Tritia obsoleta  and four trematode parasite species that obligately infect it. We examined genetic diversity, gene flow, and population structure using two molecular markers  for the host and the parasites. In the host snail, we found little to no evidence of genetic founder effects, while the trematode parasites showed significantly lower genetic diversity in the introduced versus native ranges. Moreover, the parasite's final host influenced infection prevalence and genetic diversity: Trematode species that utilized fish as final hosts demonstrated lower parasite diversity and heightened founder effects in the introduced range than those trematodes using birds as final hosts. In addition, inter‐regional gene flow was strongest for comparisons that included the putative historical source region (mid‐Atlantic populations of the US east coast). Overall, our results broaden understanding of the role that colonization events (including recent anthropogenic introductions) have on genetic diversity in non‐native organisms by also evaluating less studied groups like parasites.Species colonizations  can be associated with genetic founder effects, where founding populations demonstrate significant genetic bottlenecks compared to native populations. Yet, many successfully established free‐living species exhibit little reduction in genetic diversity—possibly due to multiple founding events and/or high propagule pressure during introductions. Less clear, however, is whether parasites may show differential signatures to their free‐living  hosts. Parasites with indirect life cycles may particularly be more prone to founder effects  because of inherently smaller founding populations and complex life cycles. We investigated this question in native (east coast) and introduced (west coast) North American populations of a host snail  More specifically, those introduction vectors with higher entrained propagule pressure can enable transfer of greater genetic variability to founding populations, lessening the depression of genetic diversity , which invaded the US west coast with cryptic lineages of trematode parasites , where host diversity and parasite diversity were used to help resolve its cryptogenic status in eastern North America  is infected by several species of trematode parasites in both its native region of eastern North America and its non‐native region of western North America. This study system allowed us to test for differences in multiple parasite species with different life cycles while controlling for the host species. The host and its parasites also reflect a relatively recent founding event, making our study particularly well suited for examining questions about the effects of introduction events on genetic diversity. Based on our expectations for differential signatures in the host versus parasites, we tested the following hypotheses: (1) T. obsoleta's trematode parasites would show a stronger genetic bottleneck than the snail host in introduced versus native regions and subregions, due to inherent differences in parasite and host propagule pressure and life cycles  is an estuarine snail (up to ~30 mm shell length) with an extensive eastern North American native range, occurring from the Gulf of Saint Lawrence, Canada, to northern Florida, USA  , California, in 1907 , Washington, in 1945; and Boundary Bay (BB), British Columbia, in 1952  or shorebirds (n = 4) as final hosts. Five of T. obsoleta's trematodes have been discovered in the introduced range: all five in SFB  from Maine to Georgia ; the North had 12 sites ; and the South had 19 sites . In the introduced region, T. obsoleta was grouped by its three discrete bays: SFB: eight sites , WB: five sites (n = 669 snails), and BB: five sites (n = 540 snails). Adult snails were collected haphazardly by hand in the low intertidal zone at daily low tide by walking parallel to the water's edge. Approximately 100 snails were collected per site and dissected in the laboratory using standard protocols . Early processing revealed little variation among individuals and populations using the 18S marker; thus, no subregional examinations were performed for this marker. For trematodes, we included the four most prevalent species in the two regions: Austrobilharzia variglandis (AV), Himasthla quissitensis (HQ), Lepocreadium setiferoides (LS), and Zoogonus lasius (ZL). The following were our trematode sample sizes for the COI marker: Source: n = 8 sites, n = 163 individuals; North: n = 4 sites, n = 43 individuals; South: n = 2 sites, n = 32 individuals; SFB: n = 4 sites, n = 58 individuals; WB: n = 1 site, n = 3 individuals; and BB: n = 2 sites, n = 18 individuals. Trematodes were inherently more difficult to detect in the introduced region, leading to unequal sample sizes; as a result, some subregional comparisons were limited. 18S was used as a second marker for the trematode (HQ) with the most equivalent sample sizes between the regions. In some analyses, trematodes were grouped by final host taxa: AV and HQ are bird‐using and LS and ZL are fish‐using  protocol , and tested significance of differentiation in Arlequin. Hierarchical analysis of molecular variance (AMOVA) was used to estimate variation for T. obsoleta between regions; between native subregions and the introduced region; between subregions across the two regions; and between subregions within regions  analysis  to look for spatial patterns between and among populations. The spatially closest populations are most genetically similar and could reveal likely source populations. Using the same regional and subregional comparisons presented in Table μ and m2/μ, where μ is the mutation rate per site) between two populations assumed to have shared a common ancestor. We performed 10 replicate runs of each comparison, which included 30 chains of at least two million steps per chain after an initial burn‐in period of 100,000 steps; we ended runs when posterior density parameter curves were stable  were only in the native region, 54 (33%) only in the introduced region, and 22 (13%) shared between regions. Haplotype richness was marginally higher  in the native versus introduced region. Interestingly, however, the opposite was found between the putative Source and the introduced region, the latter having significantly higher haplotype richness  being a single, dominant one that was ubiquitous across sites in both regions (Appendix To determine whether genetic founder effects differed between the host and parasites in the native versus introduced regions (Hypothesis 1), we examined genetic diversity using two markers (COI and 18S). In gion, 54 3% only is Figure , and hapAppendix C. Altogen = 135 haplotypes; Appendix 2 = 31.650, p < .00001) greater in the native versus introduced region and also the Source versus introduced region   native versus introduced richness, while the predicted Source richness was about 2–3× higher.In rarefaction analyses, only a fraction of the host snail's predicted genetic diversity was captured in the two ranges—likely due to the number of singleton haplotypes found in both regions and the number of unshared haplotypes detected in the introduced region Figure B. Haplot4.2North and Source subregions (Table LS and ZL) were collectively 20× less common. In contrast, bird‐using trematodes (AV and HQ) had slightly higher prevalence in the introduced region.To test how the intricacies of trematode life cycles may differentially affect diversity in the two regions (Hypothesis 2), we examined trematode prevalence in host snails and also genetic diversity in trematode species with different final hosts (fish or birds). Trematode infection prevalence was 4× greater in the native versus introduced regions for all trematode species combined, and the greatest prevalence was found in the ns Table . For fis2 = 5.070, p = .024) lower haplotype richness in the introduced versus native ranges, the pattern was stronger  for fish‐using trematodes. Haplotype frequencies demonstrated bird‐using trematodes to have relatively equal frequencies across native (25%), shared‐native (28%), shared‐introduced (30%), and introduced (17%) groupings; in contrast, fish‐using trematodes showed substantially greater frequencies in the native (67%) and shared‐native (24%) groupings compared to the shared‐introduced (5%) and introduced (5%) groupings. Moreover, in rarefaction analyses, bird‐using trematodes demonstrated less difference between predicted introduced haplotype richness and Source haplotype richness, when compared to fish‐using trematodes , we found clear associations between the introduced populations and those in the native Source  , we analyzed differentiation patterns and rates of migration for the snail and its trematodes. For both genetic markers, little genetic differentiation was detected for the snail host in regional comparisons. However, a few cases of differentiation were identified in the subregional analyses, primarily comparisons including the e Figure A. In conQ Figure B. To comn Figure  and also) Figure A. We alss Figure B.5T. obsoleta demonstrated no obvious genetic bottleneck in its introduced west coast region, especially when compared to its purported Source region . Although host and parasite were likely introduced together, they clearly demonstrate differential genetic diversity in founding populations, with the parasites showing much greater conformity to genetic founder effects, in which genetic diversity is severely depressed in the colonized range  shipments, which were transferred at a massive scale from 1869 to 1940  is associated with much lower propagule pressure  of infected hosts entrained in the introduction vector, which will depend on natural infection levels within B. attramentaria) and its trematode parasites introduced from Japan to California  and a prevalent trematode parasite . The final host stage is even less specific, typically utilizing a wide variety of hosts in a large taxonomic group, such as shorebirds or predatory fish  had a full complement of suitable hosts in all three west coast bays and were the only two trematodes detected in all three bays; in contrast, neither LS nor ZL (the fish‐using trematodes) were detected in either of the two northern bays (WB and BB)   Table . FurtherZL Table . While tZL Table . Such geZL Table . In our ZL Table .5.3T. obsoleta throughout its native and non‐native ranges on both coasts of North America, aside from some differences observed in the South subregion , along with the possibility for some present‐day gene flow among the subregions (discussed below).There was little detectable genetic structure for on Table . For theon Table . TritiaT. obsoleta's four trematode species  similarly demonstrated limited genetic structure in the native range, but lower genetic diversity than T. obsoleta   and its trematodes (n = 30) were not detected in our east coast sampling. As hypothesized in other species introductions  was actually four genetically distinct species, while Miura et al. . Most prior work on founder effects in species introductions has focused on free‐living species; thus, our study enhances understanding regarding the mechanisms that drive genetic diversity losses in founding populations in both free‐living and parasitic species. As many introduction vectors continue to operate and transport species to new locations around the world, understanding the mechanisms that influence genetic diversity in founding populations can be informative for a multitude of systems. Future work can help resolve the many unanswered and untested questions that remain in this and other host–parasite systems, and further reveal the important role that cryptic species such as parasites play in systems around the globe. Click here for additional data file. Click here for additional data file.


At birth (D0) or 7 days (D7), rats received 90 min of lung protective ventilation ; P20 ventilation ; or P24 ventilation . Prior to prolonged ventilation at D0, endotoxin-exposed rats had decreased compliance and inspiratory capacity (IC) compared to controls. At D7, endotoxin was associated with reduced compliance. High-pressure ventilation (P20 and P24) tended to increase IC and compliance in all saline-treated groups. Ventilation at D0 with P20 increased IC and compliance when applied to saline-treated but not endotoxin-exposed pups. At D7, P24 ventilation of endotoxin-exposed pups increased elastance, bronchoalveolar lavage protein content, and IL-1b and TEN-C mRNA expression in comparison to the saline group. In summary, antenatal endotoxin exposure alters lung mechanics at birth and 1 week of life and increases susceptibility to VILI as observed in lung mechanics, alveolocapillary barrier injury, and inflammatory mRNA expression. We speculate that antenatal inflammation primes the lung for a more marked VILI response, suggesting an adverse synergistic effect of antenatal and postnatal exposures.Perinatal inflammation due to chorioamnionitis and ventilator-induced lung injury (VILI) at birth is independent risk factors for the development of bronchopulmonary dysplasia (BPD). We have previously shown that antenatal endotoxin (ETX) causes abnormal lung structure and function in 2-week-old rats, but whether ETX impairs lung mechanics at birth and increases risk for VILI is unknown. Fetal rats were exposed to 10 μg endotoxin or saline  Bronchopulmonary dysplasia (BPD), the chronic lung disease that follows preterm birth, is characterized by persistent respiratory disease and strongly associated with severe lifelong co-morbidities . UnderstMultiple insults after preterm delivery have the potential to worsen lung injury, including infections, fluid overload from a patent ductus arteriosus (PDA), mechanical ventilation, and oxygen therapy . MechaniPrevious work has demonstrated that mechanical ventilation of 1-week-old rats increases lung epithelial cell apoptosis and decreases alveolar growth . In addiInflammation is a common pathway that augments the severity of acute lung injury, which can progress to BPD . While tAll animal procedures were approved by The University of Colorado Denver Institutional Animal Care and Use Committee .via cesarean section on E22 (Timed-pregnant Sprague-Dawley rats from Charles River Laboratories (Wilmington MA) were maintained in room air for at least 1 week and at embryonic day 20 (E20) received either intra-amniotic injection of saline (CTL) or 10 μg lipopolysaccharide endotoxin . Pups were then delivered n on E22 , which ivia intraperitoneal (IP) injection in ≈50 μl (D0) or ≈150 μl (D7) saline. A tracheostomy was performed using a 24 ga, ≈19 mm plastic cannula (24Gx¾” Excel Safelet Catheter) for D0 and an 18ga, ≈10 mm metal cannula for D7. The cannula was connected to the flexiVent small animal ventilator and the baseline ventilation was started consisting of a tidal volume (Vt) = 6 ml/kg (delivered volume), positive end expiratory pressure (PEEP) = 2 cmH2O, respiratory rate (RR) = 250 breaths/min, and inspiratory to expiratory ratio (I:E) = 1:1.5. These settings are based on the ARDSnet-recommend Vt for lung protective ventilation (2) = 0.21. Body temperature was maintained with a heating pad and humidified plastic dome. Proper cannulation and the structural integrity of the respiratory system was checked by performing a recruitment maneuver (RM) consisting of a 3 s ramp to 30 cmH2O and a 3 s breath hold as previously described  forced oscillation technique (FOT) measurement with onset pressure = 0, amplitude = 8 ml/kg was applied to measure pulmonary system impedance. Eight additional FOT measurements were then recorded at 16-s intervals. The constant phase model was then fit to the measured impedance (N), pulmonary system elastance (H), and tissue damping coefficient (G). The last four FOT measurements were used in the statistical analysis. Constant phase model outputs were accepted as valid if the coefficient of determination (COD) > 0.9, 0 < H < 2000 cmH2O/ml, and 0 < RN < 5 cmH2O/ml/s. Another RM was then performed followed by an 11 s trapezoidal pressure-volume loop . After 15 more seconds of ventilation, another RM was performed followed by a 16 s step-wise pressure-volume loop (maximum pressure of 30 cmH2O). A cubic spline was fit to the quasi-static points on the expiratory limb using MATLAB and the pressure-dependent compliances Cst3, Cst6, and Cst15 were calculated as the derivative of the cubic spline at pressures of 3, 6, and 15 cmH2O, respectively. Pressures of 3 and 6 cmH2O were selected to probe lung compliance in the range of tidal breathing and the effects of injury-induced derecruitment as we have reported for FOT measurements in mouse VILI  and embedded in paraffin for histological analysis  samples were obtained at D7 by instilling and withdrawing 0.2 ml saline. Protease and phosphatase inhibitors were added to each sample, and a BCA assay was performed to measure total protein concentration. Lungs from other animals were harvested and snap frozen for biochemical analysis, or inflation fixed with 4% paraformaldehyde  kit was used to isolate DNA from rat tail clips according to manufacturer instructions. PCR master mix was prepared using 1 μl of isolated DNA, as previously described . Forwardt-tests or an ANOVA with a Holm-Sidak correction for multiple comparisons. Non-normally distributed data was compared with a Mann-Whitney test or a Kruskal-Wallis test with Dunn’s correction. Changes in lung function pre-ventilation to post-ventilation were assessed with paired t-tests. Data are presented as mean ± SE unless otherwise indicated. Figures show p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), and p < 0.0001 (****).Statistical analysis was performed with GraphPad Prism. Outliers were removed using the ROUT method ; Q = 1% p < 0.0001), Quasi-static pressure volume loops (p < 0.0001), which may reflect the smaller size of the ETX pups. However, the difference in lung volume is not solely attributed to reduced body weight since inspiratory capacity normalized by body weight (IC) was significantly greater in D0 CTL (n = 27) compared to D0 ETX . By D7, ICs were approximately equal between CTL and ETX groups  was significantly greater for CTL compared to ETX (p < 0.0001). Likewise, compliance at 15 cmH2O (Cst15) was greater for CTL compared to ETX (p < 0.05). These results are not unexpected given the reduced IC in D0 ETX (p < 0.05). Taken together, these data show an ETX-induced reduction in lung volume and distensibility at birth that is nearly resolved by D7.Analysis of compliance on the expiratory limb of the quasi-static PV loops demonstrated that ETX increases pulmonary system stiffness , S4. At n D0 ETX . By D7, n D0 ETX ; howeverp < 0.05). Application of P20 to the D0 animals yielded a significantly higher Vt  than in the D7 pups. It is important to note that there was no significant difference in Vt between D7-P24 and D0-P20, suggesting that those groups are subjected to comparable degrees of tissue distension. The age‐ and endotoxin-induced alterations in pulmonary stiffness affected delivered tidal volumes as shown in PROTECT ventilation did not produce substantial qualitative changes in the PV loops compared to pre-ventilation loops in D0 and D7 animals , green. Ventilation at P20 and P24 caused significant increases in Cst6 and Cst15 in all age and treatment groups, when compared to pre-ventilation measurements, except for Cst6 in D0-ETX-P20 . Signifip < 0.01) and P20 (p < 0.01). At D7 with ETX, Cst6 was lower after PROTECT than P20 (p < 0.001); the D7-ETX-P24 group demonstrated lower Cst6 than the corresponding D7-CTL-P24 group (p < 0.01). Cst15 at D7 (p < 0.05) and P24 (p < 0.0001); the P24 group was also elevated above P20 (p < 0.0001). Likewise, Cst15 increased for D7-ETX from PROTECT to P20 (p < 0.05) and P24 (p < 0.0001); P24 was greater than P20 (p < 0.001).In D7 CTL, Cst6 was lower after PROTECT than P24 (15 at D7  with CTLp < 0.05). This increase was not present in the ETX treated mice and, as such, Cst6 for ETX-P20 was lower than in CTL-P20 (p < 0.05). The D0-CTL pups had higher Cst15 after P20 ventilation than for PROTECT (p < 0.01).At D0 with CTL treatment, Cst6  was incrp < 0.05). Likewise, IC for CTL-P20 was higher than ETX-P20 (p < 0.05) at D0. The D7-CTL rats showed increases in IC from PROTECT to P20 (p < 0.0001) and P24 (p < 0.0001). P24 was significantly greater than P20 (p < 0.05). In the D7-ETX animals, IC was lower for PROTECT than P20 (p < 0.0001) and P24 (p < 0.01). However, unlike the CTL group, ETX-P24 IC was not greater than ETX-P20 IC. As such, IC for ETX-P24 was less than CTL-P24 (p < 0.0001).Inspiratory capacity  significp < 0.01), ETX-PROTECT (p < 0.01), and ETX-P20 (p < 0.05). Among CTL groups BALF protein tended to increase with ventilation pressure, however these changes were not significant. The ETX rats ventilated with P24 showed a marked increase in BALF protein compared to CTL-P24 (p < 0.001). The mean, SE, and number of animals per group is provided in Lung airspace protein content in BALF samples of D7 rats shows a synergy between endotoxin exposure and ventilation . Among tLung histology from D0  and D7  rats demp < 0.0001) and CTL-PROTECT (p < 0.0001). Similarly, IL-6 gene expression was markedly increased in ETX-P20 rats compared to ETX-NV (p < 0.0001), and ETX-PROTECT (p < 0.0001). TNF-a gene expression increased in ETX-NV rats compared to CTL-NV (p < 0.0001). Mechanical ventilation reduced TNF-a gene expression so that ETX-NV was greater than ETX-PROTECT (p < 0.01), and ETX-P20 (p < 0.05). CXCL2 gene expression increased in CTL-P20 rats compared to CTL-NV (p < 0.001) and CTL-PROTECT (p < 0.001). TEN-C gene expression increased in CTL-P20 rats compared to ETX-P20 (p < 0.05). We did not find any significance in IL-1b gene expression among CTL or ETX groups. The mean, SE, and number of animals in each group are provided in Analysis of mRNA expression in whole lungs shows that higher ventilation pressures tend to increase inflammatory cytokine gene expression. In D0 animals , we founp < 0.05) and CTL-PROTECT (p < 0.05). TNF-a gene expression also increased in CTL-P20 rats compared to CTL-PROTECT (p < 0.05). Likewise, TNF-a gene expression increased in ETX-P24 rats compared to ETX-NV (p < 0.05) and ETX-PROTECT (p < 0.01). CXCL2 gene expression increased in CTL-P24 rats compared to CTL-NV (p < 0.01), CTL-PROTECT (p < 0.001), and CTL-P20 (p < 0.01). CXCL2 gene expression increased in ETX-P24 rats compared to ETX-NV (p < 0.01) and ETX-PROTECT (p < 0.01). TEN-C gene expression increased in ETX-P24 rats compared to ETX-NV (p < 0.001), ETX-PROTECT (p < 0.001), ETX-P20 (p < 0.01), and CTL-P24 (p < 0.01). IL-1b gene expression increased in ETX-P24 rats compared to ETX-NV (p < 0.01), ETX-PROTECT (p < 0.01), ETX-P20 (p < 0.01), and CTL-P24 (p = 0.05). IL-6 gene expression increased in ETX-P24 rats compared to ETX-NV (p < 0.001) and ETX-PROTECT (p < 0.05). IL-6 expression increased in ETX-P20 rats compared to ETX-NV (p < 0.05). The mean, SE, and number of animals in each group are provided in In D7 animals , we founLung neutrophil content was assessed by measuring myeloperoxidase (MPO) activity in lung homogenate . No signn = 5) showed a lower compliance than females .We retrospectively analyzed the differences between male and female rodents for Cst3, Cst6, Cst15, and IC. With the ventilation groups pooled, there were no sex-based differences prior to prolonged ventilation. Post-ventilation, the only significant difference was found for Cst6 in the D7-CTL-PROTECT group where males  in the absence of any postnatal injury is sufficient to stunt distal lung development with alveolar simplification, decreased pulmonary vasculature, and abnormal lung function at 2 weeks of life  and compliance with increasing ventilation pressure. In contrast, the ETX-P24 pups show substantially lower IC and Cst6 compared to CTL-P24 . The chaWhile previous studies have demonstrated injury after mechanical ventilation for 24 h in newborn mice  and incrThe VILI-induced changes in lung mechanics, we observed for D0 and D7 rat pups are strikingly different than those we have previously reported for adult rodents . We predPotential limitations of our current study include use of ETX to model the effects of chorioamnionitis on fetal lung development. While this is an established model of chorioamnionitis leading to consistent findings of lung simplification similar to what is seen with human BPD, it is unlikely to reflect the complexity of chorioamnionitis in human disease. Additionally, we used short ventilation strategies with relatively high pressures and zero PEEP to induce VILI in a short time period. While these ventilation strategies are unlikely to be used in human newborns, they provide a “stress test” for the lung and thus, this model provides insight into how antenatal inflammation influences preterm lung injury from ventilation. Furthermore, other investigations have shown that long durations of ventilation at lower pressure produce similar outcomes to short ventilation durations at higher pressures . AccordiIn conclusion, we demonstrate that antenatal endotoxin-exposed lungs demonstrate decreased inspiratory capacity and compliance at baseline and, when mechanically ventilated, are more susceptible to alveolocapillary barrier injury and atelectasis. The antenatal endotoxin-exposed lungs also demonstrate increased inflammatory mediators after mechanical ventilation as compared to control animals. Two hours of mechanical ventilation in saline-treated animals also significantly increased whole-lung inflammatory mediators, although to a lesser extent. This work highlights the importance of antenatal risk factors in the development of lung injury from postnatal mechanical ventilation. Increasing knowledge of the interactions of lung development, injury, and repair pathways will help determine development of effective treatments to mitigate BPD and late respiratory outcomes in the most vulnerable preterm infants.The original contributions presented in the study are included in the article/The animal study was reviewed and approved by University of Colorado Denver Institutional Animal Care and Use Committee.All authors contributed to conception and design. EM, CM, GS, SR, TG, AW, EB, and BS: data collection. EM, BS, SA, TG, and GS: drafting of manuscript. EM, BS, and SA: analysis and data Interpretation. All authors contributed to the article and approved the submitted version.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.


Dreissena polymorpha in the Great Lakes of North America is regarded as one of the most catastrophic ecological events in recent history. Previous studies showed a close kinship between European zebra mussels and their invasive cohorts in the Great Lakes. In this study, we repurposed and reanalyzed archived CO1 sequence data from Lake Superior and multiple sites in Europe that were collected between 1991 and 2011 to illustrate an interesting pattern of genetic isolation that was overlooked in previous studies. The results showed extreme genetic isolation of Lake Superior zebra mussels as evident by high ϕST values and strong geographic patterning of Lake Superior haplotypes.The invasion of the zebra mussel,  Dreissena polymorpha, is the most invasive freshwater mollusc in the United States, where its economic impact is estimated in the billions of dollars  genetic marker was chosen due to its overrepresentation in population genetic studies relative to other markers for this species. This was likely due to a large number of DNA barcoding projects. Sequences that were neither verified nor linked to published research and those that could not be traced to a geographic locality were discarded. To confirm collection dates, sequences were cross-referenced to their corresponding publications and in cases where no collection date was specified, authors were contacted directly for confirmation. Based on the aforementioned parameters, a total of 25 CO1 sequences were acquired from Lake Superior and 58 from Europe (Online Supplementary Table). Sequences were aligned using the MUSCLE algorithm and edited by eye in Geneious ver. 10.1.3 . All LS specimens formed a distinct haplogroup, which was extremely isolated from their European cohorts by more than 50 mutational steps. In contrast, within the European clade, parsimony networks showed a lack of any geographic patterning. For example, the haplotype EU2 was shared by individuals from nine geographically distinct regions. While Turkish zebra mussels were more related to European than North American individuals, they also clustered into a ‘mini’-haplogroup. The disjuncted haplotype distribution was corroborated by marked genetic differentiation between the two continents based on AMOVA results .Bayesian analysis showed strong population genetic signal with two major clades represented; a North American and European clade . All hapD. polymorpha and other dreissenids in Lake Superior are mainly limited to the Duluth Harbor which is regarded as the point source for D. polymorpha’s introduction to LS and also the sampling locality for where the archived sequences used in this study originated  . Despite these challenges, the current study shows that informative results can be gleaned from sequence databases but improvements in the system will be needed before invasion biologists can reliably use them.Click here for additional data file.


Picea glauca [Moench] Voss) to investigate spatial and genetic effects on tree ring width and on six xylem anatomical traits representing growth, water transport, mechanical support, and wood density. We compared different methods for estimating broad sense heritability (H2) of each trait and we evaluated the effects of spatial grouping and genetic grouping on the xylem anatomical traits with linear models. We found that the three different methods used to estimate H2 were quite robust, showing overall consistent patterns, while our analyses were unsuccessful at fully separating genetic from spatial effects. By evaluating the effect size, we found a significant effect of genetic grouping in latewood density and earlywood hydraulic diameter. However, evaluating model performances showed that spatial grouping was a better predictor than genetic grouping for variance in earlywood density, earlywood hydraulic diameter and growth. For cell wall thickness neither spatial nor genetic grouping was significant. Our findings imply that (1) the variance in the investigated xylem anatomical traits and growth is mainly influenced by spatial clustering (most probably caused by microhabitat conditions), which (2) makes it rather difficult to estimate the heritability of these traits in naturally grown trees in situ. Yet, (3) latewood density and earlywood hydraulic diameter qualified for further analysis on the genetic background of xylem traits and (4) cell wall thickness seems a useful trait to investigate large-scale climatic effects, decoupled from microclimatic, edaphic and genetic influences.The ecological function of boreal forests is challenged by drastically changing climate conditions. Although an increasing number of studies are investigating how climate change is influencing growth and distribution of boreal tree species, there is a lack of studies examining the potential of these species to genetically adapt or phenotypically adjust. Here, we sampled clonally and non-clonally growing white spruce trees ( Boreal forests are ecologically and commercially valuable ecosystems that make up almost a third of the global forest cover . They ac2, based on additive genetic variance) and a “broad sense”  is one of the most common tree species of the North American boreal forests  of growth and xylem anatomical traits of three trait groups  by comparing three different methods: (1) using raw data, (2) using data predicted with a linear mixed effects model and (3) using estimated variance extracted from a linear mixed effects model. Since vegetative reproduction in trees leads to an unavoidable spatial clustering of individuals, we additionally focused on the spatial patterns. We evaluated the results of our H2 estimations by using models to identify whether spatial grouping is the main driver for similarities in growth and xylem anatomical traits, or if genetics also influence these patterns. The advantage of this novel approach is that it combines spatial analyses with genetic analyses at an anatomical level. This informs about which xylem anatomical traits qualify for studying genetic patterns potentially leading to genetic adaptation and which qualify better for studying spatial patterns driven by the influence of microenvironment or by climatic effects.In this study, we identified and sampled naturally growing clones of white spruce at the latitudinal treeline in Alaska. We aimed at estimating broad sense heritability . The study site is a nearly monospecific white spruce stand of approximately two hectares, ranging in elevation from 860 to 940 m a.s.l. The mean annual temperature is around −7.9°C with a mean temperature of around −23.8°C in January (coldest month) and 11.1°C in July (warmest month). The annual precipitation is around 289 mm, 96 mm of which fall in July and August. The information about precipitation and temperature is taken from In 2018, we sampled all the spatially clustered groups of white spruce that were scattered throughout the study area ranging from the forest line to the treeline and appearing to be the result of vegetative reproduction see  Figure . We sampAll cores were air dried and glued onto wooden sample holders. The surface was polished with progressively finer sandpaper (up to 800 grit) and scanned with a flatbed scanner  with 3200 dpi. Ring widths (TRW) were subsequently measured using CooRecorder  and all radii were cross-dated using CDendro . We used the cross-dated tree ring chronologies solely to correctly date xylem anatomical measurements. For the analysis, we used TRW measurements obtained from the anatomical sections together with the other anatomical traits.For the xylem anatomical measurements we cut 12 μm-thick cross-sections from one radius of each tree using a rotary microtome . The cross-sections were stained with 1:1 safranin and astra blue solution, rinsed with ethanol solutions of increasing concentration , mounted on microscope slides with Euparal and dried at 60°C for 48 h. We scanned the slides with a slide scanner  at the Swiss Federal Institute for Forest, Snow and Landscape Research (WSL), Birmensdorf, Switzerland. We used the scans to quantify TRW and several xylem anatomical traits  with theEarlywood ring width (EWW) and latewood ring width (LWW) were detrended for each tree to minimize the influence of low frequency growth trends without losing too much information on the variance among the trees. For this, we compared two linear models with raw ring width (EWW and LWW separately) as a response variable and year as an explanatory variable. One model was fitted with a linear term of the explanatory variable while the other model was fitted with a linear and a quadratic term of the explanatory variable. We chose the better performing model based on the corrected Akaike Information Criterion AICc;  and calcTM Flex 96-well plate robot system  following the manufacturer protocols. For genotyping we combined 11 microsatellite loci developed by ® size standard (Life Technologies) and 12 μl HiDi Formamide (Life Technologies).To identify clones, we genotyped all sampled trees. We ground 20 mg of silica-gel dried needle tissue in a Retsch ball mill MM301 . For DNA extraction we used the Mag-Bind plant DNA DS Kit  in combination with the KingFisher® Software 5.0 (Life Technologies). To account for genotyping errors, we used the algorithm programmed by We performed fragment size determination and binning with the GeneMapperOf the 47 sampled trees in 11 spatially clustered groups, we found that 35 trees belonged to nine clonal groups, while 12 trees did not belong to any clonal group. Five spatially clustered groups consisted of clonal trees only. In four groups, clones grew spatially clustered with non-clonal individuals. Two groups consisted of non-clonal individuals only .To reduce the number of study parameters, we explored the relationship between the measured anatomical traits with a principal component analysis (PCA) using the R function prcomp  Supplem. We clasThe first principle component (PC1) of the PCA explained 41.3% of variance, the second principle component (PC2) explained 28.7% and the third principle component (PC3) explained an additional 14.7% of variance . The PCA2-estimations we only used data from trees that were growing in groups of genetically identical individuals . In order to explore the potential of using estimated data compared to raw data, we used three different methods to estimate H2. First (1), we estimated H2 using raw data of all selected xylem anatomical traits . Finally (3), we fitted a linear mixed effects model, where the investigated trait was the response variable, year was included as a fixed effect, cDBH as a covariate and clonal group as a random effect. We used the VarCorr function of the nlme R package to extract estimated genetic variance (2 according to Eq. 3 (H2mod). All models were fitted using restricted maximum likelihood estimation (REML).For the Hs H2raw; . Second s H2raw; , where ts H2raw; , we usedwhere where 2 estimations  was calculated as an error measurement for Hs Eq. 4; .where 2 values truly represent genetic effects or rather a spatial pattern caused by the spatial grouping of the clonal trees we used the full dataset of 47 sampled trees in 11 spatially clustered groups, including clonal and non-clonal individuals. We created two categorical groups, genetic group and spatial group, and each tree was assigned a level in each. For the genetic group, each tree was assigned either the clonal group ID or, in non-clonal trees, the individual tree ID; for the spatial group, all trees growing spatially clustered, with a maximum distance of 3 m, were assigned the same code, regardless of the genetic background  for each model and selected the model that performed best when the AICc was lowest with more than four units difference. Since AICc is only slightly penalizing small differences in the number of parameters, we considered the null model best in case of equal values, following the principle of parsimony , 2004.To evaluate the effect of spatial and genetic grouping and to find potential significant effects of spatial or genetic clustering that are independent of model performance we conducted an analysis of variance ANOVA; . We used2 showed overall similar results  the lowest. Traits associated with wood density and water transport  showed low to intermediate values , but for all other estimates and traits they were relatively low  and all earlywood traits  the spatial model performed better than the other models. For latewood density and latewood CWT there was no difference between the genetic and the spatial model, but in both cases the grouped models performed better than the null model. For latewood DH the null model was considered to show the best performance, as it had an AICc equal to the spatial model and lower than the genetic model .p-value < 0.05) for EWW and LWW, early- and latewood density and earlywood DH. Genetic grouping was significant for EWW and LWW, latewood density and earlywood DH. No significant grouping effect was found in early- and latewood CWT and latewood DH.The analysis of the spatial and genetic models using ANOVA showed that the spatial grouping was significant (2) of growth and xylem anatomical traits. We compared three methods to estimate H2, which overall showed consistent patterns in the resulting values. However, further analyses revealed a large influence of spatial clustering on xylem anatomy, which seemed to overlay any genetic patterns. Nonetheless, we found some evidence for a genetic influence on early- and latewood growth (ring widths), latewood density and earlywood hydraulic diameter.The rapidly changing climate conditions threaten the functioning of boreal forests. White spruce is one of the most important and abundant boreal forest tree species of North America. In order to learn more about its potential to adapt to changing environments, we explored its genetic background of xylem anatomical trait variation. The goal of our study was to investigate the broad sense heritability  of early- and latewood ring width. This inconsistency was likely caused by a high dispersion in the raw data, shown by high CVs  of xylem anatomical traits in white spruce. Though narrow sense heritability is based on additive genetic variance instead of total genetic variance, h2 is comparable to H2 because over 50% of the total genetic variance is usually additive  and ring width (reported as insignificant), respectively, Regarding growth-related traits like ring width, estimates of heritability in white spruce are quite scarce in other studies and considerably differ from our results. While 2 estimates are. Per definition, heritability can only be calculated for a specific population in a specific environment  are very short. However, other studies found significant correlations between CWT and climatic parameters in white spruce  and alsoin situ. The comparison of three methods to estimate broad sense heritability (H2) resulted in mostly consistent patterns. This suggests that in general the estimates are quite robust, independent from the method used for their calculation. However, due to spatial clustering of the trees we had to evaluate our heritability measures by testing the strength of the grouping effect. The analyses showed that spatial clustering had a strong influence on the xylem anatomy, especially in the earlywood. We assume that this strong spatial effect is related to differences in micro-environmental conditions, which implies that it is rather difficult to estimate the magnitude of genetic effects in a naturally grown population.The vegetative reproduction of white spruce at the latitudinal treeline offered the opportunity to gather data on genetically identical trees Nonetheless, we found some evidence for genetic effects in early- and latewood ring width, latewood density and earlywood hydraulic diameter. Based on previous studies we assume that results on early- and latewood width might rather be reflecting environmental conditions and individual growth patterns than actual genetics. Latewood density and earlywood hydraulic diameter, however, show a plausible significant genetic component, suggesting they are suitable traits for assessing potential local adaptation. Cell wall thickness, on the other hand, seems neither to be influenced by small-scale spatial  nor genetic patterns, potentially qualifying as a proxy for climatic conditions on a larger scale .Exploring the interacting effects of phenotypic plasticity and genetic adaptation in xylem anatomical traits related to wood density and tree hydraulics will lead to a more comprehensive understanding of the adaptation potential of tree species to global change in general, and of white spruce in particular. Yet, it is challenging to balance the reliability of experimental set-ups, which may be far from real-life conditions, with real-world studies, which may have high error potential. We believe that real-world studies dealing with clonal trees are valuable, but highlight the necessity to carefully evaluate any potential spatial effects, as they can drastically influence the growth of trees and obscure any potential genetic signal.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.TP, MW, and MZ designed the study and conducted field work and sampling. TP prepared the samples and performed xylem anatomical measurements with help from GA. MZ performed the genetic analyses. TP performed all statistical analysis with help from AA-R. TP wrote the manuscript with contributions from all authors.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.


Macadamia integrifolia, (ii) M. tetraphylla, and (iii) M. jansenii and M. ternifolia, and (iv) admixtures or hybrids. Assignment of the four species matched well with previous characterisations, except for one M. integrifolia and four M. tetraphylla accessions. Using SNP markers, 94 previously unidentified accessions were assigned into the four distinct groups. Finally, 287 accessions were identified as pure examples of one of the four species and 15 as hybrids of M. integrifolia and M. tetraphylla. The admixed accessions showed the highest genetic diversity followed by M. integrifolia, while M. ternifolia and M. jansenii accessions were the least diverse. Mantel test analysis showed a significant correlation between genetic and geographic distance for M. integrifolia  and a positive but not significant correlation for M. tetraphylla . This study provides a population genetics overview of macadamia germplasm as a background for a conservation strategy and provides directions for future macadamia breeding.Macadamia is an Australian native rainforest tree that has been domesticated and traded internationally for its premium nuts. Common cultivars rely upon a limited gene pool that has exploited only two of the four species. Introducing a more diverse germplasm will broaden the genetic base for future crop improvement and better adaptation for changing environments. This study investigated the genetic structure of 302 accessions of wild germplasm using 2872 SNP and 8415 silicoDArT markers. Structure analysis and principal coordinate analysis (PCoA) assigned the 302 accessions into four distinct groups: (i)  Macadamia belongs to the Proteaceae family and is composed of four species: Macadamia integrifolia Maiden and Betche, M. jansenii C.L. Gross and P.H. Weston, M. ternifolia F. Muell and M. tetraphylla L.A.S. Johnson. The natural distribution of the four species is in the subtropical rainforest from south-east Queensland (QLD) to north-east New South Wales (NSW), Australia [M. integrifolia and M. ternifolia occur in south-east QLD, while M. tetraphylla is distributed mainly in northern NSW, with some extension into south-east QLD [M. integrifolia and M. tetraphylla and between M. integrifolia and M. ternifolia, with natural hybridisation occurring in these zones [M. jansenii, has been found only in a single location in Bulburin (QLD) that is 180 km north from the nearest population of M. integrifolia [M. integrifolia and M. tetraphylla produce edible nuts, and hence, most of the commercial cultivars belong to either of these two species or their hybrids [M. jansenii and M. ternifolia, have not been used in directed breeding, due to their bitter inedible nuts containing high levels of cyanogenic glycoside [M. integrifolia [The genus ustralia ,2. M. ineast QLD . Overlapse zones . The fougrifolia . Of thes hybrids . The othlycoside ,8,9. Mosgrifolia ,11. Howegrifolia  ExplorinThe study of genetic diversity and population structure can determine the potential of wild germplasm in future breeding. The genetic structure is formed over time due to the multiple actions of migration, selection, mutation, and genetic drift , as wellM. integrifolia, three M. tetraphylla, and two hybrids) and three M. ternifolia accessions. Mast et al. [Macadamia species and their wild relatives, using three cpDNAs , three nDNA  genomic regions. However, this study used only one accession per species. The genetic structure of a large number of wild germplasm accessions was studied by Peace [M. integrifolia [M. tetraphylla populations [Molecular markers are considered as the most suitable tool to estimate genetic diversity, due to their polymorphic nature and independence to environmental effects . Severalt et al.  studied by Peace  using loby Peace . SSRs wegrifolia  and wildulations ,22. Howeulations , and as The rapid advancement of next generation sequencing technology (NGS) enables the discovery of high-throughput and cost-effective molecular marker systems. Using NGS technology, Diversity Array Technology (DArT) developed a marker system that facilities affordable whole-genome level genetic characterisation. DArT has been successfully applied for the genetic diversity, population structure and genetic mapping studies of many crop species ,25,26. RM. integrifolia and M. tetraphylla.In this study, for the first time, we used high-throughput DArTseq platforms for the genetic characterisation of a large number of wild accessions of macadamia. The aims were to: (1) assess the population structure of wild macadamia germplasm, (2) explore the genetic diversity among the accessions within species, and (3) determine the relationship between genetic and geographic distance within DArTseq platforms generated 13,221 SNP and 47,811 silicoDArT markers. The call rates of SNP markers varied from 0.20 to 1.00, with an average of 0.62 . Of the The K and Q values from STRUCTURE analysis were used for the assignment of individual accessions in each species/hybrid group. Population clusters in PCoA validated the species representation.M. ternifolia, two as M. jansenii, and eight as undefined species , these accessions were classified as M. ternifolia. Cluster II contained 99 predefined M. integrifolia and 36 accessions of previously undefined species. Cluster III was comprised of 94 predefined M. tetraphylla and 38 undefined species. There were 17 accessions, including one predefined M. integrifolia, four predefined M. tetraphylla, and 12 accessions from planted/unknown/mixed populations that showed the genetic admixture (hybrid) of clusters. For example, the accession “M034”, which was previously labelled as M. integrifolia, consisted of 6% of Cluster II (predominantly M. integrifolia), 16% of Cluster III (predominantly M. tetraphylla), but 78% of Cluster I (predominantly M. jansenii and M. ternifolia). Two previously labelled M. tetraphylla accessions (“M265” and “M266”) were identified as admixtures of Cluster II & III. The accession “M265” was composed of 61% of Cluster II and 39% of Cluster III, and accession “M266” was composed of 52% of Cluster III.The ΔK from the STRUCTURE analysis of SNP markers was significant when K = 2, 3 and 5, with a peak at K = 3 a. The op species . These uM. jansenii, M. ternifolia and M. tetraphylla, into one cluster, and separated M. integrifolia accessions in another cluster. The pattern displayed for K = 5  of SNP markers via distance matrix with data standardization identified three distinct groups of the four species . This reM. integrifolia, 133 M. tetraphylla, and 19 M. ternifolia/M. jansenii  ranged from 1.08 to 1.34, with the lowest in M. ternifolia/M. jansenii group and the highest in admixture. Shannon’s index (I) ranged from 0.11 (M. ternifolia/M. jansenii) to 0.33 (admixture), with a mean of 0.23. In all clusters, the observed heterozygosity (Ho) was smaller than the expected heterozygosity (He). He was highest in admixture (0.21) and lowest in M. ternifolia/ M. jansenii (0.07), with a mean of 0.15. Interestingly, accessions from the M. integrifolia group showed the highest percentage (86.53%) of polymorphism (%P), followed by admixture (74.93%), M. tetraphylla (71.5%), M. ternifolia/M. jansenii (32%) groups. Similar results were also observed for silicoDArT markers  than that of M. ternifolia/M. jansenii accessions (D = 0.23). Hence, the admixture accessions showed almost similar genetic distance with M. integrifolia (0.06) and M. tetraphylla (0.07) germplasm. Although the estimated value of genetic distance using silicoDArT markers was lower than that of SNPs, the genetic relationship between species was similar in both marker systems (Nei’s genetic distance (D), based on SNP markers , ranged  systems .The analysis of molecular variance (AMOVA) showed a higher proportion of variance detected within species than among clusters . For SNPM. integrifolia and M. tetraphylla presented in the dendrogram , while the highest (0.151) was observed between the accessions of Beenleigh and Nimbin.To explore the genetic basis of the geographic relationship we calculated the correlation between the genetic distance and geographic distances of the localities for each species. The pairwise genetic distance among the accessions of six localities of grifolia  varied fcalities  were higp = 0.05) among M. integrifolia localities   b.Population structure and PCoA of DArTseq based SNP markers facilitated the assignment of individuals in corresponding species or hybrid groups. Our species classification of each accession matches well with the field note and previous DNA study . ResultsM. integrifolia accessions were clustered together in the same group. “M034”, an accession from the “Mooloo” region of the “Gympie” locality, was identified as M. ternifolia/M jansenii, although it was previously recorded as M. integrifolia. A co-investigation of wild germplasm using 15 SSR markers identified that the same accession “M034” is a clone of another accession (X-CANB896104) from Canberra Botanic Garden. Interestingly, accession “X-CANB896104” is recorded as a cutting from wild M. ternifolia from Mary Cairncross Park, Maleny . Further phenotypic characterisation confirmed that “M034” is a M. ternifolia. Among the 99 accessions of M. tetraphylla, four accessions were assigned as hybrid of M. tetraphylla and M. integrifolia. Accessions “M265” and “M266” shared almost 50% from each species, whereas “M270” and “M277” had ~30% from M. integrifolia genotype and ~70% M. tetraphylla  and M. ternifolia (n = 16) in our study. Though M. jansenii and M. ternifolia differ in a few simple traits including leaf tip, leaf serration and flower colour, they are morphologically similar, both with small tree size, and small and bitter nuts [Macadamia species can be used to confirm the speciation of wild macadamia germplasm.This study clearly demonstrated that the accessions of  studies ,30. Thester nuts ,31. HoweM. integrifolia, 39 as M. tetraphylla, 8 as M. ternifolia and 11 as hybrids/admixtures of M. integrifolia and M. tetraphylla , accession “M054” from population 106  and five accessions from population 36 . However, these populations were noted at the time of collection as planted or uncertain populations, and it seems highly likely that their locations were the result of human activity.Some accessions we identified as He) across the wild germplasm and clusters forming “pure” species was lower than in previous genetic studies of macadamia cultivars [M. integrifolia and M. tetraphylla, which is composed of a small number of accessions (n = 15), showed greater gene diversity than previous studies on cultivars and pure wild species in the current study. This result indicates that crossing between two species can be conducted, to increase genetic diversity in the future breeding program.In this study, we developed new knowledge on genetic diversity in wild accessions by using high-throughput silicoDArT and SNPs marker. In both types of markers, the average expected heterozygosity  and M. ternifolia (n = 17) have been tested. The lower diversity of M. jansenii and M. ternifolia in our study may be the result of small populations. An investigation with larger sample numbers from diverse distributions of M. jansenii and M. ternifolia should be conducted, to define their genetic diversity more completely and accurately.Our results suggested that iversity . Generaliversity . Extincttus etc. . Certain species ,9. In thM. integrifolia germplasm. Since the gene flow for both species is restricted within a short distance (~50 km) [Geographical distance is one of the major contributing factors in species differentiation. Knowledge of the genetic structure of a species over its geographic distribution is important to develop an understanding of the evolutionary processes . In this(~50 km) , the imp(~50 km) , or a re(~50 km) , could bM. integrifolia accessions revealed a significant positive correlation between genetic and geographic distance . This correlation was higher than a previous study using RAF markers [p = 0.016), and in almond germplasm  [M. tetraphylla. Although, this correlation (r = 0.45) is non-significant, it is higher than that found by Peace [M. tetraphylla suggested that geographic distance may not be the main factor influencing the genetic distance between populations of this species, although geographical boundaries, low gene flow and genetic drift are typically key factors explaining genetic differentiation in fragmented populations [Phylogeographic analysis of  markers  (r = 0.1= 0.226) . Based oon r = 0.5 is non-ulations .M. integrifolia and M. tetraphylla provided an indication of ancestral lineage of the accessions of each species. Interestingly, the root of most of the accessions of both species originated from the population of Numinbah . These accessions originated from 75 populations  across 52 regions (one to three populations per region) from 14 localities   and Alstonville, New South Wales (NSW), Australia. Wild accessions were collected from multiple geographical regions , coverinocality) . The acchttps://www.diversityarrays.com/). DNA samples were incubated with loading dye at 37 °C for two hours and then checked for quality control on 0.8% agarose electrophoresis gel for 30 min at 100 V.Newly flushed young leaves were collected from the ex situ trials at Tiaro and Alstonville in December 2017 and placed in zipped plastic bags inside a cool box with ice blocks. The materials were stored in a cold room at 4 °C before transfer to DArT Pty Ltd. , to perform DNA extraction. A total of 2–3 mg of leaf tissue from each sample was sub-sampled into a 1.1 mL microtube containing a disposable steel ball bearing. Leaf samples were crushed using a QIAGEN Tissue Lyser . A Freedom EVO robotic Tecan 100  was used for DNA extraction following an existing protocol recommended by DArT (PstI + HhaI) of complexity reduction was selected to detect DArTseq-based markers using next generation sequencing (NGS) technology. The silicoDArT and SNP markers were scored as a binary data format in which the score was “1” for presence, “0” for absence and “-“, for failure to score of a marker in the genomic representation of each sample. The full details of methodology of developing DArTseq-based markers for macadamia were previously described by Alam et al. [Accessions were genotyped for dominant silicoDArT and co-dominant SNP markers following an established protocol developed by Kilian et al.  An approm et al.  and O’Com et al. DArTseq platforms generated SNP and silicoDArT markers. DArTsoft v7.4 software was used to automatically identify and score the polymorphic markers, using a proprietary marker calling algorithms. The quality of the markers was tested for call rate (%), reproducibility (%), one ratio, and polymorphic information content (PIC). The call rate determines the success of reading the marker sequence across the samples, and was estimated from the percentage of samples for which the score was either “0” (absence of marker) or “1” (presence of marker). The scoring of reproducibility involves the proportion of 138 technical replicates for which the marker score exhibited consistency. One ratio was determined as the proportion of samples for which genotypes were scored as “1”. PIC is the degree of diversity of the marker in the population and shows the usefulness of the marker for linkage analysis. Quality control and filtering were applied to both SNP and silicoDArT markers, including call rate (>80%), reproducibility (>95%), and one ratio (>0.05).http://taylor0.biology.ucla.edu/structureHarvester/, to determine the optimum number of populations  using ∆K value, as described by Evanno et al. [The population structure of the 302 accessions of four species was identified based on SNP markers by using the Bayesian model-based program STRUCTURE v2.3.4 ,42,43,44o et al.  The indio et al. ,47.The genetic diversity and principal coordinate analysis (PCoA) were performed using GenAlEx v6.5.2 software . Both SNM. integrifolia and M. tetraphylla are the two major species used in current breeding programs. In this study, we have identified a large number of accessions of M. integrifolia and M. tetraphylla. Therefore, a detailed study was undertaken on the genetic relationships and geographical diversity of those species. Only accessions identified as “pure” representatives of each species that were confirmed by structure analysis, including 99 M. integrifolia accessions from six localities (M. tetraphylla accessions from eight localities (The correlation between genetic distance and geographic distance was estimated. The Nei’s genetic distance matrix and the pairwise geographic distance among the localities were calculated using GenAlEx v6.5.2 . The ManM. jansenii and M. ternifolia formed a single cluster. The Nei’s genetic distance analysis clearly demonstrated that M. jansenii and M. ternifolia are related and showed greater heterozygosity in M. ternifolia than in M. jansenii. However, the limited number of accessions available in this study from M. jansenii and M. ternifolia limits the strength of our conclusion on the diversity and population structure of these species. We suggest that further analysis with more accessions from these two species should be conducted, to increase understanding of genetic diversity and clarify their classification as distinct species. We observed the significant correlation between genetic and geographic distance among M. integrifolia populations. Additionally, we were able to confirm the species identity of unknown wild accessions and suggested the use of these markers to resolve the unclear species composition of domesticated cultivars.This is the first study to investigate the genetic structure of a large collection of wild macadamia germplasm using thousands of high-throughput molecular markers. A total of 302 wild accessions were characterised, using 2872 SNP and 8415 silicoDArT markers. Our population structure and principal co-ordinate analyses identified three distinct populations, in which


Porites lobata among pairs of highly variable and high-stress nearshore sites and adjacent less variable and less impacted offshore sites on the islands of Oahu and Maui, Hawaii. Using an analysis of molecular variance framework, we tested whether populations were more structured by geographic distance or environmental extremes. The genetic patterns we observed followed isolation by environment, where nearshore and adjacent offshore populations showed significant genetic structure at both locations , but no significant isolation by distance between islands. Strikingly, corals from the two nearshore sites with higher levels of environmental stressors on different islands over 100 km apart with similar environmentally stressful conditions were genetically closer  than those within a single location less than 2 km apart . In contrast, a third site with a less impacted nearshore site  showed no significant structure from the offshore comparison. Our results show much stronger support for environment than distance separating these populations. Our finding suggests that ecological boundaries from human impacts may play a role in forming genetic structure in the coastal environment, and that genetic divergence in the absence of geographical barriers to gene flow might be explained by selective pressure across contrasting habitats.We examined genetic structure in the lobe coral  Coral reefs are centers of marine biodiversity and productivity that provide a variety of ecosystem services of substantial cultural and economic value to humankind, yet coral reefs worldwide are under serious threat as a result of human activities . AverageSuch effects are particularly pronounced in nearshore marine habitats, which are increasingly exposed to reduced water quality due to human activities . Recent What impact does an ecological landscape with such a strong gradient have on the genetics of the organisms? ‘Isolation by distance’ IBD,  predictsFavia fragum associated with strong ecological gradients. The two types are largely restricted to alternate seagrass and adjacent coral reef habitats, but retain phenotypic distinction in a narrow zone of ecological overlap , the study species, occurs over a wide geographic range in the tropical Pacific Ocean  and Wahikuli, Maui (hereafter Maui1) represent two locations with a strong environmental gradient, whereas Honokōwai, Maui (hereafter Maui2) represents a similar paired nearshore-offshore comparison site, but without a strong environmental gradient  structures have been proposed to be important for species identification, therefore, we examined the corallites of all collected samples to confirm our taxonomic identifications  of P. lobata tissue samples were collected from live colonies between February 2013 to May 2017 at the following sampling sites in Hawaii; (a) ‘Oahu’—nearshore (n = 22) and offshore (n = 21) sites at Maunalua Bay, Oahu , (b) ‘Maui1’—nearshore (n = 21) and offshore (n = 23) sites off the Hanakao’o Beach Park, West Maui , and (c) Maui2—nearshore (n = 23), and offshore (n = 20) sites off the Honokōwai Beach Park, West Maui   , Fig. S156.69°W)  , or stoP. lobata exhibits very little sequence variability  ∼400 bp coral mitochondrial putative control region (CR) with primers CRf and CO3r , (2) ∼70ic sites ) due to ic sites , and (2)2, 2 µl of 10 mM dNTPmix, 1.6 µl of each 10 mM primer, and nuclease-free water to volume. For samples with multiple bands, approximately 1500-bp PCR products were extracted from agarose gels after electrophoresis and purified using the UltraClean® 15 DNA Purification Kit  according to the manufacturer’s instruction. The rest of the PCR products were purified with UltraClean® PCR Clean-Up Kit (MO BIO Laboratories) and sequenced directly in both directions on the ABI 3730xl DNA Analyzer. Clone libraries were created for each amplified ITS region using the pGEM®-Easy Vector System (Promega). Positive inserts were verified by PCR using SP6 and T7 primers, and plasmids (2–5 per library) were treated with UltraClean® 6 Minute Mini Plasmid Prep Kit (MO BIO Laboratories) and sequenced on an ABI-3130XL Genetic Analyzer sequencer. For Maui samples, H2 was amplified and sequenced using the same method as described above.H2 was amplified under the following conditions: 96 °C for 2 min (one cycle), followed by 34 cycles consisting of 96 °C for 20 s, 58.5 °C for 20 s, and 72 °C for 90 s, and a final extension at 72 °C for 5 min. H2 amplifications (25 µl) consisted of 0.5 µl of DNA template, 0.2 µl of GoTaq® DNA Polymerase , 5 µl of GoTaq® Reaction Buffer, 1.6 µl of 50 mM MgClResulting DNA sequences were aligned using Geneious® 6.1.8 . Polymorphic sites within H2 regions were identified using Geneious® (Find Heterozygotes option) and confirmed by eye. Middle sections, as well as both ends of H2 were then trimmed to 1,352 bp (for Oahu sequences) or 1,221 bp  to minimize missing nucleotides among samples. H2 was phased using the program PHASE 2.1  and SeqPP. lobata populations, the degree of genetic differentiation was estimated using analysis of molecular variance  , which was not surprising due to its extremely low variability in corals and cnidarians in general (P = 0.15 and 0.08 respectively) (For Oahu e (AMOVA ) between< 0.001) . The mit general . The num general . The pat general . For CR,ctively) .π) were almost twice as high in the offshore population as in the nearshore one based on ITS  was clearly higher than that of the nearshore population (Richness = 13) (O), the number of heterozygous individuals, and mean gene diversity  all had marginally higher values in the offshore samples, though the differences were small (θπ (the expected heterozygosity estimated from the average nucleotide diversity) was higher than θs (the theta estimated from the number of segregating sites).The pattern of genetic diversity also differed between the nearshore and offshore populations. The degree of genetic diversity was higher at the offshore site; percent private alleles (pA), percent polymorphic sites (poly), and nucleotide diversity level (d on ITS . Standard on ITS . Rarefacss = 13) . The levre small . In bothP. lobata populations between the nearshore and offshores sites were analyzed using the novel H2 marker . At Maui1, where a strong environmental gradient exists, significant genetic differentiation was detected , but the level of genetic diversity was comparable between the two sites . The level of genetic diversity at Maui2 appeared slightly higher in the nearshore population, which had higher numbers of haplotypes (A), polymorphic sites (poly), and heterozygous individuals , but the two Maui locations showed significant differentiation  based on H2 (π) levels were relatively similar between Oahu and Maui  than to other nearshore populations of Oahu and Maui1 (FST = 0.14–0.18), suggesting collectively that Maui2 corals at both sites were genetically closer to the offshore populations . Instead, a pattern that resembles IBE was revealed, with a correlation between habitat types irrespective of geographic distance; the pairwise FST values revealed that offshore individuals from two separate islands (>100 km) were genetically closer to each other than to their geographically closest nearshore individuals (0.3–2 km), and nearshore individuals from two islands were also typically genetically closer to each other than to corals from adjacent offshore sites  within a corallite ; nearshore corals had taller and more pronounced pali than the offshore ones. Because exact functions and heritability of the traits are unknown, whether the observed morphological differences are due to divergent selection cannot be answered at this point. However, the study suggested that the differences might be due to potential beneficial roles played by larger pali in shedding sediments in turbid water . The below surface current movement seems to be more complex, and is generally towards the east (nearshore to offshore) with the presence of small eddies, at least during the summer . Eddies  gametes . For Mau gametes . While tF. fragum . Also, fine-scale water movements that the oceanographic-scale studies cannot capture  genetic structure, and environmental drivers across habitat types appear to have a stronger effect in forming such genetic structure (IBE) than geographic distances (IBD) in coastal areas that are heavily affected by anthropogenic stressors. Genetic similarity found between Oahu and Maui1 nearshore populations suggest that the observed genetic structure may be driven by similar underlying evolutionary mechanisms, including adaptation to environmentally driven factors. Our results also highlight the importance of thorough sampling among habitats at small scales; we could easily overlook such important local genetic differences, and may mistakenly conclude that populations are uniform across the landscape without thorough sampling. Although our samples are from limited locations, these results demonstrate that understanding small-scale genetic variation and diversity can provide important information on the ecological basis of genetic diversity and differentiation, which must be understood to effectively implement future coral reef conservation efforts.Our results show that the broadly distributed broadcast spawning coral 10.7717/peerj.8550/supp-1Figure S1Nearshore site is located at the mouth of the embayment around which the urban population center of Hawaii Kai has developed. This area sees intense human use and a strong anthropogenic impact gradient to Offshore site at uninhabited Koko Head. Though the offshore site locates relatively close to the shore, its conditions are closer to those of offshore due to the unique topography .Click here for additional data file.10.7717/peerj.8550/supp-2Figure S2Nearshore site is located close to the beach where run-off and human activities are concentrated. The run-off and the unique current patterns create drastic differences in water quality between the nearshore and offshore sites .Click here for additional data file.10.7717/peerj.8550/supp-3Figure S3Porites lobata  and Porites evermanni . Live polyps  and bleached corallite skeleton  of the two species. Images taken under a stereo microscope, highlighting the morphological differences to distinguish between P. evermanni  which has a smoother colony surface, whereas corallites of P. lobata  are contoured and deeper.Close-up images of polyps and corallites of Click here for additional data file.10.7717/peerj.8550/supp-4Figure S4NeighborNet phylogenetic networks generated by SplitsTree, color-coded by geographic region as shown in Click here for additional data file.10.7717/peerj.8550/supp-5Table S1The number of heterozygous sites per individual was obtained using VCFtools, and the total number of polymorphic loci was obtained using Arlequin .Click here for additional data file.10.7717/peerj.8550/supp-6Table S2AMOVA tables for (A) Individuals by geographic sampling sites, (B) Individuals sampled within habitat types across islands .Click here for additional data file.10.7717/peerj.8550/supp-7File S1Click here for additional data file.


The dog population of Southern East Asia is unique in harboring virtually the full range of the universal mtDNA gene pool, and consequently, it has the highest genetic diversity worldwide. Despite this, limited research has been performed on dog genetics within this region. Here we present the first comprehensive study of a sub-region within Southern East Asia, analyzing 528 bp of mtDNA for 265 dogs from Thailand, in the context of dogs from across the Old World. We found that Thailand was the only region in the world that has the full range of the universal mtDNA gene pool, that is, all 10 sub-haplogroups. Consequently, the statistics for diversity are among the highest, especially in North Thailand, which had high values for haplotype diversity and the number of haplotypes, and the lowest proportion of individuals with a universal type-derived haplotype (UTd) among all regions. As previously observed, genetic diversity is distinctly lower outside Southern East Asia and it decreases following a cline to the lowest values in western Eurasia. Thus, the limited geographical region of Thailand harbors a distinctly higher genetic diversity than much larger regions in western Eurasia, for example, Southwest Asia and Europe which have only five and four of the 10 sub-haplogroups, respectively. Within Thailand, diversity statistics for all four sub-regions follow the general pattern of Southern East Asia, but North Thailand stands out with its high diversity compared to the other regions. These results show that a small part of Southern East Asia harbors the full range of the mtDNA gene pool, and they emphasize the exceptional genetic status of Southern East Asia. This indicates that today’s dogs can trace a major part of their ancestry to Southern East Asia or closely situated regions, highlighting Thailand as a region of special interest. Considering the large genetic diversity found in Thailand and that many neighboring regions, e.g., Myanmar and Laos, have not been studied for dog genetics, it is possible that large parts of the dog gene pool remain undiscovered. It will be an important task for future studies to fill in these blanks on the phylogeographic map. The domestic dog is considered to be the first domesticated animal and was the only domesticate to accompany humans to every continent in ancient times. Despite this central role in human history, and considerable efforts in previous research, the question about where the dog originated remains unresolved. Previous global studies of mitochondrial DNA (mtDNA) variations among dogs have suggested they originated in East Asia , and morThus, the geographical origin of the domestic dog is under vigorous debate, but the mitochondrial data is clear in that Southern East Asia is unique in harboring virtually the full range of the universal mtDNA gene pool, and consequently, it has the highest genetic diversity worldwide for this marker. Therefore, in order to obtain a comprehensive picture of the global gene pool for mtDNA, studies of Southern East Asia and the surrounding regions are vital. Despite this, research on dog genetics in this area is limited so far.Thailand is located in the center of mainland Southeast Asia, in the southern part of Southern East Asia. It has four distinctive regions, each with unique geographic and cultural characteristics: North, Northeast, Central and South Thailand. North Thailand is the most isolated and desolate part of the country with hilly forested areas and a tropical savanna climate. Northeast Thailand is dominated by a vast and relatively arid plateau. Central Thailand is dominated by a large fertile plain. South Thailand comprises a substantial portion of the Malay Peninsula and Thailand’s coastline with major tourist industry . ThailanConsidering the exceptional genetic diversity of the mtDNA pool of Southern East Asia and the relatively sparse sampling of this large region, which is half the size of Europe, it is clear that expanded studies of Southern East Asia are a prerequisite for obtaining a full picture of global mtDNA diversity. Previously, no comprehensive genetic studies of village dogs in Thailand or any other part of Southern East Asia, or of the Thai ridgeback dog, have been reported. We therefore performed the first large-scale study of a sub-region within Southern East Asia, analyzing 528 bp of mtDNA for 265 dogs from Thailand, in the context of dogs from across the Old World. Hereby, we give the first more detailed description of the mtDNA gene pool in a sub-region of Southern East Asia, thus shedding light on the genetics in a region of central importance for studies of the origins of today’s domestic dogs, and contributing to a comprehensive picture of the global mtDNA gene pool.n = 80), Northeast (n = 60), Central (n = 32) and South (n = 61). We also included samples from one indigenous dog breed, the Thai ridgeback (n = 28). A list of all samples with information about, e.g., geographical region and mtDNA haplotype is given in A total of 265 samples for dogs from Thailand (163 new samples and 102 samples from the literature) were analyzed for a 582bp fragment of the control region of the mitochondrial genome . This region of mtDNA is the only genetic marker studied so far for a comprehensive collection of dogs from across the world. Therefore, we were able to study the Thai dogs in the context of samples from across the Old World published in previous studies ,12,13, iSamples were collected as buccal cell samples on FTA cards  according to the manufacturer’s instructions. The target sequence was amplified using a nested PCR reaction suggested by . The outThe DNA sequences were manually aligned using Mega X. A neighbor-joining (NJ) tree was constructed by Mega X , using CWe analyzed 582bp of the mtDNA control region for 265 dogs from Thailand in the context of samples from across the Old World, that is, a total of 3254 dogs. As described in previous studies, the mtDNA gene pool was, at a superficial level, remarkably homogenous among geographical regions. Phylogenetic analysis showed three principal phylogenetic groups, haplogroup A, B, and C , which aThe Thai dog population fits well in this universal phylogeographic pattern. Of the 265 dogs from Thailand, 163 were new samples, among which 17 new haplotypes were identified: 14 in haplogroup A, 2 in B, and 1 in E. In total, 65 haplotypes were found, which were assigned to haplogroups A, B, C, and E, and these haplogroups were represented by 83.0%, 9.8%, 4.9%, and 2.3% of the dogs, respectively. Among these 65 haplotypes, 13 were UTs and 32 were unique to Thailand . Thus, the Thai dog population shares the universal mtDNA gene pool, and follows the general pattern in that almost 100% of the dogs carry haplotypes belonging to haplogroups A, B and C and these three haplogroups are represented at proportions similar to most other regions.However, as it has already been reported, while the dog population of the Old World was overall strikingly homogenous at a superficial phylogenetic level in sharing the same three principal haplogroups at similar proportions, there were also very distinct differences at a more detailed phylogenetic level. Phylogenetic studies of mitochondrial genomes have shown that the three haplogroups A, B and C have a more detailed phylogenetic substructure. This divides these three haplogroups into a total of 10 distinct sub-haplogroups , which can also be distinguished for the analyzed 582 bp region based on diagnostic mutations [In contrast to haplogroups A, B and C, these 10 sub-haplogroups were not universally distributed in the Old World, instead, the distribution followed a distinct cline with a maximum in Thailand. Thus, the only region harboring all 10 sub-haplogroups was Southern East Asia, and within Southern East Asia, the Thai dog population stood out as the only subpopulation that harbored all 10 sub-haplogroups . From thThe cline for the number of sub-haplogroups was reflected by other statistics of genetic diversity: the proportion of UTs and UTds, the number of haplotypes  and haplotype diversity . The proAlso with regard to the number of haplotypes and the haplotype diversity, Southern East Asia stood out with much higher values than other regions, except for Siberia, which had the highest haplotype diversity. Within Southern East Asia, Thailand stood out also for these statistics, with higher values than South China. The only major deviation from the general trend in the data was the Nigerian population, which was obtained from a recent article by Adeola et al. . Our stuWithin Thailand, the statistics for all four sub-regions conform largely to the values generally found in Southern East Asia, e.g., concerning the proportion of individuals carrying haplogroups A, B and C, and the proportions carrying UTs and UTds, but there were also some clear differences in the genetic diversity among the four sub-regions. Most importantly, North Thailand had a higher diversity compared to the other regions, especially compared to Northeast and South Thailand, with regard to the number of haplotypes and haplotype diversity as well as the percentage of dogs carrying UTs and UTds. Especially the low proportion of dogs carrying a UT or UTd stands out, with the lowest numbers of all populations in the study. The lowest genetic diversity was found in Northeast Thailand, which had a low number of haplotypes and only 5 of the 10 sub-haplogroups, compared to 7–8 in the other regions.We also compared the sub-regions of Thailand with other parts of Southern East Asia, specifically three parts of South China: Yunnan, Guizhou and Guangxi, and Hunan and Jiangxi. These regions all had a high number of sub-haplogroups, that is, 9, 8 and 8, respectively. For the other statistics, North Thailand stood out with the highest haplotype diversity and lowest proportion of individuals carrying UTs and UTds, and second highest number of haplotypes. Notably, although Yunnan had 9 sub-haplogroups, the highest number of all sub-regions, it had the lowest haplotype diversity, possibly a result of biased sampling from related individuals . OverallThe Thai ridgeback dogs had a similar proportion of haplogroups A and B as the non-breed dogs in Thailand, with 85.7% haplogroup A, 14.3% haplogroup B, but lacked clade C. They also had a similar number of dogs carrying UTs and UTds. With 13 haplotypes among the 28 Thai ridgeback dogs, the genetic diversity was significantly higher than for European dog breeds, which normally have around 5 haplotypes .Haplogroup E was rare, both in the total sample  and in the Thai sample , and was found exclusively in East Asia: in Thailand, Vietnam, Indonesia, Korea and Japan. All four E haplotypes were found in mainland and island Southeast Asia, and three of the four were exclusively in this region; only the most frequent haplotype, E1 was found in Korea and Japan. Furthermore, three of the four haplotypes were found in Thailand while only a single one was found in any other region. It is also noteworthy that the E haplotypes were absent in the sample of 282 dogs from South China.With only 16 individuals carrying E haplotypes no certain conclusions can be drawn about the phylogeography of haplogroup E. However, the fact that all four haplotypes were found in mainland and island Southeast Asia but only a single haplotype was found outside this region, and that the haplogroup was absent in South China, indicates that haplogroup E originated from wolves somewhere in mainland Southeast Asia.This study demonstrates that the relatively small geographical region of Thailand, which is only 5% the size of Europe, uniquely harbors virtually the whole range of the universal mtDNA gene pool, that is, all 10 sub-haplogroups. This reinforces the distinct difference in the distribution of mtDNA haplotypes across the Old World, that populations in Southern East Asia have a much more complex diversity than other regions. It also, for the first time, specifically highlights the sub-regions south of China as areas of special interest. Evidence from whole nuclear-genome data has shown that dogs from Southern East Asia have the basal phylogenetic position relating to wolves and the highest genetic diversity , and thaWhile Thailand is unique in harboring all 10 sub-haplogroups, two of these, a4 and a6, are rare, and the absence of one of these in the sample from South China may reflect incomplete sampling. Thus, the difference among the sub-regions within Southern East Asia was not dramatic. However, it is notable that North Thailand and Yunnan, which are both situated in the northwestern part of Southern East Asia, had the highest haplotype diversity and lowest proportion of individuals with a UT or UTd, and the highest number of sub-haplogroups, respectively. This suggests the northwestern part of Southern East Asia as a possible source for the modern mtDNA gene pool. However, it also highlights the total lack of samples from large parts of Southern East Asia. Two specific examples are the neighboring regions, Myanmar, which borders the entire western side of Yunnan and the northwestern part of North Thailand, and northern Laos, situated between southern Yunnan and North Thailand. Considering the unique diversity in North Thailand and Yunnan, it is possible that a considerable part of the dog mtDNA gene pool remains undiscovered in Myanmar and Laos, and future studies will need to fill in these and other blanks on the phylogeographic map of southern and eastern Asia.Extended sampling is also warranted in South China. Only 282 samples from this vast region have been analyzed so far, and detailed sampling information is largely lacking detail, indicating nothing more than the province [A neighboring region of special interest is Nepal and Mongolia, referred to as Central Asia in , a studyThis study shows that, except for the 2% of the dogs that carry haplotypes belonging to haplogroups D, E and F, there are no region-specific haplogroups, which implies that dogs worldwide share a universal gene pool. This in turn implies that all dogs in today’s global domestic dog population originate from the same domesticated wolves, which were either domesticated in a single region, or in different regions for the different haplogroups followed by sufficient mixing of haplotypes. In conclusion, there is clearly a single genetic origin for today’s dog population, though not necessarily from a single geographical region.It is important to appreciate that this is a study of today’s dog population. The fact that practically the full range of today’s genetic diversity for mtDNA is found in Southern East Asia is a strong indication that the modern mtDNA gene pool originates from there, or from neighboring regions. However, this does not necessarily mean that the original mtDNA gene pool, and thus the domestic dog, also actually originated there. It is possible that the universal mtDNA gene pool experienced considerable changes during the ancient history of dogs. An indication of this is the dramatic change through time in the proportions of haplogroups A, B and C in Europe. A study of mtDNA in European pre-Neolithic dogs found all 15 investigated samples (100%) to have haplotypes belonging to haplogroup C . In contNevertheless, the fact that the full range of sub-haplogroups of haplogroups A, B and C have been found exclusively in Southern East Asia indicates this region, or neighboring regions, as a probable origin for much of the modern dog mtDNA gene pool. Together with the evidence from Y-chromosome and whole nuclear-genome data ,4,7, thiThe southern range of wolves in Southern East Asia is unclear. Western literature has incorrectly stated that wolves are absent from today´s Southern East Asia, with some articles claiming that wolves were never present in this region, even in ancient time. However, this was recently corrected in a review of the distribution of the wolf in China , showingConsequently, this study identifies two important tasks in the quest for the genetic origins of the modern as well as the ancient dog population: to describe the historical spread of the wolf in southern and eastern Asia, and to fill in large blanks on the phylogeographical map of dogs in this region. In addition, the exceptional mtDNA diversity, compared to other sub-Saharan populations, of the Nigerian dog population warrants detailed studies of mitochondrial genomes in sub-Saharan Africa.In conclusion, this study demonstrates that the relatively small geographical region of Thailand is the only region in the world that has been shown to harbor the full range of the universal dog mtDNA gene pool, all 10 sub-haplogroups. This reinforces the exceptional genetic status of the dog population in Southern East Asia, which has already been indicated in previous studies of mtDNA, Y-chromosome and whole nuclear-genome data, and highlights Thailand as a possible region from which today’s global dog population may have dispersed. However, large parts of southern and eastern Asia remain poorly studied, or are even blind spots on the phylogeographical map. Therefore, to get a comprehensive picture of the global gene pool of today’s domestic dogs, further studies in and around Southern East Asia, of mitochondrial as well as nuclear genomes, will be essential.


Panax vietnamensis Ha et Grushv. is medicinally important, endemic and endangered species of Vietnam. However, genetic diversity and structure of population are unknown due to lack of efficient molecular markers.Understanding the genetic diversity in endangered species that occur inforest remnants is necessary to establish efficient strategies for the species conservation, restoration and management. P. vietnamensis . Raw reads total of 23,741,783 was obtained and then assembled, from which the generated unigenes were 89,271 (average length = 598.3191 nt). The 31,686 unigenes were annotated in different databases i.e. Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, Nucleotide Collection (NR/NT) and Swiss-Prot for functional annotation. Further, 11,343 EST-SSRs were detected. From 7774 primer pairs, 101 were selected for polymorphism validation, in which; 20 primer pairs were successfully amplified to DNA fragments and significant amounts of polymorphism was observed within population. The nine polymorphic microsatellite loci were used for population structure and diversity analyses. The obtained results revealed high levels of genetic diversity in populations, the average observed and expected heterozygosity were HO = 0.422 and HE = 0.479, respectively. During the Bottleneck analysis using TPM and SMM models (p < 0.01) shows that targeted population is significantly heterozygote deficient. This suggests sign of the bottleneck in all populations. Genetic differentiation between populations was moderate (FST = 0.133) and indicating slightly high level of gene flow (Nm = 1.63). Analysis of molecular variance (AMOVA) showed 63.17% of variation within individuals and 12.45% among populations. Our results shows two genetic clusters related to geographical distances.In this study, we employed Illumina HiSeq™ 4000 sequencing to analyze the transcriptomes of Our study will assist conservators in future conservation management, breeding, production and habitats restoration of the species. Panax species  are medicinally important plants of North America and eastern Asia /4 /4  Polymorp) - 1]/4  Tests fo) - 1]/4 . Testing) - 1]/4 . The sigThe data were subjected to AMOVA using Arlequin 3.1 and significance test was applied on a basis of 10,000 permutations . The UPGAdditional file 1: Figure S1. The Delta K distribution graph.