Anti-cancer efficacy in vivo was performed in a CAL33 tumor murine xenograft model. UTMC with STAT3-MB caused significantly lower CAL33 cell viability compared to UTMC with STAT3-LPX . In vivo, UTMC with STAT3-MB had strong anti-tumor effects, with significantly less tumor burden and greater survival compared to that of UTMC with microbubbles loaded with a mutant control decoy and untreated control groups (p<0.05). UTMC with STAT3 decoy-loaded microbubbles significantly decreases human HNSSC tumor progression. These data set the stage for clinical translation of our microbubble platform as an imaged-guided, targeted delivery strategy for STAT3 decoy, or other nucleotide-based therapeutics, in human cancer treatment.Signal transducer and activator of transcription-3 (STAT3) is an oncogenic transcription factor implicated in carcinogenesis, tumor progression, and drug resistance in head and neck squamous cell carcinoma (HNSCC). A decoy oligonucleotide targeting STAT3 offers a promising anti-tumor strategy, but achieving targeted tumor delivery of the decoy with systemic administration poses a significant challenge. We previously showed the potential for STAT3 decoy-loaded microbubbles, in conjunction with ultrasound targeted microbubble cavitation (UTMC), to decrease tumor growth in murine squamous cell carcinoma. As a next step towards clinical translation, we sought to determine the anti-tumor efficacy of our STAT3 decoy delivery platform against human HNSCC and the effect of higher STAT3 decoy microbubble loading on tumor cell inhibition. STAT3 decoy was loaded on cationic lipid microbubbles (STAT3-MB) or loaded on liposome-conjugated lipid microbubbles to form STAT3-loaded liposome-microbubble complexes (STAT3-LPX). UTMC treatment efficacy with these two formulations was evaluated  Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide, with 49,670 cases in the United States and 600,000 cases globally in 2017 –5. Both One approach to blocking the activity of STAT3 employs a decoy oligonucleotide, which is a 15-mer double-stranded oligonucleotide that binds to activated STAT3 dimers and competitively inhibits the binding of these dimers to target gene promoters , 13. WhiMicrobubbles (1–5 μm in diameter), consisting of a gas-filled core that is stabilized by either lipid, surfactant, protein, or a polymer-based shell, could be an attractive drug delivery system . Intravein vitro to compare the efficacies of two microbubble formulations combined with ultrasound. Thereafter we tested the superior performing microbubble formulation to deliver STAT3 via UTMC in nude mice bearing CAL33 xenograft tumors.Accordingly, to further promote clinical translation, we tested the hypothesis that UTMC-mediated delivery of STAT3 decoy to human HNSCC would inhibit tumor growth. Furthermore, we sought to determine if a microbubble carrying a higher amount of STAT3 decoy than previously tested by our group, by using decoy-loaded liposomes conjugated to microbubbles, would confer greater inhibition of human HNSCC xenograft tumors. We used human HNSCC cells (CAL33) 5´-GTAAATC-18-GATTTACGGGAAATG-18-CATTTCCC-3´. A mutant decoy was used as a control and differed from the STAT3 decoy by a single nucleotide base pair with the sequence pattern: 5´-TTAAATC-18-GATTTAAGGGAAATG-18-CATTTCCC-3´.The double-stranded STAT3 decoy oligonucleotide and its mutant version were purchased from IDT Technologies . Prior to loading onto microbubbles, oligonucleotides were ligated using T4 ligase  to form cyclic decoy . The comsn-glycero-3-phosphocholine (DSPC), 1,2-distearoyl-sn-glycero-3-ethylphosphocholine  (DSEPC), and 1,2-distearoyl-sn-glycero-3-phosphoglycerol, sodium salt (DSPG) were purchased from Avanti Polar Lipids . Polyethylene glycol-40 (PEG-40) stearate was purchased from Sigma-Aldrich . All other chemicals were of reagent grade and used according to the manufacturer’s guidelines. Stock solutions of multilamellar vesicles (MLV) were prepared by dissolving DSPC, DSEPC, DSPG, and PEG-40 stearate at a molar ratio of 100:43:1:4.5 in chloroform and dried using argon gas. The dried lipid film was hydrated using isotonic saline and sonicated using a bath Ultrasonic Cleaner  until all the lipids were evenly dispersed. To prepare decoy-loaded microbubbles, 200 μL of 4X lipid stock solution was diluted with saline to 800 μL in a glass vial using saline, and 10 μg of STAT3 decoy or mutant decoy was added and sealed. The headspace of the vial was filled with perfluorobutane gas  and vortexed for 45 s using a dental amalgamator, resulting in microbubbles carrying STAT3 decoy (STAT3-MB) or mutant decoy (STAT3-MB-mut) via charge-charge interaction. The loaded microbubbles were washed three times using centrifugation at 53 g for 5 min with saline and stored at 4–8°C until further use. The maximal loading of STAT3 decoy was determined by a previously described iterative process of optimization [We have previously reported the synthesis of STAT3 decoy-loaded cationic lipid microbubbles . 1,2-dismization . The amoLipid microbubbles were prepared from DSPC, 1,2-distearoylsn-glycero-3-phosphoethanol-amine-N-[biotinyl(polyethyleneglycol)-2000 (DSPE-PEG2000-Biotin) , and PEG-40 stearate as previously described . Briefly1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) , and DSPE-PEG2000-biotin were individually dissolved in chloroform and mixed together with a molar ratio of 49:49:2. The chloroform was evaporated by flushing with argon and followed by overnight vacuum drying. The dried lipid film was rehydrated in phosphate buffered saline (PBS) for 30 min at 65°C. The crude liposomes were extruded through two stacks of 400 nm polycarbonate membrane 21 times at 65°C using a mini extruder . The free lipids were removed by passing through a Sephadex G-50 column  and the resulting biotinylated cationic liposomes were stored at 4°C until use.9 microbubbles in 5 mL of saline) was incubated with 4.15 mg of streptavidin  for 60 min. The microbubbles were then washed twice with 1 mL of PBS by centrifugation at 200 g for 3 min to remove any free streptavidin. The resulting streptavidin-microbubble suspension was slowly added to saline containing excess amounts of the biotinylated cationic liposomes. After 60 min of incubation with constant agitation, the microbubbles were washed twice with 1 mL saline by centrifugation at 200 g for 3 min to remove unbound liposomes.The biotinylated cationic liposomes were conjugated to biotinylated lipid microbubbles via biotin-streptavidin interaction. The biotinylated microbubble suspension (1×108 microbubbles) for 20 min to allow decoy loading by electrostatic interaction. The resultant microbubble complexes carrying liposomes loaded with STAT3 decoy (STAT3-LPX) or mutant decoy (STAT3-LPX-mut) were washed twice with 1 mL saline by centrifugation at 200 g for 3 min. For quantification of STAT3 decoy on LPX, STAT3 decoy-loaded LPX complexes were centrifuged at 200g for 3 min to separate LPX from unbound STAT3 decoy. Both subnatant (unbound decoy) and supernatant (containing LPX) were harvested separately. The decoy bound to LPX complexes was released by incubation with heparin for 10 min and analyzed by agarose gel electrophoresis with Gel-Red staining. Using this method, it was found that saturation of the LPX with STAT3 decoy occurred at 5 μg in the tube, equivalent to 50 μg in 1×109 microbubbles (data not shown).The liposome-microbubble complexes  were incubated with differing amounts of STAT3 decoy or mutant decoy . A sample of 5 μL was diluted in 10 mL of Isoton  and measured in triplicate. The surface charge of the particles was measured using Zetasizer Nano ZS .in vitro studies. The cultured CAL33 cells (see below) were treated with fresh media containing varying molar concentrations (25–200 nM) of lipofectamine-complexed STAT3 decoy or mutant decoy and further incubated for 48 h. Cell viability was evaluated by MTT assay (see below).Lipofectamine complexes were formulated by mixing the appropriate proportions of STAT3 or mutant decoy and lipofectamine 2000 , and incubated for 5 min prior to the 2 and 37°C ambient conditions.Human tongue squamous cell carcinoma CAL33 cells were purchased from ATCC  and were cultured in high glucose DMEM media supplemented with 10% FBS and 1% antibiotic mixture. Additionally, CAL33-STAT-Luc cells expressing the luciferase for STAT3-responsive promoter were cultured. The cells were maintained at 5% CO5 per well) and allowed to grow for 24 h. Microbubbles were added to the culture media to achieve a final ratio of 10 microbubbles/cell. The wells were covered with media containing 2% FBS, sealed with polycarbonate membrane and inverted. Ultrasound was delivered with a single-element immersion transducer , driven by an arbitrary function generator  connected to a gated radio frequency power amplifier . The ultrasound field was calibrated with a 200-μm capsule hydrophone . Ultrasound pulses were delivered at 1 MHz, 0.50 MPa peak negative pressure, 10-μs pulse duration, and pulse interval 1 ms (duty cycle 1%), for a total of 10 s. The media was removed after UTMC and cells were replenished with fresh media (10% FBS and 1% antibiotic mixture) and incubated for 48 h. Cell viability and apoptosis assays and Western blot analyses were then performed (see below).CAL33 cells were seeded in a 12-well plate (seeding density 2×105 cells per well) and incubated for 24 h. STAT3-MB or STAT3-LPX, or their corresponding mutant-loaded formulations (STAT3-MB-mut or STAT3-LPX-mut), were added to the cells. The plates were then subjected to the ultrasound protocol described above, then incubated for 20 minutes; media was then removed and replaced with fresh media, followed by incubation for an additional 48 h.Cells were seeded in 12-well plates  per well and incubated for 4 h, followed by the addition of 200 μL of DMSO to dissolve the formazan crystals. Absorbance was measured at 570 nm using a microplate reader . Cell viability was calculated by normalizing to that of untreated control cells.Knockdown of STAT3 signaling was evaluated using CAL33-STAT-Luc cells expressing luciferase gene driven by a STAT3-responsive promoter. The cells were cultured in 12-well plates, exposed to loaded microbubbles, and subjected to the UTMC protocol as described above. Luciferase activity was measured with a luciferase assay kit  according to manufacturer’s guidelines and normalized to total protein as measured by Bradford assay .A quantitative apoptosis assay was performed by flow cytometry using Annexin V-FITC and propidium iodide (PI) staining  of the CAL33 cells  were incubated for 48 h, washed twice with PBS, detached in the presence of scraping buffer, centrifuged, and lysed using lysis buffer. The extracted proteins were separated using SDS-PAGE gel and transferred to PVDF membrane . The membrane was incubated with antibodies against Bcl-xL, cyclin D1, and GAPDH  [in vivo studies described below.Balb/c nude mice  bearing human xenograft HNSCC tumors were used to determine the growth inhibitory effect of UTMC-mediated STAT3 decoy delivery. All animal experiments were approved by the University of Pittsburgh Institutional Animal Care and Use Committee. All animal welfare considerations were followed as per guidelines of the University of Pittsburgh Institutional Animal Care and Use Committee. Anesthesia was provided using 2% inhaled isoflurane. The total duration of each experiment was 15 days and a total of 22 mice were used for the study. The mouse was euthanized on the last day of the experimental protocol (Day 15), or earlier if the tumor was observed to be beyond 1000 mmr 5 min) . In the 6 CAL33 cells were subcutaneously injected into the right flank. The tumors were allowed to grow to a volume of 80–100 mm3 prior to initiation of treatment.Mice were anesthetized and 1×109 MBs formulated with 10 μg of STAT3 decoy) were infused into the jugular vein over 20 min via an infusion pump. Therapeutic ultrasound was delivered using the same single-element immersion transducer system described above for the in vitro studies. The therapy transducer was oriented directly over the tumor, tilted slightly toward the tail of the animal to avoid ultrasound delivery to the abdomen. Over a period of 25 minutes during microbubble infusion, UTMC was delivered at 1 MHz transmit frequency, 0.70 MPa peak negative pressure, 100-μs pulse duration, repeated 5 times every 1 ms, followed by a 2-sec waiting period to allow microbubble reperfusion into the treatment area . The successful destruction and replenishment of microbubbles in the treatment area was confirmed by simultaneous contrast-specific ultrasound perfusion imaging with a clinical ultrasound imaging system  at low mechanical index (0.2).On the first day of treatment (Day 0), mice were anesthetized and a chronic indwelling catheter was placed in the internal jugular vein for microbubble infusion as described previously . UTMC trV(t) = V0kte, where V0 represents the initial tumor volume and t represents time in days. Anti-tumor efficacy was evaluated by comparing normalized tumor volumes, tumor doubling time calculated as DT = (ln 2)/k, and survival rate.Tumor volume was serially measured up to Day 15 using high-resolution 3-D ultrasound , after which the mice were euthanized using anesthesia (5% of Isoflurane for 5 min) . Data foIn situ Apoptosis Detection kit  as per the manufacturer’s protocol. A similar process was followed for Ki67 analysis of proliferation using Ki67 primary antibody (ab15580) [Tumors were harvested post-mortem, fixed in 10% formalin, embedded in paraffin, and stained with Hematoxylin and Eosin (H&E). TUNEL assays were performed on tumor sections using an ab15580) .The tumor tissues in TRIzol reagent  were homogenized using an Ultrasonic Processor . Chloroform (200 μL) was added to 1 mL of TRIzol solution and centrifuged at 12,000 g for 15 min at 4°C, and 200 μL of aqueous upper layer was pipetted into an RNAse-free centrifuge tube. After subsequent treatment with isopropyl alcohol and 75% ethanol, the RNA pellet was dissolved in DEPC water. Complementary DNA was prepared from 2 μg of total RNA using TaqMan reverse transcription kit . RT-PCR amplifications were performed with the SYBR™ Green PCR Master Mix  using an Applied Biosystems 7900HT instrument . Gene expression levels were quantified using the ΔΔCt method. GAPDH RNA levels were utilized to normalize the relative gene expression levels , post hoc Student’s t-testing was performed to determine wherein the differences resided, where significant differences between means was defined as p<0.05.The data are presented as mean ± standard deviation. Analysis of variance (ANOVA) was performed to determine if there were differences among the experimental groups. If a difference was found (ANOVA 9 microbubbles/mL and a loading capacity of 10 μg of STAT3 decoy per 1×109 microbubbles. The STAT3-LPX had an average diameter of 3.50±0.29 μm with an average concentration of 2×109 microbubbles/mL and a loading capacity of 50 μg of STAT3 decoy per 1×109 microbubbles (5-fold higher than STAT3-MB).The STAT3-MB had an average diameter of 2.45±0.35 μm with an average concentration of 2×10in vitro using MTT viability assays and apoptosis assays. STAT3 decoy delivered via lipofection induced a dose-dependent cytotoxic effect in CAL33 cells (p<0.001), and the cell viability of no treatment control and mutant treated cells was similar. These lipofectamine data confirm that the STAT3 decoy was cytotoxic and the mutant decoy was not.The activity of STAT3 decoy delivered via the various formulations against CAL33 cells was evaluated 33 cells . STAT3 din vitro cytotoxicity of STAT3-MB + UTMC vs. STAT3-LPX + UTMC was compared using MTT assay. STAT3-MB + UTMC resulted in significantly lower CAL33 cell viability (56.8±8.4%) compared to that of STAT3-LPX + UTMC  (in vitro and in vivo experiments.The  p<0.05) . Based o+ cells where luciferase expression is driven by a STAT3-responsive promoter, STAT3-MB + UTMC caused a ~45% reduction in luciferase activity which was significantly (p<0.05) greater than that of mutant microbubble (<10%) (p<0.15).In CAL33 luce (<10%) . On the n = 7) reduced tumor growth compared to that in untreated control (n = 8) and STAT3-MB-mut + UTMC-treated mice (n = 7) (p<0.05) in tumor volumes were observed between the STAT3-MB + UTMC group and the two control groups starting at Day 3 and continuing until the end of study period (p<0.05) in tumor volume compared to that of the no treatment control group and a 1.5-fold reduction (p<0.05) compared to that of STAT3-MB-mut + UTMC, respectively  and mutant control decoy , resulting in reduced mortality of the STAT3 decoy treated mice. Kaplan-Meier curves (p<0.05).STAT3-MB + UTMC ( (n = 7) . Signifiy period . By Day ectively . Mice trme point . Fig 3E r curves  indicatep<0.05)  . UTMC-meOn H&E staining, STAT3 decoy treated tumors showed fragmentation of cells and apoptotic condensations . STAT3-MThe main finding of this study is that lipid microbubbles loaded with a cyclic STAT3 decoy oligonucleotide, in the presence of image-guided therapeutic ultrasound, inhibit growth of human HNSCC tumors and suppress downstream target gene expression. As the first demonstration of UTMC-mediated delivery of STAT3 decoy to cancer cells of human origin, these data set the stage for clinical translation of ultrasound-mediated delivery of STAT3 decoy for the treatment of patients with head and neck tumors.STAT3 is constitutively hyperactivated in many cancers, including head and neck cancers. Multiple signaling pathways converge at STAT3, making it a master regulator mediating molecular events that result in cancer progression. The STAT3 decoy oligonucleotide binds to activated STAT3 with a high specificity and inhibits STAT3-mediated gene transcription and cancer cell proliferation . It has in vitro and in vivo. Further, we sought to determine whether increasing STAT3 loading on the microbubbles above that previously described by us [in vitro studies using cultured human head and neck carcinoma cells (CAL33) and in vivo experiments using nude mice bearing CAL33 xenograft tumors.To this end, we developed an image-guided targeted delivery strategy employing oligonucleotide-loaded microbubbles that can be ultrasonically induced to deliver their payload exclusively to tumor as they transit through tumor microcirculation –31. We ped by us  would in9 microbubbles/mL [in vitro. We interpret this to indicate that increasing the STAT3 dose via liposomal incorporation on to the microbubble is not equivalent to higher effective bioavailability of the decoy. It is known that release of payloads from liposomal carriers may be inefficient or slow [in vitro that release of doxorubicin from liposome-microbubble constructs is increased by long tone burst ultrasound [in vitro data, we selected the STAT3-MB formulation for the remaining experiments.Our prior cationic lipid microbubble formulation (STAT3-MB) maximally loads 10 μg of STAT3 decoy on 1×10bbles/mL . To incr or slow , 33, andtrasound . In the in vitro results, we found that nude mice bearing CAL33 tumors experienced less tumor growth and longer survival when treated with STAT3-MB + UTMC compared to the negative control groups, and this was associated with downregulation of target gene Bcl-xL and Cyc-D1 expression at the RNA transcript and protein levels. The tumor immunohistochemistry data indicated more apoptosis and less proliferation activity in the STAT3-MB + UTMC treated tumors. Importantly, UTMC-mediated delivery of mutant control decoy did not result in tumor growth suppression or target gene downregulation compared to untreated control mice, indicating that the therapeutic effect was not simply due to the delivery of ultrasound.We demonstrated that compared to negative controls (no treatment control or STAT3-MB-mut + UTMC), cultured (human) CAL33 cells treated with STAT3-MB + UTMC experienced: (1) greater cytotoxicity as shown by MTT assay; (2) more apoptosis as shown by flow cytometry; and (3) a strong trend in decreased protein expression of downstream target genes Bcl-xL as shown by Western blot. Aligned with the in vitro that after a sonoporation event, junctions between confluent endothelial cells form and remain open for hours [The mechanisms for UTMC-induced enhancement of nucleic acid therapeutics are incompletely understood. UTMC capitalizes on the unique acoustic behavior of gas-filled microbubbles in an ultrasound field, whereby the microbubbles vibrate, or cavitate, in response to the mechanical perturbations of ultrasound waves. We and others , 35–37 hor hours , and othor hours , suggestin vitro studies described above), combined with the release of the decoy specifically as the loaded microbubbles transit the tumor, concentrates delivery of the decoy within the insonified tumor. The order of magnitude lower dose requirement conferred by UTMC may have clinical translational benefit by reducing the overall dose required to achieve therapeutic efficacy while reducing off-target effects.Our study demonstrated significant therapeutic efficacy of our UTMC platform compared to that of other reports using different STAT3 decoy delivery platforms and higher STAT3 decoy doses in HNSCC models. We previously reported tumor regression and inhibition of STAT3 signaling in HNSCC xenografts after intravenous administration of STAT3 decoy (5 mg/kg/day), 5 days per week for 2 weeks , 14. We Some limitations to our study bear mention. Despite the fact that UTMC-based STAT3 decoy delivery inhibited tumor growth, complete regression of tumor was not achieved and tumor growth recommenced after the treatment was terminated (Day 7). The number of UTMC treatments we could give was inherently limited because frequent general anesthesia, as would be necessary for daily microbubble intravenous infusion and UTMC in these mice, would not be well tolerated. In future clinical applications, daily peripheral intravenous microbubble administration with UTMC is highly feasible, and the opportunity to more aggressively promote tumor regression would be afforded.in vitro. Although we did not specifically test for dissociation of the decoy from the liposome in the serum-containing culture media used in our in vitro studies, previous reports have shown excellent binding and stability of nucleic acids in liposomes in a serum environment up to 50% [in vitro. Nonetheless the inferior performance of the STAT3-LPX formulation does not negate the concept that formulations with higher MB loading doses could achieve higher UTMC therapeutic efficacy. Rather, such data indicate that other factors, including the acoustic regimen and its interaction with the specific microbubble formulations, should be studied to optimize the bioavailability of decoy delivered using new, more highly loaded, microbubble species.The liposome microbubble conjugated formulation (STAT3-LPX) carried 5-fold greater STAT3 decoy than the cationic lipid formulation (STAT3-MB) yet was not more effective p to 50% –42, whilTo the best of our knowledge, this is the first study to demonstrate the efficacy of UTMC-based STAT3 decoy in human cancer cells. Our study demonstrated the ability of STAT3 decoy microbubbles and ultrasound to inhibit the growth of human HNSCC and downregulate target genes involved in signaling pathways that contribute to tumor cell survival and proliferation. Significant therapeutic effects were observed at a fraction of the STAT3 decoy dose required to achieve the same inhibitory effect from intravenously injected decoy. Taken together, our study lays important groundwork for the clinical translation of UTMC for targeted delivery of STAT3 decoy, and possibly other therapeutic oligonucleotides, for the treatment of HNSCC and other solid tumors.S1 File(DOCX)Click here for additional data file.


Ircinia campana, a common Caribbean sponge subject to mass mortalities and disease. Microsatellites were used to genotype 440 individuals from 19 sites throughout the Greater Caribbean. We found strong genetic structure across the region, and significant isolation by distance across the Lesser Antilles, highlighting the influence of limited larval dispersal. We also observed spatial genetic structure patterns congruent with oceanography. This includes evidence of connectivity between sponges in the Florida Keys and the southeast coast of the United States (>700 km away) where the oceanographic environment is dominated by the strong Florida Current. Conversely, the population in southern Belize was strongly differentiated from all other sites, consistent with the presence of dispersal-limiting oceanographic features, including the Gulf of Honduras gyre. At smaller spatial scales (<100 km), sites showed heterogeneous patterns of low-level but significant genetic differentiation (chaotic genetic patchiness), indicative of temporal variability in recruitment or local selective pressures. Genetic diversity was similar across sites, but there was evidence of a genetic bottleneck at one site in Florida where past mass mortalities have occurred. These findings underscore the relationship between regional oceanography and weak larval dispersal in explaining population genetic patterns, and could inform conservation management of the species.Understanding population genetic structure can help us to infer dispersal patterns, predict population resilience and design effective management strategies. For sessile species with limited dispersal, this is especially pertinent because genetic diversity and connectivity are key aspects of their resilience to environmental stressors. Here, we describe the population structure of  The spatial distribution of genetic diversity is influenced by both ecological and evolutionary processes, and can be used to infer a number of important characteristics of species, including dispersal, recruitment and gene flow among populations (connectivity). Understanding these processes can help in ecosystem management and conservation Baums . In addiIn Greater Caribbean marine ecosystems, sponges are abundant, diverse and serve important ecological functions, including cycling of organic material and habitat provisioning , a common species in coral reef and hard-bottom habitats. The reproduction and larval biology of I. campana has not yet been described; however, other Ircinia species are viviparous, with no true asexual reproduction (through budding or gemmules), and the vase-shaped morphology of I. campana is not susceptible to fragmentation during storms. This species is prone to disease outbreaks, and has suffered a number of mass mortality events, the most extensive having occurred in the Florida Keys  have previously been affected by mass mortalities associated with cyanobacterial blooms. We cut small fragments of tissue (~1.5 cm3) and preserved them in 95% ethanol soon after surfacing. Ethanol was replaced after 24 h to avoid dilution with the seawater held in the sponge tissue.We sampled 10–41 We dissected samples under a stereomicroscope to remove macroinvertebrates from the sponge tissue, and extracted DNA using the DNeasy® Blood and Tissue Kit (Qiagen). We genotyped the samples for 10 microsatellite loci, as described in Griffiths et al. . Brieflyp.adjust function in R 3.4.3  calculations), the full ten-loci dataset was used. In all other analyses, Icam34 and Icam10 were excluded, yielding an eight-loci dataset.We tested linkage disequilibrium between loci using Genepop on the Web v4.2 (Raymond and Rousset d Estoup . Followi01 Table . ConsequHO) and Nei’s gene diversity (HS) (expected heterozygosity) for each sampling site using GenoDive v2.0b23  cycles and 50,000 burn-in cycles. We ran the model using all combinations of parameters for possible null allele causes , and used Bayesian deviance information criterion (DIC) to infer which parameters contributed more to the observed data.We calculated probability of deviation from Hardy–Weinberg equilibrium (HWE), observed heterozygosity (HS in separate models (eight-loci datasets), and ran the models using the R package ‘lme4’  hereafter), and tested significance using 50,000 permutations (with B–Y correction applied for multiple tests). We also calculated FST between population pairs for all ten loci with ENA correction for null alleles in FreeNA (referred to as FST(10) hereafter). We tested correlation between matrices of FST(8), FST(10) and D, using Mantel tests in the ‘ade4’ package in R with 9999 permutations.The traditional measure of subpopulation differentiation, igh Jost . Becauseigh Jost , we alsoion Jost . We calcD.To visualise genetic distance relationships among sites, we carried out principal coordinate analysis (PCoA) in GenAlEx 6.503 /1–FST(10) and D/1–D) and the logarithm of oceanographic distances with Mantel tests in ‘ade4’ in R, using 9999 permutations. We calculated least-cost oceanographic distances among sites  using ‘marmap’ in R (Pante and Simon-Bouhet We tested for the presence of genetic isolation-by-distance (IBD) patterns in the data by testing the correlation between pairwise matrices of linearised genetic distances (K) and individual assignment probabilities to those clusters, using the eight-loci dataset. We first ran the model using the uncorrelated allele frequencies, spatial and null allele models with 1,000,000 iterations, 100 thinning and 500 burn-in. According to the authors’ advice, we set the maximum number of nuclei to 1320 (3 × sample size) and the maximum rate of the Poisson process to 440 (1 × sample size), and the spatial uncertainty on coordinates to 0.0005. We carried out ten independent runs of K from 1 to 19, and checked that clusters were consistent among runs. We then selected the run with the highest posterior probability to estimate allele frequencies and cluster locations to use subsequently in the admixture model. We ran the admixture model using the same parameters as above, and extracted the Q matrix of individual probability assignments to each cluster to build a bar plot in Distruct  . Two identical multilocus genotypes  were present in the dataset , one of which was removed for subsequent analyses. Average null allele frequencies were high at many loci, overall ranging from <0.001 (Icam32) to 0.323 (Icam34) (Table All loci were in linkage equilibrium after B–Y correction (4) Table . Loci we4) Table .p < 0.05) at a number of loci and sites following the correction for multiple tests  to 0.565 (Panama C) within sites, and HS ranged from 0.528 (Gray’s Reef) to 0.761 (Mayreau) . Rarefied allelic richness per site ranged from 3.258 ± 0.552 SE  to 4.667 ± 0.589 SE (Mayreau) . However, post hoc Tukey tests revealed that only three pairs of sites were significantly different: allelic richness was significantly higher at St. Lucia than at Turneffe Atoll , at Mayreau than at Turneffe Atoll  and at Mayreau than at Waltz Key . All other site-by-site comparisons were not significant (p > 0.05), although three comparisons were close to significance . Rarefied private allelic richness ranged from 0.237 ± 0.107  to 1.302 ± 0.432 (St. Lucia) (Table u) Table . Site haa) Table .p = 0.014), indicative of a genetic bottleneck.Significant heterozygosity excess with respect to allelic richness was detected only at Bamboo Key (FST(10) reaching 0.233 (between Gray’s Reef and Panama 1) and D reaching 0.598 (between Gray’s Reef and Sapodilla Cayes) (Tables FST(10) and FST(8): r = 0.921, p < 0.001; FST(8) and D: r = 0.908, p < 0.001; FST(10) and D: r = 0.825, p < 0.001).Our analyses showed strong population structure across the region, with ) Tables  and S6. FST(10)r = 0.793, p = 0.001; D: r = 0.688, p < 0.001) : p = 0.483; D: p = 0.476)  Fig.  but not 76) Fig. .Fig. 2GeGeneland analysis of the whole dataset identified four genetic clusters Fig. , with fuThe DAPC showed a clear separation of the Sapodilla Cayes from all other sites upon analysis of the full dataset Fig. . In the D among sites, the second axis represented 21.02% and the third axis represented 12.48%, giving a total of 59.12% over the three axes. The first axis separated the USA sites from the rest of the sites, while the second axis clearly separated a St. Vincent, Mayreau and Bequia cluster .Both HWE deviations and null alleles are common in sponge microsatellite studies , an area subject to highly retentive oceanographic conditions  is unexpected, given the genetic and geographic distances among these locations, and the absence of specific water circulation patterns that might cause such a grouping. However, when analyses were repeated including only these sites, the sites divided into three separate clusters. This demonstrates the utility of repeating analyses on multisite clusters to uncover further patterns of population structure that are not revealed in the overall dataset analysis . IBD is driven by distance-limited dispersal Wright , which iFST ranged between 0.006 and 0.072), and the sites formed a single cluster in Geneland, PCoA and DAPC analyses. This suggests that larvae disperse across the area with sufficient regularity to maintain gene flow . This result is interesting given the short larval duration and low dispersal capacity predicted for sponges, but concurs with other studies that show higher-than-expected levels of connectivity and dispersal  considering their proximity (15 km). In addition, the DAPC and PCoA analyses highlighted genetic separation between the sites. Diamond Rock (MAR2) is a small island located 3 km off the Martinique coast. Both the island and the channel separating it from the mainland are locally known to experience strong currents (pers. comm. G. Tollu), and early modelling of water circulation suggests that the area may be influenced by a small gyre off the southern coast of Martinique , but a bottleneck signature was not detected here. This could be because any loss in genetic diversity was rapidly regained through recruitment and gene flow from other areas. Indeed, the Long Key site is dominated by long-shore currents, whereas the Bamboo Key site sits within a bay subject to a local gyre. Alternatively, it is possible that the Long Key result is a false negative, as heterozygosity excess tests are sensitive to mutation model selection and sample-size limitations . However, we did observe a disease-like condition in individuals harbouring necrosis of various stages in Guadeloupe and Bequia during sampling for this study, and on other occasions in Martinique (pers. obs. T. Pérez and S. Griffiths). Indeed, disease outbreaks and mass mortalities have been reported multiple times in Understanding the scale and magnitude of connectivity among populations is important for the management and conservation of marine ecosystems (Almany et al. I. campana populations; this should be maintained in restored sites through the use of a large number of donor sponges, rather than extensive fragmentation of few sponges.Our results also provide insight for management of mass mortality-affected sites in Florida. Restoration through fragmenting and transplanting healthy sponges has been successfully used to repopulate barren areas (Butler et al. Supplementary Information


The existing literature has not examined how Chinese direct-to-consumer (DTC) genetic testing providers navigate the issues of informed consent, privacy, and data protection associated with testing services. This research aims to explore these questions by examining the relevant documents and messages published on websites of the Chinese DTC genetic test providers.Baidu.com, the most popular Chinese search engine, we compiled the websites of providers who offer genetic testing services and analyzed available documents related to informed consent, the terms of services, and the privacy policy. The analyses were guided by the following inquiries as they applied to each DTC provider: the methods available for purchasing testing products; the methods providers used to obtain informed consent; privacy issues and measures for protecting consumers’ health information; the policy for third-party data sharing; consumers right to their data; and the liabilities in the event of a data breach.Using 68.7% of providers offer multiple channels for purchasing genetic testing products, and that social media has become a popular platform to promote testing services. Informed consent forms are not available on 94% of providers’ websites and a privacy policy is only offered by 45.8% of DTC genetic testing providers. Thirty-nine providers stated that they used measures to protect consumers’ information, of which, 29 providers have distinguished consumers’ general personal information from their genetic information. In 33.7% of the cases examined, providers stated that with consumers’ explicit permission, they could reuse and share the clients’ information for non-commercial purposes. Twenty-three providers granted consumer rights to their health information, with the most frequently mentioned right being the consumers’ right to decide how their data can be used by providers. Lastly, 21.7% of providers clearly stated their liabilities in the event of a data breach, placing more emphasis on the providers’ exemption from any liability.Currently, the Chinese DTC genetic testing business is running in a regulatory vacuum, governed by self-regulation. The government should develop a comprehensive legal framework to regulate DTC genetic testing offerings. Regulatory improvements should be made based on periodical reviews of the supervisory strategy to meet the rapid development of the DTC genetic testing industry. Ancestry.com and 23andMe, Chinese DTC companies offers genetic testing services for both illness risk determination and lifestyle guidance purposes (Direct-to-consumer (DTC) genetic testing has gained increasing popularity internationally. The market for DTC genetic testing is estimated to reach 20 billion by 2024 . In recepurposes . For exapurposes . Accordipurposes .Notice on Strengthening the Management of Products and Technologies Related to Clinical Use of Gene Sequencing in February 2014, with the Notice suspending all genetic testing services in China  when collecting and using consumers’ personal information. Companies should inform and obtain the consent of consumers regarding the purposes and scope of collection and use of personal information (Regulation of the People’s Republic of China on Human Genetic Resources Management (the Regulation on Human Genetic Resources) on May 28, 2019 , which was promulgated by the SCNPCC in 2016, requests that DTC genetic testing providers must not steal or use other illegal means to obtain consumers’ personal information including biometric information, nor illegally sell or provide consumers’ personal information to others  launched a legislative agenda for a comprehensive data protection law, a few months after the European Union General Data Protection Regulation entered into force on May 25, 2018. The plan shows the direction of China’s data protection scheme, and the law is planned to be enacted in 2022 . Howevero others . Moreoveormation . Differempliance . For exaThe lack of effective supervision in DTC genetic testing offerings has gained increasing attention from the Chinese news media . Many neBaidu.com, the most popular search engine in China, and search keywords: “genetic testing” (in Chinese: “”) to identify and collect providers that use the DTC model to market genetic testing services and products. Based on this search, we collected 90 DTC genetic testing offerings. We then visited the websites and captured the webpages of the providers and downloaded all available documents related to the terms of service, privacy protection, and informed consent. It is essential to clarify that our study focuses on examining organizations that mainly offer genetic testing services and products.From January 17, 2019 to February 27, 2019, we used From May 7, 2019 to June 18, 2019, one of our authors analyzed the websites using a coding framework focused on the following perspectives: (1) channels provided for purchasing genetic testing products and services; (2) informed consent; (3) privacy issues; (4) strategies used to protect consumers’ personal health information; (5) data sharing with a third party; (6) consumers’ rights to their health information; (7) responsibility for data breach; and (8) specific laws or legal protections mentioned. These eight perspectives were established based on previous studies on legal and ethical issues associated with DTC genetic testing and personal data protection in the big data era in healthcare . The codRegulations on the Management of Human Genetic Resources of the People’s Republic of China. Since the collection of website information and the content analysis of consumer-related legal documents were carried out before the implementation of the new Regulation, we took advantage of the opportunity to examine how Chinese DTC genetic testing providers reacted to the new Regulation. For example, we attempted to identify if there were updates to their ToS, PP, and ICF to meet the requirements of the updated requirements 3 months after the Regulation took effect. Consequently, we revisited the websites of the companies collected in our dataset and again reviewed the content of the websites and the legal documents to determine if any changes were made to comply with the new Regulation. In this second round of collection and analysis, we found that the original links of nine websites were broken. Five of these nine problematic websites changed to new domain names, and four of the nine websites were completely invalid. As a result, our dataset in the second round consists of 83 accessible websites. We analyzed these 83 websites using the same coding framework from September 2, 2019 to September 20, 2019. We used the 83 websites as the final dataset for this research and compared the results of the two rounds of analyses.Later, on July 1, 2019, a new Decree of the State Council of China took effect, the For both rounds of content analysis, 30% of the websites were randomly selected to compare the consistency of coding results in order to verify the reliability of the coding. After obtaining the 30% of websites, an independent coder searched the URLs of the websites, reviewed the content and available privacy policy, informed consent forms, and the terms of service. We calculated the agreement between the two codes, using Cohen’s Kappa evaluation. The agreement was between 0.85 and 1.00 for all coding frame items, which indicates substantial to perfect agreement .1,2, which allowed consumers to order genetic testing products or services through a third party. In particular, we found that WeChat, the most widely used social media platform in China, became a popular vehicle for providers to promote their products and services. Thirty-two providers used WeChat to introduce their products and to offer follow-up services to consumers. Consumers could order products and make payments via providers’ WeChat stores or by transferring money after adding the providers as WeChat friends. In contrast, very few providers (n = 3) used QQ, an instant messenger that at one time was the most widely used in China, as a promotion platform. The case for the relatively few providers using QQ to reach consumers might arguably be due to the increase in WeChat users.Fifty-seven providers of DTC genetic testing services and products offered online purchase options via their websites. Twenty-six providers only accepted traditional banking transfers as payments after counseling with consultants via telephone. For providers that offered online purchases, 21 providers required consumers to register on the websites before they could order services and products online, and five websites integrated with other e-commerce platformsn = 5) that provided informed consent forms on their websites. The promulgation of the new Regulation did not make a big difference in the availability of informed consent forms. Only one provider added an informed consent form to its website after the new Regulation took effect.We only identified 6% of providers  consumers or their families may feel uncomfortable because of survey questions or genetic data results; (2) information leakage due to security breach; (3) information leakage caused by consumers sharing accounts and passwords with others; and (4) other currently unforeseeable risks. Three providers mentioned that their genetic testing reports were predictive, and which can only be used as a health consulting reference not as a clinical diagnostic basis.n = 78), although they failed in providing specific informed consent forms, 13 providers did mention the informed consent procedure on their websites. For example, 11 websites indicated that informed consent procedures would be implemented by providers during or after the purchases of the genetic testing services and products.With regard to the remaining websites . Four companies mentioned privacy issues, but they did not offer a privacy policy on their websites. For example, two websites stated in their FAQ section that they had addressed privacy issues in their privacy policies, but offered no links to the privacy policies on their websites. Similarly, two websites had privacy policy tags at the bottom of the webpages, but clicking on these tags did not lead to valid links to the privacy policies. Moreover, in our second-round investigation, we did not identify an obvious difference in the provision of the privacy policy. In fact, except for one provider who updated the privacy policy by adding one sentence addressing the liability distributions in the event of a privacy breach, the rest of the providers did not make any changes to their privacy policies.Consequently, we identified 37 websites in total that offered a privacy policy. This means, however, that more than half of the websites (55.4%) did not provide consumers with a privacy policy  of consumers, such as website registration ID, social security ID, and health information, from their genetic information. Seventeen providers proposed concrete measures for protecting consumer GPI  did not address the reuse, selling, or sharing of information gathered from consumers in their informed consent forms, privacy policies, or terms of service, 28 providers stated that with additional client permissions, the providers can reuse and share the clients’ information for non-commercial purposes. Two providers mentioned that they would not sell a client’s information unless having obtained the client’s additional permission. Only one company mentioned that it would reuse and share a client’s information for non-commercial purposes without the client’s further permission. Specifically, the company stated that: in a case where the third party agrees to assume the same responsibility of protecting users’ privacy as the company does, the company can provide the third party with users’ registration and other information without consumers’ further permissions. Moreover, 22 websites mentioned the compelled disclosure of personal health information in accordance with laws in either the ICF or PP. In regards to information disclosure, 73.5% of providers (n = 61) did not make any statements about mandatory information disclosure on their websites.While 62.7% of providers (n = 60) kept silent in this respect. In general, three types of rights have been granted by the DTC genetic testing providers to their customers. Among these, the most frequently mentioned right is the consumers’ right to decide whether providers can use their genetic data for follow-up research or provide their data to third parties (mentioned by 21 providers). Nine providers stated that consumers have the right to view and change their personal data or to remove their data from the providers’ database. Only one company mentioned that consumers have the right to be informed of follow-up use of their data, which includes: (1) using users’ genetic data to develop new products based on gene sequencing results; and (2) using genetic data for the latest interpretation of existing projects, interpreting the latest scientific literature, and recalculating existing projects more accurately.Twenty-three providers granted consumer rights to their health information, while the rest of the providers (n = 58) did not inform consumers of the risk of accidental information leakage, 30.1% of providers (n = 25) mentioned relevant risks on their websites, e.g., hacker attacks, internet errors, and other unforeseeable accidents. Among these 25 providers, all stated that the company would strive to protect customers’ privacy, preventing their health information from being disclosed arbitrarily. However, in terms of the distribution of responsibility for the breach of privacy, fewer providers (n = 18) made explicit statements regarding whether they shall bear liability. In particular, nine providers did not address who would assume responsibility in the event of a data breach, but clearly stated that providers will be exempted from liability if the leaks are due to causes beyond their control. Eight companies specified only the consumers’ responsibilities but not the obligations of providers in a breach. Just one company stated in their PP that in the event of a data leakage, that the liability would fall on the source of the breach, i.e., the source of the leak will bear the responsibility.While nearly 70% of providers (Cybersecurity Act (mentioned by two companies), the Regulation on Human Genetic Resources (mentioned by one company), and the Interim Measures for the Management of Human Genetic Resources (mentioned by one company), which had been replaced by the Regulation on Human Genetic Resources.Twenty-three Chinese DTC genetic testing websites in their ICF, ToS, or PP stated that the company would comply with relevant Chinese laws in collecting, storing, and using consumer genetic data during and after genetic testing services. However, among these 23 websites, only four companies mentioned concrete laws, e.g., the Health Insurance Portability and Accountability Act (HIPAA), the United States legislation that protects the privacy and security of Americans’ medical information in the PP, ICF, or data protection agreement for dealing with issues of privacy protection. We found that HIPAA was applied in different ways. For example, one company mentioned that the storage of consumers’ genetic data will strictly abide by the HIPAA requirements. One company stated that for international users, the process of the genetic testing services will follow HIPPA standards. Another company used the HIPAA as a reference for their practice of storing consumers’ genetic data. In addition, one company promised that it would abide by the Hong Kong Personal Data (Privacy) Ordinance to carry out genetic testing services.It is worth noting that three companies mentioned the Our research indicates that Chinese DTC genetic testing companies have begun to take action in protecting consumer genomic data privacy. For example, many providers developed specific measures for protecting the security and privacy of consumer health information. Our research particularly indicates that some DTC genetic testing providers separated the consumer general health information from their genetic information and used different measures to protect the two types of data. This may reflect the industry’s growing awareness of the sensitivity of genetic information and the need for using special measures to protect consumers’ genetic data. Moreover, consumers’ rights to their health information were recognized by many DTC genetic testing companies. For example, consumers have the right to access their personal data or have their information removed from company records. Nonetheless, our research has identified several legal concerns that Chinese regulatory bodies should immediately address.First and foremost, we found that the provision of informed consent forms is not a common practice for the Chinese DTC genetic testing providers. This finding is in agreement with previous studies on international DTC genetic testing companies, where informed consent practices were found to be inadequate and sometimes misleading . Until vRegulation on Human Genetic Resources specifies that informed consent should be obtained from providers of human genetic resources before collecting and using their genetic information, the purposes of the Regulation are mainly based on the concerns of safeguarding public health, national security, and social public interests, rather than patients or consumers’ health rights and interests in the context of genetic testing. Given the lack of regulation in China that can guide and monitor informed consent procedures for both clinical and non-health-related genetic testing, consumer rights for health and information are left without adequate protection.Moreover, we found that many Chinese DTC genetic testing companies offer both non-health-related tests and tests for health purposes. This raises further concerns about whether the same rules should be applied for regulating informed consent in both types of gene sequencing applications. In countries where regulatory measures for DTC genetic testing services are comparatively loose, they generally have strict requirements of informed consent for health-related genetic testing. For example, in the United States, the Presidential Commission for the Study of Bioethical Issues stated that if the genetic testing is prescribed due to clinical purposes, physicians have to present informed consent to patients due to their fiduciary duties to patients . In ChinPersonal Information Security Specification on privacy policy. In particular, 3 months after the Regulation on Human Genetic Resources took effect, we did not identify a significant change in the DTC genetic testing industry for improving the practice of informed consent and consumer privacy to comply with the new legal requirements. Only one genetic testing provider had given an update to its informed consent form on its website, in that they offered a link to an informed consent form and added one short sentence for the protection of consumers’ privacy in the form. This illustrates that, without an established comprehensive personal data protection law, both the Cybersecurity Law and the regulatory measure on genetic resources management had very little influence on promoting and advancing the protection of privacy and implementation of informed consent in DTC genetic testing services.Compared with the number of informed consent forms provided, more Chinese DTC genetic testing companies have addressed issues related to consumer privacy protection. For example, 37 websites offered a link to a privacy policy. This finding is consistent with existing research that privacy concerns have been increasingly addressed by international DTC genetic testing providers . That beConsumer Protection Law, Cybersecurity Law, and the Regulation on Human Genetic Resources are applicable to regulating the genetic testing market, there is no special regulatory regime covering important issues associated with the implementation of DTC genetic testing services, e.g., informed consent, privacy protection, and transparency about how consumer genetic data is used, collected, and shared. Thus, the current Chinese DTC genetic testing market operates as a self-regulated mechanism. Moreover, “soft laws,” such as best practices and a code of conduct, are also missing in the regulations of DTC genetic testing. We did not identify any established voluntary best practices guidelines for genetic testing services  demonstrated the restrictions on the use of genetic testing results in health insurance in the Measures for the Management of Health Insurance (draft for comments) – a 2017 version for the update of Measures for the Management of Health Insurance 2006 (Measures require that insurance companies protect the privacy and confidentiality of policy holders, the insured, and the beneficiaries, and that insurance companies should not add premiums to policy holders based on their genetic testing data and genetic information, other than the family genetic history of a disease. Moreover, according to Article 36 of the 2017 Measures, insurance companies should not require policy holders or the insured to take genetic tests, and that the genetic testing results are prohibited from being used as a condition for the verification of insurance. As the 2017 Measures have not been passed and entered into effect, further research is needed to examine and review the impact of the new regulations on the protection of private health information within the context of DTC genetic testing.Our research indicates that the United States nce 2006 . ArticleIn addition, our research indicates that social media has played an increasingly important role in promoting genetic testing services and products. On the one hand, social media, as previous studies indicated, could be a useful tool to increase patients’ knowledge of genetic testing and risk assessment for certain types of cancers . A recenOur study indicates that DTC genetic testing has become an emerging market in China. Eighty-three Chinese companies were identified as promoting genetic testing products directly to consumers. The existing applicable regulations on genetic testing are mainly focused on human genetic resource security and protection, and no special legislation has been developed to regulate DTC genetic testing offerings. Without an established legal regime, the availability for informed consent forms and policies for consumer data and privacy protection within the industry are self-imposed by DTC genetic testing companies. Moreover, the industry has not established any best practices guidelines for implementing DTC genetic testing services. As a result, the current DTC genetic testing business is running in a regulatory vacuum and is governed by a self-regulation mechanism. Our study indicates that the limits of this self-regulation model is obvious. Informed consent forms were generally not provided by DTC genetic testing companies, and a privacy policy was only available on less than half of all providers’ websites we examined. For the majority of DTC genetic testing companies, consumers’ autonomy for purchasing genetic testing is unable to be guaranteed, and there is a lack of transparency about how consumer genetic information is used and shared. As a result, consumers are left without adequate protection. Their genetic information might be illegally used or shared to a third party without their permission. We, therefore, urge that adequate and effective regulatory oversight over DTC genetic testing offerings should be developed. In particular, a clear and sufficient informed consent form and privacy policy should be provided on all DTC genetic testing providers’ websites . MoreoveAs the industry keeps evolving, some challenging issues associated with the provision of DTC genetic testing require further studies. For example, social media has been frequently used by DTC genetic testing companies as an alternative way to promote genetic testing services. The involvement of social media could bring opportunities for raising public awareness on potential privacy risks with DTC genetic testing. It may also trigger regulatory challenges in supervising the dissemination of truthful and balanced information on genetic testing via social media platforms. Additionally, some DTC genetic testing companies have distinguished consumers’ general health information from genetic information and used different methods to safeguard data security. This raises the question of whether different information protection rules should be developed and applied to different types of consumer health data. Given all these potential challenges and the growing industry, the development of regulations on genetic testing call for interdisciplinary perspectives, and it is essential to examine periodically the regulatory framework on genetic testing services.The datasets generated for this study are available on request to the corresponding author.LD designed the study. MW collected and analyzed the data. LD drafted the manuscript. MW made revisions to the manuscript.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.


Pterodroma baraui) on Réunion Island (Indian Ocean) corresponding to the sampled breeding colonies separated by 5 km. This unexpected result was supported by long-term banding and was clearly linked to the species’ extreme philopatric tendencies, emphasizing the importance of philopatry as an intrinsic barrier to gene flow. This implies that loss of a single colony could result in the loss of genetic variation, impairing the species’ ability to adapt to threats in the long term. We anticipate that these findings will have a pivotal influence on seabird research and population management, focusing attention below the species level of taxonomic organization.Effective conservation requires maintenance of the processes underlying species divergence, as well as understanding species’ responses to episodic disturbances and long-term change. We explored genetic population structure at a previously unrecognized spatial scale in seabirds, focusing on fine-scale isolation between colonies, and identified two distinct genetic clusters of Barau’s Petrels ( Seabirds present several challenges to the generally accepted mechanisms of population differentiation in other vagile groups4 and, despite recent advances in genetic theory and approaches, the scales at which genetic differentiation exists among their populations remains extremely difficult to predict5. Seabirds have an almost unsurpassed flight potential and have been known to cross physical features that limit dispersal in less motile groups8. Consequently, one could expect seabirds to disperse freely among their breeding sites yet growing evidence suggests that the potential for long-distance movement is not always the best indicator of gene flow10. This implies that philopatry and other intrinsic barriers to dispersal (e.g. differences in breeding phenology) may have important roles in the evolution of seabird diversity and endemism3, and that predicting genetic differentiation among populations for management or systematic purposes requires the assessment of these factors4. Despite this, little is known about the scales at which intrinsic barriers to genetic dispersal operate with few studies focusing on fine-scale inter-colony observations.A topic of critical importance in conservation biology concerns the mechanisms through which evolutionary changes occur, including the influence of population connectivity, and the implications of variation in connectivity on a species’ long-term survival and conservation needs11 as the life-history attributes of most seabirds render their populations robust in the face of fluctuations in breeding success, but highly sensitive to changes in adult mortality12. An estimated 95% of all seabird species breed in limited numbers of highly synchronous colonies13, rendering them susceptible to extreme breeding failure in the event of adverse environmental conditions or prey shortages. Nevertheless, poor reproductive output must be long-term and extensive to result in decreases in populations, whereas even slight changes in adult mortality can have lasting population or even species-level consequences12. Delayed sexual maturity and high philopatric tendencies also imply that seabird populations are typically slow to recover and that recovery after local extirpation is unlikely without interventions such as translocation or social attraction12. Thus, assuming local populations differ genetically, the loss of even a single population may result in the loss of important genetic variation that might ultimately affect the species’ ability to recolonize breeding sites, adapt to changing conditions, and possibly even to speciate15. Consequently, an understanding of the physical scales at which genetic differentiation occurs among populations is critical to the management of seabirds. This includes knowledge of their biology, ecology, and population trends including historical bottlenecks to identify population-specific management priorities.Understanding population genetic differentiation is particularly important in the case of rare, threatened, and highly localized seabirds16 assessed the population trends for the world’s seabirds and noted that as little as 19% of the global seabird population had been monitored more than five times between 1950 and 2010. Moreover, significant knowledge gaps exist with most colonies, and species in the tropics having been almost completely neglected. Among the most notable of the neglected species are the tropical petrels of the genera Pseudobulweria and Pterodroma (Procellariidae11). Most of these species breed on one or a few isolated islands and almost all remain little-known despite their poor conservation status. In this regard, Réunion Island (western Indian Ocean) is unique among tropical islands in that it supports two endemic species of petrel, the Mascarene (Pseudobulweria aterrima) and Barau’s (Pterodroma baraui) Petrels17. Both have unfavorable conservation status, suffering from threats imposed after the island was first colonized by humans in 166518. These include light-induced mortality of fledglings and the predation of adults, eggs and chicks by invasive cats and rats17. The introduction and subsequent effects of these threats have increased dramatically following the rapid expansion of human activities during the last few decades. The Mascarene Petrel remains little-known, but over the last decade, considerable scientific attention has focused on the biology and conservation needs of the Barau’s Petrel. Despite this, a critical knowledge gap concerns contemporary and historic genetic diversity, population size, and population structure.Within the past few decades, seabirds have become exceptionally well-studied and knowledge of their overall conservations status and populations trends is considered more comprehensive than for any other group of marine organisms. However, Ref.To explore the links between intrinsic barriers to dispersal and genetic population structure, we employed diversity and structure analyses based on polymorphic microsatellites to investigate the genetic relationships between two proximate breeding colonies of the Barau’s Petrel separated by roughly 5 km on Réunion Island  indicating a high proportion of rare alleles  within each of the two colonies . Most estimates of genetic diversity were higher for Piton des Neiges . Finally, the overall dataset showed no deviations from Hardy–Weinberg Equilibrium at the 99% confidence level . Thus, the proportion of rare and private alleles in both populations was notable and suggestive of fine-scale genetic drift.Successful microsatellite amplification was achieved across 14 loci in more than 94% of the sampled individuals. The fifteenth locus, PB_1030, was excluded due to a high number of false amplifications (see Primer Screening). Concerning indicators of genetic diversity across the 14 successfully amplified loci—the mean allelic diversity was roughly six alleles per locus. Both colonies contained private alleles amounting to 5% and 11% of total allelic diversity, carried by 7 and 25 individuals for the Grand Benare and Piton des Neiges colonies, respectively  indicated weak though statistically significant genetic structure between the two breeding colonies. This was supported by two similar estimates of genetic differentiation: G′ST  and RST , with 8 of the 14 loci showing p values < 0.05. The comparison between FST and RST was statistically non-significant , indicating no major contribution of stepwise mutations to the observed genetic difference but rather genetic drift.The FUnexpectedly, following multiple approaches Fig. , the besThe estimates of contemporary Ne are consistent with our evaluations of genetic diversity in the two colonies. Ne for the Piton des Neiges colony was estimated at 1148 [95% CI 434–infinite] and 1376 [95% CI 527–infinite] individuals at the cut-offs of 0.05 and 0.02, respectively. These were more than double the approximations for the Grand Benare colony where Ne was estimated as 514 (95% CI 269–2237) and 664 (95% CI 343–3541) individuals at the cut-offs of 0.05 and 0.02, respectively.The classic Bottleneck analysis based on the IAM, TPM and SMM models proved inconclusive in determining recent changes in Ne for the two breeding colonies or for the overall sampled population Table . Neverthp value = 0.03). The significant p value linked to the very low R-value indicated that any difference between the pair of breeding colonies was the result of only a very small proportion of the measurements. The overlap among birds from the two breeding colonies is displayed in Fig. Morphometric measurements for adult Barau’s Petrels sampled in each of the two breeding colonies are presented in Table The long-term banding information is consistent with our estimates of genetic differentiation, indicating extremely high colony fidelity among 2842 individuals banded as both adults (1984) and pre-fledglings (858). More precisely, among 2690 re-sightings of banded birds, all were recovered from the same colony in which they were first banded. This is indicative of extremely high natal philopatry (no birds banded as fledglings in a given colony have been re-sighted as either prospecting or breeding adults at the other colony) and breeding site-fidelity (no birds banded as a breeding adults in a given colony have been re-sighted as a breeding or prospecting adult at the other colony).19, has drawn attention to the scale of population genetic differentiation, the mechanisms driving differentiation, and the implications these factors have for seabird conservation4. The scale of population genetic structure varies extensively among studied seabird species, down to extremely small spatial and temporal distances22, although the scale to which we observed population structure in the Barau’s Petrel is finer and more robust than has previously been recognized or explored , each genetic population contains a significant proportion of the species’ overall breeding population. Moreover, though sample size limited the accuracy of our estimates of Ne, the approximations for both breeding colonies are of a similar order of magnitude to the surveyed estimates of these populations: 800–850 pairs at Piton des Neiges, and 300–400 pairs at Grand Benare .The ‘seabird paradox,’ or the contradiction between the high vagility of seabirds and their apparent reluctance to disperse among breeding sites4. Characteristics such as biometrics and coloration are often of limited use given their similarity among different populations of seabirds25 and, in the case of the Barau’s Petrels, birds from the two breeding colonies are phenotypically indistinguishable. In addition—birds from our two colonies do not differ in either their breeding phenologies or coloration26, though each of these factors has been used to explain genetic population divergence in at least a few other species28. It has been hypothesized that non-visual reproductive signals including olfactory and acoustic cues may serve as better indicators of reproductive isolation in largely nocturnal seabirds1, although these are logistically and technically difficult to explore under field conditions and remain untested for most species29. Behaviorally, Barau’s Petrels are unique among small to medium sized petrels in that they arrive back at their colonies during daylight hours17. This is believed to be an energy-saving tactic, with birds using thermals to gain in altitude. However, recent radar surveys and thermal camera observations have shown that a large proportion of birds arrive at the colonies after sunset (authors’ unpublished data), and the peak activity around the colonies happens after dark as in other related species. Thus, daytime arrival at colonies is unlikely to play a role in cross-colony interactions and colony partitioning. Moreover, several molecular studies have suggested that contemporary gene flow may not immediately override the influence of historical population genetic structure3. In the case of the Barau’s Petrel, however, there is no evidence of historical range contractions or recent bottlenecks and the major threats the species faces  are all relatively recent and linked to man’s colonization and expansion on Réunion Island18. This implies that the main threats to Barau’s Petrel populations may not yet have affected genetic diversity in this long-lived seabird. Nevertheless, the influence of small population size on genetic diversity cannot be neglected and, although our results highlight good genetic diversity with no evidence of inbreeding, both colonies contain rare and private alleles which are likely to disappear if these threats are maintained.In terms of the mechanisms of genetic population differentiation, our findings are clearly linked to the species’ extremely high philopatric tendencies, the apparent avoidance of cross-breeding between colonies and the absence of any other obvious barriers to genetic connectivity. A suite of individual, social, ecological, and historical factors are frequently invoked to explain patterns of genetic population differentiation in seabirds3. Given sufficient time, philopatry has the potential to restrict gene flow sufficiently to lead to reproductive isolation5, and has contributed to total speciation among populations of seabirds in a handful of examples31. However, philopatry alone may be insufficient to result in complete reproductive isolation as it usually acts in combination with other barriers to gene flow. In the case of the Barau’s Petrel, the genetic implications of the species’ philopatric tendencies appear to be exacerbated by high mate fidelity32. Barau’s Petrels nest in high altitude elfin forest above 2400 m a.s.l. on the two isolated peaks of Réunion Island23. The locations of the colonies are determined primarily by habitat conditions, particularly where soil conditions are suitable for burrow construction. The colony on Piton des Neiges is the largest known colony, situated in an area of optimal habitat conditions around 2400 m a.s.l., whereas the habitat on Grand Benare ranges from optimal to sub-optimal between the altitudes of 2600 m a.s.l. and 2800 m a.s.l. Thus, local environmental factors may additionally inhibit effective dispersal between the two breeding colonies, through the isolation of the nesting habitats and the habitat selectivity of the birds themselves. Importantly, however, it is not yet known whether Barau’s Petrels will engage in subadult prospecting at non-natal colonies though the limited number of band recoveries of fledglings at colonies has yet to detect such behavior and colony exchanges of breeding adults appears to be genetically negligible. Thus, although philopatry may not be universal among all seabird lineages33, the extremely fine-scale at which it obviously contributes to genetic population divergence in the Barau’s Petrel further emphasizes its role as an intrinsic barrier in the evolution of seabird diversity and endemism.Some seabirds exhibit population genetic structure in the absence of recognizable barriers to gene flow, suggesting that selective or social processes including philopatry can limit gene flow. Indeed, philopatry has been recognized as the second most obvious potential barrier to gene flow in seabirds after the physical isolation of breeding populations11. Their highly adapted biology reduces the capacity of populations to absorb additive mortality, particularly of adults, and slows their potential for recovery11. Moreover, the breeding distribution of many species is restricted to a limited number of sites, often on single islands or archipelagos, where populations can rapidly diverge25. The loss of genetic diversity within sub-divided populations can therefore have lasting impacts on the ability of the species to adapt, and possibly also to speciate. In practice, this means that seabird populations should be maintained across all conservation units to retain genetic variation at the species level and to protect the potential for future speciation. In addition to the genetic implications, the philopatric tendencies of most seabirds further imply that, without intervention , a species would be slow to recover or repopulate areas following local extirpation34. This reinforces the fact that modern molecular approaches and population-level studies provide an invaluable perspective from which to view and delimit the appropriate units for management, offering powerful approaches for the assessment of the effects of disturbance on populations to the guidance of long-term recovery programs.Our results highlight that genetic studies below the taxonomic level of species are critical to the practice of applied conservation. Seabirds are disproportionately represented among the birds that are most at risk of extinction35. A total of 259 adult birds were captured at their nests in the Grand Benare (n = 117) and Piton des Neiges (n = 142) colonies. A blood sample of approximately 0.2 ml was collected from each bird through medial metatarsal venipuncture and stored in 70% ethanol for further processing. All procedures were performed in accordance with relevant guidelines and regulations. We also received ethical approval from the Animal Ethics Committee (ZOOL-01-2013) at Rhodes University (South Africa), the Université de La Réunion (Réunion Island), Parc National de La Réunion, the Ethical Committee of Réunion Island, and the Centre de Recherche sur la Biologie des Populations d’Oiseaux . DNA was subsequently extracted from whole blood subsamples using the DNeasy Blood and Tissue extraction Kit (Qiagen).This study was carried out in compliance with the ARRIVE guidelines. Fieldwork was performed between the austral summers of 2008/2009 and 2012/2013 at two large breeding colonies of the Barau’s Petrel on Réunion Island, Indian Ocean  for the universal M13 forward primer enabling fragment analysis multiplexing37. PCR product sizes were determined using a 3730XL DNA analyzer (Applied Biosystems) by Gentyane platform  and were estimated with the LIZ(500) standard using GeneMapper version 4.0 (Applied Biosystems).A classic 3-primer Polymerase Chain Reaction (PCR) approach was used to amplify DNA at fifteen microsatellite loci following ref.38. The only examples of stuttering were at loci PB_2742 and PB_3916, indicated by the highly significant shortage of heterozygote genotypes with alleles of one repeat unit difference. Null allele frequency was low and within acceptable limits, though null alleles were present for loci PB_1890, PB_2742, and PB_4708 in both colonies, and PB_3916 only in the Grand Benare colony. All loci for which null alleles were detected were associated with homozygote excesses. Each sample-locus combination was tested for linkage disequilibrium using GenePop 4.0.1039, employing the exact probability test , and with False Discovery Rate correction40. Presence/absence of linkage disequilibrium was confirmed with the method of Index of Association41, in the R package poppr42. No linkage disequilibrium was observed among any of the loci  using either approach.Successful microsatellite amplification was achieved across the 15 loci in more than 55% of all samples from the two breeding colonies of the Barau’s Petrel. Around 3.09% of the global dataset consisted of null values. Locus PB_1030 contained an extremely high percentage of missing data with 31.62% and 47.18% missing data for Grand Benare and Piton des Neiges, respectively. Removal of this locus for all further analyses reduced the proportion of missing data to within acceptable limits (a maximum of 2.56% at any one locus in either colony). The resulting genotype data were checked for amplification errors and the presence of null alleles using MicroChecker 2.2.3A), the average number of effective alleles (AE), private allele richness (PA), and the observed (HO) and unbiased expected (HE) heterozygosities according to Ref.43 using GenALEx 6.544. The statistical significance of differences between NA and AE were assessed using non-parametric Wilcoxon Signed Rank tests implemented in PAST 3.1.545. Allelic richness , adjusted for discrepancies in sample size using rarefaction, was additionally calculated for each breeding colony using FSTAT 2.9.347, and compared between the colonies using 10,000 permutations. Deviations from the Hardy–Weinberg Equilibrium (HWE) were assessed with the R package pegas48, using the exact test based on 1000 Monte Carlo permutations and a χ2 test for global HWE. Finally, we estimated Wright’s inbreeding coefficient (FIS50) according to Ref.51 using GenAlEx version 6.544.Estimators of genetic variability were calculated across the 14 successfully amplified loci including the allele frequencies at every locus, the average number of alleles per locus (NFST49), and its associated p value, were computed over all 14 loci to assess genetic differentiation following ref.45, using the R package adegenet52. The statistical significance of differences between the pair of breeding colonies in this index, was tested using 10,000 random permutations. Mutation processes occur at relatively high rates and with stepwise changes in allele sizes at microsatellite loci, which introduces bias into classical measures of population differentiation such as FST53. Thus, to corroborate these results, we calculated two additional measures of genetic differentiation: RST using SPAGeDi53, and G′ST using GenoDive 3.0454. RST uses a stepwise mutation model to assess variances in allele sizes rather than allele frequencies (as in FST) and better reflects population differentiation among microsatellite loci in instances when stuttering is observed55. G′ST is equivalent to FST but with different statistical properties56, and with correction for bias stemming from sampling a limited number of populations57. The simple test of permutated RST (pRST), using 10,000 permutations of the genotypes, was additionally used to determine whether FST equalled RST and to assess whether stepwise-like mutations or genetic drift contributed to genetic differentiation53.Wright’s overall multi-locus fixation index  to assess convergence. The Dirichlet parameter (ɑ) was used to determine whether the standard admixture model was appropriate following Refs.59. Similarly, the r-index was used to determine the informativeness of the sampling location (LOCPRIOR), with low values of r indicating that sampling locations are informative to the overall model60. An admixture model (ɑ = 3.26 ± 0.5759), assuming sampling location , was most appropriate and provided the best levels of convergence. Correlated allele frequencies were assumed60. The number of genetic clusters (termed K) was subsequently determined following three criteria: (1) the log likelihood given K (lnP[X|K]61), (2) the second-order rate of change of mean log-likelihood (ΔK62) and (3) the median value of L(K). The first two were calculated using STRUCTURE HARVESTER online Web server63, while the third was calculated using CLUMPAK64. Ten independent simulations, at the optimal value of K, were then run using 1,000,000 iterations each . Finally, CLUMPAK64 was used to find the optimal individual alignments of replicated cluster analyses and to plot the estimates of individual membership for all genetic clusters.The multilocus genotype data were additionally used to perform a Bayesian clustering analysis implemented in STRUCTURE 2.3.465). This approach does not make any assumptions about HWE or linkage disequilibrium but is sensitive to fine genetic differences among populations. We used K-means clustering of principal components for K = 1 to K = 5 and Bayesian Information Criteria (BICs) to assess the optimal number of genetic clusters. The value of K with the lowest BIC value was considered optimal66. DAPC was applied using the Adegenet package 2.1.1 in R (52).Lastly, population structure was explored using Discriminant Analysis of Principal Components (DAPCNe) based on the 14 microsatellite markers were calculated for the two Barau’s Petrel breeding colonies using the molecular co-ancestry method of Ref.67, as implemented in NeEstimator 2.068. Ne was calculated assuming a monogamous mating pattern32. Two allele frequencies were adopted as conservative cut-offs to minimize the effect of the presence of rare alleles68: 0.02 and 0.05. Estimates of 95% confidence interval (CI) were calculated by jackknifing over loci for each estimate.Contemporary estimates of effective population size , two-phase  based on 10,000 replications and the Wilcoxon sign-rank test of significance (two tailed for heterozygosity excess or deficiency). These results were confirmed with the L-shaped method of allele mutation-drift equilibrium71, the M-ratio calculated (mean ± S.E.) across all 14 loci using the R package StrataG 2.4.90573, and the approximate likelihood MCMC approach using modelled Theta values  using the VarEff 1.2 R package74.Finally, we also used Bottleneck 1.2.0275 in R.Annual monitoring at the Grand Benare and Piton des Neiges breeding colonies began over the austral summers of 2007/2008 and 2002/2003, respectively. Subsequently, the two colonies have been visited at least twice annually up to the present for banding and control of breeding adults (November/December) and for banding of pre-fledging chicks (March). All re-sightings to the present day , were collated to quantify philopatry and levels of individual exchange between colonies. Re-sightings of birds banded at two age groups , within and between each breeding colony, were determined using dplyr 1.0.245. Barau’s Petrels are morphologically indistinguishable between the sexes32, and thus all individuals were pooled for the purposes of this analysis so as not to limit our ability to detect differences between the two colonies. Morphometric measurements were taken in the field with 1 mm precision and included the following: wing chord (AP), culmen length (LC), bill depth at the maximum gonydeal expansion (HC), maxillary unguis length (CR), and tarsus lengths (TA).Morphometric measurements of adult Barau’s Petrels were taken from a sample of individuals and compared between the two breeding colonies using One-way Analysis of Similarities (ANOSIM) based on an Euclidean distance measure and 9999 permutations in PAST 3.1.5Supplementary information.