Acanthaster solaris. In combination, these disturbances cause coral mortality that reduce the capacity of reefs to produce enough carbonate to keep up with sea-level rise. This study compared the reef-building capacity of shallow-water inner, patch, and outer reefs in the two islands of Pohnpei and Kosrae, Federated States of Micronesia. We identified which reefs were likely to keep up with sea-level rise under different climate-change scenarios, and estimated whether there were differences across habitats in the threshold of percentage coral cover at which net carbonate production becomes negative. We also quantified the influence of A. solaris on carbonate production. Whereas the northwestern outer reefs of Pohnpei and Kosrae had the highest net rates of carbonate production , the southeastern outer reefs had the lowest rates of carbonate production . The patch reefs of Pohnpei had on average higher net carbonate production rates (9.5 kg CaCO3 m-2 yr-1) than the inner reefs of both Pohnpei and Kosrae . A. solaris were common on Kosrae and caused an average reduction in carbonate production of 0.6 kg CaCO3 m-2 yr-1 on Kosraean reefs. Northern outer reefs are the most likely habitats to keep up with sea-level rise in both Pohnpei and Kosrae. Overall, the inner reefs of Pohnpei and Kosrae need ~ 5.5% more coral cover to generate the same amount of carbonate as outer reefs. Therefore, inner reefs need special protection from land-use change and local pollution to keep pace with sea-level rise under all climate-change scenarios.Coral reefs are essential to millions of island inhabitants. Yet, coral reefs are threatened by thermal anomalies associated with climate change and by local disturbances that include land-use change, pollution, and the coral-eating sea star  Coral reefs are an integral component of global marine ecosystems and are essential to millions of people that benefit from the goods and services that coral reefs provide. For example, coral reefs reduce storm-wave energy by up to 97% , reducinin situ estimates  shoAcanthaster solaris has long been known to reduce coral populations when the sea stars are in high densities  images which may be copyrighted. All PLOS content is published under the Creative Commons Attribution License (CC BY 4.0), which means that the manuscript, images, and Supporting Information files will be freely available online, and any third party is permitted to access, download, copy, distribute, and use these materials in any way, even commercially, with proper attribution. 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Please do not edit.]Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: PartlyReviewer #2: Yes**********2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: YesReviewer #2: Yes**********3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: YesReviewer #2: Yes**********4. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: Yes**********5. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: This is an overall well-written and well-executed study documenting the net carbonate production budgets for islands in Micronesia. Most significantly, the authors have pioneered spatial kriging within an excellent random, stratified plot design to determine net carbonate budgets around the perimeters of the entire islands. I believe this aspect of the study is an exceptional contribution to the field and applaud the authors for this contribution in using these methods to determine island scale carbonate production budgets. However, I have some concerns with how sedimentation and Acanthaster erosion were quantified and the language surrounding the capacity for reef accretion to keep up with sea level rise. I think this is nonetheless and important study and contribution to the field and hope the authors find the below comments helpful for improving the submitted manuscript prior to publication.Sedimentation##################################################################Lines 115–117/132–138: If sedimentation is high, how does that generate a negative term for the carbonate production equation? There’s more carbonate being dumped on the site so this should be positive… I see the clarification for this based on the influences of terrestrial sedimentation. However, if solid particles are settling on a reef then it’s adding material that will increase accretion. I agree that sedimentation could reduce calcification rates by some factor and organics could increase CaCO3 dissolution rates; however, I disagree with utilizing a baseline –0.4 kg CaCO3/m2/y as an appropriate adjustment for such affects considering that there is a material flux onto the reef. I think the sedimentation section should more accurately reflect what was actually measured in this study and the authors are welcome speculate that sedimentation decreased calcification rates nearshore if they wish to include such a conclusion in the manuscript but I do not think they should directly quantify the –0.4 kg CaCO3/m2/y in their budgets owing to sedimentation being present.Acanthaster####################################################################Lines 189-217: I appreciate the author's efforts to include Acanthaster erorion in this study however this section neglects quite a bit of the literature on the effects of Acanthaster on coral reef growth and should therefore be restructured. For example, I see no evidence in the present manuscript that Acanthaster actually consumes the coral calcium carbonate. The authors should note that Eakin (1996) found that Acanthaster digests tissue without destroying skeletons and Glynn (1973) previously assessed densities of Acanthaster that would overwhelm the ability for corals to persist. Lastly, Henderson and Walbran (1992) found that Acanthaster CaCO3 skeletal fragments were preserved in reef sediments and therefore added to reef CaCO3 production. I recognize that there is an influence of reducing coral cover owing to consumption of corals by Acanthaster that would drive decreases in CaCO3 production indirectly through the reduction in corals sensu Glynn (1973); however, the equations presented in this section make it seem as though Acanthaster is actually consuming CaCO3 in an erosional sense- a finding that the aforementioned papers would contradict and the present study does not provide sufficient quantifiable evidence otherwise. If the goal is to estimate the loss in CaCO3 production owing to Acanthaster- why not estimate a mean loss rate of corals per annual as per your equations and apply this loss in coral cover to the coral carbonate production equation to estimate the lost capacity for producing calcium carbonate by corals? However, if this is the study design then there should be some consideration of coral recruitment and whether or not the coral cover is in a quasi-steady state through time.Glynn, P. W. (1973). Acanthaster: effect on coral reef growth in Panama. Science, 180(4085), 504-506.Eakin, C. M. (1996). Where have all the carbonates gone? A model comparison of calcium carbonate budgets before and after the 1982–1983 El Nino at Uva Island in the eastern Pacific. Coral Reefs, 15(2), 109-119.Henderson, R. A., & Walbran, P. D. (1992). Interpretation of the fossil record of Acanthaster planci from the Great Barrier Reef: a reply to criticism. Coral Reefs, 11(2), 95-101.Accretion######################################################################Line 58 (and throughout): Accretion generally refers to vertical accretion (mm/y) whereas net carbonate production generally refers to (kg CaCO3/m^2/y). Because Perry et al (2013) calculates both accretion and net carbonate production rates, I think “accretion” should be switched to “net carbonate production” to be more consistent with the numbers from Perry et al (2013). It would be good to be consistent throughout the manuscript with respect to the use of accretion vs. net carbonate production terms.Line 219: What is the value for alpha in this study? I visited the previous van Woesik and Cacciapaglia (2018) publication and the alpha term derived therein referred me to the supplementary data of that manuscript where it’s a single value of -0.01949. Is this the value used in the present study? If so please just put this value in the text in place of requiring a reader to jump through so many hoops to determine a single number. Additionally, how does this calculation compare to the previously used values derived by Kinsey (1985) assuming a density of 2.9 g cm^-3 for calcium carbonate and average porosity of 50% and leveraged by Perry et al (2018) where they used 30-70% porosity with 50% reincorporation of parrotfish derived sediment back into the reef framework?Kinsey (1985) Metabolism, calcification and carbon production. Proceedings of the fifth international coral reef congress, Tahiti, Vol 4.Perry, C. T., Alvarez-Filip, L., Graham, N. A., Mumby, P. J., Wilson, S. K., Kench, P. S., ... & Januchowski-Hartley, F. (2018). Loss of coral reef growth capacity to track future increases in sea level. Nature, 558(7710), 396.Lines 331-345: Perhaps I am confused about how the authors dealt with sedimentation in this study, but if the net CaCO3 sediment transport to the inner reefs (and microbioerosion/CaCO3 dissolution) was not considered than in my opinion these statements are likely too strongly stated. I caution making strong statements on such matters when the accretion rates of the inner reefs weren’t directly quantified in this study  and the budgets presented here do not appear to fully cover all of the terms necessary to quantify net carbonate accumulation . Since accretion is estimated from the budgets and the relationship between net carbonate production budgets and accretion rate was based on just a few points, there is quite a bit of uncertainty in this value that should temper the conclusions drawn from it especially considering that such statements potentially impact human population centers associated with these reefs in Micronesia and set the precedent for the same standard elsewhere.Perry, C. T., Kench, P. S., Smithers, S. G., Riegl, B. R., Gulliver, P., & Daniells, J. J. (2017). Terrigenous sediment-dominated reef platform infilling: an unexpected precursor to reef island formation and a test of the reef platform size–island age model in the Pacific. Coral Reefs, 36(3), 1013-1021.Yates, K. K., Zawada, D. G., Smiley, N. A., & Tiling-Range, G. (2017). Divergence of seafloor elevation and sea level rise in coral reef ecosystems. Biogeosciences, 14(6), 1739.Perry, C. T., Morgan, K. M., & Yarlett, R. T. (2017). Reef habitat type and spatial extent as interacting controls on platform-scale carbonate budgets. Frontiers in Marine Science, 4, 185.Kleypas, J. A., Buddemeier, R. W., & Gattuso, J. P. (2001). The future of coral reefs in an age of global change. International Journal of Earth Sciences, 90(2), 426-437.Eyre, B. D., Andersson, A. J., & Cyronak, T. (2014). Benthic coral reef calcium carbonate dissolution in an acidifying ocean. Nature Climate Change, 4(11), 969.Coral bleaching and reef-scale calcification##########################################Line 375: There is also a growing literature on coral reef net ecosystem calcification and coral bleaching that provide critical information to the dynamics of carbonate production and thermal stress events and could be cited here. Please see the following papers:Kayanne, H., Hata, H., Kudo, S., Yamano, H., Watanabe, A., Ikeda, Y., ... & Saito, H. (2005). Seasonal and bleaching‐induced changes in coral reef metabolism and CO2 flux. Global Biogeochemical Cycles, 19(3).Watanabe, A., Kayanne, H., Hata, H., Kudo, S., Nozaki, K., Kato, K., ... & Yamano, H. (2006). Analysis of the seawater CO2 system in the barrier reef‐lagoon system of Palau using total alkalinity‐dissolved inorganic carbon diagrams. Limnology and Oceanography, 51(4), 1614-1628.DeCarlo, T. M., Cohen, A. L., Wong, G. T., Shiah, F. K., Lentz, S. J., Davis, K. A., ... & Lohmann, P. (2017). Community production modulates coral reef pH and the sensitivity of ecosystem calcification to ocean acidification. Journal of Geophysical Research: Oceans, 122(1), 745-761.Courtney, T. A., De Carlo, E. H., Page, H. N., Bahr, K. D., Barro, A., Howins, N., ... & Andersson, A. J. (2018). Recovery of reef‐scale calcification following a bleaching event in Kāne'ohe Bay, Hawai'i. Limnology and Oceanography Letters, 3(1), 1-9.McMahon, A., Santos, I. R., Schulz, K. G., Scott, A., Silverman, J., Davis, K. L., & Maher, D. T. (2019). Coral reef calcification and production after the 2016 bleaching event at Lizard Island, Great Barrier Reef. Journal of Geophysical Research: Oceans.Minor comments################################################################Lines 88–95: excellent site selection design!!!Lines 129-130: Isn’t the gross carbonate production in g/cm^2/y and the resulting units after conversion kg/m^2/y?Please maintain consistency with units as “y” and “yr” are used interchangeably throughout the manuscriptThe erosion section and equations therein are quite similar to Perry et al. (2012) and should be cited as such where appropriate.Line 199: Shouldn’t the kg/m^2 be per year?Line 256: Couldn’t this just in part be driven by the somewhat arbitrarily assigned -0.4 kg/m2/y rate applied to the high sedimentation areas? Alternatively, how did the authors account for the very large expanses of sand that typically characterize inshore lagoonal environments?Line 311: add space to “reefs,and”Line 362-364: This is an excellent point and is a feature of geological perspectives of coral reefs. For example, please see Engels (2004) and Chapter 6 Montaggioni and Braithwaite (2009) for potential citations for this.Engels, M. S., Fletcher III, C. H., Field, M. E., Storlazzi, C. D., Grossman, E. E., Rooney, J. J., ... & Glenn, C. (2004). Holocene reef accretion: southwest Molokai, Hawaii, USA. Journal of Sedimentary Research, 74(2), 255-269.Montaggioni, L. F., & Braithwaite, C. J. (2009). Quaternary coral reef systems: history, development processes and controlling factors (Vol. 5). Elsevier.Reviewer #2: This manuscript provides carbonate budgets for two coral reefs in Micronesia, a region that is likely to be highly impacted by future sea-level rise. The manuscript is well-written, the methods are sound, and the results provide much needed data from an understudied region. Most of my comments are relatively minor, but I do have a couple of more substantial points, that I would like to see the authors address before publication. Both are outlined in the document attached.**********what does this mean?). If published, this will include your full peer review and any attached files.6. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool, AttachmentReview of van Woesik and Cacciapaglia.pdfSubmitted filename: Click here for additional data file. 13 Sep 2019NAAttachmentReviewer response letter RvW & CC Sept 9 2019 Final.docxSubmitted filename: Click here for additional data file. 24 Oct 2019Carbonate production of Micronesian reefs suppressed by thermal anomalies and Acanthaster as sea-level risesPONE-D-19-15515R1Dear Dr. van Woesik,We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at onepress@plos.org.If your institution or institutions have a press office, please notify them about your upcoming paper to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact With kind regards,James R. Guest, Ph.D.Academic EditorPLOS ONEAdditional Editor Comments :I apologize for the delay in getting a final response to you on your revised manuscript. I have been through your responses to the reviewers initial comments and am completely satisfied that you have addressed all of them satisfactorily. I have no hesitation in recommending publication of the revised draft.Reviewers' comments:No additional comments 5 Nov 2019PONE-D-19-15515R1 Acanthaster as sea-level rises Carbonate production of Micronesian reefs suppressed by thermal anomalies and Dear Dr. van Woesik:I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. onepress@plos.org.If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact plosone@plos.org. For any other questions or concerns, please email Thank you for submitting your work to PLOS ONE.With kind regards,PLOS ONE Editorial Office Staffon behalf ofDr. James R. Guest Academic EditorPLOS ONE


Anopheles arabiensis and Anopheles funestus, and their physiological ages (approximated by parity and insemination states).Effective malaria surveillance requires detailed assessments of mosquitoes biting indoors, where interventions such as insecticide-treated nets work best, and outdoors, where other interventions may be required. Such assessments often involve volunteers exposing their legs to attract mosquitoes , a procedure with significant safety and ethical concerns. Here, an exposure-free, miniaturized, double-net trap (DN-Mini) is used to assess relationships between indoor–outdoor biting preferences of malaria vectors, The DN-Mini is made of UV-resistant netting on a wooden frame and PVC base. At 100 cm × 60 cm × 180 cm, it fits indoors and outdoors. It has a protective inner chamber where a volunteer sits and collects host-seeking mosquitoes entrapped in an outer chamber. Experiments were conducted in eight Tanzanian villages using DN-Mini to: (a) estimate nightly biting and hourly biting proportions of mosquitoes indoors and outdoors; (b) compare these proportions to previous estimates by HLC in same villages; and, (c) compare distribution of parous  and inseminated mosquitoes indoors and outdoors.An. arabiensis were caught outdoors as indoors (p < 0.001), while An. funestus catches were marginally higher indoors than outdoors (p = 0.201). Anopheles arabiensis caught outdoors also had higher parity and insemination proportions than those indoors (p < 0.001), while An. funestus indoors had higher parity and insemination than those outdoors (p = 0.04). Observations of indoor-biting and outdoor-biting proportions, hourly biting patterns and overall species diversities as measured by DN-Mini, matched previous HLC estimates.More than twice as many An. arabiensis more likely bite outdoors than indoors, while potentially infectious An. funestus more likely bite indoors. These observations validate previous evidence that even outdoor-biting mosquitoes regularly enter houses when young. They also demonstrate efficacy of DN-Mini for measuring indoor–outdoor biting behaviours of mosquitoes, their hourly biting patterns and epidemiologically relevant parameters, e.g., parity and insemination status, without exposure to volunteers. The trap is easy-to-use, easy-to-manufacture and affordable (prototypes cost ~ 100 US$/unit).Malaria vectors that are behaviourally adapted to bite humans outdoors also have their older, potentially infectious sub-populations concentrated outdoors, while those adapted to bite indoors have their older sub-populations concentrated indoors. Here, potentially infectious  Malaria-related deaths around the world decreased from 655,000 in 2010 to 455,000 in 2017, but these successes are fragile and could be lost if ongoing efforts are not sustained , 4 and tTo accelerate malaria elimination efforts and sustain the gains made, these important aspects of persistent malaria transmission must be measured in adequate detail, and effective interventions deployed to complement ITNs and IRS. The World Health Organization (WHO) recently outlined key indicators that should be assessed to comprehensively understand the prevailing malaria transmission and its vectors in any given area . These iFor decades, human landing catches (HLC), where adult male volunteers expose their legs to collect blood-seeking mosquitoes, have been the standard approach for sampling host-seeking mosquitoes , 10, and2) to mimic natural host odours. A reliable, cost-effective and exposure-free technique that can be used both indoors and outdoors therefore remains elusive even as countries struggle with the atypical features of residual malaria transmission.In recent years, multiple new ways of conducting HLC safely have been evaluated. Examples include the human-baited double net (HDN), which was efficacious in outdoors settings in Southeast Asia, but is considered too large and bulky to use inside houses . Anotherversus inside houses, and infectiousness of the mosquitoes. Such entomological data, if overlaid with observed human activities can help determine important indicators such as: (a) proportions of actual malaria exposure indoors and outdoors, and, (b) proportions preventable by specific interventions. Observations of increasing outdoor-biting have raised concerns that existing indoor-interventions, particularly ITNs and IRS will not be adequate for malaria control, especially in areas where the dominant vectors mostly bite outside houses [Entomologists typically assess times of night when people are most bitten by different vector species, percentage of bites occurring outside e houses . Howevere houses . Field eAnopheles arabiensis and Anopheles funestus, in relation to their physiological ages (approximated by parity and insemination states), in rural Tanzanian villages where ITNs are already widely used but malaria transmission persists. To achieve this, a new miniaturized double net trap (DN-Mini), was designed and used for exposure-free assessment and characterization of proportions of mosquitoes biting indoors and outdoors. The DN-Mini is an adaptation of the original bed-net trap design [The primary objective of this study was therefore to assess indoor–outdoor biting preferences of two major malaria vectors, p design , which wp design . It was An. arabiensis and An. funestus, the latter mediating more than 80% of transmission [The study was done in Ulanga and Kilombero districts, south-eastern Tanzania, where ITNs are already widely used but lower-level malaria transmission persists Fig. . Mosquitsmission . Long-lasmission .Fig. 1MaThe miniaturized double net trap (DN-Mini) was designed to improve comparative mosquito sampling indoors and outdoors Fig. , and wasSince its development in 2016, the DN-Mini has been used to assess outdoor-biting and indoor-biting exposures to malaria vectors in and around houses fitted with spatial repellent ribbons in rural Tanzania . It has The aim of this experiment was to determine whether varying the height above ground of the outer netting of the DN-Mini Figs.  and 3, wEight houses were identified in four of the study villages: Lupiro, Kining’ina, Kivukoni and Minepa. In each village, two houses were selected that were at least 100 m apart in each village. At each house, one DN-Mini was located indoors and another trap located outdoors, and volunteers rotated nightly between positions Fig. . The traAnopheles funestus and An. arabiensis collected were dissected following procedures described by Detinova [The DN-Mini were deployed to sample mosquitoes indoors and outdoors in four of the villages, Minepa, Mbuyuni, Mavimba, and Lupiro. At each house a DN-Mini was placed indoors and another outdoors, and each of the traps occupied by adult male volunteers. The traps operated from 18:00 to 07:00, during which the volunteers retrieved mosquitoes from the DN-Mini using mouth aspirators every hour as described above. For each hourly collection, mosquitoes were packed in separate labelled cups, sorted and recorded. Detinova , to asseDetinova , and botAnopheles bites experienced by unprotected residents indoors versus outdoors. In this current study, the DN-Mini were set indoors and outdoors in four houses per village and data collected hourly to match the previous study by Ngowo et al. [Since Ifakara Health Institute currently discourages the use of HLC in field studies, DN-Mini observations were compared to the last available HLC data collected indoors and outdoors in the same villages . The stuo et al. . The indo et al. .An. gambiae s.l. and An. funestus s.l. complexes have different biting preferences, sub-samples of mosquitoes caught indoors and outdoors in the DN Mini traps were analysed by PCR to distinguish them, using DNA extracted from hind legs. For An. gambiae s.l., PCR amplification was done for species-specific nucleotide sequences of ribosomal DNA (rDNA) intergenic spacer regions (IGS) in a 25-µl reaction volume of PCR mixture as described by Scott et al. [An. funestus, methods developed by Koekemoer et al. were used [Since sibling species in the t et al. . For An.ere used . Amplifivs outdoors), the main indicator variable, was included as fixed factor. To account for variations between collection days and pseudo-replication, experimental day and household ID (nested within village) were included as random terms in the main model. Data for each mosquito species were analysed separately. Relative rates of mosquito catches, and associated 95% confidence intervals, were reported, and were considered significant when p-value was less than 0.05. All the graphs and plots were generated using a grammar for graphic package (ggplot2) [Data were analysed in open sources statistical software, R version 3.5.0 . Descripggplot2) . The indggplot2) .Culex spp. and Mansonia spp.), while 6.2% (n = 527) were Anopheles. Among the Anopheles, 80.6% (n = 425) were An. arabiensis, 16.3% (n = 86) were An. funestus s.l and the remaining 3.1% (n = 16) were a mixture of Anopheles coustani, Anopheles ziemanni and Anopheles pharoensis. More than twice as many An. arabiensis were caught outdoors as indoors  were culicines  were An. arabiensis. An. funestus were 5.98% (n = 765), and the remaining 1.97% (n = 252) consisted of An. coustani and An. pharoensis. Similar to DN-Mini, HLC caught more than twice the number of An. arabiensis outdoors compared to indoors  age distribution of the two main malaria vector species (based on parity status as proxy) closely followed the overall biting preferences for indoors and outdoors. Data from the DN-Mini experiment showed that there was a higher proportion of parous An. arabiensis, there was higher insemination outdoors than indoors , while for An. funestus, insemination was higher indoors than outdoors   Figs.  and 9.FiAn. arabiensis caught was slightly higher compared to when the outer layer was 20 cm from the ground. There was however no statistical significance in the observed differences between the two settings  and 12 of the outdoor specimens (2%) did not amplify in the PCR tests. For An. funestus s.l., a total of 107 specimens from the outdoor collections and 137 from outdoor collections were examined by PCR. In the indoor collections, 57% were An. funestus s.s., 29% were Anopheles rivulorum and 14% were unamplified, while in the outdoor collections, 56.2% were An. funestus s.s., 30.1% were An. rivulorum, 0.7% were Anopheles leesoni, and 12.4% were unamplified.The total number of Anopheles species that are behaviourally adapted to bite either indoors or outdoors.Achieving malaria elimination in sub-Saharan African requires new mosquito surveillance approaches that can effectively capture important entomological parameters, including the key behaviours and physiologies of the dominant vectors. Such an approach must be safe, highly scalable, low-cost, and readily applicable across endemic settings without necessarily requiring specialized skills. In residual transmission settings where outdoor-biting can be an important contributor to transmission, the surveillance tools should particularly enable characterization of these outdoor-biting sub-populations in relation to indoor-biting sub-populations. This way, one can examine the extent to which specific interventions differentially impact malaria transmission as mediated by individual An. arabiensis and An. funestus, and their physiological ages . Specifically, in this study area, i.e., in rural south-eastern Tanzania, where ITNs are already widely used but malaria transmission persists, the study determined that potentially infectious sub-populations of An. arabiensis more likely bite outdoors than indoors, while potentially infectious sub-populations of An. funestus more likely bite indoors. These differences were statistically clearer for An. arabiensis for which more than twice as many biting females were caught outdoors as indoors, than An. funestus, for which the indoor–outdoor differences were marginal, with slightly more biting females found indoors. This is the first field illustration of such relationships between physiological age and biting preferences in these mosquito species, and may suggest an even stronger behavioural adaptation than previously thought [An. arabiensis) also have their older and potentially infectious sub-populations concentrated outdoors. On the contrary, those vector species adapted to bite more indoors have their older sub-populations concentrated indoors. Anopheles arabiensis was the only member of An. gambiae complex caught. However, for An. funestus, the distribution of the two common sibling species, i.e., An. rivulorum and An. funestus s.s. was similar indoors and outdoors (i.e. 56–57% An. funestus s.s. and 29–30% An. rivulorum). The observations made are therefore applicable to both An. funestus s.s., which now mediates more than 80% of all malaria transmission in the study area [An. rivulorum, which has also previously been incriminated in malaria transmission in multiple locations in East Africa [This study has provided crucial field evidence on relationships between indoor–outdoor biting preferences of two major malaria vectors,  thought –37. The udy area , and An.t Africa , 38–40.An. arabiensis mosquitoes host-seeking indoors, while their parous and inseminated con-specifics were outdoors suggests that these mosquitoes do indeed regularly make attempts at indoor biting especially when they are still physiologically immature adults. As previously concluded by Killeen et al. [An. funestus as well as young immature An. arabiensis mosquitoes, thereby accelerating malaria elimination efforts.Another clear signal from these field observations is the validation of previous theoretical observations that even outdoor-biting malaria vectors regularly enter houses when young . The obsn et al.  and Kaman et al. , the finn et al.  and IRS An. arabiensis outdoors than indoors, and slightly higher numbers of An. funestus indoors than outdoors. These DN-Mini observations match the known biting preferences of these mosquito species in the study area [Other than the primary objective, this study has also provided evidence that the newly developed exposure-free DN-Mini can effectively sample representative proportions and diversities of host-seeking mosquitoes that bite indoors and outdoors. Although the absolute catches of the different mosquito species were much lower than HLC, the actual proportions and diversities of indoor–outdoor biting females, as well as their hourly biting trends, were consistent with and representative of those most recently obtained when using HLC in the same villages. In the tests to assess biting densities indoors and outdoors, the DN-Mini caught higher number of udy area , which iudy area . AlthougCulex species indoors than outdoors, but more Mansonia mosquitoes outdoors than indoors, which matches previous findings in the same area by HLC [Culex species being the most dominant, followed by An. arabiensis then An. funestus .Malaria vectors that are behaviourally adapted to bite humans outdoors also have their older, potentially infectious, sub-populations concentrated outdoors, while those adapted to bite indoors have their older sub-populations concentrated indoors. Specifically, potentially infectious


Lacerta viridis and Lacerta bilineata are sister species of European green lizards  that, until recently, were grouped together as the L. viridis complex. Genetic incompatibilities were observed between lacertid populations through crossing experiments, which led to the delineation of two separate species within the L. viridis complex. The population history of these sister species and processes driving divergence are unknown. We constructed the first high-quality de novo genome assemblies for both L. viridis and L. bilineata through Illumina and PacBio sequencing, with annotation support provided from transcriptome sequencing of several tissues. To estimate gene flow between the two species and identify factors involved in reproductive isolation, we studied their evolutionary history, identified genomic rearrangements, detected signatures of selection on non-coding RNA, and on protein-coding genes.L. bilineata to L. viridis after their split at least 1.15 million years ago. We detected positive selection of the non-coding repertoire; mutations in transcription factors; accumulation of divergence through inversions; selection on genes involved in neural development, reproduction, and behavior, as well as in ultraviolet-response, possibly driven by sexual selection, whose contribution to reproductive isolation between these lacertid species needs to be further evaluated.Here we show that gene flow was primarily unidirectional from The combination of short and long sequence reads resulted in one of the most complete lizard genome assemblies. The characterization of a diverse array of genomic features provided valuable insights into the demographic history of divergence among European green lizards, as well as key species differences, some of which are candidates that could have played a role in speciation. In addition, our study generated valuable genomic resources that can be used to address conservation-related issues in lacertids. Drosophila [Understanding what species are and the processes driving their emergence have been two central issues in biology . During osophila . These sosophila ; (ii) inosophila , 4; and osophila –7. Howevosophila  in naturin silico tools have been developed to detect structural variation with high precision using genomic data [The advent of high-throughput sequencing together with the development of novel approaches for whole genome analyses opened new research avenues to study the origin of species, including non-model organisms . It has mic data –13, thusmic data .Krüppel-type zinc fingers (KZNFs), a family of transcription factors, were pinpointed as strong candidates to play a role in the speciation of other vertebrates [The assessment of divergence in regulatory elements and transcription factors between species further adds to a more complete understanding of the link between genotypes and phenotypes. In this respect, transcriptome sequencing offers an unprecedented resolution to investigate the general importance of divergence in gene regulation in speciation. In particular, zinc-finger genes, especially tebrates . In additebrates –18.Nevertheless, our understanding of how speciation unfolds, as well as the mechanisms involved, will remain limited without the knowledge of the demographic history between diverging taxa . Model-bIn summary, the identification of differences in genomic features between closely related species and their demographic history can now be assessed in a cost-effective manner. The resulting information is likely to provide insights about the main candidates playing a role in diversification, upon which more specific hypotheses concerning the mechanisms of divergence can be tested.Anolis, in particular, have been studied in detail, as their distribution on islands coupled with repeated adaptive radiations offer a perfect framework for evolutionary ecology studies [Anolis carolinensis (anole lizard) with the genomes of birds and mammals was pivotal in identifying accelerated evolution of egg proteins associated with amniote evolution [Gekko japonicus (gecko lizard) contributed to the understanding of evolution and adaptation of tail regeneration, clinging, nocturnal vision, and diversification of the olfactory system [Pogona vitticeps (bearded dragon lizard) and Shinisaurus crocodilurus (Chinese crocodile lizard) have recently been characterized [Lizards provide an excellent model for studying speciation due to the existing knowledge of their long-term demographics and adaptive morphologies, in addition to the ease of sample collection and experimental manipulations . Lizards studies . Not sur studies . Comparavolution . Furthervolution –31. For y system . In addicterized , 27. Howcterized .Lacerta viridis complex shows an intricate evolutionary history with secondary contact zones [Lacerta bilineata (NCBI:txid95620) and the eastern clade of L. viridis (NCBI:txid65476)  that currently occupy disjointed regions in Europe [The family Lacertidae  has been well covered in terms of phylogeographic studies, providing important information about the likely timing and geographic context of speciation . Within ct zones , 34. HerL. viridis and L. bilineata [Adult individuals from the two taxa are very similar: throat coloration of hatchlings and early juveniles is the only described diagnostic trait so far . Gene flilineata .L. viridis complex  exhibit reduced fitness under laboratory conditions [L. viridis and L. bilineata can exist in the wild due to potential genomic Bateson-Dobzhansky-Muller incompatibilities (BDMIs). Previous models have suggested that after a secondary contact, BDMIs can be maintained and further accumulate within genome rearrangements [L. viridis complex [L. viridis complex comprises a very interesting system where different genomic components can be studied to elucidate the demographic history and possible processes involved in speciation.Hybrids between different main lineages within the nditions . This sungements , 40, thungements . High kangements , also wi complex , raising complex . Finally complex , making de novo genomes for both L. bilineata and L. viridis, with annotation support from transcriptomic data. We investigated the demographic history of divergence between the two lacertid taxa and performed a broad comparison of key genomic features providing important insights about their divergence that can be tested in future studies aiming to identify the mechanisms ultimately leading to speciation between this closely related species pair.Here, we combine short Illumina and long Pacific Biosciences (PacBio) read sequencing approaches to construct high-quality L. viridis and 37x Illumina and 11x PacBio for L. bilineata aided in the construction of high-quality genome assemblies  (L. viridis had better contiguity than L. bilineata (higher N50 and fewer contigs), L. viridis was used as the reference to predict genomic variants, structural variants (SVs) and single-nucleotide polymorphisms between the two taxa.We employed a hybrid strategy of combining Illumina and PacBio sequencing data to produce separate genome assemblies for the two lacertid species. Genome sequencing coverages of 34x Illumina and 14x PacBio for semblies . The geninensis) . The BenL. viridis genome consisted of a higher number of large segmental duplications (>5 kbp) compared to L. bilineata (P = 0.35 and Wilcoxon test: P = 0.55). Hence, the occurrence of fewer large segmental duplications in L. bilineata could be the result of its more fragmented genome assembly. Synteny information was used to create unordered contig clusters (minimum size of 1 Mbp covering one-third of the L. viridis genome), which roughly represent positioning on the same chromosome  [The ilineata . Howeverromosome . The mederation) , 47.A. carolinensis confirmed the high genomic assembly quality since the HOX-clusters are highly conserved  and lacertid lizards  [A. carolinensis  , 48. Howlinensis , even thab initio methods predicted more fragmented proteins and coding sequences (CDS)  when compared to the final gene models   aminoacylation  and tRNA metabolic process  in both lacertids, indicating an expansion of tRNA-processing genes before their split. Putative Z-chromosome linked contigs consisted of few non-coding elements  (P. vitticeps (8 Mbp), but the number of identified genes were similar  [The assembled transcripts were crucial for gene annotations since the –22,500) . A majornscripts . The larl tRNAs) . The totL. viridis and L. bilineata . dxy was around 0.0123, pairwise Fst between L. viridis and L. bilineata was 0.688.Across all sites, mean heterozygosity was slightly lower in We inferred past divergence and gene flow between the two lacertid species using a composite likelihood (CL) method based on the site frequency spectrum of short sequence blocks, i.e., blockwise site frequency spectrum (bSFS) , 52. SinL. viridis and L. bilineata; heterozygous sites shared by both lacertids; fixed differences) in each block. A total of 1,965 bSFS configurations appeared more than once in the data  scores of 13 different demographic scenarios ; Table 1L. bilineata to L. viridis and fits significantly better than simpler scenarios such as divergence without gene flow (or just a single Ne parameter) or admixture  compared to its ancestor and L. viridis  (L. bilineata to L. viridis (The best of the 13 models (M3.3) supports isolation between the two lacertid species with unidirectional gene flow from  95,400)  and a mi viridis .−8 (based on cyt-b gene) or 1 × 10−9 (based on the four-fold degenerate sites), our estimate of the split between L. viridis and L. bilineata corresponds to 1.15 Mya and 20.37 Mya, respectively (Assuming a generation time of 3.5 years and a mutation rate of 1.77 × 10ectively .L. viridis genome (2.7% of the genome). Compared to L. bilineata, 10.8 Mbp (0.7%) of the L. viridis genome was covered with large rearrangements affecting genes (covering the entire length of more than one gene). These regions were enriched for RNA-directed DNA polymerase activity .We detected 20,160 genomic rearrangements or SVs longer than 50 bp between the two lacertids Fig.  coveringInsertions-deletions (indels) are the most frequent genomic rearrangements mainly affecting introns, repeat elements, and pseudo-tRNAs . This isMicroRNAs (miRNA) were the most structurally conserved family of ncRNAs followed by small nucleolar RNAs (snoRNA) . The fouL. bilineata. The human ortholog of SNORD61 occurs in the intron of a RBMX , known to be involved in the dosage compensation and cohesion regulation of sister chromatids [L. viridis: MIR6516 (mir-6516-3p)  orthologs (LiNC66 and LiNC29) overlapping genomic regions conserved across tetrapods were structurally divergent between the two species, as indicated by high selection scores and stable secondary structures  , associa6516-3p) , known tructures .G. japonicus lost two of the five functional opsin paralogs compared to diurnal anoles [A. carolinensis (22 transcripts from ENSEMBL) were also present in L. viridis and L. bilineata (20 transcript sequences), indicating conservation of genes for diurnal vision. We observed high conservation of SWS1 (opsins related to UV vision), described to be involved in sexual selection [The visual opsins are pivotal for adaptation to diurnal habitats in Squamata , 59. Forl anoles . All fivelection , 61, andelection  . Genes with different selective constraints between the two species  were related to brain and neural development, embryo and cartilage development, along with behavioral responses .L. viridis, L. bilineata, or the ancestor of L. viridis and L. bilineata) are shown in L. bilineata (STAR7) is located on the Z-chromosome. We identified two transcription factor genes, UBIP1 and RPA2, involved in gene silencing and reproductive functions [L. bilineata, both TDRD3 and UGPA with PSS in L. viridis. GPR155 is involved in cognitive functions and expressed in mice forebrain [The test for positive selection in either of the two species was performed with the branch-site model of codeml (model M2) using a subset of other lizards as background branches. The number of genes with positively selected sites (PSS) in different foreground branches  involved in regeneration through prostaglandin synthesis is positively selected in aponicus . This geL. viridis branch .We identified three paralogs of the hyaluronidases  in both the lacertid genomes. Two genes (STIK1 and HYAL2) coding for proteins in the extra-cellular matrix of the skin reacting to UV-B light (GO:0071493)  were poss branch . ArylsulKrüppel-associated box (KRAB)-ZNFs in reproductive isolation of the two lacertid species, we compared the DNA-binding domains of KZNF orthologs. From the 53 KZNF orthologs, 6 C2H2 zinc-finger proteins showed binding-specific differences between the two lacertid species . Since this can be due to abundant indels obscuring the association in other categories of SVs, we tested the association between each SV category with PSGs separately, applying independent Boschloo exact-tests . We also observed a significant association of PSGs over genes with no signs of positive selection within inversions compared to both non-rearranged regions  and collinear regions . The inversions overlapping PSGs seem to reflect independent events, since the inversions are located on different contigs in the genome with size ranges between 70 kbp and 700 kbp.Deletions are the most frequent type of SVs in the genome and occurred on both positively selected genes and those with no signs of positive selection. Duplications and insertions only occurred in genes evolving without signs of positive selection, while deletions and inversions occurred in genes irrespective of their selective regime. The ratio between number of regions with rearrangements or SVs to those with no detected rearrangements was not significantly different between genes under positive selection and those with no signs of positive selection  followed by hybrid assembly through DBG2OLC implementing removal of chimeric joins. This hybrid assembly strategy aided in generating high-quality contig-level genomes with moderate genome coverages (∼35X Illumina and ∼15X PacBio). Our lacertid genome assemblies showed higher completeness than the available lizard genomes .L. viridis and L. bilineata was estimated as at least 1.15 Mya (per generation), whereas the previously estimated mitochondrial divergence time was 2.6–3.4 Mya [Lacerta viridis and L. bilineata show a high level of genome-wide differentiation (FST = 0.688). The best demographic model (M3.3) supported unidirectional gene flow from L. bilineata to L. viridis and higher effective population size for L. viridis than L. bilineata, consistent with the difference in genetic diversity between the two lacertid species.The time of population divergence between –3.4 Mya , 69. LacL. bilineata and structural changes in SNORD61, which is involved in dosage compensation in humans [Species-specific diversity within various ncRNA classes and adaptive differences in ncRNA orthologs capable of altering their secondary structures are two important factors contributing to evolutionary divergence, since varying ncRNA structures imply functional changes . Copy nun humans .L. viridis and L. bilineata with regard to reproductive processes, i.e., spermatogenesis, fertilization, and embryo implantation [Positive selection of sites in NASP and PDLIM1 in the lacertid ancestral branch compared to distant background branches, including mammals and birds, may indicate disparate evolutionary changes in the ancestor of antation –74. In cantation –77.L. viridis has also been linked to UV-response. Males with more UV-reflective patches on the skin are preferably selected by the females [L. viridis. We speculate that differential cutaneous response to UV through changes in the chondroitin sulfate biosynthesis pathway could be driven by mating preferences, which could ultimately contribute to speciation. Further studies are needed to test this hypothesis.UV-reflectance of plumages in birds is an important trait involved in the sexual selection of morphologically similar sibling species of Passeriformes . Sexual  females , 80. We  females , 81, 82,L. viridis and L. bilineata. The behavioral differences can be related to varying ecological habitats and environmental conditions [L. viridis and L. bilineata. Selective differences in genes related to behavior and brain development have been reported to be involved in the diversification of anoles [KRAB-ZNFs or KZNFs are transcriptional regulators confined to tetrapod vertebrates  and are nditions  after thf anoles .L. viridis and L. bilineata.Genomic regions harboring inversions are known to suppress recombination in heterokaryotypes facilitating speciation in the presence of gene flow  and in mWe assembled the first high-quality genomes of two closely related species of European green lizards with a cost-effective strategy. Genes related to transcriptional regulation, behavior, and neural and reproductive development have diversified the most between the two lacertid species. Species-specific diversity of ncRNAs, adaptive evolution in regulatory elements, and transcription factors (including binding domain differences in KZNFs) indicate variation in gene regulatory networks between the two species. Adaptive evolution of genes responsible for differential cutaneous response to UV exposure, in particular, could be driven by mate choice and ultimately contribute to reproductive isolation. Altogether, we provide the first comprehensive study of the evolutionary history and genic, structural, and regulatory differences between the genomes of two closely related lacertid species. This comprises an important baseline for understanding the genomic regions and mechanisms involved in the speciation of European green lizards. In addition to a detailed analysis of the demographic history and evolutionary scenario of European green lizards, our study provides valuable resources that will help establish conservation guidelines for lacertids experiencing population declines due to habitat loss .L. viridis from Tokaj, northeastern Hungary  (September 2013) and a L. bilineata from Mâlain, France  (July 2014). There is no known morphological variation between the individuals of the two species  and 20 µg (L. viridis) genomic DNA was damage-repaired twice, end-repaired, and ligated overnight to hairpin adapters applying components from the DNA/Polymerase Binding Kit P6 from PacBio. Reactions were carried out according to the manufacturer's instructions. BluePippin Size-Selection to greater than 15 kb was performed according to the manufacturer's instructions . Conditions for annealing of sequencing primers and binding of polymerase to purified SMRTbell template were assessed with the Calculator in RS Remote, PacBio. Long-read sequencing was carried out for both genomes with 20 SMRT Cells applying P6-C4 chemistry on a PacBio RS-II sequencer. Average PacBio read lengths of 14 kb and 12 kb were retrieved for L. viridis and L. bilineata, respectively.Tail tissue from each sample was digested with proteinase K, and genomic DNA was extracted using a chloroform-based method . The whoRNA from each tissue was extracted using Trizol Reagent  and purified with the RNeasy Mini Kit . The mRNA was purified using the Dynabeads mRNA Purification Kit (Life Technologies). The purity and concentration of RNA and cDNA were checked using Nanodrop and Bioanalyzer 2100 , and fragments of length 200–250 bp were obtained using Ambion RNA fragmentation reagent. The first and second strands of cDNA were synthesized using random hexamer primers with SuperScript II reverse transcriptase (Life Technologies) and DNA Polymerase I with RNase H treatment (Life Technologies) respectively.Lacerta viridis was sequenced on a single lane for a more accurate estimate of genome size and repeat content. In order to avoid lane and run biases, sequencing was distributed over three lanes with all genomes and transcriptomes.RRID:SCR_011809) using the RFAM covariance models as input, and homologous ncRNA genes were filtered with a cutoff of 1e-06 [Small ncRNAs were annotated on the genomic contigs by performing an infernal cmscan v1.1.1  with default parameters [RRID:SCR_013048) [Additionally, ncRNA class-specific annotation methods were used for tRNAs, snoRNAs, and miRNAs. tRNAs were annotated using tRNAscan-SE v1.3.1 software   and mappFor comparison, ncRNA families (except lincRNA) were also annotated in other selected sauropsid genomes. A reference database was created using sequenced and annotated genomes from reptiles, aves, and other vertebrates. The program ePoPe  was usedRRID:SCR_015027) [de novo annotation of repeats using RepeatMasker v4.0.5  [The RepeatModeler v1.0.4 pipeline   was used_012954) . The evoL. viridis and L. bilineata, we used the blockwise composite likelihood approach. We analytically computed the probabilities of mutational configurations in blocks of fixed length using the bSFS framework [To assess the demographic history between ramework .L. viridis and L. bilineata to the L. viridis reference genome with BWA MEM v0.7.12-r1039 software [RRID:SCR_010761) [We mapped the illumina reads from software . The hom_010761)  with a m_010761) , 109. ThBlocks containing both fixed differences and shared heterozygous sites violate the four-gametes criterion, and 46,825 blocks (0.83%) were removed under the assumption of no recombination within blocks. To account for physical linkage between adjacent blocks, we assumed that every 1000th block is effectively unlinked, i.e., statistically independent, and corrected lnCL scores by a factor of 1/1000. We fitted 13 different demographic scenarios  based on the pairwise distance of 6.19% in the cytochrome b gene between L. viridis and L. bilineata [A. carolinensis (1.3% mutations per million years) [We estimated the generation length based on the mean age of the mothers of all offspring  given th1.1 eggs . Given tilineata . The lowilineata . This asn years) .Lacerta viridis was used as the reference genome since the assembly was more contiguous for this species. Genomic reads from L. bilineata were used as the query, and the reads of L. viridis mapped against the reference were used as control.Genomic rearrangements between the lacertids were detected based on both read-based methods and syntenic blocks information. L. bilineata (query) and of L. viridis (control) were separately mapped against the same reference (L. viridis).Genomic rearrangements were detected between lacertids using read mapping-based methods for Illumina paired-end reads and for PacBio-reads separately, followed by SV callers specifically developed to deal with short and long read sequences, respectively. In both approaches, reads of RRID:SCR_001799) [RRID:SCR_010821) [RRID:SCR_000560) [de novo assemblies were constructed using the ABYSS assembler for insertions. In order to maintain a high level of sensitivity and specificity (>90%) in the detection of SVs, only the rearrangements called with a minimum support of eight uniquely mapped paired-end reads were used for further analyses [The alignment of Illumina reads was carried out with BWA MEM v0.7.12 , and rea_001799)  to infer_010821)  to predi_000560)  to detecanalyses .The PacBio reads were aligned to the reference with NGMLR v0.2.1, and the alignment was fed to Sniffles v1.0.3 SV-caller  to call L. viridis and L. bilineata in our case). The events detected in both directions, i.e., L. viridis reference with L. bilineata as query and L. bilineata reference and L. viridis as query were retained. The length threshold was set to 50 bp, and the predicted rearrangements were filtered based on quality to reduce false positives  test [The predicted ncRNAs  in lacertids were tested for structural selection (selection of sites acting on secondary structure in either of the lacertids) with SS) test , a statiA ncRNA structural test to detect positively selected structures is only appropriate for structurally conserved groups. Low d-score values (d <10.0) were used to distinguish conservation chosen based on structural uniformity of the groups. This cutoff was based on the visual inspection of the secondary structures of families with d-scores of 1 to 20. Secondary structures of ncRNA sequences were predicted using RNAfold . In a siThe selection test was adapted for lincRNAs and performed only on the two lacertid species without any outgroup since lincRNA annotations of other closely related species were unavailable. Since the positive selection of secondary structure cannot be determined without outgroups, we instead detected divergence of lincRNA structure within the lacertids. Local conserved structure blocks were predicted for the orthologous lincRNA families, and these blocks were subjected to an adaptation of SSS test based on local structures. The structural selection for lincRNAs was assessed locally, since most base-pairings occur between nucleotides within a short distance , 126. LoL. viridis and L. bilineata) compared to other vertebrates, the CDS of five species, namely, anole lizard (Anolis carolinensis), chicken , frog , spotted garfish (Lepisosteus oculatus), and human (Homo sapiens) were downloaded from the Ensembl database version 83  [In order to investigate the selection pressure in the lacertid branch (ancestor of _002344) . To keep_002344) , which tL. viridis and L. bilineata) in the foreground branches. The remaining species were used as the background to test for positive selection using the branch-site model of codeml within the PAML v4.8 package  [2 distribution was used to detect genes with significant positive selection followed by multiple testing through the Benjamini–Hochberg procedure. Genes with P < 0.05 and q <0.05 were retained and referred to as being positively selected in the lacertid branch.The protein identity filtering in POTION was set to 30% in each orthologous group and sequence size limits to more than 10 times or less than 0.2 of the median size in the group. Only groups with at least four species were retained. The sequences in each orthologous group  were aligned and gap trimmed; phylogenetic trees were constructed, and groups with recombinants were excluded from the selection tests. The intermediates files from the POTION pipeline were used to generate unrooted trees with lacertids (_014932) . A likelL. viridis, L. bilineata, Anolis carolinensis, Gekko japonicus, and Pogona vitticeps. The single-copy orthologs were identified with ProteinOrtho with a minimum protein identity of 70%, e-value of 1e-06, and minimum similarity of 0.99 for additional hits. The orthologous coding sequences from the five lizard species were aligned with MACSE while accounting for frame-shifts, and the stop codon at the end of the sequence was removed. Unrooted trees were generated with three different foreground branches: (i) lacertids (L. viridis and L. bilineata), (ii) L. viridis alone, and (iii) L. bilineata alone. The rest of the workflow for detection of recombinants, removal of gaps, and codeml tests was similar to the POTION pipeline followed by filtering for significant candidate genes . In order to avoid false predictions of PSS at the beginning or toward the end of alignments, where mismatches were allowed, the candidate genes predicted to contain PSS in either species were visually inspected.To detect adaptive evolution through positive selection within either lacertids, additional tests (PAML branch-site models) were performed with less distant outgroups using a set of five lizard species, namely, GigaScience GigaDB [The assembled genomes, their annotations, transcript data, variant calls , snapshots of the code, and other supporting datasets are available in Zenodo  and in te GigaDB  repositoAdditional file 1: This supplement contains methods SM1-SM11, information SI1-SI11, Figures S1–S10, Tables S1–S17 and References.L. viridis and L. bilineata.Additional file 2: The figure for the contig clusters in lacertids generated from synteny information between Mathematica notebook containing the code used and other supporting information from the demography analysis of L. viridis and L. bilineata.Additional file 3: AIC: Akaike information criterion; BDMI: Bateson-Dobzhansky-Muller incompatibility; BLAST: Basic Local Alignment Search Tool; bSFS: blockwise site frequency spectrum; CDS: coding sequence; CL: composite likelihood; HAL: hierarchical alignment; HYAL: hyaluronidase; indels: insertions and deletions; KRAB: Krüppel-associated box; KZNF: Krüppel-type zinc finger; lincRNA: long intergenic non-coding RNA; miRNA: microRNA; Mya: Million years ago; NCBI: National Center for Biotechnology information; ncRNA: non-coding RNA; PacBio: Pacific Biosciences; PSG: positively selected gene; PSS: positively selected site; RBMX: RNA binding motif protein, X-linked gene; rRNA: ribosomal RNA; SCO: single-copy ortholog; SNORD61: small nucleolar RNA, C/D Box 61; SFS: site frequency spectrum; snoRNA: small nucleolar RNA; SSS: Selection on the Secondary Structure; SV: structural variants; tRNA: transfer RNA.The authors declare that they have no competing interests. The authors alone are responsible for the content and writing of the paper.This project was funded by the Deutsche Forschungsgemeinschaft .K.H. and A.G. collected the samples; A.W. Illumina sequenced the genome and transcriptome; C.S., B.B., and J.O. were involved in the PacBio genome sequencing; S.R.R.K. performed the assembly and annotation; S.K. and S.R.R.K. annotated the non-coding RNA; M.B.W.C. analyzed selection in non-coding RNA; S.R.R.K. and H.I. performed the positive selection analysis; S.R.R.K. and M.C. analyzed structural variants; A.S. and K.L. analyzed the population histories; S.R.R.K., R.F., K.N., P.F.S., and M.S. wrote the initial draft of the manuscript; H.I., M.C., A.S., K.L., S.K., M.B. W.C., J.O., B.B., C.B., and K.H. edited the manuscript; R.F., K.N., K.H., P.F.S., and M.S. conceived the study.GIGA-D-18-00173_original_Submission.pdfClick here for additional data file.GIGA-D-18-00173_Revision_1.pdfClick here for additional data file.GIGA-D-18-00173_Revision_2.pdfClick here for additional data file.Response_to_Reviewer_Comments_Original_Submission.pdfClick here for additional data file.Response_to_Reviewer_Comments_Revision_1.pdfClick here for additional data file.Reviewer_1_Report_ -- Ben Wielstra6/20/2018 ReviewedClick here for additional data file.Reviewer_2_Report_ -- Anthony Geneva6/22/2018 ReviewedClick here for additional data file.Reviewer_2_Report_Revision_1 -- Anthony Geneva10/28/2018 ReviewedClick here for additional data file.giy160_Supplemental_FilesClick here for additional data file.


Pinus densiflora (Korean red pine) is a species of evergreen conifer that is distributed in Korea, Japan, and China, and of economic, scientific, and ecological importance. Korean red pines suffer from pine wilt disease (PWD) caused by Bursaphelenchus xylophilus, the pinewood nematode (PWN). To facilitate diagnosis and prevention of PWD, studies have been conducted on the PWN and its beetle vectors. However, transcriptional responses of P. densiflora to PWN have received less attention. Here, we inoculated Korean red pines with pathogenic B. xylophilus, or non-pathogenic B. thailandae, and collected cambium layers 4 weeks after inoculation for RNA sequencing analysis. We obtained 72,864 unigenes with an average length of 869 bp  from a Trinity assembly, and identified 991 differentially expressed genes (DEGs). Biological processes related to phenylpropanoid biosynthesis, flavonoid biosynthesis, oxidation–reduction, and plant-type hypersensitive response were significantly enriched in DEGs found in trees inoculated with B. xylophilus. Several transcription factor families were found to be involved in the response to B. xylophilus inoculation. Our study provides the first evidence of transcriptomic differences in Korean red pines inoculated with B. xylophilus and B. thailandae, and might facilitate early diagnosis of PWD and selection of PWD-tolerant Korean red pines. Pinus that are found in the Northern Hemisphere1. They have economic, ecological, and scientific importance, as they provide timber for construction, furniture, paneling, and flooring3, habitats and food for wildlife, and (in their needles) agents with anticancer, antioxidant, and antimutagenic properties4. However, pine trees  are threatened by pine-wilt disease (PWD), a devastating disease that kills trees within a few weeks to a few months from infection, with symptoms that are characterized by wilted and brown-colored needles6. PWD is caused by the pinewood nematode (PWN) Bursaphelenchus xylophilus, which is carried by long-horned beetles (Monochamus spp.) and spread when the beetles feed on the trees5. B. thailandae is a nematode species that was first isolated from pine trees in Thailand, and which has subsequently been detected in Korea. B. thailandae differs morphologically from B. xylophilus, and is not pathogenic, so pine trees inoculated with B. thailandae can survive without showing any visible symptoms of PWD8. Therefore, examination of the transcriptome differences between B. xylophilus and B. thailandae inoculated pines might identify novel resistance mechanisms and candidate genes that play important roles in resistance specifically against B. xylophilus. Furthermore, we also might identify the pathogenesis mechanisms of B. xylophilus to cause PWD. However, comparative transcriptome analysis between trees inoculated with nematodes having different pathogenicity has not been conducted.Pines are conifers in the genus B. xylophilus and its beetle vectors11. Currently, to diagnose PWD, B. xylophilus or DNA fragments from B. xylophilus must be detected in tree samples12, or PWD symptoms must be observed. However, it is difficult to detect B. xylophilus and its DNA in pine trees at an early stage of infection, and by the time PWD symptoms can be observed, B. xylophilus has generally already spread throughout the forest.Many studies have been carried out to find ways to diagnose and block PWD, and most of them have been focused on B. xylophilus infection. Previously, we examined the transcriptome differences between trees with and without PWD symptoms in natural forest. However, we didn’t know whether trees showing PWD symptoms are indeed infected by PWN or not13. In this report, we inoculated trees with PWN and observed the PWD symptoms after inoculation of PWN. Therefore, we can assure that PWD symptoms are indeed caused by PWN inoculation, and examination of the transcriptome differences among the trees injected by water, non-pathogenic nematode, and pathogenic pine wood nematode give better understanding of the transcriptional responses against PWD in Korean red pines.The physiological symptoms of PWD have been well characterized, but there are only few studies that attempt to understand the comprehensive transcriptome of pine trees in response to Arabidopsis thaliana, thousands of genes have been shown to be differentially expressed upon nematode infection, and important regulators of defense responses against nematode infection have been discovered by transcriptomic analysis15. By extension, it might also be possible to identify genes that are differentially expressed in pine trees upon infection with B. xylophilus, and thereby to diagnose PWD infection by gene-expression analysis. In addition, it might be possible to develop PWD-resistant trees by selection of particular alleles of genes that are differentially expressed after inoculation with B. xylophilus.In the thale cress B. xylophilus infection, there is a lack of transcriptome analysis and most of them were conducted using saplings under artificial experimental conditions instead of using adult trees in a forest environment17. Next-generation sequencing (NGS) technology has been rapidly developed and widely used for research in plant biology, to enhance our understanding of plant responses under various conditions18. In addition, software developments now enable the de novo assembly of the transcriptome of an organism (such as P. densiflora) that does not have a reference transcriptome19.Despite the importance of developing a comprehensive understanding of the transcriptome of pines in conditions of de novo assembly of the P. densiflora transcriptome and quantification of transcript expression in response to inoculation with either B. xylophilus or B. thaliandae at felling age in a natural forest environment. Identification of differentially expressed transcripts in pathogenic PWN-inoculated P. densiflora could spur the development of a diagnostic method for PWD infection and aid in the selective breeding of PWD-resistant P. densiflora.Here, we report the B. xylophilus) and non-pathogenic nematode (B. thailandae) were originally isolated from Korean red pines and reared on fungal hyphae of Botrytis cinerea (de Bary) Whetzel grown on potato dextrose agar medium at 25 °C for 2 weeks. Nematodes were re-isolated from the medium by the Baermann funnel method20. Nine Korean red pines of 11–13 m height and 15–20 cm diameter at breast height in a forest in Jinju-si, Gyeongsangnam-do province, South Korea, were selected. Water, B. xylophilus, and B. thailandae were injected into three independent trees each. The stem of each tree at breast height was wounded mechanically in three places, and 1 ml sterile water or 1 ml sterile water containing 20,000 nematodes was injected into each wound site . Cambium samples of the trees were collected 4 weeks after inoculation and subjected to RNA-Seq analysis. Briefly, hard outer bark was removed and soft cambium layers were taken from the main stem at breast height by using chisel.Pathogenic PWN , and only samples with an RNA integrity number >8 were used for library preparation. Preparation of each paired-end non-directional cDNA library (2 × 101 bp) was conducted according to the TruSeq RNA Sample Preparation Guide . Sequencing of cDNA libraries was performed on an Illumina HiSeq. 2000 sequencer.Cambium samples were taken from the main stem at breast height by using chisel. Total RNA was isolated from cambium samples with an RNA isolation kit . The assessment of RNA integrity (RIN), library construction, and sequencing were performed as described previouslyet al. (2018) with minor modification (filtering of sequences <50 bp length and eliminating exact duplicates or reverse-complement exact duplicates caused by library PCR amplification with –derep 14 option)21. Reference transcriptome were generated from all clean reads using Trinity v2.5.1 with default parameters19 and de novo assembled transcriptome is available as Supplementary Data 22. Clustering of transcripts was performed using CD-HIT-EST v4.6.1 with default parameters24, and the longest transcripts in each cluster were used for subsequent analysis. Transcriptome completeness was assessed by using Benchmarking Universal Single-Copy Orthologs (BUSCO) v3 with the Embryophyta_(odb10) database25.PRINSEQ-lite v0.20.4 was used for read cleaning as described in Lee 26. Read counts were obtained using RSEM v1.3.027. The raw counts were normalized as trimmed mean of M-values (TMM)-normalized transcripts per kilobase million (TPM) values for each transcript and differentially expressed genes (DEGs) showing more than 2-fold expression change with a false-discovery rate (FDR)-adjusted P-value ≤ 0.05 among all pairwise sample comparisons were obtained using EdgeR v3.16.5, a component of Trinity28.Clean paired end reads were mapped to the reference transcriptome using Bowtie softwareA. thaliana, with an E-value ≤ 1E-7 using the Basic Local Alignment Search Tool for proteins (BLASTX) with default parameters except for max_target_seqs 129 and annotation result was presented as Supplementary Data P. densiflora transcription factors (TFs) that aligned and annotated with A. thaliana TFs were classified into TF families based on the Plant Transcription Factor Database v4.0 (http://planttfdb.cbi.pku.edu.cn/)30. Gene ontology (GO) enrichment analysis was performed with PANTHER GO classification system (http://www.geneontology.org)31. Gene ontology information files from The Arabidopsis Information Resource (TAIR) were used as a reference32. Biological process gene ontology (GOBP) with a Fisher’s exact test with FDR corrected P-value va0.05 were considered to be significantly enriched. REVIGO (http://revigo.irb.hr) were used for reducing and visualizing the genes ontology33. For MapMan analysis, homologous A. thaliana IDs and log2 fold-change values of TMM normalized TPM (B. xylophilus versus B. thailandae) were mapped to biotic-stress pathways34. Pictorial representation for the biotic-stress pathways was downloaded from the MapMan website (https://mapman.gabipd.org/)34.Translated protein sequences corresponding to assembled unigenes were compared with those of B. xylophilus relative to those inoculated with B. thailandae were used for RT-qPCR with a CFX96 Touch Real-Time PCR Detection System . Expression levels of the genes were calculated by the comparative threshold method, with EIF4A-2 as the internal control. The Pearson correlation coefficient between RT-qPCR and RNA-Seq was analyzed with the R statistical software35. Primer sequences are listed in Supplementary Table Preparation of total RNA from Korean red pines was performed using RNA isolation kit (TAESIN), and quantitative reverse-transcription PCR (RT-qPCR) was conducted using the TOPreal One-step RT qPCR Kit . 12 DEGs in trees inoculated with All the raw read sequences were deposited in the NCBI sequence read archive under the accession number SRP165817.Bursaphelenchus, we monitored the visible phenotype of selected P. densiflora trees injected with water, B. xylophilus, or B. thailandae. None of these trees showed any visible PWD symptoms 4 weeks after treatment. However, 3 months after treatment, some of the green needles turned to brown and drooped in trees inoculated with B. xylophilus, whereas trees treated with water or B. thailandae still displayed no visible PWD symptoms . Finally, 213,695,875 cleaned paired reads were used for B. thailandae inoculated tree - 1 (B. thailandae - 1) and B. xylophilus inoculated tree - 1 (B. xylophilus - 1) were not closely clustered together with their biological replicates  in all the pairwise comparisons , ons Fig. . Among tdae Fig. . Intriguees Fig. . The VenB. xylophilus inoculation, we categorized the DEGs on the basis of Gene Ontology Biological Processes (GOBPs) , the GOBP terms catabolic process, response to chemical, polysaccharide catabolic process, and cellular catabolic process were specifically enriched. Notably, the largest number of GOBPs was enriched in the DEGs identified in the comparison between trees inoculated with B. xylophilus and B. thailandae. Among these terms, response to bacterium, cell communication, oxidation–reduction process, transmembrane receptor protein tyrosine kinase signaling pathway, lignin biosynthetic process, plant-type hypersensitive response, defense response to nematode, and innate immune response were specific to this comparison. Heatmap of GOBPs enriched in the DEGs in each category of Venn diagram in Fig. To obtain comprehensive functional features associated with transcriptional programs in response to Ps) Fig. . GOBP anB. xylophilus infection, we identified differentially expressed TFs (DETFs) in the comparison between trees inoculated with B. xylophilus or with B. thailandae. We identified 34 TFs as DETFs and the most highly represented DETF families were the WRKY (7 members) followed by LBD (6 members), bHLH and MYB families (5 members each)  Fig. , and theB. xylophilus or with B. thailandae, and located each gene  in biotic-stress pathway with the MapMan visualization software  were more highly expressed in response to B. xylophilus than to B. thailandae. Oxidation-related processes make up one of the most important pathways in the control of defense responses in plants36, and genes related to oxidation–reduction processes were also differentially expressed, as were a number of TFs, including several members of both the WRKY and MYB families. These types of TFs are known to have important roles in regulation of the defense response against pathogen infection40. The MapMan metabolism and regulation overviews are also shown as Supplementary Fig. B. xylophilus infection.To investigate key signaling elements in biotic-stress pathways, we identified DEGs in the comparison between trees inoculated with are Fig. . Genes iB. xylophilus relative to those inoculated with B. thailandae. The expression changes of the DEGs from qRT-PCR and RNA-seq analyses were highly correlated  upon nematode infection, thereby activating defense responses, and ROS are closely related to the hypersensitive response49. However, ROS also function as pathogenicity factors to facilitate nematode infection in A. thaliana51. Therefore, it is also possible that B. xylophilus modulates expression of host oxidation–reduction-related genes to suppress plant defense responses.We found that the lignin biosynthetic process GOBP was enriched in the DEGs in the comparison between trees inoculated with B. xylophilus and B. thailandae, transmembrane receptor protein tyrosine kinase signaling pathway GOBP were enriched in the DEGs. An effective plant defense against pathogens is achieved through recognition of pathogen-associated molecular patterns by surface-localized receptor kinases, and by the consequent downstream signaling cascade53. In this analysis, homologues of Arabidopsis FLS2 and NILR1 were identified as DEGs in this biological process. Both of them were well known as important regulators in PAMP triggered immunity. In A. thaliana, leucine-rich repeat receptor-like kinase NILR1 is required for innate immunity against parasitic nematodes54. Therefore, interaction between nematode-associated molecular patterns and transmembrane receptor protein tyrosine kinases might be important for successful resistance to B. xylophilus in Korean red pines.In our comparison between trees inoculated with B. xylophilus infection by comparing the expression of TFs between trees inoculated with B. xylophilus and with B. thailandae. The family of DETFs with the most members represented by DEGs was WRKY, followed by theLBD, bHLH, MYB, ERF, and MIKC_MADS families. WRKY TFs are involved in many developmental processes, especially in defense and senescence37. AtWRKY6 homologue was one of the DETF genes in the present study, and AtWRKY6 is a positive regulator in the defense response against the beet-cyst nematode Heterodera schachtii, and induces expression of salicylic acid-dependent defense-response genes in A. thaliana55. In addition, AtWRKY51 homologue was also induced after B. xylophilus inoculation, and it is reported that WRKY51 mediates the defense response against Pseudomonas syringae56. Ethylene response factor (ERF) TFs are regulators of pathogenesis-related genes, as well as ethylene-, salicylic acid-, and jasmonic acid-inducible genes57. We identified an ERF9 homologue as a DETF, with induction by B. xylophilus inoculation. Results from a previous study indicate that ERF9 is a negative regulator of resistance against necrotrophic fungi and acts as a molecular brake for sustained activation of a defense response58. However, it is also plausible that B. xylophilus activates expression of ERF9 homologue to repress the host defense system. Therefore, the DETF families identified here might play roles in fine-tuning of the defense response through regulation of expression of downstream signaling components. In accordance with DETF and GOBP analysis, genes in the WRKY and MYB TF families, cell-wall biosynthesis, proteolysis, hormone signaling, and defense showed altered expression in Korean red pines inoculated with B. xylophilus in a MapMan analysis of the biotic-stress response.We explored the expression patterns of TFs that mainly govern transcriptional programs responding to B. xylophilus or B. thailandae. Our analysis defined biological processes that are involved in regulation of the defense response against inoculation of P. densiflora with B. xylophilus. These results might enable the discovery of the genes for the fast identification of B. xylophilus infected trees, as well as breeding programs to produce PWD-resistant Korean red pines.In conclusion, this is the first report that describes the differences in transcriptomes between Korean red pines of felling age inoculated with Supplementary materialsDataset 1Dataset 2Dataset 3Dataset 4


Scedosporium is a genus comprising at least 10 species of airborne fungi (saprobes) that survive and grow on decaying organic matter. These fungi are found in high density in human-affected areas such as sewage-contaminated water, and five species, namely Scedosporium apiospermum, S. boydii, S. aurantiacum, S. dehoogii, and S. minutisporum, cause human infections. Thailand is a popular travel destination in the world, with many attractions present in densely populated areas; thus, large numbers of people may be exposed to pathogens present in these areas. We conducted a comprehensive survey of Scedosporium species in 350 soil samples obtained from 35 sites of high human population density and tourist popularity distributed over 23 provinces and six geographic regions of Thailand. Soil suspensions of each sample were inoculated on three plates of Scedo-Select III medium to isolate Scedosporium species. In total, 191 Scedosporium colonies were isolated from four provinces. The species were then identified using PCR and sequencing of the beta-tubulin (BT2) gene. Of the 191 isolates, 188 were S. apiospermum, one was S. dehoogii, and species of two could not be exactly identified. Genetic diversity analysis revealed high haplotype diversity of S. apiospermum. Soil is a major ecological niche for Scedosporium and may contain S. apiospermum populations with high genetic diversity. This study of Scedosporium distribution might encourage health care providers to consider Scedosporium infection in their patients. Scedosporium is a genus of filamentous fungi with septate hyphae. These fungi are airborne saprobes that survive and grow on decaying organic matter, and thus, are frequently found in high density in human-affected areas such as sewage, contaminated water, and urban soil. The genus currently comprises 10 species, five of which, namely Scedosporium apiospermum, S. boydii, S. aurantiacum, S. dehoogii, and S. minutisporum, cause Scedosporiosis. [Scedosporium biology has been studied mainly after its isolation from the environment. In Thailand, S. apiospermum was reported in brain abscesses of near-drowning individuals [S. apiospermum [ividuals . Moreoveospermum .Scedosporium is of increasing clinical importance considering several severe Scedosporium species infections reported throughout Asia. In Japan, Nakamura et al. reported multiple brain abscesses caused by S. aurantiacum [et al. reported vertebral osteomyelitis caused by S. apiospermum in patients who nearly drowned during the Great East Japan Earthquake and Tsunami [S. apiospermum infection in a near-drowning patient. Thus, a unique feature of this environmental fungus is the infection of immunocompetent humans after near-drowning incidents. Therefore, clinicians and microbiologists should be aware of this possibility in regions with high endemicity. Furthermore, additional research on disease course and treatment is required.The distribution of antiacum , and Shi Tsunami . In Taiw Tsunami  reportedScedosporium species in their natural habitats in Thailand. In our initial study, we surveyed and isolated colonies of Scedosporium species in soil and water samples from 10 public parks in Bangkok, the capital and most populous city in Thailand. Three major Scedosporium species, namely S. apiospermum, S. aurantiacum, and S. dehoogii, were identified, but 16 sequences could not be identified, implying the possibility of additional Scedosporium species [Therefore, our team began to study  species .Scedosporium species in soil samples by investigating soil samples in Thailand from 35 public areas with high human population density and tourist popularity in six geographic regions of Thailand.In this study, we conducted a more comprehensive survey of 2 sites in each park, with sampling at four positions per site. All soil samples were collected from a depth of approximately 15 cm using a sterile metal spoon to avoid plant debris, weeds, and branches. Samples were placed in sterile plastic bags and stored at 4°C until processed.Sampling at all locations in 35 public parks was authorized by the mayor’s office of the municipality or city in 23 provinces in six geographic regions: northern , central , western , eastern (Chon Buri and Chanthaburi Provinces), northeastern  and southern  . Soils sg for 5 min, and the supernatant was discarded. The pellet was then re-suspended in 5 ml distilled water. Three 100-μl aliquots were inoculated on three separate plates of Scedo-Select III medium, which was designed and developed to minimize the growth of other rapidly growing fungal species and specifically for the isolation of Scedosporium colonies [Fungal isolation was performed according to the procedure provided by Luplertlop et al. . In briecolonies . The plaS. apiospermum species complex on the basis of their morphology were inoculated on Scedo-Select III for purification. The colonies isolated from Scedo-Select III plates were then collected and inoculated onto Sabouraud dextrose agar for detailed macroscopic and microscopic observations.Colony morphologies were visually and microscopically examined according to the procedure of Gilgado et al. –10. A si5′GGTAACCAAATCGGTGCTGCTTTC3′ and Bt2b: 5′ACCCTCAGTGTAGTGACCCTTGGC3′ [http://www.mbio.ncsu.edu/bioedit/bioedit.html). Edited sequences were compared with existing sequences in GenBank using BLASTn (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The generated nucleotide sequences were deposited in GenBank under accession numbers MF991891 and MG204345−MG204534  and amplified using PCR with the β-tubulin (Bt2) gene-specific primers Bt2a: CTTGGC3′ . Each PChttp://ipe.unsa.edu.ar/software) [http://www.splitstree.org/) [Each sequence was trimmed to the start and end of the gene. The allele number of each sequence was determined using the MLSTest v1.0.1.23 software (downloaded from oftware) . Graphicoftware) . The modee.org/) .This field study was performed under permission from the Ministry of Education (letter 0517.115/00331), as well as the following authorities concerned with protection of wildlife: Thanong Donchai, Deputy mayor; Chiangmai Province permitted by Chatri Cheuamanochan, Deputy mayor; Nan Province permitted by Arisa Boonsom, Deputy mayor; Phitsanulok Province permitted by Pongsin Sanepong, Deputy mayor; Phra Nakhon Si Ayutthaya Province permitted by Suvera Rinvet, Municipal Clerk; Samut Songkhram Province permitted by Somchai Tonpasert, Mayor; Tak Province permitted by Ananchai Taweeguekolkit, Mayor; Kanchanaburi Province permitted by Kumtorn Pusitkarnchana, Deputy mayor; Prachuap Khiri Khan Province permitted by Pichit Santimethakul, Deputy mayor; Chon Buri permitted by Jutharat Parinwachirapat, Municipal Clerk; Chanthaburi Province permitted by Pirat Atikarnkul, Municipal Clerk; Nakhon Ratchasima Province permitted by Nutcharat Chuhirunwat, Director of Public Works; Khon Kean Province permitted by Thawatchai Wanapithakkul, Mayor; Mukdahan Province permitted by Trirong Thawinprai, Deputy mayor; Nong Khai Province permitted by Winchai Waipat, Deputy mayor; Bueng Kan Province permitted by Witaya Saynjanthichai, Mayor; Ubon Ratchathani Province permitted by Kritchapol Muangnue, Deputy mayor; Surat Thani Province permitted by Suphang Saewong, Deputy mayor; Phuket Province permitted by Thavorn Jirapatsophon, Deputy mayor; Songkhla Province pemitted by Somsak Tanthiseranee, Mayor and Narathiwat Province permitted by Udom Densanthikul, Mayor. The field study did not involve endangered or protected species.Scedosporium were selected for species and strain identification based on Bt2 gene sequencing. Of the 191 isolates, 188 were S. apiospermum , and one isolate of S. dehoogii  as summarized in In this study, 350 soil samples were collected from 35 public parks that were distributed across 23 provinces of Thailand . In totaS. apiospermum sequences, we determined the number of Bt2 allele types according to sequence similarity before multiple alignment. From the 188 sequences, we found 67 alleles, with allele frequencies ranging from 1 to 34 individuals sampled , is widely used for identification of these fungi. The target gene using for sequencing are internal transcribed spacer (ITS), actin, calmodulin exons 3 and 4, the second largest subunit of the RNA polymerase II and Beta-tubulin exon 2–4, manganese superoxide dismutase, transcription elongation 1 alpha, Beta -tubulin exon 5–6 [Scedosporium species identification.There are several molecular techniques applied to identify exon 5–6 . Researcexon 5–6 –17. IsolS. aspiospermum species complex is comprised of S. apiospermum, S. boydii, S. dehoogii, S. aurantiacum, and S. minutisporum  according to the European Confederation of Medical Mycology/International Society for Human and Animal Mycology [S. cereisporum, was isolated by Rougeron et al.; the species was phylogenetically and morphologically closely related to S. aurantiacum [S. apiospermum species complex as S. apiospermum, S. boydii, and S. angusta (P. angusta) based on the phylogenetic analysis of BT2, γ-actin, transcriptional elongation factor 1α, and internal transcribed spacer of the small ribosomal protein 60sS L10 (L1), which distinguished S. minutisporum, S. aurantiacum, and S. dehoogii from the three S. apiospermum species complex. There are currently >10 species of the S. apiospermum species complex [Scedosporium species distribution in the present study.Based on molecular variation, the Mycology . More reantiacum . Chen etantiacum  defined S. apiospermum, S. boydii infection has been documented in Thailand. S. boydii was specifically isolated from the brain tissue of a renal transplant patient. In the present study, samples from only four of 23 provinces  were positive for Scedosporium species; however, at these sites, numerous S. apiospermum strains (188 from 191 isolates) were found. In addition, one isolate of S. dehoogii was identified, whereas two isolates were unidentifiable and thus could be novel species. The two unclassified strains, TMMI 275 and TMMI 293, were closely related to S. angusta and S. apiospermum, respectively. The present study confirms that S. apiospermum inhabits soil in diverse regions of Thailand , and S. aurantiacum was only isolated from a single site around Bangkok.In addition to Scedosporium distribution for guiding the further perspective in factor related fungal distribution and an impact. Our survey data indicate the prevalence of Scedosporium in different regions of Thailand, which should be useful for the clinician and medical mycologist in diagnosing fungal infections. This information may help clinicians to exclude Aspergillus as a cause of fungal infection.Therefore, this study demonstrated that Scedosporium in Thailand. The results provide valuable knowledge to assist future studies to compare genetic relatedness among pathogenic species in the clinical setting and to evaluate infection risk in specific regions.In conclusion, this study identified soil as an ecological niche of S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.