ROHF, ranged from 1.79% in ZWM to 8.62% in ACM goat populations. High ROHF values, elevated genomic coverage of very long ROH (>30 Mb) and severe decline in effective population size were recorded in ACM goat farm. The existence of a high correlation between HOMF and ROHF indicates that ROHF can be used as an alternative to inbreeding estimates in the absence of pedigree records. The eN estimates 13 generations ago were 166, 69 and 79 for ZWM, ACM and JGM goat farm, respectively indicating that these goat breeds were strongly affected by selection pressure or genetic drift. This study provides insight into the genomic relationship, levels of inbreeding and effective population size in the studied goat populations conserved in the state goat farms which will be valuable in prioritizing populations for conservation and for developing suitable management practices for further genetic improvement of these Chinese male goats.Conservation of genetic resources is of great concern globally to maintain genetic diversity for sustainable food security. Comprehensive identification of the breed composition, estimation of inbreeding and effective population size are essential for the effective management of farm animal genetic resources and to prevent the animals from genetic erosion. The Zhongwei male (ZWM), Arbas Cashmere male (ACM) and Jining Grey male (JGM) goats are conserved in three different state goat farms in China but their family information, level of inbreeding and effective population size are unknown. We investigated the genomic relationship, inbreeding coefficient and effective population size in these three breeds from three state goat farms using the Illumina goat SNP50 BeadChip. Genomic relationships and phylogenetic analysis revealed that the breeds are clearly separated and formed separate clusters based on their genetic relationship. We obtained a high proportion of informative SNPs, ranging from 91.8% in the Arbas Cashmere male to 96.2% in the Jining Grey male goat breeds with an average mean of 96.8%. Inbreeding, as measured by  In order to preserve animal genetic resources and promote sustainable livestock improvement in changing environmental conditions and demands, it is critical to assess the conservation status of genetic resources. Approximately 30% of the world’s farm animal breeds are at risk of extinction due to a lack of conservation policies . ConservF), is the probability of having pair of alleles at a locus that are identical by descent (IBD) with respect to the base population in which all the alleles are assumed unrelated [Inbreeding is of concern in the livestock industry as it has negative effects on the performance and fitness of animals. The increase in inbreeding in the livestock population tends to have higher rates of congenital disorders and may reduce the survival rates, fertility and long term viability known as inbreeding depression . The ratnrelated . The inbnrelated ,6,7.Advances in high throughput genotyping, sequencing and bioinformatic analysis have facilitated the assessment of inbreeding based on single nucleotide polymorphisms (SNPs) as an alternative to the traditional pedigree-based inbreeding coefficients. In recent times there is a growing interest to estimate the inbreeding coefficient from SNP genotyping data because of its easy estimation, accuracy and ability to trace back to many generations in the past in assessing inbreeding . MoreoveeN) is widely regarded as the most important parameter in conservation genetic because of its relationship to inbreeding, selection, migration and mutations [eN increases the chance of inbreeding and genetic drift and decreases the genetic diversity, which may compromise the genetic viability of a population and change the patterns of ROH in the long term [Effective population size , Arbas Cashmere male (ACM) and Jining Grey male (JGM) goats are conserved in three different state goat farms in China but their pedigree information, level of inbreeding and effective population size are unknown. The scope of this study was to ensure that the conservation goat farms have at least six unrelated groups of animals, a low level of inbreeding and a sufficiently large number of effective population sizes based on an accurate estimation by the SNP genotyping data.Ethical approval for animal survival was provided by the animal ethics committee of the Institute of Animal Science, Chinese Academy of Agricultural Sciences (IAS-CAAS) (Number IASCAAS-AE-03).A total of 98 male goat individuals comprising of Zhongwei male, Arbas cashmere male, Jining Grey male goats from the three Chinese state goat farms were genotyped using the Illumina Goat SNP50K Beadchip panel . The detailed sample descriptions are shown in −5.Quality control of the SNP data was implemented using Plink 1.07 . An SNP iP is the frequency of the ith allele. The genomic inbreeding coefficient for individual j is simply Gjj − 1, and genomic relationships between individuals j and the individual k, are obtained by dividing elements Gjk by square roots of diagonals Gjj and Gkk.The genomic relationship matrix (G) is an estimator of the actual proportion of the genome that is identical by descent across individuals. The genomic relationship matrix (G) was calculated using G matrix software based on the method proposed by VanRaden . The forTo further confirm the phylogenetic relationships among the individuals of each breed, we constructed a neighbor-joining (NJ) tree using MEGA 7.0 . We calcTo assess the genetic relatedness between individuals, a principal component analysis (PCA) was performed with Plink 1.90 . To evalNP) was measured as the fraction of total SNPs with MAF greater than 1% in each breed using the PLINK 1.90 software. The average heterozygosity was calculated with the command --hardy using the same software.The proportion of SNPs that displayed polymorphic (ROH(F) was estimated as the proportion of genome in ROH over the overall length of the genome covered by the involved SNPs using detectRUNS packages in R software [sn is the number of SNPs per individual, in is the number of individuals tested, and het is the proportion of heterozygosity across all SNPs.The inbreeding coefficient based on ROH software . The folsoftware :(2)l=lnαROHF) for each breed was calculated using the following method [ROHL is the total length of ROH of each individual in the genome and AUTOL is the length of the autosomal genome of the goat (2399.4 Mb). In addition, the chromosomal ROHF values were also estimated for each breed as summing the total length of an individual’s ROH in each chromosome divided by the length of each chromosome covered by the involved SNPs [The genomic inbreeding coefficient (g method :(3)     ved SNPs .ROHF, we also calculated the inbreeding coefficient as a function of the expected and observed numbers of homozygous difference (HOMF) with the following equation:L is the number of autosomal SNPs genotype, HOME and HOMO are the number of expected and observed homozygous genotypes, respectively.To test the fairness of ROHF and HOMF was calculated across the breeds.The correlation between The distribution of ROH coverage can be used to infer the recent and ancient inbreeding. Therefore, we also calculated the average ROH length for four length categories  to see the distribution of recent and ancestral inbreeding. The average ROH length for each class of each breed was estimated by adding up all ROH segments of each ROH length class per breed and dividing by the total number of individuals of that respective breed.eN) was calculated for each breed separately using SNeP v1.1 [eN estimates at different time points are based on the extent of linkage disequilibrium, using the formula proposed by Sved [eN is the effective population size, c is the genetic distance in Morgans between two markers, and E(r2) is the expected average coefficient of determination (r2) value for distance c. Each genetic distance (c) corresponds to a time point value of t generation in the past obtained as described by Hayes, Visscher [The effective population size (NeP v1.1 . Ne esti by Sved :(5)E(r2)Visscher :(6)T=12cIn this study, 53,347 SNPs were considered before quality control. After quality control, a common subset of 45,041 SNPs from 98 individuals was used for downstream analysis .The results of the genomic relationship matrix (G) are shown in Individuals of each breed clustered separately where more individuals with a close relationship in the ACM goats, followed by the JGM goats, and relatively few individuals with a close relationship in the ZWM goats. The closely related individuals of ACM goat formed 12 different families. Ten families were found among the individuals of JGM breed. The ZWM goats were genetically divergent from others due to their different pedigrees and the closely related individuals of ZWM goat clustered into 17 separate groups .A neighbor-joining (NJ) tree revealed that the individuals of studied goat populations are clustered into three major groups where the individuals of each breed formed separate groups according to their genetic relatedness. The individuals marked with the same color in the NJ tree belong to the same family. Therefore, from the cluster analysis, it can be seen that the individuals of ACM, JGM and ZWM breeds separated into 12, 10 and 17 independent sub-groups  which isThe PCA results showed three distinct clusters that were grouped according to the origin and geographical distribution of the studied goat populations a. The fiNP) and inbreeding coefficient are summarized in NP) was comparable among breeds where the highest value was observed in JGM (96.2%) and the lowest value was found in ACM (91.8%). The JGM goats presented the highest values and ACM displayed the lowest value for both OH  and EH . The ACM exhibited the highest average values for both inbreeding coefficient estimators  whereas the ZWM showed the lowest ROHF (0.017). A negative value for HOMF was observed only for JGM breed (HOMF = −0.010). A high correlation coefficient between ROHF and HOMF was noted for all the breeds ranging from 0.92 (ZWM) to 0.99 (ACM) with an overall mean of 0.92.The results of polymorphic SNPs  the highest percentage of ROHF was 0.79% presented by ACM goats.ROHF) for each chromosome across breeds. In general, the mean ROHF values followed the same pattern as those computed for the whole genome and differed between chromosomes. We observed that the ROHF values were highest for chromosome 3, 8, 19 for JGM, chromosome 27, 29 for ACM and for chromosome 25, 28 for ZWM goat breeds.To infer the ancient and recent inbreeding, the distribution of the relative numbers of ROHs was plotted in the different length categories within the breeds . The ACMeN) at different time points for the studied goat populations is presented in eN showed a declining trend over time, faster 1000 to 100 generations ago and slower since100 generations across the populations. The most severe drop in current eN was found in the ACM breed, whereas ZWM presented the highest eN across the generations (eN was obtained in ZWM (166), whereas the lowest one was observed in ACM (69) 13 generations ago  observed in this study indicated that most of the SNPs have been segregating in the breeds under investigation which was comparable to those reported in Pakistani goats [NP in ACM goat may reflect its uniqueness and narrow genetic base [OH was found in ACM (0.367 ± 0.19) goats and the highest in JGM (0.401 ± 0.20), indicating higher genetic diversity existed in JGM goats. The observed heterozygosity reported in this study was lower than the expected heterozygosity which might be due to the Wahlund effect rather than inbreeding. The JGM goats can be proposed as important goat breed for conservation due to unique genetic composition and high level of genetic diversity.The results from the genomic relationship matrix (G) and NJ tree analysis were in agreement, clustering breeds in accordance with their genetic relationship . The anani goats  and in Ani goats . More thtic base . Althougtic base . The lowROHF coefficients recorded in ZWM goats were lower than that of ACM and JGM goats which might be attributed to the absence of strong selection pressure, high gene flow, and larger effective population size resulted from broad and multiple breeding objective [ROHF = 0.086, HOMF = 0.074)  . The anare yield . Attentire yield  and Swisre yield . Strong ree data . The disROHF) in this study were consistent with those reported in Valle del Belice sheep [ROHF varied between chromosomes with the highest values obtained for chromosome 3, 8, 19 in JGM, chromosome 27, 29 in ACM and for chromosome 25, 28 in ZWM goat breeds  and severe decline in effective population size indicated strong selection pressure in the Arbas cashmere male goat farm. Thus, initiatives for sustainable conservation and utilization are imperative. The conservation farms should also be aware to maintain sufficiently large eN for ACM and JGM goats for maintaining genetic diversity. Information from this study may also assist in the reduction of unnecessary inbreeding depression or gene flow among populations in these goat farms. Further studies will be helpful to confirm and refine our results through the inclusion of more samples and other tools such as whole genome re-sequencing, high-density SNP genotyping and phenotypic data.In this study, we have investigated the pedigree background, level of inbreeding and effective population size in three goat breeds from three state goat farms using the goat SNP 50K BeadChip panel. Genomic relationship and phylogenetic analysis revealed all the populations have more than six groups of animals. High levels of inbreeding were obtained in ACM goat farm, whereas low levels of inbreeding were observed in ZWM and JGM goat farms. High


The focus of the EU regulations on the Nagoya Protocol on Access and Benefit-Sharing leaves the control of access to genetic resources up to each member state. France has chosen to control access and is going to put in place regulations for it. All the materials received should have specific documentation regarding the accession of genetic resources, where there is a National Authority to issue them. The European commission will maintain a list of biological collections with registered status proposed by each country. The member states are responsible for considering inclusion and verification of these collections. In recent years, the Collection of Institut Pasteur (CIP) staff has expressed concern over how to interact with the implementation of the Nagoya Protocol in the collection but also at the national level with the aim that the CIP will be a registered collection. The advantage of accessing resources from a registered collection is that users of genetic resources will be considered as having exercised ‘due diligence’ if they source their genetic resources from these collections. This could facilitate the process for scientists when applying for research funding. The CIP organized the accession of new deposits and the distribution of micro-organisms in connection with it. Regulations on Access to Genetic Resources and the fair and Equitable Sharing of Benefits Arising from the Utilization to the Convention on Biological Diversity (CBD) leaves the control of access to genetic resources up to each member state to establish their own national legislation with the goal of ensuring that benefits of genetic resource utilization are shared equitably with provider nations. The fair and equitable sharing of the advantages stemming from the use of the genetic resources and the associated traditional knowledge, aim to restore some equity between suppliers and users of these resources and knowledge and contributes to the preservation and the long-sustainable use of the biodiversity. Every state has sovereign rights over their natural resources and can be a supplier and user. It determines rules to obtain and use the genetic resources under sovereignty and takes measures assuring the fair and equitable benefit-sharing stemming from the use of the resources.The Nagoya Protocol (NP) on Access and Benefit-Sharing (ABS), an international agreement adopted in 2010, which came into force on 12 October 2014, was the result of several years of negotiations that started with some countries which claimed biopiracy . The most important goal of the implementation of the Nagoya Protocol is to build confidence and relationships between the North and the South countries, particularly in research and development.The European Union legislation, EU regulation no. 511/2014 on ABS, emphasizes that it is applicable to genetic resources from countries that have ratified the Nagoya Protocol, exercise sovereign rights, and have established ABS measures. All the genetic resources collected in EU members countries and used in research and development should comply with these measures. However, states can also decide whether or not to leave their resources in free access. Countries such as France and Spain are implementing regulatory control of access.https://absch.cbd.int/) [At the moment of writing, 110 states worldwide have ratified the Nagoya Protocol and various legal frameworks for implementing it may exist according to countries. The information regarding national requirements and procedures for access to genetic resources and associated traditional knowledge (TK) is managed in a central depository, the Access and Benefit-Sharing Clearing-House (ABS-CH)  has resulted in a definition of the Biological Resource Centres (BRCs) as well as the setting up of recommendations for their structuring and management. BRCs are specialized centres that collect, validate, study, secure and distribute collections of living organisms  and ‘replicable’ parts of these organisms  under rigorous conditions of quality and traceability; they also maintain the databases associated with these collections. BRCs are essential elements of the international research system, especially in the field of biotechnology. They also contribute to the preservation of biodiversity and genetic resources in the context of national policies and international conventions .Microbial Biological Resource Centres (mBRCs) are dedicated to the preservation and enhancement of microbial diversity. They group micro-organisms either generally, with a maximum representation of biodiversity, or in a more specialized way, either in the form of thematic selections, yeasts, filamentous fungi, food bacteria, human or animal pathogenic bacteria, bacteria associated with plants, entomopathogenic bacteria, etc.The main missions of mBRCs are:to ensure the sustainability of collections of micro-organisms by means of their perfect characterization, maintenance and preservation;to enrich its collections by exploring the biodiversity of micro-organisms of interest by collecting various biotypes from original strains;to scientifically and economically exploit these collections by ensuring their widest possible dissemination to the academic, scientific and industrial community by associating them with services .The Institut Pasteur hosts more specialized micro-organism collections  on a single site than any other institution in France. Most of these collections are housed in and maintained by research laboratories. Only a few of these collections are ‘open’, distributing strains to analytical, industrial and research laboratories, other laboratories and teaching establishments on request. The largest of these collections, the Collection of Institut Pasteur (CIP) contains more than 15 000 bacterial strains and almost 130 plasmids. In December 2001, the Institut Pasteur undertook a project to create a BRC, the CRBIP (Centre de Ressources Biologiques de l’Institut Pasteur) as defined by the OECD, incorporating its collections of micro-organisms.The aim of the CRBIP was to harmonize the management of collections on the campus, to enrich collections in an organized manner (e.g. with strains from collections of research laboratories), to ensure that the strains or products stored are characterized as thoroughly as possible, to improve conservation conditions  and to ensure the distribution of samples according to health and environmental safety norms, in accordance with the laws and regulations in force. Quality assurance has been an integral part of this project since its inception, to meet the criteria laid down by the OECD . The quaMost of these collections hold materials that were isolated within the sovereign boundaries of another country. In order to enable due diligence, the European Commission have proposed a list of registered collections in order to guarantee that users who source their genetic resources from these registered collections, will be considered as having exercised ‘due diligence’.In recent years, the CIP staff has expressed concern over how to deal with the implementation of the EU regulation of the Nagoya Protocol but also with the French regulatory control of access, which is not well-defined yet.The adoption of the Nagoya Protocol in October 2010 has an obvious impact for mBRCs and, therefore, for the Collection of Institut Pasteur (CIP). Since the ratification by France in August 2016, the application of the NP is binding on all users of genetic resources established on the national territory. The CIP, as mBRC, is considered to be a user of genetic resources, and for this reason, the first steps to comply with the Nagoya Protocol have been implemented.During the first stage of the implementation of the European regulation at the CIP, different tasks have been studied and others have been carried out:inventory of genetic resources linked to the NP;verification of information and documents related to genetic resources;exchanges of emails with depositors of microbial strains: requests for important information and documents on genetic resources, where necessary;drafting an explanatory sheet on general information regarding the Nagoya Protocol;archiving of information collected on genetic resources and storage for at least 20 years;verification of the procedure applicable to the CIP;verification of compliance of newly deposited genetic resources.At the European level, collections and mBRCs are obliged to respect the due diligence. Therefore, in order to establish its compliance with international provisions, the CIP must undertake the traceability of these genetic resources and then be registered in the EU Register of Collections. Generally speaking, traceability involves gathering all the essential information and documents relating to genetic resources.As part of this compliance, an inventory of all genetic resources  deposited within the CIP, has been carried out since 12 October 2014 (date the Nagoya Protocol came into force). Due to the large number of new strains received in the CIP each year, the first part of the inventory was performed with the genetic resources received between 12 October 2014 until 30 December 2015. From January 2016, the deposit form was modified with the aim of including information regarding ABS issues and compliance with the NP.The following specific information regarding the biological material was studied and if necessary, the CIP asked the depositors to complete the information regarding the Nagoya Protocol:the date of deposit in the CIP;the name of the depositor and his/her host institution;the depositor identification number of the strains as well as the CIP identification number;in situ isolation and/or ex situ isolation (where applicable);the date of in situ;the place of isolation (including country of origin) of original collecting the name of the person who isolated the strain and his/her host institutionin situ isolation or the name of the person (or institution) who isolated the strain. Thus, these resources were not considered to be in compliance with Article 4 of the 511/2014 Regulation. With the aim of respecting the due diligence, it was decided to conduct a retroactive compliance contacting the depositors of these genetic resources by e-mail in order to request the specific missing information, as well as documents such as PIC, MAT and/or the IRCC. Nevertheless, since most scientists were not aware of the Nagoya Protocol, an explanatory note on the international instrument was also included in the e-mail in order to inform them about the legal context of the NP and to explain the reasons why the CIP requested such information. At the time of writing this article, several depositors replied sending the information requested for 77% of the 36% of the strains with incomplete information. The depositors of the other 23% strains have not replied yet, despite having been contacted several times. The written reply of depositors was printed and scanned to be kept as proof of due diligence. However, none of the depositors contacted had PIC, MAT and/or the IRCC documents.In total, 36% of the strains received in the period before mentioned, had incomplete information, such as the date of isolation, the place of According to our experiences during the implementation of the Nagoya Protocol in the CIP, the procedure to be followed to verify the compliance of a deposited genetic resource is explained in Deposition of microbial resources in public mBRCs facilitates their availability, the confirmation of findings and allows continuity of research projects. Strategies have been devised to encourage researchers to deposit a higher fraction of the strains they work with and, at the same time, to implement quality measures and to comply with regulations. However, the necessary expansion of human resources and infrastructure is moving slowly.Based on a daily experience on receiving strains coming from different geographical regions, we describe here four real facts experienced during recent deposition of bacterial strains in the CIP:Deposit of strains with the necessary information, but without ABS associated documents. This may be considered as a partial compliance with the NP.At the time of writing the article, only a few countries were able to issue an IRCC . Thus, the CIP cannot request this document if one of its depositors does not have any document relating to a genetic resource originating from a country other than the countries above mentioned.Genetic resources isolated from country parties to the Nagoya Protocol. When the information and documentation, regarding genetic resources, are not completed, depositors are contacted to obtain such information and, in this way, restore compliance. However, there are cases when depositors do not reply to the email or are unable to give us the information or the associated documents, since even they do not have this information.Genetic resources from country parties of the NP may have their own legislative, administrative or policy measures. This is the case of two genetic resources isolated in Spain, which adopted the regulation 124/2017 of 24 February 2017 on access to genetic resources of wild taxa and control of its use. The Spanish regulation states that when the resource is used or distributed only for taxonomic purposes, a permit is not required. This regulation applies to biological resources isolated in Spain. Thus, in this case, depositors are not obliged to issue a PIC, MAT and/or an IRCC since the CIP has only performed taxonomy with them. However, the biological material within the CIP is not only redistributed for taxonomic purposes, but for research or industrial applications, for example. Therefore, according to the Spanish regulation, a permit must be obtained by the CIP directly with the competent Spanish authority. In order to establish compliance of the genetic resources, the Directorate General of Quality and Environmental Assessment and Natural Environment (Spanish Competent Authority) was contacted by email. However, there are difficulties in accessing information on regulations of a third country, such as when a document is written in a foreign language, without translation into English.The EU Regulation 511/2014 imposes several obligations on users of genetic resources or traditional knowledge. According to the article 4 Reg. 511/2014 users are obliged to exercise due diligence to ascertain that: (a) genetic resources and traditional knowledge have been accessed in accordance with applicable access and benefit-sharing legislation or regulatory requirements, and (b) benefits are fairly and equitably shared upon mutually agreed terms, in accordance with any applicable legislation or regulatory requirements. For these purposes, users have to seek, keep and transfer to subsequent users the PIC, MAT and/or the IRCC.Our experience in such a time-short exercise highlighted the difficulties in obtaining information related to the due diligence associated with access to genetic resources. Even more, there are specific national legislation in different countries which state other meanings for terms such as the term ‘access’. For example, Brazil has taken the position that access to all genetic resources from Brazil does not begin when they were collected, exported and deposited in collections, but when research and development activities (access) take place . On the Other concerns are focused on the possibility of involving digital sequence information in the decisions of parties of the NP. During the Second Meeting of the Parties to the NP (COP/MOP 2), held in December 2016, some issues remained undefined and a comprise proposal was adopted by COP/MOP 2 to establish a 2-year process to consider the potential implications of digital sequence information for the three objectives of the CBD. The outcome of this process was considered during the third Meeting of the Parties at the end of 2018.in situ by the state [Benefit-sharing of biological resources typically focus on the valuation of the presence of a particular micro-organism on state territory and the resulting ownership from the micro-organism he state . In thishe state .www.eccosite.org). Also, the Best Practice Manual on Access and Benefit Sharing of the Microbial Resource Research Infrastructure (MIRRI), provides guidance for implementing ABS policies and working procedures for acquisition of microbial material, accession in public collections, transfer of material to third parties and the delivery of other services (www.mirri.org) [With the purpose of dealing with the difficulties and bottlenecks that the NP brings to collections, BRCs, research laboratories, R and D institutions, etc., some best practice documents have been delivered by European Consortia such as CETAF (Consortium of European Taxonomic Facilities). Focusing on mBRCs and microbial collections, as well as, research laboratories on microbiology, some have already implemented a system in order to comply with the tracking and monitoring obligations of the NP, such as the European Culture Collections’ Organisation standard MTA (rri.org) . Thereforri.org) .The application of the Nagoya Protocol in the mBRCs and microbial collections faces several difficulties: (a) the implementing regulations can be very specific in each country; (b) the complexity in collection of retroactive data and information regarding the origin of the genetic resources could be overwhelming; (c) difficulties linked to the time in creating and writing the necessary documents in signatory countries could harm goodwill in complying with the established regulations; (d) bureaucracy in obtaining such documents could hamper accession and provision of mBRCs and collections, which in turn could hamper scientific work using microbial genetic resources.


Concerns over imminent extinction motivated a nuclear DNA study of the species in its largest continuous subpopulation in the eastern Mediterranean Sea. Despite recent evidence of partial subpopulation recovery, we demonstrate that there is no reason for complacency, as the species still shares several traits that are characteristic of a critically endangered species: Mediterranean monk seals in the eastern Mediterranean survive in three isolated and genetically depauperate population clusters, with small effective population sizes and high levels of inbreeding. Our results indicated male philopatry over short distances, which is unexpected for a polygynous mammal. Such a pattern may be explained by the species’ unique breeding behavior, in which males defend aquatic territories near breeding sites, while females are often forced to search for new pupping areas. Immediate action is necessary to reverse the downward spiral of population decline, inbreeding accumulation and loss of genetic diversity. We propose concrete conservation measures for the Mediterranean monk seal focusing on reducing anthropogenic threats, increasing the population size and genetic diversity, and thus improving the long-term prospects of survival.The Mediterranean monk seal  As a result, small and fragmented wildlife populations are more vulnerable to genetic drift and inbreeding3, which, in turn may reduce their fitness and evolutionary potential, eventually driving species to extinction4. In such populations it is particularly important to thoroughly understand basic biological traits such as genetic diversity and structure and demographic history, as they are critical for planning any immediate or long-term conservation actions3. Such a science-driven approach has been used in the development and implementation of effective conservation measures, for example, in the genetic restoration of the Florida panther (Puma concolor coryi)5; in contrast, the lack of information about demographic history and genetic diversity has hampered the development of conservation measures in the vicuña (Vicugna vicugna)6.Anthropogenic pressures in the recent past have led to severe population declinesMonachus monachus) is arguably the most endangered pinniped in the world7 and a flagship species for marine conservation. Following centuries of human persecution and habitat loss, the species has been extirpated from most of its historical range and survives in three small, isolated subpopulations: two in the northeastern Atlantic  and one in the eastern Mediterranean Sea8. The latter subpopulation currently comprises more than 90% of the species’ area of occupancy, is estimated to number fewer than 300 mature individuals9, and has been a focal point of systematic conservation efforts since the early 1990s10.The Mediterranean monk seal  study of the Cabo Blanco and the eastern Mediterranean monk seal subpopulations, using 24 microsatellite loci, recorded low intrapopulation diversity, substantial genetic differentiation between subpopulations and no obvious population structure within each subpopulation12. However, the findings for the eastern Mediterranean subpopulation should be interpreted with caution, due to the small sample size (n = 12) and the marked differences in demographics, ecology and behavior between these two subpopulations8. A more recent genetic study of the Mediterranean monk seal in the eastern Mediterranean using mitochondrial DNA (mtDNA) markers, has suggested that the species is one of the most genetically depauperate mammals on earth13.Molecular genetics has repeatedly been shown to be an invaluable tool in studying and ultimately protecting elusive and/or endangered species14, to assess the conservation status of the species in the eastern Mediterranean Sea from a genetic perspective. We analyzed contemporary genetic samples from Mediterranean monk seals in Greece  What is the current population structure of Mediterranean monk seals in Greece and how is gene flow maintained? (2) What are the levels of genetic diversity of the monk seal subpopulation in Greece and how is this diversity distributed geographically? (3) What are the levels of individual inbreeding and what is the effective population size (Ne) of the Mediterranean monk seal clusters in Greece? Based on our current understanding of the recent demographic history of the species and considering the central position of Greece within the eastern Mediterranean monk seal population, we expected limited population structure and gene flow that is mediated by male dispersal, medium to low levels of genetic diversity, and medium levels of inbreeding and small effective population sizes.The dire genetic situation of the Mediterranean monk seal, our limited understanding of its life history and the low number of surviving animals call for immediate scientific attention, and for effective, concrete conservation actions. In this study we used nDNA microsatellite markers, which have a higher resolution and reflect recent population processesOur study system provides a unique opportunity to demonstrate how hard-to-obtain information on basic genetic and demographic parameters of elusive and endangered species is indispensable in developing science-based conservation strategies for their recovery. We use our findings to develop a science-based conservation strategy with concrete management recommendations that will increase this charismatic flagship species’ chances of survival.K = 3  to the Ionian cluster. One individual in the Ionian exhibited substantial proportion of ancestry attributed to the northern Aegean cluster , with two seemingly interpretable global components, and no local structuring (P = 0.719)  Fig. . Global 9 Fig. . 19) Fig. , S7.P = 0.0172). Within this distance class, pairwise comparisons among individuals showed significantly higher values than the population’s mean kinship value  pattern until a distance of 100 km P = 0.017. Within Heterozygosity and allelic diversity were generally low, and no statistically significant differences between genetic clusters were found Table . ResultsThe northern Aegean population cluster Fig. B was cleHe) and observed (Ho) heterozygosity estimates were similar in the North Aegean, but started diverging approximately 200 km to the South along the TW axis, indicating deviations from Hardy–Weinberg equilibrium (HWE), with seven (23%) of the markers showing statistically significant departure from HWE  the animals in this cluster were most likely isolated from other Mediterranean monk seals, with genetic drift rapidly forging a unique genetic signature. Active conservation in the northern Aegean started in the early 1990s10 and likely facilitated population recovery in the area.In the Aegean Sea, the higher number of samples allowed us to explore the recent demographic and genetic history of the Mediterranean monk seal in the region in more detail. The northern cluster had low inbreeding, low 8, which likely led to a sharp population decline. This in turn resulted in reduced gene exchange with other Mediterranean monk seal populations, causing a high degree of genetic separation. The southern Aegean population started inbreeding more rapidly than the northern cluster, with genetic drift ultimately causing the allelic frequencies to change enough to detect it as a unique genetic cluster. Inbreeding and genetic difference to the northern cluster decreased towards the North, indicating that admixture between the two genetic clusters is relatively recent. This also had a clear effect on Ne, which was higher in the “admixing zone”  did not increase during the process of admixing of the clusters, indicating that the southern Aegean population is not contributing much unique genetic diversity. The pattern of deviations from HWE in the South Aegean with increasingly lower Ho compared to He as we move towards south . The Ne for the northern Aegean cluster does not meet the Ne > 50 criterion that is thought to allow a population to avoid inbreeding and it is still far below the rule-of-the-thumb minimum threshold of Ne > 500 that has been suggested to maintain genetic diversity18. Even the admixing of population clusters, which increased overall Ne, merely made the entire Aegean population marginally viable in the sense that it might be able to avoid inbreeding. However, it has been argued that for endangered species the target effective size for a conservation program should be in the range of 1000–500027. In this regard, our Ne for the entire Aegean monk seal population is more than an order of magnitude lower than the minimum effective population size conservatively recommended for the management of endangered species.28, such as the Mediterranean monk seal8. However, assessing dispersal in marine mammals is notoriously challenging. To this date, findings of sex-biased dispersal in pinnipeds has remained inconclusive. For example, female dispersal has been described in Southern elephant seals (Mirounga leonina)29 and long distance dispersal has been reported for both sexes31. The pattern indicated by our results may be explained by the unique breeding behavior of the Mediterranean monk seal, where females need to find new pupping areas due to limited space in existing ones32 and males tend to defend aquatic territories around pupping sites33. Most likely as a result of this, female Mediterranean monk seals have often been directly observed to disperse in the eastern Mediterranean8. Consequently, the fact that no dispersal of individuals from the Ionian to the Aegean has been documented so far (results of this study and13), indicates that the number of Mediterranean monk seals in the Ionian Sea is comparably low and that there is still suitable pupping habitat available in the region. Kinship coefficients in our study were moderately low, but similar to those described for e.g., the Saimaa ringed seal (Phoca hispida saimensis)34. While long-distance dispersal in adult female seals is likely29 and has been recorded in the species8, general conclusions on sex-biased dispersal in the Mediterranean monk seal cannot be drawn. We advocate for a targeted study combining movement  and genetic data to comprehensively assess the species’ dispersal behavior.Adult males appeared to be more related until a distance of 100 km, while adult females displayed no correlation between kinship and geographical distance. Thus, IBD is likely to have influenced the genetic structure among males at shorter distances. Male dispersal and female philopatry appears to be the norm in polygynous mammalsStudying Mediterranean monk seals in the wild is demanding: animals are rare, spread over a vast geographical scale , and only a small fraction of dead seals is typically recovered. This makes genetic sampling difficult, and sample sizes that are regularly obtained in many other species of conservation concern are virtually impossible to obtain in such an elusive species. On the other hand, due to the extremely small population size of this species, our current sampling represents a significant proportion of the monk seal population in the eastern Mediterranean, which has allowed a comprehensive and representative genetic assessment of the species in the region. The data that we have managed to obtain should be used to develop effective conservation policies that may as well prove to be crucial for ensuring the species’ survival.10, our genetic assessment of Mediterranean monk seals in Greece paints an alarming picture of the species’ genetic status, raising questions on how close the eastern Mediterranean monk seal subpopulation may actually have been to extinction, and highlighting the urgent need to step-up the efforts to protect it. While Greece hosts one of the largest Mediterranean monk seal subpopulations worldwide, even here the species survives in a fragmented population with low genetic diversity, small effective population size, and high levels of inbreeding. Considering that the main threats to the Mediterranean monk seal are anthropogenic8, we believe that human impacts on the species’ behavior  have played a significant role in shaping this picture. On the other hand, gene flow among previously isolated clusters is being re-established, giving ground for cautious optimism. We believe that the dispersal of Mediterranean monk seals from the northern Aegean nucleus towards the south is direct evidence of the effectiveness of the systematic conservation actions that have been in place in the area since the early 1990s, which include protecting critical pupping habitat32 and regulating human activity35.Despite recent encouraging signs of partial population recovery8). This could be supported by the establishment of a compensation system to alleviate financial losses from damages to fishing gear by Mediterranean monk seals36. (d) Since monitoring is essential for any conservation effort and considering the wealth of information that genetic monitoring of Mediterranean monk seals in Greece has provided, we believe it is essential that it is continued within the framework of the Hellenic Monk Seal Register37, i.e., a project dedicated to genetically monitoring Mediterranean monk seals in Greece. These recommendations make up the core of the newly-formulated Conservation Action Plan for the Mediterranean monk seal in Greece10, and should play an essential role in future conservation actions for the species in Greek waters.Based on our findings, we believe that reversing the downward spiral of population decline, inbreeding accumulation, and loss of genetic diversity while creating favorable conditions for a population increase must be priorities for conservation. We recommend that conservation actions for the Mediterranean monk seal in Greece should focus primarily on reducing anthropogenic threats, by: (a) Identifying and effectively protecting the most important pupping sites in the country, thus improving habitat quality and promoting effective dispersal and gene flow. (b) Increasing monitoring and protection of Mediterranean monk seals in the Ionian Sea to better understand their conservation status, but most importantly, to preserve their unique genetic diversity and encourage gene flow from the Ionian to the Aegean Sea. In a species so genetically impoverished as the Mediterranean monk seal, it is invaluable to maintain any genetic diversity still in existence. If population demographics permit, gene flow could also be artificially enhanced through the release of rehabilitated individuals found in the Ionian into the Aegean Sea and vice versa. (c) Reducing anthropogenic mortality , a rapid population increase would reduce genetic drift, loss of variability and inbreeding accumulation, improving the prospects for long-term survival40. Following the implementation of strict conservation measures, a close relative of the Mediterranean monk seal, the northern elephant seal (Mirounga angustirostris), was able to recover from a similar bottleneck despite severe loss of genetic diversity41, giving also hope for the future recovery of the Mediterranean monk seal. Our findings on the movement of Mediterranean monk seals out of the Northern Aegean show that the recipe of habitat protection and reduced anthropogenic mortality is a successful one. It remains upon humans to see this bigger picture and ensure that these effective conservation measures are continued and expanded to safeguard the future of the species.But, at the end of the day, is it really worthwhile investing in the conservation of a species that is already in such a precarious genetic state, and what are the chances of succeeding? The Mediterranean monk seal is just one species on the long list of pinnipeds whose genetic state has been severely affected by humans® Blood & Tissue extraction kit, following the manufacturer’s protocol. Tissue samples were genotyped using 30 microsatellite loci48 . Four loci were found to be monomorphic in our population and excluded from the downstream analysis .Eighty-six tissue samples were collected during the necropsies of Mediterranean monk seals found throughout Greece (1994–2016) and stored in 95% ethanol or DMSO at − 20 °C. Genomic DNA was extracted using the Qiagen DNeasy48 Table . Samples49. STRUCTURE was run five times for the optimal K value, incorporating 90% probability intervals for cluster membership (qi). The results with the highest likelihood L(K) and lowest variance were chosen. To obtain meaningful population units where only individuals with a high degree of membership were considered, individuals were assigned to a cluster k when qi ≥ 0.8 to that cluster and probability intervals excluded membership in alternative clusters .Genetic structure was explored using two approaches. First, to explore population structure and determine individual assignment to clusters, a Bayesian Markov Chain Monte Carlo (MCMC) clustering method was employed using the software STRUCTURE ver. 2.3.450 was performed, following the procedure described by54. We modelled spatial connectivity using the Delaunay triangulation51 and used the sPCA scree plot to visually determine the number of components to be interpreted, and Monte Carlo tests with 10,000 permutations to test for the existence of global and local spatial structure50. We ran the analysis in the R statistical environment52 using the adegenet package53.To further investigate and visualize population structure, a spatial Principal Component Analysis (sPCA)54, we tested for IBD of adult females and males using the software SPAGEDI 1.555. The program combines pairwise comparisons of estimated kinship coefficients56 and spatial distance between individual sampling locations, based on longitude and latitude coordinates. We obtained significant departures from the mean kinship coefficient of each distance class by 20,000 permutations and standard errors were estimated by jackknifing over loci55.Finally, to understand the drivers affecting population structure, and considering that in widely-dispersing species isolation-by-distance (IBD) likely affects population compositionadegenet53. Nuclear DNA diversity was measured as the number of alleles per locus (A), the observed heterozygosity (Ho) and Nei’s unbiased expected heterozygosity (He)57, and deviations from Hardy Weinberg Equilibrium (HWE) were tested using the likelihood ratio-based exact test described by58, as implemented in the R package HWXtest59. When a full enumeration was not feasible, the Monte Carlo method was used, with the cut-off value set to 109 tables.Genetic diversity parameters for each Mediterranean monk seal population cluster in Greece were calculated using the R package 60. The geographic northwest-southeast axis  were calculated in the R statistical environment52, using functions from the adegenet package .To understand the distribution of genetic diversity and the main drivers shaping it , a Hardy–Weinberg Dynamic Subsampling analysis (HWDS) was performedxis Fig. B was usehttps://www.zsl.org/science/software/coancestry)61. Initially, simulations were used to determine the power of the marker set and select the most appropriate method. The observed allelic frequencies at the actual markers analyzed in this study were used to parametrize simulations. We simulated genotypes with different levels of inbreeding between 0.05 and 1, in steps of 0.05, 100 individuals in each category. Inbreeding in simulated data was estimated using four of the methods implemented in the software COANCESTRY  and the estimates were compared with the simulated “true” values. The best-performing method was used for calculating the final empirical individual-level inbreeding estimates.Individual inbreeding was estimated using the methods implemented in the software COANCESTRY  estimatorSupplementary Information 1.Supplementary Information 2.


Scylla paramamosain (Decapoda:Portunidae) is a commercially important crustacean species in China, which is broadly distributed along the southeastern coastal regions of China. In previous studies, the population from Beibu Bay showed distinct genetic lineage. Here, we report the complete mitochondrial genome sequence of S. paramamosain from Beibu Bay. The mitogenome has 15,816 base pairs and made up of total of 37 genes , and a putative control region. The complete mitogenome in the Beibu Bay population was shorter than that in the Shenzhen population, because the non-coding region between tRNA-Glu and tRNA-His was shorter in the former, and there were 59 mutations sites between these two populations. This study adds a distinct mitogenomes of S. paramamosain and will provide useful genetic information for future genetic variation identification and genetic diversity evaluation of this economic valuable crab.The mud crab  Scylla is an economic valuable crustacean known as mud crab, which is an ecologically and commercially important species in the Indo-Pacific region , and the whole body specimens (#GQ0009) were deposited at Marine biological Herbarium, Guangxi Institute of Oceanology, Beihai, China. The total genomic DNA was extracted from the muscle of the specimens using an SQ Tissue DNA Kit  following the manufacturer’s protocol. DNA libraries (350 bp insert) were constructed with the TruSeq NanoTM kit  and were sequenced (2 × 150 bp paired-end) using HiSeq platform at Novogene Company, China. Mitogenome assembly was performed by MITObim , consisting of the usual set of 13 protein-coding, 22 tRNA and 2 rRNA genes and a putative control region. The overall base composition of the mitogenome was estimated to be A 34.8%, T 38.2%, C 16.9% and G 10.2%, with a high A + T content of 72.9%, which is similar, but slightly different from Shenzhen population (Ma et al. uebarica , as theyuebarica . The com


Daphnia has the capacity to respond rapidly to environmental stressors, to disperse over large geographical scales, and to preserve its genetic material by forming egg banks in the sediment. Spatial and temporal distributions of D. magna have been extensively studied over the last decades using behavioral or genetic tools, although the correlation between the two has rarely been the focus. In the present study, we therefore investigated the population genetic structure and behavioral response to a lethal threat, ultraviolet radiation (UVR), among individuals from two different water bodies. Our results show two genetic populations with moderate gene flow, highly correlated with geographical location and with inheritable traits through generations. However, despite the strong genetic differences between populations, we show homogeneous refuge demand between populations when exposed to the lethal threat solar UVR.The water flea  The study shows two genetic populations with moderate gene flow, highly correlated with geographical location and with inheritable traits through generations. Despite the strong genetic differences between populations, we show homogeneous refuge demand between populations when exposed to the lethal treat solar UVR.  At each reproduction cycle (clutch), the mother was transferred to a new culture bottle and the offspring were grown for a week prior to being transferred to separate culture bottles, thereby constituting the next generation. Only females were kept for the analyses.a Figure  were cola Figure . Lake Bya Figure , whereasa Figure . A totalAppendix  and grow2.2‐2 s‐1 to mimic nighttime and with addition of 150 mA UVR  to mimic solar radiation during daytime , incubated at 95°C for 20 min, vortexed for 5 s, and spun down. The supernatant was collected for genetic analyses. Extracted DNA was quantified using dsDNA HS Qubit Assay Kit .After tracking, organisms were individually frozen at −20°C. Their genomic DNA was extracted following a modified protocol from Richlen and Barber . Each in2.42, and 0.08 to 8 ng of DNA. Twenty‐six microsatellites markers were amplified using four multiplexes developed by Orsini et al. (https://www.geneious.com). GeneScan™ 600 LIZ™ dye Size Standard v2.0 (Thermo Fisher) was used as reference. Noise was removed from microsatellite dataset using a three‐step filtration pipeline by removing: (a) microsatellite markers with >30% of null alleles , (b) individuals with >30% of null alleles , and (c) monomorphic loci (1 locus). The final dataset was composed of haplotypes of 137 individuals (Lake Bysjön: 11 and Pond: 24 strains) over 15 microsatellite markers (Appendix Genetic amplifications using polymerase chain reaction (PCR) were performed in 12.5 µl final volume using a modified protocol from Colson et al.  with a fi et al.   and allele distribution were made using Micro‐Checker software version 2.2.3  and individuals (FIS), for small population size (G‐statistics), and with a stepwise mutation model approach (RST), with 1,000 permutations. The number of effective migrants (mN) was estimated between populations using codominant data and Equation Genetic diversity within and between populations was investigated using both Genetix and GenAlEx software version 6.51b2  for multiple‐step mutations was applied to the data with default settings recommended for microsatellites analyses  and 1,000 permutations. A two‐tailed Wilcoxon sign rank test , represented by 137 individuals, were genetically characterized over 15 polymorphic microsatellite loci, including 13 neutral and two adaptive markers Table .FIS_B = −0.0008, FIS_P = −0.0537) with little influence of inbreeding avoidance. There was a moderate gene flow between geographical locations (Fst = 0.12 and a Raufaste and Bonhomme correction (RH’) of 0.13) with few expected migrants (mN = 1.77). The number of individuals sampled per locus was variable in the two locations , but in sufficient number to perform a Wilcoxon sign rank test. A significant heterozygote excess was detected in the Pond  highlighting the existence of gene flow between populations.Population isolation by distance between POP1 and POP2 was inferred using pairwise differentiation indexes. There was a moderate genetic differentiation between POP1 and POP2 with a FIS_POP1 = −0.007, FIS_POP2 = −0.050, 1,000 permutations).Each genetic population was characterized by a pool of 37 (POP1) to 38 (POP2) alleles, a set of 2 to 4 alleles per locus , and few private alleles  Appendix . The allr2 > 5% threshold) were present in 45% (POP1) and 40% (POP2) of the locus pairwise comparisons with 1 to 8 random associations per locus in POP1 (5.4 ± 2.3) and 1 to 7 in POP2 (4.8 ± 1.9)  or the acquisition of genetic mutations in polymorphic microsatellite markers, which lead to an increase in heterozygosity in asexual lines , and the general adaptive marker (Locus B087) with four additional neutral loci   = −4.07, pPOP1 = 0.0001 and tPOP2(81) = −5.73, pPOP2 <0.0001) allowing individuals to flee away from the UVR source. Moreover, there was a significant difference in individual swimming speed between populations under UVR threat  = −2.73, p = 0.008; Figure t test, t(81) = −0.43 p = 0.67). There was also no significant difference in refuge demand nor in swimming speed under UVR threat in either POP1 or POP2 across generation  = 0.18 p = 0.84, F = 0.19, p = 0.83, respectively).The behavioral responses to UVR threat was investigated among individuals from the two genetic populations (POP1 and POP2). When exposed to UVR, there was a significant positive increase in swimming speed in both populations A‐B = 0.33 p = 0.74 and t(27)A‐B = −1.51 p = 0.14, respectively). Similarly, there was no significant difference in swimming speed over the genotypes Ab, Ba, Bb, Bc, and Bd  = 1.31, p = 0.86). However, significant difference in refuge demand was observed between the genotypes Ba and Bd  = 2.80 p = 0.034, Tukey's HSD, p < 0.05).Additionally, organismal refuge demand and swimming behavior during the Stimulation phase were examined over the adaptive genotypes. There was no significant difference in refuge demand nor in swimming speed between the genotypes A and B  .High population diversity with consequent admixture events was expected because of the vicinity of the two water systems (less than 3 km apart) and of the intensive commercial and recreational use of aquatic environments in the region of Scania, southern Sweden. Such patterns have previously been confirmed in, for example, fish  up to several kilometers and to rapidly and intensively colonize isolated pools . Solar radiation is a strong selection pressure that constrains diel vertical migration in Daphnia species. Their natural avoidance of UV radiation is a protective mechanism against the acquisition of photodamages . Low vaDaphnia individuals through the water column  to different threats  , principally associated with POP2 and the Pond, indicates strong selection for fish predation in POP1 and Lake Bysjön. Results are consistent with inferred swimming behavior and with the environmental history in Lake Bysjön  and the hand‐made pond (exempt of fish but affected by anthropogenic activities).A recent study demonstrated that D. magna adaption to different threats  occurred within three generations. Lineage analysis showed that 75% of the neutral multilocus genotypes and 71% of the adaptive genotypes were inherited within strains down to four generations. Deviation from expectation was associated with the occurrence of mutations and possibly with null alleles. Null alleles are the result of consistent amplification failure in PCR over a particular locus and can introduce substantial assessment errors , using an exhaustive set of adaptive markers ; Formal analysis (lead); Funding acquisition (lead); Writing‐original draft (lead). Yongcui Sha: Conceptualization (supporting); Formal analysis (supporting).Appendix S1Click here for additional data file.Appendix S2Click here for additional data file.Appendix S3Click here for additional data file.Appendix S4Click here for additional data file.Appendix S5Click here for additional data file.Appendix S6Click here for additional data file.Appendix S7Click here for additional data file.