Abies pinsapo and Abies marocana, both relict tree species, endemic from south Spain and north Morocco, respectively. A total of 607 samples were collected from eight nuclei: six from Spanish fir and two from Moroccan fir. A genotyping by sequencing technique called double digestion restriction site-associated DNA sequencing (ddRAD-seq) was performed to obtain a genetic matrix based on single-nucleotide polymorphisms (SNPs). This matrix was utilized to study the genetic structure of A. pinsapo populations and to carry out selection signature studies. In order to understand how Spanish fir and Moroccan fir cope with climate change, genotype–environment associations (GEAs) were identified. Further, the vulnerability of these species to climate variations was estimated by the risk of non-adaptedness (RONA). The filtering of the de novo assembly of A. pinsapo provided 3,982 SNPs from 504 out of 509 trees sequenced. Principal component analysis (PCA) genetically separated Grazalema from the rest of the Spanish populations. However, FST values showed significant differences among the sampling points. We found 51 loci potentially under selection. Homolog sequences were found for some proteins related to abiotic stress response, such as dehydration-responsive element binding transcription factor, regulation of abscisic acid signaling, and methylation pathway. A total of 15 associations with 11 different loci were observed in the GEA studies, with the maximum temperature of the warmest month being the variable with the highest number of associated loci. This temperature sensitivity was also supported by the risk of non-adaptedness, which yielded a higher risk for both A. pinsapo and A. marocana under the high emission scenario (Representative Concentration Pathway (RCP) 8.5). This study sheds light on the response to climate change of these two endemic species.Circum-Mediterranean firs are considered among the most drought-sensitive species to climate change. Understanding the genetic basis of trees’ adaptive capacity and intra-specific variability to drought avoidance is mandatory to define conservation measures, thus potentially preventing their extinction. We focus here on  At a reAbies marocana Trab. and Abies pinsapo Boiss., the westernmost circum-Mediterranean fir species, endemic from north Morocco and south Spain, respectively , a high number of repetitive sequences, and the absence of reference genomes for most species  to identify genotype–environment associations (GEAs) based on local climate variables and population genetics, and iii) to assess the vulnerability of A. marocana and A. pinsapo to future climate change scenarios based on the risk of non-adaptedness (RONA).In this work, a genotyping by sequencing (GBS) technique named double digestion restriction site-associated DNA sequencing (ddRAD-seq) was used to obtain a genetic matrix based on SNPs. Our specific aims were i) to study the genetic structure of 22.1A. pinsapo is a sub-dioicous fir with a pyramidal shape, which can grow up to 30 m. Spanish fir is located on north-facing slopes between 1,000 and 1,800 m a.s.l.  accounts for approximately 2,000 ha, and Jebel Tazaot (TZ), which is the mountain range of Talassemtane, is approximately 1,000 ha  following the manufacturer’s instructions with some modifications. DNA concentration was measured on a NanoDrop™ spectrophotometer, and the integrity of the extracted samples was determined by electrophoresis in 1% agarose gel. Only those samples that reached the requirements for the ddRAD-seq technique . To know more about the methodology, please see The extraction starts with the lyophilization of 100 mg of each fresh leaf sample. Then, total DNA extraction was carried out by using DNeasy Plant Mini Kit  and sparse non-negative matrix factorization (sNMF) analysis. PCA was performed to describe the inter- and intrapopulation structures, while sNMF was used to estimate ancestry coefficients (Two approaches were used to study the genetic structure of ficients . PCA wasficients  with theficients  package ficients . For theficients  in R to ficients .ST), to determine population differentiation, and migration rate (Nm), to describe the presence or not of gene flow among populations, were estimated. Nei’s genetic distance among populations was also determined. Heterozygosity, private alleles, and polymorphic loci were obtained too. Finally, the Shannon index was calculated to infer genetic diversity.Several statistical analyses of each genetic matrix were performed using GenAlEx v6.5  and GenoIn addition, a molecular variance analysis (AMOVA) with 9,999 permutations was carried out with the aim to determine the proportion of genetic variation attributable to differences among and within populations.2.4BayeScan 2.1 software  was used2.5The 19 bioclimatic variables from the WorldClim database  with a rThe lfmm (latent factor mixed models) function of the LEA R package v3.4.0  was used2.6The RONA value indicates the theoretical percentage of change in allele frequency at loci associated with environmental variables, which is required for a given population to cope with future changes in that variable, allowing it to survive . The estpyRona v0.3.6  for lfmm33.1de novo assembly of the Spanish fir nucleus dataset maintained 504 individuals and 3,982 SNPs. The first axis of PCA separated GR from the rest of the Spanish populations with an explanation percentage of 27.2%  and the lowest among individuals (4.3%) with a high significance . In addition, significant differences among populations were found (40.5%).The AMOVA results of ST values ranged from 0.007 to 0.062, with high significance in all the cases , indicating the presence of genetic differences among all nucleus pairs  of each population ranged from 0.467 to 0.501. The minimum appeared in PT and the maximum in CA. In this case, no private alleles were found. Finally, all Nm values are very high, which indicates that the populations of study have a strong gene flow among them. An extremely high value was found between CA and SA (35.923), and the lowest was among SA and PT (5.287).A. pinsapo nuclei are very similar. However, it is remarkable that GR showed the highest values, which indicates that this population is the most genetically different.Nei’s genetic distance showed values near 0 (from 0.006 to 0.045), indicating that all de novo assembly. The admixture results were interpreted by the PCA results, and K = 3 was the best value to explain the genetic structure of our populations. In this case, GR had its own gene pool, SA and CA conformed to other groups, and the rest of the populations were grouped. However, there are several genetic differences among populations as indicated above.The Spanish fir dataset from the reference assembly filtering retained 494 individuals and 1,642 SNPs. PCA results showed that GR separated from the rest of the populations with an explanation percentage of 24.7%  than the estimated value with the de novo assembly. In addition to Nei’s genetic distance ranging from 0.005 to 0.037, the higher value of the range was lower than the estimated value with the de novo assembly dataset.Predictably, the statistical analyses showed the same results, which were obtained with the 3.2de novo assembly of A. pinsapo nuclei showed 51 loci under selection . The complete sequence of the 10 SNPs, which had the lowest q-values, and those that showed associations with bioclimatic variables in GEA studies were queried against the databases. Homolog sequences were found for nine of these sequences, and six of them obtained protein hits , maximum temperature of the warmest month (BIO5), temperature annual range (BIO7), precipitation of wettest quarter (BIO16), and precipitation of driest quarter (BIO17). A total of 15 associations with 11 different loci were observed, with the most numerous at the maximum temperature of the warmest month (BIO5) with five loci. Some associations were shared between two or more bioclimatic variables.The genetic matrix of the A. pinsapo populations, which had the two age cohorts (adults and saplings), as follows: CA, SA, and AN. The results indicated that CA and SA populations are very similar to each other, while they are different from AN. The allele frequencies estimated by separating the individuals of these populations into old trees and saplings were slightly different within the CA and SA populations for SNP 448, within the SA population for SNP 2400 and SNP 2528, and within the AN population for SNP 3808  did not show associations with any locus. This study was performed with the same five bioclimatic variables, which were used with the de novo matrix. The highest number of associations was observed with BIO7 (seven loci). The complete sequences of all these loci hit against the nucleotide database, but only five of them matched proteins. Some of their functions were involved in ubiquitination, transmembrane transport, or stomatal closure (The GEA study with the reference assembly carried out with The GEA study with Moroccan fir populations was carried out with the 19 bioclimatic variables since Pearson’s correlation could not be performed because this genetic matrix had only two populations. The same locus (SNP 5594) was associated with all the variables, and the alignment results showed homology with a protein whose function is related to protein phosphorylation (LRK1). 3.4A. pinsapo nuclei ranged from 0.01 to 0.47, while in the high emission scenario, the range was 0.02–0.97. In the case of Moroccan fir, the change rate value was between almost 0 to 0.39 for the low emission expected scenario, ranging from approximately 0.02 to 0.90. Considering these results, the high emission scenario showed a higher risk for the Spanish and Moroccan firs’ survival than the other scenario, as expected.The obtained allele frequency change rate value in the low emission scenario for The isothermality (BIO3) showed the maximum change rates for each of the Spanish fir populations in the low emission scenario except for GR and PI populations, which displayed high values in the temperature annual range (BIO1) . Depending on the Spanish fir population, the second bioclimatic variable, which needs a higher change rate value, was related to temperature  or precipitation (PI). The second maximum value obtained with Moroccan fir populations (TA and TZ) was always related to annual range precipitation (BIO12).In terms of the high emission scenario, 4de novo assembly than with reference. This could be explained by the representation of those SNPs in the reads obtained. Due to the large-size genome of this species , the six A. pinsapo populations might behave as in complete panmixia. Nevertheless, the p-values were significant, indicating that these nuclei showed differences among them. In the light of our results, the locality of GR seems to be the most dissimilar one. Indeed, previous studies carried out with cpSSRs showed differences between GR and Sierra de las Nieves  . Thus, the mean annual temperature decreases by an average of 0.72°C for every 100 m of elevation, while annual rainfall decreased toward the east by an average of 173 mm for every 10 km and increased by an average of 81 mm for every 100 m of elevation . As a rean 3 km)  could acA. pinsapo distribution. Since the individuals of this population are under limiting climatic conditions, drier and hotter than average due to low elevation and eastern location, they should have some genetic pressure to cope with them . Generally, the expression of DREBs is induced by abiotic stresses, such as extreme temperatures, drought, and salinity. Due to this involvement in abiotic stressors responses, the DREB genes of several economically important organisms have been modified with the aim to increase their resistance, such as freezing tolerance in potatoes  Karst  Karst  has beenA. pinsapo and A. marocana  Heynh., it is expressed in the guard cells, and as a response to drought stress, this protein induced the stomatal closure as a response to ABA , is called aluminum-activated malate transporter 12-like. In e to ABA . This funditions .In the case of Moroccan fir, the identification of only one locus associated with all the bioclimatic variables, which is also under selection, gave us an insight into the relevance that its genomic region could have. The allele frequency variations found among populations and between ages could indicate that the selection pressure is acting. In addition, a protein homology was identified with a leucine-rich repeat serine/threonine-protein kinase (LRK1). This protein kinase family can be involved in the abiotic stress response . For thiFinally, the RONA studies allowed us to describe the genetic vulnerability of the species under future scenarios. Simulating the local adaptation at the population level and their adaptative potential to future conditions is very useful to determine the risk of the species. The use of this kind of study, such as RONA, allows us to integrate the current and future environment data with genetic information to obtain a robust prediction of species adaptation . ConiferA. pinsapo and A. marocana. RONA modeling supports that warming might be the main limiting factor for the survival of A. pinsapo and A. marocana, according to several studies performed on drought-sensitive species , showed that the main variables conditioning the presence of A. marocana were the average temperature of the warmest quarter and the maximum temperature of the warmest month  could change our expectations based on niche suitability and future range distribution of endangered species, such as the circum-Mediterranean firs  ranged from 0.07 to 0.38  Manetti ex Carrière .The raw data presented in the study are deposited in the NCBI’s Sequence Read Archive (SRA) repository, BioProject accesion number PRJNA1000741 (BM-C: investigation, formal analysis, writing—original draft, and writing—review and editing. IG-G: investigation and writing—review and editing. JCL: conceptualization, funding acquisition, formal analysis, and writing—review and editing. FJG: conceptualization, funding acquisition, and writing—review and editing. All authors contributed to the article and approved the submitted version.


Biodiversity loss at both species and genetic scales has been exacerbated in recent decades due to habitat fragmentation and destruction. These losses have been especially devastating to insects. Butterflies are charismatic microfauna that offer an excellent opportunity to evaluate the consequences of how fragmentation disrupts gene flow between population segments and may serve as indicators for other less visible insect species. The Florida duskywing butterfly is a rare species that is dependent on a critically endangered habitat, the Florida pine rocklands. We ran a series of genetic studies, akin to DNA fingerprinting, to assess the genetic health of the Florida duskywing as well as to evaluate the level of connectivity between the remaining populations. Despite declining population sizes, we found that populations still hold a unique genetic diversity. Genetic results also showed that these butterflies could be clustered into mainland and Florida Keys population groups, which were connected by a moderate gene flow. Additionally, no sampled individuals tested positive for a bacterial infection that has harmed other butterfly populations, meaning that individuals can be moved between populations without major concern for bacterial spread. In summary, this work supports translocations of individuals between populations to (1) facilitate an enhanced gene flow and (2) reinforce small population sizes.Ephyriades brunnea floridensis E. Bell and W. Comstock, 1948) (Lepidoptera: Hesperiidae) across a network of South Florida pine rockland habitat fragments. Based on 81 individuals from seven populations and using multiple polymorphic microsatellite loci, our analyses support the presence of mainland Florida (peninsular) and Florida Keys (island) population groupings, with a moderate, asymmetrical gene flow connecting them, and the presence of private alleles providing unique identities to each. We additionally found that despite a prevalence in many Lepidoptera, the presence of Wolbachia was not identified in any of the samples screened. Our findings can be used to inform conservation and recovery decisions, including population monitoring, organism translocation, and priority areas for management, restoration or stepping-stone creation to help maintain the complex genetic structure of separate populations.We investigated the genetic structure and diversity between populations of a rare butterfly, the Florida duskywing ( A multitude of anthropogenic drivers continue to rapidly erode and threaten global biodiversity ,2. AlthoIn the current study, we targeted a network of remnant pine rockland habitat fragments in South Florida in the United States as a model system that was ideal to investigate functional linkages between spatially structured populations. Pine rockland is a globally critically imperiled ecosystem that occurs only in South Florida and the Caribbean . Within Anaea troglodyta floridalis), Bartram’s scrub-hairstreak (Strymon acis bartrami), and Miami tiger beetle (Cicindelidia floridana) are all endemic to Florida (USA), restricted entirely to the pine rockland habitat, and listed as Endangered under the ESA. A fourth pine rockland specialist, the Florida duskywing (Ephyriades brunnea floridensis) . Of these, three insects, the Florida leafwing (idensis) , is desiWolbachia can synergistically increase this risk. Wolbachia are a diverse group of intracellular bacteria with multiple host adaptations. When Wolbachia acts as a reproductive parasite, it can cause reproductive fitness advantages in infected females [Wolbachia can create demographic challenges, especially in small populations. When considering conservation and management strategies, such as translocation and assisted breeding programs, Wolbachia screening should be taken into consideration to decrease the potential for pathogen transmission. While habitat fragmentation alone can pose the risk of extirpation for small populations, pathogens like  females . AlteredE. b. floridensis can at times be locally common, particularly in larger parcels across the existing South Florida pine rockland habitat network. Due to its relative abundance in comparison with other pine rockland specialists, E. b. floridensis represents an appropriate surrogate for other rare insects with similarly limited or presumedly limited dispersal abilities. Therefore, we conducted a multi-population assessment of the E. b. floridensis to (i) evaluate the genetic diversity, population genetic structure and gene flow at the landscape and regional levels, (ii) screen for the presence of Wolbachia infection, and (iii) identify priority conservation actions to help maintain or enhance network maintenance and gene flow.Despite this ranking, We utilized a fragmented network of South Florida pine rockland habitat parcels in Miami-Dade and Monroe Counties, Florida (USA) as our study system. This included a total of 14 conservation properties that ranged in size from <6.0 ha to over 5000 ha and of various ownership and management oversights, including county, state and federal agencies .E. b. floridensis over the past two years; (ii) a location to sample the broadest possible geographic range within Florida; and (iii) the ability to secure appropriate property access and permits for sampling.Properties targeted for sampling were selected based on several criteria. These included: (i) a documented presence of E. b. floridensis from a total of 7 of 14 conservation property locations over a four-year period from December 2016 to September 2019 for genetic studies  extraction protocol and quantified genomic DNA concentrations using a Nanodrop spectrophotometer to assess the quality and quantity of each sample. For whole genome sequencing and marker development, we extracted DNA from the legs and thorax of a voucher specimen  and treated the sample with 4 µL RNAse. We used a GenomiphiTM V2 DNA AMplification Kit to amplify genomic DNA and sequenced the whole genome on a PacBio RSII at the University of Florida Interdisciplinary Center for Biotechnology Research (ICBR). Genomic DNA was sheared into 1 kb fragments with an ultrasonicator and prepared for sequencing by annealing adaptors . We used one SMRT cell for the PacBio RSII sequencing run, completed in the circular consensus mode. We used a Globus Personal Connect client with UF-ICBR cyberinfrastructure to receive the consensus PacBio RSII data.We sampled a total of 81 individuals of  studies . For thoocols of ,18, presWe formatted input files (from over 3 billion nucleotides read) and ran them through Geneious (using the Phobos extension)  to identify microsatellite markers. We interfaced with BatchPrimer 3  to develWe followed the reaction conditions and PCR concentrations of Saarinen and Austin (2010). This protocol uses M13 primer labeling and fluorescently labeled tags and allows for multi-pooling of PCR products. PCR products were diluted 100× and multipooled for fragment size analysis on an ABI 3730 sequencer . We performed all genotyping analyses with GeneMarker v. 2.6.3  , and allWe calculated the linkage disequilibrium (LD) between loci and exact tests of the Hardy–Weinberg equilibrium (HWE) in Arelquin v.3.5.2.2 . SequentWe calculated summary statistics, observed and expected heterozygosity, and FIS (inbreeding coefficient) in each population using GenAlEx 6.5 . To quanWe used two complementary methods to evaluate genetic structuring. First, we ran principal coordinates analyses (PCoA) of genetic distances between individuals using GenAlEx 6.5  for a viE. b. floridensis for Wolbachia. We extracted genomic DNA from all individuals using a modified Qiagen® DNeasy Blood and Tissue Kit  extraction protocol and quantified genomic DNA concentrations using a Nanodrop spectrophotometer. To screen for Wolbachia, we performed two PCR reactions following the methods outlined in [Wolbachia 16S rDNA (WSpecF and WSpecR), and another reaction using universal arthropod primers for 28S rDNA to confirm negative Wolbachia results (28sF3633 and 28sR4076). We followed the PCR cycling conditions described in [Wolbachia screening. We visualized PCR amplification products on 1% agarose gels to determine the presence of Wolbachia.Here, we screened all 81 individuals of lined in : one reaThe results from the PacBio RSII run yielded 3,405,242,620 nucleotide bases read with an average read length of 27,385 bases. Of the 24 microsatellite loci we began testing, 18 had perfect repeats and no linkage to coding regions. Of these 18 loci, 5 loci consistently amplified across all templates and lacked large allele dropout and stutter problems. These 5 loci are highly informative, with 4–16 alleles per loci . After tp > 0.05 after Bonferroni correction), although the two largest populations (Big Pine Key and Zoo Miami) are in Hardy−Weinberg equilibrium at 3/5 and 4/5 loci, respectively.Individual populations are not in Hardy−Weinberg equilibrium at all loci .FST values also show similar signs of moderate connectivity between the two major groups (FST = 0.083 between the mainland and island). These values underscore the similarity between the mainland and island groups and are helpful in interpreting the next set of analyses showing two groups. This can be visualized by the PCoA analysis of samples . The AMOMultiple analyses  confirm the general presence of two-three population groupings . These gThe island group consists of the Big Pine Key and Dagny Johnson samples. Big Pine Key is an island in the lower Florida Keys and is ~141 km south of Dagny Johnson Hammock State Park on the upper keys island of Key Largo.Wolbachia in any of the 81 individuals of E. b. floridensis. Standard genomic DNA positive controls were amplified on all 1% agarose visualizations, and standard negative control amplification was absent. We verified PCR viability for all genomic DNA using 28S universal arthropod primers, and we found all 81 samples amplified at the appropriate size. Wolbachia may cause changes in the population structure, inferred by mitochondrial signatures , supporting our original hypothesis that a unique diversity persists in these populations. These also represent populations at or near the northernmost and southernmost geographic extent of Small populations are susceptible to extirpation by stochastic events. We support the continued monitoring of all small populations and, as necessary, support the translocation of individuals from other sites to augment a small population size, artificially maintain gene flow, and overcome effects of genetic drift.E. b. floridensis population abundance, host density, or connectivity measures were considered to be outside of the current project scope and not determined for each conservation property, addressing these metrics remains a focus for future research, in order to better understand the ecology and metapopulation dynamics of this increasingly at-risk butterfly.Our results also support the hypothesis that an asymmetrical gene flow connects remaining populations in mainland Florida and the southern Florida Keys. At present, we observe a moderate connectivity between the mainland and Keys populations, with similar allele frequencies uniting the groups and the presence of private alleles providing unique identities to each. Therefore, we support activities that maintain the complex genetic structure of separate populations, each maintaining a unique genetic diversity and moderate levels of connectivity. We also advocate sampling additional populations to help establish a detailed picture of the metapopulation arrangement of the Florida Duskywing across its range. Similarly, as the extent and configuration of the occupied habitat, Wolbachia infection in Florida Duskywing butterflies. As Wolbachia infection may have unintended consequences for populations  we advocate for populations to continue to be screened before any translocation or captive breeding involving the mixing of multiple populations occurs. The negative Wolbachia results found in this work allow for captive breeding, head-starting, and translocation to remain viable conservation strategies. Given the multiple threats facing this species, including increased hurricane activity, we consider that the continued monitoring of connectivity and maintenance of specific populations enhance the security of this taxon.In summary, this work supports translocations of individuals between populations in order to (1) support a discontinued gene flow and (2) reinforce small population sizes. A preliminary screening of all individuals, following  methods,


Trachidermus fasciatus) was previously a locally abundant species in China, but the habitat required for the survival and breeding of this species has been seriously damaged. Its resources have been almost depleted, and it has been listed as a national second-class protected animal. In recent years, the population of roughskin sculpin has recovered in the natural environment through enhancement programs and the release of juveniles. However, the effects of released roughskin sculpin on wild populations’ genetic structure and diversity remain unclear. In this study, the whole-genome resequencing of two cultured populations and one wild population showed that the two cultured populations have similar genetic diversity compared with the wild population. In addition, artificial feeding affected the formation of traits of roughskin sculpin. The finding provides data support for the resource recovery of roughskin sculpin.The roughskin sculpin (Trachidermus fasciatus) is an endangered fish species in China. In recent years, artificial breeding technology has made significant progress, and the population of roughskin sculpin has recovered in the natural environment through enhancement programs and the release of juveniles. However, the effects of released roughskin sculpin on the genetic structure and diversity of wild populations remain unclear. Studies on genetic diversity analysis based on different types and numbers of molecular markers have yielded inconsistent results. In this study, we obtained 2,610,157 high-quality SNPs and 494,698 InDels through whole-genome resequencing of two farmed populations and one wild population. Both farmed populations showed consistent levels of genomic polymorphism and a slight increase in linkage compared with wild populations. The population structure of the two farmed populations was distinct from that of the wild population, but the degree of genetic differentiation was low . Selective sweep analysis showed that 523,529 genes were selected in the two farmed populations, and KEGG enrichment analysis showed that the selected genes were related to amino acid metabolism, which might be caused by artificial feeding. The findings of this study provide valuable additions to the existing genomic resources to help conserve roughskin sculpin populations.The roughskin sculpin ( Trachidermus fasciatus), belonging to the Cottidae family, is a kind of small, bottom-dwelling catadromous migrating fish mainly distributed in the Gulf of Southern Japan, the west coast of the Korean Peninsula, the northeast coast of China, and the downstream of their corresponding rivers [The roughskin sculpin  populations [Salmo salar L.), the Atlantic Ocean’s eight populations in northern and southern Norway were analyzed using genome resequencing, revealing that natural selection acting on immune-related genes led to genetic differences between populations in the Norwegian Atlantic and that the plasticity of genomes may promote differences between populations [Hypophthalmichthys molitrix) was associated with disease, immune system, and environmental adaptation [C. auratus). With genome-wide variation information, genes associated with morphogenesis, pigmentation, behavior, immune response, infectious disease, energy metabolism, and hormone response have been successfully mapped [Population genomics have developed rapidly with the availability of high-throughput sequencing and the reduced costs of related technologies. Population genomics can screen thousands of genetic markers simultaneously at the genome scale, resulting in tremendous innovations in population genetics, conservation genetics, ecology, and evolutionary research ,14,15. Fulations . In a siulations . Populataptation . Whole-gy mapped .The reference genome of roughskin sculpin has been precisely assembled, laying the groundwork for subsequent population genetic analysis and resource conservation . In thisThis study was performed in accordance with the Guidelines for Care and Use of Laboratory Animals provided by the Chinese Association for Laboratory Animal Sciences (No. 2011–2). The study protocols were approved by the Animal Care and Use Committee of Beidaihe Central Experiment Station, Chinese Academy of Fishery Sciences (CAFS).n = 64, weight: 30.66 ± 10.4 g), Tianjin Municipality , and Dongying City of Shandong province , China). The detailed geographical locations of the sampling are shown in 3 MS-222. The weight was measured using and electronic balance with an accuracy of 0.1 g. The body length and height were measured with a vernier caliper, which is accurate to 0.1 cm. The measurement methods of the total length, body length, and body height of the sculpin are described as follows.A total of 192 T. fasciatus were collected from three different geographic populations  according to the kit protocol. DNA quality was checked by 1% agarose gel electrophoresis and a NanoDrop 2000  spectrophotometer. Qualified DNA samples were randomly interrupted by a Covaris crusher  with a length of 350 bp. A TruSeq Library Construction Kit  was used for library construction according to the manufacturer’s instructions. End repair, polyadenylation, the addition of a sequencing adapter, purification, and PCR amplification were conducted for the library preparation of DNA fragments. Illumina NGS technology was used to sequence the constructed libraries. After the library was constructed, Qubit3.0 was used for preliminary quantification, diluted to 1 ng/μL; then, an Agilent 2100 was used to detect the insert size of the library. After the insert size met the requirements, the effective concentration of the library was accurately quantified by qPCR (effective concentration of the library > 2 nM) to ensure the quality of the library. After the library inspection was qualified, PE150 paired-end sequencing was performed on the Illumina platform according to the effective concentration of the library and data output requirements.The raw data obtained through sequencing were filtered to remove low-quality sequencing data using fastp v0.20.0 software . The filBased on the filtered SNP variation file, PLINK  software  calculat2) measures the degree of LD between different SNPs. The observed pair-wise LD (r2) was calculated using PopLDdecay [The correlation coefficient  method to obtain the adjusted p value (Padj), and a Padj value < 0.05 was considered statistically significant.KofamKOALA  softwareprofiler  R packagThe differences in weight, body length, body height, and total length indicators between different populations are shown in All 40 fishes were genotyped for 2,610,157 high-quality SNPs and 494,698 InDels in this study. The variants were filtered with a call rate > 90% and MAF > 0.01 in each population. SnpEff annotation revealed that 52.8% of variants were located in intergenic regions, and 47.2% were located in gene regions, including exons, introns, and untranslated regions. The variants on introns accounted predominantly for variants located in the gene region (41.4%). Only 6.0% of variants were located in the exon region, containing 152,974 synonymous and 276,614 nonsynonymous substitutions .Genetic diversity among populations was assessed by estimating observed and expected heterozygosity, genetic distance, and nucleotide diversity, as shown in 2) was slower in the QHD population than in the SD and TJ populations, which were cultured and subjected to a certain degree of selection. In addition, we found that the r2 attenuation decay curves of the SD and TJ populations overlapped, indicating that the two populations exhibited a low degree of differentiation, consistent with the Fst results.By analyzing the degree of genetic differentiation among the three populations, it was found that the QHD population exhibited the highest genetic differentiation from the SD and TJ populations, with a mean Fst of 0.02. The SD population had the lowest genetic differentiation compared with the TJ population, with a mean Fst of 0.005 . BecausePrincipal component analyses were performed on the QHD, SD, and TJ populations to explore their population structures. The first three were selected as clustering factors according to the variance-explained rate of principal component factors. The QHD population could be clearly distinguished from the SD and TJ populations by three principal components . The SD Combined with Fst and the ratio of PI between different populations, strong selection signals were detected, and target genes were screened. During the comparisons of QHD and SD, QHD and TJ, and SD and TJ populations, we took the highest 5% of Fst and the window with the highest 5% (high top5%) and the lowest 5% (low top5%) Pi ratios as potential selection regions and merged adjacent and overlapping windows A. We useThe annotated genes underwent KEGG enrichment analysis to narrow the selected gene range. A total of 300 of the 500 genes in the TJ population were annotated to the corresponding KOs, and enrichment analysis showed that these genes were enriched in apoptosis, phenylalanine metabolism, and other pathways, but no significant functional clusters were identified during KEGG pathway enrichment analysis B. The enT. fasciatus) was once widely distributed in the Northwest Pacific, but its habitat has been fragmented due to human activities such as dam construction and environmental pollution, resulting in a significant decrease in its population. Nowadays, it is only found in certain river basins of the Yellow and Bohai Seas and the East China Sea in China. The endangered status of the roughskin sculpin has prompted the implementation of conservation measures, such as stocking and releasing. Research on artificial breeding technology of the roughskin sculpin, including the production and aquaculture of the species, has made breakthroughs that not only protect the population of the roughskin sculpin but also meet market demand. Due to differences in breeding methods, growth patterns, feed sources, and feeding frequencies, there are differences in growth characteristics between wild and cultured populations [Palaemonetes sinensis sollaud and Caridina nilotica gracilipes de Man are the main shrimps ingested by roughskin sculpin. The breeding population of roughskin sculpin is fed commercial compound feed. The main source of protein in commercial feed is fish meal. Given that the cultured populations are fed on a fixed basis and have access to sufficient food during the breeding process, the morphological characteristics of the cultured population are significantly better than those of the wild population. Therefore, morphological characteristics represent important and stable indicators for distinguishing sea bream and sea bass from other marine fish [The roughskin sculpin . In this respect, the population heterozygosity calculated by microsatellites was higher than that calculated by SNPs, suggesting that the number of markers affects heterozygosity. Theoretically, when the species range is limited and its effective population is decreased, its genetic diversity generally decreases due to genetic drift and inbreeding decline [Population genetic diversity refers to species’ genetic diversity within an area. Higher genetic diversity means that a species is more resilient to its environment. Current evidence suggests that changes in population size, structure, genetic mutations, and other factors can affect the population’s genetic diversity. A study by Dewoody and Avise showed that the average heterozygosity of marine fish microsatellites was 0.79 and that the average heterozygosity of anadromous fish was 0.68 . Li et apulation ,41. Howe decline . However decline ,44,45. A decline . Therefo decline . OverallPopulation structure analysis results based on the genome-wide SNPs showed the genetic structure between the populations of roughskin sculpin. Principal component analysis showed that the three geographical groups of roughskin sculpin could be divided into two genetic groups: the SD and TJ population vs. the QHD population. Population structure analysis further verified the results of the principal component analysis. Although the cross-validation values of the predicted optimal population number did not converge, at K = 3, most individuals in the SD population and the TJ population were divided into one group, and the QHD population was independent of the other group. The SD population sampling point was far away from the TJ population. Given that there is theoretically no large-scale gene exchange, we speculate that the two groups come from the same spawning ground. Many molecular markers can be obtained by genome-wide testing, and due to their wide distribution in the genome, the fine genetic structure differences between populations can be detected. Compared with the QHD population, the differentiation coefficients of the SD and TJ populations were 0.0156 and 0.0158, respectively, with weak genetic differentiation. However, the SD and TJ populations did not show apparent genetic differentiation (Fst = 0.0039), consistent with the population structure analysis and principal component analysis results. Consistently, in Li’s study, the genetic differentiation of wild roughskin sculpin from different geographical groups was not apparent .3), and hydrogen peroxide (H2O2), which involve the metabolic pathways of tryptophan, phenylalanine, and tyrosine. Because the protein source of artificial compound feed is different from that of natural feed, under long-term regular feeding and management, the selected genes related to amino acid metabolism may play an important role in the process of metabolizing artificial compound feed. It has been established that a higher standard metabolic rate can lead to increased efficiency in food intake needed for growth. In this respect, a previous study comparing transcriptomic profiles of farmed and wild Atlantic salmon showed that differentially expressed genes were highly correlated with energy metabolism [Fst and Π are effective methods for detecting selective elimination regions, especially when mining functional areas closely related to the living environment, which can yield strong selection signals . Indeed,tabolism .Overall, 2,610,157 high-quality SNPs and 494,698 InDels were identified using whole-genome sequencing, and the genetic diversity and population structure of two farmed and one wild population of roughskin sculpin were determined. The levels of genetic diversity were similar in all populations. Analysis of Fst values indicated low levels of genetic differentiation between the two farmed populations and wild populations and the lowest levels of genetic differentiation between the two farmed populations. According to population structure and principal component analysis, two clusters were detected in all the populations, and the two farmed populations could be clustered into one population, suggesting seedling exchange between the Shandong and Tianjin populations, which might affect the adaptability of roughskin sculpin. Selective sweep analysis showed that the selected genes in the two populations were related to amino acid metabolism, which might be caused by artificial feeding. These results provide essential information for the conservation of genetic diversity in roughskin sculping. Accordingly, corresponding genetic diversity conservation measures can be formulated based on to the genotyping results. Finally, this study demonstrates that whole-genome resequencing is an efficient genome-wide SNP discovery tool that can accurately evaluate population genetics in roughskin sculpin.


Centropomus undecimalis (common snook) and Centropomus parallelus (fat snook) have a wide distribution from southern Florida to southern Brazil. Due to their value as a food source, these species have been heavily exploited through predatory fishing, posing a conservation challenge. To assess their genetic diversity and population structure, we used microsatellite markers. Our findings revealed genetic differences among populations of the same species, highlighting the need for targeted conservation efforts. The microsatellite markers proved effective in assessing genetic variability, providing valuable insights for management and conservation. The parameters Ho (observed heterozygosity) and He (expected heterozygosity) were reliable indicators of genetic diversity, and specific loci showed varying allele numbers across populations. Our study contributes to understanding population genetics in these snook species and supports their conservation. Despite not being classified as endangered, genetic differences among populations emphasize the importance of considering population-level characteristics in conservation strategies. This research lays the foundation for future studies and actions aimed at preserving these valuable fish species. In summary, our study demonstrates the significance of microsatellite markers in assessing genetic variability and population structure in common snook and fat snook, informing conservation efforts for these species. Brazil has a coastal area spanning over 8000 km, accompanied by a vast volume of freshwater, making it one of the largest hydrographic basins globally. With its rich marine biodiversity, the country’s marine fish farming industry is experiencing continual expansion and economic growth ,2. In 20Centropomus spp. genus stand out. Centropomus spp. is the sole genus within the predominantly marine fish family Centropomidae (Teleostei) and comprises 12 species commonly known as snooks . These species can be found in both the eastern Pacific and western Atlantic [Centropomus undecimalis  and Centropomus parallelus  being of particular interest for economic and sport fishing. These species have a wide distribution, ranging from southern Florida (Gulf of Mexico) to southern Brazil (Rio Grande do Sul).Among the various marine species with a commercial interest and potential for exploitation, the species of the Atlantic , which wAtlantic . Among tThese species are carnivorous, primarily feeding on fish, shrimp, and other crustaceans. However, their feeding habits exhibit some seasonal variation, as they demonstrate opportunistic predation depending on the environmental availability ,7. Due tC. undecimalis, reaching sizes of up to 140 cm and weighing up to 22 kg [C. parallelus can grow up to 75 cm and weigh up to 4 kg, with females generally being larger than males of the same age [to 22 kg , is one to 22 kg . On the same age . Snook ssame age .To enhance production efficiency and profitability in aquaculture, a diverse range of genetic resources is necessary. The research focused on genomics, genetics, and breeding in aquaculture aims to improve production efficiency, sustainability, product quality, food safety, consumer protection, and overall profitability, ultimately benefiting consumers . TherefoPopulation genetics encompasses the study of allelic distribution and changes resulting from natural selection, mutation, migration, gene flow, geographic distribution, and genetic drift . Loss ofC. undecimalis and C. parallelus populations. Our aim was to determine if these species are geographically connected. We utilized 10 microsatellite loci as genetic markers, which allowed us to gather valuable data for management, sustainability, and conservation strategies for these species. Furthermore, the genetic information obtained in this study can serve as a foundation for future breeding programs focused on snook species.In this study, we employed microsatellite markers to assess the genetic variability and characterization of C. undecimalis and C. parallelus specimens were collected from artisanal fisheries in the Santa Catarina to São Paulo region of Brazil  and 13 samples from Guarujá–SP (pSP). In addition, 66 samples of C. undecimalis were collected, including 18 samples from Balneário de Penha–SC (uSC), 8 samples from Guarujá–SP (uSP), and 40 captive samples from CERES/UDESC (College of Southern Region/Santa Catarina State University) and UFSC -LAPMAR (Marine Fish Laboratory) (uCap).Samples of f Brazil . SpecifiBiological tissues from the animals  were collected for DNA extraction. The collected samples were stored in 1.5 mL microtubes containing 100% ethanol and kept in a freezer at −20 °C in the Laboratory of Genetics and Molecular Biology at CERES-UDESC in Laguna, SC, Brazil.DNA extraction was carried out using the phenol/chloroform protocol as described by Sambrook et al. . Approxi2, 2 μL of the dNTPs mixture , 1 μM of each primer (20 μg/μL), 1.25 μL of Taq Polymerase enzyme (Invitrogen), and H2O to complete the final volume. The PCR reactions were carried out in a thermal cycler with an initial denaturation step at 94 °C for 5 min, followed by 35 cycles of denaturation at 94 °C for 45 s, annealing at a temperature of 58–60 °C (depending on the primer pair) for 90 s, and extension at 72 °C for 7 min.PCR reactions were performed to amplify the loci using 10 STR markers: Cun01, Cun05B, Cun08, Cun09, Cun10A, Cun11, Cun14, Cun16, Cun21B and Cun22. The primer pairs for these markers were described by Seyoum et al. . The PCRThe sequences obtained were analyzed using the GenAlEx 6.5 program  to calcuC. undecimalis and C. parallelus. Microsatellite markers have been widely used in fish species, as they exhibit variable numbers of alleles per locus. These markers are abundant in the genomes of higher organisms, including humans [C. undecimalis, microsatellite loci were previously described by Seyoum et al. [C. parallelus. The length of the alleles in this study ranged from 80 bp for locus Cun21B to 293 bp for locus Cun09 in C. undecimalis. In the study by Seyoum et al. [The microsatellite loci used in this study exhibited high polymorphism, allowing for the assessment of genetic variability in the populations of g humans ,35. In tm et al. , and a sm et al.  also utim et al. , the varC. undecimalis. In the uSC population, the loci Cun08 and Cun11 exhibited the highest number of alleles (22), while locus Cun05B had the lowest number of alleles (7). In the uSP population, locus Cun12 had the highest number of alleles (12), while locus Cun05B had the lowest number of alleles (3). In the uCap population, locus Cun01 had the highest number of alleles (32), while locus Cun05B had the lowest number of alleles (13). For C. parallelus in the pSC population, locus Cun08 had the highest number of alleles (24), while locus Cun05B had the lowest number of alleles (4). In the pSP population, locus Cun14 exhibited the highest number of alleles (15), while locus Cun16 had the lowest number of alleles (6).The number of alleles varied among the three populations of C. parallelus and C. undecimalis. A high number of different alleles per locus is indicative of high polymorphism. However, it is important to consider the sample size of each group studied, as this can strongly influence the number of alleles detected [Although the loci with the highest number of alleles varied, locus Cun05B consistently exhibited the lowest number of alleles (ranging from 3 to 13) in all populations of detected ,38. For In addition to allele number, other parameters need to be considered in population genetics analysis. Ho (observed heterozygosity) and He (expected heterozygosity) are reliable parameters. Notably, locus Cun05B consistently exhibited the lowest Ho value. In the uSP population, this same locus had a Ho value of 0.5, while all other loci had a Ho value of 1.C. parallelus and C. undecimalis analyzed in this study, some null alleles were detected. In C. undecimalis, null alleles were observed in loci Cun5B, Cun16, and Cun21B in the uSP population; loci Cun14, Cun21B, and Cun22 in the uSC population; and loci Cun10A, Cun11, Cun14, and Cun22 in the uCap population. In C. parallelus, null alleles were detected in loci Cun10A, Cun11, and Cun16 in the pSC population; and loci Cun14, Cun21B, and Cun22 in the pSP population.The presence of null alleles in population genetics can affect estimates of genetic variability and genetic distances . DespiteIn the current study, the analysis of genotypic and genotypic differentiation based on observed allele and genotype frequencies revealed possible genetic differentiation among the populations analyzed. Studies on population differentiation in fish have been conducted to identify and manage distinct genetic pools. Population differentiation can manifest in various forms, ranging from genetically distinct populations to panmictic populations. The conservation units are typically represented by genetically homogeneous groups of individuals, such as local populations .C. undecimalis, in the fSP population, the observed heterozygosity (Ho) ranged from 0.5 to 1, with an average of 0.95, while the expected heterozygosity (He) ranged from 0.8 to 0.91, with an average of 0.82. In the fSC population, Ho ranged from 0.39 to 1, with an average of 0.89, while He ranged from 0.9 to 0.94, with an average of 0.89. In the fCap population, Ho ranged from 0.68 to 1, with an average of 0.93, while He ranged from 0.85 to 0.95, with an average of 0.92. Regarding the populations of C. paralellus, in the pSC population the HO ranged from 1 to 0.55 with an average of 0.86, while the He ranged from 0.9 to 0.94 with an average of 0.89. In the pSP population, the Ho ranged from 1 to 0.77 with an average of 0.95, whereas the He ranged from 0.9 to 0.85 with a mean of 0.84.Regarding the populations of C. undecimalis populations, according to Wright [C. parallelus, and the uSC and uSP populations of C. undecimalis. However, the uCap population showed a closer genetic relationship to the uSP population than to the uSC population (The coefficient of inbreeding (FIS) did not show negative values, indicating that there is no excess of heterozygotes in these populations. FIS varied among all loci and populations of both species, as shown in o Wright . The pripulation .The loci  did not conform to Hardy–Weinberg equilibrium across populations and species . The difC. undecimalis and C. parallelus between the coastal regions of the Brazilian states of São Paulo and Santa Catarina [The capture of these species is associated with migratory movements to freshwater ecosystems and spawning along the coast. The environmental differences found along the coast have an ecological and evolutionary impact, favoring genetic differentiation. Surprisingly, there have been no studies comparing the species and populations of Catarina ,44,45.C. parallelus. The populations also showed distinct allelic frequencies for certain alleles, indicating the presence of genetically consolidated populations. Genetically consolidated populations refer to groups of organisms of the same species that share similar genetic characteristics and have a shared evolutionary history [C. parallelus, suggesting the possibility of reproductive contact. Furthermore, the figure shows that the domesticated population of C. undecimalis is more closely related to the population of São Paulo than to Santa Catarina.Although the scientific literature lacks sources on these two species, especially regarding their genetics, some considerations can be made based on our data. In summary, our data demonstrate that these populations exhibit high genetic variability, including the recently domesticated population of  history . The figThe initial hypothesis of this study was that the populations of both species in Santa Catarina and São Paulo would exhibit little difference, given the relative proximity of these states in Brazil. However, the results of this study contradicted this hypothesis. The findings clearly demonstrate that there is no intense reproductive contact between these populations, indicating a significant degree of genetic differentiation. This is particularly interesting considering that these species have a wide distribution, spanning from the south of Florida (Gulf of Mexico) to the south of Brazil (Rio Grande do Sul).The observed genetic differentiation suggests that factors such as geographic barriers or local environmental conditions may be contributing to the distinct genetic profiles of these populations. Further studies involving other populations along the Brazilian coast could provide additional insights and help identify which populations are more closely correlated.It is important to note that low genetic variability is a concerning sign for the conservation of a species . TherefoCentropomus undecimalis and Centropomus parallelus. The results shed light on the genetic differentiation between populations and emphasize the need for further research to understand the underlying factors driving these patterns. Such knowledge is crucial for the effective management and conservation of these species, ultimately ensuring their long-term survival and sustainability.In summary, this study highlights the importance of genetic studies in assessing the populations of Centropomus parallelus and Centropomus undecimalis populations, providing valuable data on their genetic structuring. This information is essential for the management and conservation of these species. Moreover, the development of ten polymorphic microsatellite loci for both species opens possibilities for future studies and the potential for heterologous amplification. Although neither species is currently listed as endangered, the genetic differences observed among the studied populations highlight the importance of considering genetic diversity even within species. The Centropomidae species, including C. parallelus and C. undecimalis, hold significant economic and recreational value, particularly in the context of sport fishing. The findings of this study can serve as a foundation for future breeding programs and contribute to the development of tools for fish farming. It is worth noting that this study represents the first genetic characterization of populations of C. parallelus and C. undecimalis in Brazil, their primary habitat. This genetic characterization plays a crucial role in monitoring the species for conservation purposes, particularly in relation to genetic variability. The data obtained not only contribute to the understanding of the reproductive nature of these species but also shed light on their gene flow dynamics. Overall, this genetic study provides valuable insights into the populations of C. parallelus and C. undecimalis, offering a foundation for conservation efforts and informed management strategies. By understanding the genetic variability and structure of these species, we can implement effective measures to ensure their long-term survival and contribute to the sustainable use of their resources.The use of microsatellite markers proved to be highly effective in studying the genetic variability of


Camellia species, a group of habitat specialists confined to karst/non-karst habitats in southwestern China.Hybridization is generally considered an important creative evolutionary force, yet this evolutionary process is still poorly characterized in karst plants. In this study, we focus on natural hybridization in yellow C. micrantha and C. flavida, and specifically confirmed their hybrid population, C. “ptilosperma”. Ecophysiological results suggested that extreme hydraulic traits were fixed in C. “ptilosperma”, these being consistent with its distinct ecological niche, which lies outside its parental ranges.Based on population genome data obtain from double digest restriction-site associated DNA (ddRAD) sequencing, we found evidence for natural hybridization and introgression between The identified hybridization event is expected to have played a role in generating novel variation during, in which the hybrid population displays different phenological characteristics and novel ecophysiological traits associated with the colonization of a new niche in limestone karst.The online version contains supplementary material available at 10.1186/s12870-023-04164-4. Natural hybridization is known to occur in numerous lineages, leading to diverse possible evolutionary consequences –3. In maBegonia) in karst regions [Karst landforms are edaphically (soil-related) special terrestrial habitats whose floral composition is unique . In sout regions –37. Kars regions , but no Camellia species (simply referred to as ‘yellow camellias’), are a group of Camelllia plants whose flowers are yellow. According to the Flora Reipublicae Popularis Sinicae along with subsequent reports of newly described species, there are more than 20 species of yellow camellias that are confined to small areas of southwestern China [C. flavida occupies its karst valley while C. perpatua is restricted to its upper slope near to peak. In addition, several interspecific hybridization events have been inferred among the yellow Camellia plants growing in China [Yellow rn China –44. Yellin China . The higC. micrantha and C. flavida are morphologically distinguishable and favor different habitats: C. micrantha grows exclusively in non-karst forests  is restricted to a very limited area  to explore whether C. “ptilosperma” has a greater tolerance of specific habitat stresses than its parent species. The obtained data and findings provide for a possible mechanistic basis for lineage diversification in yellow camellias.To this end, here we employed double digest restriction site-associated DNA sequencing (ddRAD-seq) for genomic analysis, and investigated the ecophysiological features associated with habitat adaptation. The specific aims of this study were to: (1) test leading hypotheses about the hybrid origin of C. micrantha, C. flavida, and C. “ptilosperma”, respectively. The dataset obtained using the STACKS program contained 705 554 SNPs. After filtering, the 4422 SNPs obtained and used for subsequent analyses.A total of 2 441 209 808 , 4 004 099 068 , and 582 841 776  reads were obtained from the 53, 91, and 17 individuals of C. “ptilosperma” population showed a high degree of admixed ancestry, with ca. 68% was derived from C. micrantha and ca. 32% derived from C. flavida. Moreover, C. flavida further diverged into different lineages at K = 3 , this being especially distinct at K = 2 and K = 3  method in ADMIXTURE revealed an optimal value of K = 4 Fig. . For K == 3 Fig. j. The NG= 3 Fig. j and theC. micrantha and C. flavida were well differentiated from each other along PC1, which explained 9.31% of the variance. The individuals of C. “ptilosperma” and the NG-LR and NG-GLA populations of C. flavida that showed genetic introgression had intermediate values along PC1, yet were distinguishable along PC2, which explained 4.96% of the variance  results uncovered four major clusters, which corroborated the result obtained from the ADMIXTURE analysis Fig. a. In thiC. flavida was estimated to be higher than that of either C. “ptilosperma” or C. micrantha . The genetic differentiation was greater between C. “ptilosperma” and C. micrantha (FST = 0.268) than between C. micrantha and C. flavida (FST = 0. 225)  Fig. a.C. micrantha), N2 (C. “ptilosperma”), N3 (NG-LR and NG-GLA populations of C. flavida), N4 (C. flavida) and Na were 7.73 × 103, 3.30 × 103, 1.06 × 103, 1.25 × 104, 8.20 × 103, respectively  0.6097–0.6536), much higher than for the other nine scenarios Fig. . For sceely Fig. . The medely Fig. . Principely Fig. , suggestC. “ptilosperma” arose hybridization events between C. micrantha and C. flavida, and populations of NG-LR and NG-GLA was originated from introgression of C. micrantha genes into C. flavida  approach showed that the most supported scenario (Scenario 1) was the one considering ida Fig. b.C. “ptilosperma” vis-à-vis its putative parental species  for the C. “ptilosperma” was significantly more negative than that of either XH-NP (p = 0.01810) and XH-FL (p = 0.00002) populations of C. micrantha and likewise for the NG-LT (p = 0.00020) and NG-LR (p = 0.00041) populations of C. flavida. No significant differences were observed between the ΨTLP of C. micrantha and C. flavida , and ray and axial parenchyma (RP and AP)  Table .C. micrantha and C. flavida in narrow sympatric zone of southwest Guangxi (China), despite their strong morphological and ecological differentiation. The evidence for this came from a suite of complementary results for gene clustering, PCA, and the phylogenetic network analysis based on SNP data  is likely of hybrid origin and subsequently backcrossing with its parents, suggesting it could be a hybrid swarm. Specifically, C. micrantha served as the maternal parent while C. flavida served as the paternal parent during the initial hybridization event.Overall, population MZ-SJ (TLP) as well as vessel diameter in branches of C. “ptilosperma” differed significantly from its parental species.The ecophysiological experiments revealed that the focal taxa exhibited inherent differences in their hydraulic traits related to water availability. In particular, we find that the water potential at turgor loss point  have become well fixed in C. “ptilosperma”, conferring to it a greater ability to withstand drought, thus allowing it to grow on karst hillslopes where they must rely on shallow soil water. Moreover, C. “ptilosperma” differs in a key phenological trait from its ancestral parents. Best known for its long flowering period  [C. “ptilosperma” had been considered as the candidate for generating new cultivated varieties in yellow-flower Camellia breeding [C. micrantha plants flower from November to December (peak flowering in November), and C. flavida flowers from September to November (peak flowering in September to October). This phenological difference is genetically based, as it persists for these plants when growing in a common garden of Golden Camellia Park in Nanning, Guangxi. Accordingly, the ecological novelty and distinct phenology attribute of C. “ptilosperma” most likely arose through the combination of genetic material from those two parents.The hybrid derivative in July) , C. “ptibreeding , 78. IntC. “ptilosperma” could serve as an effective barrier to genetic exchange with its parents, contributing to its persistence, and thus promoting conditions for the establishment a new adaptive lineage. Likewise, limestone karst areas provide an essential habitat suite for the generation of hybrid plants, given that novel ecological opportunities are crucial for the origin of hybrid species [Notably, the specific traits found in  species , 17. Kar species . Soil nu species , 79, 80.C. micrantha and C. flavida that gave rise to hybrid population, C. “ptilosperma”. The novel hydraulic traits fixed in C. “ptilosperma” explain its distinct ecological niche, which lies outside its parental ranges. We highlight the role of hybridization in facilitating the evolution of hybrid population with unique phenological and ecological. This study reveals a case of natural hybridization that facilitated lineage diversification in yellow camellias.We found evidence for natural hybridization between C. flavida  and 6 populations of C. micrantha  were sampled from their natural collection sites (hereafter referred to as populations) in southwestern Guangxi, China  have been severely destroyed, the samples for it used in this study could only be collected from just 17 individuals. All the studied taxa were diploid (2n = 30) [A total of 10 populations of 2n = 30) .EcoRI and NIaIII. Paired-end 150 bp reads were sequenced using a NovaSeq 6000 (Illumina) platform. The raw reads obtained from each sample were deposited at the NCBI Sequence Read Archive (SRA) database (accession number: PRJNA770534).Fresh leaf tissues were collected from all the sampled plants and preserved on dry ice for sequencing. Total genomic DNA was extracted from each tissue sample by using a modified cetyltrimethylammonium bromide (CTAB) protocol  and thende novo loci using the STACKS v2.54  pipeline [process_radtag module of STACKS was used for quality-filtered and demultiplexing the raw fastq reads. In order to remove the tags with intact restriction enzyme cut sites and ambiguous barcode near the 3′ ends, the sequences were trimmed to a length of 135 bp using the SEQTK tool [ustacks), which were used to build a catalog (cstacks) for variant calling. In these two steps, two main parameters  were identical to those suggested previously for use in phylogenomics of yellow camellias [sstacks. Pair-end reads were associated with each single-end locus using tsv2bam and assembled into contigs using gstacks. The output SNP data files were exported using the populations in STACKS. The loci that were sequenced from at least 80% of the individuals in each population (-r 0.8) in at least 13 populations (-p 13) were retained.The sequences were assembled into pipeline  for obtaQTK tool . The cleamellias , which wamellias . All theThe original variants were filtered for generating a final SNP dataset using the VCFtools v0.1.16 software . The folhttps://www.r-project.org/). The pattern of genetic clustering was subsequently visualized by creating population splits based on the uncorrected P-distance between individuals using the Neighbor-Net algorithm of SplitTree4 program [A population genomic approach was employed for analyzing the population genetic structure in this study, and the hybrids were identified using the ADMIXTURE program . The ana program .HO), expected heterozygosity (HE), and nucleotide diversity (π) across loci, were estimated using STACKS v2.54 [FST), and the significance of the observed FST was determined using 10,000 permutations in the Arlequin program [Genetic statistics, including the average values of major allele frequency (P), observed heterozygosity , two intraspecific lineages of C. flavida that included the populations NG-LR and NG-GLA, and all remaining populations. We used approximate bayesian computation (ABC) to compare different evolutionary scenarios for the origin of population MZ-SJ and populations NG-LR and NG-GLA of C. flavida using the software package DIYABC v.2.1.0 [C. micrantha/C. flavida and hybridize with the second species. Under scenarios 5 and 6, the MZ-SJ population/C. flavida populations NG-LR and NG-GLA originate from crosses between C. micrantha and C. flavida then respectively follow introgression from C. micrantha to C. flavida. Under scenarios 7 and 8, the lineages arise from a single origin based on a hybridization event between C. micrantha and C. flavida (the MZ-SJ population and C. flavida’s NG-LR and NG-GLA populations diverge due to cladogenesis from this hybrid lineage), whereas they diverge from C. flavida under scenarios 9 and 10, respectively.Based on the ADMIXTURE, PCA and Neighbor-net tree results, four genetic group were defined. These consisted of  v.2.1.0 . A totalhttps://github.com/loire/vcf2DIYABC.py. To meet the requirements of DIYABC, SNPs were removed if they include monomorphic loci. For each prior, we set the interval for population sizes and divergence time to 10–105, and that for admixture rates to 0.001–0.999, because we lacked sufficient knowledge about the population sizes, divergence times, and admixture rate of Camellia species. A total of 25 summary statistics including genetic diversity, pairwise sample FST, and Nei’s distance as well as various admixture summary statistics were used to compare the observed versus simulated data [6 simulations were performed, from which we estimated parameter posterior distributions by taking the 1% of simulated datasets closest to the observed dataset for use in local linear regression. To compare the posterior probability of the 10 scenarios, the 10 000 (1%) simulated datasets closest to the observed dataset were selected for use in logistic regression and 500 for use with the direct approach.The variant call format (VCF) file of the unlinked SNPs was converted to DIYABC format using the Python script vcf2DIYABC.py available from ted data . For eacAfter choosing the best model, the posterior distribution of each parameter was also estimated by taking 1 000 (1%) simulated datasets closet to the observed dataset for the local linear regression and applying a logit transformation to a given parameter’s values. The goodness-of-fit of the tested scenarios was assessed implementing the model-checking function of DIYABC. The PCA was carried out to visually assess the position of the observed dataset vis-à-vis the simulated datasets.C. flavida (NG-LT and NG-LR), C. micrantha (XH-BS and BY-NP), and C. “ptilosperma” (MZ-SJ). Ecophysiological trait data were obtained from three sets of measurements. The first being leaf pressure–volume (P–V) curves obtained for each taxon to determine the relation of its leaf-level water potential at the turgor loss point to physiological drought tolerance. The second was hydraulic capacitance, assumed here to be associated with water storage strategy. The third set of measurements comprised wood anatomy traits, which are often tightly linked to the water availability of plants. We measured all the hydraulic traits of plants during the dry season (in October 2021). All measurements were performed in the laboratory of Experimental Center of Tropical Forests, Chinese Academy of Forestry . Materials were moved into the laboratory within an hour of sampling.The study site was located at Nonggang National Natural Reserve, Pingxiang Munipality and Daqing Mountain, Longzhou county, Guangxi, Southern China . The study region is profoundly influenced by the subtropical monsoon. The mean annual temperature is 22 °C; precipitation ranges from 1150 to l550 mm per year, interrupted by a dry season from October to April . The sel2) was measured using a scanner.The leaf P-V curves were generated using the bench drying method . For eacThe capacitance was measured according to the methods described by Jupa, Plavcová, Gloser and Jansen . Five olWf and Wd denote the weight of a given sample before and after its dehydration, respectively, and Ws is the weight of the sample when fully saturated. Cumulative water release  was calculated as follows:where − 3), this expressed as the ratio of dry mass of a sample to its volume. To describe the relationship between the CWR and water potential (Ψ) of each Camellia species, the data were fitted to the hyperbolic function:where a is the best-fitting parameter denoting the value of CWR in the asymptotic region of the curve, and b is the best-fitting parameter for the values of Ψ. The shape of the curve was divided into two phases according to the b parameter value: phase I (from Ψ = 0 to b MPa) and phase II (from Ψ = b to − 8 MPa). Phase I was characterized by the initial rapid release of water, concurrent with a decline in Ψ, and phase II corresponded to the period of gradual water release. Finally, the capacitance in the two distinct phases was calculated as the ratio of ∆CWR to ∆Ψ:where After measuring the hydraulic capacitance, the remaining fresh branches and roots were used to analyze the anatomical characteristics of each sample’s woody tissue. To do this, transverse sections (approximately 25 μm) were prepared using a sliding microtome . under a light microscope  and the obtained images then analyzed in ImageJ software . vessel Below is the link to the electronic supplementary material.Additional file 1Additional file 2