Sarcoptes scabiei, affecting different mammalian species worldwide including the Iberian ibex (Capra pyrenaica), in which mortalities over 90 % of the population have been reported. No efficient diagnostic methods are available for this disease, particularly when there are low mite numbers and mild or no clinical signs. In this study, three enzyme-linked immunosorbent assays (ELISA) developed for dog (ELISA A), Cantabrian chamois (Rupicapra pyrenaica parva) (ELISA B) and Alpine chamois (Rupicapra rupicapra) (ELISA C), were evaluated to detect specific antibodies (IgG) to sarcoptic mange in Iberian ibex sera.Sarcoptic mange is a contagious skin disease caused by the mite Serum samples from 131 Iberian ibexes  were collected from 2005 to 2012 in the Sierra Nevada Natural and National Parks (southern Spain). Based on visual inspection, ibexes were classified into one of three categories, namely healthy (without scabietic compatible lesions), mildly affected (skin lesions over less than 50 % of the body surface) and severely affected (skin lesions over more than 50 % of the body surface). The optimal cut-off point, specificity, sensitivity and the area under the curve (AUC) were calculated, and the agreement between tests was determined. Moreover, differences in the optical density (OD) related to scabies severity have been evaluated for the best test.ELISA C showed better performance than the two other tests, reaching higher values of sensitivity (93.0 %) and specificity (93.5 %) against the visual estimation of the percentage of affected skin, chosen as the gold standard. Significantly higher concentrations of specific antibodies were observed with this test in the mildly and severely infested ibexes than in healthy ones.Our results revealed that ELISA C was an optimal test to diagnose sarcoptic mange in the Iberian ibex. Further studies characterizing immune response during the course of the disease, including spontaneous or drug induced recovery, should follow in order to better understand sarcoptic mange in Iberian ibex populations. Sarcoptes scabiei . This parasite is worldwide distributed [Capra pyrenaica) [Rupicapra pyrenaica parva) [Ammotragus lervia) [S. scabiei are characterized by pruritus, drying, peeling, alopecia, hyperkeratosis and scab formation. In population terms, this parasite is able to cause severe demographic downturns, up to above 90 % on occasion [Sarcoptic mange is a contagious skin disease caused by the mite tributed , causingtributed . Wild untributed –6. All wrenaica) , Cantabra parva)  and aoud lervia) . The clioccasion . In factoccasion .Iberian ibex is a mountain ungulate endemic to the Iberian Peninsula . AndalusSarcoptes scabiei stimulate E and G immunoglobulin production in infested hosts [S. scabiei in dogs [ed hosts –33, incled hosts , 35. Dif in dogs , 37, pig in dogs , 39, wil in dogs  or chamo in dogs . HoweverIn this study, three IgG indirect ELISA tests were compared as diagnostic tools of sarcoptic mange in ibexes showing different lesional severity. In particular, the objectives of this study are: (i) to estimate the optimal cut-off points, specificity and sensitivity of the ELISA tests in Iberian ibex; (ii) to determine the agreement between tests; and (iii) to determine if a correlation between mange severity and the detected levels of humoral immune response (IgG) exists.Serum samples from 131 healthy and scabietic Iberian ibexes were collected from 2005 to 2012 in Sierra Nevada Natural and National Parks   [n = 117) [n = 120)  were used. ELISA B is based on a structural antigen of the mite (Ssλ20∆B3), whose encoding cDNA was identified by screening of S. scabiei var. hominis library using the sera from an infected chamois, and expressed in Escherichia coli as a unitary recombinant antigen [S. scabiei var. suis antigen  instead of the red fox (Vulpes vulpes) S. scabiei antigens originally used [The Iberian ibex sera were tested by three indirect ELISAs developed for dog  , Cantabrn = 117)  and Alpiied from ). ELISA  antigen . ELISA C antigen  by usinglly used . This telly used .Samples were analyzed in duplicate, and in triplicate in ELISA C.The optical density (OD) was read at 450 nm in ELISA A and B, and at 405 nm in ELISA C, and was expressed as Optical Density percentage (OD %) using the following formula:Ten sera from healthy Iberian ibexes  without previous contact with mange were used as negative controls, whereas ten sera from actively sarcoptic mange-infested Iberian ibexes were used as positive controls. The same negative and positive control sera were used for the three ELISA tests, and all controls consistently showed low (negative controls) and high (positive controls) values for the three ELISA tests.The optimal cut-off points between positive and negative samples was estimated using the Youden index, maximizing the difference between true positive rate (sensitivity) and false positive rate (1 - specificity). Thereby, the maximum of sensitivity and specificity is achieved , 47. Oncversus false positive rate (1 - specificity), was used to determine the diagnosis accuracy of the ELISA tests [After that, the area under a receiver operating characteristic (ROC) curve, a graph of true positive rate (sensitivity) SA tests , 50.The agreement between the ELISA tests was evaluated by the Cohen’s Kappa coefficient  and BlanSince ELISA results did not fit a normal distribution, as assayed by Agostino skewness test and Bonett-Seier test for Geary kurtosis, Kruskal-Wallis with Mann-Whitney tests with Bonferroni correction were used to evaluate whether optical density (a proxy for immunoglobulin G production) was influenced by mange severity . This analysis was performed only with the optical density values from the ELISA test showing the highest sensitivity and specificity against the gold standard .moments” package [BlandAltmanLeh” package [irr” package [OptimalCutpoints” package [Although sex and season determine sarcoptic mange severity and progression in Iberian ibex , 54, 55, package , Bland-A package , the Kap package , and the package .The cut-off points, sensitivity and specificity obtained for the three ELISA tests are shown in Table k) indicated that there is not a good strength of agreement between the three tests. The ELISA B and C showed the higher strength of agreement, which was only moderate, whereas the strength of agreement between the ELISA A and C was still lower (fair) and the ELISA A and B showed the lowest strength of agreement (slight) [Bland-Altman plots show that the three ELISA tests are not interchangeable, especially at high values of OD Fig. . Along t(slight) .Fig. 2Blχ2 = 73.99, df = 2, P < 0.0001) among the three categories of sarcoptic mange status were found. The healthy ibexes showed statistically significant lower % OD values than the mildly  and severely  infested ibexes. The difference between the higher % OD of the severely infested ibexes and the lower % OD of the mildly infested ones was close to significance .The relationship between sarcoptic mange severity and the % OD values was analyzed only for ELISA C, due to the better performance of this test as compared with the other two Table . SignifiS. scabiei in the Iberian ibex, which showed different performance in terms of specificity and sensitivity. Furthermore, the OD % varied among degrees of sarcoptic mange infestation for the best performing test.This study compared three ELISA tests for the detection of specific serum antibodies to S. scabiei in this species, showing higher sensitivity and specificity and an AUC close to 1. This test was validated for the detection of IgG in Alpine chamois lung extracts with a sensitivity of 100 % and a specificity of 98.6 % (data not published). Moreover, the ELISA C allowed the differentiation of healthy and mangy ibexes. ELISA B revealed lower specificity and sensitivity than previously reported in Cantabrian chamois and red deer, with a specificity of 97 %, and a sensitivity of 100 % and 75 % in chamois and red deer [S. scabiei in wild ungulates, and the sensitivity and specificity of the test in dog are 92.1 % and 94.6 %, respectively.To the best of our knowledge, this is the first time in which several ELISA tests are compared and evaluated for the diagnosis of sarcoptic mange in Iberian ibex. Based on results of this study, ELISA C was the best performing test to detect specific antibodies against red deer , 61, resS. scabiei strains exists, each host species responds differently to different mite varieties [The avidin-biotin detection system used in the ELISA C could be one of the reasons explaining the best performance of ELISA C as compared to ELISAs B and C. This method has the potential to increase the sensitivity of the indirect ELISA, since avidin has four binding sites for biotin, resulting in an essentially irreversible complex which is stable in later washes and incubations . Althougarieties , 64. TheS. scabiei were detected) could be explained by low levels of circulating IgG in recently infested ibexes or in chronic infestations. The clinical signs of sarcoptic mange may appear before IgG detection in different species [False negative results  [versus lung extracted). However, false positive ibexes could also be due to individuals having had previous contact with S. scabiei and having recovered from the clinical phase of the disease. Hosts infested with S. scabiei develop resistance to re-infestation [S. scabiei-positivity by ELISA in the severely infested ibexes suggests that IgGs are not protective against mange. In general, protection against mange has been associated with a cell-mediated immune response as well as with a humoral immune response [S. scabiei in the skin, but could also correlate with mite load.A low test specificity  can be explained by a lack of detection of minute skin lesions , 40, sub chamois , have be chamois , 44, 45.ula sp.)  or diffeula sp.) , 71. ELIestation  and healestation , 35. In response , 72, 73.response . The incresponse , and theresponse . MoreoveS. scabiei infestation in Iberian ibex; (ii) the correlation between parasitic load, mange severity and immune response; and (iii) the influence of sex [The evaluation of the ELISA tests carried out in this study and the identification of ELISA C as a reliable tool for sarcoptic mange diagnosis in Iberian ibex will be useful in future research. Nevertheless, the limitations in sensitivity and specificity should be considered while assessing the results, and more studies on (i) the dynamics of the antibody response after e of sex , 54 and e of sex , 55 on tIn conclusion, the ELISA developed for diagnose sarcoptic mange in Alpine chamois  has been validated in this study as an effective test to be similarly applied in Iberian ibex. This can be helpful for completing the legal requirements for the transport of wild ungulates  and for


We interrogated the genetic modulation of maternal oxytocin response and its association with maternal behavior using genetic risk scores within the oxytocin receptor (OXTR) gene. We identified a novel SNP, rs968389, to be significantly associated with maternal oxytocin response after a challenging mother-infant interaction task (Still Face Paradigm) and maternal separation anxiety from the infant. Performing a multiallelic analysis across OXTR by calculating a cumulative genetic risk score revealed a significant gene-by-environment (G × E) interaction, with OXTR genetic risk score interacting with adult separation anxiety to modulate levels of maternal sensitivity. Mothers with higher OXTR genetic risk score and adult separation anxiety showed significantly reduced levels of maternal sensitivity during free play with the infant. The same G × E interaction was also observed for the extended OXTR cumulative genetic risk score that included rs968389. Moreover, the extended cumulative OXTR genetic risk score itself was found to be significantly associated with maternal separation anxiety as it specifically relates to the infant. Our results suggest a complex montage of individual and synergistic genetic mediators of maternal behavior. These findings add to specific knowledge about genetic regulation of maternal oxytocin response in relation to maternal adjustment and infant bonding through the first few months of life. Humans are social beings who are biologically tuned to form selective and enduring bonds with other individuals. These social bonds provide protection and caregiving and offer comfort during times of distress . A plethThe involvement of oxytocin in regulating the mother's responsive engagement with her infant has been studied using the Still Face Paradigm (SFP) . SFP is Oxytocin, however, may be associated with both positive and adverse outcomes depending on the level of nurturance or adversity in the early environment of an individual . For insGenetic variability within the OXTR gene may modulate risk for psychiatric and behavioral symptoms, and this may be driven by a combination of cumulative genetic effects across the OXTR locus. Cumulative genetic risk score across OXTR, CD38, and AVPR1a, previously associated with psychopathology, sociability, and caregiving of the mother, has been shown to interact with trauma-exposure in children to predict higher levels of PTSD, anxiety disorders, conduct disorders, and ADHD , furtherThe overarching aim of the study was to investigate relations involving blood oxytocin levels, aspects of the oxytocin gene, and maternal caregiving behavior. Specifically, we aimed to evaluate (a) the genetic mediators of maternal oxytocin response following a challenging mother-infant interaction task ; test if the same genetic mediators would be associated with (b) maternal behavior following a mother-infant separation and interrogate (c) whether the genetically mediated maternal behavior was driven by separation anxiety. We hypothesized that mothers with a genetic predisposition to decreased maternal oxytocin response would be more likely to exhibit reduced maternal caregiving behavior, and this relationship would be mediated by separation anxiety.n = 668) attending a general hospital antenatal clinic, the details being provided in Eapen et al., 2014  and the Braineac database of the UK Brain Expression Consortium  revealed that rs968389 was significantly associated with gene expression of the OXTR gene within the caudate  brain tissue  as well as the frontal cortex (


Calotropis (Asclepiadaceae) is comprised of two species, C. gigantea and C. procera, which both show significant economic potential for use of their seed fibers in the textile industry, and of their bioactive compounds as new medicinal resources. The available wild-sourced germplasm contains limited genetic information that restricts further germplasm exploration for the purposes of domestication. We here developed twenty novel EST-SSR markers and applied them to assess genetic diversity, population structure and differentiation within Calotropis. The polymorphic information index of these markers ranged from 0.102 to 0.800; indicating that they are highly informative. Moderate genetic diversity was revealed in both species, with no difference between species in the amount of genetic diversity. Population structure analysis suggested five main genetic groups (K = 5) and relatively high genetic differentiation (FST = 0.528) between the two species. Mantel test analysis showed strong correlation between geographical and genetic distance in C. procera  while C. gigantea showed no such correlation . This study provides novel insights into the genetic diversity and population structure of Calotropis, which will promote further resource utilization and the development of genetic improvement strategies for Calotropis.The genus  Calotropis (Asclepiadaceae), which is native to the tropics and subtropics of Africa and Asia, has great potential for use as a fiber and medicinal plant2. Its long, fine seed hair (similar to that of cotton) is a high quality fiber, while the sap contains unique chemical compounds that have the diverse bioactive and pharmacological properties indicative of potential for new drug discovery3. In addition, Calotropis’ growth properties include drought hardiness, fast growth after establishment, a short reproductive cycle and adaptation to salty soils. This suggests that it is an important shrub which could serve as a key plant for ecological restoration in arid and semiarid regions5.The genus Calotropis procera (mainly distributed in the tropic and subtropics of Africa) and Calotropis gigantea (mainly found in tropic and subtropics of Asia). Due to their morphological similarity, with major differences occurring only on the floral structures, distinguishing them during the non-flowering season is difficult4. Taxonomically, C. procera is distinguished from C. gigantea by its flower characteristics such as subglobose flower buds, corona as long as gynostegium and erect corolla with five petals with dark purple tips4. Development of a genetic marker to distinguish the two species would facilitate species identification outside the flowering season.There are two closely related species of the genus; Calotropis products have mostly been explored from wild populations, which raises concerns regarding their sustainability and low product yield. Currently, there is a Calotropis domestication trial as a fiber source underway in Kenya . The available wild germplasm has limited genetic information, with few molecular markers pinpointed that separate the two species. This currently limits germplasm exploration and genetic improvement of Calotropis. To date, various marker systems have been used in population genetic analysis of Calotropis. In flower polymorphic (pink and white) C. gigantea, genetic analysis using random amplified polymorphic DNA markers (RAPDs) found rich genetic diversity and high genetic similarity6. In 18 accessions of C. procera studied in Egypt, RAPDs unveiled a high polymorphism level7. Moreover, RAPDs were also used to discriminate thirteen salt tolerant plant species including C. procera according to their genetic relationships8. Analysis of three populations of C. procera on the basis of their climo-geographic adaptations using zymograms of superoxide dismutase and peroxidase indicated high genetic variation9. Further, SDS-PAGE for protein and isozyme in genetic variation analysis successfully discriminated between the genotypes of the studied C. procera populations10. The analysis of C. procera from Benin using Amplified Fragment Length Polymorphism (AFLP) uncovered high genetic variation among the sampled populations11.Utilization of C. procera. In addition, AFLP and RAPDs are dominant markers which cannot distinguish between heterozygotes and homozygotes, thus their use may over-estimate genetic diversity, making them less robust for use in genetic analysis12. On the other hand, isozymes are phenotypic markers whose usefulness is limted by the requirement for fresh samples, the fact that they are influenced by environmental changes and that only a few loci are analysed13. Expressed sequence tags-simple sequence repeats (EST-SSR) are more robust and efficient in population genetic analysis based on their intrinsic characteristics such as codominance, multi-allelic, highly reproducible, transferability across taxon, hypervariable and ubiquitous distribution across the genome14. In particular, EST-SSRs target protein coded are conservative and their markers are usually transferable within species, thus they are often applied to investigate the genetic diversity and population structure across related species18.However, these previous studies were based on only a few samples within a limited sampling area and mainly focused on 19, we generated transcriptome data for C. gigantea. Using that data, in the present study, we developed 20 polymorphic and efficient EST-SSR markers. These markers were used to investigate genetic diversity and differentiation and perform population structure analysis for ten populations collected from Asia and Africa , selected at random from the sampled populations. Among the 170 selected primer pairs, 151 amplified successfully across C. gigantea and C. procera while 19 pair primers did not yield any PCR products at diverse annealing temperatures. Out of the 151 successful primer pairs, 109 yielded amplification products of the expected size, and the other 42 primer pairs generated PCR products that were larger or smaller than expected or unspecific bands. Of those successfully amplified, 20 primers (13.2%) showed polymorphisms whereas 89 were identified as monomorphic. The 20 polymorphic EST-SSR primers  to eight (CG 84), with an average of 4.85 alleles per locus, whereas in C. procera samples, 84 alleles were identified with an average of 4.2 alleles per locus (Table HO) had a mean of 0.223 in C. procera and 0.194 in C. gigantea while the mean genetic diversity (HS) was 0.487 and 0.379 for C. gigantea and C. procera respectively. PIC values ranged from 0.065 to 0.769, with the mean = 0.429 in C. gigantea and 0.045 to 0.670, with the mean = 0.338 in C. procera (Table C. procera and C. gigantea accessions display similar amount of genetic diversity (Table PIC ranged from 0.102 (CG71) to 0.800 (CG28). Inbreeding coefficient (FIS) values ranged from −0.365 to 0.879 (mean = 0.167) in C. gigantea, and from −0.597 to 0.951 (mean = 0.177) in C. procera  of the ten populations ranged from 0.157 to 0.363 with an average of 0.245. In C. gigantea the average HS was 0.249. The Nepal population showed the highest (HS = 0.363), while the Dongchuan population had the lowest genetic diversity values (HS = 0.157). In C. procera the Mali S population had the highest genetic diversity (HS = 0.339) and the lowest was the Kibwezi population (HS = 0.195) with a mean of 0.248. The average levels of observed heterozygosity (HO) across all populations was HO = 0.214 while in C. procera and C. gigantea HO was 0.223 and 0.198 respectively, while within species the level of genetic diversity (HS) was 0.249 and 0.256 for C. gigantea and C. procera, respectively. Allelic richness (AR) ranged from 2.25 (Honghe) to 3.3  in C. gigantea and 2.05 (Kibwezi) to 2.85  in C. procera. The fixation index (F) for all the ten populations ranged from 0.048 (Kibwezi) to 0.320 (Honghe). Using various genetic diversity parameters there was no noticeable difference between the two species , in concordance with the results of PCoA and STRUCTURE analyses.PCoA is often used to show genetic similarity among populations, with populations clustered according to their geographical location and species identity. Our PCoA results showed that the two species were clearly clustered and that the first two axes explained 64.62% of the total observed variation, suggesting that a distinct genetic structure exist between era Fig. . Due to era Fig. . In addiC. procera at K = 2, the populations grouped into West Africa  and East Africa populations . At K = 3 Baringo population separates from the rest of the East Africa populations, with Tanzania and Mali S populations showing admixtures  approaches respectively. Since delta K may erroneously result into K = 2 Figs  and S3 bsee Fig. . In C. goup Fig. . These roup Fig. .Figure 5C. procera and C. gigantea, with a FST value of 0.528. When AMOVA analysis was assessed within each species, C. procera had most genetic variation (55.35%) partitioned within individuals while in C. gigantea among-population divergence was highest (57.01%). The genetic differentiation (FST) for C. procera and C. gigantea were 0.366 and 0.57, respectively  and the highest differentiation (FST = 0.491) was between the Baringo (C. procera) and Hainan (C. gigantea) populations , while in C. gigantea the Dongchuan and Hainan populations were the most differentiated (FST = 0.451) (Table FST) ranged from 0.036 (CG71) to 0.707 (CG83) with a mean of 0.405 in C. gigantea whereas in C. procera the FST ranged from 0.024 (CG35) to 0.764 (CG83) with a mean of 0.291  Table . At the 91 Table .Table 2A18. Our Mantel test analysis showed a significant correlation between geographic and genetic distance among C. procera populations    Fig. , whereas21) Fig. .Figure 7Calotropis. EST-SSRs have been found to be important molecular markers for detecting genetic diversity, structure, and demography as well for applied and experimental research on plant populations20. These markers show high transferability to closely related species of same genus or even family since they are associated with transcribed genes conserved among homologous genes21. Here, 151 primer pairs (88.8%) were successfully amplified PCR fragments from 170 pairs that had been designed from unigenes generated from transcriptome data. 19 primer pairs did not yield any PCR product. This could have been a result of insertions, lack of specificity, assembly errors, chimeric primers and the presence of large introns22. The polymorphism rate of Calotropis EST-SSR primers was 13.5%, which is comparable to that reported from other plants such as Neotropteri nidus (11%)22 and sesame 11.9%23.This study represents the first attempt to develop and utilize EST-SSR markers to examine genetic diversity in the genus PIC is usually defined as low (PIC < 0.25), medium (0.5 > PIC > 0.25) or high (PIC > 0.5)24. Based on this criteria, the EST-SSR markers developed for Calotropis have a moderate level of polymorphism, with seven having high polymorphism, 12 having medium polymorphism and only one having a low PIC value  had a slightly higher genetic diversity17. Overall, the developed EST-SSR markers showed sufficient polymorphism levels across the loci to perform further analyses of genetic diversity, differentiation and population genetic structure in Calotropis. This could contribute to future Calotropis genetics and breeding research, in areas such as identification of elite germplasm and marker-assisted selection.The informativeness level of markers based on ue Table . The sliC. procera and C. gigantea. Genetic variation was lower than in previous genetic studies of Calotropis populations11. However, these studies are not directly comparable since different marker systems could result in slight differences in the results obtained.It is crucial to assess genetic diversity in order to ensure that the most diverse populations are selected to widen the genetic base of germplasm. Based on the developed EST-SSR markers the current study assessed genetic diversity in both 25, however, based on EST-SSR markers, Calotropis displays a moderate genetic diversity (HS = 0.245). This level is comparable to the genetic diversity in Blighia sapida, a woody perennial species widespread in tropics and subtropics which had an average genetic diversity of HS = 0.2926. It seems that the genetic diversity of Calotropis plants is lower than that of most outcrossing species25. The population genetic diversity did not differ between C. procera (HS = 0.248) and C. gigantea (HS = 0.249), despite the fact that C. procera was distributed widely in Afica and C. gigantea was fragmentally distributed in Asia. Generally, in fragmented populations genetic variability is expected to be low27, however, this might not be universal, and in C. gigantea there was no evidence of low variability. The European Beech has fragmented populations that nonetheless show no evidence of loss of genetic variability28. Within C. procera, the Mali S populations had the highest genetic diversity while in Nepal, C. gigantea had the highest genetic diversity  0.528 . Populations separated by greater distances were more genetically dissimilar than those populations that are geographically close - which led to stronger internal genetic differentiation. Therefore, it is likely that the genetic structure of this species is affected by geographical distance. This high isolation by distance implies that selection and use of genetically diverse genotypes are key factors in C. procera breeding program during domestication and development of varieties with a broad genetic base. However, genetic isolation by distance is not a static process and may change with time, resulting in changes in genetic composition of a given population. In C. gigantea, however, Mantel test showed no correlation between geographic distance and genetic distance pattern , which further supports our hypothesis that geographical isolation exists among studied populations of C. gigantea, which led to genetic high differentiation which is suggested to have resulted from population fragmentation, low gene flow and genetic drift, similar to that in white Jabon 33.Although Calotropis spp. Although the EST-SSR markers were of moderate polymorphism they showed power in discriminating between two closely related Calotropis species. These markers are also linked to functional genes due to their location in the coding regions of the genome; thus, may be useful for functional analysis of traits of interest. We found moderate genetic diversity between the two species of Calotropis with no difference within them. Information on genetic diversity will ensure that the maximum genetic diversity can be captured during the domestication process. We found strong interspecies and intraspecies genetic structure in this genus, thus collection of germplasm efforts should focus on sampling the maximum number of populations in order to preserve a high level of genetic diversity. These markers of genetic differentiation will give insights into incorporating the most diverse populations into breeding programs. Overall this study will be a useful resource for further genetic research in Calotropis.We developed the first EST–SSR markers, thus providing a strong impetus for genetic analysis in Calotropis from 10 natural populations from Africa and Asia, at a minimum distance of 50 meters. From each sampling point individuals’ GPS readings were taken which were then transformed into reference points and mapped  and also by running on 1.0% (w/v) agarose gel. The DNA for PCR amplification was then diluted to a final concentration of 50 ng/µL for each sample in TE buffer .We sampled 286 individuals of 16. The EST-SSR markers were screened for PCR amplification using eight individual samples selected at random, four samples from Asia (C. gigantea) and four from Africa (C. procera). Polymerase chain reactions (PCR) were performed in 12.5 µL reaction volumes containing 6.25 µL 2X easytaqPCR PAGE MasterMix , 0.5 µL of forward and reverse primers, 0.5 µL of genomic DNA (50 ng/µL) and ddH2O 4.75 µL. Amplification of the PCR products was carried out using a BIO-RAD T100TM Thermal cycler (Singapore) with the following cycle: initial denaturation at 95 °C for 5 min; 35 cycles of 30 s at 94 °C, 45 s at a range of annealing temperatures to attain the optimum , 25 mM MgCl2 (1.6 µL), 10 mM of dNTPs (0.4 µL), 5 unit/µL of Taq DNA polymerase (0.15 µL) TaKaRaTaqTM kit , 10 pmol each of forward and reverse primers (0.4 µL), 40–50 ng/µL of DNA template (1 µL) and 14 µL of sterile ddH2O. The forward primers of all the selected primers were fluorescent labeled with a 6-FAM, HEX and TAMRA . PCR amplification conditions were as follows: 5 min at 95 °C, 35 cycles of 30 s at 94 °C, 45 s annealing temperatures 54 °C–62 °C (Table Polymerase chain reaction (PCR) amplification of the 286 °C Table , 30 s atTM (Applied Biosystems), denatured and then separated on an ABI 3730xl Genetic Analyzer .The success of amplification was determined by running the PCR products on 1% agarose in 1xTAE buffer. After successful selective amplification 1 µL of PCR product was mixed with 0.5 µL of size standard GeneScanTM 500 LIZ (Applied Biosystems) and 9 µL HI-DI36 to calculate PIC, AR, number of alleles (NA), HO, HS. To determine average pair-wise between populations (FST) we used GenAlEx v. 6.537. Arlequin 3.1138 was used to determine FIS and FST per locus.The SSR allele sizes was estimated by Genemarker v4.0  for all populations, checked manually and entered in a spreadsheet. We used PowerMarker v3.2538. Principal co-ordinate analysis (PCoA) to analyze genetic structure by covariance standardised approach of pairwise Nei’s genetic distances was conducted in GenAlEx version 6.537. The genetic relationships using neighbour-joining (NJ) was determined in PHYLIP3.6939 based on Nei’s genetic distance40. Nei’s genetic distance was calculated in MICROSATELLITE ANALYSER (MSA) v4.0541.The reliability of each node was tested using 1000 resamplings. FigTree v1.3.142 was used to visualize and edit the tree.Analysis of molecular variance (AMOVA) to partition genetic variance was analysed in Arlequin 3.1143. This analysis was run at 35 independent runs per K Value (K1–10) with a burn-in period of 100,000 iterations and 100,000 Markov chain Monte Carlo (MCMC). Structure Harvester44 was used to visualize the best K value based on delta K (ΔK)45 and maximum log likelihood L (K)46. We used Mantel tests18 to determine the pattern of isolation by distance at 1000 permutations using GenAlEx version 6.537. The comparison of the ten geographic populations distance matrix was calculated according to latitude and longitude from GPS coordinates using Vincenty’s formula, http://www.movabletype.co.uk/scripts/latlong.html.In order to determine the genetic groups among populations we used Bayesian clustering method in STRUCTURE V2.3.4Supplementary file


Ixodes ricinus. Population sites were selected within and between areas separated by geographical barriers in order to evaluate the importance of tick transportation by birds in producing genetic connectivity across open sea and mountain ranges. The phylogenetic analyses of the mitochondrial control region and the cytochrome b gene revealed two distinct clades with supported sub-clades, with three genetic lineages: GB and WNo associated with Great Britain and western Norway respectively, and Eu with a wider distribution across continental Europe in agreement with much lower efficiency of tick dispersal by birds than by large mammals. The results suggest different ancestry of I. ricinus colonizing Britain and the rest of northern Europe, possibly from different glacial refuges, while ticks from western Norway and continental Europe share a more recent common ancestry. Demographic history modeling suggests a period of strong increase in tick abundance coincident with progression of the European Neolithic culture, long after their post-glacial colonization of NW Europe.Dispersal and gene flow are important mechanisms affecting the dynamics of vectors and their pathogens. Here, patterns of genetic diversity were analyzed in many North European populations of the tick,  Ixodes ricinus is a widespread and common European tick species that infests both birds and mammal hosts, and is an important vector for a wide range of pathogens. The tick-borne encephalitis virus (TBEV) complex and the Borrelia complex of spirochetes may cause serious diseases in humans [I. ricinus with associated pathogens are currently expanding in northern Europe, probably due to a combination of climate and habitat change and expansion of host species [Ticks  are bloodsucking ectoparasites of most terrestrial vertebrates, and they have a great impact on the public health and rural economy in many parts of the world . Ixodes n humans , 3 whilen humans , 5. I. r species .Dispersal and patterns of gene flow are crucial parameters for understanding the biology and infection pathways of pathogens in general . In vectI. ricinus in central and northern Europe is the result of post-glacial range expansions from southern refuges. Currently I. ricinus has a continuous distribution across most of continental Europe [Ixodid tick activity is inhibited by low temperatures, limiting their geographical distribution , 14. Thul Europe . Its norI. ricinus, studies have documented substantial genetic differences between European and North African populations, but very little phylogeographic structure at both wide and local scales across large parts of continental Europe [I. ricinus has been explained by recent range expansion and gene flow due to passive dispersal of ticks by hosts within a continuous population distribution [I. ricinus populations of Great Britain and Latvia did show marked genetic differences [Phylogenies that span large portions of a species’ range offer a powerful means to elucidate important historical or contemporary processes that shape genetic diversity and structure . Furtherl Europe , 11, 19.ribution . Howeverferences .I. ricinus in Scandinavia and how it relates to continental Europe and Great Britain has not been investigated, although distinct phylogeographical patterns could be expected from the extensive geographical features (fjords and mountains) acting as barriers to tick distributional expansion since last glacial period. Transportation across open seas depends on birds during spring and autumn migration [The genetic structure of igration , 22, andigration . Howeverigration , 24, litigration . We woulI. ricinus populations owing to reduced gene flow across open seas, and (ii) between western and south-eastern Norway owing to the south Norwegian Langfjella mountain range which separates these two areas and functions as a barrier against mammal dispersal. In addition, since the ticks of the two Norwegian areas are likely to be differentially influenced by various routes of bird migration, we also tested the hypothesis that ticks from western Norway would show more genetic resemblance to British ticks while those from southeast Norway would be more similar to ticks from south of the Skagerrak. Lastly, we also estimated the timescale of postglacial re-colonization and population expansion of I. ricinus. The two first hypotheses were confirmed. However, although we detected clear sign of a gene flow between Great Britain and western Norway, the typical west Norwegian lineages were more closely related to the continental European lineage (grouped into a common clade B). The analyses implied a fairly sudden population expansion of W European I. ricinus long after the initial postglacial colonization of the species.Our sampling was designed to evaluate the importance of transportation by migratory birds relative to mammals in producing genetic connectivity between continental Europe and the more topographically isolated island of Great Britain and the Scandinavian Peninsula. We hypothesized the existence of a genetic structure (i) between British and continental European Ixodes ricinus were collected from 22 different sites in northern Europe  or red deer (Cervus elaphus) in connection with a project led by the Norwegian Veterinary Institute. All ticks collected were immediately killed in 70% ethanol, where they were stored until analysis. Mostly adults, but also some nymphs, were sampled. Species identification was conducted according to [n Europe  with a srding to  for adulrding to  for nympI. ricinus. MtDNA is a particularly suitable marker for analyzing genetic structure in ticks due to both maternal inheritance and because long distance dispersal is more common for adult females than adult males [TM Tissue Kit (Qiagen). Two fragments of mtDNA were amplified: parts of the cytochrome b (cyt b) gene and the control region (CR) using the primers as given by Casati et al. [b gene and a 434 bp trimmed CR fragment have been deposited in GenBank  to characterize the genetic structure of lt males , 28. DNAi et al. . Polymer7 iterations, 10% burn-in and trees sampled every 5000 MCMC cycles. Log-files were inspected using TRACER and effective sample sizes (ESS) were used to evaluate MCMC convergence within chains. TreeAnnotater v.1.7.5 [www.fluxus-engineering.com).Genealogical reconstruction of haplotypes was analyzed for the amplified cyt b and CR fragments separately, and for the combined (concatenated) fragment. The best fitting model of sequence evolution of the concatenated fragment was determined using the Akaike information criterion as implemented in jModelTest . The sel v.1.7.5  was subsb region do not currently exist for I. ricinus. Here, we estimated a population-level substitution rate for the concatenated fragments in I. ricinus based on the results from our phylogenetic analysis and paleoecological evidence. Most of the British Isles and Scandinavia was covered by ice during the last glacial maximum ) and re-colonization of potential tick host mammals occurred, at the earliest after the LGM in Britain and during the first part of the current Holocene epoch in Scandinavia [Substitution rates for the mitochondrial CR and cyt ndinavia , 33. TheST differences. The program SAMOVA v. 1.0 [Genetic diversity was estimated as the number of haplotypes, haplotype diversity and nucleotide diversity calculated in DnaSP 5.10 . ARLEQUIA v. 1.0  was usedSigns of sudden demographic expansion (i.e. rapid population increase) were tested by using the mismatch distribution approach as implemented in ARLEQUIN with 10 000 bootstrap replicates. The sum of squared deviations was used to test the fit of the observed data to the model of sudden demographic expansion. We also performed two neutrality tests, Fu’s Fs and Fu and Li’s D , 38, impb). Other datasets supporting this article have been uploaded as part of the Supporting Information.DNA sequences used in this study have been uploaded to GenBank  with ancestral haplotypes possibly reflecting several sub-lineages. However, the genetic divergence of the polytomies was relatively shallow, which suggests that this cluster represents a distinct European mainland (Eu) lineage. Only 6% of the British samples and 4% of the western Norwegian samples clustered within the Eu lineage.Clade B also contained haplotypes with distinct geographic structuring. The two highly supported sub-clades, B1 and B2  with15 ab haplotypes, on the other hand, identified only two highly supported clades (both with pp of 0.99) dominated by haplotypes obtained in Great Britain and the rest of northwest Europe respectively , which differed significantly from the other west Norwegian samples . No geneocations  Tables. The three population groups showed some degree of gene flow in the proportion of shared haplotypes, being 0.12 between Great Britain and western Norway, 0.10 between continental Europe and western Norway and 0.13 between Great Britain and continental Europe. When plotting the sampled populations according to their lineage composition on a map , both a I. ricinus lineages is directly dependent on the estimated nucleotide substitution rates. Under the assumption that I. ricinus colonized Britain after the LGM and southwest Norway during the early Holocene at the earliest, we estimated a median substitution rate for the concatenated fragment of 0.283 s/s/My (substitution/site/million years) with 95% highest posterior density (HPD) intervals of 0.168–0.436 s/s/My. Separate analyses for the CR gave a median substitution rate of 0.479 s/s/My (HPD: 0.266–0.774 s/s/My) leaving an estimated rate of cyt b of 0.034 s/s/My. The analyses for the concatenated fragment yielded a genetic distance between the two main clades A and B of 0.006  and wild boar (Sus scrofa) have great potential of transporting I. ricinus owing to their generally high abundance across much of NW Europe, their great dispersal ability, and often high infestation by adult ticks [I. ricinus within landmasses, the haplotype sharing between the distributional ranges of the three genetic tick lineages also revealed effects of transportation by birds. In particular, both the British and western Norwegian tick populations appeared affected by a two-way gene flow across the North Sea, which can be mediated only by migrant birds. In contrast, the gene flow appears more asymmetrical between Great Britain and the rest of Europe, with a markedly stronger element of the GB-lineage in most European populations, than the Eu-lineage in Great Britain. This might suggest that transportation of ticks between these two areas is influenced primarily by eastward spring migration from Great Britain and less during the westward autumn migration. Nevertheless, this gene flow mediated by birds has not been sufficiently strong to erase the signal of a more basic spatial genetic structuring.The clear effect of open sea and mountain ranges on tick genetic structuring emphasizes the importance of mammals relative to birds in successful dispersal of lt ticks , 43. TraOur estimates of nucleotide substitution rates were much higher than the conventional mutation rate of 1.1–1.2% per million years per linage often applied to arthropod mitochondrial DNA, a calibration based on . The higI. ricinus haplotypes into clades A and B, with an estimated time of separation probably prior to the colonization of Great Britain and continental northwest Europe, suggests that postglacial expansion of I. ricinus into these areas came from different ancestral populations, and with clade B reaching coastal western Europe after the opening of the English Channel.During the glaciation period, temperate host species were mostly restricted to southern Europe and geographically isolated in several refuges, leading to genetic differentiation that is still traceable across the present distributions of many species. For example, east-west genetic divisions across Europe have typically been attributed to expansions from Italian/Balkan and Iberian refuges , 49. TheI. ricinus. The star-like substructures with several ancestral haplotypes suggest several repeated colonization events and isolation processes with subsequent demographic expansions. As the genetic divergence of the different sub-clades was relatively shallow, it likely reflects post-glacial colonization and isolation events. The concatenated CR/cyt b fragment suggests a common ancestor of the WNo and the Eu lineage, and thus that the founding haplotypes of western Norway came from continental Europe. This could have occurred via migrating birds or through transport of ticks by large mammals moving from the east to the west. The earliest traces of ungulates in western Norway occur after about nine kybp [The heterogeneous network of the haplotypes of clade B  tells a ine kybp . During ine kybp , and theI. ricinus through increased landscape heterogeneity, favouring a more complex fauna of natural tick hosts and introducing livestock as new host animals for I. ricinus.The mismatched distribution analyses suggested a sudden demographic expansion for all three lineages, estimated to have occurred about 7–8 kybp for the GB and Eu lineages and 4–5 kybp for the WNo lineage . These eI. ricinus is low [I. ricinus carrying Borrelia and Babesia to the coast of southern Norway [The fact that the spatial structuring of three genetic lineages has been maintained despite thousands of years of tick transportation by migratory birds across the North Sea and the English Channel , 52 is qs is low , the oven Norway , but it n Norway . Howevern Norway , which mn Norway .I. ricinus lineages and different strains of the TBEV complexInterestingly, within our study areas established occurrence of the tick-borne louping ill virus (LIV) is primarily restricted to tick populations of Great Britain, in addition to a few records of the British subtype from the most southwestern part of Norway ; i.e. whI. ricinus from NW Europe into genetically distinct clades and lineages revealed geographically structured dispersal patterns of the species, still showing signatures of differences in post-glacial dispersal routs and colonization patterns. Main dispersal of the species appears to have occurred via movements of large mammals over land, while open seas and mountain ranges act as geographical barriers. Considerable over-sea transportation of ticks by birds for thousands of years has not been sufficient to mask the ancient colonization history of I. ricinus in Great Britain and western Norway. The distinct clade A of Great Britain may reflect a post-glacial expansion from a different refuge than the more continental clade B. However, analyses of I. ricinus populations from more southern parts of Europe are needed to evaluate this further. A sudden demographic expansion of I. ricinus appears to have occurred at the time of the Neolithic Revolution, suggesting human agricultural activity as a main driver on the dynamics and post-glacial history of the species.The splitting of I. ricinus with the spatial distribution of its pathogens. A linkage between clade A and the LIV subtypes of TBEV seems possible. The most widespread zoonotic pathogen of I. ricinus, however, is the genetically diverse Borrelia burgdorferi s. l. complex [The results emphasize the potential importance of combining population genetic analyses of  complex . So far,S1 Figin European Ixodes ricinus.Bayesian consensus tree (a) and network (b) for mtDNA control region (TIF)Click here for additional data file.S2 Figb gene in European Ixodes ricinus.Bayesian consensus tree (a) and network (b) for mtDNA cyt (TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.


Micromeria (Lamiaceae). Screening 15 microsatellite markers in 289 individuals representing the eight species of Micromeria present in Tenerife, this study aims to assess the genetic diversity and structure of these species and its relation with the geological events on the island. In addition, we evaluate the extent of hybridization among species and discuss its influence on the speciation process. We found that the species restricted to the paleo‐islands present lower levels of genetic diversity but the highest levels of genetic differentiation suggesting that their ranges might have contracted over time. The two most widespread species in the island, M. hyssopifolia and M. varia, present the highest genetic diversity levels and a genetic structure that seems correlated with the geological composition of the island. Samples from M. hyssopifolia from the oldest paleo‐island, Adeje, appear as distinct while samples from M. varia segregate into two main clusters corresponding to the paleo‐islands of Anaga and Teno. Evidence of hybridization and intraspecific migration between species was found. We argue that species boundaries would be retained despite hybridization in response to the habitat's specific conditions causing postzygotic isolation and preserving morphological differentiation.Geological history of oceanic islands can have a profound effect on the evolutionary history of insular flora, especially in complex islands such as Tenerife in the Canary Islands. Tenerife results from the secondary connection of three paleo‐islands by a central volcano, and other geological events that further shaped it. This geological history has been shown to influence the phylogenetic history of several taxa, including genus  Speciation is traditionally seen as the accumulation of differences between two populations in allopatry, with geographic distance as barrier to gene flow. In general, geneflow will prevent differentiation, so continuous migration and hybridization events will counteract speciation processes  had been exchanged between two species where hybridization has been observed, but the remaining genome had been hypothesized to be stabilized by selection.This scenario might explain the comparable high levels of genetic diversity  and two to Anaga (M. glomerata and M. rivas‐martinezii). Micromeria teneriffae also grows in Anaga but its range extends toward the southeast up to Fasnia and Güímar , are less well supported in the phylogeny and further conclusions about their relationships could not be drawn , 1 μL of primer mix and 0.5 μL of template DNA (about 40 ng/μL), and 3 μL of water. PCR was performed using the following cycle profile: 95°C for 15 min; 7 cycles of 95°C for 30 sec; touchdown from 58°C to 55°C, decreasing 0,5°C per cycle for 45 sec; 72°C for 30 sec; 25 cycles of 95°C for 30 sec; 55°C for 45 sec; 72°C for 30 sec; 8 cycles of 95°C for 30 sec; 54°C for 45 sec; 72°C for 30 sec; and a final extension step of 60°C for 30 min. Amplification success was confirmed using 2% agarose gels stained with GelRed . Genotyping was performed with an internal size standard  in an ABI3130xl automatic sequencer . Alleles were called using GeneMapper ver. 4.0 . To check for reproducibility of the data, the amplification and scoring of 96 individuals were independently repeated for all primers mixes.Dried leaves were ground and DNA was extracted using the Macherey‐Nagel Plant DNA Extraction Kit  according to the manufacturer's protocol. The 289 samples were amplified with the 16 microsatellite markers developed for o et al. . Each pro et al.  and Puppo et al. . The 16 o et al.  using HoFor the population level analyses, only localities with at least four individuals sampled were considered. To better understand how the estimates vary across the island regions geographically close localities within the same habitat were considered as one population for some analyses Fig. .http://biology-assets.anu.edu.au/GenAlEx/).Microsatellite quality was evaluated by quantifying the frequency of null alleles and searching for evidence of genotyping errors such as scoring of stuttering bands and large allele drops. This was performed with the program Micro‐Checker , expected and observed heterozygosities (HE and HO), and portion of private alleles. To prevent biases due to population size, the total unbiased HE per species and regions was calculated (HT). Genetic differentiation was estimated by calculating pairwise FST, RST, and Nei distance; RST, to include the information about allele size when using microsatellites in the distance estimate. This allows to have a better perspective of the evolutionary relationships among groups  were conducted in GenAlEx 6.41 using K (unknown) genetic clusters, K was set to range from 1 to 15; the program was run as 10 iterations of 500,000 MCMC generations with a burn‐in of 100,000 generations for each K. The most likely K was selected by analyzing the second‐order rate of change of the posterior probability of the data (DK) between successive K values . Additionally, the suboptimal value of K was searched by redoing the DK test without the optimal and smaller values of K. This allowed us to investigate more detailed structure signal shown by our data. All 10 iterations were combined using the greedy algorithm from the program CLUMPP , and a third one containing only M. varia and M. hyssopifolia.Genetic structure between and within species was also investigated using the Bayesian clustering algorithm implemented in the program STRUCTURE ver. 2.3.3 . Morphological intermediate individuals were found in the field between M. densiflora and M. varia in Teno, M. rivas‐martinezii, and M. varia in Anaga, M. teneriffae and M. varia in Anaga, and M. teneriffae and M. hyssopifolia in the south coast. We performed a STRUCTURE analysis for each species pair with the parameters described above.Because some morphological hybrids were found in our sampling, we tested for the likelihood of them being real hybrids by doing a STRUCTURE analyses with the individuals from the same localities in which they were found. With this approach, we expect that hybrid individuals will show an equal assignment to the clusters from the parent species. This result is only considered to be valid if both species are clearly differentiated  and only for a few loci and single populations. No locus deviated from Hardy─Weinberg equilibrium across the majority of populations meaning that all its assumptions such as neutrality were met. The same was observed the other way around: no population deviated from HWE for most of the loci analyzed (Table S2). There was no evidence of scoring errors and none of the markers constantly showed high frequency of null alleles in the populations analyzed. The 15 loci investigated were therefore retained in the analysis.From the 16 microsatellite markers included, one (5978) presented low amplification success (<50%), so only 15 SSRs were used for further analysis. The remaining markers comprised between 11 and 25 alleles, giving a total of 273 analyzed alleles. None of the analyzed populations deviated significantly from Hardy─Weinberg equilibrium for most of the loci. A few deviations were indicated with near marginal N) varied from 4.20  to 11.27 (M. lachnophylla), HO from 0.29 (M. glomerata) to 0.62 (M. hyssopifolia), and HE from 0.28 (M. densiflora) to 0.71 (M. lachnophylla), HT from 0.32 (M. densiflora) to 0.81 (M. hyssopifolia) . In M. teneriffae, the populations from the Southern coast showed slightly lower diversity  than the population from Anaga . No differences in genetic diversity were found between the two regions (Anaga and Teno) where M. varia grows.Across all populations, mean number of alleles  suggesting that they went through a bottleneck event (Table S2). These were the populations from M. glomerata and M. densiflora, one population from M. varia from Teno, and one population from M. hyssopifolia from the Southeast.Four of the analyzed populations significantly deviated from the mutation‐drift equilibrium , varying from 0.042 to 0.500 (Table S3). FST was correlated to species age, with the older species  presenting higher pairwise FST values than the youngest (M. varia and M. hyssopifolia). The pairwise unbiased Nei (uNei) distance showed similar patterns to the FST values. RST was calculated among island regions and used to evaluate genetic distance patterns among them , the PCoA shows four clusters corresponding to each species  shows no separation of the samples  are analyzed, there is a distinction among them  are analyzed independently, this differentiation within M. hyssopifolia is clearer. For example, for K values higher than 7 one of the clusters is mainly composed of M. hyssopifolia samples from the southern coast from subdesert environments, while another cluster is mainly composed of individuals form the wet northern coast. Moreover, samples of M. hyssopifolia from Teno share the same cluster with samples of M. varia from this same region. The best K for the analysis including only the central species was also K = 3 and the suboptimum is K = 5 , while the other was assigned to the M. rivas‐martinezii cluster so it is likely a backcross. In addition to these hybrids, two individuals that were morphologically identified as M. rivas‐martinezii showed an almost complete assignment to M. varia evidencing introgression between both species. In the analysis between M. teneriffae and M. hyssopifolia, only one morphological hybrid could be confirmed with high assignment rates to both clusters . Two M. hyssopifolia individuals showed mixed assignment (41% and 52% to the M. teneriffae cluster) suggesting them as hybrids or backcrosses. For the M. varia and M. teneriffae analysis, only one of the morphological hybrids was confirmed (54% assignment to M. varia cluster). Additionally, three M. varia individuals showed a high assignment to the M. teneriffae cluster (50% to 81%).Several individuals had been determined as hybrids because they present morphologically intermediate characteristics from two species. In a STRUCTURE analysis together with the putative parental species, the hybrid status of most of these individuals were confirmed. According to the DK method, the best irs Fig. . From thM. densiflora to M. lasiophylla (17%), M. glomerata to M. rivas‐martinezii (16%), M. lachnophylla to M. hyssopifolia (25%), and M. varia from Teno to M. hyssopifolia (26%).BAYSASS and MIGRATE were used to estimate contemporary and historical gene flow among species, respectively. Contemporary gene flow as indicated by BAYSASS was generally low showing migration rates below 10% of individuals originated from other populations for most of the comparisons Table . The excMicromeria hyssopifolia showed to be the main source of interspecific gene flow because it had the highest migration rate (to M. lachnophylla). And, from the eight comparisons, five showed migration rates above 10 individuals per generation. Micromeria varia was the second main source of migrants, with both M. varia from Teno and M. varia from Anaga showing three migration comparisons above 10 individuals per generation. Micromeria rivas‐matrtinezii was the main sink population because it received more than 10 migrants per generation from six other species. The species with lowest emigration and immigration, less than 10 individuals per generation, were M. rivas‐martinezii and M. glomerata, respectively. Some loci showed higher values of migration rate than others. On average, the overall migration rate per locus varied from 4.66 individuals per generation for locus 5419 to 175.41 for locus 3963 (not shown).The historical migration rates calculated by MIGRATE varied between 1.4 and 19.1 individuals per generation . Three samples of M. varia growing in this contact zone were also assigned to M. teneriffae, which might be a consequence of introgression of ancestral alleles shared by M. teneriffae and M. rivas‐martinezii.Our analysis indicates a strong influence of historical and contemporary gene flow between the species on the genetic structure, most pronouncedly in A lower degree of reproductive isolation between island species, compared to continental ones, is generally assumed because of a potentially comparable lower effect of fitness decrease after hybridization resulting from the lower levels of interspecific competition in island systems  With exception of M. densiflora and M. lasiophylla, all other connections through gene flow were indicated between species that have contacting ranges; and (4) cluster arrangement in the structure analysis gives increased assignment probability for adjunct populations even though they belong to different species, e.g., for M. hyssopifolia and M. varia from Teno and M. hyssopifolia and M. lachnophylla. Hereby, some loci show higher values for migration than others indicating asymmetric introgression at some degree.Besides the tests for migration, the existence of hybrid zones between the allopatric ranges of the species is supported in our study by three main findings: (1) we observed and verified the status of hybrids in the contact zones of four species pairs; (2) the two species with the largest contact zones, M. varia and M. rivas‐martinezii. Here, in a very small spatial scale hybridization occurs at the transition from the range of M. rivas‐martinezii to M. varia. Micromeria rivas‐martinezii grows in a very restricted area in a small peninsula in the Anaga massif . This transition can also be seen in our SSR data, indicating a transition in the allele frequency content between M. rivas‐martinezii and adjacent M. varia populations more gradual than expected if the species were reproductively isolated.The formation of distinct hybrid zones could be observed directly between M. hyssopifolia, which is the species with the largest range. It participates in most of gene‐flow exchanges found in the island and it has one of the highest genetic diversity. Environmental conditions across the range of M. hyssopifolia are highly heterogeneous. The northern part of Tenerife is wetter due to the fog brought by the trade winds with high levels of rainfall (ca. 1000 mm precipitation per year) in the mid altitudes. Contrary to this, the southern part of the island is dry (below 100 mm precipitation per year) due to the shade effect caused by the Teide. As described below, this genetic structure might reflect these environmental differences, such as structure found between the wet northern and dry southern slopes. Like outlined above, we see the population from Adeje slightly differentiated from the remaining M. hyssopifolia populations. Besides this, at optimal (K = 3) and higher K (up to K = 9), we observed genetic structure among: (1) Teno and west Tenerife; (2) north coast, and (3) southeast and south coast, corresponding to a medium, high and very low precipitation regime. It seems likely that genotypes are locally adapted to these different habitats and genotypes from the southern part may not be able to establish in the northern part and vice versa. These different habitats correspond roughly to the subdivision of M. hyssopifolia. Three varieties are recognized within this species: var. hyssopifolia, var. glabrescens, and var. kuegleri (Pérez de Paz M. hyssopifolia (var. hyssopifolia) presents a strigose indumentum which gives the plants a grayish aspect and is distributed in the pine forest between 400–2000 m. Micromeria hyssopifolia var. glabrescens is mostly distributed in the north of the island in degraded areas between 300–600 m while var. kuegleri is the coastal form that grows in the southeast from the sea level up to 400 m. Thus, our structure pattern differentiates mostly var. glabrescens and var. kuegleri. Because the environmental conditions are not independent from geography, further work is currently being conducted to confirm the hypotheses that: hybridization after secondary contact of former paleo‐island species allowed the colonization of the whole island by one or a few species, and the genetic structure that can be observed in M. hyssopifolia is an expression of local adaptation patterns rather than geography.The formation of hybrid zones may have contributed to the increase of genetic variation of some taxa facilitating adaptation to changing conditions, shift of ecological niches, or range shift for the species after secondary contact of the paleo‐islands. An example would be The pattern of hybridization found in our study might also explain the apparent low genetic distance between the species with microsatellite datasets and with our earlier multigene analyses , some admixture between M. lachnophylla and M. hyssopifolia is found. This is probably caused by hybridization with M. hyssopifolia since M. lachnophylla is distributed from the high desert in Las Cañadas down to the border of the pine forest where M. hyssopifolia grows. Furthermore, morphologically intermediate individuals have been reported in several localities that constitute contact zones where both species occur , number of samples per locality (N), and collection information. TFC, Herbarium of the Universidad de la Laguna in Tenerife.Click here for additional data file.Table S2. Results for HWE and Bottleneck test per population. Here, we present the number of loci deviating from HWE and the P‐value for deviations from the mutation‐drift equilibrium (Bottleneck).Click here for additional data file.Table S3. List of pairwise Fst and unbiased Nei distance results for all populations with at least four individuals.Click here for additional data file.


The Cooperative Patent Classifications (CPC) recently developed cooperatively by the European and US Patent Offices provide a new basis for mapping patents and portfolio analysis. CPC replaces International Patent Classifications (IPC) of the World Intellectual Property Organization. In this study, we update our routines previously based on IPC for CPC and use the occasion for rethinking various parameter choices. The new maps are significantly different from the previous ones, although this may not always be obvious on visual inspection. We provide nested maps online and a routine for generating portfolio overlays on the maps; a new tool is provided for “difference maps” between patent portfolios of organizations or firms. This is illustrated by comparing the portfolios of patents granted to two competing firms—Novartis and MSD—in 2016. Furthermore, the data is organized for the purpose of statistical analysis. Patent data provide a primary data source for scholars interested in the development of technological knowledge  in the USA, and PatentsView (http://www.patentsview.com). PatentsView adds to the data by providing disambiguated inventor and assignee identifiers . US patenting is considered the most competitive and is therefore most relevant to technology and innovation studies , the classification system , the clustering algorithm , etc. The classifications can be used with different levels of detail by using more digits of the respective classes, i.e. main- and/or sub-classes.Kogler et al.  used IPCIn this study, we use CPC at the four digit level. CPC is similar to IPC in the first four digits, but the classification of individual patents can be changed in the process of reclassification; new classes have also been added. Furthermore, CPC contains new categories classified under “Y” that span different sections of CPC in order to indicate new technological developments such as nanotechnology and technologies for the mitigation of climate change  matrix of patents (cited) in the rows versus citing patents aggregated into the 654 CPC classes at the 4-digit level in the columns. The Jaccard index and cosine values are then computed over the 654 columns . Thus, one can also expect the two maps to be different. We explore and compare both maps.The Jaccard matrix is based on counting the cited patents as binary [A1 in Yan and Luo ],4 wherecosine can be considered as a proximity measure in the vector space and (1 − cosine) thus provides a distance measure. While the corresponding “Jaccard distance” (=1 − Jaccard) is widely accepted in the literature as well, we note that the Jaccard index in contrast is a relational measure. From the perspective of graph theory, the geodesic would be a better measure for the distance between two nodes in a network  and not the Jaccard distance.The http://www.leydesdorff.net/jcr15/program.htm). The maps based on the Jaccard indices are available (i) at http://www.leydesdorff.net/cpcmaps for each class (bottom-up) and (ii) at http://www.leydesdorff.net/cpcmaps/scope using hierarchical clustering top-down. One has the option either to access the various maps directly in the.jpg format or to webstart a map using VOSviewer. The latter option provides an analyst with follow-up options such as choosing other parameters or exporting the files in formats used by other software applications.Both the Jaccard and the cosine matrices were decomposed using the routine decomp.exe ] for the sample under study. In our opinion, these diversity measures can be considered as measures of “related variety”  that is used to label the column and row variables added. The routine is technically similar to the one defined in Kogler et al.  for IPC The global maps based on the Jaccard index Fig.  and the Reading Fig. V as a measure of Chi square between zero and one (Table p < .001 in both cases).One can compare classifications using Cramèr’s ne Table . NeitherTable n = 938 and 1030, respectively.) Eindhoven is a provincial town of approximately 220,000 inhabitants; it has long been the home town of Philips and is nowadays considered a center of technical innovation in the Netherlands. Boston is three times larger than Eindhoven in terms of its population . Considering that patenting in the USA is not necessarily the first priority for European based companies and inventors, this relative comparability of the patent portfolios was not expected. Rao-Stirling diversity is 0.80 for Boston versus 0.78 for Eindhoven.Figure ρ = .98 (p < .01). In other words, the retrieval is not incidental.Kogler et al.  used theN and Δ is 0.16 . Metropolitan cities such as Paris and Boston have the highest scores, whereas patents from Wageningen—the home town of the single agricultural university in the Netherlands—are specific. The results, however, can also be counter-intuitive and thus raise further questions for follow-up research. Rotterdam, for example, scores third on diversity behind Paris and Boston. One can envisage a systematic study of diversity in global or so-called “smart cities,” etc., as a perspective for future research.Table ρ = .42; n.s.).The values are different from the ones based on IPC-4 in 2014: Shanghai, for example, which was ranked in the first position in 2014 based on IPC, is now rated in 9th position once CPC serves as the underlying classification system. The change from IPC to CPC thus makes a difference in this evaluation; the rank-ordering differs significantly between 2014 and 2016 A matrix like the one of (20) cities versus (654) CPC-4 classes can be used for various forms of statistical analysis. For example, one can use discriminant analysis given the grouping of cities in countries. Nineteen of the twenty profiles are correctly identified by nation using the CPC classes; the exception is Beersheva, which is predicted as belonging to the French group. However, the prediction is considerably improved compared to the one based on IPC, when only 17 or the 20 (85%) were correctly predicted.Using the cosine values of CPC vectors among the 20 cities, one can generate a local map among the cities differently from the overlay on the global map for each of them Fig. . VOSviewr = .31; p ≤ .05). Major cities are grouped as factor 1; factor 2 is related to this first factor by Chinese cities , which share a pattern also with technologically oriented cities such as Grenoble, Toulouse, and Eindhoven. Factor 3 is set apart with other factor loadings of three Dutch cities  and Dalian in China. Why the grouping is in some respects different between Fig. This three-factor solution explains 69.79% of the variance. This solution is only weakly related to the corresponding three-factor matrix made on the basis of 2014 data and IPC (http://www.leydesdorff.net/cpc_cos/portfolio) allows for the further extension of comparing two portfolios with each other in a single map. (For instructions see the “http://www.leydesdorff.net/cpc_cos/portfolio.)The routine CPC2.exe  and isd/2016$$.” “AN” and “AANM” are fields code for the names of assignees and applicants, respectively, in the advanced search engine of USPTO. Note that the applicant is not always the assignee of a patent; the applicant may also be the inventor, whereas the assignee is the owner of the patent.In Fig. Table Indicators for measuring the distance or relatedness between distinct sets of technological knowledge categories are of interest in a number of disciplines; for example, management studies Almeida , economiOne of our objectives has been to show how sensitive the maps and overlays are to choices of parameters. The maps are not natural, like geographic maps, but remain constructs that originate from discursive reasoning. Consequently, one cannot infer on the basis of visual inspection that one representation is better than another; but one needs analytical arguments for the choices. For this reason, it was urgent to take Yan and Luo’s  critiqueWe did not follow Yan and Luo’s preference for the Jaccard index, but used the cosine. The Jaccard index is relational and binary, whereas the cosine is a numerical and a positional measure Burt . PortfolThe global maps were not interpretable when using the Jaccard index Fig. , but theThe multi-variate analysis confirmed the improvement. The prediction based on discriminant analysis was improved from 85 to 95%. The (varimax) rotation of the eigenvectors provided us with additional and meaningful insights in the comparisons among cities. The positions of the major Chinese cities among cities in the global domain (factor 1) and cities with strong engineering (factor 2) are suggested in the cosine-based map.Visualization is a strong instrument because maps can be provided with an interpretation more easily than numerical statistics. However, the latter are needed in the background for making analytical arguments. A direct comparison between two units  using a map, however, can provide a first orientation to their differences in terms of strengths and weaknesses.


Panulirus homarus, which has peripheral divergent lineages in the west and east. The study focuses particularly on the West Indian Ocean, which is emerging as a region of unexpected diversity. Mitochondrial control region (mtCR) and COI sequences as well as genotypes of 9 microsatellite loci were examined in 410 individuals from 17 locations grouped into 7 regions from South Africa in the west, and eastward across to Taiwan and the Marquesas Islands. Phylogenetic and population-level analyses were used to test the significance and timing of divergences and describe the genetic relationships among populations.There is increasing recognition of the concordance between marine biogeographic and phylogeographic boundaries. However, it is still unclear how population-level divergence translates into species-level divergence, and what are the principal factors that first initiate that divergence, and then maintain reproductive isolation. This study examines the likely forces driving population and lineage divergences in the broadly-distributed Indo-Pacific spiny lobster ST = 0.594, P < 0.001). Microsatellite analyses also revealed significant divergence among regions, but at a much lower level . The results reveal different patterns of mtCR v. nDNA divergence between the two distinct peripheral lineages: a subspecies in South Africa and Madagascar, and a phylogeographically diverged population in the Marquesas. The results also expose a number of other more fine-scale population divergences, particularly in the Indian Ocean.Analyses of the mtCR revealed high levels of divergence among the seven regions  contains supplementary material, which is available to authorized users. Our understanding of the origins of marine biodiversity has been greatly enhanced through investigation of the evolutionary history of biogeographic provinces. This is particularly so in the vast Indo-Pacific region, which harbours the bulk of marine diversity, and has long been of great biogeographic interest across a wide taxonomic range of species –5. MarinAlthough there are common biogeographic and phylogeographic boundaries among Indo-Pacific species that hint at common causes of divergence, there are also many discrepancies in the patterns observed among species , 16. TheIt is also unclear what the forces are that maintain reproductive isolation as diverged populations become species, and what is the relative importance of physical versus ecological boundaries in maintaining that divergence . That isAnother major gap in our understanding of Indo-Pacific biodiversity is the fine-scale pattern of population substructure across the region. The broad biogeographic patterns and provinces are now somewhat clear , but thePanulirus homarus . P. homarus is a valuable fisheries species distributed widely across the Indo-Pacific region  [P. homarus “brown”  [P. homarus homarus, which appears to inhabit all the intervening part of its distribution  , 27, P. Pacific) , and P. bspecies . P. homaiversity , 29, 30.iversity , which piversity –34.Fig. We investigate here the process of phylogeographic divergence in this species across biogeographic boundaries, to shed light on the evolutionary forces driving this. Using a greatly expanded data set that includes substantially increased sample sizes from a much wider diversity of locations, examined using two mitochondrial DNA genes and nine nuclear microsatellite loci, we address two groups of related questions: (1) What can multiple loci tell us about the processes driving divergence at the western and eastern peripheries? When did the divergences occur, have they both been driven and maintained by similar allopatric processes, do they match the predictions of the core-periphery hypothesis, and are they concordant with those of other species? (2) Do more fine-scale divergences exist between populations separated by other recognised biogeographic boundaries, the Indo-Pacific barrier (IPB), and the proposed mid-Indian Ocean barrier (MIOB ). Can weP. homarus from 16 locations during 2011–2013 throughout the distribution range of the species  [Aligned and edited 618 bp sequence fragments of mtCR (using Gious vR9 ), were uious vR9  was constraction . After ptraction  as well traction  tests ansion 3.5 . The mosions (k) .eneious using the microsatellite plugin version 1.4.0 [ST comparisons between populations and regions were carried out with Arlequin 3.5 [enepop [enAlEx [enAlEx to display the genetic relationships among sampled populations. Genalex was used to estimate allele frequency, effective number of alleles, observed and expected heterozygosity and fixation index. Allelic richness and gene diversity was analysed in Fstat 2.9.3 [dze [tructure [Microsatellite alleles were sized in Gon 1.4.0 . Of the quin 3.5 , Genepop [enepop  and GenA [enAlEx . Principat 2.9.3 . The num9.3 [dze . Spatialtructure  either utructure  method wm/mf) between the two lineages was estimated from the differences between mtDNA ФST and nDNA FST, assuming neutral divergence .east 2.4.2 [The time to most recent common ancestor (TMRCA), was calculated from the COI data using the Bayesian MCMC approach implemented in Bst 2.4.2 . To enabst 2.4.2 , the samP. h. rubellus and P. h. homarus lineages were estimated in the isolation-with-migration model using IMa2 [Gene flow between the ing IMa2  on the mThe mitochondrial control region was highly polymorphic with high haplotype diversity (mean 0.99)  and samples from the Marquesas Islands.  revealed very similar patterns to those shown in the mtCR analyses presented below, but with less discriminatory power .The mtCOI analysis of a representative subset of individuals enabled direct comparisons with other studies of  species , 53, 55.ST = 0.71, P < 0.00001). Hierarchical analysis of all 17 locations grouped into seven regions , but little divergence among locations within each region . Excluding the divergent P. h. rubellus and Marquesas clades also resulted in a significant AMOVA test among the remaining five “central” regions .Overall, the AMOVA test of mtCR sequence variation showed a very high level of differentiation among all samples (ФP. h. rubellus) and Marquesas regions are highly significantly different from all other regions. In addition, all the remaining five “central” regions are significantly different from each other, apart from the Indonesia-E. Africa and Indonesia-E. Asia pairwise comparisons .Analyses of pairwise differences between the sampled regions revealed where the main mtDNA divergences lie within the species Table . As expens Table . The NWIST values  is the most divergent region, with the Marquesas also highly divergent. Of the five “central” regions, Indonesia and E. Asia are the most similar. The significantly different mtDNA geographic clusters are also highlighted by the Baps analysis  (CI: 2.2–3.7), while the Marquesas lineage diverged around 1.25 Ma (CI: 0.9–1.6) , which closely matches the previous estimate of approximately 7 Ma [A Bely 7 Ma .Na = 15.6) and high heterozygosity (mean He = 0.83) .Of the nine most reliable microsatellite loci included in the final analyses, all loci exhibited relatively high diversity, with high numbers of alleles per locus , even after correction for high heterozygosity  [RT = 0.189, P < 0.001), but also significant differentiation among locations within each region . Excluding the divergent P. h. rubellus and Marquesas clades also resulted in a significant AMOVA test among the remaining five “central” regions .Overall, the AMOVA test of microsatellite variation showed a significant, but much lower level of differentiation among all samples compared to the mtCR F< 0.001) . Hierarc . HierarP. h. rubellus) region is less significantly diverged. Somewhat surprisingly for a recognised distinct subspecies, P. h. rubellus does not exhibit any fixed allelic differences. In contrast to the mtCR results, of the remaining five “central” regions, only the E. Africa and NWIO regions are significantly different from the other regions (Table ST = 0.026 P < 0.001) .Analyses of pairwise differences between collection regions revealed where the main microsatellite divergences lie within the species Table . Again, ns Table . Within ST values  much less so in comparison. Of the five “central” regions, Indonesia and India are the most similar, with the NWIO the most divergent of these. The significantly different microsatellite geographic clusters are also highlighted by the Structure analysis, which shows the P. h. rubellus and Marquesas samples to be distinct  were no2 = 0.826, P < 0.01), but this pattern was not significant for mtCR data , or if the samples from the Marquesas and P. h. rubellus populations were excluded .A Mantel test on the microsatellite data showed an isolation by distance pattern (Rm/mf) between the lineages was estimated from the differences between mtDNA ФST and nDNA FST [ST gave a ratio of 54.2, while using the adjusted F’ST gave a ratio of 3.6. The same ratio calculated between the Marquesas and adjacent populations was 1.1 (0.4 using F’ST). The considerable difference in mtDNA and nDNA gene flow between the lineages P. h. rubellus and P. h. homarus was highlighted by Bayesian estimates from the isolation-with-migration model in IMa2. Maximum likelihood estimates of gene flow from mtDNA peak at zero (mo>1 (migration from P. h. homarus to P. h. rubellus) = 0.0003, 95%HPD 0.0–0.53; m1>0 (migration from P. h. rubellus to P. h. homarus) = 0.0003, 95%HPD 0.0–0.47), while estimates from nDNA were significantly greater than zero .  and P. h. rubellus. This pattern is close to expectations for neutral divergence in P. h. “brown”, which has a mtDNA lineage 4% divergent, no private microsatellite alleles, a mtDNA ФST/nDNA FST ratio of 0.59/0.09 and an estimated ratio of male to female gene flow (mm/mf) [P. h. rubellus, which has a mtDNA lineage 16% divergent, and a high proportion of private microsatellite alleles, but the mtDNA ФST/nDNA FST ratio is 0.75/0.02 and the estimated ratio of male to female gene flow (mm/mf) is 54.2. This departure from neutral expectations is highlighted by the difference between mtDNA and nDNA estimates of gene flow from the isolation-with-migration model. It is unlikely that either neutral divergence with no gene flow, or high levels of current gene flow in both males and females would leave behind the pattern seen in P. h. rubellus.Potentially, the discrepancy in divergence of mtDNA and nuclear microsatellite alleles could be due simply to stochastic lineage sorting with random fixation of mtDNA alleles and/or insufficient time for nuclear alleles to diverge in frequency –64. Howe (mm/mf)  of 1.1. P. h. rubellus lineage diverged allopatrically  at around 2.9 Ma, and it is only recently that the distributions of the two lineages have begun to overlap, leading to secondary contact and introgression [homarus males with rubellus females) are substantially less fit, resulting in asymmetric male gene flow but restricted female gene flow. The fact that only rubellus father/homarus mother hybrids have so far been reported may lend some support to this hypothesis [P. h. rubellus extends the furthest south of any P. homarus population, across the recognised biogeographic boundary separating warm tropical from cooler subtropical waters along the south-westerly progress of the Agulhas current [P. h. rubellus did diverge allopatrically in the past in this region, there is every likelihood over a substantial period of time that it would adapt to the cooler temperatures of its range. If so, the resulting differential selection along this coast on the two lineages could maintain their largely non-overlapping parapatric distributions, and the degree of reproductive isolation evident today. Further exploration of this potential adaptation is worth pursuing.Given the deep mtDNA divergence, it is more likely that the gression , 66. On gression , 62. Upogression . Firstlygression –70, althpothesis . Another current . If P. hP. h. rubellus divergence likely originated with the advent of modern glacial cycles at the beginning of the Pleistocene ~2.5 Ma, when a relatively warm, stable interval of global climate transitioned to cooler, high-amplitude cycles, curtailing a warm-water corridor around southern Africa, and intermittently reducing connectivity along this coastline [Nassarius snails [Hymenosoma crabs [oastline . There is snails , Hymenosma crabs , 75), anP. h. rubellus likely diverged about 2.9 Ma (CI: 2.2–3.7), that there is a surprising discrepancy in mtDNA and nDNA divergence between these lineages, and that, despite apparently high recent nDNA gene flow, distinct lineages are able to persist in parapatry. This highlights that marine lineages may still be able to maintain divergence despite substantial gene flow, a matter of considerable interest in marine speciation and biogeography [Regardless of the exact mechanisms, the data are clear that eography , 76.P. h. rubellus, the Marquesas population (P. h. “brown”) is also a peripherally isolated unique mtDNA lineage, but the data suggest that very different processes have led to its origin and maintenance. Unlike P. h. rubellus, its mtDNA lineage is only slightly divergent, and appears to have diverged only around 1.25 Ma (CI: 0.9–1.6). Its mtDNA diversity represents just a small subset of the total diversity present in the neighbouring P. h. homarus populations. The nDNA pattern in this population is one of reduction in allelic diversity, but with no unique alleles (unlike P. h. rubellus). As such, it may best be regarded as an isolated and distinct population rather than a distinct subspecies, although it does appear to have a distinct morphology [Like rphology .Since its colonisation around 1.25 Ma (CI: 0.9–1.6), likely through a rare period of favourable current flow from the West Pacific (see below), the Marquesas population appears to have diverged genetically through random drift in allele frequencies, with no unique nDNA genotypes. The population appears to have been completely marooned through much of this time, and is now isolated by a very large distance from the main population range of the species . . The lowered levels of mtDNA and nDNA diversity, and positive value of Tajima’s D for the mtCR, all suggest that this population has a reduced effective size, although not sufficient to leave a significant signal of a bottleneck in its microsatellite allele frequencies. This genetic pattern is as predicted by the core-periphery hypothesis: lowered diversity in the periphery compared to central populations, as measured by mtDNA and nDNA allelic diversity and private alleles, and increased genetic divergence of the peripheral population .It has been recognized previously that the Marquesas Islands display a high level of endemism and genetic distinction of its marine fauna , 20, 77.P. homarus. Further, the eastern periphery matches the expectations of the core-periphery hypothesis, while the western periphery does not.Thus very different processes appear to have led to the divergence and maintenance of the western and eastern peripheral lineages of P. h. rubellus has previously indicated the Madagascan population of this lineage is genetically divergent from the South African population [Examination of mtDNA in pulation . Howeverpulation  was a COP. h. homarus, despite the long larval dispersal period (around 6 months). The NWIO region appears to be genetically isolated  from regions to the south (Kenya and Tanzania) and to the east (India and beyond). This unexpected degree of isolation observed in both marker types may be explained by the Arab Sea Gyre and east-west monsoon currents [P. homarus, and may also reflect breaks in larval connectivity. These genetic patterns do not clearly locate the existence of a single mid-Indian Ocean barrier (MIOB), as has been seen in other species [We found strong evidence for reduced gene flow across the distribution of currents , 36 whic species . The genP. ornatus  has not detected any population differentiation in mtDNA or nDNA [P. penicillatus across this region also failed to find any marked genetic differentiation [There appear to be few breaks in connectivity between the Indonesian (Aceh and Lombok samples) and East Asian (Vietnam and Taiwan samples) regions, at either mtDNA or nDNA markers. Although our sampling is less intense across this region, the continuous habitat, strong currents, and many stepping-stone islands suggest that connectivity is likely to be strong in this species across this region. Indeed, more intense sampling of this region in the closely related  or nDNA , 80, andntiation , 81.P. h. rubellus lineage strongly suggests past allopatric divergence of this lineage, with more recent secondary contact and introgression. The evidence that nDNA gene flow between the lineages is now much higher than mtDNA gene flow, suggests that a partial reproductive barrier has developed during their allopatric isolation, and that this barrier may act through a combination of male mating preferences, and ecological selection on differentially adapted phenotypes. Such a scenario may also be a common feature behind some of the marine biogeographic boundaries commonly observed across the Indo-Pacific.This study provides further evidence of the ability of marine Indo-Pacific populations to diverge genetically, despite a capacity for extensive planktonic larval dispersal. It is apparent that the levels of gene flow within the species are sufficient to maintain cohesion of the species, except at the peripheries of the distribution range. In the east, the Marquesas population appears to be an isolated remnant of past dispersal, which is now gradually diverging genetically and morphologically. In the west, the great mtDNA divergence and reduced nDNA divergence in the P. ornatus, where a very distinct mtDNA lineage has been found in the WIO [P. h. rubellus, but that a behavioural reproductive barrier did not develop in P. ornatus before secondary contact and introgression occurred. P. ornatus exhibits its greatest mtDNA lineage boundary further north than P. homarus (north of Kenya). Another related species, P. penicillatus, has its most divergent Indian Ocean mtDNA lineage in its NWIO population, specifically in the Red Sea [Panulirus populations is that they all lie at the extreme western periphery of their species’ distribution. Thus it seems that peripheral populations in the WIO may have a high probability of forming divergent lineages and perhaps species, even if there is potential for long-distance larval dispersal.A similar situation is seen in the closely related  the WIO . However Red Sea , 82, whiP. homarus in this study may also apply more widely in this region.Recent studies in other diverse species have also highlighted the importance of the WIO as a location of unique marine lineages and biodiversity. A number of finfish species have been identified as having divergent populations in the WIO , 22, 83,P. penicillatus revealed that the East Pacific lineage of that species diverged approximately 1.5 Ma (0.8–2.31), while the NWIO Red Sea lineage diverged only about 0.7 Ma (0.4–1.1) [P. homarus show that the P. h. rubellus lineage likely diverged around 2.9 Ma (2.2–3.7), while the Marquesas sub-population diverged around 1.25 Ma (0.9–1.6). Identical calculations on the P. ornatus mtDNA data estimate that the East African lineage diverged around 0.66 Ma [P. ornatus and P. penicillatus had peripheral WIO populations that genetically diverged. Around 1.5 Ma, both P. penicillatus and P. homarus had peripheral Eastern Pacific populations that genetically diverged. The P. h. rubellus lineage is by far the oldest divergence among this group, and appears to pre-date these divergences by several million years, but coincides with the estimated date of most common divergence across the MIOB in a range of fish species [Analysis of mtDNA divergence times in 0.4–1.1) . Similar 0.66 Ma , and it  species .Panulirus lineage divergences reported here fall into the most recent Pliocene period that they characterise as a period of “survival and export” of species. The present study confirms that, even within the tropical spiny lobsters with enormous larval dispersal potential, this has certainly been a period of “export” of unique genetic lineages at the peripheries of the Indo-Pacific.These estimated dates of divergence point toward the possibility of common periods of phylogeographic divergence across the Indo-Pacific, which may help to identify the potential initial drivers of divergence. The dramatic impact of sea-level changes on both sea shelf habitat availability and fluctuations in prevailing currents have previously been highlighted, especially in the Indian Ocean, where coastal shelves are generally narrow . HoweverP. homarus. The pattern of mitochondrial and nuclear divergence of the western lineage, implicates processes of parapatric isolation, secondary contact and introgression, and suggests possible maintenance through adaptation and behavioural reproductive isolation. In contrast, the eastern lineage appears to have diverged through a rare colonisation event, maintained through long-term isolation, and matches expectations of the core-periphery hypothesis. This may provide an explanation of some of the common evolutionary processes that translate phylogeographic divergence into biogeographic divergence, and continue to generate marine diversity throughout the Indo-Pacific.The data presented here show that very different evolutionary processes have led to the initial divergence and long-term maintenance of the western and eastern peripheral lineages of