Schistosoma haematobium have been investigated in West Africa, only scant data are available from Côte d’Ivoire. The purpose of this study was to analyze both genetic variability and genetic structure among S. haematobium populations and to quantify the frequency of S. haematobium × S. bovis hybrids in school-aged children in different parts of Côte d’Ivoire. Urine samples were subjected to a filtration method and examined microscopically for Schistosoma eggs in four sites in the western and southern parts of Côte d’Ivoire. A total of 2692 miracidia were collected individually and stored on Whatman® FTA cards. Of these, 2561 miracidia were successfully genotyped for species and hybrid identification using rapid diagnostic multiplex mitochondrial cox1 PCR and PCR Restriction Fragment Length Polymorphism (PCR-RFLP) analysis of the nuclear ITS2 region. From 2164 miracidia, 1966 (90.9%) were successfully genotyped using at least 10 nuclear microsatellite loci to investigate genetic diversity and population structure. Significant differences were found between sites in all genetic diversity indices and genotypic differentiation was observed between the site in the West and the three sites in the East. Analysis at the infrapopulation level revealed clustering of parasite genotypes within individual children, particularly in Duekoué (West) and Sikensi (East). Of the six possible cox1-ITS2 genetic profiles obtained from miracidia, S. bovis cox1 × S. haematobium ITS2 (42.0%) was the most commonly observed in the populations. We identified only 15 miracidia (0.7%) with an S. bovis cox1 × S. bovis ITS2 genotype. Our study provides new insights into the population genetics of S. haematobium and S. haematobium × S. bovis hybrids in humans in Côte d’Ivoire and we advocate for researching hybrid schistosomes in animals such as rodents and cattle in Côte d’Ivoire.While population genetics of  Schistosoma [S. guineensis, S. haematobium, S. intercalatum and S. mansoni in Africa and the Arabian Peninsula [S. mekongi and S. japonicum in Asia; S. mansoni mainly occurs in Africa, but is also found in Brazil and some Caribbean islands [Schistosoma haematobium causes the urogenital form of the disease. Classified as a group I carcinogen, urogenital schistosomiasis can lead to squamous-cell carcinoma of the bladder [Schistosomiasis is a chronic neglected tropical disease caused by trematodes belonging to the genus istosoma , 40. Theistosoma . More thistosoma , and theistosoma . Six speeninsula , and S.  islands . Schisto bladder . In mostS. haematobium and S. mansoni are endemic, causing urogenital and intestinal schistosomiasis, respectively [S. haematobium in school-aged children in South Côte d’Ivoire [S. bovis, a parasite of livestock and rodents. A previous study on post-mortem examinations of cattle in North Côte d’Ivoire reported a prevalence of 35% [S. bovis found in cattle in the northern parts of the country [In Côte d’Ivoire, both ectively . The forectively , 56, whiectively , 47, 59.e of 35% . These f country .Schistosoma haematobium × S. bovis hybrids are well documented in West Africa, particularly in Benin, Mali, Niger and Senegal [S. haematobium are in fact introgressed to some extent with S. bovis [S. haematobium and S. mansoni excreted by humans [S. haematobium and S. bovis being transmitted by freshwater snails of the genus Bulinus [Habitat change and migration of hosts, which is also linked to climate change, can influence the epidemiology and distribution of schistosomiasis and enhance the occurrence of interspecies hybridization between human and animal schistosomes . Schisto Senegal , 42, wit Senegal  and Cors Senegal , 10. MorS. bovis , 48. Recy humans , 41, and Bulinus  and also Bulinus .Schistosoma species [S. mansoni is the most widely studied. Several investigations have shown high genetic diversity and strong genetic structure in several countries, including Ethiopia, Kenya, Senegal and Uganda [S. haematobium have shown both less genetic diversity and less population structure compared to S. mansoni [S. mansoni and S. haematobium is also apparent at the continental scale [S. bovis are even more limited. One study has investigated the population genetic patterns of S. bovis in Cameroon revealing an intermediate pattern with high genetic diversity (i.e. like in S. mansoni), but no genetic structuration (i.e. like in S. haematobium) at the country scale [S. bovis lineages than between S. haematobium lineages [S. haematobium × S. bovis hybrids were found in cows [Population genetic structure and genetic diversity varies by  species . Among td Uganda , 61, 63. mansoni , 46. Thial scale , 62, 63.ry scale . At the lineages . More relineages . The aut in cows  and rode in cows .S. haematobium and S. haematobium × S. bovis hybrids that were obtained from human urine samples, in four sampling sites of Côte d’Ivoire. We molecularly characterized individual miracidia, using cox1 and ITS2 markers to identify S. haematobium and S. haematobium × S. bovis hybrids within the different populations, alongside microsatellite markers [The purpose of this study was to investigate the population genetic structure and genetic diversity of  markers  to analySchistosoma infection.Ethical clearance was obtained from the Ministère de la Santé et de l’Hygiène Publique en Côte d’Ivoire (reference no. 003–18/MSHP/CNER-kp). School authorities, teachers, parents/guardians and children were informed about the objectives, procedures and potential risks and benefits of the study. Written informed consent was obtained from children’s parents or legal guardians, while children provided oral assent. After sampling, a praziquantel treatment (40 mg/kg) was offered to children found with a Schistosoma miracidia from children aged 5–14 years were collected, after egg hatching, and individually stored on Whatman® FTA cards  as previously described [This study was carried out in four sampling sites in Côte d’Ivoire: (i) Agboville  and (ii) Adzopé  in the south-eastern part; (iii) Sikensi  in the south-central part; and (iv) Duekoué  in the western part of Côte d’Ivoire . The stuescribed . FTA car® beads  from 2.0 mm discs containing sample that had been punched out of the FTA card using a Harris-Micro-Punch  region, we used a restriction fragment length polymorphism (RFLP) approach , following amplification using the forward primer Sc_ITS_F: 5′–GGC TGC AGC GTT AAC CAT TA–3′ and reverse primer Sc_ITS_R: 5′–ACA CAC ACC ATC GGT AC AAA–3′, which targets 505 bp of the ITS2 [2, 0.5 μL of 10 mM dNTP mix, 1 μL of each 10 μM primer and 1 U of GoTaq Hot Start Polymerase . The reaction conditions included an activation step of 95 °C for 3 min, followed by 45 cycles of 95 °C for 40 s, 58 °C for 40 s and 72 °C for 40 s, and a final extension at 72 °C for 6 min. In the subsequent step, 5 μL of PCR products were then digested at 37 °C using 0.5 μL of restriction enzyme MboI , 2.5 μL of CutSmart buffer, and 17 μL of molecular water for 15 min, followed by inactivation at 60 °C for 20 min for a total reaction volume of 25 μL. The cut sites of the MboI enzyme for species specific SNPs were based on the following sequences positions: ↓GATC and CTAG↑. The PCR products were not cleaned or visualized before enzyme digestion. We used only samples that were positive with the cox1 multiplex PCR method. This enzymatic digestion cuts the 505 bp ITS2 fragment into four bands for S. haematobium  and three bands for S. bovis . All digested ITS2 PCR products were visualized using 2% agarose electrophoresis gels stained with GelRedTM . The nuclear ITS2 and mitochondrial cox1 identities for each individual miracidia were combined to produce a mito-nuclear genetic profile of S. haematobium (Shcox1 + ShITS2: Sh × ShSh), S. bovis (Shcox1 + ShITS2: Sb × SbSb) or S. haematobium-bovis hybrid genotypes, the latter of which is recognized by mito-nuclear discordance (Shcox1 + SbITS2: Sh × SbSb), (Shcox1 + Sb/ShITS2: Sb × SbSh), (Sbcox1 + ShITS2: Sb × ShSh) or (Sbcox1 + Sh/SbITS2: Sb × ShSb)).A ous work . For thepproach ( and Suppthe ITS2 . We perfSupplementary Tab. S1). Errors due to large allele dropout or stutter bands and evidence of the presence of null alleles at each locus were checked using Micro-checker, version 2.2.3 [Individual miracidia were further genotyped using a set of 18 microsatellite markers divided into two panels developed by Webster and colleagues . The foron 2.2.3 .cox1/ITS2 profiles obtained from each miracidium were calculated for each site and any differences using a χ2 test was observed. We also calculated the number of S. bovis and S. haematobium haplotypes for cox1 or alleles for ITS2 among sites. The mitochondrial cox1 haplotype is unique as it is inherited from the maternal line, while the ITS2 was scored as one for heterozygous (Sb/Sh) and two for homozygous (SbSb or ShSh). Differences in the relative frequencies of S. bovis vs. S. haematobium haplotypes or alleles between sites were tested using a binomial test [The number and percentages of the different ial test .p-value for 5% nominal level was performed using FSTAT, version 2.9.4 [cox1/ITS2 genetic profiles. Expected heterozygosity (He), number of alleles (A), allelic richness (Ar), and the inbreeding coefficient (FIS) of each microsatellite locus were computed per sampling site, using FSTAT version 2.9.4 [He and Ar were compared between the populations using the pairwise Friedman rank test, followed by the Nemeyi multiple comparison test implemented in the R studio PCMP plus package [For the microsatellite data, tests for deviation from Hardy–Weinberg equilibrium per locus and site were carried out using Genepop, version 4.0 . The genon 2.9.4 , 67. Genon 2.9.4 . He and  package .FST values [FST values were also calculated between samples that exhibited the different cox1/ITS2 profiles. Principal component analysis (PCA) was performed to compare either the four sites or the six cox1/ITS2 profiles, using Genetix, version 4.05 [Genotypic differentiation between sampling sites was assessed using pairwise T values , calculaT values , 67 withT values . Pairwision 4.05 .tructure, version 2.3 [K from 1 to 8 using an admixture model. The log likelihood for each K was averaged over three runs with the CorrSieve package in R  and the delta K-values were then computed to determine the most likely number among the K values tested [lumpp, version 1.1.2 [istruct, version 1.1 [The uppermost level of genetic structure for all individuals was determined by a Bayesian clustering approach using Markov Chain Monte Carlo (MCMC) permutation analysis, implemented in Ssion 2.3 , using ds tested . For theon 1.1.2  and Distsion 1.1 .rlequin, version 3.5 [Analysis of molecular variance (AMOVA) was performed to evaluate the partitioning of the overall genetic variance to the hierarchical levels at infrapopulation “within miracidia”, “among miracidia within individual children”, “between miracidia from individual children within each site” and “between sites” using Asion 3.5 . The levtructure, version 2.3 with the same procedure as described above. If no best K was found, K was set as the number of children at each sampling site . We first calculated the mean probabilities assignations of the miracidia from a given child to a given cluster. Then repartitions of the miracidia among the different clusters were tested in each participant using χ2 under the null hypothesis of random repartition (1/K).Population genetic structure between parasite infrapopulation was assessed independently for each sampling site using SSchistosoma egg-positive children at four sampling sites, 2561 were genetically analysed. cox1 and ITS2 profiles were obtained from 2164 individual miracidia  (S. bovis cox1 × S. haematobium ITS2 (Sb × ShSh) genotype was the most frequently found in Adzopé (64.6%), Duekoué (56.3%) and Sikensi (30.2%), while the S. haematobium cox1 × S. haematobium/S. bovis ITS2 (Sh × ShSb) was the most common hybrid genotype in Agboville (27.7%). We also identified 15 miracidia (0.7%), from seven children, with the S. bovis cox1 × S. bovis ITS2 (Sb × SbSb) genetic profile. Overall, 791 miracidia (36.6%) presented an S. haematobium cox1 × S. haematobium ITS2 (Sh × ShSh) genetic profile. The χ2 test showed a significant difference in the distribution of miracidia with the different cox1-ITS2 profiles between sites (S. bovis cox1 (50.7%) haplotype was similar to that of S. haematobium . The frequency of the S. bovis ITS2 allele (12.1%) was statistically lower than the S. bovis × S. haematobium allele . Analysis of the multiplicity of infection showed that 52 children (71.1%) were infected by worm-laying miracidia with at least three different cox1-ITS2 genetic profiles. One child (Id: AG122) from Agboville was infected by adult worms laying miracidia with all six possible cox1-ITS2 genetic profiles (Supplementary Tab. S2).Of the 2692 miracidia collected from the urine samples from 91 Duekoué) . Of the en sites . The frecox1/ITS2 profiles were obtained, 1966 (90.8%) also produced reliable allele data for at least 10 microsatellite loci for subsequent analyses. We found a significant genotypic disequilibrium  for 120 pairwise locus combinations across the whole dataset. Genetic diversity indices  and the probability of deviation from Hardy–Weinberg equilibrium (PHWE) for each microsatellite locus by sampling site are shown in He) was relatively stable  and that allelic richness was more variable ranging between 8.8 and 10.5 for Sikensi and Adzopé, respectively. A significant difference was observed for this last parameter , allelic richness from Duekoué being statistically higher compared to the remaining three sites . No differences in genetic diversity indices were observed according to the different cox1/ITS2 genetic profiles.Of the 2164 miracidia from which FST values between sites ranged from 4.1% to 6.9% with Duekoué being consistently most differentiated from the other sites  were not different from the other cox1/ITS2 profiles (S. bovis cox1 × S. bovis ITS2 (Sb × SbSb) genetic profile came from three sites  (Supplementary Tab. S2).The pairwise er sites . The FSTprofiles . The mirThe PCA revealed only weak structuration among the four sites with only Duekoué being partially separated from the other three sites . CompariK = 4 displayed maximal delta K at the population level suggesting that all miracidia were grouped into four genetic clusters. The Structure analysis showed a relatively strong genetic structure between sampling sites (Population genetic structure was assessed both at the population level (between sampling sites) and at the infrapopulation level (between children within each sampling site – see below). ng sites . At the tructure analysis for each population with K = number of patients showed that no best K was found for all sampling sites. However, the clusters were not uniformly distributed among children and some miracidia were assigned to a restricted number of clusters  showed that most of the miracidial genetic variance was found within sites (57.8% of the variance). There is more variance among miracidia by site rather than by host (10.4%) .S. haematobium × S. bovis hybrids among school-aged children in different parts of Côte d’Ivoire. Similar results were also reported from a large study in Senegal, where most children (88%) excreted hybrid miracidia [S. haematobium, S. bovis or hybrid genetic profiles, which indicates that all genotyped parasites most likely belong to a single genetic entity. This finding is in line with a recent genomic study indicating that most, if not all S. haematobium lineages across Africa, are likely to be introgressed to some extent with S. bovis [Our study showed a high proportion of iracidia . NotablyS. bovis .cox1-ITS2 profiles, of which four were unambiguous hybrid profiles. Hybrids with a mitochondrial cox1 haplotype from S. bovis and homozygous ITS2 from S. haematobium (Sb × ShSh) were the most commonly identified type at 41.9%. Such hybrids have sometimes been documented in Senegal and ascribed to bidirectional introgressive hybridization [S. bovis genetic profile to the genetic make-up of schistosome populations is much more evident at the mitochondrial level than at the nuclear level. The frequency of miracidia with a heterozygous ITS2, i.e. carrying both S. bovis and S. haematobium ITS alleles, was relatively low, which could be explained by the concerted evolution of ITS2 gene region [We assigned all miracidia to six distinct dization , 63. Bese region .S. bovis cox1 × S. bovis ITS2 genetic profile (SbSb) has also been reported in miracidia recovered from humans in Corsica [S. bovis parasites at the whole genome level. The genetic signature can only represent a certain introgression level between both species, and can explain some discrepancy in parasite phenotype such us the route of excretion expected to be in the feces for the S. bovis parasite.The  Corsica . We onlyS. haematobium populations are sparse [cox1 data, at the African continental scale suggested that S. haematobium is less structured than S. mansoni [S. bovis [Schistosoma challenge infection [Data on the genetic structure of e sparse , 30. Pre mansoni  and S. bS. bovis . In contS. bovis . The autnfection . Subsequnfection . The smanfection .S. haematobium and whether forms of hybrids detected in humans thus far are truly different genomic entities.No genetic differentiation was observed according to the genetic profile. This result suggests that there are no genetic mating restrictions between the six different genetic profiles, and hence, all recombinations are possible , 27. SimITS2 fragment. Even though the results we obtained are robust, the RFLP method is known to be sensitive to different factors such as enzyme used, and the time or temperature of digestion. A new method for single-nucleotide polymorphism detection adapted to large scale screening needs to be developed. Second, miracidia from livestock or rodents were not collected. This is an important point to infer the possible zoonotic capacity of these hybrid pathogens. The situations are contrasted depending on the countries without current evidence of zoonotic transmission in Senegal [The current study has some limitations, related to the reliability of the RFLP analysis, the absence of samples from animals, or the hybrid genotyping method. First, in our study we used an RFLP method based on a short 505 bp  Senegal  compared Senegal . FindingS. haematobium × S. bovis observed can lead to difficulties in accurately diagnosing schistosome hybrids by conventional techniques using light microscopy. This could also have adverse consequences on schistosomiasis control towards elimination and negatively impact disease transmission. More studies are need on population genetics of schistosomes at the human and animal interface to evaluate the parasite’s gene flow.Our study investigated the genetic diversity and population structuring of schistosomes at the population level. Each child harboured a genetic cluster of schistosomes at the infrapopulation level, which could lead to putative resistant parasite selection due to drug pressure by mass drug administration (MDA). The high frequency of hybrids between


Anisakis are among the most prevalent parasites found in fishes and marine mammals and they are the main cause of human anisakiasis. Genetic studies have described two species, A. simplex and A. pegreffii, in European waters parasitizing several hosts, being the European hake, by far, the fish with higher infection values. The aim of this study is to enhance the knowledge of the distribution and population structure of the Anisakis species infecting hakes from the major European fishing areas through the use of genetic analysis. This study provides useful information about the genetic diversity of these parasites in the different fishing areas and fish tissues, valuable findings to understand the parasite speciation to different hake tissues and how they are structured along European waters. The nematodes of the genus Anisakis. Merluccius merluccius is, by far, the most important host of Anisakis spp. at the European fishing grounds, in terms of demographic infection values, and carries the highest parasite burden. These high parasite population densities within an individual fish host offer a chance to explore new sources of variations for the genetic structure of Anisakis spp. populations. A total of 873 Anisakis spp. third-stage larvae, originally sampled from viscera and muscular sections of hake collected at ten fishing grounds, were primarily identified using ITS rDNA region as molecular marker. After that, we used mtDNA cox2 gene to reveal the high haplotype diversity and the lack of genetic structure for A. simplex. Dominant haplotypes were shared among the different fishing areas and fish sections analyzed. Results indicate a clear connection of A. simplex from European hake along the Northern North Sea to the Portuguese coast, constituting a single genetic population but revealing a certain level of genetic sub-structuring on the Northwest coast of Scotland. This study also provides useful information to advance the understanding of parasite speciation to different fish host tissues or microenvironments.The European hake, one of the most commercially valuable species in ICES fishing areas, is considered an important neglected source of zoonotic risk by nematode parasites belonging to the genus  Merluccius merluccius   is one of the most commercially important species in the North East Atlantic fishing area (FAO27). Nowadays, the International Council for Exploration of the Sea (ICES) considers two different stocks in EU Atlantic waters separated by the Capbreton Canyon. The northern hake stock comprises the North Sea, Skagerrak and Kattegat, the coast of UK, France and Ireland, while the southern stock is distributed along the Atlantic coast of Spain and Portugal [Anisakis Dujardin, 1845  for seafood consumers [Anisakis in hake fillets results in the rejection of parasitized fish products by official inspectors in the industry. Moreover, the presence of Anisakis spp. in fish edible parts gives rise to the reduction of the marketability of aesthetically unattractive fish, with significant economic losses for European hake markets [The European hake  area FAO7. Nowadaonsumers ,4,5. Fur markets ,4,6. Anisakis. To date, nine species of this genus have been identified, using different diagnostic genetic markers, showing that they can be grouped in four distinct clades. Two species of clade 1, A. simplex (s. s.)  Dujardin, 1845 and A. pegreffii Campana-Rouget & Bioca, 1955, have been reported up to now in several European fishing areas and in several intermediate and definitive hosts [A. simplex is the most prevalent species in FAO27 and coexists with A. pegreffii in Spanish and Portuguese coasts, even hybrids between them have been found in these sympatric areas [The genetic population structure studies of parasites have contributed to clarify the taxonomy of cryptic species, morphologically indistinguishable, which is common in nematodes of the genus ve hosts ,11,12,13ic areas ,15. Anisakis spp. larvae in European hakes is enhanced by their key trophic role in NE Atlantic ecosystems. Hakes feed on mesozooplankton (euphausiids and amphipods), small zooplanctivorous fish and larger demersal prey, which are all well known as intermediate and paratenic hosts of Anisakis spp. [Anisakis spp. [Anisakis spp. in any particular fish species can be affected by changes in the food-web structure, including the role of anthropogenetic impacts on the exploited fish stocks and ecosystems. The recruitment and accumulation of kis spp. ,16. Likekis spp. ,11. TherBeyond the impact on human health and on food industry, the study of the genetic population structure of parasites also provides information about their dynamics of infection as their host-specificity, their speciation to different tissues in a single host or their ability to adapt to local environments and to climate change ,18,19. IAnisakis spp. larvae collected from European hake from the most significant fishing grounds of ICES areas and (2) to establish the genetic diversity and the population genetic structure of A. simplex in relation to fish origin and site of infection.This study has a double-aim: (1) to genetically identify the Anisakis spp. third-stage larvae from Merluccius merluccius were donated by the Technical Unit of the Marine Biobank at the Institute of Marine Science . These samples consisted of Anisakis specimens originally collected from ten ICES divisions belonging to five subareas , including specimens from the muscular sections: belly flaps (BF) (N = 409), loins (LO) (N = 252) and tail (TA) (N = 72). Additionally, 140 visceral Anisakis larvae (VIS) were chosen from five hakes (over 10 parasites per host) caught in subareas VIIj, VIIIc and IXa in the period 2013–2014. A total of 873 traceable high-quality samples of subareas : IVa , 0.3 μM of each NC5/NC2 primers [A. simplex (T nucleotide in both sites), A. pegreffii (C in both of them) and heterozygotes (two overlapping C/T peaks in both positions) [Genomic DNA of each larva was obtained employing the commercial kit Wizard Genomic DNA Purification Kit (Promega), according to the manufacturer’s protocol. DNA quality and quantity was checked in a spectrophotometer Nanodrop® ND-2000 . Genetic identification of  primers  and 0.02sitions) . All genA. simplex was amplified at the mitochondrial cytochrome oxidase 2 gene (mtDNA cox2), using 211F/210R pair of primers described by Nadler and Hudspeth [Genomic DNA of all identified Hudspeth . PCR reaA. simplex populations. Three sets of mtDNA cox2 sequences were defined in DnaSP v6 as three case studies: (1) A. simplex sequences grouped by the 10 fishing divisions sampled; (2) A. simplex sequences grouped by the 3 muscular fish sections; (3) A. simplex sequences of muscular and viscera specimens from divisions VIIj, VIIIc and IXa. The number of haplotypes (Nh), the haplotype diversity (Hd), nucleotide diversity (Pi), number of segregating sites (S) and the average number of nucleotide differences (K) were calculated by DnaSP v6 for all defined sets. Median-joining haplotype networks [http://popart.otago.ac.nz (accessed on 13 December 2022)). Neutrality test, Tajima’s D [A. simplex populations was also evaluated by a hierarchical analysis of molecular variance (AMOVA). Pairwise comparisons of Fst [Multiple alignments of the sequences achieved were constructed using MEGA 7  and analnetworks  were conjima’s D  and Fu’sjima’s D  were perjima’s D  with 100s of Fst  values bAnisakis larvae collected from fish muscular sections and 140 Anisakis from fish viscera were amplified and successfully sequenced. According to the diagnostic positions, a total of 695 muscular specimens showed the homozygote pattern of A. simplex (T nucleotide in 278 and 294 sites), 12 specimens showed the profile of A. pegreffii (C in both positions) and 26 Anisakis had a heterozygote genotype (T/C in 278/294 sites). The genetic identity of Anisakis specimens from viscera by sequencing of ITS region showed the A. simplex pattern in 76 cases, those of A. pegreffii in 43 specimens and heterozygote profile in 21 larvae. A fragment of 905 bp of ITS1-5.8S-ITS2 region from 733 Distribution of Anisakis species in ICES divisions. A. simplex was the predominant species in hakes from all divisions sampled, represented the 94.82% of all specimens identified, followed by hybrids A. simplex x A. pegreffii (3.55%) and A. pegreffii (1.64%). A. simplex and hybrids were identified in the ten divisions whereas three divisions showed to be sympatric areas for A. simplex, A. pegreffii and hybrids: IXa ; VIIIa  and VIIIc  (hybrids) .species in fish sections. The predominant species A. simplex in European waters represented similar percentages 95.84% in belly flaps, 93.65 % in loins and 93.06% in tails; A. pegreffii showed lower percentages in BF (1.47%), LO (1.59%) and TA (2.78%), similar to those for hybrids in BF (2.69%), LO (4.76%) and TA (4.16%)  . Anisaki results . ParasitA. simplex collected in hakes from ten ICES divisions. The alignment of all A. simplex sequences (483 bp) contained 115 variable sites (S), which resulted in 215 haplotypes. Genetic diversity indices for all divisions are shown in A. simplex from each division showed a similar haplotype diversity for all of them ranged between 0.848 for division VIa to 0.970 for VIIIa and with pi values ranged between 0.00496 from VIa to 0.00948 from VIIc. Neutrality test, Tajima’s D and Fu’s, showed negative values statistically significant  for all divisions, except for VIIh whose Tajima’s D value was not significant (P = 0.12800), rejecting the null hypothesis, i.e. the population evolves according to the infinite-site model and all mutations are selectively neutral . Fst values (pairwise genetic differentiation) among A. simplex sequences of the 10 divisions sampled are shown in st values appeared between Via, and the other nine divisions compared with it, ranging between 0.01503 and 0.06270; all of them were significantly different (p ˂ 0.05). In contrast, the differences between the other nine populations of A. simplex were not statistically significant, and their Fst values were lower than those of VIa.A total of 168 haplotypes  were unique for some of the 10 divisions analyzed and, in all cases, were represented for only one sequence, representing more than 44.12% of total haplotypes for each division . The popA. simplex described above were grouped in belly flap (BF), loin (LO) and tail (TA), depending on where it was taken from. The genetic diversity indices calculated for A. simplex from each muscular section were similar between them and to those obtained to the 10 fishing divisions  for the three sections. A total of 510 mtDNA cox2 sequences from ivisions . The Hd A. simplex from the three muscular sections was represented  (P = 0.58065). Fst values among A. simplex sequences of the three divisions sampled showed negative low values when BF/LO (−0.00042) and LO/TA (−0.00247) and a low positive value for the comparison BF/TA (0.00020), but in all of the cases, the values were statistically non-significant.A total of 177 haplotypes  were unique for some of the three muscular sections analyzed. Remarkably, 100 unique haplotypes were found in BF, represented by between one and four sequences of each of them; 63 unique haplotypes in LO (including 1 or 2 sequences per haplotype) and 14 exclusive haplotypes in TA (with 1 sequence per haplotype) . The AMOA. simplex were compared, 156 corresponding to those of muscular sections of hakes from divisions IXa, VIIj and VIIIc (obtained in above subsection) and 45 to viscera of the hakes from the same divisions. The alignment of all A. simplex sequences (483 bp) contained 78 variable sites and 116 haplotypes. Genetic diversity indices for two groups were Hd = 0.961, Pi = 0.00773 and K = 3.73567, and they were similar between sequences of specimens from muscle and viscera. Thus, Hd values were 0.958 for muscle group and 0.971 from viscera, and Pi values of 0.00752 and 0.00846, respectively. Neutrality test, Tajima’s D and Fu’s, also showed negative values statistically significant  for the two groups  were shared between both groups . Among them, the haplotype name H2  was the more representative with 37 sequences  and with relative frequency values of 0.156 for A. simplex of viscera and 0.192 for muscle group (st of 0.00126 (P = 0.05415).Median-joining haplotype network of the 201 mtDNA cox2 sequences of le group . A totalle group /Figure 4A. simplex complex and hybrids A. simplex x A. pegreffii is crucial to understand their epidemiology, ecology and zoonotic potential. Several molecular markers have been described for this purpose, among them PCR-RFLPs profiles of ITS rDNA region [A. simplex and A. pegreffii [A. simplex, A. pegreffii and their hybrids was performed used ITS1 marker [Anisakis hybrid genotypes [The genetic differentiation between the sibling species of A region ; nuclearA region  or beta-A region  and an AA region ; mitoconA region  or even egreffii ,35. Howe1 marker . Some auenotypes ,30, but enotypes ,36. MoreA. simplex, we used those larvae that showed an unequivocal homozygote pattern of A. simplex (T nucleotide in 278 and 294 sites) in ITS1 region, according to Abollo et al. [A. pegreffii and hybrids obtained in this study were scarce to perform a robust genetic population study. Thus, mtDNA cox2 gene for all the identified A. simplex was also sequenced since it is a common marker used for studies of population structure of the genus Anisakis [To carry out the study of population structure of Anisakis ,37,38,39Anisakis ,41,42,43Anisakis larvae infecting the muscle of M. merluccius showed clearly that A. simplex is the predominant species in the 10 ICES divisions. This represents the 94.82% of all specimens identified, which is in accordance with previous studies [A. pegreffii constituted only the 1.64% of the total specimens identified, and it was found exclusively in Portuguese waters (IXa) and Bay of Biscay . Noticeably, hybrids A. simplex x A. pegreffii also appeared mainly in these fishing divisions, confirming that the Spanish and Portuguese coast are sympatric areas for both species and their hybrids [Anisakis species from belly flaps, loins and tails showed similar results since more than 93% of identified species were assigned to A. simplex in the three fish sections, whereas a lower number of A. pegreffii and hybrids were found . However, when Anisakis specimens of viscera from the sympatric areas  were analyzed, A. pegreffii doubled those values obtained for A. simplex. Likewise, a higher number of hybrids was also detected in both divisions. These results are completely different from those obtained for muscular sections from the same fishing divisions. Cipriani et al. [Anisakis species in viscera and muscle of European hake from IXa division and reported that the relative distribution of A. simplex doubled to A. pegreffii in viscera, but this proportion increased in flesh, being the prevalence almost five times higher for A. simplex than for A. pegreffii. Pascual et al. [Anisakis species from viscera of hake from the Iberian Peninsula . Our results reinforce the values obtained in previous studies. Several studies have highlighted the tissue specificity of different Anisakis species, suggesting that A. pegreffii has a lower penetration index in the fish muscle than A. simplex [A. physeteris is not capable of migrating in the flesh of the fish [The molecular identification of  studies ,44. A. p hybrids ,14,44. L simplex ,46,47 anthe fish . A. simplex from the different fishing divisions showed a high haplotype diversity but low nucleotide diversity for all populations (except for VIa) whose Hd, Pi and K value were lower than for the other populations. These values are translated at network in a high number of total haplotypes (215), with a high percentage of unique haplotypes (78.14%) with very low relative frequency. A single haplotype (H3) was clearly majority and distributed in all divisions. It indicates a clear connection of the A. simplex from Northern North Sea to Portuguese coast. Moreover, the distribution of this haplotype seems even wider since it corresponds to the same unique haplotype (H5) for A. simplex usually noted all around the North Atlantic from Sebastes mentella , and witic Sea; ). The senean Sea  and with Galicia  and Stenatic Sea . The higatic Sea . A. simplex collected in C. harengus [A. simplex from S. mentella, both from North East Atlantic fishing grounds [A. simplex is also consistent with those described for A. simplex parasitizing the Pacific sardine (Sardinops sagax) from the California Current system [Neutrality tests showed high negative values statistically significant, rejecting the null hypothesis . These results are similar to those recorded in populations of harengus  and in p grounds . The gent system . A. simplex infecting C. harengus from North East Atlantic [A. simplex from different fishing divisions. The molecular variance analysis (AMOVA) revealed a high intra-population genetic variation (99.75%) whereas a low percentage (0.25%) was explained for geographical differences among fishing divisions. This is in agreement with the results observed for Atlantic . PopulatContracaecum rudolphii A, parasitizing great cormorants  sampled in different localities from Sardinia [High genetic diversity and lack of genetic structure have also been found in other anisakid species, such as Sardinia . A high Sardinia . Two maiSardinia ,11,42,53Anisakis population of the Northwest coast of Scotland (VIa). In European waters, a similar slight geographical separation has already been described with A. simplex from C. harengus and Balenoptera acutorostrata from Norwegian Sea [C. harengus stock as an important food source for B. acutorostrata; (2) the migration pattern of B. acutorostrata, mainly distributed from the Atlantic coast of France to Greenland; and (3) the lower proportion of some species of the Delphinidae in Norwegian Sea, comparing with other European waters. Up to now, the genetic structure of Anisakis species in the Northwest coast of Scotland, including Hebrides Islands, had not been studied. A different hake stock in that VIa division could be related with the slight genetic differentiation of A. simplex. At present, ICES assumes the existence of two stocks of the European hake within the Northeast Atlantic Ocean: the northern stock  and the southern stock (ICES Divisions 8c and 9) [Orcinus orca from Hebrides Islands showed a different pigmentation pattern when compared with neighboring populations [Tursiops truncatus) in Scottish coastal waters, two of them located in the west coast. Evidences of the occurrence of hybrids between bottlenose dolphins and Risso’s dolphins (Grampus griseus) from Hebrides have also been observed [Halichoerus grypus, since UK has the 40% of the world’s population and 95% of all grey seal in Europe [A. simplex in division VIa. Despite the very limited population structure, present results suggest a certain level of genetic sub-structuring in gian Sea ,39. Threc and 9) ,2. Howevc and 9) ,55,56. Rc and 9) . Furtherulations . Cheney ulations  also sugobserved . Moreoven Europe . These cA. simplex from different fish sections of European hake was evaluated. The results showed that the genetic diversity indices for mtDNA cox 2 sequences of A. simplex of BF, LO and TA were similar for the three muscular sections and also similar to those sequences of A. simplex from viscera. Likewise, neutrality test and the population genetic variation among parasites obtained from the four different fish sections pointed out the lack of genetic structure among them. The same haplotype H3, majority in all fish divisions, is also clearly the most prevalent and it is interchangeably both in the three muscular sections and in viscera (named H2) with similar relative frequencies. The high frequency of this haplotype in the 10 fishing divisions and in the 4 fish sections shows that it is well adapted to different micro-and macroenvironmental conditions. A high number of unique haplotypes were also found for the four fish sections. A plausible explanation for the high number of unique haplotypes found for the four fish sections might be that these haplotypes play an important role for adaptation or colonization of different microenviroments. For this reason, the infection dynamic and pathogenicity of the haplotypes identified, both those predominant and minority, should be further analyzed in order to establish their hypothetical adaptative strategies related to tissue specificity. For the first time, the genetic diversity and population structure of Anisakis spp. parasitizing the European hake along the ICES fishing areas and in the different fish sections. Remarkably, A. simplex is the predominant species in the edible parts of M. merluccius in all divisions studied. However, in sympatric fishing areas for Anisakis (VIIIc and IXa), the number of A. pegreffii in viscera was similar to A. simplex, or even greater.This study provides novel insights into the distribution of A. simplex constitutes a single genetic population along the 10 fishing divisions studied although a statistically significant genetic sub-structuring was also found for A. simplex in the Northwest coast of Scotland, which could indicate the presence of a subpopulation in this area. We found a high haplotype diversity for this parasite species in hake but a clear dominant haplotype in all the divisions and fish sections studied. More experimental and analytical studies are necessary in order to test if the different haplotypes described present any difference in resilience and adaptability, which could confer a greater evolutionary fitness.Our results revealed that


Abrothrix olivacea, but none has incorporated landscape genetics analyses and ecological niche modeling, leaving a gap in the understanding of the species evolutionary history. Here, these aspects were addressed based on 186 single nucleotide polymorphisms, extracted from sequences of 801 bp of Cytb gene, gathered from 416 individuals collected at 103 localities in Argentina and Chile. Employing multivariate statistical analyses , associations between genetic differences and geographic and climatic distances were evaluated. Presence data was employed to estimate the potential geographic distribution of this species during historical and contemporary climatic scenarios, and to address differences among the climatic niches of their main mitochondrial lineages. The significant influence of landscape features in structuring mitochondrial variability was evidenced at different spatial scales, as well as the role of past climatic dynamics in driving geographic range shifts, mostly associated to Quaternary glaciations. Overall, these results suggest that throughout geographic range gene flow is unevenly influenced by climatic dissimilarity and the geographic distancing, and that studied lineages do not exhibit distributional signals of climatic niche conservatism. Additionally, genetic differentiation occurred by more complex evolutionary processes than mere disruption of gene flow or drift.Even when environmental variation over time and space is commonly considered as an important driver of population divergence, few evaluations of intraspecific genetic variation explicitly assess whether observed structure has been caused by or is correlated with landscape heterogeneity. Several phylogeographic studies have characterized the mitochondrial diversity of  These microevolutionary processes are in turn influenced by landscape features, such as climate and topographic relief that influence dispersal patterns5. This interaction can be summarized by stating that the magnitude of the effect of landscape attributes, including their spatial and temporal dynamics, on population differentiation is strongly determined by the breadth of the fundamental ecological niche of individuals; see7. This notion is not entirely new; see8, but more contemporary formulations place greater emphasis on aspects of the ecological niche and as such, on selective regimes geographically structured; see9. For example, Rundle and Nosil10, and Schluter11, described that conspecific populations occurring in different environmental configurations may become reproductively isolated due to the adaptation of individuals to local climatic conditions. This model implies that the reproductive barrier could evolve as a pleiotropic effect of niche specialization , which would impede the existence of individuals from one population in the climatic space of the other, and hence their reproduction. Conversely, conservatism of climatic niches can prevent gene flow among populations existing in equivalent environmental configurations, when these are spatially separated by climatic conditions that are unsuitable to be crossed by individuals13.Genetic differentiation of populations is largely determined by the interplay between the homogenizing effect of gene flow and the differentiation promoted by spatially varying selection and genetic drift14, is a useful approach for inferring the mechanisms by which these were molded; see16. However, without knowing the history of the spatial changes of population distributions, it is difficult to discern between alternative scenarios18. In such cases, the usage of ecological niche modeling (ENM) to infer the spatial changes underwent through time by a focal species or intraspecific lineage, can help to choose among alternative explanatory scenarios20. Under another approach, the contexts in which genetic differences are molded can be broadly summarized into two general models defined as isolation by distance (IBD) and isolation by environment (IBE)21. IBD proposes that gene flow among populations is strongly affected by the geographic distance; so, the degree of genetic differentiation between population pairs correlates with its geographic distancing22. In contrast, gene flow also can be impeded or reduced by the environmental dissimilarity, originating patterns that can depart from IBD23. Considering this fact, IBE was advanced to describe cases where genetic differentiation increases along with environmental dissimilarities, regardless of the geographic distancing populations24. Examination of both models should consider that geographic distance and environmental dissimilarity are often correlated, so to parsing their effects on genetic differentiation it is important to perform a parallel assessment of its association25.Explicitly contextualizing the study of genetic structure patterns with the geographic and environmental configuration in which populations exist . These events were also relevant for the evolution of small mammal species because its impact on demographic processes, patterns of gene flow, and changes in their geographic ranges; see30 for an early review. According to Pardiñas et al.31, the effects of these events over the structure of the mitochondrial genetic diversity of the sigmodontine rodents can be summarized in two general schemes, corresponding to a set of species that exhibit low genetic diversity and apparent absence of phylogeographic structure , is generally temperate with extreme spatial variation in seasonal temperature and precipitation. In arid regions such as the Atacama Desert and western Patagonia, the climatic conditions are strongly desertic, while in the coastal and foothills areas of central and southern Chile precipitations are abundantAbrothrix olivacea , matches with the second scheme described by Pardiñas et al.31; its mitochondrial diversity is structured in four main and mostly allopatric phylogroups originated during the last million years, which experienced independent demographic dynamics37; see also42. However, no study has explicitly evaluated whether the identified patterns of genetic structure correlate with landscape features and their temporal dynamism. Consequently, the general implications of environmental changes on the structuring of mitochondrial diversity in A. olivacea and whether genetic differentiation within this species is associated with some landscape features remain mostly unexplored. Addressing these aspects could be considered as the next step to be taken once the pattern of genetic structure has been uncovered, given that this gap impedes to fully understand the evolutionary history of this species. A. olivacea has the broadest distribution among the sigmodontine rodents of Southern South America; see43, inhabiting a wide variety of ecosystems , and exhibiting different levels of mitochondrial genetic structure across its geographic range37, making an ideal model to study the effects of the environmental variability on the genetic differentiation of small mammal species. Considering the reviewed antecedents, the aim of this study was to address the implications of dynamism and variability of the environmental-climatic features of southern South America in structuring the mitochondrial variability of A. olivacea. Past potential geographic distributions were estimated for this species and their mitochondrial lineages using ecological niche modeling, and the association between genetic differentiation and landscape features was assessed performing multivariate statistical analyses. Results obtained here suggest that gene flow among populations is unevenly influenced by the climatic dissimilarity and geographic distance, and that niche divergence observed among mitochondrial phylogroups is greater than that expected to be among intraspecific lineages. This evidence could imply that the diversification of these lineages could involve other evolutionary processes than genetic drift and/or disruption of gene flow.The olive field mouse, 37 to assess the phylogeographic structure and historical demography of Abrothrix olivacea. The dataset consists of 801 bp of the cytochrome-b gene (Cytb) from a total of 416 specimens collected from 103 localities  to summarize the dimensionality of the mitochondrial genetic data into a reduced set of principal components axes (PCs). For this and subsequent analyses, individuals were grouped according to the four mitochondrial phylogroups  described by Quiroga-Carmona et al.37, which correspond to intraspecific mitochondrial lineages distributed in northern Chile (N-Ch); Mendoza, Argentina (Men-Ar); central Chile and the Chilean and Argentinian Patagonia (CS-Ch-Ar); and Tierra del Fuego Island and southernmost Chile  and/or isolation by environment (IBE) was initially evaluated using matrices of genetic, geographic, and environmental distances calculated between pairs of localities. The genetic distances were calculated as 53). This analysis consisted in the superimposition of a genetic map constructed with the projection of 416 individuals onto the orthogonal intersection of the two first principal components of the gPCA, onto a geographic map where the 103 collection localities of the individuals are projected. Geographical coordinates were converted onto a conical projection with the R package rgdal54, and the genetic and geographical maps were superimposed with vegan. The significance of the Procrustes similarity statistic (t0) was assessed with 1 × 104 randomized permutations of locality placement, computing the similarities scores (t) between the gPCA coordinates and the geographic locations. Then, the probability of observing a similarity statistic higher than t0 under the null hypothesis  was calculated; see53. Finally, an analysis of variance (ANOVA) was performed using the magnitude of the residuals to test whether deviations from the expected pattern of genetic variation based on geography are different among the mitochondrial phylogroups.Differences among the magnitude of the IBD observed for each mitochondrial phylogroup were explored using a Procrustes analysis  was conducted. This ordination method is used in landscape genetics to evaluate the IBE model because it minimizes type I errors when used to inspect landscape genetic relationships55. This is a constrained ordination method comparable to a linear regression that can be used to model multivariate response data and distinguish the relative contribution of landscape features  and the spatial components on genetic structure; see56. The coordinates of the individuals in the first 20 PC of the gPCA were employed as genetic-response variables, and the previously identified uncorrelated bioclimatic variables were employed as landscape-predictor variables. In addition, the geographic coordinates (latitude and longitude) of each sampling locality were included as conditional variables to consider the effect of the geographic distancing on the pattern of genetic structure. The RDA was completed with a stepwise model selection to identify the climatic predictor variables that best explain the genetic differences among individuals. Subsequently, ANOVAs were performed to assess the significance of constraints and the RDA axes. These analyses were completed with vegan.Finally, to identify the climatic features that are associated with the structure of the mitochondrial variability of Abrothrix olivacea was addressed characterizing the climatic attributes of their geographic ranges. In this case, the localities corresponding to each phylogroup  were used to extract the values registered for each bioclimatic variable (employing steps described in the previous section); except for the phylogroup from Mendoza, Argentina, as it was sampled only at two localities  of Euclidean distances with 1 × 104 iterations, using vegan. Posteriorly, locality scores in the first three PC were used to construct Gaussian hypervolumes to represent the climatic niche of each mitochondrial phylogroup, and theses ellipsoid were them projected in a common multidimensional climatic space to evaluate its degree of differentiation; see58. This procedure was completed employing the R package hypervolume59, which was also used to compute the Jaccard and Sørensen indexes as measures of niche comparison and whose values of similarity range from 0 (fully different) to 1 (fully similar).The differentiation of the climatic niche of the mitochondrial phylogroups of Abrothrix olivacea. The past geographic distributions of this species and their mitochondrial phylogroups were reconstructed by estimating the geographic distribution of the climatically suitable areas available during the Last Interglacial (LIG: ∼120–140 Kay BP), the Last Glacial Maximum (LGM: ∼22 Kay BP), and the Mid Holocene (Mid-Holo: ∼6–8 Kay BP). The climatic niches were modeled using as primary presence data the 103 localities where the sequenced individuals were captured. This occurrence dataset was enriched using the records available at the Global Biodiversity Information Facility , which strictly came from specimens deposited in biological collections. This second dataset was spatially filtered with the R package spThin60 and using a threshold of 15 km of separation between localities to minimize the potential effects of spatial autocorrelation60. The filtered records were pooled with the 103 initial localities, and subsequently these records were partitioned according to the geographic distribution of each mitochondrial phylogroups , delimiting areas adjusted to the minimum convex polygon formed by the corresponding presence records, plus the addition of an enveloping geographic buffer of 50 km . Thus, the following six variables were employed for modeling climatic niches: Bio 03: isothermality ([Bio 2/Bio 7] × 100), Bio 04: temperature seasonality, Bio 06: min temperature of coldest month, Bio 09: mean temperature of driest quarter, Bio 15: precipitation seasonality, and Bio 16: precipitation of wettest quarter. These variables are good descriptors of the extreme climatic conditions and their strong seasonal fluctuations throughout the study region  and at the same time represent adequate surrogates for climatic factors limiting the dispersion and distribution of terrestrial mammal species, see64.The ecological niche models (hereafter referred as niche models) were developed to evaluate the importance of the climatic dynamism of the Quaternary on the differentiation of the mitochondrial genetic diversity of SM, Fig. . PosteriSM, Fig. . The bio67. Optimal model performance and complexity were estimated for each set of presence data, varying the values of regularization multiplier (rm) from 0.5 to 4, with increments of 0.5, and the feature classes (fc) used in each modeling instance. These procedures were completed employing the ENMeval R package68 and selecting a spatial block approach as the scheme for the geographic partition, see69. The final setting for each model was selected based on the values of the Akaike Information Criterion corrected (AICc71) obtained for each combination of rm and fc. Secondarily, the performance and realism of the models was supervised inspecting the values of omission rate under the 10-percentile threshold (OR 10), the area under the curve (avg AUC) values, the estimated response curves73, and the potential geographical distributions obtained after the spatial projection of each niche model. Constructed final niche models were spatially projected onto a study region that encompasses the geographic distribution of Abrothrix olivacea . During transferences of models, environmental differences between training and projection climate scenarios were assessed by performing Multivariate Environmental Similarity Surface  analysis, to determining the spatial distribution of analogous or non-analogous climate conditions between the climate scenarios considered in each model transference instance. These procedures were completed using the ntbox R package75.The niche models were constructed using the maximum entropy method as implemented in MaxEnt, version 3.4.476, niche model transferences were completed using an unconstrained extrapolation. The past climatic scenarios explored corresponded to the reconstructions performed by Otto-Bliesner et al.77 for climatic conditions of the LIG, Watanabe et al.78 for climatic conditions of the LGM and the Mid-Holo, and Hijmans et al.46 for the Current climatic conditions. Finally, the geographic representations of the climatically suitable areas were constructed using a continuous representation of environmental suitability values, which were spatially projected using QGIS.Following indications of Guevara et al.Cytb sequences of Abrothrix olivacea. The gPCA performed with this dataset shows complete differentiation among the four mitochondrial phylogroups ; both, for the whole sample of Abrothrix olivacea  and mitochondrial phylogroups . IBD and IBE evaluations also resulted with significant values of correlations at the spatial scale corresponding to the geographic range of A. olivacea  and TdF-SCh  , Bio 04: temperature seasonality, Bio 06: min temperature of coldest month, Bio 09: mean temperature of driest quarter, Bio 15: precipitation seasonality, and Bio 16: precipitation of wettest quarter  Table  and S5. t0 = 0.637; p-value = 1 × 10–4; SM, Fig. Abrothrix olivacea. The lines connecting individuals from the gPCA map to their geographical locations have lengths that are significantly different among the mitochondrial phylogroups  of the total mitochondrial genetic variance is explained by the geographical distancing of the localities, while 87.78% (R2 adjusted = 0.8778) is explained by the climatic variables included as model predictors. Among these, only five  (× 100)), Bio 04: ), Bio 08: Mean Temperature of Wettest Quarter, and Bio 12: Annual Precipitation; Table p-value ≤ 0.05) the pattern of mitochondrial genetic structure exhibited by Abrothrix olivacea. Together, these climatic predictors accumulate 53% of the variance  after removing the variance explained by geography, while geographic distance solely accounted for 8.61% (conditioned inertia = 0.61) after removing the variance due to climatic heterogeneity. Finally, climate and geography together have a combined effect of 38.34% (unconstrained inertia = 2.89) on genetic variance, which is the amount of variance in which due to collinearity, climate and geography cannot be separated80. The first three axes of the RDA accounted for 84.93% of the constrained genetic variation, and Bio 01, Bio 03 and Bio 08 were the variables with higher explanatory contribution in the RDA model , except for the comparison between CS-Ch-Ar and Men-Ar  could also simply be due to the inherent limitations of this analysis (see94). For instance, our data and analyses do not allow assessing the effect of seasonality and topographic variation on home range changes and of the latter in the geographic structure of the genetic variation.Differences among the internal patterns of genetic structure exhibited by each mitochondrial phylogroup of ts Table  consistees Table  and S5. 37, are not correlated to geographical distances, and significant association with climatic dissimilarity was only evidenced for the phylogroup distributed in southernmost Chile and Tierra del Fuego Island . Additionally, the combination of broader geographic and genetic sampling would allow test the relationships of the phylogroups and localities that so far have been only characterized via mitochondrial variation.On the contrary, the levels of internal genetic structure exhibited by the mitochondrial phylogroups CS-Ch-Ar and TdF-SCh, distributed along central and southern Chile and Argentina, are comparatively lowAbrothrix olivacea revealed that their realized climatic niches are markedly different. The results obtained with PCA ; a species’ property that is determined primarily by its physiological capabilities102 and whose manifestation in the environmental space represents the maximum possible expression of its ecology61. The potential ‘climatic’ niche is critically important since it largely determines where a species can occur, as well as how it has or will be able to respond to past and upcoming climatic changes104. Thus, the differences observed among the climatic niches of the mitochondrial lineages of A. olivacea are in terms of their realized climatic niches, since the comparisons performed are based on climatic attributes characterized at the localities where these lineages occur . This clarification is important because although the observed differences indicate that the realized climatic niches of these lineages are mostly distinct, these differences could be different expressions of the species potential climatic niche.The climatic differences among the geographic ranges of the mitochondrial lineages of PCA Fig. , PERMANOPCA Fig. , and thePCA Fig. , Table 5105. Geographically structured variation of biological traits, as the climatic niche, could represent environmentally-induced plastic responses12 or, alternatively, local adaptations to specific climatic conditions or other environmental aspects107. Particularly for Abrothrix olivacea, given evidence for population differences in physiological traits111 and levels of genetic expression97 among populations distributed in disparate environmental conditions have been described. The spatial differences detected are overlooked if visual inspections are limited to the projection of the niche model developed at the species level ; see37. Meanwhile, demographic inferences based on nuclear genomic variation, but with a narrower geographic and specimen sampling, indicate that Patagonian populations expanded more recently (around 13 Kay BP), and likely in response to the retreat of the ice sheet at the end of the LGM118. This pattern is consistent with the expansion of climatically suitable areas as inferred with the model projections in the climatic scenarios corresponding to the LGM and the Mid-Holocene , would likely do. Similarly, codistributed species with large phenotypic variation  are worth of analysis. Thus, future research strategies focused on these species should contemplate the possibility that different evolutionary mechanisms are acting with uneven intensities on their populations, requiring the development of methodologies and approaches that cover distinct populations simultaneously and that, in turn, consider the heterogeneity of the landscape in which they exist. A good starting point under this premise could be achieved by using genetic data generated by previous studies but analyzing them with the approaches and methodologies used here, see125, ideally those containing comprehensive and exhaustive genetic samplings118. In this line, the characterization and analysis of the dimensions that constitute the ecological niche can become a mandatory procedure when attempting to study the dynamics of species with significant levels of population differentiation126. This is important because populations or lineages within a species could have different evolutionary histories, shaped for example, by geophysical processes occurring locally, whose effect can be overlooked if ecological attributes characterized are only analyzed at the species level.The results of this study highlight patterns that will be relevant for the design of future studies aimed to address aspects related to ecological influenced genetic differentiation. On the one hand, it is important to consider that given the complex geological evolution and the large environmental heterogeneity exhibited across southern South American, patterns of genetic structure and/or ecological niche differentiation discovered in species from this region would likely be complex, and their complete unraveling will require samplings that exhaustively characterize the intraspecific genetic and ecological variation. This does not imply that all South American species have complex evolutionary histories, but potentially all those with deep phylogeographic breaks, large geographic distributions and inhabiting distinct landscape configurations as Supplementary Information.


The spread of knowledge on the important implications of a diagnosis of genetic disease does not correspond to a sharing of the knowledge and equal rights of children.It is estimated that about 5% of newborns may have a rare disease that in some cases, if diagnosed early, could have specific treatments that may be able to modify the natural history of the disease. However, in most countries the diagnosis during the first hours of life is limited to a few diseases, due to the high costs and time required for genetic investigations with classical methods. Recently, experimental projects to subject all newborns to a complete DNA analysis, with Next Generation Sequencing techniques, to detect any genetic pathologies as early as possible, have been reported in some countries. The late diagnosis of some genetic diseases that have treatment plans, such as spinal muscular atrophy, can be a serious damage, for anyone who has seen and accompanied the life of a child with this disease and his/her family, before and after, the recent availability of therapies which, if started very early, can lead to an almost normal life. Rapid sequencing and genetic diagnosis are a crucial part of directing inpatient management and this resource should be accessible not only to academic medical centers but also in community settings.It is time for a profound reflection that places in Italy, as in other countries, the use of genetic tests in neonatal and pediatric age based on principles of evidence, ethics, and democracy and on clear national guidelines, which also consider organizational aspects. The full knowledge of human genome at the beginning of present century has increased in people the hope of applying genetic testing to diagnose and predict all genetic diseases . It is nNevertheless, at the end of 2021, concrete discussions began in England and the United States of subjecting all newborns to a complete DNA analysis immediately after delivery to discover any genetic pathologies as early as possible –5. The rProponents of complete DNA mapping point out that a not small percentage of children from 5 to 7%) are born with a rare disease, sometimes treatable, or in any case well controllable if intervened early [ to 7% arProponents of genomic screening argue that with techniques known as Next Generation Sequencing (NGS), entire genomes can be “deciphered” in hours, at affordable costs. Having all the genetic information at birth—say the proponents of mass screening—could bring great benefits and translate into considerable savings, ensured by the fact of not having to treat disabilities and diseases over the course of life. Based on this principle, Genomics England  has launched a pilot study on 200,000 children, where anomalies associated with over 600 rare diseases will initially be checked, all curable, at least partially . The DNAIn the United States, plans are being made to launch a similar program to prevent or treat—when possible—the most dangerous rare diseases, but the times will certainly be longer .Science [The two projects have significant organizational differences, which could have important consequences, as reported in a dedicated issue on this new frontier in the journal Science . In GreaScience , which wScience . Some stScience .In 2018, the U.S. National Institutes of Health issued a document reiterating that there is still not sufficient justification for carrying out wide-ranging genetic tests on all children, because the effects of many mutations are not known, and why many genetic diseases lack therapy . On the The precise information aspect of what we are looking for is decisive when we need to talk to a parent about genetics and what we ask ourselves is whether we are ready for such relevant information (a consent) if we are talking about NGS which cannot be individual to moment of conception, but that must start well earlier, at the population level. The debate, on the other hand, seems very confined to laboratories and research groups, praiseworthy in scientific progress (trouble if not) but which do not have the aptitude to tackle the very serious problem of knowledge that becomes participatory and democratic, even in the method, on the impact of a diagnosis, on the risks of false positives (or negatives) and on the true possibility of cure, also knowing that for some therapies  the knowledge on the risks and benefits are not fully known. The impact of NGS on connecting patients with rare diagnoses through social media or internet-based resources can be explore more thoroughly . Rapid sThe late diagnosis of some genetic diseases that have treatment plans, such as spinal muscular atrophy (SMA), can be a serious damage, for anyone who has seen and accompanied the life of a child with this disease and his / her family, before and after, the recent availability of therapies which, if started very early, even in the forms of SMA type 1, can lead to an almost normal life . WithoutAnother illustrative example on the doubts and potential of a genetic diagnosis concerns the autism spectrum disorder (ASD). Many experts tell us that all children with a certain diagnosis of ASD (2% of newborns!) should undergo a genetic screening with comparative genomic hybridization (CGH)-array (as well as the karyotype for fragile X syndrome) . Others It is time for a profound reflection that places the use of genetic tests in neonatal and pediatric age based on principles of evidence, ethics, and democracy and on clear national guidelines, which also consider organizational aspects. What we would like as pediatricians attentive to the progress of genetics, is that a serious debate opens in Italy with very pragmatic implications (waiting for difficult and perhaps distant decisions on a genomic population screening with NGS) and that it decides on:a) The opportunity for a very defined expanded neonatal screening that is the same for all Italian newborns, and that considers those diseases that, thanks to highly sensitive and specific assessment methods, have an immediate plan of care. He will not be able to understand all of what is known, but the example of SMA is emblematic, for genetics that is indeed democratic.b) A guide to the services of public genetics laboratories, again on a national basis, which can guarantee, beyond newborn screening, rapid and competent responses to specific pathologies they have (according to the opinion of expert clinicians), a high suspicion for genetic diseases with important therapeutic implications .c) Organizational practical times, these are on a regional basis or in wider fields (but known and well organized), to provide services that are, when necessary, of rapid advice, in close interface with the pediatricians who are treating that single case and family.


Ammotragus lervia) populations in the United States are a product of multiple human‐mediated introductions. Two mitochondrial markers  and one nuclear gene  were used to determine: (1) genetic variation, (2) if genetic units correspond to taxonomic designations, (3) the number and geographic origin of translocations, and (4) divergence times. Three phylogenetic analyses  produced similar topologies with two clades (I and II). Clade I contained progeny of individuals resulting from introductions to Texas and Spain, and individuals from Algeria. Individuals in Clade II were progeny of past introductions to the United States and Europe, and northern Algeria. Clade II was subdivided into two subclades (A and B) representing two haplogroups. No genetic variation was detected in the PRNP sequences. Three haplogroups appeared to correspond to the subspecies A. l. lervia and A. l. sahariensis whose native distribution includes northwestern Africa. Network analyses assigned haplogroups to two major groups similar to those depicted in the phylogenetic analyses. Genetic distances ranged from 0.80% to 5.17% and 2.99% to 15.42% for cytb and D loop, respectively; and were higher than normally recovered for caprids, warranting a reexamination of subspecific status. Divergence dates indicated a major split between A. l. lervia and A. l. sahariensis circa 2.38 mya. Together, the high level of genetic divergences among US populations and apparent presence of two subspecies of aoudad in the United States support the hypothesis of multiple introductions from multiple sources.Translocation records indicate aoudad ( A. l. sahariensis and/or A. l. blainei and A. l. lervia. Two major clades were identified using phylogenetic, network, and divergence analyses, in which genetic distances ranged from 0.80% to 5.17% and from 2.99% to 15.42% for cytochrome b and displacement loop. Given the extensive number of free‐ranging aoudad currently in Texas, the manuscript provides baseline genetic data for future management and conservation plans being developed for a species that is considered “vulnerable” in its native range.This manuscript explains that human‐mediated translocations of aoudad into the United States were a result of multiple introductions with putative subspecies identification as  Little is known concerning genetic variation and phylogeographic patterns of diversity among these putative subspecies. In reviewing the taxonomy of Caprini, Groves and Grubb . To date, no genetic information is available for the other four subspecies, complicating the resolution of phylogenetic relationships among subspecies within their native range, as well as their role in determining source‐stock origins of introduced populations throughout the world.Aoudad, also known as barbary sheep or arrui  in the early 1900s, those that documented the movement/transfers of aoudad between zoos  were not always in agreement. Further, given that private ranches were not obligated to maintain source‐stock information or geographic history records, they generally did not provide point of origin data for imported aoudad. Consequently, the multiple imports and subsequent purchase or trading of zoo progeny by private individuals, coupled with a paucity of source/origin documentation, make it difficult to discern if more than one subspecies of aoudad was introduced into the United States during this time frame.Beyond the aforementioned zoo records and discussions presented in the Simpson,  and New s Ogren, , 1965, tOvis canadensis) , and displacement loop (D loop) and one nuclear gene  were used to determine the magnitude of genetic variation. The cytb marker is used widely as a proxy to measure genetic divergence among species and subspecies . The goals of this study were to: (1) assess genetic variation in free‐ranging populations of aoudad in Texas, California, and New Mexico, (2) determine if genetic units correspond to existing taxonomic designations, (3) ascertain the number and geographic source of introductions, and (4) provide estimates of approximate divergence times among haplogroups. Mitochondrial markers, cytochrome 22.1A total of 232 aoudad samples were collected between 2018 and 2021, these included 209 samples from free‐ranging individuals, 19 samples from pedigreed captive individuals from the Fossil Rim Wildlife Center (Texas), and four historic samples , and were used in this study  was extracted from 0.1 g ear clip, muscle, liver, or 100 µl blood (stored in standard collection tubes containing EDTA) using the Qiagen DNeasy blood and tissue extraction kit . The entire cytb gene  was amplified using the polymerase chain reaction (PCR) method . Cycle sequencing reactions were conducted with BigDye Terminator v3.1 (Applied Biosystems) using the following primers: LGL766 and LGL765  was amplified in select individuals based on resulting haplogroups identified from the cytb dataset. The differences in sequencing methods for D loop are described below. Primers utilized to amplify the full‐length D loop (1097 bp) were 2340‐4 . Primers used to amplify the complete PRNP gene (771 bp) were MD582F .Data obtained from the cytb and D loop datasets were used to direct selection of individuals to be examined for the prion protein exon 3 gene  and the nuclear gene (PRNP: n = 1) datasets were obtained from NCBI GenBank and included samples examined in Derouiche et al.  was designated as the outgroup species following Ropiquet and Hassanin  models were evaluated using jModelTest‐2.1.10  model  obtained from an estimation of divergence from the most recent common ancestor , nucleotide diversity (π), number of haplotypes (h), haplotype diversity (Hd), and Fu's test of neutrality were calculated for both the entire cytb and D loop dataset  using DNAsp v6  and heterozygous nucleotide base positions were visually determined using chromatograms. The program MEGA X . In particular, three codons known to be of importance  was obtained when identical sequences were removed. This final dataset was used for all subsequent analyses involving phylogenetics, genetic distances, and genetic indices. The three phylogenetic analyses  generated similar topologies in the cytb dataset; consequently, each analysis is discussed in detail below; however, only the topology obtained from the BI analysis is shown  of 249 individuals was used to assign haplotype affiliation of all individuals to the sampled localities . Clade I contained 52 individuals from western and southeastern Algeria; Black Gap WMA, Davis Mountains, Dolan Falls Preserve, Elephant Mountain WMA, Fawcett WMA, Fossil Rim Wildlife Center, Garza Co., Glass Mountains, Kerr WMA, Love Creek Preserve, Val Verde Co., and the Waddell Ranch (now Fawcett WMA) in Texas; Mansoura Zoo, Egypt and/or Niger; and Navalvillar de Pela region, Spain. Clade II was divided into two subclades (A and B); with Subclade II‐A being supported in the BI analysis (PPV ≥ 0.95). The three haplotypes representing the 191 individual sequences forming Subclade II‐B were not supported by the three analyses and instead formed an unresolved polytomy. Subclade II‐A contained 6 individuals from the Béchar Province, Algeria; Elephant Mountain WMA and Davis Mountains in Texas; National Museum of Natural History (MNHN), Paris, France; and Vincennes Zoo, Paris, France and/or Morocco and/or La Hoya Field Station, Almería, Spain. Subclade II‐B contained 191 individuals from northern Algeria; northern Algeria and/or La Hoya Field Station and/or Tunisia, Almería, Spain; Black Gap WMA, Caprock Canyons State Park, Carrizo Mountains, Chinati Mountains, Culberson Co., Davis Mountains, Dolan Falls Preserve, Elephant Mountain WMA, Fawcett WMA, Garza Co., Kerr WMA, Love Creek Preserve, Palo Duro Canyon State Park, Presidio Co., Scurry Co., Sierra Vieja Mountains, Van Horn Mountains, and Waddell Ranch in Texas; Cambria, California; Game Management Units near Lincoln National Forest, New Mexico; Thailand; and Tunisia.The ML analysis also produced a topology (not shown) that was nearly identical to the topology obtained from the BI analysis. The only difference between the BI and ML analyses involved nodal support for Subclade II‐A. In the ML analysis, Subclade II‐A was only moderately supported (BS = 65). Similar to the BI analysis, no support was obtained for the three haplotypes representing the 191 individual sequences forming Subclade II‐B and produced an unresolved polytomy. Bootstrap support values obtained from the ML analysis were superimposed onto the BI topology Figure .For the parsimony analysis, 379,908 equally, most parsimonious trees  were retrieved. A majority rule consensus tree was generated (not shown) that was similar in topology to the tree obtained in the BI analysis , there was no nodal support (BS < 65) for either Subclade A or B resulting in an unresolved polytomy containing seven haplotypes representing the 197 individual sequences. Bootstrap support values obtained from the parsimony analysis were superimposed onto the BI topology Figure . The 51 3.1.2n = 36) was obtained when identical sequences were removed. This final dataset then was used for all subsequent phylogenetic analyses, estimations of genetic distances, and other calculation of genetic indices. The three phylogenetic analyses  generated similar topologies; consequently, each analysis is discussed in detail below; however, only the topology obtained from the BI analysis is shown  of 70 aoudad samples was used to assign haplotype affiliation of all individuals to the sampled localities  of aoudad samples Figure . Clade IThe ML analysis of the D loop dataset also produced a topology (not shown) that was essentially identical to the topology obtained from the BI analysis. As in the BI analysis, strong support was recovered for Clades I and II  as was moderate support for Subclade II‐A and Subclade II‐B . Bootstrap support values obtained from the ML analysis were superimposed onto the BI topology  for the parsimony analysis of the D loop dataset, the heuristic search was terminated before the analysis could be completed. At the point of termination , 9,461,411 equally, most‐parsimonious trees  were retrieved. The parsimony analysis produced a topology (not shown) that was essentially identical to the topology of the BI analysis. Bootstrap support values obtained from the parsimony analysis were superimposed onto the BI topology , representing a total of 64 nucleotide substitutions, were detected in the D loop dataset Table . These i3.3Estimation of Kimura‐2 parameter Kimura,  genetic Genetic distances (see Table 3.4A. l. sahariensis) from Clade II (A. l. lervia) began approximately 2.38 mya. Radiation within A. l. sahariensis (Clade I) was estimated to have occurred at 0.99 mya. The major split between individuals assigned to Subclade II‐A and Subclade II‐B is estimated within the last 1.25 mya followed by radiation within Subclade II‐A and Subclade II‐B was estimated at 0.65 and 0.85 mya, respectively.A Molecular Clock Test  was 66, nucleotide diversity (π) was 0.01618, number of haplotypes (h) was 8, haplotype diversity (Hd) was 0.365, and Fu's test of neutrality was 42.287 for the entire cytb dataset (excluding sites with gaps or missing data) and within‐population indices are reported in Table s) was 116, nucleotide diversity (π) was 0.02296, number of haplotypes (h) was 5, haplotype diversity (Hd) was 0.182, and Fu's test of neutrality was 25.498 and within‐population indices are reported in Table p > .05) whereas Tajima's D for the D loop dataset was −1.3532 and was not significant (p = 0.068) using the program Arlequin . Given the similarity of the results of the cytb and D loop sequence analyses from all three studies, inferences from the smaller D loop dataset can be inferred using the larger cytb dataset. The PRNP gene provided negligible information relative to phylogenetic association and source‐stock determination; therefore, discussion was restricted solely to the basic description of the prion protein (PrP) genotype in aoudad.It is important to note that data used in this research were obtained from three independent studies: cytb only  produced a similar arrangement of individuals with two major clades identified . In addition, the genetic divergences of D loop observed between the two clades of aoudad (I and II) indicated a high level of genetic divergence compared to subspecies of Rupicapra  at 2.38 mya. Similar divergence estimates were reported for Ammotragus by Derouiche et al. (A. l. sahariensis (Clade I) was estimated to have occurred at 0.99 mya. The major split between individuals assigned to Subclade II‐A and Subclade II‐B was estimated to have occurred within the last 1.25 mya. Radiations within Subclade II‐A and Subclade II‐B were estimated at 0.65 and 0.85 mya, respectively. The divergence between Clades I and II and subsequent radiations within each clade were similar to that estimated for recognized subspecies of bovids  haplogroup was represented by individuals in Subclade II‐B. The second most common haplogroup represented by individuals in Clade I was restricted to populations in central and west‐central Texas. The third haplogroup (Subclade II‐A) was at a much lower frequency and was restricted to two localities in extreme western Texas.A. l. lervia, Cassinello, Based on the available information garnered from translocation records, state agencies, and scientific publications Barrett, , it appeAmmotragus lervia lervia. Second, although there are no translocation records  studbook indicated that individuals from their facility were obtained from the Khartoum Zoo, Sudan circa 1991 and therefore were most likely representative of the Kordofan subspecies, A. l. blainei . However, no samples clearly assignable to A. l. blainei were available for this study; therefore, the source‐stock at the Khartoum Zoo were not actually representative of A. l. blainei or that A. l. sahariensis and A. l. blainei are genetically identical based on the mtDNA dataset ; however, it most likely is either affiliated with A. l. lervia or one of the other subspecies that have yet to be genetically examined. Clearly, Clades I and II differ substantially in regard to levels of genetic divergence . However, when included with the D loop dataset herein, Haplotype Amle02 as well as Haplotype Amle03 grouped with samples from Algeria  contained in Clade II‐A. Additionally, Haplotype Amle01 was associated with Clade II‐B. This discrepancy suggests that Haplotype Amle04  most likely is representative of A. l. sahariensis as this haplotype groups with Clade I; whereas Haplotypes Amle01, Amle02, and Amle03 and other samples from Texas, are representative of A. l. lervia or one of the other subspecies that have yet to be genetically examined. These results imply that additional populations from Almería and surrounding regions should be examined to determine whether they are A. l. lervia or A. l. sahariensis or if they represent an undescribed genetic subspecies.Relative to Europe, Stipoljev et al.  postulat55.1n = 2) and Subclade II‐B (n = 1), respectively. Similarly, samples collected at this locality in 2020 indicated that both of these haplogroups persisted over the 40‐year interval . Third, the Dolan Falls Preserve population is highly skewed toward one haplogroup as only three individuals represented Clade I and 32 individuals were assigned to Subclade II‐B. Under this disproportional representation of haplotypes, over time, the haplogroup associated with Subclade II‐B may outcompete the haplogroup associated with Clade I, resulting in a local extinction of the second most common haplotype in United States. Fourth, if haplogroups are indicative of subspecies .Given the high level of genetic divergence ; Data curation (lead); Formal analysis (lead); Investigation (lead); Methodology (lead); Project administration (lead); Resources ; Validation ; Visualization ; Writing – original draft (lead); Writing – review & editing (lead). Rachael C. Wiedmeier: Data curation (supporting); Investigation (supporting); Resources (supporting); Writing  – review & editing (supporting). Emma K. Roberts: Data curation (supporting); Investigation (supporting); Methodology (supporting); Writing – review & editing (supporting). David R. Pipkin: Data curation (supporting); Resources (supporting); Writing – review & editing (supporting). Froylán Hernández: Data curation (supporting); Resources (supporting); Writing – review & editing (supporting). Joseph P. Bayouth: Data curation (supporting); Formal analysis (supporting); Writing – review & editing (supporting). Warren C. Conway: Conceptualization ; Data curation (supporting); Funding acquisition (lead); Project administration ; Resources ; Supervision ; Validation ; Visualization ; Writing – review & editing . Robert D Bradley: Conceptualization ; Data curation ; Formal analysis ; Funding acquisition ; Investigation ; Methodology ; Project administration ; Resources ; Software ; Supervision (lead); Validation ; Visualization (lead); Writing – original draft ; Writing – review & editing .Supplementary MaterialClick here for additional data file.


GLI1 silencing reduced the levels of MMP2/9, phosphorylated SMAD2/3, and procollagen-I, and knockdown or inhibition of GLI1 decreased fibrocyte formation and induced apoptosis of both fibrocytes and fibrocyte progenitors. Because Janus kinase (JAK)2-induced STAT3 is constitutively activated in MF and because STAT3 induces GLI1 expression, we sought to determine whether STAT3 activates GLI1 in MF fibrocytes. Imaging analysis detected phosphotyrosine STAT3 in MF patients’ BM fibrocytes, and transfection of fibrocytes with STAT3-siRNA or treatment with a JAK1/2 inhibitor ruxolitinib reduced GLI1 and MMP2/9 levels. Chromatin immunoprecipitation and a luciferase assay revealed that STAT3 induced the expression of the GLI1 gene in both MF BM fibrocytes and fibrocyte progenitors. Together, our data suggest that STAT3-activated GLI1 contributes to the induction of BM fibrosis in MF.Bone marrow (BM) fibrosis was thought to be induced exclusively by mesenchymal stromal cells (MSCs). However, we and others found that neoplastic fibrocytes induce BM fibrosis in myelofibrosis (MF). Because glioma-associated oncogene-1 (GLI1), an effector of the Hedgehog pathway, plays a role in the induction of BM fibrosis, we wondered whether GLI1 affects fibrocyte-induced BM fibrosis in MF. Multiplexed fluorescence immunohistochemistry analysis of MF patients’ BM detected high levels of GLI1 in MF fibrocytes compared to MSCs or normal fibrocytes. Immunostaining, RNA in situ hybridization, gene expression analysis, and western immunoblotting detected high levels of GLI1 and GLI1-induced matrix metalloproteases (MMP) 2 and 9 in MF patients BM-derived cultured fibrocytes. Similarly, MF patients’ BM-derived GLI1 Jak2V617F)-induced MF were fibrocytes  vector  and primers listed in Supplementary Table A modification of a previously described luciferase assay was used . In brie+ fibrocytes we used a segmentation and phenotype algorithm developed in the VIS software v2020.04 . The cultured cells were imaged using Andor Revolution XDi WD spinning disk confocal system with Zyla 4.2 sCMOS camera  and UPlanSApo 30XS silicone oil-immersion objective lens , and analyzed using Imaris v9.1.0 software . To quantitate cultured fibrocytes we imaged entire wells in duplicates using the Vectra Polaris imaging system and performed cell segmentation using an algorithm developed in the VIS software.Whole fluorescently labeled BM tissue sections were imaged using the Vectra Polaris imaging system  with a spectral library built using single-stained and unstained reference slides. Images were spectrally unmixed and single-cell GLI1 expression was quantitated using a tissue- and cell-segmentation algorithm developed in the inForm software v2.2.1 (Akoya Biosciences). To quantitate GLI1t-test, as appropriate. Post hoc multiple comparisons were performed using Tukey’s or Dunn’s method. Calculations were performed and data were visualized using GraphPad Prism software v9.0.0 . Data within each group met test assumptions of normality and equal variances. All tests were performed two-sided and a P value of <0.05 was considered statistically significant.The statistical significance of the difference between groups was assessed using the one-way ANOVA, the repeated-measures ANOVA, the Mann–Whitney test, or the paired Supplementary material 2Supplementary material 1