We demonstrate the importance of speciation of luminescent metal complexes in water on biological behaviours such as cellular uptake and subcellular localization. 1ZnL) as a case study to demonstrate that metal induced aggregation arising from the intermolecular Zn···O interaction influences its cellular uptake and subcellular localization. Comparative studies with the free bases (1L and 2L) show that 1ZnL undergoes cellular uptake through caveolae-mediated endocytosis and internalizes in endosomal/lysosomal compartments, in contrast to the localization of 1L and 2L in the mitochondria. Further studies of photophysical properties, TEM imaging and DLS analysis suggest that 1ZnL tends to form large sized fibrous structures in aqueous media. To investigate the relationship between 1ZnL aggregation and the biological behaviour, we used pyridine to tune the “aggregation-to-deaggregation” transition and found that, in the presence of pyridine, 1ZnL could localize in the mitochondria and internalize into cells through the passive diffusion pathway. Such distinctive biological behaviours resulting from the different Znsalen species clearly point out the importance of metal induced aggregation or metal speciation analysis in designing metal complexes as biological probes.Unravelling the unique effects of metal coordination on biological behaviours is of importance to design metal based therapeutic and diagnostic agents. In this work, we chose luminescent Znsalen ( Despite the tremendous progress which has been made in understanding the relationships between structural information arising from coordination, such as lipophilicity, oxidation state, charge state and coordination mode, and biological behaviours, much less attention has been given to the physical state or association of luminescent metal complexes in aqueous media.14 For metal complexes, the intermolecular metal–ligand or metal–metal interactions reinforce the aggregation process, which may influence the mobility of the metal complex, and therefore constrain the pathways of exposure to the cell and affect the subcellular distribution. Thus, the chemical and physical form of a metal complex in aqueous solution might be an important factor in designing luminescent imaging agents. To address this issue, we herein chose the Znsalen complex ethylenediamine) as a case study to demonstrate that metal induced aggregation arising from intermolecular Zn···O interaction indeed influences its cellular uptake and subcellular localization.Luminescent metal complexes have emerged as an important class of imaging agents, because of their photophysical properties such as high luminescence, good photostability, large Stokes shifts and long life-times.2+ ions possess high Lewis acidity within a planar geometry that allows for extra coordination of a Lewis base ligand or coordinating solvent, or in their absence, self-assembly through an intermolecular Zn···O axial coordination to the phenolic group of another unit.22 Such speciation of Znsalens or salophens is accompanied by changes in morphology and “switched off/on” fluorescence corresponding to the “aggregation-to-deaggregation” transition.25 This forms a chemical basis to design optical sensors to detect anions, biological alkaloids, and even metal cations.30 Extending this prominent feature to living cell imaging, Znsalens could be used as low cytotoxicity agents, and display high fluorescence intensity in specific organelles, in which the environment is more hydrophobic than in cytosol or cell culture media.34 Since Znsalen tends to aggregate in aqueous media, it is feasible to investigate whether and how Znsalen aggregation affects its biological behaviours such as cellular uptake and subcellular location. This would be important to further design luminescent Znsalen complexes as bioprobes,36 combined with the insights gained from the coordination chemistry of Znsalens.Znsalens were selected because Zn1ZnL, which is conjugated with the mitochondria-targeting triphenylphosphonium cation (TPP), and its free base congeners 1L and 2L  and the free bases 1L and 2L (mitochondria) indicated the significant biological effect arising from Zn coordination. Using 1L and 2L as controls, we excluded the effects of factors such as lipophilicity and charge state on biological behaviours. Then, we hypothesized that the intermolecular Zn···O interaction driving 1ZnL aggregation plays an important role in its distinctive biological behaviours, according to the photophysical properties and morphology of 1ZnL and 1L in aqueous media. To confirm this hypothesis, we used pyridine as an extra ligand to dissociate the aggregates by breaking the intermolecular Zn···O interaction, and found that 1ZnL undergoes the passive diffusion pathway and mainly localizes in mitochondria  show two major absorption bands between 350–450 and 500–600 nm. The free bases 1L and 2L display broad absorption bands from 360 to 470 nm, whereas 1ZnL shows a sharp band centered at 385 nm with a low-energy shoulder at 435 nm. The low-energy bands are likely due to an internal charge transfer (ICT) transition. 1ZnL, 1L and 2L exhibit red emission  with fluorescence quantum yields of 0.26, 0.29 and 0.21  NMR to estimate the molecular weight of 1ZnL in d6-DMSO  according to Leo's method.43 This suggests that Zn coordination increases the lipophilicity of 1L, by an amount comparable to the methoxylation of the phenolic group of 1L . FYVE-EGFP, EHD1-EGFP, LysoTracker® Green DND-26 and MitoTracker Green served as markers for the early endosome, late endosome, lysosome and mitochondria organelles, respectively. As shown in 1ZnL showed good cell permeability and was mainly distributed in the lysosomal/endosomal compartments. It exhibited a co-localization level of approximately 0.56 with LysoTracker® Green DND-26. Interestingly, 1L and 2L particularly stained mitochondria with Pearson's correlation coefficients of 0.92 and 0.83 ,46 genistein (inhibitor of caveolae-mediated endocytosis),48 or cytochalasin D (inhibitor of macropinocytosis).53 As –1 genistein resulted in a drastic decrease of 80% in the cellular uptake of 1ZnL, suggesting that 1ZnL is mainly internalized via caveolae-mediated endocytosis, which plays a general role in the internalization of large sized particles into cells.55 In addition, treatment with cytochalasin D also led to a 40% decrease in cellular uptake, suggesting that a macropinocytosis-mediated pathway might also be partly involved  as mitochondria models following Pielak's method  showed a logistic regression with P values of 2.71 and 1.04  in the mixtures of D2O and d6-DMSO solvents  NMR for 1ZnL in d6-DMSO solution containing 30% D2O showed an average molecular mass of 1602  experiments were performed. As shown in 1L (1 μM) has a smaller hydrodynamic diameter  and narrower distribution than 1ZnL . No change of the 1ZnL or 1L aggregates were observed in cell culture media  and scanning electron microscopy (SEM) by drop-casting the aqueous solutions of 1ZnL or 1L onto Cu–C (200) substrates. As shown in 1ZnL, while 1L shows a distinct globular morphology with dimensions ranging from 20 nm to 70 nm, which are consistent with the DLS results. To exclude the effect of lipophilicity, we used 2L as a control, and 2L exhibited a similar particle size and distribution to those of 1L in both aqueous solution and cell culture medium . The flexible spacer (C4 chain and triazole linker) promotes the aggregation of the Znsalen moiety and the triphenylphosphonium cations as head groups located in the interfaces, which is in agreement with the changes of the proton signals in the 1H-NMR spectra. Thus, the hydrophobic effect is another important factor that determines 1ZnL aggregation in water.Although the aggregation of Znsalen complexes through the intermolecular Zn···O interaction in non-coordinating solvents is a usual feature, such studies in water have much less been reported. There are several examples of water soluble Znsalens that were found to be monomeric species in aqueous solutions,1ZnL aggregation, we carried out competitive pyridine binding experiments. According to previous studies,19 the competitive binding of Lewis bases such as pyridine would dissociate the aggregates if the intermolecular Zn···O interaction was the main driving force for the self-assembly process in non-coordinating solvents. As shown in 1ZnL, the bands at around 380 and 550 nm were red shifted to 387 and 590 nm, indicating the dissociation of the 1ZnL aggregates. As expected, the fluorescence intensity increases along with the increasing amount of pyridine , Br– (nBu4NBr) and HBSS buffer (containing various inorganic anions), which are relevant to biological studies. As shown in nBu4NBr and HBSS from 0 to 50 mM, the UV-vis spectra showed increasing bandwidths and blue-shifted with decreasing intensity at 500–600 nm, indicating increased aggregate formation. To understand this, we proposed that the high concentration of anions promoting the 1ZnL aggregation is due to a salt effect arising from PBS, nBu4NBr and HBSS. To test this, we measured the UV-vis spectra of 1ZnL in the presence of NaCl (0–49 mM). As shown in 1ZnL might be affected by the ionic strength of these salts. This also could be used to explain why pyridine is very effective to disassociate 1ZnL for the high binding constant to Zn ion and negligible ionic strength of pyridine. Although the “aggregation-to-deaggregation” process for Znsalen/salophens has been extensively studied,21 few studies on the stability of such aggregates in aqueous media have been performed. This is important to verify whether the aggregates are stable enough to affect the biological behaviours. Following Kleij's method,20 we also estimated the stability constants of the 1ZnL aggregates in aqueous media. The relationship of the overall aggregation constant Kagg, stepwise aggregation constant Kn/n+1, and the aggregation number n can be described using the following equation:Kpy represents the coordination constant of pyridine to the Zn center of 1ZnL. To evaluate Kpy, we carried out pyridine titration on the solution of 1ZnL in dichloromethane (DCM)  reported by Kleij,26 and the value of 4.7 × 106 reported by Di Bella.62 The lower coordination constant was probably due to the interference of the large triphenylphosphonium groups. As in the pyridine titration in aqueous solution, the increase at 590 nm was monitored to calculate the total reaction constant K. Kagg and Kn/n+1 depend on the aggregation number n. Table S3Kagg and Kn/n+1 with increasing aggregation numbers. Each addition of monomer to a previously formed oligomer has a high association constant, in the order of about 1010 M–1, which is about 2 orders higher than for the dimerization of Znsalens in organic solvents.18 This clearly demonstrates that the solvent effect reinforces the intermolecular Zn···O interaction and enhances the stability of the 1ZnL aggregates.In addition to pyridine, we attempted to test the effect of anions on  Fig. S14. The coo1ZnL aggregation affected its biological behaviours, we performed cell imaging experiments using 1ZnL in the presence or absence of pyridine. According to the above results, 1ZnL aggregates can be dissociated by the addition of pyridine, which would exhibit different cellular uptake pathways and subcellular distributions from the sole use of 1ZnL. The intracellular luminescence and subcellular distribution of 1ZnL were investigated in HeLa cells using confocal laser scanning microscopy (CLSM). As shown in 1ZnL exhibited punctate red fluorescence in the lysosomal/endosomal compartments, which were mainly co-localized with LysoTracker® Green DND-26 (Pearson's correlation coefficient 0.56). In contrast, in the presence of pyridine (about 6000 equiv.), 1ZnL was mainly localized in the mitochondria and co-localized with commercial MitoTracker Green (Pearson's correlation coefficient 0.60) (To confirm the hypothesis that nt 0.60) . These r1ZnL in aqueous solution influences the internalization mechanism, we examined the temperature-dependent cellular uptake using flow cytometry. As shown in 1ZnL in the presence of pyridine at 4 °C than that at 37 °C, indicating that the 1ZnL species were internalized by cells via a temperature-dependent process. Then, we employed the endocytosis inhibitors chlorpromazine (inhibitor of clathrin-mediated endocytosis),46 genistein (inhibitor of caveolae-mediated endocytosis),48 and cytochalasin D (inhibitor of macropinocytosis)53 as described above, to investigate whether 1ZnL internalizes into the cell through endocytosis. As shown in 1ZnL in the presence of pyridine is almost not correlated to endocytosis, which is markedly different from the endocytosis pathway of 1ZnL in the aggregated state.To understand whether the deaggregation of 1ZnL with pyridine may be facilitated by organic cation transporters (OCT) or driven by the plasma membrane potential .66 When using tetrabutylammonium bromide as the OCT inhibitor,67 HeLa cells showed no obvious intracellular fluorescence change 68 or nigericin-HBSS .69 As shown in +-HBSS-treated cells displayed a remarkable decrease in intracellular fluorescence (80%), while the nigericin-treated cells showed an intracellular fluorescence increase of nearly 2 fold. This clearly demonstrated that 1ZnL in the presence of pyridine is internalized mostly by membrane potential dependent passive diffusion. Since we have demonstrated that PBS, HBSS, and Br– cannot lead to 1ZnL deaggregation and couldn't disturb pyridine coordination, combined with its photophysical properties and morphology in aqueous media, we hypothesized that the distinctive internalization pathways may be due to the different morphologies between the “aggregation/deaggregation” transition of 1ZnL, because the caveolae-mediated or macropinocytosis-mediated endocytosis is related to the uptake of large sized particles into cells.68Given that the TPP conjugate possesses a positive charge, the internalization of e change . To inve1ZnL and the free bases 1L and 2L, Zn coordination was found to lead to a distinctive cellular uptake pathway and subcellular distribution. More importantly, the photophysical and morphology studies on 1ZnL and the free bases in aqueous media suggest that the different aggregation states arising from the intermolecular Zn···O interaction play a critical role in influencing the biological behaviours. This hypothesis was confirmed by cell imaging experiments using 1ZnL in the presence or absence of pyridine, which clearly demonstrated the effect of tuning the “aggregation/deaggregation” transition of 1ZnL in aqueous media on the cellular uptake pathway and subcellular distribution. These results point to a new factor, “metal induced aggregation”, which effectively influences cellular uptake and subcellular distribution, and which should not be overlooked in designing luminescent metal complexes as biological probes.Taking our results together, we demonstrated that the intermolecular Zn···O interaction between Znsalen played an important role in determining its cellular uptake pathway and subcellular distribution. Through comparative studies between


Dypsis ambositrae and D. decipiens, are known to be threatened with extinction and conservation management for these species is a priority for the newly created protected area in the region.The need to incorporate genetic data into conservation management decisions is increasingly recognised. However, many published studies represent a ‘gold standard’ of sampling, techniques, and analyses. Such rigour is often not possible with limited funding and resourcing available for developing plans for the increasing number of threatened species requiring conservation management. Two endemic palm species of the Itremo Massif in central Madagascar, The genetic diversity of these two species was studied using the relatively low-cost and rapid AFLP technique. DNA fragments generated using three primer combinations were analysed for 20 and 50 individuals of the two species, respectively, from across their ranges.D. ambositrae grows were found to be genetically distinct . Despite having a much wider distribution and relatively large population, D. decipiens did not show clear geographical nor genetic groupings and had similarly low genetic heterozygosity to D. ambositrae.Genetic diversity was relatively low for both species. The two sites where the highly restricted D. ambositrae are conserved and that seed are collected from both for ex situ conservation and potential future reintroduction. It may be less important to focus resources on conserving or collecting ex situ material from all sites where D. decipiens is found, as the genetic diversity represented by each subpopulation is limited and increasing sampling may not protect significantly higher levels of genetic diversity. This study provides data that inform and support conservation decisions taken for both species within this region, and in the management of the newly designated Itremo Massif Protected Area, which covers most of the sites where these two species remain in the wild.With so few individuals remaining in the wild and two genetically distinct subpopulations, it is recommended that both sites of  The flora of Madagascar exhibits remarkably high diversity and endemism, more than 80% of the approximately 14,000 known vascular plant species being unique to the country . Many plin situ all single-site species that are assessed using IUCN Red List categories and criteria as either Critically Endangered or Endangered  in Madagascar comprises 204 species in 17 genera , 98% of Beccariophoenix madagascariensis by Voanioala gerardii has extremely small populations in the wild, the species contains an unexpectedly high level of genetic diversity, contrasting with the known populations of the Endangered Lemurophoenix halleuxii which exhibit a low level of genetic variation.Many studies of extinction risk and genetic factors in palms have been undertaken in recent years, including several in Madagascar . A studyin situ protection of sites, the collection of seeds and plants for ex situ seedbanks, and the cultivation of species in botanic gardens and field gene banks. Other studies consider how representative ex situ collections such as botanic gardens and seed banks are of species’ genetic diversity  Beentje & J Dransf., D. decipiens (Becc.) Beentje & J Dransf., and Ravenea madagascariensis Becc. Two of these species, D. ambositrae and D. decipiens, have been assessed as Critically Endangered and Endangered respectively, using IUCN Red List categories and criteria (version 3.1)  . Dypsis is study .Dypsis decipiens is found across the Central High Plateau of Madagascar between Andilamena and Fianarantsoa. In the Itremo Massif Protected Area, the species occurs in large stands of hundreds of individuals, along riverine valleys and hillsides, in open grasslands, and it has been estimated that at least 2,700 individuals may grow here . Samples of leaf tissue were dried in silica gel to preserve their DNA. The majority of specimens were collected from the Itremo Massif region, where the population sizes of the two species are largest; other known sites for the two species visited and sampled were Ankazobe, Ilaka, Ambalamanakana, and Ankafina Tsarafidy . There ao Massif , a 1 km2DNA was extracted from the silica-dried leaf samples using a modified CTAB method —washing EcoRI and MseI restriction enzymes were used to digest each DNA sample, and double stranded EcoRI and MseI adaptors were ligated onto the ends of the digested fragments. A primer trial was conducted using 12 primer pairs, to select three combinations that would provide a level of variation appropriate for this study. Primer combinations MseI-CTC + EcoRI-AC, MseI-CTG + EcoRI-AAG, and MseI-CAC + EcoRI-AAC, were used in the analysis, the selective EcoRI primer being labelled with a fluorescent dye, and a 250 ROX size ladder being used. Resulting fragments were analysed using an ABI 3730 Genetic Analyser. GeneMapper .D. ambositrae and D.decipiens datasets were calculated using AFLPsurv   in MVSP The significance of the partitioning of AFLP variation among populations was tested using Analysis of Molecular Variance (AMOVA) in GenAlEx using 999 permutations, and a Mantel test was implemented in GenAlEx to test for Isolation by Distance (IBD), comparing genetic and geographic distance matrices for both species.D. ambositrae and 50 D. decipiens individuals. Descriptive statistics of the results for both species are shown in AFLP profiles with data from all three primer combinations were produced and analysed for 20 D. decipiens individuals, from seven geographically distinct populations. A total of 135 loci were produced using the primers MseI-CTC + EcoRI-AC, 113 loci from MseI-CTG + EcoRI-AAG, and 122 loci from MseI-CAC + EcoRI-AAC.Altogether a total of 370 loci were scored from 50 D. ambositrae individuals, from 2 geographically distinct populations. 87 loci were produced using the primers MseI-CTC + EcoRI-AC, 67 loci from MseI-CTG + EcoRI-AAG, and 76 loci from MseI-CAC + EcoRI-AAC.A total of 230 loci were scored from 20 Descriptive statistics for both species can be seen in D. decipiens, with the higher levels in populations D2 and D3, where more samples were collected.The lowest levels of diversity are seen in the samples from the smaller size subpopulations of D. ambositrae separate into two separate clusters using the first two PCoA axes . The third axis for each species (not shown) adds an additional 9.5% and 4.3% of the variation but reveals no more clustering of the individual points. Oddly, for both species, there is a single outlying individual. The outlying individual for D. ambositrae is a sample collected from the A2 population, outside the Itremo protected area. The outlying individual of D. decipiens is one of just two individuals collected from the D7 population north of Antananarivo .The PCoA results using Euclidean genetic distances between individuals show that there is some clear separation of the genetic variation in the two species into clusters. The two populations of CoA axes . The clu 1 and 2  do not sD. ambositrae and D. decipiens respectively); thus, more of the genetic variation in D. ambositrae is related to among population variation than in D. decipiens (21% compared with 7%), though it is recognised that the number of subpopulations studied for each species differ substantially. The pairwise genetic distances among sample sites (ϕPT) was much higher for D ambositrae (0.211) compared with those for D. decipiens (0.074).The AMOVA test  shows thP < 0.001). As visualised in the PCoA results  is found within the Itremo Protected Area, in a relatively undisturbed area of humid gallery forest. The second population (A2) occurs close to a major road , in a fragment of forest that has been heavily degraded in recent years . Degradation has been particularly due to the decline in habitat quality, through the extraction of large  trees for in situ charcoal production, and the felling of the palms to facilitate access to these trees.We now believe that Dypsis decipiens is found across a much larger area than previously thought; we now estimate that 2,500–3,000 individuals grow across the open grassland of the Itremo Massif region, and are grouped into the subpopulations shown in It is worth noting that not all of the silica-dried samples collected across the region yielded DNA of adequate quality for analysis; this is likely to have been due to problems with drying some samples sufficiently quickly in the field. Other researchers have anecdotally reported problems with extracting DNA from palm leaf material, possibly due to the presence of mucilage and secondary metabolites that are thought to either interfere with the extraction process or lead to the degradation the DNA after collection in the field.D. decipiens actually has lower genetic diversity than D. ambositrae, the species with the dramatically smaller and geographically restricted population.This study has shown that overall genetic diversity and levels of heterozygosity are fairly low in both of the study species. Comparatively higher levels of genetic diversity are known to enhance species’ resilience and persistence in the wild, so these results raise concerns about the future ability of these two palms to survive in the future in the face of climate change and encroaching human-induced habitat destruction. Interestingly, the more widespread species The low proportion of polymorphic loci revealed in the two palm species (46.5% and 36.6%) is comparable with the low proportion found in a similar AFLP study of Madagascan legume tree species, where an average of 40% of loci were found to be polymorphic, and the more threatened species were found to have fewer polymorphic loci than the less threatened taxa . Low genBeccariophoenix madagascarienis  is much higher , and similarly, the expected heterozygosity (He) is 0.595–0.691 for B. madagascariensis, versus 0.233 and 0.239 for D. ambositrae and D. decipiens. In the Madagascan legume AFLP study of D. ambositrae and D. decipiens). A study of two Madagascan palms, Voanioala gerardii and Lemurophoenix halleuxii , being revealed by the AFLP analysis. In D. decipiens, there seems to be some genetic structuring dividing the sampled individuals into two main groups, but these do not correspond completely with the geographically distinct populations. There could be said to be a core Itremo genetic group, comprising individuals from D1 (west Itremo), D2 (north Itremo), D3  and D4 (south Itremo), and a separate genetic group that follows the road network through Itremo from the west to the centre, and northward along routes RN 4 and 7. The distribution of this latter group could reflect the anthropogenic movement of the species along the road system.There is clear evidence for a degree of genetic and geographic clustering in both species. Clustering is more clearly defined in The population sizes and genetic and geographic groupings revealed in this study can be used to inform conservation decision making for the two species studied, and already are being taken into account in current conservation efforts.The overall genetic diversity represented in both species is so low that extensive, time- and funding-consuming efforts to try to conserve representatives from across their entire distributions  would not be the best use of available resources, as most of the limited genetic diversity would be captured by relatively few individuals. Although we might predict threatened species to represent low genetic diversity compared with less threatened species, and the results of this study are congruent with this theory, the results of these other studies suggest that this rule does not always apply and cannot be assumed for all species. Further study of biological and ecological traits of threatened taxa, including life history traits, population distributions and trends, geographic and habitat preferences, may help to interpret these conclusions further.D. ambositae populations in order to capture the maximum genetic diversity. This should result in ex situ seed and living collections being that which represent as many unique alleles as possible, improving the genetic basis upon which future re-introduction and supplementation work would be based. In situ efforts should aim to protect both of the remaining populations from future degradation—although one population is protected within Itremo NPA, the population near route RN7 is highly threatened.The results of this study suggest that it should be a priority to collect seed from both D. decipiens is perhaps less urgent than is the case with D. ambositrae, which has a much larger total population size. Moreover, most of the plants are found within the Itremo NPA, a legally designated site managed for species and habitat conservation objectives by RBG Kew with strong community engagement and development work. Interestingly, this study has shown that the genetic groupings of the species are limited to just two main clusters, and these do not correspond well with the geographic sites from across the full distribution of the species, from which the plants were sampled. Resource-intense efforts could in theory be made to collect D. decipiens seed from across its full distribution in order to capture as much genetic diversity as possible, but the results of this study show that increased sampling effort may well result in limited gains in terms of genetic diversity represented in the collected material.The situation with D. decipiens occurring outside the Itremo Massif area do not represent significant genetic diversity beyond that represented in the main populations within Itremo NPA, and so are less important for conservation either in situ or ex situ. We hypothesis that these small populations, reliably found near main roads, may be the result of humans deliberately moving these aesthetically attractive plants.The small populations of D. decipiens grow within the Itremo NPA, and therefore are conserved in situ, our main recommendation would be ensure that the species is well represented in ex situ collections, ideally by progeny from a range of mother plants. As with other horticulturally desirable species, D. decipiens is widely grown around the world. However, in the case of species such as this, confined in the wild to remote islands like Madagascar, cultivated plants are likely to be the result of just a small number of original exported seed collections–and therefore represent a very limited gene pool from the species in the wild. This certainly applies to the Critically Endangered palm Tahina spectabilis, although represented in many ex situ collections around the world, virtually all plants are progeny of a single seed collection taken from just one individual in the wild  be gathered from across the entire geographic distribution of the species, and less structured and more opportunistic collecting may well result in the collection of as much genetic diversity as a more systematic, geographically even but labour-intensive approach.Since most individuals of The utility of employing population genetic techniques in conservation situations where population sizes are extremely small and/or threatened may, in some cases, be limited when resources (including time) are severely restricted. In the situation presented in this study, the amount of information obtained from assessing the genetic diversity and structure of the two study species proved to be relatively limited. It is also unclear how far one can extrapolate from these results to other palm species in Madagascar, or more generally to other island species.D. ambositrae population located outside the Itremo Protected Area), with less focus on additional conservation measures for species already enclosed within protected areas and/or with larger and more continuous populations (as for D. decipiens). In situ species conservation should continue to be the priority, but in terms of ex situ conservation, the priority when resources are limited should be to sample small populations across their distribution, thereby capturing as much genetic diversity in collections as possible. Less effort need be expended on sampling extensively across larger, and less fragmented, populations.The results of this work do usefully support current priorities of conservation organisations to concentrate specific resources on conserving otherwise unprotected small populations and fragmented sites (such as the 10.7717/peerj.3248/supp-1Supplemental Information 1Dypsis ambositrae samples, 3 primer combinations, 230 loci.AFLP coded data for 20 Click here for additional data file.10.7717/peerj.3248/supp-2Supplemental Information 2Dypsis decipiens samples, 3 primer combinations, 370 loci.AFLP coded data for 50 Click here for additional data file.


Previous seascape genetics studies have emphasized the role of ocean currents and geographic distances to explain the genetic structure of marine species, but the role of benthic habitat has been more rarely considered. Here, we compared the population genetic structure observed in West Pacific giant clam populations against model simulations that accounted habitat composition and configuration, geographical distance, and oceanic currents. Dispersal determined by geographical distance provided a modelled genetic structure in better agreement with the observations than dispersal by oceanic currents, possibly due to insufficient spatial resolution of available oceanographic and coastal circulation models. Considering both habitat composition and configuration significantly improved the match between simulated and observed genetic structures. This study emphasizes the importance of a reefscape genetics approach to population ecology, evolution and conservation in the sea. The dispersal of individuals between populations is the foundation of meta-population dynamics . In a meRecent advances in population genetics and the identification of hypervariable genetic markers provide new opportunities to infer demographic connectivity by estimating gene flow among populations from allele frequencies . Indeed,Trying to relate genetic connectivity with environmental factors expected to influence dispersal has been the focus of several recent studies. This approach was termed “landscape genetics” in terrestrial ecosystems, by a fusion of “landscape ecology” with “population genetics” . LandscaThe simplest form of landscape genetics relates genetic distances to geographical distances , a model termed “isolation by distance” (IBD). Isolation by distance is the process by which geographically restricted gene flow and random genetic drift generate genetic differentiation among populations on different habitat patches . In termThe translation of landscape genetics to the marine realm was termed “seascape genetics” , 15, butMore recently, however, several modelling and demographic studies have suggested that seascape features beyond oceanographic distance may influence demographic connectivity. This includes, for example, the spatial configuration of favourable habitat for mature adults of benthic species . In coral reef ecosystems, the spatial configuration of benthic habitat is referred to as the “reefscape configuration.” Indeed, habitat patchiness may drive the relative influence of local and non-local offspring, with higher self-recruitment expected in fragmented habitats compared to continuous habitat patches . At timeDespite these recent conceptual advances, few marine studies have tried to relate genetic structure to the spatial configuration and composition of adult habitats . ImproviThe hypothesis to test in this study is that reefscape configuration and composition shapes genetic patterns. We propose explicitly quantifying the influence of reefscape configuration and composition on genetic structure with a multi-disciplinary modelling approach  that incElacatinus lori on the Mesoamerican Barrier Reef from genetic parental data while considering the spatial distribution of habitat patches. For the same species and in the same area, D’Aloia et al. [Macrocystis pyrifera obtained from microsatellite empirical data, as a function of geographic distance and habitat continuity (i.e. similar to habitat fragmentation). They found a significant influence of habitat continuity on genetic distance. Nanninga et al. [Acropora hyacinthus and A. digitifera) in Micronesia. They found a strong influence of geographic distance and oceanographic distance on genetic patterns and concluded that Micronesia may serve as a stepping stone pathway between the Coral Triangle in Asia (Philippines and Indonesia in particular) and the Central Pacific islands. Johansson et al. [Macrocystis pyrifera. They found a significant effect of habitat continuity on genetic distance, but we are not aware of equivalent studies in coral reef ecosystems. Kool et al. [Developing such an approach that integrates habitat suitability maps, biophysical modelling, gene flow modelling and empirical genetic data is, however, complicated by the range of spatial and temporal scales inherent to these processes . To datea et al.  tested ta et al.  modelleda et al.  highligha et al.  tested bn et al.  investigl et al. , 28 usedl et al.  used a cl et al. , 28, 29.Tridacna maxima) in New Caledonia and Vanuatu, South-West Pacific. The originality of our approach is about using a quantified, spatially explicit view of the benthic reef habitat of our targeted species coupled with the biophysical model. Specifically, we simulated the expected genetic divergences among giant clam populations across the study area given the observed reefscape composition and configuration and under various scenarios of larval dispersal. These simulated genetic structures were compared to the genetic structure observed from empirical genetic data. Then, sensitivity analyses explicitly quantified the importance of habitat patchiness and habitat suitability on genetic structure. The giant clam T. maxima is our model species, but our results remain relevant for a wide range of species of similar life history traits .In this study, we followed the multi-disciplinary seascape modelling approach proposed above see  to test Permits for mantle biopsies of giant clams were from Direction de l'environnement de la Province Sud , Direction du Développement économique et de l'environnement, Province Nord , and Direction du développement économique de la Province des Iles Loyauté (n° 6161-37/PR). No permissions were required for Vanuatu.2 lagoon. Around Grande-Terre, several satellite reef systems of various sizes are found. The most prominent is further west: the “Chesterfield” archipelago includes a vast and open lagoon  with several highly exposed intertidal reefs and islands [The study area extended from 158°E to 171°E and from 24°S to 12°S. It covered Vanuatu’s oceanic islands and New Caledonia . Reefs o islands . The LoyThe main features and variability in regional circulation are reviewed and described in Cravatte et al. . The wesT. maxima densities and from 30 m Landsat satellite imagery from the Millennium Mapping Project [T. maxima densities came from Gilbert et al. [T. maxima. By contrast, level L2 distinguished shallow reefs from variable depth reefs, the latter being characterized by hundred-fold lower densities of T. maxima. Since no quantitative information on giant clam density on variable depth reefs was available in the literature, we arbitrary fixed T. maxima density on variable depth reefs at the density reported in shallow reefs divided by one hundred. Level L3 dissociated “Outer barrier reef of continental islands”  from “Intermediate reef of continental islands” , “Fringing reef of continental islands” , “Oceanic patch reef of continental islands” , “Oceanic islands” , and “Atolls/banks” . Densities of all L3 categories falling in the “Variable depth reefs” L2 category were fixed at the densities of the corresponding L3 category falling in the “Shallow reed” L2 category, divided by 100. For example, the density of giant clams in Shallow Fringing reef of continental island was estimated as 256 ± 272 ind.ha-1 from field data, so we calculated the density of giant clams in Variable depth Fringing reef of continental island to be 2.56 ± 2.72 ind.ha-1  and different spatial relations to each other (seascape configuration) .i to habitat patch j (Pij) as a decreasing function of geographic distance d , providing 60 scenarios of IBD. Geographical distance between habitat patches was defined as the shortest path to reach one cell from another without crossing land. Land maps for New Caledonia, Vanuatu, and Chesterfield were rasterized (0.1 degree resolution) using the rasterize function of the raster package in R 3.1.0, and the shortest path between each pair of cells was determined using the costDistance function of the gdistance package.A large panel of values was tested for i to habitat patch j as a function of oceanic currents. Larval dispersal between habitat patches was simulated with Lagrangian particle tracking models. We modelled the drift of larvae for 9 days, as that is the typical time for T. maxima’s Larval Precompetency Duration (LPD) [Second, we tested the IBOD model by considering the probability for larvae to travel from habitat patch on (LPD) , 38. Duron (LPD) , 40. We on (LPD)  and Neo on (LPD)  , which is expected to produce robust estimations of dispersal [Dij containing the probability of transport for larvae between patches i and j through oceanic waters.The drift of particles through oceanic waters was simulated using the Roms OFfline Floats  model bispersal . For eact as a 243 row matrix containing allele frequency among the 243 habitat patches  distributed among habitat patches.To evaluate the expected genetic structure across the study area under a given hypothesis of larval dispersal, we modelled gene flow among populations with a matrix projection model following Kool et al. . For a g patches . Model sT. maxima density per habitat. The density per habitat was randomly generated from a Gaussian distribution with mean and standard deviation estimated from our literature review .Q) at t+1 is the result of migration progeny (AQt) added to the original parent generation (NQt). As Q refers to a matrix of allelic frequencies, a row normalisation among alleles was performed at each time step (indicated by top bar in The interpretation of i to habitat patch j (A)ij during a generation was considered time invariant and was calculated as the number of clams in habitat patch i (Ni), multiplied by a fertility rate f  to obtain the progeny (fNi). The number of progeny was then multiplied by Dij (see previous section) to determine their distribution among habitat patches.The number of migrants transferring from habitat patch Under the influence of migration  and in the absence of drift, the allele frequencies between pairs of sites were progressively homogenised. The aim of the model was thus to identify which pairs of sites were homogenised more quickly than others to test our hypothesis about dispersal and test the relative importance of geographic distance, oceanographic currents, giant clam abundance, and habitat patchiness.T. maxima spread over 23 sampled sites were conducted from 2008 to 2014  and Nei’s unbiased expected heterozygosity (Hexp) in GENETIX version 4.03 [FIS were estimated using Weir & Cockerham’s [http://genepop.curtin.edu.au/). Significance levels for multiple comparisons of loci across samples were adjusted using a standard Bonferroni correction [Genetic diversity within samples was estimated using observed  using a direct estimation of pairwise values for the Cavalli-Sforza & Edwards [P-value of the log-likelihood (G) based on the exact test [Since null alleles were detected in our samples, genetic divergences among samples were estimated in FREENA  (i) usinockerham  and the  Edwards  genetic  Edwards  that decact test  was estiact test .ds) were compared with the observed genetic distances (do), using Mantel’s correlation coefficient for matrices.Among the dispersal kernels tested (IBD and IBOD), we identified those most similar to the observed genetic structure by comparing the observed and simulated standardized Cavalli-Sforza & Edwards’s  genetic T. maxima density  to coarser habitat maps (down to L2 and L1). This provided three maps of habitats, each characterized by a more or less accurate spatial distribution of  density .To evaluate if reefscape configuration (i.e. habitat continuity) and composition need to be taken into account for modelling genetic structure accurately, we compared the correlation between the observed and the simulated genetic structures obtained when considering the original habitat maps with the correlation obtained when degrading the original habitat map. Degradation of the original habitat map was performed by progressively reducing habitat area between populations surveyed for genetic data. For this, we respectively tested decreases from 20% to 80% of the initial habitat area. Patches that were removed were randomly chosen among patches not surveyed for genetic samples. These scenarios represented the process of habitat fragmentation that generates habitat loss and increases geographical distance between remaining habitat patches. All scenarios tested in this study are summarized in https://figshare.com/s/a00f8be352aae916c147. Over all samples, the number of alleles per locus ranged from 11 to 40 (mean 26.8). The observed heterozygosities ranged from 0.493 to 0.610, and the expected heterozygosities ranged from 0.752 to 0.831 .Raw microsatellite data can be found at .831 See . SignifiP-values < 0.01, see The Efate sample (Vanuatu) was significantly differentiated from other sampled sites with pairwise Cavalli-Sforza & Edwards’ genetic distance values ranging from 0.388 to 0.467  tended to be exported northwestward by the North Vanuatu Jet (NVJ) where no patch of suitable habitat exists. The occurrence of successful exports of larvae from Vanuatu to New-Caledonia progressively increased for release points further south, but successful exports were restricted to the Loyalty Islands, New Caledonia’s East coast, and Entrecasteaux reefs through the East Caledonian Current (ECC), the North Caledonian Jet (NCJ) and the South Caledonian Jet (SCJ). By contrast, a significant fraction of larvae could cross the distance between the north part of New Caledonia and the Chesterfield Islands using eddies issued from the NCJ and the Sub-Tropical Counter Current (STCC). This provided a connectivity matrix with higher connections among habitat patches of New Caledonia than between Vanuatu, New Caledonia, and Chesterfield .a = 0.95 in The dispersal kernel that provided the best match with genetic data was the IBD scenario with When considering an IBD pattern for dispersal, the gene flow model adequately isolated Vanuatu and Chesterfield from New-Caledonia, but did not adequately mimic the genetic structure among New-Caledonia’s reefs . By contDegrading the original habitat map by reducing habitat area significantly decreased the correlation between simulated and observed genetic distances . Other evolutionary forces  were neglected, but their influence on the results should be assessed in future research programs. Mutation is likely unimportant at the small spatial and temporal scales that are addressed in this study. Selection may affect parts of the genome including neutral genomic regions , but it t values . Then, gversus observed genetic distances, but the Mantel test has been recently criticized due to low robustness to spatial autocorrelation [versus observed genetic distances, it is unlikely to have changed the conclusions of our study which are based on a relative comparison of correlation coefficients between scenarios and not on the absolute value of correlation.Finally, we used the Mantel correlation coefficient to compare simulated relation , 61. Whia = 0.95 in Only by considering IBD with limited dispersal  the movement of gametes before fertilization, which last several to tens of hours for giant clams [2) larval swimming speed, which may also affect the path of larvae and is low for T. squamosa  factors inducing settlement, including crustose coralline algae [4) spatial differences in larval survival, such as from spatial variation in predation or food availability. Finally, it is worth noting that despite the recent advances in characterizing the circulation around New Caledonia [Our biophysical model (IBOD) considered the PLD, larval survival, and ability for larvae to settle when crossing a suitable substrate. Available computer time limited our ability to run dedicated sensitivity analyses for each of these parameters, but all are expected to influence the dispersal kernel  and warrnt clams  and may  μm.s-1, ) but remwimming, ); 3) facne algae  or conspne algae ; and 4) aledonia , ROMS modemographic and genetic connectivity patterns has strong foundations in the terrestrial literature [seascape genetics” only recently emerged in marine ecology as studies began linking connectivity patterns for geographic features. Thus far, seascape genetics has mostly used meso-scale oceanic features  to descurrents, . In thisPopulation connectivity often enhances resilience to climate change and other anthropogenic impacts on ecosystems . Loss ofSeascape features can provide useful spatial surrogates for population genetic structure that are potentially relevant for a wide range of marine organisms. Our study concluded that habitat composition and configuration significantly influenced genetic patterns of giant clams. Further investigation is needed to assess the robustness of this result to species traits , but habitat maps and other reefscape features may provide a new path for marine ecology in regions where more detailed information on ocean currents are lacking.versus island reef) should also be investigated. It would also be helpful to compare results from reefscape genetics among taxa and identify proxies of connectivity robust enough to be related to particular species traits, including PLD. Over the long run, this could help elevate reefscape genetics from a population to community scale and help characterize the link between reefscape features and biodiversity broadly. Finally, it will be useful to characterize how reef use, reef degradation, and other aspects of environmental change affect genetic connectivity, and to assess the consequences for reef management and for the identification of priority areas for conservation. These three recommendations are not exhaustive, but would help reinforce reefscape genetics as a new path for marine ecology and conservation science.We suggest future research to investigate the following directions. First, one important advance will be to quantify the relative influence of a wider variety of reefscape features on genetic structure. We here demonstrated that reefscape composition and configuration can enhance our understanding of population connectivity through a reduction of habitat area and a degradation of habitat map, but the influence of reefscape features like reef enclosure  estimate of Wright’s (1951) fixation index . Full names of abbreviated sampled locations are given in n: number of sampled individuals; (PDF)Click here for additional data file.S2 TableFST; below diagonal: Cavalli-Sforza and Edwards (1967) genetic distance used for all comparisons with simulated genetic structures. Significant genotypic differentiations are indicated in italic for P-value < 0.01 and in bold for significant after sequential Bonferroni correction. Full names of abbreviated sampled locations are given in Above diagonal: pairwise (PDF)Click here for additional data file.S1 FigCompetency period considered is from 9 to 19 days, and the relative probability of survival was considered for calculation of dispersal probability in the IBOD model.(PDF)Click here for additional data file.S2 FigA) Results obtained when using the mantel coefficient of matrix correlation. B) Results obtained when using the linear R-squared. Points are medians and error bars are quantile 5% and 95%.(PDF)Click here for additional data file.S3 Fig(PDF)Click here for additional data file.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file.S3 File(PDF)Click here for additional data file.S4 File(PDF)Click here for additional data file.


Ambystoma annulatum, A. maculatum, and A. opacum) and the Central Newt  on the same landscape in Missouri, USA. While all four salamander species are forest dependent organisms that require fishless ponds to reproduce, they differ in breeding phenology and spatial distribution on the landscape. We use these differences in life history and distribution to address the following questions: (1) Are there species‐level differences in the observed patterns of genetic diversity and genetic structure? and (2) Is dispersal influenced by landscape resistance? We detected two genetic clusters in A. annulatum and A. opacum on our landscape; both species breed in the fall and larvae overwinter in ponds. In contrast, no structure was evident in A. maculatum and N. v. louisianensis, species that breed during the spring. Tests for isolation by distance were significant for the three ambystomatids but not for N. v. louisianensis. Landscape resistance also contributed to genetic differentiation for all four species. Our results suggest species‐level differences in dispersal ability and breeding phenology are driving observed patterns of genetic differentiation. From an evolutionary standpoint, the observed differences in dispersal distances and genetic structure between fall breeding and spring breeding species may be a result of the trade‐off between larval period length and size at metamorphosis which in turn may influence the long‐term viability of the metapopulation. Thus, it is important to consider life history differences among closely related and ecologically similar species when making management decisions.Understanding metapopulation dynamics requires knowledge about local population dynamics and movement in both space and time. Most genetic metapopulation studies use one or two study species across the same landscape to infer population dynamics; however, using multiple co‐occurring species allows for testing of hypotheses related to different life history strategies. We used genetic data to study dispersal, as measured by gene flow, in three ambystomatid salamanders ( Similarly, differences in fecundity, larval period length, and population size were hypothesized to drive differential phylogeographic structure in two sympatric marine gobies  have more limited gene flow across the same spatial scale than spring breeding species (A. maculatum). The authors posited that differences in breeding phenology and the presence of suitable water sources on the landscape during breeding events shape genetic structure as spring breeding species can utilize a wider variety of breeding habitats  and as a result are more adept dispersers  and spring breeding (N. v. louisianensis) salamander species. We first assessed the patterns of genetic diversity and genetic structure for all four species across the same study landscape. We then tested the effect of the interpond landscape matrix on genetic differentiation using a resistance modeling approach.Our study investigates whether the genetic structure and diversity of four co‐occurring salamander species is influenced by breeding phenology. We test this by extending the work of Peterman et al.  by colle22.1Quercus spp. and Carya spp. canopy; Rhus aromatic and Cornus florida understory) with intermixed short‐leaf pine plantations (Pinus echinata). There are approximately 500 constructed or unintentional bodies of water  on the FLW landscape with varying hydroperiods and aquatic communities; however, the majority are small (<0.04 ha), fishless, manmade ponds  military training facility in Pulaski County, Missouri, USA Figure . This 24A. maculatum samples from ponds of varying hydroperiod across the entire landscape, whereas we collected A. annulatum, A. opacum, and N. v. louisianensis from ponds over a 7,140‐ha subset of FLW . We sized PCR products on an ABI 3730xl DNA Analyzer  using Liz 600 size standard at the University of Missouri DNA Core Facility and scored genotypes using GeneMarker v.1.97 . Samples that amplified at <80% of loci were removed from the dataset. We tested for the presence of full siblings with COLONY v2.0.5.9  following the protocol outlined in Peterman, Connette, Spatola, Eggert, and Semlitsch . We genoci Table , A. opacci Table , A. macuci Table , and N. ci Table . All forci Table . With tho et al. , Petermao et al. , or TablWe tested for deviations from expected heterozygosity values under Hardy–Weinberg Equilibrium (HWE) and linkage disequlibrium among pairs of loci with Genepop on the Web  account for differences in sample sizes. We calculated rarefied allelic richness in the program HP‐Rare . We tested for isolation by distance (IBD) using the “vegan” package . As in STRUCTURE, we tested for 2–15 potential clusters using ten replicates for each potential cluster number within each species. In both STRUCTURE and BAPS, we tested for hierarchical substructure within each putative cluster in a separate analysis for K = 2–15 with the same criteria used in the initial analyses.We did not correct for sample sizes across species because the metrics we used for genetic diversity  and differentiation . All other resistance surfaces were derived from 90‐m resolution USGS DEM layer in ArcGIS (ESRI) following the methods outlined in Peterman and Semlitsch  to test our hypothesis that the species differ in their response to the landscape. We obtained our land cover data (30 × 30 m resolution) and digital elevation model  from the U.S. Geological Survey National Map server  from which we did not have at least nine fully genotyped individuals.We successfully genotyped 472 of 488 A. annulatum, A. maculatum, or N. v. louisianensis. However, for A. opacum two loci (Aa‐29 and Aa‐312) deviated significantly from expectations under HWE due to heterozygote deficiency and were removed from further analyses. We did not observe evidence of null alleles at any loci or of linkage disequilibrium among pairs of loci in any species. Our final dataset included 421 A. annulatum from 26 sites at 19 loci, 123 A. opacum from eight sites at 11 loci, 332 A. maculatum from 17 sites at 18 loci, and 108 N. v. louisianensis from 11 sites at nine loci.When testing for independence of loci in our dataset, no population or locus significantly deviated from HWE following Bonferroni's corrections for A. annulatum was 0.682 . The slopes of genetic distance and geographic distance for both A. annulatum  and A. opacum  were not significantly different from each other but both species have significantly greater slopes than A. maculatum  and N. v. louisianensis . In addition, we observed strong support in A. annulatum and marginal support in the other three species for isolation by resistance being a better predictor of genetic structure than distance alone  with distance from ravines also being strongly supported . Ambystoma opacum genetic differentiation was best predicted by the eastness resistance surface (Rm2 = .30); moreover, TWI, distance from ravine, and percent slope resistance surfaces all had ΔAICC < 2.00 . For A. maculatum, our best supported model was northness (Rm2 = .43) with eastness (Rm2 = .42) and percent slope (Rm2 = .46) both having ΔAICC < 2.00 , although it was not identified as the top model  and these significant relationships were observed over a smaller spatial scale for A. annulatum and A. opacum than for A. maculatum . Similarly, the lack of an IBD effect in N. v. louisianensis is not surprising as this species has been found to be capable of dispersing at least 3 km (Gill, A. maculatum and N. v. louisianensis are likely a result of the ability and or propensity of these organisms to disperse over greater distances than either A. annulatum or A. opacum.At the scale of our study, we observed a strong and significant signal of IBD in three of the four study species (m Figure . This sue et al.  found thMadison,  and singOsbourn,  and 53.4km Gill, , 1979. TA. annulatum, we observed the strongest support for our composite resistance surface that combined TPI and distance from ravines. For this, surface ridges, flat areas, and upper slopes had lower resistance values than mid‐slopes, lower slopes, and valley bottoms. As ridges and higher slopes in the Missouri Ozarks tend to be warmer and drier than lower slopes and valley bottoms (Peterman et al., A. annulatum show lower resistance to movement on ridge tops could be an artifact of the pond configuration on our landscape. Additionally, A. annulatum have been observed moving through old field and pasture habitats toward breeding ponds (Briggler, Johnson, & Rambo, Ambystoma likely due to increased desiccation risk and predator abundance (Rittenhouse & Semlitsch, Across all species, we found support for landscape resistance describing genetic differentiation better than distance alone. With A. opacum and N. v. louisianensis, eastness emerged as the top model and northness emerged as the top model for A. maculatum. These surfaces approximate the temperature and soil moisture of a landscape as north‐ and east‐facing aspects are cooler and moister than south‐ and west‐facing aspects. Given that amphibians are prone to desiccation (Peterman, Locke, & Semlitsch, A. opacum, resistance was lower on western slopes, which are typically warmer and drier, than on slopes with easterly aspects. Similarly, A. maculatum resistance was higher on southerly aspects than on northerly aspects. The result for both species suggests that, like A. annulatum, A. opacum, and A. maculatum may exhibit compensatory movement through dry areas in the study landscape. In contrast, westerly aspects were estimated to have greater resistance for N. v. louisianensis than the typically moister easterly aspects. However, N. v. louisianensis overall F’ST was extremely low and it exists as one genetic cluster across our study landscape. Thus, N. v. louisianensis are less prone to desiccation than the ambystomatids due to their granular skin (Gill, For in Gill, , which mA. annulatum and eastness describing genetic differentiation for A. maculatum extends previous work with A. annulatum and A. maculatum on this landscape. Peterman et al. (A. maculatum samples were collected from a larger extent and we used TPI as a discrete variable with a 90 × 90 m cell size instead of using TPI as a continuous surface with a 30 × 30 m cell size. Although inference in “ResistanceGA” does not substantially change based on grid cell size (Peterman, The support for a composite surface of TPI and distance from ravines explaining the genetic differentiation for n et al.  found thn et al.  in that A. annulatum and A. opacum, and a lower degree of genetic differentiation and a lack of genetic clustering in the spring breeding species, A. maculatum and N. v. louisianensis. In Missouri, A. annulatum and A. opacum breed and oviposit from September to early November and their larvae overwinter in the ponds before metamorphosing in late April to early June; thus, they require ponds that are continuously inundated and large enough to not freeze solid during that 6–9‐month period for successful reproduction (Anderson, Ousterhout, Peterman, Drake, & Semlitsch, A. maculatum and N. v. louisianensis breed and oviposit in February and March and larvae metamorphose and disperse in a large pulse between early June and late July, although metamorphosis can continue into October, meaning that these two species can utilize more ephemeral ponds on the landscape as larvae can metamorphose in as few as 3 months (Gill, In addition to landscape resistance predicting genetic differentiation, a likely explanation for the observed patterns of genetic diversity in our study is the effect of breeding phenology. We observed higher degrees of genetic differentiation and the presence of genetic clusters for the fall breeding species, hs Gill, . Althoughs Gill, .A. annulatum had higher levels of genetic differentiation than the spring breeding A. maculatum on the same landscape. Similarly, Whiteley et al. (A. opacum has stronger genetic differentiation than A. maculatum among the same ponds in Massachusetts. From an evolutionary standpoint, the observed differences in dispersal distances and genetic structure between fall breeding and spring breeding species may be a result of the trade‐off between larval period length and size at metamorphosis (Petranka, Our observation of differential patterns of genetic structure between spring and fall breeding salamander species concurs with previous genetic studies of ambystomatid species that investigated patterns of genetic differentiations in species with different life histories. In Missouri, Peterman et al.  found thy et al.  observedHad our study only used one caudate species as a surrogate for all of the others, our inference would have differed as we would not have had the ability resolve the influence of breeding phenology on genetic differentiation in our system. Similar comparative landscape genetic studies support this idea that genetic inference can vary substantially, even among closely related and widely distributed species, when there are subtle differences in life history (Engler, Balkenhol, Filz, Habel, & Rödder, None declared. Click here for additional data file.


The development of genetic profiles of invading plants can provide insight towards a better understanding of niche utilization. Recent advances in sequencing techniques have greatly facilitated genetic assessment and profiling. Here we review our recent studies on selected Australian Cucurbitaceae and Boraginaceae to demonstrate the application of genetic sequencing across a selection of invasive Australian plants. Cucumis myriocarpus) and camel melon (Citrullus lanatus) were represented by only a single genotype in Australia, implying that each was probably introduced as a single introduction event. In contrast, a third invasive melon, Citrullus colocynthis, possessed a moderate level of genetic diversity in Australia and was potentially introduced to the continent at least twice. The Boraginaceae study demonstrated the value of comparing two similar congeneric species; one, Echium plantagineum, is highly invasive and genetically diverse, whereas the other, Echium vulgare, exhibits less genetic diversity and occupies a more limited ecological niche. Sequence analysis provided precise identification of invasive plant species, as well as information on genetic diversity and phylogeographic history. Improved sequencing technologies will continue to allow greater resolution of genetic relationships among invasive plant populations, thereby potentially improving our ability to predict the impact of these relationships upon future spread and better manage invaders possessing potentially diverse biotypes and exhibiting diverse breeding systems, life histories and invasion histories.Part of the challenge in dealing with invasive plant species is that they seldom represent a uniform, static entity. Often, an accurate understanding of the history of plant introduction and knowledge of the real levels of genetic diversity present in species and populations of importance is lacking. Currently, the role of genetic diversity in promoting the successful establishment of invasive plants is not well defined. Genetic profiling of invasive plants should enhance our understanding of the dynamics of colonization in the invaded range. Recent advances in DNA sequencing technology have greatly facilitated the rapid and complete assessment of plant population genetics. Here, we apply our current understanding of the genetics and ecophysiology of plant invasions to recent work on Australian plant invaders from the Cucurbitaceae and Boraginaceae. The Cucurbitaceae study showed that both prickly paddy melon ( Chrysanthemoides monilifera), is associated with population decline in 63 rare and threatened native plant species in New South Wales alone  and camel melon (Citrullus lanatus) to the more heterogeneous populations of Citrullus colocynthis composed of two major introduced genotypes in Australia (C. myriocarpus) and (C. lanatus) are annuals, whereas C. colocynthis is perennial. The experience of working with these two different plant families in Australia using similar genetic analysis methods enables us to draw some general conclusions on the value of such analyses in characterizing continental invasions by a variety of taxa. Thus, our overall objective is to examine how recent advancements in genetic characterization and sequence analysis can be applied successfully to invasive plants with varying life histories, breeding systems and invasion histories.In this review, we compare and contrast the genetic diversity of two models; Australian congeneric invaders representing the Cucurbitaceae and Boraginaceae. The Boraginaceae model compares two congeric invaders introduced to Australia in a similar time frame; one highly successful invader and the other a niche colonizer with similar morphological, chemical and biological features  can be used for species identification’  and ribulose-bisphosphate carboxylase gene  recommended a two-marker-based system as a barcode for flowering plants, i.e. maturase K , may also be required for increased resolution , Amplified Fragment Length Polymorphism (AFLP), Restriction Fragment Length Polymorphism (RFLP), Random Amplified Polymorphic DNA (RAPD) and Inter Simple Sequence Repeat (ISSR). Compared with DNA barcoding, these markers can be cheaper and sometimes more polymorphic. However, they can also be impracticable because of erroneous results in scoring  Matsum. and Nakai], prickly paddy melon (Cucumis myriocarpus L.) and colocynth melon [Citrullus colocynthis (L.) Schrad.] are currently distributed across Australia  and a nuclear gene (G3pdh intron region) based on The two annual wild melon species share similar vegetative growth and produce yellow flowers; therefore, they are often confused. This is particularly so before fruit formation in the case of C. lanatus and C. myriocarpus were each represented by a single genotype and haplotype, indicating that the populations present were derived from a single introduction event or multiple introduction events of a single genotype (and subsequently selfing). Moderate levels of genetic diversity were present among Australian C. colocynthis, and this species sorted geographically into separate haplotypes found in eastern and western regions, suggesting at least two separate introductions from two different source populations . The more successful invader, E. plantagineum, showed significantly higher levels of genetic diversity than did the less invasive E. vulgare, which supports the hypothesis that a certain level of genetic diversity is associated with success of invasion in herbaceous plants , Gladstone  and Western Australia in 1880, 1889 and 1881, respectively . These findings support the hypothesis that multiple introductions of E. plantagineum occurred across Australia. However, to unravel the pathway of E. plantagineum introduction to Australia further, additional investigation is required and is currently ongoing through evaluation of a global collection of samples.The relative pattern of introduction of Australian ectively . SpatialE. vulgare . Echium vulgare is restricted in its spread across Australia and is mainly found in the southeastern highlands .This study also highlights the limited genetic diversity found in Australian specimens of nds Fig. . As a pelia Fig. , is predModes of reproduction and dispersal play a vital role in determining the genetic structure of a population of a particular species , includiCitrullus lanatus and C. myriocarpus reproduce by selfing and form large infestations of a single haplotype in the invaded range. The breeding system in Echium species features protandrous individual flowers, which cannot self-pollinate, but the possibility of fertilization by other flowers on the same plant renders them self-compatible .For the Australian weeds discussed above, genetic characterization provided helpful insights into invasion history, although breeding systems varied widely in each case. Aegilops triuncialis; Macfadyena unguis-cati; Lepidium latifolium; Arundo donax; M. unguis-cati showed 27 chloroplast DNA haplotypes in its native range and only one haplotype in its invaded range  in its introduced range  showed higher epigenetic variation (leading to phenotypic variation) than genetic variation  populations were diverse in the introduced European range, suggesting multiple introductions from their source populations in Japan . For the other three species, control tactics should logically be designed to account for regional or local differences, especially if it is shown that responses to specific controls vary among genotypes. For example, different ecotypes of a biological agent currently being tested for use against knotweed  in North America have been shown to favour particular Fallopia species, and there are three closely related target species, including Fallopia × bohemica, which form a hybrid swarm (E. plantagineum, the success of biocontrol agents in Australia was clearly associated with long-term regional adaptation of each biocontrol organism; however, genetic differences among regional plant populations may also influence biocontrol (In the case of five invasive Australian weeds described above, genetic characterization allows managers to approach each of the species differently based on the degree of variation present. Chemical control is generally used for all of these species, except Comparisons among the five taxa evaluated in these case studies Table  reveal a


Curcuma longa L., Zingiberaceae) that has been extensively used in a traditional medicine over the centuries. In the present study, we have found that curcumin inhibits STAT3 signaling that is persistently overactivated in H-Ras transformed breast epithelial cells (H-Ras MCF10A). Specific cysteine residues present in STAT3 appear to be critical for the activity as well as conformation of this transcription factor. We identified the cysteine residue 259 of STAT3 as a putative site for curcumin binding. Site-directed mutation of this cysteine residue abolished curcumin-induced inactivation of STAT3 and apoptosis in H-Ras MCF10A cells. The α,β-unsaturated carbonyl moiety of curcumin appears to be essential in its binding to STAT3 in H-Ras MCF10A cells. Tetrahydrocurcumin that lacks such electrophilic moiety failed to interact with STAT3 and to induce apoptosis in the same cell line. Taken together, our findings suggest that curcumin can abrogate aberrant activation of STAT3 through direct interaction, thereby inhibiting STAT3-mediated mammary carcinogenesis.Signal transducer and activator of transcription 3 (STAT3) is a transcription factor that is latent but constitutively activated in many types of cancers. It is well known that STAT3 plays a key role in inflammation-associated tumorigenesis. Curcumin is an anti-inflammatory natural compound isolated from the turmeric ( STATs exist as latent monomers in the cytoplasm. However, upon ligand stimulation, STATs become phosphorylated at a single carboxyterminal tyrosine by receptor-intrinsic, receptor-associated, or nonreceptor tyrosine kinases2. Subsequently, phosphorylated STATs undergo dimerization through reciprocal interaction between the Src Homology 2 (SH2) domain and the phosphorylated tyrosine residue5. STAT dimers then translocate to nucleus where they bind to the promoters of target genes6. STAT3 is one of the STAT family members and known to play a key role in inflammation-associated tumorigenesis7. In normal cells, STAT3 signaling is transiently activated, and its activation is tightly regulated by extracellular stimuli. However, aberrant STAT3 activation leads to cancer cell proliferation, survival and resistance to apoptosis, thereby accelerating tumor development and progression8. Conversely, disruption of STAT3 signaling by antisense oligonucleotides or STAT3 mutants results in growth inhibition and induction of apoptosis in various types of cancer cells9. Therefore, STAT3 is considered to be an attractive target for cancer prevention and therapy.Transcription factors that belong to the family of Signal Transducer and Activator of Transcription (STAT) are activated by various cytokines, growth factors and hormonesCurcuma longa), a spice that has been widely used in a traditional medicine for many centuries10. This compound is known to exert anti-cancer effects through regulation of a variety of intracellular signaling pathways and protein-protein interactions11. Although, curcumin has the ability to inhibit JAK/STAT signaling14, the molecular mechanism by which curcumin inactivates STAT3 remains elusive. Notably, curcumin possesses an α, β unsaturated carbonyl moiety and can act as an electrophile15. This prompted us to investigate whether curcumin could effectively inhibit aberrantly activated STAT3 and induce apoptosis in H-Ras transformed human mammary epithelial (H-Ras MCF10A) cells by directly interacting with this transcription factor.Curcumin is a natural polyphenolic compound isolated from turmeric  present in STAT3 may be covalently modified by the electrophilic moiety of curcumin. In support of this speculation, the thiol reducing agents, dithiothreitol (DTT) and N-acetylcysteine (NAC), blunted curcumin-mediated inhibition of STAT3 phosphorylation and Bcl-xL expression Fig.  and tran19. Moreover, mutation of STAT3 at cysteine 259 has been reported to abolish the effect of S-nitrosoglutathione on interleukin (IL-6)-induced STAT3 phosphorylation and transactivation20. In silico modeling reveals that curcumin has great potential to bind to STAT3 on cysteine 251 as well as cysteine 259 residue  had not been reported. STAT3 activation has also been found to be sensitive to redox regulation27. In line with this notion, cysteine modification, such as S-glutathionylation of STAT3 at cysteine 328 and 542, was found to modulate STAT3 signaling28.Owing to the presence of an electrophilic α,β-unsaturated carbonyl moiety, curcumin tends to react with a variety of electron donors. Especially, cysteine thiol residues ubiquitously present in cellular proteins are subject to covalent modification by electrophiles. One of the well-characterized electrophilic and oxidative stress sensing proteins is Kelch-like ECH-associated protein 1 (Keap1). Covalent modification of some highly reactive regulatory cysteine residues in Keap1 drives the activation of Nrf2 signalingConsidering electrophilic nature of curcumin, thiol modification of STAT3 could account for its inactivation of this transcription factor. Our results show that the thiol reducing agents DTT and NAC restored the phosphorylation and transcriptional activity of STAT3 which were suppressed by curcumin, lending support to the above supposition. In line with this notion, curcumin abrogated phosphorylation, dimerization, DNA binding and transcriptional activity, while tetrahydrocurcumin lacking the electrophilic moiety was incapable of inhibiting all these events.30. Interestingly, one study suggests the possibility that curcumin can directly interact with the SH2 domain of STAT3, thereby disrupting STAT3 dimerization4. However, targeting the dimer interface of STAT3 is practically challenging due to the planarity of the large surface area30. Mass spectrometry analysis and in silico modeling data identified cysteine 251 and 259 present in the coiled-coil domain of STAT3 as the putative binding sites of curcumin. Although DNA binding and SH2 domains are generally targeted by STAT3 inhibitors, we could not find sites modified by curcumin other than cysteine 251 and 259. Similar to our findings, Stattic, a non-peptidic STAT3 inhibitor, was found to react with cysteine 251, 259, 367 and 426 of STAT3  which are not located in protein-protein or protein-DNA interfaces31. Thus, it is plausible that cysteine 251 and 259 residues to which curcumin predominantly binds are allosteric modification sites. We then further confirmed the specificity of the curcumin binding. It was observed that Sepharose-conjugated curcumin bound to wild type and to a slightly lesser extent, cysteine 251-mutated STAT3, while cysteine 259-mutated STAT3 barely interacted with curcumin. Furthermore, curcumin-induced apoptosis in PC3 cells harboring cysteine 259-mutated STAT3 was abrogated compared with that in cells transfected with wild type or cysteine 251-mutated STAT3. These may suggest that cysteine 259 is more likely to be involved in curcumin-induced apoptosis than cysteine 251. Consistent with our findings, it was found that methyl-2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oate (CDDO-Me) suppressed STAT3 dimerization by alkylating cysteine 25932. Therefore, cysteine 259 of STAT3 is considered essential in its function and a specific binding site of curcumin  which is known as a STAT3 repressorRas MCF10A cells were grown in DMEM/F-12 supplemented with 5% horse serum, 100 ng/ml cholera toxin, 20 ng/ml human epidermal growth factor, 10 μg/ml insulin, 0.5 μg/ml hydrocortisone, 2 mM L-glutamine and 100 units/ml penicillin/streptomycin. Human prostate cancer (PC-3) cells were grown in RPMI 1640 supplemented with 10% fetal bovine serum, and P-STAT3-luc stably expressing human cervical cancer (HeLa) cells were grown in Dulbecco’s modified Eagle’s medium (DMEM). Curcumin was purchased from LKT Laboratories, Inc. . Tetrahydrocurcumin was prepared by catalytic hydrogenation of curcumin. DTT and NAC were purchased from Sigma-Aldrich . Recombinant human IL-6 and OSM were purchased from R&D Systems, Inc. . Primary antibodies for P-STAT3Y705, STAT3, HA-tag (anti-rabbit), Myc-tag (anti-mouse), and 10× lysis buffer were purchased from Cell Signaling Technology . Primary antibodies against P-STAT3Y705, STAT3, phosphorylated extracellular signal-regulated protein kinase (P-ERK) and phosphorylated JAK3 (P-JAK3) were purchased from Santa Cruz Biotech . Primary antibody against STAT3 was purchased from BD Biosciences . FITC-conjugated Annexin v. STAT3 recombinant protein was purchased from Active Motif .Normal human mammary epithelial (MCF10A) and H-STAT3 knockdown was achieved using STAT3 siRNA with the following target sequence: sence: 5′-CUAUCUAAGCCCUAGGUUUdTdT-3′; antisense: 5′-CCUAGGGCUUAGAUAGdTdT-3′.Ras MCF10A cells were treated with vehicle, curcumin or tetrahydrocurcumin for 12 h and digested with lysis buffer. Protein A/G agarose beads were washed with the same lysis buffer 5 times. The cell lysate of each group was immunoprecipitated with protein A/G agarose beads and STAT3 antibody (Santa Cruz Biotech) at 4 H-Ras MCF10A cells was immunoprecipitated with anti-STAT3 antibody. The binding of STAT3 at promoter regions of Survivin, Bcl-2 or Bcl-xL was detected using primers with the following sequence. Survivin forward primer, 5′-CAGTGAGCTGAGATCATGCC-3′; reverse primer, 5′-TATTAGCCCTCCAGCCCCAC-3′. Bcl-2 forward primer, 5′-CTTCATTTATCCAGCAGCTT-3, reverse primer, 5′-GAGGGGACGATGAAGGAG-3′. Bcl-xL forward primer, 5′-CTGGGTTCCCTTTCCTTCCA-3′, reverse primer, 5′-TCCCAAGCAGCCTGAATCC-3′.Chromatin prepared from H-4) in a 6 well plate and pretreated with curcumin for 12 h and then stimulated with 10 ng/ml oncostatin M for 6 h. The cells were lysed with a reporter lysis buffer , and the cell lysate (20 μl) was gently mixed with 100 μl luciferase assay reagent (Promega). The luciferase activity was measured using a luminometer .HeLa/P-STAT3-luc reporter cells were cultured  was used to make a docking model of curcumin binding to STAT3 protein. The structures of STAT3 are from Protein Data Bank (PDB ID: 3CWG and PDB ID: 1BG1). Curcumin coordinates were obtained from the GlycoBioChem PRODRG2 Server (http://davapc1.bioch.dundee.ac.uk/prodrg/)34. The grid maps for the docking model was set to have 74 × 126 × 126 points with 1.0 Å spacing to cover all surface of STAT3. Other parameters were set in a default value in AutoDock Vina program. Binding mode analysis of curcumin was carried out using a covalent docking module implemented in Maestro program v9.5 . The STAT3 crystal structure (PDB code: 1BG1) was downloaded from PDB bank. STAT3 protein was prepared with neutralization and energy minimization using ProteinPrep Wizard. Curcumin was prepared with protonation at pH 7.4 and energy minimization using LigPrep module. In a covalent docking module, the reaction type of ligand was set to Micheal addition with cysteine 259 of STAT3 protein. Grid box was generated with residues within 5.0 Å of the cysteine 259. The 10 initial output of covalent binding pose was energetically minimized with residues within 3 Å of curcumin, and their binding energy was calculated by Prime MM-GBSA module. The covalent binding pose of curcumin was selected with low PrimeΔGbind for binding mode analysis.AutoDock Vina program2 plates were prepared. H-Ras MCF10A cells (1 × 105) were added to media containing 3.3% agarose and layered onto the previously prepared agar plates. DMSO, curcumin or tetrahydrocurcumin was given every 3 days. The colonies were allowed to grow for 3 weeks and then detected under a microscope.Underlayers consisting of DMEM/F-12 media containing 0.5% agar in 60 mm3/0.5 M NaCl in 40% DMSO) and curcumin or tetrahydrocurcumin and rotated overnight at 4 °C. The beads were then washed with the same coupling buffer 5 times and again incubated with 0.1 M Tris.HCl overnight. After overnight incubation, beads were rinsed with a buffer containing 0.1 M acetate/0.5 M NaCl and a buffer containing 0.1 M Tris.HCl/0.5 M NaCl 3 times each and then with PBS.To generate CNBr-activated-Sepharose-4B-conjugated curcumin and tetrahydrocurcumin, Sepharose-4B beads  were activated with 1 mM HCl. The activated beads were transferred into a 15 ml conical tube containing coupling buffer  5 times. The protein was detected with anti-STAT3 antibody . PC-3 cells were transfected with GFP-tagged wild type STAT3, GFP-tagged C251A STAT3 or GFP-tagged C259 A STAT3 vector and lysed with a 1×lysis buffer. The cell lysates were precipitated with curcumin-conjugated Sepharose 4B beads. The protein was detected with anti-HA antibody.Curcumin-conjugated Sepharose-4B and tetrahydrocurcumin-conjugated Sepharose-4B beads were incubated with the lysates of H-Ras MCF10A cells were seeded in a 6 well plate and treated with curcumin or tetrahydrocurcumin for 12 h. Cells were permeablilized with 0.1% Triton X-100 and stained with FITC-conjugated annexin v and PI. FITC positive cells were detected using a FACSCalibur flow cytometer (BD Biosciences).Induction of apoptosis was measured by flow cytometry. H-The recombinant STAT3 protein (Active Motif) was treated with curcumin, and the solution mixture was added to the urea buffer . The sequencing grade modified trypsin (250 ng/μl) was then added to the above mixture for the digestion of protein followed by incubation at 37 °C for 8 h. The resulting tryptic peptides were desalted using Pierce® C-18 spin columns . Thereafter, they were dried using Speed-Vac and then reconstituted in 200 μl of mobile phase for LC-MS analysis.min/% solution of B): 0/5, 60/50, 61/80, 70/80, 72/1, 90/1. The flow rate of the mobile phase was increased gradually throughout the gradient run . The column temperature was maintained at 60 °C and the injection volume was 10 μl. For LC-MS analysis, Easy-nLC 1000 system (Thermo Fisher Scientific Inc.) was coupled to Q-Exactive mass spectrometer (Thermo Fisher Scientific Inc.) equipped with an EASY-SparyTM source. The data acquisition was under the control of Xcalibur software. The typical operating source conditions for MS scan in positive mode were optimized as follows: spray voltage, 2.1 KV; capillary temperature, 320 °C; and nitrogen was used as damping gas. All the spectra were recorded under identical experimental conditions, and are average of 20–25 scans. The scan range was set from m/z 300–1800. For Higher energy Collision Dissociation (HCD) experiments, keeping MS1 static, the precursor ion of interest was selected using the orbitrap analyzer and the product ions were analyzed. The normalized collision energies used were 30 NCE. Resolution of precursor ions was set at 70,000 and was set at 17,500 for HCD experiments. The top 8 precursor ions in the MS scan were selected by orbitrap analyzer for subsequent MS/MS analysis. The raw data were analyzed using Thermo Proteome Discoverer 1.3 (Thermo Fisher Scientific Inc.) against STAT3 database . The search parameters were optimized as follows: Precursor ion tolerance was set at 10 ppm, fragment ion tolerance was set at 0.8 Da, and two miss cleavage was allowed. Variable modification options were used for the modification of curcumin (+368.126 Da) and carbamidomethylation (+57.021) on cysteine and oxidation (+15.995 Da) on methionine.The HPLC analysis was performed on Easy-nLC 1000 system  equipped with a trap column (C18, 75 μm t-test. P values less than 0.05 were considered significant.The data were presented as means ± SD. Difference between groups was analyzed by Student’s unpaired The data sets generated and/or analysed during this study are included in this publication and in its Supplementary Information file.Supplementary Information