AbstractApioceridae (apiocerid flies) of the Afrotropical Region is provided. Each taxon entry includes links to open-access, online repositories such as ZooBank, BHL/BioStor/BLR, Plazi, GBIF, Morphbank, EoL, and a research web-site to access taxonomic information, digitized literature, morphological descriptions, specimen occurrence data, and images. Cybercatalogs as the one presented here will need to become the future of taxonomic catalogs taking advantage of the growing number of online repositories, linked data, and be easily updatable. Comments on the deposition of the holotype of Apiocerabraunsi Melander, 1907 are made.A cybercatalog to the  Cybertaxonomic tools enable us to utilize web-based databases and data repositories to store and retrieve information on taxon names, publications, digitized literature, morphological descriptions, molecular sequences, occurrence data, or images. The availability of these kinds of data in an open-access, online framework allows scientists to test and support taxonomic and phylogenetic hypotheses readily as well as link data in support of biodiversity research across taxon boundaries. Furthermore, future research programs are enhanced by re-using and re-purposing available data in analyses and syntheses. The open-access movement encourages researchers to share primary data , the numEoL) strives to present a species page for each known taxon summarizing diverse data from disparate online sources. As a data aggregator, it relies on information being either stored elsewhere and harvested regularly or entered directly into the EoL database.There is currently no central online gateway to deposit all of the above kinds of data. However, the Encyclopedia of Life  edited by Ashley Kirk-Spriggs and Bradley Sinclair will be an outstanding resource on the status of research on Diptera occurring in the Afrotropical Region. The taxon-specific chapters will address each of the 109 families known to occur in this zoogeographic region and provide identification keys to the genera and a synopsis of each genus. The manual will cover the Afrotropical Region as proposed by The forthcoming Apioceridae, for example, DipteraManual of Afrotropical  cannot serve as an updated catalog to genera and species, which is outside of the scope of the project, there is an opportunity to publish taxonomic catalogs in open-access electronic format as data papers prior to  the publication of MAD volumes. Because MAD will be published as a traditional book and the inclusion of unique identifiers  and URLs to data repositories harboring a wealth of information will not be possible, data papers can be cited and the reader will have access to more detailed information on taxa that cannot be included in the general taxon-specific chapters in the MAD.The last comprehensive catalog to the genera and species of Afrotropical flies was published 35 years ago by Apioceridae in the Afrotropical Region is small, currently only three species are known, this catalog should be seen as an example highlighting the usefulness of how online, open-access resources can provide valuable taxon and specimen information. Furthermore, it highlights the need to organize research information and unique identifiers locally (see below) in order to export them for inclusion in data papers. The BDJ Checklist template, which has been employed here, allows for the easy import of a taxonomic catalog for publication as well as an update of a previously published catalog.While the species diversity of Biodiversity Data Journal (BDJ) publishing platform in that this catalog can be updated easily in the future and re-published under a new Digital Object Identifier (DOI) should a new species be described or other taxonomic changes be made. This feature will facilitate the continuous update of the taxonomic information not possible through traditional publishing means in book or article form.The present cybercatalog includes a novel feature available in the LSIDs (Life Science Identifier), GUIDs , UUIDs , and URLs (Uniform Resource Locator) to access data in the various repositories listed below are locally stored in a custom FileMaker Pro database  or web addresses  to data repositories.The ase Fig. . This sBiodiversity Data Journal Checklist template. The resulting Excel file needs minor adjustments to comply with the column names in the template before upload into the Pensoft ARPHA Writing Tool can be achieved.A custom layout Fig.  has beenGUID) whereas all other identifiers are unique within their respective data storage system. However, they should not be seen as less permanent because the identifiers will provide a permanent and persistent way to link to the resource (as long as funding of the data storage system is continued). GUIDs, Digital Object Identifiers (DOIs) or Archival Resource Keys (ARKs), would be preferable  Zoobank issues a UUID as part of the LSID, which is minted. Zoobank and Plazi (see below) share the UUID allowing the retrieval of the related treatment (see below) by adding the respective treatment-specific prefix.The present catalog includes the ZooBank GUID to all taxa, which have been registered for the purpose of this catalog. For each nomenclatorial act  it can be directly accessed.a species-group name: the record will show the original work in which the name was established along with the page number and any figures in which the species is depicted. Furthermore, information on the number and depository of the primary and secondary type specimens as well as the type locality with geographic co-ordinates, if available, are included. Should the original publication be available online, it can be directly accessed.BHL) is a digital archive of natural history literature and works collaboratively to make biodiversity literature openly available as part of a global biodiversity community. It digitizes any natural history literature that is out of copyright and published prior to 1923. In copyright books and journals can also be digitized by BHL after an agreement with a publisher has been signed. The community can propose titles to be digitized by BHL free of charge by entering information on the BHL scanning request form.The Biodiversity Heritage Library (BioStor (BioStor  works wiBLR) is part of CERN’s digital Zenodo archive for scientific data. The BLR is focused on biodiversity literature, specifically articles and illustrations. The items stored are at article- or subarticle-level  for which a DataCite Digital Object Identifier (DOI) is provided, which allows for a citation of the item in a standardized way. Furthermore, it has the potential to assign DOIs to all legacy literature and with that make these articles first-class citizens. BLR is currently administered by Plazi (see further information below) and Pensoft. Upload of articles is open and free to anybody. Generally, any article published before 2000 without a DOI can be made accessible. BLR is mainly focused on providing access to collections covering specific taxa, regions or subjects such as all ant, proctotrupine or drosophilid taxonomy.The Biodiversity Literature Repository the BHL page on which a particular taxonomic name was established to read the original description and obtain other information , article PDF) .a variety of digital archives, for example, BHL (first page of an article not yet itemized by BioStor), BioStor (article single page PDFs), BLR (article PDF), publisher (article PDF), or other digitization archive  by lication . TreatmeGoldenGATE editor, taxonomic articles can be marked-up in XML making the underlying information  accessible in machine-readable form for harvest by aggregators. Both, previously (retroactive) or newly (proactive) published articles/books can be marked-up to extract the species descriptions  that focuses on easy and consistent retrieval of content so that comparison of multiple descriptions dealing with the same or different taxa can be achieved. Plazi treatments can also be used for quantitative comparative studies of published taxonomic research , called TaxPub, has been developed that includes treatments and other elements specific to taxonomic publications and which is now underlying Pensoft journals such as Biodiversity Data Journal or ZooKeys (In collaboration with Pensoft and the  ZooKeys . This woThe Plazi persistent identifiers/queries provide access to:Plazi")the original description treatment  of a given taxon (labeled "Plazi taxon query").all treatments (GBIF) is a data aggregator that gathers specimen occurrence data from numerous natural history collections and herbaria around the world. It provides free and open access to biodiversity data on all types of life on Earth.The Global Biodiversity Information Facility  using an IPT instance. A third way to upload data to GBIF is through articles in which occurrence/observation records are either marked-up a priori  or a posteriorly (Plazi treatments) and made accessible via a Darwin-Core Archive (DwC-A) to GBIF. Through these institutional, personal, or publication-based uploads to GBIF the global distribution maps are enhanced regularly and will provide a more complete picture of the distributional range of taxa based on explicit specimen data.An increasing number of entomological collections have started to digitize their holdings by databasing the specimen occurrence data and submitting them to GBIF. Once the collecting localities have been geo-referenced, GBIF will include the specimens in global distribution maps. Data can also be directly submitted by a researcher or a natural history collection to GBIF through the use of an Integrated Publishing Toolkit IPT. For exaThe GBIF URLs provide access to:the respective taxon page with the global distribution.Morphbank :: Biological Imaging is an image repository for scientific images of organisms and parts thereof. It provides permanent, open-access to the "published" images and the user can access the original image files. This, for example, allows users to zoom in to see more detail than is available when an image is published in a traditional book or journal article. Furthermore, the user can access specimen-level information, e.g. collecting locality, unique specimen identifier, and institutional depository of the photographed specimen as well as imaging technique, and specifics of the view presented. When submitting images to Morphbank, the user has the option to allow the images to be harvested by the EoL (see below) and so the same image can be available on both the Morphbank platform and on the EoL species page. However, a separate entry to Morphbank is advantageous here as not every user provides access to their images on the EoL. Since the number of images of insect specimens from museum collections and publications of a particular taxon will only increase, Morphbank can be an ever-increasing resource for digital photographs.The Morphbank URLs provide access to:Morphbank (by taxon)").the respective taxon page summarizing all images available under that specific name (labeled "EoL) is a data aggregator that harvests information such as scientific names, images, descriptions, digitized literature, and others pertaining to species or higher taxa. It attempts to provide a summary page, the so called species page, for every single species and higher taxon known to science and provides all information in an open-access framework.The Encyclopedia of Life  KE EMu specimen-level database will be shown on the EoL species page (compare images of the USNM record of the holotype of Apiocerabraunsi to the EoL species page for that taxon).Some of the data provided on the EoL species page is duplicated from other individual data sources employed here, such as GBIF and Morphbank. Similar to GBIF, the EoL can also directly receive data, such as images, from museum collection databases or treatments from Pensoft journals, The EoL URLs provide access to:the respective taxon page.asiloidflies.si.edu) provides access to various data on Apioceridae, Asilidae, and Mydidae flies using the Drupal content management system.The research web-site of the senior author a distribution map (labeled "asiloidflies.si.edu specimen table")a table  of that institution is provided to disambiguate the institutional acronym.To facilitate access to specimens for further research, the notes section includes a list of institutions that have specimens of the respective species in their holdings. It should be noted that additional museum collections might have specimens available as this list is based on visits by the senior author to numerous institutions including those holding the majority of Afrotropical Westwood, 1835urn:lsid:zoobank.org:act:B653DD23-4F34-4E6A-90EF-3A781066A923http://biodiversitylibrary.org/page/2548917http://dx.doi.org/10.1080/14786443508648642http://biodiversitylibrary.org/page/2548916http://treatment.plazi.org/id/DFE567BB-19D7-90B4-F081-A45EF7A98A33http://plazi.cs.umb.edu/GgServer/search?taxonomicName.isNomenclature=true&taxonomicName.exactMatch=true&taxonomicName.genus=Apiocerahttp://www.gbif.org/species/1611607http://www.morphbank.net/Browse/ByImage/?tsn=132326http://eol.org/pages/55333/overviewhttp://asiloidflies.si.edu/specimen-map-Apioceridae?genus=Apiocerahttp://asiloidflies.si.edu/specimen-table-Apioceridae?genus=ApioceraApioceraArgentina, Australia, Canada, Chile, Mexico, South Africa, USAHermann, 1909urn:lsid:zoobank.org:act:7822B44A-767F-4B67-9BE3-19F53894CABBhttp://biodiversitylibrary.org/page/33103604http://dx.doi.org/10.5281/zenodo.30975http://biostor.org/reference/101352http://treatment.plazi.org/id/D74EC1E3-A0DD-6EB1-6269-4F121D8FAC36http://www.morphbank.net/Browse/ByImage/?tsn=999022154http://asiloidflies.si.edu/specimen-map-apioceridae?genus=Apiocera&subgenus=Ripidosyrmahttp://asiloidflies.si.edu/specimen-table-apioceridae?genus=Apiocera&subgenus=RipidosyrmaRipidosyrmaApiocera , new junior synonymy by Apiocera (Ripidosyrma) , new subgeneric rank by South Africaurn:lsid:zoobank.org:act:C4D38C42-45E5-48A0-B55A-29BC06FFB72Ahttp://biodiversitylibrary.org/page/38706155http://dx.doi.org/10.5281/zenodo.30972http://www.biodiversitylibrary.org/item/119308http://www.gbif.org/species/1611657http://treatment.plazi.org/id/03EF3D32-FFB6-5C2C-FFF5-FAD25186B0F7http://plazi.cs.umb.edu/GgServer/search?taxonomicName.isNomenclature=true&taxonomicName.exactMatch=true&taxonomicName.order=Diptera&taxonomicName.species=alastorhttp://www.morphbank.net/Browse/ByImage/?tsn=999022155http://eol.org/pages/718862/overviewhttp://asiloidflies.si.edu/specimen-map-Apioceridae?genus=Apiocera&species=alastorhttp://asiloidflies.si.edu/specimen-table-Apioceridae?genus=Apiocera&species=alastorAsilusalastorApiocera (Ripidosyrma) alastor , new combination by Apiocera (Ripidosyrma) africana Paramonov, 1950, new junior synonymy by Apiocera (Ripidosyrma) alastorB745D2C2-8CB6-4C95-970E-B3C3246B7A73 Plazi original treatment GUID B745D2C2-8CB6-4C95-970E-B3C3246B7A73 ZooBank GUID South Africa BMNH, SAMC, ZSMC.Institutions with specimens: Yeates, 1994urn:lsid:zoobank.org:act:0335A2B2-8650-419C-A645-7FD4A441B690http://dx.doi.org/10.5281/zenodo.15791http://hdl.handle.net/10520/AJA03040798_231http://treatment.plazi.org/id/0335A2B2-8650-419C-A645-7FD4A441B690http://plazi.cs.umb.edu/GgServer/search?taxonomicName.isNomenclature=true&taxonomicName.exactMatch=true&taxonomicName.genus=Apiocera&taxonomicName.species=badipeniculatahttp://www.gbif.org/species/1611753http://eol.org/pages/424464/overviewhttp://asiloidflies.si.edu/specimen-map-Apioceridae?genus=Apiocera&species=badipeniculatahttp://asiloidflies.si.edu/specimen-table-Apioceridae?genus=Apiocera&species=badipeniculataApiocerabadipeniculataTankwa Karoo National Park, straddles the border of the Northern and Western Cape provinces.South Africa (Northern Cape/Western Cape). The type locality of Tankwa Karoo, which can be interpreted to overlap with the SAMC.Institutions with specimens: Melander, 1907urn:lsid:zoobank.org:act:5C0A5C13-EC67-428B-9712-4734564EA06Dhttp://biodiversitylibrary.org/page/47141510http://dx.doi.org/10.5281/zenodo.16194http://biodiversitylibrary.org/page/47141509http://treatment.plazi.org/id/5C0A5C13-EC67-428B-9712-4734564EA06Dhttp://plazi.cs.umb.edu/GgServer/search?taxonomicName.isNomenclature=true&taxonomicName.exactMatch=true&taxonomicName.genus=Apiocera&taxonomicName.species=braunsihttp://www.gbif.org/species/1611644http://www.morphbank.net/Browse/ByImage/?tsn=999022156http://eol.org/pages/718861/overviewhttp://asiloidflies.si.edu/specimen-map-Apioceridae?genus=Apiocera&species=braunsihttp://asiloidflies.si.edu/specimen-table-Apioceridae?genus=Apiocera&species=braunsiApiocerabraunsiSouth Africa AMGS, BMNH, NMSA, SAMC, SANC, SMNS, TMSA, USNM, ZSMC.Institutions with specimens: A.braunsi. Asilusalastor to Apiocera, notes that he has seen "part of the type series of braunsi", but he does not indicate where these specimens are deposited. However, A.braunsi, states that he has only seen a single specimen, a male. What appears to have happened is that J. Brauns collected three specimens on 1 January 1905 of an Apiocera species and dispersed them shortly thereafter to two individuals. One specimen was sent along with other Asilidae and Mydidae to A.L. Melander in the USA and two specimens ended up in the Transvaal Museum  in Pretoria where Brian Stuckenberg must have studied them. The entire Transvaal Museum Diptera collection was donated to the KwaZulu-Natal Museum (NMSA) in 1974 . Therefore, we regard the USNM specimen to be the holotype and the NMSA specimens  as non-type specimens.Two museum collections claim to house the holotype of  in 1974  and thisholotype . Yeates Apioceridae by utilizing freely available digital resources from numerous data repositories. While the initial work to locate information in or upload images, treatments, and publications to data repositories might seem daunting, the long-term effects of using openly accessible digital content through persistent identifiers and linked data far outweighs the time spent. Open-access science, in which taxonomists share data freely and electronically, will on the one hand advance the discipline within the biological sciences and on the other hand make taxonomic hypotheses easily testable through the re-use and re-purpose of previously gathered data with the addition of new data. A taxonomist's dream of having access to all previously published descriptions, images, illustrations, and notes of a particular taxon can become true if the community supports sharing research results in machine-readable form and structured data repositories as exemplified above.The present cybercatalog summarizes taxonomic information on Afrotropical At the same time, this approach is opening up taxonomy to the widest possible community and only this step allows to position taxonomy as a central resource within the life sciences, applied fields of study, and beyond to the public and society at large. We taxonomists have always claimed to fulfill this role, but never understood why it did not happen.


Plasmodium vivax are rare causes of HLH. We report the first ever case of a young man who developed fatal HLH that complicated Dengue Hemorrhagic Fever (DHF) and Plasmodium vivax infection.Hemophagocytic lymphohistiocytosis (HLH) is a rare disorder. Dysfunction of cytotoxic T and natural killer (NK) cells causes uncontrolled activity of lymphocytes and histiocytes which leads to HLH. Infections, malignancies, and autoimmune disorders are associated with development of HLH. Dengue and  Hemophagocytic lymphohistiocytosis (HLH) is a rare disorder which can be familial or acquired. Acquired HLH complicates a number of viral, bacterial, and parasitic infections. It is also associated with autoimmune disorders and certain malignancies like T cell lymphoma. Familial HLH has autosomal recessive transmission and is seen in infants less than 18 months old. HLH is associated with high mortality . Plasmodium vivax (P. vivax) malaria infections.HLH is characterized by prolonged fever and sepsis-like syndrome. It presents with nonspecific clinical features like fever and hepatosplenomegaly. Laboratory findings include cytopenias, raised serum aminotransferase levels, hypofibrinogenemia, hypertriglyceridemia, hyperferritinemia, and increased lactic dehydrogenase (LDH) levels. Dengue and malaria are important but rare causes of HLH , 4. We rA 19-year-old, previously healthy, motor mechanic was admitted with 13-day history of high grade fever, headache, retroorbital pain, and myalgias. One day before admission he became restless and irritable. On examination his pulse was 104/minute, temperature 101°F, respiratory rate 24/minute, and blood pressure 110/70 mmHg (pulse pressure 40 mmHg). He was jaundiced. Abdominal examination revealed tender hepatomegaly with liver span of 18 cm. Chest examination was suggestive of right sided mild to moderate pleural effusion. He was noted to be drowsy, disoriented, and confused. His Glasgow Coma Scale (GCS) was 12/15 . No focal neurological deficit or signs of meningeal irritation were noted. The rest of the clinical examination was unremarkable.Ultrasonographic examination at admission showed thick walled gall bladder , hepatomegaly (17 cm), splenomegaly (13.6 cm), mild to moderate ascites, and mild to moderate pleural effusion. CT scan brain without contrast showed brain edema. Respiratory alkalosis was noted on arterial blood gas (ABG) analysis. ECG was normal and chest X-ray showed right pleural effusion. Other investigations are shown in Day 1. An initial assessment of DHF complicated by dengue fulminant hepatic failure was made with differential diagnosis of encephalitis, cerebral malaria, sepsis, and multiorgan dysfunction. Injectable artesunate, piperacillin/tazobactam, acyclovir, and dexamethasone  were started. Standard DHF management was commenced. Lactulose and N-acetyl cysteine were also administered per nasogastric tube.Day 2. Clinical condition remained the same. Investigation showed positive dengue markers  and positive smear examination for malarial parasite (gametocytes and schizonts of P. vivax). Markers for hepatitis A, hepatitis B, hepatitis C, and hepatitis E turned out negative. Ferritin  and triglycerides levels  were elevated. Serum fibrinogen levels were on lower side ( P. vivax coinfection was considered. Dexamethasone was replaced with methyl prednisolone (30 mg/Kg/day for 3 days). PCR for dengue was also sent along with Congo fever markers.wer side . DiagnosDay 3. Patient's conscious level, restlessness, and irritability improved. Two episodes of melena and epistaxis occurred. Intravenous proton pump infusion was started and fresh whole blood was transfused.Day 4. Patient became breathless. Chest examination revealed bilateral wheeze and crepitations. Oxygen saturation dropped to 74%. On ABGs, PO2 was 48 mmHg. CXR showed bilateral infiltrates. Diagnosis of ARDS was considered and patient was started on ventilatory support.Day 5. Patient's condition remained the same. Mechanical ventilation was continued. ECG showed sinus tachycardia. Dexamethasone was restarted at a dose of 10 mg/m2 per day.Day 6. Fever and hypotension developed. Antibiotics were modified to Imipenem and Vancomycin considering the diagnosis of hospital acquired infection. Ultrasound scan revealed right sided moderate to massive pleural effusion. Thoracocentesis was done and straw-colored 1-liter fluid was aspirated which was transudative on laboratory evaluation. Echocardiography showed dilated left ventricular (LV) and globally reduced LV systolic function. Ejection fraction was 25%. Diagnosis of dengue myocarditis was also considered and digoxin was added to treatment regimen.Day 7. Patient's condition did not improve and management continued. Repeat ultrasound scan showed mild bilateral effusion. Bone marrow biopsy was done; it was suggestive of HLH . Congo fever markers were negative.HLH is an uncommon inflammatory disorder which is characterized by activation of macrophages that cause phagocytosis of blood cells in bone marrow. HLH is diagnosed when 5 out 8 diagnostic criteria are fulfilled , 5–7. Crα and IFN gamma which possibly contribute to development of HLH syndrome [Dengue is an uncommon cause of HLH –12. In dsyndrome . Most ofsyndrome . Our patsyndrome . HLH duesyndrome .P. vivax malaria is another unusual cause of HLH syndrome [ P. vivax detection confirms diagnosis of malaria.syndrome , 11. Malsyndrome . Fever, γ monoclonal antibodies, and hematopoietic stem cell transplantation are additional available modalities for HLH treatment [Treatment of the cause, supportive therapy, and suppressing immune response are main stay of HLH management . Specifireatment . P. vivax associated HLH is generally good if appropriate antimalarials are used [Complexity of clinical situation, difficulty in differentiation from sepsis, and multiorgan dysfunction lead to delayed diagnosis in HLH . Untreatare used –19. 0–10are used , 20. Denare used . P. vivax. Malaria and dengue coinfection can occur in countries where these are endemic; however HLH complicating the two infections has never been reported according to our knowledge. Both these diseases come in the differential diagnosis of acute febrile illness. The question is what predominantly caused HLH in our patient. P. vivax was not detected after artesunate administration on smear and bone marrow examinations of our patient making it less plausible etiological factor for HLH development.Our patient had dual infection with dengue virus and P. vivax infection may have additional variable contribution.Persistence of fever > 8 days in dengue alerts towards diagnosis of HLH, as duration of dengue febrile phase is generally 3–7 days , 23. AddOur patient had cardiac involvement. Troponin T was negative twice however. It is known that Troponin autoantibodies can result in false negative Troponin T results . Was it  P. vivax, corticosteroids for HLH, cover for infection, and supportive therapy. He however did not receive etoposide, methotrexate, and immunoglobulins. HLH related neurological, respiratory, hepatic, and cardiac involvement lead to multiorgan dysfunction which caused death in our patient. Infection possibly contributed to it as well, which can complicate the scenario because of HLH pathophysiology and immunosuppressive medications like steroids used in treatment. HLH protocol was not employed in our patient that may have altered the outcome. Remembering that HLH can occur in similar settings can help in early diagnosis and institution of HLH protocol.Our patient received focused DHF management, antimalarials for


Apolygus lucorum (Meyer-Dür) host plant use in successive spring, summer and winter seasons at one single location . Data were used to quantify changes in host plant breadth and host fidelity between seasons. Host fidelity of A. lucorum differed between seasons, with 87.9% of spring hosts also used in the summer and 36.1% of summer hosts used in winter. In contrast, as little as 25.6% host plant species were shared between winter and spring. Annual herbaceous plants are most often used for overwintering, while perennial woody plants are relatively important for initial population build-up in the spring. Our study contributes to an improved understanding of evolutionary interactions between A. lucorum and its host plants and lays the groundwork for the design of population management strategies for this important pest in myriad crops.In herbivorous insects, host plant switching is commonly observed and plays an important role in their annual life cycle. However, much remains to be learned about seasonal host switching of various pestiferous arthropods under natural conditions. From 2006 until 2012, we assessed  Rhopalosiphum padi (L.) and the cotton aphid, Aphis gossypii Glover use woody host plants as primary hosts, and switch to herbaceous plants as secondary hosts in the summer [Nezara viridula L. adults frequently moving from one plant species to another [Due to temporal and spatial variation in host plant availability, and specific nutrition and/or habitat requirements of insects, most phytophagous insects exploit multiple plant species . Also, me summer . Host sw another . Hence,  another ,8.As plant species temporally and spatially differ in nutritional quality, host plant switching can remediate qualitative nutritional impediments associated with certain host species  and helpIn their exploitation of host plants, specialist herbivores exhibit high host fidelity , whereas generalists show low fidelity . Also, oLygus lineolaris , frequently alter host plants and habitats to locate suitable food sources [Apolygus lucorum (Meyer-Dür) has become the primary pest of several key agricultural crops and fruit trees over the past decade [A. lucorum adults exploit a broad range of host plants, including crops, fruit trees and common weed species [A. lucorum has been recorded on a total of 66 and 86 plant species during respective spring and winter seasons, and their host species greatly differed [A. lucorum in summer season. We hypothesized that polyphagous A. lucorum would exhibit host switching and have low host fidelity between seasons. However, the degree to which A. lucorum relies on certain plant species, families or growth forms (inc. herbaceous and woody plants) during successive seasons remains to be quantified.Mirid bugs (Hemiptera: Miridae) are found in natural and agricultural ecosystems throughout the world, and many of them are generalists, exhibiting diverse feeding habits or preferences  . Several sources ,26,27. It decade ,29. This species ,31. To ddiffered ,33. HencA. lucorum host plant use [A. lucorum exploitation of certain plant species, families or growth forms, and determine the degree of host fidelity and temporal changes in host breadth of this mirid bug. These findings will provide further insights into seasonal host plant use of polyphagous herbivores and contribute to the design of area-wide management tactics for this key pest in Chinese agro-landscapes [In this study, we analyzed multi-year data on lant use ,33. Our ndscapes ,35,36.No specific permits were required for the described field studies.A. lucorum host plant use were carried out from December to May 2006–2009 for winter hosts [A. lucorum adults or nymphs were found were defined as ‘host plants’ of this species [Field surveys of er hosts , from Aper hosts , and froer hosts  or with  species .2 screen-cage with about 10 horsebean plants (Vicia faba L.). Horsebean is an important host plant and suitable food source of A. lucorum [2 plant material of one plant species; and each plant species had 1–5 cages per year, depending on the amount of plant material. From April to May, when A. lucorum over-wintering eggs hatch and emergent nymphs move to surrounding horsebean plants for feeding, we surveyed nymphal density on the plants within each cage, using a standard white pan beating method [A. lucorum nymphs indirectly indicated presence of its overwintering eggs, which are too small to be counted with the naked eye [For the survey of winter hosts, above-ground parts of different plant species were collected in late March and subsequently placed in a 3 m lucorum ,31. Withg method . Emergenaked eye .A. lucorum individuals in the pan was recorded [A. lucorum with a given plant species, we only selected uniform patches or carefully picked single stems of a given plant species for sampling. Surveys were conducted every 3–5 days, and A. lucorum individuals were counted for each sample. As part of each sampling activity, the exact area covered by each sampled plant was recorded. For common plant species, 10–20 samples were randomly selected per survey date, and a single sample consisted of a total area of 2–5 m2, while for uncommon species, all plants at a given site were sampled. Sampling was repeated 11–16 times per year [For the survey of spring host plants, we examined different plant species using a standard white pan beating method from mid-April to mid-June. More specifically, entire specimens of most herbaceous plants or young branches of fruit and other trees were immediately shaken over a 40 x 26 x 11 cm white pan, and the number of dislodged recorded ,38. To uper year .A. lucorum from July to September. A total of 3 sampling events (once per month) were conducted per year, with 10–20 random samples taken per plant species and event. During the successive seven summers, a total of 252 species of plants were sampled and each plant species was sampled for at least 2 years.For the survey of summer host plants, we used the above method to sample A. lucorum per plant species during each season was computed on a yearly basis, i.e., by dividing the total number of individuals on a given plant species by the total sample area covered by this respective plant. A. lucorum density was compared between different plant species using a two-way un-replicated ANOVA with year and plant species as fixed factors, if the data met normality and homogeneity of variance; otherwise, the data were analyzed using a non-parametric test (Friedman’s test) instead. Its average density per plant group  during each season was also computed on a yearly basis. The density of A. lucorum was compared between different plant groups using a one-way ANOVA with a Tukey test. A Chi-square test was performed to compare between-season host use ratio . All statistical analyses were performed using SAS [The average density of sing SAS .A. lucorum  V. Krecz, Cicer arietium L., Codonopsis pilosula (Franch.) Nannf., Echinochloa crusgalli (L.) Beauv., Eleusine indica (L.) Gaertn., Equisctum ramosissimum Desf., Ginkgo biloba L., Liquidambar formosana Hance, Pinellia pedatisecta Schott, Platycladus orientalis (L.) Franco, Polygala tenuifolia Willd., Setaria viridis (L.) Beauv., Syringa vulgaris L., Trigonella foenum-graecum L., Vaccaria segetalis (Necr.) Gracke., and Zanthoxylum bungeanum Maxim   plants and 76 herbaceous weeds, were identified as host plants of  lucorum . Most imum Maxim . Through 0.0001) .A. lucorum eggs successfully hatched on 86 plant species, among which 16 weeds and fruit trees were considered as key overwintering hosts [In the winter, ng hosts .A. lucorum. Among these species, 6 hosts were identified as dominant host species due to their wide distribution and high population densities of A. lucorum [In the spring, 66 plant species were found to be hosts of  lucorum .Host plant species. Over the course of a year, A. lucorum exhibited a highly variable host breadth, using 66 host plants in spring, 233 in summer and 86 in winter  (n winter . Of the  0.0001) .Natural plant death led to 58 (of 66) spring host plants being monitored during summer, and 134 (of 233) summer host plants monitored during the winter. Lastly, only 63 (of 86) winter hosts were alive during spring, of which 35 plant species were at seedling stage .Plant growth forms. Among all different species of host plants, 149, 70 and 23 species were annual herbaceous plants, perennial herbaceous plants and perennial woody plants, respectively. The extent to which distinct plant growth forms were used as A. lucorum hosts was significantly different between successive seasons . Annual herbaceous plants accounted for 47.0%, 61.4% and 69.8% of all host plants in spring, summer and winter, respectively; perennial herbaceous plants being 28.8%, 29.2% and 22.1%, and perennial woody plants occupying 24.2%, 9.4% and 8.1% in the distinct seasons , Sp-Su (for spring and summer), Sp-Su-Wi , and Wi-Sp (for winter and spring) . More specifically, seasonal density of A. lucorum on Wi-Sp during the spring was significantly higher than in the three other categories. In summer, its densities also significantly differed between the following plant categories: Sp-Su, Sp-Su-Wi, Su (only for summer) and Su-Wi (for summer and winter) . More so, A. lucorum density on Sp-Su-Wi and Su-Wi was significantly higher than on Sp-Su and Su. In winter, A. lucorum densities on Sp-Su-Wi, Su-Wi, Wi (only for winter) and Wi-Sp varied within the range of 0.3–1.6 individuals per square meter and did not differ between plant categories  ( 0.3698) .A. lucorum densities significantly differed in summer season , but not for spring  and winter . In summer, its densities on perennial herbaceous plants, annual herbaceous plants, and perennial woody plants significantly decreased in turn ,  in turn .A. lucorum overall density showed considerable variation, with the highest population levels recorded during summer and the lowest densities during winter. Among 21 year-round hosts, 7 species were found with relatively high A. lucorum population. On Artemisia annua, Artemisia argyi and Artemisia lavandulaefolia, A. lucorum attained high population densities in the summer and winter, and low density in the spring. Its population levels were high in the summer on H. scandens and M. sativa, whereas its density peaked in the winter on Vitis vinifera L. and Ziziphus jujuba Mill.  [Lygus lineolaris Palisot de Beauvois  [Lygus rugulipennis Poppius  [Pseudatomoscelis seriatus (Reuter)  [L. lineolaris [P. seriatus [Rapistrum rugosum L. Allioni and Ratibida columnifera  Wooton and Standley were primary weed hosts of L. lineolaris during the early season, Conyza canadensis L. Cronquist and Ambrosia trifida L. were primary weed hosts during the midseason and late-season, respectively. Sisymbrium irio L. and Lamium amplexicaule L. sustained L. lineolaris populations during the overwintering period [A. lucorum. Combining these data with literature records [A. lucorum to be associated with 288 plant species in 54 families and noted important seasonal changes in its host plant range. Although the possibility of individual transfer between different plant species was not fully evaluated and limited sampling might lead to underestimate its host range, the results from this 7-year study definitely provide a comprehensive set of information on host plant range of A. lucorum, especially for identifying the dominant ones. This pattern of multiple-host plant use of A. lucorum may be one of the reasons why A. lucorum quickly attained pest status in several agricultural crops throughout China [Host plant use and host switching play an important role in the evolutionary success of herbivorous insects ,40,41. Tamilies) , Lygus lamilies) , Lygus ramilies) , and Pseamilies) ; and seaneolaris ,26,27, aseriatus . For exa records ,32,33, wut China .A. lucorum to be likely determined by seasonal density of the insect and local availability of its host plants. Possibly, high A. lucorum density during summer months could explain its broader host breadth in this season [A. lucorum diet over summer , its spring hosts appear to be largely employed during summer. Also, as little as 12.1% of its spring hosts  get abandoned in summer due to their absence during that season. Similar food requirements of A. lucorum populations and identical host plant phenology may further explain similarities in host plant use between these two successive seasons [Host plant diversity and food quality have been identified as the major drivers of host switching in herbivorous insects ,46,47. Os season . Despite seasons .A. lucorum winter host repertoire. Similar A. lucorum feeding requirements between summer and fall/winter may lead to high plant fidelity between seasons. However, especially for herbaceous annuals, plant senescence lead to 99 plant species being abandoned as hosts  winter hosts during spring [A. lucorum is found during spring season [On the other hand, host plant use between winter and spring greatly differs, with only 25.6% of winter hosts used in spring. We suspect that winter hosts mainly act as refuge and (over-winter) oviposition sites for d nymphs . The dist plants ,49. Femat plants . Contrart plants ,51. Durit plants , with adt plants ,54. As hg spring . Such phg season .A. lucorum use of Asteraceae and Fabaceae is not necessarily related to specific feeding preferences, but rather to the species richness and wide distribution of both plant families in northern China [Between different seasons, the ratio of host plant use among the two leading families  and three plant growth forms was similar. We suspect that high rn China . SimilarA. lucorum, but its population levels on a given host greatly fluctuated over time. For example, A. lucorum eggs overwinter on Z. jujube and V. vinifera [A. lucorum varies between seasons [Vicia faba L.), in summer  or winter . Additionally, the role of particular host plants/habitat patches on population dynamics of insect pests is greatly affected by landscape composition, (crop) management and other factors [L. hesperus in cotton, but high insecticide application can change it into a sink; alfalfa also is a primary host plant, however it can divert L. hesperus adults away from nearby cotton depending upon the management of the former plant species [A. lucorum population dynamics need to be further investigated, especially at the landscape level.A total of 21 plant species  served as year-long hosts of vinifera ,33,55, b seasons ,56. Othe factors ,56. For  species ,57. Lu e species  showed t species . Hence, A. lucorum [Humulus scandens (Lour.) Merr.,Z. jujuba, and V. vinifera, could be central in devising ways to prevent rapid A. lucorum population buildup in summer [A. lucorum population levels on some plant species  were much higher than on others (A. lucorum host plant location [A. lucorum choice of its preferred host species. Preferred host plants could then be used as trap crops or for incorporation in push-pull strategies [Host breadth during spring and winter is relatively limited, and its spring density is low. This provides an opportunity for regional population suppression of  lucorum ,33. Sprin others , hintinglocation ,58, follrategies ,59,60,61A. lucorum seasonal host plant use and host switching behavior helps elucidate the interaction between this polyphagous insect and its host plants, and improve our understanding of its meta-population dynamics in agricultural landscapes.In conclusion, our assessment of


Leishmania (Viannia) braziliensis complex is responsible for most cases of New World tegumentary leishmaniasis. This complex includes two closely related species but with different geographic distribution and disease phenotypes, L. (V.) peruviana and L. (V.) braziliensis. However, the genetic basis of these differences is not well understood and the status of L. (V.) peruviana as distinct species has been questioned by some.The L. (V.) peruviana isolates (LEM1537 and PAB-4377) using Illumina high throughput sequencing and performed comparative analyses against the L. (V.) braziliensis M2904 reference genome. Comparisons were focused on the detection of Single Nucleotide Polymorphisms (SNPs), insertions and deletions (INDELs), aneuploidy and gene copy number variations.Here we sequenced the genomes of two L. (V.) peruviana isolates  against the L. (V.) braziliensis M2904 reference genome while only 26,853 variants separated both L. (V.) peruviana genomes.We found 94,070 variants shared by both L. (V.) peruviana isolates against L. (V.) braziliensis M2904 and revealed two L. (V.) braziliensis pseudogenes that are likely to have coding potential in L. (V.) peruviana. Chromosomal read density and allele frequency profiling showed a heterogeneous pattern of aneuploidy with an overall disomic tendency in both L. (V.) peruviana isolates, in contrast with a trisomic pattern in the L. (V.) braziliensis M2904 reference.Analysis in coding sequences detected 26,750 SNPs and 1,513 indels shared by both L. (V.) peruviana, and the likely absence of expanded amastin gene arrays.Read depth analysis allowed us to detect more than 368 gene expansions and 14 expanded gene arrays in L. (V.) peruviana and L. (V.) braziliensis and the presence of different gene and chromosome copy number variations support the classification of both organisms as closely related but distinct species.The greater numbers of interspecific SNP/indel differences between L. (V.) peruviana suggests the possibility that these may contribute to some of the unique features of its biology, including a lower pathology and lack of mucosal development.The extensive nucleotide polymorphisms and differences in gene and chromosome copy numbers in The online version of this article (doi:10.1186/s12864-015-1928-z) contains supplementary material, which is available to authorized users. Leishmania. It is transmitted by the bite of an infected female phlebotomine sand fly belonging to the genus Lutzomyia in the New World and Phlebotomus in the Old World [Leishmaniasis is a neglected tropical disease caused by a group of digenetic protozoan belonging to the genus ld World . Leishmald World , 3.Leishmaniasis presents a wide spectrum of clinical manifestations that ranges from cutaneous leishmaniasis (CL) that affects tissues near the sand fly bite to mucosal leishmaniasis (ML) that is characterized by a progressive ulceration at the nares and nasal septum to the lethal visceral leishmaniasis (VL) that disseminates to visceral organs causing hepatomegaly, splenomegaly and even death , 4.L. (V.) braziliensis complex is one of the most important Leishmania group in the New World [L. (V.) peruviana and L. (V.) braziliensis) [L.(V.) peruviana is an endemic species in Peru with a limited distribution range within the Andean and inter-Andean valleys with some narrow areas of sympatry with L. (V.) braziliensis [The ew World . It compliensis) , althougliensis) . As curriliensis , 8.L. (V.) peruviana causes CL and has been isolated from peridomestic mammals including dogs, mice and opossums, revealing its zoonotic status [L. (V.) braziliensis is widely distributed in South America, although primarily in the Amazon Basin, and is referred as an anthropozoonosis [L. (V.) braziliensis infections have a substantially higher potential to manifest as ML than any other new world leishmaniasis species, including L. (V.) peruviana [c status . L. (V.)zoonosis . L. (V.)eruviana , 11. HowLeishmania it has allowed to rapidly and comprehensively analyze a wide range of mutation types, including gene copy number variations (CNV) and aneuploidy [Leishmania presents a mosaic structure that may contribute to gene expression changes in response to environmental alteration modulating parasite phenotypes [Next generation sequencing has provided several advantages for characterizing species-specific traits across the genomes of several organisms. In euploidy . Recentleuploidy . These seuploidy . Moreoveenotypes , 14.. (V.) peruviana isolates against the reference genome M2904 of L. (V.) braziliensis. Comparative assessments have shown important differences in chromosome and gene copy number between both species. These analyses may serve to improve our understanding of parasite variation between these two closely related species that could be linked to their different disease phenotypes and to provide further insights into their status as distinct species.In this study, we have conducted a comparative genomics analysis of two Lde novo and reference based assembly approach  to generate a draft genome for each strain. L. (V.) peruviana mapped reads showed an overall 92.51 % mapping rate for PAB-4377 and 95.87 % for LEM1537 against L. (V.) braziliensis. Median genome coverage estimated from mapped reads into 6,899 single copy genes was of 59.1 and 35.0 for PAB-4377 and LEM1537, respectively.We used a combined L. (V.) peruviana assemblies resulted in 28.51 and 25.27 megabases that were generated from 11,504 and 29,816 contigs in PAB-4377 and LEM1537, respectively. The resulting ordered assemblies consisted of 37 pseudo-chromosomes, due to the split of chromosome 20 in the L. (V.) braziliensis M2904 reference genome (LbrM.20.1 and LbrM.20.2) and a pseudo-chromosome containing unordered scaffolds (Chromosome 0).The L. (V.) braziliensis and L. (V.) peruviana calculated with MUMmer [L. (V.) braziliensis and L. (V.) peruviana (identity of 87.58 % for PAB-4377 and 77.1 % for LEM1537), and a closer relationship between the two L. (V.) peruviana isolates (99 %).The overall identity between h MUMmer  confirmeVariants were identified following filtering for quality, read depth and haplotype score as described in the methods.L. (V.) braziliensis and L. (V.) peruviana PAB-4377  and L. (V.) peruviana LEM1537 , respectively. Of these; 94,070 variants were shared between the two L. (V.) peruviana isolates. In contrast, the two L. (V.) peruviana isolates showed fewer variants among them . This finding is consistent with the high similarity obtained with MUMmer3 between both L. (V.) peruviana isolates and the greater difference with L. (V.) braziliensis.Comparisons identified 144,079 and 136,946 variants between L. (V.) braziliensis and L. (V.) peruviana while intra L. (V.) peruviana variation is substantially lower. For comparison, a previous comparative study between L. (L.) infantum and L. (L.) donovani reference genomes found that 156,274 nucleotide changes differentiate between these closely related species [L. (V.) braziliensis and L. (V.) peruviana.Our results show that there is significant genetic differentiation between  species , comparaL. (V.) braziliensis that were shared by the two L. (V.) peruviana lines. Of these; 26,750 SNPs were located in 6,114 coding DNA sequences (CDS)  peruviana from L. (V.) braziliensis resulted in 270 genes with high SNP density regions between both species  braziliensis pseudogenes  appeared to be functional in L. (V.) peruviana. LbrM.28.2130 codes for an X-pro, dipeptidyl-peptidase, serine peptidase and has orthologs in other Leishmania species from the Old and New World suggesting that it could be functional in L. (V.) peruviana. Peptidases have an important role in parasite survival, invasion, metabolism and host-parasite interaction [L. infantum. It has been shown that Leishmania are pteridine auxotrophs and rely on a network of folate and biopterin transporters. Pteridine levels have a strong influence on metacyclogenesis in L. (L.) major [Two eraction , highlig.) major .L. (V.) braziliensis M2904 strain is mostly trisomic [Chromosome numbers were estimated by the average read density to each chromosome, and normalized to an assumed overall genome ploidy of 2n. Normalized chromosome copy number clustered around “disomy” although with significant departures from non-integral values evident for some chromosomes Fig. . This fitrisomic .Fig. 1ChThe most pronounced departure from disomy occurred in chromosome 31, which presented a read depth between tetrasomy to hexasomy in PAB 4377 and trisomy in LEM1537 Fig. .Fig. 2ChL. (L.) mexicana [In both samples, chromosomes 1–5 and 7 appear to be closer to monosomy. This characteristic has also been estimated for chromosomes 1 and 3 of mexicana . InteresLeishmania remains to be investigated [It has been suggested that mosaic aneuploidy could be a mechanism of rapid parasite adaptation in response to environmental changes within its host  and it hstigated .A second approach for assessing chromosome number is based upon allele frequencies. For disomic chromosomes, heterozygous SNPs should cluster around 50 %, while trisomic chromosomes should show two peaks at 33 and 67 % and tetrasomics at 25, 50 and 75 %, .Allele frequency counts for each predicted heterozygous SNP further confirmed the overall disomic tendency Fig.  and the Chromosomes with discordance between read depth analysis and their allele frequencies included chromosome 5, 7, 13, 17 and 19 that presented a tetrasomic or a mixture of trisomic and disomic patterns in PAB-4377  mexicana [Interestingly, chromosome 31 that has been identified as supernumerary in both isolates appears to have disomic pattern  braziliensis reference genome  braziliensis M2904 resulted in 18 tandem gene arrays from which only three arrays were shared with L. (V.) peruviana  braziliensis seems to be not expanded in L. (V.) peruviana.The same analysis in Amastins have been shown to be highly expressed in the amastigote life stage and appear to mediate host-parasite interactions allowing infection and survival . While tWe found 398 and 942 dispersed duplicated genes in PAB-4377 and LEM1537 with 360 expansions in common  peruviana as has been previously shown in L. (V.) braziliensis [We did not detect an increase in copy number in GP63 genes in iliensis  reinforciliensis .Leishmania presenting different roles in the vector and mammal host that aim to protect parasites from host immune responses and promote infection [L. (V.) peruviana could affect parasite-host interactions and influence its distribution and clinical manifestation with lack of mucosal development.The zinc-metalloprotease GP63 stands out as a major virulence factor in nfection . TherefoThe marked intra-species difference in dispersed duplicated genes shows that extensive variation in gene copy number can occur between isolates belonging to the same species and supports the hypothesis that chromosome and gene CNV act as a mechanism of rapid parasite adaptation , 26.Leishmania and were proposed as a mechanism of rapid parasite adaptation to different environments and pressures in the host. Our study shows that there are major differences regarding gene copy number variations and aneuploidy even between closely related Leishmania species.Extensive chromosomal and gene copy number variations have been described in L. (V.) braziliensis, L. (V.) peruviana presents a different set of expanded gene arrays that can result in different expression profiles between both species. Moreover, high SNP and indel counts as well as extensive variation in chromosome and gene copy numbers between L. (V.) peruviana and L. (V.) braziliensis support maintaining the classification of both organisms as closely related but distinct species.Although highly similar to L. (V.) peruviana and L. (V.) braziliensis isolates and the use of transcriptomic data are needed to assess if these differences are conserved across isolates of L. (V.) peruviana and reveal how tandem gene arrays and CNV affect genome expression.Further analysis including a greater number of L. (V.) peruviana isolate PAB-4377 was kindly provided by the U.S. Naval Medical Research Unit No. 6 (NAMRU-6) and the LEM1537 (MHOM/PE/84/LC39) isolate was obtained from the Montepellier reference center.L. (V.) peruviana by Multilocus Enzyme Electrophoresis (MLEE) and sequencing of the Manose Phosphate Isomerase and 6-phosphogluconate dehydrogenase genes. LEM1537 is a L. (V.) peruviana reference strain (MHOM/PE/84/LC39) and has been widely characterized by MLEE.PAB-4377 was confirmed as L. (V.) braziliensis M2904 genome was obtained from the Tritryp database (http://tritrypdb.org/) to serve as a reference for comparative analysis.Libraries consisting of 350 bp fragments were obtained and 100 bp paired end reads were generated at the Genome Technology Access Center (GTAC) at Washington University in St. Louis by Illumina HiSeq 2000. The version 6 of the L. (V.) peruviana reads were filtered by quality using Trimmomatic [mmomatic  with a mDe novo and reference based assembly approach  was used to generate a draft assembly for each sample. Briefly, De Novo assemblies were generated using the Velvet optimizer perl script under Velvet version 1.2.10 [A combined n 1.2.10 . Draft an 1.2.10  and corrn 1.2.10 .L. (V.) braziliensis M2904 genome using Bowtie2 [For reference-based assembly, reads from each sample were mapped against the  Bowtie2 . Redunda Bowtie2  using baDe Novo and referenced based sequences of each sample were combined using the ZORRO hybrid assembler as previously described [L. (V.) braziliensis M2904 genome as a reference sequence.escribed . The finescribed . Scaffolescribed  using thL. (V.) peruviana genomes and the reference L. (V.) braziliensis. Briefly, identity scores and number of bases from best local alignments among assembled and reference genomes were retrieved and normalized with the total number of bases in the draft genome in order to compute a global identity score.MUMmer3  was usedRead and assembly files are available through the European Nucleotide Archive under the project number PRJEB7263.L. (V.) peruviana and L. (V.) braziliensis and determine their potential effects on coding sequences, L. (V.) peruviana reads were mapped onto the L. (V.) braziliensis M2904 reference genome using Bowtie2 and analyzed using the recommended parameters of GATK [L. (V.) peruviana isolates.To detect SNPs between  of GATK . BrieflyL. (V.) peruviana genome, we filtered variant calls of PAB-4377 and LEM1537 selecting only SNPs shared by both isolates to limit the potential impact of within-species SNP variability and minimize incorrect SNP calling. The combined variant called was used as input for SnpEff [To analyze the effects of SNPs in coding regions of the L. (V.) peruviana from L. (V.) braziliensis we employed a custom Perl script to screen the genes with variant calls. These genes were analyzed using a sliding window of 1000 nucleotides to report the region with the highest SNP density and the number of SNP that it presented. Genes with significant SNP calls were detected using the ROUT test under Graph Pad Prism V5 [To find potential targets sequences to accurately discriminate Prism V5 L. (V.) braziliensis pseudogenes from the Tritryp database and compared them against L. (V.) peruviana to detect potential pseudogenes that remained functional in L. (V.) peruviana. Briefly, L. (V.) peruviana amino acid fasta sequences were generated using SAMtools Mpileup and translated into amino acids for sequence alignment against L. (V.) braziliensis pseudogenes in ClustalΩ [We downloaded ClustalΩ .Allele frequencies for PAB-4377 and LEM1537 assemblies were obtained from filtered SAMtools Mpileup results as described elsewhere . BrieflyL. (V.) braziliensis and L. (V.) peruviana. First, OrthoMCL was used to select single copy genes from the proteomes of L. (V.) braziliensis, L. (L.) mexicana and L. (L.) major, L. (L.) infantum, L. (L.) donovani and L. (Sauroleishmania) tarentolae  values for each chromosome by the median FPKM of the whole genome. We plotted the mean and standard deviations from the three approaches using Graph Pad Prism V5.We normalized haploid copy numbers with the average chromosome ploidy calculated from the allele frequency analysis to estimate chromosome ploidy. Plots for each chromosome were generated in R using a sliding window of 10 kilo bases.Gene Ontology codes that were significantly overrepresented in the genes of supernumerary chromosomes were detected using the hypergeometric distribution analysis in BiNGO  with BenWe defined tandem gene arrays as groups of genes that are located contiguously in a chromosome and that share a homology relationship. Dispersed gene duplications are defined as genes that are duplicated and do not belong to any tandem array.L. (V.) braziliensis M2904 and a coding sequence (CDS) GFF file were used as input for Cufflinks2 to determine FPKM for each CDS and chromosome. Haploid copy number for each CDS was estimated by a proportion of their respective FPKM and the median FPKM of all CDS in the respective chromosome. We employed OrthoMCL [Dispersed and tandem gene duplications were identified using a combination of Bowtie2 and Cufflinks2 . BrieflyOrthoMCL  to identOrthoMCL . Gene duOrthoMCL .L. (V.) braziliensis genome using reads from the M2904 reference strain.We employed this same approach to detect expanded gene arrays in the


Staphylococcus aureus (MRSA) colonization among imprisoned populations. However, there are no data on that prevalence in Brazilian correctional institutions.Previous studies report high prevalence of Methicillin-resistant Staphylococcus aureus from February 2009 through April 2010. The overall isolation rate of S. aureus was 16.5% (50/302). Men who had sex with men, users of inhalatory drugs and those with previous lung or skin diseases were more likely to be colonized with S. aureus. MRSA was isolated from 0.7% of subjects (2/302). The two Community-associated (CA)-MRSA belonged to ST5 but were unrelated based on the PFGE results. Both harbored SCCmec IV, and did not possess the Panton-Valentine Leukocidin gene.We tested 302 male prisoners for nasopharyngeal colonization with S. aureus and CA-MRSA among prisoners. MRSA isolates ST5 from two subjects harboured SCCmec IV and presented different PFGE patterns.We found low prevalence of  Staphylococcus aureus emerged as relevant agents of community-associated infections in the past decades [S. aureus carriage among special populations.Methicillin-resistant  decades . Recent  decades -4. This Previous studies emphasize the relevance of MRSA among imprisoned populations . In someS. aureus (including MRSA) among male prisoners form a Brazilian correctional facility. We were especially interested in identifying colonization with CA-MRSA. For that purpose, we assumed the classical definition of CA-MRSA, implying no exposure to health care . Coherently, we excluded subjects who could harbour healthcare-associated (HA)-MRSA as a consequence of exposure to hospitals or other healthcare services. Our study also included testing for methicillin resistance with disk diffusion and molecular methods, alongside with identification of genes coding for the Panton-Valentine Leukocidin (PVL) and with molecular typing.Our study aimed to identify the prevalence of nasopharyngeal colonization with °S, 48.9°W) during the period from February 2009 through April 2010.The study had a cross-sectional design. It was carried out on the Avaré Center for Re-socialization, in Avaré City, Brazil  and ATCC 33591  were used as controls in all experiments. DNA isolation previous to Polymerase chain reaction (PCR) was performed using Illustra kit , according to the manufacturer’s instructions. PCR was performed for detection of the genes related to methicillin resistance (mecA) [mec (SCCmec) [SmaI restriction enzyme, Fermentas . International clones kindly provided by Dr. Antonio Carlos Campos Pignatari, Laboratório Especial de Microbiologia Clínica, Disciplina de Infectologia, Universidade Federal de São Paulo/Escola Paulista de Medicina, and Dr. Agnes Marie Sá Figueiredo, Universidade Federal do Rio de Janeiro, Instituto de Microbiologia Prof. Paulo de Góes, Brazil, were used as controls.This study was approved by the reference committee for ethics in research. Nasopharyngeal swabs were obtained from all study subjects. The swabs were manufactured with natural fibers not treated with chemical additives, whitening agents or bleach. The samples were transported in Stuart media . Cultures were performed in agar Baird-Parker . Species identification and disk diffusion susceptibility tests (with oxacillin 1 μg and cefoxitin 30 μg) followed standard procedures from the Clinical Laboratory Standards Institute (CLSI) . S. auree (mecA)  and to Pe (mecA) . Methici(SCCmec)  and to m(SCCmec)  and Mult(SCCmec) . ReactanS. aureus. Data were obtained though interviews with subjects and review of their medical charts. We recorded demographic information, as well as data on sexual orientation, habits, use of illicit drugs and comorbidities. Statistical analysis was performed in SPSS 19.0 . Data were initially submitted to univariate analysis. Dichotomous data were analyzed using the Chi-square or Fisher Exact test. For continuous variables we used the Student T or Mann-Whitney U test. Subsequently, data were included in multivariable models of logistic regression. A backward selection process for variables was applied, using p-values of 0.05 and 1.0 as requirements for entering and staying in the models, respectively. A final p-value of 0.05 was stated as the limit for statistical significance.We also analyzed risk factors for colonization with S. aureus and MRSA colonization were 16.5%  and 0.7% , respectively. Identification of the two MRSA isolates was confirmed by both phenotypic and genotypic methods. In the disk diffusion tests, methicillin resistance was confirmed according to CLSI breakpoints for both oxacillin and cefoxitin [mecA gene by the PCR assay. The two MRSA isolates harbored SCCmec type IV and belonged to ST5 , use of inhalatory illicit drugs  and recent lung  or skin  diseases  [Presently there are 494 thousand people living in correctional institutions in Brazil - the world’s 3.jus.br) . TherefoInterestingly, the prevalence of MRSA colonization in our study was similar to that reported in population-based surveys . Our finS. aureus and MRSA carriage among prisoners, when compared to results from a general population-based survey conducted in the same region of Brazil. In that study, 32.7% and 0.9% of persons carried S. aureus and MRSA, respectively [Even more surprising was the lower prevalence of First of all, one should notice that both the general population and the prison studies were conducted simultaneously in the same research laboratory, applying the same techniques and protocols. Thus, we can rule out methodological disparities. Other important detail is that a significant proportion of the carriers in the population-based survey were aged less than 18 years. That is the threshold for penal adulthood in Brazil, so all the prisoners belonged to higher age groups, though most were young adults.S. aureus and/or MRSA spread: overcrowding, poor sanitation and high prevalence of HIV infection and tuberculosis [S. aureus in our study. Sex with men, a common risk factor for infection with community-associated MRSA (CA-MRSA) [S. aureus colonization. It is worth noting that one of the two MRSA carriers reported frequent sexual intercourse with other men.There are many different kinds of correctional institutions in Brazil. Most of them present characteristics that are usually associated with greater likelihood of rculosis . In thatCA-MRSA) , was a pmec type IV. Both belonged to ST5, which seems to be widespread in Brazil [mec IV-harboring clones and PVL production, not a single S. aureus strain from our study tested positive for PVL-related genes. Since PVL is related to severe skin diseases [mec insertions into various methicillin-susceptible S. aureus strains in different regions of the world, not to the global spread of a single ST5-MRSA clone [The two MRSA strains, though unrelated on PFGE, harbored SCCn Brazil ,24. Thisdiseases , this fiSA clone . Even thTo date, outbreaks of CA-MRSA among Brazilian prisoners were not reported. Given the wide variation of sanitary condition and population among prisons in Brazil, we can only guess that prevalence of MRSA colonization and infection may be higher in other correctional facilities. Our study is a first step, one that should be followed by further research, enrolling several different institutions.The data set supporting the results of this article is included within the article.Staphylococcus aureus; CA-MRSA: Community-associated methicillin-resistant Staphylococcus aureus; ST: Sequence type; PFGE: Pulsed-field gel electrophoresis; PVL: Panton-valentine leukocidin; SCCmec: Staphylococcal chromosome cassette mec; CC: Clonal complex; HA-MRSA: Healthcare-associated Methicillin-resistant Staphylococcus aureus; PCR: Polymerase chain reaction; mecA: Methicillin resistance gene; MLST: Multilocus Sequence Typing; CLSI: Clinical laboratory standards institute.MRSA: Methicillin-resistant All authors declare they have no competing interests regarding the present study.CLW: participated in the conception and design of the study, carried out the microbiological tests, and participated in the writing of the paper. CMCBF: participated in the conception and design of the study, analyzed the clinical data, performed the statistical analysis and wrote the paper. CSMS: helped with the microbiological tests and performed the MLST. DFMR: performed the PFGE and analyzed the data. MLRSC: responsible for the conception and design of the study, coordination of laboratory work, data analysis and manuscript writing. All authors read and approved the final manuscript.


Identifying trace amounts of bacteria rapidly, accurately, selectively, and with high sensitivity is important to ensuring the safety of food and diagnosing infectious bacterial diseases. Microbial diseases constitute the major cause of death in many developing and developed countries of the world. The early detection of pathogenic bacteria is crucial in preventing, treating, and containing the spread of infections, and there is an urgent requirement for sensitive, specific, and accurate diagnostic tests. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is an extremely selective and sensitive analytical tool that can be used to characterize different species of pathogenic bacteria. Various functionalized or unmodified nanomaterials can be used as affinity probes to capture and concentrate microorganisms. Recent developments in bacterial detection using nanomaterials-assisted MALDI-MS approaches are highlighted in this article. A comprehensive table listing MALDI-MS approaches for identifying pathogenic bacteria, categorized by the nanomaterials used, is provided. AccordiThere are challenges associated with identifying various types of pathogenic bacteria in a wide range of samples. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has been used to analyze various biomolecules, including peptides, proteins, DNA, RNA, oligonucleotides, oligosaccharides, and polymers –16. This4 cells per well) are required to generate detectable MALDI-MS ion signals. However, samples obtained from infectious biological fluids or food poisoning samples are difficult to characterize directly by MALDI-MS because the ions generated from the bacterial cells may be seriously suppressed by the complex sample matrices. This led to the idea of using nanoparticles as affinity probes, to enhance the ability of MALDI-MS to detect bacteria [2) nanoparticles [A sufficient number of bacterial cells . In 2010, a protocol for direct analysis of blood was introduced by Stevenson et al. [et al. [3–108 cfu. Sepsityper™ Kit  was used to for the testing milk sample and then analyzed by the Bruker BioTyper database. For a slightly contaminated (104 cfu/mL bacteria) milk sample, bacterial identification could be performed after initial incubation at 37 °C for 4 h. The detection limits for bacteria were in the range of 106–107 cfu/mL.MALDI-MS is a very sensitive method where a single bacteria colony is sufficient for analysis, while other methods typically require culturing or enrichment of bacteria to obtain sufficient materials. Therefore, in clinical microbiological laboratories, the MALDI-MS is increasingly used for bacterial identification through the determination of the exact molecular masses of numerous peptides and small proteins, many of which are ribosomal. Conventional biochemical differentiation methods  have alret al.  introducn et al. , who sep [et al.  inoculat3.Nanoparticles are clusters of a few hundred to a few thousand atoms, and range from 1 to 100 nm in diameter. The chemical and physical properties of nanoparticles depend on their surfaces; therefore, these properties are highly dependent on the sizes, shapes, and compositions of the nanoparticles –58. Nano3.1.et al. [3O4 nanoparticles through covalent bonding (Staphylococcus aureus (S. aureus) and Staphylococcus saprophyticus (S. saprophyticus), from sample solutions. The bacteria were then characterized using MALDI-MS. The lowest bacterial concentration detected in an aqueous sample solution (0.5 mL) was 3 × 105 cfu/mL, for both S. aureus and S. saprophyticus, and the lowest detectable S. saprophyticus concentration in a urine sample was 3 × 107 cfu/mL.Ho et al.  immobili bonding . The fun3O4) nanoparticles were used as affinity probes to selectively bind to the surface walls of Gram-positive bacteria (S. aureus and S. saprophyticus), as shown in D-Alanine (Ala) (D-Ala) moieties on the cell walls of Gram-positive bacteria. The lowest cell concentrations that could be detected in a urine sample (3 mL) were 7.4 × 104 cfu/mL for S. aureus and 7.8 × 104 cfu/mL for S. saprophyticus.Vancomycin-modified 11 nm magnetic  15–20 nm nanoparticles has been used to selectively concentrate Gram-positive bacteria from tap water and reservoir water, allowing the bacteria to be rapidly analyzed using whole-cell MALDI-MS [4, giving a limit of detection of 5 × 102 cfu/mL for Bacillus cereus (B. cereus), Enterococcus faecium (E. faecium), and S. aureus in water samples (2 L).A combination of membrane filtration and vancomycin-modified magnetic (FeMALDI-MS . The cap3.2.Escherichia coli (E. coli) and Serratia marcescens (S. marcescens), by Gopal et al. [E. coli and 0.5 mL/L in the case of S. marcescens. Ag nanoparticle concentrations higher than these values showed pronounced bactericidal activities.The bifunctional properties of Ag nanoparticles allowed them to be used as affinity probes for l et al. , to incr42−) and 0.035 mg of Ag nanoparticles could be used to capture yogurt bacteria (Bifidobacterium lactis (B. lactis), Lactobacillus acidophilus (L. acidophilus), Streptococcus thermophilus (S. thermophilus), and Lactobacillus bulgaricus (L. bulgaricus) from AB yogurt and L. acidophilus, Bifidobacterium longum (B. longum), L. bulgaricus, and S. thermophilus from Lin yogurt), improving the sensitivity achieved for detecting bacteria in yogurt samples [The same research group also observed that an ionic solution  peaks using an incubation time of up to 3 h. The authors confirmed that CdS QDs can function as more than just affinity probes, being able to degrade EPSs. CdS QDs can, therefore, be used to inactivate pathogenic E. coli and also inhibit the growth of E. coli biofilms.Gopal et al.  has repoSaccharomyces cerevisiae (S. cerevisiae) and Candida utilis (C. utilis). From their MALDI-MS results, the authors proposed the mechanism involving the CdS QDs interacting with the EPSs and removing small molecules from the EPS layers. The MALDI-MS protein signals were enhanced at all of the CdS QD concentrations that were tested (10–30 mg).Manikandan and Wu  observedPseudomonas aeruginosa (P. aeruginosa) and S. aureus cells were detected at low concentrations, 200 and 150 cfu/mL, respectively, after an extremely short time (1 min). MALDI-MS and transmission electron microscopy were used to confirm the interactions and the biocompatibility of the chitosan-modified CdS QDs with bacterial cells.Chitosan-modified CdS QDs have been used as effective bacterial biosensors because of the strong affinities between chitosan molecules and bacterial membranes . In that3.4.E. coli and S. marcescens at concentrations as low as 106 cfu/mL.Ahmad and Wu  employedet al. [Bacillus thuringiensis (B. thuringiensis) and Bacillus subtilis (B. subtilis) isolated from rhizospheric soil and carrot plant roots. This study proved that bacteria can be directly detected even at low concentrations.A rapid method for detecting bacteria associated with plants by an on-particle ionization and enrichment approach using IgG-functionalized Pt nanoparticle-assisted MALDI-MS was reported by Ahmad et al. . The appS. marcescens and E. coli. This approach enabled the rapid detection of bacterial proteins, at a high resolution and with good sensitivity, without the need for tedious washing and separation procedures, and can be used to detect approximately 105 cfu/mL of S. marcescens and E. coli.A rapid and sensitive approach to studying interactions between an affinity probe and a bacterial wall was introduced by Ahmad and Wu . IgG was3.5.et al. [E. coli, Klebsiella pneumonia (K. pneumoniae), P. aeruginosa, pandrug-resistant Acinetobacter baumannii (A. baumannii), S. aureus, E. faecalis, and vancomycin-resistant E. faecalis, can be concentrated by lysozyme-encapsulated gold nanoclusters (AuNCs) that photoluminesce red, and distinguished by the results combining MALDI-MS and principal component analysis. E. coli J96 in urine samples containing different concentrations of the E. coli. Photographs of the control experiment sample tubes are shown in E. coli J96, in which the peaks at m/z 6177 and 6237 correspond to E. coli J96. The lowest E. coli concentration that could be detected using this approach was approximately 106 cfu/mL. The advantages of this method include speed (without cell culturing) and simplicity, and it can be used as universal affinity probes for Gram-positive/negative and antibiotic-resistant bacteria.Chan et al.  demonstrP. aeruginosa and S. aureus). The GMCS were observed to act as efficient separation and preconcentration nanoprobes for SALDI and enhance the ionization of bacterial biomolecules. GMCS have been used in the direct detection of low concentrations of P. aeruginosa and S. aureus in blood samples, demonstrating their practical applicability. This approach offers many advantages such as robustness, simplicity, and the capability for fluorescence based real-sample monitoring.Abdelhamid and Wu  demonstrE. coli (at 107 cfu/mL) at different temperatures has been studied using nickel oxide (NiO) nanoparticle-assisted MALDI-MS by Hasan et al. [E. coli under heat stress at temperatures between 40 and 80 °C in the absence or presence of NiO nanoparticles. Dramatic decreases in the viability of E. coli in the presence of NiO were confirmed from the MALDI-MS results. This technique is a rapid, sensitive, and efficient approach for bacterial detection under extremely harsh conditions.The heat stress response of n et al. . MALDI-Met al. [S. aureus isolated from the human nasal passage can be directly detected using MALDI-MS assisted by TiO2 nanoparticles, without any culturing steps or sample pretreatment being required. TiO2 nanoparticles were used to enhance the bacterial signals in the direct MALDI-MS analysis.Gopal et al.  demonstrE. coli cells by zinc oxide (ZnO) nanoparticles [MALDI-MS has been used to evaluate bactericidal activity, by detecting proteins produced because of the inactivation of articles . The reset al. [2, and ZnO) and two bacteria (S. aureus and P. aeruginosa) from studies using transmission electron microscopy, ultra spectrometry, and MALDI-MS. Two mechanisms  as affinity probes. The nanomaterials described here act as concentration probes for the selective capture of unique biomarkers from microorganisms, and as surfaces to absorb energy from the laser irradiation, thereby inducing desorption and ionization of the analytes.2 and ZnO, also exhibit bactericidal activity, and therefore might not be good affinity probes at higher nanoparticles concentrations. Controlling of the nanoparticle concentration will be a key factor. All these nanomaterials-assisted MALDI-MS methods also encounter challenges with respect to the enrichment of unknown target bacterial species from the urine, blood, and cerebrospinal fluid. Thus, a limiting factor in MALDI-MS analysis is insufficient database entries. The addition of certain species to the database has been demonstrate significantly improve MALDI-MS precision in bacterial identification.As mentioned above, the most important advantage of the affinity-based nanoparobe methods is their ability to selectively concentrate and purify microorganisms from complex samples, such as urine and blood, and allow the further identification of microorganisms without microbial culturing using MALDI-MS. For the nanomaterials-assisted MALDI-MS, Direct analysis of microorganisms at low microbial levels can be performed using the nanomaterials-based MALDI-MS. Numerous nanomaterials have been demonstrated to be useful as affinity probes for targeting bacteria. However, some nanoparticles such as Ag, TiOThe broad adoption of nanomaterials-assisted MALDI-MS methods for bacterial identification will require a substantial improvement in performance compared with the existing methods, such as conventional MALDI-MS and biochemical tests. Thus, the standardization of terminology is required. Advances in nanomaterials-assisted MALDI-MS methods will support the simple and accurate means of bacterial identification for food safety, environmental monitoring and clinical diagnosis.


Australes (genus Pinus). Support for phylogenies reconstructed based on all data was generally weak and inconsistent among methods. Subsequent analysis revealed that certain topologies were supported by genes with common putative functionalities. Therefore, each set of genes was analysed independently. Recovered alternative topologies were highly supported and the results were consistent among methods. Multiple evolutionary hypotheses could potentially explain the observed patterns, although incomplete lineage sorting seems to be the simplest one.Eleven nuclear genes and multiple phylogenetic techniques were used to study relationships among four of North American pines from subsection  Pinus (pines) is one of the most abundant and most studied groups among gymnosperms, and a good example of a lineage where these phenomena have been observed. Pines are among the most ecologically and economically important plant species. Some, such as the pines of the southeastern USA , are subjects of intensive breeding programmes. Despite numerous published studies, the evolutionary history of Australes remains ambiguous and often controversial. We studied the phylogeny of four major southern pine species: shortleaf (Pinus echinata), slash (P. elliottii), longleaf  and loblolly (P. taeda), using sequences from 11 nuclear loci and maximum likelihood and Bayesian methods. Our analysis encountered resolution difficulties similar to earlier published studies. Although incomplete lineage sorting and introgression are two phenomena presumptively underlying our results, the phylogenetic inferences seem to be also influenced by the genes examined, with certain topologies supported by sets of genes sharing common putative functionalities. For example, genes involved in wood formation supported the clade echinata–taeda, genes linked to plant defence supported the clade echinata–elliottii and genes linked to water management properties supported the clade echinata–palustris. The support for these clades was very high and consistent across methods. We discuss the potential factors that could underlie these observations, including incomplete lineage sorting, hybridization and parallel or adaptive evolution. Our results likely reflect the relatively short evolutionary history of the subsection that is thought to have begun during the middle Miocene and has been influenced by climate fluctuations.Adaptive evolutionary processes in plants may be accompanied by episodes of introgression, parallel evolution and incomplete lineage sorting that pose challenges in untangling species evolutionary history. Genus  Plant genome structure and evolution are subjects of intensive investigations with recent milestone advances spanning whole-genome sequencing of some of the largest plant genomes, such as those of pines and spruces  comprise a group of coniferous tree species occurring almost exclusively in the Northern Hemisphere, with populations often dominant across areas of the North Temperate Zone , slash (P. elliottii), longleaf  and loblolly (P. taeda) . They are widely cultivated, greatly dominating the southern US forest inventory (P. elliottii), spruce (P. glabra), longleaf , pond (P. serotina) and loblolly (P. taeda) pines in the southeastern USA; shortleaf (P. echinata), Table Mountain (P. pungens) and pitch (P. rigida) pines in the eastern USA; Cuban pine (P. cubensis) in Cuba; West Indian pine  in the West Indies; and Caribbean pine (P. caribaea) in both the West Indies and adjacent Central America. Attempts to refine the phylogeny of Australes are ongoing, but typically have been approached in the broader context of the Pinaceae family or along with other subsections.Four major pines of the southeastern USA were investigated in this study: shortleaf  markers, P. echinata, P. elliottii and P. palustris within the same clade, where P. elliottii and P. palustris were sisters, and P. taeda was in a separate clade, albeit with low bootstrap support (BS). Consequently, the monophyly of the four species was placed under question.Several previous investigations have provided insights to the phylogenetic relationships of the four species on which we focus in our study   taxa that could be used as appropriate outgroup species. Eleven putative orthologous genes were identified in P. pinaster   and mergSupporting Information—Table S1][see . We, therefore, clustered genes into three groups following similarity in topologies. The genes 4cl, c4h-2 and cesA3 (Group A) supported the clade echinata–taeda, the genes agp-4 and sod-chl (Group B) supported the clade echinata–elliottii and the genes dhn-2 and erd3 (Group C) supported the clade echinata–palustris. Group C genes disagreed in the placement of the species P. elliottii and P. taeda; however, we decided to focus on the similarity. Additional analysis in the R environment (ver. 2.15.2)  1.00 and the level of support for these clades became consistent between GARLI and MrBayes  and BI (MrBayes). The two approaches produced consistent results for each gene, although the difference in clade support varied among the genes  2.15.2)  supporte 2.15.2)  placed techinata−taeda, supported by Group A genes, was previously recovered by palustris–taeda, supported by the gene agp-4 (Group B), were placed in one clade by echinata–palustris and elliottii–taeda supported by the Group C genes corresponded to grouping in The discordance among various approaches applied in this study mirrors the conflicting results from previously published work (see Introduction). Comparison of the topologies supported by Group A vs. Group B vs. Group C genes shows highly consistent results within each group across different methods, yet the conclusions contradict each other among the groups. The clade Australes. Interestingly, however, the genes forming the three groups are also connected at the putative functional level  by agp-4 and sod-chl) appear to contribute to plant defence, water management and other functions. Proteins from the arabinogalactan family play signalling and protective roles, and participate in xylem development, cell growth and expansion, programmed cell death and other processes (Sod-chl has antioxidative properties (Dhn-2 and erd3 (Group C) are also associated with water stress recognition and response. Dhn-2, a dehydrin, is responsive to dehydration stress and also plays other protective functions , long-distance gene flow through pollen, hybridization events that still occur today and large (but variable in the past) population sizes of the investigated species, incomplete lineage sorting could be the simplest and most straightforward explanation of the pattern observed in our study. It is a known phenomenon in pines , although the split between the ancestors of Ponderosae and Australes might have happened as early as 15 MYA  Cooperative State Research, Education, and Extension Service (CSREES) and Texas Agricultural Experiment Station (TAES) McIntire-Stennis Project (TEX09122-0210381). The Pine Integrated Network: Education, Mitigation, and Adaptation Project (PINEMAP), a Coordinated Agricultural Project funded by the USDA National Institute of Food and Agriculture, Award #2011-68002-30185, provided support during preparation of this manuscript.The project was supported by the T.E.K., M.M. and K.V.K. conceived and designed the study. T.E.K. arranged the data and ran the software. T.E.K., M.M. and K.V.K. interpreted results. T.E.K. drafted the manuscript. T.E.K., M.M. and K.V.K. edited, read and approved the final version of the manuscript.None declared.The following additional information is available in the online version of this article —Dataset S1. Multiple alignment in NEXUS format.Figure S1. Separate analysis of each gene for the four Australes species: P. echinata, P. elliottii, P. palustris and P. taeda. Cladograms are shown. From top to bottom: 4cl, c4h-2 and cesA3 (A); agp-4 and sod-chl (B); dhn-2 and erd3 (C); cad, glyhmt, pp2c and comt-2 (D). Numbers at nodes correspond to clade support: GARLI  and MrBayes .Figure S2. Principal coordinate analysis run on Robinson–Foulds distance matrix for the individual gene trees. Group A gene names  are in green, Group B gene names (agp-4 and sod-chl) are in orange and Group C gene names (dhn-2 and erd3) are in blue.Figure S3. Joint analysis of the genes from Group A , Group B  and Group C  in P. echinata, P. elliottii, P. palustris and P. taeda using GARLI and MrBayes. From top to bottom: GARLI (AIC), GARLI (BIC), MrBayes (AIC) and MrBayes (BIC). Numbers at nodes correspond to clade support.Figure S4. Joint analysis of the genes from Group A  and comt-2 in P. echinata, P. elliottii, P. palustris and P. taeda using GARLI and MrBayes. From top to bottom: GARLI (AIC), GARLI (BIC), MrBayes (AIC) and MrBayes (BIC). Numbers at nodes correspond to clade support.Table S1. Partitioning schemes and software settings.Table S2. Linkage information for the studied genes.


AbstractAfrotremex Pasteels, 1951 is a rare genus of wasps endemic to the Afrotropical region, containing 6 species represented by 14 specimens. Specimens were previously only recorded from central Africa: Cameroon, Congo, Democratic Republic of Congo, Gabon and Uganda.A.xylophagus Goulet, 2014 collected in Ghana (previously Gold Coast). This record extends the known distribution of the genus into west Africa, and represents the second known male specimen for the genus. The other BMNH specimen is a female paratype of A.violaceus Pasteels, 1951 collected in the Democratic Republic of Congo. We provide high quality photographs of these additional two specimens. Images of all six known species are openly available online on WaspWeb. In addition we have developed interactive online Lucid Matrix and Lucid Phoenix identification keys to the species, which are openly available on WaspWeb at: http://www.waspweb.org/Siricoidea/Siricidae/Keys/index.htmHere we record two additional specimens housed in the Natural History Museum in London (BMNH), one of which is a male of  AfrotremexPasteels 1951 is the only indigenous genus representative of Siricidae in the Afrotropical region ; Musée Royal de l’Afrique Centrale, Tervuren, Belgium (MRAC); National Museum of Natural History, Smithsonian Institution, Washington, DC, USA (USNM); Museum für Naturkunde der Humboldt Universität, Berlin, Germany (ZMHB)  . This isy (ZMHB) . Subsequ WaspWeb . In addiImages were acquired using the Leica LAS 4.4 multi-stacking imaging system. The Leica system comprised a Leica® Z16 microscope and a Leica DFC450 Camera. Leica Application Suite V 4.4 software was used to manage image acquisition using an automated Z-stepper and merging of the image series into a single in-focus image. Images were post-processed in Adobe Photoshop and plates produced using Adobe illustrator. Specimens were imaged using the EntoVision multiple-focus imaging system to illustrate diagnostic characters.Online interactive keys were produced using Lucid 3.5 Builder and Lucid Phoenix Builder meeting the requirements of publishing both static and dynamic interactive keys under an open access model . CharactBMNH: The Natural History Museum, London. Curators David Notton & Gavin Broad.Afrotremex was published in http://www.waspweb.org/Siricoidea/Siricidae/Afrotremex/Afrotremex_hyalinatus.htmCongo; GabonGoulet, 2014http://www.waspweb.org/Siricoidea/Siricidae/Afrotremex/Afrotremex_opacus.htmDemocratic Republic of CongoGoulet, 2014http://www.waspweb.org/Siricoidea/Siricidae/Afrotremex/Afrotremex_pallipennis.htmDemocratic Republic of CongoPasteels, 1951http://www.waspweb.org/Siricoidea/Siricidae/Afrotremex/Afrotremex_violaceus.htmDemocratic Republic of CongoGoulet, 2014http://www.waspweb.org/Siricoidea/Siricidae/Afrotremex/Afrotremex_xylophagus.htmCameroon, GhanaA.xylophagus Goulet, 2014 collected in Ghana (previously Gold Coast). This record extends the known distribution of the genus and this species into west Africa. This is only the second male specimen known for the genus. The other BMNH specimen is a female paratype of A.violaceus Pasteels, 1951 from the Democratic Republic of Congo. Both specimens are damaged with missing wings, legs and antenna. The head and pronotum of both specimens have, at some point, been glued back onto the rest of the body.Of the two additional specimens located in the collections of the Natural History Museum in London (BMNH), one is a male of Afrotremex species is poorly known. The two ZMBH specimens of A.xylophagus were reared from a common widespread central African tree species, Antrocaryonklaineanum Pierre (Anacardiaceae), and since species of the related genera Tremex and Eriotremex are recorded as utilizing a range of angiosperm trees as hosts (Afrotremex are not necessarily highly host specific either (The life history and biology of as hosts  it is poc either . The genc either , has not