masked by a return to random selection. Although the DLA region is only a small part of the genome, the importance of these haplotype imbalances, small haplotype numbers, and increased homozygosity should not be underestimated , 42. The % haplotIn order to gauge the extent to which humans shaped the phenotype of English bulldogs from typical dogs, we decided to compare the English bulldog with a breed that was similarly inbred based on the number of homozygous SNPs identified by Illumina 170 K canine SNP arrays and IR scores, but outwardly similar in appearance to the ancestral dog. The Standard Poodle, which has been similarly studied , met theFGFR3, which is responsible for achondroplasia in humans and presumably linked with related genes responsible for canine chondrodysplasia.We did not associate runs of homozygosity in English bulldogs with characteristic phenotypic traits but are confident based on previous studies that the greater genomic variation in English bulldogs compared to Standard Poodles reflected stronger human-directed selection in the former breed than in the latter. Pollinger and colleagues  also conIt can be assumed from this and other studies that the small founder population of the English bulldog, estimated at 68 individuals,7,8 The one region of the genome of English bulldogs that is least amenable to reverse genetics may be the DLA, which has very low diversity based on DLA class I and II haplotypes and a high level of homozygosity. The nine DLA class II haplotypes recognized in this group of English bull dogs were only a fraction of the 88 haplotypes that were reported for dogs in 2007 [Assuming that there is a will to improve the overall health of English bulldogs, the question raised by this study is whether or not there is sufficient genotypic diversity remaining in the breed to allow “reverse genetics” to correct phenotypic abnormalities that have major impacts on health. There are certainly phenotypic differences that still exist between various lines of English bulldogs and among individuals, and many English bulldogs enjoy much better health than others. The regions flanking consensus ROH vary greatly in size in individual English bulldogs, suggesting that some of these regions may contain “hidden” genetic diversity that may prove critical for reversing the degree of brachycephaly or chondrodysplasia. The existence of phenotypic variation within the breed is evident from photographs of modern English bulldogs. in 2007 . Genes wPopulations that have lost genetic diversity through small founder numbers and artificial genetic bottlenecks are more likely to accumulate deleterious traits , 52. A lA low level of breed-wide genetic diversity also limits the ability to rapidly introduce desired traits, usually in the form of simple recessive mutations. There is increased demand for smaller and more compact English bulldogs, dogs with wrinkled coats and rare coat colors. Such refinements in the breed create popular sire effects and yet more artificial genetic bottlenecks that will cause the loss of more genetic diversity if not properly managed. The negative effects of the rapid introduction of new genetic traits on the health have been best documented in the “The Rare Color Bulldog Craze”.18,12,13In conclusion, English bulldog breeders differ widely on their perception of health problems in their breed and what do about them. Some breeders blame disreputable or backyard “commercial” breeders for the unhealthy dogs that are being sold.19The feelings of individual English bulldog breeders about the health of their breed and what if anything should be done about it may ultimately be taken out of their hands. English bulldog breeders across the world must take seriously constitutional amendments on the rights of animals. The European Union has recently updated their rules on animal welfare in 2015.Breeding of the English bulldog for extremes of brachycephaly, chondrodysplasia, skin folding and child-like appearance and personality has required a level of human-directed positive selection that has made the English bulldog both one of the most popular and unhealthiest of dog breeds. A DNA-based assessment of the breed along a number of parameters has confirmed that the breed is greatly lacking in genetic diversity, which may preclude or minimize the ability of breeders to recreate healthier phenotypes from existing genetic stock, to eliminate deleterious mutations, and to add in new phenotypic traits.AKC, American Kennel Club; DLA, dog Leukocyte antigen; IR, internal relatedness; STR, short tandem repeat; VGL, veterinary genetics laboratory, UC Davis