Bemisia tabaci is a cryptic whitefly-species complex that includes some of the most damaging pests and plant-virus vectors of a diverse range of food and fibre crops worldwide. We combine experimental evidence of: (i) differences in reproductive compatibility, (ii) hybrid verification using a specific nuclear DNA marker and hybrid fertility confirmation and (iii) high-throughput sequencing-derived mitogenomes, to show that the “Mediterranean” (MED) B. tabaci comprises at least two distinct biological species; the globally invasive MED from the Mediterranean Basin and the “African silver-leafing” (ASL) from sub-Saharan Africa, which has no associated invasion records. We demonstrate that, contrary to its common name, the “ASL” does not induce squash silver-leafing symptoms and show that species delimitation based on the widely applied 3.5% partial mtCOI gene sequence divergence threshold produces discordant results, depending on the mtCOI region selected. Of the 292 published mtCOI sequences from MED/ASL groups, 158 (54%) are low quality and/or potential pseudogenes. We demonstrate fundamental deficiencies in delimiting cryptic B. tabaci species, based solely on partial sequences of a mitochondrial barcoding gene. We advocate an integrative approach to reveal the true species richness within cryptic species complexes, which is integral to the deployment of effective pest and disease management strategies. Evidence of both reproductive and genetic isolation is necessary for practical and accurate species identification while providing the opportunity to develop species-specific molecular markers. Ideally, an integrated approach involving the study of multiple factors  is required to solve the problem4.Accurate species identification underpins our understanding of biodiversity and enables clear scientific communication. In addition, if the organism is a pest having a negative impact on humans or the environment, it is essential for developing targeted and effective pest management strategies. Adequate species identification, however, is often difficult, especially when the organism belongs to a complex of cryptic species that cannot be distinguished by morphology alone. In such cases, it is logical to study populations of such organisms using the biological species concept. This concept defines a species as a group of individuals that can breed together (panmixis), but cannot with other groups, 5. The process involves sequencing of a standardised region in the organism’s DNA and comparing it to an established ‘DNA Barcode’ database to enable species identity confirmation. For the Insecta, the commonly used region for DNA barcoding is a partial sequence of the mitochondrial cytochrome oxidase I (mtCOI) gene. Although this approach is attractive, simple and relatively fast, it has some fundamental problems, including: (i) the overlap between intra- and inter-species sequence divergences7, (ii) the large number of misidentified and erroneous sequences in online databases10, (iii) the arbitrary approach to selecting a ‘preferred’ barcoding gene region, i.e. either the 5′ region of mtCOI5 or its 3′ region11, and (iv) difficulties differentiating nuclear mitochondrial DNA segments (NUMTs) from genes in mitochondrial DNA (mtDNA)12.A popular and widely used technique for species assignment is DNA barcodingBemisia tabaci cryptic species complex of whiteflies (Hemiptera: Aleyrodidae), some of whose members are regarded to be amongst the worst invasive pests and plant-virus vectors14. A considerable effort, therefore, has already been invested in attempting to resolve the systematics of this species group. The former system of classifying B. tabaci populations into biotypes and host races based on various biological and biochemical markers has been superseded by molecular analyses based on the 657 bp partial sequence of the 3′ end of the mtCOI gene17. In addition, a genetic distance threshold of 3.5% was identified based on a gap in the distribution of pairwise sequence divergences amongst unique mtCOI partial sequences of B. tabaci16. In subsequent analyses, patterns of clusters of putative species were recognised which could be defined by sequence divergence equal to or higher than 3.5%. Mating experiments among some of these phylogenetic species have reported either complete or partial reproductive isolation21.The practice of submitting DNA sequences generated for barcoding purposes to public data repositories such as GenBank has made them readily available for further analyses including phylogenetic analyses. That is the case for the B. tabaci populations collected worldwide29. Of these, two species are especially of significant economic importance as highly invasive pests: Mediterranean (MED) and Middle East-Asia Minor 1 (MEAM1)14. These two species form the “Africa/Middle East/Asia Minor” clade together with the non-invasive Indian Ocean (IO) species, which was found to have asymmetric mating interactions with MEAM1 while showing 7–8% divergence at the mtCOI level30. Until recently, a Middle East-Asia Minor 2 (MEAM2) species also formed part of the clade, but its species status was based exclusively on pseudogene sequences originating from MEAM1 nuclear DNA12. A “MEAM2” population was recently reported from Uganda31, however, this is a new putative species that was named on the basis of 97.1% similarity (i.e. within the 3.5% species delimitation boundary) to a published MEAM2 pseudogene sequence and should not be confused with the MEAM2 pseudogene clade12.At least 39 putative species have now been proposed in Bayesian and maximum likelihood phylogenetic analyses of the mtCOI sequenced from Aleurodes tabaci originally collected by Gennadius in 1889, which indicates that MED represents the original B. tabaci32. MEAM1 was controversially named B. argentifolii33 due to its ability to induce silver-leafing symptoms when feeding on squash34. However, this trait was later also observed in IO35, and reportedly also in the “African silver-leafing” group of MED16.The mtCOI partial sequence of MED corresponded to a syntype of 16. An increased availability of new mtCOI sequences, however, led to the distinction of two subclades that were called Q1 and Q236, or MedBasin1 and MedBasin237, followed by further subdivision into four subclades 38) or five subclades called Q1–Q539, without an indication of how the group names corresponded to those previously published. Microsatellite analyses of sympatric field populations suggested that Q1 and Q2 were reproductively compatible42, while evidence of gene flow was not detected between Q1 and ASL in the field43, suggesting that the ASL population is a reproductively isolated entity. However, the model-based Bayesian methods for detecting hybrids that were implemented in the microsatellite field studies are biased towards the detection of F1 hybrids44. To ascertain reproductive compatibility between populations it is important to test the fertility of F1 hybrids.MED was proposed to be a single species after grouping multiple biotypes , based on the 3.5% species boundary threshold39) constitutes a single biological species. We performed two-generational reciprocal crossing experiments in laboratory conditions associated with nuclear-gene based molecular markers to verify hybrid progeny. We then contrasted our findings with the mtCOI-based classification of MED and compared the sequence divergence among two different barcoding regions and 15 mitochondrial genes. Full mitogenomes were assembled from high-throughput sequencing (HTS) data, which enabled distinction between true mtDNA sequences and potential nuclear pseudogenes in the MED mtCOI datasets.In this study, we tested the hypothesis that the MED putative species  × ♂Q2 cross.Fertility of the F2 offspring counts, depending on the direction of the cross  toward female offspring produced by hybrid females with a Q2 maternal origin  compared to their counterparts with a Q1 maternal background (59.5% and 65.3%).The proportion of females also differed between F1 females from the Q1 and Q2 cross was analysed by Sanger sequencing of the GC1 nuclear DNA marker. Its sequence enabled the Q1 and Q2 populations to be distinguished based on a 4 bp insertion/deletion (INDEL) and two single nucleotide polymorphisms (SNPs)  were separated by molecular cloning and Sanger sequencing. The final sequence alignment of unique GC1 haplotypes showed that each of the four F1 females contained sequences specific for both Q1 and Q2 populations  and significant amino acid changes (criterion iv) occurred in all five groups Table . No premBemisia species or chloroplast genome sequences in BLAST search. Criterion (iv) failed 47 (22.2%) Q1 haplotypes based on the alignment with a reference set of COI protein sequences, which revealed amino acid changes in positions that are fully conserved across the B. tabaci complex and the Bemisia “JpL” species.Within the Q1 dataset, 67 31.6%) of the 212 unique haplotypes failed criterion (i), out of which 12 overlapped with criterion (iii) , failing criterion (i). Four Q2 haplotypes were detected by criterion (iii). Three of them contained clusters with high density of non-synonymous mutations , including multiple INDELs. The fourth haplotype contained a high number of SNPs throughout the sequence . In addition, ten haplotypes (27.0%) showed non-synonymous substitutions resulting in amino acid changes in positions that were conserved across the reference set of protein sequences, failing criterion (iv).et al.39, were 99.2–99.5% identical to the partial mtCOI of Israel Q2 and one of them contained a non-synonymous mutation in a conserved site. The group of haplotypes called Q338 or Q539, all from Burkina Faso, contained one haplotype with an INDEL and three haplotypes with one significant amino acid change. Finally, within the ASL38 or Q439 group, 7/33 (21.2%) haplotypes contained INDELs and additional 10/33 (30.3%) haplotypes showed amino acid changes in conserved sites.The two haplotypes from Croatia, called Q3 in Chu A sliding window analysis was performed to investigate and compare the level of nucleotide divergence in a 657 bp window across the full length of mtCOI gene . Levels of nucleotide diversity in the 5′ and 3′ barcoding regions were compared.16 showed that the distribution of sequence divergence is not uniform across the gene length .The divergence between ASL and Q1 + Q2 groups ranged from 2.48 to 4.77% , 249,142 (Israel Q2) and 149,343 (Uganda ASL), with mean base coverage 4606.5 ± 3502.6, 2231.5 ± 1576, and 1351 ± 666 reads, respectively.The numbers of mapped Illumina HiSeq reads to the reference MED mitogenome JQ906700B. tabaci species complex for three regions: (i) the 3′ mtCOI barcoding region  thion Fig.  and , while at the 5′ the divergence rose to 3.81–3.96%. The divergence among the Q1, Q2, and ASL populations across 15 mitochondrial genes was more closely reflected in the 3′ region for Q1 and Q2 populations, while for the ASL population it was the 5′ region.B. tabaci complex were reconstructed in a phylogenetic analysis based on the concatenations of 15 mitochondrial genes , K3Pu + F + I (atp8), HKY + F + G4 (cox1 and cox2), K3Pu + F + I + G4 (cox3), TIM + F + I + G4 (cytb), TPM3 + F + I (nd1 and rrnL), TPM2u + F + I + G4 (nd2), TPM3 + F + G4 (nd3), TIM3 + F + I + G4 (nd4), TPM3u + F + I (nd4l), TIM3 + F + I + G4 (nd5), TPM2 + F + I (nd6), HKY + F + G4 (rrnS). The UltraFast bootstrap values for all branches were 80–100%, which unlike the normal bootstrap corresponds roughly to 80–100% probability that the clades are true46. Uganda ASL was placed outside the Q1 and Q2 clade with 100% bootstrap support, between MED and MEAM1 species.The relationships between MED populations, Uganda ASL population and six other species of nes Fig. . The topThe ability of the Ugandan “ASL” population to induce squash silver-leafing was tested in a bioassay with MEAM1 as a positive control. Squash plants infested with MEAM1 whiteflies developed silver-leafing symptoms within two weeks after whitefly infestation Fig. . The “AS15, “okra biotype”47 and “Ug4”48  tested, exceeded the theoretically possible number of alleles per individual. For example, two Q1-specific (Hap2 and Hap3) and two Q2-specific haplotypes (Hap7 and Hap8) were detected in the hybrid female f1, while in reality she would have received only one allele from each parent. The 12 GC1 haplotypes differed from one another by 1–4 SNPs that occurred outside the diagnostic region shown in Fig. Taq DNA polymerases is 1–20 × 10−551, which is 6- to 50-fold higher than more precise DNA polymerases such as Pfu or Phusion Hot Start52. Despite these introduced errors, the cloned sequences proved unequivocally that each hybrid female contained a copy from both parental types as shown by the INDEL region. This method of checking for genuine hybrid female progeny could be improved by using a high-fidelity DNA polymerase and a reduced number of PCR cycles for amplification of the marker sequence prior to cloning. Alternatively, the hybrids could be detected using microsatellite markers53 or evidence of hybridisation between species could also be demonstrated by using genome-wide SNPs54.Sequences of the cloned inserts of the B. tabaci (Gennadius) from Greece corresponds to MED Q132, we conclude that the Q1 and Q2 both belong to B. tabaci sensu stricto. These two groups are differentiated in allopatry, but retained the ability to interbreed when occurring sympatrically in the field. The gene flow between Q1 and Q2 MED populations was suggested to be asymmetric, because the nuclear background associated with Q1 individuals introgressed into, or completely replaced, the nuclear background of individuals with Q2 mitochondrial type, but not vice versa41. However, a different population genetic study based on microsatellites showed evidence for both directions of hybridisation55. Samples from France and Canary Islands had “Western Mediterranean” nuclear background and Q2 mitochondrial type, while sample from Greece had Q1-type mitochondria and “Eastern Mediterranean” DNA introgressed into its nuclear genome55. The interaction also did not appear asymmetric in Terraz et al.40, as the sympatric populations in France and Spain had homogeneous nuclear backgrounds regardless of the mitochondrial haplotype. In our study, no significant differences were observed between F1 progeny from the two directions of the cross. In the F2 generation, however, the fertility was unequal between hybrid females with different parental origin. Albeit the nuclear DNA of our samples was not studied, F2 females with Q1 mitochondria were more fecund than their Q2 counterparts, which could make hybrids with Q1 mitochondrial type more prevalent and thus more likely to be detected in the field.From the biological species concept definition, the Q1 and Q2 subclades belong to the same species. As the mtCOI of the original specimen of 57 or affect the host’s fitness59. Cytoplasmic incompatibility could explain the mechanism behind the lack of gene flow between Q1 females and Q2 males in Gauthier et al.41. Alternatively, the asymmetry could be caused by the difference in fecundity between hybrids which emerged from the opposite directions of the same cross. The effect of endosymbiotic bacteria on the reproductive compatibility and fitness of MED whiteflies remains to be established.We hypothesise that these mating interactions are influenced by secondary endosymbiotic bacteria. Some endosymbionts residing within insects are known to induce cytoplasmic incompatibility39, revealed the extent of NUMTs or erroneous sequences included in previous molecular studies. In all five subclades, a half or more (50–55%) of the unique haplotypes contained mutations that are unlikely to occur in a functional protein-coding sequence. It is important to note that the FaBox tool assigned identical sequences with different lengths as unique haplotypes. Thus, the percentages in Table The analysis of 292 unique mtCOI haplotypes deposited in GenBank as assigned to MED subclades Q1–Q5 and ASL60 and confound phylogenetic analyses62, as well as create confusion and inconclusive results in the research community. Pseudogene sequences have recently been shown to cause an overestimation of species richness within the Africa/Middle East/Asia Minor clade (the case of MEAM212). This indicates that unrecognised NUMT sequences might commonly occur across multiple clades of the B. tabaci species complex and interfere with efforts to resolve its systematics. It is therefore important to recognise this issue and to analyse critically the quality of putative mitochondrial sequences amplified with universal primers. The pseudogene sequences are not immediately obvious if they do not contain stop codons or significant amino-acid changes within the analysed partial sequence. We emphasise the utility of HTS and acquiring mitochondrial sequences by assembly from a large number of reads. Such sequences can be then used as a reference for all the Sanger-sequenced marker sequences (e.g. from a field collection), to identify sequencing errors and potential pseudogenes and exclude them from downstream analyses.The inclusion of NUMT sequences in phylogenetic analyses can overestimate the species richnessB. tabaci occurred in sympatry on the same host plants43. In addition, our phylogenetic tree shows the genetic distinctness of the ASL population from the MED Q1 and Q2 populations with a high statistical support. The tree also supports the consensus that Indian Ocean occupies a basal phylogenetic position within the Africa/Middle East/Asia Minor clade16.The sub-Saharan ASL population is reproductively isolated from Q1 and Q2 groups as demonstrated by our laboratory crossing experiments and the field microsatellite study in Burkina Faso, where Q1 and ASL et al.15. These two clades, however, were later merged to form one “Mediterranean” putative species16. This placement was in accordance with the 3.5% species-level boundary in the 3′ barcoding region of mtCOI. We show here, however, that the identity values vary considerably across the full length of mtCOI gene. Results from the sliding window analysis demonstrate that the sequence divergence between ASL and Q1 + Q2 populations in a 657 bp long window ranged from 2.48 to 4.77% (a difference of 2.29%), depending on the region chosen for barcoding. Such variability is not apparent between Q1 and Q2, divergence of which remains low across the full gene length . A similar pattern was observed in the comparison at the mitogenome scale. Interestingly, both mtCOI barcoding regions quite accurately reflected the divergence across 15 mitochondrial genes, but each for a different group .The “Sub-Saharan Africa silver-leafing” clade was originally placed separately from the “Mediterranean invasive” clade in the first global Bayesian phylogenetic study by Boykin et al.63 showed symptoms within two weeks. This is not surprising, as the original paper by Sseruwagi et al.48 clearly stated that only groups Ug6 and Ug7  induced squash silver-leafing, while Ug4 (similar to “okra biotype” from Ivory Coast) did not. The “silver-leafing” group of MED was linked to the former J biotype14 reported in Nigeria, Ghana, Cameroon, Ivory Coast and Zimbabwe64. However, De la Rúa et al.64 did not study the capacity to induce silver-leafing and in earlier studies the J biotype failed to induce this phytotoxic disorder66. Despite all this evidence, multiple subsequent studies about African silver-leafing populations of MED referred either to Sseruwagi et al.48 or De la Rúa et al.64, or to other papers that have not demonstrated the silver-leafing capacity16. It is possible that the results of Sseruwagi et al.48 were misinterpreted due to the mixed “Ug4/Ug6” population reported to induce silver-leafing, and/or a confusion caused by the naming system in the study, and this misinterpretation was then cited in subsequent literature.Our study also shows that the “African silver-leafing” population from Uganda does not induce silver-leafing. None of the plants infested with the ASL population developed silver-leafing symptoms five weeks after whitefly infestation, while the positive control with MEAM1 in our study and the one of Yokomi 15, “okra biotype”67 and “Ug4”48 indicates that these groups might belong to the same species, but it remains to be confirmed. A list of these potential synonyms of the “ASL” species from previous literature is summarised in Supplementary Table We propose that our “ASL” population from Uganda belongs to a non-MED species within the Africa/Middle East/Asia Minor clade. The 98.87–100% identity among 3′ mtCOI sequence across samples of “African silver-leafing”B. tabaci (Russell 1957). The synonymised species collected in sub-Saharan Africa include B. gossypiperda var. mosaicivectura Ghesquière , B. vayssierei Frappa , B. goldingi Corbett , B. nigeriensis Corbett  and B. rhodesiaensis Corbett 68. DNA sequencing from museum syntypes of these species will be attempted in the near future to establish whether any of the above names apply to ASL/okra/Ug4. If not, a formal description of this new species will be required to accompany a new species name.It is possible that “ASL” has already been described and was later synonymised with sensu Dinsdale et al.16) comprises at least two biological species. One is the original B. tabaci (Gennadius), which includes the MED populations Q1 and Q2 from the Mediterranean Basin; the other is the sub-Saharan “MED ASL” species, awaiting a new binomial name. This division is based on the biological species concept, as “ASL” and MED are reproductively isolated both in the laboratory and in the field43. The reproductive compatibility of Q1 and Q2 was demonstrated in both directions, the fertility of F1 hybrids was verified and for the first time the combined genetic material from both parents in the F1 hybrids was confirmed using a specific nuclear DNA marker. Furthermore, by using HTS-derived mitogenomes, we were able to compare the genetic divergence among the MED populations and seven other Bemisia species, using true mitochondrial sequences (i.e. no pseudogenes) and multiple genes. This approach contrasts previous studies inferring phylogenetic relationships and sequence divergence among MED populations from 3′ partial mtCOI sequences, out of which 54% in the public databases contain errors and potentially are NUMTs.In conclusion, we gathered multiple lines of evidence that the MED species  will offer greater power for inference of B. tabaci cryptic species phylogenetic relationships. We also suggest that understanding the species diversity in pest-species complexes is the first step towards designing novel and targeted management strategies. It shall also bring consistency and clarity to communication within the scientific community, as well as to the wider public.The previous inclusion of the “ASL” population in the MED species demonstrates a fundamental problem with species delimitation based exclusively on an arbitrarily chosen barcoding sequence in mitochondrial DNA, because the results varied depending on the selected region of comparison. Furthermore, we showed a case of serial miscitation that led to the perpetuation of the erroneous assignment of the diagnostic trait of squash silver-leafing to the “ASL” species. Overall, we advocate the importance of an integrative approach to cryptic species identification and delimitation, most importantly including the biological species concept and using multiple genes or genomic data to infer B. tabaci colonies were reared on aubergine (Solanum melongena ‘Black Beauty’). Plants were grown from seeds in a whitefly-free room at 28 ± 2 °C, 50–60% relative humidity (r.h.) and a 14:10 L:D photoperiod. Whitefly colonies were maintained in rectangular 45 × 44 × 44 cm cages  at 28 ± 2°C, 30% r.h. and 14:10 L:D. Colonies were established from field populations collected in Spain , Israel  and Uganda . The purity of core whitefly colonies was monitored periodically by sequencing of the partial mtCOI gene (detailed below).69. At least five individual adults from each core colony were used for the species identity verification. Whiteflies were homogenised individually using sterile pestles in 50 µl of 10% Chelex® 100 Resin solution (Bio-Rad). The mixture was incubated at 56 °C for 20 min, subsequently at 95 °C for 5 min and centrifuged for 5 min at 13,500 rcf. The supernatant was used as a template for amplification by polymerase chain reaction (PCR).Genomic DNA was extracted from adult whiteflies stored in 90% ethanol using the Chelex method31. PCRs  were carried out using DreamTaq DNA polymerase  following the manufacturer’s instructions. The 867 bp amplicons were purified with reSource  or GeneJET  PCR purification kits prior to Sanger sequencing by Source Bioscience .Whitefly identification was based on the 3′ barcoding region of mtCOI gene. The segment was amplified and sequenced with primers 2195Bt and COI2-BtSh270, unless stated otherwise. The mtCOI sequences were trimmed to 657 bp, corresponding to nucleotide positions 782–1,439 of the complete mtCOI gene of MED45. Sequence-based comparisons were done using two reference datasets: (i) consensus sequences for 24 putative species of B. tabaci16 to verify MED species, and (ii) MED mtCOI haplotypes38 to place our populations in a recognised naming framework. In addition, the partial mtCOI sequences of Uganda ASL sample were compared to published sequences of “African silver-leafing”15, “okra biotype”67 and “Ug4”48 groups.All DNA sequence analyses were carried out in Geneious version 10.0.8Portiera) and five secondary endosymbionts  was tested by conventional PCR. Genus-specific primers targeting the 23 rDNA (Hamiltonella) or 16 S rDNA genes (the remaining bacteria) from Ghosh et al.71 were used. Total genomic DNA (extracted as above) from ten individual females per each colony was used as a template. Positive and negative controls were included in screening for each endosymbiont. PCR products were visualised by agarose gel electrophoresis, with results summarised in Supplementary Table The presence/absence of the primary bacterial endosymbiont .Control crosses were carried out with one female and three males from the same population with 8.7 replications on average. For the reciprocal crosses, the female and males originated from different populations and were used (average n = 8.3). The four adults were released onto aubergine plants in three to seven true-leaf stage, rooted in soil and enclosed in Lock&Lock whitefly-proof cages1 hybrids was tested in a separate set of reciprocal crossing experiments. A female and three males per replicate (average n = 8.8) were used as above, but the F1 hybrid female nymphs were harvested from a plant onto which 20 + 20 virgin females and males from populations of interest were given the opportunity to reproduce for 14 days. Virgin hybrid females from both directions of the cross were then backcrossed with males from either parental population, resulting in four types of crosses to produce F2 generation. The parents were collected after seven days and progeny collected and sexed until all emerged.The fertility of F74. Counts of offspring generated in crossing experiments were analysed by a generalised linear model with negative binomial error distribution and a log link using the MASS library75. For the proportion of female progeny, a generalized linear model with quasibinomial error distribution and logit link of the proportional data was used. Multiple comparisons of offspring counts and female proportions were performed by Tukey’s test76 using the multcomp package77 and significant differences were demonstrated by compact letter display. Reciprocal crosses that produced only male offspring were omitted from the multiple comparison. A separate analysis was also carried out with results from the F1 and F2 generations relevant only to the reproductively compatible populations.Statistical analyses were performed using R1 females. We targeted nuclear genes encoding mitochondrial proteins, because of their potentially faster mutation rate78. Candidate genes were extracted from transcriptome sequences of adults from Spain MED Q1 and Israel MED Q2 populations (unpublished data) based on Drosophila melanogaster homologues (www.flybase.org) and searched for variable regions. The marker chosen for this study was a segment of 3′ untranslated region of the predicted Glutamate Carrier 1 (GC1) gene . Primers were developed in Primer 3 software80 .The parentage of female progeny was studied using a nuclear DNA marker. Mitochondrial DNA marker was not used due to its maternal mode of inheritance, rendering it ineffective for discerning the paternal origin of F1 females produced in reciprocal and control crosses. Trace files were analysed using the Pregap and Gap4 programs within the Staden molecular analysis software81. The GC1 amplicons from F1 females were cloned into pGEM®-T Vector System I (Promega) with a 2:1 insert:vector ratio. Colonies of transformant E. coli cells JM109 were selected randomly and individually lysed in 200 µl sterile water by brief vortexing, then used as a template for PCR with vector-specific T7 and SP6 primers. Selected amplified inserts were sequenced at the ACRF Biomolecular Resource Facility at the Australian National University in Canberra and analysed as described above. The insert sequences were collapsed into unique haplotypes in FaBox DNA Collapser version 1.4182 and mapped to reference sequences from control crosses.The amplicon was sequenced from representative F83 and quantified using a Qubit® 2.0 fluorometer (Invitrogen). Genomic DNA (~30 ng in 100 µl of TE buffer) was sheared for 10 min using an ultrasonic cleaner (VGT-1620QTD) and size-selected (300–500 bp) using Blue pippin (Sage Science). Sequencing libraries were prepared separately using each size-selected DNA pool using the NEBNext® Ultra DNA Library Prep Kit for Illumina® with NEBNext Multiplex Oligos for Illumina (New England BioLabs). Individual libraries were checked by capillary microchip electrophoresis , pooled in equimolar amounts, and then purified using Agencourt AMPure XP Beads (Beckman Coulter). The multiplexed library was sequenced on a single lane of an Illumina HiSeq. 4000 platform  and 150 bp paired-end reads were generated.Genomic DNA from single male whiteflies representing each pure, inbred laboratory colony, was isolated using the silica spin-column method84. The reads trimmed with a quality threshold of 40 and minimum length of 18 bp were used in subsequent analyses. Draft mitogenomes of the Q1, Q2 and ASL males were assembled by iterative reference-guided assembly. The trimmed reads were mapped to the reference MED mitogenome JQ90670045 using Geneious 10.0.8 mapper set to medium-low sensitivity with up to 5 iterations. The draft mitogenomes were annotated in MITOS284 and manually checked for start and stop codons in all protein-coding genes by translating into amino acid sequences using the invertebrate mtDNA genetic code. As the sequence was assembled from short reads, the accuracy of the low complexity intergenic region between cox3 and tRNA-Ile represents only an estimate. Nevertheless, the lengths of these AT-rich regions among our mitogenomes  were similar to the one in the reference MED mitogenome JQ906700 (972 bp), which was obtained by long PCR and Sanger sequencing45.Quality control was carried out in FastQC version 0.11.5 before and after trimming the reads with Skewer 0.2.2 version for Linux39 were downloaded from GenBank (accessed 14th November 2017) and collapsed into unique haplotypes in FaBox  non-synonymous mutations resulting in amino-acid substitutions in positions conserved across other Bemisia species. For the fourth criterion, only sequences that passed the first two criteria were analysed. Such DNA sequences were translated and aligned by Geneious alignment tool using Blosum62 matrix to a reference set of mtCOI amino acid sequences translated from 12 mitogenomes: (1–3) populations from this study, (4) China MED Q1 (JQ906700)45, (5) Burkina Faso MED Q1 (KY951447), (6) Peru MEAM1 (KY951450), (7) Australia I (KY951541), (8) Indian Ocean (KY951448)12, (9) B. emiliae , (10) Bemisia “JpL” (KX714968)85, (11) Asia I (KJ778614)86 and (12) New World (AY521259)87. The likelihood of the non-synonymous substitutions occurring in MED haplotypes was estimated by comparing the intra- and inter-species variability in respective triplet positions. Sequences with high nucleotide divergence from the group were compared against the NCBI database using BLASTn88 to investigate the origin of the sequence.The criteria used to categorise a partial mtCOI sequence as a potential pseudogene included either: (i) the presence of INDELs, (ii) the presence of premature stop codons inside the protein-coding sequence, (iii) anomalous polymorphisms  in a sliding window analysis in DnaSP version 5.10.0189. The size of commonly used partial mtCOI sequence (657 bp) was used for the window size, sliding along the sequence in 5 bp steps while plotting the divergence  between the compared samples. Sequences included in the sliding window analysis were the three populations from this study, Burkina Faso MED Q1 (KY951447), Peru MEAM1 (KY951450) and Indian Ocean (KY951448)12.Intra- and inter-specific sequence divergence levels within the Africa/Middle East/Asia Minor cladeBemisia “JpL” due to incomplete sequence data. The genes were orientated in 5′−3′ direction, individually aligned by MUSCLE alignment tool in Geneious and trimmed to the same length prior to being manually concatenated into 12,595 bp sequences. The multiple sequence alignment in fasta format was submitted to IQ-tree web server90 along with a manually created nexus file partitioning the data91. Three different partition schemes were analysed: (i) no data partitioning, (ii) partitioning the protein-coding genes into 1st + 2nd codon position, 3rd codon position, and rRNA genes grouped in the third partition; and (iii) partitioning into 15 individual genes. No substitution saturation in the protein coding sequences was detected using DAMBE692, indicating that all three codon positions could be used in the phylogenetic analysis. The best-fitting substitution model for each partition was identified using ModelFinder93. Ultrafast Bootstrap94 with 1,000 replications was performed and the tree was visualised in Dendroscope 395.Concatenations of 13 protein-coding genes and two rRNA genes were generated from 11 mitogenomes listed above, excluding Cucurbita pepo ‘All Green Bush’, individually enclosed in Lock&Lock cages in five replications. As a positive control, two replicates with 60 MEAM1 adults from Peru were set up using the same protocol. After two weeks the adults were collected and plants were visually assessed for leaf silvering symptoms, for up to five weeks post-infestation.The capacity of Uganda ASL to induce the squash silver-leafing symptoms was tested by releasing 30–60 adults on squash plants Draft mitogenomes assembled in this study are available under GenBank accession numbers MH205752 (Spain Q1), MH205753 (Israel Q2) and MH205754 (Uganda “ASL”). The partial 3′ mtCOI gene sequences (657 bp) of the whitefly colonies used in this study correspond to positions 782–1,439 of the draft mitogenomes.GC1 marker acquired from molecular cloning can be found under accessions MH205738–MH205749. Accessions for the GC1 marker from Spain Q1 and Israel Q2 controls are MH205750 and MH205751, respectively.Haplotypes of the Supplementary Information


Specifications TableValue of the data•The data presents the specific experimental process.•The details of the experiment, test methods and instrument models.•The data shows that the temperature dependence of the lattice thermal conductivity.1x contents. x contents.The dataset of this article provides the sample preparation process information. The 2x samples with different x values  were prepared by using the In, Sb, (99.9% in purity) powders. We put the powders of In and Sb (both is 200 mesh) with a certain proportion of InSbx  and sealed them in different crucibles(23 mm × 18 mm). Then delicately placed the crucibles into quartz tubes. The samples were healed from room temperature to 973 K with the pressure at 5 × 10−2 Pa under purified argon atmosphere in 30 min. Then samples were melted at 973 K for 3 h and the furnace was cooling down to room temperature at the rate of 20°C/min. Finally, we obtained the cylindrical specimens with the size of 16 mm × 5 mm.The InSbρ) of the samples were determined by Archimedes method. The fractographs were measured by a field emission scanning electron microscope , and the composition identification was conducted by electron probe microanalysis  on a JEOL JXA-8230 microscope. To measure electrical conductivity and Seebeck coefficient, the samples were cut from the sintered pellets into rectangular bars with a dimension of 1 × 3 × 10 mm. The Seebeck coefficient and electrical resistance were measured using a Seebeck Coefficient/Electrical Resistance Measuring System  in a helium atmosphere. The thermal diffusivity (D) was measured by laser flash method . The thermal conductivity (κ) was calculated by the density (ρ), specific heat and thermal diffusivity using the equation κ = pρDC. These thermoelectric properties were measured in the temperature range of 323–773 K.The speciments were characterized by X-ray diffraction  and the density (L is the Lorentz number The temperature dependences of This work mainly adopted the strategy of eutectic melting to prepare InSb type alloy thermoelectric material. Using solid reaction to prepare eutectic compounds not only can prepare lamellar InSb alloy, but also this method is efficient and simple. Furthermore, the thermoelectric properties of the alloy could be improved by doping and changing the experimental conditions.


Volumetric quantification of mean and fluctuating velocity components of transient and turbulent flows promises a comprehensive characterization of valvular and aortic flow characteristics. Data acquisition using standard navigator-gated 4D Flow cardiovascular magnetic resonance (CMR) is time-consuming and actual scan times depend on the breathing pattern of the subject, limiting the applicability of the method in a clinical setting.We sought to develop a 5D Flow CMR framework which combines undersampled data acquisition including multipoint velocity encoding with low-rank image reconstruction to provide cardiac- and respiratory-motion resolved assessment of velocity maps and turbulent kinetic energy in fixed scan times.Data acquisition and data-driven motion state detection was performed using an undersampled Cartesian tiny Golden angle approach. Locally low-rank (LLR) reconstruction was implemented to exploit correlations among heart phases and respiratory motion states. To ensure accurate quantification of mean and turbulent velocities, a multipoint encoding scheme with two velocity encodings per direction was incorporated. Velocity-vector fields and turbulent kinetic energy (TKE) were obtained using a Bayesian approach maximizing the posterior probability given the measured data. The scan time of 5D Flow CMR was set to 4 min.5D Flow CMR with acceleration factors of 19 .0 ± 0.21 (mean ± std) and velocity encodings (VENC) of 0.5 m/s and 1.5 m/s per axis was compared to navigator-gated 2x SENSE accelerated 4D Flow CMR with VENC = 1.5 m/s in 9 subjects. Peak velocities and peak flow were compared and magnitude images, velocity and TKE maps were assessed.While net scan time of 5D Flow CMR was 4 min independent of individual breathing patterns, the scan times of the standard 4D Flow CMR protocol varied depending on the actual navigator gating efficiency and were 17.8 ± 3.9 min on average. Velocity vector fields derived from 5D Flow CMR in the end-expiratory state agreed well with data obtained from the navigated 4D protocol . On average, peak velocities assessed with 5D Flow CMR were higher than for the 4D protocol (3.1 ± 4.4%).Respiratory-motion resolved multipoint 5D Flow CMR allows mapping of mean and turbulent velocities in the aorta in 4 min. The hemodynamic assessment of blood flow through heart valves and in the great vessels using 4D Flow cardiovascular magnetic resonance (CMR) has received increasing attention and a number of important applications have emerged . While sA number of clinical studies have indicated the added value of measuring turbulent velocities alongside mean velocities using 4D Flow CMR , 6, 7, eA key challenge of encoding mean and turbulent velocities relates to achieving appropriate measurement sensitivity to both quantities. For the expected ranges of mean and turbulent velocities in-vivo, multiple velocity encodings per spatial direction are preferably obtained and combined during image reconstruction . This apSignificant scan time reductions in 4D Flow CMR have been achieved using combinations of parallel imaging and compressed sensing , k-t metInstead of using respiratory gating, respiratory-motion resolved imaging –24 has bData correlations lead to low-rank properties of the decoding problem to be solved in image reconstruction. Various studies have demonstrated that these low-rank characteristics are most efficiently exploited using local rather than global low-rankedness using patch-based decomposition of the multi-dimensional data , 27.The objective of the present work was to implement and test respiratory-motion resolved Bayesian multipoint 5D Flow CMR for mapping both mean and turbulent velocities in the aorta with a fixed scan time.The data acquisition and motion binning process is illustrated in Fig. d = d as part of the pseudo-radial Cartesian sampling pattern. Upon inverse Fourier transform of d, the projection X of the excited volume resolved along the frequency encode direction x and over time t was obtained, showing signal modulation depending on respiratory and cardiac motion. Given TR = 3.3 ms and with one in 21 readouts through the k-space center, a respiratory sampling rate of ca. 14 Hz was obtained. The signal of each coil channel was treated as a separate signal Nx being the number of samples in readout dimension and Nt the number of readouts through the k-space center. Only the magnitude was considered, as the main variation in signal phase was found to be related to the different velocity encodings which were alternated per heart beat (beat-interleaved) and which led to phase variations which were too large to be eliminated by lowpass filtering.Data-driven respiratory motion detection  was perfXTX) was selected, as it provided the predominant motion for each coil channel  [Ω, Fourier transform dkv and regularization weight λ. As illustrated in Fig. b-th out of Nb blocks of size nx × ny × nz in the image from all Nhp heart phases and Nrs respiratory motion states and transforms them into a Casorati matrix with dimensions nxnynz × NhpNrs. By penalizing the nuclear norm of this local Casorati matrix, low-rankedness is enforced locally.Accordingly, the final data vector reads nk (LLR) , 27 apprλhp and λrs denote the regularization weights along cardiac phases and respiratory motion states, respectively.For comparison, respiratory-motion resolved data were also reconstructed by penalizing total variation (TV) along the respiratory motion states and heart phases :2\documeσ [kv is related to the first gradient moment of a bipolar velocity encoding gradient by T being the time of application of gradient waveform G(t) and determines the encoding velocity as Assuming a Gaussian distribution of intra-voxel velocities, the following signal model of mean velocity entclass1pt{minimavenc to avoid phase wraps [venc~σ. It has been demonstrated that this trade-off may lead to insufficient sensitivity to fluctuating velocity components and hence a multipoint encoding scheme using two instead of one kv encoding point per axis was implemented [σ given the model S and the measured image data D were obtained [kv′ s were combined by choosing the values for σ maximizing the posterior probability of the measured data:The signal model implies a trade-off between a sufficiently high encoding velocity se wraps , and a sse wraps  such thalemented . Using tobtained . Data acAn illustration of multipoint acquisition and Bayesian decoding is provided in Fig. 3, TE = 3.3 ms, TR = 4.9 ms, flip angle = 8 and 25 cardiac phases. The different velocity encodings were alternated per heart beat. The image acquisition matrix was Nreadout = 100 ± 9, Nphase = 101 ± 8 and Nslice = 20 ± 1 (mean ± std). Using the proposed method, velocities were encoded with venc = 50 cm/s and 150 cm/s per axis and an additional venc = 0 cm/s measurement. Scan time was fixed to 4 min. After sorting the data into 4 respiratory motion states, acceleration factors were 19.0 ± 0.21 (mean ± std). On average the view-sharing approach led to 61% of the total number of acquired samples being shared among motion bins in the study cohort.All in-vivo work was performed upon written informed consent of the subjects and according to local ethics regulations. Data of the ascending aorta of nine healthy subjects  were acquired on a 3 T CMR system  using the proposed 5D Flow CMR and a standard, navigator-gated 4D Flow CMR approach [venc = 150 cm/s) per axis (4D Flow Ref). Two-fold acceleration using parallel imaging [Standard 4D Flow CMR was recorded with a single venc  [λ = 0.01 for Equation λhp = 0.04 and λrs = 0.05 for Equation Equation ts, USA) . ResultiUpon image reconstruction, concomitant gradient terms were corrected  and thirIn order to avoid noise at the vessel boundaries the segmentation masks were eroded by one pixel for MIP projections.ROI| corresponds to the number of voxels in the segmentation mask.The normalized root-mean-square error (nRMSE) of the velocity magnitude in the segmented aorta was calculated according to:TKE was calculated voxel-wise as:j and ρ is the fluid density (here we assumed 1060 kg/m3 for blood).where www.vmtk.org) and peak flow and peak through-plane velocities (i.e. the maximum velocity component in the corresponding cross-section of the aorta) were assessed for each plane. Peak through-plane velocities were determined upon application of a 3x3x3 median filter to reduce contributions from noise.Moreover, multi-planar reslicing along the aorta was conducted using VMTK (The effectiveness of respiratory-motion resolved 5D Flow CMR was assessed by comparing it to reconstructions using only data from the end-expiratory bin (EXP) and to reconstructions using no respiratory binning (NG).To assess the performance of navigator gating in the standard 4D Flow CMR protocol, the positions measured by the navigator on the diaphragm were interpolated and each readout was attributed its corresponding navigator position. Then, mean and standard deviations of the acceptance window per heart phase were calculated. Due to the coarse temporal resolution of the navigator signal (one navigator per heart beat), linear interpolation was chosen in order to provide a lower bound on the width of the acceptance window for different cardiac phases.Bland-Altman analysis  of peak Scan durations for 4D Flow CMR were 17.8 ± 3.7 min compared to 4 min for the 5D Flow protocol.Figure Reconstructions exploiting respiratory motion states (5D Flow LLR) are compared to using only data in end-expiration (EXP LLR) and without gating (NG LLR) in Fig. Magnitude and velocity magnitude images for the end-expiratory motion state obtained with 5D Flow LLR and 5D Flow TV are compared relative to the 4D Flow reference in Fig. Exemplary slices in the ascending and descending aorta show through-plane velocity profiles to be in agreement among the 4D Flow reference and 5D Flow LLR whereas 5D Flow TV shows artifacts in the descending aorta.Exemplary magnitude and velocity magnitude images for different respiratory bins reconstructed with 5D LLR are shown in Fig. Figure In this work, a respiratory-motion resolved Bayesian multipoint 5D Flow CMR approach has been implemented based on pseudo-radial tiny Golden angle Cartesian sampling in conjunction with locally low-rank image reconstruction to map mean and turbulent velocities in the aorta in a fixed scan time of 4 min.By exploiting data from all respiratory motion states, the duration of 5D Flow CMR becomes independent of the individual respiratory motion patterns of the subjects. In comparison to standard 4D Flow CMR protocols , 5D FlowIt has been demonstrated that the LLR model allows to improve reconstruction accuracy relative to using data from end expiration only, as shown in Fig. When comparing the 5D Flow LLR reconstruction with the 5D Flow TV approach, both methods showed good agreement of velocity data relative to the 4D Flow reference. However, TKE maps and magnitude images exhibit less noise when using the 5D Flow LLR approach. The inferior performance of the TV approach is associated with the piecewise constant solutions favored by TV which in turn leads to smoothing of peak values. To reduce underestimation of peak velocities, hyperparameters may be determined for best agreement with reference velocities. However, this led to non-optimal results for magnitude images which therefore still showed a high degree of aliasing noise.In-vivo, good agreement of peak velocities and peak flow values of 5D Flow LLR with data derived from standard 4D Flow CMR was found. A small bias towards higher velocities was observed for 5D Flow LLR compared to the 4D Flow reference. This can be related to previous findings that respiratory motion leads to blurring of the image , 51 whic2 and turbulence mainly occurring in the region of the flow jet in the proximal aorta. In comparison, TKE maps derived from the 4D Flow reference with 4-point encoding appeared noisy, due to only using a single encoding velocity of 150 cm/s. Moreover, the 4D Flow reference showed TKE values in the descending aorta which were as high as in the ascending aorta. This is considered unrealistic as transient/turbulent flow typically occurs in the region downstream of the aortic valve and not in the descending aorta in healthy, young volunteers.A multipoint encoding scheme  was incoThe acquisition time for 7-point encoded 5D Flow was fixed to 4 min. For 10-point encoding, as it has been used when assessing stenotic valves [Image quality of data in inspiration as shown in Fig. The present work assessed mean and fluctuating velocity vector fields in healthy subjects only. When applying the proposed 5D Flow method in patients with aortic stenosis, an extension to 10-point encoding  needs toWhile the present study was focused on turbulence encoding, the proposed technique can be readily applied for the acquisition of standard 4D flow CMR data in the aorta and in the heart. For different applications the scan time will change as a function of the size of the acquisition matrix, but respiratory motion is still expected to be accurately modelled as a low-rank problem. Therefore, fixing scan time independently of respiratory motion should still be feasible. Moreover, the suggested multipoint encoding approach provides improved accuracy for lower velocities, similar to other multi-VENC approaches , 53. In A limitation of the present study is the lack of a ground truth data. The 4D Flow reference data were acquired in a separate scan and consistency of flow conditions cannot be expected over the entire scan session. In addition, the 4D Flow reference data showed considerable respiratory-motion related artifacts in later cardiac phases since the pencil-beam navigator was played out right after R-wave detection and only once per cardiac cycle (acceptance rates varied between 42 and 72%). Accordingly, the quantitative comparisons with the 4D Flow data were limited to systole in order to avoid respiratory-motion corrupted cardiac phases as much as possible.Finally, a drawback of both 5D Flow LLR and 5D Flow TV are considerably longer reconstruction times when compared to standard 4D Flow CMR . In the future, this relative disadvantage may be addressed by employing variational neural network based reconstructions and their deployment on dedicated hardware .Respiratory motion resolved multipoint 5D Flow CMR allows for breathing-pattern independent mapping of mean and turbulent velocities in 4 min. The reduction in scan time allows for integration of the sequence into standard clinical workflows. Further in-vivo studies are now warranted to assess the performance of the method in relevant patient populations.


West Africa was subjected to deformation and exhumation in response to Gondwana break-up. The timing and extent of these events are recorded in the thermal history of the margin. This study reports new apatite fission track (AFT) data from Palaeoproterozoic basement along the primary NE-SW structural trend of the Bole-Nangodi shear zone in northwestern Ghana. The results display bimodality in AFT age (populations of ~210-180 Ma and ~115-105 Ma) and length distributions (populations of 12.2 ± 1.6 and 13.1 ± 1.4 µm), supported by differences in apatite chemistry (U concentrations). The bimodal AFT results and associated QTQt thermal history models provide evidence for multiple cooling phases. Late Triassic – Early Jurassic cooling is interpreted to be related with thermal relaxation after the emplacement of the Central Atlantic Magmatic Province (CAMP). Early to middle Cretaceous cooling is thought to be associated with exhumation during the Cretaceous onset of rifting between West Africa and Brazil. Late Cretaceous – Cenozoic cooling can be related with exhumation of the Ivory Coast – Ghana margin and NNW-SSE shortening through western Africa. Furthermore, our data record differential exhumation of the crust with respect to the Bole-Nangodi shear zone, preserving older (CAMP) cooling ages to the south and younger (rifting) cooling ages to the north of the shear zone, respectively. This suggests that the Palaeoproterozoic BN shear zone was reactivated during the Cretaceous as a result of deformation in the Equatorial Atlantic region of Africa. Geochronological and thermochronological studies have revealed that the ICG marginal ridge formed during the Cretaceous in response to rifting between the WAC and Brazil during the opening of the equatorial Atlantic Ocean8. Recent studies9 proposed that NE-SW oriented onshore faults in Ghana and the Ivory Coast are linked to an oceanic fracture zone 90 km South of the St Pauls transform zone in the Atlantic Ocean and that the eastern limit of the St Pauls Transform Zone can be linked with onshore faults in Ghana10. It is further suggested that the pre-existing shear zone may have controlled the orientation of the present-day oceanic transform zone9, however the theory remains inconclusive. Hence, this study hypothesises that the BN shear zone shares a common low-temperature thermo-tectonic history with the ICG marginal ridge. We present Apatite Fission Track (AFT) thermochronological data and associated thermal history models to constrain the low-temperature thermal and exhumation history of the BN shear zone, aiming to provide temporal constraints on the rifting history of the WAC from Brazil in response to the opening of the Equatorial Atlantic Ocean. Furthermore, we attempt to constrain the timing of shear zone reactivation, which has economic relevance to the exhumation and preservation of Ghana’s gold deposits12.South of, and parallel to, the BN shear zone, the Ivory Coast-Ghana (ICG) continental margin forms a NE-SW trending basement ridge, which represents the eastern prolongation of the Romanche Transform Zone in the equatorial Atlantic Ocean Fig. 4. Geochr12. The WAC covers a large area across much of western Africa and consists of two Archean terranes in the north-western and south-western parts of the craton1. These Archean terranes are juxtaposed against an array of Palaeoproterozoic terranes that are made up of greenstone belts, contemporaneous volcano-sedimentary depocentres and regions of extensive tonalite-trondhjemite-granodiorite plutons, collectively known as the Tamnean plutonic suite, the Eburnean plutonic suite and the Birimian plutonic supergroup (emplaced between 2250 Ma and 1980 Ma)4. These terranes are overlain by Mesoproterozoic and younger sedimentary basins2, such as the Neoproterozoic to lower Palaeozoic Volta Basin13.The study area in northern Ghana forms part of the south-eastern section of the WAC Fig. 12. The W9. The time period between the end of the Triassic and the early Cretaceous is characterised by two stages of rifting and related magmatism that precede the opening of the Equatorial Atlantic Rift System: (1) break-up within the Central Atlantic transpired at ca. 200 Ma, contemporaneous with the emplacement of the Central Atlantic Magmatic Province (CAMP)15 and (2) break-up within the South Atlantic occurred synchronously or marginally preceded the emplacement of the Paraná – Etendeka Large Igneous Province (LIP) at ca. 137 Ma as dated by 40Ar-39Ar methods16. Furthermore, magmatism in the Benue Trough between 147 and 106 Ma (as dated by 40Ar-39Ar methods) preceded the final stages of break-up in in the equatorial Atlantic Ocean and overlaps in time with Paraná – Etendeka magmatism17. Rocks related to those igneous events are limited within Ghana and are mostly constrained to Nigeria, Guinea, Mali, Morocco, Mauritania and Liberia within the African continent19. Crossing the Atlantic from the Ghana study area, the Maranhão and Parnaíba Basins in north-eastern Brazil  At the start of the Aptian, the first stages of continental rifting and transform faulting occurred and the Deep Ivorian Basin was initiated as a result of E-W to ENE-WSW oblique extension10 (2) Intra-continental transform faulting between the African and South American plates led to other rapidly forming isolated rift basins (eg. Tano Basin) north of the transform during the late Aptian to Cenomanian10. During this time, the ICG margin experienced a shift from an overall transtensional to a transpressional tectonic setting as a result of a change in the orientation of drift in the South American plate from SW to SSW25. The change in drift direction is recorded in a series of folds and flower structures in offshore basins north of the margin10. This short period (between the late Albian and Cenamonian) of intense rifting and deformation is marked by a pronounced angular unconformity associated with basin inversion across the southern edge of the margin and exhumation of the outer ridge. Deformation during this time is thought to mark the final stages of contact between the rifting continents10. (3) During the Turonian, continued transform faulting resulted in the transition to seafloor spreading and caused newly created oceanic crust to come into contact with the continent. The mechanism for the cooling of the ICG margin is debated.6 interprets the late Cretaceous cooling to be related with exhumation in response to the bathymetric step between the continental margin and the oceanic crust, while5 interpret the cooling signal to be a flexural response to unloading during continental separation. Since the Santonian, the ICG margin became passive8. Furthermore, at the end of the Cretaceous (~65 Ma), western Africa was experiencing NNW-SSE shortening, folding and strike-slip faulting, which induced an angular unconformity in the Benue Trough27.The ICG marginal ridge is the modern expression of the Romanche transform fault associated with rifting and break-up in the equatorial Atlantic during the Early Cretaceous29. Later deformation events in the area are described as localised E-W to NE-SW striking brittle deformation associated with dextral strike-slip shearing under a transcurrent regime3. Prior to this study, the timings of these fault events are only relatively constrained.Within the study area, seven relative deformation events are described, for which absolute ages are only recorded related with D1, D2 and D3. These early deformation events are interpreted to be associated with the Palaeoproterozoic Eburnean OrogenyA total of 17 samples were chosen based on apatite quality and on their proximity to the BN shear zone in northern Ghana Fig. . All samGroup 1 (referred to as SE of BN shear zone) yield the oldest AFT results for this study. The pooled AFT ages obtained for individual samples in this Group range between 193 ± 16 Ma to 279 ± 31 Ma Fig. . Samples2 test and yield single grain dispersion values of >25%, suggesting a complex thermal history record. For those samples, two statistical age populations were derived (defined as peak ages P1 and P2). P1 ages range between 112 ± 8 and 122 ± 14 Ma. P2 ages cluster between 183 ± 10 and 217 ± 11 Ma, with one older exception (270 ± 55 Ma) (Table Group 2 (referred to as ‘within BN shear zone’) yields younger central ages and relatively large single-grain age dispersion. The pooled AFT ages for this Group range between 134 ± 10 Ma and 170 ± 18 Ma Fig. . BN-270 a) Table . Dpar vaa) Table . The plo2 test and RadialPlotter discriminates two statistically probable age peaks (P1 and P2) for these samples, with P1 ranging from 75 ± 10 Ma to 106 ± 6 Ma and P2 ranging from 171 ± 6 Ma to 206 ± 35 Ma. Sample BN-127 acts as an exception and yields a P1 age of 56 ± 10 Ma and P2 age of 118 ± 7 Ma  samples exhibit pooled AFT ages ranging between 96 ± 9 Ma and 127 ± 13 Ma. Individual sample pooled AFT ages and MTL data can be found in Table  Ma Fig. . MTL dat2 test and RadialPlotter discriminates two statistically probable age peaks with a P1 of 39 ± 15 Ma and a P2 of 95 ± 21 Ma . Sample BN-132 fails the χ Ma Fig. . SimilarThermal history (tT) modelling was conducted on eleven individual samples that yield sufficient confined lengths for modelling purposes Table . Given tThe modelled samples display three distinct cooling periods Fig. , which aIn summary, a Late Triassic-Jurassic cooling signal and a middle Cretaceous cooling signal were modelled, consistent with the two distinct age populations. In addition, late Cretaceous - Palaeogene cooling is recorded for several thermal history models, confirmed by the youngest AFT age peaks in the study area. However, this modelled Palaeogene cooling signal could represent a modelling artefact, induced by constraining the model to present-day ambient temperatures, and therefore caution is required during interpretation in further discussion.The thermal history models display a rather complex thermal history record Fig.  with eviFive samples from within and NW of the shear zone were used for this exercise, where confined tracks were only measured in AFT dated grains. All samples yield high single grain dispersions and low P(χ2) values and exhibit clear open-jaw signatures in the radial plots . For each of those samples, confined track lengths related to AFT age populations P1 are displayed in a blue frequency plot and those related to AFT age populations P2 are displayed in a red frequency plot Fig. . The MTL238U concentration was evaluated  ages correlate with shorter track lengths and higher 238U concentrations. There are no clear correlations observed between AFT ages and Cl concentrations30  and Basin in southern Africa formed36, which initiated the development of the so-called African erosion surface37. We suggest that the emplacement of LIPs such as CAMP caused a temporary increase in the geothermal gradient and the AFT ages in Ghana record the subsequent cooling  ages of approximately 200 Ma and interpret slow cooling related to the decline of magmatic activity in the region. The study by39 reports AFT ages of approximately 200 Ma and relate the data to crustal heating, induced by CAMP. Alternatively,40 describe substantial Permian – Jurassic plateau exhumation in NE Brazil to be related with erosion of topography that was generated during Gondwana amalgamation.We propose that the Late Triassic – Early Jurassic cooling of the Ghanese upper crust is related to thermal relaxation after emplacement of the Central Atlantic Magmatic Province (CAMP) Fig. . Geochroica Fig.  indicate41. Extension in western Africa is generally regarded as occurring since the Late Permian and driven by far field tectonics during Pangea break-up with the aid of the emplacement of magmatic provinces (such as CAMP) through the Jurassic and Early Cretaceous  thermal eventous Fig. 7,14,15,443. The Early to middle Cretaceous cooling event is therefore interpreted in terms of exhumation in response to the two initial geodynamic stages of break-up. (1) The onset of rifting, intracontinental transform faulting and the initiation of the Deep Ivorian Basin during the Aptian and (2) the shift in the overall tectonic setting as a result of the change in drift orientation (from SW to SSW) of the South American plate with respect to the African plate from the Aptian to the Cenomanian24  of Neoproterozic shear zones in the Paraíba Basin in northeastern Brazil induced exhumation of the region, which indicates that a similar circumstance could occur on the opposite margin, within West Africa.The thermal history models for samples NW of the BN shear zone reveal associated fast cooling during the Early and middle Cretaceous Fig. , coincidn24 Fig. . There a3. Published off-shore AFT data from9 reveal cooling ages ranging between 65 Ma and 92 Ma, which are interpreted to constrain the final stages of break-up and exhumation of the ICG marginal ridge  low temperature thermochronology, both the Gombe Fault and ICG margin were active and exhumed during the Late Cretaceous  as the equatorial margin became passive27. Previous studies conducted by45 have noted similar styles of reactivation along pre-existing shear zones in the South Tien Shan . In light of these relationships, we suggest that the youngest region of the study area  thermal history through the apatite fission track partial annealing zone (APAZ)47. For this study, apatite grains were picked, mounted in epoxy resin, ground and polished to expose internal apatite sections. The apatite mounts were etched in 5 M HNO3 for 20.0 ± 0.5 s at 20.0° ± 0.5 °C to expose the spontaneous fission tracks and subsequently imaged on a Zeiss AXIO Imager M2m Autoscan System at The University of Adelaide. Fission track densities, Dpar and lengths were measured using the FastTracks software. Where possible, a minimum of 20 grains were analysed per sample. The objective was to measure at least 100 confined fission track lengths, a target that was not always achieved. The 238U and 35Cl concentration of each counted grain were measured using spot analyses on a New Wave UP213 laser connected to an Agilent-7500cs ICPMS48. U concentrations were determined by measuring 238U/43Ca ratios, calibrated against NIST610, NIST612 and Madagascar apatite standards (U-Pb age of 473.5 ± 0.7 Ma49). Age calculations were carried out following50 using a zeta approach51 with Durango as secondary age standard (40Ar/39Ar age of 31.44 ± 0.18 Ma52). A total of 102 measurements on Durango apatite were carried out for this study, spread over 3 different (well-characterised and homogeneous in U) crystals with mean U concentrations of 15.54 ± 0.08 ppm, 12.67 ± 0.07 ppm and 9.80 ± 0.10 ppm. The resulting mean AFT age of 31.65 ± 0.7 Ma for Durango, obtained in this study, is in excellent agreement with the published age. Accuracy of the applied methodology against the traditional external detector method is demonstrated in5335Cl measurements were carried out following54, who were able to demonstrate that electron probe and LA-ICP-MS data are the same when analysing a number of different apatites with different 35Cl compositions. Durango was used as accuracy check, producing a mean Cl concentration of 4.4 ± 0.1 ppm, which is in good agreement with published values. Data reduction was performed using the Trace_Element_IS data reduction scheme in the Iolite software55. More details on the methodology, can be found in50.Apatite Fission Track thermochronology is based on the spontaneous fission decay of 56 and represent the apparent AFT cooling age of the analysed samples. This central age is not necessarily a good estimate of a cooling event and therefore subsequent thermal history modelling is needed to derive the tT history preserved in the sample. Furthermore, it is possible to  preserve multiple AFT cooling ages or ‘peak ages’ in samples which fail Pearsons χ2 test or where single-grain ages show significant dispersion  which is regarded as a beyond natural spread57. In more detail, radial plots often show an open-jaw display of an older event and younger age component that can be related to distinct cooling events58. This is especially the case when two apatite populations have significantly different chemistries. In this regard, chlorine concentrations are often used as a chemical discriminator30. Elevated chlorine concentrations are known to slow fission track annealing but often show only little differences between apatite populations. This study uses uranium concentrations in addition to chlorine concentrations as a discriminator, which is thought to have a similar effect on fission track annealing. In this regard34, described a model of radiation-enhanced annealing for apatites with elevated uranium concentrations from old cratonic rocks. More recent observations35; confirm that alpha-particle induced annealing of radiation damage occurs in apatite and should be considered alongside with the better understood thermal annealing.AFT central ages were calculated with RadialPlotter59. The input parameters required in our modelling approach include individual AFT ages, their standard deviations, confined track lengths, sample elevations and Dpar used as a proxy for apatite chemistry as kinetic parameter59. The final output of each modelling attempt resolves three different models  and generates a weighted mean model or ‘Expected’ model based on a probability function at each point in the time-temperature space. The probability function is visualised in a colour spectrum ranging from blue (low probability) to red (high probability) where the absolute numerical probability value varies per model  modelling was performed using the software package QTQt which uses the Bayesian trans-dimensional Markov Chain Monte Carlo statistical method to derive the most likely thermal history modelsSupplementary Information


Blood clotting at the vascular injury site is a complex process that involves platelet adhesion and clot stiffening/contraction in the milieu of fluid flow. An integrated understanding of the hemodynamics and tissue mechanics regulating this process is currently lacking due to the absence of an experimental system that can simultaneously model clot formation and measure clot mechanics under shear flow. Here we develop a microfluidic-integrated microclot-array-elastometry system (clotMAT) that recapitulates dynamic changes in clot mechanics under physiological shear. Treatments with procoagulants and platelet antagonists and studies with diseased patient plasma demonstrate the ability of the system to assay clot biomechanics associated with common antiplatelet treatments and bleeding disorders. The changes of clot mechanics under biochemical treatments and shear flow demonstrate independent yet equally strong effects of these two stimulants on clot stiffening. This microtissue force sensing system may have future research and diagnostic potential for various bleeding disorders. Blood clotting is a complex process involving platelet adhesion and clot stiffening. Here the authors present a microfluidic system to recapitulate the dynamic changes in clot mechanics under physiological shear. In hemostasis, the interplay of these mechanical factors leads to strong clots that stem bleeding, but in clotting disorders such as von Willebrand disease (VWD), disrupted biomechanical interactions lead to weak clots and bleeding5. Although different aspects of the clotting problem such as shear flow-mediated platelet adhesion and single platelet mechanics have been studied in the past7, an integrated understanding of the interplay between clot mechanics and hemodynamic shear is still missing. This is mainly due to the lack of combined capacity to model and measure clotting mechanics in the presence of shear flow in existing research models.Blood clotting at the vascular injury site is a complex biomechanical process mediated by several key factors including platelet deposition and contraction, clot stiffening and hemodynamic forces8. These systems are powerful tools to study the effects of various flow conditions, surface chemistries and coagulation factors on platelet adhesion and aggregation13; however, they are not equipped with the ability to measure clot mechanics. Furthermore, matrix proteins are coated on glass or relatively rigid surfaces, which do not mimic the physiological stiffness of exposed matrix at vascular injury sites. In the study of platelet mechanics, atomic force microscopy and flexible micropost arrays have been used to measure single platelet-generated contractile forces under static conditions14, and matrix protein microdot arrays have been used to study the effects of substrate stiffness and mechanosensitive signaling pathways on platelet-generated contractile force16. Single platelet-mediated fibrin fiber remodeling has also been studied using confocal microscopy17. While these studies provide important information on platelet mechanics, they do not include the effect of hemodynamic forces and offer limited insight into clot mechanics at the tissue level. In the clinic, cup and cone type of rheometers, also known as thromboelastography (TEG) or rotation thromboelastometry (ROTEM), have been developed to measure clot stiffness at different stages of the coagulation process19. Although these systems provide useful information that can assist the diagnosis of coagulation disorders, their consideration of hemodynamic flow and platelet−matrix interactions is inadequate. As a result, their diagnostic power is limited and they are seldom applied for disorders of primary hemostasis, like VWD19. Together, the lack of an integrated system that can follow clot formation and mechanical properties in the presence of shear flow has hindered an integrated understanding of the biomechanical events occurred during blood clotting.Recent technological advancement has resulted in the evolution of independent approaches that focus on different parts of the clotting problem. For example, microfluidics systems create well-controlled blood flow in matrix protein-coated microchannels, and thus they have become a widely used vascular injury model for the study of platelet adhesion, aggregation and thrombus formation22. In this system, cell contraction-mediated self-assembly of matrix proteins such as collagen and fibrin leads to the formation of submillimeter microtissues that anchor on top of multiple flexible poly(dimethylsiloxane) (PDMS) micropillars. These micropillars serve as mechanical sensors that can report in-situ tissue mechanical properties. The composition and stiffness of these microtissues, contributed both by preseeded and cell-secreted matrix proteins, mimic those of native vascular tissue. By virtue of their small dimensions, the microtissues offer orders of magnitude scale-up advantages over conventional bulk hydrogel models and open up the possibility to integrate with microfluidics. This system has been used to study the mechanics of cardiac and skeletal muscles, fibrotic tissues and wound healing27; however, it has not been integrated with the shear flow.With recent advent of microfabrication technology, we and others have created microtissue array-based mechanical sensing platforms that allow simultaneous control of tissue formation and measurement of tissue mechanicsIn the current study, we develop a microfluidic-integrated microclot array elastometry (clotMAT) system by integrating a collagen microtissue array-based mechanical sensing platform with platelet flow. This system recapitulates the dynamic process of platelet adhesion and clot formation under various flow conditions and reports changes in clot mechanical properties in real time. Through platelet agonist (ADP and thrombin) treatments, we dissected the contributions of hemodynamic and tissue/cell forces to clot stiffening. Treatments with platelet antagonist ) further enabled the examination of the effects of platelet adhesion and cytoskeletal tension on clot mechanics. The utility of the clotMAT system in bleeding disorders was demonstrated through the study of clotting mechanics of VWD. Integrated analysis of results show strong correlation between clot stiffness, clot contractile force and the level of clot retraction (volume shrinkage) under both biochemically treated conditions and shear flow conditions, suggesting these two stimulants independently yet equally strongly affect clot remodeling and stiffening. This finding unveils the tissue remodeling/stiffening mechanism in a previously unexplored shear flow regime, and thus it significantly expands the boundary of previously established tissue remodeling theory. Together, this work describes a physiologically relevant microtissue force sensing system for the study of normal and abnormal clot biomechanics under shear flow, which may have future research and diagnostic potential for various bleeding disorders.28. An array of submillimeter-size microwells (13 columns by 6 rows) were cast in the middle PDMS layer, and each microwell contains a pair of flexible PDMS micropillars that serve as force sensors to report clot-generated contractile forces  wide and 80.7 ± 5.9 µm (16 replicates) thick. The microfluidic channels in the top PDMS layer were aligned with individual rows of collagen microtissues in the middle layer , flowing platelets were arrested on a row of individual micro-collagen matrices contained by the microchannel Fig. . The adh29. To detect the minute change in microclot-generated contractile forces, micropillars were engineered to sense force in nanoNewton range  were used to calculate contractile force according to cantilever bending theory /2, and the microtissue tensile strain was determined based on image tracking of the displacement of fiducial markers in the microtissue before and after stretch  was less than that of bare collagen microtissue (δ1′collagen + δ2′collagen)  Fig. , indicatces Fig. . The sti−1 shear rate and measured the microclot properties . Here, we observed more platelet binding and higher contractile force generation in the early stage (first 10 min) of flow experiment in the PPACK group compared to the citrate group  rescued the decrease in fluorescence intensity during mid to late stage of the flow experiment in the clotMAT system but had no effect on contractile force , we also tested the utility of the clotMAT system under several other common preparation conditions, including the use of whole blood, recalcified PRP samples and different anticoagulants. Since red blood cells (RBCs) interfere with optical detection, we washed away unbounded RBCs at the end of 30 min of whole blood flow at 500 s−1, and microtissue contractile force, fluorescence intensity, stiffness and tissue volume were measured over 30 min of citrated PRP flow  compared to thrombin (MW 37 kDa) in this setting may be due to its smaller size which enables greater penetration into the platelet-dense clot, unlike thrombin which would be diffusion limited33. Indeed, when thrombin or ADP was added to citrated PRP just prior to flow, thrombin caused the formation of larger, fibrin-rich clots that blocked the flow channel within minutes  and arterial (1500 s−1) shear rates. Here, antagonist treatment significantly inhibited platelet binding to the collagen matrix and associated fluorescence intensity  that recognizes von Willerbrand factor (VWF), and also a GpIIb-IIIa receptor antagonist (Abciximab) at both venous  treatment. ASA treatment of citrated PRP resulted in significant reductions in microtissue fluorescence  type 2A patient plasma due to the prevalence of this disease and the role of VWF on platelet adhesion in this system Fig. . In thesgen Fig. . Microtigen Fig. . TogetheR2 = 0.97, Fig. R2 = 0.93, Fig. −1 shear rate), strong correlations between these mechanical parameters were also observed , we observed strong correlations between microclot stiffness and its contractile force  analysis of the microclot retraction process by representing the contractile platelet population using contractile 3D elementsion Fig. , recapit15, little is known about the origin of tissue-level clot mechanical properties29. Commonly used tissue mechanics models such as cell-laden bulk hydrogel models do not recapitulate the dynamic flow environment that is critical to clot formation and are low-throughout and time and resource intensive43. We described herein a medium-throughput clotMAT system with integrated flow and mechanical measurement capacity. Using a panel of agonist and antagonist used in classical vascular injury models, we demonstrate the ability of this system to integrate the biomolecular players and shear stresses that are known to be critical in vivo. The system recapitulates the evolution of clot morphology and mechanical properties under normal and abnormal clotting conditions. Since clot mechanical properties are widely used in the clinic as indicators for blood coagulation functions, the knowledge gained in this study may help to improve the diagnosis and treatment of coagulation disorders.The mechanical properties of blood clots are critical to stem bleeding upon vessel injury, and may also contribute to embolism. Despite numerous studies on platelet mechanics37. Fibrin has an indirect role in clot retraction as it stabilizes the platelet thrombi and prevents embolization. Addition of thrombin or ADP to the flowing PRP results in higher amounts of fibrin formation and larger platelet filled clots  and blebbistatin, was performed using healthy blood samples collected from more than 20 healthy donors, yet the significant differences in the results were not masked by donor variation, which demonstrates the detection sensitivity of the system. Together, these data provide preliminary confirmation of the potential clinical utility of the clotMAT system. Additional comprehensive tests in a clinical setting are needed in the future to fully validate the clotMAT system. These tests may include reagents often used in TEG and ROTEM assays such as thromboplastin, heparinase and ellagic acid, different VWD/disease populations, and hyper reactive platelets from individuals suffering from cardiovascular diseases. It would be also interesting to compare the detection sensitivity of the clotMAT system directly with TEG and ROTEM. Finally, the development of the clotMAT system was enabled by the advancement in microfabrication technology; due to its low-cost, medium-throughput and the potential for scale-up, there exists a high potential to translate this technology into an improved point-of-care system for coagulation diagnosis43. However, due to the difficulty to incorporate flow condition and the inherent limitations in solute diffusion and experimental throughput, bulk hydrogel models are not suited for the study of clot mechanics. Previous studies using these models have shown that stiffening of the fibrin gels can be induced through either externally applied mechanical strain or myosin-mediated cellular contraction that aligns and bundles fibrin fibers and compacts matrix meshwork45. However, the applicability of this strain stiffening theory under shear flow condition has not been examined. Here, through analyzing results from coagulation conditions regulated by either biochemical treatments or shear flows, we observed equally strong correlations in the microclot mechanical properties  was added when washed platelets were prepared. PRP was obtained by centrifuging blood at 180 × g for 15 min47. To obtain healthy plasma or platelet-poor plasma (PPP), the remaining blood was again spun at 1200 × g for 12 min and the plasma supernatant was collected. Washed platelets were similarly obtained by further centrifuging PRP containing PGE1, at 1200 × g for 12 min. In this case, the pellet was washed once using HEPES buffer (30 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 110 mM NaCl, 10 mM KCl, 1 mM MgCl2, 10 mM glucose, pH 7.4) containing 2 μM PGE1 before resuspension in HEPES buffer lacking PGE1 35. 1 µM BCECF -5-(and-6)-Carboxyfluorescein, Acetoxymethyl Ester) (ThermoFisher Scientific) was added to PRP/washed platelets for 30 min at room temperature to stain platelets with green fluorescence.Human blood was obtained by venipuncture from healthy adult volunteers, following protocols approved by the UB Health Science Institutional Review Board . Volunteers of either sex were used and studies were performed within 3 h of blood draw. Blood donors self-reported absence of coagulation defects and use of medication. Majority of the samples were prepared using sodium citrate (1:9) as anticoagulant with a few exceptions that were prepared using PPACK . In some cases, PRP was recalcified by addition of 10 mM CaCl47. ADAMTS13 activity was measured in terms of FRET ratio using the XS-VWF FRET substrate48. Briefly, this involved addition of citrated plasma VWF to 1 µM XS-VWF FRET for 1 h at room temperature. FRET ratio was then quantified using a Synergy 4 BioTek fluorescence plate reader, based on the ratio of XS-VWF emission intensities at 541/25 nm vs. 485/20 nm following excitation at 420/50 nm. A flow cytometer-bead sandwich assay determined VWF concentration49. Washed platelets  from healthy donor blood were mixed with either healthy plasma from normal donor to obtain reconstituted healthy PRP  or VWD plasma to obtain reconstituted VWD PRP (rVWD PRP).VWD type 2 A patient plasma from a single donor was obtained from CoaChrom Diagnostica GmbH (Austria). 0.7% agarose gel electrophoresis was used to compare VWF multimer distribution in patient and healthy plasma21. Briefly, multiple layers of SU-8 (bottom layer for the leg section and top layer for the head section) were successively deposited on the silicon wafer (University Wafer), exposed to UV light through transparency masks printed by laser plotting (CAD/Art Services Inc.), and baked and developed according to the manufacturer’s protocols. Softlithography was then used to transfer the micro-patterns to polydimethylsiloxane  molds made with 10:1 ratio of dimer to curing agent  = 42 μm, depth (T) = 81 μm, and height (L) = 291 μm  of the micropillar is 120 nN μm−1. The micropillar head was designed to stick out of the microwell so that when microtissues form on the heads, they can directly face and interact with the platelet flow in the microchannel to increase the platelet capture efficiency  and 20 µg mL−1  and mixed with HUVECs . This mixture was seeded into sterilized micropillar device through centrifugation, polymerized and maintained under standard culture condition for 2 days  were obtained from Lonza  and cultured in Endothelial Cell Growth Medium 2 . To perform microtissue seeding, unpolymerized collagen type I were neutralized and diluted to final concentrations of 3 mg mL−1 and the corresponding shear rate was 3~1500 s−1, calculated by Q is the volume flow rate, w and h are the width and the height of the channel, respectively. In the study of the effect of shear rate on microtissue formation, the experiments at each shear rate were performed on at least three donors with at least seven samples per donor.The PDMS microchannels measuring 1189 µm in width and 498 µm in height were fabricated using standard photolithography and soft-lithography methods. The microchannels were aligned with individual rows of trypsinized microtissues under stereomicroscope, and the PDMS slab containing microchannels was bond with the PDMS substrate containing microtissue array through custom clamps /2 of the two micropillars and k = 120 nN µm−1 is the spring constant of the micropillar  was recorded before platelet flow. During platelet flow, bright field images of the micropillars were taken every 10 min to monitor platelet-generated contractile forces in real time. Micropillar deflection was determined by the travel distance of the micropillar head relative to the bottom of its leg and was used to calculate the microtissue contractile force according to the cantilever bending theory d2–d1), and the force needed to develop such elongation was reported by micropillar deflection as F = k (δ1′ + δ2′)/2. This force was divided by the cross-sectional area of microtissue to calculate the tensile stress /d1, by tracking the change in distance between two fiducial markers in the microtissue before and after stretch  of the microtissue for cross-sectional area calculation was determined by confocal microscopy imaging  that is mounted on a custom-made uniaxial stretching frame , 10 μM blebbistatin (Sigma), 20 μg mL−1 anti-CD42b antibody , and 15 μM acetylsalicylic acid . Here, mAbs and ASA were incubated with citrated PRP for 30 min before flow and blebbistatin was incubated for 5 min before flow. In some cases, 15 µM TRAP-6  was added to PRP prepared using PPACK just before flow. ADP (10 μM) and thrombin (5 U mL−1) were diluted in Dulbecco’s phosphate-buffered saline and infused at 500 s−1 shear rate for 10 min over microclots previously formed under citrated PRP flow  were directly added to citrate PRP before flow to study their effects on clot formation  and PE-labeled anti-CD62P mAb AC1.2  for 5 min at room temperature. This mixture was then stimulated with either 15 µM TRAP-6 , 5 U mL−1 Thrombin , 20 µM ADP  or 5 µgmL−1 collagen . A portion of the sample was diluted 200-fold at various times and analyzed using a BD Fortessa X-20 flow cytometer  as anticoagulant. In some cases, PRP was recalcified by addition of 10 mM CaCl−1 fibrinogen  with 20-fold molar excess Alexa flour-594  in PBS (pH = 8.0) for 1 h at room temperature. Following quenching of the reaction using 100 µL 1 M Tris-HCl (pH = 8.0) for 10 min, labeled protein was desalted into PBS using a Zeba Spin Column according to the manufacturer’s instructions .Alexa fluor-594-conjugated fibrinogen (Fb-594) was prepared by mixing 10 mgmL−1 Fb-594 was added to PRP (20 µL volume). Following this, various stimuli were added: 15 µM TRAP-6, 5 U mL−1 Thrombin, 20 µM ADP or 5 µg mL−1 collagen. Such assays were performed using citrated PRP, recalcified citrated PRP and PPACK anticoagulated PRP under static condition. Following 10 min, 150 µL PBS containing 5 mM EDTA was added to stop the reaction. Fibrin clot was then immediately pelleted using centrifugation at 14,000 × g for 10 min, and Alexa 594 fluorescence in supernatant was measured using a plate reader in order to determine % Fb-594 incorporated into fibrin clot  or VWD type 2A plasma. The mixture was sheared in a cone-plate viscometer at 9600 sMicrotissues were fixed using 4% (v/v) paraformaldehyde (PFA)  for 10 min at room temperature, permeabilized by 0.1% (v/v) Triton X-100 for 5 min, and blocked with 3% BSA (Sigma) for 30 min at 37°C. Samples were incubated with collagen type I antibody  overnight and labeled with Alexa Fluor™ 594 secondary antibody  for 1.5 h. Hoechst 33342 (Invitrogen) was used at 1:1000 ratio to label cell nuclei in the microtissue. Confocal images of the microtissue were taken using an Andor Technology DSD2 confocal unit coupled to an Olympus IX-81 motorized inverted microscope. Plan-Apochromat ×10 objective was used to record the stack with 1 μm optical slices for all channels. The stack of images was then processed using the Z stack tool in ImageJ (NIH) to obtain the projected 2D views. To characterize the microclot volume change after flow, topview area and thickness of the microtissue were analyzed based on bright field topview images and confocal stacks in ImageJ  for 1.5 h, and dehydrated through a series of ethanol treatment at 15, 30, 50, 70, 90 and 100%. Finally, hexamethyldisilazane was added to the sample to prepare them for imaging on a HITACHI SU-70 SEM system.t test with Welch’s correction method. Significance difference for multiple groups was determined by one-way analysis of variance (ANOVA). Correlation between groups was analyzed using Pearson correlation coefficients. The significant difference of slopes and intercepts for two linear regressions was measured using GraphPad Prism, using a method equivalent to analysis of covariance (ANCOVA).For all the shear flow conditions and all the agonist and antagonist treatment conditions, at least three donors with at least seven samples per donor were measured. Data are presented as mean ± standard deviation unless otherwise stated. Significance difference between dual comparison was verified by unpaired Further information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Movie 2Supplementary Movie 3Reporting Summary