IS values varied from 0.047 (Lavender) to 0.257 (Albino), reflecting a deficit of heterozygotes, which suggests low to moderate inbreeding levels. The genetic distances between phenotypes are low, ranging from 0.0068 (Bonaparte-Pearl grey) to 0.1559 (Lavender-Albino), unlike the strong genetic identities that reflect a strong genetic similarity between the seven phenotypes of indigenous guinea fowl studied. These results indicate the existence of a single indigenous guinea fowl population, derived from three probable parental populations, with a high within population genetic diversity . These results could be of use to conservation and improvement programs aiming at the maintenance and sustainable exploitation of this important socio-cultural and economic resource in Togo.Indigenous guinea fowl is an important animal resource for improving rural household income. In order to provide molecular data for a sustainable management of this poultry resource, an assessment of the genetic diversity and phylogenic relationships was undertaken on seven guinea fowl phenotypes from two agroecological zones (Dry Savannah and Atakora) of Togo. Genotyping was carried out using 18 microsatellite markers on 94 individuals from Dry Savannah (59) and Atakora (35) zones. The results obtained showed a high genetic diversity, with six as an average alleles per locus and an observed heterozygosity of 0.512. However, the F Numida meleagris) belonging to the order Galliformes, domesticated in West-Africa, probably in Mali and Sudan  prefectures of the two agroecological zones (Dry Savannah and Atakora) in Northern Togo . With ab –2. The human population, mainly rural, was estimated to be 985,900 inhabitants in 2013 , primer annealing at 55 °C or 60 °C (1 min) according to optimal temperature of the primers, extension at 72 °C (1 min) and a final extension at 72 °C for 15 min. An automated thermal cycler Veriti TM 96-Well Thermal cycler, Applied Biosystems was used. The amplified products generated by PCR were electrophoresed on an AB3500 automatic DNA sequencer using 8 capillaries (Applied Biosystems) at the CIRDES genotyping platform in Bobo-Dioulasso (Burkina Faso). The GeneMapper version 5.0 software (Applied Biosystems) was used to analyse the microsatellite markers electrophoretic profiles and estimate the size of the fragments  in base pairs.A set of eighteen (18) microsatellite markers were chosen for this study: six microsatellite markers  developed by FIS, FST and FIT . The analysis was performed with a burn-in period of 50,000 iterations and 100,000 Markov Chain Monte Carlo (MCMC) based on the assumption of uncorrelated allele frequencies between parental populations and an admixture model. The optimal value of ‘K’ determining the most likely number of genetic clusters was identified on the basis of ΔK, the second order rate of change of LnP(D) proposed by http://taylor0.biology.ucla.edu/structureHarvester/)  and expected (He) heterozygosities. The factorial correspondence analysis (FCA), to visualise the differences between the studied subpopulations, and the Wright’s F-statistics  and FIT  proposed and FIT  and FSTA and FIT  software and FIT . In addi and FIT . In orde and FIT . The gen and FIT . The disvester/) .All the 18 microsatellite loci selected for genotyping the populations studied were polymorphic. A total of 108 alleles were identified from the 94 animals genotyped with an average number of 6.0 ± 5.1 alleles per locus . The numFIS values significantly higher than zero  ranged from 0.203 (GUJ0001) to 0.816 (GF37), with an average of 0.512 ± 0.154, while the expected heterozygosity (He) varied from 0.226 (GUJ0001) to 0.811 (GUJ0066), with an average of 0.604 ± 0.137. With the exception of GUJ0084, GF12 and GF37, all the other loci showed a heterozygote deficit compared to the expected according to the Hardy-Weinberg equilibrium (Ho < He) with han zero . The obshan zero . Apart fFactorial Correspondence Analysis (FCA) showed a high degree of genetic similarity between the seven guinea fowl phenotypes studied  and betwFIT values ranging from −0.122 (GF37) to 0.347 (GUJ0059) with an average value of 0.171. The pairwise genetic distances between the seven indigenous guinea fowl subpopulations ranged from 0.0068 (Bonaparte-Pearl grey) to 0.1559 (Albino-Lavender). These genetic distances were inversely proportional to the genetic identities between pairs of subpopulations  of individuals from the Dry Savannah, Cluster 2 predominated individuals from Atakora (43%) and Cluster 3 included 27% and 24% of individuals from the Dry Savannah and Atakora, respectively. This structuring shows the same trend as the FCA results.The most consistent gain in information was obtained with a number of clusters K = 3 . Among tThe aim of this study was to assess the diversity and genetic structure of the indigenous guinea fowl population in northern Togo using seven phenotypes from two agroecological zones as subpopulations. The present study is the first investigation on indigenous guinea fowl population in Togo using molecular markers. All loci were polymorphic with an average PIC value of 0.556, considered to reflect reasonably informative loci . Indeed,FIS value. The Wright’s fixation indices  found in indigenous guinea fowl population of Togo were low. Similar results were reported in indigenous guinea fowl from Benin, Burkina Faso, Ghana and Sudan  means that only 1.2% of the total genetic variability could be ascribed to differences between subpopulations, suggesting a low sub-structuring of the studied population. The low difference of the genetic variability between the studied phenotypes is corroborated by the low genetic distances and the high values of genetic identity between phenotypes and between agroecological zones.A total of 108 alleles were found for the 18 microsatellite loci studied, with an average number of 6.0 alleles per locus. This value is similar to the findings of nd Sudan . The lowa posteriori grouping from the STRUCRURE software. It also appears that the indigenous guinea fowl in northern Togo seem to come from three parental populations with some individuals specific to the Dry savannah zone or to the Atakora zone and others with a very heterogeneous genetic structure. It would be interesting to include in future studies, subpopulations from other agroecological zones of Togo but also guinea fowl populations from border countries such as Burkina Faso, Benin and Ghana. It would be also interesting to investigate on the genes underlying colour variation, most notably EDNRB2, in order to understand how these genes can affect a phenotype. EDNRB2 gene was selected as the most likely candidate gene due to its functional importance in melanocyte development and would explain various phenotypes found in domesticated animals .Traditionally, indigenous guinea fowl classification is based on the colour of the plumage. The present study showed a low genetic structuring in the seven phenotypes of indigenous guinea fowl in northern Togo. It also showed a heterozygotes deficit in the overall population and in the subpopulations represented by the phenotypes. The traditional rearing system, which is particularly based on wandering, allows gene flow between subpopulations of the indigenous guinea fowl population of Togo and no specific isolated genetic group was identified among the phenotypes studied. Despite a low level of differentiation, the high heterozygosity rates obtained show that there is an important reservoir of genetic diversity in the indigenous guinea fowl of Togo. This study provides a good genetic basis for establishing genetic improvement and conservation strategies for guinea fowl in Togo. The information generated here is useful for setting up programs of selection, genetic improvement and adaptation to tropical environmental conditions in the study area, as well as for a sustainable use of the indigenous guinea fowl of Togo. Such strategies and programs should also involve the indigenous communities in order to exploit their interesting endogenous knowledge and combine them with scientific knowledge for a better exploitation and conservation of the valuable economic and food resource that the guinea fowl is to the people of the studied area.10.7717/peerj.12637/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.12637/supp-2Supplemental Information 2Click here for additional data file.


Genetic counseling services have only recently been introduced in most Arab countries, and their utilization is increasing. Prenatal genetic counseling is essential, particularly in the Arab context, which is characterized by high rates of consanguinity. Nevertheless, little is known about the decisions faced by parents and the factors underlying the complex decision making that must occur when accessing these services in Arab countries. Herein, we performed a narrative review to discuss the reported experiences of parents accessing genetic counseling in the prenatal setting in the 22 Arab countries. We also highlight the different types of decisions encountered and the factors influencing them. We report that: (i) utilization of genetic counseling services varies across different Arab countries; (ii) many factors affect decision making and service utilization, especially religion; and (iii) parents are faced with an array of decisions in the prenatal setting, partly driven by increased utilization of prenatal diagnosis and preimplantation genetic testing in some countries. Our work is the first to highlight the different factors and decisions influencing genetic counseling in Arab countries. Understanding these factors is essential for improving genetic counseling services in the region and helping counselors facilitate informed decision making. The characterization of the “Arab genome” has renewed scientific interest in its unique features and complexities . Up to 5Genetic counseling is a core part of the management of families with suspected or confirmed genetic conditions . Many ofPrenatal genetic counseling services have therefore been implemented in some Arab countries. There is relatively little evidence concerning the factors affecting delivery, evaluation, and outcomes of genetic counseling or concerning user and counselor experiences of these services in the Arab region. We therefore reviewed the types of decisions and the factors affecting decision making in the prenatal setting in the Arab context. Box 1Two independent searches of the PubMed, Embase, Scopus, and Cochrane Li-brary databases were conducted between October 2020 and December 2020. The first search included the following terms: “genetic counseling” AND “Arab/middle east” AND “preimplantation” OR “PGT” OR “PGT”. The second search included the combination of the following terms: “genetic counseling” AND “Arab” AND “pre-natal”. Only papers published in English were reviewed. The final reference list was collected based on originality and relevance to the scope of this review.Genetic counselors are needed in every health system due to their integral role in increasing knowledge and awareness about hereditary conditions in high-risk/affected individuals, their mode of inheritance, and management options. Any information provided ultimately helps patients to make informed decisions. The Arab region is characterized by large families and high consanguinity rates, with first-cousin marriages being the most common. For example, in the Kingdom of Saudi Arabia (KSA), the rate of consanguinity is 57.7%, with first-cousin marriages being the most common form (28.4%) . ConsangIn no small part due to consanguinity, the Arab region is characterized by a high incidence of genetic and congenital disease and hemoglobinopathies . Some ofBox 2What is known about this topicThere is abundant literature on the factors affecting decision making and the types of decisions made by parents accessing prenatal genetic counseling ser-vices around the world.Most of the 22 Arab countries still do not provide genetic counseling services, despite a significant need, given the elevated consanguinity rates in the region.It is known that social and cultural factors influence decision making around access to and uptake of prenatal genetic counseling services, although the fac-tors specific to the Arab world are less well documented.What this paper adds to the topicUtilization of genetic counseling services varies across different Arab countries.Many factors affect decision making and service utilization in the region, not least religion.Knowledge of these factors, especially religious drivers of acceptance of prena-tal genetic counseling services, can help to improve informed decision making in the Arab world and beyond.Prenatal options vary based on several factors, including the stage of family planning (prior to or during pregnancy) and the existence of a molecular diagnosis in the parents. Nevertheless, these options can be classified into four general themes. The first is prenatal genetic screening, such as non-invasive prenatal testing (NIPT), which is used in the following circumstances: high-risk pregnancies, abnormal fetal ultrasound findings and positive family history . Second A screening test, NIPT, offers the significant advantage of avoiding miscarriage risk associated with invasive diagnostic procedures, such as chorionic villus sampling (CVS) and amniocentesis . In NIPTIn the Arab context, the option of not having children is not always considered when parents are carriers or affected by a genetic condition. Having children is integral in family dynamics , especiaPGT usually results in successful pregnancy outcomes and is a convenient choice for people with a religious opposition to TOP. PGT is used to select healthy non-carrier embryos and, if not available, healthy carrier embryos, delivering an embedded risk of 4% of a carrier to be affected . In KSA,A cohort of Saudi students reported contradicting attitudes when TOP was discussed as an option for untreatable genetic conditions . HoweverThe decision to undergo TOP depends on the time of diagnosis. This is a crucial factor in decision making and highlights the importance of early genetic counseling for at-risk couples conceiving a child . In a coIn a study assessing the acceptance of TOP among Arab Muslim females with fetuses affected by congenital anomalies, TOP was not offered in all cases . FemalesIn the Arab world, there are many barriers to the acceptance and delivery of prenatal genetic counseling services. These include lack of national databases, scarcity of population-specific genetic information, relatively new service provision, moderate experience in delivering those services, ethical and legal considerations, and the availability of testing and interpretation of test results ,71. GeneSocial factors, such as socioeconomic status, health insurance, and accessibility of the service, are known to influence the uptake of genetic counseling services ,73,74. FIslam is the most common religion in the Arab world, so it is essential to consider how Islamic views tackle some of the issues arising in the setting of prenatal services. The most obvious example is decisions related to TOP, in which couples take into consideration the Islamic Fatwa. This states that TOP can be considered before 120 days from gestation, i.e., before ensoulment, and for severe conditions only . Some adThe literature seems to suggest that in Arab countries, religion—particularly Islam—is a common factor affecting decision making and utilization of genetic services. Therefore, Islamic beliefs must be considered when counseling Muslim couples or communities, particularly when discussing reproductive options, in order to better understand them, provide legitimacy, and support decision making . MoreoveReligious beliefs are also a significant factor affecting service utilization and decision making. Among pregnant Israeli Arabs, 92.7% of women view themselves as religious, and 40% of that cohort of women do not favor attending genetic services because they believe that counselors would be dismissive of their religious beliefs . In KSA,Culture is another critical factor that affects decision making. In a study comparing perceptions about prenatal genetic counseling between native Palestinians and American Palestinians, differences were noted across many levels. American Palestinians perceived genetic counselors as having a nondirective approach and favoring female choice concerning prenatal options in the face of disagreements between couples. By contrast, native Palestinians viewed the counselor as having a directive approach and favored male decision making . AnotherSome factors, such as the education level of the parents, have only been discussed in a few countries. For example, in Palestine, educated parents had greater knowledge about genetic disorders, which resulted in them having a positive attitude toward the genetic counselor . The impOther factors that cause low utilization of PGT include its high cost, lack of knowledge about the technology, and overall lack of information regarding reproductive options for couples ,93,94. IThere has been very little research exploring experiences of prenatal genetic counseling services from a patient perspective in Qatar. However, based on the literature and sociocultural similarities, Qatar is likely to share the same ethical and legal problems concerning prenatal services as other CGG countries, for example, the window of legal abortion (prior to 19 weeks of gestation). Prenatal genetic counseling services in Qatar are provided in the Women’s Wellness and Research Center, which belongs to Hamad Medical Corporation (HMC). The service is managed by the Medical Genetics Department of HMC. Referrals to prenatal genetic counseling services are made for several reasons, including a positive family history for a genetic disease, abnormal fetal ultrasound, abnormal screening results, and advanced maternal age.In the clinic, it is usually preferable to see couples  to ensure that a proper and equal amount of information is delivered to both parents, which can help them to reach a mutually agreed-upon decision. For example, the decision to undergo TOP might depend on the religious adherence of the couple and the severity of the condition. Prenatal genetic counseling services in Qatar are growing, as reflected by the enormous number of referrals made to HMC, the main healthcare-providing institution in Qatar. In prenatal genetic counseling sessions, patients experience different emotions when receiving a diagnosis of a genetic disease, ranging from acceptance of the diagnosis to denial. The counselor needs to have the necessary skills to address these varied and often difficult emotions.Offering a prenatal genetic diagnosis or PGT is vital for reducing the incidence of genetic conditions. One of the most important roles for the counselor is to facilitate decision making. There is currently a lack of evidence discussing genetic counseling from the legal and ethical perspective in the region. Nevertheless, the factors discussed in this review should help counselors in the region to maintain cultural sensitivity by being aware of the couple’s motivating factors and critical concerns. Decisions faced in the prenatal setting are variable and have different implications for the individual, family, and society. Thus, the role of genetic counselors is particularly critical and sensitive when facilitating decision making. Further work is needed to explore counselors’ perspectives in this region.We recommend that counselors in the region start to ask patients for feedback after the session. This can be achieved by providing patients with a questionnaire to assess, for example, patient satisfaction about the quality and quantity of information supplied, opinions about the delivered information, issues that should be discussed in future sessions, and patients’ willingness to discuss religion and its implications for service provision. Indeed, engaging religious scholars, such as imams and rabbis, to address some misconceptions related to prenatal genetic services and other healthcare issues has been implemented in the Western world ,97. This


Genetic characterization is one of the tools to assess the genetic diversity of livestock breeds towards the goals of conservation and sustainable use. This research aimed to assess the genetic diversity, population, genetic relationship, and structure of the Malaysian indigenous Katjang goat breed, which has been reported to be at risk of extinction by the Food and Agriculture Organization of the United Nations (FAO). Through assessment from microsatellite DNA markers, this breed was found to have low genetic diversity and showed evidence of high inbreeding. This breed might also have undergone population bottlenecks in the past. Through combined data analysis with other breeds and populations, available through data from published research, the Katjang goat population was found to have interconnection and form the centre of the network; it was also found to be the centroid of the multidimensional scaling plot. The findings of this research help in the understanding of the current genetic diversity of this breed and the need for its conservation.IS, was high, at 0.46. Significant (p < 0.01) deviations from the Hardy Weinberg equilibrium were noted for all loci. The bottleneck analysis using the Wilcoxon Rank test under the two-phase model of mutation was significant (p < 0.01) for heterozygosity excess, which suggested that the Katjang breed had undergone significant population reduction in the past. Through combined analysis of data from publicly available research, almost the entire population of Katjang goats represent the centroid and are grouped together on a multidimensional scaling plot, except for the Terengganu population. Network analysis revealed that the goat population from Pahang formed the centrality of the network. The Katjang goat is the only indigenous domestic goat breed in Malaysia. Following a national baseline survey from 2001 to 2002, this breed was reported to the FAO as being at risk of extinction. In this study, 36 microsatellite markers were screened, and 25 polymorphic markers were used to analyze the genetic structure of the Katjang goat breed in Peninsular Malaysia. A sample set of data derived from another 10 populations from three published research studies was used as an outgroup for an inter-population genetic study. The analysis showed that the mean value of the observed heterozygosity was 0.29 ± 0.14, and the expected heterozygosity was 0.72 ± 0.14, which indicated low genetic diversity. The inbreeding coefficient, F In Malaysia, the total goat population was estimated to be 312,571 heads in the year 2019 [Goat breeds in Malaysia can be classified as indigenous, crossbreed, and introduced or imported breeds. Among all the domesticated goat breeds available in Malaysia, the Katjang goat is the only indigenous breed . This brThe Katjang goat is also called the Kacang  or pea and bean goat due to its small size  and prefThe Katjang goat is a hardy animal that can utilize a wide range of vegetation. It is better suited to the rich bush and tree growth of the wet tropics. It can also survive on the very poor browse of the secondary jungle, on scrub, and by scavenging the village area . As an iDuring a national baseline survey of livestock breeds from 2001 to 2002, the Katjang goat breed in Malaysia was classified as being at risk for extinction  and giveIn this study, the genetic characterization of the Katjang goat was done using DNA microsatellite markers. Microsatellite markers were chosen due to their high variability, co-dominant inheritance, and relative ease of detection, making them very useful for detecting differences among populations and between individuals . MicrosaTo date, genetic characterization of the Katjang goat in Malaysia has been reported using protein loci for West Malaysia (Sabah and Sarawak) populations . Geneticn = 28), Pahang (n = 35), Johor (n = 8) and Terengganu (n = 8) . A total of 79 purebred Katjang goats were randomly sampled from various farms of four different states in peninsular Malaysia. The purebred individuals were randomly selected based on their phenotypic features, as described by historical data ,11,13. T (n = 8) . Since tDNA was extracted from the whole blood using the DNEasy Blood & Tissue Kit . The concentration and purity of the genomic DNA were analyzed using a Biophotometer , and the quality of the genomic DNA was tested using gel electrophoresis. A total of 36 microsatellite markers were used in this study. Thirty microsatellite loci were based on the list recommended by ISAG & FAO’s Domestic Animal Diversity Information System-Measurement of Domestic Animal Diversity , and ano2, and 1X polymerase buffer. PCR was done in a thermal cycler . DNA amplifications were set up with the initial denaturation at 94 °C for 5 min, followed by 35 cycles of denaturation (94 °C for 30 s), annealing (53–67 °C for 30 s) and elongation (72 °C for 30 s), followed by a final extension of 72 °C for 3 min. DNA amplifications were done using Polymerase Chain Reaction (PCR), with optimized protocol for each locus. PCR was carried out on 50 ng/µL genomic DNA in a 50 µL reaction volume using primers, template DNA (50 ng/µL), double distilled water and reagents  consisting of 200 mM dNTP, 1 unit of Taq DNA Polymerase, 1.5 mM of MgClMicrosatellite genotypings were done using the MetaPhor agarose  gel electrophoresis technique with 1X Tris borate EDTA (TBE) buffer . Electrophoresis was conducted at 70 V for two hours. Alleles were observed under ultraviolet (UV) light using the Gel Documentation System . Allele sizes generated from the microsatellite markers were estimated in comparison to 25bp DNA ladder  using the Gel Analyzer software, version 2010a . The sizIS) according to Nei [The CONVERT software, version 1.31 , was useg to Nei  for overg to Nei , was useg to Nei  and one-g to Nei . Mode-shg to Nei .A genetic distance [ST values [For inter-population genetic relationships and structure of the five populations, the data were analyzed by combining allele frequency data of other breeds and populations from publicly available data repositories and literature ,44,45 todistance  using POdistance  with thedistance  was useddistance  for popuT values . EDENetwT values , was useT values . The netT values . From the 36 microsatellite markers examined, one microsatellite locus (BM6444), which was suggested by the ISAG & FAO’s Domestic Animal Diversity Information System-Measurement of Domestic Animal Diversity , failed From the thirty-one polymorphic markers, the number of alleles observed across the polymorphic loci varied between two to fifteen alleles. Another six markers  generated low allele numbers of between two and four.According to Barker , each miThe values of observed heterozygosities ranged from 0.08 at locus TCRVB6 to 0.59 at the MAF065 locus. The mean number of observed heterozygosity was 0.29 ± 0.14, which was lower than the value of the effective heterozygosity, at 0.72 ± 0.14. All markers showed higher effective numbers of alleles than the observed numbers.IS, which indicated heterozygote deficiencies occurred in 24 loci. Overall, the mean value of FIS for the Katjang breed across the polymorphic loci was 0.46, which indicated heterozygote deficiencies in this breed. All loci significantly deviated from HWE, with a significance level of p < 0.01.All loci, except the P19 (DYA) locus, showed positive values of the inbreeding coefficient, Fp < 0.01) for heterozygosity excess, which suggested that the Katjang breed had undergone a population bottleneck. However, the test of bottleneck using allele frequency data of the mode-shift distribution produced a normal L-shaped distribution, which suggested that the allele frequency was not significantly distorted.Heterozygosity and allele frequency data were utilized for testing the possible occurrence of bottlenecks. The Wilcoxon Rank test gave the probability of 0.00171 under the two-phase model of mutation for detecting bottlenecks in this breed. The probability was significant  plot based on Reynold’s FST values  was usedNetwork analysis was used to assess connectivity of all populations and identify central populations. Microsatellite polymorphism assessment is useful to evaluate the suitability of the microsatellite panel chosen for the diversity analysis, such as heterozygosity, inbreeding estimates and genetic distances between different populations. The allele diversity obtained reflected the differences in DNA sequences and consequently reflected the genetic diversity . From thOf the other 35 microsatellite loci, four loci were found to be monomorphic in this study. Three of the monomorphic loci  were among the list of polymorphic microsatellite loci for goat studies suggested by the FAO . INRABERAccording to Barker , each miIS) suggested that this breed had high inbreeding, with the value of 0.46, which manifested in the decrease in the total number of heterozygous genotypes or an increase in the number of homozygous genotypes. This may seem to suggest that the Katjang goat breed has suffered genetic erosion due to indiscriminate crossbreeding and has been left with little genetic variation. In this study, purebred individuals could only be obtained from farms maintained by small-scale farmers, who used the purebreds only for personal use or sold them in niche markets. Therefore, there was minimum immigration and emigration occurring between farms and between populations.The genetic diversity of the Katjang goats can be assessed by the estimation of heterozygosity, which is the average proportion of individuals that exhibit heterozygous alleles in a population . The KatBottleneck analysis showed that significant heterozygosity excess was historically found in this breed; this suggests population size reduction has occurred. The test method used in this study exploits the fact that rare alleles are reduced faster than heterozygosity at the mutation-drift equilibrium during a bottleneck event . HoweverEven though the DVS Farm’s conservation herd has been subjected to inbreeding in the past decade, with no new individuals being brought into the herd to infuse genetic variation, the farm still contains the highest genetic variation as compared to the other populations. This might be attributed to the mixed original geographical distribution of the founder group of individuals initially incorporated into the herd. However, the value of this herd’s heterozygosity was still considered low. Populations from the other four states also contained low genetic variations, even though the individuals were sampled from various farms in each state.As revealed by the dendogram tree of genetic distance, the Katjang goat population from Pahang had the closest distance to Negeri Sembilan, and they showed a high level of betweenness with each other in the network analysis; thus, it is assumed they had the most amount of gene flow from all populations studied. Notably, almost all Katjang populations grouped together in the MDS plot, except for Terengganu, which can be assumed to be due to its lowest heterozygosity amongst all populations. The connectivity of the network, which was represented by the flow of genes, suggested that Katjang goats have substantial gene flow among all populations studied. However, since this breed is mostly reared by small-scale farmers, thus assuming limited gene flow in current time, it can be suggested that the Katjang goats in Malaysia were historically admixed with various populations from other countries, which influences their genetic make-up. Historical literature suggests that the Katjang breed might have entered Malaysia from India through the Mediterranean, Red Sea and Nile Delta  as well This finding also highlights that, through assessment of genetic structure and connectivity measures that may indicate gene flow among populations, there may not be positively correlation with genetic diversity. The population that emerged as the centroid of the multidimensional scaling, along with centrality of the network, might still have the lowest genetic diversity. Comparatively, even though the Katjang population showed higher betweenness in the network system, the Katjang goat exhibits the lowest heterozygosity from other studied populations ,45,54. AThrough measures of genetic relationship and structure, the Katjang goat showed the highest similarity with China, which, in accordance with the previous study of microsatellite and protein loci, suggested that there was possible dispersion of this breed from Western Asia via China . HoweverIS (inbreeding coefficient). This result might be influenced by the low availability of the purebred samples, since almost all the samples were obtained from small-scale farmers, and there was minimum genetic drift to permit exchange of genetic variation. This research also proved that a conservation herd set up with the injection of individuals from various geographical areas could lead to success in increasing the genetic variation of the population, even though the population might later go through closed breeding. This study provides insight into the genetic diversity of the purebred Katjang goat in Malaysia as well as into genetic relationships and structures. The indigenous Katjang goat in Malaysia has low genetic variation as compared to other breeds reported in various goat studies outside Malaysia. Bottleneck events might have occurred from 25 to 250 generations ago, although there was no detailed historical event that could give insight into the reduction in numbers of this breed. This breed also suffered high inbreeding as indicated by FThrough the study of population structure, it is suggested that this breed shared substantial gene flow with other breeds from various countries in the past, possibly during ancient migration of this breed into Malaysia. It is also suggested that the genetic make-up of this breed is molded by its ancient spatial originality, and it might have gone through admixture during migration and possible dispersion in the past. Although Katjang goats form the centre of the goat population network analysis and centroid of the multidimensional scaling plot, the genetic diversity of this breed is comparatively low.Since this breed is the only indigenous domesticated goat in Malaysia and has proven to adapt suitably to the local environment, having low genetic diversity is alarming and might cause possible challenges in the future if this breed is not properly maintained and conserved. In addition, with the indiscriminate practice of crossbreeding and the unknown conservation status of this breed in Malaysia, sustainable conservation is necessary to prevent this breed from further genetic erosion. Hopefully, the characterization of this breed will facilitate the conservation, improvement and maintenance of this Malaysian national asset.


Amorphophallus albus occurs naturally and is in cultivation throughout the downstream region of the Jinshajiang River in Southwest China. This species is characterised by high konjac glucomannan content, and has been cultivated in China for nearly 2,000 years. To study genetic differentiation and local adaption of A. albus, we sampled 13 distinct local cultivated populations of this species. Restriction site-associated DNA sequencing was conducted with 87 samples, resulting in 24,225 SNPs. The population structure analyses suggest two main genetic groups: one in the relatively upstream region, and one downstream. We found evidence of additional sub-structure within the upstream group, demonstrating the statistical power of genomic SNPs in discovering subtle genetic structure. The environmental and geographic factors were all identified as significant in shaping the genetic differentiation of this species. Notably, the proportion of environmental factors was larger than geographic factors in influencing the population genetic patterns of A. albus. We also discovered loci that were associated with local adaptation. These findings will help us understand the genetic differentiation of this newly domesticated species, thereby informing future breeding programs of A. albus.Domesticated species represent unique systems in which the evolutionary genomic consequences of intensive selective breeding and adaptation can be thoroughly investigated.  Understanding the genetic basis of adaptation has been a long standing goal of evolutionary biology . DomestiAmorphophallus species are characterised by a single leaf and an underground stem   . A. albu content . The cul content .A. albus is an asexually reproduced crop that can be harvested 2–3 years after planting. A. albus can propagate several cormels in one growth cycle, and farmers usually harvest swollen corms and leave cormels in the soil as the “seed” for next year   . AlthougA. albus has formed stable variations in different cultivation areas, leaving local germplasm with specific leaf colors, leaf surface spots, and content of the KGM  has revolutionised genetic studies of population differentiation and adaptive selection, making it possible to study these phenomena in a wide range of species  to generate genome-wide SNPs and conducted population genetics analyses as well as genome-environment associations (GEAs) of A. albus. The goal of our study was to: (1) describe the genome-wide genetic diversity pattern in A. albus; (2) evaluate the relative effects of geographic and environmental factors in shaping genetic differentiation of A. albus populations; (3) find genomic regions which might be associated with local adaption in A. albus. These results will shed light on the domestication history and patterns of selection during the early stages of domestication in A. albus, which could accelerate crop improvement in the future breeding programs.The agricultural domestication process, which allows for the crop’s widespread cultivation, often requires substantial adaptation, including for traits related growth, water use, and climate . A deep  the KGM . Our preribution . However species . In ordeA. albus landrace germplasms  were collected during 2016 and 2017  or 10% unidentified nucleotides (Ns). Stacks v2.3b was then used to assemble pair-ended reads and identify SNPs , expected heterozygosity (HE), nucleotide diversity (π), and inbreeding coefficient (FIS) were calculated for variant loci as well as all nucleotide positions using the “populations” program in Stacks v2.3b (FST) among 13 populations of A. albus was analysed with the “populations” program and the significance was inferred by Arlequin 3.5 with 1,000 permutations  ranging from 1 to 10, and 10 independent calculations were run for each K number. The simulations were conducted with 100,000 burn-ins as well as 500,000 Markov Chain Monte Carlo (MCMC) chains after burn-ins. The most likely cluster number was determined by the ΔK value calculated by Structure Harvester 0.6  implemented in Arlequin 3.5 was used to detect the genetic variance among genetic clusters, among populations within clusters and within populations. The significance was calculated with 9,999 permutations  and isolation by environment (IBE) were conducted. Geographic distance between each pair of populations was calculated with the geographical coordinates of each population using GenAlex 6.5 , implemented in the “vegan” package in R, to simultaneously estimate the effects of geography and environment on genomic variation . The genA. albus germplasms  with 10,000 SNPs was simulated according to parameters estimated from the real data set and used to calibrate the XtX statistics  within populations was highest in DGZB and lowest in YPHG. The largest number of private alleles was found in LBMH  to 0.352 (YFYT versus YPHG), and significant genetic differentiations were detected between most population pairs  (P < 0.05)  .K = 6 was the best supported number of genetic clusters, with the second largest ΔK value detected when K = 2 . When the populations were assigned into three groups, a higher percentage of variation (29.90%) among groups was revealed (P < 0.0001) (To quantify the genetic variance distribution in  0.0001) .The first three PCs summarised 58.52, 32.22, and 8.45%, respectively, of the total variation in 19 climatic variables downloaded from WorldClim . Both IBXtX statistics (XtX = 19.62) was robust to identify outliers under genomic selection. Through this genome scan for outlier loci, 20 outliers were detected by the XtX calibration in BayPass  . This in BayPass . GEA ana BayPass . In tota BayPass .A. albus germplasms with genome-wide SNPs. Overall, in our study the highest genetic diversity indices, such as the number of private alleles, expected heterozygosity and nucleotide diversity, were all detected in the LBMH population. Future breeding strategies may seek to give a high priority to this diverse population. We found that the genetic diversity revealed by SNPs (HE: 0.083–0.142) is lower than that evaluated from SSRs (HE: 0.294–0.487)  . While S4–0.487) , which lstimates . In ligh tackled .A. albus genetically differentiated into two geographic groups, one from the upstream region and another (Group C) in the downstream region  all suggested that populations of A. albus germplasms should be divided into three genetic groups  can be found below: YG, LT, and DD conceived and designed the study. YG, HChu, HChen, and SY collected the samples. SY and YZ carried out the laboratory work and performed the analyses. YG drafted the manuscript. All authors contributed to writing and editing and read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Olives are one of the most important fruit and woody oil trees cultivated in many parts of the world. Olive oil is a critical component of the Mediterranean diet due to its importance in heart health. Olives are believed to have been brought to the United States from the Mediterranean countries in the 18th century. Despite the increase in demand and production areas, only a few selected olive varieties are grown in most traditional or new growing regions in the US. By understanding the genetic background, new sources of genetic diversity can be incorporated into the olive breeding programs to develop regionally adapted varieties for the US market. This study aimed to explore the genetic diversity and population structure of 90 olive accessions from the USDA repository along with six popular varieties using genotyping-by-sequencing (GBS)-generated SNP markers. After quality filtering, 54,075 SNP markers were retained for the genetic diversity analysis. The average gene diversity (GD) and polymorphic information content (PIC) values of the SNPs were 0.244 and 0.206, respectively, indicating a moderate genetic diversity for the US olive germplasm evaluated in this study. The structure analysis showed that the USDA collection was distributed across seven subpopulations; 63% of the accessions were grouped into an identifiable subpopulation. The phylogenetic and principal coordinate analysis (PCoA) showed that the subpopulations did not align with the geographical origins or climatic zones. An analysis of the molecular variance revealed that the major genetic variation sources were within populations. These findings provide critical information for future olive breeding programs to select genetically distant parents and facilitate future gene identification using genome-wide association studies (GWAS) or a marker-assisted selection (MAS) to develop varieties suited to production in the US. Olea europaea L.) are one of the economically important fruit and oil trees contributing to the Mediterranean food diet. Often referred to as ‘liquid gold’ [Olives  is an NGS-based genome-wide SNP discovery and genotyping technique with cost- and time-effective characteristics . It can In the present study, we used GBS technology to genotype a collection of olive accessions assembled from the USDA-ARS National Plant Germplasm System (NPGS). The objectives of this study were to: (1) generate SNP markers by GBS technology and evaluate their characteristics; (2) determine the population structure of the USDA germplasm collection; and (3) measure the genetic relationships and sources of the genetic variations. The knowledge of the genetic diversity and relationships within and among the accessions in the US olive repository would serve as a resource for effective conservation, management, and utilization of these accessions as well as developing superior cultivars that fulfil the needs of the US market.The study comprised 90 olive accessions obtained from the National Clonal Germplasm Repository (NCGR) at the University of California-Davis (UC-Davis) along with samples of 6 regionally popular olive varieties. For efficient rooting, the basal end of the cuttings was dipped in 1000 ppm indole butyric acid  for 10 s® Plant Mini Kit  as per the manufacturer’s protocol and treated with RNase A. The purity of the DNA was analyzed using a NanoPhotometer spectrophotometer . An ApeKI restriction enzyme was used to construct the DNA libraries for the GBS. The library construction and sequencing by NovaSeq 6000  were performed in the Bioinformatics Resource Center, University of Wisconsin–Madison. For the sequence analysis, low-quality reads and adapter sequences were removed from the raw fastq files using computational pipelines developed at the Bioinformatics Resource Center (BRC) at the University of Wisconsin–Madison  using a trimming software, Skewer [https://tassel.bitbucket.io/, accessed on 23 February 2021) are shown in Leaf samples were collected from the different accessions in 2 mL centrifuge tubes and flash-frozen in liquid nitrogen. The frozen leaf tissue was homogenized to a fine powder in a Harbil model 5G-HD paint shaker  using 3 mm Demag stainless steel balls . The total DNA was extracted using a DNeasy, Skewer . The raw, Skewer . The detOlea europaea var. europaea L. reference genome [The raw reads were quality trimmed to remove the adapters and low-quality bases (Phred ≥ 20) using Skewer software . Once the genome  and genee genome . The SAMTo conduct the genetic diversity analysis, 54,075 biallelic SNP markers were finally selected based on the following filtering criteria using TASSEL 5 : missingThe summary statistics including the minor allele frequency (MAF), gene diversity (GD), and polymorphic information content (PIC) for all SNP markers were calculated using the snpReady package in .A model-based (Bayesian) method implemented in STRUCTURE 2.3.4 software  was usedAn analysis of molecular variance (AMOVA) was carried out to determine the sources of the genetic variance within and among the populations detected by the STRUCTURE software. The Poppr R package  was usedA total of 96 olives accessions were sequenced and genotyped using GBS. After filtering out the raw reads, the total demultiplexed reads for all the genotypes were 418.78 M with the average reads per accession being 4.36 M. The lowest and highest number of reads was 0.21 M and 11.35 M, respectively . After pA total of 54,075 SNP markers were selected, satisfying the filtering criteria mentioned in the Method section, to conduct a genetic diversity analysis of the 96 olive germplasm used in this study. To explain the total variability of each marker, the minor allele frequency (MAF), gene diversity (GD), and polymorphic information content (PIC) were used. Although markers with a MAF < 0.05 were removed, the average MAF value was 0.160 with a minimum of 0.05 and a maximum of 0.50. About half (~44%) of the total markers  had a MAF less than or equal to 0.1 b. The meTo understand the pattern of the genetic structure, a Bayesian clustering analysis in STRUCTURE was performed a. A popuThe principal coordinate analysis (PCoA)  agreed wThe genetic diversity among the subpopulations identified by the structure analysis and PCoA was computed using the average nucleotide diversity (π) and fixation index (Fst). The highest average π was observed for the whole population (0.246), followed by the admixture group and Pop5. The lowest genetic diversity was found for Pop6 a. We alsWe conducted a genotype-based phylogenetic analysis using the neighbor-joining method implemented in MEGA7  The genoAn AMOVA analysis was performed to understand the underlying sources of the genetic variation in the germplasm. When the accessions were divided based upon the population structure as of the first level of stratification, the results of the AMOVA indicated that the genetic variation mainly occurred within a population (67%) whereas 33% of the variation was attributed to the difference among populations . HoweverFor the minor allele frequency (MAF), gene diversity (GD), and polymorphic information content (PIC), the admixture group had the highest MAF, GD, and PIC values overall, followed by Pop5 and Pop7, among the populations. In contrast, Pop6 had the lowest value for each of the characteristics. The remaining Pop1, Pop2, Pop3, and Pop4 had almost similar MAF, GD, and PIC values .Olea europaea L. originating from 18 countries across four climatic zones.Knowing the genetic variability in the collection of the available pre-adapted olive genotypes is a prerequisite for the US olive improvement program. Despite the availability of genetic studies of numerous European or Mediterranean germplasm accessions, little is known about the population structure or genetic diversity of the existing USDA collection of olives. An accurate molecular documentation is critical for germplasm curators, breeders, and geneticists as well as plant pathologists. The olive germplasm collections at several centers have been largely influenced by natural dissemination and human migration as well as multilocal selection, breeding, and propagation ,44,45. TOlea europaea cv.; “Farga” (version Oe9) [The average number of 3.99 million sequence reads per sample obtained in this study was much higher than other similar studies of olives ,11. We oion Oe9)  to a pubTransition SNPs were more available than transversions, consistent with the previous studies of olives and other plant species ,49,50,51The population structure analysis revealed seven subpopulations (K = 7) with admixture groups in the US olive germplasm collection based on a 0.70 membership probability. This analysis coincided well with the PCoA and genotype-based phylogenetic analysis. The structure analysis outcomes of the study were similar to a previous study , which iThe US accessions were grouped into Pop1, 3, 4, 6, and 7 as well as the admixture group. Interestingly, most accessions of these subpopulations were from temperate and dry climatic zones, signifying the restrictive selection of the accessions in these regions. Similarly, the accessions from the non-native areas  were grouped into the admixture groups with accessions from the Mediterranean regions . It may be due to a shared ancestry, outcrossing genotypes from diverse backgrounds, or selection pressure to adapt to the local environment.p ≤ 0.001) genetic variation existed among and within the subpopulations. Although most of the genetic variation (67%) came from within the population, among the subpopulations was the source of a 33% genetic variation. After including the climatic zone as a sub-source of the genetic variation, the climatic zones within the subpopulations accounted for 26 and 4% of the genetic variation among the populations. In both AMOVA analyses, the genetic variation within the subpopulations was the highest and the geographical location also explained a major source of genetic variability. It is believed that the domestication of the olive started 6000 years ago in the eastern Mediterranean basin, from where it migrated to different regions across the world during classical civilization [The Fst values describe the genetic differentiation between two subpopulations . This stlization ,56. The lization ,55. In tThis study used high-throughput GBS technology for SNP genotyping to explore genetic diversity and structures in the olive accessions within the US repository. The study identified and examined the features of the SNP markers to facilitate future efforts of US olive genetic improvements. The SNP markers performed well in terms of the polymorphism, genetic diversity, and population structure analysis. A total of 33% of the total SNPs used in the study showed half of their maximum PIC value (0.5), indicating their suitability for future marker-assisted breeding. Overall, our germplasm was genetically diverse with seven subpopulations. This genetic diversity could be helpful for future breeding programs through the selection of suitable parents for developing new olive cultivars with desirable agronomical characteristics adapted to US climatic challenges. The seven subpopulations identified in this study did not align with the geographical origin or climate zones, possibly due to regional selection and domestication. The subpopulations Pop5 and Pop7were genetically diverse whereas Pop6 was less diverse. This information could be helpful to select parents from various groups to widen genetic diversity during olive improvement and breeding programs. The overall findings of this study will help to conduct genetic mapping, association mapping, genomic selection, and marker-assisted breeding.


Rhipicephalus sanguineus group encompasses at least 12 validated species of Palearctic and Afrotropical hard ticks, which are relevant in veterinary medicine and public health. The taxonomy of R. sanguineus s.s., has been particularly intensely debated, due to its wide geographic distribution, morphological variants, parasite-host associations, and its capacity and vectorial competence for the transmission of several pathogens. By sequencing mitochondrial markers, it was possible to identify the existence of multiple lineages, among which the Tropical and the Temperate lineages stand out, particularly in America. However, the northern limit between these lineages is not clear due to the lack of extensive sampling across Mexico. For this reason, the aim of the present study was to determine the genetic diversity and structure of the R. sanguineus group in Mexico and to compare it with the populations reported in the Americas, in order to propose the northern limit of the R. sanguineus Tropical lineage and the potential regions of sympatry with R. sanguineus s.s. The findings of this study now confirm the presence of R. sanguineus s.s. in Mexico, showing a subtle genetic structure and high genetic diversity throughout its distribution in the Americas. In contrast, the Tropical lineage seems to be genetically less diverse in its overall distribution in the Americas. The genetic diversity of these two independent lineages could have important epidemiological implications in the transmission of tick pathogens.The  Rhipicephalus sanguineus group encompasses at least 12 validated species  of Palearctic and/or Afrotropical hard ticks widely distributed worldwide by several historical colonization processes, which exhibit a high relevance in veterinary medicine and public health due to direct damage caused by infestation in the hosts and by the transmission of pathogens  and CMT-30 [MZ618826]), four sequences from the US , and two from France (MH630343 and MH630344). The second group was formed by two sequences from Brazil (MF477856 and KX533942), Argentina (KU498302 and MT152825), and Chile (MW509950 and KX632155), as well as the single sequence from Uruguay (GU553084) [Finally, with the Nei’s distances, the group of U553084) .R. sanguineus group from the Americas. A total of 156 (77.3%) corresponded to the Tropical lineage and the remaining 46 records (22.7%) were from R. sanguineus s.s. The Tropical lineage is widely distributed across the Neotropical Region; meanwhile, R. sanguineus s.s. is restricted to North America, including the US and the US-Mexico border, and is also present in the South American Cone, where it is restricted to Argentina, Chile, and Uruguay , the cytochrome b (cytB), or the second internal transcribed spacer of the nuclear ribosomal gene cluster (ITS2) to further characterise the subpopulations [Specifically, this gene fragment revealed subtle genetic differentiation across the distribution of both lineages. Regarding ulations ,33,34.R. sanguineus s.s. were lower than those from the Tropical Lineage, when these two lineages were compared with the haplotype network, R. sanguineus s.s. seems to be more diverse than the Tropical lineage, with six haplotypes of the total 10. This was also supported by the genetic distances, where a subtle genetic structure could be shown within this species in North and South America. This obvious genetic diversity could be due to an isolation-by-distance effect or by two independent colonization events, that may have been influenced by multiple factors related to radiation across several new hosts and climate niche partitioning, as was recently proposed by Bakkes et al. [Although the numbers of available sequences from s et al. . HoweverR. sanguineus s.s., the use of the 16S rDNA gene fragment seems to be less informative for the Tropical lineage, containing only four haplotypes and without an obvious genetic structuring. However, it is important to note that in this case, a single haplotype (H2) concentrates most of the diversity and could be inferred as the differentiation centre, since all mentioned countries are included in this haplotype. This has been proposed for other biological systems, with some unique haplotypes from South America (Colombia and Brazil) as tip haplotypes [Amblyomma maculatum within the US [Contrary to the information obtained with plotypes ,36,37. Tn the US .R. sanguineus group to transmit, for example, E. canis in Brazil [This differentiation might be epidemiologically relevant due to variability in the vectorial capacity and the competence of some of the populations of the n Brazil .R. sanguineus s.s. and that of the Tropical lineage. In the Northern limit, the two sympatric locations for both lineages are highly crowded border crossings that allow a constant exchange between the populations of R. sanguineus of the US and Mexico through the movement of domestic animals.Based on the biogeographic regionalisation, no correlation could be evidenced between the presence of R. sanguineus s.s. and Tropical linage only share the Chacoan province in Argentina. In this context, the use of the distribution proposed in this study must be applied with caution until future efforts allow us to complete the distribution analysis in this area.On the other hand, in South America, where a more complex biogeographic regionalisation is found, due to the complexity of the biota and fauna, R. sanguineus s.s. corresponds to the previously known “Temperate Lineage”, containing a subtle genetic structure and high genetic diversity throughout its distribution in the Americas. In contrast, the 16S rDNA region seems to be less informative for inferring the genetic diversity of the Tropical lineage in its overall distribution in the Americas. Yet caution is warranted, since the analysis of the 16S rDNA gene fragment does not permit us to untangle the phylogenetic taxonomy of this complex group formed by other Palearctic and/or Afrotropical species, such as R. turanicus, R. sanguineus Southeast Lineage, R. guilhoni, R. leporis, and R. camicasi. To conclude, the findings of this study confirm that R. sanguineus group in the Americas, showing different biogeographical distributions. Finally, the genetic diversity shown by these two independent lineages could have important epidemiological implications in the transmission of tick pathogens.We also now propose the first fine scale map with the boundaries of these two lineages of the Rickettsia Surveillance’, we received brown dog ticks (R. sanguineus group) from the National Network of Public Health Laboratories of the Mexican states of Aguascalientes, Baja California, Campeche, Chihuahua, Coahuila, Guanajuato, Guerrero, Hidalgo, Morelos, Oaxaca, Puebla, Sonora, Veracruz, and Yucatan. Additionally, we analysed several ticks from previous tick-borne surveillance studies carried out in Chihuahua, Sonora, and Veracruz [As a part of the ‘National Program for Veracruz ,39,40. WVeracruz : the Nea® tube and cut into small pieces using a sterile scalpel blade for each specimen to avoid cross contamination between tick samples. The samples were then macerated with a 1.5-pellet pestle.All ticks were fixed and preserved in 70% ethanol and morphologically identified with an Olympus SZX7 stereoscopic microscope, using the specialised taxonomic keys of Keirans and Litwak , Direcci®, implementing the “Purification of Total DNA from Animal Tissues (Spin-Column Protocol)”. For molecular identification of the tick species, a segment of approximately 400 bp of the mitochondrial 16S rDNA gene was amplified in all samples, using the primers 16S + 1/16S − 1 and the cycling conditions reported by Norris et al. [® Green Master Mix, 2X Promega Corporation , the pair of primers (100 ng each), 6.5 μL nuclease-free water, and 30 ng DNA in a final volume of 25 μL. Negative (reaction mix without the DNA template) and positive  controls were included. The PCR products were resolved in 2% agarose gels using SmartGlow™ Pre-Stain of Accuris Instruments®  and visualised by UV-transillumination. Purified amplification products were submitted for sequencing at Macrogen Inc., Seoul, Korea.The DNA extraction was carried out using the Qiagen “DNeasy Blood & Tissue Kit”s et al. . The reahttps://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome). Recovered sequences were edited using FinchTV 1.5 , conducting a visual inspection. The final sequences were compared to each other to identify inter/intra species variation, using the BLASTn tool from NCBI (16S rDNA structure, using the Q-INS-i algorithm in the MAFFT version. The set of sequences included all available sequences of R. sanguineus s.l. from the Americas in GenBank, with the sequences of the neotype of R. sanguineus from France. Following the recommendations of Guglielmone et al. [Rhipicephalus sanguineus group, R. turanicus, R. guilhoni, R. leporis, R. camicasi, and R. sanguineus tropical lineage from the Australasian, Oriental, and Afrotropical regions according to Chitimia–Dobler et al. [R. sanguineus s.l. clade III [according to Nava et al. [Rhipicephalus microplus, as an outgroup. The complete list of these sequences and their geographical distribution are presented in We then performed multiple alignments according to the secondary structure of the r et al. , Hornok r et al. , and Slar et al. . AdditioTwo phylogenetic analyses were performed, and the best substitution model was evaluated differentially, following the recommendations by Colunga-Salas and Hernández–Canchola . The firmrbayes models and the greedy scheme search [The second approach was performed using MrBayes 3.2 . To calce search ,53 were e search .R. sanguineus Tropical Lineage and R. sanguineus s.s. from the Nearctic and Neotropical regions, without the outgroup. To provide a more detailed account of the genetic diversity and to detect possible populations and subpopulations in the R. sanguineus Tropical Lineage and R. sanguineus s.s., an ordination PCoA of the eigenvalue scores was performed in PAST 4.04 [For all following analyses, we only used the multiple alignment of the AST 4.04 , based oAST 4.04 .adegenet R package [Haplotype networks with mutational step estimations were constructed using the median-joining network in POPART 1.7 . Additio package , conside


Here, we present the first high-resolution characterisation of both subaerial and submarine components of the collapse. Combined Synthetic Aperture Radar data and aerial photographs reveal an extensive subaerial failure that bounds pre-event deformation and volcanic products. To the southwest of the volcano, bathymetric and seismic reflection data reveal a blocky landslide deposit (0.214 ± 0.036 km3) emplaced over 1.5 km into the adjacent basin. Our findings are consistent with en-masse lateral collapse with a volume ≥0.175 km3, resolving several ambiguities in previous reconstructions. Post-collapse eruptions produced an additional ~0.3 km3 of tephra, burying the scar and landslide deposit. The event provides a model for lateral collapse scenarios at other arc-volcanic islands showing that rapid island growth can lead to large-scale failure and that even faster rebuilding can obscure pre-existing collapse.As demonstrated at Anak Krakatau on December 22 The authors here present a detailed reconstruction of the landslide mass following the 2018 eruption of Anak Krakatau. Bathymetry data reports the volume of the collapsed submarine flank to be much larger than previously reported. Detailed flank collapse reconstructions are key to understanding their mechanisms, timing and associated tsunami generation, which are essential to better identify the factors controlling failure and improving global monitoring of volcano instability hazards. Although characterising volcanic island flank collapses in the geological record (often 10 s kyr old) and numerically modelling their tsunamis have provided valuable insights into tsunamigenesis8, there are uncertainties towards determining pre-event bathymetry, precise volumes, failure and emplacement processes, island geometry and tsunami magnitudes are all subject to uncertainties4. As a result, forecasting collapses and mitigating their associated cascading hazards are a major challenge5. The December 22nd, 2018 flank collapse and tsunami at Anak Krakatau , lack accurate pre-collapse edifice and seafloor data, and have only limited tsunami observations, which restricts the resolution of numerical tsunami modelsHere, we first provide new evidence of processes that preconditioned the 2018 failure. Using comprehensive, time series of satellite Synthetic Aperture Radar (SAR) images, field observations and aerial photographs we then define a new geometry of the subaerial component of the collapse scar. This is applied to a digital elevation model (DEM) of a pre-slide Anak Krakatau to provide a more accurate volume calculation of the subaerial landslide. We then present results from a new high-resolution seafloor and sub-seafloor hydroacoustic survey offshore Anak Krakatau, completed in August 2019, which provides a comprehensive view of the submarine landslide deposits. We compare these data to pre-event hydroacoustic data to resolve a total landslide volume, and insights into the submarine landslide emplacement dynamics.5. Strombolian ejecta were distributed equally on all flanks of the pyroclastic cone, but restricted to within the confines of the mapped failure area  during the period of flank collapse. However, a near-daily time series of constellation SAR images ) captures the major topographic changes at Anak Krakatau  CSK SAR image from December 23rd, 2018, is a bright, curvilinear feature and is consistent with a 0.18 km13 gives a subaerial failure volume of 0.098 ± 0.019 km3. There is some uncertainty in the subaerial gradient of the failure plane, which from the CSK imagery is estimated at 30–40°, although up to 61° has been suggested5. The subaerial volume estimate is similar to other interpretations (e.g. 0.094 km3) that extend the failure plane east of the pre-collapse cone13, but between two and twenty-two times larger than estimates where the failure plane limit is interpreted further to the west29.Comparison of the newly-mapped subaerial failure area with a pre-collapse 2018 DEM11 has a lower (100 m) resolution than the 2019 post-event survey (5 m). From it we interpret a simple, steep-sided pre-collapse caldera basin, with the steepest slopes adjacent to the SW flank of Anak Krakatau. There are no landslide blocks on the seafloor SW of the volcano  and maximum (deeper-seated) failure scenario can be constrained (Supplementary From the available marine data we suggest two possible scenarios for the failure geometry of either: (1) a shallow failure propagating to a maximum −100 to −120 m water depth, which is limited to the depth of the intra-island shelf and slope parallel features on the SW flank (Supplementary 14). Subtraction of the failure surface from a combined 1990 bathymetry and 2018 DEM topography yielded a shallow landslide volume of around 0.175 ± 0.015 km3  and 0.313 ± 0.043 km3 (deep-seated failure scenario)  that 0.098 ± 0.019 km3 of this volume was from the subaerial edifice. This means that the subaerial edifice contributed at least 45% of the landslide volume, with the remainder sourced from the submarine flank as well as bulking by incorporating seafloor material and expansion during transport.Subtracting the post-collapse blocky deposit surface from the 1990 bathymetry gives a volume of 0.236 km3 , buried beneath the post-collapse sediments observed on seismic reflection profiles Figs. . We inte3 Fig. . We suggOn the 2019 bathymetry there is an older, blocky deposit N of Rakata Island not related to the recent landslide. The landslide blocks are similar in dimensions to those of 2018, but show extensive gullying, assumed to be from erosion by strong bottom currents, known to be present in the caldera. They contrast with the ‘fresh’ appearance of the 2018 landslide blocks Fig. . These b32. Although the 2018 flank collapse reduced the area of Anak Krakatau from 3.19 km2 to 1.7 km2, volcaniclastic deposits from the Surtseyan post-collapse eruptions led to the island exceeding its pre-collapse area within weeks, reaching 3.27 km2 33. An interpolated submarine thickness  of post-collapse eruptive deposits across the caldera basin gives a deposit volume of 0.154 ± 0.023 km3. Post-collapse submarine deposits must also have infilled much of the scar on Anak Krakatau’s submarine flank, enabling the subaerial island to extend west of the collapse scar during the regrowth period, eventually enclosing the vent, and also resulting in the submarine failure scar being largely obscured, as described above. Thus, from the volume of landslide deposit, less the subaerial collapse component, we estimate that the submarine scar volume (and thus the infilling sediment volume) is ~0.116 ± 0.025 km3. In a previous study, the subaerial volume of post-collapse pyroclastic materials deposited on the island was calculated at ~0.029 km3 13. From these volumes, we estimate a present total post-collapse eruptive volume of 0.299 ± 0.05 km3.The acoustically transparent or chaotic 2018 debris flow deposit in the caldera basin (lower unit) is blanketed by a parallel-bedded (upper) unit, similar in seismic character to the pre-collapse caldera infill. We interpret this upper unit to be volcanic sediment generated between the failure in December 2018 and the survey in August 2019 by the vigorous post-collapse activity Fig. . HoweverInterpretations of satellite SAR backscatter for Anak Krakatau have implications for positioning the 2018 failure plane, determining the failure plane geometry, and calculating the subaerial landslide volume. During the event, eruption plumes Fig.  made dis3, Supplementary 3, Supplementary 3 , that bulked by sediment erosion and expansion during transport to form a deposit volume of 0.214 ± 0.036 km3. Because elements of submarine failure geometry cannot be constrained, it is out deposit volume that provides the best guide to the total collapse volume, with the lowest uncertainties.From our two end-member failure plane geometries, the deep-seated scenario  modelled a range of landslide volumes (0.22–0.3 km3) for the Anak Krakatau flank collapse and found that a 0.27 km3 source volume was most consistent with the recorded tsunami on the surrounding islands and coasts. Based on a finer-resolution grid simulation, these authors showed that the tsunami observations could also be explained by a shallower failure surface and a 0.22 km3 collapse volume31. Our new volume calculation is close to the lowest end of this volume range, similar to the authors’ revised model. The landslide volume presented here is also supported by tsunami modelling estimates in other studies37; thus, indicating that our mapped landslide volume can explain the recorded tsunami without the need to invoke any additional source mechanisms.Ye et al. (2019) calculated the landslide volume independently from seismic wave inversion methods to be up to 0.2 km39. This 2.4–4.2 km3 volcanic collapse was highly erosive, with the distal deposits dominated by remobilized seafloor sediment39. Other volcanic island flank collapses, such as the 165 ka Icod landslide on Tenerife40, disintegrate much less rapidly, forming block-rich deposits with strong elements of cohesive flow. Here, the collapse formed tongue-shaped debris lobes with large blocks rafted towards its periphery39. Alternatively, as at the 15 ka El Golfo landslide on El Hierro42, even less block interaction and disintegration formed of randomly scattered large blocks spread across a broad, fan-shaped, debris apron. Finally, slides, such as Nu’uanu and Wailau on Oahu and Molokai islands, Hawaii, comprise much larger, kilometer-sized, blocks that did not disintegrate or scatter, but fractured and translated directly downslope, forming coherent block trains44.The submarine and satellite observations illuminate the landslide emplacement processes and make the 2018 Anak Krakatau flank collapse dataset the most complete volcanic landslide-tsunami yet studied. Previous volcanic island flank collapses display a variety of mechanisms and emplacement dynamics. That of Ritter Island, 1888, resulted in a highly disintegrative mass, that transformed into debris flows running out for 70 km5 of 90 s implies emplacement velocities of at least 16.5 m s−1; this is consistent with modelled slide velocities that range from 12 to 45 m s−1 45. The Anak Krakatau deposit has morphological similarities with the  Nu’uanu and Wailau landslides, with two important implications. First, that deposits are dominated by coherent, large blocks, with almost purely translational transport and relatively short travel distances into the adjacent basin, can originate via rapid en-masse failure and emplacement, and are an efficient tsunami mechanism. Second, that the style of fragmentation and transport may be difficult to predict and, at island-arc volcanoes, are just as varied as those observed in ocean-island settings, albeit on a smaller scale.The 2018 Anak Krakatau landslide deposit is dominated by large  angular blocks, distributed in distinctive block trains. These blocks travelled only 1.5 km into the basin with limited erosion Figs. , and yet45. Although a superficially similar edifice, the pattern of disintegration at Anak Krakatau is in strong contrast to Ritter, suggesting that the bulk properties, fragmentation and pre-collapse pattern of deformation preconditioning failure, may not be easily predictable. Nevertheless, for Anak Krakatau, assuming either a simple dense Newtonian fluid or homogeneous granular slide rheology, numerical models can reproduce, the near- and far-field tsunami observations from our estimated failure volume46. While translational subaerial landslides are reported at other volcanic island sites, the detailed pre- and post-event surveys at Anak Krakatau and direct monitoring of the tsunami makes this an important benchmark event.Here, we propose a dichotomy between translational slides, represented by the event at Anak Krakatau, and disintergrative and long-runout landslides, such as at Ritter in 1888. In many respects, the Anak Krakatau edifice is comparable to Ritter, being dominated by well-bedded mafic volcaniclastic deposits with interbedded lavas. At Ritter, however, this clastic failure mass appears to have been relatively weak and disintegrated rapidly, forming a highly mobile flow47. While the Anak Krakatau debris flow unlikely contributed to the tsunami50, it illustrates the potential for disruption of the seafloor beyond the termination of the primary landslide.The Anak Krakatau landslide also resulted in an associated (now buried) debris flow Figs. , 8, whic5, and our observations suggest that this activity preconditioned failure by loading the SW flank with lavas and Strombolian ejecta  using high-resolution marine survey and satellite data. This is the first event volume estimated from the deposit, rather than from the projected failure plane, which is subject to greater uncertainties. Our landslide volume includes 0.098 km3 (45%) from the subaerial failed edifice, and thus implies 0.116 km3 (55%) is derived from the submarine flank. This favours a relatively shallow failure plane that cuts the submarine flank at −110 to −120 m water depth.We identify volcanism, fumaroles and faulting that preceded, and could have preconditioned, the flank collapse, which highlights processes that could be monitored at this and other islands to indicate potential future events. Our identification of the scale, geometry and emplacement of the 2018 Anak Krakatau flank collapse is important to understanding flank collapses at other volcanic islands, thereby underpinning improved numerical tsunami modellingThe landslide triggered a period of intense volcanism that generated a greater volume of material than was lost during the flank collapse, but that 90% of those materials were deposited offshore. Furthermore, the active modification of the seafloor during this time highlights that major volcanic environments can remain highly dynamic months after a flank collapse and illustrating the potential for collapse to initiate vigorous eruptive activity, which then rapidly obscures evidence of the collapse itself. This has implications for how easily we may identify, or not, evidence of prehistoric volcanic collapses in general.52. The flank collapse of Anak Krakatau is the first recent volcanic island lateral-collapse tsunami observed by modern instrumentation, in particular, from a post-caldera collapse cone. It is also the first event to be studied by state-of-the-art, multiple disciplines, at high-resolution . It therefore provides another event, like Mt St Helens, that can form the basis for a similar step-change in understanding the mechanisms and hazards from rapid volcanic-island growth and destruction.The May 18th, 1980 Mt St Helens flank collapse and eruption resulted in a step-change understanding of explosive lateral blasts, edifice collapse and debris-avalanche emplacement processes54. True-colour  images showed the addition of lavas and outbuilding of the island, as well as the presence of steam venting and ash clouds , ALOS-2, TerraSar-X and Cosmo-SkyMed (CSK) acquisitions spanning the collapse. We geocoded the images using a DTM clipped to the new island shoreline found from satellite imagery. SAR backscatter depends on (1) local slope angle relative to the satellite look direction, (2) roughness of the reflecting surface with respect to the incident radar wavelength and (3) dielectric constant of the surface . It is therefore sensitive to both major changes in topography and surface cover. The CSK SAR image was acquired in Spotlight mode on December 23rd, 2018 (10:28 UTC) with a descending geometry at pixel spacing of 0.3 m in range direction and 0.7 m in azimuth direction Fig. . This imThe flank failure on December 22nd, 2018 left a steep cliff with a summit that has a high backscatter contrast associated with a change in local slope Fig. . The faiThe NW side of the collapse scar correlates with the part of the scar that we can see in the oblique aerial photos Fig.  and videAerial photographs during the eruptions that followed the December 22nd, 2018 flank collapse on Anak Krakatau allow us to validate our interpretations of the SAR data by identifying distinctive physical features to act as tie points Fig. . These p5).We constrain the slope angle of the subaerial failure plane from combining observations from satellite imagery and DTMs. The main scar surface has been reconstructed by creating a post-collapse DTM whose shorelines have been identified on the CSK image and whose elevations have been taken from the pre-collapse topography reconstructed in Gouhrier and Paris (2019). We estimate the slope of the collapse scar by interpolating between sea level at the new shoreline and the updated summit of the scar (max 150 m a.s.l.). The resulting sliding plane for the subaerial failure has a slope angle up to 60 degrees with median value of 35 degrees, comparable to alternative estimates  of the Krakatau caldera basin by the R/V Baruna Jaya III (Supplementary Fortuna was equipped with a Teledyne Reson T20-P multibeam echosounder (10–160° swaths) and R2Sonic 2026 bottom detection range finder (6 mm resolution). A Teledyne TSS-DMS 05 motion sensor was mounted on the multibeam echosounder transducers for post-processing corrections of vessel heave. Navigation was recorded from a Trimble SPS 462 GPS and data logger in a Hydropro data format. Track line artefacts were removed or smoothed in CARIS software. Digital data was analysed using ESRI ArcGIS software.The post-event bathymetry of the Krakatau islands, including the Krakatau caldera basin was collected 12–19th August 2019 as part of a NERC-funded Urgency Grant  and EG & G triggered capacitor bank (model 231). The system operated with a 1200 Kva power source and fired at a 500-millisecond rate with a 250-millisecond sweep rate. The system released energy in 400–600 Ws−1 pulses with a frequency range of 300–10000 Hz. A neutrally buoyant streamer (EG & G eight-element hydrophone) was deployed 5 m from the acoustic source and 23–28 m behind the vessel. Signals received by the streamer were channeled to a Khron Hite 3700 band pass filter (30–3000 Hz) and then recorded using Chesapeake SonarWiz Shuttle with SonarWiz 4.0 recording software. SEG-Y data was analysed in Petrel software. The 2017 seismic reflection survey was collected under the same parameters but with a 360-millisecond sampling rate. The vertical resolution is <10 m, in the range 4–6 m with the working frequency towards the lower end of the range. The lateral resolution is 2 m based upon shots every 500 ms and a boat speed of no more than 4 knots. Lateral resolution away from the central track line suggests than features as great as 80 m away may influence the seismic reflection profile, potentially imaged as sideswipes.The 2019 seismic reflection survey of the Anak Krakatau landslide deposits was completed from 20th August to 3rd September 2019. Here, a high-resolution Sparker methodology was applied. Acoustic pulses were generated by a multi-electrode Sparker using an 5. The 2018 DEM was combined with the existing 1990 bathymetric survey to provide a pre-collapse surface from which two subaerial-submarine geometries were modeled. These scenarios were limited laterally by ridges to the W and SE and then (1) slope features at −100 to −120 m, and (2) base of slope at −230 m. Failure surfaces were then contoured to provide appropriate concave spline fits between a steep and shallow end-member for each scenario.The new extent of the subaerial tip-line of the failure plane was applied to an existing pre-collapse 2018 DEM of Anak Krakatau to delineate the volume of the subaerial slide. The slope angle of the failure plane was limited by the new post-collapse coastline (slope angles 30–40°) and from calculations of the dip-angle of the shear failure (~60°)With a pre-event 1990) and post-event (2019) bathymetric dataset it is possible to perform a volumetric analysis of the submarine landslide deposits in the caldera basin. There are resolution differences between the 1990 and 2019 bathymetric surveys that are considered  to depth. Modern deposits represented by those in the caldera basin, especially volcaniclastic sediments, likely have varied consolidation and may have equally varied seismic P-wave velocities. Furthermore, the landslide blocks are intact and comprise older volcanic strata that may have much higher seismic P-wave velocities compared to unconsolidated sediments. Measurements of P-wave velocities in materials from the accretionary toe in the Nankai trough, which may be comparable to the compressive nature of deposits here range from 1600 to 2000 m/s−1 (average 1500 m s−1). This horizon was compared to seismic reflection profiles to estimate the amount of sedimentation between 1990 and 2018. This method was also applied to convert the TWT of the first reflector in the 2017 seismic reflection profiles to depth to provide estimates of pre-event basin depths.To convert the 1990 bathymetry to TWT, in order to compare profiles of the bathymetry to the seismic reflection profiles required depth to be converted only using the P-wave of seawater between 1440 and 1570 m s8 and regional reviews and report summaries57. These were georeferenced and digitised to generation new DEMs of Anak Krakatau for the available past surveys. Archived photographs were also collated for Anak Krakatau, including several from the National Gallery of Australia that show evidence of past mass wasting on the island. Topographic maps (Fig. 29. While attributed to wave action at the time, the scale of the topographic change and reduced awareness of flank collapses in 1949 may mean that this event should have been attributed to a landslide. Photographs of Anak Krakatau in 1950 (Fig. Topographic survey data was collated from publications of the Bulletin of the East Indian Volcanological Survey (Supplementary aps Fig.  show a paps Fig. , whereby950 Fig.  show sim950 Fig. , which pSupplementary InformationPeer Review File