Ruditapes philippinarum, a major cultured shellfish species, is threatened by infection with the microparasite Perkinsus olseni, whose prevalence increases with high water temperatures. Under the current trend of climate change, the already severe effects of this parasitic infection might rapidly increase the frequency of mass mortality events. Treating infectious diseases in bivalves is notoriously problematic, therefore selective breeding for resistance represents a key strategy for mitigating the negative impact of pathogens. A crucial step in initiating selective breeding is the estimation of genetic parameters for traits of interest, which relies on the ability to record parentage and accurate phenotypes in a large number of individuals. Here, to estimate the heritability of resistance against P. olseni, a field experiment mirroring conditions in industrial clam production was set up, a genomic tool was developed for parentage assignment, and parasite load was determined through quantitative PCR. A mixed-family cohort of potentially 1,479 clam families was produced in a hatchery by mass spawning of 53 dams and 57 sires. The progenies were seeded in a commercial clam production area in the Venice lagoon, Italy, where high prevalence of P. olseni had previously been reported. Growth and parasite load were monitored every month and, after 1 year, more than 1,000 individuals were collected for DNA samples and phenotype recording. A pooled sequencing approach was carried out using DNA samples from the hatchery broodstock and from a Venice lagoon clam population, providing candidate markers used to develop a 245-SNP panel. Parentage assignment for 246 F1 individuals showed sire and dam representation were high , indicating a very limited risk of inbreeding. Moderate heritability (0.23 ± 0.11–0.35 ± 0.13) was estimated for growth traits , while parasite load showed high heritability, estimated at 0.51 ± 0.20. No significant genetic correlations were found between growth-associated traits and parasite load. Overall, the preliminary results provided by this study show high potential for selecting clams resistant to parasite load. Breeding for resistance may help limit the negative effects of climate change on clam production, as the prevalence of the parasite is predicted to increase under a future scenario of higher temperatures. Finally, the limited genetic correlation between resistance and growth suggests that breeding programs could incorporate dual selection without negative interactions.The Manila clam  Infectious diseases continue to be a major limit in fish and shellfish aquaculture, with production losses estimated to cost over 6 billion USD per annum, and far more in treatments such as vaccinations, cleaning, and antibiotics , 2. The Over the past 20 years, a number of commercial and experimental breeding programs have been introduced for mollusks –18, manyVibrio tapetis and shell repairing has also been reported after three generations of mass selection by SATMAR hatchery . In addition, an important genotype-by-environment (GxE) interaction appeared to have been induced by highly variable ranges of salinity and water temperature between sites.The Manila clam is a bivalve species of particular commercial interest as it represents 25% of global mollusk production worldwide, and although it was introduced to Europe in the early seventies, European production currently accounts for only about 2% of global production –33. To dhatchery . In anotr growth  and resiathogens . In M. polyticus , and difolyticus . Howeverelection –44. As iP. olseni has been identified as a major pathogen of the Manila clam, responsible for mass mortality of up to 70% in experimental infections, and affects clam production worldwide, including in the clam production zones of northern Italy , Legnaro (PD)].An experimental population of clams was produced by mass spawning in May 2016 at the French hatchery SATMAR  from 109 adult clams of hatchery broodstock, according to a two-factorial mating design: 31 dams × 25 sires (775 putative families) and 22 dams × 32 sires (704 putative families), representing potentially 1,479 F1 families. Soon after fertilization, the larvae from the two pools were mixed together and reared at the SATMAR hatchery in a common tank in order to avoid confounding between genetic and common environment effects. After spawning, the parents were sacrificed and a piece of mantle from each of the 109 parent clams was kept in 99% ethanol for future DNA extraction and genotyping. In September 2017 a batch of 10,000 progenies was seeded in Chioggia at 1.1 g , a commercial grow-out site in the Venice lagoon, Italy, where there is a high prevalence of the pathogen P. olseni in the gill tissues of the F1 clams was evaluated monthly since the time of seeding. Each month, about 30 clams were sampled for whole weight, shell weight, and shell length, and the gills were dissected and frozen in liquid nitrogen for subsequent DNA extraction. Biometric measurements allowed us to follow growth parameters of the clam experimental population [Over the course of 1 year, the prevalence and intensity of P. olseni infestation and, having spent 1 year in grow-out conditions, had reached commercial size. This threshold of infestation was chosen according to Chapuis et al., allowing better settings with which to maximize differences between families and thus increase the precision of the genetic parameter estimates  to stimates . The numr parent . This isr parent  demonstrP. olseni, and thus it was possible to carry out total DNA extraction from the same sample for both parasite quantification and host genotyping (see below: P. olseni quantification). All of the individual phenotype data was recorded in the INFAQUA database system dedicated to data storage and provided by SYSAAF. A description of all the traits measured can be found in In our study, 1,000 clams were individually weighed whole, then, meat and shell were separated and the shell was weighed after draining. Sex was determined, where possible, by visualization under a microscope of a gonad smear. Both gills were collected from each individual clam and stored in 1.5 ml 70% ethanol. Gill tissue was selected for the DNA extraction as it is the first tissue to become infected with P. olseni load in individual clams was estimated by real-time PCR using specific primers for P. olseni to amplify 1μl total DNA extracted from one whole clam gill . PCR cycles were performed using a 7,300 Real Time PCR system from Applied Biosystems as follows: 95°C for 10 min; 40 cycles at 95°C for 30 s and 60°C for 1 min followed by 72°C for 5 min and a melting curve at 95°C for 15 s; 60°C for 30 s and 95°C for 15 s  of the 109 Manila clams used as broodstock by the SATMAR hatchery were sent to the BCA laboratory for DNA extraction in March 2018.Fifty individuals of Extractions were carried out individually using Invisorb® DNA Tissue Kit . Total DNA was quantified by Qubit® 2.0 fluorometer and quality was evaluated on a 1% agarose gel. For the Venice lagoon samples, two equimolar pools of 25 individuals/pool were prepared, and the DNA was fragmented using Covaris and prepared for sequencing in 2017. For the broodstock individuals, samples were allocated to equimolar “lower quality” (LQ) and “higher quality” (HQ) pools based on molecular weight as visualized by electrophoresis.R. philippinarum draft reference genome  were prepared in triplicate using TruSeq® DNA Library Prep (Illumina). After Qubit 2.0 quantification and quality evaluation using Agilent 2100 Bioanalyzer, the triplicate libraries were pooled (equimolar) and sent for Illumina HiSeq 4000 for 150-bp paired-end sequencing at the Genome Center of the University of California—Davis. Reads were trimmed to remove low-quality reads using TRIMMOMATIC, and CLC Genomics Workbench was used for aligning raw reads to the e genome . SNP idee genome .SNPs detected by the PoPoolation pipeline in both Venice lagoon clams and hatchery broodstock DNA were selected based on the criteria listed in The 100 bp flanking sequences for the SNPs that matched all of the selection criteria were shared with Labogena Genotyping Company, which designed probe sequences using Illumina DesignStudio® Sequencing Assay Designer, following supplier recommendations. From the initial 250 probes designed, single probes for 245 SNPs were placed on an Illumina Infinium® genotyping chip able to genotype 96 samples/chip.−1) using the Infinium chip and analyzed on a fully automated Illumina iScan® platform, following supplier recommendations. Genotype quality control was carried out using GenomeStudio® software, according to the following standard Labogena clustering procedure: run of Illumina automated clustering algorithm  was included in the software parameters in order to correct assignments lying outside of the mating plan .Ne) were used to estimate the number of parents necessary to maintain genetic diversity, or allele frequencies, within a population. Ne can be calculated assuming random mating and equal parent representation among the F1 population, according to Kimura and Crow  and female (dam) parents, respectively.where Ne can give an indication of the risk of inbreeding, and in order to increase the accuracy of inbreeding estimations, the variability of family representation can be considered when calculating Ne, according to Harney et al.  can be calculated for a population and a number of generations (t) based on the Ne, according to Falconer  was included in the model. Genetic parameters were estimated using pedigree information with the following mixed animal model . a is a vector of additive animal genetic effects distributed following a normal distribution NA is the pedigree relationship matrix and p × p) genetic additive variance-covariance matrix. X and Z are incidence matrices for fixed and genetic additive effects, respectively, and ε is the residual component distributed following a normal distribution NI is the identity matrix and p × p) residual variance-covariance matrix.where h2) was calculated according the following formula:Heritability (p = 1), genetic additive covariance was estimated and genetic correlations (Rg) between two traits were calculated as follows:where Cova is the genetic additive covariance between trait 1 and trait 2, a(trait1)σ and a(trait2)σ are the standard deviations of additive genetic effect for trait 1 and trait 2, respectively.where Sampling occurred monthly over the course of 1 year, from the time of seeding (T0) to the final sampling event. Time-points and days post-seeding (dps) are detailed in At the time of seeding in the grow-out area, average shell length was 1.75 cm (SD = 0.33) and average whole weight was 1.115 g (SD = 0.626). At the final sampling date, 355 days post seeding (dps), average shell length was 3.48 cm (SD = 0.37) and average whole weight was 11.918 g (SD = 3.577). Growth, represented as average shell length over time, is reported in P. olseni (>10 copies.ng−1). Prevalence continued to increase, reaching 36.7% at T10 (322 dps), 1 month prior to the final sampling event (355 dps). The rise in P. olseni prevalence follows a trend similar to that of the average monthly water temperature, as it is well-known that the parasite is more virulent at higher temperatures. Final sampling was carried out when clams reached the commercial size for harvesting and at the end of the warmest summer months, when it was expected the prevalence would rise considerably. After diagnosis of all the sampled F1 individuals, the final prevalence was 51.5%.At each time-point over the course of the year-long monitoring, gill samples were collected from each of the clams and used for DNA extraction. Real-time PCR was carried out at the IZSve to quantify the parasite DNA for all samples from T4 (130 dps) to the final sampling (355 dps). Diagnosis was not carried out on samples from T0 to T3 (0 to 93 dps) as parasite levels were below the limit of detection (LOD). Prevalence (considered as the percent of samples above LOD) began to increase at T5 (187 dps), where 4.5% of the samples tested positive for Perkinsus-positive individuals above the limit of quantification (Perkinsus-free (negative).During the mass sampling event in Chioggia, clams were measured for total weight, shell weight, and shell length. Sex was recorded whenever it was possible to unambiguously assess gonadal sex, and final male to female sex ratio was 47:53 . Parasitfication . PositivP. olseni DNA, 268 showed detectable parasite DNA, but below the limit of quantification (LOQ: > 30 threshold cycle (Ct); equivalent to 100 copies of P. olseni DNA), and 544 samples contained a quantifiable amount of P. olseni DNA . Parentage assignment to a single parent pair was successful for 246 (41%) of the genotyped samples.n = 246), 163 families (or parent-pairs) containing 42 sires and 45 dams were observed out of the 1,479 putative full-sib families from the two-factorial mating design.Of the 56 sires and 53 dams used to produce the F1 clams, parentage assignment showed that most were represented in the F1 clams . DespiteNe), based on number of parents that were used in the mass spawning, was 108.9. After parentage assignment of the F1 clams, the observed Ne  was 86.9. When considering the variability in representation of the parents, i.e. the variance of the number of offspring per sire and dam,  is 1.28%.The initial expected effective population size (h2) was estimated for each trait, and genetic correlations (Rgtrait) between traits were calculated pairwise  and 0.97 ± 0.03 (SW vs. TW). The average measured values for biometric traits between negative, detectably (<LOQ), and quantifiably infected clams  tended tShifts in global temperature levels associated with climate change and other anthropogenic pressures are having increasingly negative impacts on marine ecosystems, namely with regard to pathogens. The geographical ranges of hosts and pathogens are being modified and their respective resistance capacity and virulence altered, leading to devastating mortality events due to both emerging and previously described pathogens. The viability of bivalve production will largely depend on the ability of the species to adapt to the future environmental conditions, through either natural or artificial selection of those individuals that are genetically more resistant to pathogens and stressors associated with global warming . To asseP. olseni under commercial conditions. Analysis of the growth trajectory confirmed previous findings for on-growing sites in Adriatic Sea lagoons, including the Venice lagoon (P. olseni was first detected in 4.5% of the F1 clams in March (T5), increasing steadily with the seasonal rise in seawater temperature. Optimal temperatures for proliferation of P. olseni have been shown to range between 20°C and 34°C , excluding reproductive success variance, was close to the expected Ne of 108.9), indicating that high genetic variability may be conserved when using DNA-parentage assignment to manage broodstock and limit over-representation of certain parents. When considering variance in reproductive success  Ne dropped to 39.0. When the observed Ne values were used to estimate inbreeding rate after one generation (ΔF = 1/(2*Ne), estimated ΔF was lower (0.006) or slightly higher (0.013) than the internationally recommend threshold (ΔF < 0.01) to avoid excessive increase of inbreeding rate in farmed animals  appeared to have contributed to the offspring at the time of analysis, over 2 years after the reproductive event. Comparable values of parent representation were reported in four hatchery populations of Pacific oyster, ranging between 64 and 100% for sires and 68–96% for dams. The clam progeny used here were obtained using mass spawning, a technique where all sires and dams are mixed together and release sperms and eggs in the same tank. In aquatic species such as bivalves, where a large number of animals participate in the spawning event, often only a limited number of sires and dams significantly contribute to the offspring, strongly reducing the effective population size , 77. The animals . In otheneration , while ineration . Even coiability , 82. Maiiability .h2 = 0.26 in the Manila clam . These heritability estimates fall within the range of realized heritability previously reported by mass selection for shell length of ila clam . While tTotal weight is highly correlated with shell weight (0.97) and shell length (0.9). This indicates that rapid assessment of growth can be done on the field by selecting only shell length. Similarly, as shell weight is highly correlated to total weight, it means that measurement of growth on sibs could be delayed and assessed by considering shell weight. However, as shell weight and length are less genetically correlated (0.78), and genetic correlation is lower than 0.8, these two last traits have to be considered as separate traits.When considering genetic correlations between traits, the correlated response to selection of one trait on another trait highlights the possible benefit the 52% higher heritability of SW compared to TW (0.35 vs. 0.23). Based on the Falconer (1960) formula  to estimP. olseni was estimated at 0.51 ± 0.20, indicating high potential for genetic improvement of this trait, though results should be interpreted with caution due to the low number of assigned individuals. Infections due to Perkinsus, with time and given the right environmental conditions, are likely to affect up to 100% of a Manila clam population. In the present study parasite load was recorded after 1 year on the field. In the past, natural spat was routinely used to seed on-growing areas, therefore clams were exposed to potential infection with P. olseni for a longer period (1.5–2 years) as the entire life-cycle occurred in the field. In recent years a dramatic drop in natural recruitment has forced producers to use hatchery-produced spat of larger size (12–15 mm), with a reduced potential exposure to P. olseni, much more closely resembling the experimental protocol that was followed in this study. It is important to consider that individual parasite load 1 year post-seeding was used a as a measure of resistance, which, in the case of chronic infections, may be an indirect assessment for resistance. In Penaeus monodon a limited genetic correlation was reported between gill-associated virus (GAV) load and survival (C. virginica regarding infection with P. marinus (see Introduction), evaluations for resistance to disease should integrate potential GxE effects brought on by variations in salinity or temperature between sites. Our results would thus greatly benefit from validation through future evaluations of response to realized selection experiments and larger data sets.Regarding disease resistance, heritability for parasite load with survival . MoreoveInterestingly, there was no genetic correlation between parasite load and growth-associated traits. Because of the limited number of analyzed individuals, however, the standard error for these estimates is large and caution should be exerted when considering genetic correlation. If the evidence of no genetic correlation between growth and parasite load will be confirmed using a large set of animals, then selection for one of these two traits may have no direct effect on the performance of the other, indicating that dual genetic selection could be implemented without running the risk of inducing a negative effect.P. olseni indicate there is a significant possibility of limiting the impact of this disease through genetic selection, not only in European clam aquaculture but also more broadly, as mass infestation and mortality due to this pathogen has also been frequently reported in Asia (P. olseni infection intensity and prevalence, it is likely that selected families that perform best in this specific environment may not perform as well for any of the selected traits in another clam production are such as along the Asian coast.Understanding the genetic correlations between various phenotypes is essential, as focusing selection on certain traits of interest can provoke devastating effects on other health-related traits, as demonstrated extensively in terrestrial livestock selection , 86. Ove in Asia –90. ThatP. olseni. Being a chronic parasitic disease, directly challenging selection candidates might lead to introducing the parasite in the broodstock population, while sib testing would allow to maintain a Perkinsus-free broodstock. In turn, this should guarantee the production of parasite-free spat.While the Manila clam is one of the most produced bivalve species in the world, the availability of genetic tools necessary to initiate selective breeding lag far behind those developed for most other cultured fish and shellfish. Here we showed that it is possible to design and carry out field challenges for one of the major clam pathogens, simulating commercial growing conditions. Likewise, we demonstrated that using mass spawning, the routine method for reproduction of clams in hatcheries, yields a sufficiently high number of full-sib families to reliably estimate genetic parameters and to provide a sufficient number of selection candidates. The development of a SNP panel allowed for partial parentage assignment, albeit further improvement in successful genotyping and assignment rate is required for greater efficiency and more accurate estimation of genetic parameters. The possibility of reconstructing pedigrees is important not only for estimating genetic parameters and managing inbreeding, but it will be required to use sib testing for measuring phenotypes that either cannot be recorded in selection candidates or could be risky to measure on them, as in the case of resistance to Finally, these preliminary results indicate that the high heritability for resistance against perkinsosis and the apparent lack of genetic correlation with growth-traits hold the promise to mitigate the effects of this climate-related parasitic disease without affecting growth performance.https://doi.org/10.5061/dryad.rr4xgxd6d.The dataset can be found at: MS, CP, LB, and FE designed the experiment. EV and J-FA produced the experimental population. GA and GD carried out the Perkinsus analysis. SF and MS contributed to the bioinformatic analysis. LG and RM carried out the genotyping. FE, MS, and PH carried the quantitive genetic analysis. MS and LB drafted the manuscript. All authors contributed comments to the draft.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Lavandula angustifolia Miller is a Mediterranean aromatic shrub widely cultivated for its high quality essential oil used in perfumery and phytotherapy. Despite its economic importance, the intra-specific diversity among wild, non-cultivated plants remains poorly understood. We analyzed the structure of the chemical and genetic diversity of plants from 14 sites sampled over the entire native range of the true lavender. Volatile organic compounds of inflorescences were analyzed using gas chromatography coupled to mass spectrometry. Genotyping was performed with fingerprinting genetic markers. To limit the influence of environmental variability on chemical composition, plants were grown in the same conditions in a common garden. Without prior knowledge, discriminant analysis of principal component identified unambiguously four distinct chemotypes among three genetic populations. Co-inertia analysis and supervised analysis which integrated multiple datasets indicated a strong congruency between chemical and genetic patterns. Two distinct genetic units were located at the edge of the distribution area in the south of Italy and in the northeast of Spain, and were associated with two distinct chemotypes. Our results confirmed the existence of three genetically distinct entities, suggesting speciation. All French populations and the Italian Piedmontese population were genetically homogeneous but separated in two distinct chemotypes. The dominant chemotype was present in the center of the native range in southeastern France and was at the origin of the current most cultivated French varieties. Its main compounds were linalyl acetate, linalool, and caryophyllene oxide. The second French chemotype was found in south of Massif Central and presented high abundance of valuable linalyl and lavandulyl acetates. Linalool, eucalyptol, β-caryophyllene, borneol, camphor, and cis-sabinene-hydrate were significantly associated with southern latitudes and their role would be worth exploring.The true lavender  Lavandula angustifolia Miller) is a medicinal and aromatic plant of the Lamiaceae family. The plant grows in an open arid habitat and is endemic to the Mediterranean climate. The distribution of the species is determined by the occurrence of calcareous sedimentary rocks and schists. The natural range includes the south of France, the French and Spanish Pyrenees, and the Italian Alps [L. angustifolia sp. angustifolia in southern France and northern Italy, and L. angustifolia sp. pyrenaica in the Pyrenees. The Pyrenean lavender is characterized by broader bracts, larger flowers with a darker hue, and a felt-like indumentum of the calyx due to short highly branched hairs. More recently, based on morphological and phytochemical data, Passalacqua et al. [pyrenaica to species level according to the phylogenetic study of Moja et al. [The true lavender  used in perfumery, cosmetics, and therapeutics since antiquity [L. angustifolia sp. angustifolia was domesticated in the early twentieth century [https://www.iso.org and NF ISO 3515:2004 (T75-301), https://www.boutique.afnor.org). Lavender EO is rich in mono- and sesquiterpenes that are volatile organic compounds (VOCs) [https://www.boutique.afnor.org).The subspecies ntiquity . In Prov century . Seeds fs (VOCs) . The two15:2004 T5-301, htLavandula essential oils contribute largely to this growing industry with approximately 1500 tons produced annually [Lavandula such as L. stoechas and L. multifida. In L. stoechas, different studies identify a high level of variability in EO composition within wild populations collected in different areas . In L. ic belts . Populatic belts ,15 or de tobacco .Thymus vulgaris, six chemotypes  were identified and related to allelic variants using crossing experiments [Genetic inheritance of different chemical signatures within a species introduces the concept of chemotype, which is a chemically distinct entity within the same species. In eriments . These seriments . High ineriments . InheritIn this study, we analyzed the structure of chemical and genetic diversity of plants grown from 14 wild locations of true lavender over its native range, including Spanish and south Italian sites at the edge of the range. To disentangle phenotypic plasticity from local adaptation, seeds were collected in the wild and were grown in the same conditions in a common garden. Samples were collected at the same date on plants of the same age to limit the effect of ontogeny and weather conditions. For comparison with domesticated lavender, four different cultivars amongst the most cultivated in France  were included in the study. Plants were genotyped using amplified fragment length polymorphism (AFLP) fingerprinting markers and the content of terpenes in the inflorescences was obtained using gas-chromatography coupled with mass spectrometry. We inferred independently the structure of the chemical and genetic diversity using an unsupervised discriminant approach based on principal components, called DAPC . This mWe identified a total of 63 repeatable VOCs from our entire data set (14 sampled sites × 4.6 replicate plants per site + 25 plants from cultivated fields), which varied per site in the mean between 10.7 (Fam) and 55.2 (Ica) optim.a.score and xvalDapc) to determine the optimal parameters to construct the discriminant model. We ran the final discriminant analysis on the 4 first PCs (76% of inertia) to assign the individuals to the four groups. Stratified cross-validation with 1000 repetitions gave 99.2% of good assignment with two discriminant axes. The We first ran the unsupervised clustering method of DAPC on the chemical composition of sampled plants. We retained 20 principal components with 98% of inertia. The optimal number of clusters that maximized the inter-group variance and minimized the BIC was 4. We used the optimization and validation procedures (2 is high (0.60) and significant , indicating a good concordance between the two datasets. The DIABLO analysis is a complementary approach to co-inertia analysis , at the southeastern limit of the species range. Cluster 2 corresponds to the individuals of the Spanish areas . In France, two distinct chemical profiles are found. Cluster 1 groups individuals from the south of Massif Central , while cluster 3 covers all samples located in the center of lavender native range  together with all cultivars. Two individuals from the Spanish site Sar2 and three individuals from the south of Massif Central (3/5 Fav) are also assigned to the cluster 3 . In a second step, we run DAPC on the genetic data. The optimal number of genetically distinct groups is unambiguously evaluated to 3. Indeed, the cross validation process finds a success of assignment of 100%, whatever the number of PCs selected. One group corresponds to the isolated Calabrese plants (Ica), another contains the Pyrenean plants  and the last group clusters all French individuals and the Italian Piedmontese plants . The prostudies, ). It comPT between genetic clusters indicates a high level of differentiation with PHIPT ranging from 0.552, between French and Spanish clusters, to 0.752 between Calabrese and Spanish clusters  and the highest total amount . On the contrary, the Provencal chemotype (cluster 3) is the poorest. The mean percentage of monoterpenes ranges from 84.5 (cluster 3) to 90.7 (cluster 1) and only differs significantly between the two French chemotypes . A pairwise post-hoc comparison indicates that the chemical composition of all clusters differs significantly from each other . The redundancy analysis RDA detected significant differences in chemical composition between clusters , eucalyptol , β-caryophyllene , borneol , camphor , and cis-sabinene hydrate . These negative correlations may indicate a higher abundance of these compounds in southern populations. Terpinen-4-ol is positively correlated with latitude  and negatively correlated with altitude . Camphor is positively correlated with altitude  but the correlation is essentially driven by the high amount of camphor in the two Spanish populations located at 1000 and 1450 m  was suca priori known groups of samples  than toe oxide; ). This fc marker . The autLavandula angustifolia, constitutes a second genetically homogeneous unit. Chemical and genetic data show an important divergence from all other populations, suggesting the absence of gene flow and local adaptation. The differentiation is even stronger than with L. angustifolia sp. pyrenaica and the Calabrese population is probably a relict that has been isolated for a long time. Médail and Diadema [L. angustifolia sp. angustifolia and L. angustifolia sp. pyrenaica .The Calabrese population, the southernmost population of  Diadema  indicate Diadema , our resAll individuals in the center of the native range around Provence, including the Italian Alps, belong to the same genetic population and express the typical chemotype of the cultivated lavenders . Indeed,Among the largest genetic units including all French plants and the Italian Piedmontese population, a second chemotype was clearly distinct from the traditional chemotype. Constituted by individuals from south of Massif Central , in Lot,L. latifolia, Herraiz-Peñalver et al. [p < 0.001) and camphor , and associated with southern populations. The enantiomeric analysis of linalool should provide an explanation to these incongruent results. In Bulgarian crops, the major form is R-linalool with a ratio 95/5 [The composition in secondary metabolites of the inflorescence of true lavender was highly variable with significant differences for the number of compounds and the total amount of VOCs between clusters . The pror et al.  found thtio 95/5  but envitio 95/5  indicateThymus vulgaris, climate change has already modified the natural repartition of the six chemotypes: freezing sensitive chemotypes (with high contents in carvacrol and thymol) are now present in populations that were exclusively composed of freezing tolerant plants 30 years ago  [In the context of climate change, pinpointing compounds potentially involved in plant tolerance to high temperature or water stress is of particular interest for a sustainable production. In the case of motypes) . These cmotypes) . ClimateLavandula angustifolia Miller) was conducted over its natural range by Bernard Pasquier, director of the French national conservatory of fragrant, medicinal, aromatic, and industrial plants  and expert botanist of the genus Lavandula. Two hundred and eighty-two sites were identified as wild lavender sites (not recently escaped from cultivation). This work allowed us to update the map of natural distribution of true lavender  according to the manufacturer’s instructions. The AFLP procedure was carried out with restriction enzymes EcoRI and MseI following the protocol of Nicolè et al. . Three p−1 of cis-3-hexenol as internal standard (Sigma-Aldrich). After an overnight incubation at 4 °C, supernatant was collected and stored at −20 °C. Supernatant was analyzed in gas chromatography-mass spectrometry (GC-MS) with an Agilent 6850N GC, equipped with a 5973N mass selective detector and a DB-5 MS column (30 m × 0.25 mm with a 0.25 µm film-Agilent 122-5532). The initial temperatures of injector and detector were at 250 and 260 °C, respectively. The gas carrier was helium at a flow rate of 1 mL min−1. The injection volume was 2 µL with a split ratio of 5:1. The oven’s initial temperature was maintained at 40 °C during 6 min after injection; then ramped at 1 °C min−1 to 60 °C, 2 °C min−1 to 140 °C, and 12 °C min−1 to 240 °C. The oven temperature would finally remain at 240 °C for 5 min. Finally, we could obtain reliable profiles for 90 samples . A relative area for each compound was thus calculated per dry mass unit and by correcting for differential evaporation during sample handling using the internal standard. A commercial range of n-alkanes C8–C20 (Fluka) was co-injected to calculate retention indices. Identification of compounds was ultimately based on the comparison of retention time and mass spectra to commercial standards when available (lab bank of 80 standards). Identification was otherwise performed by using reference libraries of mass spectra [For quantification purposes, we checked the quasi perfect linearity between the range of concentration from 0 to 300 mg dry inflorescence per mL and the areas of peaks  implemelusters, ). We theIn a second step, for chemical data, discriminant analysis ran on the optimal number of clusters to identify discriminant compounds among chemotypes. Discriminant analysis requires the construction of an assignment model, associated with conditions of application. Linear discriminant analyses require the number of variables (compounds) to be less than the number of observations  to avoid overfitting problems. This condition is rarely met with chemical data and fingerprinting genotype. Likewise, abundance of compounds is rarely normally distributed for minor compounds and this prevents us from using parametric methods. However, partial least square discriminant analysis (PLSDA ) and disfunction xvalDapc). Redundancy analysis (RDA) was achieved to test for difference of the overall chemical composition between the chemical groups identified by DAPC [Similar to the classification procedure of DAPC, the discriminant function of DAPC used the first principal components as the new variables. These new synthetic variables were thus independent, normally distributed, and less numerous than the number of samples, allowing the application of a linear discriminant model. This discriminant model was evaluated by repeated stratified cross-validation  was performed using GenAlex 6.5  to estim2 which varies between 0 (no concordance) and 1 (perfect concordance). The significance of this statistic was tested using the function protest  was performed. Contrary to the bidimensional Mantel test, PCIA provides the capacity to deal with higher dimensional spaces. Indeed, PCIA fitted a set of points to another based on the ordination of distance matrices. Euclidean distance was calculated for chemical data and the Jaccard index on AFLP band sharing was used on the genetic dataset. These distance matrices were thus ordinated by principal coordinate analysis (PCoA) and the concordance of the two PCoAs was evaluated by a statistic called mge vegan ). We appge vegan  because


Nerodia spp.) are known to occur in saltwater habitat and represent a useful system for studying speciation by providing an opportunity to investigate gene flow and evaluate how species boundaries are maintained or degraded. We use double digest restriction-site associated DNA sequencing to characterize the migration-selection balance and test for evidence of ecological divergence within the Nerodia fasciata-clarkii complex in Florida. We find evidence of high intraspecific gene flow with a pattern of isolation-by-distance underlying subspecific lineages. However, we identify genetic structure indicative of reduced gene flow between inland and coastal lineages suggesting divergence due to isolation-by-environment. This pattern is consistent with observed environmental differences where the amount of admixture decreases with increased salinity. Furthermore, we identify significantly enriched terms related to osmoregulatory function among a set of candidate loci, including several genes that have been previously implicated in adaptation to salinity stress. Collectively, our results demonstrate that ecological differences, likely driven by salinity, cause strong divergent selection which promotes divergence in the N. fasciata-clarkii complex despite significant gene flow.The migration-selection balance often governs the evolution of lineages, and speciation with gene flow is now considered common across the tree of life. Ecological speciation is a process that can facilitate divergence despite gene flow due to strong selective pressures caused by ecological differences; however, the exact traits under selection are often unknown. The transition from freshwater to saltwater habitats provides strong selection targeting traits with osmoregulatory function. Several lineages of North American watersnakes ( Once thought to be rare, speciation with gene flow is now considered exceedingly common across the tree of life . SpeciatStrong selection caused by ecological differences between populations can lead to local adaptation and promote speciation . If seleNerodia spp.) lineages are known to occur in high-salinity environments  predominantly occupying—but not limited to—freshwater habitats and N. clarkii  occupying coastal, mostly saltwater habitats  identify population structure and infer the evolutionary history of the group, 2) characterize the role of gene flow and hybridization in shaping these patterns, 3) test for ecological niche divergence and determine if genetic differentiation or admixture is related to niche differentiation, 4) identify loci under selection and determine if they are related to osmoregulatory function, and 5) evaluate the implications of our findings for conservation in light of the threatened status of N. c. taeniata. To do this, we use a combination of Sanger sequencing and double digest restriction-site associated DNA sequencing (ddRADseq) to infer population structure and phylogenetic history. We use the ddRADseq data and multiple analytical methods to provide evidence of gene flow, including hybrid lineage formation, and search for outlier loci associated with osmoregulatory function. We use publicly available environmental data, including soil cation exchange capacity as a proxy for salinity, to test for differences in ecological niche and ask whether salinity may be a driver for genetic differentiation or admixture between the species. Finally, we utilize several morphological characters to explore their utility in diagnosing species or subspecies within this complex. For ease, we utilize the existing nomenclature to communicate and discuss our results.Given the ecologically divergent nature of the  clarkii . If adapThamnophis saurita). Our final filtered data set consisted of 119 individuals (including the outgroup) and 7,057 single nucleotide polymorphisms (SNPs). Excluding the outgroup, we identified population clusters using a discriminant analysis of principal components  for downstream analyses. In addition, we examined the geographic structure at K = 2 to assess if genetic clustering accurately represents the distribution of the two species  and found that the majority of the data are explained with two layers, suggesting that there are two significantly different IBD clines in our data  and was similar to the species ranges of N. clarkii and N. fasciata , two nuclear genes (PRLR and TATA), and two anonymous loci (M and E) for 85 individuals and 11 outgroups confirmed these results. Overall, single-gene sequencing provided little resolution within the N. fasciata-clarkii complex with large polytomies, low support, and the only clear distinction occurring between continental and peninsular populations  ranging from 0.44 to 0.75 . The results of Humboldt were inconclusive and prone to Type I error due to niche truncation or observations being located close to the edge of the available environmental space, leading to the inability to obtain appropriate background data . If IBE plays a role in species divergence, we expect a significant correlation when comparing all individuals, but not within each species. Mantel tests confirm that IBD strongly influences our data both across and within a single species . Partial Mantel tests confirm that genetic dissimilarity is associated with differences in salinity when looking across species , but not within N. fasciata  or N. clarkii .Therefore, we further explored the six most informative variables identified by  clarkii  online.  clarkii —was much clarkii  online. N. fasciata and N. clarkii, we sought to identify genes potentially contributing to their divergence. We used BayeScan with a reduced data set, only including N. clarkii with <50% admixture proportion from N. fasciata. BayeScan identified 62 outlier SNPs between N. fasciata (n = 41) and N. clarkii (n = 60) which corresponded to 41 loci , we identified 31 potential genes of interest , we found that conStruct admixture proportions (K = 2) was the best predictor of PC1, demonstrating general agreement between the morphological and genetic differentiation of N. fasciata and N. clarkii, with genetically intermediate individuals also having intermediate phenotypes . However, the amount of morphological difference between species was small and even less between subspecies. The main difference between species is related to the number of postocular scales, with a difference of only one scale between the two species.To evaluate our findings in light of current taxonomy and morphological variation, we measured 11 meristic and dimensional features for 137 specimens across the range fig.  online amclust  identifiN. fasciata-clarkii complex. Specifically, we find that IBD explains patterns of population structure, but IBE—caused by differences between inland and coastal habitats and subsequent selection on osmoregulatory genes—better explains broader genetic structure despite introgression from N. fasciata into N. clarkii and putative hybrid formation of some lineages.The formation and maintenance of lineages are governed by the homogenizing effects of gene flow and reinforcement of genomic divergence due to ecological and environmental selection . We provN. fasciata-clarkii complex . Nonetheless, a similar pattern is seen in Andean Speckled Teals (Anas flavirostris) where low-altitude populations migrate at a higher rate into high-altitude populations despite positive selection in hemoglobin genes within high-altitude populations  relative to N. clarkii . An alternate explanation may be exclusion, where N. clarkii alleles are less able to permeate into N. fasciata due to potential trade-offs with fitness and subsequent inability to compete with the dominant N. fasciata populations. Under this scenario, N. fasciata likely functions as a generalist capable of moving across the landscape and N. clarkii as a coastal specialist.Reconciling the pattern of asymmetric gene flow from ulations . The mosto admix . Future N. fasciata-clarkii complex, the nomenclature has undergone frequent revision with lineages being listed as subspecies of the Northern Watersnake (Nerodia sipedon), then the Southern Watersnake (N. fasciata), and later being re-elevated to species or subspecies based on morphology, allozymes, and ecological differentiation  to quantify extractions, requiring 500–1,000 ng of DNA for ddRADseq. Our ddRADseq protocol follows ipyrad v0.7.28 (Trimmomatic v0.38 (We used the  v0.7.28  toolkit ic v0.38  prior toVCFtools v0.1.15 (We used  v0.1.15  to furthDAPC (adegenet package in R (k-means clustering and models compared with Bayesian Information Criterion (BIC) for K = 1–20 with all principal components retained  contributes to the explanation of the data, we also performed an analysis of layer contributions. Specifically, we ran a spatially aware construct analysis for K = 2–7 with three Markov chain Monte Carlo (MCMC) chains and 10,000 iterations per MCMC. Layer contributions from each model were then loaded and plotted  and the DAPC clusters, respectively. A majority rule consensus tree was inferred from the bootstrap replicates.To infer a phylogeny from the SNP data, we ran quartets  with a rTreemix . To estimate the optimal number of migration edges for each value of K, we used the R package OptM  based on the DAPC clusters at K = 5, 6, and 7. We then ran 200 bootstrap replicates on the significant HyDe results and plotted the resulting bootstrap values of gamma (ɣ) using ggplot2  as an indicator of effective population size. Nucleotide diversity was calculated in VCFtools and compared using a t-test in R.To determine if the direction of gene flow might be due to differences in effective population size, we calculated nucleotide diversity (π) for each species . For the input environmental data, we used the BioClim data set from the WorldClim database with spatial resolution of 30 arcsec  for both the NOT and NDT and mixed significance for the background statistics (D = 0.37); however, the tests are inconclusive and prone to Type I error (N. fasciata (PNTI >0.73). In other words, because N. fasciata occupies nearly all of Florida and N. clarkii is near the edge of available environmental space , Humboldt is unable to sample appropriate background data to provide conclusive results.To assess ecological niche divergence between 0 arcsec , cation 0 arcsec , and lan0 arcsec . Importa0 arcsec . Environectively . Humboldatistics  online.  I error  online.  I error  and founHumboldt for both GBIF data and assigned genetic clusters by extracting environmental data for each point and comparing species with ggpubr  and cation exchange capacity using ggpubr. Finally, we performed Mantel and partial Mantel tests to disentangle the influence of IBD and IBE caused by salinity. Specifically, we tested for correlations between genetic dissimilarity , geographic distance (km), and environmental distance . To test for IBD, we performed a Mantel test between genetic dissimilarity and geographic distance. To test for IBE, we performed a partial Mantel test between genetic dissimilarity and environmental distance, controlling for geographic distance. These tests were performed on three data sets: 1) all individuals, 2) N. fasciata only (DAPC K = 2), and 3) N. clarkii only (DAPC K = 2).To circumvent these issues, we looked for differences in the most important environmental variables identified by h ggpubr . In addiN. fasciata and N. clarkii, we searched for outlier SNPs using BayeScan . The resulting data were converted to BayeScan format with PGDSpider . The loci with SNPs that were identified as significant  using blastn returning a maximum of five hits and hsps with a stringent e-value of 1e−50  and genes that were within 2,000 bp of a hit within the genome were extracted as potential genes of interest. To search for enriched GO terms, we used ShinyGO  are less complete and do not have functional information matching many of the identified genes. We used the R package igraph , 95 N. fasciata (21 N. f. fasciata and 71 N. f. pictiventris), and 21 with uncertain assignment to a taxon. Of the 11 features, we measured several diagnostic characters including the number of ventral scales, subcaudal scales, and tail length  and when only using samples with corresponding genetic data (n = 21) we used DAPC assignment and conStruct admixture proportion to assess agreement between morphological and genetic data sets. We also performed a Mantel test between genetic dissimilarity  and the Euclidian distance of PC1.We counted or measured a total of 11 meristic and dimensional features for 137 specimens representing 24 l length . We mease mclust . To detee mclust  using tht (bbmle . SpeciesMolecular Biology and Evolution online.msaa266_Supplementary_DataClick here for additional data file.


Hipposideros larvatus from mainland China and Hainan Island. Using microsatellites and mtDNA to analyze genetic differentiation, we found an obvious genetic structure between the mainland and the island in H. larvatus. Integrating the genetic data, geography and climatic factors, we uncovered the combined effects including geography, environment and colonization history, on the genetic variation in H. larvatus. Our results are valuable in understanding the complex evolutionary processes among bat populations and provide implications for the conservation of island bat populations.It is vitally important to unravel the evolutionary processes and potential drivers of genetic differentiation among populations in evolutionary biology. However, it remains unexplored how those drivers act on the genetic divergence among bat populations. In this study, we studied the genetic structure and evolutionary history of a bat species, Hipposideros larvatus is a common bat species in Southern China, including Hainan Island. We examined genetic variation in H. larvatus using mitochondrial DNA and nuclear microsatellites. We found a population structure on both markers with a geographic pattern that corresponds well with the structure on mainland China and Hainan Island. To understand the contributions of geography, the environment and colonization history to the observed population structure, we tested isolation by distance (IBD), isolation by adaptation (IBA) and isolation by colonization (IBC) using serial Mantel tests and RDA analysis. The results showed significant impacts of IBD, IBA and IBC on neutral genetic variation, suggesting that genetic variation in H. larvatus is greatly affected by neutral processes, environmental adaptation and colonization history. This study enriches our understanding of the complex evolutionary forces that shape the distribution of genetic variation in bats.Determining the evolutionary history and population drivers, such as past large-scale climatic oscillations, stochastic processes and ecological adaptations, represents one of the aims of evolutionary biology.  Uncovering the evolutionary processes that drive genetic differentiation among populations is a central goal of evolutionary biology ,2. NeutrSelective or adaptive processes can also drive genetic divergence among populations. Selection can be involved in driving differentiation at neutral DNA markers ,6. In caHistoric events, such as glacial vicariance or founder events, are additional factors that can affect population differentiation, which is termed “isolation by colonization (IBC)” ,5,11. HoThe importance of geography, the environment and colonization history in driving genetic divergence is of great interest ,5,14,15.Bats (Chiroptera) are the second largest order of mammals (ca. 1400 species). They are distributed globally  and are Hipposideros larvatus, the intermediate leaf-nosed bat, is widely distributed in Asia, including China, Bangladesh, Indonesia, Malaysia, Myanmar and Northeast India [H. larvatus is widespread and occurs in heterogeneous environments, even in different climatic zones across the south of mainland China and Hainan Island [Hipposideros armiger) [Apis cerana) [Quercus championii) [H. larvatus, it is necessary to combine multiple factors to reveal genetic differentiation.st India . This spn Island . For exaarmiger) , the Asi cerana)  and the mpionii) . Thus, tH. larvatus, (ii) reconstruct the recent demographic history of this species and (iii) test for correspondence between genetic differentiation, the environment, geography and colonization history. To do this, we sampled populations across the range of H. larvatus in mainland China and Hainan Island and examined genetic variation in both nuclear microsatellites and mitochondrial (mt) DNA. We tested for IBD and IBA by comparing two neutral genetic markers with geographic and climate differences among the sampled populations. To disentangle these effects from IBC, maternal mitochondrial markers were incorporated to describe the historic genetic signatures and colonization history [H. larvatus.Our research goals were to (i) describe levels of genetic diversity and the current genetic structure in  history . ColonizH. larvatus across the continent and Hainan Island in China  and downloaded 19 bioclimatic variables, including a variety of temperature and precipitation data from CHELSA  ,25 by thH. larvatus were deposited in GenBank with accession numbers MW670581-MW670910 . For each individual, the complete cytochrome gene  and partial control region  fragments were sequenced using primers L14724 vs. H15915 ,29 and PSeven microsatellite loci  were amplified using fluorescent-labeled primers for 165 individuals. Primer sequences and PCR conditions followed those of Guo et al.  and Liu R) per locus were assessed for each population in Fstat 2.9.4 [Haplotype diversity (h) and nucleotide diversity (π) were calculated using DnaSP v5  for eachat 2.9.4 .−8 per year for cytb based on previous studies on bats [Tajima’s D  estimate on bats ,41 and a on bats .−8 subs/site/year. The HKY + I substitution model was used, as determined by Akaike information criterion (AIC) implemented in jModelTest [The time to the most recent common ancestor (TMRCA) among clades was estimated in BEAST v.2.4.6  based onodelTest . No outgodelTest . ESS valHipposideros armiger (GenBank accession JX849178 and JX014191) and Hipposideros turpis (GenBank accession JN247028), were selected as outgroups. Phylogenetic reconstruction using maximum likelihood (ML) in PhyML [For the mitochondrial analyses, two closely related species, in PhyML  on T-REXin PhyML  on CIPREFor the microsatellite analyses, we used a Bayesian approach by Structure v2.3.4  using 500,000 iterations after a 100,000-iteration burn-in. The admixture model without a priori designation for populations was used and the number of tested clusters (K) was based on the numbers of populations. For K = 1 to 18, 10 runs were performed for each K value. A structure harvester  was usedH. larvatus populations: (i) mitochondrial genetic distances (given by Fst/(1 − Fst)) between populations were assessed based on the concatenated cytb and CR sequences, with the FST values calculated by Dnasp; (ii) nuclear genetic distances between populations were calculated using Slatkin’s linearized Fst (given by Fst/(1 − Fst)) based on microsatellites; (iii) geographic distance from the latitude and longitude of each sampling site was calculated by Geographic Distance Matrix Generator online tool (http://biodiversityinformatics.amnh.org/open_source/gdmg); and (iv) climate differences between populations were calculated as the Euclidian distance based on the first three principal components from the PCA using 19 climatic variables. First, we used simple and partial Mantel tests, one of the most widely used approaches to assess spatial processes that drive population structure, to test the correlations between different matrices. Four matrices were built to describe differences between p values were corrected by the Benjamini and Hochberg [Genetic divergence was compared to climate difference and geographic distance using simple Mantel tests, with significance determined by 999 permutation tests. To test for IBD directly, we assessed the correlation between genetic and geographic differences when controlling for climate difference by partial Mantel tests. To test for IBA directly, we assessed correlation between climate and genetic differences when controlling for geographic distance. In addition, we selected mtDNA representing isolation by colonization from glacial refugia and assessed the correlation between nuclear genetic differences and mitochondrial differences when controlling for geographic and climatic differences to test for IBC directly. All of the simple and partial Mantel tests were implemented using the R package “vegan”, with significance determined via 999 permutation tests. To control for multiple tests, Hochberg  criteriaH. larvatus. We performed PCA on the allele frequency matrix. PCs 1–10, which cumulatively explained 87.6% of the variation in the microsatellites, were retained for RDA. Three independent matrices were included: (i) the first three principal components (PCs 1–3) of climatic variables (representing IBA), (ii) geographic variables (IBD), and (iii) mtDNA variables . For the geography matrix, we calculated second-order polynomials and combinations of the coordinates of sampling locations  to ensure linear gradients in the data [Second, a redundancy analysis (RDA) was performed to partition the among-population genetic variation into three components, IBD, IBA and IBC. RDA as a multiple linear regression method performed between a matrix of dependent variables and matrices of independent variables is more appropriate than Mantel tests because RDA does not require distance-based metrics and can violate the underlying assumptions of the Mantel test. The dependent matrix contained population allele frequencies of microsatellites of the data . For thethe data . SignifiH. larvatus sampled from 18 localities across the entire Chinese range of the species, we examined genetic variation in cytb (1140 bp) and the control region (473 bp). Totals of 53 and 72 haplotypes of cytb and CR were defined, respectively. The alignment of the concatenated cytb and CR (1613 bp) resulted in 94 haplotypes. For cytb, haplotype diversity (h) of each population ranged from 0 (JX and YN2) to 0.956 (HN5) and the nucleotide diversity (π) ranged from 0 (YN2) to 0.01649 (YN1)  . For CR,49 (YN1) .All of the individuals formed a highly supported monophyletic lineage  and two clades were recovered by BI and ML phylogenetic reconstruction based on the concatenated mtDNA data . Clade AH. larvatus, Clade A and Clade B1 . RDA axis 2 was most related positively to the mitochondrial variable (PC3 and PC4) and the climatic variable (PC2)  and climatic variables  significantly explained the greatest fraction of the nuclear divergence, however, geographic variation explained a smaller fraction with no significant pure effects on nuclear genetic divergence   suggested that different variables contributing to the nuclear divergence were variable a. RDA axle (PC2) a. The re= 0.186) b.H. larvatus in mainland China and on Hainan Island. We found divergent lineages and a strong genetic structure for nuclear and mitochondrial markers between Hainan Island and mainland China in this bat species   and some individuals from Yunnan Province (subclade B2) (Our demographic simulations suggested the TMRCA of the entire group of 2.14 Ma) . During lade B2) . For theHowever, interestingly, subclade B1 with all individuals from Hainan Island showed high support , suggestH. larvatus, with an estimated time of the expansion occurring 41 Ka (thousand years ago) (95% CI: 15–68 Ka). This was probably driven by the higher precipitation and the warmer climate in China that appeared from 40 to 30 Ka [We also detected a rapid population expansion of subclade B1 in to 30 Ka . This poH. larvatus  is an important factor involved in genetic differentiation among populations ,59 reporlarvatus . Intereslarvatus . This miIsolation by adaptation (IBA) is another important factor that drives genetic divergence among populations . IBA wasAnthus berthelotii) [Daphnia magna) [H. larvatus. Here, we utilized the information from maternally inherited mtDNA as a proxy for colonization, similar to studies on plants such as Pinus tabuliformis [Ficus hirta [adj2 = 0.568, p = 0.004) play more important roles in nuclear divergence than geography and the environment (H. larvatus driven by climatic oscillations were observed across mainland China and Hainan Island in this study (H. larvatus.The isolation by colonization (IBC) pattern is more complex than the IBD and IBA patterns and it is very difficult to dissect IBC from IBD and IBA . Howeverhelotii)  and the a magna) . We obtaliformis  and Ficuus hirta . Signifiironment . Indeed,is study , which mH. larvatus across mainland China and Hainan Island. Incorporation of microsatellites, mtDNA, geography and climate data in Mantel tests and RDA analyses offered a better understanding of the complex interactions of IBD, IBA and IBC, as well as their impacts on population genetic structure. The results showed that the observed genetic structure in H. larvatus is affected by IBD, IBA and IBC. The combined effects among multiple drivers contribute to the genetic variation and complex evolutionary processes in H. larvatus. Furthermore, the genetic differentiation between mainland and the island suggests that it is necessary to integrate the phenotypic, environmental and genetic data to assess the cryptic taxa in H. larvatus in further research, like some successful cases [In conclusion, we studied the genetic diversity and evolutionary history of ul cases ,71,72. A


Acropora aspera (n = 534) and the brooding coral Isopora brueggemanni (n = 612) across inter‐archipelago (tens to hundreds of kilometres), inter‐reef (kilometres to tens of kilometres) and within‐reef (tens of metres to a few kilometres) scales. Initial analysis of A. aspera identified four highly divergent lineages that were co‐occurring but morphologically similar. Subsequent population analyses focused on the most abundant and widespread lineage, Acropora asp‐c. Although the overall level of geographic subdivision was greater in the brooder than in the spawner, fundamental similarities in patterns of genetic structure were evident. Most notably, limits to gene flow were observed at scales <35 kilometres. Further, we observed four discrete clusters and a semi‐permeable barrier to dispersal that were geographically consistent between species. Finally, sites experiencing bigger tides were more connected to the metapopulation and had greater gene diversity than those experiencing smaller tides. Our data indicate that the inshore reefs of the Kimberley are genetically isolated from neighbouring oceanic bioregions, but occasional dispersal between inshore archipelagos is important for the redistribution of evolutionarily important genetic diversity. Additionally, these results suggest that networks of marine reserves that effectively protect reefs from local pressures should be spaced within a few tens of kilometres to conserve the existing patterns of demographic and genetic connectivity.Management strategies designed to conserve coral reefs threatened by climate change need to incorporate knowledge of the spatial distribution of inter‐ and intra‐specific genetic diversity. We characterized patterns of genetic diversity and connectivity using single nucleotide polymorphisms (SNPs) in two reef‐building corals to explore the eco‐evolutionary processes that sustain populations in north‐west Australia. Our sampling focused on the unique reefs of the Kimberley; we collected the broadcast spawning coral  We first tested for cryptic diversity within samples identified as A. aspera or I. brueggemanni. We then measured the spatial distribution of genetic diversity at inter‐archipelago (tens to hundreds of kilometres), inter‐reef (kilometres to tens of kilometres) and within‐reef (hundreds of metres to kilometres) scales to determine the relative strength of genetic connections. Finally, we explored key seascape drivers of metapopulation structure by testing whether heterogeneity in environmental factors such as temperature, turbidity and tide was associated with genetic differentiation and diversity of local coral populations.Here, we investigated the eco‐evolutionary processes that sustain the metapopulations of 22.1We sampled a range of spatial scales Figure . At the Acropora aspera samples from 15 sites . We did not filter for Hardy–Weinberg or gametic‐phase disequilibrium at this stage of the analysis because large  divergence would be associated with such disequilibrium, and removal of such markers would likely limit power of the analyses. Seven individuals with more than 15% missing data were removed.We extracted genomic DNA from coral specimens using a salting‐out protocol modified from Cawthorn, Steinman, and Witthuhn  and puriJombart,  and the Jombart,  to filteA. aspera samples that often occurred in sympatry (see results). Due to low sample sizes in three of the four lineages, we only conducted population‐level analyses on the most common and widespread lineage (Acropora asp‐c). To this end, we recalculated the descriptive statistics across all SNP loci for those samples identified as Acropora asp‐c with the same filters and methods as for the entire A. aspera collection except we relaxed the reproducibility (>0.98) and call rate (>0.90) thresholds. This filtering resulted in 3,472 loci. We then filtered out loci that significantly departed from Hardy–Weinberg equilibrium and gametic‐phase equilibrium with R packages dartR, SNPassoc . For Hardy–Weinberg testing, we removed 343 loci that showed departures from expectations at p < .05 in three or more (out of five) sites. For gametic‐phase disequilibrium, we removed 294 loci with r values >0.8 at three or more sites. In the remaining 2,898 SNPs, we identified loci possibly affected by selection with OutFLANK v0.1 . Four loci were identified as outliers, and these were removed from subsequent analyses resulting in final data set of 2,894 loci.We identified four distinct lineages in the I. brueggemanni, and we filtered the primary data set  using the same criteria as for Acropora asp‐c. This resulted in 2,946 loci. We then filtered out loci that exhibited significant Hardy–Weinberg and linkage disequilibrium at each sampling site (n = 21). For Hardy–Weinberg disequilibrium, we removed 133 loci that showed departures from expectations at p < .05 in five or more of the 21 sites. For linkage disequilibrium, we removed 681 loci with r values >0.8 among five or more sites. These filters resulted in 2,132 SNPs. Six I. brueggemanni individuals with more than 15% missing data were removed. We identified putative loci affected by selection as for the Acropora asp‐c analysis. Initial analysis using the entire data set did not detect any outliers, but when OutFLANK was applied to the inshore data only, seven loci were identified as outliers and were removed from subsequent analyses resulting in final data set of 2,125 loci.There was no indication of cryptic diversity in N), genotypic richness , observed heterozygosity (HO), gene diversity measured as unbiased expected heterozygosity (HE) and fixation index (FIS) at each site and averaged across sites (±standard error).After removal of clones Appendix , we calc2.3K) and membership coefficients (q) of each colony to a range of clusters with the Bayesian software STRUCTURE v2.3 , and all other parameters were set as default values. CLUMPAK was used to summarize and graphically present the STRUCTURE results as well as to calculate optimal K using the ΔK method of Evanno, Regnaut, and Goudet  values to identify the k for which Pr(K = k) is highest . We gauged the relative divergence among versus within these lineages by estimating the genetic relationships among individuals with principal coordinate analysis (PCoA) in GenAlEx v6.5. PCoA takes a simple multi‐ordination approach calculated from a codominant genotypic distance among pairs of samples and does not incorporate any a priori information or assumptions of equilibrium. Therefore, PCoA provides a complimentary analysis to estimate the number and membership of clusters to the sophisticated Bayesian approach of STRUCTURE. We used the standardized distance option for the PCoA. We also calculated pairwise FST between lineages and the number of private alleles (PA) in each lineage in GenAlEx to further estimate the magnitude of differentiation among lineages.The initial analysis in STRUCTURE identified four divergent and sympatric lineages of 2.4Acropora asp‐c lineage and I. brueggemanni. STRUCTURE and PCoA were run with the same parameters as in the tests for cryptic diversity. However, we ran STRUCTURE from K = 1 to 10 for I. brueggemanni because geographic clusters continued to segregate at K >8. We assessed the genetic relationships among corals that were obscured by divergent samples by repeating the STRUCTURE and PCoA in hierarchical analyses that excluded those divergent samples  of Ashmore Reef, the central Kimberley, Buccaneer Archipelago and Dampier Peninsula; among sites within archipelagos (FSR); and among all sites (FST). We also calculated pairwise FST between all sites. We tested for statistical significance in all AMOVAs using 999 random permutations. Some Acropora asp‐c sites had small sample sizes, but because we employed thousands of SNPs, estimation of FST for samples sizes >4 . Spatial autocorrelation uses the spatial position and genetic identity of each individual. This analysis is therefore well‐suited to establishing the finest scale of genetic structure, is sensitive to recent dispersal processes and is robust to most natural characteristics of plant or animal populations  from within the set of pairwise comparisons for a specific distance class. We inferred significant spatial genetic structure when the confidence interval did not straddle r = 0.We investigated population genetic structure at the inter‐reef to within‐reef scale in the FRT_REEFS); between sites within these reefs (FSR_SITES); and among all these sites (FST_SITES) for I. brueggemanni. This analysis was only possible in this species because we sampled replicate sites at these three reefs. We calculated pairwise FST between all sites and tested for statistical significance with 999 random permutations.We also estimated fine‐scale genetic structure with AMOVA among Bathurst, Pope and Mermaid Islands reefs , 10 pilot runs with a length of 5,000 and an additional burn‐in of 50,000. We included six environmental factors: latitude, longitude, the 90th percentile in tidal height (m), range in sea surface salinity (PSS), range in sea surface temperature (°C), dissolved oxygen (mL/L), nitrate (μmol/L) and water clarity (the diffuse attenuation coefficient at 490 nm/m). The latter four factors were assembled from several sources of remotely sensed and in situ measured oceanographic data specific to our sites. Tidal height was sourced from model output of the Renewable Energy Atlas of Australia . Sea surface salinity and temperature were sourced from the MARSPEC ecological archives  at 5‐arc‐minute resolution one kilometre offshore of the sites and collected from 2002 to 2009. The environmental factors were normalized and transformed into the mean absolute difference between values at the focal population and all the other sampled populations.We quantified the effect of the environment on the population genetic structure and diversity of the A. aspera by testing whether sites with greater environmental heterogeneity were associated with greater genetic diversity in these corals. To this end, we used a simple linear regression to correlate gene diversity (unbiased expected heterozygosity) with the same environmental factors used in the GESTE analysis at each site in the Acropora asp‐c lineage.We also investigated the seascape influences on the diversification of lineages within 33.1A. aspera revealed four sympatric genetic lineages . STRUCTURE indicated ΔK was the highest at K = 4  were very strong  plot indicated the presence of additional finer level of structure with optimal K = 8, q‐values were much weaker at this K and likely reflect population geographic structure within lineages. Further, the PCoA supported the ΔK results, distinguishing four discrete lineages within the entire collection of A. aspera, two of which (asp‐c and asp‐d) were relatively closely related . Pairwise FST between lineages was very large, ranging from 0.480 to 0.704  of 4 Figure , and mem04 Table . Further04 Table .A. aspera lineages  occurred side by side at many sites but exhibited pairwise FST of 0.480 (Table Acropora asp‐a (0.108) and lowest in Acropora asp‐d , and Kooljaman in the far west was the lowest  method , the Buccaneer Archipelago  or the Dampier Peninsula , the remainder exhibited intermediate ancestry (q ~ 0.50) between the Buccaneer and Dampier Peninsula clusters . However, small and significant differences were detected among sites within archipelagos . Consequently, overall subdivision among all sites was moderate but highly significant . The largest pairwise differences were between the Ashmore site and all the other sites, with average FST of 0.380  of all corals to either an offshore Ashmore cluster or an inshore cluster . However, at K > 2, clusters continued to segregate according to geography, and the Ln (Pr(X|K) method identified optimal K = 10. An additional cluster was formed by Kooljaman at K = 3  or among individuals  and among sites within archipelagos , yielding a large overall level subdivision among all sites . Pairwise FST between Ashmore Reef and the inshore reefs was very high and averaged 0.450 (±SE 0.015), but was lowest with West Montalivet .The geographic structuring observed across a range of spatial scales in 4 Figure . At K = r Figure . This ger Figure , as werer Figure . Strong 3.3Acropora asp‐c and I. brueggemanni. The positive correlation coefficient (r) in both plots was significant and relatively constant up to 500 m , over distances of approximately 500m. Pairwise FST comparisons indicated the significant differences between sites occurred at reefs of Pope Island  and Mermaid Island , but not Bathurst West . Despite this subdivision within reefs, much more of the variation was attributed by AMOVA to subdivision among the three reefs .The AMOVA of 3.4Acropora asp‐c (p = .556) and for I. brueggemanni . The slope of regression was negative for both corals (Table FST) decreased with increasing tidal magnitude. However, the deviation from the regression was moderate for both corals , and no relationship with the other environment factors.Tidal magnitude also exhibited a strong association with gene diversity at each site within the 4Acropora aspera, and the brooding coral, Isopora brueggemanni. This consistency between species with different life histories indicated that the heterogeneous seascape and powerful oceanographic currents of this region have important influences on their metapopulation dynamics. Environmental influences not only promote strong genetic differentiation between bioregions and archipelagos (tens to hundreds of kilometres) and regular local recruitment within reefs (tens of metres to a few kilometres), but also rare longer‐distance connectivity between reefs within archipelagos (kilometres to tens of kilometres). Underlying this spatial genetic structure, we discovered several highly divergent and cryptic lineages in A. aspera that co‐occur on the same reef patch.Strong genetic divergence and restricted population connectivity characterized the distribution of genetic diversity in two reef‐building corals from north‐west Australia. These characteristics were evident across a wide range of spatial scales in both the spawning coral, 4.1A. aspera that were not distinguished by macro‐morphological characteristics. Pairwise FST between the four lineages was large (FST ≥ 0.469), and private alleles were observed at more than half the loci analysed. These results indicate inter‐breeding between lineages is rare  history of coral reefs of the inshore Kimberly  is the best explanation for the maintenance of reproductive isolation among sympatric lineages. Indeed, direct evidence of species‐level genetic differences has been observed in Acropora lineages that appear identical but spawn in either spring or autumn  was half that of the brooder I. brueggemanni (FST = 0.230). This result reflects the potential for greater dispersal by spawned larvae through longer precompetency periods compared with brooded larvae . This result is consistent with offshore studies in north‐west Australia from other brooding species . Therefore, strong tidally driven currents appear to have increased the likelihood of occasional larval dispersal between local populations. Conversely, the deep‐water tidal current at Sunday Strait appears to be a semi‐permeable barrier to dispersal of larvae between the genetically distinct Dampier Peninsula and Buccaneer Archipelago. This result is consistent with other studies that have shown strong oceanic currents often act as “leaky” barriers to larval dispersal that override the influence of biological factors on genetic structure such as planktonic period  and were more diverse (in I. brueggemanni sites and is probably small, isolated and close to the limits of its south‐western range. Therefore, Kooljaman may be more vulnerable to local extinction compared with other reefs studied here. Lastly, the genetic signatures at Tide Rip and Mermaid islands were admixed between the Dampier Peninsula and Buccaneer Archipelago, indicating these reefs provide stepping stones for occasional genetic exchange between the archipelagos important for the adaptive capacity of the metapopulation. We suggest these islands should be considered conservation priorities.The congruent patterns among two species with very different modes of reproduction suggest that a single spatial marine management strategy may be used to aid resilience of all coral populations in this region. We recommend that multiple sanctuary networks be spaced at distances no greater than a few tens of kilometres. More specifically, the Dampier Peninsula and Buccaneer Archipelago should be managed as demographically independent systems that sustain their populations through production of local recruits. Further, the population at Kooljaman was the most genetically divergent and depauperate of the A. aspera also has implications for management. Such unrecognized diversity is probably common in these systems (Richards et al., Acropora asp‐d may reflect a limited adaptive capacity. Such lineages are likely vulnerable to silent extinction.The discovery of cryptic lineages within Coral reefs worldwide are threatened by the increased frequency and severity of marine heatwaves (Hughes et al., None declared.Appendix AClick here for additional data file.Appendix BClick here for additional data file.Appendix CClick here for additional data file.Appendix DClick here for additional data file.Appendix EClick here for additional data file.


Salmonella spp., Listeria spp., and Listeria monocytogenes were not detected in 25 g samples. Generally, less than 102 cfu/g of Staphylococcus aureus, Escherichia coli, Pseudomonas spp., Enterobacteriaceae, and molds were present in the fermented foods. Bacillus cereus group members (<102 to 2.3 × 104 cfu/g), lactic acid bacteria (<102 to 1.1 × 107 cfu/g), halophilic and halotolerant bacteria (<102 to 8.9 × 106 cfu/g), sulfite-reducing Clostridium spp. (<102 to 3.5 × 106 cfu/g), and yeasts (<102 to 1.1 × 106 cfu/g) were detected in this study. Still, the presence of pathogenic and spoilage microorganisms in these fermented foods was within the acceptable ranges. Putrescine, cadaverine, tyramine, and histamine were detected in 100%, 89%, 81%, and 75% of the tested products, respectively. The concentrations of histamine (>500 ppm) and tyramine (>600 ppm) were higher than the recommended maximum levels in respectively four and one of 57 fermented foods, which represents a potential health risk. The results suggest that the production process, distribution, and domestic handling of fermented foods should be re-evaluated. Further research is needed for the establishment of applicable preservation techniques in Cambodia. Naturally fermented foods are an important part of the typical diet in Cambodia. However, the food safety status of these products has not been widely studied. The aim of this study was, therefore, to provide an overview of the quality of these foods in relation to microbiology and biogenic amines. Additionally, the obtained results were compared to the habits and practices of Cambodians in handling this type of food. A total of 57 fermented foods (42 fishery and 15 vegetable products) were collected from different retail markets in the capital of Cambodia. Pathogenic  Due to higher ambient temperatures and moisture, food spoils faster in the tropics. As a result, it is not surprising that food security issues are reported in densely populated tropical cities [Cambodia is an agricultural country that has a tropical climate with two distinct monsoon seasons (dry and rainy seasons). Thus, the availability of certain products is not stable through the year, and food preservation and storage are required to maintain the food supply . Since fl cities . Howeverl cities . Fermented foods are an important part of the typical diet in Cambodia . Since CCambodian fermented foods are produced through knowledge that is passed on from generation to generation and from person to person . The greEscherichia coli, Cronobacter sakazakii, Enterobacter spp., opportunistic non-Enterobacteriaceae, Staphylococcus spp., and Listeria spp. have already been detected in fermented vegetables in Cambodia [Bacillus, Clostridium, and Staphylococcus were found in traditional Cambodian fermented fish products [N-nitrosamines when nitrite is present. These two BAs also potentiate the effects of HIS and TYR by inhibiting their metabolizing enzymes [Cambodia ,12. Furtproducts . Next toproducts . The mosproducts . BAs areproducts . Beside products . The mosproducts ,17. Low products . BAs witproducts . PUT and enzymes . HIS is  enzymes . In addi enzymes . wa, and salt content) as well as the presence of certain microorganisms (spoilage and pathogenic bacteria) and the concentrations of the BAs HIS, TYR, PUT, and CAD were determined in 57 Cambodian fermented food samples within this study. The main purpose of this manuscript is to give an overview of the quality of Cambodian fermented foods, to correlate physicochemical parameters with BA contents, and to describe the prevailing habits and practices of Cambodians in dealing with this type of food.Baseline surveillance data are essential to monitor the disease burden of fermented foods in Cambodia. To obtain such data, the physicochemical properties , fish paste , shrimp paste , fermented fish , sour fermented fish , and salted fish . Fermented vegetables such as salty fermented radish , sweet fermented radish , fermented melon , fermented mustard , and fermented papaya  were bought on the next day. Detailed information about each fermented product is provided in Fifty-seven samples of naturally fermented foods (42 raw fermented fish and 15 RTE fermented vegetable products) were randomly purchased from wet markets in Phnom Penh, the capital city of Cambodia. These products originated from various provinces of the country. Fermented fishery samples included fish sauce  into the samples. The pH values were then measured using a digital pH meter . wa value was measured using the digital water activity meter Rotronic Hygropalm HP23-AW-A  after equilibration at room temperature (~25 °C).The 3 was added. The samples were then titrated with 0.1 M AgNO3. With the obtained results, the salt content of the original samples was calculated as % (w/w) NaCl = V × M × 0.0584 × 100/m; where V and M are the volume and molarity of the AgNO3 standard solution used. The initial sample weight is m, which was calculated considering the sample preparation: m = a × c/b. The test was conducted in duplicate.The salt content was analyzed by potentiometric precipitation titration of chloride-ions with the 877 Titrino plus-Titrator equipped with a calomel electrode . The protocol was performed according to the producer with minor modifications. Depending on the expected salt content, 1 to 10 g ± 0.01 g (a) of the samples was weighed into a glass beaker and filled with distilled water to 200 g ± 0.01 g (b). Subsequently, the samples were homogenized for 2 min at 9500 rpm using an Ultra Turrax T25 . Fifty grams ± 0.01 g (c) of the homogenized samples was weighed into a new glass beaker, and 50 mL of distilled water was added. Afterwards, 2 mL (2 M) HNOPseudomonas spp. were detected by plating appropriate dilutions on Cephalothin-Sodium Fusidate-Cetrimide agar  and incubation at 25 °C for 44 h based on ISO 13720 [Staphylococcus aureus was enumerated on Baird Parker agar , which was incubated at 37 °C for 24 h based on ISO 6888-1/AMD 1 [Bacillus cereus group members was investigated by spreading dilutions on Mannitol Yolk Polymyxin agar  and incubating plates at 30 °C for 18–48 h. The evaluation was also performed after 18 h and 48 h of incubation. Colonies were confirmed by endospore staining [E. coli was enumerated by pour plating on Tryptone Bile Glucuronic medium  with an incubation at 44 °C for 18–24 h based on ISO 16649-2 [Clostridium spp. (SRC) was analyzed by pour plating with an additional overlay on Sulfite-Cycloserin agar . Plates were anaerobically incubated at 37 °C for 20 h. Confirmation tests were performed using Lactose-Gelatine medium  and Motility-Nitrate medium  according to ISO 7937 [Salmonella spp. was investigated using the VIDAS UP  Salmonella (SPT) system, whereas that of Listeria spp. and L. monocytogenes was tested by the VIDAS LDUO  system. VIDAS SPT and VIDAS LDUO are based on an enzyme-linked fluorescent immunoassay. The preparation of the samples was similar to the previous method, but 25 g of the sample was weighted in instead of 10 g. After pre-enrichment (Listeria spp.) and enrichment  steps, which were carried out according to the manufacturer’s manual, the assay steps were performed automatically by the instrument. From each fermented product, a 10 g sample was aseptically collected, transferred to a stomacher bag, and homogenized  with 90 mL of buffered peptone water for 45 s at 230 rpm. Appropriate dilutions of the samples were prepared using the same diluent, and 0.1 or 1 mL aliquots of each dilution were applied on various selective media using the spread plate method or pour plate method. Lactic acid bacteria (LAB) were anaerobically grown on DeMan Rogosa Sharpe agar  at 30 °C for 72 h according to ISO 15214 . EnterobSO 13720 . Yeasts SO 13720 . HalophiSO 13720 . Staphyl-1/AMD 1 . The pla-1/AMD 1 . The num 16649-2 . The preISO 7937 . The preThe concentrations of BAs in the supernatant were analyzed by reverse-phase HPLC  according to the method of Šimat et al. and Saarinen et al. ,34. Briep value of less than 0.05 was considered statistically significant. The least-squares difference (LSD) test was used to determine the significance of differences in the physicochemical parameters and BA contents among the samples. The relationship value was determined using the Pearson correlation coefficient. Result units of quantitative microbiological analyses were cfu/g. The physicochemical parameters and BA concentrations results were analyzed with statistical analyses using the Statistical Package for the Social Sciences . Data were analyzed for the degree of variation by calculating the mean and standard deviations (SDs) of the results. The significance of differences was evaluated using analysis of variance (ANOVA). A wa, and salt content were measured and compared to discuss possible causes for the different BA levels. wa values of the fermented fishery products ranged from 0.69 to 0.84. The lowest wa value (0.69) was detected in kapi (shrimp paste) and the highest (0.84) in paork chav (fermented fish). The salt contents were in the range of 6% to 34%, with the lowest value (6%) found in trey proheum  and the highest (34%) in prahok (fish paste). In the fermented vegetables, the pH values were between 3.6 and 5.5. Lower pH values were found in spey chrourk  and mam lahong , while the highest value was detected in chaipov brey  (4.6). The wa values in these fermented products were between 0.75 and 0.97. The highest salt concentration (25%) was found in chaipov brey , while the lowest (2%) was detected in spey chrourk (fermented mustard)  .p < 0.05) between the physicochemical parameters of teuktrey (fish sauce) and those of paork chav (fermented fish) and trey proheum . There was no statistically significant difference (p > 0.05) among the samples of teuktrey (fish sauce) and prahok (fish paste), and of paork chav (fermented fish), paork chou (sour fermented fish), and mam trey (fermented fish). Statistical analysis of fermented fish and vegetables samples was conducted separately. Regarding the fermented vegetable products, the physicochemical values of chaipov brey  were significantly different from that of spey chrourk (fermented mustard) and mam lahong (fermented papaya) (p < 0.05), while no significant difference was found among chaipov paem (sweet fermented radish) and trasork chav (fermented melon) (p > 0.05)  of fermented fishery products, there were significant differences ( > 0.05) .2 to 1.1 × 106 cfu/g), halophilic and halotolerant bacteria (<102 to 8.9 × 106 cfu/g), Enterobacteriaceae (<102 cfu/g), Pseudomonas spp. (<102 cfu/g), yeasts (<102 to 1.1 × 106 cfu/g), and molds (< 102 to 2.3 × 102 cfu/g) from the different types of fermented fish tested are indicated in B. cereus group members (<102 to 2.3 × 104 cfu/g), SRC (<102 to 3.5 × 106 cfu/g), S. aureus, and E. coli . Counts of LAB (<102 to 1.1 × 107 cfu/g. The highest LAB counts were detected in spey chrourk (fermented mustard) and mam lahong (fermented papaya). Halophilic and halotolerant bacteria were found in numbers ranging from 2 × 102 to 5.5 × 104 cfu/g. The counts of B. cereus group members ranged from <102 to 1.2 × 104 cfu/g. SRC and yeasts were detected in the range of < 102 to 1.5 × 103 cfu/g and <102 to 2.6 × 105 cfu/g in the tested vegetable samples, respectively  were found among the detected concentrations of PUT, CAD, HIS, and TYR in each product type. The statistical analysis of 15 fermented vegetable samples showed no statistical difference (p > 0.05) among concentrations of PUT, CAD, and HIS, while TYR concentrations were statistically different (p < 0.05)  .wa, and salt content (%) in 42 fermented fishery products, a weak positive relationship between total BAs and wa values , and a weak negative with pH values  were found. There was no correlation among total BAs and salt content   and salt content  were characterized by a moderate and strong negative correlation coefficient, respectively, while a strong positive correlation between total BAs and wa value  were determined in fermented vegetable products  . Furtherproducts . w values of fermented fish products were comparable to fermented fish products from other countries, for example, Thai shrimp paste (0.65–0.72) and Indonesian fermented fish (0.75–0.93) [Based on the physicochemical results, types of fermented fishery products were more different than those of fermented vegetables. Nevertheless, the pH values of this study are in good agreement with those of fermented fish products in Thailand, Vietnam, Laos, Myanmar, China, Korea, Japan, Malaysia, and Taiwan ,36,37,3875–0.93) ,40. wa, the lowest wa values were also determined in these samples (0.75–0.76)  .wa and pH [wa value [Growth of microorganisms in foods are mainly influenced by the w and pH . The addaw value . Microorganisms associated with fermented foods are commonly present on the external surface and in the pre- and post-harvest environment of raw materials. Additionally, they exist in the gill and gut of seafood .Bacillus spp. and Clostridium spp., our results are comparable to those of Chuon et al., who also analyzed Cambodian traditional fermented fish sauce, fish paste, and shrimp paste [C. botulinum to ensure food safety is not recommended [C. botulinum [C. botulinum, C. perfringens—the most important of the SRC—poses a frequent problem and challenge in fish industry [5 to 108 CFU/g C. perfringens are capable of generating toxinfection [C. perfringens as causative agent are related to inadequate storage, processing, and food service operations [C. perfringens could be confirmed within this study. It is known that >105 cfu/g B. cereus group members are potentially harmful for human consumption [B. cereus in low numbers in several fermented products, including those based on fish and vegetables, has already been described [S. aureus could be quantified (<102 cfu/g) in the tested samples, which is in contradiction to a previous study [S. aureus is uniquely resistant to adverse conditions such as low wa values (0.83), high salt contents, and pH stress. Thus, most strains can grow over an wa and pH range of 0.83 to >0.99 and 4.5–9.3, respectively [wa value in the range specified above, 10 of them , spey chrourk (fermented mustard), and mam lahong (fermented papaya), and one chaipov paem (sweet fermented radish) product had a pH < 4.5  of most food samples tested (90%). Also, less than 102 cfu/g of Pseudomonas spp., Enterobacteriaceae, and E. coli were detected in all products examined , and the fermentation process itself [Regarding mp paste . Such trmp paste . Howeverommended . Insteadotulinum . In addiindustry . It is enfection . Foodborerations . Neverthsumption . None ofescribed . The inaescribed . Moreoveectively . AlthougpH < 4.5 . Overallexamined . Furthers itself . LAB ares itself , produces itself . Howevers itself . Forty-tSince typical spoilage bacteria are generally non- or only slightly halotolerant , the extensive use of salt is another technological process for food preservation besides fermentation . Up to 2wa) may result in higher yeasts and mold numbers. These microorganisms are quite salt-tolerant [3 cfu/g [6 cfu/g of yeasts are acceptable in RTE foods placed on the market [6 cfu/g) was only found in one paork chav (fermented fish) sample, which could lead to spoilage by acid and gas production [The unfavorable conditions for bacterial growth , Engraulidae for paork chou (fermented fish) and Clupeidae for mam trey (fermented fish). Hence, these products contained higher HIS amounts , LAB, Bacillus spp., and Clostridium spp. were said to be capable of HIS formation [Bacillus strains [Lactobacillus, Enterococcus) during fermentation [Clostridium, Pseudomonas, and Staphylococcus, LAB  are also involved in the production of PUT. It should be kept in mind that decarboxylase activities are often related to strains rather than to species or genera [The possible involvement of molds and yeasts in BA  accumulation is still discussed . Howeverormation ,57,67. Aormation . Thus, Eentation . Next tor genera . The capr = −0.22 and −0.57) were respectively found between the total BA contents and pH values for fermented fishery and fermented vegetable products. A strong negative linear fit (r = −0.81) could be detected between total BAs and salt content in fermented vegetables, whereas there was no correlation between these parameters in fermented fishery products  and vegetable (r = 0.79) products, respectively. The main factors affecting microbial activities in food are temperature, salt concentration, and pH . Most feproducts . In geneter loss . CorrespThe ability of microorganisms to produce BAs is limited by low temperature . Within TYR concentrations from <0.4 to 270.6 ppm in commercially Chinese fish sauces , from 77It has also been reported that the acute toxicity levels of TYR and CAD are respectively greater than 2000 ppm and the oral toxicity level of PUT is 2000 ppm . It is kThe HIS content alone may be a reliable indicator of food safety, but not of food quality. TYR and CAD are used as spoilage index . Other aIn view of these results, the production process, distribution, and domestic handling of fermented products should be re-evaluated under strict hygienic practices together with the hazard analysis critical control point (HACCP) approach in order to minimize the content of BAs and microbiological contamination. The storage of food by cooling or freezing requires electricity that is not available to all Cambodians. Therefore, preservation techniques, such as the use of antimicrobial substances and/or autochthonous starter cultures, which are characterized by the absence of any BA formation ability or the presence of a BA detoxification activity, should be tested for their possible application on an industrial and small scale. The presence of microorganisms in the examined fermented samples presented no health risk since pathogenic and spoilage microorganisms were in acceptable ranges. Nevertheless, one paork chou (sour fermented fish), one mam trey (fermented fish), and two paork chav (fermented fish) products represent a health risk because of the high level of HIS (>500 ppm). One of the paork chav (fermented fish) samples additionally exceeded the recommended TYR maximum (>600 ppm) per meal. The totals of all BAs tested were higher than the recommended corresponding BAI values in about 69% of the tested fermented fishery and 13% of the vegetable products. This may indicate a poor hygienic quality for these products. Hence, the production process, distribution, and domestic handling of fermented products in Cambodia should be re-evaluated in order to minimize the content of BAs and microbiological contamination. Further research is required to establish preservation techniques that could be applied on an industrial and small-scale in Cambodia.