Because of the influence of genetics on animal production and the risk of losing genetic diversity of naturally adapted breeds, this study evaluated the genetic diversity of sheep of the Morada Nova breed belonging to an animal science institute in Brazil. The herd in question is one of the country’s most representative of the breed. Samples of DNA extracted from the plasma of 61 animals were used for later analysis of the genotypes using microsatellite molecular markers.The polymorphic information content was 0.66, the observed heterozygosity was 0.65 and the fixation index was 0.048. According to the results, there is moderate genetic diversity in the studied population, suggesting the implantation of breeding programs aimed at conservation of the observed genetic diversity.The results obtained in this study will be of great importance to decisions on herd structure, besides contributing to other work to be carried out at the research center. Morada Nova is one of the main hair sheep breeds in Brazil. They are small animals that stand out among naturalized sheep breeds sheep due to the good production of meat and hides. Because of traits such as high prolificacy , parasitCurrently, small herds of Morada Nova sheep can also be found in the Southeast and Midwest of Brazil, populations are distributed mainly in research centers, most frequently in São Paulo state. The Instituto de Zootecnia in the municipality of Nova Odessa has one of the main herds representative of the breed, assisting and monitoring producers them in addition to developing research in areas like nutrition, health, reproduction and breeding, among others. Since genetics exerts an influence on production indexes, either positive or negative, so the best use of this potential depends on selection, which in turn requires a diversified number of alleles that can favor larger numbers of combinations.In addition to support for genetic improvement programs, genetic diversity is a determining factor for the conservation of genetic resources and the evolutionary persistence of species. Naturally adapted breeds may be a source of genetic material able to improve resistance of other breeds to unfavorable conditions . The abiGenetic diversity of indigenous breeds is a major concern considering the need to preserve what may be irreplaceable richness regarding new productive demands . A speciThe aim of obtaining higher animal production rates is impaired by loss of diversity and the introduction of exotic breeds in crosses carried out indiscriminately, modifying the population structure and compromising a breed’s existence. Genetic depression due to inbreeding is also an extremely important factor, also impairing herd performance .To maintain diversity, studies need to be conducted to learn the genetic structure of populations. Microsatellite markers are used to estimate parameters of genetic diversity and herd structure in genetic resource conservation programs due to their dominance and high sensitivity .Considering this, the objective of this work was to evaluate the genetic diversity of sheep of the Morada Nova breed belonging to the Instituto de Zootecnia in Nova Odessa, São Paulo. The results obtained in this study will serve as an aid to future research and conservation of the breed.Of the 15 microsatellite markers tested, just INRA63 did not amplify loci and 14 were polymorphic, for a total of 100 detected alleles. We did not observe the presence of null alleles, that is, all samples amplified the 14 microsatellite loci tested. The mean number of alleles (Na) was 7.14 and the mean number of effective alleles (Ne) was 3.68. The locus ILSTS08 presented the lowest polymorphism with two alleles and Ne of 1.44, while locus OARFCB304 was the most polymorphic, with Na 11 and Ne of 7.728.According to the polymorphic information content (PIC) test, all markers presented PIC above 0.5, considered highly informative, except for the OARAE129 marker, which presented a low PIC of 0.19.p-values: p < 0.001 ; p < 0.01 ; and p < 0.05 . Another seven were not in equilibrium, with p > 0.05 .Of the 14 microsatellites tested, seven were in Hardy Weinberg equilibrium with significant In the evaluation of allelic richness , the mean value of all markers in this population was 4.51, showing the long-term potential of adaptation and persistence of the population. When observed individually by marker, the lowest values of allelic richness were for the loci ILST08 (1.95), OAR129 (2.168) and MCMX42 (3.49), the highest value was in the OAR304 locus (7.06), and the other markers obtained medium values ranging from 4.15 to 5.30.The mean observed heterozygosity (Ho) in the population was 0.65 while the expected (He) was 0.66. The diversity estimates applied for each locus studied are presented in Table The fixation index (F) reflects the probability that two alleles within the same individual are identical in offspring. Six of the 14 markers tested had negative indices, that is, they were not fixed in the population. For the other markers, two presented moderate indices  and one a high index (OARAE129). The overall mean considering all the microsatellite markers was low (F = 0.048).The 14 microsatellite loci analyzed showed 91 possible combinations, a total of 16 combinations of markers, the genotypes in one locus are not independent of the genotypes in the other locus. Of the 16 combinations in linkage disequilibrium, only one combination was syntenic Table .Table 2TThe statistical cluster analysis by ascending hierarchical ordering, grouped the 61 animals into two clusters  technique also presented highly polymorphic loci and has been used previously in genetic diversity studies . These mCurrently, breeding values in genomic selection are generally predicted based on SNP markers (single nucleotide polymorphism). These have also been used in analyses of genetic diversity . There aIn this study, the microsatellite markers used reflected the genetic diversity evaluated in the population. The 14 markers were highly informative, with PIC above 0.50. PIC values above 0.50 are considered highly, values between 0.50 and 0.25 are moderately informative and values below 0.25 are considered slightly informative . This hiThe number of alleles within a population as one of the parameters of genetic diversity and is a major factor in animal production, since a greater number of alleles within a population allows for greater possibilities of recombination. It is common to note that in commercial or geographically isolated herds, selection pressure exerts forces contrary to diversity, leading to genetic drift. However, the herd studied, although new and composed of small sheep, had a high Na value compared to other studies conducted in different regions analyzing the same breed.We found 100 alleles, with average number of alleles per locus of (Na) 7.14. The highest number of allele markers were in OARFCB304, with 11 alleles, and INRA05 with 10 alleles, while least number were in ILST08 and OARAE129, with 2 and 3 alleles respectively. This result indicates that even though the population studied is isolated, the Na was higher than that described in other studies evaluating Morada Nova herds. In Ceará, 89 alleles  were observed using 17 microsatellite markers in a study performed in 13 different farms . FerreirOur study revealed the presence of low allelic richness. The decrease of allelic richness may lead to a reduction in the population’s potential for adaptation to future environmental changes, since this diversity is the raw material for evolution through natural selection .The value of 4.51 identified for allelic richness in this study suggests unequal distribution of allelic frequencies with the presence of low-frequency alleles, such as ILST08 (1.96) and OAR129 (2.17). The average of allelic richness observed by GebreMichael  was 6.79Like the AR values, the heterozygosity indices also estimate genetic variation within a population and are one of the most widely used parameters of genetic diversity . HeterozValues of Ho (0.53) and He (0.59) were attributed to the low diversity of the Morada Nova breed . In thatEstimates of diversity in populations of sheep from Colombia presented mostly low heterozygosity, with values of Ho below the values of He, with means of 0.68 and 0.77 respectively . In thatOne of the main factors that causes loss of heterozygosity is the fixation of alleles of commercial interest and mating directed to obtain higher production rates, common in industrial production. This heterozygosity loss increases with rising number of alleles in homozygosis, resulting in allele fixation. According to the FAO (1998), the estimated value through the fixation index (F) should not exceed 1%. In this study, the estimated value of F was 4.8%. The ETH152, ILSTS08 and OARAE markers presented values of 14.6, 19.6 and 37.5, respectively. These large values indicate high homozygous for these loci and possible loss of alleles. Five markers presented values below 6% and another five negative values, with these also being the loci with highest Ho. This fact can be attribuited to the lack of gene flow and rigorous mating control. However, the low number of Morada Nova sheep in this geographic region makes it hard to introduce new genes to increase the diversity.Likewise, in a study of diversity study of Morada Nova herds located in five Brazilian states including São Paulo, the authors found F values between 4 and 12% and reported consanguinity between the herds . InbreedThe Bayesian group factor analysis performed by the Structure software determined the structure of the sheep population studied and presented a population structure clearly divided into two representative groups, with some individuals presenting a mixture of the two gene groups, possibly due to the miscegenation of the main cluster present. This structure is possibly due to the origin of the herd, which was formed of individuals coming from different states.The ability of the Bayesian algorithm to detect the most probable number of clusters that explain the distribution of the genetic groups of the population studied was based on a model that correlates the allelic frequencies within the population (admixture model). This contributes to the detection of subpopulations or genetic subgroups in a single population when individuals may be particularly related and presents the same detection ability as the independent allele frequency model, such as the absence of high levels of relationship between the individuals or populations evaluated .The structure of the population presented here is important for the maintenance of genetic diversity through the direction of mating. The dendrogram allows observing the animals according to their genetic distance, which facilitates the identification of the best genetic combinations to avoid endogamous mating.Diversity is conserved by the maintenance of individuals with few genetic relationships. Knowledge about these relationships helps in the management herds with better use of existing genotypes in breeding programs, besides being fundamental in programs for conservation of genetic resources.The herd of Morada Nova of the Instituto de Zootecnia presented moderate genetic diversity. Strategies to maintain or increase genetic diversity should be implemented considering the need to preserve what may be irreplaceable richness and the importance of the herd in the conservation of the breed regarding new production demands, mainly as a maternal lineage in industrial mating.The study was carried out on 61 animals of the Morada Nova breed from the Sheep Unit of the Center for Research and Development of Diversified Animal Husbandry, of the Instituto de Zootecnia, part of the Paulista Agribusiness Technology Agency of the Department of Agriculture, located in Nova Odessa,SP (22 ° 42 ‘S and 47 ° 18’ W).DNA was extracted from blood plasma using the commercial NucleoSpin Blood kit  following the manufacturer’s instructions, with subsequent quantification of DNA concentrations measured with a spectrophotometer (NanoDrop™ 2000-Thermo Science). The extraction and purification steps were performed at the Animal Genetic Improvement Laboratory of Norte Fluminense State University in Rio de Janeiro.Genotyping was performed using a capillary sequencer . We used 15 microsatellite markers recommended for identification of kinship and paternity in sheep by the International Society for Animal Genetics (ISAG): CSRD24, ETH152, ILSTS08, ILSTS87, INRA05, INRA06, INRA172, MASF65, MCM42, MCM527, OARAE129, OARCP49, OARFCB20, OARFCB304 AND INRA 63.The GenAlEx software version 6.502 was used to evaluate population genetics, to estimate: number of alleles (Na), effective number of alleles (Ne), observed heterozygosity (Ho), expected heterozygosity (He), fixation index (F); and polymorphic information content analysis (PIC). Linkage disequilibrium was estimated for all marker pairs using GenePop software version 1.2 .R) measure of the 14 markers was estimated using the HP-RARE 1.0 software. Using Nei’s genetic distance matrix with standardized data the dendrogram was constructed from the unweighted pair group method with arithmetic mean using the software MEGA-5 .The distribution of genetic diversity was studied using the genetic distance analysis of Nei. For the Hardy-Weinberg equilibrium (HWE), Fisher’s exact test was applied. The population allele richness  and correlating the allelic frequency in the population. The burn-in period was 200,000 rounds followed by 500,000 Monte Carlo Markov Chain interactions. To observe numbers of possible clusters in the population, the independent “K” test was performed from 1 to 10 clusters with 20 replicates, thus verifying the consistency of the results. The ideal “K” value was evaluated after the analysis of the result file by the Evanno method using the web-software STRUCTURE Harvester version 0.6.94.2012 software. After identification of the number of subpopulations, a test was performed to select the optimal “K” to generate the illustrative population plot.


Vitis vinifera ssp. sylvestris). The wild grapevine arises in the Eastern Adriatic region in a contact zone of the EU-Mediterranean and the sub-Mediterranean characterized by typical karst relief. This study focuses on the chloroplast DNA (cpDNA) analysis of wild grapevines and the biodiversity of accompanying woody species to better understand the genetic variation of the sylvestris populations of the Eastern Adriatic region and to investigate how this variation fits within today’s wild grapevine distribution in the European continent. The allelic variation at nine cpDNA microsatellite loci of wild individuals was used to characterize haplotype diversity in 53 individuals from four population sites. All individuals were grouped into two chlorotypes: A and D, D being the rare haplotype among wild populations on the European continent. In total, 52 woody plant species were identified. However, the studied vegetation structures have been affected by permanent human pressure on natural resources and the preservation status of the collection sites. Based on our results, we conclude that the investigated areas were probably shelter zones for wild grapevine preservation during the unfavorable glaciation era.The Eastern Adriatic region, encompassing Croatia and Bosnia and Herzegovina, is considered an important area of natural populations of wild grapevines ( Vitis vinifera ssp. sylvestris can be found throughout the northern hemisphere, from the Atlantic coast of Europe and northern Africa to the Western Himalayas. It is believed to be a living ancestor of modern grapevine cultivars [Wild grapevines , parentage, domestication events and the history of wild grapevine migration as well as how to find resistance genes for breeding purposes [“Reading”the grapevine genome at the molecular level presents a good opportunity to clarify several major issues, such as the relationships between the two subspecies; wild and cultivated grapevine . The extracted DNA was quantified and used as a working DNA concentration of 1 ng/μL. A set of 9 microsatellite loci were analyzed to study the genetic diversity of cpDNA: cpSSR3, cpSSR5, cpSSR10, NTCP8, NTCP12, ccSSR5, ccSSR9, ccSSR14, ccSSR23 , 26.The KAPA Fast Multiplex PCR Kit (2x)  was used to create reaction mixtures containing the master mix, 100 pmol of each primer and approximately 1 ng of template DNA. Amplification was performed in ABI 9700 thermal cyclers (Applied Biosystems) using the following program: 15 min initial denaturation at 95°C, followed by 30 cycles of denaturation at 94°C (30 s), annealing at 60°C (90 s) and extension at 72°C (60 s). A final extension was performed at 60°C for 40 min.The amplified products were resolved using capillary electrophoresis on an ABI 3130xl Genetic Analyzer  using GeneScan-LIZ 500 as an internal standard. Peaks were identified by size and height with GeneMapper 5.0 software (Applied Biosystems).At the collection sites, all woody species  were collected to assess the forest vegetation that supports wild grapevine growth and to estimate the (abiotic) conditions at the sites. Herbaceous plants, mosses and ferns were not considered. All species were brought to the laboratory for definitive identification and a herbarium encompassing all the collected plants was made. The names of the woody species follow the Euro+Med PlantBase .For each location average Ellenberg indicator value for light, temperature, moisture, soil reaction and soil nutrients availability was calculated. Average Ellenberg indicator values –29 were Different measures of genetic variability among 53 unique genotypes at 9 cpSSR loci were calculated. The number of effective alleles (Ne), Shannon's information index (I), and genetic diversity (h) were calculated for each locus using GenAlEx 6.5 . HaplotyThe floristic composition was elaborated using Non-Metric Multidimensional Scaling (NMDS) and Bray-Curtis dissimilarity measure. Twenty iterations were used. The Ellenberg indicator values were passively projected as vectors plotted on the bi-dimensional space on the NMDS plot. Analysis was performed in R  using thFifty-three unique individuals of wild grapevine were analyzed with cpDNA microsatellite markers. DNA amplifications with the 9 primer pairs used for the chloroplast SSR analysis revealed that 8 analyzed loci were polymorphic . ShannonAt all four collection sites, a total of 52 woody species, i.e., trees and shrubs, were identified. The largest variability of species was found in the Neretva study area: 39 . Thirty-The main supporters of wild grapevine populations were typical woody species from Mediterranean karst forests .Fraxinus ornus, Carpinus orientalis, Rubus sp., Colutea arborescens and Cornus maswith 20, 18, 13, 12 and 11 occurrences, respectively. The following species were the most commonly supporting plants for grapevine climbing lianas: Prunus mahaleb, Ficus carica, F. ornus and C. orientalis with 8, 6, 6 and 5 occurrences, respectively.As shown in Quercus ilex and Q. pubescens, appeared a short distance from the wild grapevine populations. In addition to the preceding two species, two basic conifer species also occurred in these areas: Pinus halepensis and P. nigra. The forests contained a number of deciduous tree species that accompany Quercus: Acer monspessulanum, A. campestre, Fraxinus ornus, Carpinus orientalis. In the higher parts of Paklenica, Fagus sylvatica was present but only in a small part of the area.Components of the climax vegetation, such as Arbutus unedo, Asparagus acutifolius, Colutea arborescens, Cornus mas, Crataegus monogyna, Paliurus spina-christi, Phillyrea latifolia, Rubus sp and Viburnum tinus. Climbing species included Clematis vitalba and Hedera helix in addition to wild grapevines. Ficus carica and Prunus mahaleb were observed relatively frequently  show that forest have reached a steady state. As a consequence of the best preserved forests in these locations the largest number of wild grapevine individuals was also identified: Neretva, 20; Paklenica, 19 , invasive species  are the major hazard to be controlled. Vertical structure of vegetation and presence of water will also de essential to provide a sustainable environment. These wild grapevine populations constitutes a rare endangered plant in the Eastern Adriatic region, ant it is necessary to start activities addressed to its in situ and ex situ protection. The most efficient way to conserve endangered plant species is to protect their natural habitats and ecosystems.Out results shows that different stage of area protection has an impact on biodiversity and number of wild grapevine individuals. During the period of time, human impact, grazing, wildfire, competition of other plants: lianas (V. vinifera ssp. sylvestris habitats on Croatian and Bosnian and Herzegovinian territory and on the Italian Peninsula lead us to conclude that these areas were once connected and shelter areas for wild grapevines. Filling knowledge gaps regarding the chloroplast genetic diversity of present-day wild grapevines but also of cultivated grapevines, and their relationship with domesticated grapes can contribute to a better understanding of grapevine domestication and its evolution in general. Investigating wild grapevines, we investigate history and processes that occurred millennia ago. However, the advanced fragmentation and erosion of wild genetic pools should be prevented for further research on wild grapevines to remain plausible.The haplotype diversity of wild grapevines in the Eastern Adriatic region and the diversity of the associated woody plant species were presented. The presence and frequency of chloroplast haplotypes A and D in


Habia rubica).The effects of geographic and environmental variables on patterns of genetic and phenotypic differentiation have been thoroughly studied. Ecological speciation involves reproductive isolation due to divergent natural selection that can result in a positive correlation between genetic divergence and adaptive phenotypic divergence . If the phenotypic target of selection is unknown or not easily measured, environmental variation can be used as a proxy, expecting positive correlation between genetic and environmental distances, independent of geographic distances . The null model is that the amount of gene flow between populations decreases as the geographic distance between them increases, and genetic divergence is due simply to the neutral effects of genetic drift . However, since phenotypic differentiation in natural populations may be autocorrelated with geographic distance, it is often difficult to distinguish IBA from the neutral expectation of IBD. In this work, we test hypotheses of IBA, IBE, and IBD in the Red‐crowned Ant tanager  and South America.Habia rubica .H. rubica at both phylogroup and population levels.We compiled genetic data, coloration, and morphometric data from specimens from collections in Mexico and the United States. We used the Multiple Matrix Regression with Randomization (MMRR) approach to evaluate the influence of geographic and environmental distances on genetic and phenotypic differentiation of H. rubica. We did not find evidence that climate variation is driving population differentiation in this species across a widespread geographic region.Our results provide strong evidence that geographic distance is the main driver of genetic variation in H. rubica and suggest that climate is not playing a major role in genetic differentiation within this species.Our data point to geographic isolation as the main factor structuring genetic variation within populations of  Habia rubica. Our data point to geographic isolation as the main factor structuring genetic variation within populations of H. rubica and suggest that climate is not playing a major role in genetic differentiation within this species.Our study provides strong evidence that geographic distance is the main driver of genetic variation in  Within Habia rubica) a good model for performing tests of IBD and IBE. It is a highly polytypic species that is distributed from central Mexico to northeastern Argentina and southeastern Brazil  and ventral (from the throat to the lower belly) brightness. However, both hue and saturation also present some variation, ranging from pale pink in the populations from the Mexican Pacific to dark red in populations from eastern South America  from phyDat, modelTest, and dist.ml functions of the “phangorn” package in R v3. 5. 0  was based on the affiliation to a given phylogroup as defined in Ramírez‐Barrera et al.  to generSchliep, .2.2H. rubica  and males ,  as a proxy for body size  with an Ocean Optics PX‐2 pulsed xenon light source, connected to a bifurcated fiber‐optic probe. The probe was fitted with a rubber stopper to exclude ambient light and maintain a constant distance and 90° angle between the probe tip and the plumage. Measurements were taken following standard procedures Eaton,  to recorvisual model function to determine the relative stimulation levels of the four avian cones using the Sturnus vulgaris (Common starling) visual model. The Common starling is the closest relative of H. rubica for which a spectral sensitivity function was available, and however, it is unlikely that changing the species on which the visual model is based would affect our analysis because the sensitivities of the avian cones are highly conserved  into a vector of three angles, which locates the color in the avian tetrahedral color space. We obtained three main measurements as a result of this processing: (a) the total volume occupied by the points across all body patches (color volume), (b) the mean of the hue span, and (c) mean saturation (chroma). The chroma measurement was included to avoid the underestimation of color variation in uniformly colorful birds , and the third used only the ventral patches . We tested the relationships between hue span and latitude to explore the possibility of latitudinal color trends.Three distance matrices were generated from the color measurements  using the 2.3H. rubica is a species with evident sexual dimorphism in coloration and we found significant differences in body size between males and females  whenever possible. When the two types of data were not available for the same individual, we matched phenotypic data to the genetic data from the closest individual available in terms of geographic proximity and membership in the same phylogroup. This association was conducted based on the georeferenced collection location of each sample . Finally, since 2.4distm function from “geosphere” in R , and estimated the geographic centroid for each group , the overall fit of the model (R2 = coefficient of determination), and the significance of each variable . We used the MRM function implemented in the R package “ecodist”  all distance matrices using the scale function in R to obtain comparable standardized linear regression coefficients.We used a MMRR approach to estimate the independent effects of environment and geography on genetic and phenotypic variation Wang, . This apTo explore the relative importance of geographic and environmental distances as predictors of genetic and phenotypic  divergence, we constructed both a multivariate model and univariate models. In each model, the geographic and environmental distance were the linear predictors of the pairwise genetic or phenotypic difference between phylogroups or individuals.H. rubica in Mexico  could have on the phylogroup that, according to its distribution, belongs. Therefore, this analysis is limited to the distribution of 33.1The corrected pairwise genetic distances (expressed in percentages) between phylogroups (124 sequences) and individuals  ranged from 1% to 7%, showing a clear signal of geographic population structure. In the pairwise comparisons by phylogroup, the largest genetic distances were between the South American phylogroups and those distributed from Panama to Mexico. The Panama phylogroup had the smallest genetic distance from the Northern Central America and Gulf of Mexico phylogroups. In addition, we observed that phylogroups from the Northern Mexican Pacific and Southern Mexican Pacific exhibited the shortest genetic distances and were most closely related with the Gulf of Mexico and Southeastern Mexico phylogroups Appendix .3.2R2 = 0.10 and 0.06, body size: R2 = 0.05 and 0.07 for males and females, respectively). See Appendix Within‐individual correlation coefficients for each pair of morphometric measures ranged between 0.2 and 0.6 Appendix  and S3.23.3H. rubica , than when data were grouped by phylogroup (β = 0.6). Climate and body size were significant only for the individual data, although their contribution was notably lower than geographic distance in both sexes , .The results of univariate MMRR analyses showed that geography was the best predictor of genetic distance in 3.4R2 = 0.66) and individual levels . However, the environmental difference also proved to be an important factor . In the case of the regressions made with data grouped by phylogroups, the contribution of the factors was not significant.In the comparison between the univariate and multivariate results obtained through the data matching Tables  and 2 an4H. rubica. We did not find evidence that climate variation or phenotypic variation  is driving population differentiation in this species complex over its large geographic distribution.Our results provide strong evidence that geographic distance is a major driver of genetic variation in 4.1H. rubica  and southern (SP) regions of the Mexican Pacific coast, on the slope of the Gulf of Mexico (GM), from Southeastern Mexico to Costa Rica (SE), and Panama (PA). Mesoamerica has been described as a highly fragmented topographic complex where the composition of flora and fauna has been strongly influenced by both climatic and geological events  and Eastern/Northwestern (ES) South America , the MMRR analysis does not provide enough evidence to support ecological speciation.Habia rubica has considerable plumage color differentiation, but this appears to be a result of neutral processes . We found little support for the role of plumage divergence in explaining genetic divergence .Even though plumage differentiation is often considered a relevant character for species delimitation in avian taxonomy, in some cases it does not provide enough evidence for the correct discrimination of species. H. rubica and, in practice, body size is generally not ultimately an important character in avian species delimitation (Price, Body size clines that are correlated with temperature gradients are common in nature, particularly in birds (Friedman & Remeš, n Price, . Howevern Price, .Plumage and vocal differences are expected to play a more important role in conspecific recognition and mate choice in birds than body size Hilty,  and thusNone declared.SM Ramírez‐Barrera conceived the ideas, designed and performed the research, analyzed data, prepared figures and/or tables and reviewed drafts of the paper. JA Velasco analyzed, interpreted data and reviewed drafts of the paper. T Orozco‐Tellez collected part of the data. AM Vázquez‐López collected the data. BE Hernández‐Baños conceived the ideas, designed the research, contributed reagents/materials/analysis tools and reviewed drafts of the paper. Click here for additional data file.


Lagopus muta). Within our study area, we defined the largest contiguous mountain area as the mainland, while smaller mountains surrounding the mainland were defined as islands. We found that the observed heterozygosity (Ho) was significantly higher, and the inbreeding coefficient (Fis) significantly lower, on the mainland compared to islands. There was a positive significant relationship between the unbiased expected heterozygosity (Hn.b.) and island size (log km2), but a negative significant relationship between Ho and the cost distance to the mainland. Our results are consistent with the equilibrium model of island biogeography and show that the model is well suited for investigating genetic diversity among islands, but also on islands located on the mainland.The island biogeography theory is one of the major theories in ecology, and its applicability to natural systems is well documented. The core model of the theory, the equilibrium model of island biogeography, predicts that species diversity on an island is positively related to the size of the island, but negatively related by the island's distance to the mainland. In recent years, ecologists have begun to apply this model when investigating genetic diversity, arguing that genetic and species diversity might be influenced by similar ecological processes. However, most studies have focused on oceanic islands, but knowledge on how the theory applies to islands located on the mainland  is scarce. In this study, we examined how the size and degree of isolation of mountain islands would affect the genetic diversity of an alpine bird, the rock ptarmigan ( We chose islands of various size and distance to the mainland  to see whether there was (a) a difference in genetic diversity between the mainland and the mountain islands, (b) a relationship between genetic diversity and the size of an island, and (c) a relationship between genetic diversity and the distance to the mainland. We expected that genetic diversity would be higher on the mainland compared to the islands, and that genetic diversity would be higher on larger islands and on islands closer to the mainland.Of the many studies investigating the relationship between genetic diversity and the different parameters of the island biogeography theory, few have been conducted on population living on the continent , every sample was amplified with the species‐specific mtDNA primers Lagsp3F, Lag3R, and Mut3R , while DNA from fecal pellets was extracted with the “QIAamp DNA Stool Mini Kit” . To maximize the final yield of extracted DNA, the “DNeasy Blood & Tissue” procedure was modified with the addition of 5 µl of DTT 1 M to the ATL buffer during the first step of the protocol. We also modified the “QIAamp DNA Stool Mini Kit” protocol, where we adjusted the quantity of buffer ASL from 1.4 to 1.6 ml. Because fecal pellets of the rock ptarmigan are impossible to differentiate from fecal pellets of the willow grouse  with 1.5 µl of PCR product added in a mix containing 9.7 µl of formamide  and 0.3 µl of GeneScan 500LIZ dye Size Standard . Allele scoring was performed using Genemapper software V5.0 (Applied Biosystem) and visually controlled.For this study, we used 14 of 16 microsatellite markers previously developed for the rock ptarmigan  and a genotyping error rate was calculated. Individuals with more than 10 missing loci were removed from analysis. The multitube approach, which is generally used on noninvasive samples  above the tree line.To measure the distance between the mainland and the different islands, we used the Euclidian distance and cost distance. The Euclidian distance was measured as the shortest straight‐line distance between the mainland and an island, while cost distance was measured using the function “cost distance” in the Spatial Analyst Tools in ArcMap 10.4 ESRI. We used2.5Ho), expected heterozygosity (He), unbiased expected heterozygosity (Hn.b.), and allelic richness (Ar.). We also calculated the inbreeding coefficient Fis as a supplement to the four indices. Ho, He, Hn.b., and Fis were calculated with the software Genetix V4.05 . For each index, six candidate models were created with the independent variables: log(area), log(cost distance), log(Euclidian distance), log(area) + log(cost distance), log(area)  + log(Euclidian distance), and finally, an intercept model using 1 as an independent variable. A model selection based on Akaike's information criterion corrected for sample size  lower than 2, only the most parsimonious model was selected to avoid the “pretending variable” issue  to 2,492 km2 . Average microsatellite error rate over all loci was 2.6% (range: 0.76%–7.4%). The total dataset had less than 5% of missing data. For IL4 data were missing in 14 and 11 individuals (of 16) for Mut17 and Mut24, respectively, and for IL5 in 15 individuals (of 17) for Mut24. The genetic indices Ho, Hn.b, Ar., and Fis were found to be highly correlated when calculated with 12 or 14 loci , and we therefore decided to use 14 loci for the rest of the analysis. We found no significant relationship between Ho, Hn.b, Ar., Fis and sampling effort , or between Ho, Hn.b, Ar., Fis, and the average altitude per site .After removing samples from the willow grouse, poor quality samples, and duplicates (66 samples), 616 individual rock ptarmigan samples were available for analyses. Of these, 491 came from islands and 125 came from the mainland. We found no Hardy–Weinberg disequilibrium or linkage disequilibrium after Bonferroni's correction , and we therefore decided to keep these individuals for further analyses  and 0.993 for the willow grouse (range: 0.980–0.998). When analyzing relatedness among individuals, we found that overall sites 1.99% comparisons showed potential full siblings (range: 0.83% for site M2%–14.71% for site IL11). Sites with more than 5% full siblings  did not significantly affect allele frequencies  and the number of samples per site , we replaced He by the unbiased expected heterozygosity (Hn.b.) to compensate for differences in sample size .Because we found a significant relationship between expected heterozygosity (3.2Hn.b.) ranged from 0.53 (IL8) to 0.67 (IL1 and IL4), observed heterozygosity (Ho) ranged from 0.45 (IL12 and IL8) to 0.66 (IL10), and the inbreeding coefficient (Fis) ranged from −0.03 (IL10) to 0.27  and 23 (Mut17). Average allelic richness (Ar.) per site over all loci ranged from 2.16 (IL8) to 2.56 (IL1), unbiased expected heterozygosity (Fst: 0.008), and significantly lower  than between islands (average pairwise Fst: 0.041), as well as between islands and the mainland  was best explained by the cost distance to the mainland, while the unbiased expected heterozygosity (Hn.b.) was best explained by area , but the difference in AICc score corrected for sample size (ΔAICc) was not significantly different from the intercept model , and that individuals from islands of larger size were more likely to have a high Hn.b.  and distance to the mainland, and between the unbiased expected heterozygosity (Hn.b.) and island size. This shows that the theory is well suited for not only analyzing species diversity among oceanic islands, but also genetic diversity within a fragmented mountain landscape. It seems that for isolated populations, the immigration rate is insufficient to compensate for genetic drift, and suggest that the future viability of such populations could be imperiled by extinction vortex processes.The main results of this study support the predictions of the equilibrium model of island biogeography, in which genetic diversity was found to be significantly higher on the mainland compared to the surrounding islands. We found a significant relationship between observed heterozygosity , while the opposite was true for the level of inbreeding (Fis). These results are in line with the meta‐analysis carried out by Frankham  that might have had a potential bottleneck in the past.A relationship between genetic diversity and island area has been documented in several studies (Hänfling et al., Contrary to what was expected we did not find a significant relationship between Ar. and area size (e.g., results from Hill et al., 4.3Ho and distance to the mainland. This makes sense since Ho is more influenced by changes in genotype frequencies than in allele frequencies (Keller & Waller, Ho observed among the severely isolated islands is probably the result of a higher genetic drift than gene flow, resulting in the loss of genetic diversity (Allendorf et al., Fis, although a positive trend was observed.Several studies have found a significant relationship between distance to the mainland and genetic diversity (Francisco et al., 5The negative effect of either isolation or area size on genetic diversity has been documented in a large number of studies, but surprisingly few have combined the two landscape parameters or related it to the island biogeography theory. This study is an attempt to increase our knowledge on how genetic diversity is affected within such a system, but also if the theory can be applied to mountain islands rather than oceanic islands. Our results support the island biogeography theory, and they show that even for a highly mobile species such as the rock ptarmigan, isolation appears to be the negative factor in maintaining a high genetic diversity. The rock ptarmigan is probably not exceptional in that regard, and any species living above the tree line within a fragmented system are likely to experience similar consequences of isolation. Alpine species are also particularly vulnerable to climate change, in which an upward shift of the tree line is predicted, and also observed, to occur (Harsch, Hulme, McGlone, & Duncan, None declared.Ø.S. designed the study; J.M.C. and Ø.S. collected the samples; J.M.C. conducted the laboratory and data analyses; J.M.C. and Ø.S. wrote the manuscript. Click here for additional data file.


We seek to develop a plausible conception of genetic parenthood, taking a recent discussion by Heidi Mertes as our point of departure. Mertes considers two conceptions of genetic parenthood—one invoking genetic resemblance and the other genetic inheritance—and presents counter‐examples to both conceptions. We revise Mertes’ second conception so as to avoid these and related counter‐examples. One such development is the successful application of mitochondrial replacement therapy (MRT) in human reproduction. The aim of MRT is to avoid transmission of mitochondrial DNA (mtDNA) mutations; a baby is created through in vitro fertilization (IVF) using a man’s sperm and a woman’s egg, but shortly before or after fertilization, the egg’s disease‐linked mtDNA is replaced with healthy mtDNA from a donor egg. The first baby resulting from MRT was born in 2016. Such babies have been referred to as “three‐parent babies.”Another technique that may enable a significant departure from “normal” human genetic inheritance is the creation of stem cell‐derived gametes. In the not too distant future, sperm and eggs derived in the laboratory from pluripotent stem cells may be used to create embryos and eventually children. The pluripotent stem cells needed to generate these gametes could be extracted from embryos left over following fertility treatments, from embryos created through IVF for the specific purpose of gamete‐derivation, or from embryos cloned from a person’s somatic (body) cells, either via a process known as somatic cell nuclear transfer SCNT or via tQuestions arise as to whether the children created through these techniques would have genetic parents, and, if so, who, precisely, those genetic parents would be. Bioethicists have already begun to address these questions,if the genetic parenthood relation is morally significant, as some have suggested, then it is obviously important that we are able to determine when the relation is present and when it is not. Second, a fuller understanding of genetic parenthood may help to inform discussions of whether genetic parenthood is indeed morally significant. For example, if, on the correct understanding of genetic parenthood, the presence or absence of this relation depends on factors that are clearly morally irrelevant, this will cast serious doubt on the suggestion that genetic parenthood is morally significant.Despite this growing interest, however, there is little agreement on the nature of genetic parenthood, and indeed, few conceptions of genetic parenthood have been proposed. We believe a better understanding of genetic parenthood is needed for two reasons. First, In this article, we seek to develop a plausible conception of genetic parenthood. We take as our point of departure a recent discussion by Heidi Mertes,However, before turning to these tasks, we must offer three preliminary comments.The first concerns the relationship of the concept of genetic parenthood to other concepts of parenthood. The concept of genetic parenthood that we seek to capture is, of course, distinct from the concepts of gestational and social parenthood. But we believe that it is also, though less obviously, distinct from the concepts of biological and causal parenthood. Biological and causal parenthood are, we take it, concepts that were without error deployed even before the details of genetic inheritance were understood.humans, and we discuss only cases involving humans. There is plausibly a concept of genetic parenthood that applies to other species; perhaps it applies to all other species that reproduce sexually. But we do not wish to commit ourselves to the view that this is the same concept as the concept that applies to humans.The second preliminary comment concerns the scope of our analysis. We assume throughout that genetic parenthood is a relation that obtains between two Finally, the third clarification concerns the aim of our analysis. There are various aims one might have in formulating a conception of genetic parenthood. First, one might aim to capture the concept of genetic parenthood that is implicit in common usage of the term. What are the necessary and sufficient conditions of genetic parenthood as that concept is employed in everyday discourse? That is, under what conditions do people tend (not) to ascribe genetic parenthood? Second, one might seek to capture what people care about when they, for instance, pursue forms of assisted reproduction in preference to adoption. What sort of genetic relation do prospective social parents want to have with their social children?Our aim will be to develop a conception of the first kind; we aim to formulate a conception of genetic parenthood that captures the concept of genetic parenthood implicit in everyday usage.2a child is my genetic child when it has 50% of my DNA or when it has 23 of my chromosomes. This 50% overlap of genetic material is, for example, what is looked into when performing a paternity or maternity test.As noted above, we take as our starting point Mertes’ instructive discussion of genetic parenthood. Mertes distinguishes two candidate conceptions of genetic parenthood. According to the first of these conceptions,inherited from P, for, as we shall see, she criticizes this account of genetic parenthood on the basis that it is insensitive to the direction of inheritance. Rather, the thought simply seems to be that 50% of C’s DNA resembles 50% of P’s DNA. More precisely the thought appears to be:Informational Overlap: P is C’s genetic parent if and only if 50% of C’s genetic material encodes the same information as 50% of P’s DNA.What does it mean to say that C “has 50% of P’s DNA” or that there is a “50% overlap” between C and P’s DNA? The thought cannot be that 50% of C’s DNA is numerically identical to 50% of P’s DNA, for P and C’s DNA are in different locations, and have different survival conditions (one could destroy P’s DNA without destroying C’s). Nor does Mertes mean that 50% of C’s DNA is One difficulty with this conception is that it is too inclusive. For instance, as Mertes notes, it would arguably classify one’s sibling as one’s parent (or child), or one’s parent as one’s child: “My siblings also share 50% of my DNA, as do my parents, and yet, the relation I have with my parents and siblings differs profoundly from the one I have with my children.”A second difficulty is generated by the fact that “almost all (99.9%) of nucleotide bases are exactly the same in all humans.” This leads Mertes to question “whether it even makes sense to say that we share 50% of DNA with our genetic children.”sense to say that we “share 50% of our DNA with our children,” in the sense specified by Informational overlap, it is just that this is false: we in fact share much more of our DNA with them than that, since we share more than 99% of our DNA even with other humans to whom we are not closely related. Moreover, it seems possible to modify Informational overlap to avoid this problem; we simply need to increase the percentage of informational overlap that is required. After all, whatever share of our DNA overlaps with that of all other humans, an even higher share overlaps with that of our putative genetic parents. Suppose that 99.9% of our DNA overlaps with that of all other humans, but 99.95% overlaps with that of our putative genetic parents. In that case, we should modify Informational overlap such that “50%” is replaced by “99.95%.”We take issue with Mertes’ second concern. It makes perfectly good Informational overlap does not distinguish parent–child relations from child–parent relations, some sibling–sibling relations, and instances in which genetic strangers by chance share the same proportion of genetic information as putative genetic parents and their putative genetic children. This is clearly a problem.Mertes’ first concern, however, would remain. which information in an individual’s genome must resemble that of the putative genetic parent. The nuclear genome of most humans is composed of 23 pairs of chromosomes, where in most cases one member of each and every chromosome pair resembles a corresponding chromosome possessed by the putative genetic parent. Perhaps we should revise Informational Overlap to require this specific form of genetic overlap. This might seem to help prevent siblings from falling within the scope of Informational Overlap, since any genetic overlap between siblings will not normally be distributed across chromosome pairs in the same way. Still, siblings could, by chance, possess the same sort of genetic overlap as that existing between putative genetic parents and their putative genetic children, as indeed could strangers. In addition, the proposed revision to Informational Overlap does not help to prevent the genetic parenthood relation from running in both directions, such that P is C’s genetic parent, and vice versa.Moreover, this problem cannot be resolved by being more precise about 3How should we solve these problems? One option that Mertes considers involves shifting to a second conception of genetic parenthood according to which “X is a genetic child of Y if X is directly derived from Y’s genes.”grandparenthood or some similarly or more distant genetic descent—that is, relations of genetic descent that are mediated by other individuals. We take it that the “directly” is intended to rule out cases in which the genetic relation is one of genetic genes derive from the putative genetic parent’s genes. Suppose a sperm from P1 is used to fertilize an egg from P2. The resulting zygote then has its DNA removed and replaced by DNA from some other individual T. This zygote is then carried to term and eventually a child, C, is born. There is a sense in which C derives directly from P1 and P2’s genes; those genes governed the development of gametes, which created a zygote from which C developed. But C’s genes do not derive from P1 and P2’s genes, they derive instead from T’s genes, and this surely prevents P1 and P2 from qualifying as C’s genetic parents.We take it also that the kind of derivation in question is one in which the putative genetic child’s Direct Genetic Descent: P is C’s genetic parent if and only if (a) C’s genes derived from P’s genes, and (b) not through deriving from the genes of some third, intervening individual, M.Taking these two qualifications into account, we could formulate Mertes’ second proposed conception of genetic parenthood as follows:Cloned Child) in which P1 and P2 have a child C1 whom they then clone to create C2. According to Direct genetic descent, C2 is C1’s genetic child, but Mertes thinks it seems more plausible to say that C2 is the genetic child of P1 and P2. Sparrow would likewise deny that C2 is the child of C1, for he holds that “clones share too much genetic material with their donors to be their genetic children.”What should we make of this conception? Mertes holds that it is susceptible to a number of counter‐examples. For instance, she asks us to imagine a case , P1 and P2 decide to directly clone P1 instead, creating a child C3. Direct Genetic Descent implies that P1 is C3’s genetic parent, but Mertes questions whether this is so; she thinks C3 may bear too much genetic resemblance to P1, and again, Sparrow concurs, suggesting that the relation between P1 and C3 is closer to that between identical twins than to that between genetic parents and their genetic children.In the second case , a child (C4) derives all of her nuclear DNA in the usual way from P1 and P2, but her mitochondrial DNA, which constitutes about 0.15% of her total genome, comes from a third individual (P3). Direct Genetic Descent implies that P3 is C4’s genetic parent, but Mertes thinks that, in many circumstances, this is not intuitively plausible.Finally, in the third case , it appears that there is also a grey area in which some people may be more or less of a genetic parent than others. There is no fixed, scientific, everlasting criterion of genetic parenthood that everyone can agree upon. Quite on the contrary, the concept is increasingly challenged by new and hypothetical interventions in reproductive medicine.We do not ourselves have clear intuitions about genetic parenthood in the  Genetic parenthood comes in degrees or at least has vague boundaries. There is disagreement about the nature of genetic parenthood. Genetic parenthood is a subjective concept that depends on the views of people about what sorts of genetic relation matter. Those views may change as a result of scientific developments.This passage advances a number of claims. Among them, plausibly, are the claims that:Informational Overlap and Direct Genetic Descent. In particular, we think it would be premature to conclude from the rejection of these views that genetic parenthood is a subjective concept in the sense specified by 3 (and assuming that we are interested in the concept of genetic parenthood implicit in common usage), for there are other objective conceptions of genetic parenthood available. In the next section, we explore some of these available conceptions and present one of these as a plausible alternative to Informational Overlap and Direct Genetic Descent.We believe that these claims are all somewhat plausible, but we do not think they follow from the rejection of 4Direct Genetic Descent so as to avoid the putative counter‐examples that Mertes and Sparrow discuss. Here is one possible refinement:Direct Gametic Genetic Descent: P is C’s genetic parent if and only if (a) C’s genes derived from P’s genes, (b) through a gamete produced by P, and (c) not through deriving from the genes of some third, intervening individual, M.One promising route to a more plausible conception of genetic parenthood would involve refining Cloned Child and P1 is the genetic parent of C3 in Cloned Parents. It also allows us to evade the conclusion that P3  is the genetic parent of C4  in Mitochondrial Donation.The requirement that genetic derivation occurs via a gamete prevents cloning (whether through SCNT or an iPS cell‐based technique) from generating a relation of genetic parenthood between the donor individual and the clone. Thus, it avoids the putatively implausible implications that C1 is the genetic parent of C2 in Direct Gametic Genetic Descent may allow us to avoid implausible attributions of genetic parenthood in these cases, it arguably does entail implausible denials of genetic parenthood. It implies that the clones created in Cloned Child and Cloned Parent lack any genetic parents, since their genomes were not inherited via gametes. Yet, Mertes and Sparrow believe that these individuals do have genetic parents: on their view, P1 and P2, the genetic parents of the donor individual (C1), are also the genetic parents of the clone (C2) in Cloned Child. In Cloned Parent, the genetic parents of the donor (P1) are presumably again the genetic parents of the clone (C3). In denying that the clones created in these cases have genetic parents, Direct Gametic Genetic Descent may seem too stringent in the conditions that it imposes on genetic parenthood.However, though Direct Gametic Genetic Descent—cases in which humans would reproduce without the creation of gametes, but in which we would still want to ascribe genetic parenthood. Suppose it were possible for adult humans to reproduce through a process that we might call Two‐Donor Genome Transplantation. In this process, DNA from a zygote would be replaced with genetic material extracted directly from body cells taken from two individuals, P1 and P2, who would each contribute roughly 50% of the zygote’s (new) genome. If the embryo derived from this zygote were carried to term, we would want to say that P1 and P2 were the genetic parents of the resulting child, even though no gametes were involved in the reproductive process. Direct gametic genetic descent cannot accommodate this verdict.Moreover, it is possible to imagine circumstances closer to those of normal human reproduction that also create difficulties for via gametes with a requirement for genetic inheritance of some specified proportion of the putative genetic child’s genome from the putative genetic parent. Consider:Direct Proportionate Genetic Descent: P is C’s genetic parent if and only if (a) some proportion X of C’s genes derived from P’s genes and (b) not through deriving from the genes of some third, intervening individual, M.This second problem could be avoided by replacing the requirement for genetic inheritance Direct gametic genetic descent, this formulation avoids the implication that donor individuals are the genetic parents of the clones created from them (the proportion of genes inherited by the clones is too high) as well as the implication that mitochondrial donors are the genetic parents of the mitochondrial recipients (the proportion of genes inherited by the recipients is too low). Yet, unlike Direct Gametic Genetic Descent, this formulation also gets the right result in Two‐Donor Genome Transplantation; it implies that the two genetic donors in this process are the genetic parents of the resulting child.Suppose we set “proportion X” to “20–80%.”Direct Proportionate Genetic Descent remains too stringent in the cases of Cloned Child and Cloned Parent, for it continues to imply that the clones produced in these cases have no genetic parents—an implication that Mertes and Sparrow find implausible. On Direct Proportionate Genetic Descent, the donor does not count as the genetic parent of the clone because she passes on too much of her genetic material. But the genetic parents of the donor seemingly also do not count as the parents of the clone, because their genetic contribution is mediated by another individual: the donor. Thus, the directness requirement rules out their genetic parenthood of the clone.However, Cloned Child and Cloned Parent. Our suggested refinement consists in qualifying how the intervening individual should genetically relate to his or her descendant in order to break or maintain a presumed genetic parenthood relation between the ancestor and the descendant. In Cloned Child, Mertes and Sparrow share the intuition that P1 and P2, not C1, qualify as the genetic parents of C2 (who is cloned from C1). We propose that the intervening individual (C1) does not break the genetic parenthood relation in this case because she passes on too much of her genetic information to C2. This appears to be the result that Mertes and Sparrow favour. In Cloned Parent, Mertes and Sparrow think it is counterintuitive that C3 (a clone of P1) has no genetic parents; they appear to believe that P1’s genetic parents count as C3’s genetic parents too. We propose that this is because P1 again passes on too much of her genetic information to C3 and so does not qualify as an intervening individual for the purposes of breaking the genetic parenthood relation. We stipulate, then, that, when C derives genetic information from P via some intervening individual M, this disqualifies P from being C’s genetic parent only if C derived some specified proportion of his genetic material from M. This specified proportion will most plausibly, as with “proportion X”, be a range of proportions, and it may be a range with fuzzy boundaries. But so long as the range is set so as to exclude cases of cloning—that is, cases where  100% of C’s genes derive from M’s genes—we can maintain the result that, if C is a clone of M, the genetic parenthood relation between P and C is preserved.To avoid this implication, it is necessary to weaken the directness requirement. There are various plausible ways in which it could be weakened; we shall make what we think is the most minimal plausible revision required to yield the result that Mertes and Sparrow prefer in Modified Direct Proportionate Genetic Descent: P is C’s genetic parent if and only if (a) some proportion X of C’s genes derived from P’s genes and (b) not through deriving from the genes of some third, intervening individual M from whom C derived proportion Y of his genes.This suggested revision yields the following conception of genetic parenthood:Modified Direct Proportionate Genetic Descent is our favoured conception of genetic parenthood. We do not claim that it is certainly correct; it may well be susceptible to counter‐examples that we have not identified, and in any case, it is not clear that there is any single concept of genetic parenthood implicit in everyday usage. But we do believe that Modified Direct Proportionate Genetic Descent is an initially plausible conception of genetic parenthood, and that it is more plausible than any of the alternative conceptions described above. We thus propose that it could serve as a helpful starting point for future analyses.organisms can fill these roles, but this does not settle all of the interesting questions here, since there is, for example, disagreement about whether zygotes and early embryos qualify as human organisms.Of course, this conception needs to be fleshed out in various ways. Most obviously, more would need to be said about what ranges of proportions are picked out by X and Y, and whether these ranges of proportions have sharp boundaries. Similarly, more needs to be said about what types of entities can fill the roles of P, M and C on our conception. For example, must these be live born humans, or can they be filled by, for example, human zygotes, embryos or foetuses? One plausible view would be that only human Modified Direct Proportionate Genetic Descent invokes properties that are most naturally understood as threshold (or “range”) properties: namely, the property of deriving proportion X (or Y) of one’s genes from another individual. These properties are most naturally understood to consist in lying between two (possibly fuzzy) thresholds on some scale of genetic inheritance. Yet, it is difficult to see why these thresholds should mark anything of moral significance. Perhaps it matters morally that we derived some proportion of our genes from another individual. Perhaps it even matters that we derived a greater rather than a lesser proportion from that other individual. But even if the proportion of our genetic inheritance matters in these ways—a point on which we remain neutral—it is difficult to see why it should matter that this proportion lies between two thresholds marking the boundaries of our commonsense conception of genetic parenthood. Those thresholds seem arbitrary from a moral point of view.Then there is the question of moral significance. It is not our intention to explore in detail the moral implications of this conception of genetic parenthood here—our goal in this article has been conceptual, not ethical. However, it is perhaps worth briefly noting that our conception does not appear helpful to those who wish to hold that the genetic parenthood relation is of moral significance. The chief reason for this is that The author declares no conflict of interest.


The first stage in both breeding and programs for the conservation of genetic resources are the identification of genetic diversity in the relevant population. The aim of the present study is to identify genetic diversity of six brown layer pure chicken lines  with microsatellite markers. Furthermore, the study aims to employ its findings to discuss the possibilities for the conservation and sustainable use of these lines that have been bred as closed populations for a long time.In the present study, a total number of 180 samples belonging to RIRI (n = 30), RIRII (n = 30), BARI (n = 30), BARII (n = 30), L-54 (n = 30), and COL (n = 30) lines were genotyped using 22 microsatellite loci. Microsatellite markers are extremely useful tools in the identification of genetic diversity since they are distributed throughout the eukaryotic genome in multitudes, demonstrate co-dominant inheritance and they feature a high rate of polymorphism and repeatability.IS (inbreeding coefficient) values in the range between 0.16 (L-54) and 0.46 (RIRII). The FIS values obtained in this context points out to a deviation from Hardy-Weinberg equilibrium due to heterozygote deficiency in six different populations. The Neighbour-Joining tree, Factorial Correspondence Analysis and STRUCTURE clustering analyzes showed that six brown layer lines were separated according to their genetic origins.In this study, we found all loci to be polymorphic and identified the average number of alleles per locus to be in the range between 4.41 (BARI) and 5.45 (RIRI); the observed heterozygosity to be in the range between 0.31 (RIRII) and 0.50 (BARII); and FThe results obtained from the study indicate a medium level of genetic diversity, high level inbreeding in chicken lines and high level genetic differentiation between chicken lines. Genetic diversity, shaped in the process extending from the past to the present in populations, is of paramount importance for the sustainability of species. In years following the domestication of livestock, the processes of mutation, selection, adaptation, isolation and migration created a large body of genetic diversity in local populations. Nevertheless, intensive breeding processes implemented for the maximisation of various yields among livestock or the preference of high yield breeds to local breeds, have led to decreases in genetic variation in the past 50 years ,2. This The conservation of genetic diversity among livestock and their sustainable use represents one of the extremely interesting and current items on the agenda . StudiesMicrosatellite markers are extremely useful tools in the identification of genetic diversity since a multitude are distributed throughout the eukaryotic genome, are present in both introns and exons, demonstrate co-dominant inheritance and feature a high rate of polymorphism and repeatability ,3,5.In Turkey, there are six brown layer pure chicken lines  and four white layer pure chicken lines  under Ankara Poultry Research Institute. These populations were imported from Canada in 1995 and have been made subject to selection based on various characteristics by Ankara Poultry Research Institute in Turkey since then. L-54 is synthetic chicken line obtained in 1974 year and it has approximately 15 percent Leghorn blood. Therefore, body weight is lower than other lines and the eggshell colour is quite light brown. Studies undertaken on pure lines resulted in the development of three hybrid materials, i.e. two brown  and one white (ATABEY) layers. RIRI and RIRII are used as sire lines, while BARI and L-54 are used as dam lines to obtain the brown layer hybrids.The aim of the present study is to identify genetic diversity among six brown layer pure chicken lines using 22 microsatellite loci. Moreover, the study further aims to discuss the possibilities for conservation and sustainable use of these lines bred as closed flocks for a long period, as well as the relationship among the existing chicken lines.A total of 180 blood samples including 30 samples from each brown layer pure chicken line in Ankara Poultry Research Institute were taken into tubes with K3 ethylenediaminetetraacetic acid (approximately 1 mL). The blood samples thus collected were stored at −20°C until DNA isolation. DNA was isolated from each line using the protocol reported by Miller et al . DNA conThe sizes of PCR products were identified using 96 automated capillary electrophoresis systems . The system is able to differentiate among PCR products up to 2 bp without fluorescent-labelled primers. This system detects size of PCR products by using a sensitive intercalating dye coupled with a powerful LED light source. The gel, inlet buffer, capillary conditioning solution and 35 to 500 bp marker were prepared according to the manufacturer’s instructions using dsDNA 900 Reagent Kit (35 bp/500 bp). Every well of the 96 PCR plate was filled with a 25-μL mixture including 3 μL PCR product and 22 μL 1X dilution buffer and only the last well (H12) was loaded with 25 μL ladder without dilution. After capillary electrophoresis separation, the data was opened and band sizes calculated by using PROSize 2.0 version 1.3.1.1  (AdvanceIS) and genetic differentiation among the lines (FST) the loci under examination. Additionally, factorial correspondence analysis (FCA), Neighbor-Joining tree (NJ) and STRUCTURE clustering analysis were conducted to identify the relationship between lines and individuals.The identification of genetic diversity within the chicken lines was calculated using allele range (AG), number of alleles (Na) and number of effective alleles (Ne), observed heterozygosity (Ho), and expected heterozygosity (He). Moreover, calculations were undertaken for polymorphism information content (PIC), inbreeding coefficient for the determination of inbreeding among the lines  and 23 (LEI0228) .In this study, the null allele frequency was detected above 0.2 in only four loci . The null allele frequency on LEI0196 locus for RIRI, RIRII, BARI, and BARII populations 0.244, 0.329, 0.276, and 0.224, respectively. For MCW0020 locus, RIRII, BARI, and BARII lines as to be 0.319, 0.212, and 0.272, respectively. In RIRI, RIRII, and COL lines, for MCW0287 and MCW0330 loci were calculated as 0.267, 0.255; 0.417, 0.257 and 0.295, 0.242, respectively.IS and PIC value were determined to be 0.47 and 0.76, respectively. FIS values were found positive in all loci and varied in the range of 0.196 to 0.654. The PIC value is higher than 0.5 in all loci except for MCW0111.The values for observed and expected heterozygosity in all populations were calculated as 0.42 and 0.79. The FThe genetic diversity parameters obtained in six different pure chicken lines are summarised in ST value is an indicator of genetic differentiation in sub-populations. Genetic differentiation among populations is high level when the FST value is higher than 0.25. The lowest pairwise FST value (0.11) along the lines under examination was found between BARI and BARII lines and the highest pairwise FST value (0.35) from L-54 and COL lines. The average pairwise FST value was identified to be 0.29 as obtained for 22 microsatellite markers in all population  is higher than the values reported by Zanetti et al  for fourThe observed heterozygosity value established for the six brown layer pure chicken lines (0.42) is lower than the value reported by Tadano et al  for browIS value calculated for all loci in all populations (0.47) is higher than the values reported by Muchadeyi et al [The average Fyi et al  for whityi et al  for browyi et al  for eighyi et al  for fouryi et al  for nineyi et al  for fiveIS coefficient is much higher. We observed that the PIC value is higher than 0.50 for all loci except for MCW0111 and that these loci are quite useful for genetic diversity studies. There may be multiple reasons underlying the higher average number of alleles per locus when compared to those in other studies. These reasons can include the six different pure lines used in the present study come from different genetic origins  and have been as closed flocks and subjected to selection for a long period of time. These populations have been selected in Turkey since 1995. According to various resources maintained at Ankara Poultry Research Institute, the lines were bred as closed flocks and selected for various characteristics 45 to 50 years before they entered to Turkey. The selection procedure applied on these lines for 70 years may have led to the emergence of different alleles.Comparing the present values with those in the other studies in the literature the number of alleles per locus is higher and the heterozygosity values are at medium levels; but the FIS values identified in the present study. If sub-populations are bred as closed flocks in isolation from each other, as is the case in the present study, a deviation from Hardy-Weinberg equilibrium is an expected consequence. However, the FIS values obtained here are much higher than expected. The reason behind the high FIS value is considered to be the small population size.All loci are considered to suffer from a heterozygote deficit arising from inbreeding as a basis for the positive FThe number of alleles and effective alleles obtained from pure layer lines in the present study  are loweIS value results to be positive. Similar findings were reported for various pure lines et al [IS value is an indicator of inbreeding within a population and is used to determine the deviation from Hardy-Weinberg equilibrium. At the same time, it is also an important indicator for the designation of conservation priorities for populations.The Ho values in the present study varied in the range of 0.31 (RIRII) and 0.50 (BARII); He values in the range of 0.54 (RIRII) and 0.64 (RIRI); and FIS in the range of 0.16 (L-54) and 0.46 (RIRII). All observed heterozygosity values obtained for the lines in the present study are lower than expected heterozygosity values. This finding is an indicator of excess homozygosity in the populations and has led Fes et al ,28. The IS value was below 0.05; they are under potential danger if this value is in the range between 0.05 and 0.25; they were at the minimum level of danger if this value is in the range between 0.15 and 0.25; and the danger reached a critical level if this value is higher than 0.40. Breeds in such a situation needed to be kept under conservation. The FIS values obtained for pure lines utilised in our study indicate that the populations are in serious danger. Specifically, the danger has exceeded the critical level in the RIRII line (0.46) and emergency measures should be taken for this population. The reason for an excess of homozygosity in all populations might be null alleles or subpopulation structure (Wahlund effect) may induce deviation from random mating which leads to higher FIS values. According to Mahammi et al [According to the report by Simon and Buchenauer  breeds ami et al , if the The present study found the average number of alleles per locus and the number of effective alleles to be higher than most of the figures reported by similar studies. In terms of observed and expected heterozygosity values, the population features a medium level of genetic diversity. Nevertheless, the level of inbreeding in the population is higher than that in all relevant studies performed along similar lines in the literature in terms of the FIS values.The number of private alleles determined in the present study vary between 7  and 17 (L-54) . PrivateThe high number of private alleles was expected, because there is a negative correlation between the number of private alleles and migrating individuals in populations. The breeding of the six populations in isolation is the main reason behind the high number of private alleles. Another reason behind this high number of private alleles is the selection procedure performed on these populations for various purposes. Selection performed in different directions for a long period of time may be considered to have led to the emergence of different alleles in the populations.Tadano et al  reportedST value (0.35) being identified on COL and L-54 lines is considered to be that the L-54 line carries Leghorn blood at 15%. L-54 is a synthetic line and was created from COL line by adding Leghorn blood at 15%. As can be seen in ST values are statistically significant at the level of 0.001. The lines under study are subjected to such a high rate of differentiation as 29% (0.29).The reason behind the highest pairwise FNei’s genetic distance values ranged from 0.28 (BARI-BARII) to 1.14 COL-L-54). Muchadeyi et al  reported4. MuchadWhen K = 6 where the highest ΔK value was obtained, bayesian clustering analysis showed every line was in a separate cluster with only slight transitions among lines. The applied selection and breeding system have lead to high genetic differentiation in chicken lines. In the light of all of these findings, BAR lines may be stated to have diverged from each other later than the other lines. The situation in COL and L-54 lines is considered to have stemmed from the presence of 15% Leghorn blood in the L-54 line.Consequently, the findings obtained from the study indicate a medium level of genetic diversity and a high level of inbreeding on the chicken lines. Furthermore, genetic differentiation among the populations is also at high levels. Our results show that microsatellite markers are extremely useful in the demonstration of genetic variations even in populations closed for a long period of time and the identification of conservation priorities in populations. For the continued maintenance of activities undertaken at Ankara Poultry Research Institute, various measures are recommended specifically with a view to minimising inbreeding in populations.


Urban sustainability is a crucial part of the United Nations (UN) Sustainable Development Goals (SDGs) and one of the core objectives of China’s national strategy to promote new urbanization and achieve integration in the Yangtze River Delta (YRD). This paper mainly focused on the 11th SDG, which is a universal call to make cities and human settlements inclusive, safe, resilient and sustainable. The full permutation polygon synthetic indicator (FPPSI) method was applied to synthetically evaluate the sustainable level of 26 cities in the YRD urban agglomeration from 2007 to 2016. The results showed that: (1) the synthesis indicators were increasing year by year, which implied that the sustainable development of the YRD has shown obvious progress in recent years. However, each city faced its own challenges to achieving the sustainable development goals. The sustainability level for the majority of cities was restricted by obstacles such as the per capita green area, air quality and commercial housing sales area; (2) Among the 26 cities, small and medium-sized cities were subject to the traditional strong sustainability indicators while large and mega cities were more affected by weak sustainability indicators; (3) Spatial differences were found for the overall sustainable development level of the YRD. The diffusion and assembly effect among cities had not yet been formed; however, the strong spillover effect of developed cities might influence the ability of other cities to achieve sustainable development goals in many aspects of the environment, economy and society. The results suggest the need for a stronger focus on improving regional developing patterns and strengthening coordination in the process of achieving the sustainable development goal of urban agglomeration in the YRD. Furthermore, according to the conditions of different cities, integrated policies are required to address all aspects of sustainability and to avoid unintended consequences.  In September 2015, the United Nations (UN) adopted a list of SDGs  to aid in the creation of framework arrangements for the three aspects of global economic development, social equity and ecological protection across the period of 2015–2030. Specifically, the SDGs are 17 universal goals that meet the urgent environmental, political and economic challenges facing our world (United Nations 2015). The document provides a framework for global sustainable development. SDGs are mainly concentrated on cities, energy, health, climate and poverty eradication. The latter is regarded as a key component of MDGs , emphasizing sustainable and inclusive development in multiple dimensions with a higher degree of universality and flexibility. Based on the SDG-13 (Climate action), Raheem counseled urgent action throughout the world to address climate change and its impacts. Taking Africa as an example, regional differences and diversified development needs are the main obstacles in achieving this goal  Despite More than half of the world’s population now live in urban areas, which means urban has become a major component of human civilization as well as a key concern for sustainable development . TherefoTo date, research on urban sustainability is still in the early stages; especially compared with the qualitative exploration research, the quantitative empirical research is quite limited ,16. In aSince its reform and opening up in 1978, China has been faced with the overlapping and interweaving of urban and rural space as well as the high concentration of population and economy. The solid erosion, the heat island effect, air, water and solid waste pollution result into greater environmental pressures and economic loss in the process of rapid urbanization ,22,23,24The YRD is one of the regions with the most diversity, openness, innovation and the absorption of the largest numbers of immigrants in China. The study area comprises 26 cities, including Shanghai, Nanjing, Wuxi, Changzhou, Suzhou, Nantong, Yancheng, Yangzhou, Zhenjiang, Taizhou, Hangzhou, Ningbo, Jiaxing, Huzhou, Shaoxing, Jinhua, Zhoushan, Taizhou, Hefei, Wuhu, Maanshan, Tongling, Anqing, Zhangzhou, Chizhou and Xuancheng , with a With the rapid economic and social development of the YRD, there are potential problems, such as the increasing prominence of urban issues like energy consumption pressure, cultivated land loss, water shortage, serious environmental pollution, aging population and increasing population burden ,32. ThesReferencing to the preceding interpretation of SDGs-11 and the accessibility of research data, a total of 11 indicators were selected to construct the index system in six themes: Housing, traffic, land use and participatory planning, environmental impact, public space and relationship between urban and rural areas. With respect to Urban sustainability science, the ‘‘weak’’ and ‘‘strong’’ substitutability indicators can comprise environmental, political and economic challenges in achieving SDGs. What’s more, the strong sustainability at a broad scale may not be achieved without a proper combination of weak sustainability on smaller scales, more attention should be paid to emphasize the difference between artificial and natural attributes of the capital resources, we developed and grouped 11 urban-related SDG-11 indicators into two categories—weak and strong sustainability indicators ,34. 2.5 annual average concentration and days of good air quality. Meanwhile, the weak indicators include commercial housing sales area, buses, urbanization rate, urban green space area, per capita park and green space area and household registered population. The data were collected from the China City Statistical Yearbook and the National Bureau of Statistics of China (2007–2016). In order to achieve vertical comparability of the indicator system, the indicators are processed in a positive and negative manner. When the index has a “+” indicator, the datapoint is taken as a positive value and vice versa. The final list of 11 indicators considered in the present analysis is reported in n−1)! /2 different irregular center n-gons, which can be used to evaluate each objective function:The full permutation polygon synthetic indicator method was used for data standardization and indicator synthesis . It inclF(x) satisfies F(L) = −1, F(T) = 0, F(U) = 1, where L is the lower limit of the index x, U is the upper limit of the index x and T is the threshold of the indicator x. According to the above three conditions, we can obtaini-th indicator isThe standardized calculation formula for calculating the The formula for calculating the composite index based on fully arranged n-gons isiS and jS are the indicators for i and j and S is the value of the synthetic indicator for each city. The value of S is between  and larger value that is considered, the better. F(x) takes a value between  and the theoretically largest value is the farthest from the center of the n-gon, which is normalized to have a value set to 1. In contrast, the center of then-gon has a value of −1. Since setting different weights for the indicators of each type of city might bring about great uncertainty and end in results that are not comparable, all the related indicators were all equally weighted [In Formula (4), weighted ,34.The empirical analysis of this paper includes an evaluation of the sustainable development performance in the YRD from 2010 to 2016, which examined the changes in the related indicators and synthesis indicators of SDG-11 before and after the launch of the UN Sustainable Development Goals. The results show that the synthesis indicators in the YRD increased year by year. The urban planning tended to be reasonable and several urban-related SDG-11 indicators fluctuated slightly in different years. To provide a preliminary indication of potential trajectories for the next few years, we assessed each urban-related SDG-11 indicator from 2007–2016 .Concerning city size, the urbanization level of the YRD municipality and the provincial capital city had slowed down quite markedly. The main growth areas of the growth point included Shanghai, which grew with an urbanization rate of over 80%; Jiangsu, which grew by 55.6%; and Zhejiang, which grew by 57.9%. The urban agglomeration network system centered on Shanghai, Nanjing and Hangzhou, as sub-level centers had been initially formed. In the past 10years, the number of registered households in Anhui Province dropped significantly with a large number of people flowing out; in contrast, other cities were relatively stable. As the mega city, Shanghai was the only one with a registered population of over 10 million. The growth of housing sales area was still concentrated in Shanghai, Hangzhou, Hefei and other municipalities directly under the central government and provincial capitals. Only the housing sales area of Suzhou was larger than that of the provincial capital city, Nanjing, as it attracted many investments and achieved great development in the last decades.As for the urban environmental impact, the overall urban sewage discharged from major cities in the YRD showed a steady and small upward trend in the past decade. The discharge of solid waste experienced a process of increasing at first, then decreasing and then increasing again throughout the period of 2007 to 2016. The cities that contributed a lot to the emission indicators in the early period were Shanghai, Suzhou and Maanshan. In recent years, since the standardized management systems and standards for various types of waste have been gradually implemented, the values of emission indicators have declined. The domestic garbage disposal rate has reached 100% for most cities since 2010. The average annual air quality was maintained in good condition between 2007 and 2009. Because of the introduction and implementation of a more severe national air quality standard, a significant deterioration took place after 2010 but the air quality gradually returned to good condition over the last two years. The air pollution in Hefei, Anqing, Chizhou, Yangzhou and Tongling is still relatively severe. Zhoushan, which has good coastal diffusion conditions and low industrialization, was the only city that had performed well all year round in terms of the maintenance of air quality. In general, industrial structure has an important impact on environmental pollution. With the continuous strengthening of environmental policies in the YRD, environmental pollution was greatly relieved, and the conditions of the coastal areas were found to be in relatively better conditions than those inland.The area of urban green space in the YRD increased slightly in the past decade. Due to the population density, the per capita park and green space area of Shanghai was at the bottom of all the cities in the YRD. In recent years in the YRD, due to the strict control of urban development boundaries and the optimization of urban and rural spatial structures, the central government implemented a new greening action plan by building a number of urban parks and national parks. However, the high density of the urban population still causes tensions in public space resources.The synthetic indicator for the sustainability of 26 cities from 2007–2016 exhibited a descending trend . The pioThus, there existed a polarized phenomenon in the sustainable development in the YRD agglomeration. The sustainability level for those cities was divided into four types (levels I–IV).According to which level the highest score belonged to, some of the credit for the excellent performance of cities (level I) like Hangzhou, Shanghai, Suzhou and Nanjing must go to their coastal location, developed transportation network and high level of public services. As representatives of level II, Wuxi, Zhoushan, Hefei and Changzhou had one or two indicators perform badly and the others performing well or excellently. Meanwhile, the synthetic scores of those cities experienced the trend of rapid development and obvious decline during the last five years of 2007 to 2016. This indicated the emergence of hidden dangers from rapid development and the rise of competitors among urban agglomerations. Moreover, the air pollution problems generated in the short term caused those cities to perform poorly in the sustainability index. The level III cities exhibited so-called neutral development, with almost no indicators performing excellently or very badly. In terms of time span, cities like Zhenjiang, Chizhou, Shaoxing and Xuancheng, which are rich in natural resources but lacking in technological development, faced a lot of pressure in recent decades. The increasing industrialization level of cities could also aggravate the risk and instability of sustainable development. To be specific, those cities suffered from a low quality of life, which was caused by the rapid expansion of the city’s size and a large number of emigrants. In the future they can try to maintain a good level of development through technology innovation, the introduction of new talent and the construction of infrastructure. Cities (level IV) represented by Maanshan and Anqing—with several scores less than 0 and some reaching −1—were representative of those cities that performed poorly or very poorly in the majority of the indicators. Different from the cities of level I, these cities are located in the hinterlands and have poor natural resources, population emigration, traffic problems and fierce competition within the agglomeration. In this context, it can be predicted that it was a great challenge for local governments to improve their level of sustainability.Based on the results from single indicators analysis above , ShanghaAs shown in Through the global spatial autocorrelation analysis of the spatial autocorrelation coefficient Moran’s I from 2007 to 2016 , it was This study introduced the FPPSI method to assess both the single and synthesis indicators for sustainable development of the YRD agglomeration in 2006–2016. The key factors and obstacles of urban sustainable development were identified from the viewpoint of urban agglomeration integration in order to assist in problem diagnosis and policy guidance for the central government. The results implied that:(1) The FPPSI method is an objective and quantitative method to evaluate the sustainability level and understand the overall effect of policies on SDGs, especially over time. It could not only evaluate the overall level but also analyzed the impact of single indicators by sorting out the superior and inferior indicators. This method has advantages such as ease of calculation and the ability solve multiple indicator evaluation problems, improving the comparability of the valuation scores in different years and grading the comprehensive level of sustainable urbanization.(2) During the studied years, the synthesis indicators for most cities in the YRD were significantly relieved but each city still showed a unique trend due to their different positions and functions. Meanwhile, there were also some common problems to be solved urgently in the YRD agglomeration. Judging from the carrying capacity of each subsystem, the sustainable development level of small and medium-sized cities was still dominated by the traditional strong sustainability indicators. The sustainable levels of large and mega cities were affected by weak sustainable indicators. (3) The spatial distribution and structure of sustainable development in the YRD agglomeration showed difference and imbalance among the cities. The inter annual standard deviations of Shanghai, Yancheng, Xuancheng, Huzhou and Taizhou seemed to increase over the years, which indicated the unbalanced development and instability among the indicators. It is necessary to pay attention to the strong spillover effects of developed cities in the YRD, such as Shanghai, Hangzhou and Ningbo. Therefore, while rationally developing the YRD, regional developing patterns need to be improved, especially in terms of the innovation of internal and external systems in the northern cities of the YRD. This can be achieved by strengthening various forms of integrated cooperation and coordinated development among regions ,40. Judging from the comprehensive evaluation of typical cities, the YRD has realized the initial sustainable development of resource-based cities but the degree and stage of development among those cities vary. The low score indicators should be seen as a wake-up call; those cities should take further action to promote the concept of green and sustainable development to achieve so-called sustainable transition and development in the process of the YRD integration. It is worth noting that new actions in relation to one indicator are very likely to affect scores for other indicators, how they relate to big issues need further study to confirm, such as climate change, loss of biodiversity, poor air quality, poverty and so forth. In the process of the integrated development of the YRD, it is necessary to differentiate between cities according to their background and level, especially to overcome the restrictions and obstruction factors for each city.The rising trend in the coastal areas is obvious and the sustainability level of large cities is gradually developing as well. However, the developing pressure is proportional to the size of the city and paying scant attention can bring about serious impacts and cause “urban mass.” The less developed cities from inland areas have just set out on the way towards “positive changes;” meanwhile, the pressure to achieve sustainable development has gradually increased. The main reasons for this situation might be the protection of various localities and the existence of competition among neighboring cities. Some regions have higher profits, while other regions have sacrificed their own interests. Therefore, cities call for closer cooperation and more effective policies to strengthen inter-cooperation and to build their capacity to realize sustainable development. In the future, it will be necessary to establish a unified benefit sharing and interest compensation mechanism within the YRD ,41,42.OnWe recognize that the FPPSI method has both practical and operational significance in the assessment of the sustainable development level for cities and agglomerations. However, as a result of the spatial differences of observed indicators, the weight of the single indicators needs to be taken into consideration and adjusted appropriately based on the consultation of experienced experts in future research. This would make the results more reasonable and strengthen the practical guiding significance for regional sustainable development. Due to data gaps in the SDGs, more indicators are needed in order to be able to display all different aspects of sustainability. Since the traditional statistical data is short in real-time performance and lacks sufficient spatial completeness, new perspectives and pathways for realizing real-time dynamics, spatial fineness and the quantitative evaluation of sustainability ought to be further explored and addressed.