Determining the value of livestock breeds is essential to define conservation priorities, manage genetic diversity and allocate funds. Within- and between-breed genetic diversity need to be assessed to preserve the highest intra-specific variability. Information on genetic diversity and risk status is still lacking for many Creole cattle breeds from the Americas, despite their distinct evolutionary trajectories and adaptation to extreme environmental conditions.A comprehensive genetic analysis of 67 Iberoamerican cattle breeds was carried out with 19 FAO-recommended microsatellites to assess conservation priorities. Contributions to global diversity were investigated using alternative methods, with different weights given to the within- and between-breed components of genetic diversity. Information on Iberoamerican plus 15 worldwide cattle breeds was used to investigate the contribution of geographical breed groups to global genetic diversity.Overall, Creole cattle breeds showed a high level of genetic diversity with the highest level found in breeds admixed with zebu cattle, which were clearly differentiated from all other breeds. Within-breed kinships revealed seven highly inbred Creole breeds for which measures are needed to avoid further genetic erosion. However, if contribution to heterozygosity was the only criterion considered, some of these breeds had the lowest priority for conservation decisions. The Weitzman approach prioritized highly differentiated breeds, such as Guabalá, Romosinuano, Cr. Patagonico, Siboney and Caracú, while kinship-based methods prioritized mainly zebu-related breeds. With the combined approaches, breed ranking depended on the weights given to the within- and between-breed components of diversity. Overall, the Creole groups of breeds were generally assigned a higher priority for conservation than the European groups of breeds.Conservation priorities differed significantly according to the weight given to within- and between-breed genetic diversity. Thus, when establishing conservation programs, it is necessary to also take into account other features. Creole cattle and local isolated breeds retain a high level of genetic diversity. The development of sustainable breeding and crossbreeding programs for Creole breeds, and the added value resulting from their products should be taken into consideration to ensure their long-term survival. Today, it is estimated that throughout the world, there is one cow for every five people. The most recent report of the Food and Agriculture Organization (FAO) on farm animal genetic resources  illustraThe risk status of each breed is defined on the basis of both demographic and cultural aspects . For exaDetermining the conservation value of breeds is essential to define priorities, including allocation of funds, and to manage extant genetic diversity. Several methods based on the establishment of conservation priorities have been developed to maintain high levels of neutral genetic variation ,12. The Notwithstanding these limitations, it is important to consider that the genetic diversity of a livestock species results from the genetic variation both within- and between-breeds in order to preserve the highest intra-specific variability ,12,24. WGiven the limitations of the above methods, alternative approaches have been proposed for breed prioritization, which aim at minimizing global molecular coancestry (i.e. inbreeding) considering both within- and between-breed kinship coefficients ,23,29,30ST[ST. It has been argued that higher weights should be given to the between-breed dimension [Assuming that both within- and between-breed contributions to genetic diversity should be taken into account to define conservation priorities, various weights applied to each component have been proposed ,34-37. FST, combineimension , but no imension  carried Several studies have aimed at characterizing the genetic diversity of worldwide cattle (for a review see ). HoweveWe analyzed 3383 animals belonging to 82 populations of Iberoamerican cattle that included British, Continental European and Indicine breeds from 12 countries. Animals were sampled within the framework of the BIOBOVIS consortium  accordinBos indicus, 168; 6), widely spread zebu breeds.Based on previous results ,43,44, rBM1818, BM1824, BM2113, CSRM060, CSSM066, ETH003, ETH010, ETH185, ETH225, HAUT027, HEL009, ILSTS006, INRA032, INRA063, MM12E6, SPS115, TGLA053, TGLA122, and TGLA227. These loci are distributed across 17 cattle autosomes and are recommended by the International Society for Animal Genetics (ISAG) / Food and Agriculture Organization of the United Nations (FAO) Advisory Committee for genetic diversity studies. The genotyping and allele standardization procedures have been validated and are described in detail in [We used a microsatellite dataset previously generated by the BIOBOVIS research consortium , which ietail in .o) and unbiased expected (He) heterozygosities, mean number of alleles (MNA) per breed and geographical breed group. This software was also used to estimate F statistics according to Weir and Cockerham [t) over all loci per breed was calculated with FSTAT v. 2.9.3 software [Estimates of within-breed genetic diversity were obtained with GENETIX 4.05.2 software , namely ockerham . Allelicsoftware .Analysis of conservation priorities depends on how the metapopulation is defined to investigate partial contributions to global genetic diversity. Also, breed prioritization will vary considerably according to the relative importance of the within- and between-breed components of genetic diversity in each breed and to the genetic relationships among breeds. Given the extensive collection of cattle specimens available for this study and for the sake of simplicity, we took into consideration two dimensions that were analyzed separately i.e., (1) the 27 Creole cattle populations and (2) the 13 major geographical breed groups defined above. From the perspective of practical applications, we chose to consider geographical breed groups with their specificities, rather than the complete worldwide distribution of cattle included in a single metapopulation.For the conservation analyses, we followed the methods described by Cañon et al.  and outlWe applied the Core Set methods of Eding et al.  to invesfm) based on allele frequencies [fii) and between (fij) each Creole breed and geographical breed group were calculated with the MOLKIN3 software [fm) were carried-out with a FORTRAN program developed and kindly shared by Eding and Meuwissen.In the absence of genealogical data, kinships were estimated from molecular data with different methods: (1) marker-estimated kinships (MEK) obtained from individual genotypes, as described by Eding and Meuwissen ; (2) a vquencies . MEK werquencies , whereassoftware . Analysei, j) = fii + fjj - 2fij. Kinship genetic distances were used to construct the neighbor-net phylogenies of the Creole breeds and geographical breed groups with the SPLITS TREE4 4.12.6 software [fm) with the MATLAB® software .We derived pairwise kinship distances from the MEK coefficients following Eding et al.  as: d.In order to assess within-breed genetic diversity directly, the partial contributions of each Creole breed and each geographical breed group were also calculated as the proportional variation in expected heterozygosity of the metapopulation after removal of each breed or breed group (PCWeitz) of each Creole breed and each geographical breed group to the total genetic diversity using the Weitzman method [Weitz were calculated with the FORTRAN program developed by Garcia et al. [We calculated the partial contributions  [st to weight the between- and (1-Fst) to weigh the within-population components of diversity i.e., PCFst = PCWeitz*Fst + PCHe*(1-Fst); (2) the approach of Piyasatian and Kinghorn [5:1), such that PC5:1 = PCWeitz*0.833 + PCHe*(1–0.833); and (3) the method proposed by Caballero and Toro [Ideally, analyses of conservation priorities should take into account both within- and between-population genetic variability in order to make more accurate management decisions. We used three approaches to calculate contributions that combine these two levels of the global diversity of the metapopulation: (1) aggregate diversity (PCFst) , which uKinghorn , which gand Toro  and Fabuand Toro , which gand Toro  were useand Toro .Results are organized according to the two dimensions defined above i.e., the 27 Creole cattle populations and the 13 geographical breed groups [see Additional file o = 0.719±0.053, He = 0.739±0.049, MNA = 6.95±1.52 and Rt = 4.71±1.1). Among the Creole cattle, the Creole1 and Creole3 groups of breeds that are related to Iberian or British breeds and also to African cattle, showed intermediate to high levels of genetic diversity , while the Creole breeds admixed with zebu cattle (Creole5 and Creole6) had high values across all estimates .Summary statistics that describe the genetic diversity within the Creole cattle breeds and the geographical breed groups are in Tables The neighbor-net phylogeny of kinship distances Figure a shows tThe neighbor-net graph of geographical breed clusters in Figure fm) estimated from allele frequencies. Because of the poor quality of the extracted DNA, possibly due to inappropriate hair collection (e.g. insufficient hair roots and/or inadequate preservation), no results were obtained for BM2113, ETH003, ETH185 and ETH225 loci in the 35 Cr. Casanareño specimens. Therefore, this breed was excluded from the calculation of allelic richness and from average coancestry analyses because absence of genotyping data for one breed prevents the MOLKIN software from generating complete results for the other breeds (namely between-breed coancestries of Cr. Casanareño and the other Creole populations). In order to visualize both within- and between-breed kinships, contour plots were drawn in which populations were sorted according to their genetic proximity defined in the phylogenetic neighbor-net graph ; Blanco Orejinegro (MEK = 0.191 and fm = 0.319); Cr. Patagonico (MEK = 0.215 and fm = 0.339); Cr. Uruguayo (MEK = 0.205 and fm = 0.335); Cr. Argentino (MEK = 0.209 and fm = 0.330); Romosinuano (MEK = 0.206 and fm = 0.344); and Costeño con Cuernos (MEK = 0.210 and fm = 0.344), while yellow areas represent breeds with intermediate kinship values i.e. Caracú (MEK = 0.163 and fm = 0.300), Lucerna (MEK = 0.162 and fm = 0.302) and Sanmartinero (MEK = 0.152 and fm = 0.300).The levels of within-breed diversity can also be assessed using kinship coefficients with either the MEK obtained from individual genotypes or average coancestries  are clearly separated from all others.fm = 0.297), which includes the threatened Colombian breeds Costeño con Cuernos and Romosinuano, and Iberian3 (MEK = 0.172 and fm = 0.271), which includes two isolated breeds from the Canary Islands. Interestingly, the group of Indicine breeds, mainly composed of widely spread commercial zebu cattle, also had high kinship coefficients (MEK = 0.167 and fm = 0.272), which suggests the existence of a substructure within this group.The geographical breed clusters with the highest within-group kinship values  and Iberian2 (mostly Spanish breeds), between the Continental European group and Iberian2, and between Iberian3 (Canary Islands) and Creole3 (Colombia), as shown by the yellow areas in Figure fm and WLM, resulted in a considerable number of Creole breeds with a null contribution to the overall genetic diversity . Consequently, only highly prioritized breeds can be easily identified i.e. Siboney, Cr. Cubano, Pampa Chaqueño and Cr. Poblano  and the results were similar with the three methods applied. These may reflect the high within-breed genetic diversity of these breeds, possibly due to past crossbreeding. The WLMM method, which allows only one null contribution (in this case the Creole Blanco Orejinegro), separated breeds more effectively and prioritized for conservation the Casanareño, Siboney, Cr. Cubano, Velasquez and Cr. Ecuatoriano breeds (0.56 < WLMM < 0.107). Nevertheless, the contrasting results for the Casanareño breed  must be interpreted with caution, because of the lack of results for four loci in this breed, as explained above. The breeds Pampa Chaqueño, Cr. Nayarit, Caracú, and Cr. Chiapas had intermediate conservation values (0.042 < WLMM < 0.048).The results of the analyses of conservation priorities of Creole breeds are in Tables He values (between -0.411 and -0.357). This method ranked breeds with greater He values at a higher level, such as Cr. Nayarit, Caqueteño, Hartón del Valle, Cr. Chiapas and Cr. Chihuahua (0.196 < PCHe < 0.252). The breeds Chino Santandereano, Cr. Poblano, Cr. Ecuatoriano, Pampa Chaqueño, Velasquez and Cr. Pilcomayo had intermediate contributions (0.152 < PCHe < 0.192). Although Cr. Casanareño was excluded from the coancestry analysis, it resulted in a null MEK contribution and was highly prioritized by the WLM and WLMM as a consequence of having a significant effect (~14%) in the overall heterozygosity of Creole cattle.The proportional contribution of each breed to the average heterozygosity of the metapopulation, resulted in many negative values (10 breeds), which, if these breeds were removed, would lead to a 'gain’ in diversity. In accordance with their inbred status (high within-breed kinship coefficients), the breeds Guabalá, Romosinuano, Cr. Patagonico and Costeño con Cuernos had the most negative PCWeitz < 9.88) were Guabalá, Romosinuano, Cr. Patagonico, Siboney and Caracú followed by Costeño con Cuernos, Cr. Uruguayo and Cr. Argentino (5.38 < PCWeitz < 4.97). Mexican breeds had the lowest contributions amongst all breeds (< 2%).The Weitzman approach prioritizes highly differentiated breeds  based only on their contribution to between-breed genetic diversity. In this case, breeds with the highest contributions (5.39 < PCFst), which takes into account both within- and between-breed components of the genetic diversity, seems to provide more balanced solutions. In this case, the between-breed component (i.e. PCWeitz) was weighted by the overall Fst value of 0.087 obtained for the metapopulation of Creole breeds. The PCFst approach prioritized Creole breeds that also ranked high with the kinship-based methods (i.e. with high within-breed diversity), namely Siboney, Cr. Cubano, Velasquez and Cr. Nayarit (0.385 < PCFst < 0.579). Nevertheless, some of the breeds prioritized by PCWeitz (i.e. with greater genetic distances) also had high PCFst estimates, particularly Guabalá, Caracú and the above mentioned Siboney (0.337 < PCFst < 0.579). Creole breeds Chino Santandereano, Cr. Ecuatoriano, Cr. Chiapas, Caqueteño, Pampa Chaqueño, Hartón del Valle and Guaymí had intermediate conservation values (0.310 < PCFst < 0.378). The PC5:1 method gave the same results in terms of breed ranking for conservation priorities as the Weitzman approach.The combined approach of Ollivier and Foulley  (PCFst),f, 0.019) because its within-breed coancestry was quite high (fii = 0.266) and its distance from all the other populations was among the lowest observed (DNei = 0.064). The breeds Cr. Argentino, Caracú and Cr. Uruguayo also had high contributions to f  as a consequence of their high fii values (between 0.300 and 0.329). Although the Cr. Patagonico, Guabalá, Costeño con Cuernos and Romosinuano breeds had high fii values (between 0.339 and 0.354), their mean genetic distances were also rather large (between 0.106 and 0.127) and thus their contributions to f, obtained from the difference between fii and DNei, were less significant . The Texas Longhorn breed had the highest contribution to D (0.073) and to proportional contribution estimates , but these estimates can be biased as a consequence of the rather large sample size of this population (N = 80), which is taken into account in all calculations. This could also be valid for the PC estimates of the breeds Siboney (6.123), Cr. Cubano (5.999), Pampa Chaqueño (5.884) e Cr. Argentino (5.430), which also had large samples sizes. Indeed, when the proportional contributions to genetic diversity are estimated by ignoring sample sizes (PCunweighted), only the Siboney and Cr. Cubano breeds maintain their high ranking , followed by Cr. Chiapas (4.001), Cr. Nayarit (3.987) and Velasquez (3.981). The Caracú, Cr. Poblano, Cr. Uruguayo, Cr. Pilcomayo, Guaymí and Cr. Patagonico breeds had quite high proportional contributions to genetic diversity (3.813 < PCweighted <5.321), whereas Cr. Ecuatoriano had the lowest contribution (PCweighted = 1.414), perhaps because of its low representation (N = 12). The proportional contribution of each breed to a pool of maximum genetic diversity showed very little variation among Creole breeds, but removal of the Siboney and Cr. Cubano breeds from the metapopulation of Creole cattle caused the greatest loss in total genetic diversity .The results of the combined approach of Caballero and Toro  and Fabufm, WLM and WLMM), the breed groups Creole6 (zebu-influenced), Indicine, British and Iberian4 (most Spanish breeds) were highly prioritized  because of the rather high within-group genetic diversity which reflects their heterogeneous genetic make-up. Based on the MEKs and fm estimates, Creole3 (Costeño con Cuernos and Romosinuano breeds from Colombia) had an intermediate conservation value . Similarly, with WLM and WLMM, the Iberian3 (breeds from the Canary Islands) and Continental European  breed groups had an intermediate position. As expected, PCHe estimates resulted in negative contributions for eight of the 13 geographical breed groups and prioritized Creole6, Creole2, Creole5, Iberian4 and Continental European cattle (0.041 < PCHe < 0.694) for conservation.The results of the analyses of conservation priorities of geographical breed-groups are in Tables Weitz = 27.16) which reflected their wide phylogenetic differentiation. This approach also prioritized Creole3 (16.53) and Creole4 (14.22), whereas Iberian3 and Creole1 had more intermediate values . The strong differentiation of these geographical breed groups is confirmed by their large genetic distances, as shown [see Additional file Indicine cattle had a particularly high Weitzman estimate  to calculate conservation values. With this method, well-differentiated breed groups such as Indicine and Creole6 were prioritized , but Creole4, Creole5 and Creole2 also ranked high (0.372 < PCFst < 0.567). According to this method, Iberian2 (a group of Iberian breeds with a recognized admixed background) had the lowest conservation value (0.097). Both Piyasatian and Kinghorn [The strategy based on the analysis of breed groups produced an overall F Foulley  (PCFst =Kinghorn  and Weitweighted < 22.623). Indicine and Creole6 had the most negative impact on overall genetic diversity when they were removed from the metapopulation . When the proportional contributions to genetic diversity were estimated ignoring sample sizes (PCunweighted), Indicine, Creole6, Creole5, Iberian4, Creole2 and British breed groups ranked highest (7.715 < PCunweighted < 8.174).The results of the combined approach of Caballero and Toro  and FabuThe need for conservation of genetic resources for food and agriculture is widely recognized , but thein situ or ex situ in vivo preservation, and cryoconservation, which differ in their ability to address the different aspects considered in the rationale for conservation [In many cases, a conservation program will concern many breeds while financial resources are limited, thus the need to set priorities. Factors that need to be considered when defining such priorities include the importance of a breed in terms of genetic uniqueness but also its own genetic diversity, and other aspects such as adaptation to specific environments, possession of unique traits, cultural and historical value, contribution to environmental sustainability, etc. . Once prervation .Although other factors that can potentially impact conservation decisions are important, knowledge of the population structure of a livestock species in terms of distribution of genetic variability within- and between-breeds is a key factor to establish conservation priorities and strategies , that aiOur results indicate that under different conservation perspectives, the groups of Creole breeds generally had a higher conservation priority than Iberian, British or Continental European breed groups Tables  and 6. Te). This is in line with previous findings [The difficulties associated with the choice of the best method to prioritize breeds for conservation decisions were recently discussed in several papers ,24,27,39findings ,17 that findings . When anOverall, the choice of the most appropriate method to prioritize breeds for conservation decisions is determined by whether it is important to maintain genetic diversity for the short- or long-term. For example, if the focus is on short-term objectives, the emphasis should be placed on maintaining high levels of heterozygosity, while if it is on long-term objectives, the emphasis should be placed on allelic richness and breed differentiation . In the One key issue, which is well-exemplified in the case of Creole cattle, is how misleading the exclusive consideration of high heterozygosity might be, and how it should be carefully assessed and interpreted. For example, several of the Creole breeds included in our study are known to have been admixed with zebu cattle in the beginning of the 20th century, and show strong signs of this influence ,61. As eB. taurus animals imported from Iberia since the 15th century [in vivo preservation of AnGR [The establishment of conservation priorities based only on 'neutral’ genetic markers, such as microsatellites, can fail to take into account important genetic information associated with phenotypic variation (e.g. morphology or production traits), disease resistance, and other adaptive traits. Starting with a few  century , Creole  century ,64, grea century ,66 and t century , lower i century , better  century , greater century , higher  century , and hig century . This re century . Whole-g century -75. Beca century . Thus, a of AnGR , it is i of AnGR , Creole Prior to applying the conservation priority principles discussed here on a large scale, it is essential that a better and more extensive sampling is carried out, particularly for more endangered breeds, and that a general agreement is reached on the specific criteria to be used in the definition of priorities. Besides factors directly associated with genetic diversity, which have been the subject of our study, other aspects such as the contribution of a breed to food security and economic return, the demography and risk status, the existence of unique traits or specific adaptation features, the historical and cultural values, the contribution to sustainable development and environmental balance, etc., should also be taken into account . The endThe contributions to within- and between-breed genetic diversity based on 'neutral’ genetic markers were evaluated in a large sample of Iberoamerican cattle breeds, to provide the basis for establishing conservation priorities. Our results indicate that Creole cattle breeds retain considerable levels of genetic diversity and that several local isolated breeds are important reservoirs of genetic diversity. Conservation priorities depended on the approach used, i.e., on whether the emphasis was placed on the within- or the between-breed component of genetic diversity. In general, if the focus was on between-breed diversity, the Creole breeds classified as top priority were the most clearly differentiated with smaller census and higher levels of inbreeding, while if the focus was on within-breed genetic diversity, they failed to be considered. Thus, besides its contribution to the overall genetic diversity, other features of a breed should also be considered when considering a conservation program, such as its adaptation to specific environments, possession of unique traits, cultural and historical value, and contribution to environmental sustainability, among others. Other types of genetic markers, including novel SNPs, may detect other genetic factors related to breed differentiation, especially those underlying adaptation and production traits, and should be investigated for use in conservation applications.The authors declare that they have no competing interests.Participated in the design of the study and performed the statistical analysis: CG, LTG, OC, JVD, DG, JC. Conceived and designed the experimental assays: CG, SD, VL, IM-B, AM-M, MCTP, PZ. Carried out the assays: CG, SD, VL, IM-B, AM-M, CR. Drafted the manuscript: CG, LTG, OC, JC. Reviewed and edited the manuscript: JVD, SD, AM-M, MCTP, PZ. Members of the BIOBOVIS Consortium provided biological samples and logistic support. All authors read and approved the final manuscript.http://biobovis.jimdo.com/investigadores/.Members of the BioBovis Consortium, BioBovis Consortium members : Atzel Acosta, Centro Nacional de Sanidad Agropecuaria, Cuba; Luz A Álvarez, Universidad Nacional de Colombia, Colombia; Esperanza Camacho, IFAPA, Centro Alameda del Obispo, Spain; José R Marques, EMBRAPA Amazônia Oriental, Brazil; O Roberto Martínez, Centro Multidisciplinario de Investigaciones Tecnológicas, Universidad Nacional de Asunción, Paraguay; Ruben D Martínez, Facultad de Ciencias Agrarias, Universidad Nacional de Lomas de Zamora, Argentina; Guillermo Martínez-Velázquez, Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias, Mexico; Lilia Melucci, Universidad Nacional de Mar del Plata and Instituto Nacional de Tecnología Agropecuaria, Argentina; Jaime E Muñoz, Universidad Nacional de Colombia, Colombia; E. Armstrong, Facultad de Veterinaria, Universidad de la República, Uruguay; Jorge Quiroz, Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias, Mexico; Philip Sponenberg, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Tech, United States of America; Odalys Uffo, Centro Nacional de Sanidad Agropecuaria, Cuba; José Luis Vega-Pla, Laboratorio de Investigación Aplicada, Cría Caballar de las Fuerzas Armadas, Spain; Axel Villalobos, Instituto de Investigación Agropecuaria, Panama; Delsito Zambrano, Universidad Técnica Estatal de Quevedo, Ecuador.o) and unbiased expected (He) heterozygosities, mean number of alleles (MNA), allelic richness corrected for sample size (Rt), and the respective standard deviation (SD).Details on the cattle breeds and geographical breed groups included in this study. Description: Breed names axnd acronyms, sample origins and sizes, and number of genotyped microsatellite loci are provided. Summary statistics of within-breed genetic diversity are also shown, namely: observed (HClick here for filefm). Description: Creole cattle breeds (a) and geographical breed groups (b) were sorted according to the respective kinship phylogenies. Breed acronyms are defined as follows: CRA, Cr. Argentino; PAT, Cr. Patagonico; CAR, Caracú; CRU, Cr. Uruguayo; BON, Blanco Orejinegro; HVA, Hartón del Valle; LUC, Lucerna; PCH, Pampa Chaqueño; CCC, Costeño con Cuernos; RMS, Romosinuano; GUA, Guabalá; GUY, Guaymí; SMA, Sanmartinero; CBC, Cr. Baja California; CHU, Cr. Chihuahua; CNY, Cr. Nayarit; CPO, Cr. Poblano; TLH, Texas Longhorn; CAQ, Caqueteño; CHS, Chino Santandereano; CAS, Cr. Casanareño; VEL, Velasquez; CUB, Cr. Cubano; SIB, Siboney; ECU, Cr. Ecuatoriano; CHI, Cr. Chiapas; PIL, Cr. Pilcomayo.Contour plots of average coancestries (Click here for fileDescription: Neighbor-net graph of Reynolds distances showing the genetic relationships among the 13 geographical breed groups studied.Click here for fileVariation of breed-group contributions to Weitzman diversity using the approximation algorithm of Garcia et al. . DescripClick here for file


Cocos nucifera L.) played a fundamental role in human migrations and the development of civilization across the humid tropics. Here we investigated the coconut's domestication history and its population genetic structure as it relates to human dispersal patterns. A sample of 1,322 coconut accessions, representing the geographical and phenotypic diversity of the species, was examined using ten microsatellite loci. Bayesian analyses reveal two highly genetically differentiated subpopulations that correspond to the Pacific and Indo-Atlantic oceanic basins. This pattern suggests independent origins of coconut cultivation in these two world regions, with persistent population structure on a global scale despite long-term human cultivation and dispersal. Pacific coconuts show additional genetic substructure corresponding to phenotypic and geographical subgroups; moreover, the traits that are most clearly associated with selection under human cultivation  arose only in the Pacific. Coconuts that show evidence of genetic admixture between the Pacific and Indo-Atlantic groups occur primarily in the southwestern Indian Ocean. This pattern is consistent with human introductions of Pacific coconuts along the ancient Austronesian trade route connecting Madagascar to Southeast Asia. Admixture in coastal east Africa may also reflect later historic Arab trading along the Indian Ocean coastline. We propose two geographical origins of coconut cultivation: island Southeast Asia and southern margins of the Indian subcontinent.As a portable source of food, water, fuel, and construction materials, the coconut ( Cocos nucifera L.) on the history of human dispersal in the humid tropics is unparalleled in the plant kingdom. As a portable source of both food and water, the coconut played a critical role in the ability of humans to voyage, establish trade routes, and colonize lands in the Pacific Rim and regions throughout the Old World tropics The impact of the coconut palm , has served as the primary source of materials for genetic characterizations. Together with a polymorphic microsatellite marker kit In the last decade, a worldwide coconut germplasm collection, coordinated through the International Coconut Genetic Resources Network (COGENT) and the French Agricultural Research Centre for International Development (CIRAD), with further support through the Generation Challenge Programme  Geographical locations of genetically admixed populations are consistent with human introductions of Pacific germplasm along the ancient trading routes connecting Asia to Africa.In the present study, we have employed ten polymorphic loci from the GCP/CIRAD microsatellite kit to examine genetic variation in a worldwide collection of >1300 coconuts, representing GCP/CIRAD germplasm plus collections from key under-sampled regions of the western Indian Ocean: Madagascar, Comoros, and Seychelles islands. We use population structure analyses, together with ethnographic and archaeobotanical evidence, to examine the impacts of human-mediated dispersal and domestication on this important tree crop. Our analyses suggest the following: 1) Despite the widespread movement of coconuts by humans, both historically and today, the species has retained clear population structure on a global scale; 2) Present-day cultivated coconuts arose through independent domestications in the Indian and Pacific Ocean basins; however, the definitive domestication traits — dwarf habit, self-pollination, and With new sample collections that fill an important gap in an already extensive worldwide data set, we have examined variation at ten microsatellite loci in a global collection of coconut germplasm. Genotypes were successfully obtained for 1322 samples, representing 1210 individuals from the GCP/CIRAD collection and 112 samples from the western Indian Ocean . For gere = 0.271 and 0.579 for the two growth forms, respectively. Dwarfs also show greater evidence of inbreeding , consistent with the low within-cultivar genetic heterogeneity characterizing these self-pollinating varieties, most of which are pure-breeding lines. This overall pattern of reduced genetic variability in Dwarfs has been reported previously . To explicitly evaluate the relative contributions of the two subpopulations to the genomes of the putative admixed populations, we calculated a composite introgression index . In the Seychelles, outside the Austronesian trade route, no evidence of introgression is observed (iT = −0.065≈0).Introgression from Pacific coconuts into the western Indian Ocean is further reflected in the distributions of individual microsatellite alleles whose frequencies differ between the two major subpopulations and which can therefore serve as subpopulation-diagnostic markers. We identified six such alleles using Shannon's mutual information index see . Their dndex Ti; . This mead hoc statistic  and Jost's D)  for these five subpopulations. Both measures highlight the genetic isolation of the Indian Ocean from the Pacific populations, consistent with long-term evolutionary divergence between the two lineages. The main interest of Jost's measure is that differentiation and diversity represent structurally independent between- and within-population diversity components. As a result, the range of variation of D between the Indian and Pacific populations (0.800–0890) is much narrower than in the distances (0.365–0.566), which are, by construction, correlated with heterozygosity  sity see . Jost's To assess the geographical distribution of the five population components, we assigned accessions to one of seven categories based on population membership coefficients at K = 5: accessions with membership coefficients of Q>80% were assigned to each of the five subpopulations ; those with 20–80% Indo-Atlantic membership were defined as ‘admixed’; and remaining accessions  were assigned to a generic ‘Pacific’ class. As is observed at K = 2, the Indian Ocean component predominates in South Asia and the Seychelles (A), as well as in West Africa (B), the Caribbean (C) and Brazil (D) . HistoriAdmixture from the Indo-Atlantic subpopulation is evident at a low frequency in the Pacific coconuts of continental Southeast Asia (H), especially in Thailand, Malaysia, and Cambodia . This paPacific coast ‘Panama Tall’ coconuts (K) are characterized predominantly by the ‘Panama’ population component. This component is absent elsewhere, except in the Philippines (J) where it occurs at a low frequency . This pa‘niu leka’ (‘Fiji Dwarf’), which represents an independent origin of the dwarf habit, distinct from the widely-cultivated self-pollinating Dwarfs of Southeast Asian origin (In Papua New Guinea (M) and in Micronesia (N), the ‘PNG’ population component predominates. The apparent presence of Indo-Atlantic admixture in Micronesia N,  may refln origin , S1.Cocos nucifera is a native species of both the Indian and Pacific Oceans, with a long-standing evolutionary presence in both ocean basins. Fossil data from the Palaeocene also support the long-term presence of coconuts (or coconut-like species) in both the Indian and Pacific basins A striking observation from our worldwide analysis of coconuts is the high level of genetic differentiation between Pacific and Indian Ocean samples ; Fig. S2In addition, the clear genetic differentiation between the Pacific and Indian Ocean lineages allows us to conclude definitively that coconuts were brought into cultivation independently in each of these regions. In the Pacific, the phenotypic diversity and population heterogeneity associated with a region extending from the Malay peninsula to New Guinea , Fig. 2 niu kafa’ type. This group also remained confined within the Indian Ocean basin until the European colonial era. In contrast, the Pacific group has higher levels of gene diversity (niu leka’ (‘Fiji Dwarf’) variety, also arose in the Pacific group  and the Indian subcontinent (indica rice), with admixture in Madagascar The genetic distinctness of the Indo-Atlantic and Pacific coconut lineages facilitates our ability to track the genetic footprints of human introductions around the world. Most striking is the genetic admixture in the western Indian Ocean reflecting Pacific coconut introgression. Our analyses suggest that admixed coconuts predominate in the region corresponding to the ancient Austronesian trade route connecting Southeast Asia to Madagascar and coastal east Africa; in contrast, no admixture is evident in the more northerly Seychelles, which fall outside the trade route ; Fig. 2.buahniu /voanio (Madagascar), not to mention vanu in the Loyalty Islands, but also from narikela (Sanskrit)/nargil /mnazi (Bantu), a double linguistic pathway for the same tree, the one directly across the Indian Ocean, the other via the north of the same ocean.” Recent observations of genetically admixed coconut populations in Oman Admixture between Pacific and the Indian Ocean coconuts was likely further promoted by the later presence of Arabo-Persian merchants who regularly visited East Africa, trading coconut and favoring its cultivation Within the Pacific basin, human influence on coconut population structure is most readily detectable in the pre-historic introduction of Southeast Asian coconuts to the New World coast. This introduction is estimated to have occurred ∼2,250 years ago, and our analyses are consistent with previous findings suggesting a Philippine origin ; the lowIn the most extensive genetic analysis of coconuts to date, we find evidence for independent origins of coconut cultivation in the Pacific and Indian Ocean basins. Interestingly, despite the long-term, extensive movement of coconuts by humans both within and between these oceanic basins, most contemporary coconuts do not show evidence of substantial genetic admixture between the two major genetic subpopulations ; Fig. S2Besides revealing basic insights into the cultivation and dispersal history of this iconic tropical species, our findings may also facilitate efforts to protect the viability of the coconut as a crop species. Coconut lethal yellowing, a phytoplasma infection, has reached epidemic levels in the Caribbean and other regions of the Neotropics; susceptible trees typically succumb within a year of infection. Knowledge of the worldwide genetic structure of the coconut, including regions where genetic admixture has generated augmented levels of genetic diversity ; growth form, variety name, source country, and germplasm group assignment are indicated in GCP/CIRAD accessions correspond to those in the GCP database . Genotyping of the GCP/CIRAD collection is described in ref GENALEX 6Structure 2.3 InStructInStruct is similar to Structure but relaxes assumptions of Hardy-Weinberg equilibrium within subpopulations. For Structure analyses, the number of subpopulations, K, was set at values ranging from 1–10, with 20 replicate runs apiece . An admixture ancestry model was selected with allele frequencies correlated. For the optimal inferred K value (K = 2), we employed CLUMPP version 1.1.2 Structure runs . InStruct analyses were performed using the Cornell University BioHPC web portal (http://cbsuapps.tc.cornell.edu/InStruct.aspx). The program DISTRUCTCLUMPP and InStruct analyses.Analyses of genetic diversity and AMOVA were performed with BAPSGENELANDBecause Dwarf accessions are highly homozygous and show little genetic diversity, clustering analyses were performed both with and without Dwarfs to test for potential artifacts created by their inclusion; excluding these accessions did not substantially alter inferences. In additional analyses, we applied explicit spatial clustering as implemented in a in population A3 as a function of aAp, its frequency in population A3: Ah(a) = −aAplog aAp−(1−aAp)log(1−aAp). Likewise, we calculated Bh(a) based on aBp its frequency in population B1 and Th(a), based on aTp = ½(aAp+aBp). The mutual information quantity between a  and G (the group) is thus I = Th(a)−½[Ah(a)+Bh(a)]. Expressed in Shannon units , the mutual information quantity may range from 0 (same frequencies in A3 and B1) to 1 . We retained alleles corresponding to the six top values.To test for Pacific introgression into the Indian Ocean populations, we defined ‘diagnostic alleles,’ i.e., alleles that are differentially represented in GCP/CIRAD subgroup A3 (a representative Pacific subgroup) relative to subgroup B1 (representative Indo-Atlantic), and we selected them using Shannon's mutual information iaT = (iaZ−aX)/(aY−aX) where aX, aY, and iaZ are the respective frequencies in B1, A3, and the four other groups. Indices i and a refer to group and allele, respectively. The mean of the index over all alleles (iT) is an estimation of the percentage of alleles from Southeast Asia in each group. Finally, we assessed the consistency of the introgression model by calculating the coefficient of determination R2 of the regression of the frequencies of each allele on iT (excluding groups B1 and A3).Based on the frequencies of these alleles in six groups , we then calculated ‘introgression indices’ for each allele: H) and the associated STG are not adequate measures of diversity and differentiation, respectively. He suggests instead using the reciprocal of Nei's identity as a measure of diversity, and he derives absolute and relative measures of differentiation. These measures are, respectively, ΔST = ΔT/ΔS = JS/JT and D = (TJ/SJ −1)/[(1/n)−1]. In these formulae, J = 1−F refers to Nei's identity and is the expected proportion of homozygotes in a population. SJ is the average of Nei's identities in the sub-populations. The within-population component of diversity is ΔS = 1/SJ.The total diversity is ΔT = 1/TJ where TJ is calculated based on the allele frequencies in the pooled population. We derived these parameters from the Structure outputs (heterozygosities and distances).Jost Figure S1Structure analyses.Assessment of subpopulation number in (DOC)Click here for additional data file.Figure S2InStruct output at K = 2 subpopulations.(DOC)Click here for additional data file.Table S1Structure analysis.Information on coconut accessions used in analyses and assignment probabilities at K = 2 and K = 5 using (DOC)Click here for additional data file.Table S2Allele frequencies for each locus for Pacific and western Indian Ocean populations.(DOC)Click here for additional data file.Table S3Analysis of Molecular Variance (AMOVA) for all coconut accessions .(DOC)Click here for additional data file.Table S4Language roots associated with the coconut in proto-South-Dravidian and proto-Telugu.(DOC)Click here for additional data file.Text S1Early evidence of coconut use in the southern Indian subcontinent and neighboring islands.(DOC)Click here for additional data file.


Mean LD decay distance for the landraces at the whole genome level was <5 cM, while a higher LD decay distance of 5–10 cM in modern varieties. LD decay distances were also somewhat different for each of the 21 chromosomes, being higher for most of the chromosomes in modern varieties (<5∼25 cM) compared to landraces (<5∼15 cM), presumably indicating the influences of domestication and breeding. This study facilitates predicting the marker density required to effectively associate genotypes with traits in Chinese wheat genetic resources.Two hundred and fifty bread wheat lines, mainly Chinese mini core accessions, were assayed for polymorphism and linkage disequilibrium (LD) based on 512 whole-genome microsatellite loci representing a mean marker density of 5.1 cM. A total of 6,724 alleles ranging from 1 to 49 per locus were identified in all collections. The mean PIC value was 0.650, ranging from 0 to 0.965. Population structure and principal coordinate analysis revealed that landraces and modern varieties were two relatively independent genetic sub-groups. Landraces had a higher allelic diversity than modern varieties with respect to both genomes and chromosomes in terms of total number of alleles and allelic richness. 3,833 (57.0%) and 2,788 (41.5%) rare alleles with frequencies of <5% were found in the landrace and modern variety gene pools, respectively, indicating greater numbers of rare variants, or likely new alleles, in landraces. Analysis of molecular variance (AMOVA) showed that A genome had the largest genetic differentiation and D genome the lowest. In contrast to genetic diversity, modern varieties displayed a wider average LD decay across the whole genome for locus pairs with r Triticum aestivum L.) is one of the most important cereal crops worldwide, including China. Wheat is grown in 30 of China's 31 provinces in 10 major agro-ecological zones based on wheat type, growing season, and varietal response to temperature and photoperiod http://icgr.caas.net.cn/cgris_english.html]. Recently, a candidate wheat core collection  was established based on geographical regions, ecotypes, and 21 agronomic and botanic characters of the basic collections 5 SSR data-points Bread wheat (Zea mays L.) Oryza sativa L.) Hordeum vulgare L.) Sorghum bicolor L. Moench) T. turgidum L. var. durum) Glycine max L. Merr.) Linkage disequilibrium (LD), or nonrandom association of alleles between loci (linked or unlinked), is becoming increasingly important for identifying genetic regions associated with agronomic traits Based on genotyping of 250 accessions mostly from the Chinese bread wheat mini core collection  using 512 microsatellite loci distributed over all 21 chromosomes, the objectives of this study were: 1) to evaluate the allelic diversity within the Chinese wheat collection; 2) to analyze the population structure and compare the diversity level between landraces and modern varieties; 3) to investigate genetic differentiation of wheat genomes within the two gene pools; and 4) to examine the extent and genomic structure of LD between pairs of SSR markers on both genome-wide and chromosome scales. The results of this study should describe the level of genetic diversity and linkage disequilibrium decay of a representative Chinese collection for breeding and genetic research, and provide a molecular basis to enrich genetic diversity of bread wheat worldwide.The genetic characteristics of the 250 member Chinese wheat mini core collection based on 512 microsatellite loci are listed in Population structure of whole collection was investigated using a Bayesian clustering approach, to infer the number of clusters (populations) with STRUCTURE v2.2 software Principal coordinate analysis also indicated two major sup-groups within the mini core collection . A largePIC per locus  and genetic diversity indices (0.640) of the landraces were higher than those for modern varieties at 9.8 and 0.628, respectively. Consistent with landraces having higher diversity than modern varieties at the whole genome level, these relationships were retained when the three genomes were individually compared. To eliminate the influence of sample size on evaluations of genetic diversity, allelic richness calculated following rarefaction on samples of 68 accessions per sub-group likewise indicated that landraces had higher genetic diversity than modern varieties  on all individual chromosomes, as well as the distribution of rare alleles in the two gene pools. The mean stF value for all chromosomes was 0.021 ranging from 0.010 to 0.035 between landraces and modern varieties. Chromosome 3A (0.035) provided the highest genetic differentiation and 1D (0.010) the lowest  and genetic distance (GD), the A genome was the most diverse and the D genome was the least. Gene flow estimated from stF (mN) and genetic identity (GI) placed the A genome lowest among the three genomes, while the D genome ranked first. This indicated that the largest genetic differentiation between the two sub-sets was within the A genome with the least differences in the D.Comparisons of the landrace and modern variety sub-groups on the basis of genomes are shown in P<0.001) and more than 95% of the variance was explained by differences within the A, B and D genomes, whereas only a small part of the overall variance (less than 5%) could be attributed to differences between landraces and modern varieties. The AMOVA analysis also revealed similar structures of genetic differentiation consistent with the basic statistics  between the landraces and modern varieties by genomes was also carried out . All souatistics  when comP<0.001 level for landraces, whereas there were 275 (4.46%) of 4,736 in significant LD among modern varieties. In addition, the numbers of locus pairs in LD with r2>0.1 or r2>0.2 in modern varieties were also relatively higher than those in landraces. Furthermore, the mean r2 for all significant LD (P<0.001) in modern varieties  was still larger than for landraces . Although the landraces possessed more significant unlinked locus pairs (P<0.001) than modern varieties, i.e. 1,509 (1.30%) vs 1,019 (0.88%), modern varieties had higher r2 value in other parameters. LD comparisons on a genome basis showed similar trends of higher LD in modern varieties than in landraces, even though there were very low genome-wide LD levels in both sub-groups. Plots of significant r2 values (P<0.001) between locus pairs in different genomes of the two sub-groups  in both sub-groups, 495 loci were chosen to evaluate the extent of linkage disequilibrium (LD) on a whole genome level in the two wheat gene pools . There wb-groups  further P<0.001) LD and mean r2 among distance intervals for each gene pool (P<0.001) LD decreased as genetic distance increased, and there were higher scales of significant LD within 10 cM generally. However, mean r2 along distance intervals presented an uneven distribution, i.e. there were some points with relatively higher mean r2 at larger intervals. Considering lower LD values for our samples  LD and mean r2 values between landraces and modern varieties  of fitting either sub-group included Lianglaiyoubaipixiaomai (Inner Mongolia), Bihongsui (Inner Mongolia), Mingxian 169 (Shanxi), Shite 14 (Hebei), Fuzhuang 30 (Shaanxi), and Jingyang 60 (Shaanxi). Even though they were arbitratrily classified into modern varieties, most of them were selections of landraces or were from hybrid progeny of landraces To address possible limitations in the number of loci used in above-mentioned studies, we employed 512 microsatellite loci identifying 6,724 alleles to obtain a genetic structure of Chinese wheat genetic resources using principal coordinate analysis and Bayesian clustering approaches. The larger number of alleles identified in 512 SSR loci also indicated that individual microsatellite loci have higher information content Allelic diversity analysis in this study revealed that the total number of alleles amplified at 512 SSR loci in 250 accessions was up to 6,724 , and polymorphism information content values ranged from 0 to 0.967 (mean 0.650). These values were higher than the previously reported estimates of SSR marker diversity in wheat The amounts of genetic diversity in the two gene pools and PIC values were significantly different at both the genome  and indiI), genetic distance (GD), genetic differentiation coefficient (stF), and analysis of molecular variance (AMOVA) between the landrace and modern variety sub-groups for different genomes suggested that the A genome (4.73%) was significantly more variable than the B (4.25%) and D (3.05%) genomes, indicating stronger selective pressure on the A genome during Chinese wheat breeding. However, a selection sweep imprinted across genomes suggested that some important loci or chromosomal intervals rather than whole genomes (or chromosomes) were responsible for the differences  on a whole genome level, but only 0.02% and 0.03% were in LD based on r2>0.2 within the two gene pools  were from the Chinese wheat mini core collection constructed in our group http://wheat.pw.usda.gov/ggpages/SSRclub/], 10 GDM http://www.shigen.nig.ac.jp/wheat/komugi/top/top.jsp]  was used to capture amplification products by a fluorescence detection system for microsatellite markers. More detailed experimental procedures are given in Hao et al., Genomic DNA of all materials was extracted using lyophilized pooled young leaves of ten seedlings following Sharp et al., top.jsp] . In totatop.jsp] . More inPopulation structure analysis for the 250 Chinese wheat accessions was performed using the molecular datasets of 512 whole-genome SSR markers with STRUCTURE v2.2 software Genetic dissimilarities between accessions were calculated using the simple matching coefficient in DARwin software PIC) at each SSR locus according to the formula PIC = 1-∑i2pip is the frequency of the ith allele, were carried out with PowerMarker v3.25 mN), genetic distance (GD), genetic identity (GI), Shannon's information index (I) and coefficient of gene differentiation (stF). The genetic variation within and among populations of wheat accessions for different genomes was evaluated using analysis of molecular variance (AMOVA) implemented in Arlequin v3.11 software Basic statistics of genetic diversity including total number of alleles, and polymorphism information content (2) and significance of each pair of loci Linkage disequilibrium (LD) between markers, including the pairwise estimated squared allele-frequency correlations Click here for additional data file.Figure S2Comparative PIC distributions of SSR loci in the landrace and modern variety sub-groups for all 21 wheat chromosomes. Blue curves show PIC trends in the modern variety sub-group, and red curves show trends for the landrace sub-group. Blue broken line means average PIC value of all SSR loci for the modern variety, and red broken line for the landrace. The detailed mean PIC values are listed at the bottom of each broken line. Genetic positions (cM) of SSR loci are from the Komugi wheat genetic resources database [http://www.shigen.nig.ac.jp/wheat/komugi/top/top.jsp].(DOC)Click here for additional data file.Table S1Detailed information of 250 accessions used in the study.(XLS)Click here for additional data file.Table S2List of 512 SSR loci used in the study.(XLS)Click here for additional data file.Table S3Chromosomal distribution of SSR loci used in the study.(XLS)Click here for additional data file.Table S4Molecular database of 512 SSR loci in 250 Chinese wheat genetic resources.(XLS)Click here for additional data file.


Buthus scorpions using mtDNA sequences. We sampled 91 individuals of the genus Buthus from 51 locations scattered around the Atlas Mountains . We sequenced 452 bp of the Cytochrome Oxidase I gene which proved to be highly variable within and among Buthus species. Our phylogenetic analysis yielded 12 distinct genetic groups one of which comprised three subgroups mostly in accordance with the orographic structure of the mountain systems. Main clades overlap with each other, while subclades are distributed parapatrically. Geographic structures likely acted as long-term barriers among populations causing restriction of gene flow and allowing for strong genetic differentiation. Thus, genetic structure and geographical distribution of genetic (sub)clusters follow the classical theory of allopatric differentiation where distinct groups evolve without range overlap until reproductive isolation and ecological differentiation has built up. Philopatry and low dispersal ability of Buthus scorpions are the likely causes for the observed strong genetic differentiation at this small geographic scale.The immense biodiversity of the Atlas Mountains in North Africa might be the result of high rates of microallopatry caused by mountain barriers surpassing 4000 meters leading to patchy habitat distributions. We test the influence of geographic structures on the phylogenetic patterns among  The Mediterranean region comprises a highly diverse geographic area characterized by a variety of peninsulas and islands. This underlying geographic structure is reflected in biogeographical patterns of the region already described by de Lattin (1949) De Lattin (1949) Buthus, which has undergone extensive speciation within NW Africa Buthus scorpions are widely distributed over large parts of Africa and show a circum-Mediterranean stronghold including the Mediterranean islands, with extraordinarily high diversity in North Africa In order to test for the influence of orographic structures on differentiation and speciation processes, we selected the scorpion genus Buthus species from 51 locations over major parts of the Antiatlas, High Atlas, Middle Atlas and Jebel Sahro during spring 2008 and 2009. Three specimens of the related genus Androctonus from two locations served as outgroup. We analysed a fragment of the mitochondrial Cytochrome Oxidase I (COI) gene, which has been shown to be highly informative at species level and consequently is one of the most commonly used markers for phylogenetic and phylogeographic studies. Due to its high potential for species identification it is often chosen as the barcoding gene in zoological studies. Here we use its properties to address the following questions:For this study we collected a total of 91 individuals belonging to 4 described Buthus exhibit geographic structuring?Does genetic variation within Do genetic structures follow any geographical and/or evolutionary pattern?Are genetic lineages in accordance with current taxonomy?Androctonus mauritanicus (JF820097) was obtained from genbank as an additional outgroup. Details about analyzed individuals, including genbank accession numbers, are given in We generated 94 sequences (including three outgroup specimens) with a total length of 452 base pairs. One sequence from The second dataset included 84 additional sequences obtained from genbank . In totaButhus lineages, which were recovered in all analyses  Several lineages are composed of different species: four of the five lineages with ten or more individuals analysed contain two to three species; even lineage H  includes two different morpho-types.The 12 lineages A through L do not match with current taxonomy (as reflected by morphology based identification keys): (i) The two tree hypotheses (ML and BI) constructed from our original dataset distinguish 12 genetic lineages. One of these clusters (lineage K) is further structured into three sub-clades. The genetic clusters exhibit a clear geographic pattern with all the lineages being confined to relatively restricted areas. None of the lineages is widely distributed. Most lineages are separated from each other by geographic barriers such as high mountain ranges or steep valleys. Only some basal lineages show range overlap (e.g. K) while all younger groups are strictly allopatric or parapatric (e.g. the three sub-groups of K). Four groups (A–D) are situated around the western and north-western parts of the High Atlas. One group is located in the westernmost Antiatlas (E), three others at the northern foothills of these mountains (F–H) and one at the southern foothills of the Antiatlas (J). The largest cluster (K) is distributed south of the High Atlas, east of the Draa valley, and splits further into three subclades, which form distinct strictly parapatric groups following the orographic structures of this region . Another cluster (I) extends from the Draa valley to the south western edge of the Antiatlas, probably including parts of the distribution ranges of clade G and H.Buthus scorpions mostly does not exceed elevations higher than 2500 m asl Buthus individuals. A similar pattern was observed for the same taxonomic group using various molecular markers such as allozymes, nuclear and mitochondrial DNA all of which showed significant genetic differentiation patterns even among local populations Buthus lineages also include their last centre of differentiation. Thus, our study area may comprise 12 allopatric refugia during the last ice age revealing a much more fine grained biogeographical sub-structure than previously observed in the Maghreb e.g. Buthus in Morocco is much more complex than the structures observed in other Mediterranean sub-centres Buthus individuals  gene was amplified using standard PCR procedures with the following primers: forward 5′ GGT CAA CAA ATC ATA AAG ATA TTG G 3′, reverse 5′ TAA ACT TCA GGG TGA CCA AAA AAT CA 3′For all individuals, DNA was isolated from leg, caudal segment or telson muscle tissue using the Qiagen DNeasy kit with standard protocols for tissue samples. A 452 bp fragment of the mitochondrial Cytochrome Oxidase I ). Sequences were inspected and aligned using Geneious 5.0.3 We included 91 sequences of General alignment statistics  were calculated using DNAsp v. 5.10 The best fitting model of sequence evolution for phylogenetic tree reconstruction was chosen using MrModeltest 2.3 Buthus scorpions obtained from genbank  In a second analysis, we included 84 sequences from bank see  resultinbank see . When thAppendix SIDNA sequences obtained from NCBI genbank.(DOC)Click here for additional data file.


Phascolarctos cinereus) is an arboreal marsupial that was historically widespread across eastern Australia until the end of the 19th century when it suffered a steep population decline. Hunting for the fur trade, habitat conversion, and disease contributed to a precipitous reduction in koala population size during the late 1800s and early 1900s. To examine the effects of these reductions in population size on koala genetic diversity, we sequenced part of the hypervariable region of mitochondrial DNA (mtDNA) in koala museum specimens collected in the 19th and 20th centuries, hypothesizing that the historical samples would exhibit greater genetic diversity.The koala (The mtDNA haplotypes present in historical museum samples were identical to haplotypes found in modern koala populations, and no novel haplotypes were detected. Rarefaction analyses suggested that the mtDNA genetic diversity present in the museum samples was similar to that of modern koalas.Low mtDNA diversity may have been present in koala populations prior to recent population declines. When considering management strategies, low genetic diversity of the mtDNA hypervariable region may not indicate recent inbreeding or founder events but may reflect an older historical pattern for koalas. Phascolarctos cinereus) was apparently a rare animal at the time of the first European settlement of Australia. Many of the relatively large mammals of eastern Australia  were first documented by Europeans almost immediately after the settlement of modern-day Sydney in 1788, but the first European sighting of a koala was not reported until 1798. The first live specimen was secured and brought to Sydney in 1803, and very few detailed accounts of the animal appeared in the decade that followed. Remarkably, it was not until 1816, nearly three decades after the arrival of Britain’s First Fleet to Botany Bay, that the koala was studied in sufficient detail to be given its original scientific name, Lipurus cinereus[th century, and koala numbers are believed to have risen drastically, such that the koala was apparently a common animal by the middle to late 1800s across much of eastern Australia [Though distributed widely across eastern Australia, the koala  ,3.Canis lupus) lost diversity due to widespread extirpation of local populations. Leonard et al. [A severe decrease in population size has been shown to lead to loss of genetic diversity in other species . For exad et al.  demonstrd et al. ,6.Macrotis lagotis). Genetic differentiation among the koala populations of Queensland and New South Wales was found to be high, while koala populations from Victoria were little differentiated [Several molecular markers have been used to examine the genetic variability of modern Australian koala populations; these have shown that genetic diversity within and gene flow between koala populations is low ,7-9. Houentiated , presumaentiated  examinedentiated  from 96 entiated  but stilThe historical DNA diversity present among koalas can be examined using museum specimens, as mtDNA analyses from museum samples have been reported from many species including Australian wildlife -14. We hThe koala has an 860 bp hypervariable mtDNA region that is adenine and thymine rich. Primer sets were designed to amplify fragments of 73 bp and 112 bp (excluding primer lengths) of this mtDNA region and were tested on a modern koala DNA dilution series. The primers were designed to target small amplicons to accommodate the potential degradation that has often been observed in archival museum samples. We attempted DNA extractions and PCR on 29 different museum samples originally collected from the 1870s to the 1980s  co-existed in Australia, and the modern koala had a wider distribution that extended to south-western Western Australia [P. cinereus. Further, if the rarity of koalas at the time of European arrival to Australia provides an indication of a historical baseline for koala abundance under the impacts of Aboriginal hunting, it can be hypothesized that the limited genetic diversity seen in modern koalas might reflect a long history of relatively low population numbers throughout the range of koalas during the Late Pleistocene and Holocene, another demographic factor that may have strongly limited koala genetic diversity in recent millennia.This provides an indication that older events may have been more important in shaping modern genetic diversity in koalas. Late Pleistocene extinctions in Australia, attributed to the impacts of Aboriginal arrival, climate change, or their combination, resulted in the extinction of most of the continent’s larger animals , as well as the extinction or decline of some medium-sized species, including koalas . Prior tustralia ,17. The Sarcophillus harrisii). A recent study by Miller et al. [Thylacinus cynocephalus) demonstrated low genetic diversity, which may have been a consequence of the separation of Tasmania from mainland Australia [Lynx pardinus) has maintained extremely low genetic diversity for at least 50,000 years [Historically low mitochondrial diversity is not unique to Australian koalas. Among Australian marsupials, low mtDNA diversity has also been reported in the Tasmanian devil . Cycling conditions were: 94°C for 4 min; 60 cycles at 94°C for 30 s, 55°C for 30s, 72°C for 30s; and at 72°C for 10 min, with the samples then held at 4°C . PCR proThe positive samples were amplified in duplicate or triplicate to control for DNA damage induced errors that almost universally characterize ancient or degraded DNA samples. A subset of 8 samples from 7 individual koalas, representing the range of collection dates that provided positive results, was selected for GS FLX sequencing using a specific multiplex identifier (MID) according to manufacturer’s instructions (Roche Life Sciences) Table . The seqGiven that DNA damage induced change appeared to be low across the koala museum specimens, we hypothesized that direct Sanger sequencing would be as effective for sequencing as the GS FLX method. Four of the seven high throughput sequenced koala samples were Sanger sequenced directly  to allow for comparison of the two methods; among them were two of the koalas that showed a higher percentage of damage induced change for certain substitutions  [GS FLX sequence reads were first sorted by MID and .fna and .qual files were transformed into fastq format using perl scripts. Primer regions were removed from the analysis by trimming both ends of each read, and only fragments 36 bp and longer were considered for mapping assembly. Each one of the seven datasets was then mapped to a reference corresponding to the n 0.5.9)  and a peAlignment and comparison of the archival sample data with modern sequences were performed using Clustal X and Bioedit, respectively. The modern koala sequences were obtained from Genbank . Minimum spanning networks of the haplotypes were generated using TCS 1.21 software at the default 95% connection limit . RarefacThe authors declare no competing interests.KT performed ancient DNA extractions, PCR and sequencing experiments. YI performed all modern koala DNA work. KT and MCAA performed bioinformatics and phylogenetic analyses for the study. KMH provided koala samples and curatorial information and wrote parts of the manuscript. KT, ALR and ADG designed the study, contributed to the experimental work and analysis and wrote the manuscript. All authors read and approved the final manuscript.FASTA formatted files of the consensus haplotype sequences for the 73 bp and 112 bp museum koala amplicons.Click here for fileFASTA formatted files of the consensus haplotype sequences for the 73 bp and 112 bp museum koala amplicons.Click here for file


Manihot esculenta) landraces from selected regions in Brazil, and examine how this variety is distributed according to origin in several municipalities in the states of Minas Gerais, São Paulo, Mato Grosso do Sul, Amazonas and Mato Grosso. High diversity values were found among the five above-mentioned regions, with 3.3 alleles per locus on an average, a high percentage of polymorphic loci varying from 88.8% to 100%, an average of 0.265 for observed heterozygosity and 0.570 for gene diversity. Most genetic diversity was concentrated within the regions themselves (HS = 0.52). Cluster analysis and principal component based scatter plotting showed greater similarity among landraces from São Paulo, Mato Grosso do Sul and Amazonas, whereas those from Minas Gerais were clustered into a sub-group within this group. The plants from Mato Grosso, mostly collected in the municipality of General Carneiro, provided the highest differentiation. The migration of human populations is one among the possible reasons for this closer resemblance or greater disparity among plants from the various regions. Based on nine microsatellite loci, the aim of this study was to appraise the genetic diversity of 42 cassava ( Based on previous studies ; 1.0 μL 10x Buffer ; 1.0 μL MgCl studies . PCR rea studies ; 2 min aGenetic diversity parameters, such as the number of alleles per locus, allelic frequency, percent of polymorphic loci, observed average heterozigosity and gene diversity (expected heterozygosity) obtained per locus and per group of accessions, were estimated using GDA software . AllelicCluster analysis with the 42 landraces was performed with NTSYS software, by using binary data whereby alleles were transformed into the presence or absence of an SSR band, as well as with the Jaccard similarity coefficient and UPGMA (Unweighted Pair Group Method with an Arithmetic Mean) method. A principal component analysis with binary data was also carried out using the A total of 46 alleles were amplified with nine SSR loci analyzed in the 42 landraces, the number of alleles observed per locus varying from 3 to 6 alleles . The numThe groups MS, AM and MT revealed 100% polymorphism, while those of MG and SP presented 88.8%. The observed heterozygosity, 0.265 on an average, varied from 0.233 to 0.288, while higher values, varying from 0.427 to 0.677, were estimated for gene diversity, with an average of 0.570.HT = 0.635) was observed in all the 42 cassava landraces, thus confirming the high variability which can be found in this cross-pollinated and vegetatively propagated crop. However, most of this SSR variability was concentrated within ethno-variety groups (HS = 0.552), while lower values were due to the proportion of diversity distributed among the groups themselves (GST' = 0.131)  .The dendrogram in Similar results were observed through principal component analysis when examining the distribution of the five groups in the four quadrants of a scatter plot compounded with the two principal components, and which explained 25.8% of total variation (data not shown), besides indicating the high genetic variability of the material.et al., 2000; In our study, high genetic diversity was detected in all the five regions in Brazil, with an average of 5.0 alleles per locus, which is in agreement to similar studies with cassava  on the contrary to group diversity (GST' = 0.131). A larger portion of diversity in this study was found to be concentrated within the groups themselves (In addition to measuring genetic diversity, one of the objectives in our study was to verify how landraces originating from five different regions in Brazil were mutually related. Results showed a greater proximity of landraces from the states of São Paulo, Mato Grosso do Sul and Amazonas. The landraces from Minas Gerais were clustered into a sub-group in the dendrogram , within The most differentiated landraces were those from General Carneiro, Mato Grosso. In Mato Grosso, traditional farming is undertaken by local populations, particularly by indians, “quilombolas” and “pantaneiros”. After the 50's more recent settlers began coming from diverse regions of the country, mainly from the south, northeast, Goiás and Minas Gerais . GeneralBoth sweet and bitter varieties of cassava are grown by farmers in the States of São Paulo  and Matoin situ conservation by traditional farmers, also known as on farm in situ conservation, of an important genetic and autochthon resource.In this study we intended to contribute to a better knowledge of cassava genetic diversity and distribution within and among different regions in Brazil, and promote


The aetiology of congenital clubfoot is unclear. Although studies on populations, families, and twins suggest a genetic component to the aetiology, other studies have identified environmental factors. The purpose of this study was to calculate heritability in order to determine to what extent genetic and/or environmental factors contribute to the aetiology of congenital clubfoot and to asses whether there was a change in the prevalence over time.The Odense based Danish Twin Registry is unique as it contains data on all the approximately 85,000 twin pairs born in Denmark over the last 140 years. All 46,418 twin individuals born from 1931 through 1982, who had earlier consented to contact, received a 17-page Omnibus questionnaire in the spring of 2002. Data were analysed with structural equation models to identify the best fitting aetiological model based on a balance of goodness-of-fit and parsimony and to estimate heritability.We found an overall self-reported prevalence of congenital clubfoot of 0.0027 . Fifty-five complete (both twins answered the question) twin pairs were identified representing 12 monozygotic, 22 same-sex dizygotic, 18 opposite-sex dizygotic, and 3 with unclassified zygosity. The model with only environmental factors (CE) was best fitting based on AIC, and the model with an additive genetic factor (ACE) came in second. Due to the small statistical power, we hypothesise that the model with both genetic and environmental effects (ACE) was the better model. Choosing the ACE-model we found a heritability of clubfoot of 30 %. Regression coefficient for age was −0.002 (−0.011 to 0.005), indicating that there has been no change in prevalence of clubfoot over the 50-year age span we examined.We conclude that non-genetic factors must play a role, and a genetic factor might contribute, in the aetiology of congenital clubfoot. The aetiology of congenital clubfoot is speculative and the influence of genetic and environmental factors remains unclear. Although studies on populations, families, and twins suggest a genetic component, the mode of inheritance does not fit the classic inheritance patterns –6.Twin studies are useful when determining whether or not a disorder is genetic in aetiology. Monozygotic (MZ) twins inherit identical genes, while dizygotic (DZ) twins, like siblings, share 50 % of their genes. All twins share an identical environment before birth, and regarding congenital disorders, it is therefore possible to study the effect of genetic factors. Increased rates of double-affected MZ twins compared to DZ twins (concordance) indicate a genetic aetiology of a disorder. In clubfoot patients, the higher concordance in MZ twins (33 %) compared to DZ twins (3 %) is a strong indication of a genetic component . StudiesVarious environmental influences have been shown to increase the risk of clubfoot in that they may affect the developing foetus in different ways including via genetic alterations, deformation, or growth arrest. The incidence has been reported to rise with increased maternal alcohol consumption , smokingIn animal studies a clubfoot-like deformity has been induced in different ways, and with the human studies in mind the most interesting are: the raise of body temperature of pregnant hamsters , irradiaDespite previous studies, the aetiology of congenital clubfoot and the role of genetic and environmental factors remains unclear. The purpose of this study was to establish a congenital clubfoot twin cohort that enables us to estimate heritability, in order to determine to what extent genetic and/or environmental factors contribute to the aetiology of congenital clubfoot and to asses whether there was a change in the prevalence over time. Furthermore, we estimate the best fitting aetiological model for the phenotype.The Odense-based Danish Twin Registry (DTR) is quite unique, as it contains data on all approximately 85,000 twin pairs born in Denmark over the last 140 years. It was the first population-based twin registry and one of the largest existing today . All twiThe information on zygosity in the DTR is based on a validated method consisting of four questions of similarity with an accuracy of 95 % .The prevalence of self-reported congenital clubfoot was calculated using the total number of answers as the general population and stratified according to gender.Heritability is defined as the proportion of the total phenotypic variance that is attributable to genetic variance. Heritability was estimated in a generalized structural equations model, which combines a structural part with a liability threshold part. A liability threshold model assumes that the dichotomous distribution of clubfoot (affected versus non-affected) reflects an underlying normally distributed liability in the population. When a threshold value is exceeded, an individual is affected; otherwise not. Different thresholds in different groups, e.g. the two sexes or siblings versus unrelated individuals, reflect different loads of genetic and environmental risk factors in the groups, and thereby also the prevalence of the trait. Adjustment for age and sex effects on clubfoot was done through a probit regression model on the thresholds. These are standard assumptions in quantitative genetic analysis of categorical traits .The structural equation model quantifies sources of individual variation by decomposing the total phenotypic variance into genetic and environmental variances . The genTetrachoric correlation is a special case of the polychoric correlation, applicable when the observed variables are dichotomous. The tetrachoric correlations are used to decide which etiological model to fit. Selection of the best-fitting submodel is based on a balance between goodness of fit and parsimony . The AkaA total of 46,418 twins received and 34,944 (75 %) returned the questionnaire. 34,485 (99 % of the responders) answered the question ‘Were you born with clubfoot?’ The gender distribution in these was 15,731 (46 %) males and 18,754 (54 %) females.p = 0.29).Ninety-four answered ‘Yes’ to the above question, giving an overall self-reported prevalence of congenital clubfoot of 0.0027 (95 % c.i. 0.0022–0.0034), with male and female prevalences of 0.0031 (95 % c.i. 0.0022–0.0041) and 0.0025 (95 % c.i. 0.0018–0.0033), respectively (Fifty-five complete (both twins answered the question) twin pairs were identified, representing 12 monozygotic (MZ), 22 same-sex dizygotic (DZss), 18 opposite-sex dizygotic (DZos), and 3 pairs with unclassifiable zygosity. Only twins with a classifiable zygosity were used in the subsequent analysis.Tetrachoric correlations were 0.81 (0.49–0.95) for MZ twins and 0.56 (0.25–0.77) for DZ twins; the difference in correlations indicates a possible genetic component. Variance component models ACE, ADE, and submodels were fitted to the data. Results are presented in Table Choosing the ACE model, we found a heritability of clubfoot of 30 %.Regression coefficient for age was −0.002 (−0.011 to 0.005), indicating that there has been no change in prevalence of clubfoot over the 50-year age span we examined.The present study is based on one of the largest twin cohorts in the world. In the study we propose that the best model was the ACE, suggesting both a genetic and an environmental component in congenital clubfoot.We calculated a regression coefficient of age. It tells us whether there is a tendency for the diagnosis to become more or less frequent over time. Since it is very close to 0 (−0.002), we believe that there has been no change in prevalence of clubfoot over the 50-year age span we examined.Twin studies are accepted as unique in discriminating the contribution of genetic and environmental factors in the phenotypic variance. This discrimination is purest in a congenital disease, since any influence of differences in the postnatal environment is avoided. It is important that the twin cohort is population-based, since compared to population-based studies the disease-based studies have a tendency toward overrepresentation of MZ and concordant pairs , 28. HowThe cardinal challenge in questionnaire-based studies is the identification of the patients. In the present study on congenital clubfoot we consider this to be a reliable method, since this distinct disorder is not easily overlooked or mistaken for other frequent disorders. However, some patients may have other foot deformities such as metatarsus varus, pes cavus, etc. The treatment of clubfoot in these cohorts was splinting and/or operation. The final results of the treatment were inferior to the present-day Ponseti technique. We do not expect patients to be unaware of having had clubfoot, however well corrected. Less than 50 % of clubfeet will be corrected in one operation, around 80 % in two operations, and approximately 90 % after three operations . Long-teThe prevalence of 0.27 % is higher than the 0.1–0.2 % reported for other Scandinavians. This might be due to an over-estimation in the self-reported “diagnosis”. If not, we must conclude that in this large and comprehensive twin cohort, there is an increased risk of clubfoot .Previous studies have demonstrated that genetic factors might play a significant role; we found evidence of an environmental component and a probable genetic component in the aetiology of congenital clubfoot. Because of the incidence of congenital clubfoot and despite the size of this big twin cohort, the number of twins with clubfoot only yielded a relatively small amount of data. Due to this and the very similar AIC, we have chosen the largest model to explain heritability of clubfoot. Based on the findings presented here, we conclude that environmental factors must play a significant role in the aetiology of congenital clubfoot. And there is a possible genetic factor as well and a heritability of 30 %. Our finding lends no strong support for the theory of a major genetic factor in clubfoot.To clarify the role of inheritance in congenital clubfoot, further studies are necessary, and the focus should be on interacting factors, thus rendering a certain genetic constitution more or less susceptible to environmental influences and epigenetic changes.


AbstractCarabus violaceus s. str. and the subspecies Carabus violaceus purpurascens, which is ranked as a distinct species by some authors. We studied 12 Carabus violaceus populations from a 30 km transect of ancient and recent forests in north-western Germany. We analyzed three polymorphic enzyme loci, classified the elytron sculpture and measured the shape of the aedeagus tip of the specimens. Carabus violaceus showed secondary gradients both in allozyme markers and morphometric characters in our study area. A genetic differentiation of 16% between the populations is high but lies within the range of intraspecific variability in habitat specialists of the genus Carabus. Populations had no significant deficit of heterozygotes. We found many hybrid populations in terms of morphological properties. This study highlights the conservation value of PageBreakancient woodland and the consequences of landscape connectivity and defragmentation on the genetic setting of a ground beetle. Moreover, it shows that differences in the external shape of male genitalia do not prevent gene flow within the genus Carabus. Thus, the establishment of species status should not exclusively be based on this property.Only very few cases have documented that an increase in connectivity after a period of fragmentation in ecological time has had an effect on the distribution, genetic structure and morphology of stenotopic species. In this study we present an example of clinal variability in a woodland ground beetle as a result of changes in the connectivity of a landscape during the last two centuries. The study area hosts both the nominate form  The history of a landscape has a tremendous effect on both the species composition of communities and assemblages on the one hand and the genetic variability of species on the other. This is especially true for woodlands, which have become highly fragmented since the Middle Ages in large areas of north-western Europe including Britain, southern Scandinavia, Belgium, the Netherlands and the lowlands of northern Germany . AgainstAt the genetic level some studies have shown a strong differentiation between remnants of stenotopic woodland species in ancient woodlands that were at least in former times isolated from one another e.g. . Only veCarabus violaceus Linné 1758. Due to its flightlessness, this ground beetle has a low power of dispersal. We used two sets of markers for thePageBreak analysis, morphometric characters and allozymes, in order to determine typical features of the source populations and their geographic distribution within the contact zone. The results are of increased significance, as the study area hosts not only the nominate form but also Carabus violaceus purpurascens Fabricius, 1787, which is ranked as a distinct species by some authors  from colonizing neighbouring forests, so that the beetle is regarded as highly sensitive to fragmentation of forests  were homogenized in 600µl 0.15M Tris-Citrate buffer . After centrifugation, the homogenates were applied to vertical polyacrylamide slab gels and electrophoresis was run at 3°C. Both the mixture of the polyacrylamide slab gels and the staining was performed according to The abdomina of HO) and mean gene diversity (HE)   were estity (HE) . Tests fity (HE) . Data wePoecilus lepidus by Data were investigated for the occurrence of clinal variation by spatial autocorrelation analysis implemented in SGS ver. 1.0 d . Male genitalia were prepared and mounted on cards. The remains of the exoskeletons were pinned to dry and deposited in the entomological collection of Thorsten Assmann, Bleckede .Carabus violaceus were analyzed. Since measurements of the aedeagus tips provide suitable characteristics to distinguish Carabus violaceus s. str. and Carabus violaceus purpurascens  to nine (GPI). Allele frequencies, expected and observed heterozygosities and FIS values are shown in Table 1. No significant deviations from Hardy-Weinberg equilibrium were observed for any of the populations or loci after correcting for multiple tests . There seems to be a tendency of populations to display a positive FIS, i.e. a deficit of heterozygotes. However, after correction for multiple testing by Fstat , no significant deficit or excess of heterozygotes were found. No significant linkage disequilibrium was found, thus the studied loci can be interpreted as independent markers.The overall FST value was 0.160 and ranged from 0.127 (GPI) to 0.201 (EST-X). Pairwise population differentiation in FST between the 78 pairs in our study ranged between 0.011 and 0.501, with a significant differentiation for 54 population pairs after standard Bonferroni corrections .Spatial genetic structure analysis revealed gradients in allele frequencies in the EST-X locus, in the MPI locus, and in the whole sample , Table 3Carabus violaceus purpurascens  showed the elytron sculpture typical of urascens . No specCarabus violaceus violaceus, and smallest in one of the populations close to the southern ancient woodland Gehn, which is characteristic of Carabus violaceus purpurascens. Several population comparisons between these two groups display significant differences  = 71.157 p < 0.001; AedMax/AedMin: H = 22.737 p = 0.019). The pattern of AedMax is very similar to that of the elytron properties. The maximum width of the genital tip is generally highest in populations adjacent to the northern ancient woodland Börsteler Wald, which is characteristic of ferences . HoweverCarabus violaceus shows clear gradients both in allozyme markers and morphometric characters in our study area. Clinal variability can be caused by adaptation to geographically changing environmental conditions and by secondary contacts. In the first case, selection is the driving force to create specific patterns of genetic differentiation  that are inhabited by Carabus violaceus are located several kilometres south and southeast of Bramsche . Such evidence is strongly dependent on landscape connectivity and history  the northern and southern edge of the study range. Our results suggest that Carabus violaceus s. str. survived in the north, while Carabus violaceus purpurascens survived in the south of the study area. These refuges correspond to the overall geographic distribution of the two subspecies  within the hybrid zone and at the same time a strong differentiation of the postulated source populations and (2) the concordance of different clines concerning different markers and including numerous individuals with intermediate characters, indicate that the populations are real hybrid populations and not co-occurring species which hybridize occasionally or up to the level of some percentages such as Carabus auronitens and Carabus splendens in the eastern Pyrenees and Carabus glabratus and Carabus hortensis in north-western Germany  where, in some years, the proportion of hybrids exceeds 40% .The shape of the aedeagus tip is not only used for taxonomic distinction between different carabids, but also for a justification of species rank . Some fo Germany . At leaseeds 40% ; 1990. HCarabus: differences in the male genitalia  do not prevent excessive gene flow and should thus not be used as a character to establish species’ status of forms  prevents the detection of species with genitalia differentiated at the subspecies or population levels  despite strong differences in the shape of the aedeagus of both forms has consequences for taxonomy within the genus orms cf. . Moreoven levels . TherefoOhomopterus demonstrates that (1) copulatory pieces can reduce cross-breeding and (2) the fitness costs of interspecific matings are high in the given species (e.g. Carabus. Moreover, morphological characters to differentiate Carabus species should be clear without ambiguity as is the longitudinal striation in Carabus violaceus purpurascens in comparison to the smooth elytra of the nominate form.The strong differentiation of the endophallus in some species of the subgenus ies e.g. . In thesCarabus violaceus has the potential to be an important modelPageBreak species in the fields of conservation genetics and evolutionary biology at the interface to systematics.Numerous questions that are important for a better understanding of the investigated hybrid zone are still unanswered, e.g. if the contribution to gene flow into the hybrid zone is the same for both sexes or if the diffusion rate of markers differs. These and others can only be studied when further molecular, both mitochondrial and nuclear markers are analysed. mtDNA analysis, which is presently being conducted at our institute, may enable us to further estimate evolutionary divergence time between the two investigated subspecies with the help of phylogenetic analysis. The results of the present study reveal that