Despite multiple conservation efforts of the Mexican government, the leatherback turtle is at serious risk of extinction. In this study, we investigated the possible presence of a genetic bottleneck that could prevent the recovery of this species and compared these findings with those of the olive ridley turtle, which is in true recovery. Our results confirmed that a demographic change occurred in the past and the presence of two different leatherback turtle lineages that diverged approximately 13.5 million years ago. Local ecological knowledge (LEK) also described the presence of these two lineages and warned that one is at higher risk of extinction than the other. Genetic analysis confirmed 124 mutations between the two lineages, and much lower genetic diversity in one lineage than the other. Our study highlights and substantiates the power of mixing LEK, environmental history, and genetics to better understand conservation challenges of highly threatened species such as the leatherback turtle. Moreover, we report a new lineage of the leatherback turtle which may represent a distinct species. Future studies should focus on morphological, ecological, biogeographical, evolutionary and conservation perspectives for the analysis of the new lineage. Dermochelys coriacea Vandelli, 1761) populations have been in sharp decline for almost three decades, and the species is currently on the verge of extinction2. Over recent decades, key nesting sites in the Mexican Pacific have been identified and heavily protected to prevent poaching of both the eggs and nesting females, and laid eggs are often moved to protected hatcheries3.Despite multiple and ongoing efforts of the Mexican and Costa Rican governments in the eastern tropical Pacific, leatherback turtle  call into question whether the barrier to the recovery of Mexican leatherback turtle populations is in fact oceanic fishing activities.In addition to the ever-present threats to nesting females and eggs on beaches, mortality due to high rates of bycatch in the ocean, which mainly occurs as a result of entanglement in longlines meant for swordfishLepidochelys olivacea, Eschscholtz 1829) from the beaches of Oaxaca  has been proven to be a powerful tool to speed our understanding of species, ecological life histories, and population trajectories, especially in the marine realm17. In addition, population genetics has proven to be a powerful tool to uncover some biological and ecological information, particularly for rare and endangered species20. Although combining genetic information with local ecological knowledge has been an approach rarely used in marine science21, it appears to have great potential to better understand barriers that are limiting the recovery of marine animals threatened for their conservation in moderns times. Here we propose to undertake this combined approach to better understand the demographic trajectories for the leatherback and olive ridley turtles.During the nineteenth century, two different species of leatherback turtles were considered to be distributed in the eastern Pacific OceanIn our study, we collected local ecological knowledge to identify threats that could be preventing the recovery of the leatherback turtle that could have passed unnoticed in western literature. To our surprise, the first barrier identified by local people regarding the conservation of this species was that modern ecological monitoring did not recognize what was pretty obvious to local people—that there were two different types of leatherback turtle Table  and that24. A total of 124 and 125 polymorphic sites were found in Barra de la Cruz and Cahuitán, respectively. A total of five haplotypes were found for both localities (three in Cahuitán and four in Barra de la Cruz). The nucleotide diversity was higher in Cahuitán (π = 0.091) than in Barra de la Cruz (π = 0.0748), although the same tendency was observed with haplotype diversity (Hd) and Watterson’s theta  showed diversity levels similar to those of the olive ridley turtle  and were similar to those of the olive ridley turtle Table . Lineagele Table .π) was 0.00109 while θW was 0.00166. A total of six haplotypes were found with an Hd value of 0.584. We found a higher level of diversity in olive ridley turtles than in lineage A of leatherback turtles but lower than in lineage B based on the Hd and π data , lineage B (0.457), or the olive ridley turtle (− 0.901).Fu’s The haplotype network for the leatherback turtle showed two lineages separated by 124 mutational steps. One lineage was made up of H1 and H5 while the other was made up by H2, H3, and H4 ; this haplotype was found in both beaches and in the Mexican Turtle Center. The second-most frequent haplotype was H1 (N = 4), which was also found in these three locations. Haplotype H4 (N = 3) and haplotypes H3 (N = 2) and H5 (N = 2) were only found in Escobilla. Haplotype H6 (N = 1) was private to Morro Ayuta  between lineages A and B of Dermochelys coriacea found in the present study; 7.95 Mya  between N. depressus and C. mydas; and 4.75 Mya  between L. kempii and L. olivacea.The independent runs made with Beast2 exhibited posterior values of − 2756.42 and − 2756.09 with ESS values of each parameter > 350. This suggests that the runs converged and that the results are robust. Also, the Maximum Clade Credibility tree  reported and described having seen a turtle that was distinct but similar to the leatherback turtle Table . The intIn the present study, we used a LEK and population genetics approach to better understand the demographic trajectories of the leatherback and the olive ridley turtles. Our combined approach allowed uncovering an undescribed lineage for the leatherback turtle, which may be in high risk of extinction. Thus, our study highlights the importance of considering local knowledge in the study of biodiversity and corroborating this information with appropriate methods.π, and Hd for L. olivacea compared to those of other studies25. In contrast, we found high genetic diversity levels for D. coriacea. This was not expected given that the population of D. coriacea is currently decreasing while that of L. olivacea is increasing8. The values of nucleotide diversity for the leatherback turtle (π = 0.07917) were 72 times higher than the value for L. olivacea (π = 0.00109) and higher than those reported by Dutton et al.22 (π = 0.0015), Vargas et al.24 (π = 0.0011), and Dutton et al.23 (π = 0.0006–0.0032). These high levels of genetic diversity were clearly caused by nucleotide differentiation between the two groups of haplotypes (124 polymorphic sites). When we considered each lineage separately, the nucleotide diversity values were consistent with those obtained for the olive ridley turtle and with those that have been previously reported for the leatherback24.We registered lower levels of genetic diversity in terms of polymorphic sites, π = 0.714) is very similar to the value reported by Dutton et al.22 (π = 0.712) for Mexiquillo beach in the Mexican Pacific. The nucleotide diversity of Lineage A (0.00034) was an order of magnitude lower than that of lineage B (0.00176), which concurs with what has been reported previously24. These values suggest that lineage A may be less diverse than lineage B, and thus individuals from lineage A may be more vulnerable than leatherback turtles from lineage B.The haplotype diversity value for lineage B  and π (0.00109) for L. olivacea are lower than those that have been previously reported in other locations of the Mexican Pacific 25. The lower levels of genetic diversity found in this study could be related to the exploitation of this species in the region. Even if the populations of this species are currently increasing, the recovery of genetic diversity after a bottleneck would depend on the magnitude and duration of a bottleneck26. As marine turtles have long generation times, their effective population size, and their genetic variation, would take a longer time to recover.The high and surprising genetic diversity of Fs values for leatherback lineage A (0.177) and lineage B (0.671) are consistent with a signal from a population bottleneck. In contrast, the olive ridley turtle value (− 1.75) agrees with those expected for a species experiencing a recent population expansion. and is further supported by a starlike shape in the haplotype network. Tajima’s D on the other hand was not significant for either leatherback lineage or for the olive ridley turtle. Nevertheless, Tajima’s D is not as sensitive to demographic changes as Fu’s Fs, thus a positive value (even if not significant) for both leatherback lineages supports the existence of a historic bottleneck, while the negative value for the olive ridley turtle suggests population expansion27.Fu’s In the case of the haplotype networks for the leatherback turtle, large genetic distance was observed between lineage A (reported for the first time in this study) and lineage B . The molecular clock analysis also revealed that the haplotypes of lineage B in the eastern Pacific are closely related to those found in the Italian (Ity), South Korean, and Colombian Caribbean (CC) areas, whereas lineage A has evolved as an independent group  and S. schlegelii, although no descriptions were given for S. schlegelii. Later, one S. schlegelii specimen was included in a herpetological list of the Isthmus of Tehuantepec11, which is in the same region as our study area.In his “Natural History of the Reptiles of Bermuda,” Garman12 proposed a new species of Sphargis nominated S. angusta. The specimen was collected near Tocopilla, Chile. The description of this new species indicated that the shell was narrower and darker than that of S. coriacea with spots that were yellowish or hardly visible. The description also indicates that the hind flippers were proportionally smaller than those of S. coriacea while the neck and tail were longer and the back flippers pointier. The reptile collection of the National Museum of Natural History of Chile has embalmed specimens of D. angusta and D. coriacea that were preserved in 191629. Until the first half of the twentieth century, D. schlegelii was described as a valid species31.In 1899, Philippi13 compiled taxonomic descriptions of both species and considered that there was not enough information to continue referring to them as separate species and thus advised that they should collectively be classified as D. coriacea. More recently, Eckert et al.32 noticed the taxonomic ambiguities that have arisen since 1884 and concluded that no author had made a sufficiently valid claim to either confirm or reject the existence of two different species or subspecies of leatherback turtle. Some authors argue that the morphological differences may have been the result of adaptations to environmental conditions or simple variations between populations32. Future research should search for preserved specimens in herpetological scientific collections, so that morphological and genetic data can be correlated, and to confirm the taxonomic status of this taxon.Pritchard12. Thus, it is urgent to conduct a taxonomic reassessment of the leatherback turtle that includes both population genetics analyses and morphological descriptions of each specimen. Sufficiently robust sampling efforts are needed that cover large areas to determine whether we are currently dealing with one highly endangered leatherback turtle species or two different species. Nuclear genetic analyses are also needed to understand whether both groups have remained separate over 13.5 million years or there has been secondary contact or introgression.The results of the genetic diversity analysis, haplotype network, molecular clock analysis, and interviews suggest the existence of two variants of leatherback turtles. However, it is still too early to define if they are two separate species. The morphological characteristics described by local people of both variants agree with older accounts that describe what were considered to be two different species of leatherback turtle in the past Table 10–12. Th5 potentially poses to the entire eastern tropical Pacific leatherback turtle population appears to be only one component of a complex network of factors that currently influence the potential recovery of these sea turtle lineages. The results of our study clearly highlight the need to invest more time and resources to study the ecology, demography, evolutionary history and population genetics of these sea turtles. With better information, we will be able to infer the evolutionary trajectories of both leatherback lineages with more confidence and the potential capacity of the olive ridley turtle to increase its population size even with apparently low levels of genetic diversity.Regarding current population trends and population recovery of these turtles, the threat that the Chilean swordfish fishery35 along with local ecological knowledge36. However, combining local ecological knowledge with genetics appears to be an untapped tool to better understand the dynamics that are impeding the recovery of highly endangered marine animals such as marine turtles. However, global studies have also found similar agreement. For example, Dao et al.21 identified that local people understand the potential genetic connectivity of marine species through ocean currents.Conservation genetics/genomics have been largely recognized as a key tool to improve conservation strategiesOur study shows, how genetic analyses coupled with LEK, can help us speed our understanding of the complex drivers that are behind the recovery of vulnerable species such as the leatherback turtle in the eastern Pacific. We are aware there is still work to be done to determine if there are indeed two separate species of leatherback turtle. Nonetheless, the information collected in our study indicates that the conservation strategies aimed at the recovery of this species in the Eastern Pacific Ocean will benefit from understanding its evolutionary trajectory and the observations made by local people.37. For this study, we collected tissue samples from turtles in four beaches of Oaxaca: Cahuitán, Barra de la Cruz, Escobilla, and Morro Ayuta . All ecological methods were performed in accordance with the relevant guidelines and regulations.All samples were collected following the rules and procedures established in the Mexican Norm (NOM-162-SEMARNAT-2012) to manage marine turtles in their nesting sites, which includes all ethical and conservation standards for handling sea turtle species considered to be at risk in Mexican waters. A sampling permit was expedited by El Colegio de la Frontera Sur (ECOSUR). Samples were stored at − 20 °C until DNA extraction. We used existing registration numbers to identify each female before sampling and to ensure that no female was sampled twice.The skin of each turtle was cleaned with an antibenzil solution before and after taking each sample. We used a 6-mm diameter biopsy punch to collect each tissue sample from the hind flipper. Tissue samples were stored in 1.5 ml microcentrifuge tubes with 90% alcohol and transported to the genetics laboratory of 38. The mtDNA control region (D-loop) was amplified via PCR with primers H950g (5′-GTCTCGGATTTAGGGGTTTG-3′) and LCM15382 (5′-GCTTAACCCTAAAGCATTGG-3′) for the leatherback turtle and H950 (5′-GTCTCGGATTTAGGGGTTTG-3′) and LTEi9 (5′-AGCGAATAATCAAAAGAGAAGG-3′) for the olive ridley turtle that were designed by Abreu-Grobois et al.39. For the PCR, the tubes were prepared with 12 μl of Taq PCR Master Mix , 10 μl of nuclease free water, 2 μl of each primer at 10 nM, and 2 μl of DNA (~ 20 ng/μl). We added nuclease free water instead of DNA to one of the reactions in each batch as a negative control.DNA extraction was performed using the phenol:chloroform: isoamyl alcohol method23 with an initial denaturation step at 94 °C for 5 min, followed by 35 cycles with denaturation at 94 °C for 30 s, annealing at 53 °C for 1 min, extension at 72 °C for 2 min, a final extension step at 72 °C for 10 min, and hold at 4 °C ad infinitum. For the olive ridley turtle samples, the program consisted of 94 °C for 5 min, followed by 35 cycles with denaturation at 94 °C for 45 s, annealing at 52 °C for 45 s, extension at 72 °C for 45 s, a final extension step at 72 °C for 5 min, and hold at 4 °C40. DNA extraction and amplification success were assessed by agarose gel electrophoresis and visualized with ethidium bromide.We modified the amplification protocol proposed by Dutton et al.In all, 24 leatherback turtle samples (10 from Cahuitán and 14 from Barra de la Cruz), and 27 olive ridley turtle samples  were successfully amplified. Nucleotide sequences were obtained from PCR products, using the forward primer, through the Sanger method in a ABI 3730xl DNA analyzer  at Macrogen Korea (dna.macrogen.com).41. Sequence lengths were 732 and 625 bp for leatherback and olive ridley turtles, respectively. Basic genetic diversity measures were calculated, namely the number of haplotypes (h), haplotype diversity (Hd), nucleotide diversity (π), and Watterson’s theta (θW). We also evaluated neutrality with Fu’s Fs and Tajima’s D in DnaSP v. 642 to infer the historical demography for each species; negative values suggest population expansion while positive values suggest a demographic bottleneck27.All sequences were evaluated for quality, and those with poor quality were discarded. Subsequently, a BLAST analysis was conducted to confirm that the correct region was amplified. The sequences were aligned and edited with BioEdit43. To estimate the divergence time and the phylogenetic relationship of the new lineage found in this study, we obtained the substitution model that best fit with our data with jModelTest44 based on the Akaike criterion. The model with the best fit was HKY.The haplotype network was obtained with Network 10 using the median joining method45. We downloaded three more sequences from GenBank for the leatherback turtle with distinct geographic origins in South Korea , Colombia , and Italy . We also included sequences for loggerhead , green , hawksbill , Kemp’s ridley , and flatback  turtles in this analysis. For the olive ridley turtle, we used the most frequent haplotype obtained from this study. As an external group, we used the terrestrial common snapping turtle  and alligator snapping turtle , as suggested by Duchene et al.20. These sequences were also obtained from GenBank.We conducted a molecular clock analysis to estimate the divergence time with our sequence data. This analysis was carried out with Beast247 and used four calibration points based on the fossil record, which were previously used to date the phylogeny of sea turtles: Cheloniidae and Dermochelyidae families (100–150 Mya49), Cheloniidae species (50–75 Mya50), Caretta and Lepidochelys (12–20 Mya51), and Lepidochelys (4.5–5 Mya53). All calibration points were set with normal distributions, and we performed preliminary runs to adjust the parameters of the model. Finally, we conducted two independent runs with 100 million MCMC chains and a 10% burnin. We implemented a relaxed log normal clock model and a calibrated Yule model. The results of both runs were combined with LogCombiner45, and we obtained the MCC with median height with TreeAnotator45. The MCC was visualized with Figtree v. 1.4.4 (available at http://tree.bio.ed.ac.uk/software/figtree/).To define calibration points, we followed the methodology of Bowen et al.Comité de Ética en la Investigación of ECOSUR on 24 February 2021. All social methods were performed in accordance with the relevant guidelines and regulations.We conducted interviews with local people that were actively involved in sea turtle conservation or exploitation or that had been in the past. When our first interviewee mentioned a leatherback turtle with differences in size, color, and egg characteristics, we decided to interview different local experts who would have collected eggs before the activity became illegal. We asked these individuals if they were familiar with the variety of leatherback turtle described by the first interviewee. In all, we conducted nine interviews in the communities of Cahuitán, Chacahua, Mazunte, Barra de la Cruz, and Río Seco /Morro Ayuta beach in Oaxaca and Paredón in Chiapas Fig. . MethodsAll the interviews were conducted in the homes of the interviewees or in landing sites. Prior to each interview, we clearly explained the objective of our research. In addition, we ensured the anonymity of any information collected, and informed consent was obtained from all interviewees before the interview formally started. We included questions to assess changes in leatherback abundance and questions regarding the importance of beaches and the leatherback in the dietary habits of local people. The interviews followed a semi-structured format (Appendix Supplementary Information.


Clematis acerifolia (C. acerifolia) plants belonging to nine populations were collected from the Taihang Mountains in Beijing, Hebei, and Henan. Twenty-nine simple sequence repeats (SSR) markers developed based on RAD-seq data were used to analyze the genetic diversity and population structure of C. acerifolia. The mean PIC value for all markers was 0.2910, indicating all SSR markers showed a moderate degree of polymorphism. The expected heterozygosity of the whole populations was 0.3483, indicating the genetic diversity of both C. acerifolia var. elobata and C. acerifolia were low. The expected heterozygosity of C. acerifolia var. elobata (He = 0.2800) was higher than that of C. acerifolia (He = 0.2614). Genetic structure analysis and principal coordinate analysis demonstrated that C. acerifolia and C. acerifolia var. elobata showed great genetic differences. Molecular variance analysis (AMOVA) demonstrated that within-population genetic variation (68.31%) was the main contributor to the variation of the C. acerifolia populations. Conclusively, C. acerifolia var. elobata had higher genetic diversity than C. acerifolia, and there are significant genetic differences between C. acerifolia and C. acerifolia var. elobata, and small genetic variations within the C. acerifolia populations. Our results provide a scientific and rational basis for the conservation of C. acerifolia and provide a reference for the conservation of other cliff plants.Investigating the genetic diversity and population structure is important in conserving narrowly distributed plants. In this study, 90  Clematis acerifolia Maxim. (C. acerifolia) belongs to the genus Clematis L. and the family Ranunculaceae, and is a diploid shrub with white or pink petals [Fig 1) in the Mentougou and Fangshan districts of Beijing, with sporadic distributions in Henan [C. acerifolia var. elobata is a varietas of C. acerifolia, and is mainly distributed in the Taihang mountains in Henan province. C. acerifolia is one of the rare and precious plant species that grow on the cliffs of Taihang mountains. Their vertical or nearly vertical growth habit limits water, nutrient, and soil availability, leading to the situation that such plants are usually narrowly distributed, fragmented, and susceptible to environmental influence [C. acerifolia has been listed as a second-class protected plant species in China in 2021. Research on the genetic diversity of C. acerifolia would provide suggestions on the conservation of this species, maintenance of ecological diversity, and development of new varieties for garden landscaping.k petals ,2. It flin Henan  and Hebein Henan . C. acernfluence –7. Thesenfluence . As a raGenetic diversity is a key factor for the survival of a species , playingClematis L. For example, randomly amplified polymorphic DNA (RAPD) and single nucleotide polymorphisms (SNPs) were used for identifying the hybrid origin of progenies derived by crossing Clematis tubulosa with Clematis brevicaudata [Clematis L. [Clematis species [Clematis finetiana, Clematis chinensis, and Clematis heracleifolia [Genetic diversity analysis providing suggestions on species conservation and management . As an eicaudata . Inter-smatis L. –28. Simpmatis L. ,29. SSR  species , and perleifolia –33. Restleifolia –36.C. acerifolia. Allozyme marker analysis showed that C. acerifolia had low genetic diversity in Beijing and Hebei, and revealed that heterozygosity was not significantly associated with adaptation [C. acerifolia var. elobata in the genus Clematis L., and found that C. acerifolia was relatively isolated within the genus Clematis L. [C. acerifolia.Several studies have investigated the genetic diversity of aptation ,38. DNA matis L. ,39. ConsC. acerifolia and C. acerifolia var. elobata sampled from different locations along the Taihang Mountains, (2) use SSR markers to distinguish C. acerifolia from C. acerifolia var. elobata, and (3) propose effective conservation measures for C. acerifolia based on the results of genetic diversity analysis.The aim of this study was to (1) develop SSR markers that can assess the genetic diversity and genetic structure of C.acerifolia only grows in the crevices of cliffs in the Taihang Mountains, showing an extremely narrow and fragmented distribution. Its core distributions are in the south-western part of Beijing and a small part of Hebei (close to Beijing). The C. acerifolia var. elobata is mainly distributed in the south of Taihang Mountains in Jiaozuo, Henan, and was chosen as the southernmost representative population. Nine C. acerifolia populations, each including ten individuals, were obtained in the study (C. acerifolia var. elobata) was sampled from Jiaozuo, Henan. In order to avoid taking samples from the same crevice, individual samples were taken from distances of at least 3 meters. Samples were collected in 2019, and C. acerifolia was recognized as a protected plant in 2021. Therefore, there was no need to get permission from any owners to take the samples. Specific information, including location, region, numbering, longitude, latitude, and altitude, is presented in -1.he study . PopulatWe prepared the library for RAD sequencing according to a previous study . Brieflyhttps://bioinfo.ut.ee/primer3-0.4.0/) based on a length of PCR amplifying product ≤ 200 bp. Primer synthesis was performed by the Shanghai Biotechnology Company.Sequencing data preparation was performed according to the method used in a previous study . Briefly2O. PCR was conducted at 94°C for 5 min for pre-denaturation, followed by 30 cycles at 94°C for 30 s, with annealing at 58–62°C for 30 s, elongation at 72°C for 30 s, and finally an extension at 72°C for 7 min. Polyacrylamide gel electrophoresis was performed as stated in a previous study [Each 10-μL PCR reaction contained 5 μLof Taq Master Mix , 1 μL of DNA solution, 0.3 μL of reverser primer, 0.3 μL forward primer, and 3.4 μLof ddHus study  to displFIX) was calculated as (He-Ho)/He. F-statistics [FIS, FIT, and FST were calculated using POPGENE Version 1.32. At the same time, we counted the numbers of alleles, polymorphic alleles, common alleles and rare alleles of SSR primers.We firstly analyze the genetic diversity using the 29 SSR markers. The polymorphic information content (PIC) of each marker was computed with PowerMarkerV3.25 , whereasatistics  includinFST and gene flow (Nm) calculation, and principal coordinate analysis (PCoA) were performed using GenALEx6.5 [We also analyzed the population structure of the samples. Nei’s genetic distance  and the nALEx6.5 . MoleculnALEx6.5 . The gennALEx6.5 ,52.After removing low-quality sequences, ambiguous barcodes and orphan paired-end reads, 161.49 GB of sequences containing 1914 million clean reads were obtained. The average Q30 ratio was 92.9%, ranging from 91.7% to 93.2%, indicating high-quality clean data. The average guanine-cytosine (GC) contents were 39.1% . The lenTwo samples from each location  were used for screening the polymorphisms of the 1600 SSR primers. To screen reliable SSR markers, we required that at least one of the six samples had a different band from the other five samples. We selected 29 pairs of markers in the end. Among the 29 SSR markers, the number of markers that had 4, 5, 6, 7, 10, and 13 repeats of tandem sequences were 21, 2, 3, 1, 1, and 1 respectively, and the number of markers that contain 2, 3, and 5 nucleotides in their tandem repeats were 25, 3, and 1, respectively. “AT” was the most common tandem repeat, followed by “TA”, “TC”, “GA”, “AG”, “CA”, “CT”, and “TG” . The lenFIS) in each population was less than 0 (-0.3442) and that the mean value of inbreeding coefficient (FIT) in the whole population was greater than 0 (0.0851), indicating that these loci were mostly heterozygous in each population, and homozygous across the whole population. The FST variation ranged from 0.064 to 1, with a mean value of 0.3194, indicating a greater degree of genetic diversity across populations.PIC is an important indicator of marker quality and varied from 0.0994 to 0.4888, with a mean value of 0.2910. The PIC values of 12 SSRs were in the range of 0–0.25, and those of 17 SSRs were in the range of 0.25–0.50 . F-statiFIX was less than 0, indicating that there were more heterozygous loci in these populations. PPL ranged from 51.72–65.52%, with a mean value of 58.62%, and Shannon’s diversity index varied from 0.2972 to 0.4011, indicating little variation in the genetic diversity of these populations.Seven parameters were used to measure the genetic diversity: number of observed alleles, number of effective alleles, observed heterozygosity, expected heterozygosity, PPL, Shannon’s diversity index, and fixation index. The results demonstrated  that theC. acerifolia var. elobata population, suggesting that the genetic diversity of C. acerifolia var. elobata. is higher than that of C. acerifolia. of the C. acerifolia populations in Beijing and Hebei, populations 1  and 4  had higher expected heterozygosity, Shannon’s index, and the number of effective alleles, suggesting higher genetic diversity for these populations. The value for Shannon’s diversity index, expected heterozygosity, and PPL of population 2 and population 3 were similar, and both had comparable levels of genetic diversity, which were lower than those of populations 1 and 4. The lowest number of observed alleles and PPL were found in population 7 (Jingxi Ancient Road), and the observed heterozygosity, expected heterozygosity, and Shannon’s diversity index were low in populations 6 and 8, implying low genetic diversity in populations 6, 7, and 8. The genetic diversity in population 7 was higher than that of populations 6 and 8. Population 9, which is located in Xinghuang Village, had the highest number of observed alleles, suggesting that a higher number of genotypes may have been retained in this population.Comparison of the genetic diversity indices among different populations indicated that these genetic parameters were highest in the C. acerifolia populations from different regions were analyzed at both the species and the population level  [Camellia huana, He = 0.466) [Rosa odorata var. gigantea (He = 0.569) [Taihangia rupestris var. ciliata  [Opisthopappus taihangensis  [C. acerifolia var. elabata and C. acerifolia.Compared with widely distributed plants, endemic species that are narrowly distributed in specialized and limited environments have relatively small populations and are susceptible to fragmentation due to external disturbances. Therefore, it is challenging to conserve the narrowly distributed endemic plants. Compared to other narrowly distributed plants in China, y Tables  and 4, w= 0.395) , Guizhou= 0.466) , and Ros= 0.569)  all show= 0.569) . Taihang= 0.787)  and Opis= 0.472) , which aT. rupestris and Oxyria sinensis [C. acerifolia populations with strongly branched rhizomes can grow along stone crevices [The living environment and reproduction of plants are closely related to the genetic structure of a population. As a habitat for many rare plants, cliffs are a special habitat with fragmented distribution and high heterogeneity . Cliff psinensis , C. acercrevices . Small ncrevices .C. acerifolia population observed in this study (92.19%) was higher than that across populations (7.81%). This intra-genetic variation is derived from selection due to the cliff environment and is complemented by new sporadic seedlings. First, the environmental selection of particular ecotypes contributes to genetic diversity. During the long evolutionary process, plants adapt to and are selected by environments, especially species in heterogeneous microenvironments, where competition can be highly aggressive [C. acerifolia seedlings is infrequent, the occasional and irregular complementation with seedlings can also provide a source of genetic variation and prevent genetic drift, as found by Araki [Genetic variation can be divided into inter- and intra-population differences. The variation within the gressive . Plants ations 7.1%. This by Araki , Zhao [6by Araki , Watkinsby Araki .C. acerifolia seeds spread mainly by gravity, while spider webs on the cliffs retain seeds in the same area [C. acerifolia seeds are more likely to spread within their original or adjacent populations, with a smaller probability of dispersal between Beijing and Henan, which are at a greater distance. The biological mechanism by which pollination takes place in C. acerifolia remains unclear. Flower-visiting organisms such as bees and beetles observed in the wild may be potentially pollinators for this plant. The large genetic variation between C. acerifolia and C. acerifolia var. elobata suggests that seed or pollen flow transmission is small, and the low genetic variation among the populations of C. acerifolia indicates that seed or pollen transmission is high.Genetic variation among populations is largely dependent on gene flow, with seeds and pollen acting as vehicles. Studies have shown that ame area . The seeame area . The taiC. acerifolia in Beijing and Hebei. The results of the present study using SSR molecular markers demonstrated low genetic diversity for C. acerifolia. The degree of genetic diversity obtained in the two studies differs. According to the FST values, the results of the former study showed large genetic differences between the C. acerifolia populations in Beijing and Hebei, while the present study demonstrated only a moderate degree of genetic variation. Such differences may be caused by temporal changes in the genetic diversity of the populations, the use of different molecular markers, and variation in the samples from various sampling sites. Inbreeding decline is a problem commonly faced by cliff plants and is closely related to the production of homozygous offsprings. Contrary to the previous assessments of inbreeding in C. acerifolia populations, the inbreeding coefficient (FIS) was less than 0 and the Ho is greater than He in this study, indicating a more heterozygous state in these loci. The possible reason for this situation is the harsh environment of cliff plants, with less interspecific competition and more intraspecific competition [A related study in 2005 used allozyme to analyze the genetic diversity of petition . Relatedpetition . In contC. acerifolia plants may also live for long periods. Habitat changes, intra-population competition, and random plant mortality may be responsible for the reduced genetic diversity of this population.Previous studies have shown that the life span of cliff plants is generally long, probably ranging from 45 to 324 years . The indC. acerfolia in this study. First, attention should be paid to protecting habitats. C. acerifolia has a unique habitat and can only grow in the cliff environments of the Taihang Mountains, thus requiring protection via actions such as lowering human interference and minimizing the reduction of population genetic diversity due to habitat destruction. Second, in situ conservation is important to protect existing plants. C. acerifolia populations are renewing and evloving slowly, and the individuals live for long periods; therefore, in situ conservation can maintain or delay decreases in genetic diversity. At the same time, the conservation of wild seedlings is particularly critical because of their contribution to genetic diversity. Thirdly, various methods are required to increase the number of plants. A seedling establishment can be maintained by artificially raising seedlings, which can then be returned to their native habitat. It may also be possible to place seeds directly into the crevices of cliffs. The survey also showed that sheep climb the cliff and eat the plants; therefore, grazing should be prohibited in areas where the plant is concentrated.The following conservation measures are recommended based on the genetic diversity assessment and cluster analysis of C. acerifolia and C. acerifolia var. elobata are genetically different and that each is an important component of the total genetic diversity. The Beijing and Hebei groups have lower genetic diversity and are thus more vulnerable, and the conservation of these groups should be prioritized. Genetic diversity should be maximized as much as possible during conservation. Populations 4  and 1  had high genetic diversity, and populations 6 , 7 , and 8  had low genetic diversity. Therefore, for populations in Nanshiyang Grand Canyon, Jingxi Ancient Road, and Xiayun Hill, measures should be taken to protect the habitats and propagate seedlings for re-entry into the ecosystem. Population 9 in Xinghuang Village has the highest number of observed alleles, and conserving this species in this location can significantly protect the diversity of all genotypes. At the same time, introducing populations with distinct genetic characteristics from the south of Taihang mountains (such as Henan) to Beijing is an advisable way to improve the genetic diversity of C.acerifolia. Artificial crossing of Henan and Beijing populations will accelerate the improvement of genetic diversity in Beijing.Lastly, the results of this study indicate that the C. acerifolia and C. acerifolia var. elobata respectively at the DNA level, and evaluated the genetic diversity of C. acerifolia populations sampled from different locations. Our results showed that the nine populations had low genetic diversity and that C. acerifolia var. elobata had higher genetic diversity than C. acerifolia. The molecular markers developed from the nuclear genome enabled a more comprehensive evaluation of genetic differences.In this study, we used SSR markers to genotype plants belonging to S1 FigThe numbers under the gel represent the genotype numbering.(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.


Jacobaea. There are many naturally occurring interspecific hybrids in this genus, including those between Jacobaea vulgaris and its relatives. Although Jacobaea erucifolia and J. vulgaris often co‐occur and are considered closely related, apart from the few reports of German botanists on the existence of such hybrids, there is no information on research confirming hybridization between them. Morphologically intermediate individuals, found in the sympatric distributions of J. vulgaris and J. erucifolia, were hypothesized to be their hybrids. Two molecular marker systems (nuclear and chloroplast DNA markers) were employed to test this hypothesis and characterize putative hybrids. Nuclear and chloroplast DNA sequencing results and taxon‐specific amplified fragment length polymorphism (AFLP) fragment distribution analysis confirmed the hybrid nature of all 25 putative hybrids. The AFLP patterns of most hybrids demonstrated a closer relationship to J. erucifolia, suggesting frequent backcrossing. Moreover, they showed that several individuals previously described as pure were probably also of hybrid origin, backcrosses to J. erucifolia and J. vulgaris. This study provides the first molecular confirmation that natural hybrids between J. vulgaris and J. erucifolia occur in Poland. Hybridization appeared to be bidirectional but asymmetrical with J. vulgaris as the usual maternal parent.Progressive changes in the environment are related to modifications of the habitat. Introducing exotic species, and interbreeding between species can lead to processes that in the case of rare species or small populations threatens their integrity. Given the declining trends of many populations due to increased hybridization, early recognition of hybrids becomes important in conservation management. Natural hybridization is prevalent in  J. vulgaris and J. erucifolia, were hypothesized to be their hybrids. Two molecular marker systems were employed to test this hypothesis and characterize putative hybrids. This study provides the first molecular confirmation that natural hybrids between J. vulgaris and J. erucifolia occur in Poland. Hybridization appeared asymmetrical and bidirectional with J. vulgaris as the usual maternal parent.Morphologically intermediate individuals, found in the sympatric distributions of  HybridiJuglans hopeiensis, Athrotaxis laxifolia, and Actinidia zhejiangensis) actually consist mainly of first‐generation hybrids and advanced generation backcrosses. Despite their distinct morphological traits and long recurrent hybrid origination, they do not initiate speciation.  as well as solutions  to global change challenges  Rottenst (=S. × whitwellianus Murr) in Austria and Liechtenstein  habitats throughout Europe and western Asia  represent subsp. erucifolia. The remaining populations found in the southern part of the country are currently classified as subspecies tenuifolia , with the leaves with narrow lobes (as J. erucifolia) but not tucked up at the edges and covered with thicker hair layer. The underground part was a thick, horizontal root not resembling a typical rhizome of J. erucifolia. The individuals were found in the typical habitats for both species—abandoned farmlands and xerothermic grasslands.J. vulgaris and J. erucifolia, are often confused , clustering‐based methods (fingerprinting – multilocus genotype data) such as amplified fragment length polymorphism  Is it possible that native species are being displaced by hybrids, which threatens the extinction of pure parental genomes? (2) What consequences may result from the spread of hybrid individuals in the future? and (3) In the light of the reports on the impact of hybridization on biodiversity discussed above, can we expect similar consequences  in the case of a rare taxon such as J. erucifolia in Poland? We will attempt at least a preliminary assessment of these threats. We hope to get a more complete answer once the ongoing studies on other J. erucifolia populations are completed.Initially, in this study, we aimed to test the hypothesis that specimens characterized by intermediate morphology are natural hybrids of two closely related 22.1J. vulgaris, 20 specimens of J. erucifolia, and 25 putative interspecific hybrids of J. vulgaris × J. erucifolia. All putative hybrids were collected from the two localities: 15 individuals from Mięćmierz (Mm1–Mm15) and 10 individuals from Pęczelice (Pc1–Pc10). Material of both supposed parental species came from various regions of Poland. Specimens of J. vulgaris were collected in Golęcin  and the remaining samples  near Puławy, relatively close to the occurrence of hybrids. Because of the genetic variation within populations of Jacobaea species, at least in the case of J. erucifolia seems to be higher than between populations , four individuals were taken from each of the four presently existing J. erucifolia subsp. erucifolia populations in the Western Pomerania. In addition, four samples were collected from individuals previously described as J. erucifolia subsp. tenuifolia, in the region of origin of the hybrids (near Pęczelice); thus, a greater number of different populations will allow for covering most of the intraspecific variation. For details concerning examined specimens, see Table Fresh plant material for molecular studies was collected in natural populations from 19 specimens of 2.2Young leaves collected in the field were dried with silica gel. The total genomic DNA of all 64 specimens was extracted following the method described by Doyle and Doyle . The con2.3J. vulgaris and pure J. erucifolia were initially tested with 15 different selective primer combinations. From this group, primer pairs producing bands that seemed to distinguish the two species were selected, and these primers were further screened over all individuals. For selective amplification, seven EcoRI/MseI (+3/+3) primer pairs  were tested to find specific diagnostic markers for each parental species. Each reaction was repeated at least two times (repeat runs were made to check for reproducibility). Amplification products were separated and detected on an ABI Prism 3500 capillary sequencer. GeneScan 600 LIZ‐labeled size standard (Applied Biosystems) was used for fragment sizing. The fluorescent AFLP patterns were evaluated using GeneMarker version 1.6 (Softgenetics LCC). Fragments ranging from 50 to 500 bp were automatically scored as present (1) or absent (0) and manually corrected after visual inspection of the electrophoretograms.Sixty‐four individuals representing two taxa and putative hybrids were included in the AFLP analysis. All sequences of primers and AFLP adapters were based on Vos et al. . Detaile2.4The entire ITS region  was amplified using the universal ITS1 and ITS4 primers developed by White et al. . Reactio2.5trnL (UAA) 3′ exon and trnF (GAA) was amplified using universal primers  described in Taberlet et al. (J. vulgaris × J. erucifolia were compared with those of its respective parents. The four different cpDNA sequences generated in this study have been deposited in GenBank (accession numbers: OP806269–OP806272).As the mode of inheritance of cpDNA in Senecioneae, according to Harris and Ingram  is strict et al. . PCR rea2.6The distribution of bands across taxa as well as the number of polymorphic bands, the average number of bands per primer, and bands shared among species were examined. Apart from monomorphic , polymorphic , group of unique bands which were present in at least one individual in a given taxon and not present in any other were also defined  dendrogram and phylogenetic network based on the Neighbor‐Net algorithm using the SplitsTree4 v.4.13.1 software , adjusted visually where needed and automatically aligned using the program MEGA 11  were visualized and analyzed using SnapGene Viewer 6.0.2. (33.1EcoACA/MseCAT) to 142 (EcoAGG/MseCAG) with an average of 131.4 bands. However, in particular groups of species , the percentage of polymorphic bands was variable and dependent on the primer combination used. Generally, populations of J. vulgaris showed lower genetic variation than that of J. erucifolia and hybrids  for each parental species. Fourteen of the 32 such bands detected in J. vulgaris were fully diagnostic (present in 100% of the plants and completely absent in J. erucifolia), while eight others were found in most J. vulgaris individuals. In the J. erucifolia group, 24 diagnostic bands were detected. Only two of them were present in the profiles of almost all accessions. Most of the remaining 22 bands showed significant differences in the frequency of occurrence across individual profiles. Almost all of the DNA bands amplified from the putative hybrids co‐occurred in either J. vulgaris or J. erucifolia banding patterns. Furthermore, some bands unique for putative hybrids also were observed.Seven of the 15 initial AFLP primer combinations produced clear and reproducible fragments and yielded a total of 920 amplification products sized from 50 to 500 bp, all were polymorphic across all 64 accessions. The number of polymorphic bands per primer combination ranged from 114  and putative hybrids  and was located between the group of J. erucifolia and the group encompassing samples of J. vulgaris and three putative hybrids . The Neighbor‐Net network  formed one cluster, while J. vulgaris individuals and the remaining tree hybrids  clustered in other. The analysis also showed that individuals of all surveyed species collected in particular localities clustered together and may reflect some genetic distinction related to the geographic isolation of populations . PCoA Figure  divided K = 2). The detected differentiation pattern agreed with the sample partition into three groups. Two were represented by tentatively parental, pure species populations (except for population K). They were quite homogenous and consisted of individuals assigned nearly entirely to a single cluster . The third group, encompassing populations Mm and Pc  and population K , showed a distinct overlap of the gene pools of the two previous groups. In the latter two populations, the cluster typical of J. erucifolia dominated clearly. The Mm population was the most diverse because it included specimens with the pattern of pure J. erucifolia (or with the domination of its gene pool), and those in which the cluster present in J. vulgaris predominated. On the other hand, one sample of “pure‐species” population S (J. erucifolia) seemingly combined genetic material from the other parental species . A slight admixture of the J. erucifolia gene pool was also present in several individuals of J. vulgaris population Pu . Another three plants (0.061–0.119) represented nearly pure parental plants. As in the above case, 17 specimens identified as J. erucifolia (index = 1 or index = 0.966–0.986) were classified as pure. Six others (index = 0.855–0.948) comprising individuals of J. erucifolia (population K) and three plants, tentatively identified as hybrids, were also interpreted as nearly pure. Among the putative hybrids, none exhibited perfect marker additivity as expected in F1 (index = 0.5); however, several of them had an index very close to this value  and were therefore interpreted as F1‐type. Three putative hybrids (index = 0.234–0.309) were likely to be backcrosses toward J. vulgaris while the other 12 (index = 0.649–0.841) were diagnosed as probable backcrosses towards J. erucifolia , sequences of J. vulgaris obtained in this study, and that deposited in GenBank (accession number: GU818567) were also identical. This within‐individual polymorphism may indicate that J. vulgaris contains two separate ITS copies that differ by three substitutions. Position 607 was also polymorphic in the sequences of most hybrids.Forty ITS sequences from the putative hybrids (some had two ITS sequence variants) and the parental species were aligned and compared. Sequence analysis of several parent specimens, both in the J. vulgaris . The remaining sequences showed significant sequence similarity to J. erucifolia. Nineteen of these sequences were almost identical to J. erucifolia: haplotypes J‐II (97% match), J‐III (98.5% match), and J‐IV (95.5% match), found in 12, 4, and 3 accessions, respectively. Haplotype II differs from the other two mainly by the lack of a 1‐bp indel (position 60). Other haplotypes were found in two: J‐VI (93.9% match) or in only one accession (J‐V and J‐VII–J‐XIV). Apart from the J‐VII haplotype (97% match), in others, the sequence similarity to J. erucifolia was in the range of 92.4%–93.9%. Their sequences were similar to each other and the differences concerned only a few single nucleotides. The additive sites of two putative parental species were detected in the hybrid sequences proving that all putative hybrids were indeed hybrid derivatives of J. vulgaris and J. erucifolia. In addition, within the sequences of some hybrids, the presence of single nucleotides and one deletion, absent in the sequences of any of the parent taxa, was also observed. These data are summarized in Figure The total length of ITS sequences varied from 685 to 691 bp. The parental sequences differed in 58 variation sites, including 52 substitutions and six indels (insertion/deletion). As a result of comparing the ITS sequences of hybrids, 66 variation sites, including 59 substitutions and seven indels, were found. These 66 variation sites divided the ITS sequences of hybrids into 14 distinct haplotypes (defined here as haplotypes J‐I–J‐XIV). Seven of the sequences derived from the putative hybrids were identical to 3.3trnL (UAA) 3′ exon and trnF (GAA) were 396 bp in length. Sequences of 25 putative hybrids, one individual of J. vulgaris  and four individuals from populations of J. erucifolia were compared. We took into account previous results, showing that small admixture of the J. vulgaris gene pool was also present in individuals of “pure‐species” populations of J. erucifolia (S and K). Two individuals from each of these groups were selected for sequencing, while next two were randomly selected.The aligned sequences of the chloroplast intergenic spacer between J. vulgaris and J. erucifolia. All, except one different nucleotide (position 45), had the same sequence as J. vulgaris (haplotype H1). The remaining two individuals (Mm9 and Mm10) had an identical sequence with J. erucifolia on these four fixed sites (haplotype H2). Moreover, within individuals of J. erucifolia, two genotypes divided by one variation (position 45) were detected. S1, P1, and P10 individuals had a G nucleotide, while K3 individual had a T nucleotide at this position  and one 10‐bp indel (positions 268–277). Twenty‐three of the 25 morphologically intermediate individuals exhibited chromatogram additivity on these differentially fixed sites between 4Hybridization is generally considered a natural phenomenon in both the plant and animal worlds. Morphological intermediates are usually suggested to be derived from direct hybridization or introgressive hybridization of the two distinguished species. However, many examples show that distinguishing hybrids is not so easy, especially if no single morphological feature allows you to clearly distinguish the analyzed plants. The definition of the boundaries between “typical” and ‘intermediate’ units is often more or less arbitrary Aas, . MoreoveJ. vulgaris and J. erucifolia were recorded but not confirmed by any research. Moreover, Benoit et al. (J. vulgaris and J. erucifolia.Hybridization within Senecioneae species is well known. They hybridize with other congeners when they co‐occur. As mentioned above (introduction), hybrids between t et al.  challengJ. vulgaris and J. erucifolia is possible. Both species are closely related according to their phylogeny and morphology  in one reaction and is highly reproducible.Since 25 specimens showed morphological traits intermediate between Jacobaea species and their putative hybrids. An AMOVA revealed that the main part of AFLP variation was due to differences between individuals within populations (77%), while 12% accounted for the inter‐population component. Similar results for J. vulgaris and J. aquatica populations have been reported previously  were in the clade of J. vulgaris.All analyses based on the AFLP data clearly distinguished J. erucifolia. A slight admixture of the J. erucifolia gene pool was also present in several individuals of the J. vulgaris population Pu. This might indicate the repeated backcrossing of an interspecific hybrid with J. vulgaris. Both parental gene pools contributed to the genotypes in the hybrid population, but J. erucifolia generally had a more considerable share in them. Nevertheless, the highly diverse hybrid population Mm also included specimens in which the cluster present in J. vulgaris predominated, as well as those with the pattern of nearly pure J. erucifolia.Bayesian analyses using STRUCTURE clustered the accessions similarly confirmed by previous results  and showed the admixture of parental gene pools in several individuals tentatively classified as “pure species” based on morphology. Population clustering demonstrated a consistent pattern, including independent clusters formed by individuals from pure populations with hybrids exhibiting an admixed genotype. Moreover, a distinct overlap of the gene pools of the parental groups was also observed in all analyzed specimens from population K and one from population S, which were preliminary assigned to “pure‐species” J. erucifolia or the J. vulgaris genome. On the other hand, more than half of all individuals (63.3%) had a hybrid index larger than 0.6, which confirms the results of other analyses, indicating that most individuals had a more significant fraction of J. erucifolia in their genome.Finally, AFLP provided a measure of the nuclear composition of 30 putative hybrids . It showed that 20%  of all putative hybrids might be classified as F1‐type with a hybrid value of 0.466–0.580. The remaining 24 plants in the hybrid group had lower or higher hybrid indices. Such values suggest that they are rather later‐generation backcrosses containing a high proportion of the J. erucifolia contributing more to the genomic composition of the hybrids than J. vulgaris. Such hybrids demonstrated a closer relationship to one parent, usually suggesting frequent backcrossing .Summing up, the results of the Bayesian admixture analyses using STRUCTURE indicated that introgression was asymmetric, with J. vulgaris and J. erucifolia differed significantly. Both haplotypes were found in most putative hybrids with several substitution or indel differences . Such minor differences in ITS sequences in the remaining haplotypes were also observed. Again, evidence from both the ITS sequence comparison and the ITS phylogenetic tree (not shown) supported the significant similarity of most hybrids to J. erucifolia. At the same time, additive sites of two putative parental species were also detected, providing evidence that all putative hybrids were indeed hybrid derivatives. In addition, single substitutions and one deletion were observed in the hybrid sequences, which were not found in the sequences of either parent species. As with bands found only in AFLP profiles of some hybrids, interpretation is difficult. Perhaps we are dealing with earlier involvement of another species or ancient hybridization, but confirmation of either hypothesis requires research covering larger areas of the genome.Some authors  or J. erucifolia (three accessions). It indicated that the hybridization was asymmetrical and bidirectional because chloroplast DNA is maternally inherited in most angiosperms, including Senecio‐Jacobaea complex  may raise some doubts. They clustered in Nj and PCoA with other hybrids, while having J. erucifolia chlorotype and being pure or almost pure J. erucifolia according to STRUCTURE and hybrid index analysis. At the same time, they had additive sites in the ITS sequence and diagnostic markers of the other parent. The presence of unique markers from both parents is believed to identify the presence of a hybrid  and J. vulgaris , probably were also of hybrid origin, being backcrosses to J. erucifolia) and J. vulgaris, respectively.On the other hand, hybridization can counter extinction by maintaining or increasing population sizes. This phenomenon is known as the rescue effect of hybridization ; investigation ; methodology (lead); writing – original draft (lead). Małgorzata Marszałek: Investigation . Piotr Kosiński: Formal analysis ; writing – review and editing . Marek Podsiedlik: Resources . Leszek Bednorz: Resources . Joanna Zeyland: Funding acquisition (lead); writing – review and editing .The authors declare that they have no conflict of interest.In this case, Nagoya Protocol is not applicable, so there are no benefits to report.


To describe indications, test types, and results of prenatal diagnostic genetic amniocentesis among Ethiopian pregnant women.This study was a descriptive study on prenatal diagnostic genetic testing among Ethiopian pregnant women with certain indications and it was conducted at St. Paul’s Hospital Millennium Medical College  from January 2017 to April 2023. Data on sociodemographic characteristics, genetic testing indications, types, and results were collected electronically. Data were analysed using SPSS version 23.A total of 159 cases were analysed. The commonest indication for genetic testing among the study subjects was major fetal structural anomalies identified on specialized prenatal anatomic scanning of the index pregnancy detected in 71(44.7%) cases. Down syndrome and Edward syndrome were the commonest genetic aberrations detected accounting for 6.3% (10/159) and 4.4% (7/159), respectively. Among the rare genetic aberration detected were Di-George syndrome (0.6%) and Duchenne muscular dystrophy (0.6%).Findings of our study underscore the importance of diagnostic prenatal testing in a Sub-Saharan Africa setting, as common (trisomy 21&18) and rare genetic defects were identified using this important prenatal diagnostic testing. Considering the implications of detecting chromosomal abnormalities for future counselling and care, carrier state in parents for some chromosomal anomalies, and planning post-natal management of some abnormalities that are associated with aneuploidies , initiation of diagnostic prenatal genetic testing service at tertiary public health facilities should be acted up on. Prenatal genetic testing is classified as invasive and non-invasive prenatal genetic testing (NIPT) . NIPT haTraditionally, fetal chromosome analysis has been performed using karyotyping with samples taken through chorionic villus sampling (CVS) or amniocentesis. However, this traditional method has a disadvantage of being time-consuming and labor intensive –12. As oIn General, genetic testing in Africa is limited to some academic centers or National Health Laboratory centers and it is mostly the invasive type. For example, in south Africa, there are well organized but small genetic services, based mostly in academic centers, provincial health departments and the National Health Laboratory Service .Currently, there is scare literature regarding diagnostic prenatal genetic testing and patterns of chromosomal abnormalities in high-risk mothers in an African setting. In Ethiopia, there are no well-established prenatal genetic counselling and testing canters. In Ethiopia, clinicians have been using first-trimester combined screening to assign the genetic risk to the patients in the first trimester. Recently, prenatal diagnostic genetic testing (genetic amniocentesis) has been practiced at our Hospital, St. Paul’s Millennium Medical College , in concert with a an in-country private genetic testing centre. Our study aimed to describe indications, test types, and results of prenatal genetic testing among Ethiopian pregnant women.This was a retrospective descriptive study on prenatal diagnostic genetic testing in a population of Ethiopian pregnant women and was conducted from May 5, 2023 to August 5, 2023. The study setting is St. Paul’s Hospital Millennium Medical College, is a tertiary leading referral hospital in Ethiopia for advanced patient diagnostics and treatment including advanced maternal-fetal medicine diagnostic and therapeutic interventions. It nests the biggest Maternal-fetal medicine center in Ethiopia, which is fully engaged in sub-specialty level care for Ethiopian pregnant women as well as delivering a high-quality sub-specialist training for fellows enrolled in the institution, who come from different parts of Ethiopia.St. Paul’s Hospital Millennium Medical College (SPHMMC) has recently improved their ability to prenatally screen for and diagnose congenital anomalies and genetic diseases. This has been accomplished through training of maternal-fetal medicine (MFM) specialists with prenatal ultrasound expertise, genetic counselling education of nurses and availability of in-country diagnostic genetic testing  able to detect pregnancies affected with aneuploidies and select microdeletion/microduplication syndromes . In our Data were extracted from amniocentesis registry at SPHMMC and genetic testing registry at MRC-ET Advanced Laboratory, where all the study subjects had their prenatal genetic testing, over a period of 6 years (from January 2017 till April 2023). The medical records of the study subjects were accessed from May 5, 2023, to June 5, 2023. Data on sociodemographic characteristics, genetic testing indications, types, and results were collected electronically, retrospectively. Non-random sampling technique was used during data collection, in which consecutive cases with complete data were included in the study.A formal Ethical clearance letter was obtained for this study from St. Paul’s Hospital Millennium Medical College Institutional Review Board (IRB) on May 2, 2023. Data were fully anonymized before we accessed them and the IRB waived the requirement for informed consent. Hence, informed consent was not obtained from the study participants.Data were analysed using SPSS version 23. Simple descriptive analysis were used to analyse the data. Indications, test types, and chromosomal changes of prenatal genetic testing were analyzed. Frequency and proportions were used to present the results.Out of 186 pregnant women who had prenatal genetic testing, 159 were included in the final analysis after 27 cases were excluded due to incomplete data. The commonest indication for genetic amniocentesis and testing among the study subjects was major fetal structural anomalies identified on detailed sonography of the index pregnancy . SeventyFrom those who had major structural anomalies, 29 (18.2%) cases had fetal ventriculomegaly,11(6.9%) cases had multiple congenital anomalies, 8 (5%) cases had renal anomalies, and another 6(3.8%) fetal cardiac anomalies. Eighteen (11.3%) study subjects had their prenatal genetic testing for sonographic soft markers of aneuploidy . Two pregnant women (1.3%) had history of previous affected child with Duchene’s muscular dystrophy as an indication for the prenatal genetic testing. Chromosomal analysis was the predominant genetic test type employed during the study period, accounting for 64.8%(103/159),followed by peripheral chromosomal analysis  and Karyotype/trisomy.Analysis of genetic testing results showed that Down syndrome and Edward syndrome were the commonest genetic aberrations detected accounting for 6.3% (10/159) and 4.4% (7/159), respectively. Patau sIn this study, the most common indications for prenatal genetic testing were sonographic findings of major fetal structural anomalies, high risk for trisomy 21 and 18, and findings of soft markers of fetal aneuploidy. The most frequently detected genetic aberrations were Trisomy 18(Edward syndrome), trisomy 21(Down syndrome). Among the rarest genetic abnormalities were Patau syndrome, Di-George syndrome, Turner syndrome, and carrier for Duchenne muscular dystrophy.Genetic counselling has been shown to be an effective intervention in decreasing the negative impact of congenital anomalies and genetic conditions. However; this intervention is not available in most sub-Saharan Africa countries , and litCentral nervous system malformations detected on ultrasound are strongly associated with and predictive of chromosomal abnormalities, especially trisomy 13 and 18 . Among tAmong the health problems that impose a heavy burden on families and a society in general are chromosome aneuploidies. The most common chromosome aneuploidies among fetuses are trisomy 21 , trisomy 18 , trisomy 13  . From thAlthough non-invasive prenatal genetic testing is being practiced worldwide, currently, invasive prenatal testing is still the most commonly practiced in low-income countries that includes Sub-Saharan Africa (SSA) . VariatiWe recommend initiation of diagnostic prenatal genetic testing service at a tertiary public health facility with Maternal-fetal medicine sup-speciality care in Sub-Saharan Setting including Ethiopia, for two reasons. One, considering the importance of detecting chromosomal abnormalities in the index pregnancy for future counselling and care, and the fact that it will open a window for testing carrier state in parents for some chromosomal anomalies. Secondly, it’s importance neonatal screening and planning for management of some abnormalities that are associated with aneuploidies .Strengths of our study include being among the first studies from Africa to document the indications, test types, and test finding of prenatal genetic testing. The main limitations of this study are missing analysis of management approaches (pregnancy termination vs conservative management) and birth outcomes for those that had confirmed diagnosis of genetic defects. Retrospective data collection and lack of cost-benefit analysis are the other limitation of our study.In conclusion, our study substantiates the practicality of diagnostic prenatal testing in a Sub-Saharan Africa setting with further evidence. It shows common (trisomy 21&18) and rare genetic defects (such as Di-George syndrome and Duchene’s Muscular dystrophy) were identified by sending amniocentesis samples for prenatal genetic testing at the private center after proper screening and evaluation by our MFM experts, though the final management outcomes are lacking. We recommend further prospective studies inclusive of cost-benefit analysis of invasive prenatal genetic testing, with a goal of making it affordable for pregnant women with limited resources and at high risk of fetal genetic diseases.


Variation among host-associated microbiomes is well documented across species, populations, and individuals. While numerous factors can contribute to this variation, understanding the influence of host genetic differences on microbial variation is particularly important for predicting co-evolutionary dynamics between hosts and their microbiota. Functional understanding of host genetic and microbial covariation is also of biomedical relevance, for example, providing insights into why some humans are more susceptible to chronic disorders like inflammatory bowel diseases. Unfortunately, disentangling the relative contribution to microbiome variation of host genetics from the environment has been difficult, particularly in humans where confounding environmental effects cannot be completely controlled experimentally. While isogenic laboratory models can be used in controlled environments, the effects on microbiomes of induced large-effect mutations may not recapitulate those of genetic variation observed in nature. Few studies have tested for the direct influence of natural host genetic variation on microbiome differences within a highly controlled environment in which hosts interact freely. To fill this gap, we performed a common garden experiment using families of genetically divergent populations of threespine stickleback fish—an outbred model organism commonly used for determining the genetic basis of complex traits in the context of natural genetic variation. Using germ-free derivation of divergent lines and hybrids between them in this experimental framework, we detected a clear, positive association between stickleback genetic dissimilarity and microbiome dissimilarity. With RAD-seq data, we identified regions of the genome that contributed most significantly to this relationship, providing insight into the genomic architecture of gut microbiome variation.A major focus of host-microbe research is to understand how genetic differences, of various magnitudes, among hosts translate to differences in their microbiomes. This has been challenging for animal hosts, including humans, because it is difficult to control environmental variables tightly enough to isolate direct genetic effects on the microbiome. Our work in stickleback fish is a significant contribution because our experimental approach allowed strict control over environmental factors, including standardization of the microbiome from the earliest stage of development and unrestricted co-housing of fish in a truly common environment. Furthermore, we measured host genetic variation over 2,000 regions of the stickleback genome, comparing this information and microbiome composition data among fish from very similar and very different genetic backgrounds. Our findings highlight how differences in the host genome influence microbiome diversity and make a case for future manipulative microbiome experiments that use host systems with naturally occurring genetic variation. Multicellular organisms harbor and interact with a diverse array of microbes on and in their bodies –3. ComSubstantial variation in microbiome composition has been documented among many different host species, populations, and individuals –11. StDisentangling confounding factors from host genetic influence on the microbiome has proven challenging. Microbiome variation across human populations has been documented previously, pointing to a possible role for host genetic differences , 17. HowWork in model organisms has helped address some challenges as environmental factors can often be more easily controlled. Common garden designs, in which replicated studies are performed in a common controlled environment, provide a particularly powerful approach for isolating and quantifying the influence of host genetic vs environmental factors , 24. TheOther factors beyond environmental influence can also be difficult to control in animal models. Parental transmission  of microbes has often not been controlled because of a lack of gnotobiotic protocols that allow a common set of microbes to be re-introduced , 33. GenGasterosteus aculeatus) have all these characteristics and have proven to be an exceptional model for studying natural genetic variation contributing to other complex phenotypes. This small fish has been widely studied in its natural marine and freshwater habitats, can be reared in the laboratory, and has extensive genomic resources. More recently, stickleback has been developed as a model for host-microbe interactions through the development of tools, such as gnotobiosis, for microbial experiments  have natural genetic variation that has been previously linked to microbiome differences in the wild, (ii) are amenable to controlled gnotobiotic studies in the laboratory, (iii) can be analyzed using a true unrestricted co-housed common garden design, and (iv) can take advantage of genomic tools to precisely study host genetic effects. Threespine stickleback fish . We find evidence of a causal relationship between host genetic and microbiome variation. We also find that the strength of association between host genetic variation and microbiome attributes varies regionally across the genome, providing insight into the genomic architecture of the gut microbiome as a complex trait. Importantly, we characterize this relationship in a broader phenotypic context and document that at least some of the genetic variation associated with microbiome variation is also related to body size, highlighting the need to account for such intermediate morphological traits in future studies of host-associated microbiomes.Here, we fill this gap by using genetically divergent, laboratory-raised populations of threespine stickleback fish—and hybrids between these populations—in a controlled-and-replicated common garden experiment (Gasterosteus aculeatus) fish derived from wild-caught Alaskan populations previously maintained in the laboratory for at least 10 generations with periodic within-population outcrossing. These included three families originating from the freshwater population Boot Lake , three families from the anadromous population Rabbit Slough , and two F1 hybrid families generated from Rabbit Slough females and Boot Lake males (https://figshare.com/s/e40c984d26187d4d5fe7). These families each have their own genetic variants segregating in the offspring and vary in their relatedness . All experimental families were generated on the same day in a 2-h period via in vitro fertilization. Embryos were incubated overnight in 0.1 µM filter-sterilized antibiotic medium containing 1 µL/mL Ampicillin, 0.1 µL/mL Kanamycin, 0.312 µL/mL Amphotericin, and four parts per thousand (ppt) artificial seawater (Instant Ocean)  . Fin clips from the parents were saved and flash frozen in liquid nitrogen (LN2) to be used later for DNA-based parentage assignments. All procedures involving animals were in accordance with University of Oregon institutional animal care and use committee (IACUC) protocols.We generated eight genetically distinct families of threespine stickleback (n = 40) from each family (n = 8) were randomly assigned to one of two groups (n = 20 each) and pooled to comprise two replicate groups of 160 embryos (https://figshare.com/s/11c61208acba7777b117). To remove microbes from the fertilization process and prevent vertical transfer, we followed germ-free derivation protocols for threespine stickleback that have been validated using 16S PCR and fluorescent in situ hybridization (FISH) , fish were removed from the tanks for dissection following procedures to standardize the dissecting process (see Supplementary Methods Text S1 at https://figshare.com/s/ff4e981a6adacaf06ada). An empty Eppendorf tube accompanied the samples throughout processing and was later used as a negative control to capture environmental microbial contamination. Some mortality is common in clutches of stickleback embryos, and at the end of rearing, tank replicate 1 contained 69 surviving fish while tank replicate 2 contained 80 surviving fish .Embryos (n (FISH) . Specifihttps://figshare.com/s/351b3938a2d47c5acffa). Tail clips were flash frozen in LN2 and stored at −80°C. Guts were dissected following sterile procedures and tracked to account for introduced microbes (see Supplementary Methods Text S1 at https://figshare.com/s/ff4e981a6adacaf06ada). Guts were then flash frozen in 1.5 mL Eppendorf tubes previously sterilized via UV irradiation in a Stratalinker  at 1,000 J/cm2 and stored at −80°C . The order of processing was random with respect to experimental factors of interest for all samples, throughout dissection, library preparation, and sequencing.After euthanasia, the offspring were imaged using confocal microscopy to capture phenotypic variation including standard length and tail clipped for DNA-based parentage assignment , and DNA was then extracted following the Qiagen DNeasy spin column protocol described by Small et al.  and quantified using the Qubit Fluorometer Broad Range Kit . Sex was determined using PCR amplification of the male-specific GA1 region . Gut saml et al. .Genomic DNA from each tail clip was standardized to 10 ng/µL and digested with the restriction endonuclease SbfI-HF  and was used to generate RAD-seq libraries –48. Thhttps://figshare.com/s/ff4e981a6adacaf06ada) (Gut DNA isolations were standardized to 20 ng/µL and submitted to the University of Oregon Genomics Core Facility (GC3F) for 16S rRNA (V4 region) amplicon library construction and sequencing (paired-end 150 nt reads) in a single lane on an Illumina HiSeq 4000 (see Supplementary Methods Text S1 at af06ada) . Also seprocess_radtags program in the Stacks suite , vsearch (join-pairs), quality-filter (q-score-joined), and deblur (denoise-16S) to define amplicon sequence variants (ASVs). To assign taxonomy to the ASVs, we used feature-classifier (extract reads and fit-classifier-naive-bayes) to train a classifier based on the GreenGenes 13_8 99%-clustered OTU database, followed by application of the classifier using feature-classifier (classify-sklearn). We filtered out any remaining ASVs of mitochondrial or chloroplast origin using taxa (filter-table). The ASV count tables and ASV sequences were then exported for further analysis using version 3.6.1 of the R statistical language  but found no strong candidates that would warrant removal or affect the outcome of any downstream analysis. We also excluded from downstream analysis individual libraries with fewer than 40,000 total ASV counts. In the case of subsequent multivariate and random forest analyses, ASV count data were normalized to account for depth differences among libraries using the cpm function  from the edgeR R package  sources. Specifically, we excluded one ASV  that was in high abundance in the no-tissue negative control library and a likely reagent contaminant, and we excluded ASVs that were present in only one fish library. We conducted a  package , whereasAlignSeqs from the R package DECIPHER  using principal coordinates analysis (PCoA) based on Bray-Curtis dissimilarity, as implemented via the R package phyloseq . We alsoDECIPHER , optimizphangorn , and genadonis2 function from the R package vegan  models  using thge vegan . Both vi package  to explistrata and sampsize arguments set to minimize bias arising from class imbalance during training.To test whether gut microbiome structure as quantified by ASV relative abundances provides any predictive potential with respect to host population of origin, we built three random forest (RF) classifiers. The first was trained based on ASV data from tank 1 individuals and applied to tank 2 individuals, the second was based on tank 2 training and tank 1 evaluation, and the third was based on a training set of 80 individuals randomly selected regardless of tank, and application to the remaining 54. We implemented RF models using the randomForest R package and function , with thTo evaluate whether average classification accuracy of the RF models was higher than chance expectations, we performed 999 permutations for each model, across which population labels for the training set were randomly shuffled. The accuracies obtained from the original models with unshuffled training data were compared to the null distributions of accuracies from the permutations to infer statistical significance.glmm.zinb or glmm.nb, including tests for model terms, using type-II analysis of variance as implemented by the R package car , tank, and their interactions by fitting zero-inflated and standard negative binomial mixed models, implemented via the R package NBZIMM . We deemkage car . P-valueery rate .mantel and mantel.partial functions from the vegan R package  from the R package poppr . We obtained a total of 2,306 ASVs for final analyses, and the mean number of ASV-assigned reads among the 134 individuals was 238,706.4  (see Data Set S1 at https://figshare.com/s/1fe12c74e01708e7cc7d).We obtained 16S amplicon sequencing profiles from the guts of 149 juvenile threespine stickleback, including 69 from the first tank replicate and 80 from the second tank replicate . We exclhttps://figshare.com/s/1fe12c74e01708e7cc7d). One Boot Lake family did not contain any surviving progeny in either tank, so we inferred that this family sustained complete mortality at an early stage of development. This was consistent with our monitoring of extra embryos (siblings of the 40 embryos per clutch included in the experiment), which also sustained 100% mortality early in development. Remaining for analysis were approximately uniformly distributed family sizes for two Boot Lake families, three Rabbit Slough families, and two F1 Boot Lake—Rabbit Slough hybrid families .Using RAD-seq data generated for the 149 progeny and all 16 of the potential parents, we assigned parentage with 100% confidence to all progeny , followed by tank , and then by standard length . It should also be noted that in a model including first-order interactions , suggesting that fish size influences the gut microbiome (or vice versa) but to different degrees depending on the host’s genetic background . Beta diversity of the gut microbiome  may differ subtly among the three genetic backgrounds , with Boot Lake individuals being less dispersed, on average, than Rabbit Slough or hybrid individuals (https://figshare.com/s/ff4e981a6adacaf06ada) had a minor effect on overall microbiome dissimilarity .We found that three factors in our experiment reliably influenced overall microbiome dissimilarity among individual fish: population of origin (“genetic background”), host size, and rearing tank (“environment”) . With reractions , we deteividuals . In conthttps://figshare.com/s/0d139faa133de0a2afde and S5 at https://figshare.com/s/422bc5f6f3737c682c51). However, standard length explained the most dissimilarity , followed by population of origin , followed by rearing tank . Again, we noted an effect of interaction between population and standard length . We did not find a significant difference in beta diversity among the three genetic backgrounds .We found similar trends when analyzing phylogenetic community dissimilarity using weighted UniFrac , relative to family effects .To evaluate whether the effect of host genotype in the PERMANOVA models was driven by overall population differences, as opposed to idiosyncratic family effects within populations, we also performed nested analysis based on the Bray–Curtis dissimilarity matrix. We found that population-level variation significantly explained microbiome variation . We found no evidence for such a relationship between host genomic distance and community dissimilarity as measured by weighted UniFrac .We calculated pairwise genomic dissimilarity among individual fish, based on genotypes from 2,408 RAD-seq loci. Closely related host pairs, on average, tended to have more similar gut microbiomes as measured by Bray-Curtis community dissimilarity . We testP = 0.04, P = 0.073, and P = 0.004, respectively; see Fig. S6 at https://figshare.com/s/bc99ee67fb3d46dcfb55). In all cases, the class-wise accuracy was consistently low for F1 (Hy) individuals  but higher for Bt  and RS individuals .We applied three random forest models using ASV relative abundances, first by training on fish from Tank 1 to classify population of origin for fish from Tank 2, second by training on Tank 2 fish to classify Tank 1 fish, and third by randomly selecting (regardless of tank) 80 fish for training and 54 fish for testing. For two of these models, total accuracy was significantly better than a null distribution generated by permutation , which belonged primarily to Phyla Proteobacteria and Actinobacteria, with representation from diverse classes, including Actinobacteria, Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, and Bacilli. One ASV (from Genus Agrobacterium) was consistently important for all three models, and several others assigned at least genus-level taxonomy, including Luteibacter rhizovicinus, Pseudonocardia halophobica, Sphingomonas spp., Bacillus spp., and Edaphobacter spp., showed high saliency for at least two of the classifiers (see Data Set S2 at https://figshare.com/s/fe7ff5a0127e7fdd3244).Each model included at least 10 ASVs with relatively high feature importance (see Data Set S2 at https://figshare.com/s/a80fd5df2e3f8c38541f), class (https://figshare.com/s/a80fd5df2e3f8c38541f) levels, but we found statistical evidence of association with population of origin for many ASVs (see Data Set S3 at https://figshare.com/s/1eadd006fa1063417ef2). Among the 20 ASVs with the largest test statistics (for the effect of population), Class Alphaproteobacteria (Phylum Proteobacteria) dominated, with substantial representation also from Class Chlamydia (Phylum Chlamydiae). Many (48%) of the ASVs subject to a significant population effect also showed evidence for an effect of interaction between population and tank (see Data Set S3 at https://figshare.com/s/1eadd006fa1063417ef2). For example, the ASV with the fifth ranking population effect test statistic (assigned to Genus Bradyrhizobium) was in low relative abundance among Boot Lake families, but this effect was more pronounced in Tank 2 . Another ASV, assigned to Genus Agrobacterium and mentioned above as demonstrating consistent random forest feature importance, was also subject to population and population-by-tank interaction effects but with consistent low abundance in Boot Lake individuals . Other ASVs, for instance one assigned to Genus Perlucidibaca, showed strong tank differences without population or population-by-tank interaction effects .We fit negative binomial or zero-inflated negative binomial mixed models to test whether relative abundances of individual ASVs  differed among stickleback populations of origin. Among-individual variation was high at the ASV , class , and phyhttps://figshare.com/s/4a6a3e22f857861fb981). In a parallel analysis using Weighted UniFrac to quantify community dissimilarity, we identified at least five genomic regions , and all except one overlapped with the nine strongest candidate regions from the Bray-Curtis analysis. These four intersecting candidate regions lie on chromosomes 1, 14, 16, and 20, in marker position intervals of 1.923, 0.406, 3.077, and 3.372 Mb, respectively. The strength of the relationship is clear between gut community (Bray-Curtis) and host genetic dissimilarity for these regions (https://figshare.com/s/6f8e5af4c698dd347851) relative to all markers (https://figshare.com/s/6f8e5af4c698dd347851).Based on a sliding window approach using partial Mantel tests to account for overall relatedness and size differences among fish, we identified at least nine regions of the stickleback genome at which genetic dissimilarity is positively associated with gut microbiome (Bray-Curtis) dissimilarity  and fish size (standard length). These statistical interactions between host genotype and other factors are not surprising, as other studies of host–microbiome associations have revealed similar patterns. For example, a tightly controlled, recent study of influences on the stickleback gut microbiome revealed that host genotype effects were more pronounced during infection with the cestode parasite controls . Additiocontrols .sensu stricto make significant contributions to microbiota. Indeed, traits such as body size in killifish and cod  is the identification of host genetic microbiome determinants. Importantly, our ability to document the interactions among these variables further highlights the strength of the common garden experimental design.Daphnia galeata hatched from different sediment layers  or pure anadromous (Rabbit Slough) populations. Accuracy declined when predicting membership for F1 individuals from the hybrid crosses. Deeper investigation, including greater sampling of progeny from a more diverse panel of crosses, is needed to better understand what aspects of genetic variation and forms of inheritance  are driving the patterns observed here.https://figshare.com/s/540694b8f9a180402fae), suggesting for this metric of community dissimilarity anyway, a comparable genomic architecture in the broad sense.Our sliding window analyses of the relationship between host genetic and microbiome dissimilarity suggest that several regions of the stickleback genome contribute large effects to gut microbiome variation among individual fish, relative to the genome average. The distribution of effect sizes for the analysis based on Bray–Curtis community dissimilarity is similar to that for an analysis in which we tested for correspondence between genetic dissimilarity and standard length (SL) differences  of association between genomic variants and gut microbiome composition in threespine stickleback from two British Columbia lake populations . AlthougP-value of 0.001, and the contribution of any particular genomic region should be considered preliminary in light of the possibility of false positives inherent in genomic multiple testing frameworks. As in other genomic association studies, the effects of candidate regions here should be confirmed in the future via additional approaches such as independent replication, fine-scale mapping, and genome editing.Given our finding of an overall association between genetic dissimilarity and microbiome dissimilarity, as well as genomically localized peaks of extreme test statistic values, we conclude that there is an overall polygenic host contribution to microbiome beta diversity. However, our permutation-based association tests carried a lower bound We conclude from our carefully controlled common garden experiment that host genomic diversity is a definitive facilitator of beta diversity for gut microbiomes among individual stickleback fish and that inheritance of structural microbiome traits is not strictly additive. Future studies that both leverage metagenomic interrogations of the microbiome and precise manipulation of host genomes will be required to understand mechanistic bases of the associations reported here. The replicated tank element of our common garden design also demonstrated that rearing environment is an important factor and that the interaction between genetic and environmental variation (G-by-E) influences the microbiome as well. Our results indicate that future studies which harness standing genetic variation measured across the host genome, as compared to gross population or inbred line membership, permit much more nuanced understanding of genomic architecture and possible mechanistic connections between host genes and microbes. In addition, for those studies with inherent reliance on inferences from host genomic data , efforts should be made to limit the impact of variables that confound genetic effects, through tools like gnotobiotic protocols and unrestricted co-rearing. These tools can be implemented now for powerful outbred fish models such as threespine stickleback and can be developed for many other organisms in future studies to better understand the general principles and mechanisms by which host genetic variation influences microbiome diversity.